Biorefineries - Industrial Processes and Products: Status Quo and Future Directions

Biorefineries – Industrial Processes and Products Edited by Birgit Kamm, Patrick R. Gruber, and Michael Kamm Biorefine...

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Biorefineries – Industrial Processes and Products Edited by Birgit Kamm, Patrick R. Gruber, and Michael Kamm

Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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Biorefineries – Industrial Processes and Products Status Quo and Future Directions Volume 1

Edited by Birgit Kamm, Patrick R. Gruber, and Michael Kamm

The Editors Dr. Birgit Kamm Research Institute Bioactive Polymer Systems biopos e.V. Kantstr. 55 14513 Teltow Germany Dr. Patrick R. Gruber President and CEO Outlast Technologies Inc. 5480 Valmont Road Boulder, CO 80301 USA Michael Kamm Biorefinery.de GmbH Stiftstr. 2 14471 Potsdam Germany

n All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library. Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Typsetting K+V Fotosatz GmbH, Beerfelden Printing Betz-Druck GmbH, Darmstadt Binding Litges & Dopf Buchbinderei GmbH, Heppenheim Printed in the Federal Republic of Germany Printed on acid-free paper ISBN-13: 978-3-527-31027-2 ISBN-10: 3-527-31027-4

V

Contents Editors’s Preface

XXI

Foreword XXIII Henning Hopf Foreword XXV Paul T. Anastas List of Contributors

XXVII

Volume 1 Part I

Background and Outline – Principles and Fundamentals

1

Biorefinery Systems – An Overview 3 Birgit Kamm, Michael Kamm, Patrick R. Gruber, and Stefan Kromus Introduction 3 Historical Outline 4 Historical Technological Outline and Industrial Resources 4 The Beginning – A Digest 5 Sugar Production 5 Starch Hydrolysis 5 Wood Saccharification 5 Furfural 6 Cellulose and Pulp 6 Levulinic Acid 6 Lipids 7 Vanillin from Lignin 7 Lactic Acid 7 The Origins of Integrated Biobased Production 8 Situation 11

1.1 1.2 1.2.1 1.2.2 1.2.2.1 1.2.2.2 1.2.2.3 1.2.2.4 1.2.2.5 1.2.2.6 1.2.2.7 1.2.2.8 1.2.2.9 1.2.3 1.3


VI

Contents

1.3.1 1.3.2 1.3.3 1.3.4 1.4 1.4.1 1.4.2 1.4.3 1.4.3.1 1.4.4 1.5 1.5.1 1.5.2 1.5.3 1.5.4 1.5.5 1.6

2

2.1 2.2 2.2.1 2.2.2 2.2.3 2.3 2.3.1 2.3.2 2.3.3 2.4 2.4.1 2.4.2 2.4.3 2.5 2.5.1

Some Current Aspects of Biorefinery Research and Development 11 Raw Material Biomass 12 National Vision and Goals and Plan for Biomass Technology in the United States 14 Vision and Goals and Plan for Biomass Technology in the European Union and Germany 15 Principles of Biorefineries 16 Fundamentals 16 Definition of the Term “Biorefinery” 19 The Role of Biotechnology 20 Guidelines of Fermentation Section within Glucose-product Family Tree 21 Building Blocks, Chemicals and Potential Screening 22 Biorefinery Systems and Design 23 Introduction 23 Lignocellulosic Feedstock Biorefinery 24 Whole-crop Biorefinery 26 Green Biorefinery 29 Two-platform Concept and Syngas 31 Outlook and Perspectives 32 References 33 Biomass Refining Global Impact – The Biobased Economy of the 21st Century 41 Bruce E. Dale and Seungdo Kim Introduction 41 Historical Outline 42 Background and Development of the Fossil Carbon-processing Industries 42 The Existing Biobased Economy: Renewable Carbon 43 Toward a Much Larger Biobased Economy 44 Supplying the Biorefinery 45 What Raw Materials do Biorefineries Require and What Products Can They Make? 45 Comparing Biomass Feedstock Costs With Petroleum Costs 48 How Much Biomass Feedstock Can be Provided at What Cost? 50 How Will Biorefineries Develop Technologically? 53 Product Yield: The Dominant Technoeconomic Factor 53 Product Diversification: Using the Whole Barrel of Biomass 54 Process Development and a Technical Prerequisite for Cellulosic Biorefineries 55 Sustainability of Integrated Biorefining Systems 56 Integrated Biorefining Systems: “All Biomass is Local” 56

Contents

2.5.2 2.5.3 2.6

3

3.1 3.2 3.3 3.3.1 3.3.2 3.3.3 3.4 3.4.1 3.4.1.1 3.4.1.2 3.4.2 3.4.2.1 3.4.2.2 3.4.2.3 3.5

4

4.1 4.2 4.2.1 4.2.2 4.2.3 4.2.3.1 4.2.3.2 4.2.3.3 4.2.4 4.2.4.1 4.2.4.2 4.2.4.3

Agricultural/Forestry Ecosystem Modeling: New Tools for an Age of Sustainability 57 Analyzing the Sustainability of Integrated Biorefining Systems: Some Results 60 Conclusions 64 Acknowledgements 65 References 65 Development of Biorefineries – Technical and Economic Considerations 67 Bill Dean, Tim Dodge, Fernando Valle, and Gopal Chotani Introduction 67 Overview: The Biorefinery Model 68 Feedstock and Conversion to Fermentable Sugar 68 Sucrose 70 Starch 70 Cellulose 71 Technical Challenges 74 Cellulase Enzymes 74 Improved Cellulase Production Economics 74 Improved Cellulase Enzyme Performance 76 Fermentation Organisms 77 Biomass Hydrolyzate as Fermentable Carbon Source 78 Production Process as a Whole 79 Emerging Solutions 80 Conclusions 81 Acknowledgments 82 References 82 Biorefineries for the Chemical Industry – A Dutch Point of View 85 Ed de Jong, René van Ree Rea, Robert van Tuil, and Wolter Elbersen Introduction 85 Historical Outline – The Chemical Industry: Current Situation and Perspectives 86 Overview of Products and Markets 86 Technological Pathways 87 Biomass-based Industrial Products 87 Carbohydrates 89 Fatty Acids 90 Other 91 International Perspectives 92 Production 92 Integration 92 Use and Re-use 93

VII

VIII

Contents

4.3 4.3.1 4.3.2 4.4 4.4.1 4.4.2 4.4.3 4.4.4 4.4.5 4.4.6 4.4.6.1 4.4.6.2 4.4.6.3 4.5 4.5.1 4.5.2 4.5.3 4.5.4 4.5.5

Part II

Biomass: Technology and Sustainability 93 Transition to a Bio-based Industry: Sectoral Integration in the Netherlands 93 Can Sustainability Drive Technology? 96 The Chemical Industry: Biomass Opportunities – Biorefineries 97 Biomass Opportunities 97 Biorefinery Concept 98 Biomass Availability 100 Primary Refinery 101 Secondary Thermochemical Refinery 102 Secondary Biochemical Refinery – Fermentative Processes 104 Feedstocks 105 Product Spectrum 105 Side Streams and Recycling 106 Conclusions, Outlook, and Perspectives 106 Biomass – Sustainability 106 Biomass Refining and Pretreatment 107 Conversion Technology 108 Chemicals and Materials Design 108 Dutch Energy Research Strategy (“EOS”) 109 References 109 Biorefinery Systems Lignocellulose Feedstock Biorefinery

5

5.1 5.2 5.2.1 5.2.2 5.2.3 5.2.4 5.3 5.3.1 5.3.2 5.3.3 5.3.4 5.3.5 5.3.6 5.4 5.5

The Lignocellulosic Biorefinery – A Strategy for Returning to a Sustainable Source of Fuels and Industrial Organic Chemicals 115 L. Davis Clements and Donald L. Van Dyne The Situation 115 The Strategy 115 A Strategy Within a Strategy 116 Environmental Benefits 117 The Business Structure 117 Cost Estimates 118 Comparison of Petroleum and Biomass Chemistry 118 Petroleum Resources 118 Biomass Resources 119 Saccharides and Polysaccharides 121 Lignin 121 Triacylglycerides (or Triglycerides) 121 Proteins 122 The Chemistry of the Lignocellulosic Biorefinery 122 Examples of Integrated Biorefinery Applications 125

Contents

5.5.1 5.5.2 5.5.3 5.6

6

6.1 6.2 6.2.1 6.2.2 6.3 6.3.1 6.3.2 6.4 6.4.1 6.4.2 6.4.3 6.5

7

7.1 7.2 7.2.1 7.2.2 7.3 7.3.1 7.3.2 7.3.3 7.3.3.1 7.3.3.2 7.3.3.3 7.3.3.4 7.3.3.5 7.3.3.6 7.3.3.7 7.3.4 7.3.5

Production of Ethanol and Furfural from Lignocellulosic Feedstocks 125 Management of Municipal Solid Waste 125 Coupling MSW Management, Ethanol, and Biodiesel 126 Summary 127 References 127 Lignocellulosic Feedstock Biorefinery: History and Plant Development for Biomass Hydrolysis Raphael Katzen and Daniel J. Schell Introduction 129 Hydrolysis of Biomass Materials 129 Acid Conversion 129 Enzymatic Conversion 130 Acid Hydrolysis Processes 130 Early Efforts to Produce Ethanol 130 Other Products 133 Enzymatic Hydrolysis Process 134 Early History 134 Enzyme-Based Plant Development 134 Technology Development 135 Conclusion 136 References 136

129

The Biofine Process – Production of Levulinic Acid, Furfural, and Formic Acid from Lignocellulosic Feedstocks 139 Daniel J. Hayes, Steve Fitzpatrick, Michael H. B. Hayes, and Julian R. H. Ross Introduction 139 Lignocellulosic Fractionation 139 Acid Hydrolysis of Polysaccharides 141 Production of Levulinic Acid, Formic Acid and Furfural 142 The Biofine Process 144 Yields and Efficiencies of the Biofine Process 145 Advantages over Conventional Lignocellulosic Technology 146 Products of The Biofine Process 147 Diphenolic Acid 148 Succinic Acid and Derivatives 149 Delta-aminolevulinic Acid 149 Methyltetrahydrofuran 150 Ethyl Levulinate 152 Formic Acid 153 Furfural 154 Biofine Char 155 Economics of The Biofine Process 158

IX

X

Contents

7.4

Conclusion 161 References 162 Whole Crop Biorefinery

8

8.1 8.2 8.2.1 8.2.2 8.2.3 8.2.3.1 8.2.3.2 8.2.4 8.2.4.1 8.2.4.2 8.3 8.3.1 8.3.2 8.3.2.1 8.4

A Whole Crop Biorefinery System: A Closed System for the Manufacture of Non-food Products from Cereals 165 Apostolis A. Koutinas, Rouhang Wang, Grant M. Campbell, and Colin Webb Intro 165 Biorefineries Based on Wheat 167 Wheat Structure and Composition 167 Secondary Processing of Wheat Flour Milling Byproducts 169 Advanced Wheat Separation Processes for Food and Non-food Applications 173 Pearling as an Advanced Cereal Fractionation Technology 173 Air Classification 176 Biorefinery Based on Novel Dry Fractionation Processes of Wheat 176 Potential Value-added Byproducts from Wheat Bran-rich Fractions 178 Exploitation of the Pearled Wheat Kernel 180 A Biorefinery Based on Oats 183 Oat Structure and Composition 183 Layout of a Potential Oat-based Fractionation Process 183 Potential Value-added Byproducts from Oat Bran-rich Fractions 185 Summary 187 References 187 Fuel-oriented Biorefineries

9

9.1 9.2 9.3 9.3.1 9.3.2 9.3.3 9.3.4 9.4 9.4.1 9.4.2 9.4.3

Iogen’s Demonstration Process for Producing Ethanol from Cellulosic Biomass 193 Jeffrey S. Tolan Introduction 193 Process Overview 193 Feedstock Selection 194 Feedstock Composition 194 Feedstock Selection 196 Ethanol from Starch or Sucrose 197 Advantages of Making Ethanol from Cellulosic Biomass 197 Pretreatment 198 Process 198 Chemical Reactions 198 Other Pretreatment Processes 199

Contents

9.5 9.5.1 9.5.2 9.5.3 9.6 9.6.1 9.6.2 9.6.3 9.7 9.7.1 9.7.2 9.8

Cellulase Enzyme Production 201 Production of Cellulase Enzymes 201 Enzyme Production on the Ethanol Plant Site 202 Commercial Status of Cellulase 202 Cellulose Hydrolysis 202 Process Description 202 Kinetics of Cellulose Hydrolysis 203 Improvements in Enzymatic Hydrolysis 205 Lignin Processing 205 Process Description 205 Alternative Uses for Lignin 206 Sugar Fermentation and Ethanol Recovery 206 References 207

10

Sugar-based Biorefinery – Technology for Integrated Production of Poly(3-hydroxybutyrate), Sugar, and Ethanol 209 Carlos Eduardo Vaz Rossell, Paulo E. Mantelatto, José A. M. Agnelli, and Jefter Nascimento Introduction 209 Sugar Cane Agro Industry in Brazil – Historical Outline 209 Sugar and Ethanol Production 209 The Sugar Cane Agroindustry and the Green Cycle 210 Biodegradable Plastics from Sugar Cane 212 Poly(3-Hydroxybutyric Acid) 212 Biodegradable Plastics and the Environment 212 General Aspects of Biodegradability 213 Poly(3-Hydroxybutyric Acid) Polymer 214 General Characteristics of Poly(3-hydroxybutyric Acid) and its Copolymer Poly(3-hydroxybutyric Acid-co-3hydroxyvaleric Acid) 214 Processing of Poly(Hydroxybutyrates) 215 Poly(3-Hydroxybutyric Acid) Production Process 217 Sugar Fermentation to Poly(3-Hydroxybutyric Acid) by Ralstonia eutropha 217 Downstream Processing for Recovery and Purification of Intracellular Poly(3-Hydroxybutyric Acid) 218 Processes for Extraction and Purification of Poly(hydroxyalkanoates) 218 Chemical Digestion 218 Enzymatic Digestion 219 Solvent Extraction 219 Integration of Poly(3-Hydroxybutyric Acid) Production in a Sugar Mill 221

10.1 10.2 10.2.1 10.2.2 10.3 10.3.1 10.3.1.1 10.3.1.2 10.3.2 10.3.2.1

10.3.2.2 10.4 10.4.1 10.4.2 10.4.2.1 10.4.2.2 10.4.2.3 10.4.2.4 10.4.3

XI

XII

Contents

10.4.4 10.5

Investment and Production Cost of Poly(3-Hydroxybutyric Acid) in a Sugar Mill 222 Outlook and Perspectives 223 References 225 Biorefineries Based on Thermochemical Processing

11

11.1 11.2 11.2.1 11.2.2 11.3 11.3.1 11.3.2 11.3.2.1 11.3.2.2 11.3.2.3 11.3.3 11.3.4 11.4 11.4.1 11.4.2 11.4.3 11.4.4 11.5

Biomass Refineries Based on Hybrid Thermochemical-Biological Processing – An Overview 227 Robert C. Brown Introduction 227 Historical Outline 228 Origins of Biorefineries Based on Syngas Fermentation 228 Origins of Biorefineries Based on Fermentation of Bio-oils 229 Gasification-Based Systems 230 Fundamentals of Gasification 230 Fermentation of Syngas 233 Production of Organic Acids 234 Production of Alcohols 235 Production of Polyesters 236 Biorefinery Based on Syngas Fermentation 239 Enabling Technology 240 Fast Pyrolysis-based Systems 241 Fundamentals of Fast Pyrolysis 241 Fermentation of Bio-oils 244 Biorefineries Based on Fast Pyroylsis 246 Enabling Technologies 248 Outlook and Perspectives 249 References 250 Green Biorefineries

12

12.1 12.2 12.2.1 12.2.2 12.2.3 12.3 12.3.1 12.3.2 12.3.3

The Green Biorefiner Concept – Fundamentals and Potential Stefan Kromus, Birgit Kamm, Michael Kamm, Paul Fowler, and Michael Narodoslawsky Introduction 253 Historical Outline 254 The Inceptions 254 First Production of Leaf Protein Concentrate 254 First Production of Leaf Dyes 257 Green Biorefinery Raw Materials 258 Raw Materials 258 Availability of Grassland Feedstocks for Large-scale Green Biorefineries 259 Key Components of Green and Forage Grasses 260

253

Contents

12.3.3.1 12.3.3.2 12.4 12.4.1 12.4.2 12.5 12.5.1 12.5.1.1 12.5.2 12.5.2.1 12.5.3 12.5.3.1 12.5.3.2 12.5.3.3 12.5.3.4 12.5.3.5 12.5.3.6 12.5.3.7 12.5.4 12.5.4.1 12.5.4.2 12.5.4.3 12.6 12.7

Structural Cell Wall Constituents 260 Cell Contents 265 Green Biorefinery Concept 269 Fundamentals and Status Quo 269 Wet Fractionation and Primary Refinery 271 Processes and Products 273 The Juice Fraction 273 Green Juice 273 GJ Drinks/Alternative Life 275 Silage Juice 276 Ingredients and Specialties 277 Proteins/Polysacharides 277 Cholesterol Mediation 277 Antifeedants 277 Silica 277 Silicon Carbide 278 Filter Aids 278 Zeolites 278 The Press-Cake (Fiber) Fraction 278 Fibers 280 Chemicals 282 Residue Utilization 283 Green Biorefinery – Economic and Ecological Aspects 283 Outlook and Perspectives 285 Acknowledgment 285 References 285

13

Plant Juice in the Biorefinery – Use of Plant Juice as Fermentation Medium 295 Margrethe Andersen, Pauli Kiel, and Mette Hedegaard Thomsen Introduction 295 Historical Outline 295 Biobased Poly(lactic Acid) 296 Fermentation Processes 296 The Green Biorefinery 296 Lactic Acid Fermentation 298 Brown Juice as a Fermentation Medium 298 Materials and Methods 299 Analytical Methods 299 Sugar Analysis 299 Analysis of Organic Acids 299 Analysis of Minerals 299 Analysis of Vitamins 299 Analysis of Amino Acids 299 Analysis of Protein 299

13.1 13.2 13.3 13.3.1 13.3.2 13.3.3 13.3.4 13.4 13.4.1 13.4.1.1 13.4.1.2 13.4.1.3 13.4.1.4 13.4.1.5 13.4.1.6

XIII

XIV

Contents

13.4.2 13.4.3 13.4.4 13.5 13.5.1 13.5.2 13.5.3 13.5.4 13.6 13.6.1 13.6.2 13.7 13.8 13.9

Fed Batch Fermentation of Brown Juice with Lb. salivarius BC 1001 299 Pilot Scale Continuous Fermentation with Lb. salivarius BC 1001 300 Study of Potato Juice Quality During Aerobic and Anaerobic Storage 300 Brown Juice 300 Chemical Composition 300 Seasonal Variations 302 Lactic Acid Fermentation of Brown Juice 305 The Green Crop-drying Industry as a Lactic Acid Producer 306 Potato Juice 309 Potato Juice as Fermentation Medium 309 The Potato Starch Industry as Lactic Acid Producer 310 Carbohydrate Source 311 Purification of Lactic Acid 312 Conclusion and Outlook 313 Acknowledgments 313 References 313

Part III

Biomass Production and Primary Biorefineries

14

Biomass Commercialization and Agriculture Residue Collection 317 James Hettenhaus Introduction 317 Historical Outline 318 Case Study: Harlan, Iowa Corn Stover Collection Project 319 Case Study: Bagasse Storage – Dry or Wet? 321 Dry Storage 321 Wet Storage 323 Biomass Value 324 Soil Quality 324 Farmer Value 325 Processor Value 327 Sustainable Removal 328 Soil Organic Material 328 Soil Erosion Control 329 Cover Crops 331 Innovative Methods for Collection, Storage and Transport 332 Collection 332 Baling 333 One-pass Collection 333 Storage 334 Density 335 Storage Area 335

14.1 14.2 14.2.1 14.2.2 14.2.2.1 14.2.2.2 14.3 14.3.1 14.3.2 14.3.3 14.4 14.4.1 14.4.2 14.4.3 14.5 14.5.1 14.5.1.1 14.5.1.2 14.5.2 14.5.2.1 14.5.2.2

Contents

14.5.2.3 14.5.2.4 14.5.2.5 14.5.3 14.5.3.1 14.5.3.2 14.6 14.6.1 14.6.1.1 14.6.1.2 14.7

Storage Loss 335 Foreign Matter and Solubles 337 Storage Investment 337 Transport 337 Harvest Transport 338 Biorefinery Supply 338 Establishing Feedstock Supply 339 Infrastructure 340 Infrastructure Investment 340 Organization Infrastructure 340 Perspectives and Outlook 341 References 342

15

The Corn Wet Milling and Corn Dry Milling Industry – A Base for Biorefinery Technology Developments 345 Donald L. Johnson Introduction 345 Corn – Wet and Dry Milling – Existing Biorefineries 345 The Corn Refinery 346 Wet Mill Refinery 346 Dry Mill Refinery 346 Waste Water Treatment 347 The Modern Corn Refinery 348 Background and Definition 348 Technologies and Products 348 Refinery Economy 350 Refinery Economy of Scale and Location Considerations 350 Carbohydrate Refining 351 Outlook and Perspectives 352 References 352

15.1 15.1.1 15.2 15.2.1 15.2.2 15.2.3 15.3 15.3.1 15.3.2 15.3.3 15.3.3.1 15.4 15.5

Part IV

Biomass Conversion: Processes and Technologies

16

Enzymes for Biorefineries 357 Sarah A. Teter, Feng Xu, Glenn E. Nedwin, and Joel R. Cherry Introduction 357 Biomass as a Substrate 359 Composition of Biomass 359 Cellulose 359 Hemicellulose 360 Lignin 360 Starch 360 Protein 361 Lipids and Other Extracts 361 Biomass Pretreatment 361

16.1 16.2 16.2.1 16.2.1.1 16.2.1.2 16.2.1.3 16.2.1.4 16.2.1.5 16.2.1.6 16.2.2

XV

XVI

Contents

16.2.2.1 16.2.2.2 16.2.2.3 16.2.2.4 16.3 16.3.1 16.3.2 16.3.3 16.3.4 16.4 16.4.1 16.4.1.1 16.4.1.2 16.4.1.3 16.4.2 16.4.2.1 16.4.2.2 16.4.2.3 16.5 16.6 16.6.1 16.6.2 16.6.3 16.7 16.7.1 16.7.2

Dilute Acid Pretreatment 362 Ammonia Fiber Explosion 362 Hot-wash Pretreatment 362 Wet Oxidation 363 Enzymes Involved in Biomass Biodegradation 363 Glucanases or Cellulases 364 Hemicellulases 364 Nonhydrolytic Biomass-active Enzymes 365 Synergism of Biomass-degrading Enzymes 365 Cellulase Development for Biomass Conversion 366 Optimization of the CBH-EG-BG System 366 BG Supplement 366 Novel Cellulases with Better Thermal Properties 367 Structure–Function Relationship of EG 370 Other Proteins Potentially Beneficial for Biomass Conversion 371 Secretome of Cellulolytic Fungi 371 Hydrolases 373 Nonhydrolytic proteins 374 Expression of Cellulases 374 Range of Biobased Products 375 Fuels 376 Fine/Specialty Chemicals 378 Fuel Cells 378 Biorefineries: Outlook and Perspectives 380 Potential of Biomass-based Material/Energy Sources 380 Economic Drivers Toward Sustainability 381 References 382

17

Biocatalytic and Catalytic Routes for the Production of Bulk and Fine Chemicals from Renewable Resources 385 Thomas Willke, Ulf Prüße, and Klaus-Dieter Vorlop Introduction 385 Renewable Resources 385 Products 386 Bulk Chemicals and Intermediates 386 Fine Chemicals and Specialties 386 Historical Outline 387 Processes 388 Immobilization 389 Biocatalytic Routes from Renewable Resources to Solvents or Fuels 390 Ethanol Production with Bacteria or Yeasts? 390 Biocatalytic Route from Glycerol to 1,3-Propanediol 393 Introduction 393 The Process 393

17.1 17.1.1 17.1.2 17.1.2.1 17.1.2.2 17.2 17.3 17.3.1 17.3.2 17.3.2.1 17.3.3 17.3.3.1 17.3.3.2

Contents

17.3.4 17.3.4.1 17.3.4.2 17.3.4.3 17.3.4.4 17.3.4.5 17.3.4.6 17.3.5 17.3.5.1 17.3.5.2 17.3.5.3

Biocatalytic Route from Inulin to Difructose Anhydride Introduction 397 Enzyme Screening 398 Genetic Engineering 398 Fermentation of the Recombinant E. coli 399 Enzyme Immobilization and Scale-up 400 Summary 401 Chemical Route from Sugars to Sugar Acids 402 Introduction 402 Gold Catalysts 403 Summary 405 References 405 Subjcet Index

397

407

Volume 2 Part I

Biobased Product Family Trees Carbohydrate-based Product Lines

1

The Key Sugars of Biomass: Availability, Present Non-Food Uses and Potential Future Development Lines 3 Frieder W. Lichtenthaler

2

Industrial Starch Platform – Status quo of Production, Modification and Application 61 Dietmar R. Grüll, Franz Jetzinger, Martin Kozich, Marnik M. Wastyn, and Robert Wittenberger

3

Lignocellulose-based Chemical Products and Product Family Trees 97 Birgit Kamm, Michael Kamm, Matthias Schmidt, Thomas Hirth, and Margit Schulze Lignin Line and Lignin-based Product Family Trees

4

Lignin Chemistry and its Role in Biomass Conversion Gösta Brunow

5

Industrial Lignin Production and Applications E. Kendall Pye

165

151

XVII

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Contents

Protein Line and Amino Acid-based Product Family Trees 6

Towards Integration of Biorefinery and Microbial Amino Acid Production 201 Achim Marx, Volker F. Wendisch, Ralf Kelle, and Stefan Buchholz

7

Protein-based Polymers: Mechanistic Foundations for Bioproduction and Engineering Dan W. Urry

217

Biobased Fats (Lipids) and Oils 8

New Syntheses with Oils and Fats as Renewable Raw Materials for the Chemical Industry 253 Ursula Biermann, Wolfgang Friedt, Siegmund Lang, Wilfried Lühs, Guido Machmüller, Jürgen O. Metzger, Mark Rüsch gen. Klaas, Hans J. Schäfer, Manfred P. Schneider

9

Industrial Development and Application of Biobased Oleochemicals 291 Karlheinz Hill Special Ingredients and Subsequent Products

10

Phytochemicals, Dyes, and Pigments in the Biorefinery Context George A. Kraus

11

Adding Color to Green Chemistry? An Overview of the Fundamentals and Potential of Chlorophylls Mathias O. Senge and Julia Richter

Part II

Biobased Industrial Products, Materials and Consumer Products

12

Industrial Chemicals from Biomass – Industrial Concepts Johan Thoen and Rainer Busch

13

Succinic Acid – A Model Building Block for Chemical Production from Renewable Resources 367 Todd Werpy, John Frye, and John Holladay

14

Polylactic Acid from Renewable Resources Patrick Gruber, David E. Henton, and Jack Starr 381

15

Biobased Consumer Products for Cosmetics Thomas C. Kripp

409

347

315

325

Contents

Part III

Biobased Industry: Economy, Commercialization and Sustainability

16

Industrial Biotech – Setting Conditions to Capitalize on the Economic Potential Rolf Bachmann and Jens Riese Subject Index

463

445

XIX

XXI

Editor’s Preface In the year 2003 when the idea for this set of books “Biorefineries, Biobased Industrial Processes, and Products” arose, the topic of biorefineries as means of processing industrial material and efficient utilization of renewable products had been primarily a side issue beyond the borders of the United States of America. This situation has changed dramatically over the last two years. Today in almost every developed and emerging nation much work is being conducted on biorefinery systems, driven by the rising cost of oil and the desire of to move away from petrochemical-based systems. In these books we do not claim to describe and discuss everything that belongs or even might belong to the topic of biorefineries – that would be impossible. There are many types of biorefinery, and the state of the technology is changing very rapidly as new and focused effort is directed toward making biorefineries a commercial reality. It is a very exciting time for those interested in biorefineries – technologies for bio-conversion have advanced to a state in which they are becoming practical on a large scale, economics are leaning more favourably to the direction of renewable feedstocks, and chemical process knowledge is being applied to biobased systems. As the editors of the first comprehensive biorefinery book we saw it as our duty to provide, first of all, a general framework for the subject – addressing the main issues associated with biorefineries, the principles and basics of biorefinery systems, the basic technology, industrial products which fall within the scope of biorefineries, and, finally, technology and products that will fall within the scope of biorefineries in the future. To provide a reliable description of the state of biorefinery research and development and of industrial implementations, strategies, and future developments we asked eighty-five experts from universities, research and development institutes, and industry and commerce to present their views, their results, their implementations, and their ideas on the topic. The results of their contributions are thirty-three articles organized into seven sections. Our very special thanks go to all the authors. We are especially indebted to Dr. Hubert Pelc from Wiley-VCH publishing, who worked with us on the concept and then, later, on the development and implementation of the book. Thanks go also to Dr. Bettina Bems from Wiley-


XXII

Editor’s Preface

VCH publishing, who managed with admirable professionalism and very much patience, and to the three editors and eighty-five authors from three different continents. We are also indebted to Hans-Jochen Schmitt, also of Wiley-VCH publishing, who had the not always easy task of arranging the manuscripts in a form ready for publication. Maybe in 2030, when a biobased economy utilizing biorefinery technology has become a fundamental part of national and globally connected economies, someone will wonder what had been thought and written about the subject of biorefineries at the beginning of the 21st century. Hopefully this book will be highly representative. Until then we hope it will contribute to the promotion of international biorefinery developments. Teltow-Seehof (Germany) Boulder, CO (USA) Potsdam (Germany) November 2005

Birgit Kamm Patrick R. Gruber Michael Kamm

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Foreword One-hundred-and-fifty years after the beginning of coal-based chemistry and 50 years after the beginning of petroleum-based chemistry industrial chemistry is now entering a new era. In the twenty-first century utilization of renewable raw materials will gain importance in the chemical conversion of substances in industry. Partial or even complete re-adjustment of whole economies to renewable raw materials will require completely new approaches in research, development, and production. Chemical and biological sciences will play a leading role in the building of future industries. New synergies between biological, physical, chemical, and technical sciences must be elaborated and established and special requirements will be placed on raw material and on product-line efficiency and sustainability. The necessary change from chemistry based on a fossil raw material to biology-based modern science and technology is an intellectual challenge for both researchers and engineers. Chemists should support this change and collaborate closely with their colleagues in adjoining disciplines, for example biotechnology, agriculture, forestry, and the material sciences. The German Chemical Society will help direct this necessary development by supporting within its structure new kinds of organization for chemists to work on this subject in universities, research institutes, and industry. This two-volume book is based on the approach developed by biorefinery-systems – transfer of the logic and efficiency of today’s petrochemical product lines and product family trees into manipulation of biomass. Raw biomass materials are mechanically separated into substances for chemical conversion into other products by different methods, which may be biotechnological, thermochemical, and thermal. Review of biomass processes and products developed in the past but widely forgotten in the petroleum age will be as important as the presentation of new methods, processes, and products that still require an enormous amount of research and development today. Henning Hopf President of the German Chemical Society Frankfurt (Germany) November 2005


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Foreword On October 5, 2005, the Nobel Prize Committee made an interesting and important statement with regard to the prize in chemistry. It said, “This represents a great step forward for ‘green chemistry’, reducing potentially hazardous waste through smarter production. [This research] is an example of how important basic science has been applied for the benefit of man, society and the environment.” By making this statement, the Nobel committee recognized what a new generation of scientists has known for quite some time, that by working at the most fundamental level – the molecular level – we are able to design our products, processes, and systems in ways that are sustainable. There is general recognition that the current system by which we produce the goods and services needed by society is not sustainable. This unsustainability takes many forms. It would be legitimate to note that in our current system of production we rely largely on finite feedstocks extracted from the Earth that are being depleted at a rate that cannot be sustained indefinitely. It is equally legitimate to recognize that our current production efficiency results in more than 90% of the material used in the production process ending up as waste, i.e. less than 10% of the material ends up in the desired product. Yet another condition of unsustainability is in our current energy use; this not only relies largely on finite energy sources but also results in degradation of the environment that cannot be continued as the growing population and demands of the developing world emerge over the course of the twenty-first century. Finally, the products and processes we have designed since the industrial revolution have accomplished their goals without full consideration of their impact and consequence on humans and the biosphere, with many examples of toxic and hazardous substances being distributed throughout the globe and into our bodies. If we are to change this unsustainable path, it will need the direct and committed engagement of our best scientists and engineers to design the future differently from the past. We will need to proceed with a broader perspective such that when we design for efficiency, effectiveness, and performance, we now must recognize that these terms include sustainability – a minimized impact on humans and the environment. An essential part of meeting the challenge of designing for sustainability will be based on the nature of the materials we use as starting materials and feedstocks. Any sustainable future must ensure that the materials on which we base Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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our economic infrastructure are renewable rather than depleting. The rate of renewability is also important because certainly one could argue that petroleum is renewable if you have a few million years to wait. Serious analysis would, however, necessitate that the rate of renewability is connected to the rate of use. There are options for how to approach this technological challenge, for example using waste products from one process as a feedstock for another, that are well thought through in industrial ecology models. There is, however, recognition that an essential part of a sustainable future will be based on appropriate and innovative uses of our biologically-based feedstocks. This book addresses the essential questions and challenges of moving toward a sustainable society in which bio-based feedstocks, processes, and products are fundamental pillars of the economy. The authors discuss not only the important scientific and technical issues surrounding this transition but also the necessary topics of economics, infrastructure, and policy. It is only by means of this type of holistic approach that movement toward genuine sustainability will be able to occur where the societal, economic, and environmental needs are met for the current generation while preserving the ability of future generations to meet their needs. While it will be clear to the reader that the topics presented in this book are important, it is at least as important that the reader understand that these topics – and the transition to a sustainable path that they address – are urgent. At this point in history it is necessary that all who are capable of advancing the transition to a more sustainable society, engage in doing so with the level of energy, innovation, and creativity that is required to meet the challenge. Paul T. Anastas Director of the Green Chemistry Institute Washington, D.C. November, 2005

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List of Contributors (Volume 1 and 2) José A. M. Agnelli Universidade Federal de São Carlos Departamento de Engenharia de Materiais Rodovia Washington Luis (SP-310) São Carlos, São Paulo Brazil

Robert C. Brown Center for Sustainable Environmental Technologies Iowa State University 286 Metals Development Building Ames, IO 50011 USA

Margrethe Andersen AgroFerm A/S Limfjordsvej 4 6715 Esbjerg N Denmark

Gösta Brunow Department of Chemistry University of Helsinki A. I. Virtasen aukio 1 00014 Helsinki Finland

Rolf Bachmann McKinsey and Company Inc Zurich Office Alpenstrasse 3 8065 Zürich Switzerland Ursula Biermann Fachbereich Chemie Carl von Ossietzky Universität Oldenburg Postfach 2603 26111 Oldenburg Germany

Stefan Buchholz Degussa AG Creavis Projecthouse ProFerm Rodenbacher Chaussee 4 63403 Hanau-Wolfgang Germany Rainer Busch Dow Deutschland GmbH & Co. OHG Industriestrasse 1 77836 Rheinmünster Germany


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Grant M. Campbell Satake Centre for Grain Process Engineering School of Chemical Engineering and Analytical Science The University of Manchester Sackville Street Manchester M60 1QD UK Joel R. Cherry Novozymes Biotech Inc 1445 Drew Ave Davis, CA 95616 USA Gopal Chotani Genencor International 925 Page Mill Road Palo Alto, CA 94304 USA L. Davis Clements Renewable Products Development Laboratories 3114 NE 45th Ave. Portland, OR 97213 USA Bruce E. Dale Department of Chemical Engineering and Materials Science Michigan State University East Lansing, MI 48824 USA Bill Dean Genencor International 925 Page Mill Road Palo Alto, CA 94304 USA

Tim Dodge Genencor International 925 Page Mill Road Palo Alto, CA 94304 USA Donald L. Van Dyne Agricultural Economics University of Missouri – Columbia 214c Mumford Hall Columbia, MO 65211 USA Wolter Elbersen Agrotechnology and Food Innovations B. V. P.O. Box 17 6700 AA Wageningen The Netherlands Steve Fitzpatrick Biofine 245 Winter Street Waltham, MA 02154 USA Paul Fowler The BioComposites Centre University of Wales Bangor Gwynedd LL57 2UW UK Wolfgang Friedt Institut für Pflanzenbau und Pflanzenzüchtung 1 Justus-Liebig-Universität Giessen Heinrich-Buff-Ring 26–32 35392 Giessen Germany


John Frye Pacific Northwest National Laboratory P.O. Box 999/K2-12 Richland, WA 99352 USA

James R. Hettenhaus CEA Inc 3211 Trefoil Drive Charlotte, NC 28226 USA

Patrick R. Gruber President and CEO Outlast Technologies Incorporated 5480 Valmont Road Suite 200 Boulder, CO 80301 USA

Karlheinz Hill Cognis Deutschland GmbH & Co. KG Paul-Thomas-Straße 56 40599 Düsseldorf Germany

Dietmar R. Grüll Südzucker Aktiengesellschaft Mannheim/Ochsenfurt Wormser Strasse 11 67283 Obrigheim/Pfalz Germany Daniel J. Hayes Department of Chemical & Environmental Sciences University of Limerick Limerick Ireland Michael H. B. Hayes Department of Chemical & Environmental Sciences University of Limerick Limerick Ireland David E. Henton Nature Works LLC (former Cargill Dow LLC) 15305 Minnetonka Blvd Minnetonka, MN 55345 USA

Thomas Hirth Fraunhofer-Institut Chemische Technologie Joseph-von-Fraunhoferstraße 7 76327 Pfinztal Germany John Holladay Pacific Northwest National Laboratory P.O. Box 999/K2-12 Richland, WA 99352 USA Franz Jetzinger Zuckerforschung Tulln Gesellschaft mbH Josef-Reither-Strasse 21–23 3430 Tulln Austria Donald L. Johnson Biobased Industrial Products Consulting 29 Cape Fear Drive Hertford, NC 27944 USA Ed de Jong Agrotechnology and Food Innovations B.V. P.O. Box 17 6700 AA Wageningen The Netherlands

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Birgit Kamm Research Institute Bioactive Polymer Systems (biopos e.V.) Research Centre Teltow-Seehof Kantstraße 55 14513 Teltow Germany Michael Kamm Biorefinery.de GmbH Stiftstraße 2 14471 Potsdam Germany and Laboratories Teltow Kantstraße 55 14513 Teltow Germany Raphael Katzen 9220 Bonita Beach Road Suite 2000 Bonita Springs, FL 34135 USA Ralf Kelle Degussa AG R & D Feed Additives Kantstrasse 2 33790 Halle/Westfalen Germany Pauli Kiel Biotest Aps Gl. Skolevej 47 6731 Tjæreborg Denmark Seungdo Kim Department of Chemical Engineering and Materials Science Michigan State University East Lansing, MI 48824 USA

Apostolis A. Koutinas Satake Centre for Grain Process Engineering School of Chemical Engineering and Analytical Science The University of Manchester Sackville Street Manchester M60 1QD UK Martin Kozich Zuckerforschung Tulln Gesellschaft mbH Josef-Reither-Strasse 21–23 3430 Tulln Austria George A. Kraus Department of Chemistry Iowa State University 1605 Gilman Hall Ames, IA 50011-3111 USA Thomas C. Kripp Wella AG Abt. FON Berliner Allee 65 64274 Darmstadt Germany Stefan Kromus BioRefSYS-BioRefinery Systems Innovationszentrum Ländlicher Raum Auersbach 130 8330 Feldbach Austria


Siegmund Lang Institut für Biochemie und Biotechnologie Technische Universität zu Braunschweig Spielmannstraße 7 38106 Braunschweig Germany

Achim Marx Degussa AG Creavis Projecthouse ProFerm Rodenbacher Chaussee 4 63403 Hanau-Wolfgang Germany

Frieder W. Lichtenthaler Institute of Organic Chemistry Darmstadt University of Technology Petersenstraße 22 64287 Darmstadt Germany

Jürgen O. Metzger Fachbereich Chemie Carl von Ossietzky Universität Oldenburg Postfach 2603 26111 Oldenburg Germany

Wilfried Lühs Institut für Pflanzenbau und Pflanzenzüchtung 1 Justus-Liebig-Universität Giessen Heinrich-Buff-Ring 26–32 35392 Giessen Germany

Michael Narodoslawsky Graz University of Technology Institute of Resource Efficient and Sustainable Systems (RNS) Inffeldgasse 21 B 8010 Graz Austria

Guido Machmüller FB 9 – Organische Chemie Bergische Universität GH Wuppertal Gaußstraße 20 42097 Wuppertal Germany

Jefter Nascimento PHB Industrial SA Fazenda da Pedra s/n – C. Postal 02 CEP 14150 Servana São Paulo Brazil

Paulo E. Mantelatto Centro de Tecnologia Canavieira (formerly Centro de Tecnologia Copersucar) Fazenda Santo Antonio CP 162 13400-970 Piracicaba Brazil

Glenn E. Nedwin Novozymes Biotech Inc 1445 Drew Ave Davis, CA 95616 USA

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Ulf Prüße Federal Agricultural Research Centre (FAL) Institute of Technology and Biosystems Engineering Bundesallee 50 38116 Braunschweig Germany

Carlos Eduardo Vaz Rossell Centro de Tecnologia Canavieira (formerly Centro de Tecnologia Copersucar) Fazenda Santo Antonio CP 162 13400-970 Piracicaba Brazil

E. Kendall Pye Lignol Innovations Corp. 3650 Westbrook Mall Vancouver, BC V6S 2L2 Canada

Mark Rüsch gen. Klaas Department Technology University of Applied Sciences Neubrandenburg Brodaer Straße 2 17033 Neubrandenburg Germany

René van Ree Rea Energy research Centre of the Netherlands (ECN) – Biomass Department P.O. Box 1 1755 ZG Petten The Netherlands Julia Richter Institut für Chemie Universität Potsdam Karl-Liebknecht-Str. 24–25 14476 Golm Germany Jens Riese McKinsey and Company Inc Munich Office Prinzregentenstraße 22 80538 München Germany Julian R. H. Ross University of Limerick Department of Chemical & Environmental Sciences Limerick Ireland

Hans J. Schäfer Organisch-Chemisches Institut Universität Münster Corrensstraße 40 48149 Münster Germany Daniel J. Schell National Bioenergy Center National Renewable Energy Laboratory 1617 Cole Blvd. Golden, CO 80401-3393 USA Matthias Schmidt Biorefinery.de GmbH Stiftstraße 2 14471 Potsdam Germany Manfred P. Schneider FB 9 – Organische Chemie Bergische Universität GH Wuppertal Gaußstraße 20 42097 Wuppertal Germany


Margit Schulze FB Angewandte Naturwissenschaften FH Bonn-Rhein-Sieg Grantham-Allee 20 53754 Sankt Augustin Germany

Robert van Tuil Agrotechnology and Food Innovations B.V. P.O. Box 17 6700 AA Wageningen The Netherlands

Mathias O. Senge SFI Tetrapyrrole Laboratory School of Chemistry Trinity College Dublin Dublin 2 Ireland

Dan W. Urry BioTechnology Institute University of Minnesota Twin Cities Campus 1479 Gortner Avenue Suite 240 St. Paul, MN 55108-6106 USA and Bioelastics Inc. 2423 Vestavia Drive Vestavia Hills, AL 35216-1333 USA

Jack Starr Cargill Dow LLC 15305 Minnetonka Blvd Minnetonka, MN 55345 USA Sarah A. Teter Novozymes Biotech Inc 1445 Drew Ave Davis, CA 95616 USA Johan Thoen Dow Europe GmbH Bachtobelstrasse 3 8810 Horgen Switzerland Mette Hedegaard Thomsen Risø National Laboratory Biosystems Department Frederiksbovgvej 399 4000 Roskilde Denmark Jeffrey S. Tolan Iogen Corporation 8 Colonnade Road Ottawa Ontario K2E 7M6 Canada

Fernando Valle Genencor International 925 Page Mill Road Palo Alto, CA 94304 USA Klaus-Dieter Vorlop Federal Agricultural Research Centre (FAL) Institute of Technology and Biosystems Engineering Bundesallee 50 38116 Braunschweig Germany Rouhang Wang Satake Centre for Grain Process Engineering School of Chemical Engineering and Analytical Science The University of Manchester Sackville Street Manchester M60 1QD UK

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Marnik M. Wastyn Zuckerforschung Tulln Gesellschaft mbH Josef-Reither-Strasse 21–23 3430 Tulln Austria Colin Webb Satake Centre for Grain Process Engineering School of Chemical Engineering and Analytical Science The University of Manchester Sackville Street Manchester M60 1QD UK Volker F. Wendisch Institute of Biotechnology 1 Research Center Juelich 52425 Juelich Germany Todd Werpy Pacific Northwest National Laboratory P.O. Box 999/K2-12 Richland, WA 99352 USA

Thomas Willke Federal Agricultural Research Centre (FAL) Institute of Technology and Biosystems Engineering Bundesallee 50 38116 Braunschweig Germany Robert Wittenberger Zuckerforschung Tulln Gesellschaft mbH Josef-Reither-Strasse 21–23 3430 Tulln Austria Feng Xu Novozymes Biotech Inc 1445 Drew Ave Davis, CA 95616 USA

Biorefineries – Industrial Processes and Products Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm © 2006 WILEY-VCH Verlag GmbH & Co.

Part I Background and Outline – Principles and Fundamentals


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1 Biorefinery Systems – An Overview Birgit Kamm, Michael Kamm, Patrick R. Gruber, and Stefan Kromus

1.1 Introduction

The preservation and management of our diverse resources are fundamental political tasks to foster sustainable development in the 21st century. Sustainable economic growth requires safe and sustainable resources for industrial production, a long-term and confident investment and finance system, ecological safety, and sustainable life and work perspectives for the public. Fossil resources are not regarded as sustainable, however, and their availability is more than questionable in the long-term. Because of the increasing price of fossil resources, moreover, the feasibility of their utilization is declining. It is, therefore, essential to establish solutions which reduce the rapid consumption of fossil resources, which are not renewable (petroleum, natural gas, coal, minerals). A forward looking approach is the stepwise conversion of large parts of the global economy into a sustainable biobased economy with bioenergy, biofuels, and biobased products as its main pillars (Fig. 1.1).

Fig. 1.1 3-Pillar model of a future biobased economy. Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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1 Biorefinery Systems – An Overview

Whereas for energy production a variety of alternative raw materials (wind, sun, water, biomass, nuclear fission and fusion) can be established, industry based on conversion of sustainable material, for example the chemical industry, industrial biotechnology, and also the fuel generation, depends on biomass, in particular mainly on plant biomass. Some change from the today’s production of goods and services from fossil to biological raw materials will be essential. The rearrangement of whole economies to implement biological raw materials as a source with increased value requires completely new approaches in research and development. On the one hand, biological and chemical sciences will play a leading role in the generation of future industries in the 21st century. On the other hand, new synergies of biological, physical, chemical, and technical sciences must be elaborated and established. This will be combined with new traffic technology, media- and information technology, and economic and social sciences. Special requirements will be placed on both the substantial converting industry and research and development with regard to raw material and product line efficiency and sustainability. The development of substance-converting basic product systems and polyproduct systems, for example biorefineries, will be the “key for the access to an integrated production of food, feed, chemicals, materials, goods, and fuels of the future” [1].

1.2 Historical Outline 1.2.1 Historical Technological Outline and Industrial Resources

Today’s biorefinery technologies are based (1) on the utilization of the whole plant or complex biomass and (2) on integration of traditional and modern processes for utilization of biological raw materials. In the 19th and the beginning of the 20th century large-scale utilization of renewable resources was focused on pulp and paper production from wood, saccharification of wood, nitration of cellulose for guncotton and viscose silk, production of soluble cellulose for fibers, fat curing, and the production of furfural for Nylon. Furthermore, the technology of sugar refining, starch production, and oil milling, the separation of proteins as feed, and the extraction of chlorophyll for industrial use with alfalfa as raw material were of great historical importance. But also processes like wet grinding of crops and biotechnological processes like the production of ethanol, acetic acid, lactic acid, and citric acid used to be fundamental in the 19th and 20th century.

1.2 Historical Outline

1.2.2 The Beginning – A Digest 1.2.2.1 Sugar Production The history of industrial conversion of renewable resources is longer than 200 years. Utilization of sugar cane has been known in Asia since 6000 BC and imports of cane sugar from oversea plantations have been established since the 15th century. The German scientist A. S. Marggraf was a key initiator of the modern sugar industry. In 1748 he published his research on the isolation of crystalline sugar from different roots and beet [2, 3]. Marggraf’s student, F. C. Achard, was the first to establish a sugar refinery based on sugar beet, in Cunern/Schlesien, Poland, in 1801.

1.2.2.2 Starch Hydrolysis In 1811, the German pharmacist G. S. C. Kirchhoff found that when potato starch was cooked in dilute acid the starch was converted into “grape sugar” (i.e. d-glucose or dextrose) [4]. This was not only a very important scientific result but also the starting point of the starch industry. In 1806 the French emperor Napoleon Bonaparte introduced an economic continental blockade which considerably limited overseas trade in cane sugar. Thus, starch hydrolysis became of interest for the economy. The first starch sugar plant was established in Weimar, Germany, in 1812, because of a recommendation of J.W. Döbereiner to grand duke Carl August von Sachsen-Weimar. Successful development of the sugar beet industry, however, initially obstructed further development of the starch industry [5]. In 1835, the Swedish Professor J. J. Berzelius developed enzymatic hydrolyses of starch into sugar and introduced the term “catalysis”.

1.2.2.3 Wood Saccharification In 1819 the French plant chemist H. Braconnot discovered that sugar (glucose) is formed by treatment of wood with concentrated sulfuric acid. 1855, G. F. Melsens reported that this conversion can be carried out with dilute acid also. Acid hydrolysis can be divided into two general approaches, based on (1) concentrated acid hydrolysis at low temperature and (2) dilute acid hydrolysis at high temperature. Historically, the first commercial processes, named wood saccharification, were developed in 1901 by A. Classen (Ger. Patent 130 980), employing sulfuric acid, and in 1909 by M. Ewen and G. Tomlinson (US Patent 938 208), working with dilute sulfuric acid. Several plants were in operation until the end of World War I. Yields of these processes were usually low, in the range 75–130 liter per ton wood dry matter only [6, 7]. Technologically viable processes were, however, developed in the years between World War I and World War II. The German chemist Friedrich Bergius was one of the developers. The sugar fractions generated by wood hydrolyses have a broad spectrum of application. An important fermentation product of wood sugar of increasing interest is ethanol.

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Ethanol can be used as fuel either blended with traditional hydrocarbon fuel or as pure ethanol. Ethanol is also an important platform chemical for further processing [8].

1.2.2.4 Furfural Döbereiner was the first to report the formation and separation of furfural by distillation of bran with diluted sulfuric acid in 1831. In 1845 the English Chemist G. Fownes proposed the name “furfurol” (furfur – bran; oleum – oil). Later the suffix “ol” was changed to “al” because of the aldehyde function [9, 10]. Treatment of hemicellulose-rich raw materials with dry steam in the presence of hydrogen chloride gave especially good results [11]. Industrial technology for production of furfural from pentose is based on a development of an Anglo-American company named Quaker Oats. The process was been developed in the nineteentwenties [E. P. 203 691 (1923), F. P. 570 531 (1923)]. Since 1922 Quaker Oats Cereal Mill in Cedar Rapids/Iowa, USA, has produced up to 2.5 tons of furfural per day from oat husks. Since 1934 the process had been established as an industrial furfural plant. Furfural was the cheapest aldehyde, at 16–17 cents per lb (lb = pound; 1 metric ton = 1000 kg = 2204.62442 lb; 1 kg = 0.453592 lb) [12]. Until approximately 1960 DuPont used furfural as a precursor of Nylon-6.6. Furfural has since been substituted by fossil based precursors.

1.2.2.5 Cellulose and Pulp In 1839 the Frenchman A. Payen discovered that after treatment of wood with nitric acid and subsequent treatment with a sodium hydroxide solution a residue remained which he called “les cellules”, cellulose [13]. In 1854 caustic soda and steam were used by the Frenchman M. A. C. Mellier to disintegrate cellulose pulp from straw. In 1863 the American B. C. Tilgham registered the first patent for production of cellulose by use of calcium bisulfite. Together with his brother, Tilgham started the first industrial experiments to produce pulp from wood by treatment with hydrogen sulfite. This was 1866 at the paper mill Harding and Sons, Manayunk, close to Philadelphia. In 1872 the Swedish Engineer C. D. Ekman was the first to produce sulfite cellulose by using magnesium sulfite as cooking agent [14]. By 1900 approximately 5200 pulp and paper mills existed worldwide, most in the USA, approximately 1300 in Germany, and 512 in France.

1.2.2.6 Levulinic Acid In 1840 the Dutch Professor G. J. Mulder (who also introduced the name “protein”) synthesized levulinic acid (4-oxopentanoic acid, c-ketovaleric acid) by heating fructose with hydrochloride for the first time. The former term “levulose” for fructose gave the levulinic acid its name [15]. Although levulinic acid has been well known since the 1870s when many of its reactions (e.g. esters) were


established, it has never reached commercial use in any significant volume. In the 1940s commercial levulinic acid production was begun in an autoclave in the United States by A. E. Staley, Dectur, Illinois [16]. At the same time utilization of hexoses from low-cost cellulose products was examined for the production of levulinic acid [17]. As early as 1956 levulinic acid was regarded as a platform chemical with high potential [18].

1.2.2.7 Lipids From 1850 onward European import of tropical plant fats, for example palm-oil and coconut oil, started. Together with the soda process, invented by the French N. Leblanc in 1791, the industrialization of the soap production began and soap changed from luxury goods into consumer goods. The developing textile industry also demanded fat based products. In 1902 the German chemist W. Normann discovered that liquid plant oils are converting into tempered fat by augmentation of hydrogen. Using nickel as catalyst Norman produced tempered stearic acid by catalytic hydration of liquid fatty acids [19]. The so called “fat hardening” led to the use of European plant oils in the food industry (margarine) and other industries.

1.2.2.8 Vanillin from Lignin In 1874 the German chemists W. Haarmann and F. Tiemann were the first to synthesize vanillin from the cambial juice of coniferous wood. In 1875 the company Haarmann and Reimer was founded. The first precursor for the production of vanillin was coniferin, the glucoside of coniferyl alcohol. This precursor of lignin made from cambial juice of coniferous trees was isolated, oxidized to glucovanillin, and then cleaved into glucose and vanillin [20]. This patented process [21] opened the way to industrial vanillin production. It was also the first industrial utilization of lignin. Besides the perfume industry, the invention was of great interest to the upcoming chocolate industry. Later, however, eugenol (1-allyl-4-hydroxy-3-methoxybenzene), isolated from clove oil, was used to produce vanillin. Today, vanillin production is based on lignosulfonic acid which is a side product of wood pulping. The lignosulfonic acid is oxidized with air under alkaline conditions [22, 23].

1.2.2.9 Lactic Acid In 1895 industrial lactic acid fermentation has been developed by the pharmaceutical entrepreneur A. Boehringer. The Swedish pharmacist C. W. Scheele had already discovered lactic acid in 1780 and the conversion of carbohydrates into lactic acid had been known for ages in food preservation (e.g. Sauerkraut) or agriculture (silage fermentation). Because of the activity of Boehringer the German company Boehringer-Ingelheim can be regarded as the pioneer of industrial biotechnology. Both the process and the demand for lactic acid by dyeing

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factories, and the leather, textile, and food industries made the company the leading supplier. In 1932 W. H. Carothers, who was also the inventor of polyamide-6.6, developed, together with van Natta, a polyester made from lactic acid, poly(lactic acid) [24]. In the late 1990s this poly(lactic acid) was commercialized by the company NatureWorks (Cargill, the former Cargill Dow) [25]. 1.2.3 The Origins of Integrated Biobased Production

In the year 1940 the German chemist P. von Walden (noted for his “Inversion of configuration at substitution reactions”, the so-called “Walden-Reversion”) calculated that in 1940 Germany produced 13 million tons of cellulose leaving 5 to 6 million tons of lignin suitable only as wastage. He then formulated the question: How long can national economy tolerate this [26]? Approaches to integrated production during industrial processing of renewable primary products have a long tradition, starting from the time when industrial cellulose production expanded continuously, as also did the related waste-products. Typical examples of this will be mentioned. As early as 1878 A. Mitscherlich, a German chemist, started to improve the sulfite pulp process by fermentation of sugar to ethyl alcohol – it should be mentioned that sugar is a substance in the waste liquor during sulfite pulp production. He also put into practice a procedure to obtain paper glue from the waste liquor. Both processes were implemented in his plant located in Hof, Germany, in the year 1898 [27]. In 1927 the American Marathon Corporation assigned a group of chemists and engineers to the task of developing commercial products from the organic solids in the spent sulfite liquor from the Marathon’s Rothschild pulp and paper operations close to Wausau, Wisconsin, USA. The first products to show promise were leather tanning agents. Later, the characteristics of lignin as dispersing agents became evident. By the mid 1930s, with a considerable amount of basic research accomplished, Marathon transferred operations from a research pilot plant to full-scale production [28]. One of the most well known examples is the production of furfural by the Quaker Oats Company since 1922, thus coupling food, i.e. oat flakes, production and chemical products obtained from the waste [10] (Section 1.2.2). On the basis of furfural a whole section of chemical production developed – furan chemistry. Agribusiness, especially, strived to achieve combined production from the very beginning. Modern corn refining started in the middle of the 18th century when T. Kingsford commenced operation of his corn refining plant in Oswego, New York [29]. Corn refining is distinguished from corn milling because the refining process separates corn grain into its components, for example starch, fiber, protein and oil, and starch is further processed into a substantial number of products [30]. The extensive usage of green crops has been aim of industry for decades, because there are several advantages. Particularly worthy of mention is the work


of Osborn (1920) and Slade and Birkinshaw (1939) on the extraction of proteins from green crops, for example grass or alfalfa [31]. In 1937 N. W. Pirie developed the technical separation and extraction methods needed for this use of green crops [32, 33]. By means of sophisticated methods all the botanical material should have been used, both for production of animal feed, isolated proteins for human nutrition, and as raw material for further industrial processes, for example glue production. The residual material, juices rich in nutrients, had initially been used as fertilizer; later they were used for generation of fermentation heat based on biogas production [34, 35]. These developments resulted in market-leading technology, for example the Proxan and Alfaprox procedures, used for generation of protein–xanthophyll concentrates, including utilization of the by-products, however, predominantly in agriculture [36]. In the United States commercial production of chlorophyll and carotene by extraction from alfalfa leaf meal had started in 1930 [37, 38]. For example Strong, Cobb and Company produced 0.5 ton chlorophyll per day from alfalfa as early as 1952. The water-soluble chlorophyll, or chlorophyllin, found use as deodorizing agent in toothpastes, soaps, shampoos, candy, deodorants, and pharmaceuticals [39]. A historical important step for today’s biorefinery developments was the industry-politics-approach of “Chemurgy”, founded in 1925 in the US by the Chemist W. J. Hale, son-in-law of H. Dow, the founder of Dow Chemical, and C. H. Herty, a former President of the American Chemical Society. They soon found prominent support from H. Ford and T. A. Edisons. Chemurgy, an abbreviation of “chemistry” and “ergon”, the Greek word for work [40], means by analogy “chemistry from the acre” that is the connection of agriculture with the chemical industry. Chemurgy was soon shown to have a serious industrial political philosophy – the objective of utilizing agricultural resources, nowadays called renewable resources, in industry. There have been common conferences between agriculture, industry, and science since 1935 with a national council called the “National Farm Chemurgic Council” [41]. The end of Chemurgy started with the flooding of the world market with cheap crude oil after World War II; numerous inventions and production processes remained, however, and are again highly newsworthy. One was a car, introduced by Henry Ford 1941, whose car interior lining and car body consisted 100% of bio-synthetics; to be specific it had been made from a cellulose meal, soy meal, formaldehyde resin composite material in the proportions 70% : 20% : 10%, respectively. The alternative fuel for this car was pyrolysis methanol produced from cannabis. Throughout the thirties more than 30 industrial products based on soy bean were created by researchers from the Ford company; this made it necessary to apply complex conversion methods [42]. Hale was a Pioneer of ethyl alcohol and hydrocarbon fuel mixture (Power Alcohol, Gasohol) [43]. This fuel mixture, nowadays called E10-Fuel, consisting of 10 percent bioethanol and 90 percent hydrocarbon-based fuel, has been the national standard since the beginning of this millennium in the United States.

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Associated with the work of Bergius, 1933 [44], and Scholler, 1923 and 1935 [45, 46], wood saccharification was reanimated at the end of WW II. Beside optimization of the process, use of lignocelluloses was of great interest. The continuously growing agribusiness left behind millions of tons of unused straw. Two Americans, Othmer and Katzen, were the main pioneers in the field of wood saccharification [47]. Between the years 1935 and 1960 several hydrolysis plants were built in Germany and the United States; in these deal, wood flour, surplus lumber, and also straw were hydrolyzed [48]. One of the most well known plants are those of Scholler/Tornesch located in Tornesch, Germany, with a production rate of 13,000 tons per year, in Dessau, Germany, production rate 42,000 tons per year, based on wood, in Holzminden, Germany, production rate of 24,000 tons per year, also based on wood, in Ems, Switzerland, with a production rate of 35,000 tons per year, also wood based, and the plants in Madison and Springfield, United States, and the Bergius plants in Rheinau, Germany (Rheinau I, built 1930, with a production rate of 8000 tons per year, based on surplus lumber; Rheinau II, built 1960 with a production rate of 1200 tons per year, based on wood) and the plant in Regensburg, Germany, with a production rate of 36,000 tons per year [49]. During WW II the plant in Springfield, Oregon, US (using the Scholler-Tornesch process as modified by Katzen) produced 15,000 gallons of ethyl alcohol per day from 300 tons wood flour and sawdust, i.e. 50 gallons per ton of wood [50]. The plant in Tornesch, Germany, has been producing approximately 200 liters of ethyl alcohol, purity 100%, per ton wood and approximately 40 kg yeast per ton of wood. In 1965 there were 14 plants in what was then the Soviet Union, with a total capacity of 700,000 tons per year and an overall annual wood consumption of 4 million tons [6]. During the nineteen-sixties wood chemistry had its climax. Projects had been developed, which made it possible to produce nearly all chemical products on the basis of wood. Examples are the complex chemical technological approaches of wood processing from Timell 1961 [51], Stamm 1964 [52], James 1969 [53], Brink and Pohlmann 1972 [54], and the wood-based chemical product trees by Oshima 1965 [55]. Although these developments did not make their way into industrial production, they are an outstanding platform for today’s lignocellulose conversions, product family trees, and LCF biorefineries (Section 1.5.2). Most of the above mentioned technologies and products, some of which were excellent, could not compete with the fossil-based industry and economy; nowadays, however, they are prevailing again. The basis for this revival started in the seventies, when the oil crisis and continuously increasing environmental pollution resulted in a broad awareness that plants could be more than food and animal feed. At the same time the disadvantages of intensive agricultural usage, for example over-fertilization, soil erosion, and the enormous amounts of waste, were revealed. From this situation developed complex concepts, which have been published, in which the aim was, and still is, technological and economical cooperation of agriculture, forestry, the food-production industry, and conventional industry, or at least consideration of integrated utilization of renewable resources.

1.3 Situation

Typical examples of this thinking were: · integrated industrial utilization of wood and straw [56]; · industrial utilization of fast growing wood-grass [57, 58]; · complex utilization of green biomass, for example grass and alfalfa, by agriculture and industry [59–61]; · corn wet-grinding procedures with associated biotechnological and chemical product lines [62]; · modern aspects of thermochemical biomass conversion [63]; · discussion of the concept “organic chemicals from biomass” with main focus on biotechnological methods and products (white biotechnology) [64–66] and industrial utilization of biomass [67]. These rich experiences of the industrial utilization of renewable resources, new agricultural technology, biotechnology, and chemistry, and the changes in ecology, economics, and society led inevitably to the topic of complex and integrated substantial and energy utilization of biomass and, finally, to the biorefinery.

1.3 Situation 1.3.1 Some Current Aspects of Biorefinery Research and Development

Since the beginning of the 1990s the utilization of renewable resources for production of non-food products has fostered research and development which has received increasing attention from industry and politicians [68–70]. Integrated processes, biomass refinery technology, and biorefinery technology have become object of research and development. Accordingly, the term “biorefinery” was established in the 1990s [1, 71–80]. The respective biorefinery projects are focused on the fabrication of fuels, solvents, chemicals, plastics, and food for human beings. In some countries these biorefinery products are made from waste biomass. At first the main processes in the biorefinery involved ethanol fermentation for fuels (ethanol-oriented biorefineries) [81–85], lactic acid (LA) fermentation [25, 86], propanediol (PDO) fermentation [87], and the lysine fermentation [88] especially for polymer production. The biobased polymers poly(lactic acid) [25], propandiol-derived polymers [89], and polylysine [88] have been completed by polyhydroxyalkanoates [90] and polymerized oils [91]. Many hybrid technologies were developed from different fields, for example bioengineering, polymer chemistry, food science and agriculture. Biorefinery systems based on cereals [92, 93], lignocelluloses [94, 95], and grass and alfalfa [35, 96], and biorefinery optimization tools are currently being developed [97, 98]. The integration of molecular plant genetics to support the raw material supply is currently being discussed intensely [99, 100].

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Broin and Associates has begun the development of a second generation of dry mill refineries and E. I. du Pont de Nemours has developed an integrated cornbased biorefinery. In 2001 NatureWorks LCC (to Cargill, former Cargill Dow LLC) started the industrial production of PLA (PLA-oriented Biorefinery) on the basis of maize. Biorefineries are of interest ecologically [101], economically, and to business, government, and politicians [102–107]. National programs [108, 109], biobased visions [110], and plans [111] have been developed and the international exchange of information is increasing, for example as a result, among others, of series of international congresses and symposia: 1. BIO World Congress on Industrial Biotechnology and Bioprocessing [112]; 2. biomass conferences [113, 114]; 3. The Green and Sustainable Chemistry Congress [115]; and 4. the Biorefinica symposia series [116, 117]. Currently, biorefinery systems are in the stage of development world-wide. An overview of the main aspects, activity, and discussions is the content of this book. An attempt to systematize the topic “Biorefinery” will be presented below. 1.3.2 Raw Material Biomass

Nature is a permanently renewing production chain for chemicals, materials, fuels, cosmetics, and pharmaceuticals. Many of the biobased industry products currently used are results of direct physical or chemical treatment and processing of biomass, for example cellulose, starch, oil, protein, lignin, and terpenes. On one hand one must mention that because of the help of biotechnological processes and methods, feedstock chemicals are produced such as ethanol, butanol, acetone, lactic acid, and itaconic acid, as also are amino acids, e.g. glutaminic acid, lysine, tryptophan. On the other hand, only 6 billion tons of the yearly produced biomass, 1.7–2.0 ´ 1011 tons, are currently used, and only 3 to 3.5% of this amount is used in non-food applications, for example chemistry [118]. The basic reaction of biomass is photosynthesis according to: nCO2 nH2 O

!

CH2 On nO2

Industrial utilization of raw materials from agriculture, forestry, and agriculture is only just beginning. There are several definitions of the term “biomass” [118]: · the complete living, organic matter in our ecological system (volume/non-specific) · the plant material constantly produced by photosynthesis with an annual growth of 170 billion tons (marine plants excluded) · the cell-mass of plants, animals, and microorganism used as raw materials in microbiological processes

1.3 Situation

Biomass is defined in a recent US program [108, 109]: “The term “biomass” means any organic matter that is available on a renewable or recurring basis (excluding old-growth timber), including dedicated energy crops and trees, agricultural food and feed crop residues, aquatic plants, wood and wood residues, animal wastes, and other waste materials.” For this reason it is essential to define biomass in the context of the industrial utilization. A suggestion for a definition of “industrial biomass” [108, 109] is: “The term “industrial biomass” means any organic matter that is available on a renewable or recurring basis (excluding old-growth timber), including dedicated energy crops and trees, agricultural food and feed crop residues, aquatic plants, wood and wood residues, animal wastes, and other waste materials usable for industrial purposes (energy, fuels, chemicals, materials) and include wastes and co-wastes of food and feed processing.” Most biological raw material is produced in agriculture and forestry and by microbial systems. Forestry plants are excellent raw materials for the paper and

Fig. 1.2 Products and product classes based on biological raw materials [78].

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cardboard, construction, and chemical industries. Field fruits are an pool of organic chemicals from which fuels, chemicals, chemical products, and biomaterials are produced (Fig. 1.2), [69]. Waste biomass and biomass of nature and agricultural cultivation are valuable organic reservoirs of raw material and must be used in accordance with their organic composition. During the development of biorefinery systems the term “waste biomass” will become obsolete in the medium-term [119]. 1.3.3 National Vision and Goals and Plan for Biomass Technology in the United States

Industrial development was pushed by the US President [108] and by the US congress [109], initially in 2000. In the USA it is intended that by 2020 at least 25% of organic-carbon-based industrial feedstock chemicals and 10% of liquid fuels (compared with levels in 1994) will be produced by biobased industry. This would mean that more than 90% of the consumption of organic chemicals in the US and up to 50% of liquid fuel needs would be biobased products [1]. The Biomass Technical Advisory Committee (BTAC) of the USA in which leading representatives of industrial companies, for example Dow Chemical, E. I. du Pont de Nemours, Cargill Dow LLC, and Genencor International, and Corn growers associations and the Natural Resources Defense Council are involved and which acts as advisor to the US government, has made a detailed plan with steps toward targets of 2030 with regard to bioenergy, biofuels, and bioproducts (Table 1.1) [110]. Simultaneously, the plan Biomass Technology in the United States has been published [111] in which research, development, and construction of biorefinery

Table 1.1 The US national vision goals for biomass technologies by the Biomass Technical Advisory Committee [110]. Year

Current

2010

2020

2030

BioPower (BioEnergy) Biomass share of electricity and heat demand in utilities and industry

2.8% (2.7 quad) a)

4% (3.2 quad)

5% (4.0 quad)

5% (5.0 quad)

BioFuels 0.5% Biomass share of demand for (0.15 quad) transportation fuels

4% (1.3 quad)

10% (4.0 quad)

20% (9.5 quad)

BioProducts 5% Share of target chemicals that are biobased

12%

18%

25%

a)

1 quad = 1 quadrillion BTU = 1 German billiarde BTU; BTU = British thermal unit; 1 BTU = 0.252 kcal, 1 kW = 3413 BTU, 1 kcal = 4.186 kJ

1.3 Situation

demonstration plants are determined. Research and development are necessary to: 1. increase scientific understanding of biomass resources and improve the tailoring of those resources; 2. improve sustainable systems to develop, harvest, and process biomass resources; 3. improve efficiency and performance in conversion and distribution processes and technologies for development of a host of biobased products and 4. create the regulatory and market environment necessary for increased development and use of biobased products. The Biomass Advisory Committee has established specific research and development objectives for feedstock production research. Target crops should include oil and cellulose-producing crops that can provide optimum energy content and usable plant components. Currently, however, there is a lack of understanding of plant biochemistry and inadequate genomic and metabolic information about many potential crops. Specific research to produce enhanced enzymes and chemical catalysts could advance biotechnology capabilities. 1.3.4 Vision and Goals and Plan for Biomass Technology in the European Union and Germany

In Europe there are already regulations about substitution of nonrenewable resources by biomass in the field of biofuels for transportation [120] and the “Renewable energy law” of 2000 [121]. According to the EC Directive “On the promotion of the use of biofuels” the following products are regarded as “biofuels”: (a) “bioethanol”, (b) “biodiesel”, (c) “biogas”, (d) “biomethanol”, (e) “biodimethyl ether”, (f) “bio-ETBE (ethyl tertiary-butyl ether)” on the basis of bioethanol, (g) “bio-MTBE (methyl tertiary butyl ether)” on the basis of biomethanol, and (h) “synthetic biofuels”, (i) “biohydrogen”, (j) pure vegetable oil Member States of the EU have been requested to define national guidelines for a minimum amounts of biofuels and other renewable fuels (with a reference value of 2% by 2005 and 5.75% by 2010 calculated on the basis of energy content of all petrol and diesel fuels for transport purposes). Table 1.2 summarizes this goal of the EU and also those of Germany with regard to establishment of renewable energy and biofuel [122, 123]. Today there are no guidelines concerning “biobased products” in the European Union and in Germany. After passing directives relating to bioenergy and biofuels, however, such a decision is on the political agenda. The “biofuels” directive already includes ethanol, methanol, dimethyl ether, hydrogen, and biomass pyrolysis which are fundamental product lines of the future biobased chemical industry.

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1 Biorefinery Systems – An Overview Table 1.2 Targets of the EU and Germany with regard to the introduction of technologies based on renewable resources. Year

2001

2005

2010

2020–2050

Bioenergy 7.5% Share of wind power, photovoltaics, biomass and geothermal electricity and heat demand in utilities and industry Biofuels 1.4% Biomass share of demand in transportation fuels (petrol and diesel fuels)

–

12.5%

26% (2030) 58% (2050)

2.8%

5.75%

20% (2020)

Biobased Products 8% Share of target chemicals that are biobased

–

–

–

In the year 2003, an initiative group called “Biobased Industrial Products” consisting of members from industry, small and middle-class businesses, and research, and development facilities met and formulated a strategy paper, called “BioVision 2030” [124]. This strategy paper has been included in the resolution of the German Government (Deutscher Bundestag) on the topic “Accomplish basic conditions for the industrial utilization of renewable resources in Germany” [125]. An advisory committee consisting of members of the chemical industry, related organizations, research facilities, and universities has been established to generate a plan concerning the formulation of the objectives for the third column, bio-products in Europe (Table 1.2) [126].

1.4 Principles of Biorefineries 1.4.1 Fundamentals

Biomass, similar to petroleum, has a complex composition. Its primary separation into main groups of substances is appropriate. Subsequent treatment and processing of those substances lead to a whole range of products. Petrochemistry is based on the principle of generating simple to handle and well defined chemically pure products from hydrocarbons in refineries. In efficient product lines, a system based on family trees has been built, in which basic chemicals, intermediate products, and sophisticated products are produced. This principle of petroleum refineries must be transferred to biorefineries. Biomass contains the synthesis performance of the nature and has different C : H : O : N ratio from

1.4 Principles of Biorefineries

petroleum. Biotechnological conversion will become, with chemical conversion, a big player in the future (Fig. 1.3). Thus biomass can already be modified within the process of genesis in such a way that it is adapted to the purpose of subsequent processing, and particular target products have already been formed. For those products the term “precursors” is used. Plant biomass always consists of the basic products carbohydrates, lignin, proteins, and fats, and a variety of substances such as vitamins, dyes, flavors, aromatic essences of very different chemical structure. Biorefineries combine the essential technologies which convert biological raw materials into the industrial intermediates and final products (Fig. 1.4). A technically feasible separation operation, which would enable separate use or subsequent processing of all these basic compounds, is currently in its initial stages only. Assuming that of the estimated annual production of biomass by biosynthesis of 170 billion tons 75% is carbohydrates, mainly in the form of cellulose, starch, and saccharose, 20% lignin, and only 5% other natural compounds such as fats (oils), proteins, and other substances [127], the main attention should first be focused on efficient access to carbohydrates, and their subsequent conversion to chemical bulk products and corresponding final products. Glucose, accessible by microbial or chemical methods from starch, sugar, or cellulose, is, among other things, predestined for a key position as a basic chemical, because a broad range of biotechnological or chemical products is accessible from glucose. For starch the advantage of enzymatic compared with chemical hydrolysis is already known [128]. For cellulose this is not yet realized. Cellulose-hydrolyzing enzymes can only act effectively after pretreatment to break up the very stable lignin/cellulose/

Fig. 1.3 Comparison of the basic-principles of the petroleum refinery and the biorefinery.

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Fig. 1.4 Providing code-defined basic substances (via fractionation) for development of relevant industrial product family trees [78, 79].

Fig. 1.5 Possible schematic diagram of biorefinery for precursor-containing biomass with preference for carbohydrates [78, 79].

hemicellulose composites [129]. These treatments are still mostly thermal, thermomechanical, or thermochemical, and require considerable input of energy. The arsenal for microbial conversion of substances from glucose is large, and the reactions are energetically profitable. It is necessary to combine degradation


processes via glucose to bulk chemicals with the building processes to their subsequent products and materials (Fig. 1.5). Among the variety of microbial and chemical products possibly accessible from glucose, lactic acid, ethanol, acetic acid, and levulinic acid, in particular, are favorable intermediates for generation of industrially relevant product family trees. Here, two potential strategies are considered: first, development of new, possibly biologically degradable products (follow-up products from lactic and levulinic acids) or, second, entry as intermediates into conventional product lines (acrylic acid, 2,3-pentanedione) of petrochemical refineries [78]. 1.4.2 Definition of the Term “Biorefinery”

The young working field “Biorefinery Systems” in combination with “Biobased Industrial Products” is, in various respects, still an open field of knowledge. This is also reflected in the search for an appropriate description. A selection is given below. The term “Green Biorefinery” was been defined in the year 1997 as: “Green biorefineries represent complex (to fully integrated) systems of sustainable, environmentally and resource-friendly technologies for the comprehensive (holistic) material and energetic utilization as well as exploitation of biological raw materials in form of green and residue biomass from a targeted sustainable regional land utilization” [73]. The original term used in Germany “complex construction and systems” was substituted by “fully integrated systems”. The US Department of Energy (DOE) uses the following definition [130]: “A biorefinery is an overall concept of a processing plant where biomass feedstocks are converted and extracted into a spectrum of valuable products. Based on the petrolchemical refinery.” The American National Renewable Energy Laboratory (NREL) published the definition [131]: “A biorefinery is a facility that integrates biomass conversion processes and equipment to produce fuels, power, and chemicals from biomass. The biorefinery concept is analogous to today’s petroleum refineries, which produce multiple fuels and products from petroleum. Industrial biorefineries have been identified as the most promising route to the creation of a new domestic biobased industry.” There is an agreement about the objective, which is briefly defined as: “Developed biorefineries, so called “phase III-biorefineries” or “generation III-biorefineries”, start with a biomass–feedstock-mix to produce a multiplicity of most various products by a technologies-mix” [74] (Fig. 1.6). An example of the type “generation-I biorefinery” is a dry milling ethanol plant. It uses grain as a feedstock, has a fixed processing capability, and produces a fixed amount of ethanol, feed co-products, and carbon dioxide. It has almost no flexibility in processing. Therefore, this type can be used for comparable purposes only. An example of a type “generation-II biorefinery” is the current wet milling technology. This technology uses grain feedstock, yet has the capability to pro-

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Fig. 1.6 Basic principles of a biorefinery (generation III biorefinery) [78].

duce a variety of end products depending on product demand. Such products include starch, high-fructose corn syrup, ethanol, corn oil, plus corn gluten feed, and meal. This type opens numerous possibilities to connect industrial product lines with existing agricultural production units. “Generation-II biorefineries” are, furthermore, plants like NatureWorks PLA facility [25] (Sections 1.2 and 1.3.1) or ethanol biorefineries, for example Iogen’s wheat straw to ethanol plant [132]. Third generation (generation-III) and more advanced biorefineries have not yet been built but will use agricultural or forest biomass to produce multiple products streams, for example ethanol for fuels, chemicals, and plastics.

1.4.3 The Role of Biotechnology

The application of biotechnological methods will be highly important with the development of biorefineries for production of basic chemicals, intermediate chemicals, and polymers [133–135]. The integration of biotechnological methods must be managed intelligently in respect of physical and chemical conversion of the biomass. Therefore the biotechnology cannot remain limited to glucose from sugar plants and starch from starch-producing plants. One main objective is the economic use of biomass containing lignocellulose and provision of glucose in the family tree system. Glucose is a key chemical for microbial processes. The preparation of a large number of family tree-capable basic chemicals is shown in subsequent sections.


1.4.3.1 Guidelines of Fermentation Section within Glucose-product Family Tree Among the variety of chemical products, and derivatives of these, accessible microbially from glucose a product family tree can be developed, for example (C-1)-chemicals methane, carbon dioxide, methanol; (C-2)-chemicals ethanol, acetic acid, acetaldehyde, ethylene, (C-3)-chemicals lactic acid, propanediol, propylene, propylene oxide, acetone, acrylic acid, (C-4)-chemicals diethyl ether, acetic acid anhydride, malic acid, vinyl acetate, n-butanol, crotonaldehyde, butadiene, 2,3-butanediol, (C-5)-chemicals itaconic acid, 2,3-pentane dione, ethyl lactate, (C-6)-chemicals sorbic acid, parasorbic acid, citric acid, aconitic acid, isoascorbinic acid, kojic acid, maltol, dilactide, (C-8)-chemicals 2-ethyl hexanol (Fig. 1.7). Guidelines are currently being developed for the fermentation section of a biorefinery. The question of efficient arrangement of the technological design for production of bulk chemicals needs an answer. Considering the manufacture of lactic acid and ethanol, the basic technological operations are very similar. Selection of biotechnologically based products from biorefineries should be done in a way such that they can be produced from the substrates glucose or pentoses. Furthermore the fermentation products should be extracellular. Fermenters should have batch, feed batch, or CSTR design. Preliminary product recovery should require steps like filtration, distillation, or extraction. Final product recovery and purification steps should possibly be product-unique. In addi-

Fig. 1.7 Biotechnological sugar-based product family tree.

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tion, biochemical and chemical processing steps should be advantageously connected. Unresolved questions for the fermentation facility include: 1. whether or not the entire fermentation facility can/should be able to change from one product to another; 2. whether multiple products can be run in parallel, with shared use of common unit operations; 3. how to manage scheduling of unit operations; and 4. how to minimize in-plant inventories, while accommodating necessary change-overs between different products in the same piece of equipment [95]. 1.4.4 Building Blocks, Chemicals and Potential Screening

A team from Pacific Northwest National Laboratory (PNNL) and NREL submitted a list of twelve potential biobased chemicals [98]. A key area of the investigation as biomass precursors, platforms, building blocks, secondary chemicals, intermediates, products and uses (Fig. 1.8). The final selection of 12 building blocks began with a list of more than 300 candidates. A shorter list of 30 potential candidates was selected by using an iterative review process based on the petrochemical model of building blocks, chemical data, known market data, properties, performance of the potential candidates, and previous industry experience of the team at PNNL and NREL. This list of 30 was ultimately reduced to 12 by examining the potential markets for the building blocks and their derivatives and the technical complexity of the synthetic pathways. The reported block chemicals can be produced from sugar by biological and chemical conversions. The building blocks can be subsequently converted to several high-value biobased chemicals or materials. Building-block chemicals, as considered for this analysis, are molecules with multiple functional groups with the potential to be transformed into new families of useful molecules. The twelve sugar-based building blocks are 1,4-diacids (succinic, fumaric, and malic), 2,5-furandicarboxylic acid, 3-hydroxypropionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, and xylitol/arabinitol [98]. A second-tier group of building blocks was also identified as viable candidates. These include gluconic acid, lactic acid, malonic acid, propionic acid, the triacids citric and aconitic, xylonic acid, acetoin, furfural, levuglucosan, lysine, serine, and threonine. Recommendations for moving forward include: · examining top value products from biomass components, for example aromatic compounds, polysaccharides, and oils; · evaluating technical challenges in more detail in relation to chemical and biological conversion; and · increasing the suites of potential pathways to these candidates.

1.5 Biorefinery Systems and Design

Fig. 1.8 Model of a biobased product flow-chart for biomass feedstock [98].

No products simpler than syngas were selected. For the purposes of this study hydrogen and methanol comprise the best short-term prospects for biobased commodity chemical production, because obtaining simple alcohols, aldehydes, mixed alcohols, and Fischer-Tropsch liquids from biomass is not economically viable and requires additional development [98].

1.5 Biorefinery Systems and Design 1.5.1 Introduction

Biobased products are prepared for a usable economic use by meaningful combination of different methods and processes (physical, chemical, biological, and thermal). It is therefore necessary that basic biorefinery technologies are developed. For this reason profound interdisciplinary cooperation of various disciplines in research and development is inevitable. It seems reasonable, therefore, to refer to the term “biorefinery design”, which means: “bringing together well founded scientific and technological basics, with similar technologies, products, and product lines, inside biorefineries”. The basic conversions of each biorefinery can be summarized as follows. In the first step, the precursor-containing biomass is separated by physical methods. The main products (M1–Mn) and the by-products (B1–Bn) will subsequently be subjected to microbiological or chemi-

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cal methods. The follow-up products (F1–Fn) of the main and by-products can also be converted or enter the conventional refinery (Fig. 1.6). Currently four complex biorefinery systems are used in research and development: 1. the “lignocellulosic feedstock biorefinery” which use “nature-dry” raw material, for example cellulose-containing biomass and waste; 2. the “whole crop biorefinery” which uses raw material such as cereals or maize; 3. the “green biorefineries” which use “nature-wet” biomasses such as green grass, alfalfa, clover, or immature cereal [78, 79]; and 4. the “biorefinery two platforms concept” includes the sugar platform and the syngas platform [98]. 1.5.2 Lignocellulosic Feedstock Biorefinery

Among the potential large-scale industrial biorefineries the lignocellulose feedstock (LCF) biorefinery will most probably be pushed through with the greatest success. On the one side the raw material situation is optimum (straw, reed, grass, wood, paper-waste, etc.), on the other side conversion products have a good position on both the traditional petrochemical and future biobased product market. An important point for utilization of biomass as chemical raw material is the cost of raw material. Currently the cost of corn stover or straw is 30 US$/ ton and that of corn is 110 US$/ton (3 US$/ bushel; US bushel corn =25.4012 kg = 56 lb) [136]. Lignocellulose materials consist of three primary chemical fractions or precursors: · hemicellulose/polyoses, sugar polymers of, predominantly, pentoses; · cellulose, a glucose polymer; and · lignin, a polymer of phenols (Fig. 1.9). The lignocellulosic biorefinery-regime is distinctly suitable for genealogical compound trees. The main advantages of this method is that the natural structures and structural elements are preserved, the raw materials are inexpensive, and large product varieties are possible (Fig. 1.10). Nevertheless there is still a demand for development and optimization of these technologies, e.g. in the field of separation of cellulose, hemicellulose and lignin, and utilization of the lignin in the chemical industry.

Fig. 1.9 A possible general equation for conversion at the LCF biorefinery.


Fig. 1.10 Lignocellulosic feedstock biorefinery.

An overview of potential products of an LCF biorefinery is shown in Fig. 1.11. In particular furfural and hydroxymethylfurfural are interesting products. Furfural is a starting material for production of Nylon 6,6 and Nylon 6. The original process for production of Nylon-6,6 was based on furfural (see also Section 1.2.2). The last of these production plants was closed in 1961 in the USA, for economic reasons (the artificially low price of petroleum). Nevertheless the market for Nylon 6 is huge.

Fig. 1.11 Products of a lignocellulosic feedstock biorefinery (LCF-biorefinery, Phase III) [78, 79, 95].

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There are, however, still some unsatisfactory aspects of the LCF, for example the utilization of lignin as fuel, adhesive, or binder. Unsatisfactory because the lignin scaffold contains substantial amounts of mono-aromatic hydrocarbons, which, if isolated in an economically efficient way, could add a significant increase in value to the primary processes. It should be noticed there are no natural enzymes capable of splitting the naturally formed lignin into basic monomers as easily as is possible for natural polymeric carbohydrates or proteins [137]. An attractive process accompanying the biomass-nylon-process is the already mentioned hydrolysis of the cellulose to glucose and the production of ethanol. Some yeasts cause disproportionation of the glucose molecule during their generation of ethanol from glucose, which shifts almost all its metabolism into ethanol production, making the compound obtainable in 90% yield (w/w; with regard to the chemical equation for the process). On the basis of recent technology a plant has been conceived for production of the main products furfural and ethanol from LC feedstock from West Central Missouri (USA). Optimal profitability can be achieved with a daily consumption of approximately 4360 tons of feedstock. The plant produces 47.5 million gallon of ethanol and 323,000 tons of furfural annually [74]. Ethanol can be used as a fuel additive. It is also a connecting product to the petrochemical refinery, because it can be converted into ethene by chemical methods and it is well-known that ethene is at the start of a series of large-scale technical chemical syntheses for production of important commodities such as polyethylene or poly(vinyl acetate). Other petrochemically produced substances, for example hydrogen, methane, propanol, acetone, butanol, butandiol, itaconic acid, and succinic acid, can also be manufactured by microbial conversion of glucose [138, 139]. 1.5.3 Whole-crop Biorefinery

Raw materials for the “whole crop biorefinery” are cereals such as rye, wheat, triticale, and maize. The first step is mechanical separation into corn and straw, approximately 10 and 90% (w/w), respectively [140]. Straw is a mixture of chaff, nodes, ears, and leaves. The straw is an LC feedstock and may further be processed in a LCF biorefinery. There is the possibility of separation into cellulose, hemicellulose, and lignin and their further conversion in separate product lines which are shown in the LCF biorefinery. The straw is also a starting material for production of syngas by pyrolysis technology. Syngas is the basic material for synthesis of fuels and methanol (Fig. 1.13). The corn may either be converted into starch or used directly after grinding to meal. Further processing may be conducted by four processes – breaking up, plasticization, chemical modification, or biotechnological conversion via glucose. The meal can be treated and finished by extrusion into binder, adhesives, and filler.


Fig. 1.12 Whole-crop biorefinery based on dry milling.

Fig. 1.13 Products from a whole-crop biorefinery [78, 79].

Starch can be finished by plasticization (co- and mix-polymerization, compounding with other polymers), chemical modification (etherification into carboxymethyl starch; esterification and re-esterification into fatty acid esters via acetic starch; splitting reductive amination into ethylenediamine, etc., hydrogenative splitting into sorbitol, ethylene glycol, propylene glycol, and glycerin), and biotechnological conversion into poly-3-hydroxybutyric acid [69, 76, 92, 93, 141].

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An alternative to traditional dry fractionation of mature cereals into grains and straw only was been developed by Kockums Construction (Sweden), which later became Scandinavian Farming. In this crop-harvest system whole immature cereal plants are harvested. The whole harvested biomass is conserved or dried for long-term storage. When convenient, it can be processed and fractionated into kernels, straw chips of internodes, and straw meal (leaves, ears, chaff, and nodes) (see also green biorefinery). Fractions are suitable as raw materials for the starch polymer industry, the feed industry, the cellulose industry, and particle board producers, gluten can be used by the chemical industry and as a solid fuel. Such dry fractionation of the whole crop to optimize the utilization of all botanical components of the biomass has been described [142, 143]. A biorefinery and its profitability has been described elsewhere [144]. One expansion of the product lines in grain processing is the “whole crop wet mill-based biorefinery”. The grain is swelled and the grain germ is pressed, releasing high-value oils. The advantages of whole-crop biorefinery based on wet milling are that production of natural structures and structure elements such as starch, cellulose, oil, and amino acids (proteins) are kept high yet well known basic technology and processing lines can still be used. High raw material costs and, for industrial utilization, the necessary costly source technology are the disadvantages. Some of the products formed command high prices in, e.g., the pharmaceutical and cosmetics industries (Figs. 1.14 and 1.15). The basic biorefinery technology of corn wet mills used 11% of the US corn harvest in 1992, made products worth $7.0 billion, and employed almost 10,000 people [1 a]. Wet milling of corn yields corn oil, corn fiber, and corn starch. The starch products of the US corn wet milling industry are fuel alcohol (31%), high-fructose corn syrup (36%), starch (16%), and dextrose (17%). Corn wet milling also

Fig. 1.14 Whole-crop biorefinery, wet-milling.


Fig. 1.15 Products from a whole-crop wet mill-based biorefinery.

generates other products (e.g. gluten meal, gluten feed, oil) [62]. An overview about the product range is shown in Fig. 1.15. 1.5.4 Green Biorefinery

Green biorefineries are also multi-product systems and furnish cuts, fractions, and products in accordance with the physiology of the corresponding plant material, which maintains and utilizes the diversity of syntheses achieved by nature. Most green biomass is green crops, for example grass from cultivation of permanent grass land, closure fields, nature reserves, or green crops, such as lucerne, clover, and immature cereals from extensive land cultivation. Green crops are a natural chemical factory and food plant and are primarily used as forage and as a source of leafy vegetables. A process called wet-fractionation of green biomass, green crop fractionation, can be used for simultaneous manufacture of both food and non food items [145]. Scientists in several countries, in Europe and elsewhere, have developed green crop fractionation [146–148]. Green crop fractionation is now studied in approximately 80 countries [149]. Several hundred temperate and tropical plant species have been investigated for green crop fractionation [148, 150, 151] and more than 300,000 higher plants species have still to be investigated. The subject has been covered by several reviews [73, 146–148, 151–155]. Green biorefineries can,

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by fractionation of green plants, process from a few tonnes of green crops per hour (farm scale process) to more than 100 tonnes per hour (industrial scale commercial process). Careful wet fractionation technology is used as a first step (primary refinery) to isolate the contents of the green crop (or humid organic waste goods) in their natural form. Thus, they are separated into a fiber-rich press cake (PC) and a nutrient-rich green juice (GJ). The advantages of the green biorefinery are high biomass profit per hectare, good coupling with agricultural production, and low price of the raw materials. Simple technology can be used and there is good biotechnical and chemical potential for further conversion (Fig. 1.16). Rapid primary processing or use of preservation methods, for example silage production or drying, are necessary, both for the raw materials and the primary products, although each method of preservation changes the content of the materials. In addition to cellulose and starch, the press cake contains valuable dyes and pigments, crude drugs and other organic compounds. The green juice contains proteins, free amino acids, organic acids, dyes, enzymes, hormones, other organic substances, and minerals. Application of the methods of biotechnology results in conversion, because the plant water can simultaneously be used for further treatment. In addition, the lignin–cellulose composite is not as intractable as lignocellulose-feedstock materials. Starting from green juice the main focus is directed toward products such as lactic acid and its derivatives, amino acids, ethanol, and proteins. The press cake can be used for production of green feed pellets, as raw material for production of chemicals, for example levulinic acid, and for conversion to syngas and hydrocarbons (synthetic biofuels). The residues of substantial conversion are suitable for production of biogas combined with generation of heat and electricity (Fig. 1.17). Reviews have been published on the concepts, contents, and goals of the green biorefinery [73, 75, 119].

Fig. 1.16 A “green biorefinery” system.


Fig. 1.17 Products from the green biorefinery. In this illustration a green biorefinery has been combined with a green crop-drying plant [78, 79].

1.5.5 Two-platform Concept and Syngas

The “two-platform concept” is one which uses biomass consisting, on average, of 75% carbohydrates which can be standardized as a “intermediate sugar platform”, as a basis for further conversion, but which can also be converted thermochemically into synthesis gas and the products made from this. The “sugar platform” is based on biochemical conversion processes and focuses on fermentation of sugars extracted from biomass feedstocks. The “syngas platform” is based on thermochemical conversion processes and focuses on the gasification of biomass feedstocks and by-products from conversion processes [63, 98, 131]. In addition to the gasification other thermal and thermochemical biomass conversion methods have also been described – hydrothermolysis, pyrolysis, thermolysis, and burning. The application chosen depends on the water content of biomass [156]. The gasification and other thermochemical conversions concentrate on utilization of the precursor carbohydrates and their intrinsic carbon and hydrogen content. The proteins, lignin, oils and lipids, amino acids, and other nitrogen and sulfur-containing compounds occurring in all biomass are not taken into account (Fig. 1.18). The advantage of this concept is that the production of energy, fuels, and biobased products is possible using only slightly complex and low-tech technology,

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Fig. 1.18 The sugar platform and the syngas platform [131].

for example saccharification and syngas technology. The sugar platform also enables access to a huge range of family tree-capable chemicals (Figs. 1.7 and 1.8). In-situ conversion of biomass feedstock into liquid or gas could be one way of using existing infrastructure (developed pipe network), but with the disadvantages of the need to remove hetero-atoms (O, N, S) and minerals present in the biomass and the highly endothermic nature of the syngas process [157]. Currently, production of simple alcohols, aldehydes, mixed alcohols, and FischerTropsch liquids from biomass is not economically viable and additional developments are required [98] (Fig. 1.19).

1.6 Outlook and Perspectives

Biorefineries are the production plants in which biomass is economically and ecologically converted to chemicals, materials, fuels, and energy. For successful development of “industrial biorefinery technologies” and “biobased products” several problems must be solved. It will be necessary to increase the production of substances (cellulose, starch, sugar, oil) from basic biogenic raw materials and to promote the introduction and establishment of biorefinery demonstration plants. Ecological transport of biomass must also be developed, for example utilization of already developed pipe networks. Another important aspect is committing chemists, biotechnologists, and engineers to the concept of biobased products and biorefinery systems and promoting the combined biotechnological and chemical conversion of substances. Last, but not least, the development of systematic approaches to new synthesis and technologies is required to meet the sustainable principles of “ideal synthesis” and “principles of green chemistry and process engineering” [159–161].

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Fig. 1.19 Syngas-based product family tree [157, 158].

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2004; http://www.bio.org/ind/pubs/ cleaner2004/cleanerReport.pdf Dti Global Watch Mission Report: Impact of the industrial biotechnology on sustainability of the manufacturing base – the Japanese Perspective, 2004 Dale, B.; Encyclopedia of Physical Science and Technology, Third Edition, Volume 2: (2002) 141–157 Ringpfeil M.; Biobased Industrial Products and Biorefinery Systems – Industrielle Zukunft des 21. Jahrhunderts? [2001, www.biopract.de] Zeikus, J. G.; Jain, M. K.; Elankovan, P.; Biotechnology of succinic acid production and markets for derived industrial products. Appl. Microbiol. Biotechnol., 51 (1999) 545–552 Vorlop, K.-D.; Wilke, Th.; Prüße, U.; Biocatalytic and catalytic routes for the production of bulk and fine chemicals from renewable resources: [in this book], 2005 Wurz, O.; Zellstoff- und Papierherstellung aus Einjahrespflanzen [Eduard Roether Verlag, Darmstadt, 1960] Rossel C. E. V.; Mantellato, P. E.; Agnelli, A. M.; Nascimento, J.; Sugar-based Biorefinery – Technology for an integrated production of Poly(3-hydroxybutyrate), Sugar and Ethanol, in this book, 2005 Rexen, F.; New industrial application possibilities for straw. Documentation of Svebio Phytochemistry Group (Danish) [Fytokemi i Norden, Stockholm, Sweden, 1986-03-06, 1986] 12 Coombs, J.; Hall, K.; The potential of cereals as industrial raw materials: Legal technical, commercial considerations; In: Cereals – Novel Uses And Processes [G.M. Campbell, C. Webb, and S.L. McKee (eds.), Plenum Publ. Corp., New York, USA, 1997] 1–12. Audsley, E.; Sells, J. E.; Determining the profitability of a whole crop biorefinery; In: Cereals – Novel Uses and Processes [G. M. Campbell, C. Webb, and S. L. McKee (eds.), Plenum Publ. Corp.; New York, USA; 1997] 191–294 Carlsson, R.; Sustainable primary production – Green crop fractionation: Effects of species, growth conditions, and

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physiological development; In: Handbook of Plant and Crop Physiology [M. Pessarakli (ed.), Marcel Dekker Inc., N.Y., USA, 1994] 941–963. Pirie, N. W.; Leaf Protein – Its agronomy, preparation, quality, and use [Blackwell Scientific Publications, Oxford/Cambridge, UK, 1971] Pirie, N. W.; Leaf Protein and Its ByProducts in Human and Animal Nutrition [Cambridge Univ. Press, UK, 1987] Carlsson, R.; Status quo of the utilization of green biomass. In: The Green Biorefinery, Proceedings of 1st International Green Biorefinery Conference, Neuruppin, Germany, 1997 [S. Soyez, B. Kamm, M. Kamm (eds.), Verlag GÖT, Berlin, 1998, ISBN 3-929672-065] Carlsson, R.; Food and non-food uses of immature cereals; In: Cereals – Novel Uses and Processes [G. M. Campbell, C. Webb, S. L. McKee (eds), Plenum Publ. Corp., New York, USA, 1997], pp. 159–167 Carlsson, R.; Leaf protein concentrate from plant sources in temperate climates. In: Leaf Protein Concentrates [L. Telek, H. D. Graham (eds.), AVI Publ. Co., Inc., Westport, Conn., USA, 1983] 52–80 Telek, L.; Graham, H. D. (eds.); Leaf Protein Concentrates [AVI Publ., Co., Inc., Westport, Conn., USA, 1983] Wilkins, R. J. (ed.); Green Crop Fractionation [The British Grassland Society, c/o Grassland Research Institute, Hurley, Maidenhead, SL6 5LR, UK, 1977] Tasaki, I. (ed.); Recent Advances in Leaf Protein Research, Proc. 2nd Int. Leaf Protein Res. Conf. [Nagoya, Japan, 1985] Fantozzi, P. (ed.); Proc. 3rd Int. Leaf Protein Res. Conf., Pisa-Perugia-Viterbo, Italy, 1989 Singh, N. (ed.); Green Vegetation Fractionation Technology [Science Publ. Inc., Lebanon, NH 03767, USA, 1996] Okkerse, C.; van Bekkum, H.; From fossil to green. Green Chemistry, 4 (1999) 107–114

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1 Biorefinery Systems – An Overview 157 Lancaster, M.; The Syngas Economy;

160 Lancaster, M.; The Biorefinery. In:

In: Green Chemistry [The Royal Society of Chemistry, Cambridge, UK, 2002, ISBN: 0-85404-620-8] 205 158 Matlack, A. S.; The Use of Synthesis Gas from Biomass; In: Introduction to Green Chemistry [Marcel Dekker, New York, 2001, ISBN: 0824704118] 369 159 Clark, J. H.; Green Chemistry. Challenges and opportunities. Green Chemistry, 1 (1999) 1–8

Green Chemistry [The Royal Society of Chemistry, Cambridge, UK, 2002, ISBN: 0-85404-620-8] 207 161 Anastas, P. T.; Warner, J. C.; Green Chemistry. Theory and Practice [Oxford University Press, New York, 1998]


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2 Biomass Refining Global Impact – The Biobased Economy of the 21st Century Bruce E. Dale and Seungdo Kim

2.1 Introduction

We are in the early phases of a truly historic transition – from an economy based largely on petroleum to a more diversified economy in which renewable plant biomass will become a significant feedstock for both fuel and chemical production. The development of the petroleum refining industry over the past century provides many instructive lessons for the future biobased economy and also many reasons for supposing that the new biobased economy will be different from the hydrocarbon economy in crucial ways. This paper explores the similarities and differences between the petroleum refining and biorefining industries in a historical context and the implications of these similarities and differences for the biobased economy in the long term. We assume a mature biobased economy – as the petroleum economy is mature today – and from that assumption we extrapolate likely features of the mature biobased economy. Among the technical, social, and economic forces that will drive the mature biobased economy are: 1. yield (using the whole “barrel of biomass”); 2. gradual diversification of biobased products, probably starting with higher-value chemical products and trending toward fuels over time; 3. the great diversity of biomass resources combined with their considerable compositional similarity; 4. possible/likely limits on agricultural productivity; 5. integration of biorefining and agricultural ecosystems in a local social and political context (the “all biomass is local” paradigm); and 6. the sustainability of the mature biobased economy and its most important underlying resource – productive soils.


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2 Biomass Refining Global Impact – The Biobased Economy of the 21st Century

2.2 Historical Outline 2.2.1 Background and Development of the Fossil Carbon-processing Industries

Materials that contain carbon, including the primary commercial fuels, virtually all food and fiber products, and most commodity chemicals, pharmaceuticals, and nondurable manufactured goods, play an integral role in the world economy [1]. Carbon-rich raw materials originate through the process of photosynthesis in which plants and some bacteria use solar energy to convert atmospheric carbon dioxide into organic substances including simple sugars, polysaccharides, amino acids, proteins, lipids, and aromatic compounds such as lignin. Some carbon-rich raw materials are derived from fossil sources such as petroleum, coal, and natural gas. Fossil sources result from photosynthesis in ancient times and comprise a large, but nonrenewable, reserve. In contrast, present day photosynthesis provides a potentially renewable source of carbon. Renewable agricultural and forestry resources have been used since ancient times as fuels and raw materials for numerous products. Starting in the late 1700s, at the beginning of the Industrial Revolution, coal began to displace wood as a fuel. In the mid 1800s the large-scale processing of petroleum to fuel and chemical products began, taking away some markets from coal and many more from renewable carbon sources. In the last half of the 20th century, uses of natural gas began to expand greatly, both as a fuel and as a feedstock for chemical production [Ref. 1, Chapter 1]. Currently petroleum provides 40% of the United States’ and 35% of the world’s direct primary energy supply whereas plant material in all forms provides approximately 10% of the world’s energy supply. Although remaining supplies of petroleum, coal, and natural gas are very large, it is, nonetheless, obvious that the world is using these essentially nonrenewable resources at a huge and growing rate. Natural processes are simply not replacing fossil carbon at even a minute fraction of the rate at which we are using it. For example, some experts believe that the peak rate of production of conventional oil will occur within this decade whereas others predict this will occur before mid-century [2, 3]. After that point, conventional, inexpensive oil production will irreversibly decline. Natural gas production will peak later than conventional oil, but will still begin permanent decline within the next few decades. Although other sources of petroleum exist (e.g., tar sands, deep-water oil), they will be more difficult and much more expensive to produce. Whatever the exact date of peak oil production, we are approaching a major change in the way we must provide energy and other services to the world’s population. This paper addresses two related questions: 1. is it realistic to believe that renewable sources of carbon can provide a large share of the energy and other services currently provided by fossil carbon, particularly petroleum?


2. what might be the salient characteristics of a mature biobased economy producing not only food and fiber but also fuels and chemicals? We will touch somewhat on question 1 but will treat question 2 more extensively. An extensive ongoing project called the Role of Biomass in America’s Energy Future (RBAEF Project) sponsored by the US Department of Energy and the Energy Foundation is attempting to address the first question with considerable breadth and depth. The RBAEF Project is led by Professor Lee R. Lynd of Dartmouth College (Hanover, New Hampshire, USA), Dr John Sheehan of the National Renewable Energy Laboratory (Golden, Colorado, USA), and Mr Nathanael Greene of the Natural Resources Defense Council (New York, New York, USA). We encourage all readers of this article to make themselves aware of the findings of the RBAEF team as these findings become available. Dr Lynd provides an outline of some of the expected results in another article in this volume. 2.2.2 The Existing Biobased Economy: Renewable Carbon

Renewable carbon-based raw materials are produced in agriculture, silviculture and microbial systems, including managed and unmanaged systems. Although estimates are necessarily imprecise, the total amount of new carbon-based plant material fixed yearly by terrestrial plants is of the order of 100 ´ 1012 kg (assuming biomass is on average 50% by weight carbon) [4, 5]. This amount of plant material has an energy content (via heat of combustion) roughly five times the energy value of all forms of energy used worldwide and over ten times the energy content of all petroleum used worldwide [6]. Although this plant resource is dispersed, has competing uses, and, as a solid, is not in an ideal form for easy transporting and processing, the size of the renewable carbon resource and its associated energy content clearly suggest significant potential to provide raw material and energy services. The amount of new plant biomass dwarfs the use of fossil carbon to produce organic chemicals. For example, the United States produces about 100 ´ 109 kg fine, specialty, intermediate, and commodity organic chemicals each year, or approximately 0.1% of total world biomass production [7]. Less than 10% of this total is produced from renewable carbon [1]. The total mass of these organic chemicals is roughly equal to 40% of US production of corn grain, about twice the grain that the United States exports each year. In fact, there is pressure on the US (and the EU) from developing countries to reduce export and other subsidies of their agricultural products. As this occurs, more US grain may rather quickly find its way into fuel and chemical production. Already approximately 9 billion kg (3 billion gallons) annually of fuel ethanol are produced primarily from corn in the US, consuming approximately 12% of domestic corn production. There is, however, no reasonable expectation that grain will be able to pro-

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vide the approximately 750 billion kilograms per year of refined petroleum products used in the United States [8]. That quantity of renewable carbon must come from crop and forestry residues and energy crops (crops grown specifically to produce energy products) – not from food/feed grain. In addition to existing uses of renewable carbon to produce organic chemicals and fuels, renewable carbon sources provide about 90% of organic materials such as lumber and paper, natural fibers, and composites, cellulosics, and some proteins [Ref. 1, Chapter 2]. Finally, renewable carbon provides nearly all of our food and animal feed. There is no reasonable alternative to using renewable carbon to meet food/feed demand, although the efficiency with which this demand is met is certainly subject to change and innovation. We briefly explore some possible innovations for food and feed production in this paper – a much more complete discussion will be available in the RBAEF reports. In total, the organic chemical, fuels (solid, liquid, and gaseous), carbon-based materials, food, and feed components of the US domestic economy exceed US $ 2 trillion per year. Much of this economic activity is already based on renewable carbon. The question which naturally arises is: could the fraction of fuels and chemicals from renewable carbon be significantly increased without interfering with other essential uses of plant material? Before addressing this issue, we wish to point out two important facts that we will explore in more detail below. First, there are several largely unexplored alternatives for coproducing animal feed and human food with fuels. Doing so would make the whole enterprise more efficient, and very probably more economically competitive. Second, both US and European agriculture are currently constrained by demand, not by ability to produce. Respective national governments have been paying farmers not to produce to capacity for many years. If large new demands for renewable carbon were to arise, there is every reason to believe that much more plant material could be produced on the same or similar acreages. 2.2.3 Toward a Much Larger Biobased Economy

Given the amount of grain available in the US and the EU and the ability to supply very pure dextrose in large quantities for around US $ 0.20 per kg at corn wet mills, we suggest that material supply, convenience, and purity considerations favor dextrose derived from corn (and perhaps other grain) as a feedstock for organic chemical manufacture. The supply of grain dextrose is more than adequate to produce all organic chemicals that conceivably can be made from dextrose. Furthermore, corn yields are tending to increase with time. Given historical yield increases, each year US agriculture produces an additional 3.5 ´ 109 kg new dextrose equivalents. The biobased products industry will need to grow very rapidly even to consume the additional dextrose made available from new corn production. A switch from fossil carbon to renewable carbon for organic chemicals will occur as conversion technologies improve, conversion costs decline, and various barriers to entry are overcome.

2.3 Supplying the Biorefinery

In most situations it will be difficult for plant biomass to compete economically with coal as a solid fuel for stand alone electricity generation and we do not consider this case. However, electricity generation from biomass processing residues in an integrated processing facility producing liquid fuels and other products is very attractive. Biomass gasification to produce a natural gas substitute is also a much more attractive possibility than direct combustion, but likewise we do not treat it here because of space limitations. Gasification (and even direct combustion) seem most useful when applied to forest products and residues. US forest growth has exceeded harvest since the 1940s. Harvested timber, pulp, and paper have increased from private forestlands at the same time interest in preserving public forests in more or less “unmanaged” conditions has reduced the timber, pulp, and paper available from these public lands. Thus the land and other agricultural resources of the US seem more than adequate to satisfy current domestic and export demands for food, feed, and fiber and still produce ample raw materials for a much larger biobased economy. Because the US consumes a disproportionate amount of fuel and chemicals compared with the rest of the world, there is reason to hope that other countries can also provide much more of their fuel and chemical needs from renewable carbon sources. We offer further evidence below that this is so. The only situation for which biobased feedstock supply adequacy for materials and chemicals does not seem to be the obvious conclusion is a massive increase of liquid fuel production from renewable carbon. The remainder of this paper deals with the concept of liquid fuel production from renewable carbon (specifically agricultural crops and crop residues) in mature, integrated processing facilities called “biorefineries”.

2.3 Supplying the Biorefinery 2.3.1 What Raw Materials do Biorefineries Require and What Products Can They Make?

Although petroleum feedstocks vary somewhat in composition, their compositional variety is much less than for biomass feedstocks. Biomass compositional variety is both an advantage and disadvantage. Biomass feedstocks consist of grain, crop residues, oilseeds, sugar crops, forage crops, and a wide variety of woody crops. Figure 2.1 depicts typical compositions of some of these biomass feedstocks. The major components of biomass include carbohydrates (cellulose and hemicellulose for crop residues, forage crops and woody crops – called lignocellulosic materials, starch for grain, and primarily sucrose for sugar crops), lipids (fats, waxes and oils), proteins of many types, aromatic compounds (primarily lignin), and ash (non carbon minerals such as calcium, phosphorus, potassium, etc.). An advantage of biomass compositional variety is that biorefineries can make more classes of products than can petroleum refineries, thus providing addi-

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Fig. 2.1 Composition of several biomass species.

tional economic stability and opportunities for new product development. Biorefineries can also use a wider range of raw materials than can petroleum refineries. Among the products that might be produced from biomass are liquid transportation fuels (including both gasoline and diesel substitutes), electricity and steam, a tremendous variety of chemicals containing carbon, oxygen, hydrogen, and nitrogen and combinations of these elements, monomers and polymers, lubricants, adhesives, fertilizers and, significantly, animal feeds and human foods. Some of these products are summarized in Fig. 2.2 in their life cycle context as possible replacements for petroleum-based or petroleum-dependent products [9]. A disadvantage of biorefineries compared with petroleum refineries is that a relatively larger range of processing technologies is needed. This is particularly true for conversion and/or separation of the wider range of components of the renewable feedstock raw materials, as shown in Fig. 2.1. It is important to note in Fig. 2.2 that biorefineries will probably operate by first preprocessing (separating and reacting) the inlet raw materials to a relatively small range of intermediate products including carbohydrates, protein, syngas (mixtures of carbon monoxide, hydrogen and carbon dioxide) and a few other products. These intermediate products will then be upgraded by further reaction and separation steps to a very wide variety of final products. Some of these reaction and separation steps are well developed, others remain to be developed. In particular, the processing technologies to convert lignocellulosic materials economically and in high yield to carbohydrates and other products are not yet fully available. Once these and other processing technologies are developed and deployed, however, they will find use for a much wider variety and much more geographically dispersed set of renewable raw materials than is true for petroleum.

Fig. 2.2 Life cycle overview of biobased products [9].

2.3 Supplying the Biorefinery 47

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Prototype biorefineries already exist, including corn wet and dry mills, pulp and paper mills, and other renewable carbon-based processing facilities. Corn wet mills perhaps best exemplify many of the features that are likely to be found in mature biorefineries for large-scale fuel production. Wet mills are large, highly integrated facilities producing a wide range of chemical, biochemical, feed, food, and fuel products, as outlined in Fig. 2.2. Over 90% of the inlet corn leaves as value-added products (selling price per kilogram greater than corn feedstock) [Ref. 1, Chapter 3]. Corn wet mills have continued to add products with time, particularly higher value chemical/biochemical products. Similarly to petroleum refineries, wet mills alter their product mix to meet changing market conditions, including seasonal variations in demand. 2.3.2 Comparing Biomass Feedstock Costs with Petroleum Costs

Of course, the widely dispersed and renewable nature of biomass feedstocks is of little practical importance unless we can reasonably expect to convert these feedstocks to products that will compete economically with petroleum-derived products. Fortunately, this is an entirely reasonable and feasible goal. To support this statement, we note that competitive pressures and continually improving conversion technologies gradually force many high margin new products to eventually become mature, commodity products with narrow margins. This process has occurred with products as diverse as penicillin, Nylon and, of course, gasoline. Experience shows that approximately 60–70% of the cost of manufacturing commodity products depends on the cost of the raw materials from which these commodities are made [10, 11]. This is true for petroleum-derived commodities in particular, and explains the large swings in gasoline prices as crude oil prices change. We envisage a mature biorefining industry producing liquid biofuels to replace gasoline and diesel fuel. In such a mature biorefining industry the cost of manufacturing biofuels will also depend very highly on raw material cost. The question therefore arises: how does the cost of renewable plant biomass compare with the cost of petroleum? I am indebted to Professors Lee Lynd and Charles Wyman of Dartmouth College (Hanover, New Hampshire, USA) for suggesting the approach outlined in Fig. 2.3 below. Figure 2.3 shows the cost of plant biomass relative to the cost of petroleum on two different bases – cost per kilogram of material and cost per unit of energy. Three different horizontal lines are drawn representing different classes of plant biomass available at three different prices. Crop residues are valued at $ 20 per ton (US) in Fig. 2.3, hays and forage crops such as low quality alfalfa are valued at $ 50 per ton, and corn grain at $ 120 per 1000 kg is roughly equivalent to current US Corn Belt prices of corn at about $ 2.75 per bushel. Historically, corn grain has been priced at closer to $ 2.00 per bushel in constant dollars, see Fig. 2.4 below. Figures 2.3 and 2.4 teach several important lessons. First, corn grain at $ 3 per bushel is roughly equivalent to petroleum at $ 35 per barrel on an energy basis


Fig. 2.3 Relative costs of biomass and petroleum by mass and energy content.

Fig. 2.4 Real and historial corn prices.

(arrow #1 in Fig. 2.3). On a mass basis, however, corn is less than half the cost of petroleum (arrow #2) – giving corn starch and other corn components real potential as a feedstock for chemical production to replace petroleum-derived chemicals. Corn wet millers often use net corn cost to reflect the cost of starch available for chemical and fuel production after coproduct credits (e.g. for protein and oil) are subtracted. Typical net corn costs are roughly 70% of the purchase cost of corn, further reducing the actual cost of corn starch for chemical or fuel production. Ethanol production from corn grain currently requires various financial incentives to be competitive with gasoline from oil. No such incentives are required for chemicals from corn starch. Very large efforts are currently in progress to

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2 Biomass Refining Global Impact – The Biobased Economy of the 21st Century Table 2.1 Ten required biomass feedstock properties. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Economical Stable price and availability Consistent composition Low cost Favorable co-products and by-products Multiple product opportunities Inexpensive Environmentally benign or beneficial Storable Inexpensive

produce chemicals (e.g. lactic acid, 1,3 propanediol, etc.) from corn starch by either wet milling or dry milling. This is precisely how the petroleum refining industry developed. Additional valuable products such as plastics were added to the refinery over a period of decades as these products and processes were invented or improved and economics became attractive for the new products. Second, lignocellulosic biomass such as crop residues and herbaceous species (grasses, hays, forage crops) is available at prices that are a fraction (one fifth to one half) of petroleum costs (at $ 35 per barrel) on an energy basis, and even less on a mass basis (arrows #3 and #4 in Fig. 2.3). Therefore, given processing technology that economically and efficiently converts the energy content of lignocellulosic biomass to liquid fuels, we can reasonably expect to derive fuel products that are available at prices similar to current gasoline and diesel prices. Sugar fermentation to ethanol is one such process. Over 90% of the energy content of glucose is captured in the ethanol product from high-yield fermentations. Feedstock costs are absolutely critical to commodity chemicals and fuels, and biomass feedstocks are already much less expensive than petroleum on both a mass and energy basis. It is impossible to overstate the importance of low cost renewable carbon feedstocks to the eventual commercial success of large-scale, integrated biorefineries. Dr Paul Roessler of Dow Chemical (US) makes this point in a humorous and effective way in his list of ten required biomass feedstock properties, given as Table 2.1. In line with the historical development of other processing industries, as the biomass processing industry develops and the related technology matures, raw material costs will become dominant in the cost of manufacture. 2.3.3 How Much Biomass Feedstock Can be Provided at What Cost?

Renewable carbon feedstock prices are critical to the economic success of biorefineries. Although we believe data on likely biomass prices are encouraging, the ultimate possible scale of the industry, and hence its ability to displace petro-


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leum, will also be determined by the amount of biomass available at these prices. We now briefly examine this subject using Table 2.2 to frame our discussion. Table 2.2 summarizes world production of crop residues. In all, approximately 1.55 ´ 1012 kg residue are produced annually, the equivalent of approximately 600 billion liters of bioethanol at expected ethanol yields for mature technology of about 400 liters per 1000 kg residue [12]. The figure of 600 billion liters is close to the volume of gasoline and diesel consumed each year in the US, a very significant figure indeed. Rice straw must be removed from fields before planting of the next crop and is probably available close to collection costs, given the demand for the straw. Currently most rice straw is field-burned to remove it, a practice becoming less and less tolerated everywhere. Worldwide, over 700 ´ 109 kg rice straw are produced annually, mostly in Asia. Almost 200 ´ 109 kg sugar-cane bagasse is collected annually in many locations worldwide. Bagasse is probably available at its fuel cost or below, because it has only limited value to provide energy for the sugar mill. Thus nearly one thousand billion kilograms of rice straw and sugar cane bagasse are probably available at nominal costs. Considering other residues, approximately 100 billion kg per year corn stover and wheat straw in the Corn Belt and Great Plains regions of the United States are probably available at delivered prices of $ 20 per 1000 kg and less [14]. Similar amounts of residue, mostly wheat straw and barley straw, are available in Europe, probably at costs comparable with those in the United States. At $ 50 per 1000 kg, crop residue availability increases significantly. Approximately 150 billion kg US crop residues are available at this price compared with 100 billion kg at $ 20 per 1000 kg [14]. Other countries should also experience increased incentives to collect and utilize crop residues at higher prices. At about $ 50 per 1000 kg, farmers will also begin to produce hays and grasses specifically for biorefineries [15], and lignocellulosic biomass supplies will expand greatly. The extent to which supplies expand at a price of $ 50 per 1000 kg depends

Table 2.2 Worldwide availability of crop residues [13]. Material (billion kg)

Africa

Corn stover 0.0 Barley straw 0.0 Oat straw 0.0 Rice straw 20.9 Wheat straw 5.3 Sorghum 0.0 straw Bagasse 11.7 Subtotal 38.0

Asia

Europe

North America

Central America

Oceania

South America

33.9 2.0 0.3 668 145 0.0

28.6 44.2 6.8 3.9 133 0.4

134 9.9 2.8 10.9 50.1 6.9

0.0 0.2 0.0 2.8 2.8 1.2

0.2 1.9 0.5 1.7 8.6 0.3

7.2 0.3 0.2 23.5 9.8 1.5

74.9 924

0.0 217

4.6 219

19.2 26.1

6.5 19.7

63.8 106

Subtotal

204 58.5 10.6 731 354 10.3 181 1549

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largely on the productivity of crop and pasture lands. The RBAEF Project will treat this subject of productivity in great detail. We simply observe here that the US has nearly 16 million ha in its Conservation Reserve Program (CRP), land which is removed from grain crop production but which would be very suitable for grass and hay production. In the US, pasture lands (including crop land used as pasture) average about 5600 kg of biomass per ha per year [16]. There is little incentive to increase production on these lands, because there are no markets for increased hay production. Biomass production for energy would provide increased incentive to use these lands efficiently and a severalfold increase in average yield over time seems entirely likely. For example, some lands supporting dairy cattle in Michigan are managed intensively for maximum biomass production, including the use of winter cover crops and corn harvested as silage. Such lands are currently yielding 20 000 kg dry biomass per ha per year. Double these yields (40 000 kg ha–1 per year) of other species such as sugarcane and elephant grass have also been achieved on degraded lands [17]. If all the CRP lands and one half of crop land used as pasture (15 million ha) achieved 20 000 kg ha–1, an additional 600 ´ 109 kg biomass would be produced annually in the US. We believe the large scale conversion of cellulosic biomass to fuel will be based first on crop residues, given their low cost and availability. There is more than enough low-cost crop residue in the US and elsewhere to begin such an industry. As the industry expands, and processing economics improve with research and experience, the “demand pull” for additional biomass will cause the agricultural research and production sector to learn how to produce much larger amounts of herbaceous biomass profitably at costs approximating $ 50 per 1000 kg on lands that compete only modestly or not at all with food crop production. In addition, biorefineries producing fuels will also produce both protein and energy feeds for animals, just as corn wet and dry mills producing ethanol fuel do today. Coproduction of animal feeds with fuel and chemical products in biorefineries will increase feed supplies and reduce pressure on cropland. Before concluding this treatment of biomass feedstock costs, we note that the wide geographic availability, abundance and variety of biomass will tend to reduce the risks of raw material supply availability and reduce price swings. Uncertain availability and price volatility are major features of the current petroleum economy. As an essentially fixed world endowment of easily recoverable petroleum is gradually consumed, these risks will only grow and an increasing price paid in terms of national security and stability [2, 18]. Therefore, this economic and energy security issue can only grow in importance. Furthermore, many developed and developing nations which lack petroleum can grow large quantities of plant biomass, and thereby begin to escape the “development trap” that petroleum dependence brings in an era of decreasing petroleum supplies and high and volatile prices [18].

2.4 How Will Biorefineries Develop Technologically?

2.4 How Will Biorefineries Develop Technologically? 2.4.1 Product Yield: The Dominant Technoeconomic Factor

Yield (kg salable products per kg purchased raw materials) is usually the dominant factor governing the economics of a given reaction/separation system to produce commercial products. Because, essentially, all chemical and biological reactions produce multiple products, the yield of salable products influences the economics of the system in the following ways. 1. Raw material cost increases per unit of product as yield declines. For example, at $ 0.10 per pound of glucose and 90% yield of lactic acid produced by fermentation of this glucose, the glucose raw material cost is $ 0.11 per pound of lactic acid. At 50% yield, the raw material cost is $ 0.20 per pound of lactic acid. Because the cost of manufacturing commodity fuels and chemicals is very dependent on raw material costs, a lower yield significantly increases the cost of manufacture. 2. The cost of the reaction system increases. If a fixed total annual production rate is required, then at 50% yield almost twice the total reactor volume is needed compared with 90% yield to provide that amount of product. 3. The cost of the separation system increases even more rapidly than the reaction system. The cost of separation is proportional to the volume of fluid handled. Under similar reaction conditions, a lower yield means lower concentration of product and, therefore, a greater volume of reaction fluid must be handled for a given production rate. The cost of separation also increases with the number of components needing separation. Decreasing yield usually means there are more components that must be separated. 4. The cost of waste treatment increases. Either markets for byproducts must be found, which is not always possible, or the resulting waste streams must be treated before disposal, adding to both the capital and operating expense of the overall system. Given the dominance of yield in process economics for commodities, several conclusions regarding biorefinery development for fuels and commodity chemicals arise. First, fuel production in biorefineries will tend to be performed first in existing facilities where the yield of products can be improved by adding fuel production and where substantial capital investment has already been made, reducing the risk to innovators. Second, given the mild conditions (moderate pH and temperature) of biological catalysts, bioprocessing and biotechnology will tend to be used in biorefineries instead of harsh chemical or thermal processing to avoid degradation (and thereby the loss of value) of sugars, proteins and other labile biomolecules. Third, there will be continuing pressure to use all of the components of the biomass feedstock at their highest possible value. Thus the

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number of products will increase over time as will the yield of salable products per unit of raw material(s) consumed. This is precisely the trend that has occurred historically in the petroleum refining industry. Because we have pointed out several probable similarities between petroleum refineries and biorefineries, it is worth pointing out here a very significant difference between them. Both genetic tools and conventional plant breeding can be used to develop biomass feedstocks that are particularly designed for processing in the biorefinery. For example, lignin content can be altered to make lignocellulosic biomass easier to process. Also, valuable products such as enzymes can be produced in plants and recovered in the biorefinery [Ref. 1, Chapter 1]. This capability has no parallel in petroleum refining and is a major advantage for biorefineries. Continuing advances in the life sciences virtually guarantee that this advantage will grow with time. 2.4.2 Product Diversification: Using the Whole Barrel of Biomass

The importance of finding valuable uses for all biomass components is illustrated by the following example based on the composition outlined in Fig. 2.1. Assume a corn stover-based biorefinery producing 378 million liters per year of fuel ethanol at a yield of 420 liters of ethanol per 1000 kg stover. At a 50% removal rate for stover, approximately 200 000 ha corn will be required to provide the stover. Such a biorefinery will also produce nearly 21 ´ 106 kg minerals (Ca, K, Mg and P), 20 ´ 106 kg lipids, fats, and waxes, 52 ´ 106 kg protein (equivalent to the protein produced from nearly 70 000 ha soybeans), electricity from burning the lignin residue and probably residual sugars for animal feeding. These conclusions arise directly from the chemical nature of the components of biomass and the realities of chemical and biological processing outlined in Section 2.4.1, above. A biorefinery will be in many businesses (fuels, chemicals, power, feed, etc.) simultaneously. This fact cannot be evaded, but must be faced and dealt with, hopefully to the benefit of the overall biorefining system. One significant potential benefit that arises from this analysis is that the net requirement for agricultural land to provide feed protein decreases by 70 000 ha, or about 1/3 of the land from which stover is harvested. Put another way, 3 ha of corn production are now able to replace 1 ha of soybean production, while still providing all the grain these corn acres produced before and 900 ´ 106 kg stover for liquid fuel production. If herbaceous biomass species, for example switchgrass, are grown for energy production, they will probably be higher yielding and will also contain significantly more protein than corn stover. Thus such crops should provide even greater net savings of crop land required to meet protein needs. For example, if switchgrass or another herbaceous species such as coastal Bermuda grass is produced at dry matter yields of 20 000 kg ha–1 per year (about 9 tons acre–1 year–1) and contains 10% protein of which 80% is recovered [19, 20], the system will produce 1,600 kg ha–1 protein, over twice the amount of protein produced per ha of soybeans. Because the United States cur-

2.4 How Will Biorefineries Develop Technologically?

rently devotes approximately 30 million ha to soybean production, this emphasis on using the whole barrel of biomass could lead to substantial coproduction of energy and protein, but without devoting any new acreage to energy crop production. As described in Section 2.4.1 above, if the various components of biomass are not used in salable products, they must be disposed of at a cost. If they are not used in products, they must also be carried along with all the other streams in the process, adding to the capital and operating costs of all the related processing equipment. Likewise, if these components are not used in products, the remaining products must bear a larger portion of the overall costs, particularly the crucial feedstock costs. Thus many forces converge on a single objective: utilizing the entire “barrel of biomass”. The overall result of this convergence is quite simple: there is strong and unrelenting pressure to increase the yield and value of the multiple products of biomass and not to waste even a fraction of the raw materials. Because of this driving force, continuous, incremental process improvement is a key feature of both oil refineries and existing biorefineries. There are, however, many improvements and innovations that cannot occur until a refinery is actually operating and producing products and will not result from laboratory discoveries. We expect this pattern of continuous, incremental improvement in existing facilities will continue both with existing, starch-based biorefineries. It will also occur with the next generation of cellulose biorefineries that will come when key breakthroughs occur. We now briefly discuss breakthroughs required for cellulose biorefineries. 2.4.3 Process Development and a Technical Prerequisite for Cellulosic Biorefineries

If plant raw material is already inexpensive compared with petroleum and sufficiently abundant to support a large scale biorefining industry, one may legitimately ask: “Why has such an industry not already emerged?” We note that such industries have arisen: corn wet and dry mills and pulp and paper mills are examples. For these industries, inexpensive raw materials are coupled with well developed and efficient processing technology to convert the plant raw materials to products. For large scale liquid fuel production from cellulosic materials, however, the missing part of the equation is demonstrated, inexpensive processing technology to convert inexpensive and abundant cellulosic raw materials to fuels. While we believe that the entire barrel of biomass must be used effectively, use of the carbohydrate component (cellulose and hemicellulose represent approximately 70% of cellulosic biomass) is the sine qua non for cellulosic biorefineries [Ref. 1, Chapters 4 and 21]. The accessibility (reactivity) of cellulose and hemicellulose must be increased without significantly degrading these components. These carbohydrate polymers must somehow be converted into chemically reactive intermediate species, as shown in Fig. 2.2. Achieving this objective

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requires some sort of pretreatment before enzymatic, biological, or chemical upgrading of these sugar polymers to more valuable products such as ethanol fuel. Overcoming the recalcitrance of cellulosic biomass is therefore arguably the single most important research/process development obstacle confronting biorefineries processing cellulosic biomass to fuels. A closely related research objective for cellulose biorefineries is significantly reducing the cost of enzymes (called “cellulases”) required to convert pretreated biomass into fermentable sugars. An even more powerful impact on the economics of cellulose biorefineries than inexpensive enzymes would occur given the high yield, one step biological conversion of pretreated biomass to useful products. This process by which cellulase production, cellulose hydrolysis, and fermentation of soluble sugars to desired products occur in a single process step is called “consolidated bioprocessing” [21]. If viable, inexpensive pretreatments to overcome the recalcitrance of cellulosic biomass are developed, it seems likely they will first be demonstrated in existing facilities where a cellulose/hemicellulose-containing stream is available for upgrading to fuels and chemicals. Such existing facilities include pulp and paper mills, corn wet and dry mills, and perhaps flour mills. When this key piece of technology is available, the first generation of cellulose biorefineries will be launched. These pioneer plants will become the loci for further improvements, both incremental and transformational, in converting cellulosic biomass to a wide variety of fuels, chemicals, animal feeds, and other products.

2.5 Sustainability of Integrated Biorefining Systems 2.5.1 Integrated Biorefining Systems: “All Biomass is Local”

Because biomass feedstocks are solids and tend to be bulky, there is considerable incentive to refine them close to where they are grown. Likewise the waste streams from biorefineries will tend to be high in organic material and therefore biological oxygen demand. But these biorefinery wastes will not be particularly toxic. Many of these waste streams are therefore suitable candidates for returning to agricultural land. Given the capital and operating costs associated with waste handling in the biorefinery, there is a strong economic incentive to apply these wastes to land. Furthermore, agriculture is, by nature, a regional or local activity because of differences in soil and climate. Therefore crops grown for biorefineries can be or will be adapted to local conditions. The relatively smaller scale of biorefineries (compared with petroleum refineries) also enhances the likelihood that the farmers who produce the crops will have some sort of formal or informal “ownership” of the biorefinery. This pattern of local ownership of the biorefinery, or at least participation in ownership, is observed in many corn dry mills in

2.5 Sustainability of Integrated Biorefining Systems

the US. Corn wet mills are, however, much larger than dry mills (the largest wet mills approach the mass throughput rate of petroleum refineries) and are not farmer-owned. Local ownership of the biorefinery will give farmers an additional incentive to utilize animal feeds, mineral fertilizers, and organic waste streams from the biorefinery. We call this framework the “All Biomass is Local” concept; it is illustrated in Fig. 2.5. We envisage farmers using locally appropriate agricultural systems growing biomass specifically for the biorefinery. In the biorefinery, biomass is separated into its major components. Some of these components (e.g., protein and minerals or “ash”) may be salable or usable without further upgrading. The ash is returned as fertilizer to the land. Protein is fed to animals, preferably in a nearby location to avoid costs of drying and transportation. Animal wastes and organic waste streams from the biorefinery are used in the agricultural system. The nature of these waste streams makes them particularly appropriate for land application to perennial grass systems, where the potential for runoff and leaching to groundwater is minimized. Therefore, the convergence of these factors: · local ownership/participation in biorefineries, · the widespread geographical distribution of these refineries, · the fact that biorefineries will be intimately involved with land use practices, and · society’s continuing concern with the environment virtually guarantee that biorefineries will be conceived, designed, built, and operated with an unprecedented emphasis on their local and global environmental impact. Petroleum refineries largely avoided environmental/social issues when that industry was in its infancy, but now are being forced to do so at great cost. Biorefineries that do not adequately address appropriate environmental and social issues will be at substantial risk of failure. We believe these environmental and social issues surrounding biorefineries are best addressed using the concept of sustainability as an organizing framework and life cycle analysis as a powerful analytical methodology [22]. As we illustrate below, we believe that thoughtful, intelligent design and implementation of integrated agricultural production and biorefining systems can do much more than simply maintain the environmental status quo. Rather, we believe it is possible to effect significant improvements in the local, regional, and global environment by using life cycle analysis of integrated crop production and refining systems. 2.5.2 Agricultural/Forestry Ecosystem Modeling: New Tools for an Age of Sustainability

Sustainability is a very broad subject. In this paper we do not have the scope to treat all aspects of sustainability in relation to crop production and biorefining systems such as those represented in Fig. 2.6. To illustrate an approach that

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Fig. 2.5 All biomass is local: integrating agriculture and the biorefinery.

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Fig. 2.6 Carbon flows in the CENTURY model [25].


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might be taken, however, we consider the dynamics of carbon and nitrogen flow in agricultural ecosystems. We then analyze how those flows might be manipulated to enhance the sustainability of the agricultural production and biorefining system when taken as an integrated whole. We also point out what seem to be some emerging principles for understanding the sustainability of these systems. Analyzing and modeling carbon and nitrogen flows in agricultural ecosystems has been an active area of research for over thirty years [23, 24]. Researchers have studied a variety of biological and nonbiological processes connected with plant growth. These processes occur in the soil, on the surface and above the surface of the ground. Researchers have estimated the rates of these processes based on local factors such as temperature, soil type, rainfall, plant genetic potential and existing pools of carbon such as microbial carbon, standing dead plant, carbon, etc. Tillage and harvesting practices have also been taken into account, as have other human inputs such as fertilizers and irrigation. As a result of such work, flexible, powerful agricultural ecosystem models such as the CENTURY model have been developed [25, 26]. To illustrate the breadth of processes considered in such ecosystem level studies, a diagram for carbon flow in the CENTURY model is given below. Similar agricultural ecosystem models have been developed for nitrogen-containing species and are being developed for phosphorus-containing compounds. When these ecosystem models are combined with models of the biorefinery processes, we can use them to evaluate and then improve the sustainability of integrated biorefining systems. We illustrate this approach below. 2.5.3 Analyzing the Sustainability of Integrated Biorefining Systems: Some Results

We envisaged an integrated biorefining system based on corn grain, soybeans, and corn stover, i.e. the cellulosic residue remaining after grain is harvested. We assume that a second harvesting trip through the field is required to cut, bale, and remove the corn stover. Fuel ethanol is assumed to be produced from the corn grain by wet milling and additional ethanol from corn stover by acid hydrolysis and fermentation [27]. Residual fermentation solids are burned to produce electricity and steam. Steam is used in the plant and excess electricity is exported to the grid. Soybean oil is converted to biodiesel. To satisfy soil erosion prevention guidelines, a minimum of 1070 kg ha–1 corn stover are left behind. None of the soybean stover is removed. Several different agricultural scenarios are investigated to determine the effect of cropping system management on environmental performance. The cropping system assumptions for sustainability analysis are summarized below in Table 2.3. The acronyms given to each scenario in Table 2.3 are used in subsequent figures to identify simulation results. In all, six different scenarios are modeled and analyzed. Because “all biomass is local” we choose a particular location, Washington County, Illinois, USA, for our analysis. Climate and soil data and cropping practices are available for this

2.5 Sustainability of Integrated Biorefining Systems Table 2.3 Assumptions for cropping system sustainability analysis. Basic cropping system – Corn (plow till) – soybean (no-till): CPSN (grain) Effect of winter cover crop under no-till corn continuous cultivation – 0% of corn stover removed: CC (grain) (No winter cover crop) – Average 56% of corn stover removed: CC (56%) (No winter cover crop) – Wheat and oat as winter cover crops with 70% corn stover removal: CwCo (70%) Effect of winter cover crop under no-till corn–soybean rotation – Wheat and oat as winter cover crops after corn cultivation with 70% corn stover removal: CwCo (70%) – Average 54% of corn stover removed: CS (54%) (No winter cover crop) Cover crop not harvested

location. The agricultural base is conventional midwestern US corn–soybean rotation with only corn grain and soybean harvested, using plow till for corn and no till cultivation for soybeans (CPSN in Table 2.3). For all other scenarios both corn and soybeans are grown under no-till cultivation practices, in which soil is left undisturbed from harvest to planting. A second scenario is the continuous cultivation of corn (CC), again with only corn grain harvested. A third scenario includes the harvest and removal of 56% of the corn stover and all the grain under continuous corn cultivation (CC 56%). A fourth scenario is rotation of corn with soybeans and removal of 54% of the corn stover and all the corn grain and soybeans (CS 54%). We explore the use of cover crops in the two final scenarios. In some areas it is common to plant another crop such as wheat or oats in the late fall after the corn is harvested. These young plants grow a few centimeters, survive over the winter and then resume growth in the spring once conditions permit. Cover crops can be harvested, killed with herbicide or plowed under to increase soil organic matter. In this analysis we assume that the cover crop is not harvested by rather is killed with herbicide and the corn or soybean is planted in the dead cover crop. Cover crops help eliminate wind and water erosion and are also effective in removing excess nitrogen fertilizer left behind in the previous cropping cycle. This excess nitrogen is vulnerable to conversion by various processes to soluble nitrogen species that leach through soil and are transported to streams, lakes, rivers and the ocean. This excess nitrogen can also be converted by anaerobic soil bacteria to nitrous oxide, a potent greenhouse gas. The fifth scenario therefore considers continuous corn with 70% removal of stover plus winter wheat or oats as a cover crop (CwCo 70%). The sixth and last scenario assumes a corn–soybean rotation from which 70% of the corn stover is harvested and winter cover crops are grown (CwSo 70%). Soil organic carbon levels were predicted over time using the six scenarios modeled. Depending on conditions chosen, either static or increasing soil or-

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ganic carbon levels are possible as shown in Fig. 2.7, below, as long as cultivation is no till. The use of winter cover crops enables very substantial removal of corn stover for industrial uses while still enhancing soil quality over time. The total amount of protein, lipids, lignin and carbohydrate (including starch, cellulose and hemicellulose) produced by each of the cropping systems above was estimated over a 40 year period. Based on data from biorefinery operations and crop production databases, greenhouse gases were also calculated in kilogram carbon dioxide equivalents per kilogram of each of these species produced. Figure 2.8 shows greenhouse gas production per kilogram of carbohydrate for each of the six scenarios. Once again, the cropping system chosen has a significant effect on the results. The results range from a net production of 560 g of CO2 kg–1 carbohydrate for conventional corn–soybean rotation to 9 g (net carbon sequestration) kg–1 carbohydrate for continuous corn cultivation under no-till conditions with winter wheat and 70% stover removal. A similar life-cycle approach is taken to estimate the greenhouse gas reduction when ethanol produced using the biorefinery systems outlined above is consumed in a mid-sized passenger vehicle. Figure 2.9 summarizes these results. All of the cropping systems result in net greenhouse gas reduction, but there are substantial differences between cropping systems. Aggressive corn stover removal when coupled with winter cover crops can result in over 70% reduction in greenhouse gas formation compared with a gasoline fueled vehicle. But as Fig. 2.7 shows, soil health can continue to improve even when large amounts of stover are removed. Finally, even more dramatic reductions in leached nitrogen (inorganic nitrogen species escaping the root zone of plants) are possible by judicious choice of cropping systems and agricultural system management. Using the nitrogen flow submodel of the CENTURY model to predict the effects of different agricultural practices, it seems possible to reduce inorganic nitrogen losses more

Fig. 2.7 Soil organic carbon trends under different agricultural practices.


Fig. 2.8 Greenhouse gas emissions per kg carbohydrate.

Fig. 2.9 Greenhouse gas reductions under different cropping systems.

than a factor of ten by use of conventional corn production as the base case. These results are summarized in Fig. 2.10. The results are for a 40 year period in which these practices are used in a specific geographic location – Washington County, Illinois, USA. As expected, the use of winter cover crops greatly reduces nitrogen leaching. It is also interesting to note that stover removal seems to reduce nitrogen leaching – compare CC with CC (56%). One mechanistic explanation of these results is that the nitrogen content of the corn stover (approx. 1% by weight) is not available for conversion to soluble inorganic nitrogen species when the stover is removed, thereby reducing leaching. Also, the carbohydrates in harvested stover are not then available to provide metabolic energy for microbial processes that

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Fig. 2.10 Inorganic nitrogen losses over 40 years – Washington Country, Illinois.

convert organic nitrogen into inorganic nitrogen. Although stover removal must be carefully managed to maintain soil fertility, stover removal has powerful environmental benefits. These benefits include providing renewable carbon for biorefining to fuels and chemicals and reduced inorganic nitrogen leaching. On the basis of these and other results, we believe that a combination of creative system design, careful planning and use of powerful ecosystem and biorefinery modeling tools can help us achieve very significant environmental improvements as we develop the biobased economy of the 21st century. We need not be content with maintaining the environmental status quo or even with modest improvements. Instead, very significant improvements are possible if we are both wise and informed.

2.6 Conclusions

The biobased economy will grow rapidly during the 21st century. A combination of low-cost plant raw materials and gradually improving biorefinery process technology for converting these raw materials into a variety of fuels, chemicals, materials, food, and feed will drive the adoption of the biobased economy. The biological sciences will have a particularly powerful impact on both the raw materials and the processing technologies underlying the biobased economy. The biobased economy, and its associated biorefineries, will be shaped by many of the same forces that shaped the development of the hydrocarbon economy and its refineries over the past century. These similarities include the importance of yield (using the whole “barrel of biomass”), continuing diversification of products, and gradual process improvement in functioning biorefineries.

References

However, significant differences between the biobased economy and the hydrocarbon economy are also apparent. Among these are the great compositional variety of plant raw material, requiring a greater range of processing technologies to add value to the basic components, and the much wider geographic distribution of both raw materials and the associated refineries. This wide geographic distribution of both raw materials and biorefineries will promote greater economic/national security and more equitable distribution of wealth. We believe that supposed limits on agricultural productivity to support the biobased economy are mostly illusory. There is no “food vs. fuel” conflict. Economic profitability and process efficiency will force the adoption of “food and fuel” scenarios. Biorefineries and their associated crop production systems will be highly integrated. Furthermore, integrated biorefining systems will be designed to achieve not only economic profitability but also environmental benefits. Truly transformational environmental benefits can be achieved by creative design of these integrated biorefining systems.

Acknowledgements

The authors gratefully acknowledge support from Cargill Dow, LLP, from DuPont Biobased Materials, Inc., and from the Center for Plant Products and Technology at Michigan State University.

References 1 C. J. Arntzen, B. E. Dale (Eds.), Biobased

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Industrial Products: Research and Commercialization Priorities, National Academy Press, USA, 2000, 26–54. C. J. Campbell, J. H. Laherrere, Scientific American. 1998, 278 (3), 78–83. L. F. Ivanhoe, World Oil 1996, 217(11), 91–94. J. T. Houghton, Ency. Energy Tech. Environ. 1995, 1, 491–504. M. Graboski, R. Bain, Biomass Gasification: Principles and Technology, T. B. Reed (Ed.), Noyes Data Corporation, USA, 1981, 41–71. Energy Information Agency, http:// www.eia.doe.gov/pub/international/iealf/ table18.xls, United States Dept. of Energy, 2002. Anon., Chem. Eng. News 2002, 22, 55–63. Statistical Abstract of the United States, Table #958 (http://www.census.gov/prod/ 2001pubs/statab/sec19.pdf), 2000.

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7(3/4), 147–162. 10 B. E. Dale, TIBTECH. 1987, October

287–291. 11 G. E. Tong, Chem. Eng. Prog. 1978, April

70–83. 12 C. E. Wyman, Ann. Rev. Energy Environ.

1999, 24, 189–226. 13 S. Kim, B. E. Dale, Biomass & Bioenergy

2004, 26, 361–375. 14 P. Gallagher, et al., Biomass from Crop

Residues: Cost and Supply Estimates, US Dept of Agriculture Economic Report 819, USA, 2003. 15 United States Dept. of Agriculture Hay Reporter http://www.ams.usda.gov/ mnreports/DC_GR310.txt. 2004. 16 United States Dept. of Agriculture Agricultural Statistics Table 6.1 http:// www.usda.gov/nass/pubs/agr04/ 04_ch6.pdf. 2003.

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agement of Biomass/Energy Crops on Phosphatic Clay in Central Florida, Univ. of Florida Cooperative Extension Service Circular 1084, USA, 1993. R. G. Lugar, R.J. Woolsey, Foreign Affairs. 1999, 78(1), 88–102. M. E. Ensminger, C. G. Olentine, Jr., Feeds and Nutrition – Complete, The Ensminger Publishing Company, USA, 1978. L. B. de la Rosa, et al., Appl. Biochem. & Biotech. 1994, 45/46, 483–497. L. R. Lynd, C. E. Wyman, T. U. Gerngross, Biotech. Prog. 1999, 15, 777–793. R. Anex (Ed.), J. Ind. Ecol. 2004, 7(3/4). See articles on pages 75–92, 93–116, 117–146 and 147–162. L. Kristen, et al., Parameterizing Century to model cultivated and noncultivated sites in the loess region of western Iowa, US Geological Survey Report 00-508, US Department of the Interior, USA, 2000.

24 P. Smith, et al., Quantifying the change in

greenhouse gas emissions due to natural resource conservation practice application in Indiana, Final report to the Indiana Conservation Partnership, Colorado State University Natural Resource Ecology Laboratory and USDA Natural Resources Conservation Service, USA, 2002. 25 A. K. Metherall, L. A. Harding, C. V. Cole, W. J. Parton, CENTURY Soil organic matter model environment. Technical documentation. Agroecosystem version 4.0. Great Plains System Research Unit Technical Report No. 4. UDSA-ARS, USA, 1993. 26 R. H. Kelly, et al., Geoderma 1999, 81, 75–90. 27 J. Sheehan, et al., Is ethanol from corn stover sustainable?, National Renewable Energy Laboratory Draft Report, USA, December 2002.


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3 Development of Biorefineries – Technical and Economic Considerations Bill Dean, Tim Dodge, Fernando Valle, and Gopal Chotani

3.1 Introduction

As the world’s population and economy continue to grow, so does the demand for goods to sustain that growth. Growth has come at the expense of our nonrenewable resources. It is only a matter of time before the price of petroleum, upon which the world economy is heavily dependent, will rise to a point where we will be forced by economic factors to find alternatives. We suspect this time is not far off in the future and the time is now to explore viable alternatives. Our nation is fortunate to have abundant agricultural and forest renewable resources and a climate that is amenable to their productive use. If we put our mind to it, these renewable assets can go a long way to replace our heavy dependence on petroleum and other non-renewables. That process has, in a way, already begun, as we have been in the business of “bio-refining” in the broader sense for quite some time. Our forests are harvested to produce a host of products including paper, solvents, building materials, and many more. Our agriculture industry can produce large quantities of grain and other crops, which can and are being used to produce a host of materials. Starch from grain crops and sucrose from beet, sugar cane, and other materials are being converted to an increasing number of products and chemicals. Some of the chemicals now being produced include ethanol, 1,3-propanediol, lactic acid, and ascorbic acid. As the technology of pathway engineering advances, one can expect many more chemicals (and polymers) to be produced from sugar as a carbon source [1, 2]. Increasing demand for sugars will eventually result in increasing sugar prices and, ultimately, supply problems. It is logical to assume that as more chemicals and materials are produced from fermentable sugars as the carbon source, market forces will inevitably drive fundamental changes in the agriculture and chemical sectors. Genencor International has been active in developing technologies that have affected the evolution of biorefineries. As part of our efforts in this area, we have actively explored the concept of using cellulosic biomass to provide the carBiorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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bon source for such future refineries. In this pursuit, we have focused on the conversion of cellulosic biomass into fermentable sugars and on the challenges of biocatalysis in utilizing those sugar streams [3, 4]. Advances in pathway engineering will result in commercially viable and competitive processes based on renewable feedstocks which in many cases will provide the low-cost route to production. The cost of carbon to feed biocatalysis is often more than 50% of the total direct cost of production, and therefore efforts to reduce this cost play an important role in the overall development of the biorefinery concept. It is not surprising that many have focused on cellulosic biomass (e.g., agricultural waste) as the ultimate low cost source of the fermentable sugar in the biorefinery. Much knowledge has been gained and progress made on the understanding of cellulosic biomass saccharification to fermentable sugars to be used for bioconversion to chemicals. The challenge before us is to develop the technologies that will be required to enable this upcoming industry. We will focus here on two of these technologies: 1. enzymes required for conversion of cellulosic biomass to fermentable sugars within an enabling cost structure; and 2. engineered organisms to produce chemicals competitively.

3.2 Overview: The Biorefinery Model

Biorefineries, often referred to as integrated biorefineries, are processing facilities that use renewable plant materials as feedstocks. Plant materials, comprising carbohydrates and associated oil, protein, lignin, and other components, are converted in the biorefinery into higher-value chemicals and materials. Our broad definition of biorefineries includes a initial process that utilizes renewable carbon feedstocks containing sucrose, starch, and cellulose as shown in Fig. 3.1. Essential elements of a biorefinery are: 1. multiple feedstock capability and a tolerance of wide variation in those feedstocks; 2. feedstock-processing by enzymes to fermentable sugars (and by-product streams); 3. biocatalyst, which converts sugars to desired product(s), and 4. co-products which are used in the process, recycled through the process, or sold.

3.3 Feedstock and Conversion to Fermentable Sugar

Three basic biorefinery approaches, as portrayed in Fig. 3.2, are evolving on the basis of the nature of the feedstock, i.e. sucrose, starch, or cellulose. On a fermentable-carbon-cost basis, the sucrose-based Biorefinery I is currently the most cost-competitive. In recent years, however, the starch-based Bio-


Fig. 3.1 The overall biorefinery model.

Fig. 3.2 Biorefinery evolution.

refinery II has become more cost competitive as a result of innovations in farming and milling grain like corn. In principle, the cellulose-based Biorefinery III will become more competitive as technical and economic challenges are addressed. As illustrated in Fig. 3.3, fermentable sugars, whether derived from sucrose, starch, or cellulose, will become cost competitive with petroleum-derived carbon for production of fuel, chemicals, polymeric materials, and specialty intermediates.

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Fig. 3.3 Change of cost of fermentable carbon with time for different carbohydrate sources.

3.3.1 Sucrose

The simplest biorefinery system uses sucrose as feedstock (sugar beet, cane, etc.). Sugar is first extracted from the feedstock. Lignin and cellulosic residuals, if sugar cane is used, are utilized separately or burned for energy to run the operation. The sucrose-based industry is a significant biorefinery opportunity today and into the future. Sucrose is second only to cellulose in availability and current output far exceeds all other commercial carbohydrates combined. It is estimated that only 1.7% of annual sucrose production goes to non-food uses. It is possible to imagine a sugar biorefinery as an integrated producer, not only of sucrose and ethanol, but of important high-value products whose manufacture could be scaled up or down depending on circumstances, economics, and demand [5]. Not surprisingly, more than 50% of ethanol volume produced in the world today has sucrose as feedstock. 3.3.2 Starch

The grain wet-milling industry as it exists today is an excellent example of the biorefinery concept. The grain kernel, e.g. corn, is processed through a series of steps resulting in a glucose (or multimers of glucose) stream and co-products such as oil, protein, fiber, and nutrients (e.g. carotenoids and corn steep liquor). The glucose can be further converted to ethanol, 1,3-propanediol, lactic acid, etc., by fermentation, and to sweeteners, for example high-fructose syrup, by enzymatic conversion. The starch-based biorefinery has been enabled by the development of highly efficient and thermostable amylases and the development of isomerization processes to convert glucose to fructose by immobilized glucose isomerase enzyme systems. The corn (and other grain) dry mill is a highly specialized abbreviated version of the wet mill whereby many of the initial steeping and extraction steps have been removed and starch is enzymatically converted to glucose and fermented


to ethanol in a process called simultaneous saccharification and fermentation (SSF). The capital cost of building these less complex bio-refineries is significantly lower than for wet mills, but the trade-off is that dry mill co-product streams (distillers dried grains and solubles, or DDGS, sold into select animal feed markets) have relatively lower value. Thermal energy consumption is significant in the milling process. A wet corn mill is very effective in extracting maximum value, because of the multiplicity of products and co-products mentioned above. In general, however, a wet grain mill is capital-intensive, and therefore new ethanol production plants coming on-line in recent years are mainly of the dry grain mill type. New enzyme technology developments hold considerable promise for impact here and these refineries will continue to evolve. 3.3.3 Cellulose

Considering the amount of cellulosic biomass available for saccharification to fermentable sugar, there is a clear opportunity to develop commercial processes that could generate products that are needed at very high volumes and low selling price. Most of such products are now being made from non-renewable resources, mainly through oil refineries. These refineries, starting from a complex mixture (petroleum), use a wide range of unit operations to generate an impressive variety of products that are sold directly or transformed into value-added products like plastics, fibers, etc. Approximately 17% of the volumes of products derived from petroleum in the US are classified as chemicals. If these chemicals could be obtained from renewable resources like biomass in a biorefinery, it would reduce our petroleum dependence and also have a positive environmental impact. Supplies of starch and sucrose feedstock will probably not be sufficient to meet the feedstock needs of future biorefineries. As an example, the gasoline market in the US alone is 150 billion gallons per year. MTBE (methyl tertiarybutyl ether) replacement at 6% would result in approximately 10 billion gallons per year of ethanol, which would require about 30% of the US farmland currently growing corn. Given that ethanol is only one of many compounds that can flow out of a corn biorefinery, it is likely that other sources of carbon feedstock will also be required. Brazil and a few other tropical and sub-tropical countries use sucrose in biorefineries that produce ethanol. Because it is widely believed that it is not feasible to produce enough sugarcane (and other sugar crops) to meet potential fuel ethanol volume requirements, there has been a 50plus year effort to develop technologies to convert cellulose from biomass into a carbon feedstock for the biorefinery of the future. Cellulosic biomass conversion to fermentable sugars has been explored as the potentially lowest cost feedstock for the biorefinery of the future. Unlike sugar energy storage compounds like starch, which can be converted to fermentable sugar with relative ease, cellulosic biomass is a complicated structure of cellu-

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lose (b1–4 glucose linkage), hemicellulose (linked C5 sugars including mannose, galactose, xylose, and arabinose), lignin, and numerous minor components. This structure has evolved as a support element of plants, and therefore is not meant to be readily accessible as a carbon source. Currently, the most promising approach for using this feedstock is enzymatic hydrolysis of the cellulose content after pretreatment of the fiber to make the cellulose more accessible to enzymatic attack. The process for conversion of cellulosic biomass to fermentable sugars faces major technical and engineering challenges that have so far prevented large-scale commercial use of cellulose as a source of fermentable sugar. A significant cost component in the overall process to break down cellulosic biomass has been the cost of cellulase enzymes required to carry out the process. As much as 100-fold more native cellulase protein (compared with amylase protein for breakdown of starch) is required for conversion of pretreated substrate, e.g. corn stover, into fermentable sugars. Given the relatively high cost of the enzymes and the amount required to produce fermentable sugars, the process has not been viable. In addition, the pretreatment of cellulosic biomass, making cellulose available for the enzymatic hydrolysis, has been a significant challenge. Many approaches have been explored; however, all suffer from their capital intensity because of the extreme conditions of the process. The cost impact of processing differences between corn and stover is reflected in Fig. 3.4. The cost of cellulose-hydrolyzing enzymes, or cellulases, is not included in this analysis. Through a three-year DOE funded program administered by the National Renewable Energy Laboratory (NREL), and a one year extension of that program, major advances have been made toward reducing the cost of enzymes in ethanol production from pretreated corn stover. The impact of these improvements on cellulase cost, estimated as $ per gallon of ethanol (EtOH) produced in a bench-scale NREL assay, can be seen in Fig. 3.5. This improvement has come from reducing the cost of producing the enzymes, enhancing the mix of enzymes, and altering, or recruiting, key enzymes to enable operation at elevated temperatures. However, enzyme requirement remains high, even under the higher-temperature operating conditions engineered into the multi-enzyme system. It is anticipated that enzymatic hydrolysis cost will be further reduced by continued improvement in the enzymes and new processes that use elevated temperatures and more effective pre-treatment processes. One strategy to minimize ethanol production cost is to run simultaneous saccharification and fermentation, or SSF, which would use ethanologens engineered to operate in high temperature environments. Also, the fermentation organism’s ability to utilize C5 sugars derived from the hemicellulose component, and have acceptable productivity in the presence of numerous byproducts of the biomass pretreatment process, would lead to lower overall production cost.


Fig. 3.4 Cost components production of fuel ethanol from corn and corn stover [6].

Fig. 3.5 Cellulase enzyme cost estimate based on the NREL assay [6].

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3.4 Technical Challenges 3.4.1 Cellulase Enzymes

The challenge of improving cellulase enzymes is significant. Lignocellulose is not a pure compound on which cellulase activity can be optimized. The composition and structure of the biomass substrate can vary dramatically depending on the source of the lignocellulose. Corn stover structure is different from sugarcane bagasse, which in turn is different from municipal solid waste. In addition, corn stover from one part of the country can differ significantly from that from another, because of soil, weather, and other conditions. Finally, different pretreatment processes affect biomass in unique ways, resulting in even further differences between structure and composition. Thus the enzyme system required may need to be optimized for each specific combination of substrate and pretreatment that would be employed. In 2000, Genencor International was awarded a three-year US Department of Energy (DOE) subcontract through the National Renewable Energy Laboratory (NREL) to reduce the cost of cellulase, on a “per gallon of ethanol produced by NREL process and assay basis”, by a factor of ten. That goal was achieved within the three-year timeframe, and the program was extended to further reduce costs to a factor of twenty. These aggressive goals were approached from two directions by making significant improvements in cellulase production economics (reduced cost/gram enzyme) and in cellulase enzyme performance (reduced grams of enzyme needed). Enzyme cost in assay

$ $ enzyme cost gallons EtOH g enyzme g enzyme enyzme loading gallons EtOH

1

3.4.1.1 Improved Cellulase Production Economics An understanding of the components of production cost is a necessary prerequisite for effectively reducing the cost. A simplistic, but highly useful, model would identify fixed and direct costs for each of the three major production processes fermentation, enzyme recovery, and formulation:

Total cost Fermentation cost (fixed direct) Recovery cost (fixed direct) Formulation cost (fixed direct)

2

3.4 Technical Challenges

Most current commercial cellulase products are sold as cell-free, stabilized concentrates. Although these formulations meet market needs with regard to application and cost, for many enzyme products it is not uncommon for the recovery and formulation costs to be a major portion of the overall cost. It then follows that reduced post-fermentation processing would possibly lead to reduced cost, with no post-fermentation processing being the lowest cost. This is, however, only practical if the resulting product still meets the needs of the application. Fermentation broth with no processing was tested in saccharification of pretreated corn stover. Performance in the saccharification was indistinguishable among fresh whole fermentation broth, fresh fully-recovered and formulated product, and 28-day old fermentation broth. These results suggest that typical recovery and formulation costs can be eliminated for use in biorefinery operations, especially in an integrated plant that both makes and uses the cellulase enzymes. This leaves the fermentation costs that can be broken down into fixed costs, for example depreciation and labor, and direct costs, for example utilities and raw materials. Fermentation cost Fixed cost (labor depreciation) Direct cost (utilities other raw materials carbon/energy source)

3

The single largest cost component for the fermentation process was estimated to be the carbon/energy source for the culture. For Trichoderma cellulase, the carbon source has historically been lactose. The cost per unit enzyme produced is a function of the yield of enzyme on the carbon source and the cost of the carbon source itself. When performed in a similar manner, the other costs are proportional to the rate of enzyme production, as measured by volumetric productivity. An integrated plan of action was taken to affect both enzyme expression and the fermentation process. Improvements were made on the production organism, the production process, and in their interactions. A conventional mutagenesis and screening approach was applied to the existing production strain. The work had several objectives. One was to find strains capable of fast and efficient growth on cellulose. A useful screening method was adapted from the method of Toyama [7]. In this method, mutated spores are poured in large numbers with agar in the bottom of a Petri dish. A second layer is added on top containing cellulose as the sole carbon source. The spores germinate and grow through the agar eventually emerging on top of the cellulose-agar layer. Those that erupt first are more likely to grow quickly and efficiently on cellulose. Another goal was to disrupt existing regulatory mechanisms that result in catabolite repression and the need for induction of cellulase expression. Several plate screening methods were developed that used a high glucose concentration to overcome catabolite repression or glycerol as the carbon source to find expression without induction. A third goal was to improve the secretion of cellulases. Resistance to different chemical agents and selection for hyper-branched morphological mutants were both employed.

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Through successive rounds of mutagenesis and selection with different screening methods, an improved host strain was developed. Several new traits were recruited that positively affected fermentation cost. First, the specific growth rate of the strain was increased by 50%. This reduced the growth time needed for the fermentation, positively affecting enzyme productivity. The specific productivity of the strain, enzyme per unit cell mass per unit time, was also improved. This impacted both key metrics, i.e. yield of enzyme from the carbon source and volumetric productivity. The improvement in yield helped reduce cost of the sugar lactose per unit enzyme produced. Lactose, a by-product of the dairy industry, typically costs significantly more than glucose, a product from sucrose, starch, or cellulose processing. Lactose is a mild inducer of cellulase expression with little to no catabolite repression. Glucose, on the other hand, does not induce cellulase expression and exhibits high catabolite repression. It would thus seem improbable to replace relatively expensive lactose with relatively inexpensive glucose. The disaccharide sophorose (b-1,2 linkage of two glucose units) is one of the most potent inducers of cellulase expression known [8]. It was found that treating a concentrated glucose solution with whole cellulase at elevated temperatures leads to the formation of numerous higher sugars, among them sophorose. This treated solution can then be fed to a cellulase-producing fermentation with performance equal to or better than that obtained using lactose. The small amount of enzyme product that must be recycled to produce sophorose is more than offset by the reduced cost of glucose relative to lactose and by the improved fermentation performance. Traditional fermentation optimization was performed on the improved strain by adjusting the level of salts and other nutrients, temperature, pH, and glucose/sophorose mixture. The cumulative effect of all these improvements was very significant, and the production cost, per unit of enzyme, was reduced by approximately a factor of seven. These improvements went far beyond what had been deemed probable for an established and mature product such as whole cellulase. Investigations were also made to evaluate pretreated corn stover as a carbon feed for cellulose production; this will be discussed below.

3.4.1.2 Improved Cellulase Enzyme Performance Reducing enzyme production cost was only part of the solution. As shown in Eq. (1), improving the performance of the cellulase enzymes, resulting in reduced enzyme loading per gallon of ethanol can be equally effective. Several of different approaches have been used to improve enzyme performance, including increasing thermal stability, recruitment of novel cellulolytic activity, and increasing specific activity [9–12]. A major focus was on increasing the thermal stability of the cellulase enzymes. Cellobiohydrolase I (CBH I) is the major component of whole cellulase. It also happens to be the component with the lowest thermal stability, as indicated by Tm, the characteristic “melting” temperature. Successful engineering of


CBH I to improve thermal stability also required several strategies. CBH I structure analysis suggested sites that could affect thermal stability, as did comparing the structures of CBH I homologues. Random mutagenesis and screening were also used. An important part of this effort was the development of a small-scale screening method to quickly and accurately measure the effect of the changes made. By the incorporation of a large number of specific site changes, Tm of CBH I was improved significantly. Placing the engineered CBH I into a production strain in which the native CBH I gene was deleted did not have a negative effect on enzyme production levels. Along with the ability to engineer proteins for increased thermal stability, it is imperative that the engineered proteins can be expressed at high levels in production strains without negatively affecting the other cellulase components. This has proven to be the case for both T. reesei-engineered CBH I and for homologues from other fungi. The homologue from Humicola grisea var thermoidea was expressed at levels similar to the wild-type CBH I without negatively affecting the production of the other cellulase components. By analysis of the CBH I structure, several sites were selected that were hypothesized to affect binding of substrate and products in the active site cleft. Several mutants were made, with significant effects on both Km, the MichaelisMenten constant and kcat, the catalytic rate constant. Many of these improved enzymes have been produced effectively in whole cellulase production strains. As discussed above, improved cellulase production has been maintained with the improved enzymes incorporated. The resulting products have been tested in the NREL process scheme and have resulted in an approximately threefold reduction in enzyme loading. The work discussed above was performed by Genencor International and collaborators as part of a subcontract funded by the DOE administered by NREL. A similar subcontract was also awarded to Novozymes. The improvements reported to date by both the groups have been quantitatively very similar. The approaches taken have also been similar. Together, it is clear that the cost of enzymes for biomass saccharification has been dramatically reduced from the baseline in 2000. Enzyme cost can no longer be regarded as the largest barrier to commercialization of biorefineries. 3.4.2 Fermentation Organisms

A biorefinery would be a processing factory that would separate biomass into component streams, and transform them into a wide range of products, using enzymatic and/or fermentation processes. This concept has been around for some time, but has not been implemented to its full potential. The utilization of biomass and the development of biorefineries are enormous technological challenges that no doubt require the solution of multiple problems.

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3.4.2.1 Biomass Hydrolyzate as Fermentable Carbon Source The use of live cell fermentation to convert sugars into commercial products or intermediates has been exploited successfully for many years. These fermentation processes are based on the use of relatively clean sugar streams that contain few impurities. However, the full use of all the carbon components in biomass is more complicated. After biomass pretreatment, and enzymatic hydrolysis of cellulose (and hemicellulose), a mixture of hexose and pentose sugars and several degradation by-products, for example furfural, hydroxymethylfurfural, phenols, and formic, acetic and other acids is obtained. Several of these pretreatment and hydrolysis by-products are well known inhibitors of fermentation processes. The extent of inhibition depends on the fermentation process. Occasionally it is severe and inhibits cell growth completely. To avoid this, the hydrolyzates must be diluted or fed during fermentation, complicating the fermentation processes and reducing the total amount of hydrolyzed biomass that can be used per unit of time or volume. In other circumstances the impact of the byproducts is less severe, because they do not inhibit growth but instead have a negative impact on performance of the cells. For example, at certain concentrations, some organic acids, for example formic or acetic, have a negative effect on cell physiology and increase the amount of energy that cells use to perform their normal functions (cell maintenance). This increase in energy consumption is, in turn, reflected in higher consumption of carbon from sugars, with a consequent decrease in the overall yield of product per unit of biomass [13]. Besides the effects of by-products present in hydrolyzed biomass, the highly concentrated stream of carbohydrates obtained can also be a problem. When exposed to high concentrations of sugars and salts, cells tend to undergo osmotic stress that, depending on its magnitude, can inhibit cell growth or cause an increase in cell maintenance, leading to problems described above. Another well-known response of cells to the presence of high concentrations of carbon sources is that they tend to use the sugars in a particular order and in an inefficient manner. Cells tend to utilize first the carbon source that is easiest to metabolize and that provides more energy. When this carbon source is completely consumed or reaches a particular concentration, cellular metabolism is re-adjusted to utilize another carbon source present in the mixture. This sequential use of the carbon sources and the associated metabolic adjustments complicates fermentation process design because optimum cell-performance is obtained when some of the growth media components, mainly carbon and nitrogen, are present in certain ratios. From the engineering design perspective of the fermentation process, these ratios are calculated on the basis of total carbon present in the mixtures. From the cells’ perspective, however, only one or a few carbon sources at a time are “sensed”, and a carbon-to-nitrogen ratio calculated on the basis of total carbon content is detected as an unbalanced mixture by the cells. Very often this leads to poor cell and fermentation process performance.


3.4.2.2 Production Process as a Whole In addition to addressing problems related to the use of mixtures of complex sugars and inhibitors, development of commercially viable fermentation processes requires proper process integration of the different unit operations to reduce costs and disposal of fermentation by-products. Enzymatic hydrolysis of biomass is currently performed with a mixture of enzymes that work in the 50– 60 8C temperature range and 3–5 pH range. However, most of the production strains developed so far to utilize biomass hydrolyzates perform better in the 30–40 8C temperature range and the 6–8 pH range. This means temperature and pH must be adjusted before starting fermentation. pH adjustment is not only expensive but also produces salts that will have an osmotic effect during fermentation and must be disposed of properly at the end of the process. Temperature adjustments also increase fermentation costs. For these reasons it is highly desirable to develop production strains that can utilize biomass hydrolyzates satisfactorily at 50–60 8C and pH 3–5 [14]. This would enable relatively inexpensive SSF processes. Furthermore, low pH and high-temperature fermentation conditions would retard contamination. On the other hand, it is important to note that at high temperatures the inhibition effects of toxic compounds in biomass hydrolyzates are also magnified. An SSF process may not always be the best means of obtaining specific products from biomass hydrolyzates, however. Occasionally the physiochemical properties of the product(s) must be considered when designing a process. For example, a study on production of twelve top value-added chemicals from biomass identified eight as acids, three of which must be produced in the free-acid form directly during fermentation. If these are produced in the salt forms first, the cost impact on product purification and disposal of by-product salts is high [15]. As shown in Table 3.1, pK values of several commercially relevant acids are in the 3–5 range. For production of these, therefore, the fermentation pH necessary for a product in the free-acid form must be in the 2–4 range, making SSF an unlikely process choice.

Table 3.1 pK values of some commercially important organic acids that are or potentially can be produced by fermentation. Acid

pK Value

Pyruvic Fumaric Malic Itaconic Lactic Aspartic Succinic Glutamic Oxalic

2.50 3.03 3.40 3.84 3.86 3.90 4.19 4.20 4.21

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3.4.2.3 Emerging Solutions On the basis of the challenges discussed above it is evident that future efforts to develop commercial processes utilizing biomass hydrolyzates for fermentation will require production strains different from traditional organisms like yeast, Bacillus, E. coli, Pseudomonas, Corynebacterium, etc. [14]. Instead, we will need to find or develop microorganisms capable of performing better at low pH and/or higher temperatures, and under high concentration of carbohydrates. Furthermore, these new microorganisms would also need to be able to resist the byproducts generated during biomass pretreatment and hydrolysis. In the last few years there has been an explosion in our knowledge of nontraditional microorganisms. Today, the genomes of hundreds of bacteria and Archaea are available [16]. In this collection of newly characterized microorganisms we can find almost any physiological trait that we want. These strains can, furthermore, often be manipulated genetically. Some examples of these are shown in Table 3.2. From these microorganisms and many others we can learn the strategies that Nature has selected to deal with high temperatures, high concentrations of toxic compounds, sugars, salts, pH, etc., and in some of the genomes we can also find new enzymes that would enable the use of lignocellulosic materials for fermentation. On one side, genomics is providing an immense number of physiological solutions. Likewise, advances in other research areas are providing possible solutions to some of the problems mentioned. For example, some mutations have been designed to eliminate the sequential utilization of carbohydrates present in complex mixtures [17–19]. Some of these mutations reduce the ability of the production strains to induce hundreds of genes that could produce undesired phenotypes during fermentation. Also, use of genomic arrays is helping us un-

Table 3.2 Some examples of the sequenced genomes from microorganisms with properties relevant for the development of biorefineries. Organism name

Relevant properties

Thermoplasma acidophilum Thermatoga maritima

Optimum growth: 59 8C, pH 2.0 Optimum growth temp. 80 8C. Capable of using starch, cellulose, xylan as carbon sources Requires 4 m NaCl to grow Grows and produces glutamic acid above 40 8C Extracellular oxidation of a wide range of carbohydrates and alcohols. The corresponding products (aldehydes, ketones and organic acids) are secreted almost completely into the medium Optimum growth at 60 8C and pH 0.7. Its membrane has very low proton permeability Grow optimally at 80 8C and pH 2–4

Halobacterium sp. NRC-1 Corynebacterium efficiens Gluconobacter oxydans

Picrophilus torridus Sulfolobus sulfataricus P2 and Sulfolobus tokodaii strain 7

3.5 Conclusions

derstand the types of physiological response that occur when cells are exposed to stressful conditions. For example, comparison of E. coli strains capable of producing ethanol at different levels has shown that increased ethanol tolerance results from a combination of multiple changes affecting different aspects of cell physiology [20].

3.5 Conclusions

The importance of the biorefinery concept is growing and the recent increased demand for fuel ethanol has in large part driven that growth, as shown in Fig. 3.6. New products such as the lactic acid and 1,3-propanediol produced from sugar in engineered organisms are recent examples of the biorefinery concept progressing toward reality. Apart from co-product streams resulting from the processing of grain or biomass, the resulting sugars are being used as carbon feed to furnish an ever-growing list of products including fuel and the building blocks for the synthesis of chemicals and polymers. Commercial production of 1,3-propanediol for Sorona, based on technology developed in a close collaboration between DuPont and Genencor, will mark the emergence of commercial viable application of the biorefinery concept for production of basic chemical building blocks in competition with petrochemical-derived materials [21]. Many of the biorefinery elements required for financial success seem to be currently present, for example: · high-volume, low-cost application (fuel ethanol); · multiple alternate product streams; · ability to shift to different products quickly when required; and · acceptable cost structure (at least for sucrose, starch).

Fig. 3.6 World demand for fuel ethanol over the years.

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Several financial requirements will enable the biorefinery concept to become a reality in the near future: · parts of the overall biorefinery scheme that can operate in a stand-alone manner from a financial perspective, and the ability to evolve from there; · the potential for multiple product or co-product streams – note that this may ultimately drive efforts to engineer crops to maximize their potential; · the ability to make use of a variety of feedstocks as a hedging strategy and to take advantage of pricing opportunities; · manageable capital requirements and favorable investment environment; and · government enablement (funded demonstration/development pilot plants; reasonable regulation of biorefinery operations; favorable tax treatment; strategic market support to give new biorefinery products a head start). In summary, the concept of the biorefinery is not new. In its simplistic form, several pieces have been in operation for thousands of years. Biorefineries continue to evolve depending on market needs, and growth now is being driven by the rising demand for fuel ethanol. It will continue to evolve in a manner similar to the petroleum refineries in the 1900s. Ultimately, as technological developments expand and the range of products grows, biorefineries will utilize a wide variety of feedstocks, including cellulosic biomass.

Acknowledgments

This work was supported in part by a subcontract from The Office of Biomass Program, within the DOE Office of Energy Efficiency and Renewable Energy. The authors thank Colin Mitchinson and Mike Knauf of Genencor International for their guidance of the work presented.

References 1 G Chotani, T Dodge, A Hsu, M Kumar,

3 Mitchinson, Colin. Improved Cellulases

R LaDuca, D Trimbur, W Weyler, and K Sanford. Commercial Production of Chemicals using Pathway Engineering. Biochemica Biophysica Acta, 1543 (2), 434–455, 2000. 2 Manoj Kumar, Jeff Pucci, Gopal Chotani, and Karl Sanford. Biocatalytic Conversion of Renewable Feedstock to Industrial Chemicals. In Lignocellulose Biodegradation (ACS Symposium Series 889) Badal C. Saha and Kiyoshi Hayashi, editors, Oxford Univ. Press, pp 363–376, 2004.

for the BioRefinery: A review of Genencor’s progress in the DOE subcontract for cellulase cost reduction for bioethanol. Stanford GCEP Biomass Energy Workshop, April 2004. 4 Michael Knauf and Mohammed Moniruzzaman. Lignocellulosic Biomass Processing: A Perspective. International Sugar Journal, 106#1263, 147–150, April 2004. 5 Mary Ann Godshall, Future Directions For The Sugar Industry, 2003. http:// www.spriinc.org/buton10a.html

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nol from corn starch and lignocellulosic feedstocks. NREL/TP-580-28893. Oct 2000. Toyama et al. 2002. Applied Biochemistry and Biotechnology, 98–100:257–263. Mary Mandels, Frederick W. Parrish, and Elwyn T. Reese, J. Bacteriol. 1962 February; 83(2): 400–408. Foreman PK, Brown D, Dankmeyer L, Dean R, Diener S, Dunn-Coleman NS, Goedegebuur F, Houfek TD, England GJ, Kelley AS, Meerman HJ, Mitchell T, Mitchinson C, Olivares HA, Teunissen PJ, Yao J, Ward M. Transcriptional regulation of biomass degrading enzymes in the filamentous fungus Trichoderma reesei. J. Biological Chemistry, 278(34): 31988–31997, 2003. M. Sandgren, P. Gualfetti, A. Shaw, A. G. Day, L. Gross, T. Alwyn Jones and C. Mitchinson. Comparison of Homologous Family 12 Glucosyl hydrolases and Recruited Variants Important for Stability. Protein Science, 12(4): 848–860, 2003. F. Goedegebuur, J. Phillips, WAH van der Kley, P. van Solingen, L. Dankmeyer, S.D. Power, T. Fowler. Cloning and Relational Analysis of 15 Novel Fungal Endoglucanases from Family 12 Glycosyl Hydrolase. Current Genetics, 41:2, 89–98, 2002. Sandgren M, Shaw A, Ropp T, Wu S, Bott R, Cameron A, Stahlberg J, Mitchinson C, Jones A. The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3 (Cel12A) at 1.9 Å J. Molecular Biology, 308(2):295–310, 2001. Zaldivar, J., Nielsen J. and Olsson L. Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Appl. Microbiol. Biotechnol. (2001) 56: 17–34. Hettenhauss, J. Ethanol fermentation strains: Present and future requirements

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for biomass to ethanol commercialization. http://www.afdc.nrel.gov/pdfs/ 4957.pdf. Werpy, T. and Petersen G. Eds. Top value added chemicals from biomass. Volume I. Results of screening for potential candidates from sugars and synthesis gas, NREL, August 2004. http:// www.osti.gov/bridge TIGR Microbial Database: a listing of microbial genomes and chromosomes in progress. http://www.tigr.org/tigr-scripts/ CMR2/CMRGenomes.spl. Hernandez-Montalvo, V., Valle, F., Bolivar F. and Gosset G. Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system. Appl. Microbiol. Biotechnol. (2001) 57: 186–191. Chatterjee, R., Sanville-Millard, C., Champion, K., Clark D. and Donnelly M. Mutation of the ptsG gene results in increased production of succinate in fermentation of glucose by Escherichia coli. Appl. Environ. Microbiol. (2001) 67: 148–154. Nichols, N., Dien, B. and Bothast R. Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol. Appl. Microbiol. Biotechnol. (2001) 56: 120–125. Gonzalez, R., Tao, H., Purvis J., York, S., Shanmugam K. and Ingram L. Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant). Biotechnol. Prog. (2003) 19: 612– 623. C. Nakamura and G. Whited. Metabolic engineering for the microbial production of 1,3 propanediol. Curr. Opin. Biotechnol. 2003 Oct; 14(5): 454–459.

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4 Biorefineries for the Chemical Industry – A Dutch Point of View Ed de Jong, René van Ree, Robert van Tuil, and Wolter Elbersen

4.1 Introduction

As a major policy goal for 2020, the Dutch government has stipulated that 10% of its energy use should be provided by renewable sources to meet its Kyoto objectives. Biomass is expected to be a major contributor with an expected share of more than 50% of the policy goal mentioned. Further, the Ministry of Economic Affairs has defined some very ambitious policy targets for biomass in the longer term (2040), namely 30% fossil fuel substitution in the power and transport sector and 20–45% fossil-based raw material substitution in the industrial sector. It has been calculated that the energy-substitution policy goal corresponds to a long-term required biomass substitution volume of about 600– 1000 PJth annum–1, in a scenario in which severe energy savings have also been taken into account (Ministry of Economic Affairs 2003). Adding the very ambitious raw material policy goal an additional biomass substitution volume of several hundreds of PJth annum–1 will be required. Biomass in the Netherlands that is not currently used for food applications is mainly used as animal feed or as fuel for power (and heat) production. Biomass is converted mainly by means of direct/indirect cofiring in conventional coal-fired power plants and also by stand-alone combustion plants (Cuijk, Lelystad). To meet the longer-term policy ambitions biomass must be applied in additional market sectors of the Dutch economy, using new thermochemical and (bio)chemical conversion/production processes, for example advanced gasification and fermentation technology. A current disadvantage of these processes is that final products will be produced that are more expensive than their fossil-based alternatives. Prolonged financial governmental support (e.g. investment subsidies, fiscal measures) necessary to support successful market implementation is currently lacking in the Netherlands. Further, to meet the longer-term policy ambitions, the use of potentially available relatively cheap organic side- and waste streams will not be sufficient. The use of dedicated, relatively expensive “energy” crops grown both in and outside the Netherlands (imports) is therefore inescapable. Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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4 Biorefineries for the Chemical Industry – A Dutch Point of View

Within this framework it is believed biorefineries will play a major role in the transition to a more sustainable Dutch economy. Realization of high-efficiency biorefining processes at places where biomass can be gathered, grown, and/or imported and where the “green” products can be sold to a cluster of chemical and material industries are believed to be key technologies to meet the longer-term policy goals. The chemical and material industries are founded on innovation. Because of the emerging interaction between chemistry, biology, and process engineering the industries of 2020 will be significantly different from those of today. Not only does technological development encourage changes in the design of processes and products, however, pressure from consumers and the general public also fuels a transition to a more sustainable industry. This stimulates discussion on if and how the use of renewable resources can add to a future scenario of a continuing innovative chemical industry taking into account the wishes and constraints of all current and future stakeholders. This paper first provides the societal and institutional context for the transition to sustainability in the evolution of the chemical industry. Second, it reviews different perspectives on the future of a modified chemical industry, partly resulting from emerging technological opportunities. Third, taking into account these emerging technological opportunities, this paper discusses the potential of the use of biomass in the chemical industry of today and tomorrow. Special emphasis will be given to the potential that “biorefineries” offer the chemical industry.

4.2 Historical Outline – The Chemical Industry: Current Situation and Perspectives

The objective of this section is to present an overview of the chemical products that the current industry produces. It also gives an overview of the technological pathways involved in the production of these chemical products. This overview covers the scope of chemical products and chemical intermediates that need to be produced from biomass and also provides an overview of currently produced biomass-based chemical products. 4.2.1 Overview of Products and Markets

The oil industry may be divided into several important refinery sectors and products including: gas for commercial energy supply, heavy gasoline for car fuels, naphtha for the petrochemical industry, kerosene for aviation fuel, and oil residues used in bitumen and lubricating oils. This review focuses on the naphtha fraction in the petrochemical industry and the gamut of chemicals, products, applications, and markets that can be derived from it. The current chemical industry’s most important feedstock is naphtha which can be cracked to obtain a range of olefins, for example ethylene and butanes, and other small (un)saturated hydrocarbons and aromatic compounds, for ex-


ample benzene and alkyl benzenes. These simple hydrocarbons form the backbone of the possible products that are generated in the chemical industry today. The scope of different chemicals (and transformations) that can be achieved from naphtha for the chemical industry is illustrated schematically in Fig. 4.1. In principal these materials can be transformed to the bulk of chemicals produced by two initial key pathways: · they may be directly isolated, used, and transformed by a variety of chemical techniques to a range of compounds; or · they may undergo a gasification process to form synthesis gas (CO and H2) which on recombination enables access to another branch of alternative chemicals and technologies. The chemicals described in Fig. 4.1, derived from the small (un)saturated hydrocarbons and synthesis gas, enables an array of chemicals of technological and economically important products, applications, and markets to be obtained. For example: · vinyl monomers for plastics used in pipe, packaging, and rubber applications; · monomers for polyester and amide synthesis used in fibers (for textiles), engineering materials, and some container materials; · solvents for, among others, the paint industry; · chemicals for the pharmaceutical industry; and · chemicals for the insecticide and herbicide industry. 4.2.2 Technological Pathways

Closer examination of the chemical transformation steps involved in converting one substance into another (Fig. 4.1), reveals some generic approaches to the types of chemistry and technology used in the chemical industry: · oxidative and reductive techniques are most prolific; · introduction of nitrogen into chemical structures is most frequently achieved by initial amination or amidation with ammonia; · carbonylation reactions are frequently used to make small incremental changes in chain length and for introduction of new functionality; · extensive use of gaseous reagents; · high selectivity of chemical steps by utilization of catalytic materials, therefore reducing the need for chemical derivatization for transformation; and · high conversion in chemical steps by use of catalytic materials 4.2.3 Biomass-based Industrial Products

Although most chemicals are of petrochemical origin an important example of a chemical produced in bulk from a non-petrochemical source is ethanol. Ethanol, produced by fermentation of molasses, etc., has been most extensively used

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Fig. 4.1 Schematic representation of chemical transformation steps in the (petro)chemical industry.

4.2 Historical Outline – The Chemical Industry: Current Situation and Perspectives Table 4.1 Estimated EU potential of major biomass-based products (Ehrenberg 2002). Market sector

Total consumption market (1998) (kton)

Renewable consumption market (1998) (kton)

Potential of renewables in 2010 (kton)

Potential share in 2010 (%)

Polymers Lubricants Solvents Surfactants

33 000 4240 4000 2260

25 100 60 1180

500 200 235 1450

1.5 5 12.5 52

in the food and beverage industry, although increasing amounts are also used in bio-based transportation fuels and to prepare non-food industrial chemical products. Although ethanol is perhaps the most widely known example of a biobased chemical product both in the Netherlands and worldwide, a range of other bio-based chemical products is produced and used in a variety of industrial applications. These fall into several generic categories: · naturally occurring carbohydrate polymers; · fats and oils of plant origin (and, to a lesser extent, of animal origin); · terpene-based materials; · chemical products of carbohydrate-containing materials; and · fermentation products of carbohydrate-containing sources. A recent study coordinated by the European Renewable Resources and Materials Association (ERRMA) has evaluated the current situation of biomass use as an industrial feedstock for chemicals and materials (Ehrenberg 2002). Table 4.1 shows the potential of biomass to replace petrochemical-based products in the areas of polymers, lubricants, solvents, and surfactants. Many of those applications, especially lubricants, solvents, and surfactants, can be achieved by direct extraction of the components from the biomass without additional (bio)conversion steps.

4.2.3.1 Carbohydrates Today’s bio-based products include commodity and specialty chemicals, fuels, and materials. Some of these products result from the direct physical or chemical processing of biomass cellulose, starch, oils, protein, lignin, and terpenes. Most biomass consists of natural polymers and most biomass is carbohydrate in nature. This means that most biomass is in the form of carbohydrate polymers (polysaccharides). These natural polymers can be used both as nature provides them and as the skeletal framework of other derived polymers. By far the most abundant of these carbohydrate polymers is cellulose, the principal component of the cell walls of all higher plants. It is estimated that 75

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billion tons of cellulose are biosynthesized and disappear each year, most of the disappearance being through natural decay. Cellulosic plant materials are used as fuel, lumber, mechanical pulp, and textiles. Purified cellulose is currently used to make wood-free paper, cellophane, photographic film, membranes, explosives, textile fibers, water-soluble gums, and organic-solvent-soluble polymers used in lacquers and varnishes. The principal cellulose derivative is cellulose acetate, which is used to make photographic film, acetate rayon, a variety of thermoplastic products, and lacquers. The world’s annual consumption of cellulose acetate is about 750,000 tons. Cellulose acetate products are biodegradable. Commercial production of lyocell, a cellulosic fiber made from a solvent spinning process, has also started recently. Lyocell, which, unlike rayon, does not require dry cleaning but is washable and very strong, is the first new textile fiber to be introduced in 30 years. Cotton is currently the most important non-wood fiber crop. It is mainly used for weaving and spinning into cloth. Advances in biotechnology and genetic engineering are now enabling development of cotton cultivars with improved pest resistance, yield, and quality, thereby potentially reducing production costs and better matching cotton characteristics to specific applications. Natural fibers other than cotton occupy a variety of niche markets, for example specialty fabrics, fiber-reinforced composites, papers, cordage, and horticultural mulches and mixes. Heightened environmental concerns are also helping jute, hemp, flax, sisal, abaca, coir fibers, and products derived from these fibers to find their way into new markets. The use of natural fibers in fiber-reinforced composites, especially, has seen tremendous growth in Europe in recent years (van Dam et al. 2004).

4.2.3.2 Fatty Acids Fatty acids, readily available from plant oils, are used to make soaps, lubricants, chemical intermediates such as esters, ethoxylates, and amides. These three important classes of intermediate are used in the manufacture of surfactants, cosmetics, alkyd resins, polyamides, plasticizers, lubricants and greases, paper, and pharmaceuticals (Ahmed and Morris 1994). Of the approximately 2.5 million tons of fatty acids produced in 1991, about 1.0 million tons (40%) were derived from vegetable and natural oils; the remaining 1.5 million tons were produced from petrochemical sources. Twenty-five percent of all plant-derived fatty acids used in the coatings industry comes from tall oil (a byproduct of kraft paper manufacture). Surfactants are, currently, by far the most important outlet for fatty acids (Table 4.1). In Europe most raw materials used for surfactant production are derived from tropical oils, because of their more suitable chemical structure. Besides oil-based surfactants there is also a relatively small market (< 5%) for starch-derived surfactants.


4.2.3.3 Other Terpenes, derived from woody materials, also give rise to a variety of chemicals and products. Crude turpentine, isolated from the pulping industry, may be used to isolate “pine oil” commonly used in cleaning products, alternatively its components may be isolated and chemically transformed to materials such as dipentene, which can be polymerized to prepare tacky polymers and used in chewing gum and food packaging coatings. Although not widespread in Western Europe and North America, several Eastern European, Asian, and South American countries use carbohydrate-containing agricultural residues as a raw material in the chemical industry. For example, hydrolysis of starchy materials to glucose with concomitant severe acid-catalyzed degradation can result in oxalic acid, which is used in the leather industry. Alternatively, pentoses, found in bagasse and corncobs, for example, readily undergo acid-catalyzed dehydration to furfural. Furfural is a flexible chemical raw material which can be used as a solvent itself in several applications or can be used to prepare furfuryl alcohol used in resin materials, and tetrahydrofuran, a common organic solvent. Although many other chemical transformations are possible, their current commercial status is unclear. Carbohydrates remain a flexible raw material, and beside “classical” chemical transformations, biotechnological transformations have also been explored. For example lactic acid can be used to prepare a biodegradable polymer with interesting properties and has a wide range of potential applications including fibers and packaging materials (Sreenath et al. 2001). Other biotech products include citrates to prepare additive chemicals (dyeing, cleaning, and polymer) and fumaric acid in preserving agents and as a component of unsaturated polyesters. Specialty chemicals can be made using fermentation and enzymatic processes or directly extracted from plants (or aquatic biomass). It has been shown that plants can be altered to produce molecules with functionality and properties not present in existing compounds (e.g. chiral chemicals). Examples of bio-based specialty chemicals include bioherbicides and biopesticides, bulking and thickening agents for food and pharmaceutical products, flavors and fragrances, nutraceuticals (e.g. antioxidants, noncalorific fat replacements, cholesterol-reducing agents, and salt replacements), chiral chemicals, pharmaceuticals (e.g. Taxol), plant-growth promoters, essential amino acids, vitamins, industrial biopolymers such as xanthan gum, and enzymes. Specialty chemical markets currently represent a wide range of high-value products. These chemicals usually sell for more than 4 1 kg–1. Although the worldwide market for these chemicals is smaller than for bulk and intermediate chemicals, the specialty chemicals market now exceeds $ 3 billion. It is expected that advances in biotechnologies will have significant impacts on the growth of the specialty chemicals market.

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4.2.4 International Perspectives

In several industrialized countries (i.e. USA, UK, and the Netherlands) government, business, society, and science have been engaged in outlining future developments in the material and chemical industry (American Chemical Society 1996; Molendijk et al. 2004; National Research Council 2000; Okkerse and van Bekkum 1997; Parris 2004; Sims 2004; UK Foresight program Chemicals Panel 2000; US Department of Energy 1998, 1999; Weaver 2000). Most of these foresight exercises are led by the view that an increased demand for chemicals and materials will place additional pressure on the use of resources and on the environment. Accordingly, the thrust is in finding new technologies and creating novel materials, processes, and capabilities to bring this growth in line with society’s demand for sustainability. The perspectives indicate a shared interest in shifting from sole dependence on fossil resources to a chemical and material industry founded on the application of plant-based resources, derived either from secondary streams (i.e. waste and recycling) or from primary streams (i.e. dedicated production). The discussion suggests that changing the resource base of the chemical and material industries will induce cleaner processes, safer products and a more effective use of scarce resources. The shift to a bio-based chemical and material industry will alter the technological basis of the industry quite radically. To substantiate the sustainable credentials of new products and processes, further research and actual implementation will indicate what specific technological routes in fact contribute to sustainability. This will be necessary for communication with NGO, the general public, regulators, and policy makers about, for example, CO2 emissions. From the perspective of the chemical industry striving for sustainability with sound economic foundations draws the attention to three key areas.

4.2.4.1 Production The actual production process has a major environmental impact both on efficient use of energy and resources and on emission and waste production; this is especially so in bulk industries. This links the provision of multi-quality biomass and the industrial production process. Because of the large volumes used, it may have a farreaching impact on the environment. Cost reduction, because of cheaper raw materials or processes with less extreme conditions, will be an important consideration.

4.2.4.2 Integration Implementing a strategy for sustainability requires coordination between different levels of a supply chain, product portfolio, and fine-tuning between distributed technological capabilities. Key technologies in conversion, extraction, and separation will lay the foundation for further improvement in bulk production and the development of products with well defined functionality.

4.3 Biomass: Technology and Sustainability

This requires an integrated view on resource use and a strategic view on technology development. Linking of life sciences, chemistry, energy technology, and process engineering is required for taking up such a challenge.

4.2.4.3 Use and Re-use In terms of specific functionality, life cycle and recycling or safety, the actual performance of end-products importantly defines the shape of a market-oriented strategy for sustainable resource use. Increasing revenues by generating highvalue products will also be an important consideration. This links production process with product design and defines new terrain for innovative business enterprises. Communicating the sustainability benefits to consumers or users in the (new) end-use market will enable them to make more informed choices. To develop a sustainable perspective for the chemical industry a combinatorial approach integrating functionality provided by new molecules or materials, improved efficiency and safety of production processes, and use and re-use of materials must be applied. Integrating criteria such as design for functionality or recycling properties of new materials will encourage the search for new sustainable solutions in the chemical industry.


The chemical industry undergoes rapid and important changes inherent to the turmoil resulting from transitions at the end of an industry’s current lifecycle. These changes require new technological, organizational, and commercial answers from the industry. Moreover, the business strategy of the chemical industry will become increasingly dependent on the acceptance or rejection by society and consumers of its activities and its conduct. This section introduces a perspective on a chemical industry that combines technological innovation with a socially acceptable business strategy. 4.3.1 Transition to a Bio-based Industry: Sectoral Integration in the Netherlands

Products from the chemical, material, and power industries have become an integral part of our daily lives and demand for these products is projected to increase. A general concern is the intensive use of finite resources, in particular fossil resources, and, consequently, the industry is in the midst of reconsidering its current resource use. Two major Dutch companies communicated its transition to the general public – the chemical company DSM advertised its transition process to a specialty company while building an image of sustainability and, in 2002, Shell, the energy company, launched a nation-wide advertisement campaign highlighting the future of natural and renewable resources. In this pro-

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cess, industrial research and development seems to (re)discover the variation of quality and specific functionality in renewable resources; this offers the opportunity to meet the demand for healthy and environmentally friendly end-products. The industry must, however, contend with both established paths of research and development and its extensive infrastructure in terms of production facilities and equipment. As a result, transition to a bio-based industry requires the development of new chains and persistent crossing of boundaries between disciplines, departments, and sectors. Innovation in the energy sector is an important driver of technology development in the chemical and material industries. Hence, petrochemistry is a constant factor in industrial development, although the use of biomass or renewable resources comes to the fore anticipating rises in oil prices or directive measures related to international agreements, for example EU directive 2003/30/EC on promotion of the use of biofuels or other renewable fuels for transport. There are several political drivers for enforcement of such directives. The most important include: · the widely accepted threat of global warming as a result of the emission of greenhouse gases; · the unwanted dependency on oil-producing countries; · the recent enlargement of the EU with associated political issues especially related to agriculture; and · sustainable development of rural areas and creation of employment. The question is whether an integrated energy sector and petrochemical industry is the right or only venue for a transition to a bio-based industry. Two non-exclusive scenarios can be distinguished: 1. The energy sector and the petrochemical industry integrate further and the oil price remains the major driving force in business and in innovation. 2. The chemical and materials industry dissociate themselves from the petrochemical sector and seeks new potentials of bio-based industrial processes using renewable resources supplied by the agro-sector, leading to environmentally friendly modes of production and to healthy and sustainable end-products with specific qualities (Coombs 1995). Most likely vertically integrated companies, including Sabic and Shell, will tend to stick to the first strategy, whereas companies not involved in exploration, e.g. Dow, DSM, might tend to follow the second strategy. The selling of the polyolefins division of DSM to Sabic and the acquisition of part of Roche can be seen as an example of this strategy. These scenarios (represented in Fig. 4.2), discussed on a number of occasions in the Netherlands, draw the attention to the linking of the agro-sector and the food and feed-processing industry with the chemicals and materials industry (de Klerk et al. 2002). Although the Dutch economy still has a strong base in manufacturing, both of food and non-food products, the agro-sector and the chemicals sector operate remotely and lack of synergy between these two sectors may hinder progress in


Fig. 4.2 New synthesis between economic clusters.

developing biorefineries with new manufacturing processes and new products. In the 1980s and 1990s the agro-sector was involved in so-called “agrification”, i.e. producing industrial crops in rotation with food crops or potatoes, but failed to create ventures with end-users for its resources in the chemical and manufacturing industries (van Roekel and Koster 2000). Pushing the use of renewable resources without identifying a clear demand, either by industry-based endusers or consumers, thus seemed to be unproductive and even counterproductive. Fine-tuning across sectoral boundaries seems to be an important condition for changing current resource use. In the following discussion we will identify the consequences of this condition for the position of the Dutch agrosector and food industry. The Dutch agro sector and the food and feed industry are founded not only in primary production of agricultural crops but equally in processing, distributing, and transporting primary and secondary flows of agricultural resources. The combination of intensive agricultural production, the transit of commodities in Rotterdam harbor and an extensive food processing industry is a specific quality of the Dutch economy. Import of resources for the food-processing in-

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dustry, including starch, sugar, and vegetable oils, results in a high “biomass intensity”. A related factor is the opportunity to direct secondary flows, side, and waste streams, to other industries; without the absorption of these secondary flows, mainly by the feed and alcohol producers, the continuity of the processing industry would be in danger (Rabobank International 2001). This sketches a landscape wherein a new synthesis between sectors is possible. This contrasts, however, with the current low-value use of renewable resources, mainly in the feed and power industry. A driver for change in the feed industry might originate in recent scares about the use of slaughter residues and of potentially hazardous fats and oils in feed, bringing these undesired components in the cycle for human consumption. The European debate on genetically modified crops might also result in stricter regulation of the use of these crops in feed and food. Consequently, the composition of feed is affected by public pressures and stricter regulation which, in combination with the tendency to reduce the intensive livestock industry in the Netherlands, might lead to the disappearance of existing markets for secondary flows. This will place high pressure on the food processing industry, because secondary or waste streams will become a cost rather than an income generator (Rabobank International 2001). The short-term question is where the food processing industry and the agrosector can market their secondary resource flows and/or plant-based resources; this is an environmental and economic problem. Therefore, companies, research institutes, and government try to address the issue of creating new chances for biomass, which includes finding alternative markets for residues and waste streams. A related question is how to find high-value utilization and application of these renewable resources, in addition to low-value energy and bulk products. This will require fine-tuning of the quality, price, and quantity of renewable resources with demand in the end-user market and functionality requirements of new products. We must, therefore, turn round and move from demand and functionality to processing and, eventually, to the production or supply of the raw material. The next section briefly identifies how different social actors perceive healthy and sustainable products and processes, to provide a guide for formulating technology agendas for the chemicals and materials industry. 4.3.2 Can Sustainability Drive Technology?

One of the more important societal driving forces is the drive for sustainability. Directing innovation and technology development from the perspective of a sustainable, bio-based society is one of the major challenges for the chemicals and materials industries. In response to this widely conceived public concern, companies try to focus their business strategies both on sustainability, including environmental concerns, and on consumer demands for safe products. Most companies trying to address the three Ps (planet, people, profit) are, however, well aware that profit is always the ultimate driving force.

4.4 The Chemical Industry: Biomass Opportunities – Biorefineries

To develop the technology supporting such an ambition still requires a substantive effort, both in terms of innovative research and in terms of bringing together different disciplines, such as chemistry, biology, energy technology, and engineering, and different professional fields, such as design, bulk production of chemicals, and the supply and storage of renewable or plant-based resources. Hence, both from a sustainability and business viewpoint an integral approach to chemical, material, product, and energy outlets must be addressed in biorefineries creating maximum added value from the selected biomass resources. A sustainable chemical industry must strive for a business strategy that integrates social, safety, health, and environmental objectives with the technological and economic objectives of its activities. Assembling industry, science, and government is one aspect of developing foresight; consulting a wider range of stakeholders, i.e. consumers and citizens, about technology strategies is another. In doing so, interdisciplinary research may be able to contribute to fine-tuning business strategies and technology strategies. Before formulating the shape of such a new perspective, a selected number of international perspectives on technological change in the materials and chemicals industry will be discussed.


The preceding paragraphs have shown the scope of chemical products that are currently produced and therefore the targeted product portfolio of biomass-based chemical products. It was also shown that drivers for a transition to a bio-based economy lie not in technological opportunities alone but are a complex combination of societal, economic, and technological opportunities, challenges, and constraints. This section will focus in more detail on the main technological opportunities to transfer biomass, either directly or via chemical intermediates, into chemical products. 4.4.1 Biomass Opportunities

In the realization of a bio-based chemical industry two distinct approaches can be identified. In the first approach, the value chain approach, value-added compounds in biomass are identified and isolated in different processing and (bio)conversion steps. The remaining biomass is then transformed into a universal substrate from which chemical products can be synthesized. In this approach it is thought that it is technologically and economically beneficial to extract valuable chemicals and polymers from biomass rather than building these compounds from universal building blocks. It can be concluded that the main technological challenges to aid the economic feasibility of this approach lie in the area of biomass refining, separation technology, and bioconversion technology. Far-reaching integration of the food, feed, and chemical industries is, moreover, required, as is a major investment in infrastructure.

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The second approach, the integrated process chain approach, follows the analog of the petrochemical industry. In this scheme a “universal” substrate is first transformed into universal building blocks, based on which chemical products are produced. In this approach it is thought that it is economically and technologically beneficial to build chemicals in highly integrated production facilities. The main technological challenges for this approach lie in the high-efficiency transformation of biomass into commonly known building blocks for the petrochemical industry (van Tuil 2002). The main technologies producing chemicals from biomass are: · biomass refining or pretreatment; · thermochemical conversion (gasification, pyrolysis, hydrothermal upgrading (HTU)); · fermentation and bioconversion; and · product separation and upgrading. Five main categories of building block can be identified as intermediates in the production of chemical products from biomass. · Refined biomass, i.e. biomass in which the valuable components have been made accessible by physical and/or mild thermochemical treatment. These components are extracted from the refined biomass. The remaining biomass then undergoes further transformation. · BioSyngas. This gas (mainly CO and H2) is a multifunctional intermediate in the production of materials, chemicals, transportation fuels, power, and/or heat from biomass; it can easily be used in existing industrial infrastructures to substitute the conventional fossil-based fuels and raw materials. · Mixed sugars. These C5 and C6 sugars are further refined substrates for chemical and bioconversion. These substrates mainly originate from side streams in the food industry and potentially from ligno-cellulosic biomass streams. · Pyrolysis oil. This oil is produced in fast and flash pyrolysis processes and can be used for indirect cofiring for power production in conventional power plants, for direct decentral heating purposes, and potentially as high energy density intermediate (important for long-distance transportation) bio-based intermediate for the final production of chemicals and/or transportation fuels. · Biocrude. This material is a fossil oil-like mixture of hydrocarbons with low oxygen content. Biocrude results from severe hydrothermal upgrading (HTU) of (relatively wet) biomass and can, potentially, like its petroleum analog, be used to produce materials, chemicals, transportation fuels, and power and/or heat. 4.4.2 Biorefinery Concept

A biorefinery is a facility that integrates biomass conversion processes and equipment to co-produce fuels, power, and chemicals from diverse biomass sources (Fig. 4.3). The biorefinery concept is analogous to today’s petroleum refineries, which produce multiple fuels and products from petroleum (Fig. 4.4).


Fig. 4.3 Schematic overview general integrated biorefinery process.

Fig. 4.4 Detailed overview integrated biorefinery process.

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Industrial biorefineries have been identified as the most promising routes to the creation of a bio-based economy (Realff and Abbas 2004). Partial biorefineries already exist in some agricultural and forest products facilities (e.g. pulp mills, corn wet milling, starch and sugar beet refining). These systems can be improved by better utilization of residues and optimization of total added-value creation; new biorefineries can be enhanced by applying the lessons learned from existing facilities to comparable situations. By producing multiple products, a biorefinery can take advantage of the natural complexity and differences between biomass components and intermediates and therefore maximize the value derived from the biomass feedstock. A biorefinery might, for example, produce one or several low-volume, but high-value, chemical products and a low-value, but high-volume, platform chemical and/or liquid transportation fuel, while generating power and heat for its own use, and probably enough for sale of electricity. The high-value products enhance profitability, the high-volume chemicals and/or transportation fuels help meet European energy needs and CO2 emission-reduction goals; whereas the power and/ or heat both reduce overall production costs and greenhouse gas emissions. 4.4.3 Biomass Availability

The Netherlands is a small country where land is scarce. Although the options for primary crop production are limited, the biomass flux of utilized biomass (organic material) is very high (partially imported). An estimated 42 million dry tons of biomass (13 ton ha–1) (van Dam et al., in preparation) are used and produced in different sectors of the economy (compare with a biomass flux some 5 tonnes for Germany). As markets change this high turnover of biomass does generate many opportunities for reallocating streams toward biorefinery feedstocks. One of the largest biomass streams is produced in the agri-industrial complex. Many of these streams can be regarded as byproducts. Approximately 10 million tonnes (as received) of byproducts are currently mostly (90%) used for animal feed (Vis 2002; Elbersen et al. 2002). These byproducts vary from slaughterhouse wastes to discarded frying oil, potato peel, etc. As traditional markets change (diminish) and markets for biobased energy and products increase, a large part of these streams can become partially available for biorefinery. This change in market demand for feed is already apparent from the decline in livestock (from 4.6 in 1995 to 3.6 million in 2003), and pigs (14.5 million in 1995 to 10.7 million in 2003), which leads to a smaller demand for feed. Another estimate has been made of the potentially available (ligno)cellulose biomass feedstocks in The Netherlands that could be used for ethanol production (de Jong et al. 2003). The total amount of these technically suitable feedstocks is approximately 12 million tons (dry weight) per year (about 220 PJth annum–1), excluding import and biomass energy crops. The potential feedstocks are highly variable, however, and include a range of agro-industrial residues, agricultural wastes, forestry residues, and household organic waste, etc.


As already mentioned, the domestic (primary) biomass energy crop potential is limited with some 2 million ha of agricultural land of which less than 1 million ha is arable land. It is hard to estimate the potential for biomass energy crops as competing demands for land are uncertain. Estimates range from 0 to a maximum of 3 million tonnes (300.000 ha at 10 ton dry weight ha–1 annum–1, 18 GJ ton–1 dry weight) equivalent to 50 PJth annum–1 (Minnesma 2003). In the short term, arable agriculture could be an important source of biorefinery feedstocks with more than 1 million tonnes of crop field residues available. The maximum total Dutch biomass availability (organic residues and crops) will therefore amount to 300 PJth annum–1 (approx. 15 million tonnes dry weight). Taking the longer-term (2040) policy ambitions into account, requiring a biomass substitution volume of about 600–1000 PJth annum–1, it can be concluded that The Netherlands will have to import at least half, if not more, of its long-term biomass requirements. The potentially available feedstocks for biorefinery in The Netherlands are highly variable and most streams are dispersed over the country. Though many streams currently have other applications the potential is more than 15 million dry tonnes of biomass. Large-scale biorefinery systems will have to use a variety of feedstocks to secure feedstock availability (“multi-feedstock plant”). The option to import biomass over longer distances should also be available. For medium-term development (< 10 years) a focus on feedstocks which have the desirable characteristics (for example homogeneous streams low in lignin and/or low in ash) are desirable. For the longer term (> 10 years), as the scale of production increases, the feedstock range should be broadened with additional residues and (woody) energy crops (e.g. willow). The worldwide average net available biomass potential for non-feed and nonmaterial purposes is expected to amount 200–700 EJth annum–1 (maximum 1100 EJth annum–1) in 2050 (Lysen 2000). Worldwide, enough biomass will be available to fulfill the needs. Because other countries will claim the same biomass, the market price will be internationally settled. Timely participation in the developing international market is a requirement to become an important global player. 4.4.4 Primary Refinery

Deriving a raw material stream with desired specifications (i.e. amount of ash, fermentable sugars, lignin) while simultaneously extracting valuable components from the heterogeneous biomass streams is one of the major biorefinery research and development issues. The main research and development areas which must be addressed before an efficient biomass pretreatment chain can be established are: · characterization and standardization of raw materials and products; · development of a cost-effective infrastructure for production, collection, characterization, storage, identity preservation, pre-processing activities, import

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and transportation of feedstocks for bio-based products and bioenergy applications; and · development of economically viable pretreatment processes for commercial use of a range of current and new bio-based feedstocks. Biorefineries could potentially use complex processing strategies to efficiently produce a diverse and flexible mixture of conventional products, fuels, electricity, heat, chemicals, and material products from all available, environmentally appropriate biomass feedstocks. To achieve economically viable biorefineries it is important that: · separation and fractionation technologies for high-throughput systems are developed that produce value-added products and no waste streams; and · generic solutions are identified that will apply across multiple feedstocks while simultaneously achieving a zero-waste production system with either direct use or recycling of all components.

4.4.5 Secondary Thermochemical Refinery

Thermochemical-based refinery processes usually consist of the following interconnected unit operations: pretreatment (i.e. drying, size reduction), feeding, conversion (gasification, pyrolysis, HTU), product clean-up and conditioning, and product end-use. In this subsection only gasification-related aspects are discussed, because it is expected that gasification will be the key technology in (secondary) thermochemical refinery processes. Atmospheric air-blown gasification processes, based on fixed bed, (circulating) fluidized bed, or indirect dual reactor technology, are commercially available for product gas (mainly CO, H2, N2, impurities) production. After gas clean-up this – so called – fuel gas can potentially be used for heat, power, or CHP production in a variety of prime movers. At the moment only direct heat production by coupling of these technologies to conventional (natural gas or diesel fired) furnaces, and power production by indirect cofiring in coal-fired power plants is technically and economically feasible. The market implementation of fully integrated gasification-based systems for stand-alone power or CHP production is delayed by the high power/CHP production costs, mainly caused by the relatively high investment costs of these new and emerging technologies, and insufficient financial support from the government. Oxygen-blown gasification processes, especially applicable for the production of BioSyngas (mainly CO and H2), based on both bubbling fluidized bed and entrained-flow technology, are technically not yet available for biomass applications. Within the framework of the EU Chrisgas-project, TPS et al. are now trying to modify the air-blown pressurized Varnamo gasification plant (Sweden) for oxygen-blown operation.


Within the sixth Framework Program of the EU, ECN et al. are currently (October 2004) preparing a large STREP proposal concerning the modification of existing coal-based slagging oxygen-blown entrained-flow-based gasification technology for 100% biomass use. Main research areas are biomass feeding, gasification/slag behavior, product gas cooling, and the commercial applicability of produced solid waste streams. The final goal is to design, build, and operate several MWth pilot plants within 4 years, so that large commercial implementation can become feasible around 2010. The gasification processes mentioned all require size-reduced and relatively dry (about 15–20% moisture maximum) biomass fuels. “Wet” biomass fuels require rigorous drying before they can be used. Alternatively these fuels can potentially be converted by means of sub/supercritical gasification processes (or fermentation processes, see Section 4.4.6). Supercritical biomass gasification is performed at conditions above the critical point of water (374 8C, 221 bar), mostly in the temperature range 500–700 8C. Under these conditions an H2-rich product gas is produced. Under subcritical conditions (temperature range approx. 350–400 8C) a methane-rich gas will be produced. Under these conditions for full carbon conversion very low dry matter concentrations and a catalyst are required. Although bench/pilot facilities are available – FZK (D) 100 L h–1 (since 2003), University of Twente (NL) 5–30 L h–1 (since 1998) – the ECN opinion is that some years of laboratory-scale PhD work will be required before the potential commercial implementation of this technology. The technology is expected to be developed for “green natural gas” (SNG) production; for the production of hydrogen, ECN has the opinion that this technology will not become financially competitive in the longer term. Some main research items are: feeding, heat exchange and catalyst behavior. Within the framework of the biorefinery concept, two gasification-based pathways can be distinguished. · Application of the biorefinery concept to increase the financial yield of “conventional” gasification processes. By separating highly added-value components from raw biomass fuels before conversion, or afterwards from the raw “products” in product gas clean-up/conditioning, the overall plant economics could be improved, simplifying market implementation (Fig. 4.5). · Development of highly efficient advanced gasification-based thermochemical secondary biorefining processes. By developing advanced catalyst-supported staged or subcritical gasification processes it is expected that a variety of “products” could be separated from biomass in such a way that the overall process will be market competitive, without the need for substantial governmental support.

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4.4.6 Secondary Biochemical Refinery – Fermentative Processes

This section focuses on fermentation as the main form of bioconversion. A large number of chemicals are currently produced by fermentation. These range from bulk chemicals, for example ethanol (for food and fuel purposes) (Reith et al. 2002) and lactic acid (as food ingredient or as monomer of polylactic acid) (Datta et al. 1995), via compounds as amino acids, gluconic acid, and citric acid, to high-value products such as antibiotics. The efficient conversion of sugars into ethanol and lactate by fermentation increases interest in fermentation technology as a means of production of bulk chemicals. Fermentation has several advantages over conventional chemical reactions. · Fermentation processes are usually one-step syntheses which could reduce investment costs. · Microbial biosynthesis offers control over chemical reactions that is unchallenged by state-of-the-art chemical synthesis resulting in highly functional compounds. Currently, however, fermentation has several disadvantages which have to be solved before it will be able to compete with conventional chemical reactions.

Fig. 4.5 Thermochemical biorefinery concept of ECN to increase the financial yield of “conventional” gasification processes (Boerrigter et al. 2004).


· The costs of fermentation processes for bulk processes are higher than those for the corresponding chemical process. · The natural product spectrum of microorganisms is limited. · In fermentation processes several side streams are formed which can be coped with on the small scale but will cause a severe burden on the bulk scale.

4.4.6.1 Feedstocks Most feedstocks currently used for fermentation processes are based on sugar beet, sugar cane, and corn. To reduce feedstock costs other substrates must be used. Several alternative feedstocks are being considered, for example fruit waste, wood, straw, agricultural waste streams, dung, oils, and fatty acids, etc. Among these the lignocellulosic materials are the most abundant polysaccharide-containing biomass available in the world and are therefore an extensively studied feedstock for fermentation processes. For almost all microorganisms this lignocellulosic material must be hydrolyzed into its component saccharides by mechanical pretreatment, followed by acid, base, or heat treatment, use of organic solvents or wet-oxidation to open the matrix, and, usually, enzymatic hydrolysis of the cellulose. The costs of the hydrolytic enzymes are the main expense in feedstock pretreatment and hydrolysis. Hydrolysates from lignocellulosic materials contain, beside C6 sugars such as glucose, also C5 sugars, for example xylose, and inhibitors. The relative amount depends on the type of feedstock and the process used. Bakers’ yeast, used in the production of ethanol, cannot use these C5 sugars. It has been calculated that for a competitive process these C5 sugars also must be converted into ethanol. This is probably also true for other, future, processes.

4.4.6.2 Product Spectrum Microorganisms can produce an extremely wide variety of chemical compounds. Most of these compounds are, however, only intermediates in the overall metabolism and will not be produced in significant amounts. Many of the platform chemicals used in the chemical industry are, furthermore, not produced by natural microorganisms. The new fermentation technology must interface directly with existing processes and installations in classical chemistry to reduce investment costs, indicating that the biochemical pathways of the microorganisms must be modified to be able to produce current platform chemicals. Several physiological aspects (yield, productivity, toxicity of product, use of GMO, byproduct formation, etc.) must be taken into account if this is to be achieved successfully. This shows that the production of non-natural compounds by microorganisms can be a complex task. Some successful examples of the modification of metabolic pathways, however, are the production of 1,3-propanediol for the production of Sorona by DuPont, the synthesis of muconic acid as a precursor for adipic acid, and the optimization of succinic acid production (Biebl et al. 1999; Chotani et al. 2000; Zeikus et al. 1999).

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4.4.6.3 Side Streams and Recycling Fermentation processes are usually regarded as “clean” processes. During fermentation, however, some side-streams are created which could be a severe environmental burden on the bulk scale, for example: · During ethanol production by bakers’ yeast glycerol and fusel oils are produced. · Lactic acid is removed from the fermentation broth by complexing with calcium ions resulting in the formation of insoluble calcium lactate. This eventually results in the formation of equimolar amounts of gypsum. · In all fermentations microbial biomass is formed. This can be a considerable fraction. For example in the experimental production of polyhydroxyalkanoates (biopolymers) 20–50% of the product is biomass.

Because gypsum production could also be a problem in the synthesis of other organic acids, much research has been devoted to in the development of other down-stream-processing processes, for example membrane electrodialysis. For the problem of microbial biomass formation another solution must be sought in which the high-added-value components of the microbial biomass, for example pigments, vitamins, and antioxidants are extracted and the remaining material could then be recycled as feedstock in the fermentation process.

4.5 Conclusions, Outlook, and Perspectives

The objective of this section is to draw conclusions from previous sections by presenting an agenda for further activity to facilitate the transition toward the production of chemicals and chemical products from biomass. We believe that if these agendas are addressed the sustainable production of chemicals from biomass will become a realistic option in the future. 4.5.1 Biomass – Sustainability

A transition towards increased use of biomass originates from its possible contribution to sustainable production of energy, fuels, chemicals, and materials. Several chemicals can, moreover, be more easily or energy-efficiently produced from biomass than from other feedstock. Many of these products can be extracted directly from the biomass. The importance of biomass use towards the sustainability of the production of chemicals is directly linked to the scale of production. It is, moreover, often questioned whether the use of biomass for energy, fuels, and chemicals can form a symbiosis with the use of the same biomass for the production of food, feed, and materials, for example paper and wood. Also mentioned is the uncertainty of the implication of a change in the main feedstock for energy and fuels industry on the chemical industry.

4.5 Conclusions, Outlook, and Perspectives

What will be the main feedstock for energy and fuels in 50 years time – water, sunlight, natural gas, or biomass? Are the conversion technologies mentioned in this study elegant ways of waste disposal in the food, feed, and cellulose/paper industry? Is biomass mainly of interest for extraction of valuable chemicals? What will be the impact of a biotechnological revolution on the opportunities and pitfalls of biomass? Some of these questions have been introduced in this paper. It is, however, suggested that further insight into the role of biomass in chemicals production in the Netherlands will be obtained by scenario evaluation. The result of this evaluation will make it possible to choose feedstock/conversion technology combinations that maximize the potential of biomass use for chemicals and chemical products. 4.5.2 Biomass Refining and Pretreatment

A range of standards is needed to verify performance in the industry and to help improve marketability. These include standards for environmental quality of feedstocks and conversion technologies, and accreditation and standards for energy content and the quality of feedstocks and products. Much of the technology and many of the products developed have not been proven under “real world” conditions and/or face significant certification challenges. These certification challenges are often the result of systems that set standards based on the physical characteristics of petroleum products, rather than on the performance of the end product. Improved practices in agriculture, silviculture, and aquaculture can play a significant role in increasing yields while reducing required inputs. To achieve great increases in biomass feedstock availability many issues must be resolved in harvesting, collection, storage, import, and transport. Current methods result in low densities of desired components, high transportation costs, and potential storage stability issues. Pre-processing might be done “on the farm” or even during harvesting or transportation “en route” to densify, dry, and perhaps initially separate biomass components. New transportation schemes might include pumping a fluid slurry, torrefication, pyrolysis, or “pelletizing” biomass locally. All of these advances must be made while maintaining biodiversity and ensuring the safety and sustainability of the technologies utilized. Most biomass is solid, requiring improved material handling systems at the front end of conversion operations. Breakthroughs in fractionation and separation technology will be required to produce higher value-added products, to reduce processing costs, waste, and environmental impact.

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4.5.3 Conversion Technology

Next to already known conventional thermal, chemical, and bioconversion technologies this report has shown that the potential in conversion technology is enormous, partly as a result of ever-increasing knowledge about thermochemical and biotechnological pathways for the conversion of biomass into chemical products. It is suggested that research efforts be focused on the following areas: · Optimization of thermochemical conversion technologies. This paper has shown that different technologies have been proposed for thermochemical conversion of biomass. Further improvements lie in: (1) optimization of efficiency and cost reduction of currently employed conventional technologies, and (2) development of new advanced technologies, for example catalytic supported staged or subcritical gasification processes. · (Bio)catalyst development and bioreactor engineering. Metabolic pathway engineering enables synthesis of very specific catalysts (both whole-cell and enzymes) for transformation of refined biomass (mainly mixed sugars, fatty acids, and syngas) into a variety of chemical intermediates and chemical products. This will lead to the controlled, safe and efficient production of new and existing chemicals and polymers. Important issues for the efficient use of biocatalysts include immobilization, reactor kinetics and design, and separation of the chemical product from the reaction mixture. As in the pretreatment of biomass, separation technology also plays an important role in efficient and cost-effective biocatalytic production processes. 4.5.4 Chemicals and Materials Design

The approaches to the production of chemicals from biomass presented in this section are resource-based forward-integrated approaches. A design approach can also be suggested in which a backward-integrated chemical industry designs chemicals and production methods that fit the application of the chemical product. It is thought that whereas the resource-based approach fits current (petro)chemical business and production models, the design approach will lead to a real transition in the production and design of chemicals and materials. This backward-integrated approach will initially be applicable in specialty chemical markets but there are also long-term opportunities in bulk chemical markets. It is thought that with the envisaged development in advanced thermochemical and bioconversion technologies the role of biomass and a biomimetic approach to the design of chemicals and materials may add to sustainable production, use, and reuse or disposal of chemicals and materials. Further examination of the role that chemicals and materials design can have on the sustainable use of biomass and biomass-based resources is therefore suggested.

References

4.5.5 Dutch Energy Research Strategy (“EOS”)

In The Netherlands the Ministry of Economic Affairs has defined a national subsidy program “Energie OnderzoeksStrategie (EOS)” for co-financing longterm (> 10 years) technology developments that support the transition process to a more sustainable society. In this program (2004–2008) an annual subsidy budget of 35 Mio 1 is available for technology development in pre-defined areas. Biomass-based technology development in direct/indirect cofiring in conventional power plants, gasification, and biorefineries have been selected for co-financing. It is expected that for biorefinery technology development about 10 M 1 subsidy will be available for project co-funding for the next four years. The Energy Research Center of the Netherlands (ECN) – Biomass Department, with Agrotechnology and Food Innovations B.V. (A&F), Wageningen University and Research center (WUR), University of Twente (UT), Utrecht University (UU), and Groningen University (RUG), have defined an integral biorefinery-based research and development program for the coming four years. Within this program joint projects will be defined on the basis of a common vision and submitted for co-funding. Research items that will be addressed are: integral chain analysis and scenario studies to identify platform chemicals and provide the framework for technological developments; primary refining processes, including pretreatment; secondary thermochemical and biological refining processes, including product separation and upgrading; and some site-specific case studies to encourage real market implementation.

References Ahmed, I. and Morris, D. J. (1994) Replacing Petrochemicals with Biochemicals: A Pollution Prevention Strategy for the Great Lakes. Minneapolis: Institute for Local Self-Reliance. American Chemical Society, American Institute of Chemical Engineers, Chemical Manufacturers Association, Council for Chemical Research, Synthetic Organic Chemical Manufacturers Association (1996) Technology vision 2020; the U.S. chemical industry (downloaded at http:// www.acs.org) Biebl, H., Menzel, K., Zeng, A. P., and Deckwer, W. D. (1999) Microbial production of 1,3-propanediol, Appl. Microbiol. Biotechnol., 52, 289–297. Boerrigter H. et al. (2004). Thermal Biorefinery; high-efficient integrated production of renewable chemicals, (transportation)

fuels, and products from biomass, 2nd World Conference and Technology Exhibition on Biomass for Energy, Industry and Climate Protection, Rome, Italy, 10–14 May 2004. Chotani, G., Dodge, T., Hsu, A., Kumar, M., LaDuca, R., Trimbur, D., Weyler, W. and Sanford, K. (2000) The commercial production of chemicals using pathway engineering. Biochim Biophys Acta, 1543 (2): 434–455. Coombs, R. (1995) Firm strategies and technological choices. In: Rip et al. (eds) Managing technology in society: the approach of constructive technology assessment. Pinter Publishers: London, New York, 331–345. Dam, J. E. G. van, de Klerk-Engels, B., Struik, P. C. and Rabbinge, R. (2004) Securing renewable resources supplies for

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4 Biorefineries for the Chemical Industry – A Dutch Point of View changing market demands in a biobased economy. Industrial Crops and Products, in press. Dam, van J. E. G., C. Boeriu, and J. P. M. Sanders – Sustainable plant production chains for “green chemicals”. ACS Symposium: Feedstocks for the Future: Renewables for the Production of Chemicals and Materials, Anaheim, CA, March 28–April 1, 2004 (in preparation). Datta, R., Stai, S.-P., Bonsignore, P., Moon, S.-H., and Frank, J. R. (1995) Technological and economic potential of poly(lactic acid) and lactic acid derivatives. FEMS Microbiol. Rev., 16, 221–231. Diamantidis, N. D., and Koukios E. G. (2000) Agricultural crops and residues as feedstock for non-food products in Western Europe. Ind. Crops Products, 11, 97–106. de Jong, E., R. R. Bakker, H. W. Elbersen, R. A. Weusthuis, R. H. W. Maas, J. H. Reith, H. den Uil, and R. van Ree (2003) Perspectives for Bioethanol Production in the Netherlands: feedstock selection and pretreatment options, Poster presentation at the 25th Symposium on Biotechnology for Fuels and chemicals, Breckenridge, Colorado. de Klerk, B., Vellema, S. and van Tuil, R. (2002) Chemie: de weg naar duurzaamheid? Chemisch2Weekblad, 16 Feb. 2002, 14–15. Ehrenberg, J. (2002) Current situation and future prospects of EU industry using renewable raw materials, (coordinated by the European Renewable Resources & Materials Association (ERRMA), Brussels, February 2002. Elbersen, H. W., F. Kappen and J. Hiddink (2002) Quickscan value-added applications for byproducts and wastes generated in the Food Processing Industry (Hoogwaardige Toepassingen voor Rest- en Nevenstromen uit de Voedings- en Genotmiddelenindustrie). ATO, Arcadis IMD. For the Ministry of Agriculture. Lysen, E. H. (2000) GRAIN: Global Restrictions on Biomass Availability for Import to the Netherlands, E2EWAB00.27, Utrecht, The Netherlands, August 2000 (partly in Dutch). Ministry of Economic Affairs (2003) The Netherlands: Biomass in 2040, The Green

Driving Force Behind a Knowledge Economy and Sustainability ... A Vision Ministry of Economic Affairs (2001) Catalysis: key to sustainability (Technology Roadmap Catalysis initiated by Ministry of Economic Affairs and facilitated by PricewaterhouseCoopers Management Consultants) (available at http:// www.technologyroadmapping.com). Minnisma, M. and Hissemoller, M., Biomassa – Een Wenkend Perspectief, IVM, Februari 2003 (in Dutch). Molendijk, K. G. P., Venselaar, J., Weterings, R. A. P. M. and de Klerk-Engels, B. (2004) Transitie naar een duurzame chemie. TNO-rapport R2004/179. National Research Council (2000), Biobased industrial products: priorities for research and commercialization, Washington D.C.: National Academic Press. Okkerse C. and van Bekkum, H. (1997) Towards a plant-based economy? In: Van Doren H. A. and van Swaaij A. C. (eds), Starch 96 – the book. Parris, K. (2004) Agriculture, Biomass, Sustainability and Policy: an Overview OECD Workshop on Biomass and Agriculture: Sustainability, Markets and Policies, 10–13 June 2003, Vienna, Austria. OECD Code 512004011P1, pp 27–36. Rabobank International (2001) De Nederlandse akkerbouwkolom: het geheel is meer dan de som der delen. Rabobank Food and Agribusiness Research: Utrecht. Realff, M. J. and Abbas, C. (2004) Industrial Symbiosis, refining the biorefinery. Journal of Industrial Ecology 7:5–9. Reit, J. H., den Uil, H., van Veen, H., de Laat, W. T. A. M., Niessen, J. J., de Jong, E., Elbersen, H. W., Weusthuis, R., van Dijken, J. P. and Raamsdonk, L. (2002) Coproduction of bio-ethanol, electricity and heat from biomass residues. Proc. of the 12th European Conference and Technology Exhibition on Biomass for Energy, Industry and Climate Protection, June 17–21 2002, Amsterdam, The Netherlands. Roekel, G. J. van and Koster R. (2000) Succes – en faalfactoren van de agrificatie in Nederland. ATO: Wageningen (report for the Dutch Ministry of Agriculture). Sims, R. E. H. (2004) Biomass, Bioenergy and Biomaterials: Future Prospects. OECD

References Workshop on Biomass and Agriculture: 10–13 June 2003, Vienna, Austria. OECD Code 512004011P1, pp 37–62. Sreenath, H. K., Moldes, A. B., Koegel, R. G. and Straub, R. J. (2001) Lactic acid production from agriculture residues. Biotechnol. Lett. 23:179–184. UK Foresight Programme Chemicals Panel (2000) A chemicals renaissance (available at (http://www.foresight.gov.uk) US Department of Energy (1998) Plant/ Crop-based Renewable Resources 2020: A vision to enhance U.S. economic security through renewable plant/crop-based resource use (available at http://www.oit.doe.gov/ agriculture/). US Department of Energy (1999) The Technology Roadmap for Plant/Crop-based Renewable Resources 2020: research priorities

for fulfilling a vision to enhance U.S. economic security through renewable plant/cropbased resource use (available at http:// www.oit.doe.gov/agriculture/). Vis, M. (2002) Beschikbaarheid van reststromen uit de voedings – en enotmiddelenindustrie voor energieproductie. A report for Novem, Utrecht. DEN nr 2020-01-23-030003/4700001071 (in Dutch). Weaver, P., Jansen, L., van Grootveld, G., van Spiegel, E. and Vergragt, Ph. (2000) Sustainable technology development. Greenleaf Publishing: Sheffield. Zeikus, J. G., Jain, M. K. and Elankovan, P. (1999) Biotechnology of succinic acid production and markets for derived industrial products. Appl. Microbiol. Biotechnol. 51, 545–552.

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Part II Biorefinery Systems


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Lignocellulose Feedstock Biorefinery 5 The Lignocellulosic Biorefinery – A Strategy for Returning to a Sustainable Source of Fuels and Industrial Organic Chemicals L. Davis Clements and Donald L. Van Dyne

5.1 The Situation

The current, historically high, prices of crude oil are causing economic hardship for families and businesses worldwide, because of the resulting high energy prices. The estimated impacts of increasing energy costs for farmers, truckers, and airlines exceed US $ 13 billion per year for the United States alone. At the same time, it is clear that increasing demand and a finite supply of petroleum will sustain the rising prices and increase competition for secure petroleum supplies. The strategy outlined here not only addresses these issues, but also provides major benefits to the environment and to farm incomes. This is all accomplished by using known, proven chemical processes (no new research needed) and at very attractive rates of return on investment (ROI) that will not require long-term government subsidies when this new industry is established.

5.2 The Strategy

At the most basic level, the strategy is to reduce then, essentially, eliminate dependence on petroleum as the primary source of liquid fuels and industrial organic chemicals (where “industrial organic chemicals” includes those made from both petroleum and biological resources). This is accomplished by replacBiorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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5 The Lignocellulosic Biorefinery

ing petroleum, the current dominant hydrocarbon source, with hydroxycarbons derived from plant and animal biomass sources. The biomass carbon resources are processed in biorefinery complexes that are analogs of present day petrochemical complexes. These biorefineries will produce the same, or functionally equivalent, fuels and industrial chemicals that are currently obtained from petroleum. The difference is that the biomass resources are renewable and, as will be shown in examples based upon the United States, the biomass resources are largely derived from materials currently regarded as waste. The United States annually discards more tonnes of biomass carbon as “trash” than we consume from petroleum carbon resources. Technologies are, however, available for converting agricultural and forestry residues and municipal solid wastes (MSW), which we now discard, into the fuels and chemicals that we consume. Each year the United States consumes approximately 10 million tonnes of industrial organic chemicals and 325 million tonnes of liquid transportation fuels. Currently, the United States converts approximately 15 million tonnes of agricultural products into liquid fuels (ethanol and biodiesel) and discards approximately 270 million tonnes of agriculturally derived residues in the form of harvestable crop residues, animal manure, forest residues, and the organic fraction of municipal solid wastes. Since 1933, an average of 15 million hectares of crop land have laid idle annually in the US, representing an additional 75 million tonnes of potential agriculturally produced biomass. There is no technical or economic reason why the US demand for carbon resources could not be met by biomass replacing petroleum as the primary carbon source. The functional equivalent of the petrochemical complex for processing agriculturally derived raw and residue materials and MSW processing is a biorefinery. Here the term “biorefinery” is defined as “a production facility that uses multiple renewable, agriculturally derived raw materials, in combination with multiple processing methods, to provide the most profitable mix of higher value chemicals and energy products possible at a given location.” 5.2.1 A Strategy Within a Strategy

An essential component of the structural shift from petroleum to biomass as the source of carbon is a “two-use” ethic. Everything that grows or is derived from organic sources (even plastics) should have at least two uses. MSW is collected and recycled to the biorefinery. Other organic materials, for example agricultural residues, used tires and plastics, and human and animal wastes, are converted into new chemicals or fuels in biorefineries. In this “two-use” ethic, carbon is recovered and recycled in much the same way that aluminum, iron, and lead are recycled today. In contrast with the huge petrochemical complexes of today, biorefineries will probably be limited in total capacity to 1000 to 2000 tonnes of biomass per day.

5.2 The Strategy

This is because the distributed nature of agricultural production, forestry production, and municipal solid-waste management strategies limit the amount of material that can be economically assembled in one area, on a continuing basis, at an acceptable cost. This means that biorefineries will be regional, and dispersed. This regionality not only means greater domestic security through dispersion and redundancy, but also wider distribution of new jobs and economic activity in rural areas. 5.2.2 Environmental Benefits

The use of biomass as a replacement for petroleum in the production of liquid fuels and industrial organic chemicals would have immediate and far-reaching environmental benefits. · First, biomass resources are renewable. Most are annually renewable. This means that the carbon exhausted into the atmosphere as carbon dioxide when liquid fuels are burned would be recycled into new plant growth in the following years’ crops. This factor alone will greatly improve air quality worldwide, and directly address the issue of global warming as a result of greenhouse gas emissions. · Second, entire biomass resource needs are available domestically for many countries. No imports are needed. In the United States we currently have approximately 360 million tonnes per year of non-fossil carbon raw materials available to replace some, if not all, of the demand for petroleum for liquid fuels and industrial organic chemicals. · Third, the assembly and refining of these resources will create a large number of jobs, primarily in rural areas. · Fourth, the production and/or conversion of biomass resources to liquid fuels and organic chemicals involves processing steps that usually reduce the toxic burden associated with the petrochemical production of these products. · Fifth, the required tonnage of carbon-rich biomass can be derived largely through the implementation of the “two uses” policy, i.e. the recycling of biomass. This provides a very economical input for the biorefinery. 5.2.3 The Business Structure

Because a major source of the biomass needed for this transition is “2nd use”, this provides a unique opportunity for new business partnerships. Municipal solid wastes (MSW), agricultural crop residues, and forestry residues comprise the bulk of the 2nd-use sources. The biomass input resources are the major component of the operating cost of production of liquid fuels and industrial organic chemicals for the biorefinery. Because these inputs are now considered to be of low or even negative value, the owners of these 2nd-use resources can very profitably move these bio-

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mass materials into the refining process, but delay taking their share of the profit until the final products are produced and sold. Thus, the 2nd-use producers would receive their fair share of the generated profits after the increased value has been added. This provides a considerably better return than the current negative, zero, or near zero waste value. The 2nd-use strategy leads directly to the concept of a business partnership comprising the biorefinery owner(s) and the biomass production owner(s). For agricultural wastes and residues, assembly of these inputs could be achieved by use of already existing cooperatives and/or farmer organizations. Similarly, MSW inputs could be supplied through existing waste-management companies or municipal waste-management operations. It is envisaged that the biomass suppliers would participate equally with the biorefinery companies in the direction and operation of the partnership. Essentially, the business model is one of vertical integration through a collaborative partnership or a joint venture business. 5.2.4 Cost Estimates

A biorefinery complex is not cheap. The approximate cost of a 1000 to 2000 tonnes per day multi-product plant is approximately $ 0.5 billion (US). In the United States, example, the biorefinery will draw raw materials from a radius of 150 to 300 km. This means, allowing for desert and inland waters, that from 300 to 500 biorefineries would eventually be built in the US. This represents an approximate $ 200 billion investment for the US. This is, however, approximately the same investment that will be required to replace approximately 200 petroleum refineries and petrochemical complexes that are now more than 30 years old and nearing the end of their useful lives.

5.3 Comparison of Petroleum and Biomass Chemistry 5.3.1 Petroleum Resources

Petroleum is a mixture of hundreds of hydrocarbon compounds. The dominant chemistry of petroleum is that of linear hydrocarbons, with relatively little unsaturation or branching. The chemical structure is polymers of the –(CH2)– mer, with the hydrocarbon chains typically from 4 to 30 units long. Lesser quantities of aromatic compounds (benzene derivatives), naphthenic compounds (benzene dimers), and anthracenes (benzene trimers) are present. Additional elements are incorporated into some molecules, for example oxygen, nitrogen, sulfur, or phosphorus, but the essential elements of petroleum are carbon and hydrogen.

5.3 Comparison of Petroleum and Biomass Chemistry

Utilization of petroleum for the production of liquid fuels and organic chemicals involves both physical separation of the numerous different compounds and chemical synthesis. Fuels production is primarily a separation process, with additional synthesis needed for higher-quality products, for example reformulated gasoline, and for removal of sulfur and nitrogen. Crude petroleum is separated into different fractions according to molecular size by distillation. Distillation processes account for approximately three percent of the US total energy budget. The largest-volume fuel produced from petroleum is gasoline. Gasoline is a mixture of smaller (four to eight carbon) straight-chain hydrocarbons recovered by distillation and synthetically created branched chain hydrocarbons with a similar number of carbon atoms. Diesel fuel consists of larger (nine to fifteen carbon) hydrocarbons that are recovered largely by distillation. Nitrogen, sulfur, and phosphorus are elements that occur naturally in petroleum and must be removed from liquid fuels, largely because of the detrimental environmental effects of their combustion products. The removal processes typically involve catalytic reactions under extreme operating conditions. Petrochemical products are, in general, based on chemical addition of organic functional groups such as hydroxyl, aldehyde, acid, ester, etc., or other elements, such as oxygen, nitrogen, and halides. Much of the synthetic chemistry used is based on addition of functional groups to olefin hydrocarbons such as ethylene, propylene, and butylenes. Ethylene, propylene, and butylene are derived by high-temperature processing of ethane, propane, and butane recovered from petroleum crude oil by distillation. Benzene occurs naturally in petroleum, but most of the benzene family of hydrocarbons is produced synthetically by catalytic reforming of hexane and/or alkylation reactions. A representation of the petrochemical products families is given in Fig. 5.1. 5.3.2 Biomass Resources

Although a tremendous variety of biomass resources is available, only four basic chemical structures present in biomass are of significance for production of fuels and industrial products: · saccharides and polysaccharides (sugars, starches, cellulose, hemicellulose); · lignins (polyphenols); · triacylglycerides or lipids (vegetable oils and animal fats); and · proteins (vegetable and animal polymers made up of amino acids). In addition to these basic structural resources, there are hundreds of specific organic compounds of biomass origin that have commercial uses ranging from medicinal materials, nutrients and natural products, to industrial products.

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Fig. 5.1 Fossil sources of industrial organic chemicals [1].

5.3 Comparison of Petroleum and Biomass Chemistry

5.3.3 Saccharides and Polysaccharides

Saccharides and polysaccharides may be characterized as hydroxycarbons because their basic chemical structure is CH2O. Most hydroxycarbons occur naturally as either five- or six-membered ring structures. This ring structure may include only one or two connected rings (sugars) or they may be very long polymer chains (cellulose and hemicellulose). The basic six-sided saccharide structure is exemplified by glucose. Long-chain polymers of glucose, or other hexoses, may be categorized as either a starch or a cellulose. The categorization depends on the configuration of the bonds formed across the oxygen molecule that joins two hexose units. Starch is an energy-storage compound found in the seeds of many plants and is readily hydrolyzed enzymatically. Cellulose is found in association with two other polymers, hemicellulose and lignin, and is much more difficult to hydrolyze. Hemicellulose is a polysaccharide that has a large fraction of pentose sugars, with some hexoses, and is relatively easy to hydrolyze with acid. Current industrial uses of starch and hexose sugars include the production of ethanol and other fermentation products, and the use of starch derivatives for polymers, absorbents, and adhesives. The pentose sugars from hemicellulose are a source of furfural and its derivatives and numerous xylose products. 5.3.4 Lignin

Lignin is a network polymer made up of multi-substituted, methoxy, arylpropane, and hydroxyphenol units. The resulting thermosetting polymer serves as the glue that holds the strands of cellulose and hemicellulose together in plant fibers to provide structure and strength. Together, the three polymers make up the largest biologically derived resource on earth – “lignocellulosics”. The “lignocellulose” structure is the basis of the semi-rigid fibers found in all multi-cellular plants. The structural difference between a corn stalk, a tree, a flower stem, and a piece of waste paper is the difference between the relative amounts of hemicellulose, cellulose and lignin present and the shape and length of the fibers formed by the intertwined lignocellulosic chains. The most significant current industrial uses for lignocellulosics are for construction materials and for pulp and paper products. The abundance of lignocellulosics is the basis for regarding them as the key input resources for the biorefinery strategy. 5.3.5 Triacylglycerides (or Triglycerides)

Triacylglycerides (or triglycerides) are the primary component of vegetable oils and animal fats. Irrespective of origin, these materials have identical chemical structures and very similar chemical compositions. The basic structure of a tri-

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glyceride looks like a comb with three long tines. The backbone of the comb is a three-carbon hydroxycarbon, dehydrated glycerol, with three medium to longchain fatty acids attached. The fatty acid part of the molecule is a 7 to 31-carbon hydrocarbon chain with an organic acid group at one end. Triglycerides are relatively easily reacted with water or alcohols to form free fatty acids or fatty acid esters, respectively, with glycerol as co-product. Vegetable oils and animal fats are an essential part of our diet. As such, they are available as recyclable materials in the form of recycled cooking oils and as the “float-grease” fraction recovered in municipal water treatment plants. In the United States an average of 10 kg of “waste” triglyceride materials is produced per person annually [2]. Derivatives of fats and oils have extensive use in non-food/feed applications. Current uses of triglyceride derivatives range from latex paints, high performance lubricants and polymers, to biodiesel fuel and personal care products. The hydrocarbon structure of the fatty acid chain has facilitated the acceptance and use of triglycerides and their esters in conjunction with traditional petrochemical products. In fact, use of fats and oils for the production of industrial chemical products (oleochemical industry) is the largest contribution to the current industrial chemical industry made by biomass resources today. 5.3.6 Proteins

Proteins are long-chain polyamides based solely upon amino acid units. The –(NH)– (peptide) bonds characteristic of proteins are the same bonds found in industrial Nylons. The primary non-food/feed uses for proteins currently are as leather products, protein glues, and personal-care products. There are opportunities for the creation of “designer proteins”, for example synthetic spider silk for light-weight, high strength cables or for polymer films and adhesives, but progress in this area has been somewhat slow.

5.4 The Chemistry of the Lignocellulosic Biorefinery

The production of liquid fuels and industrial chemicals from biomass will rely most heavily on the utilization of polysaccharide, lignocellulosic, and triacylglyceride feedstocks. These materials are available, easily assembled, and storable in the large quantities. In addition, these materials may be converted into products that are identical, or functionally equivalent, to current petroleum-based liquid fuels and industrial chemicals. Petroleum is predominantly a liquid resource, with some gases and a small fraction of solid materials (waxes and asphalts). As such, every petroleum refinery begins with a massive distillation system to separate the crude oil into a large number of “cuts” that are further manipulated into the desired products.

5.4 The Chemistry of the Lignocellulosic Biorefinery

Chemical manipulation in a petrochemical refinery usually can be characterized as the synthesis of structure – the conversion of linear hydrocarbons into more structured and substituted materials, or the creation of very large, synthetic polymer molecules. Biomass materials are solids that include a sizable aqueous component. In general, initial treatment of biomass materials includes steps of drying and physical size reduction. The biomass materials already have a richly varied chemical structure. This means that the goal of biomass utilization is, in part, preservation of the intrinsic functional structures while depolymerizing the original material. The process chemistries used in the depolymerization of biomass materials and their products are depicted in Fig. 5.2. These processes include: · Pyrolysis – Treatment of biomass at moderate temperatures (300 to 600 8C) in the absence of oxygen to cause partial depolymerization of the material. Slow heating rates tend to favor production of volatile gases (CO, CO2, hydrogen, methane, ethylene), organic acids and aldehydes, mixed phenols, and char. High heating rates tend to minimize liquid production and maximize gas production. · Gasification – High-temperature (> 700 8C) treatment of biomass in the absence of oxygen but with addition of steam, and possibly CO2, to maximize the production of synthesis gas (syngas), a mixture of H2, CO, CO2, and CH4. Syngas can be used directly as a fuel or as a chemical intermediate in the production of ammonia, methanol, and higher alcohols, organic acids and aldehydes, synthetic gasoline (using Fischer-Tropsch processing), and isobutene and isobutane. · Thermochemical Liquefaction – Pyrolytic processing with addition of H2, CO, CO2, and selected catalysts to convert the biomass into hydrocarbons, mixed phenols (from the lignin fraction), and light gases. The key to commercial utilization of liquefaction processing is the separation and recovery of the multiple products created during the liquefaction process. · Hydrolytic Liquefaction – This processing includes the use of acids, alkalis, or enzymes to depolymerize polysaccharides into their component sugars. These aqueous-phase reactions are used to provide basic polymers, for example cellulose and fermentable sugars for further processing, or hexose and/or pentose sugars for chemical conversion into other organic compounds. · Fermentation – Biochemical processing uses microorganisms or/and enzymatic reactions to convert a fermentable substrate into recoverable products. Hexoses, particularly glucose, are the most frequently used fermentation substrates, but pentoses, glycerol, and other hydroxycarbons are also used. Fermentations are most commonly performed in aqueous solution, with the final products present in modest concentration. · Chemical Synthesis – Cellulose recovered from a lignocellulosic matrix using hydrolytic liquefaction can be treated with several reagents to make materials such as cellulose acetate, nitrocellulose, and rayon. Xylose from the hydrolytic depolymerization of hemicellulose can be further reacted in the same process to

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Fig. 5.2 Biomass-derived industrial organic chemicals.

make furfural. Furfural is the starting point for a large family of derivative chemical and polymer products. The adipic acid and the hexamethylene diamine used in the original synthesis of Nylon-6,6 were made by acid hydrolytic depolymerization of hemicellulose from oat hulls, followed by chemical synthesis of the monomers and condensation polymerization to form the Nylon-6,6. There are several possible ways of using lignocellulosic and other categories of biomass for production of fuels and chemicals. Because the initial production steps involve solid materials, preparation and handling of the raw materials is more complicated and costly than using petroleum liquids. Also, as noted

5.5 Examples of Integrated Biorefinery Applications

above, biomass materials are more disperse and less dense than petroleum. This means that assembly and storage of the raw materials is more complicated than for petroleum. Finally, some of the biomass resources, notably crops and crop residues, are cyclical in production. This means that storage and integration with other resources are necessary in the design and management of a biorefinery complex.

5.5 Examples of Integrated Biorefinery Applications 5.5.1 Production of Ethanol and Furfural from Lignocellulosic Feedstocks

An approach to implementation of a biorefinery using lignocellulosic feedstocks (LCF) that has been given much attention in the United States, via USDOEsponsored programs, is the use of chemical and enzyme treatments to depolymerize the LCF to produce fermentable sugars and lignin. The primary goal has been the production of ethanol. The basic process is: Lignocellulosics + Water ? Xyloses + Cellulose/Lignin (Acid Process) Cellulose + Water ? Glucose (Enzyme Process) Glucose Fermentation ? Ethanol + CO2 + Biomass Xylose Fermentation ? Ethanol + CO2 + Biomass Lignin + Biomass ? Heat + Steam Unfortunately, with ethanol as the sole product, and no subsidies, this process facility does not make a profit. If, however, the xyloses are diverted from the fermentation process into the production of furfural, a highly versatile intermediate chemical, the overall operation can be highly profitable. Van Dyne et al. [3] showed that the optimum capacity for the integrated ethanol/furfural facility is 4360 tonnes per day of LCF. The discounted cash flow rate of return, after taxes, for this facility is approximately sixteen percent. At capacities below approximately 750 tonnes per day, the combined ethanol–furfural operation is not profitable, because the plant capital and operating costs are too large (economy of scale). On the other hand, the operation becomes increasingly less profitable for capacities above 4400 tonnes per day because the costs of assembling sufficient feedstock are too high. 5.5.2 Management of Municipal Solid Waste

Municipal solid wastes (MSW) are the largest single source of lignocellulosic materials available for utilization in modern society. Most localities have systems for collection and “disposal” of these materials. Too often the “disposal” consists of burial of the materials. Typically approximately 25% of the waste is

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either recyclable materials or inorganic materials such as stones, cement, and wallboard (gypsum) materials. The remaining 75% is non-recyclable organic solid waste materials (NROSW) that are available for production of a wide range of fuels and industrial chemicals. There are numerous locations where the NROSW is burned, either to reduce the volume or, hopefully, to recover the energy value of the wastes. Using the biorefinery approach and the “Two-uses” paradigm, the following options should be considered for the NROSW: A. Gasification ? Syngas + Ash ? Electrical power + CO2 B. Gasification ? Syngas + Ash ? Chemical synthesis + CO2 C. Gasification ? Syngas + Ash ? Fermentation ? Ethanol D. Liquefaction ? Mixed organic liquids ? Specialty chemicals and fuels In many locations the use of the NROSW materials for gasification and the cogeneration of electrical power is not competitive with other power sources, without special incentives. The production of a variety of specialty chemicals from syngas is commercial technology [4], although the easiest fuel source to use is often natural gas. Liquefaction processing of coal has long been commercially used for fuels and chemicals, with South Africa being the prime example. Liquefaction of biomass is beginning to be used in commercial applications in some countries, including the US. The liquefaction strategy can be a very profitable alternative. As an example, a NROSW liquefaction plant handling 140 tonnes per day of MSW and 90 tonnes per day of waste tires costs about US $ 30 million. The facility produces a range of liquid fuels and a number of higher-value industrial organic chemicals. Using a 10 year project life, the facility produces a before-tax cumulative net profit of more than US $ 100 million. 5.5.3 Coupling MSW Management, Ethanol, and Biodiesel

Conventional ethanol production uses corn as the source of fermentable sugars to produce ethanol. The corn typically is dry milled and the entire milled kernel is added to the saccharification tank before fermentation. The process requires a sizable amount of process steam and electricity. Biodiesel production requires modest amounts of energy, but requires an inexpensive source of fats or oils to be competitive. The oil can be, for example recycled restaurant grease, a virgin vegetable oil, or rendered animal fat. The corn that goes into the fermentation process contains approximately 3% by weight corn oil, which passes through the fermentation process as an inert material. Biodiesel also requires a source of alcohol to make the biodiesel from oil. MSW, or other lignocellulosics, such as seed hulls, waste paper, etc., can be burned directly to provide steam and heat, but it is more compatible with the production of ethanol if the LCF is first converted to syngas to be used as a fuel gas. Also, the syngas can be converted into methanol – the most commonly used alcohol for making biodiesel.

References

The integrated process scheme for the biorefinery facility is: Corn ? Milling ? Starch + Germ ? Glucose ? Ethanol Germ ? Corn oil + Defatted Germ Meal Gasification + LCF ? Syngas + Ash ? Steam + Electricity Syngas ? Methanol Corn Oil + Methanol ? Biodiesel + Glycerol In this process system, corn and LCF are the inputs and ethanol, biodiesel, defatted corn germ meal, and glycerol are the outputs. Economies of scale in the biodiesel plant may require additional sources of fats/oils, but this only makes the processes more profitable. The ethanol product is not used in the biodiesel because it is more valuable as an intermediate chemical or as a fuel on its own.

5.6 Summary

There is already significant use of renewable biomass resources for industrial chemicals [5, 6] and recognition of the opportunities for greatly expanded energy and fuel uses for biomass materials [7–11]. The biorefinery strategy advocated here is combining of the utility of multiple feedstocks with the utility of a wide array of conversion technologies to create a new, sustainable strategy to meet the world’s needs for industrial chemicals and liquid fuels.

References 1 Morris, D. and I. Ahmed, The Carbohy-

drate Economy: Making Chemicals and Industrial Materials from Plant Matter, The Institute for Local Self Reliance, Washington, D.C., 1992. 2 Wiltsee, G., “Urban Waste Grease Resource Assessment,” NREL/SR-57026141, November, 1998. 3 Van Dyne, D. L., M. G. Blaise, and L. D. Clements, “A Strategy for Returning Agriculture and Rural America to LongTerm Full Employment Using Biomass Refineries,” Perspectives on New Crops and New Uses, J. Janick, ed., ASHS Press, Alexandria, VA, 1999. 4 Spath, P. L. and D. C. Dayton, “Preliminary Screening – Technical and Economic Assessment of Synthesis Gas to Fuels and Chemicals with Emphasis on the Potential for Biomass-Derived Syngas,” NREL/TP-510-34929, December, 2003.

5 Szmant, H. H., Industrial Utilization of

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Renewable Resources: An Introduction, Technomic Publishing Company, Lancaster, PA, 1986. Johnson, R. W. and E. Fritz, Fatty Acids in Industry, Marcel Dekker, New York, 1989. Donaldson, T. L. and O. L. Culberson, “Chemicals from Biomass: An Assessment of the Potential for Production of Chemical Feedstocks from Renewable Resources,” ORNL/TM-8432, June, 1983. Wise, D. L., Organic Chemicals from Biomass, The Benjamin/Cummings Publishing Company, Inc., Cambridge, MA, 1983. Clements, L. D., S. R. Beck and C. Heintz, “Chemicals from Biomass Feedstocks,” Chem. Engr. Prog., 79, 59–62 (1983).

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5 The Lignocellulosic Biorefinery 10 Lowenstein, M. Z., Energy Applications

of Biomass, Elsevier Applied Sciences Publishers, New York, 1985. 11 Thames, S., R. Kleiman and L. D. Clements, “How Crops Can Provide Raw Materials for the Chemical Industry,” in

New Crops, New Uses, New Markets – 1992 Yearbook of Agriculture, US Department of Agriculture, Washington, D.C., 1992.


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6 Lignocellulosic Feedstock Biorefinery: History and Plant Development for Biomass Hydrolysis Raphael Katzen and Daniel J. Schell

6.1 Introduction

The current high level of interest in lignocellulosic biomass conversion technology is driven by the potential to produce fuels and chemicals to reduce dependence on petroleum, improve air quality, and reduce greenhouse gas emissions. Research and development efforts and attempts to commercialize biomass hydrolysis technology began in the early 1900s. This chapter discusses some of this early work which set the stage for current efforts to bring to fruition the lignocellulosic biorefinery. We highlight early efforts to pilot and commercialize lignocellulosic biomass conversion technology using acid hydrolysis processes and enzyme-based cellulose hydrolysis.

6.2 Hydrolysis of Biomass Materials

Producing ethanol from lignocellulosic biomass depends on converting the complex cellulosic and hemicellulosic carbohydrates into simple sugars which are then fermented to ethanol by a variety of microorganisms. This section briefly reviews both acid- and enzyme-based methods for hydrolyzing the polymeric carbohydrates into their constituent sugars. 6.2.1 Acid Conversion

Hydrolysis of cellulose and hemicellulose (primarily xylan) to sugars can be catalyzed by a variety of acids, including sulfuric, hydrochloric, hydrofluoric, and nitric acids. The hydrolysis process is represented by the following simple expressions:


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6 Lignocellulosic Feedstock Biorefinery: History and Plant Development for Biomass Hydrolysis

cellulose ? glucose ? HMF ? tars xylan ? xylose ? furfural ? tars in which HMF is 5-hydroxymethylfurfural. If hydrolysis conditions are severe (e.g. high temperatures or acid concentrations) a large fraction of the sugars is degraded to other products, e.g. HMF, furfural, and tars. Because dilute sulfuric acid is inexpensive, use of this acid is the most studied in acid conversion processes and these processes are most often used in plants based on acid conversion technology. Biomass is impregnated with a dilute sulfuric acid solution and treated with steam at temperatures ranging from 140–260 8C. At lower temperatures of 140–180 8C, xylan is rapidly hydrolyzed to xylose with little cellulose degradation. At higher temperatures, cellulose is also rapidly hydrolyzed to glucose and xylan is quickly converted to furfural and tars. Concentrated acids are also used to hydrolyze cellulose and hemicellulose to sugars. Because low temperatures (100–120 8C) are typically used, high yields of sugars are obtained with little production of degradation products. The economic viability of this process depends, however, on the successful recovery of acid at low cost. 6.2.2 Enzymatic Conversion

Sugar yields are limited during acid hydrolysis because sugars are also converted to degradation products. Cellulase, a multi-component enzyme system, catalyzes cellulose hydrolysis and is 100% selective for conversion of cellulose to glucose; high yields are, therefore, possible. This enzyme is produced by a variety of microorganisms, most commonly, the fungus Trichoderma reesei. Cellulose conversion rates are limited by the ability of the enzyme to access the cellulosic substrate. To increase accessibility, biomass is subjected to physical and chemical treatments that disrupt the biomass structure, usually by removing a fraction of the hemicellulose and/or lignin. Effective pretreatment is necessary to achieve good cellulose-to-glucose conversion yields.

6.3 Acid Hydrolysis Processes 6.3.1 Early Efforts to Produce Ethanol

Before World War II, research was conducted in the United States on hydrolysis of biomass to produce sugars for production of ethanol with emphasis on the use of forest and wood-processing wastes; no operation achieved true commercial success, however. Sherrard and Kressman [1] highlight developments in acid-based hydrolysis technology before World War II. In the early 1900s, a

6.3 Acid Hydrolysis Processes

plant was built in Hattiesburg, MS, USA, to process wood waste using sulfurous acid, but never operated successfully [2]. A commercial operation was also set up in Georgetown, SC, USA in the 1910s to process wood waste using dilute sulfuric acid, but eventually failed because of low ethanol yields [3]. Process development was being investigated in Germany at the same time. One process used concentrated hydrochloric acid [4] to hydrolyze the carbohydrate fraction of wood waste. The treated material was then neutralized with caustic soda to yield a mixture of wood-based sugars and salt (sodium chloride), used primarily as cattle feed. In another process, a 7300 dry metric ton year–1 plant hydrolyzed wood chips in countercurrent diffusers by contact with 42% (w/w) HCl to produce a concentrated sugar solution [5]. After World War II the plant was extensively rebuilt to use a modified process known as the Udic–Rheinau process [6]. Work on sulfuric acid processes was also conducted in Germany at the same time, leading to the development of the Scholler process [7]. The Scholler process used a percolation reactor to hydrolyze wood waste, producing glucose as the primary product; this was then fermented to ethanol. Many such plants were built in Germany and Russia before World War II. The extensive use of water required for this process, however, produced a rather dilute (4%) sugar stream that was more costly to process. In the late 1930s, a continuous wood-hydrolysis pilot plant was built in the United States to produce lignocellulosic plastics, and wood sugars, furfural, and acetic acid as by-products [3]. The plant was designed to hydrolyze wood slurries continuously with dilute sulfuric acid by pumping the mixture through heated hydrolysis tubes and then recovering the solid product. The plant produced 180–260 kg day–1 hydrolyzed product. At the beginning of World War II molasses was fermented to ethanol as a raw material for synthesis of butanediol, which in turn was polymerized to produce synthetic rubber. Because German submarines were sinking transports from Cuba containing shipments of molasses and ethanol, the US government made the decision to develop technology for producing ethanol from wood waste for the synthetic rubber program. The Defense Plant Corporation, an organization of the US Government, awarded a contract to the Vulcan Copper and Supply Company (later known as Vulcan Cincinnati) to design and to construct a facility to produce ethanol. The author (Raphael Katzen) was involved as the senior process engineer and later as the project manager for design and construction of this facility in Springfield, OR, USA. The plant was designed to process 270 metric tons (dry basis) day–1 of softwood sawdust trucked from sawmills in the area, with the goal of producing 208 L/dry metric ton (50 gal/dry ton) ethanol. This plant was based on modifications to the Scholler process, as a result of work at the Forest Products Laboratory (FPL) of the US Department of Agriculture in Madison, Wisconsin, USA. Based on early work by Ritter [8] and Sherrard [9], FPL built a pilot facility that improved upon the Scholler technology, yielding the Madison-Scholler process [10, 11].

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Vulcan Cincinnati’s design for their plant was based on the Madison-Scholler process and used information and data produced during test runs of the FPL pilot plant. Equipment was designed and fabricated at Vulcan’s shops in Cincinnati, OH, USA, and construction was carried out in Springfield, OR, USA, with the assistance of contractors. Although construction was stopped at the end of World War II, the Defense Plant Corporation decided to complete the facility and test the technology. Plant construction was completed in 1946 and the plant was started up under management of the Willamette Valley Wood Chemical Company, a group of individuals in the local forest products industry. Test runs were then initiated with Vulcan providing technical management under Raphael Katzen’s supervision. Despite problems with tar formation and calcium sulfate deposits from neutralization of sulfuric acid used to hydrolyze the cellulose, the design production rate was achieved. The process proved too costly to compete with petroleum-derived synthetic ethanol which appeared on the scene at the end of World War II, however. Efforts were made to utilize the Springfield facility for production of waxy products, but it was not designed for this operation and the plant was shut down and dismantled. With the prevalence of cheap petroleum-derived ethanol and other petrochemical products after World War II, there was little economic incentive to pursue cellulose hydrolysis technology further. In Germany many of the plants shut down and most of the Scholler process plants in Russia were converted to single-cell yeast production, because it was a more profitable product than ethanol. Research did continue, however, in various laboratories and pilot plants in different parts of the world. In the 1950s the Tennessee Valley Authority (TVA) built a dilute-sulfuric-acid-hydrolysis-based pilot plant using percolation reactors at Muscle Shoals, AL, USA [12]. The New Zealand Forest Products Laboratory built a similar plant in the 1980s [13]. Several pilot facilities were also built in the 1980s that performed continuous dilute-sulfuric-acid hydrolysis of cellulose and hemicellulose and were able to process 1–2 metric dry tons of biomass per day. Plug-flow reactor systems processing dilute biomass streams were constructed by the American Can Company [14] and at the Solar Energy Research Institute (SERI), now the National Renewable Energy Laboratory (NREL), in Golden, CO, USA [15]. Systems for processing high-solids biomass streams were constructed using twin-screw extruders at New York University [16] and in Canada by Bio-hol/St Lawrence Reactor [17], and TVA installed a new system using a modified pulp digester [18]. Except for the new reactor at TVA and a new but similar reactor recently installed at the NREL [19], none of these systems is currently operational. In the mid-1980s researchers at the SERI proposed a “progressing batch reactor system” [20] that retained the simplicity of percolation reactors but achieved countercurrent flow of liquors to reduce sugar losses due to degradation reactions even further compared with percolation reactors. Further testing of the system, however, did not produce substantial sugar yield improvements [21]. In the late 1990s, the idea of a countercurrent shrinking bed reactor was also pro-

6.3 Acid Hydrolysis Processes

posed for dilute-acid total cellulose hydrolysis [22], but operational difficulties limited the effectiveness of this system. Several economic studies performed by engineering companies in the early 1980s to evaluate dilute-acid total hydrolysis processes for production of ethanol [23, 24] showed the economics to be favorable. The potential of enzymatic cellulose hydrolysis to achieve better yields was shifting emphasis away from acid hydrolysis to enzymatic-based processing, however [25]. Although there is no current active effort to build dilute-acid based cellulose plants, two companies are pursuing concentrated acid hydrolysis processing. Arkenol has examined the ability of using recombinant Z. mobilis to ferment acid hydrolysates produced by its concentrated-acid hydrolysis process [26] and is pursuing international opportunities to build a large-scale plant. Masada Resources Group plans to build a waste-handling facility in Middetown, NY, USA, that will use its patented OxyNol process to recycle or convert municipal solid waste. Profitable economics for both companies rely on achieving cost-effective recovery of the acid catalyst. 6.3.2 Other Products

Both glucose and xylose produced by acid-catalyzed hydrolysis of lignocellulosic biomass will, under the same conditions, further degrade to HMF and furfural, respectively. Furfural has been produced commercially from biomass but there has been no commercial interest in producing HMF. The Quaker Oats Company began production of furfural in the United States in the 1920s [27] and until recently was the major world producer of furfural from agricultural residues [28]. Furfural is produced from agricultural sources including corncobs, oat hulls, rice hulls, cereal grasses, and sugar cane bagasse. Early plants used batch production technology, but in 1965, at the recommendation of the Katzen Company [29], Quaker Oats agreed to pilot a continuous process in an existing leased pulping facility operated by Katzen. Successful results led to the design of the largest furfural plant in the world by the Katzen Company and the Black Clawson Company of Middletown, OH, USA. The plant is based on a highly modified Pandia digester system, originally used to pulp wood and agricultural residues. The plant was constructed in Belle Glade, FL, USA and began operation in 1966. The feedstock for the plant was sugar cane bagasse from nearby sugar mills. When successful operation was demonstrated, the plant capacity was expanded to process 1800 dry metric tons day–1 bagasse to yield 137 metric tons day–1 furfural. By-product methanol and HMF were burned, with the lignocellulose residue, to provide steam and electrical energy for the plant. The plant was sited adjacent to a sugar mill operated by the Sugar Cane Growers Company of Florida, which provided transport and bulk storage of up to 227 000 metric tons (dry basis) of bagasse from nearby sugar mills, thereby enabling year-round operation. After 31 years of continuous operation this plant was shut down in 1997 because of the availability of lower cost crude furfural from China.

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6.4 Enzymatic Hydrolysis Process 6.4.1 Early History

A new and exciting development in biomass conversion technology began when reports from US troops in the South Pacific during World War II described how “the green fungus among us” [30, 31] was destroying uniforms and other cotton gear. Reese, at the Natick Massachusetts Laboratory of the US Army Materials Command, analyzed samples of cotton items affected by this fungus in the 1950s [32]. He identified the fungus as Trichoderma viride and later named T. reesei QM 6a as the organism responsible for producing a cellulase complex that hydrolyzed cellulose to glucose. Serious studies of the use of this enzyme for biomass conversion began in the early 1970s with the pioneering work of Mandels and coworkers [33, 34]; this resulted in the development of improved T. reesei strain QM 9414. In the late 1970s and early 1980s much research effort was devoted to mutating the wild T. reesei strains QM 6a or QM 9414 to enhance production of cellulase [34]. One such effort was conducted by researchers at several university laboratories in a cooperative program coordinated by Eveleigh at Rutgers University. This cooperative research program resulted in substantial improvement of the wild-type fungal strain and produced the widely known T. reesei strain RUT C30 [35]. Many other efforts from around the world have produced modified T. reesei strains. 6.4.2 Enzyme-Based Plant Development

In the late 1970s several industrial organizations became interested in enzymatic conversion of cellulose to sugars and ethanol. One of the first efforts to develop and pilot this technology was a collaboration between Gulf Oil Chemicals and Nippon Mining in a program carried out by the Bio-Research Corporation of Japan. This led to the building of a 900 kg day–1 pilot plant in Pittsburg, KS, USA. In addition to producing cellulase, the plant was the first to use a novel technology combining cellulase saccharification with glucose fermentation in the same reactor system, called simultaneous saccharification and fermentation (SSF) [36, 37]. The plant successfully processed paper mill waste, producing dilute beer streams containing 30–35 g L–1 ethanol, although problems with contamination in the SSF fermentors were reported [38, 39]. Although no commercial plants have been built for enzymatic cellulose conversion, several pilot-scale facilities were built in the 1980s throughout the world to test a variety of technology and feedstocks. For example, a pilot plant located in Soustons, France, utilized a Stake process [40] to pretreat biomass, followed by enzymatic saccharification. Ralph Katzen Associates International, with the

6.4 Enzymatic Hydrolysis Process

University of Arkansas and Procter and Gamble, erected a pilot plant at a pulp mill in Pennsylvania, USA, to process pulp mill waste through disc refiners followed by SSF in a 9500-L fermentor [30]. In 1987, a 3000 kg day–1 pilot plant that processed wheat straw through a batch digester (no catalyst) and used the washed pretreated solids to produce cellulase that was subsequently used to saccharify the remaining washed solids was constructed in the Voest-Alpine Biomass Technology Center [41]. Two pilot plants were also constructed in Japan. One operated from 1983 to 1987 and processed 500 kg day–1 bagasse or rice straw. It used mild alkaline pretreatment then enzymatic saccharification of the pretreated biomass with cellulase produced on Avicel as the carbon source, followed by sugar concentration using reverse osmosis and subsequent fermentation to produce ethanol [42]. The other plant operated from 1986 to 1990 and processed 1000 kg day–1 cedar wood or white birch chips. It used steam explosion to treat the wood that was then fermented with a strain of Clostridium [43]. In the early 1990s, the US Department of Energy (DOE) and NREL constructed a fully integrated 900 kg day–1 pilot plant to produce ethanol from a variety of lignocellulosic biomass sources [19] that used a modified pulp digester for pretreatment that has already been discussed. The plant includes unit operations for feedstock handling, pretreatment in a modified pulp digester, seed culture production, SSF in 9000-L fermentors, feed tanks for enzyme and nutrient addition, ethanol stripping in a sieve-tray distillation column, and solid–liquid separation. The plant has also extensive instrumentation for process control and data collection. The plant was operated on a corn fiber feedstock during a 15day run [44] and was later operated continuously for up to six weeks [45]. The Iogen Company of Ottawa, Canada, recently built a 983,000-L-ethanol-peryear demonstration-scale plant processing nearly 5 metric tons day–1 agricultural residue. Because Iogen is a cellulase producer, they can supply the cellulase for the plant from an adjacent enzyme-production facility. Although little has been publicly disclosed about this facility and its performance, it has been reported to produce a 4% alcohol stream from conversion of lignocellulosic biomass [46]. 6.4.3 Technology Development

The Biomass Research and Development Technical Advisory Committee, a group of biomass industry and academic experts, recently issued a “roadmap” document [47] outlining bioconversion research needs. The recommendations most relevant to enzyme-based processing are: 1. the need to improve physical and chemical pretreatments before fermentation; 2. the need for cost-effective chemical/enzymatic conversion; and 3. the need to overcome barriers associated with inhibitory substances in hydrolysate sugar streams. Engineering catalyst and microorganisms to improve their tolerance are methods used to achieve the third goal.

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Advances in all three areas will be required to achieve economic viability and thus success in the marketplace. The Consortium for Advanced Fundamentals and Innovation (CAFI), a group of independent academic researchers, is collaborating in an effort to identify and develop new pretreatment technology. The most promising technology is uncatalyzed steam explosion, liquid hot water, pH controlled hot water, flow-through liquid hot water, dilute acid, flow-through acid, lime, and ammonia-based processing [48]. Development of new and improved enzymes for biomass conversion has been a focus of the US DOE for the last few years. They have funded cost-shared efforts with the two largest cellulase producers, Genencor International and Novozymes, to produce cost-effective enzymes with a goal of achieving a tenfold cost reduction that would bring cost down to an estimated $ 0.50 gallon–1 ethanol. Both companies report success in reaching this goal, but further cost reduction is required and the DOE’s goal is to achieve an effective enzyme cost of $ 0.10 gallon–1 ethanol. Efficient conversion of all sugars derived from biomass to desired products is required for this technology to become economically viable. Development of genetically modified microorganisms able to utilize other sugars beside glucose has been an ongoing effort in many laboratories [49, 50]. As also emphasized by the last recommendation above, the ability to tolerate inhibitory substances is also a highly desirable characteristic. Currently, however, no microorganisms can tolerate inhibitory substances in dilute-acid pretreated biomass substrates without some type of conditioning process that removes inhibitors.

6.5 Conclusion

Early efforts to commercialize acid-based processes for producing products from cellulose hydrolysis were ultimately unsuccessful, because of competition from lower-cost petroleum-derived materials. These efforts, and ongoing work on enzyme-based processes, are, however, providing valuable knowledge and experience. The future is the multi-product biorefinery that can utilize all biomass components. We need to build upon past efforts and use new advances in technology to ensure the commercial success of the lignocellulosic feedstock biorefinery.

References 1 E. Sherrard, F. Kressman, Ind. Eng.

Chem. 1945, 37, 5. 2 Ruttan, J. Soc. Chem. Ind. 1909, 28, 1291. 3 R. Katzen, D. Othmer, Ind. Eng. Chem. 1942, 34, 314.

4 F. Bergius, Trans. Inst. Chem. Engrs.

(London) 1933, 11, 162. 5 F. Bergius, Ind. Eng. Chem. 1937, 29,

247. 6 J. Wright, A. Power, P. Bergeron, Evalua-

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Solar Energy Research Institute, SERI/ TR-231-2074, Golden, CO 1984. H. Scholler, French Patent # 777,824, 1935. G. Ritter, Ind. Eng. Chem. Anal. Ed., 1932, 4, 202. E. Sherrard, Ind. Eng. Chem. 1923, 15, 1164. E. Harris, E. Berlinger, G. Hajny, E. Sherrard, Ind. Eng. Chem. 1945, 37, 12. E. Harris, E. Berlinger, Ind. Eng. Chem. 1945, 38, 890. N. Gilbert, A. Hobbs, J. Levine, Ind. Eng. Chem. 1952, 44, 1712. R. Burton, Proc. of the Royal Soc. of Canada Internat. Symp. on Ethanol from Biomass, Royal Society of Canada, Ottawa, Canada, 1983, 247. J. Church, D. Wooldridge, Ind. Eng. Chem. Res. Dev. 1981, 20, 371. A. Brennen, W. Hoagland, D. Schell, Biotechnol. Bioeng. Symp. No. 17, John Wiley & Sons, New York, 1987, 53. B. Rugg, P. Armstrong, A. Dreiblatt, D. Wise, Liquid Fuel Developments, CRC Press, Baco Raton, FL, USA, 1983, 139. R. Lawford, R. Charley, R. Edamura, J. Fien, K. Hopkins, D. Potts, B. Zawadzki, H. Lawford, Fifth Canadian Bioenergy R&D Seminar, National Research Council of Canada, 1984, 503. M. Bulls, J. Watson, R. Lambert, J. Barrier, Energy from Biomass and Waste XIV, Institute of Gas Technology, Chicago, 1991, 1167. Q. Nguyen, J. Dickow, B. Duff, J. Farmer, D. Glassner, K. Ibsen, M. Ruth, D. Schell, I. Thompson, M. Tucker, Bioresource Technol. 1996, 58, 189. J. Wright, P. Bergeron, P. Werdene, Ind. Eng. Chem. Res. 1987, 26, 699. D. Schell, Personal Communication. Y. Lee, W. Zhangwen, R. Torget, Bioresource Technol. 2000, 71, 29. Badger Engineers, Inc., SERI Subcontract Report No. ZX-3-03096-1, 1982. Stone & Webster Engineering Corp., SERI Subcontract Report No. ZX-303096-1, 1982. J. Wright, Chem. Eng. Prog. 1988, 84, 62. T. Yamada, M. Fitigati, M. Zhang, App. Biochem. Biotechnol. 2002, 98-100, 899.

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Chemical Technology, Supplement to 4th Ed., John Wiley & Sons, NY, 1998, 155. J. Levy, K. Yokose, Chemical Economics Handbook, Report 660.5000, SRI Consulting, Menlo Park, CA, USA, 2004. R. Katzen, Personal Communication. R. Katzen, D. Monceaux, App. Biochem. Biotechnol. 1995, 51/52, 585. J. Sheehan, M. Himmel, Biotechnol. Prog. 1999, 15, 817. D. Ryu, M. Mandels, Enzyme Microb. Technol. 1980, 2, 91. M. Mandels, L. Hontz, J. Nystrom, Biotechnol. Bioeng. 1974, 16, 1471. I. Persson, F. Tjerneld, B. Hahn-Hagerdal, Proc. Biochem. 1991, 26, 65. B. Montenecourt, D. Eveleigh, App. Environ. Microbiol. 1977, 34, 777. F. Gauss, S. Suzuke, M. Takagi, US Patent 3 990 944, 1976. G. Huff, N. Yata, US Patent 3 990 945, 1976. D. Becker, P. Blotkamp, G. Emert, Fuels from Biomass and Waste, Ann Arbor Science Publishers, Ann Arbor, MI, USA, 1981, 375. K. Bevernitz, S. Gracheck, D. Rivers, D. Becker, K. Kaupisch, G. Emert, Energy from Biomass and Waste XIV, Institute of Gas Technology, Chicago, 1982, 897. J. Heard, W. Schabas, Chem. Eng. 1984, 91 (6), 49. M. Hayn, W. Steiner, R. Klinger, M. Sinner, H. Esterbauer, Bioconversion of Forest and Agricultural Plant Residues, CAB International, Oxon, U.K. 1993, 33. Y. Shirasaka, H. Ishibashi, H. Etoh, H. Michiki, H. Miyakawa, S. Moriyama, Energy from Biomass and Waste XIII, Institute of Gas Technology, Chicago, 1989, 1311. S. Matsui, Conference on Alcohol and Biomass Energy Technologies, NEDO-OS-9106, New Energy and Industrial Technology Development Organization, Tokyo, 1991, 27. D. Schell, C. Riley, N. Dowe, J. Farmer, K. Ibsen, M. Ruth, S. Toon, R. Lumpkin, Bioresource Technol. 2004, 179. D. Schell, Personal Communication. B. Foody, From Presentation at the 27th Symposium on Biotechnology for Fuels

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der, Y. Lee, M. Holtzapple, M. Ladisch, Bioresource Technol. 2005, 96, 673. 49 R. Bothast, N. Nichols, B. Dien, Biotechnol. Prog. 1999, 15, 867. 50 A. Aristidou, M. Penttila, Current Opinions in Biotechnol. 2000, 11, 18.


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7 The Biofine Process – Production of Levulinic Acid, Furfural, and Formic Acid from Lignocellulosic Feedstocks Daniel J. Hayes, Steve Fitzpatrick, Michael H. B. Hayes, and Julian R. H. Ross

7.1 Introduction

The energy needs of the developed world are currently over-dependent on the utilization of finite mineral resources. Although renewable-power technologies, for example wind and photovoltaics, may, in the future, have major roles in the production of electricity, provision must still be made for the supply of industrial chemicals and motor fuels that are currently produced predominately from oil. In fact, of the approximately 170 chemical compounds produced annually in the US in volumes exceeding 4.5 ´ 106 kg, 98% are derived from oil and natural gas [1]. The vast majority of modern synthetic products are also derived from oil. Emerging biorefinery technologies offer a sustainable alternative by utilization of carbohydrates, the most abundant organic chemicals on the surface of the earth. This chapter will focus on the Biofine Process [2, 3], biorefinery technology that transforms carbohydrate feedstocks into products that include the platform chemicals levulinic acid, furfural, and formic acid in high yields. The process involves high-temperature acid-hydrolysis in two reactors and is one of the most advanced and commercially viable lignocellulosic-fractionating technologies currently available. The process involves the hydrolysis of polysaccharides to their monomeric constituents, and these are then in turn continuously converted into valuable platform chemicals.

7.2 Lignocellulosic Fractionation

The major polysaccharides of importance in biomass are the glucans and hemicelluloses. Of the glucans (carbohydrate homopolysaccharides consisting of repeating d-glucopyranose units), starch and cellulose are the most abundant. Technologies utilizing starchy feedstocks (e.g. maize) for production of ethanol, by fermentation of the liberated glucose monomers, are well-established and Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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7 The Biofine Process

Fig. 7.1 Physical structures of cellulose and of starch amylose and amylopectin.

run at relatively high efficiencies. This is because of the comparative ease of starch hydrolysis, using mainly a-amylase and gluco-amylase enzymes [4]. In 1999 a total of 1.48 billion gallons (ca 5.3 ´ 109 L) of fuel ethanol was blended with gasoline for use in motor vehicles in the United States. About 94% of this was produced by fermentation from maize; most of the remainder was from other grain [5]. Cellulose is much more abundant in nature than is starch, and its annual production is estimated at 100 ´ 109 tonnes [6]. Furthermore, cellulosic feedstocks tend to be more productive and require less energy to produce than starch crops. Technologies for hydrolysis of the cellulosic feedstocks are currently not commercially developed at a scale approaching that for starch, however. This is because cellulose (Fig. 7.1) is of the order of 100 times more difficult to hydrolyze than starch [7]. The d-anhydroglucopyranose units in cellulose are linked through b-(1 ? 4)-glycosidic bonds, as opposed to the a-(1 ? 4)-linkages in the amylose component of starch and the a-(1 ? 6) amylopectin branches in starch. The structure of cellulose enables intimate intermolecular associations that do not occur in starches, and this explains the relative resistance to degradation in cellulose fibrils and microfibrils compared with starch macromolecules.


7.2.1 Acid Hydrolysis of Polysaccharides

Cellulose is hydrolyzed in pure water by attack by the electrophilic hydrogen atoms of the H2O molecule on the glycosidic oxygen (Fig. 7.2). This is a very slow reaction, because of the resistance of the cellulose to hydrolysis. The rate of the reaction can be increased by use of elevated temperatures and pressures or can be catalyzed by acids (concentrated or dilute), or by highly selective enzymes such as cellulases. The steps involved in the acid-catalyzed hydrolysis of cellulose are illustrated in Fig. 7.2. The H+ ions equilibrate between the O atoms in the system, including those of water and the glycoside, with the consequence that there is an equilibrium concentration of protonated glycoside. This equilibrium tends towards the protonated form of the glycoside with increasing temperature. The protonated conjugate acid then slowly breaks down to the cyclic carbonium ion, which adopts a half chair conformation (while the other glucopyranose residue retains the OH at C-4). After rapid addition of water, free sugar is liberated. Because the sugar competes with the water, small amounts of disaccharides are formed as reversion products. There is a time/temperature relationship whereby lower acid concentrations require more extreme conditions and longer times for cellulose degradation. The use of stronger acid may reduce the costs associated with higher-pressure vessels, but the costly effects of equipment corrosion and of acid loss may be excessive. Rates of cellulose hydrolysis may differ according to the degree of crystallinity of the cellulose (i.e. the proportions of crystalline and amorphous cellulose present), a factor which varies between feedstocks. The mechanism of hydrolysis of hemicellulose polysaccharides is similar to that illustrated for cellulose in Fig. 7.2 and usually involves protonation of the glycosidic oxygen. Process conditions do not need to be as severe, however, given the lower degree of polymerization (formation of the carbonium ion occurs more rapidly at the end of a polysaccharide chain) and a tendency for the occurrence of less intermolecular bonding in most hemicelluloses. The rate of hydrolysis of hemicelluloses with a higher uronic acid content may be lower than for other hemicelluloses, however, as a result of the steric effects of the carboxyl groups.

Fig. 7.2 Steps involved in the acid hydrolysis of cellulose [8].

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The ash content of feedstocks is important because ash tends to lower the acidity of the mixture – the catalytic hydrogen ion is a function of the concentration of the acidic solution applied and the neutralizing power of the ash [9]. It is therefore useful to measure the titratable alkalinity of feedstocks to ascertain what acid levels may be necessary for their hydrolysis. 7.2.2 Production of Levulinic Acid, Formic Acid and Furfural

The Biofine Process involves the use of dilute sulfuric acid as a catalyst but differs from other dilute-acid lignocellulosic-fractionating technologies in that free monomeric sugars are not the product. Instead, the 6-carbon and 5-carbon monosaccharides undergo multiple acid-catalyzed reactions to give the platform chemicals levulinic acid (C5H8O3) and furfural (C5H4O2) as the final products. Hydroxymethylfurfural (HMF) is an intermediate in the production of levulinic acid (4-oxopentanoic acid) from 6-carbon sugars in the Biofine Process. The series of consecutive reactions involved in its production are illustrated in Figs 7.3 and 7.4. These reactions have been established by numerous studies aimed at identification of intermediate products and analyses of pathways for their further transformation [10]. The enediol (1), obtained by enolization of dglucose, d-mannose, or d-fructose, is the key compound in the formation of HMF. Further dehydration of the enediol (1) yields the product (2); which is

Fig. 7.3 Dehydration of the enediol (1) of D-glucose, D-mannose and D-fructose.


Fig. 7.4 Formation of hydroxymethylfurfural from 3,4-dideoxyglucosulosene-3.

further dehydrated to give 3,4-dideoxyglucosulosene-3 (3). 3,4-dideoxyglucosulosene-3 (3) is readily converted (Fig. 7.4) to the dienediol (4), which eventually results in the formation of 5-hydroxymethylfurfural (6) via the intermediate cyclic compound (5). Humic-type compounds can also be produced as side products in this reaction [11]. If the CH2OH group of the hexoses is, instead, a hydrogen (as with the pentoses) a similar procedure occurs, but furfural is now the product.

Furfural Hydration of HMF, i.e. addition of a water molecule to the C-2–C-3 olefinic bond of the furan ring, leads to an unstable tricarbonyl intermediate (7) which decomposes to levulinic acid (LA) (8) and formic acid (HCOOH). A possible reaction process is shown in Fig. 7.5 [11]. The steps in the brackets in the mechanism below have not been proven and include several assumptions; these inter-

Fig. 7.5 A possible process for formation of LA from HMF [11].

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mediates were proposed by Horvat et al. [12, 13] based on analysis of spectra of the reaction mixture formed in the hydration of HMF.

13

C NMR

7.3 The Biofine Process

Feedstock materials for a Biofine plant must be of appropriate particle size (ca 0.5 to 1 cm) to ensure efficient hydrolysis and optimum yields. The feedstock is therefore initially shredded before the biomass particulates are conveyed by high-pressure air injection system to a mixing tank. Here the feedstock is mixed with recycled dilute sulfuric acid (1.5–3%, depending on feedstock and titratable alkalinity). The Biofine Process then consists of two distinct acid-catalyzed stages (Fig. 7.6) that are operated to give optimum yields with a minimum of degradation products and tar formation. The objective in the first reactor is the dominant, first-order, acid hydrolysis of the carbohydrate polysaccharides to their soluble intermediates (e.g. HMF). This reaction is favored by the use of a plug-flow reactor, at a temperature of 210–220 8C and a pressure of 25 bar. The rapid nature of the hydrolysis reaction means that a residence time of only 12 s is required. Given that the products are removed continuously, such a small residence time requires that the diameter of the reactor is kept small. The completely mixed conditions of the second reactor favor the first-order reaction sequence leading to LA (Fig. 7.5) rather than higher-order tar-forming condensation reactions. Although the acid concentration remains the same as

Fig. 7.6 Chemical conversion of cellulose to LA (major product), formic acid (byproduct), and tars (minor condensation products) in the two Biofine reactors.


in the first reactor, operating conditions are less severe (190–200 8C, 14 bar). This reactor is considerably larger than the first, however, because of the need for a residence time of approximately 20 min. Furfural and other volatile products tend to be removed at this stage while the tarry mixture of LA and residues are passed to a gravity separator. From here the insoluble mixture goes to a dehydration unit where the water and volatiles are boiled off. The heating of the mixture to boil off the LA is conducted under reduced pressure and results in the tarry material being “cracked”, to give a bone-dry powdery substance (“char”). The crude 75% LA product can be purified up to a purity of 98%. The acid is recovered in the final recycle stage, enabling it to be re-used in the system. In a complete Biofine plant, additional processing may then occur, depending on the final products required. For example, syngas production from the Biofine char (a dry, powdery material of calorific value comparable with that of bituminous coal, and composed of the residual materials in the Biofine Process which has value as a fuel and as a soil additive) can be conducted in a gasification unit or the LA can be esterified with ethanol to produce ethyl levulinate. The downstream conversions will be discussed further below. 7.3.1 Yields and Efficiencies of the Biofine Process

The maximum theoretical yield of LA from a hexose is 71.6% w/w and formic acid makes up the remainder [14]. How close to this theoretical yield is achieved in the conversion process will depend on the degradation reactions involved. In addition to cellulose and LA there are likely to be many intermediates other than those presented above. Some authors [12] have estimated there are over 100. These intermediates tend to cross-react and coalesce to form an acid-resistant tar which incorporates many insoluble residues such as humins. Previously developed technologies that attempted to produce LA from lignocellulosics were expensive because of low LA yields (approx. 3% by mass) and significant tar formation. The Biofine Process, because of its efficient reactor system and the use of polymerization inhibitors that reduce excessive char formation [2, 3], achieves from cellulose LA yields of 70–80% of the theoretical maximum. This translates to conversion of approximately 50% of the mass of 6-carbon sugars to LA, with 20% being converted to formic acid and 30% to tars. The mass yield of furfural from 5-carbon sugars is also approximately 70% of the theoretical value of 72.7%, equivalent to 50% of the mass, the remainder being incorporated in the Biofine char. These claims have been supported by process data from a pilot plant located in Glens Falls, New York State. This processes one dry tonne of feedstock per day and has been operational for several test-run periods since 1996. Its construction followed successful laboratory-scale demonstrations of the viability of the process at the National Renewable Energy Laboratory in Golden, Colorado. In the latter experiments, paper sludges from nearby paper mills were initially used as pilot plant feedstocks and gave LA yields ranging from 0.42 to

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0.595 kg per kilo of cellulose (between 59 and 83% of the theoretical maximum yield). The acid-insoluble ligneous and ash components of the feedstock become incorporated in the Biofine char with 100% mass conversion, although the properties of the resulting materials are likely to be altered under the “cracking” conditions of high-temperature and pressure. For most lignocellulosic feedstocks that may be processed in a Biofine unit, the dry mass balance of structural polysaccharides, lignin, and ash is likely to be close to 100%. Some feedstocks may have a relatively high proportion of extractives (extraneous components that may be separated from the insoluble cell wall material as a result of their solubility in water or neutral organic solvents). Bark, for example, may contain up to 25% by mass of extractives (predominately fats, waxes and terpenes) [15] whereas some grasses may contain a significant proportion (e.g. 20%) of watersoluble carbohydrates (WSC), depending on the time of year and environmental conditions. Although these WSC are also potential LA precursors, their fate in the Biofine process (as with other acid-hydrolysis schemes [16]) is likely to tend towards tar/residue formation because the process conditions are geared towards the conversion of cellulose and hence may be too strong to give LA as an end-product from WSC. Other extractive components are also likely to be incorporated in the Biofine char. That may be advantageous in instances where the char is to be combusted, given the relatively high heating values of these impurities [17]. 7.3.2 Advantages over Conventional Lignocellulosic Technology

The Biofine Process is entirely chemical and does not rely on the use of any form of microorganism, as in enzymatic hydrolysis and in conventional dilute/ concentrated acid hydrolysis technologies. The use of biological agents is often responsible for poor yields and a lower range of feasible feedstocks. Most dilute acid hydrolysis technologies utilize microorganisms in the fermentation of the fully hydrolyzed monomers (e.g. Saccharomyces cerevisiae [18]). Some of the more recently developed schemes also utilize microorganisms in the hydrolysis of cellulose after hemicellulose extraction (simultaneous saccharification and fermentation, SSF). Even in the most advanced SSF technology the fermentation process takes a substantial time. After pretreatment, the cellulase enzyme and fermentation organisms require about 7 days to bring about the conversion to ethanol, compared with approximately 2 days for conversion of starch and approximately 30 min for conversion of cellulose to levulinic acid in the Biofine Process. Ethanol yields are also reduced as a result of the formation of sugar degradation products that inhibit the organisms/enzymes used for fermentation [19]. There are also significant problems associated with the fermentation of nonglucose sugars, particularly xylose. Although these sugars can be converted to ethanol by the genetically engineered yeasts that are currently available, for ex-


ample Pachysolen tannophilus [20], ethanol yields are not sufficient to make the process economically attractive. It also remains to be seen whether the yeasts can be made “hardy” enough for production of ethanol on a commercial scale [21]. The inefficient utilization of nonglucose monosaccharide residues is a major disadvantage in fermentation schemes because these residues may be a significant proportion of the total polysaccharide mass (e.g. xylose makes up approximately 20% of the total dry mass in much woody and herbaceous biomass). The 50% (by mass) conversion of C5 sugars to furfural in the Biofine Process looks particularly attractive in such instances. In avoiding the use of microorganisms, Biofine also enables use of a wider range of heterogeneous lignocellulosic feedstocks, including those (e.g. cellulosic municipal solid waste, sewage) that contain contaminants that might inhibit fermentation. The flexibility of the technology for a variety of feedstocks has been demonstrated over a four-month evaluation period during which the highly heterogeneous organic fraction of municipal solid waste (from the Bronx district of New York City) was successfully fractionated [22]. Furthermore, the lignin content of biomass has no inhibiting effect on the Biofine Process and this contrasts with enzymatic hydrolysis in which steric hindrance, caused by lignin–polysaccharide linkages, limits access of fibrolytic enzymes to specific carbohydrate moieties, this resulting in lower yields or the need for steam-explosion pretreatment [23]. 7.3.3 Products of the Biofine Process

LA is a valuable platform chemical because of its particular chemistry – it has two highly reactive functional groups that enable many synthetic transformations. LA can react both as a carboxylic acid and as a ketone. The carbon atom of the carbonyl group is usually more susceptible to nucleophilic attack than that of the carboxyl group. Because of the spatial relationship of the carboxyl and keto groups, many of the reactions proceed with cyclization forming heterocyclic molecules (for example methyltetrahydrofuran). LA is readily soluble in water, alcohols, esters, ketones, and ethers. The worldwide market for pure LA at a price of $5 kg–1 has been estimated to be about only half a million kilograms. The key to an increased potential marketability for LA is the vast range of derivatives possible from this platform chemical (e.g. Refs. [24–26]) and its economical production via the Biofine Process. Figure 7.7 lists some of the sectors that offer markets for the products of the Biofine process. The following subsections will discuss some of the more promising products which potentially have the largest markets and hence the greatest potential for significantly replacing oil as a source of industrial chemicals and transport fuels.

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Fig. 7.7 Possible markets and saleable products from the Biofine process.

7.3.3.1 Diphenolic Acid Diphenolic acid [4,4-bis-(4'-hydroxyphenyl)pentanoic acid] is prepared by reaction of levulinic acid with two molecules of phenol [27]. It may be a direct replacement for bisphenol A (BPA) in polycarbonates, epoxy resins, polyarylates, and other polymers. The acid also has numerous other uses including applications in lubricants, adhesives, and paints [28]. It can also copolymerize with BPA or can replace it in a variety of formulations. It contains a carboxyl group, absent from BPA, which confers additional functionality useful in polymer synthesis.

Diphenolic Acid

Bisphenol A

Diphenolic acid (DPA) was used commercially in various resin formulations before it was replaced by the petrochemically-derived BPA which could be supplied at a lower price. The reduced cost of LA production made possible with the Biofine Process may enable DPA to recapture some market share. Extensive research into near-term applications of DPA, particularly those that displace currently marketed BPA products, has been conducted at the Rensselaer Polytechnic Institute in New York State [29]. In the longer term DPA could be a viable alternative to oil in the production of plastics. The cost of LA produced by other technologies is the principle reason for the high price of DPA (approx. $\phi 6 kg–1). On the basis of Biofine estimates, the production of DPA from LA from the Biofine Process could result in a market price of $ 2.40 kg–1. That price, based on Biofine estimates, could result in DPA capturing 20% of the US market (2.5 ´ 108 kg year–1 for BPA). It may also result in DPA recapturing some of the 2.5 ´ 106 kg year–1 market it held for its old use as a coating material.


7.3.3.2 Succinic Acid and Derivatives

Oxidation of levulinic acid can lead to the production of succinic acid (Fig. 7.8). Currently, succinic acid is produced using a hydrocarbon-based process. A fermentation process using glucose derived from corn syrup can also produce succinic acid but this is not economically competitive. The most important uses of succinic acid are in food additives, soldering fluxes, and pharmaceutical products. The US market for succinic acid is approximately 4.50 ´ 108 kg year–1, with a market price of approximately $ 2.8 kg–1. Succinic acid can be used to produce tetrahydrofuran (THF), 1,4-butanediol, and c-butyrolactone (GBL). THF is formed by cyclization of succinic acid to give succinic anhydride which is then reduced and dehydrated to provide tetrahydrofuran. THF is a cyclic ether whose major use is as a monomer in the production of poly(tetramethylene ether glycol) (PTMEG), a component of, among other things, polyurethane stretch fibers (Spandex). A smaller amount of THF is used as a solvent in poly(vinyl chloride) (PVC) cements, pharmaceuticals, and coatings and as a reaction solvent. The Western European market for tetrahydrofuran is estimated to be approximately 7.5 ´ 107 kg, valued at $ 2.6 kg–1. Almost 80% of production is used captively, mostly for PTMEG [30]. c-Butyrolactone (C4H6O2) is used as a chemical intermediate in the manufacture of the pyrrolidone solvents. It can be used in the production of pesticides, herbicides, and plant-growth regulators. Mechanisms for the production of GBL are currently being refined – catalysts have been identified for the selective reduction of succinic acid to GBL in the presence of acetic acid [31]. Although high GBL yields have been successfully demonstrated, catalyst productivities are currently still below commercially attractive rates [31]. The market price for 1,4butanediol, another possible derivative of succinic acid, is approximately $2.30 kg–1 [30].

Fig. 7.8 The production of succinic acid in base (e.g. NaOH).

7.3.3.3 Delta-aminolevulinic Acid d-Aminolevulinic acid (DALA) is a naturally occurring substance present in all plant and animal cells [32–34]. It is the active ingredient in a range of environmentally benign, highly selective, broad-spectrum herbicides. It has high activity against dicotyledonous weeds and little activity against monocotyledonous crops such as corn (maize), wheat, or barley [35]. DALA also has use as an insecticide [36] and in cancer treatment [37].

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DALA Difficulties experienced in production of DALA from LA involve the selective introduction of an amino group at the C5-position. The most common approach for activating the C5 position toward amination is bromination of LA in an alcohol medium to give mixtures of 5-bromo- and 3-bromoesters that are separated by distillation [38]. The 5-bromolevulinate is then aminated using a nucleophilic nitrogen species [39]. These conventional mechanisms give low yields at very high cost. The National Renewable Energy Laboratory (NREL) process (Fig. 7.9), significantly improves yields and reduces costs. It also results in the production of two moles of formic acid per mole of DALA, the resulting DALA being obtained at a purity of greater than 90%. During initial testing in a greenhouse environment, NREL found that this crude DALA was active as a herbicide. A significant amount of research is still being conducted on the formation of DALA from LA. The complexity and low yields of conventional DALA synthesis techniques mean that it is currently a very expensive product, being used only for highly selective herbicidal treatment and some cancer therapies. There is a large potential in the agricultural and horticultural sector for lower-cost Biofinederived DALA; however, quantification of this area is not possible at present because specific commercial formulations must be developed.

7.3.3.4 Methyltetrahydrofuran The production of fuel additives via renewable feedstocks offers perhaps the greatest potential for mass-market penetration of LA. Methyltetrahydrofuran (MTHF) can be added to petroleum in amounts up to 30% by volume with no adverse effects on performance, and engine modifications are not required. Some important properties of MTHF are listed in Table 7.1. Although it has a

Fig. 7.9 NREL mechanism for the production of DALA from LA. Taken from [28].

7.3 The Biofine Process Table 7.1 Selected properties of MTHF and ethyl levulinate [42–44].

Boiling point (102 mmHg), 8C Boiling point (Atm.), 8C Flash point, 8C Reid vapor pressure, psig Lower heating value, kJ kg–1 Specific gravity Octane rating Cetane number Lubricity (HFRR micros)

MTHF

Ethyl levulinate

20 80 11 5.7 32 000 0.813 80 – –

93 206.2 195 < 0.01 24 300 1.016 < 10 287

lower heating value than regular petroleum, it has a higher specific gravity and hence mileage from MTHF blended fuel would be competitive. MTHF substantially reduces the vapor pressure of ethanol when coblended in gasoline. This has led to the development of “P-Series” fuels, i.e. fuels miscible with petroleum designed for vehicles with flexible-fuel engines and containing “pentanes-plus” hydrocarbons from natural gas, ethanol (preferably from biomass), and methyltetrahydrofuran as a co-solvent for ethyl alcohol (high-octane) [40]. P-Series fuels can be used alone or may be mixed in any proportions with petroleum. Vehicle tailpipe and evaporative emissions tests have been conducted on three P-Series formulations by the Environmental Protection Agency [41] and the results have been compared with those obtained from reformulated gasoline (RFG). It was found that the formulations had a reduced ozone-forming potential (OFP) and resulted in reduced emissions of nonmethane hydrocarbons and total hydrocarbons – approximately a third of that formed with Phase 2 RFG. It has been estimated that when the MTHF and ethanol are derived from biological materials, the full fuel-cycle greenhouse gas emissions will be between 45 and 50% below those of reformulated gasoline [41]. These successful emission and performance tests have recently resulted in the P-Series formulations being approved by the US Department of Energy as an alternative gasoline, meeting the requirements of the Energy Policy Act for automobile fleet usage. It should be noted, however, that P-Series fuels can only be used in “flexible fuel engines” and have to be distributed at gasoline stations supplied with pumps specially modified for alcohol-based fuels. Their short-term markets may therefore be limited to captive fleets (e.g. city buses). Direct conversion of LA to MTHF occurs in low yield, hence indirect routes are utilized (Fig. 7.10). One possible mechanism involves the catalytic hydrogenation of LA to c-valerolactone (GVL) which, on further hydrogenation, yields 1,4-pentanediol and, finally, MTHF [28]. An efficient application of this mechanism was devised by scientists at the Pacific Northwest Laboratory (PNL) in the US [45]. The process is conducted at elevated temperatures and pressures using a continuous-flow catalytic reactor. Levulinic acid is pumped into a tube where it is warmed to approximately 40 8C, then mixed with hydrogen. Both com-

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Fig. 7.10 Possible mechanisms for formation of MTHF from LA [28].

pounds are then pumped through a reactor filled with a catalyst in which a series of chemical reactions occurs at approximately 240 8C and 100 atmospheres pressure to create MTHF. The procedure requires three moles of hydrogen per mole LA. Laboratory tests indicated the yield was 83% on a theoretical (molar) basis [45]. This would be equivalent to a yield of approximately 63 kg (71 L) MTHF for every 100 kg of LA, or 81 L of MTHF for every 100 L of LA. The yield of the PNL process is significantly greater than that of other processes. These usually used mechanisms in which MTHF was a merely a byproduct, with final yields of approximately only 3%. The extra costs involved in producing MTHF from LA are minimal – for a Biofine plant processing 1000 dry tonnes of biomass per day, the estimated extra capital cost for an MTHF production facility would be $ 10 Mio. The required 6 kg of hydrogen for every 100 kg of LA could be supplied from the residual process char (via a syngas production unit, see below) with an estimated cost of hydrogen production of 5 c kg–1. In addition to transport, MTHF has value in other markets – it is, for example, an excellent general solvent that is, in many regards, superior to tetrahydrofuran. It should also be noted that catalytic production of MTHF from furfuryl alcohol is possible, as is the production of dimethyltetrahydrofuran (DMTHF) from hydroxymethylfurfural (the product of the first Biofine reactor). It is hypothesized that the additional methyl group in DMTHF may afford superior performance and mileage over MTHF, although vehicle tests have yet to be carried out.

7.3.3.5 Ethyl Levulinate Esters of LA produced from either methanol or ethanol have significant potential as blend components in diesel formulations. LA esters are similar to the biodiesel fatty acid methyl esters (FAME) that are used in some low-sulfur diesel formulations but they do not have their principal drawbacks (cold flow prop-


erties and gum formation [46]). Addition of ethyl or methyl levulinate to FAME would be expected to alleviate both these problems. The most studied of the LA esters is a low-smoke diesel formulation developed by Biofine and Texaco that uses ethyl levulinate (made by esterifying LA with fuel-grade ethanol) as an oxygenate additive. The 21 : 79 formulation consists of 20% ethyl levulinate, 1% co-additive, and 79% diesel and can be used in regular diesel engines. The oxygen content of ethyl levulinate (EL) is 33%, w/w, giving a 6.9%, w/w, oxygen content in the blend, resulting in a significantly cleaner burning diesel fuel [44]. The ethyl levulinate blend gives lower sulfur emissions than does regular diesel. This is because ethyl levulinate contains no sulfur. Lower sulfur emissions can also be attributed to the high lubricity of EL blends. Fuel lubricity is used to determine the amount of wear that occurs between two metal parts covered with the fuel as they come into contact. Fuels of higher lubricity result in less wear and prolong engine component life. The sulfur level of diesel is reduced in the refinery using a hydro-treating process; this results in undesirable removal of some of the lubricity components from the fuel and hence a decrease in diesel lubricity. Addition of EL, with high lubricity, will therefore mean that diesel blend-stocks of low lubricity, and hence lower S content, can be used without reducing the all-over lubricity of the end product. In Europe lubricity is measured by use of the high frequency reciprocating rig (HFRR) test with lower values indicating higher fuel lubricity. Biofine has shown that addition of 20% EL to a standard No 2 base fuel improves the HFRR from 410 to 275 [44]. Importantly, the significant losses of engine efficiency (a decrease of up to 15% in the distance driven per unit volume is found with other diesel oxygenates, for example ethanol) do not occur with ethyl levulinate. This is because of the high energy content of the 21 : 79 formulation (selected properties of EL are listed in Table 7.1). The levulinate esters also have potential as replacements of kerosene as a home heating oil and as a fuel for the direct firing of gas turbines for electrical generation [47]. The production of levulinic acid esters from LA formed in the Biofine Process has the added advantage over conventional bioesters that there is no coproduction of glycerol which would have to be disposed of.

7.3.3.6 Formic Acid Formic acid (HCOOH) is a byproduct in the production of levulinic acid from cellulose. It can be purified by distillation and sold directly as a commodity chemical. It is conventionally produced, usually as a byproduct of acetic acid production, by liquid phase oxidation of hydrocarbons. It is used extensively as a decalcifier, as an acidulating agent in textile dying and finishing, and in leather tanning [48]. It is also used in the preparation of organic esters and in the manufacture of drugs, dyes, insecticides, and refrigerants. Formic acid can also be converted into calcium magnesium formate for use as a road salt. In Europe, the largest single use of formic acid is as a silage additive. For example,

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the AMASIL additive produced by BASF Ireland contains 85% formic acid which is bought at a price of 1 1.35 per liter. The catalyst-preparation sector is a very large potential future market for formic acid. As well as being used in the manufacture of many catalysts, formic acid is also used in the regeneration of catalyst metals poisoned with sulfur. The increasing demand for low-sulfur fuels will result in increased demand for the catalysts produced using the formates and, consequently, will require formic acid. Additionally, esters of formic acid (e.g. methyl and ethyl formate) may also have value as fuel components and as platform chemicals. In 2000, world consumption of formic acid amounted to approximately 4.15 ´ 108 kg [48], roughly half of which was consumed in Europe. A Biofine plant processing 300 dry tonnes of feedstock per day would produce approximately 9 ´ 106 kg formic acid per year (assuming a cellulose content of 40%). Where supply of formic acid exceeds demand for its conventional uses, the merchant market price may fall to around $ 0.16 per liter, which is the price needed to open up other markets such as its use as a road salt or for formaldehyde production [49]. If even these markets are not available, the formic acid byproduct still has value, because it can provide energy via gasification or anaerobic digestion.

7.3.3.7 Furfural Furfural is produced from the hemicellulosic pentose fractions of biomass. Xylose is the predominant pentose in most feedstocks, with hemicellulosic arabinose found to a lesser extent. Furfural can be sold as a solvent or used in the production of furfuryl alcohol, tetrahydrofuran (THF), and LA. Furfuryl alcohol (Fig. 7.11) is a monomer for furan resins, these being used mainly as foundry binders. It is prepared by hydrogenation of furfural. THF is produced by decarbonylation of furfural to furan, then catalytic hydrogenation [50]. LA is produced by first converting furfural to furfuryl alcohol. Figure 7.11 shows the mechanism involved – furfuryl alcohol, when boiled in ethyl methyl ketone in the presence of HCl, gives rise to 90–93% levulinic acid, the reaction occurring via hydroxy derivatives [11]. Global production of furfural in 2001 amounted to 22.5 ´ 107 kg year–1 [51]. Approximately 4 ´ 107 kg furfural was consumed in Europe in 2000, furfuryl alcohol being the major market. Most furfural is now produced in China, whose total capacity is 15–20 ´ 107 kg year–1 [51]. Low labor and feedstock prices in Chi-

Fig. 7.11 Production of LA from furfuryl alcohol.


na, coupled with increasing Chinese capacity, have resulted in prices falling over the last decade – the current market price of furfural is approximately $ 1 kg–1 compared with prices in 1990 of $ 1.74 kg–1 for furfural and $ 1.76 kg–1 for furfuryl alcohol [50]. EU and US import tariffs are placed on furfural from China, these being designed to lessen this effect of this price differential, but market prices are still highly dependent on Chinese supply. (A significant rise in price between 1995 and 1998 was attributed to a drought in China during that period). A Biofine plant processing 300 dry tonnes of feedstock per day would produce, from hemicelluloses, approximately 1.3 ´ 107 kg furfural per year (assuming 25% pentosans by mass). This represents 32.5% of the total consumption of furfural/furfuryl alcohol in Europe in 2000. Furfural conversion products, whether THF or LA and their subsequent downstream products, may therefore be more marketable final products than furfural itself in large biorefinery schemes, especially if the fuel additive market is explored. 7.3.4 Biofine Char

The quantity of residual char from the Biofine process and its calorific value will depend on the acid-insoluble lignin content of the biomass, the ash content, any insoluble proteins present, and the amount of degradation and reversion products formed from the cellulose and hemicellulose fractions. The boiling off from the char of volatiles and LA gives rise to a “cracking” of the char. It is, therefore, difficult to predict the final composition of the char from the mass compositions of virgin biomass. The composition would be predictable with greater certainty if each potential feedstock was tested in the Biofine Process. Biofine char has much promise as a fuel. Residual char from the processing of paper sludge in the Biofine pilot plant was found to have a heating value of approximately 25.6 MJ kg–1 (with 15% ash content), a value that was significantly larger than that of the original feedstock (18.6 MJ kg–1). It was found that combustion of the dry Biofine char (the mass of which was 15% of that of the original paper sludge) yielded more energy than combustion of the entire feedstock (at its initial 50% moisture content). It has been estimated that the energy provided by the residual char is greater than that needed to completely fuel the steam and electric power needs of the biorefinery when the scale of operation is equal to or greater than approximately 270 dry tonnes of feedstock per day (assuming a feedstock lignin content of approximately 25%). Appropriately sized plants may therefore expect to gain significant revenue from the marketing of surplus electricity. Research is ongoing for alternative uses and markets for Biofine char. It is believed it may have value as a soil conditioner. Figure 7.12 compares the FTIR spectra of lignin with those of chars obtained from paper sludge and straw feedstocks. It can be seen that straw char retains many of the functionalities of lignin, characterized by absorbance in the 1000–1800 cm–1 region of the spectrum.

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Fig. 7.12 FTIR spectra for lignin and Biofine chars from paper sludge and straw feedstocks.

The chars from straw and from paper have significant carbonyl/carboxyl functionality and the presence of significant acidic functionality was confirmed by titration data. Solid-state NMR will indicate the nature of the aromatic functionality and whether or not the Biofine chars have the fused aromatic structures characteristic of charcoals. We have performed thermogravimetric analyses on several feedstocks and on the chars from straw and paper. In thermogravimetry, sample weight loss is monitored as a function of increasing temperature. Thermograms, and their first derivatives (rate of weight loss with respect to temperature) for the renewable energy crop Miscanthus, for a lignin extract, and for the Biofine chars from paper sludge, and straw feedstocks are shown in Fig. 7.13. Weight loss in the range 50–120 8C is associated with volatilization of water whereas that resulting from loss of low-molecular-weight compounds and other volatile organic compounds (extractives in woods/grasses) occurs from 120–250 8C; degradation of hemicelluloses occurs in the 250–300 8C range, and cellulose degrades very sharp at approximately 300–340 8C in air (higher in nitrogen). The thermogram for Miscanthus is strong evidence for hemicellulose, cellulose, and lignin components. The two thermograms for the chars are similar and clearly show that the hemicellulose and cellulose components are greatly diminished – indicated by a significant shift of the peaks to the right compared with the Miscanthus thermogram. This shift reflects a predominance of the ligneous type components, this being indicated by a similarity between the char thermograms and those of the lignin extract. Another possible use of the char is steam gasification (thermochemical production of hydrogen from a feedstock, for example biomass materials, or, in this context, Biofine char) followed by upgrading of the synthesis gas produced. Although little work has yet been done along these lines using the Biofine char,


Fig. 7.13 Thermograms and their first derivatives for lignin, Miscanthus, and Biofine chars from paper and straw feedstocks.

there has been much work over the years on the gasification of equivalent materials, for example peat or coal [52, 53]. Coal gasification was originally used for the production of syngas for use in, for example, the Fischer-Tropsch Process. More recently, interest in connection with that process has shifted to the reforming of natural gas as a source of the syngas for the process (e.g. Ref. [54] and other articles in the same volume). The steam gasification of carbon can be represented simply by a combination of the gasification reaction: C + H2O ? CO + H2 followed by the water-gas shift reaction: CO + H2O = CO2 + H2 The exothermic water-gas shift reaction is favored by operation at low temperatures whereas high temperatures favor the reverse reaction. Hence, a product gas containing largely hydrogen is produced if the temperature is low and a syngas with a CO/H2 ratio of 1 is obtained if the temperature is high. The syngas can be used as a fuel (e.g. to give the energy needed for the Biofine process) but it can also be used in a variety of reactions such as methanol synthesis:

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CO + 2H2 ? CH3OH Alternatively, it can be used in the Fischer–Tropsch process mentioned above; this can be depicted simplistically as: nCO + mH2 CnH2m in which the product hydrocarbons are usually aliphatic in nature and can be used as diesel substitutes. Much work has recently been performed on so-called GTL technology (GTL = Gas to Liquids – technology that converts synthesis gas (from gasification of biomass or biorefinery process chars, for example) into liquid fuels), and many of these developments have been summarized recently [55]. 7.3.5 Economics of the Biofine Process

The Biofine technology is commercially viable. A commercial plant (Fig. 7.14) processing 50 dry tonnes of feedstock per day has been constructed in Caserta, Italy (with joint funding from the EU and private investment) and is expected to be operational in 2005. The primary feedstocks will be paper sludge, agricultural residue, and waste paper with the major products being LA and EL (for use as a fuel). The process char will be gasified to produce a fuel gas for the process boilers. The modular nature of the technology means that the capacity can easily be upgraded, and there are plans for a supplemental 250 tonne per day reactor system to be installed eventually – bringing the total capacity to 300

Fig. 7.14 Commercial plant in Caserta, Italy. Recovery vessels (top); outside of building (bottom left); mixing tank (top middle); second reactor (bottom middle); arial view (right).


tonnes per day. Indeed, the Biofine process is extremely compact – a feasibility study conducted by a marine architectural company concluded that a self-contained 1000 tonne per day facility could be accommodated on an ocean-going “Panamax” barge [56]. Significant economies of scale can accrue with increasing plant size, as shown in Fig. 7.15 a and b. Figure 7.15 c shows, for various plant sizes, the trend in the cost of ethyl levulinate production with feedstock cost. Table 7.2 shows a detailed breakdown of these costs and of byproduct revenues for a plant processing 1000 dry tonnes of feedstock per day. It is assumed that the feedstock is of composition, by mass, 50% cellulose, 20% hemicellulose, 20% lignin, and 5% ash (comparable with many woody and herbaceous energy crops). It is also assumed that the plant is operational for 350 days per year, that all of the furfural

Fig. 7.15 (a) Capital cost of Biofine plants at different scales of operation; (b) Operating cost ($ per dry tonne of feedstock processed) at different scales of operation; (c) Production cost of ethyl levulinate with three plant sizes (1000, 500 and 300 dry tonnes of feedstock processed per day) and varying feedstock cost.

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7 The Biofine Process Table 7.2 Data provided by Biofine on estimated operating costs and byproduct revenues from a plant processing 1000 dry tonnes of feedstock per day for the production of ethyl levulinate. Scale of Operation – 1000 dry tonnes per day (350 000 dry tonnes/year) Capital Cost – $ 150 million (Grassroots, fully self-contained and integrated) Production Output – 133 000 tonnes per year ethyl levulinate Raw materials: Feedstock Sulfuric acid Caustic soda Ethanol Hydrogen Others

350,000 dt/y 3,500 t/y 500 t/y 35,000 t/y 120 t/y allow.

@$ 40/t @$ 100/t @$ 120/t @$ 350/t @$ 1,500/t

Subtotal Utilities: Steam Electric Electric usage Electric sold Water Gas (boiler)

$ 27,000,000/r 250,000 lb/h 14.4 MW 11.3 MW 3.1 MW 250 gpm 294 m BTU/h

gen. on-site gen. on-site

gen. on-site

Subtotal Labor and Maintenance: Operators Supervision Maintenance

17 per shift 2 per shift

Gross Production costs Byproducts: Formic acid Electric sold

$ 500,000/y 0

@$ 20 per hr $ 2,800,000/y @$ 24 per hr $ 400,000/y @4% of cap cost/yr $ 6,000,000/y $ 9,200,000/y

@30% of labor @25% of Lab. & Maintenance allow.

Subtotal Disposal Costs: Ash: Subtotal

0 0 0

$ 500,000/y

Subtotal Overheads Direct General Taxes and Insurance

$ 14,000,000/y $ 350,000/y $ 60,000/y $ 12,250,000/y $ 180,000/y $ 160,000/y

$ 1,000,000/y $ 2,300,000/y $ 3,700,000/y $ 7,000,000/y

17,500 t/y

@$ 35/t

38,500 t/y 26,000 MWhr/y

@$ 110/t @$ 60/MWhr

$ 600,000/y $ 600,000 $ 44,300,000 $ 4,200,000 $ 1,500,000

Subtotal

$ 5,700,000

NET PRODUCTION COST Production cost per to Ethyl Levulinate (no capital charges) Equivalent Energy price

$ 38,600,000 $ 291 per tonne 12 per GJ

7.4 Conclusion

is converted to LA, and that formic acid is sold for 10 c kg–1 and electrical power for 6 c kw h–1. It can be seen that ash is the only waste product, incurring a disposal charge ($ 35/tonne). Disposal need not be a problem, however. The Caserta plant will supply an adjacent tile factory with the ash, avoiding such costs. The ash can also be used as a fertilizer on agricultural land. The ethyl levulinate production cost of $ 291 tonne–1 is equivalent to a price of $ 12 GJ–1, competitive with the $ 16 GJ–1 of gasoline at $ 2 gallon–1 or $ 10 GJ–1 for crude oil at $ 50 per barrel.

7.4 Conclusion

Lignocellulosic fractionating technology offers the potential for inexpensive production of a range of chemicals and fuels that are currently competitive only from petrochemical reserves. The Biofine Process is among the most advanced of this technology and provides high yields of levulinic acid (LA), furfural, and formic acid. Furthermore, unlike most other processes aimed at harvesting added value from carbohydrate feedstocks, the Biofine process is continuous, compact, easily expandable and entirely chemically based. Feedstocks that contain reasonable concentrations of carbohydrates can be utilized. Thus heterogeneous biomass reserves such as cellulosic municipal solid waste and animal manures may be processed as well as the more conventional lignocellulosic feedstocks such as sugar cane bagasse and high-yielding energy crops. Maximum value is targeted from the diverse chemical constituents of biomass – the moist nature of the biomass is exploited in the acid hydrolysis of polysaccharides to provide platform chemicals such as LA (unlike in combustion/gasification schemes where moisture is a barrier). The dry char residue has a significantly higher fuel calorific value than the feedstock and it has potential for syngas production and as a soil conditioner. The technology offers a potentially sustainable “bio-recycling” solution in a world where rising and erratic oil prices and unpredictable oil supplies are coupled with growing levels of waste production. The extent to which this potential is realized will depend on the size of the market for LA, furfural, and their derivatives. Mechanisms already exist for the production of numerous saleable industrial chemicals from these, and promising research indicates avenues for expansion into the potentially huge transport, agricultural, and plastics sectors. It is therefore feasible that such a technology can stimulate a transition from a hydrocarbon to a carbohydrate-based economy (i.e. economy in which chemical, energy, fuel, and consumables requirements are provided by carbohydrate rather than hydrocarbon feedstocks) where local selfsustainability is possible. This concept is supported by the range of commercialscale Biofine plants planned for Ireland, the UK, and the US.

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7 The Biofine Process 49 Idriss, H., Lusvardi, V. S. and Barteau,

53 Sutton, D., Kelleher, B. P. and Ross,

M. A. (1996), Two routes to formaldehyde from formic acid on TiO2(001) surface. Surface Science 348(1/2): 39–48. 50 Gravitis, J., Vedernikov, N., Zandersons, J. and Kokorevics, A. (2001), Furfural and levoglucosan production from deciduous wood and agricultural wastes. In: J. J. Bozell (eds), Chemicals and Materials from Renewable Resources. American Chemical Society, Washington DC, 110– 122. 51 Levy, J. and Sakuma, Y. (2001), CEH Report: Furfural. SRI, Menlo Park, CA. 52 Rieche, A. (1964), Outline of Industrial Organic Chemistry. Butterworths, London.

J. R. H. (2001), Review of Literature on Catalysts for Biomass Gasification. Fuel Processing Technology 73(3): 155–173. 54 Bakkerud, P. K., Gol, J. N., Aasberg-Petersen, K. and Dybkjaer, I. (2004), Preferred Synthesis Gas Production Routes for GTL. (eds), Studies in Surface Science and Catalysis. Elsevier, New York, 147: 13–18. 55 Bao, X. and Xu, Y. (2004), Natural Gas Conversion VII, Studies in Surface Science and Catalysis, 147. Elsevier, New York. 56 Seaworthy Systems Inc. (1997), SSI Report #403-01-01.


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Whole Crop Biorefinery 8 A Whole Crop Biorefinery System: A Closed System for the Manufacture of Non-food Products from Cereals Apostolis A. Koutinas, Rouhang Wang, Grant M. Campbell, and Colin Webb

8.1 Introduction

Selection of the appropriate renewable raw material to supply sustainable processes is dependent on infrastructural, economical and technological factors (e.g. availability, skilled workforce, pretreatment technology and costs, transportation). Cereals meet most of these prerequisites and have the potential to be used for the production of not only traditional foods but also novel functional foods and non-food products (e.g. biodegradable plastics, chemicals, fuels). However, cereal-based processes are currently more expensive than petroleumbased ones. The reduction of processing costs is strongly dependent on restructuring, integrating and optimizing current processes. To achieve this, the introduction of contemporary low-cost unit operations, the reduction of utilities costs and capital investment, and the creation of added-value byproducts in line with core end-products is imperative. The starting point would be to evaluate current cereal fractionation processes as the basis for a biorefinery and to identify focus areas for optimization, operating/capital cost reduction and end-product/byproduct production. The concept of the whole crop biorefinery is inextricably linked to cereals as one of the most energy intense and chemically rich groups of agricultural crops. Cereals are also amongst the most developed crops having been progressively ’improved’ throughout the past 10 000 years. They have been continuously optimized in terms of yield, with many now exceeding 10 tonnes per hectare. However, it is still the case that for every tonne of readily processable, starch-rich cerBiorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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8 A Whole Crop Biorefinery System

eal grain there is approximately another tonne of rather less accessible lignocellulosic material such as straw and other residues. How to deal with this material is one of the prime challenges of the whole crop biorefinery. Not only is there less to be had from the straw component of the crop than from the grain but it is also more difficult to get at; requiring tedious and often costly preprocessing. In addition, there is a major impediment to transportation of this fraction of the crop because it has a bulk density of only about a fifteenth of that of the grain, thereby requiring vastly more voluminous vehicles to move it around. Ideally, of course, the whole crop biorefinery would involve harvesting the whole of the crop and transporting it directly to the refinery where fractionation would begin. Unfortunately, this is unlikely to be the reality and the more pragmatic approach of separation in the field followed by utilization of the straw as a primary energy source, through combustion, or incorporation into building composites, is more likely to prevail for some time to come. It must also be conceded that traditional outlets for straw such as re-incorporation into the soil, animal feed, and animal bedding, will continue to be “first call” uses in the foreseeable future [1]. Thus one of the principal conclusions of a European study to determine the profitability of a wholecrop biorefinery [2] was that “Combine harvesting with grain processing in a biorefinery is more profitable than wholecrop.” In view of these observations, this chapter will consider the biorefinery concept as one in which on-site separation of grain is carried out prior to fractionation and conversion of the whole grains at a remote site. Should it become feasible to process straw and other residues alongside the grain, such processing could readily be integrated within the biorefinery. Current cereal fractionation processes (CFP) break down the grain into macro and micro components that are used either as end-products (e.g. gluten, oil) or as raw materials (e.g. starch) for secondary processing in many industries (e.g. food, pharmaceuticals, textiles, cosmetics, fermentation). The term macro component incorporates any high molecular weight compound (e.g. starch, protein, cellulose, hemicellulose, oil, gums), while micro components are defined as relatively low molecular weight molecules (e.g. lipids, vitamins, minerals). Traditional CFP can be categorized into dry and wet milling operations. Dry milling involves the use of successive grinding and sieving steps aiming at the maximum economic separation of bran from endosperm. Dry milling operations are relatively inexpensive and result in incomplete macro component separation. Wet CFP can be generally categorized into wet–aqueous and wet–nonaqueous processes resulting in selective separation of one or more cereal components. Wet fractionation processes could be applied to the end-products of a primary dry milling operation. Traditional CFP have been developed to suit the needs of the food industry and do not exploit the potential of cereal grains for non-food applications. The development of viable whole-crop biorefineries depends on the constructive integration of physical, chemical, thermal and biological processing resulting in various products, such as functional proteins, oils, antioxidants, polysaccharides,

8.2 Biorefineries Based on Wheat

fine and bulk chemicals, biofuels and biodegradable plastics. Novel cereal fractionation plants could be of three kinds depending on their production capacity: · Small-scale plants that will not focus directly on the market outlets of the traditional cereal processing plants but will target specialty industries (e.g. cosmetics, pharmaceuticals) by extracting value-added minor constituents (e.g. antioxidants). Such plants will utilize only one cereal grain and will leave the majority of the raw material unprocessed, creating the need to find market outlets for this bulk quantity of material. The use of the remaining material for the production of fine or platform chemicals through microbial bioconversion would be an attractive option. · Intermediate-scale plants may have wider flexibility in terms of the cereal grains that they can process. They will be able to market more end-products. Their commercial survival though will be dependent on continuous research and development and process improvements. · Large-scale plants would be able to utilize any cereal grain for the simultaneous commercialization of traditional end-products, value-added minor components as well as biofuels and biodegradable plastics. Extensive technical and market research should certify high efficiency, cost-competitiveness and customer demand for all the end-products. In this chapter, potential whole-crop biorefineries based on wheat and oats are presented. Future biorefineries based on cereals should aim to exploit the vast complexity of cereal grains by extracting valuable macro and micro components and converting the starch fraction into platform chemicals, biodegradable plastics and biofuels via microbial bioconversions. This approach targets waste and cost reductions and the creation of more market outlets.

8.2 Biorefineries Based on Wheat 8.2.1 Wheat Structure and Composition

The structure of the wheat grain consists of several layers with varying composition and functions (Fig. 8.1 and Table 8.1). The two main parts of the kernel are the pericarp and the seed. The pericarp covers the entire wheat kernel and is divided into the outer and the inner pericarp. Beneath the inner pericarp, the seed coat and the pigment strand provide a complete covering around the seed. The nucellar epidermis and the nucellar projection are located underneath the seed coat and surround the endosperm and embryo. One of the most nutritionally important wheat layers, the aleurone layer, is situated below the nucellar tissues. This is a one-cell-thick layer that encompasses the entire endosperm and part of the embryo. The embryo lies on the lower dorsal side of the wheat kernel and its two major components are the embryonic axis and the scutellum.

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Fig. 8.1 Morphology of the wheat kernel [3].

Table 8.1 Wheat nutrients and their location in the kernel. Constituent Pericarp Seed coat, pigment strand and nucellus Aleurone layer Endosperm Embryo Scutellum a)

Function

)

Protect the grain

Mass a) 5 3

Composition

Fiber, K, P, Mg, Ca

Bran Encases endosperm Stores food Root and shoot Stores food

7 82 1 2

Niacin, phytic acid, minerals (especially P) Starch, protein, pantothenic acid, B2, minerals Fats, lipids, sugars P, B vitamins (especially thiamine)

Mass fraction of the constituent within the kernel as % on a dry basis (db)


The scutellum is a storage organ that is considered a cotyledon. The embryo, known as germ to a miller, when separated by traditional milling processes, consists principally of the embryonic axis. A detailed analysis of the chemical composition of wheat grain is given by Pomeranz [4] and MacMasters et al. [5]. Each wheat layer has a unique composition in certain micro and/or macro components. In addition, wheat chemical composition and distribution in grain varies between varieties. The average chemical composition of pericarp is crude fiber (20–21%), cellulose (23.5–24%), and arabinoxylan (25–28%). Small amounts of protein (2.5–4%) and fat (< 1%) are also present. In comparison with the pericarp, the seed coat contains much more protein (10–17%), less arabinoxylan (12–13.5%), much less crude fiber (ca. 1.0%), and no cellulose. The aleurone layer contains high levels of total phosphorus (2.7%), phytate phosphorus (2.4%), and niacin (B3) (530–640 lg g– 1 ). The niacin in the aleurone layer represents about 80% of the total amount in the entire grain. Pyridoxine (B2) occurs in a pattern similar to that of niacin, with over 60% of all pyridoxine in the aleurone layer (30 lg g–1). The aleurone layer is also an important contributor of pantothenic acid (B5), containing more than 40% of the total in the wheat grain (40 lg g–1) [6]. Total free sugar accounts for 10% of the aleurone materials, including sucrose 42%, raffinose 31%, neokestose 20%, and fructosyl raffinose 6% [7]. Monosaccharides, disaccharides (maltose) and higher oligosaccharides which occur in other parts of the grain are absent from the aleurone cells. The content of total lipids in aleurone cells accounts for between 8 and 11% [8], of which 70–80% are nonpolar. Endosperm contains mainly starch (55–65%) and protein (7–11%). Peripheral cells have the lowest starch content and the highest protein content. Values as high as 54% protein have been found in subaleurone cells [9]. The main constituents of endosperm cell walls include polysaccharides (75%) and protein (15%). Arabinoxylan contributes 85% of the total polysaccharide content in endosperm cell walls and b-glucan and b-glucomannan account for the remainder in equal amounts [10]. Non-starch lipids and starch lipids contribute nearly evenly to the 1.5–2.5% total lipids in wheat endosperm. Wheat germ is rich in lipids (25– 30%), protein (21–23%), phosphorus (3%) and B vitamins including B1, B2, B3, B5, B6. Sucrose (10%) and raffinose (7%) are the two sugars abundant in wheat germ and no substantial amount of the other oligosaccharides has been located. 8.2.2 Secondary Processing of Wheat Flour Milling Byproducts

In the traditional wheat flour milling process, wheat is milled into various flour fractions involving a large number of milling and sifting operations developed originally to serve the needs of the food industry. In particular, wheat grains are initially cleaned from impurities and tempered with water for an average of 12 hours to detach the outer bran layers from the endosperm, facilitating their separation. The tempered grains are subsequently processed through a series of break and reduction roller mills and sifting stages. The main aim of the conven-

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tional dry milling process is to produce flour fractions with the lowest possible bran impurities. However, the nature of this process will never produce any flour fraction 100% free from germ and bran. Current wheat flour mills operate at 70–80% grain to flour conversion efficiency where the remaining 20–30% constitutes various byproduct streams that contain predominantly bran as well as lower amounts of germ and flour. The byproduct streams are mixtures of all coarse streams from each break and reduction roll and are usually used as animal feeds or commercial bran. As a whole, the dry milling process of wheat produces many flour streams of varying particle sizes and compositions in endosperm, bran and germ. This means that the separation of certain wheat layers enriched in specific valueadded chemical components is not possible. This way of processing results in high capital investment, high operating costs for milling, conveying and sifting, and products of low purity. Thus, traditional dry milling of wheat cannot be considered as the basis for the development of a viable biorefinery for non-food applications. The most realistic option to use traditional mills for non-food applications is through bioconversion of the significant amounts of byproduct produced. In 1999/2000, 5624 ´ 103 tonnes of wheat (83% of which was home grown) was processed by UK flour millers resulting in 4481 ´ 103 tonnes of flour and 1148 ´ 103 tonnes of byproducts. The majority of this byproduct stream, known as wheatfeed or middlings, contains 15–19% protein (75% of which is degradable), 20–35% starch and around 1% phosphorus. However, the high crude fiber content (7–11%), low essential amino acid content (0.6% Lys, 0.5% Thr, 0.2% Try, 0.4% Met) and the presence of phytic acid (containing about 60–80% of the total phosphorus) reduce its nutritional value. Consequently, its use as animal feed is restricted only to pigs and cattle. Phytic acid, in particular, has been identified as a certain antinutritional compound that forms complexes with iron and zinc ions and makes these metal ions less accessible for assimilation by humans and other monogastric animals [11]. Nonruminants excrete most of it as they do not have an efficient system for making phosphate available from phytate. This forces producers to add inorganic phosphate to animal feed as a supplement, leading to excessive phosphate excretion, which worsens water pollution and eutrophication [12]. The addition of enzyme preparations such as phytase, xylanase and protease could increase the overall digestibility of flour milling byproducts, allowing animals to consume phosphorus from the phytic acid and better assimilate the iron and zinc ions. For this reason, the animal feed industry is currently developing large-scale enzyme applications [12]. The development of an integrated biorefinery that leads to the production of high quality wheat flour and upgrades the byproduct stream into a nutrient-enriched animal feed and value-added chemicals through microbial bioconversions is an attractive option. Figure 8.2 shows such a process based around the bioproduction of succinic acid as a major potential platform chemical. The byproducts from a traditional wheat flour mill could be treated with successive washing and screening stages to separate the majority of starch and some of the pro-

Fig. 8.2 Schematic diagram of a possible biorefinery utilizing traditional wheat flour milling byproducts.

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tein, as well as other soluble nutrients. The starch-rich suspension would be used for production of the bioconversion feedstock, while bran will be used in fungal solid state bioprocessing (SSB) for enzyme production. The crude enzymatic mixture produced by SSB would be used to hydrolyze wheat macromolecules (e.g. starch, protein) contained in the starch-rich suspension. In-situ production of enzymes through SSB would reduce operating costs by eliminating the purchase of unnecessarily purified commercial enzymatic solutions. SSB would also provide a potential use for residual solids as a value-added animal feed, enhanced by the presence of enzymes that increase digestibility and fungal cells that contain high levels of essential amino acids (3.3% Lys, 2.82% Thr, 1.18% Try, 1.46% Met) [13, 14]. The solids could also be used for the production of nutrient supplements similar to yeast extracts by exploiting the natural degradation of fungal hyphae via the excretion of autolytic enzymes under oxygen limiting conditions [15]. Optimization of bran utilization for enzyme production could lead to the exploitation of the excess bran as raw material for gasification. The synthesis gas produced in this way could be used either for energy production to minimize the non-renewable energy requirements for the whole process or as crude feedstock for the production of value-added chemicals via microbial bioconversion or chemical synthesis. The generic nature of the microbial feedstock produced from wheat flour milling byproducts can be used in various microbial bioconversions for the production of value-added chemicals. This specific biorefinery should target the production of low-to-medium volume and medium-to-high value chemicals because of the relatively small amount of raw materials (milling byproducts contain 20–35% starch) available for microbial bioconversion. It should be taken into consideration that the main activity of this biorefinery is the production of wheat flour, while only an average of 25% of the initial grain will be used for non-food applications. In the case of high-volume, low-value chemicals, fuels and plastics, economies of scale apply and small plants are not profitable. However, an economic analysis is necessary as the revenue derived from the primary market outlet may lead to the creation of an overall viable biorefinery even for commodity materials. For this biorefinery, a potential market opportunity could be the production of succinic acid, which has the potential to be a future intermediate molecule for the production of a wide variety of commodities and specialties [16]. Succinic acid is currently produced petrochemically; maleic anhydride produced via butane oxidation is hydrated to maleic acid and then hydrogenated to succinic acid. Global production via this route exceeds 15,000 tonnes per annum. At a selling price between $ 6–8.8 kg–1, succinic acid is a valuable commodity, but is too expensive to be considered a feasible bulk chemical [16]. A preliminary material balance based on reaction stoichiometries (eqs 1–3) demonstrates the potential commercial and environmental impact of using wheat flour milling byproducts as the raw material for succinic acid production:


C6 H10 O5 n nH2 O ! nC6 H12 O6

1

C6 H12 O6 2CO2 4H ! 2C4 H6 O4 2H2 O

2

C6 H12 O6 ! 2C2 H5 OH 2CO2

3

The total amount of wheat flour milling byproducts (1.1 ´ 106 tonnes) available per annum in the UK contains on average 30% starch, i.e. equivalent to 3.66 ´ 105 tonnes of glucose (eq. 1). The theoretical yield from glucose to succinic acid is 1.31 kg acid kg–1 glucose (eq. 2), giving a theoretical maximum of 4.8 ´ 105 tonnes succinic acid per annum for the UK. This is far greater than the current world production of 1.5 ´ 104 tonnes. At the same time, succinic acid production would result in 1.8 ´ 105 tonnes per annum CO2 sequestration (eq. 2) because mass production of the acid requires CO2 fixation in the metabolism of the microorganism involved. This is equivalent to the CO2 released from the production of 2.4 ´ 105 tonnes of bioethanol (eq. 3). In addition, current bioprocessing practices could reduce the succinic acid production cost to lower than $ 0.6 kg–1 [17]. Thus, by utilizing a low-cost agro-industrial raw material succinic acid could be transformed into a low-cost platform intermediate. Such alternative use of wheat flour milling byproducts would also give flexibility to farmers and industrialists in exploring new market opportunities. 8.2.3 Advanced Wheat Separation Processes for Food and Non-food Applications

Traditional wheat processing for food (excluding breadmaking) and non-food applications concentrates mainly on the extraction of the bulk macromolecules, starch and gluten. However, the extraction of a number of value-added components from individual wheat layers could upgrade wheat processing into a novel whole-crop biorefinery producing both low-volume, high-value and high-volume, low-value products. In recent years, there has been a growing interest in the utilization of advanced cereal fractionation technologies to reduce operating/capital costs and to separate/purify value-added macro and micro components from cereal grains. The desired functional component may be concentrated in a particular layer in the cereal grain (e.g. aleurone layer, pericarp, germ). For this reason, novel CFP involve the selective separation of the outer bran layers including the aleurone layer and the germ.

8.2.3.1 Pearling as an Advanced Cereal Fractionation Technology In the 1990s, a new technology was commercialized for wheat dry milling, which involves gradual removal of the outer layers (e.g. pericarp, aleurone cells) of the wheat kernel as a means to increase wheat milling efficiency [18]. In this process, the wheat bran layers are removed sequentially by friction and abrasion operations, while the byproducts of pearling hold great promise as novel food

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ingredients with physicochemical and nutritional properties that differ from those of previously available cereal products [18, 19]. There are a number of pearling devices for sequential removal of cereal outer layers [20–22]. Wheat pearling can be used as a preprocessing stage before conventional dry milling or non-food applications. The Tkac and the PeriTec model Satake VBW5A pearling systems have been presented in a previous publication [18]. The fourth generation Satake pearling system VCW5A is presented in Fig. 8.3 [23]. Wheat is fed to the top of the pearling equipment into the abrasion chamber where the wheat kernels are evenly distributed by a rotating scroll. The abrasion chamber contains abrasive wheels, a slotted screen and four vertically adjusted resistance bars. The wheat kernels are rubbed between the abrasive wheels and the resistance bars resulting in the removal of the outer kernel layers. The bran is collected through the slotted screen by an air stream that is blown through the holes of a hollow central shaft. The degree of debranning in the abrasion chamber is set by a weighted flap on the outlet of the chamber. The partly pearled wheat kernels are transferred through a screw conveyor into the friction chamber. This section contains a cast steel cylinder with two vertical agitator bars on it, which rotates around a slotted screen. The wheat kernels are further debranned by rubbing against the slotted screen. The friction stage is efficient only when the tough and oily testa

Fig. 8.3 The Satake VCW5A debranning apparatus.


layer has been disrupted during the abrasion stage. The bran separated during the friction stage is removed through the screen by an air stream blown into the center of the chamber via a perforated main shaft. Research and commercial implementation of pearling has revealed the numerous potential uses of this advanced CFP. When pearling is used prior to conventional milling, it can improve flour quality and reduce operating costs [24]. The application of pearling can lead to the production of bran-rich streams that can be used as food ingredients. Cui et al. [19] reported that non-starch polysaccharides (NSP) from a wheat bran fraction produced by the Tkac and Timm pearling technology exhibited novel rheological properties. The NSP extracted from a pearling fraction that contained 13.4% starch, 29.2% insoluble dietary fiber, 8.5% soluble dietary fiber and 2.6% b-glucan, exhibited shear-thinning flow behavior at low concentrations in water (0.5%, 25 8C) and formed a thermally reversible gel upon cooling at 4 8C [19]. The bran-rich fractions produced by pearling are also rich in value-added components (e.g. antioxidants, b-glucan) in comparison to the bran-rich fractions produced from conventional milling operations. This occurs because the latter contain higher quantities of starchy endosperm, diluting the overall amounts of added-value components [25]. In the case of wheat, Dexter and Wood [18] used the Tkac debranning system, which produced friction fractions rich in pericarp and enriched in dietary fiber, while the abrasion fractions were rich in aleurone cells and enriched in protein, b-glucan and soluble fiber in comparison to whole wheat. Marconi et al. [26] used barley pearlings enriched in b-glucan and dietary fiber for the production of functional pastas with the aim of meeting the Food and Drug Administration (FDA) requirements of 5 g of dietary fiber and 0.75 g of b-glucan per serving (56 g in the US and 80 g in Italy). It has been reported that pearling fractions from oats may contain significant quantities of specific antioxidants [27]. It has also been reported that byproducts that are produced from the pearling stage prior to dry milling of barley are enriched in b-glucans, tocopherols, and tocotrienols [28]. Cereal constituents with antioxidant properties can be used in pharmaceutical and cosmetic applications. Using pearling before wheat milling could also lead to increased gluten extraction yield from pearled wheat grains [29]. The gluten extraction yield from ground, pearled wheat (11.4% of bran and germ was removed during pearling) was 20–25% higher than from conventionally milled flour in a process achieving 70% extraction of flour from wheat. Apart from milling operations that produce flour streams for food, pharmaceutical and cosmetic purposes, the usefulness of pearling has also been recognized in the case of non-food applications. Wang et al. [30] used whole grain flour and pearled grain flour as fermentation medium for bioethanol production and the latter increased ethanol yields by 6.5–22.5%. This occurred because the use of pearling reduced the bran content in the fermentation feedstock, while at the same time the starch throughput per batch increased by an average of 12% (w/v, db). By integrating the grain pearling technology in rye and triticale

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with very high gravity fermentations (0.30 g solids mL–1 supernatant) the ethanol concentration was increased to around 16% (v/v) [31]. Partial removal of outer grain solids in an alcohol plant would also improve plant efficiency and decrease energy requirements for mash heating, mash cooling and ethanol distillation. In addition, the bran byproduct fractions containing pure grain layers or mixtures of them may be used as functional food ingredients or as raw materials to extract value-added products (e.g. enzymes, phytic acid, tocopherols, oil, proteins, wheat germ glycerides, agglutinin, arabinoxylans, b-glucan), contributing significant revenue for the development of cost-competitive biorefineries.

8.2.3.2 Air Classification Air classifiers are rotary machines that are used in dry milling operations to separate a particulate feed into a fine and coarse fraction, using mainly air to entrain the fine product and a rotor to reject any airborne coarser particles [32]. The application of air classification in dry CFP can lead to the enrichment of milling fractions with specific components. The main advantage of air classification is that the procedure can easily be scaled up using a commercially available large air classifier without the clogging of screens by fine particles in a sieving method [33]. Letang et al. [34] reported that the combination of jet milling with air classification of soft wheat could produce a starch-enriched stream with only 2% protein content and reduced lipid and pentosan contents. Wu and Doehlert [33] enhanced the b-glucan content (200 g kg–1) in oat bran fractions by applying a combination of pin milling, air classification and sieving. Similar results were obtained when air classification was applied to enrich the b-glucan content in milled fractions of barley [35] and oat groats [36]. 8.2.4 Biorefinery Based on Novel Dry Fractionation Processes of Wheat

A potential wheat-based biorefinery (Fig. 8.4) could utilize a pearling system to produce a bran-rich fraction containing pericarp, seed coat, aleurone cells and germ layers from the wheat kernel. This fraction could be significantly enriched in bran by separating flour particles for further processing by the application of air classification. The bran-enriched fraction could be then used for the production of various coproducts, including natural polymers (e.g. arabinoxylans, b-glucans), monomers (e.g. glucose, xylose, arabinose, ferulic acid) or oil components (e.g. triglycerides, sterols). The selection of coproducts that will be eventually derived from the bran-rich fraction would be dependent on process economics, available technologies and existence of markets for their disposal. Pearled wheat grains could be ground in a hammer mill and the resulting bran-free flour could be used for gluten extraction by the Martin, the Batter or a modified version of these processing schemes. The bran-free and gluten-free flour suspension resulting from the gluten extraction stage could be used for

Fig. 8.4 Schematic diagram of a wheat-based biorefinery employing pearling and air classification.

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the production of a glucose-rich solution to provide the carbon and energy source in subsequent microbial bioconversions. The enzymes required to hydrolyze this stream could be produced by submerged or solid state fungal bioconversions on whole wheat flour and pearlings, respectively. Remaining fermentation solids could be used as animal feed, glucosamine/N-acetyl-d-glucosamine extraction or for the production of nutrient supplements by exploiting fungal autolysis that occurs under oxygen-limiting conditions. Unhydrolyzed solids could be gasified for the production of energy or other value-added products. The generic feedstock formulated by mixing the glucose-rich solution with nutrient supplements produced by fungal autolysis can be used in microbial bioconversions for the production of biofuels, platform chemicals and biodegradable plastics.

8.2.4.1 Potential Value-added Byproducts from Wheat Bran-rich Fractions Arabinoxylans Wheat bran and especially the aleurone layer contain high amounts of arabinoxylans and lower quantities of b-glucans. Arabinoxylan is a polysaccharide comprised of a backbone formed by xylose residues connected together with b-(1 ? 4) bonds. The xylose residues are highly substituted with primarily single a-(1 ? 3) and/or a-(1 ? 2)-linked l-arabinose residues as well as short arabinooligosaccharides, d-galactose, d-4-O-methylglucuronic acid and ferulic acid residues. Phenolic acids, such as ferulic acid, play a significant role in the linkage of hemicelluloses with other cell wall components, especially lignin, through ester and ether bonds [37]. In addition, ferulic acid assists in the formation of covalently crosslinked arabinoxylans with well developed networks that exert an interesting water-holding capacity [38]. Different concentrations of ferulic acids would correspond to varying gelling potential of arabinoxylans. Arabinoxylan properties in solution are also dependent on the degree of polymerization of the xylan backbone. The use of enzymatic oxidizing systems such as laccase, horseradish peroxidase or manganese peroxidase could dimerize the esterified ferulic acids causing gelation of the water-extractable arabinoxylans from wheat flour [39]. The high viscosity of aqueous solutions containing water-extractable arabinoxylans that have undergone oxidative gelation after they were treated with laccase and/ or manganese peroxidase has a positive effect in breadmaking [40]. In particular, water-soluble arabinoxylans can absorb water, influence dough rheology and affect such bread characteristics as loaf volume, crumb firmness and staling events [41]. Arabinoxylans could also be potential ingredients for wound-care applications [42]. Sterigel is an arabinoxylan-based product that is used as wound management aid. Ferulic Acid Ferulic acid could be released from bran-rich fractions by the synergistic action of various enzymes, such as esterase and xylanase [43]. Extrac-


tion of arabinoxylans located at the outer bran layers, seed coat and pericarp, may be more difficult than the ones located in the aleurone and nucellar layers [44]. The aleurone-free wheat bran is more resistant to enzymatic hydrolysis because it contains higher amounts of lignin, cellulose and glucuronoarabinoxylans than the aleurone layer. Increased crosslinking among cell wall components creates a rigid cell wall network and decreases the effectiveness of enzymatic hydrolysis. Ferulic acid could be used as a natural food preservative due to its ability to inhibit peroxidation of fatty acids. The acid and its derivatives, steryl ferulates, have antioxidant properties. A commercial product, c-oryzanol, with cholesterol lowering properties containing steryl ferulates has been extracted from rice bran [45]. Wheat bran fractions may contain up to 0.34 mg g–1 total steryl ferulates, which is significantly higher that the 0.063 mg g–1 content in the whole wheat grain [45]. Ferulic acid is currently used as an ingredient in many skin lotions, sunscreens and wound dressings [43]. The antioxidant potential of ferulic acid could be enhanced by converting it enzymatically into cafeic acid by using a microbial cell extract from the anaerobic bacterium Clostridium methoxybenzovorans SR3 [46]. Another commercial option is the bioconversion of ferulic acid into vanillin by Streptomyces setonii. Muheim and Lerch [47] reported that shake flask cultures of S. setonii resulted in the production of 6 g L–1 vanillin at a molar conversion yield from ferulic acid to vanillin of 68%. Hwang et al. [48] patented a process separating ferulic acid and arabinoxylan from cereal brans by extrusion and subsequent treatment with plant cell wall hydrolyzing enzymes. Wheat Germ Most wheat germ currently produced by flour mills is used as animal feed contributing a very low revenue. Wheat germ could be upgraded into a useful byproduct if it is used for the extraction of various value-added materials, such as proteins, oils, vitamins and enzymes [24]. Oil extraction could be achieved simply by mechanical pressure or solvent extraction. The germ oil could be used as a specialty food or gourmet cooking oil. A commercial application of oil extracts has been introduced by the French company Bertin, which extracts these oils by cold pressing at 4,000 bars in sunflower oil as solvent [49]. The purified wheat germ oil (104 kg year–1) is called heliogerme and is sold as an ingredient in cosmetics at a price ranging between £ 5–8 kg–1. In the case that wheat germ is processed by solvent extraction, the functional properties of germ protein are retained enabling its commercialization as food grade protein. The extraction of micro components (e.g. enzymes, vitamins, flavonoids) from wheat germ may open additional market opportunities but additional research and market/process evaluation is required [24]. The bran-rich fraction produced by wheat pearling would contain a large amount, if not all, of the wheat germ. Thus, the utilization of the pearling technology in a wheat biorefinery may decrease the cost currently required by traditional flour mills to isolate the wheat germ fraction.

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Monosaccharide Production Another potential market outlet would be the fractionation of pearlings into oil components and polysaccharides, which can be subsequently converted enzymatically into a spectrum of monomers, the most important of which would be glucose, xylose, arabinose and ferulic acid. The monosaccharides could be either utilized as a carbon source in microbial bioprocesses or converted catalytically into various chemicals currently derived from petrochemicals. For instance, glucose could be hydrogenated to sorbitol, which in turn could be catalytically converted to propylene glycol. Xylene and arabinose could also be catalytically converted to ethylene glycol, propylene glycol and xylitol. Functional Foods Supplementation of human diets with wheat bran may enhance the prevention of a range of cancers due to the presence of various functional components, such as dietary fiber, phytic acid, lignans, oligosaccharides, antioxidants, phytoestrogens [50]. Reddy et al. [51] reported that the lipid fraction of wheat bran has strong colon tumor inhibitory properties and further studies are required to identify biologically active constituents of wheat bran lipid fractions and their relative role in colon tumor inhibition. The insoluble dietary fiber content of wheat bran may be responsible for their significant bile acid binding properties that cause decrease in the plasma cholesterol level in humans [52]. In turn, Cui et al. [19] reported that bran-rich fractions enriched in soluble fiber could be superior to AACC standard wheat bran, which contains 46.85% insoluble fiber and 2.8% soluble fiber [19]. Pearling may be employed to produce bran-rich fractions enriched in either soluble or insoluble dietary fiber depending on the requirements of end-product composition. Pearling could be exploited for the sequential removal of individual wheat layers. Fenech et al. [53] reported that pearling could lead to the production of an aleurone-rich fraction that contains high amounts (5 lg g–1) of folate. Branand germ-rich fractions produced by pearling could potentially be used to enrich the mineral content of pan breads [54]. In addition, the quality of wheat bread, in terms of loaf volume, crumb structure, shelf life, starch structure and flavor, can be significantly improved when it is supplemented with wheat bran pre-fermented with yeast and/or lactic acid bacteria [55].

8.2.4.2 Exploitation of the Pearled Wheat Kernel Vital Wheat Gluten Pearled wheat kernels could be used for the extraction of vital wheat gluten as a valuable co-product. Gluten can be separated from wheat flour by mixing endosperm particles with water that initiates the formation of protein microfibrils. The formation of dough from the mixture of flour and water is dependent on the water to flour ratio used and mixing. The main processes for the separation of vital wheat gluten from flour (e.g. Posner, Alfa-Laval/Raisio, Hydrocyclone, Pillsbury Hydromilling, Far-Mar-Co., High-Pressure Disintegration) could be categorized into those based on either the Martin or


the Batter process [56, 57]. Such processes utilize a spectrum of raw materials (e.g. wheat, flour), solvents, equipments and wheat flour to water ratio. Weight for weight, gluten is more valuable than starch and could therefore improve the economics of biorefineries based on wheat. In general, 10 tonnes of wheat will give 6–7 tonnes of starch and 1–1.5 tonnes of gluten. The major impediments in gluten separation processes are the operational effectiveness of processing, the high cost required for drying vital gluten and waste treatment costs. Gluten has many applications particularly in the food industry, such as in breadmaking, specialty baked goods, pet foods, breakfast cereals, meat and cheese substitutes and pizza [57]. Other industrial uses include production of hydrolysates, wheat protein isolate and deaminated gluten. The gradual decrease of gluten price in the last 10 years has shown that a potential large-scale utilization of wheat for non-food applications will saturate the current gluten market reducing its current price even lower. Identifying novel uses for gluten could provide more commercial outlets and higher revenue for this value-added co-product. Gluten could be used for the production of biodegradable or edible films and packaging materials [58]. It could also be used for the manufacture of biodegradable high-performance engineering plastics and composites when thiol-terminated, star-branched molecules are incorporated directly into the protein structure [59]. Microbial Feedstock Production and Bioconversion Processes After gluten extraction, the remaining aqueous suspension is rich in starch. Starch has many applications in numerous industries such as paper sizing/coating, thickening/gelling agent in food applications, industrial glues and pastes, dusting agent, slipping agent in oil drilling, water retention agent, biodegradable plastics, building panels, cosmetic applications, encapsulating polymers, packing material, edible packaging and feedstock for fermentation processes. This study will concentrate on the utilization of starch as a raw material for the production of fuels, chemicals and plastics by microbial bioconversion. To produce a nutrient-complete medium for microbial fermentations, three steps are required (Fig. 8.4): 1. on-site enzyme production via submerged or solid state fermentations; 2. enzymatic hydrolysis of gluten-free and bran-free flour suspensions; and 3. production of nutrient supplements (fungal extract) via fungal autolysis.

In many existing microbial fermentations, commercial (purified) starch is enzymatically hydrolyzed into directly assimilable glucose by unnecessarily pure commercial enzyme formulations. Cost reduction could be achieved by utilizing a much less refined process involving on-site production of crude enzyme mixtures via fungal fermentations conducted by single or combined fungal strains belonging to the genus Aspergilli (A. awamori, A. oryzae). The medium for the fungal fermentations could be whole cereal flour or pearlings depending on the bioprocess employed, which might be carried out in submerged or solid state, respectively. The solid residue from the enzyme producing fermentation, which contains mainly fungal cells and any undigested wheat particles, could be converted into nutrient supplements via fungal cell autolysis. A similar process that

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generates a generic microbial feedstock from whole wheat is described and cost evaluated in previous publications [60–62]. The generic microbial feedstock can be used for the bioproduction of several organic chemicals, including platform intermediates for chemical synthesis, ingredients for a spectrum of commercial products, biofuels, biodegradable plastics and solvents. This process could lead to the production of both commodities and specialties depending on market outlets and cost competitiveness. Potential bioconversion products, the large-scale bioproduction of which have or will incur low environmental and high societal and industrial impacts are [16]: 1. lactic acid, succinic acid and 3-hydroxypropionic acid as platform molecules and industrial ingredients; 2. 1,3-propanediol as a monomer for the production of novel plastics; 3. butanol as platform molecule, solvent, industrial ingredient and biobased transport fuel; 4. PHA as biodegradable plastics; 5. bioethanol as a platform intermediate and biofuel; 6. l-lysine as animal feed additive and industrial ingredient. Remaining solids from fungal fermentation could also be used as animal feed or for the extraction of value-added coproducts, such as glucosamine and N-acetyl-dglucosamine. The fungal cell walls contain chitin, which is an unbranched homopolymer of N-acetyl-d-glucosamine. Bohlmann et al. [63] used enzymatic and chemical methods for the recovery of N-acetyl-d-glucosamine from fungal biomass (A. niger) that was produced from a citric acid fermentation. The efficient degradation of chitin and glucan required the synergistic action of chitinases, bN-acetylglucosaminidases and glucanases. Fan et al. [64] have also used various fungal species (Aspergillus, Penicillium, Mucor) for the production of glucosamine. Fungal autolysis might evolve into an effective and economic processing route for releasing glucosamine or/and N-acetyl-d-glucosamine from fungal cell walls. It is strongly believed that glucosamine provides joint health benefits and pain relief. The US National Institute of Health is currently conducting research in order to assess the effectiveness of glucosamine on patients with osteoarthritis. The US retail market for nutritional supplements containing glucosamine is more that $ 109/year [65]. Demand for bulk glucosamine has been growing in excess of 20% annually and global consumption exceeds 5 ´ 106 kg [65]. Apart from glucosamine, N-acetylglucosamine is used as health supplement as well as in cosmetic and pharmaceutical applications. Unhydrolyzed solids from the fungal autolysis stage and solids from the microbial bioconversion could be gasified at various combinations of high temperatures, pressures and oxygen for the production of synthesis gas that is constituted of various compositions of CO, CO2, H2 and CH4 depending on the conditions applied. Synthesis gas can be subsequently used either for energy generation or biological/chemical conversion into a spectrum of chemicals. Potential bioderived products from synthesis gas are bioethanol [66], biodegradable plastics [67], acetic acid and methanol [16].

8.3 A Biorefinery Based on Oats

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8.3 A Biorefinery Based on Oats 8.3.1 Oat Structure and Composition

Like all cereal grains, oats have a complex structure and constitution (Table 8.2). The oat groat, consisting of endosperm, germ and bran, is encompassed in a fibrous hull structure that contains approximately 50% hemicellulose, 40% crude cellulose and 10% crude lignin. The main sections of oat groats are the trichomes (hairs), pericarp, nucellar tissues, aleurone, endosperm, embryo and scutellum. The composition of each layer varies significantly. In addition, the location and concentration of components in oats depend upon cultivar and environmental conditions. The chemical composition of oat groats and respective layers has been given by Webster [68] and Wood [69]. 8.3.2 Layout of a Potential Oat-based Fractionation Process

The high value of oat bran necessitates its separation prior to further processing of the rest of the grain. Figure 8.5 presents a schematic diagram of a proposed biorefinery based on oat groats, employing pearling and air classification. Significant information in the formulation of this processing scheme was taken from Paton et al. [70] who used pearling to remove outer oat layers (1–15% of the oat kernel). Pearlings were used to produce the following products: 1. a highly concentrated anti-irritant and a light oat oil extracted successively by a volatile aqueous polar solvent and a nonpolar solvent; 2. a dark high quality oil and a highly stable lipase-active powder extracted by a nonpolar solvent;

Table 8.2 Oat nutrients and their location in the kernel. Constituent

)

Hull Pericarp Seed coat and nucellus Aleurone layer

Endosperm Embryo Scutellum a) b) c)

Function

Bran

Mass a)

30 b) – – Encases endosperm –

Composition

Encases oat groat Protect the grain

Fiber, some protein, some antioxidants Fiber, protein, antioxidants, K, P, Mg, Ca

Stores food Root and shoot Stores food

Protein, b-glucan, niacin, antioxidants, lipids, phytin, aromatic amines, minerals (especially P) Starch, protein, b-glucan, lipids, minerals Similar to bran excluding niacin and aromatic amines

55–70 c) 2.8–3.7 c)

Mass fraction of the constituent within the kernel as % on a dry basis (db) Content as related to dry weight of the complete oat kernel (hulls plus groat) Content as related to the dry weight of the oat groat

Fig. 8.5 Schematic diagram of a biorefinery based on oat groats employing pearling and air classification.

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8.3 A Biorefinery Based on Oats

3. a b-glucan-rich fraction extracted by successive aqueous steeping of bran, starch removal by enzymatic hydrolysis, bran removal by screening/centrifugation and alcoholic extraction. Pearled oat kernels could be either converted into a feedstock for subsequent microbial bioconversions or fractionated into various coproducts. Steeping the pearled oat groats in water would facilitate the maceration of bran and flour fractions accomplished by aqueous ethanol extraction. The bran fraction is used for the extraction of b-glucan, while the flour fraction is divided into starch and protein. From the remaining pearled oat groat extract, an oat anti-irritant can be recovered. Flour from pearled oat kernels or starch could be used in some fungal bioconversions for the production of chemicals without hydrolysis. For instance, Rhizopus oryzae can utilize starch for the production of lactic acid [71].

8.3.2.1 Potential Value-added Byproducts from Oat Bran-rich Fractions Oat Gum: b-Glucan b-Glucans are linear homopolysaccharides of glucose units linked via a mixture of (1 ? 4)- and (1 ? 3)-b-d-glycopyranosyl units. Water-soluble b-glucans have potential nutritional and health benefits. b-Glucan preparations exhibit blood serum cholesterol lowering capabilities when consumed daily [72]. A potential mechanism for b-glucan cholesterol lowering capability is their high molecular weight, which increases the luminal viscosity in the gastrointestinal tract [73]. Viscous water-soluble b-glucan preparations have hypoglycemic effects assisting in the metabolic control of diabetes [74]. It was recently reported that b-glucans could potentially be used in biomedical applications because of their abilities to: 1. activate host defense mechanisms against microbial and parasitic infections [75]; and 2. reduce the glycemic index, making them a useful food ingredient for decreasing postprandial glycemia while, at the same time, maintaining the food palatability [76].

Research has also focused on the investigation of the rheological properties of b-glucan extracts aiming at the creation of novel products for food purposes, such as stabilizers, thickeners, textural agents and fat replacers in food products, and medical as well as cosmetic applications [73]. In addition, oligosaccharides from b-glucan hydrolysis exhibit prebiotic properties and are considered as food ingredients in probiotic and synbiotic preparations. Malkki and Virtanen [77] stressed that the health effects of b-glucans on cholesterol reduction, improved gastrointestinal function and glucose metabolism would require a daily consumption of 10 g oat b-glucan. This means that the daily digestion of adequate amounts of b-glucans requires consumption of substantial amounts of conventional oat products, which contain 35–50 g kg–1 b-glucan. Thus, the commercialization of concentrated oat bran products is necessary

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in order to provide consumers with the daily requirements for b-glucan. The presence of b-glucan in the pericarp, aleurone and sub-aleurone layers indicates that the application of a pearling technology would be ideal for the separation of b-glucan rich fractions [24]. However, distribution of b-glucan in the oat kernel is dependent on the oat cultivar used. Thus, oat cultivars should be screened so as to identify the ones that will provide b-glucan enriched fractions through pearling. The market price, functional properties and concentration of b-glucan commercial preparations will be dependent on the final application and the fractionation process employed. For instance, purified b-glucan could reach a value of $ 55 kg–1, while its use as thickening agent would compete with guar and xanthan gums, the market price of which is $ 0.78 kg–1 and $ 12 kg–1, respectively [24]. Antioxidants Oats contain various compounds with antioxidant activities, such as tocols, phytic acid, phenolic acids, avenanthramides, flavonoids and sterols. A comprehensive review of these oat antioxidants has been published by [78]. Antioxidants are mainly located in the outer groat layers but each compound with antioxidant properties can be predominantly located in a specific outer layer, in the whole bran fraction or even in the hull. Some problems that are related to the extraction of antioxidants from cereal grains have to do with: 1. heat treatment to deactivate lipase prior to milling; 2. the localization of specific components with high antioxidant activities; 3. the production of cereal fractions containing botanical constituents enriched in antioxidants; and 4. the selection of analytical assays that can realistically measure the antioxidant activities of cereal fractions.

The selection of analytical assays is crucial in the detection of antioxidant activities because of the presence of both antioxidants and pro-oxidants in any cereal fraction and the fact that it measures the inhibition of oxidation of case-specific reactions depending on the combination of the various micro components with antioxidant properties [79]. Several researchers have shown that pearling could be an important tool in the production of bran-rich fractions enriched in compounds with antioxidant activities. Handelman et al. [25] fractionated oats by either pearling or conventional dry milling, with further enrichment by air classification to identify their antioxidant activities. Pearlings exhibited the highest antioxidant content in comparison to flour, trichome and bran fractions. Even the trichome fraction, that is the outer (hairy) surface of the groat, exhibited relatively high antioxidant activity. Peterson et al. [27] compared the antioxidant activity of bran-rich fractions produced through pearling for between 5 and 180 s, concluding that the antioxidant activity of 80% ethanol extract and the concentration of total phenolic compounds and specific phenolic acids (e.g. ferulic acid, q-coumaric acid) was higher in short-pearling-time fractions, and decreased at prolonged pearling

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8.4 Summary

This chapter presents some potential cereal-based biorefineries that can be employed for the production of fuels, chemicals and plastics, as well as many value-added byproducts derived from cereal bran. Cereals are complex biological entities and will represent a major category of the core photosynthetic raw materials that will replace petroleum in the production of commodities as well as specialties. The creation of viable biorefineries based on cereals will require the exploitation of all the botanical constituents of the grain as well as, if possible, the non-grain components such as straw, stalks and hulls. Novel processing routes for starch and protein in the production of a generic feedstock for microbial bioconversions have been presented and various products that could be derived from wheat and oats have been identified. The cost-competitiveness of such cereal-based biorefineries will depend on the range of high value end products and their integration with the much larger volume low to mid-value products, and could be further improved by exploiting straw, hulls and other nongrain components of the crop, either for the production of more value-added byproducts or simply for the generation of energy.

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Fuel-oriented Biorefineries 9 Iogen’s Demonstration Process for Producing Ethanol from Cellulosic Biomass Jeffrey S. Tolan

9.1 Introduction

The production of fuel alcohol from cellulosic biomass is of growing interest around the world. Cellulosic biomass can be used to produce transportation fuel, with the overall process having little net production of greenhouse gases. Biomass is available as agricultural residues or as a byproduct of many processes, or can be potentially produced from dedicated energy crops. The technology for biomass conversion has many significant technical and economic challenges that have delayed its commercialization. However, significant progress has allowed Iogen Corporation of Ottawa, Canada to produce up to 2000 gallons day–1 of ethanol from wheat straw since April 2004 to demonstrate the technology.

9.2 Process Overview

The basic process steps of Iogen’s Ottawa plant are shown in Fig. 9.1. This is a demonstration plant that produces up to 2000 gallons day–1 of ethanol from wheat straw. A full scale plant would produce about 170 000 gallons of ethanol per day (60 million gallons year–1). A cellulosic feedstock material such as wheat straw or other straws, corn stover, or grass is subjected to pretreatment, that is, cooked in the presence of acid to break down its fibrous structure. After pretreatment, the material has a muddy texture. Cellulase enzymes are added to the pretreated material to hydroBiorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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9 Iogen’s Demonstration Process for Producing Ethanol from Cellulosic Biomass

Fig. 9.1 Iogen’s process for converting wheat straw to ethanol.

lyze the cellulose to the simple sugar glucose; this is known as cellulose hydrolysis. The cellulase enzymes are made at the plant site by using a wood-rotting fungus in large fermentation vessels. This is known as cellulase enzyme production. After cellulose hydrolysis, the sugars are separated from the unhydrolyzed solids, which include lignin and residual cellulose. These solids are burned to provide energy for the entire process (lignin processing). The sugars are fermented (sugar fermentation) to ethanol using recently developed recombinant Saccharomyces yeast from Purdue University to ferment the glucose and the more difficult sugar to ferment, xylose. In ethanol recovery, the ethanol is recovered by conventional distillation, denatured, and blended into gasoline. The remainder of this chapter describes the process steps and related technologies in more detail.

9.3 Feedstock Selection 9.3.1 Feedstock Composition

The term “cellulosic biomass” refers to potential feedstocks that have cellulose as a primary constituent. Other major constituents of these materials include hemicellulose and lignin. Minor constituents include proteins, ash, starch, and various other organic compounds that are not carbohydrates.

9.3 Feedstock Selection

Cellulose comprises 35% to 50% of most plant material. Cellulose is a polymer of glucose, of dp (degree of polymerization, or chain length) of 1000 to 10 000. Cellulose is a linear, unbranched polymer, with glucose joined together by beta-1,4-linkages. Individual polymer chains run parallel to each other and form hydrogen bonds with each other, up to three per monomeric glucose unit. Several such chains form a microfibril. A microfibril region that has the full degree of hydrogen bonding forms a roughly cubic, 3-dimensional lattice. Such a region is crystalline cellulose and is very stable against attack by enzymes or acid. Other regions are not hydrogen-bonded to nearly this extent, and in the extreme are simply random configurations of glucose polymers. This is amorphous cellulose. Most natural cellulose is primarily crystalline cellulose. The main source of ethanol is from the glucose, originating from the cellulose. However, a second source of ethanol is from the simple sugars that comprise the hemicellulose. Hemicellulose is a mixture of linear and branched polymers of the five-carbon sugars xylose and arabinose and (less importantly) the six-carbon sugars glucose, mannose, and galactose. Hemicellulose is readily dissolved and hydrolyzed to its simple sugars in dilute acid at moderate temperatures, for example, 120 8C. Hemicellulose comprises 15–25% of most plant material. Lignin differs from cellulose and hemicellulose in that lignin is not comprised of carbohydrates, but rather consists of a complex three-dimensional matrix of phenolic propane units. Lignin confers water resistance and stiffness to the fiber and protection against microbial attack. Lignin does not participate in the pretreatment or hydrolysis processes except with a decrease in the degree of polymerization. The burning of lignin is the mode of energy generation for the process. Lignin comprises 15–30% of most plant materials. In normal operation, the minor constituents exert only a minor impact on the process. The protein is degraded by pretreatment to the point where it cannot be recovered economically. The ash must not be at too high a level as to be abrasive to the process equipment. Such can be the case if large amounts of silica (sand) are present due to the harvest practices or the natural silica uptake by the plant. The starch is easily hydrolyzed to glucose and increases the overall

Table 9.1 Feedstock composition (mg g–1 total solids) (from Foody et al. 1999 [1]). Feedstock

Cellulose

Starch

Xylan

Arabinan

Lignin

Ash

Protein

Barley straw Wheat straw Wheat chaff Switch grass Corn stover Maple wood Pine wood

406 455 391 399 408 500 648

20 9 14 3 3 4 1

161 165 200 184 128 150 33

28 25 36 38 35 5 14

168 204 160 183 127 276 320

82 83 121 48 60 6 0

64 64 33 54 81 6 2

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ethanol yield. Table 9.1 shows the composition of several typical lignocellulosic materials. 9.3.2 Feedstock Selection

The scope of feedstocks considered by Iogen for ethanol production are listed in Table 9.2. These potential feedstocks are evaluated based on the desired feedstock properties. The desired qualities of a feedstock are: 1. Low cost. Naturally, the cost of the feedstock is an important part of the overall cost. A desired feedstock can be obtained and transported to the plant at low cost. This rules out primary and secondary tree growth, sawdust, and waste paper, all of which have existing markets and high cost. 2. Availability. A feedstock must be in sufficient quantity to supply a commercial plant. This requires perhaps 800 000 tons year–1, which is not available from bagasse in many locations. 3. Uniformity. For a high-speed production process, foreign matter present in municipal waste is unacceptable. 4. Cleanliness. High levels of silica can abrade equipment, as stated above. A high degree of microbial contamination is unacceptable. High levels of toxic or inhibitory materials are not acceptable. 5. High potential ethanol yield. The main constituents, cellulose and hemicellulose, must be present in high enough levels to produce ethanol. This is a disadvantage of forestry waste, which is high in bark that is mostly lignin and phenolic acids. 6. High efficiency of conversion. The efficiency of conversion to glucose (following Iogen’s pretreatment process) is proportional to the arabino-xylan content

Table 9.2 Potential feedstocks. Material

Subclass

Wood

Native forest Tree farms

Agricultural residues Energy crops Waste cellulose

Comments

Difficult to process, especially softwood Too expensive due to demands of other markets Forest waste (bark) Cellulose/hemicellulose content is too low Mill waste (sawdust) Too expensive due to pulp and paper market Straws (wheat, barley, oat, rice) Leading candidate feedstocks Bagasse (cane) Localized feedstock of interest Corn stover Leading candidate feedstock Grass Possible second generation feedstock Municipal waste Not uniform enough to process Waste paper Too expensive due to paper demand

9.3 Feedstock Selection

of the feedstock [1]. The arabino-xylan content is roughly the sum of the arabinan and xylan content listed in Table 9.1. Feedstocks with a low arabino-xylan content, such as softwood, demand unacceptably high levels of enzyme for conversion to cellulose. Matching the feedstock list with the desired properties results in the feedstocks used by Iogen, which are agricultural residues such as straws and stover and energy crops such as grasses. The energy crops, such as grass, are not currently harvested in a large scale, as this will require large scale demonstration of the technology before people commit to these as crops. The grasses are therefore second-generation feedstocks. 9.3.3 Ethanol from Starch or Sucrose

Starch-based or sucrose-based processes are already widely used to make ethanol. The leading starch-based material is corn, which is widely used to make ethanol in the US. Starch is converted to glucose by grinding corn kernels (in a dry milling process) or by steeping kernels in dilute sulfurous acid (in a wet milling process), then using starch-degrading enzymes known as amylases. The glucose is then fermented to ethanol. Sucrose-based feedstocks include sugar cane (Brazil) and sugar beets (Europe). These feedstocks are pressed and washed with water to extract the sucrose, which is then fermented to ethanol by yeast. Other feedstocks used to make small amounts of fuel ethanol in some regions include potatoes and Jerusalem artichokes. Many cellulosic materials, including straw and grass, contain up to 10% starch. Wheat straw fed to Iogen’s plant is 3–4% starch. This is converted to glucose during pretreatment and carried through to sugar fermentation, where it is converted to ethanol. 9.3.4 Advantages of Making Ethanol from Cellulosic Biomass

The conversion of cellulosic biomass to ethanol is more difficult than starch or sucrose, and this has limited commercialization of the technology. However, cellulosic biomass is available in much greater quantity and offers the potential for much greater ethanol production than the other feedstocks. In addition, ethanol production from starch and sucrose faces competition for the feedstock from the food and cattle feed industries, which exerts pressure on the price of the ethanol. Most cellulosic biomass is free of competition from other uses. Cellulosic biomass can be grown in a wider variety of climates and soils than starch and sucrose and therefore represent a potential new agricultural opportunity in many areas. The most important advantage of making ethanol from cellulosic biomass is that the production and use of the ethanol does not add to the emission of greenhouse gases. Producing ethanol from corn, sugar cane, or sugar beets requires

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large amounts of energy-intensive fertilizers. The use of these feedstocks results in significantly less net energy generation from the lignin byproduct than that from using cellulosic biomass. These two areas (fertilizer and process energy) require significant input of fossil fuels in ethanol plants using corn, sugar cane, or sugar beets. By contrast, ethanol made from cellulosic biomass is not a net user of fossil fuels. The neutral fossil fuel usage is why ethanol from cellulose is expected to be neutral relative to the production of greenhouse gases [2]. An additional benefit of biomass conversion arises from the fact that corn, sugar cane, and sugar beets all contain 5–15% cellulose and hemicellulose. Cellulose conversion technology represents an opportunity to improve the ethanol yields and decrease the wastes from these processes.

9.4 Pretreatment 9.4.1 Process

Pretreatment is the process by which surface area of the feedstock is opened up for the subsequent enzymatic attack. In the absence of pretreatment, the requirement for cellulase enzymes is too high to be practical. The basis for Iogen’s pretreatment is steam explosion [3]. In the Iogen demonstration plant, wheat straw in bales is chopped and milled, then conveyed to the pretreatment reactor. High pressure steam and sulfuric acid are added to the feedstock to reach 180–260 8C with 0.5–2% sulfuric acid. The material is maintained at this condition for 0.5–5 min. The pressure is then released rapidly. As a result of pretreatment, the fibrous structure of the feedstock is destroyed. The pretreated material has a muddy texture and a slightly sweet smell, with a dark brown color. Among different feedstocks, the cooking time varies while most of the other conditions are maintained relatively constant. The appearance of pretreated material is similar among different feedstocks. 9.4.2 Chemical Reactions

Of the major components, the first to react is the hemicellulose. The xylan portion is depolymerized and solubilized, and then hydrolyzed to xylose by the reaction: C5 H8 O4 n H2 O ! C5 H8 O4 n

1

C5 H10 O5

1

The presence of exogenous sulfuric acid is particularly important in the formation of monomeric xylose. In the absence of exogenous acid, xylose oligo-

9.4 Pretreatment

mers are formed. Further, the added acid improves the uniformity of the process, because natural acid levels vary considerably among feedstocks or batches of a given feedstock. If the pretreatment reaction proceeds further, the xylose is dewatered to produce furfural (Eq. 2), which is undesirable. C5 H10 O5 ! C5 H4 O2 3H2 O

2

Arabinose undergoes analogous reactions, but more slowly than xylose. Acetic acid is released from the hydrolysis of acetyl groups attached to the xylan. Removing hemicellulose from the feedstock accomplishes two objectives. First, it makes the simple sugars that can potentially be fermented to ethanol. Second and more importantly, it opens up surface area on the feedstock, thereby allowing the cellulase enzyme to digest the cellulose more efficiently. A small amount of cellulose reacts to form glucose, which is degraded to hydroxymethylfurfural, by Eqs (3) and (4): C6 H10 O5 n H2 O ! C6 H10 O5 n C6 H12 O6 ! C6 H6 O3 3H2 O

1

C6 H12 O6

3 4

Only a small amount of cellulose hydrolysis (< 20%) is desired in the pretreatment. More than this results in a decrease in glucose yield and accessibility of the enzyme to the cellulose. The older acid hydrolysis process carried out a much harsher acid hydrolysis and will be described below. The lignin undergoes a depolymerization during pretreatment, but remains insoluble in water or acid. Protein is destroyed and starch is hydrolyzed to glucose in the pretreatment. One can draw an analogy to cooking a turkey to describe the trade-off between time and temperature in the pretreatment: the longer the time, the lower the temperature. Acid also acts to decrease the temperature or time required for pretreatment. The choice of reactor for this pretreatment is only important insofar as the desired chemistry must be delivered to the system. Numerous guns, vessels, and tubes have been proposed and built to carry this out. 9.4.3 Other Pretreatment Processes

Katzen et al. [4] published a detailed review of pretreatment that will only be summarized here. Pretreatment processes may be divided into (1) those that produce a stream directly for fermentation to ethanol and (2) those that are followed by enzymatic hydrolysis. The former are necessarily harsher and have a longer history. Direct sugar production in pretreatment has been carried out using concentrated chemicals and dilute acid.

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Concentrated chemical pretreatment represents the cellulose conversion technology with the longest history, dating back to 1890 or earlier in Germany. The concentrated chemicals include acids, bases, and salts. The principle behind the use of concentrated chemicals is to disrupt the crystalline cellulose structure, thereby dissolving and depolymerizing the cellulose. Among the chemicals used are 72% sulfuric acid, 40% hydrochloric acid, 40% sodium hydroxide, 65% zinc chloride, 40% calcium chloride. These methods have very high yields and low operating temperatures. Unfortunately, the economics of the process dictate that recovery and reuse of the pretreatment chemicals is critically important. The inability to obtain the necessary recoveries of order 99.9% has hindered the commercialization of these processes. A second problem is the exotic materials required to handle these streams. Dilute acid hydrolysis represents the cellulose conversion technology with the most commercial experience. In dilute acid hydrolysis, the feedstock is treated at perhaps 180–200 8C for 1–4 h with 1 to 4% sulfuric acid. A glucose yield of 50% of the cellulose or higher is obtained. This process was used for ethanol production by Germany during World War II, Russia in the late 1940s, and pilot plants in the 1950s in Switzerland and Springfield, Oregon. All of these plants have been plagued by corrosion, low yields, high investments, and overall poor returns. Several plants of up to 10 t day–1 built in Russia in the 1950s still operate with this process. Pretreatment followed by enzymatic hydrolysis is newer than direct pretreatment, as cellulase enzymes were only identified and worked with after World War II. The milder pretreatments carried out in preparation for enzymatic hydrolysis include mechanical action, solvent-based pretreatments, alkali treatments, and acid prehydrolysis. Mechanical action was first tried at the U.S. Army laboratory in Natick, Massachusetts in the 1960s. The principle behind mechanical action is to increase the surface area of the feedstock particles. However, beyond producing small particles for uniform distribution of acid, there is no real advantage to further milling of the feedstock. In solvent-based pretreatments, organic solvents such as ethanol and methanol are used to dissolve a portion of the lignin, thereby freeing up the cellulose for enzymatic attack. However, recovery of the solvent is difficult, and partial delignification is not of significant benefit in the hydrolysis. In alkali pretreatments, such as with sodium hydroxide or ammonia, the crystalline cellulose is converted to a different form, cellulose II or III respectively. These forms of cellulose can be more easily hydrolyzed than native cellulose. However, destruction of the hemicellulose is reported in these systems, and they are not yet used commercially. Acid prehydrolysis is preferred by Iogen because it has fewer of these problems than the other methods. The levels of acid are low enough that recovery is not needed and corrosion is not a problem. The process provides a selective hydrolysis of hemicellulose and produces a cellulosic substrate with a high surface area suitable for enzymatic hydrolysis.

9.5 Cellulase Enzyme Production

9.5 Cellulase Enzyme Production 9.5.1 Production of Cellulase Enzymes

Cellulase enzymes convert cellulose to glucose, which can then be fermented to ethanol. Cellulase enzymes are made by a wide variety of microbes, but those best suited to cellulose hydrolysis are made by the wood-rotting fungus Trichoderma. This fungus was isolated during World War II in rotted U.S. Army cotton tents in the South Pacific. Researchers led by Elwyn Reese and Mary Mandels at the U.S. Army laboratory in Natick, Massachusetts determined that the microbe responsible for the destruction of the cotton was secreting a mixture of enzymes that hydrolyzed the cotton. Reese and Mandels determined cultivation conditions for production of cellulase in liquid culture. Selection of Trichoderma strains with higher productivities of cellulase was successfully carried out by Montenecourt at Lehigh University in the 1970s. Despite research and development of cellulase production from other microbes, Trichoderma remains the organism of choice to produce cellulase for ethanol production. Cellulase is made at Iogen’s commercial enzyme plant in Ottawa in a submerged liquid culture, as is used by Genencor International, Novozym Biotech, Rohm AB, and other commercial cellulase manufacturers. The fermentation vessels are similar to those used for producing antibiotics. The fermenters are 50 000 gallons and are maintained free of contaminating microbes. The liquid broth contains carbon source, salts, complex nutrients such as corn steep liquor, and other nutrients in water. The most important nutrient is the carbon source. Glucose promotes growth of the organism but not cellulase production. The carbon source must include an inducing sugar to promote cellulase production. Well known inducers of cellulase include the sugars cellobiose, lactose, sophorose, and other low molecular weight oligomers of glucose. The nutrient broth is sterilized before the start of the fermentation by heating with steam. The fermenter is inoculated with the enzyme production strain once the liquid broth has cooled down. The operating conditions are 30 8C, pH 4 to 5, and these are maintained by the addition of cooling water in external coils and by alkali, respectively. Trichoderma is highly aerobic and a constant stream of air or oxygen is used to maintain aerobic conditions. A cellulase enzyme production run lasts about one week. At the end of the run, the broth is filtered across a cloth to remove cells. The spent cell mass is destroyed and disposed of by landfilling. The resulting enzyme broth is a clear, light brown liquid, similar in appearance to weak tea.

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9.5.2 Enzyme Production on the Ethanol Plant Site

Iogen has the unique advantage of operating an ethanol plant on a cellulase production site. In this case, the crude fermentation broth containing cellulase is simply added to the cellulose hydrolysis tanks. If the cellulase is to be stored for long periods before use, it must be stabilized against (1) microbial contamination, which uses preservatives such as sodium benzoate, and (2) protein denaturation, which uses compounds such as glycerol. The production and use of cellulase on the ethanol plant site therefore has the advantage that the cost of preservatives and stabilizers, as well as transportation of the enzyme, is avoided. This is a big cost advantage for on-site enzyme production. 9.5.3 Commercial Status of Cellulase

There is an ongoing business in cellulase enzymes besides that used for cellulose hydrolysis. Cellulase sales are roughly $ 100 million annually to the textiles, detergent, animal feed, beverage, and pulp and paper industries [5]. These commercial cellulase enzymes are made by several microbes, including Humicola, Aspergillus, and Penicillium fungi, and Bacillus bacteria in addition to Trichoderma. Ethanol production requires aggressive action of cellulase to destroy cellulose, for which Trichoderma cellulase is superior. The other industries often require milder action and/or specific conditions better suited to other cellulases. For example, the textile industry uses cellulase to soften denim blue jeans. Humicola cellulases can be less aggressive in this application than Trichoderma cellulases, though Trichoderma cellulases can be modified to improve their performance. Detergents require alkaline conditions that are not easily accessible to Trichoderma enzymes.

9.6 Cellulose Hydrolysis 9.6.1 Process Description

In cellulose hydrolysis, cellulase enzymes convert the cellulose to glucose. The pretreated feedstock is conveyed to the hydrolysis tanks in a slurry that is 5– 15% total solids (as high as can be handled). The slurry is adjusted to pH 5 with alkali and maintained at 50 8C. A single hydrolysis tank has a volume of 200,000 gallons. Crude cellulase broth is added as a liquid at a dosage of 100 liters per tonne of cellulose. The contents of the tank are agitated to move the material and keep it dispersed, but not nearly as agitated as a fermentation vessel.

9.6 Cellulose Hydrolysis

The hydrolysis proceeds for 5–7 days. As it proceeds, the viscosity of the slurry drops, and the remaining insoluble particles, which are lignin in increasing proportion, diminish in size. At the end of the hydrolysis, 90% to 98% of the cellulose is converted to glucose. The remainder is insoluble and contained within the unhydrolyzed particles, which are mostly lignin. 9.6.2 Kinetics of Cellulose Hydrolysis

Trichoderma cellulase is a mixture of three types of enzymes: cellobiohydrolase(CBH), endoglucanase (EG), and beta-glucosidase (BG). CBH enzymes act sequentially along the cellulose. Trichoderma cellulase includes two CBH enzymes, CBHI and CBHII, which together account for 80% of the total cellulase protein. EG enzymes act to cut random locations on the fiber. Trichoderma makes at least 4 different EG enzymes: EGI, EGII, EGIII, and EGV. The EG enzymes account for about 20% of the cellulase protein. The third type of enzyme, betaglucosidase, hydrolyzes the glucose dimer cellobiose to glucose. The enzymatic hydrolysis of cellulose proceeds as two consecutive reactions: C5 H10 O5 n H2 O ! C5 H10 O5 n C12 H22 O11 H2 O ! 2C6 H12 O6

2

C12 H22 O11

5 6

Eq. (5) depicts the hydrolysis of cellulose to its soluble dimer, cellobiose. This reaction is catalyzed by the CBH and EG enzymes. The CBH and EG enzymes work synergistically to hydrolyze cellulose. Reaction (6) is the hydrolysis of cellobiose to glucose. This reaction is catalyzed by the soluble enzyme beta-glucosidase and proceeds according to standard Michaelis-Menten kinetics. BG accounts for less than 1% of the total cellulase protein. Hydrolysis of the cellobiose is important because glucose can be readily fermented to ethanol while cellobiose is not. In addition, cellobiose is a potent inhibitor of CBH and EG, so the accumulation of even 5 g L–1 cellobiose slows down the hydrolysis significantly. The properties of the Trichoderma cellulase enzymes are summarized in Table 9.3. There are several inherent difficulties in cellulose hydrolysis that have been the focus of much research. The first is the inherent shortage of BG, both because of its low concentration and because it is inhibited by its product glucose. With a shortage of BG, cellobiose accumulates, thereby inhibiting the action of CBH and EG in hydrolyzing cellulose. Three approaches have been proposed to overcoming the shortage of BG. The first is to produce BG in a separate fermentation by Aspergillus spp. The disadvantage of this is the added process cost of a second fermentation. The second and most widely discussed approach is to carry out a simultaneous saccharification and fermentation (SSF) process. In SSF, the enzymatic

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9 Iogen’s Demonstration Process for Producing Ethanol from Cellulosic Biomass Table 9.3 Trichoderma cellulase enzymes. Enzyme

Mol. wt.

Isoelectric point

Family

Concn. (%)

Ref.

CBHI CBHII EGI EGII EGIII EGV

63 000 58 000 53 000 50 000 25 000 23 000

4.3 6.0 4.6 5.3 7.4 3.7

7 6 7 5 12 45

50–60 15–18 12–15 9–11 0–3 0–3

6 7 8 9 10 11

hydrolysis and glucose fermentation are run simultaneously, with the notion that the yeast consumes the glucose to prevent inhibition of BG, which can then hydrolyze cellobiose and lift the inhibition of CBH/EG. In Iogen’s research, SSF systems have achieved poor results, because the enzyme’s optimum temperature is 50 8C and the yeast runs best at 28 8C, so a compromise temperature of 37 8C is used. In addition to the loss of rate, microbial contamination is observed at 37 8C. We have put this approach aside. The third option is to develop Trichoderma strains with high beta-glucosidase production included in the CBH/EG. This has been carried out at Iogen [12] and has largely overcome the shortage of BG. Other important difficulties in the hydrolysis are the decrease in rate as hydrolysis proceeds and the diminishing returns with enzyme dosage, i.e. doubling the amount of enzyme does not double the extent of conversion achieved. These issues, which are probably linked, are not well understood and differ substantially from Michaelis-Menten kinetics. These effects can, however, be characterized empirically. The enzyme components initially must adsorb to the surface of the insoluble substrate cellulose. An equilibrium corresponding roughly to a Langmuir adsorption isotherm is reached within a few minutes. The adsorbed enzyme then acts on the cellulose at a rapid initial rate. The rate declines significantly after the first few minutes of hydrolysis, and after 24 h is less than 2% of the initial rate. The hydrolysis continues over several days at ever decreasing rates. Depending on the enzyme dosage used and the duration of the hydrolysis, the final cellulose conversion is 90% to 98%. The reason the rate slows down is not fully known. Speculation in the literature has centered on: end-product inhibition; an increasingly difficulty in hydrolyzing the substrate (substrate recalcitrance); and denaturation of the cellulase protein over time. However, straightforward experiments demonstrate that none of these factors account for more than a small fraction of the drop in rate. Further research continues in this area.

9.7 Lignin Processing

9.6.3 Improvements in Enzymatic Hydrolysis

Despite many years of research, cellulose hydrolysis remains the least efficient part of the process. To illustrate the problem, hydrolysis of pretreated cellulose requires 100-fold more enzyme than hydrolysis of starch. The enzyme manufacturing cost is still sufficiently high that the trade-off between enzyme dosage and hydrolysis time favors longer times and lower dosages. One approach to potentially decrease the cost of enzymatic hydrolysis is to recycle enzyme and/or substrate. These ideas have not been fully explored. The cellulase enzyme adsorbs to the substrate, so recycle of unconsumed cellulose would be one way to reuse the enzyme. Reuse of the enzyme in solution is also a possibility. The current configuration of Iogen’s plant does not carry out these recycles, but upgrades are under way to allow the demonstration plant to evaluate these options. Another approach to reducing the cost of hydrolysis is to use fed-batch of substrate or of enzyme. Again, these are ideas with a sound basis that have not been explored. Fed batch systems are especially interesting because they might allow high cellulose concentrations to be maintained at all times. The development of superior cellulase enzymes has been explored extensively at several research labs, with as yet no cellulases found that are superior to Trichoderma cellulase. However, the new tools of molecular biology, such as protein engineering, can be brought to bear on this problem and might lead to success. Large development efforts are under way at Iogen and at Genencor International, Novozym Biotech, and Diversa to address this area. Another area that has been widely explored is novel hydrolysis reactors, such as trickling bed, high shear, etc. Some improvements in efficiency have been reported, but not easily generalized across feedstocks and enzymes. A better understanding of the nature of the enzyme’s action will be helpful in evaluating these reactors. Taking a wider point of view, better hydrolysis would be a direct benefit of better pretreatment. Pretreatment has been widely studied, but there are always new ideas on the horizon.

9.7 Lignin Processing 9.7.1 Process Description

The hydrolysis slurry contains glucose, xylose, arabinose, and other compounds dissolved in the aqueous phase, and insoluble lignin and unconsumed cellulose. The insoluble particles are separated by a plate and frame filter, with the cake washed with water to obtain a high sugar recovery. The sugar stream is pumped to the fermentation tanks. The lignin cake is disposed of. In a full-scale ethanol

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plant, the lignin would be burned on site to generate power and steam to run the plant, with excess electricity sold to the power grid. 9.7.2 Alternative Uses for Lignin

Lignin is a lattice of phenolic propane units. Lignin has been the object of much study, and a good review of applications of lignin is provided by Chum et al. [13]. The potential applications of lignin fall into the broad categories of insoluble and chemically modified. Insoluble lignin is limited to high-volume, low value applications such as an ingredient in roads or cement. In these applications, lignin is a filler and competes with corn cobs, gravel, and ground bark. Chemically modified lignin has a much wider variety of potential markets. The major types of chemical modifications are to solubilize the lignin, by reaction with i.e. sulfurous acid, or to crosslink the lignin, by reaction with i.e. phenol. Crosslinked lignin is suitable for resins, glues, and other such materials, where it competes with phenol–formaldehyde resins. Solubilized lignin, i.e. sodium lignosulfonate, is used in surfactants, detergents, and biocides.

9.8 Sugar Fermentation and Ethanol Recovery

The hydrolysis sugars, which consist of glucose, xylose, arabinose, and various organic impurities in aqueous solution, are pumped to the sugar fermenters for ethanol production. The fermenters are large tanks with gentle agitation, sufficient to keep the contents moving. The fermenters are inoculated with Saccharomyces yeast, which readily ferment the glucose to ethanol, and have been genetically modified for metabolism and fermentation of xylose. These yeast strains have been developed at Purdue University [14]. The advantage of these strains over others is that the plant operations (contamination control, cell recycle, and markets for spent cells) involving Saccharomyces yeast are well developed, and the ethanol tolerance of the strains are good. The areas for further improvement include developing the inability to ferment arabinose to ethanol and increasing the yields and rates of xylose fermentation. In addition to Saccharomyces yeast, other microbes are under development to ferment xylose to ethanol, which is not carried out naturally in high efficiency. Pichia yeast have a natural ability for xylose uptake, but require genetic modification to ferment the xylose to ethanol. This strategy is carried out at the University of Wisconsin [15]. In addition, Zymomonas bacteria have been genetically modified for xylose uptake and metabolism. This strategy is carried out by the National Renewable Energy Laboratory in Boulder, Colorado [16]. Two other strategies for pentose fermentation include genetically modified enteric bacteria such as E. coli and Klebsiella, carried out at the University of Florida [17–19], and thermostable Bacillus strains developed at Imperial College.

References

After fermentation, the broth containing ethanol and unfermented sugar is pumped to a distillation column. The ethanol is distilled off the top and dehydrated. The ethanol yield is about 75 gallons per tonne wheat straw. The ethanol is denatured with 1% gasoline. The denatured ethanol is then blended into gasoline in 10% or 85% ethanol mixtures. The still bottoms are disposed of. Tests are under way to determine whether the still bottoms can be recovered for byproducts.

References 1 Foody, B., Tolan, J. S., Bernstein, J., Pre-

2

3

4

5 6

7

8

9

treatment process for conversion of cellulose to fuel ethanol, US Patent 5,916,780 issued June 29, 1999. Singh, L., Scenarios of U.S. Carbon Reductions: Potential Impacts of Energy Technologies by 2010 and Beyond, Interlaboratory Working Group on Energy Efficient and Low-Carbon Technologies (ORNL, LBNL, PNNL, NREL, ANL), 1997. Foody, P., Method for Increasing the accessibility of cellulose in lignocellulosic materials, particularly hardwoods, agricultural residues, and the like, US patent 4,461,648 issued July 24, 1984. Katzen, R., Madsen, P. W., Monceaux, D. A., Bevernitz, K., Use of Cellulosic Feedstocks for Alcohol Production, Chapter 5 of The Alcohol Textbook, edited by T. P. Lyons, D. R. Kelsall, and J. E. Murtagh, 1990. Godfrey, T., Industrial Enzymology, Chapter 1, 1996. Shoemaker et al., Molecular cloning of exo-cellobiohydrolase I derived from Trichoderma reesei strain L27, Bio/technology, 1983, 1, 691–696. Chen et al., Nucleotide sequence and deduced primary structure of cellobiohydrolase II from Trichoderma reesei, Bio/technology, 1987, 5, 274–278. Penttila et al., Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene, Gene, 1986, 45, 253–263. Saloheimo et al., EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme, Gene, 1988, 63, 11–21.

10 Ward, M., Clarkson, K. A., Larenas, E. A.,

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13

14

15

16

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Lorch, J. D., Weiss, G. L., DNA Sequence Encoding Endoglucanase III Cellulase, US patent 5,475,101 issued Dec. 12, 1995. Saloheimo et al., A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast, Molecular Microbiology, 1993, 61, 1090–1097. White, T. C., Hindle, C., Genetic constructs and genetically modified microbes for enhanced production of beta-glucosidase. US patent 6,015,703 issued January 18, 2000. Chum, H. L., Parker, S. K., Feinberg, D. A., Wright, J. D., Rice, P. A., Sinclair, S. A., Glasser, W. G., The Economic Contribution of Lignins to Ethanol Production from Biomass, National Renewable Energy Lab, Golden, Colo., 1985. Ho, N. W. Y., Chen, Z.-D., Stable Recombinant Yeasts for Fermenting Xylose to Ethanol, PCT patent application WO 97/ 42307, 1997. Cho, J., Jeffries, T. J., Pichia stipitis Genes for Alcohol Dehydrogenase with Fermentative and Respirative Functions, Appl. Environ. Microbiol., 1998, 64(4), 1350–1358. Mohagheghi, A., Evans, K., Finkelstein, M., Zhang, M., Cofermentation of Glucose, Xylose, and Arabinose by Mixed Cultures of Two Genetically Engineered Zymomonas mobilis Strains, Applied Biochem. Biotechnol., 1998, 70–72, 285. Ingram, L. O., Conway, T., Clark, D. P., Sewall, G. W., Preston, J. F., Genetic Engineering of Ethanol Production in Escherichia coli, Appl. Environ. Microbiol., 1987, 53(10), 2420–2425. Ohta, K., Alterthum, F., Ingram, L.O., Effects of Environmental Conditions on Xy-

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9 Iogen’s Demonstration Process for Producing Ethanol from Cellulosic Biomass lose Fermentation by Recombinant Escherichia coli, Appl. Environ. Micro., 1990, 56(2), 463–465. 19 Ohta, K., Beall, D. S., Mejia, J. P., Shanmugam, K. T., Ingram, L. O., Genetic Improvement of Escherichia coli for Ethanol

Production: chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase and alcohol dehydrogenase II, Appl. Environ. Micro., 1991, 57(4), 893–900.


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10 Sugar-based Biorefinery – Technology for Integrated Production of Poly(3-hydroxybutyrate), Sugar, and Ethanol Carlos Eduardo Vaz Rossell, Paulo E. Mantelatto, José A. M. Agnelli, and Jefter Nascimento

10.1 Introduction

Poly(3-hydroxybutyric acid) polymer (PHB) and related copolymers such as poly(3-hydroxybutyric-co-3-hydroxyvaleric) are natural polyesters synthesized by a wide range of organisms, particularly some bacterial strains. They have very interesting properties, for example they are totally and rapidly biodegraded to carbon dioxide and water by many different microorganisms and are biocompatible. These polyesters can be compounded to thermoplastic resins whose physicochemical and mechanical properties are quite similar to petrochemical-based polymers such as polyethylene and polypropylene. PHB can be produced in an environmentally safe way integrated to a sugar mill. This context, with its large quantities of readily available and comparatively low-cost sugar, and accessible thermal, mechanical, and electrical energy obtained from renewable agricultural sources, could be the optimum place to introduce a large-scale facility for its production.

10.2 Sugar Cane Agro Industry in Brazil – Historical Outline 10.2.1 Sugar and Ethanol Production

During the last quarter of the 20th century, Brazil’s sugar cane agroindustry underwent major changes. This initially traditional sector, previously devoted specifically to the production of sugar and occasionally to by-products, recovering or producing very few new products from available sugar or molasses, shifted its scope to diversify its production lines. In 1975, after the international oil crisis and the rise of oil prices, the Brazilian government launched the National Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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Fuel Ethanol Program, with the objective of replacing oil imports with sugar cane-based ethanol as an alternative fuel. Many mills began to produce aqueous ethanol, for direct use in converted car engines, or anhydrous ethanol to add to gasoline as a fuel enhancer. The scale of production increased substantially, agricultural and industrial productivity grew, and production costs dropped. This new situation, which led to a favorable change in the country’s energy balance, caused the production of an excess of bagasse, which has been used as primary fuel by the mills or has been sold to third parties. Today, Brazil is the largest worldwide producer of sugar and sugar cane-based ethanol and, on the basis of cultivated area and income, sugar cane is one of the most important agricultural activities. This statement is confirmed by figures from the 2003/ 2004 season – sugar cane production was 338 316 619. metric tons. Ethanol production was 14 107 873 m3, 8 577 410 m3 of which was anhydrous fuel grade and the remaining 5 530 468 m3 aqueous fuel grade. Sugar production rose to 23 404 703 tons, of which 12 914 468 tons were exported, accounting for revenues of US $ 2 140 002 217 [1]. 10.2.2 The Sugar Cane Agroindustry and the Green Cycle

The modern sugar agroindustry, which tends to operate as a “green cycle”, can achieve a unique condition as a source of renewable raw materials if a continuous effort is made to keep its production practices environmentally friendly. The basis of this sector is sugar cane (Saccharum officinarum), a subtropical gramineous plant cultivated on a large scale in Brazil’s south central region with productivity averaging 85 tonnes per hectare per year and for which production costs are low. Sugar cane captures solar energy and converts atmospheric carbon dioxide into biomass. Macedo [2] produced an inventory of carbon dioxide released during sugar cane processing into final products and, after fuel ethanol combustion, concluded that the net balance was positive and that atmospheric carbon dioxide is effectively removed by cane crops. Macedo also demonstrated that the balance between energy available from final products such as fuel ethanol, bagasse and electrical power generated versus energy consumption during cane culture and industrial processing is positive [3, 4]. The sugar mills operating in Brazil today are very large units functioning as autonomous industrial complexes. Their main products are sugar and fuel ethanol, and bagasse – the fibrous fraction of sugar cane – is the primary fuel. Bagasse burned in high-pressure boilers provides mechanical power for driving mill tandems, electrical power to meet the requirements of sugar and ethanol production, and the heat demand involved in processing cane into final products. Mills whose energy production is well planned and optimized exceed their energy requirements, generating excess electrical power and bagasse. Modern mills have large facilities for processing and cooling water, and systems for recycling liquid and effluent to irrigate and/or fertilize crops. Vinasse from ethanol distillation, condensed water from sugar processing, filter sludge resulting from

10.2 Sugar Cane Agro Industry in Brazil – Historical Outline

Fig. 10.1 Sugar cane processing to sucrose, ethanol, by-products, and new products.

juice treatment, and ashes and carbon residue captured in the treatment of boiler exhaust gases are disposed of in this way. The idea of integrating the production of new products with that of sugar is not new. Sugar cane is a renewable agricultural resource and its processing yields sugar, cane juice, syrup, and molasses, a very rich substrate that is convertible by fermentation into higher value chemicals such as organic acids, amino acids, and biopolymers, etc. Sugar, a highly reactive molecule, can be chemically converted into valuable products, as indicated by Kahn [5]. Ethanol, the other main product of Brazilian sugar mills, is the starting molecule of ethanol chemistry, whereby important basic chemicals such as aldehyde, acetic acid, and acetates are produced. Paturau [6] describes some successful examples of integration at sugar mills whose processing involves products such as citric acid, fodder yeast and yeast products, acetic acid, vinegar and cellulose, paper, and bagasse-based fiberboard. The block diagram in Fig. 10.1 illustrates the context of green cycle production from renewable agricultural raw materials.

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10.3 Biodegradable Plastics from Sugar Cane 10.3.1 Poly(3-Hydroxybutyric Acid) 10.3.1.1 Biodegradable Plastics and the Environment A wide variety of synthetic plastics have been applied in many products, replacing metal, wood, natural rubber, and other materials, and developing into a base industry in industrialized countries since the early 20th century. Today, the production of plastics amounts to 150 million tonnes year–1 and the trend is still upward [7, 8]. The main polymeric materials are oil-derived, and the world’s growing population has led to increasing oil consumption, which may lead to its depletion as a natural resource [7]. Another problem is the environmental pollution resulting from the disposal of polymeric materials, which may take hundreds of years to decompose. This, with the world’s growing environmental awareness, renders the characteristic of nondeterioration desired for a material’s use an inconvenience during its disposal. Although attempts have been made to solve this environmental problem by recycling techniques, despite its wide acceptability, recycling alone has proved insufficient to solve environmental problems, because it is impossible to recover all discarded plastic by this process. The recycling of loaded materials and multicomponents is usually a more complex process. One relevant aspect is that such processes usually consume substantial amounts of energy [7, 8]. Another means of treating solid residues (discarded plastics) is by incineration; this also generates environmental problems, for example air and water pollution, because it releases aggressive chemical agents and causes global warming by release of carbon dioxide. These effects are not restricted to the places where such techniques are employed but are spread around the globe [7, 8]. In this context, the search for a material that is durable while in use and degradable after disposal has led to the emergence of biodegradable plastics – materials that decompose into low molar mass compounds as a result of the action of microorganisms (bacteria, fungi). Biodegradable materials were initially used in medical applications such as sutures, prostheses, controlled drug-release systems, vascular grafts, etc., owing to their biocompatibility, their ability to dissolve and be absorbed by the body, and because their mechanical properties were appropriate for such applications. More recently, biodegradable plastics have been applied elsewhere, including packaging and agriculture (plant containers, controlled release of chemical substances, etc.) [10, 11]. According to U. J. Hänggi [12], because of their higher costs, biodegradable plastics should not be used as a substitute for traditional materials but in applications where traditional plastics are inappropriate. It is worth noting that biodegradable plastics cannot yet compete with traditional plastics because of their higher cost, and that some types are limited to products which are stored for less than one year [12].

10.3 Biodegradable Plastics from Sugar Cane

10.3.1.2 General Aspects of Biodegradability It is important to clearly define the concepts of degradation and biodegradation. According to the ASTM D-833 there are [13]: · degradable plastic which, under specific environmental conditions, undergoes significant changes in chemical structure, resulting in loss of some of its properties, and · biodegradable plastic, degradation of which also results from the action of naturally occurring microorganisms such as bacteria, fungi, and algae.

Biodegradable plastic must initially be broken down into fragments of low-molecular-mass by chemical reactions, after which they can be absorbed by microorganisms, because they are inert to microorganism attack in their original form [8]. These reactions can be induced by oxidative enzymes, which cause superficial erosion, or by abiotic mechanisms (without the presence of living organisms), through hydrolytic or oxidative reactions. In the former process a bacterial or fungal colony on the surface of the material releases an extracellular degrading enzyme which breaks down the polymer into smaller units (monomers or oligomers) which are then absorbed by the microorganisms’ cell walls and metabolized as a source of nutrients (carbon). It has been proposed that this mechanism first hydrolyzes the chains of the amorphous phase of PHB and then proceeds to attack the chains in the crystalline state. The enzymatic degradation rate decreases as the crystallinity increases [14, 15]. In the latter situation, hydrolytic and oxidative mechanisms occur in the absence of living microorganisms and are restricted to the amorphous phase and the borders of the crystals, because the crystalline regions are almost impermeable to water and oxygen. The action of the microorganisms begins after the polymeric chains have been broken down. Although the phenomenon of biodegradation seems simple, it is actually quite complex, for it is affected by several factors which are often interrelated. The rate of biodegradation of these materials may vary over time and depends on the material and on environmental factors, for example the type of repeating unit (nature of the functional group and its complexity), morphology (crystallinity, size of the spherulites), hydrophilicity, surface area, presence of additives, and environment (humidity, temperature, pH, etc.) [9, 15]. A complete understanding of the degradation process will enable us to optimize the entire life cycle of the materials obtained [16]. The products resulting from the biodegradation process are carbon dioxide and water in aerobic environments and carbon dioxide and methane in anaerobic environments [17].

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10.3.2 Poly(3-Hydroxybutyric Acid) Polymer 10.3.2.1 General Characteristics of Poly(3-hydroxybutyric acid) and its Copolymer Poly(3-hydroxybutyric Acid-co-3-hydroxyvaleric Acid) PHB is an environmentally degradable material belonging to the polyhydroxyalkanoates (PHA) family, alkanoic acid polyesters which were first described in 1926 by Lemoigne [18]. Figure 10.2 shows the general chemical structure of the PHA and the biodegradable PHB plastic. This is a material with a unique characteristic among thermoplastics because it presents a complete cycle starting from sugar cane and bacterial fermentative synthesis. PHB is produced, molded, and, after a period of use, discarded and transformed into compost, thus completing the natural cycle. In the absence of microorganisms biodegradability the hydrolysis of PHB in aqueous environments is slow, because of its hydrophobicity. Thus, in principle, the lifetime of a stored PHB product is unlimited; after its disposal, however, PHB becomes clearly biodegradable in domestic effluent-treatment systems [19]. PHB is a biocompatible, biodegradable, thermoplastic, hydrophobic, and stereospecific material. It has a high molecular mass, high crystallinity (55 to 75%), good chemical resistance, and its barrier properties enable practical packaging applications [20, 21]. Table 10.1 lists the physical and thermal characteristics of this polymer. This material has unit cells with an orthorhombic structure, with the lattice parameters a = 5.76 Å, b = 13.20 Å, c = 5.96 Å [22]. Many of the physical and mechanical characteristics of PHB are similar to those of polypropylene (PP), among them [14]:

Fig. 10.2 Chemical structure of the repetition unit (mer) present in macromolecules of (a) PHA and (b) biodegradable PHB plastic.

Table 10.1 Physicochemical properties of PHB [14, 18, 21, 22]. Properties

Units

Reference values

Mean weighted molar mass Crystalline melting temperature (Tm) Vitreous transition temperature (Tg) Theoretical density of 100% crystalline PHB Density of amorphous PHB

Dalton (Da) 8C 8C kg m–3 kg m–3

200 000 to 600 000 165 to 175 0 to 5 1260 1180

10.3 Biodegradable Plastics from Sugar Cane

· Young’s modulus 2.5–3.5 GPa (comparable with PP or PET) · tensile strength 20–40 MPa · Elongation at rupture 3–5%. The processibility, high crystallinity, and high Tm value of PHB can be altered by bacterial fermentation or use of polymer blends. The various copolyesters (PHBV) include random copolyester (R)-3-hydroxybutyrate with (R)-3-hydroxyvalerate, P(3HB-co-3HV), and random copolyester (R)-3-hydroxybutyrate with (R)-4-hydroxybutyrate, P(3HB-co-4HB). The copolyesters are characterized by their lower crystalline melting point, hardness, tensile strength, and crystallinity (hence, greater ductility and elasticity) than pure PHB. Their physical and thermal properties can be adjusted by varying the HV content of P(3HB-co-3HV) and the 4-HB content of P(3HB-co-4HB). Simply varying the copolymer content [15, 21] enables production of a material with elastomeric characteristics from one which is rigid and highly crystalline. For P(3HB-co-3HV) the crystallinity usually decreases with increasing HV content. The Tm of PHB of approximately 175 8C drops to 71 8C for P(3HB-co-3HV) with a 3HV molar mass of 40%. The Tg of P(3HV) varies from –10 to –12 8C and the Tm varies from 107 to 112 8C [14]. Although copolymers such as PHBV can be used to prevent thermal degradation (lower melting point), the comonomer must be inserted homogeneously into the polymeric chains. For example, for a comonomer content of 16 mol% HV inserted homogeneously into the polymeric chains the corresponding Tm is approximately 140 8C and a single melting peak is obtained for the copolymer in differential scanning calorimetry (DSC) thermal curves. If the comonomer is not inserted uniformly into the PHB chains, however, multiple melting peaks will appear, and the highest-melting peak among these multiple peaks will be located at a temperature exceeding that at which the material is thermally stable (below 160 8C). The HV content is therefore important for the reduction of the system’s Tm; equally important is reduction of the heterogeneity of the HV content in the copolymer [23]. The physical properties of copolymers can be altered by varying the comonomer content, but the use of polymeric blends and incorporation of plasticizers are preferable alternatives, because they enable greater versatility in the modification of properties. The synthesis of copolymers is also a more complex process.

10.3.2.2 Processing of Poly(Hydroxybutyrates) Poly(hydroxybutyrate) can be processed as a conventional thermoplastic in most industrial transformation processes, including extrusion, injection, and thermopressing. By extrusion, PHB can be transformed into rigid shapes (for example pipes) and films for packaging. PHB can also be modified by extrusion by incorporation of additives (stabilizers, plasticizers, and pigments), immiscible additives (e.g. wood and starch powder), or by mixing with other plastics. Because poly(hydroxybutyrate) undergoes thermal degradation at temperatures above 190 8C, the extruder’s temperature profile must be as low as possible (approx.

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150 8C), and the screw speed must be appropriate. Because of the need for strict control, the processing window of PHB is narrow in comparison with that of conventional plastics. It should be pointed out that virgin PHB is supplied in the powder form, requiring an appropriate screw profile to enable it to be processed efficaciously. Poly(hydroxybutyrate) is highly versatile when used in the injection process and is easily molded into pieces of varying shapes and sizes, which may range from a few grams to several kilograms. The main uses of this material include injectable packaging for cosmetics and food, agrotoxic packaging and tubettes, and medical and veterinary implants. As in extrusion, injection of poly(hydroxybutyrate) should be conducted with a nonaggressive temperature profile and a low injection pressure to prevent the appearance of burrs. PHB can also be thermopressed into sheets or other flat shapes. Because characteristics that affect the speed of biodegradation (for example crystallinity, physical shape, molar mass distribution, and compaction of the material) can be altered during processing, PHB samples that have undergone different types of processing may not biodegrade at the same rate. When using most types of conventional equipment, without inclusion of special modifications such as screws or matrixes with specific designs, several operating procedures must be observed if products with satisfactory end quality are to be obtained. Extrusion or injection processes can use typical polyolefin processing screws (L/D 20 : 1) designed to minimize the exposure time of the melted biopolymer. After use the equipment can be purged using low-density polyethylene resin as vehicle. To prevent marked thermal degradation poly(3-hydroxybutyric acid) should not be exposed to temperatures exceeding 160 to 170 8C for more than 5 min. The polymer’s “window of processibility” is relatively narrow compared with that of olefinic polymers, so precise control of the processing temperature is required to prevent temperature peaks in the heating resistances, which would lead to rapid degradation of the material. The “window of processibility” can be monitored by observation of the surface characteristics of the molded component, for example rugosity and shine, which are highly indicative for this polymer. When using multistage injection equipment, it is advisable to fill the mold cavity rapidly, using high injection velocities combined with high pressures, and ensuring that the injection pressure applied does not lead to the appearance of defects such as burrs and rugosity, because the fluidity index of poly(3-hydroxybutyric acid) drops rapidly when the mass is subjected to high shearing rates. Poly(3-hydroxybutyric acid) does not usually require long cooling times in the mold in comparison with polyolefins. To minimize PHB-molding “cycle times” it is advisable to use injection molds heated to approximately 50 8C rather than injection molds with cold water systems at approximately 10 8C; this enables faster crystallization, with direct consequences on the thermomechanical properties of the molded component. This mold temperature is appropriate, because it is approximately 50 8C higher than the polymer’s vitreous transition temperature. The use of this procedure improves the characteristics of ejectability of the molded component, resulting in a gain in “cycle times”.

10.4 Poly(3-Hydroxybutyric Acid) Production Process


Poly(3-hydroxybutyric acid) is produced in an aerobic fermentation process in which the sugar carbon source is converted into biopolymer by means of the microorganism Ralstonia eutropha. Biopolymer stored in cells as a carbon reserve is recovered by an extraction and purification process described by Derenzo et al. [24]. A description of the fermentation process, microorganism strains, culture media, and the fermentation procedure has been given by Braunegg [25]. 10.4.1 Sugar Fermentation to Poly(3-Hydroxybutyric acid) by Ralstonia eutropha

The fermentation process follows the procedure described by Bueno et al. [26]. A strain of Ralstonia eutropha is grown under aerobic conditions to furnish a high-cell-density culture. After this step the culture conditions are altered and the metabolic pathway is shifted to biosynthesis and intracellular storage of poly(3-hydroxybutyric acid). The block diagram in Fig. 10.3 illustrates the fermentation process; its main characteristics are listed in Table 10.2. Industrial strains of the Ralstonia genus, for example those described by Braunegg [25], are employed in fermentation. Some varieties are highly productive and can store up to 80% of their dry weight as biopolymer. When cultured in a medium containing sugar, ammonium, phosphorus, and salts with a low carbon-to-nitrogen ratio they produce mainly biomass; when cultured with a high carbon to nitrogen ratio, however, their growth phase stops and they begin synthesizing biopolymer.

Fig. 10.3 Fermentation for PHB production.

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10 Sugar-based Biorefinery Table 10.2 Fermentation process data. Biomass concentration in final culture (dry basis)

150 kg m–3

Poly(3-hydroxybutyric acid) content of biomass Fermentation yield Fermentation productivity Remaining reducing sugars in culture Fermentation temperature Fermentation pH Molecular weight of poly(3-hydroxybutyric acid) obtained

75% 98% 1.89 kg m–3 h–1 10 kg m–3 32–34 8C 6.8 1200 kDa

Fermentation is by a fed-batch procedure at temperatures of 32–34 8C and pH 6.8. Vigorous aeration and stirring must be provided to accommodate the high oxygen uptake of the biomass. The cell growth stage is highly exothermic and requires the assistance of an efficient cooling system. The growth phase lasts from 24 to 28 h and is followed by the polymer biosynthesis stage, which lasts 30–35 h. At the end of fermentation, when the poly(3-hydroxybutyric acid) content has reached 75% of the dry biomass weight, the microorganisms are inactivated by heating to 105 8C for 5 min to prevent polymer loss, because starved Ralstonia use it as a carbon source in the absence of reducing sugars. The final culture is then ready for the polymer recovery stage. 10.4.2 Downstream Processing for Recovery and Purification of Intracellular Poly(3-Hydroxybutyric Acid) 10.4.2.1 Processes for Extraction and Purification of Poly(hydroxyalkanoates) Recovery of poly(hydroxyalkanoates) in biomass from fermentation involves several complex steps, for example microorganism cell breakdown, removal of impurities, and purification of the final product. These steps are critical from the standpoint of production cost and polymer quality [28]. Use of organic solvents, some of them chlorinated, therefore is unavoidable, at least during the final steps of purification. These solvents are usually hazardous to human health and the environment. The large-scale production of a biodegradable and biocompatible product using environmentally aggressive procedures is contrary to common sense. Poly(hydroxyalkanoate) production should be conducted according to a green cycle, using renewable materials and energy, to avoid impact on the environment [27]. The product recovery process can follow two main routes – biomass digestion and solvent extraction [29].

10.4.2.2 Chemical Digestion Chemical digestion of microorganism cells using strong oxidants such as sodium hypochlorite was one of the initial procedures for recovering intracellular poly(hydroxyalkanoates) [31]. The granules obtained were washed with solvents such as


diethyl ether or methanol to increase their purity. A drawback of this procedure is the possibility of partial breakage of the polymer, reducing its molecular weight and the purity of the final product. Improvements introduced in this procedure, mainly an organic solvent wash of the polymer granules [32–34], yielded a 95% pure product with a molecular weight of 600 kDa. The main disadvantage of chemical digestion is the resulting harmful and heavily polluting effluents.

10.4.2.3 Enzymatic Digestion This procedure, which was introduced by Imperial Chemical Industries [35–37] for commercial production of poly(3-hydroxybutyrate-co-valerate), consists of enzymatic digestion of biomass to recover polymer granules. Biomass cells are subjected to thermal treatment at 100–150 8C and pH 6.8 to dissolve the nucleic acid and denature proteins. After this pretreatment, the material is digested with a mixture containing enzymes such as phospholipidase, proteinase, and alkalase. The granules thus obtained are 90% pure, containing 6–7% of proteinaceous material and 3–4% of peptidoglycan as impurities. To obtain a polymer with the required purity, the procedure must include a solvent-treatment step. The solvent step increases production costs and requires the use of environmentally harmful solvents.

10.4.2.4 Solvent Extraction Many extraction and purification procedures using solvents have been reported [30]. The dry biomass is treated with a chlorinated solvent such as chloroform [38–43], dichloroethane [45, 46], 1,1,2-trichloroethane [46], dichloromethane [47, 48], and/or propylene carbonate [48]. The polymer is dissolved by the solvent, suspended insoluble material is then removed by filtration, and the poly(hydroxyalkanoates) are recovered from the solution by precipitation with methanol, diethyl ether, or hexane. This procedure yields a highly pure final product, but requires the use of undesirable toxic compounds. Extraction Process in Brazilian Patent PI 9302312-0 Extraction using a renewable, biodegradable solvent is described in the Brazilian patent PI 9302312-0. Figure 10.4 illustrates the solvent extraction and purification of poly(3-hydroxybutyric acid) with isoamyl alcohol, a byproduct of ethanol fermentation [50]. Thermally inactivated fermentation liquor is diluted with water and then coagulated by adding phosphoric acid and lime. A polyelectrolyte is added to flocculate particles, which are recovered by settling and centrifugation. A sludge containing 25 to 35% solids is recovered and sent to the extraction stage. Extraction is performed in a set of multistage continuous stirred-tank reactors coupled with hydrocyclones. Vaporized solvent and liquid are fed continuously countercurrent to the biomass flow. Extraction is performed at the boiling point of the binary mixture. This condition makes it possible to remove excess water, break the cell wall, and remove the polymer with the aid of the solvent.

219

220


Fig. 10.4 Downstream processing of PHB.

Raw extract of Poly(3-hydroxybutyric acid) leaving the extraction stage contains suspended material such as cell debris and calcium salts, which are removed by deep filtration in a bed of diatomaceous material. The clarified extract is cooled to precipitate the polymer and the debris cake is sent to the solvent-recovery section. The solvent-free cake, consisting of biomass debris and filter aid, is composted and applied to cane crops. The poly(3-hydroxybutyric acid) suspended in isoamyl alcohol is preconcentrated by membrane microfiltration, raising the concentration from 1.5 to 4%. The solvent permeate is recovered by distillation and the main stream of the polymer suspension is concentrated in a multistage evaporation system to which steam and water are added to distil the solvent as a binary mixture. A final stripping step completes the removal of the solvent. The poly(3-hydroxybuty-


ric acid) is washed, dewatered, and carefully dried to avoid thermal breakage of the polymer. The moisture content after drying and cooling is less than 0.3%. A white powder of 100 to 200 mesh size and 500 to 800 kDa molecular weight is obtained. The fraction of degraded polymer of molecular weight < 300 kDa is less than 3%. This procedure yields a highly pure polymer by solvent extraction, avoiding the negative environmental impacts of other processes. 10.4.3 Integration of Poly(3-Hydroxybutyric Acid) Production in a Sugar Mill

Production of poly(3-hydroxybutyric acid) is integrated with that of sugar and ethanol and with the generation and consumption of energy at the sugar mill. The production of poly(hydroxybutyrates) from sugar cane is conceived by us as a process to be integrated into sugar mill operations, using not only sugar substrate but also all the facilities the mill can advantageously offer, for example heating and cooling, electric power, water, and effluent treatment and disposal. Figure 10.5 shows a flow diagram for simultaneous processing of sugar cane to sugar, ethanol, and PHB in a typical Brazilian sugar mill in which a milling capacity of 12 000 tons of sugar cane per day is used for production of sugar (55% of total milling) and the rest for fuel ethanol, and where PHB production is to be installed. The milling season last 180 days whereas PHB facilities work all year round, a total of 330 working days.

Fig. 10.5 Mass and energy balance for sugar, ethanol, and PHB.

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222


Sugar cane processing begins with extraction of cane juice by mill tandems, leaving behind bagasse, the fibrous material that is sent to the boiler house and to storage. The cane juice is treated physically and chemically to purify it. Most of this juice is used for production of sugar and the rest goes to the fermentation plant to produce ethanol. In the sugar-processing sector, this juice is concentrated in multiple effect evaporators, yielding thick syrup consisting of nearly 65% soluble solids. The syrup is then boiled in vacuum pans until sugar crystals are produced by evaporation–crystallization, which is usually performed in two stages. White sugar of various standards is recovered in this step; the molasses by-product, left over noncrystallizable thick impure sugar syrup, is normally fermented and converted into ethanol. Part of the sugar production will be used to produce PHB, and sent to the PHB plant for direct use or stored for use during the off-season. At the cane juice fermentation plant, the molasses and sugar syrup are blended to formulate the fermenting must. The must then undergoes ethanol fermentation, yielding the final liquor, which is distilled into aqueous and anhydrous fuel ethanol. Byproducts such as fusel oil and yeast are recovered in the ethanol production sector. Meanwhile, at the PHB production facility, medium quality standard sugar is processed into PHB by the aforementioned procedure. Sugar cane milling and ethanol and PHB processing are energy-intensive processes that require mechanical and electrical power and thermal energy in the form of low-pressure steam. Recovered bagasse is burned in a high-pressure boiler, producing superheated steam at 60 bar and 450 8C. This primary steam is expanded in high efficiency multistage turbines equipped with electricity generators, providing the electricity consumed by the manufacturing complex. Medium pressure steam at 20 bar is extracted from multistage turbines and used as the primary mover for mechanical power generation in the milling step involving cane cutters, defibrators, and extraction tandems. Low-pressure steam extracted from the turbines is used as the source of thermal energy for sugar, ethanol, and PHB processing. The solid effluents from the PHB process will be composted and spread over sugar cane fields as filtering mud from the juice treatment. Liquid effluents, for example the final fermentation liquor and the washing water left behind after removal of microorganism cells containing PHB, will be sprayed on the cane crops like vinasse from ethanol distillation. 10.4.4 Investment and Production Cost of Poly(3-Hydroxybutyric Acid) in a Sugar Mill

A preliminary analysis has been made of the investments required for, and the production costs involved in, sugar-derived PHB. The purpose of the work was to determine the economical feasibility of biodegradable production integrated with an existing sugar mill. The analysis was based on two scenarios – an autonomous unit producing 10 000 tons of PHB per year, located outside the mill site, and an integrated unit having the same production capacity. Construc-

10.5 Outlook and Perspectives Table 10.3 Investment and production cost of the PHB process.

Buildings and civil works Fermentation unit Downstream processing unit Utilities Total Production cost breakdown Depreciation over buildings and equipment Sugar substrate Other raw materials and chemicals Bagasse for energy needs Salaries Maintenance Others

Investment in US $

%

3 000 000.00 15 000 000.00 15 000 000.00 5 000.00 38 000 000.00

7.9 39.5 39.5 13.2

11 29 15.5 4.9 12.3 5.1 8.6

tion of a manufacturing unit for a production capacity of 10 000 tons per year requires a large investment. Table 10.3 gives a breakdown of investments and costs, with installed equipment totaling US $ 38 000 000. The production cost of PHB is highly dependent on the price of sugar, which is the major factor, accounting for almost 29% of the final cost. The production cost for a basic 99% pure poly(3-hydroxybutyrate) chemical is estimated at US $ 2.25 to 2.75 kg–1, depending on the price of sugar, other chemicals, and bagasse. It is worth noting that a similar calculation for an autonomous PHB unit shows the production cost rises to US $ 2.50 to 3.00 kg–1, indicating the advantages of integrating PHB production with an existing mill. Comparing our cost data with Lee’s [51] estimate of $US 2.65 kg–1 and Bertrand’s [52] of US $ 5.85 kg–1, we conclude that our proposed scenario is feasible.


Expectations of the development of industrial poly(3-hydroxybutyric acid) production by the sugar cane agroindustry are high. There is a large margin for improvement of the current production process, which will result in lower capital and production costs, less generation of solid and liquid effluents, and lower consumption of energy. Poly(hydroxyalkanoate) production technology will undergo significant improvements when novel microorganisms are obtained by selection or by genetic engineering. One target is microorganisms that can directly assimilate more complex carbon sources, for example sucrose, or even metabolize pentose sugar. Other targets are simultaneous growth and polymer production steps and larger percentages of intracellular stored PHB. Efficient synthesis of heteropolymers other than poly(3-hydroxybutyrate) and fermenta-

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tion at higher temperatures and lower medium pH will also affect the fermentation technology favorably. Steinbuchel [53] reviewed bacterial strains and possible genetically modified organisms to improve PHB production. Vicente [54, 55] gave examples of the construction of genetically modified bacteria for poly(3-hydroxybutyric acid) biosynthesis. Poly(3-hydroxybutyric acid) production costs could be significantly reduced by optimizing production processes. Moreover, fermentation could be improved to increase the PHB content of the cells to 80%. There is also a margin for increasing the concentration of biomass in the final liquor to 200 kg m–3, assuming the oxygen transfer rate in fomenters is improved. The technology available today limits fermenter capacity to less than 500 m–3; overcoming this obstacle will lead to increasing gains resulting from scale. The fermentation cycle can be optimized by reducing the content of reducing sugars in the final liquor from the current 1 to 0.2%. There is a broad range of possibilities for optimizing and reducing the cost of technology available for the extraction and purification of poly(3-hydroxybutyrate). The consumption of thermal energy and the power requirements in downstream processing could be reduced. Polymer extraction and purification should be reviewed to improve the processing stages and to obtain a purer product of higher molecular weight and a lower poly dispersion index. A survey of new extraction solvents is required to identify nontoxic and environmentally friendly solvents that can dissolve more polymer and less undesirable impurities. A milestone in poly(hydroxyalkanoate) production will be reached when it becomes possible to replace sucrose with carbohydrates contained in the lignin– cellulose biomass component of sugar cane. The bagasse or trash left behind after cane harvesting are lower-cost substrates than sugar and, according to Macedo [3], will be available in large quantities. Depending on the extent of optimization of energy production and available consumption cycles, the amount of bagasse will range from 39 to 67 kg ton–1 sugar cane. Biomass availability in trash is estimated to be 56 to 98 kg ton–1 cane, depending on the harvesting method employed. In the near future, when the hydrolytic process is commercially ready, this lignin cellulose-based material will be transformed into hexose and pentose sugars, replacing sucrose in PHB fermentation. Da Silva [56] reports on the biosynthesis of poly(3-hydroxybutyric acid) by strains of Bhukolderia Spp, using hydrolysis liquor from an organosolve process that yields 65 to 75% total reducing sugars from bagasse. The Bhukolderia strain can ferment the pentose and hexose sugar contained in bagasse. Other poly(hydroxyalkanoates) unlike poly(3-hydroxybutyric acid) and with superior functional properties will lead to improvement of biodegradable plastics. Long chain hydroxyacids and heteropolymer alkanoates can be produced by dosing specific carbon substrates in the polymer-biosynthesis step. Thus, the biological process could be engineered to produce new polymers with desirable properties.

References

References 1 ÚNICA, www.unica.com.br, 2004. 2 I. Macedo, São Paulo State Government 3 4 5

6

7 8 9

10 11 12

13 14 15

16

17

18

19

Report. March, 2004. I. Macedo, Copersucar Technical Bulletin. 1985, 31:22–27. I. Macedo, Biomass and Bioenergy. 1996, 14:77–81. R. Kahn, H. F. Jones, Chemistry and processing of sugar beet and sugar cane. M. A. Clarke, M. A. Godshall editors, 1988, Elsevier Science Publishers B. V., Netherlands. J. M. Paturau, By-products of the cane sugar industry. 1989, Elsevier Science Publishers B. V., Netherlands. M. Okada, Progress in Polymer Science. 2002, 27, 88–103. W. M. Pachekoski, Ms Sc. Thesis. UFSCAR, SP, Brazil, 2001 S. Karlsson, A. C. Alberton, Polymer Engineering and Science. 1998, 38, 1251– 1254. R. Chandra, R. Rutsgi, Progress in Polymer Science. 1998, 23, 1273–1335. K. Van de Velde, P. Kiekens, Polymer Testing, 2002, 21, 433–442. U. J. Hänggi, International Symposium on Natural Polymer and Composites, 2. Proceedings, Embrapa, SP, Brazil. 1998, 309–310. ASTM. D883: Terminology relating to plastics. Filadélfia, 2002, vol. 08.01. C. Ha, W. J. Cho, Progress in Polymer Science. 2002, 27, 759–809. M. K. Cox, International Scientific Workshop on Biodegradable Polymers and Plastics, 2. Proceedings, Royal Society of Chemistry, Montpellier. 1992, 95–100. P. Gatenholm, A. Mathiason, Journal of Applied Polymer Science 1994, 51, 1231– 1237. G. Swift, International Scientific Workshop on Biodegradable Plastics and Polymers. Proceedings, Tokio, Japan. 1994, 228–236. Y. Doi, International Scientific Workshop on Biodegradable Polymers and Plastics, 2. Proceedings, Royal Society of Chemistry, Montpellier. 1992, 139–148. G. J. M. Koning, Prospects of Bacterial Poly(R)-3-Hydroxyalkanoates, 1993, 5–26.

20 M. K. Cox, International Scientific Work-

21 22 23 24 25 26 27 28

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31 32 33 34 35 36 37 38 39 40 41

shop on Biodegradable Polymers and Plastics, 2. Proceedings, Royal Society of Chemistry, Osaka, Japan, 1994, 120–135. H. Sudesh, Progress in Polymer Science 2000, 25, 1503–1555. A. El-Hadi. Polymer Testing, 2002, 21, 665–674. M. Yamaguchi, Yokkaichi Research Laboratory Report. 2004. S. Derenzo, Brazilian Patent PI 93023120, 1993. Braunegg, J. Biotechnol. 1998, 65, 127– 161. C. L. Bueno, Brazilian Patent PI 9103116-8, 1993. R. V. Nonato, Appl. Microbiol. Biotechnol. 2002, 57, 1–5G. A. J. Asenjo, Separation Processes in Biotechnology, Marcel Dekker, Inc., New York, USA, 1990. G. J. L. Griffin, Chemistry and Technology of Biodegradable Polymers, Blackie Academic and Professional, Glasgow, Scotland, 1994. E. Berger, Dr. Sc. Thesis, Ecole Polytechnique, Universite de Montreal, Canada, 1990. D. H. Williamson, J. F. Wilkinson, J. Gen. Microbiol., 1958, 19, 198. M. P. Nuti, Can. J. Microbiol., 1972, 18, 1257. E. Berger, Biotechnol. Tech., 1989, 3 (4), 227. B. A. Ramsay, Biotechnol. Technol. 1989, 4 (4), 221. J. M. Merrick, M. Dourdoroff, J. Bacteriol. 88(1), 60–71, 1964. P. A. Holmes, European Patent Application EP 46 335, 1985. P. A. Holmes, G. B. Lim, European Patent Application EP 145 233, 1985. Y. Inoue, N. Yoshie, Prog. Polym. Sci, 1992 17, 517–610. M. Lemoigne, C. R. Acad. Sci. 1925, 180,1539. M. Lemoigne, Ann. Inst. Pasteur. Sci. 1925, 39, 144. M. Lemoigne, Bull. Soc. Chim. Biol, 1925, 8, 770.

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10 Sugar-based Biorefinery 42 M. Lemoigne, Ann. Inst. Pasteur Sci. 43 44 45 46 47 48

1925, 41 ,148. J. Walker, European Patent Application EP 0 046 017, 1982. Solvay and Co, European Patent Application EP 14 490, 1979. P. J. Barham, A. Selwood, US Patent Application 4 391 766, 1982. P. J. Barham, A. Selwood, European Patent Application EP 0 058 480, 1982. J. N. Baptist, US Patent Application 3 036 959, 1962. J. N. Baptist, US Patent Application 3 044 942, 1962.

49 J. N. Baptist, US Patent Application

3 044 942, 1962. 50 S. Y. Lee, J. Choi, Polym Deg Stab, 1998,

59, 387–393. 51 J. L. Bertrand, Dr. Sc. Thesis, Université

de Montreal, Montreal, Canada, 1992. 52 A. Steinbuchel, B. Fuchttenbusch, Tib-

tech. 1998, 16, 419–427. 53 E. J. Vicente, Brazilian Patent PI

9806581-5, 1998. 54 E. J. Vicente, Brazilian Patent PI

9805116-4, 1998. 55 L. F. Da Silva, J. Ind. Microb. Biotech-

nol., 2004, 31, 245–254.


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Biorefineries Based on Thermochemical Processing 11 Biomass Refineries Based on Hybrid Thermochemical-Biological Processing – An Overview Robert C. Brown

11.1 Introduction

The Biomass Research and Development Technical Advisory Committee (2002) of the US Departments of Energy and Agriculture defines a biorefinery as: “A processing and conversion facility that (1) efficiently separates its biomass raw material into individual components and (2) converts these components into marketplace products, including biofuels, biopower, and conventional and new bioproducts.” Implicit in this definition is the assumption that grain will be fractionated into starch, oils, proteins, and fiber and lignocellulosic crops will be fractionated into cellulose, hemicellulose, lignin, and terpenes before these components are converted into market products. Certainly, this is the approach of modern wet corn milling plants and wood pulp and paper mills. Another possibility for high fiber plant materials is, however, thermochemical processing into a uniform intermediate product that can be biologically converted into a biobased product. This alternative route to biobased products is known as hybrid thermochemical-biological processing or simply hybrid processing of biomass. There are two distinct approaches to hybrid processing: · gasification followed by fermentation of the resulting gaseous mixture of carbon monoxide (CO), hydrogen (H2) and carbon dioxide (CO2), and · rapid pyrolysis followed by hydrolysis and/or fermentation of the anhydrosugars found in the resulting bio-oil.


228

11 Biomass Refineries Based on Hybrid Thermochemical-Biological Processing – An Overview


The history of hybrid thermochemical-biological processing is brief. The concept only emerged in the 1980s and no such processes have been commercially introduced. Although hybrid refining has potential for overcoming some of the shortfalls of conventional fractionation of biomass, it has not drawn wide attraction, possibly because it crosses the disparate fields of high-temperature thermochemistry and biology. The following paragraphs give brief histories of the gasification and fast pyrolysis approaches to hybrid refining. 11.2.1 Origins of Biorefineries Based on Syngas Fermentation

Traditionally, the feedstocks for the manufacture of biotechnology products have been carbohydrates. Several anaerobic bacteria use C1 compounds such CO, CO2, and methanol (CH3OH) and hydrogen as sources of carbon and energy for growth and metabolite production, however. Syngas, a mixture rich in CO, CO2, and H2, is produced by heating carbon-rich solids under high-temperature (600–1000 8C), oxygen-starved conditions. Its name derives from the fact that this gas mixture is used to synthesize a variety of industrially important organic compounds, for example acetic acid and methanol, by the application of metal catalysts and elevated temperatures and pressures. Zeikus and his colleagues (1985) at the Michigan Biotechnology Institute were among the first to propose substituting biocatalysts for the metal-based catalysts currently used to convert syngas into industrial chemicals. A few years later Gaddy and coworkers at the University of Arkansas published a series of papers detailing how a variety of products, including methane, acetic acid, and ethanol, might be fermented from syngas (Ko et al. 1989; Vega et al. 1989 a, b, c, d). Because of the US Department of Energy’s interest in developing alternative transportation fuels from biomass, much of the early work in syngas fermentation focused on alcohol production. By 1991, the Michigan Biotechnology Institute had identified the Gram-positive, nonmotile, rod-shaped anaerobe Butyribacterium methylotrophicum as a possible candidate for production of alcohol from syngas (Worden et al. 1991) whereas the University of Arkansas focused on the Gram-positive, motile, rod-shaped anaerobe Clostridium ljungdahlii (Vega et al. 1989 d). These two groups published several papers in the 1990s although by 1993 Gaddy had started a company to commercialize syngas fermentation technology (Tobler 1994) and his group ceased publishing the results of their investigations. Only a few other groups have explored syngas fermentation. Elmore and coworkers at Louisiana Tech University (Madhukar et al. 1996) isolated three unidentified rod-shaped, Gram-positive cultures that used mixtures of CO, CO2, and H2 (that is, simulated syngas) as their primary carbon source to produce acetate, ethanol, methanol, and smaller quantities of other alcohols and organic acids. Maness and Weaver at the National Renewable Energy Laboratory (1994)


opened up new opportunities for syngas fermentation by exploring the conversion of CO and H2 into poly(3-hydroxybutyrate) by photosynthetic bacteria. More recently, a team at the University of Oklahoma (Datar et al. 2004) has demonstrated the production of ethanol from clean syngas derived from a biomass gasifier and Iowa State University (Brown et al. 2003) is exploring the production of both hydrogen and polyesters from the purple non-sulfur bacteria Rhodospirillus rubrum under dark reaction conditions. 11.2.2 Origins of Biorefineries Based on Fermentation of Bio-oils

Bio-oil is a liquid mixture of oxygenated organic compounds produced by heating finely divided biomass in the absence of oxygen. The process, known as fast pyrolysis, involves lower temperatures and shorter reaction times than gasification with the result that it produces mostly liquid product (as much as 70% w/w) whereas gasification yields essentially all gas. The technology was developed in the 1980s with the goal of using bio-oil as a substitute for (petroleum-derived) fuel oil (Bridgwater and Peacocke 2000). Scott and his coworkers at the University of Waterloo, Ontario (1989) discovered that the amount of anhydrosugars, particularly levoglucosan, in bio-oil could be dramatically increased by demineralizing the biomass before pyrolyzing it. Because levoglucosan is readily hydrolyzed to glucose, they suggested that fast pyrolysis be used as an alternative to acid or enzymatic hydrolysis for recovery of sugars from lignocellulosic biomass. Recognizing that elimination of the hydrolysis step would improve the prospects for using bio-oil as substrate, Zhuang and coworkers (2001 a) at the Chinese Academy of Sciences adapted a mutant of A. niger CBX-209 to directly ferment levoglucosan to citric acid. They demonstrated that levoglucosan was the sole source of carbon and energy for this fermentation. So and Brown at Iowa State University (1999) performed an economic assessment that found the cost of ethanol from this fast pyrolysis route to be, within the uncertainty of the analysis, comparable to the cost of ethanol from acid hydrolysis or enzymatic hydrolysis of woody biomass. The prospects of producing both power and chemical products by fast pyrolysis of fibrous biomass, which would constitute a biorefinery, was recently evaluated by Sandvig and his collaborators (2004). One manifestation of this biorefinery included recovery, hydrolysis, and fermentation of levoglucosan. Although fast pyrolysis is a commercial technology, it has yet to be employed as part of a hybrid thermochemical-biological biorefinery.

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11.3 Gasification-Based Systems 11.3.1 Fundamentals of Gasification

Gasification is the high-temperature (750–850 8C) conversion of solid, carbonaceous fuels into flammable gas mixtures, sometimes known as synthesis gas or syngas, consisting of CO, H2, CO2, methane (CH4), nitrogen (N2), and smaller quantities of higher hydrocarbons (Reed 1981). Not only can syngas be used for generation of heat and power, it can serve as feedstock for production of liquid fuels and chemicals. Because of this flexibility of application, gasification has been proposed as the basis for refineries that would provide a variety of energy and chemical products, including electricity and transportation fuels. Gasification consists of several distinct processes: heating and drying of the fuel; pyrolysis of solid fuel to gases, condensable vapors, and char; solid-gas reactions that consume char; and gas-phase reactions that adjust the final chemical composition of the syngas. Pyrolysis, which begins between 300 8C and 400 8C, may convert up to 80% w/w of solid biomass into gases and vapors. The pyrolytic gases include CO, CO2, H2, H2O, and CH4 and the condensable vapors include a variety of hydrocarbons and oxygenated organic compounds. The solid-gas reactions produce CO, H2, and CH4 by the following reactions (Reed 1981): Carbon-oxygen reaction: 1 C O2 $ CO 2

DHR

110:5 MJ kmol

1

DHR 172:4 MJ kmol

1

Boudouard reaction: C CO2 $ 2CO Carbon-water reaction: C H2 O $ H2 CO

DHR 131:3 MJ kmol

1

Hydrogenation reaction: C 2H2 $ CH4

DHR

74:8 MJ kmol

1

Two important gas-phase reactions also influence the overall gasification process: Water-gas shift reaction: CO H2 O $ H2 CO2

DHR

41:1 MJ kmol

1

11.3 Gasification-Based Systems

Methanation: CO 3H2 $ CH4 H2 O

DHR

206:1 MJ kmol

1

The final gas composition is highly dependent on the amount of oxygen and steam admitted to the reactor and the time and temperature of reaction. For sufficiently long reaction times chemical equilibrium is achieved and the products are essentially limited to the light gases CO, CO2, H2, and CH4 (and nitrogen if air was used as a source of oxygen). Gasifiers are usually classified according to the method of contacting fuel and gas. The three main types suitable for biomass gasification are illustrated in Fig. 11.1 (Brown 2003). Updraft gasifiers are the simplest, consisting of little more than a grate with chipped fuel admitted from above and insufficient air for complete combustion entering from below. This countercurrent flow of fuel and air results in producer gas with large quantities of undesirable tars. In downdraft gasifiers, fuel and gas move in the same direction with contemporary designs usually adding an arrangement of tuyeres that admit air or oxygen directly into a region known as the throat where combustion forms a bed of hot char. This design assures that condensable gases released during pyrolysis are forced to flow through the hot char bed, where tars are cracked. A disadvantage is the need for tightly controlled fuel properties (particles sizes between 1 and 30 cm, low ash content, and moisture less than 30%) and an upper bound on gasifier size of approximately 2 MW thermal. In fluidized bed gasifiers a gas

Fig. 11.1 Common types of biomass gasifier: (a) updraft, (b) downdraft, (c) fluidized bed (Brown 2003).

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stream passes vertically upward through a bed of inert particulate material to form a turbulent mixture of gas and solid. They are able to process a wide variety of fuels and are easily scaled to large sizes. Disadvantages include moderate tar loadings, high particulate loadings, and relatively high power consumption to run the air blower. Overall, gasification is endothermic and requires either simultaneous burning of part of the fuel or an external source of heat to drive the process. Addition of air is called air-blown gasification and has the disadvantage of admitting nitrogen to the syngas, which dilutes the concentration of reactive compounds in the syngas and reduces its chemical enthalpy. Substitution of oxygen for air, known as oxygen-blown gasification, eliminates nitrogen as a diluent but is an extremely expensive solution to the problem. Several researchers have developed methods for bringing sufficient heat to the gasifier without admitting air or oxygen, a process known as indirectly heated gasification (Bridgwater 1995). Chemical enthalpy from such gasifiers is typically 200% higher than from air-blown gasifiers. Often gasifier temperatures and reaction times are not sufficient to achieve chemical equilibrium and the raw syngas contains various amounts of light hydrocarbons such as C2H2 and C2H4 and up to 10% w/w heavy hydrocarbons that condense to a black, viscous liquid known as “tar.” Furthermore, two kinds of particulate matter often contaminate syngas. The first, known as ash, is mineral matter in the raw biomass that remains on completion of gasification. The second is char formed during pyrolysis but not consumed by solid-gas reactions. Taken together, ash and char are sometimes referred to as ash or gasification residue. Typical gas concentrations and chemical enthalpies for syngas are compared in Table 11.1 for air-blown and indirectly heated gasifiers. Clearly, the higher concentrations of CO and H2 from an indirectly heated gasifier reduce the size of bioreactors needed to convert these gases into organic compounds. Table 11.1 Syngas composition from various kinds of gasifiers (Brown 2003). Gasifier type

Gaseous constituents (% v/v, dry)

HHV (MJ m–3)

H2

CO

CO2

CH4

N2

Air-blown updraft

11

24

9

3

53

5.5

Air-blown downdraft

17

21

13

1

48

5.7

9

14

20

7

50

5.4

Oxygen-blown downdraft

32

48

15

2

3

10.4

Indirectly-heated fluid bed

31

48

0

21

0

17.4

Air-blown fluidized bed

Gas quality Tars

Dust

High (*10 g m–3) Low (*1 g m–3) Medium (*10 g m–3) Low (*1 g m–3) Medium (*10 g m–3)

Low Medium High Low High


11.3.2 Fermentation of Syngas

Traditional fermentations rely on carbohydrates as the source of carbon and energy in the growth of microbial biomass and the production of commercially valuable metabolites. Several microorganisms, however, can use less expensive substrates for growth and production. These include autotrophs, which use C1 compounds as their sole source of carbon and hydrogen as their energy source, and unicarbonotrophs, which use C1 compounds as their sole source of both carbon and energy. Among suitable C1 compounds are CO, CO2, and methanol (CH3OH), all of which can be produced by thermochemical processing of biomass. Although both aerobic and anaerobic microorganisms can use C1 substrates, anaerobes offer the most promising route to chemicals and fuels from syngas, because they employ very energy-efficient metabolic pathways – most of the chemical energy of the substrate appears in the products of fermentation. Organisms that form the metabolic intermediary acetyl-CoA from carbonyl or carboxyl precursors are known as acetogens. Although many acetogens consume alcohols or fatty acids to produce acetate, CO2, and H2, some are able to utilize CO2 and hydrogen (autotrophic acetogens) or CO (unicarbonotrophic acetogens) as substrates for growth and production of organic acids and, occasionally, alcohols (Grethlein and Jain 1993). As illustrated in Fig. 11.2, metabolism of CO begins with the reaction of CO and H2O via CO dehydrogenase to yield CO2 and H2; this is the biologicallymediated water-gas shift reaction. Subsequent steps include production of formate from CO2 via formate dehydrogenase, several tetrahydrofolate-mediated dehydrogenase transformations resulting in a methyl-corrinoid complex, reaction of CO with CO dehydrogenase to form an enzyme-bound carbonyl moiety, and synthesis of acetyl-CoA from methyl and carbonyl groups bound to the CO dehydrogenase complex (Zeikus et al. 1985). Metabolism of H2 and CO2 occurs by the same mechanism. As shown in Fig. 11.2, H2 reduces CO2 to a bound form of CO in a ferredoxin-dependent reaction. The resulting carbonyl group reacts with the methyl-corrinoid complex described previously via CO dehydrogenase to form acetyl-CoA (Zeikus et al. 1985). Acetyl-CoA is the chemical intermediate in the subsequent formation of biomass (growth) and metabolites (production), as subsequently described. The autotrophs and unicarbonotrophs that convert single-carbon compounds into higher-molecular-weight products are dependent on enzymes and co-enzymes that contain nickel, cobalt, iron, tungsten, molybdenum, selenium, zinc, or combinations of these metals (Zeikus et al. 1985). Similarly, the petrochemical industry is dependent on metal-based catalysts to convert syngas into products. This includes nickel catalysts for steam reforming of hydrocarbons, iron-chromium and copper-zinc catalysts to produce hydrogen via the water-gas shift reaction, copper catalysts to produce methanol, and iron or chromium catalysts to produce hydrocarbons via the Fischer-Tropsch reaction (Spath and Dayton 2003).

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Fig. 11.2 Metabolic pathways for acetogenic bacteria that synthesize acetate or butyrate during growth on C1 substrates or H2 and CO2. The symbols [CH3OH] and [HCOOH] represent two oxidation states of C1 units bound to tetrahydrofolate (THF) carriers whereas the chemical nature of [CO] remains undetermined. Numbers indicate the

following enzymic activities: 1, hydrogenase; 2, CO dehydrogenase; 3, formate dehydrogenase; 4, formyl-THF synthetase; 5, other THF enzymes; and 6, one or more enzymes required for the synthesis of acetyl-CoA from [CO] and a methyl-corrinoid (Zeikus et al. 1985).

Biological routes to syngas fermentation have several potential advantages over conventional catalytic routes (Grethlein and Jain 1993). Most catalysts used in the petrochemical industry are readily poisoned by sulfur-bearing gases whereas syngas-consuming anaerobes are sulfur-tolerant; thus, expensive sulfur-gas clean-up can be eliminated by using biological catalysts. In conventional catalytic processing the CO/H2 ratio of the syngas is critical to commercial operations. Because CO/H2 ratios depend on the quality of the gasified feedstocks, water-gas shift reactors are required to make this adjustment. Biological catalysts are not sensitive to this ratio; indeed, the water-gas shift reaction is implicit in the metabolism of autotrophic and unicarbonotrophic anaerobes. Gas-phase catalysts typically employ temperatures of several hundreds of degrees Centigrade and at least ten atmospheres of pressure whereas syngas fermentation proceeds at near ambient conditions. Finally, biological catalysts tend to be more product specific than inorganic catalysts.

11.3.2.1 Production of Organic Acids As illustrated in Fig. 11.2, the primary metabolites from the conversion of C1 compounds by autotrophic and unicarbonotrophic anaerobes are organic acids. The chemical intermediate acetyl-CoA can produce either acetate via acetyl


phosphate or butyryl-CoA and subsequently butyrate. The relative yields of these two organic acids depend on the type of organism and the substrate. For example, in studies with Butyribacterium methylotrophicum Worden et al. (1989) showed that the fraction of electrons going from CO into butyrate production could be increased from 6 to 70% at the expense of acetate production by reducing the pH from 6.9 to 6.0. Representative species of acidogenic (acid-forming) anaerobes include Clostridium thermoaceticum, Clostridium ljungdahlii, Peptostreptococcus productus, Acetobacterium woodii, Eubacterium limosum and Butyribacterium methylotrophicum (Grethlein and Jain 1993), with some of these also forming alcohols, as subsequently described. The metabolism of B. methylotrophicum is given as an example of the molar stoichiometric yields that can be expected (Bredwell et al. 1999). For CO substrate and acetate production, the molar stoichiometry, balanced for carbon and available electrons, is: 4CO ! 2:17CO2 0:74CH3 COOH 0:45Cell

1

where Cell indicates C-moles (carbon equivalents) in the cell mass produced. At least half of the CO must be oxidized to CO2 to provide enough electrons to reduce the remaining CO to acetate and cell mass. For CO2 and H2 substrate and acetate production, the molar stoichiometry is: 2H2 1:03CO ! 0:43CH3 COOH 0:13Cell

2

11.3.2.2 Production of Alcohols Although production of organic acids seems to dominate the metabolites from wild strains of autotrophic and unicarbonotrophic anaerobes, alcohols have also been produced from some organisms. For example, the wild strain of Clostridium ljungdahlii, a Gram-positive, motile, rod-shaped anaerobe, originally furnished ethanol/acetate ratios of only 0.05 with a maximum ethanol concentration of 0.1 g L–1 (Vega et al. 1989 d). Adjustment of the fermentation conditions, notably reducing the pH, was reported by Gaddy and coworkers to essentially eliminate acetate production and produce ethanol concentrations as high as 48 g L–1 (Phillips et al. 1993). Similarly, in continuous-culture experiments with B. methylotrophicum, Worden and coworkers (Grethlein et al. 1990) found increasing quantities of ethanol and butanol in the fermentation products as pH decreased. More generally, they found a trend toward more reduced products (acids with longer chain lengths and alcohols) as fermentation pH decreased. For example, at pH 6.8 the molar stoichiometry of the continuous fermentation of B. methylotrophicum was:

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4CO ! 2:09CO2 0:63CH3 COOH 0:043C3 H7 COOH 0:027C2 H5 OH 0:43Cell 3 whereas reducing the pH to 6.0 changed the molar stoichiometry to: 4CO ! 2:27CO2 0:30CH3 COOH 0:161C3 H7 COOH 0:032C2 H5 OH 0:029C3 H7 OH 0:31Cell

4

Worden et al. (1991) noted that acetone, butanol, and ethanol were once produced commercially from glucose by use of a biphasic process consisting of an acidogenic phase, which produced organic acids and H2, followed by a solventogenic phase, in which the organic acids were reduced to alcohols. In this process acid production generates ATP but consumes no electrons while alcohol production consumes electrons but produces no ATP. They propose a similar scheme for syngas fermentation, with the first phase producing acetate and butyrate from CO (acidogenic phase) followed by alcohol production in a second phase, with reducing equivalents to convert the acids to alcohols coming from hydrogen in the syngas.

11.3.2.3 Production of Polyesters Acetyl-CoA is the chemical intermediate not only for production of organic acids and alcohols by anaerobes but for growth of cell mass, including polyesters that serve as energy stores for the organism. Under conditions of stress, such as an imbalance in the supply of nutrients, many microorganisms synthesize polyhydroxyalkanoates (PHA), which are stored in the cells as discrete granules, as illustrated in Fig. 11.3, that can accumulate to levels as high as 30 to 80% of their cellular dry weight (Kim and Lenz 2001). Most known polyhydroxyalkanoates are polymers of 3-hydroxyalkanoic acids, the monomeric unit of which is illustrated in Fig. 11.4. Although a wide range of alkanes of different carbon chain length between 4 and 14 carbon atoms can

Fig. 11.3 Photomicrograph showing the accumulation of PHA in Rubrivivax gelatinosus (Maness and Weaver 1994).

11.3 Gasification-Based Systems Fig. 11.4 Monomeric unit of poly(3-hydroxybutyrate).

be incorporated into PHA, the most commonly occurring in nature is poly(3-hydroxybutyrate) (PHB). PHB can be synthesized by a variety of prokaryotes, including Gram-positives and Gram-negatives, aerobic and anaerobic chemo-organo-heterotrophs, chemo-litho-autotrophs, and aerobic and anaerobic phototrophs (Babel et al. 2001). Although glucose is commonly used as the substrate for PHA production, a variety of carbon and energy sources, including CO and CO2 and H2, have been exploited by bacteria in production of PHA. The key enzyme in CO utilization, CO dehydrogenase, functions to oxidize CO to CO2, synthesize acetyl-CoA, and cleave acetyl-CoA in a variety of energy-yielding pathways, depending on the microorganism (Ferry 1995). Ralstonia eutropha is an autotrophic bacterium that produces PHB from CO2, H2, and O2 (Schlegel et al. 1961). Synechococcus sp. MA19 is a cyanobacteria capable of producing up to 20% PHB when cultivated in a nitrogen-free inorganic medium aerated with CO2 (Miyake et al. 1996). The photosynthetic bacterium Rubrivivax gelatinosus has produced similar yields of PHA copolymers from syngas (Maness and Weaver 1994). Rhodopseudomonas gelatinosa can utilize CO as a sole carbon and energy source in the dark to produce PHA (Uffen 1983) and Rhodospirillum rubrum can accomplish this in either the presence or absence of light (Kerby et al. 1995). Irrespective of the carbon source, synthesis of PHA is initiated from acetylCoA. The process is illustrated in Fig. 11.5 for synthesis of PHB (labeled poly(3HB)). The route from acetyl-CoA to PHB is thought to involve three steps (Babel et al. 2001). The first step, catalyzed by 3-ketothiolase, links two acetylCoA moieties to acetoacetyl-CoA. The second step produces d-(–)-3-hydroxybutyryl-CoA either through a single reaction catalyzed by reductase or a sequence of three reactions involving reductase and two hydratases. The final step, mediated by a polymerase, adds hydroxybutyryl monomer to the growing polymer chain to form PHB. Other steps shown in Fig. 11.5 are associated with the decomposition of PHB. PHB was thought to be the only polyester produced by microorganisms. In 1974, however, other 3-hydroxyalkanoates, including 3-hydroxyvalerate (PHV) and 3-hydroxyhexanoate, were isolated from microorganisms in sewage sludge (Wallen and Rohwedder 1974). Since then a variety of polyesters containing 3-, 4-, and 5-hydroxyalkanoate units have been found to be synthesized by bacteria (Steinbuchel 2001). Most of these are obtained only if precursor substrates structurally related to the resulting PHA are provided to the bacteria as carbon sources, however. Although this might seem to preclude the synthesis of all but a few PHA from syngas, the volatile organic acids that can be generated from syngas by some anaerobes might prove suitable substrates for production of longer-chained hydroxyalkanoates.

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Fig. 11.5 Metabolic pathways to PHB synthesis and degradation (1) ketothiolase; (2) NADPH-dependent acetoacetyl-CoA reductase; (3) poly(3HB) synthase; (4) NADH-dependent acetoacetyl-CoA

reductase; (5), (6) enolases; (7) depolymerize; (8) D-(–)-3-hydroxybutyrate dehyrogenase; (9) acetoacetyl-CoA synthetase; (10) succinyl-CoA transferase; (11) citrate synthase (Babel et al. 2001).

Yields of PHB and cell biomass from a given substrate can be determined by experiment or by calculation based on the known metabolic pathways involved. Recovery of this water-insoluble polymer can proceed by one of several methods. Solvent extraction gives high recovery but requires high capital investment and large quantities of solvent. Non-solvent alternatives disintegrate cells by heat shock followed by enzymatic and detergent digestive processes to solubilize the non-PHA components (Anderson and Dawes 1990). PHB is structurally similar to polypropylene and has similar crystallinity and glass transition temperature. Their chemical properties are completely different, however, and PHB is stiffer and more brittle than polypropylene. These physical properties can be changed by forming copolymers from monomeric units of PHB and PHV. Thus, a range of properties can be engineered from copolymers, ranging from hard and brittle to soft and tough (Anderson and Dawes 1990). Polyhydroxyalkanoates are attractive as biobased and biodegradable polymers. Specialty applications of PHA include hydrophobic coatings, specialty elastomers, medical implants, functionalized polymers for chromatography, microgranules for use as binders in paints or in blends that incorporate latexes, and as sources of chiral monomers (Kessler et al. 2001).


11.3.3 Biorefinery Based on Syngas Fermentation

One possible manifestation of a biorefinery based on syngas fermentation is illustrated in Fig. 11.6. Fibrous feedstock, for example switchgrass, woodchips, or cornstover, is fed into an oxygen-blown or indirectly heated gasifier followed by gas clean-up to remove particulates and char from the gas stream. Removal of trace contaminants, for example sulfur, chlorine, ammonia, and alkali, is probably unnecessary because they are not thought to poison the anaerobes used in the fermentation process. The cleaned gas is cooled and passed through a bioreactor where CO is dissolved in the fermentation media and taken up by a suitable unicarbonotroph, for example Rhodospirillum rubrum. In the analysis that follows, it is assumed that 20% w/w CO is used for growth (formation of cellular biomass) and 80% w/w of CO goes to metabolite production (H2 generation via the biologically-mediated water-gas shift reaction). It is further assumed that 40% w/w of the cellular biomass is in the form of storage polymer (PHA). The actual values for these yields are not well known but these assumed values are within the expected ranges (Dispirito 2004). Although the diagram shows gas recycle, it is not clear whether this will be necessary, because the process may not be thermodynamically limited as it is for Fischer-Tropsch and other types of syngas-to-chemicals catalytic reactors. PHA is recovered and the hydrogen-rich gas-stream leaving the reactor is used for either on-site or distributed power production via fuel cells. Brown et al. (2003) have performed a preliminary economic assessment of a biorefinery producing 20 tpd of PHA and 50 tpd of hydrogen. The capital costs for this biorefinery, detailed in Table 11.2, are estimated to be $103 million, with 60% of the cost associated with the fermentation equipment. The operating costs for this plant are detailed in Table 11.3. In this analysis, PHA is taken to be the primary product with hydrogen a co-product, providing a credit of

Fig. 11.6 Conceptual schematic diagram of biorefinery to produce hydrogen and PHA coproducts from fibrous biomass (Brown et al. 2003).

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11 Biomass Refineries Based on Hybrid Thermochemical-Biological Processing – An Overview Table 11.2 Estimated capital costs for a biorefinery to produce hydrogen and PHA coproducts from fibrous biomass (Brown et al. 2003). Gasifier Fermenter

$ 18.6 million $ 59.1 million

Separation equipment

$ 25.3 million

Basic Capital

$ 103 million

Estimated from: Larson and Svenningsson (1990) Estimated as 25% of total cost of an ethanol plant Estimated as 30% of total costs of a fermentation plant

Table 11.3 Estimated operating costs for biorefinery to produce hydrogen and PHA coproducts from fibrous biomass (Brown et al. 2003). Annual H2 output

16.4 ´ 106 kg

Annual PHA output Annual input (switchgrass) Total Capital

6.6 ´ 106 kg 192 ´ 106 kg $ 119 million

Raw materials (switchgrass) Credit for H2

$ 9.6 million ($ 42.8 million)

Labor, utilities, maintenance Indirect costs Annual capital charges Annual operating costs PHA Production costs

$ 16.0 million $ 11.5 million $ 13.9 million $ 8.2 million $ 1.24 kg–1

Assumes 20% CO to cell mass; 40% cell mass to PHA 90% capacity factor

Purchased at $ 0.05 kg–1 Assumed to sell for $ 2.60 kg–1 (DOE target price)

10% interest, 20 years

$ 2.60 kg–1, which is based on a US Department of Energy target price for this fuel. The cost of producing PHA through syngas fermentation is estimated to be $ 1.24 kg–1, which is in the range of many petroleum-derived polymers and considerably cheaper than the production cost of PHA from glucose, which may be as much as $ 5–7 kg–1. Until more complete information is available on yields of H2 and PHA from syngas, however, this cost of production should be considered an approximate estimate. 11.3.4 Enabling Technology

In a comprehensive review on the prospects of obtaining ethanol from cellulosic biomass, Lynd (1996) noted that syngas fermentation represents an “end run” with regard to acid or enzymatic hydrolysis of biomass, because it avoids the costly and complicated steps of extracting monosaccharide from lignocellulose. Syngas fermentation, by virtue of reducing all feedstocks to a common set of

11.4 Fast Pyrolysis-based Systems

low-molecular-weight building blocks, is able to accept a wide variety of biomass feedstocks, irrespective of their chemical composition. It also has the potential for being more energy-efficient, because it effectively utilizes all the constituents of the feedstock, whether cellulose, hemicellulose, lignin, starch, oil, or protein. Nevertheless, as described by Grethlein and Jain, syngas fermentation has several barriers to overcome before it can be commercialized (1993). Among these are relatively low rates of growth and production by anaerobes, difficulties in maintaining anaerobic fermentations, product inhibition by acids and alcohols, and difficulties in transferring relatively insoluble CO and H2 from the gas phase to the liquid phase, where the anaerobes can utilize the gas. Of these, mass-transfer limitations are probably the main bottleneck to commercializing this technology. Studies by Worden and coworkers (1997), however, give encouragement that the use of non-toxic surfactants and novel dispersion devices can enhance mass transfer through the generation of microbubbles to carry syngas into bioreactors.

11.4 Fast Pyrolysis-based Systems 11.4.1 Fundamentals of Fast Pyrolysis

Fast pyrolysis is the rapid thermal decomposition of organic compounds in the absence of oxygen to produce liquids, gases, and char (Bridgwater and Peacocke 2000). The distribution of products depends on the biomass composition and the rate and duration of heating. The yield of pyrolytic liquid, also known as bio-oil, depends on pyrolysis conditions, including relatively short residence times (0.5–2 s), moderate temperatures (400–600 8C), and rapid quenching at the end of the process. Rapid quenching is essential if high-molecular-weight liquids are to be condensed rather than further decomposed to low molecular weight gases. Typical product yields for two kinds of wood are given in Table 11.4. Bio-oil from fast pyrolysis is a low viscosity, dark-brown fluid containing up to 15 to 20% water, which contrasts with the black, tarry liquid resulting from slow pyrolysis or gasification (Piskorz et al. 1988). As indicated by Table 11.4, the biooil is a mixture of many compounds although most can be classified as acids, aldehydes, sugars, and furans, derived from the carbohydrate fraction, and phenolic compounds, aromatic acids, and aldehydes, derived from the lignin fraction. The liquid is highly oxygenated, approximating the elemental composition of the feedstock, which makes it highly unstable. Despite the high water content of bio-oil, no appreciable phase separation is apparent (Scott et al. 1999). If an equal volume of water is added to the liquid, however, the high-molecular-weight, largely aromatic compounds, are precipitated. Because most of the aromatic compounds can be traced to the lignin content of the biomass, this precipitate is widely known as “pyrolytic lignin”.

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11 Biomass Refineries Based on Hybrid Thermochemical-Biological Processing – An Overview Table 11.4 Analysis of products from fast pyrolysis (Piskorz et al. 1988). White Spruce

Poplar

Moisture content, % w/w Particle size, lm (max) Temperature Apparent residence time

7.0 1000 500 0.65

3.3 590 497 0.48

Product yields, % w/w, m.f. Water Char Gas Pyrolytic liquid (bio-oil)

11.6 12.2 7.8 66.5

12.2 7.7 10.8 65.7

Gas composition, % w/w, m.f. H2 CO CO2 CH4 C2 hydrocarbons C3+ hydrocarbons

0.02 3.82 3.37 0.38 0.20 0.04

– 5.34 4.78 0.41 0.19 0.09

66.5 – 0.99 2.27 3.96 – 2.49 2.47 – – – 7.67 0.30 0.05 1.24 1.11 0.89 3.86 7.15 34.5 20.6 11.4

65.7 0.70 0.41 1.32 3.04 2.43 1.30 2.18 0.65 1.16 0.02 10.03 – – 1.40 0.12 1.05 5.43 3.09 34.3 16.2 15.2

Pyrolytic liquid composition, % w/ w, m.f. Organic liquid Oligosaccharides Glucose Other monosaccharides Levoglucosan 1,6-anhydroglucofuranose Cellobiosan Glyoxal Methylglyoxal Formaldehyde Acetaldehyde Hydroxyacetaldehyde Furfural Methylfurfural Acetol Methanol Ethylene glycol Acetic acid Formic acid Water-soluble – Total above Pyrolytic lignin Amount not accounted for (losses, water soluble phenols, furans, etc.)

Type of compound

Saccharides

Anhydrosugars

Aldehydes

Furans Ketones Alcohols Carboxylic acids


Bio-oil has several undesirable characteristics (Oasmaa and Czernik 1999). The low pH of bio-oil, which arises from organic acids derived primarily from the hemi-cellulosic content of the feedstock, makes the liquid highly corrosive. The oil contains large quantities of non-volatile carbohydrates and oligomeric phenolic compounds, which prevents complete distillation of the bio-oil. The highly oxygenated product is chemically unstable, with polymerization of double-bonded compounds and etherification and esterification reactions proceeding over time. The liquid contains fine-particulate char, which is thought to promote polymerization. The higher heating values of pyrolysis liquids range between 17 MJ kg–1 and 20 MJ kg–1 with liquid densities of about 1280 kg m–3. Assuming conversion of 72% of the biomass feedstock to liquid on a weight basis, yield of pyrolysis oil is approximately 560 L ton–1. The mechanism by which cellulose, hemicellulose, and lignin in biomass are converted into liquids is not fully understood. Rapid pyrolysis of pure cellulose yields levoglucosan, an anhydrosugar with the same empirical formula as the monomeric building block of cellulose: C6H10O5 (Evans and Milne 1987). Addition of a small amount of alkali inhibits the formation of levoglucosan and promotes the formation of hydroxyacetaldehyde (glycolaldehyde). Pyrolysis of pure cellulose at slower heating rates and lower temperatures favors the formation of char rather than liquids. These observations suggest the multiple reaction pathways for pyrolysis of cellulose illustrated in Fig. 11.7 (Bridgwater and Peacocke 2000). Low temperatures and slow heating rates favor dehydration reactions that ultimately convert the cellulose to char and water. At higher temperatures, depolymerization dominates, yielding levoglucosan as the primary product. The presence of alkali, however, catalyzes the dehydration route but yields hydroxyacetaldehyde instead of char and water if reaction products are removed fast enough. Similarly, hemicelluloses form furanoses and furans as primary reaction products whereas lignin forms monocyclic aromatics and non-condensed bicyclic aromatic materials with high phenolic content. The mechanism by which reaction products are transported from the reaction zone and recovered as liquids is also uncertain (Daugaard and Brown 2004). Many of the reaction products, including levoglucosan, have very low vapor pressures, making vapor transport problematic, but this possibility has not been definitively disproved. Alternatives include transport of low-molecular-weight compounds that condense to higher molecular weight compounds outside the reactor and the elutriation of fine liquid droplets (aerosols) from the reactor.

Fig. 11.7 Reaction pathways in fast pyrolysis.

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Fig. 11.8 Schematic diagram of fast-pyrolysis plant (Brown 2003).

Production of pyrolysis oils and its co-products is illustrated in Fig. 11.8 (Brown 2003). Lignocellulosic feedstocks, such as wood or agricultural residues, are milled to fine particles of less than 1 mm diameter to promote rapid reaction. The particles are entrained in an inert gas stream, which transports the material to the pyrolysis reactor, shown here as a fluidized bed, which provides the prerequisite high heat transfer rates. Within the reactor, the particles are rapidly heated and converted into condensable vapor, non-condensable gas, and solid charcoal. These products are transported out of the reactor into a cyclone operating above the condensation point of pyrolysis vapor where the charcoal is removed. Vapor and gas is transported to a direct-contact quench vessel where a spray of pyrolysis liquid cools and condenses the vapor. The non-condensable gas, which include the flammable gases CO, H2, and methane (CH4), are burned in air to provide heat for the pyrolysis reactor. A number of schemes have been developed for indirectly heating the reactor, including transport of solids into fluidized beds or cyclonic configurations to bring the particles into contact with hot surfaces. There are several problems with bio-oil. Phase-separation and polymerization of the liquids and corrosion of containers make storage of these liquids difficult. The high oxygen and water content of bio-oil makes it incompatible with conventional hydrocarbon fuels. Furthermore, bio-oil is of much lower quality than even Bunker C heavy fuel oil, so upgrading is highly desirable. 11.4.2 Fermentation of Bio-oils

One possibility for upgrading bio-oil is to change the processing conditions to yield a product that is compatible with biochemical processing. Scott and coworkers (1989) at the University of Waterloo in Ontario, Canada recognized that alkali and alkaline earth metals in the biomass serve as catalysts that degrade lignocellulose to char. If these cations are removed by soaking the feedstock in dilute acid before pyrolysis, the lignocellulose is depolymerized to anhydrosu-

11.4 Fast Pyrolysis-based Systems Fig. 11.9 Chemical structure for 1,6-anhydro-beta-D-glucose.

gars at very high yields. Anhydrosugar is a sugar from which one or more molecules of water have been removed, resulting in the formation of an internal acetal structure. The prevalent anhydrosugar from the fast pyrolysis of biomass is 1,6-Anhydro-beta-d-glucose. The chemical structure of this compound, commonly known as levoglucosan, is illustrated in Fig. 11.9. A dimer of levoglucosan, cellobiose, is also produced during fast pyrolysis, but in much lower concentrations. Anhydrosugar has prospects as a platform for chemical synthesis or as a substrate for fermentation. In studies on woody biomass with and without cation removal Piskorz et al. (1997) found that levoglucosan increased from 3.04% in pyrolysis liquid from untreated poplar wood to 30.42% in bio-oil from pretreated poplar wood. Increases were more modest for cellobiose. Brown and his collaborators (2001) evaluated the effect of alkali removal on the pyrolytic products of cornstover, an important herbaceous biomass. Three pretreatments were evaluated: acid hydrolysis, washing in dilute nitric acid, and washing in dilute nitric acid with addition of (NH4)2SO4 as a pyrolytic catalyst. Although alkali compounds in plant materials are generally water-soluble, attempts to remove alkali by water washing did not prove effective in this study. On the other hand, all three acid treatments were able to substantially increase the yield of anhydrosugars, as shown in Table 11.5. Acid hydrolysis of this anhydrosugar yielded 5% solutions of glucose and other simple sugars. The resulting glucose solutions can be fermented, as demonstrated by Prosen et al. (1993). However, the substrate derived from the bio-oil contains fermentation inhibitors that must be removed or neutralized by chemical or biological methods. Chemical methods that have been evaluated on bio-oil-derived substrate include solvent extraction, hydrophilic extraction, and adsorption extraction (Brown et al. 2000). The most effective of the chemical methods employed activated carbon. As a less expensive alternative, Khiyami (2003) explored biological treatments. He found that biofilms of Pseudomonas putida and Streptomyces setonii were able to remove toxins from substrates derived from bio-oil. As an alternative to hydrolyzing levoglucosan to glucose, several microorganisms have been identified that directly ferment levoglucosan (Kitamura et al. 1991; Nakahara et al. 1994; Zhuang et al. 2001 a, b). This would eliminate the hydrolysis step and probably improve the economics of producing fermentation products from bio-oil.

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11 Biomass Refineries Based on Hybrid Thermochemical-Biological Processing – An Overview Table 11.5 Products of pyrolysis for several different pretreatments of cornstover (Brown et al. 2001). No pretreatment

Acid hydrolysis

Demineralization

Demineralization with catalyst

Pyrolysis products (% w/w maf) Char 15.8 Water 2.57 Organics 59.1 Gases 22.6

13.2 10.6 67.2 9.02

13.2 10.4 68.5 7.88

15.9 7.96 67.7 8.44

Organics (% w/w) Cellobiosan Levoglucosan Hydroxyacetaldehyde Formic acid Acetic acid Acetol Formaldehyde Pyrolytic lignin

4.55 17.69 5.97 Trace 1.51 Trace 1.63 16.89

3.34 20.12 3.73 Trace 1.26 Trace trace 17.74

4.97 23.10 3.93 0.73 0.40 Trace 0.70 20.08

Trace 2.75 11.57 2.61 3.40 4.53 2.75 33.40

11.4.3 Biorefineries Based on Fast Pyrolysis

One manifestation of a biorefinery based on fermentation of bio-oil is illustrated in Fig. 11.10. Fibrous biomass is pretreated with dilute acid to simultaneously remove alkali and hydrolyzes the hemicellulose fraction to pentose. The remaining fraction, containing cellulose and lignin, is pyrolyzed at 500 8C to yield char, gas, and bio-oil. The bio-oil is separated into pyrolytic lignin and levoglucosanrich aqueous phase. The char, gas, and lignin are burned to generate steam for distillation and other process heat requirements of the plant and the levoglucosan is hydrolyzed to hexose. The pentose and hexose are fermented to ethanol. So and Brown (1999) compared the cost of producing ethanol from cellulosic biomass using fast pyrolysis combined with a fermentation step to acid hydrolysis and enzymatic hydrolysis technologies. The azeotropic ethanol production capacity used in this case study was 95 million L year–1 and the assumed cost for biomass was $ 46 ton–1 (1997 US dollars). As summarized in Table 11.6, total capital investment for a plant based on fermentation of bio-oil was estimated to be $ 69 million, while the annual operating cost was about $ 39.2 million, resulting in an ethanol selling price of $ 0.42 L–1. This is about 23% higher than ethanol from plants based on acid hydrolysis and enzymatic hydrolysis of biomass, but well within the uncertainty of the analysis (30%). A more advanced concept for a biorefinery based on fast pyrolysis is illustrated in Fig. 11.11 (Sandvig et al. 2004). This biorefinery integrates pyrolysis with combined cycle (IPCC) power. Biomass is first washed to remove alkali before it is pyrolyzed.


Fig. 11.10 Schematic diagram of cellulosic biomass-to-ethanol based on fast pyrolysis.

Table 11.6 Comparing production cost of ethanol from cellulosic biomass for three conversion technologies (1997 US $). Fast pyrolysis

SSF a)

Acid hydrolysis

Annual ethanol output Annual biomass input Total capital

95 million L 240 ´ 106 kg $ 69 million



Raw materials Labor, utilities b), maintenance Indirect costs Annual capital charges Annual operating costs

$ 11.1 $ 6.18 $ 8.07 $ 13.8 $ 39.2

$ 11.3 million $ 0.9 million $ 7.13 million $ 12.8 million $ 32.1 million

$ 11.0 $ 2.13 $ 7.21 $ 13.3 $ 33.7

Production cost of ethanol

$ 0.42 L–1

$ 0.34 L–1

$ 0.35 L–1

a) b)

million million million million million

million million million million million

Simultaneous saccharification and fermentation (enzymatic hydrolysis) Includes credit for steam generation for SSF and acid hydrolysis processes

Pyrolytic char is recovered for additional processing to activated carbon or marketed as a soil amendment. Pyrolytic gas is used to heat the pyrolysis reactor. Bio-oil is recovered in a specially designed quencher intended to fractionate the bio-oil into different chemicals, including levoglucosan. The levoglucosanrich fraction is either purified or used as substrate for fermentation to additional chemical products (not illustrated). The other fractions of bio-oil are used to fire the gas turbine cycle to produce electricity. The waste heat from the gas turbine is directed to a waste heat recovery generator, which provides power to a Rankine steam turbine bottoming cycle. Advantages of the biomass-fueled IPCC system include: cycle efficiency exceeding that of biomass-fired Rankine cycles; avoids need for high-pressure thermal

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Fig. 11.11 Biorefinery based on fast pyrolysis incorporating combined cycle power and chemical recovery (Sandvig et al. 2004).

reactors; reduces the strong coupling between fuel processing and power generation typical of integrated power systems; and provides opportunities for recovering value-added products. The proposed IPCC system is estimated to have a total project cost of between $ 2300 and $ 2500 kW–1 based on a net combined cycle output of 7655 kW. This cost does not include the additional equipment required for production of value-added chemicals. This capital cost compares favorably with that of conventional biomass power systems in this range, which cost around $ 2000 for basic systems to over $ 3000 kW–1 for systems designed for higher efficiency and reliability. The cost of electricity for the IPCC plant is projected to be similar to the costs for conventional biomass power systems, approximately $ 0.02 kWh–1. 11.4.4 Enabling Technologies

When Scott and coworkers (1989) first proposed a pyrolytic route to cellulosic ethanol, it was offered as a way of leapfrogging the barriers to fractionating biomass. In a simple, rapid process, fast pyrolysis was able to separate lignocellu-


lose into a pyrolytic lignin and a carbohydrate-rich aqueous phase. The process introduces its own set of technical barriers that have yet to be fully solved, however. Fast pyrolysis of pure cellulose produces, in principle, levoglucosan as its sole reaction product. In practice, the presence of lignin and a variety of inorganic compounds in fibrous biomass results in more than a hundred chemical products, many of which are not only unsuitable as a carbon and energy source for fermentation but are actually toxic to the microorganisms to be cultivated. Improved selectivity of pyrolytic reactions will be important to achieving high yields of fermentable carbohydrate. Understanding reaction pathways will be the key to success in this endeavor. Like many of the pre-treatment processes used to facilitate fractionation by acid or enzymatic hydrolysis, fast pyrolysis generates biological inhibitors that must be removed before the bio-oil is used as a fermentation substrate. Methods that are more cost-effective than adsorption with activated carbon must be developed. Like any process for the production of ethanol from biomass, efficient use of the hemicellulosic and lignin fractions of the lignocellulose will be essential to economic viability. In principle, the pentoses released during demineralization of the biomass can be fermented and the pyrolytic lignin can be used in the production of process steam. Effective use of these coproducts will require more attention to integrating the individual processes making up a system for pyrolytic production of cellulosic ethanol.


Thermochemical processing of biomass to produce substrates suitable for fermentation is a relatively new and unexplored approach to biobased products. Two distinct routes for hybrid thermochemical-biological processing have been offered in this paper: (1) gasification then fermentation of the syngas, and (2) fast pyrolysis then hydrolysis and/or fermentation of the anhydrosugars in the resulting bio-oil. The syngas route, by transforming all the plant constituents into CO and H2, is attractive for its efficient use of biomass. The fast pyrolysis route, by yielding a storable carbohydrate-rich liquid, enables processing of the solid biomass to be decoupled from fermentation and offers prospects for distributed processing of widely dispersed biomass resources. Both have an advantage over hydrolytic methods in that they are able to process a wider variety of feedstocks, although this is especially true for the gasification route. Compared with acid and enzymatic hydrolysis, relatively few resources have been devoted to developing hybrid thermochemical-biological routes to biobased products. The reason for this circumstance is easy to understand – the original feedstocks of the fermentation industry were naturally occurring sugars and starches that were easily hydrolyzed to sugar. The fact that starch and cellulose

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are both polymers of glucose encouraged similar approaches to depolymerizing these two carbohydrates. In fact, cellulose is not only more recalcitrant than starch but it is imbedded in a matrix of lignin, which makes the process of releasing sugar from lignocellulose much more difficult than for starch. Considering these difficulties, hybrid thermochemical-biological approaches to biobased products deserves increased attention.

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Brown, R. C., D. Radlein and J. Piskorz (2001). Pretreatment Processes to Increase Pyrolytic Yield of Levoglucosan from Herbaceous Feedstocks. Chemicals and Materials from Renewable Resources: ACS Symposium Series No. 784. Washington, D.C., American Chemical Society: 123–132. Datar, R. P., R. M. Shenkman, B. G. Cateni, R. L. Huhnke and R. S. Lewis (2004). “Fermentation of biomass-generated producer gas to ethanol.” Biotechnology and Bioengineering 86(5): 587–594. Daugaard, D. E. and R. C. Brown (2004). The transport phase of pyrolytic oil exiting a fluidized bed reactor. Science in Thermal and Chemical Biomass Conversion Conference, Victoria, British Columbia, Canada. Dispirito, A. (2004). Personal communication. Evans, R. J. and T. A. Milne (1987). “Molecular characterization of the pyrolysis of biomass.” Energy and Fuels 1: 123–137. Ferry, J. G. (1995). “CO dehydrogenase.” Annual Review of Microbiology 49: 305–333. Grethlein, A. J. and M. K. Jain (1993). “Bioprocessing of coal-derived synthesis gases by anaerobic bacteria.” Trends in Biotechnology 10: 418–423. Grethlein, A. J., R. M. Worden, M. K. Jain and R. Datta (1990). Applied Biochemistry and Biotechnology 24/25: 875. Kerby, R. L., P. W. Ludden and G. P. Roberts (1995). “Carbon monoxide-dependent growth of Rhodospirillum rubrum.” J. Bacteriology 177: 2241–2244. Kessler, B., R. Weusthuis, B. Witholt and G. Eggink (2001). “Production of Microbial Polyesters: Fermentation and Downstream Processes.” Advances in Biochemical Engineering/Biotechnology and Bioengineering 71: 159–182.

References Khiyami, M. A. (2003). Biological methods for detoxification of corn stover and corn starch pyrolysis liquors, Ph.D. Thesis, Iowa State University. Kim, Y. B. and R. W. Lenz (2001). “Polyesters from Microorganisms.” Advances in Biochemical Engineering/Biotechnology 71: 51– 79. Kitamura, Y., Y. Abe and T. Yasui (1991). “Metabolism of levoglucosan (1,6-Anhydro-b-d-glucopyranose) in microorganisms.” Agric. Biol. Chem. 55: 515–521. Ko, C. W., J. L. Vega, E. C. Clausen and J. L. Gaddy (1989). “Effect of high-pressure on a co-culture for the production of methane from coal synthesis gas.” Chemical Engineering Communications 77: 155–169. Larson, E. D. and P. Svenningsson (1990). Development of biomass gasification systems for gas turbine power generation. Energy from Biomass and Wastes XIV, Lake Buena Vista, FL, USA. Published by Inst Gas Technology, Chicago, FL, USA, pp. 1–19. Lynd, L. R. (1996). “Overview and evaluation of fuel ethanol from cellulosic biomass: technology, economics, the environment, and policy.” Annu. Rev. Energy Environ. 21: 403–465. Madhukar, G. R., B. B. Elmore and H. K. Huckabay (1996). “Microbial conversion of synthesis gas components to useful fuels and chemicals.” Applied Biochemistry and Biotechnology 57/58: 243–251. Maness, P. C. and P. F. Weaver (1994). “Production of Poly-3-Hydroxyalkanoates from CO and H2 by a Novel Photosynthetic Bacterium.” Applied Biochemistry and Biotechnology 45/46: 395–406. Miyake, M., M. Erata and Y. Asada (1996). J. Fermentation Bioengineering 82: 512. Nakahara, K., Y. Kitamura, Y. Yamagishi and H. Shoun (1994). “Levoglucosan dehydrogenase involved in the assimilation of levoglucosan in Arthrobacter sp. I-552.” Biosci. Biotech. Biochem. 58: 2193–2196. Oasmaa, A. and S. Czernik (1999). “Fuel oil quality of biomass pyrolysis oils – State of the art for the end users.” Energy and Fuels 13: 914–921. Phillips, J. R., K. T. Klasson, E. C. Clausen and J. L. Gaddy (1993). “Biological production of ethanol from coal synthesis gas.”

Applied Biochemistry and Biotechnology 39/40: 559–571. Piskorz, J., P. Majerski, D. Radlein, D. S. Scott, Y. P. Landriault, R. P. Notarfonzo and D. K. Vijh (1997). Economics of the Production of Fermentable Sugars from Biomass by Fast Pyrolysis. Third Biomass Conference of the Americas, Montreal, Ontario, Canada. Piskorz, J., D. S. Scott and D. Radlein (1988). Pyrolysis Oils from Biomass. ACS Symposium Series 376. E. J. Soltes and T. A. Milne. Washington, DC, American Chemical Society: 167–178. Prosen, E. M., D. Radlein, J. Piskorz, D. S. Scott and R. L. Legge (1993). “Microbial utilization of levoglucosan in wood pyrolysate as a carbon and energy source.” Biotechnol. and Bioengineering 42: 538–541. Reed, T. (1981). Biomass Gasification: Principles and Technology. Park Ridge, N.J., Noyes Data Corp. Sandvig, E., G. Walling, D. E. Daugaard, R. J. Pletka, D. Radlein, W. Johnson and R. C. Brown (2004). “The prospects for integrating fast pyrolysis into biomass power systems.” International Journal of Power and Energy Systems 24(3): 228–238. Schlegel, H. G., G. Gottschalk and R. von Bartha (1961). Nature 191: 463. Scott, D. S., S. Czernik, J. Piskorz and D. Radlein (1989). Sugars from biomass cellulose by a thermal conversion process. Energy from Biomass and Wastes XIII, New Orleans, LA, USA, Published by Inst of Gas Technology, Chicago, IL, USA, pp. 1349– 1362. Scott, D. S., P. Majerski, J. Piskorz and D. Radlein (1999). “A second look at fast pyrolysis of biomass – the RTI process.” J. Analytical and Applied Pyrolysis 51: 23–37. So, K. and R. C. Brown (1999). “Economic analysis of selected lignocellulose-to-ethanol conversion technologies.” Applied Biochemistry and Biotechnology 77–79: 633– 640. Spath, P. L. and D. C. Dayton (2003). Preliminary Screening – Technical and Economic Assessment of Synthesis Gas to Fuels and Chemicals with Emphasis on the Potential for Biomass-Derived Syngas. National Renewable Energy Laboratory, Technical Re-

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Green Biorefineries 12 The Green Biorefinery Concept – Fundamentals and Potential Stefan Kromus, Birgit Kamm, Michael Kamm, Paul Fowler, and Michael Narodoslawsky

12.1 Introduction

Green biorefineries are integrated technologies and technology systems for production of materials and energy processing of green plants and parts of green plants. Above all, green biorefinery technologies are based on traditional technologies of green forage preservation, leaf-protein extraction, chlorophyll production, and modern biotechnological and chemical conversion methods. The main raw material of green biorefineries are green plants, for example grass, alfalfa, and immature (green) grain or green plant parts, for example leaves. Green plant parts are a virtually inexhaustible raw material reservoir, which is fast-growing, available world-wide, and may have ecological advantages. Considering alfalfa alone, 32 million hectares are currently cultivated and converted into green pellets or forage flour worldwide. By means of primary photosynthesis green C3 plants can yield up to 20 tons dry matter with up to four tons of proteins in temperate climates each year. C4 plants in tropical zones can, however, produce up to 80 tons dry matter with six tons of proteins per hectare per year. Green plants are, moreover, rich in carbohydrates, proteins, lipids, lignin, and a group of secondary plant substances and phytochemicals. Green plants and green plant parts may therefore be seen as a chemical plant with a huge potential. In comparison with other vegetable biomass green plants are characterized by a high content of aqueous cell juice with carbohydrates of low molecular weight, a large amount of enzymes (proteins) for photosynthesis and a relatively low content of lignin in the cell walls. For these reasons all technological concepts of green biorefineries include the separation of the cell juice from the plant framework. Both fractions, the cell Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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juice and the cellulose-containing leaf cells are subjected to different biotechnological and physicochemical conversion methods.

12.2 Historical Outline 12.2.1 The Inceptions

The green parts of plants have been used by the human being for ages. Primarily ruminants use green plants as fodder. The relevance of green crop cultivation was already apparent from the occupation of the sons of Adam and Eve, Cain and Abel – Abel became a shepherd, Cain a farmer [1]. The direct use of green plants in the diet of humans is ancient. We are able to distinguish between use as nutrition or for salvation or as a natural stimulant. In Europe the consumption of spinach (Spinacia oleracea), stinging-nettle (Urtica), sorrels (Rumex acetosa), curly kale (Brassica oleraceae var. Sabellica), and leek (Allium ampelobrasum) is very common. The chemical and biochemical scientific exploration of green plant leaves can be traced back to the 18th century. The French chemists G. F. and H. M. Roulle reported obtaining protein extracts from alfalfa leaves in 1773. Hillaire Marin Roulle in particular, a demonstrator at the royal garden in Paris since 1770, proved that the juices obtained by pressing the vegetable alfalfa contain a substance which coagulated when heated into a “cheesy” substance similar to animal material. The new coagulate, found by Roulle in the heated juice obtained by pressing different plants, was therefore called “vegato-animale” substance (today it is called leaf protein) [2]. Mild warming yielded a green colored fraction, further heating delivered an almost colorless precipitate. Chemical analysis of the colorless substance revealed it contained a larger amount of that new substance than the green coagulate. Also, by extraction of the green pigments with alcohol the green precipitate could be removed [2]. It is impressive that key procedures of modern protein separation of green biomass in green biorefineries were researched as early as 1773 [3–7]. The functional characteristic of thermal coagulation of proteins was, moreover, the first criterion for later definition of proteins as a class of substance [3]. 12.2.2 First Production of Leaf Protein Concentrate

In the 20th century research on leaf protein concentrate (LPC) focused on use of the proteins for human nutrition [4–6], because of the widespread availability, high nutritional value, and high protein productivity in green plants, with its valuable spectrum of essential amino acids and the intensive growth of green


plants under climatically favorable conditions. Technical manipulations are necessary to separate the leaf protein in a form digestible by humans, because the human being is not equipped with the opportunity to digest leaf cells and, particularly, their cellulose membrane as ruminants do. Of special economic interest is a fraction expressed in the chloroplasts of green plants. The so called fraction-I-protein is the photosynthesis enzyme ribulose-1,5-biphosphatecarboxylase/oxygenase or rubisco, which can be regarded as the most widespread protein in the world. In spinach leaves, rubisco accounts for 75% of soluble proteins; in wheat and barley it is 53–75% and in corn and sweet sorghum (sugar millet) (Sorghum dochna) 15% [8, 9]. It must, however, be stressed that by means of electrophoresis approximately 250 to 300 different proteins and polypeptides can be detected in green-plant extracts [10, 11]. In alfalfa (Medicago sativa L.) rubisco amounts for 30–70% of the soluble proteins, depending on genotype and vegetation cycle [9, 12]. On the basis of the protein harvest from one hectare of arable land, alfalfa provides a 3 to 10-fold higher yield than oilseeds, grain legumes, or grain [9]. In times of crises, especially, the topic of leaf protein for nutrition has repeatedly been put on the nutrition agenda. For example, in 1917 the use of alfalfa flour for bread manufacture was reported in the “Literary digest”. In 1920 and 1921 Osborne and Chibnell published their results of examinations of proteins in green leaves. Ereky proposed the utilization of leaf proteins for public nutrition in 1925 and Slade renewed this suggestion in 1937. Slade and Birkinshaw were the first to patent the utilization of grass and other green plants in 1939 [13]. These developments were favored by results from examinations which proved that green forage supplies ruminants with more proteins and essential amino acids than they can actually utilize. Ruminants like cows, oxen, and sheep need, on a dry matter basis, only 16% of crude protein in their feed. Alfalfa and grasses, however, contain 22–28% crude protein on dry matter basis [14]. The opportunity to simultaneously provide benefits for both animals and humans from green plants would result from removal of the “surplus” protein before feeding the green fodder to ruminants. This idea evolved for the first time during World War II. Because of the manure-nitrogen problem, due to the industrialization of livestock farming, the idea underwent a revival. During World War II, researchers and developers stressed the importance of leaf protein concentrate for providing the population with sufficient protein because of food-supply shortages in Europe. After the occupation of France by the Germans in 1940, Great Britain in particular, was cut off from the food supply on the continent. Thus, the United Kingdom enforced large-scale developments and put priority on nutrition by use of green-plant proteins. All large-scale developments revealed technical problems and were not very profitable, and development was stopped because of the American-British Land Lease Agreement in 1941. Nevertheless, young Pirie, Scientist at the Rothamsted Experimental Station in Hertfordshire, UK, was able to accomplish important pioneering work for later industrial production of leaf protein [15–17].

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In the sixties development of production plants utilizing green leaves began again. Five main reasons can be identified for the relaunch of leaf protein plants. First, forecasts suggested a lack of protein-rich products worldwide. Second, the industrial livestock farming which was developing demanded standardized and metered feeding. Third, the interest of the industrialized world in the nutritional problems of the developing countries was increasing. Fourth, the member states of the Warsaw Pact wanted to separate themselves from the world market and generated their own supply of food and feed protein. Finally, the rising cost of energy (the later oil price shock) focused interest on extraction of leaf protein as alternative to other means of green forage conservation. The first modern industrial process for leaf protein extraction was called the Rothamsted process, developed by Pirie. The procedure based on heat coagulation of green plant juice at 70 8C, resulted in leaf protein concentrates with 60%

Fig. 12.1 Flow chart of the fractionation of green plant parts for extraction of leaf protein concentrate.


protein content and a lipid content of 20 to 25% (lipid–protein concentrate) [4, 18]. Later, processes were developed which were based on heat coagulation at 80 8C [19, 20]. Finally, procedures based on two-step heating of the green press juice enabled fractionated extraction of proteins, leading to products of different composition [21, 22]. The basic fractionation process is the same for all concepts, as shown in Fig. 12.1. Delays between harvesting in the field and processing in the factory should be reduced to a minimum. After grinding and crushing in mills the green matter is pressed. Wet fractionation (pressing) leads to two fractions – the press cake and the press juice. The press cake is rich in crude fiber and the press juice contains proteins, water-soluble sugars (WSC), ash and other interesting substances, for example lutein. The developments mentioned above resulted throughout the seventies in market-leading technologies such as the Proxan procedure and the Alfaprox procedure, which are used for generation of protein–xanthophyll concentrates, including utilization of the by-products, although predominantly in agriculture [14, 23]. 12.2.3 First Production of Leaf Dyes

Chlorophylls, often termed the “pigments of life”, are green colored macrocyclic pigments which are the primary photosynthetic pigments in nature. The term chlorophyll, coined by Berzelius in 1838, is derived from Greek roots and indicates the green of leaves [24]. In fact, as green pigments they are responsible for the primary biochemical energy generation in nature and give the only indications of life on earth visible from outer space. Reduction of the chlorophyll (leaf green) gives the xanthophylls (leaf yellow) and carotenes that are not dissolved and remain in the leaf, resulting in showy yellow and orange tinges. The red color pigments are derived from anthocyan, created by metabolic alteration of the leaves [25]. Although scientists had previously studied the green plant pigment it was only in 1913 that the first significant research on its structure, separation, and properties was reported. This work, which won the 1915 Nobel Prize for the German chemist Willstätter, serves as the basis for subsequent production of chlorophyll [26, 27]. In the United States, commercial production of chlorophyll and carotene by extraction from alfalfa leaf meal has conducted since 1930 [28, 29]. Chlorophyll, in various forms, was reported to be present in 1000 products that consumed 10 000 pounds of the green material per month in 1952, with a market value of 50 Million USD [30]. For example, Strong, Cobb and Company obtained 0.5 ton of chlorophyll per day from alfalfa in 1952. The water-soluble chlorophyll, or chlorophyllin, found use as a deodorizing agent in toothpastes, soaps, mouthwashes, shampoos, chewing gums, candies, deodorants, and pharmaceuticals [31].

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Since 1990 chlorophyll has also been used for conversion of light energy into electric energy. Electrochemical solar cells, e.g. the Graetzel cell a TiO2–chlorophyll-SnO2 solar cell, use organic dyes (not a semiconductor material), for example the leaf dye chlorophyll, for absorption of light [32].

12.3 Green Biorefinery Raw Materials 12.3.1 Raw Materials

The major raw material of green biorefineries is “green biomass”, including the large group of green plant materials (green grass from meadows, willow, extensive willow management, other natural resources), wild fruit and crops, alfalfa and clover, and immature cereals and plant shoots. The green plant material contains complex natural and valuable materials in the form of carbohydrates, proteins, fibers, fragrances, dyes, fats, hormones, amino acids, enzymes, and other important substances [5, 7, 33, 34]. Ecologically friendly agriculture is based on primary production by photosynthesis in green plants during the whole growth season. During a vegetation period successive harvest and re-growth of one and the same crop can give a maximum yield of dry matter and protein per area. Green grasses and immature cereals are excellent for this purpose. Especially, grasses can be grown on most types of soil in most types of climate, on both normal agricultural land and marginal land. Thus C3 species in temperate climates can yield up to 20 tonnes of dry matter and 4 tons of protein per ha (hectare = 10 000 m2) per year whereas C4 species in tropical climates can produce 80 tonnes of dry matter and 6 tonnes of protein [33, 34]. The yield of dry matter and protein from grass and the quality of the leaf protein concentrate (LPC) obtained is affected by the type of photosynthesis [35]. Leaf anatomy and cell structure are different for the two types of plant adapted to different climates. The C4 species have a more efficient carbon dioxide fixation mechanism and are grown on soils poor in nitrogen. Thus C4 species have a very high dry-matter production per soil area, but have a low protein content of the dry matter. The C3 species lose fixed carbon dioxide by a process called photorespiration. Thus C3 plants produce less dry matter per unit area. In C3 species more leaf cells are rich in protein (FI protein/rubisco protein). Therefore, a relatively high proportion of the dry matter of C3 species consists of protein. Subsequently, LPC from C3 species is rich in protein. Both temperate grass species, including green cereals [34, 35] and tropical grasses [36], have been investigated for LPC production (Table 12.1). The second important raw material source is the green harvesting residue material from agricultural cultivated crops. In particular the vegetables of importance are those with green foliage. This includes, e.g., not insignificant

12.3 Green Biorefinery Raw Materials Table 12.1 Grasses and green cereals investigated for green crop fractionation [34, 35]. Avena sativa Bromus arvensis Cynodon dactylon Dactylis glomerata Festuca arundinaceae Festuca pratensis

Holcus lanatus Hordeum vulgare Lolium multiflorum Lolium perenne Paspalum dilitatum

Pennisetum purpureum Phalaris arundinacea Secale cereale Tricum aestivum Zea mays

amounts of sugar beet leaves (sugar beet for the sugar industry), hemp scrapes and leaves (hemp for fiber production), residues from flax processing, and residues from the fresh vegetable production. Further potential refinery raw materials are the little standardized juice-rich waste biomass. This should contain moisture and mainly natural and valuable materials or have a substantial conversion grade. According to coupling effects of material and energy use, the constitution can strongly vary. Such waste biomass is not yet standardized, but is a renewable natural waste resource that must be managed. Such biomass can be residues from plant production (mixed and ripe harvest residuals), potato juice, hydroxycarboxylic acid-rich waste as silage seepage, juices from the canned food industry, or residues from the sugar industry or animal production. The fourth large group is the little standardized dried biomass and waste biomass. These often contain a large amount of plant cellulose and will therefore be supplied as raw material to press-cake-using production lines. This can be residual straw, hay, and all kinds of dried foliage (e.g. maize hay). Residues from in-plant waste paper and wood, e.g. for energy production or cardboard production, are also included in this category. This group also includes modern concepts of dry crop fractionation, for example immature cereals [37]. It should be mentioned that transitions between raw material types will and should be fluid [38]. 12.3.2 Availability of Grassland Feedstocks for Large-scale Green Biorefineries

In Europe, grassland amounts to about 45 Mio ha, which is approximately 35% of the arable land (basis: 15 member states without new member states). A large part of this grassland is regarded as absolute grassland habitat which cannot legally be converted into plain arable land. Based on an average yield of 10 tons dry matter per ha per year, however, the European grassland produces about 450 Mio tons dry matter each year [39]. The main purpose of grassland cultivation is still the production of forage for animal farming. The use of grassland for feed production is dropping, because limitation of production quotas and the increasing efficiency of animal breeding (especially dairy farming) is leading a decrease in livestock numbers. Other uses for grassland must there-

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12 The Green Biorefinery Concept – Fundamentals and Potential Table 12.2 Production of green pellets or powder in Europe. Country

Amount dry matter (t a–1)

Country

Amount dry matter (t a–1)

Germany Austria Belgium Denmark Spain Finland France Greece

320 000 1 292 3 600 170 868 2 100 678 1518 1 398 445 48 848

The Netherlands Ireland Italy Portugal Great Britain Sweden Czech Republic Europe

214 300 5 337 704 000 3 734 56 539 11 571 29 158 5 069 888

fore be found, for example the supply of raw material for the bio-industry. This trend is apparent throughout Europe [40]. Because of its ability to fix nitrogen from the air and enrich the soil with this element, alfalfa is the most important forage crop in the world, cultivated on approximately 32 million hectares. The plant contains the protein rubisco as approximately 20% of the total dry matter [41]. Green forage drying plants, especially, offer a very good possibility for use in biorefinery systems. These plants can be seen as agro–industrial knots in grassland farming. More than 300 green forage drying plants are used to produce over 5 Mio tons of dried pellets and powder (Table 12.2) [42]. In the USA the Alfalfa New Products Initiative (ANPI) has the objective of extending the cultivation and utilization of alfalfa. The ANPI consists of five states: North Dakota, South Dakota, Minnesota, Wisconsin, and Michigan. Prominent technology in this context is dehydration and fractionation (dry or wet) [43]. 12.3.3 Key Components of Green and Forage Grasses

The research literature on forage grasses is mainly concerned with their nutritive aspects as fodder grass, hay, or silage. From a biochemical perspective the composition of forage grasses is well described. Thus, a comprehensive inventory of forage grass chemical/material constituents is available in the literature. The components can be conveniently categorized according to their location within the grass, either as a cell wall constituent or as a component within the cell.

12.3.3.1 Structural Cell Wall Constituents Cell wall constituents comprise structural polysaccharides (hemicellulose, cellulose), lignin, and pectin substances. The qualitative and quantitative composition of constituents within the cell walls varies during the growing season.

12.3 Green Biorefinery Raw Materials

Hemicellulose, Cellulose, and Lignin The hemicellulose, cellulose, lignin, and crude fiber content of fresh herbage, hay, and silage from meadow grasses has been compared [44]. The crude fiber content of grass harvested as fresh herbage was 24.0–35.5% of the dry matter (DM). The crude fiber content increased with delay in harvesting and was higher in hay and silage than in the fresh herbage. The combined total hemicellulose, cellulose, and lignin content was twice that of crude fiber in grasses. During ensiling of grasses the hemicellulose content was reduced by an average of 3–11%, the cellulose remained unchanged, and the lignin content increased by 23%. With advancing maturity, the concentrations of cellulose, hemicellulose, and lignin, in grasses increase. In general, the digestibility of cellulose decreases during the growing season; this commonly attributes to an increasing lack of accessibility of the polymer to be attacked by microorganisms [45]. This variation should be considered when assessing grasses as feedstock for industrial processes, particularly when a decision to harvest a forage grass specifically for its fiber content must be made. Chemical composition and digestibility were studied in vivo and in vitro [45]. The main finding was that although the grass species studied had similar gross chemical composition, the digestibility varied substantially at comparable stages of maturity. Thus a rapid decrease in digestibility was observed between the two first cuts whereas only small changes were observed between the two times of harvesting the re-growth. Although digestibility in this study was studied with reference to ruminants, variation in this property may become significant when considering the use of grass as an industrial fermentation feedstock, for example xylitol or lactic acid production. The more digestible rye grasses have two to three times more (1 ? 4)-linked d-xylose units without branch points at the O-2 and O-3 positions, the proportions of those branch points being substantially reduced. The changes were greater in the early cut samples [46]. Similarly, it has been noted that re-growth has a lower nutritional value than the first cut at a comparable stage of growth. Dactylis glomerata and Lolium perenne were cut 1 to 3 times and analyzed chemically. The material from the first cutting had the highest total digestible nutrient content, 55.96%. Protein utilization value was lowest in the third cut grass [47]. The amounts of d-galactose and other carbohydrates were much lower in the re-growth [48]. Åman and Lindgren studied [49] the change in the chemical composition and degradability of six grasses including Festuca pratensis, Festuca arundinacea, and Dactylis glomerata which were harvested at two stages of maturity in both the first and second cuts. The results are shown in Tables 12.3 and 12.4. Composition studies have also been driven by recognition of the effect of covalent binding between the cell wall polymers on utilization of the cell wall as a nutrient source. Morrison investigated [45] variations in the hemicellulose and lignin composition of grasses over the growing season. It is known that these two cell components are covalently bonded and it is believed that the lignin has a substantial

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12 The Green Biorefinery Concept – Fundamentals and Potential Table 12.3 Composition of grasses harvested at early first cut and late first cut (% of DM of unextracted material, sugar residues given as anhydrosugars). Dactylis glomerata

Festuca pratensis

Festuca arundinacea

31.5 16.6 37.5 0.1 2.6 11.2 0.2 1.4 19.1 2.9 1.4 7.7 10.0 51.4 28.5 4.2

29.2 14.2 38.8 0.1 2.6 10.6 0.1 1.0 21.3 3.1 1.6 10.3 10.2 55.4 31.4 5.2

28.2 14.3 40.5 0.1 2.8 13.1 0.2 0.9 20.0 3.4 1.3 9.1 10.0 55.4 30.8 4.8

Residual organic matter in vitro 12.1 in vivo 25.8 Leaf percent 48.0

15.8 22.8 41.0

16.5 27.1 42.0

Late first cut 80% Ethanol extract Crude protein Polysaccharides Rhamnose Arabinose Xylose Mannose Galactose Glucose Uronic acids Glu/Xyl + Ara Klason lignin Ash NDF ADF Permanganate lignin

28.1 10.6 44.2 0.1 2.8 12.1 0.2 0.8 25.3 3.0 1.7 13.2 8.6 57.9 32.6 6.0

24.9 9.8 48.3 0.1 3.1 15.5 0.2 0.9 25.4 3.1 1.4 13.8 8.5 62.3 35.6 6.2

26.6 9.8 44.9 0.1 2.5 14.4 0.2 0.9 22.9 4.0 1.4 15.0 8.5 62.4 34.3 5.7


24.6 32.2 34.0

28.9 35.7 25.0

Early first cut 80% Ethanol extract Crude protein Polysaccharides Rhamnose Arabinose Xylose Mannose Galactose Glucose Uronic acids Glu/Xyl + Ara Klason lignin Ash NDF ADF Permanganate lignin

12.3 Green Biorefinery Raw Materials Table 12.4 Composition of grasses harvested at early second cut and late second cut (% of DM of unextracted material, sugar residues given as anhydrosugars). Dactylis glomerata

Festuca pratensis

Festuca arundinacea

22.2 9.9 47.2 0.1 3.2 11.9 0.2 1.2 26.3 4.2 1.7 13.1 9.7 65.1 40.0 8.3

22.7 10.0 44.8 0.2 3.2 10.2 0.2 1.5 26.2 3.2 2.0 13.8 11.5 60.5 37.0 5.4

25.6 10.0 44.1 0.2 3.1 12.5 0.2 1.1 24.0 3.1 1.5 13.8 10.9 61.2 34.0 5.0


19.4 28.4 73.0

18.4 29.2 77.0

Late second cut 80% Ethanol Extract Crude Protein Polysaccharides Rhamnose Arabinose Xylose Mannose Galactose Glucose Uronic acids Glu/Xyl + Ara Klason lignin Ash NDF ADF Permanganate lignin

24.3 9.0 45.8 0.3 2.9 10.5 0.2 1.2 26.7 4.0 2.0 16.0 9.8 63.5 41.2 8.6

22.0 8.7 46.9 0.2 3.2 11.8 0.4 1.7 26.4 3.3 1.8 19.0 10.6 62.3 39.2 7.4

24.8 9.7 43.3 0.1 2.9 11.2 0.2 1.3 23.4 4.2 1.7 12.9 11.5 60.6 34.7 5.7


19.9 29.6 81.0

16.8 30.1 77.0

Early second cut 80% Ethanol Extract Crude Protein Polysaccharides Rhamnose Arabinose Xylose Mannose Galactose Glucose Uronic acids Glu/Xyl + Ara Klason lignin Ash NDF ADF Permanganate lignin

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effect on the digestibility of the hemicellulose moiety. In this study, ten varieties of temperate grass were studied by harvesting at five stages of maturity, taking only a first cut. The lignin and hemicellulose content were measured, with the hemicellulose being further fractionated into linear and branched hemicellulose by iodine treatment. The hemicelluloses were analyzed for the neutral sugars larabinose, d-xylose, d-galactose and d-glucose. The results are shown in Table 12.5. Lignin–carbohydrate complexes from Lolium perenne contained high proportions of d-glucose residues (ca 50%). Leaf tissue complexes had the highest dglucose content, whereas stem and leaf sheath were very similar. The other neutral sugar residues present in these complexes were mainly l-arabinose and dxylose. The polysaccharide components of the lignin-hemicellulose complexes contained mainly d-xylose (63–77%) and l-arabinose (19–28%) [50]. Forage grass lignin was more extensively solubilized by acid detergent than forage legume lignin. Forage plant lignins were characterized by guaiacyl–syringyl lignin with p-hydroxyphenylpropane units. The number of ferulic acid crosslinkages in the cell wall matrices of forage grasses increased with plant maturation [51]. Two classes of phenolic–carbohydrate complexes were purified from the watersoluble products obtained from digestion of Lolium perenne cell walls with a cellulase preparation [52]. They contained d-glucose, d-xylose, l-arabinose, d- galactose, and d-mannose in the ratios 3.6 : 10 : 6.3 : 1.4 : 2.3 and 5.3 : 10 : 3.0 : 1.1 : 2.1, respectively. The complexes were based on (1 ? 4)-b-d-xylan chains to which were attached residues of l-arabinofuranose and d-galactopyranose. Mixed linkage (1 ? 3),(1 ? 4)-b-d-glucan chains also seemed to be integral components of these complexes. The principle outcomes of these studies were: 1. The quantities of hemicellulose increased with increasing maturity with the increase being larger in stem tissue compared with leaf tissue. For example, the hemicellulose content of Lolium perenne leaf tissue increased from 7.6 to

Table 12.5 Hemicellulose concentrations (g kg–1 DM) in the leaf and stem tissue of forage grasses. Leaf Cut no.

1

Lolium perenne S24 83 Lolium perenne Reveille 79 Lolium perenne S23 76 Lolium perenne Barpastra 78 Festuca pratensis 113 Festuca arundinacea S170 124

Stem 2 114 104 120 111 159 172

3

4

167 153

162 140 183 180 202 194

5

1

2

211 199

101 97 99 89 147 162

133 136 137 136 177 201

3

4

5

204 183

244 211 228 194 270 193

291 272


21.1% of dry matter over the study period. The d-xylose content of the linear hemicellulose increased concomitantly from 69 to 85%. 2. The hemicellulose content of stem tissue increased from 9.9 to 29.1% with the linear hemicellulose increasing from 74 to 91%. 3. The linearity of hemicelluloses tended to increase with crop age. 4. Higher lignin content was associated with hemicellulose of a higher linear: branched ratio. 5. Hemicellulose also had higher d-xylose : l-arabinose ratios. In summary: 1. Time of harvesting has a significant effect on the sugar composition. 2. Hemicellulose sugars increase during the growth season. 3. Stem tissue contains greater amounts of hemicellulose (xylans) than leaf tissue. 4. Digestibility of grasses decreases with age, which may affect yields of fermentation-derived products such as xylitol and lactic acid. Although, overall, at higher maturity, the absolute quantities of potentially fermentable sugars (e.g. d-xylose, present as hemicellulose) are greater, their accessibility to fermentation media may be reduced. Pectin Substances Extraction of mesophyll cell walls from the leaves of Lolium perenne afforded 25 mg of a uronic acid polymer per gram of material [53]. The polymer was identified as a 1,4-linked homogalacturonan, essentially free from neutral sugar residues, with a low degree of acetylation (3.6%) and methyl esterification (3.3%). Thus, the pectin was similar to the pectins of dicotyledons but the amounts found were substantially lower than in most dicotyledonous plants. On that basis there seems little scope for industrial end uses of forage grass pectins.

12.3.3.2 Cell Contents The cell contents of forage grasses contain sugars, fructans, amino acids, proteins, silica, alkanes, starches, minerals, nucleic acids, lipids, and alkaloids. Protein and sugars are the most abundant components. The concentration of nonstructural carbohydrates in leaves and stems is highest in winter for Lolium perenne – 13% of dry matter. Seasonal variations in element concentration are small [54]. Festuca pratensis and Dactylis glomerata are characterized by high cell wall contents. Sugars d-Glucose, d-fructose, d-sucrose, and fructans are the main nonstructural carbohydrates in Lolium perenne tissues [55]. The d-glucose, d-fructose, dsucrose, and d-xylose, d-mannitol, d-sorbitol, glycerol, and d-maltose content of Dactylis glomerata, Lolium perenne, and Festuca pratensis cut three times on different dates without interim harvesting have been recorded [56]. The results are reported in Table 12.6. Significant findings were:

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12 The Green Biorefinery Concept – Fundamentals and Potential Table 12.6 Changes in water-soluble carbohydrate content and mono- and disaccharide content (% on dry matter basis) of Lolium perenne and Festuca pratensis.

Species

Monosaccharides

Sugar alcohol

Glc

Disaccharides

Mono + WSC Disacch.

Fru

Xyl

Mann

Sorb

Glyc

Sucr

Malt

Lolium perenne June 6 3.11 June 21 3.82 July 6 2.89 Aug 6 4.36 Aug 21 2.43 Sep 3 1.46 Sep 30 5.00

5.43 4.62 3.92 6.25 5.10 1.87 7.47

0.00 0.05 0.08 0.26 0.23 0.11 0.14

0.05 0.00 0.04 0.13 0.08 0.00 0.07

0.05 0.00 0.04 0.00 0.00 0.00 0.00

0.24 0.24 0.20 0.46 0.32 0.19 0.40

0.15 0.14 0.12 0.26 0.00 0.11 0.27

0.10 0.14 0.16 0.26 0.16 0.08 0.20

9.12 9.02 7.45 11.98 8.32 3.83 13.53

27.0 16.1 18.1 9.2 9.8 7.8 16.0

Festuca pratensis June 6 2.38 June 21 2.93 July 6 2.13 Aug 6 2.07 Aug 21 1.74 Sep 3 1.53 Sep 30 4.56

4.91 3.48 3.33 3.30 2.8 1 0.47 6.56

0.00 0.00 0.07 0.15 0.07 1.80 0.13

0.00 0.00 0.00 0.25 0.07 0.19 0.13

0.00 0.00 0.00 0.00 0.00 0.12 0.06

0.24 0.19 0.18 0.20 0.34 0.19 0.44

1.42 0.12 0.00 0.30 0.00 0.19 0.19

0.10 0.12 0.21 0.40 0.20 0.27 0.19

9.04 6.85 5.95 6.66 5.22 4.78 12.25

13.8 9.0 12.5 4.9 7.6 6.6 10.3

Glc: d-glucose; Fru: d-fructose; Xyl: d-xylose; Mann: d-mannitol; Sorb: d-sorbitol; Glyc: glycerol; Sucr: d-sucrose; Malt: d-maltose; Mono + disacch: monosaccharides + disaccharides; WSC: water-soluble carbohydrate

· Lolium perenne contained the most water-soluble carbohydrate (27%) in the early season, compared with Festuca pratensis (13.8%). · This figure decreased steadily through the growing season to 7.8% in early September although it increased to 16% at the end of the month. · Lolium perenne also contained the most xylose (0.26%) in mid-season. · In Lolium perenne, glucose levels peaked in early August and again in late September. Although xylose levels similarly peaked in early August, there was no corresponding peak in late September. Similar trends were observed for Festuca pratensis. In analogous work, Fales and colleagues [57] reported results for stems of Festuca arundinacea. The stems were extracted with 95% ethanol and water to afford d-glucose, d-fructose, d-sucrose, and fructans. The fructan extract was hydrolyzed with sulfuric acid and shown to contain d-glucose and d-fructose. A hemicellulose fraction was hydrolyzed and found to contain d-xylose, l-arabinose and small amounts of d-glucose.


Fructans In grasses, fructan reserves are mobilized from vegetative plant parts during seasonal growth, after defoliation during grazing. In expanding leaves, fructans are accumulated in cells of the elongation zone [58]. Fructan structures have been characterized in Lolium perenne as belonging to essentially three series: the inulin series, the inulin neoseries, and the levan neoseries [59]. Festuca arundinacea contains an inulin and neokestose based series of oligosaccharides [60]. Fructans are an important class of carbohydrate of substantial biotechnological importance [61]. First, they are a potential source of d-fructose, for which there is a growing market in the food industry as a sweetener. The use of fructans has been reviewed by Fuchs [62]. Fructans are mainly used in the food sector. More pertinently, fructans could be chemical feedstocks from which a variety of chemicals can be produced. Hydrolysis to d-fructose and subsequent dehydration leads to hydroxymethyl furfural which, like lactic acid, is regarded as key chemical intermediate for chemistry based on renewable raw materials. Similarly, hydrolysis of inulin to d-fructose followed by catalytic hydrogenation yields d-mannitol/d-sorbitol mixtures from which d-mannitol can be easily crystallized. d-Mannitol, like xylitol, is a valuable, non-cariogenic low-calorie sweetener. Other chemicals that could be derived from fructans include ethanol, other organic solvents, and chemicals such as furans [63]. Inulin is the best known sub-class of the fructans. Inulin is colorless and odorless, and has a pleasant slightly sweet taste; it is moderately soluble in water and acts as a gel-forming agent at concentrations > 30%; it is also a foam stabilizer and texturing agent. Its calorific value is 4 kJ g–1, but it acts as dietary fiber. It suppresses putrefying bacteria and selectively supports bifidobacteria and lactobacilli in the colon. In food applications its main functions are to replace fat and sugar, to enrich with dietary fiber, to activate bifidobacteria, and to reduce cariogenicity. It is classified as a foodstuff [64]. Amino Acids The amino acid composition of Festuca pratensis, Dactylis glomerata and Lolium perenne has been studied. There were no significant differences between the species. As the grasses aged, amounts of aspartic acid, glutamic acid, alanine, tyrosine, and phenylalanine decreased and amounts of threonine, serine, and proline increased. Lysine, histidine, arginine, glycine, valine, methionine, isoleucine, and leucine did not change with plant age nor did the total amino acid content [65]. In amino acid analysis of six crops and the corresponding juice, the amino acid composition of the juice deviated only slightly from that of the crop but the amounts of glutamic acid and aspartic acids were somewhat higher and correspondingly the amounts of other amino acids, particularly arginine, glycine, alanine, tyrosine, and phenylalanine were somewhat lower [66]. Degradation of protein and amino acids in juice extracted from ryegrass can be reduced by adding hydrochloric acid. For complete preservation, the pH must be less than 3. Heating to 80 8C also has a preservative effect [67].

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Proteins Leaf protein concentrate is obtained by green crop fractionation. The acceptability of leaf protein concentrate in the human diet has been discussed by McDougall [68]. Lesnitski has also advocated [69] the manufacture of high protein feeds from green mass for partial replacement of, for example, soybean protein and dried skim milk. Silica In comparison with elements commonly associated with the nutrition of higher plants, silicon has received relatively little attention. Biogenic amorphous silica (BAS) is a natural constituent of living matter. In some plants some of the BAS occurs externally as pointed or irregularly shaped fibers, and these have been implicated as human toxicants [70]. Silica deposits commonly called phytoliths occur in cell walls, cell lumens or in extracellular locations. Silicification occurs in roots and shoots, including leaves, culms, and in grasses, most heavily in the inflorescence. Biogenic silica structures provide support and protection [71]. Grasses are heavy accumulators, but substantial variation occurs between and within species. Deposition is heaviest in inflorescence bracts [72]. Silica has been detected in the leaf mesophyll of Lolium multiflorum at an estimated concentration of 1–2%. Samples were also subjected to a range of techniques for removal of organic matter, to confirm the presence of silica throughout the cell walls [73]. In Lolium perenne, only the epidermal cell walls of the leaf edges and the trichomes contained silica [74]. The Lolium perenne variety Fortis, which has some resistance to stem borer, had many silica bodies between the veins of the leaf sheath [75]. The silica content of each of four cuts of 3 Dactylis glomerata, 4 Festuca pratensis, 5 Lolium perenne, 5 Lolium multiflorum are reported by Puffe and colleagues [76]. Silica content is lower in legumes than in grasses. Alkanes The total n-alkane (C27–C35) content of Dactylis glomerata, Lolium multiflorum, and Lolium perenne was found to be 143, 681, and 531 mg kg–1 dry matter, respectively. The concentrations of C29 and C31 were always highest [77]. These levels do not seem to be high enough for commercial exploitation. Starch The starch content of forage grasses is low – a maximum of 3% starch is accumulated in field-grown grasses. Cocksfoot contains more starch than Lolium perenne or Lolium multiflorum [78]. This low level rules out industrial use of forage grass starches. Minerals The dry matter of extracted juice of Lolium perenne has a high mineral content [66] that may be exploitable as plant fertilizer. Alkaloids Perloline, perlolidine, and loline are alkaloids of the Lolium spp. The toxicity of lolium species is because of to a symbiotic fungal infection of the plants [79].

12.4 Green Biorefinery Concept

Antifreeze Protein A plant antifreeze protein from Lolium perenne has been reported [80]. Present in organisms enduring freezing environments, antifreeze proteins have the ability to inhibit damaging ice-crystal growth. The macromolecular antifreeze protein present in Lolium perenne has superior ice recrystallization inhibition activity compared with fish and insect antifreeze proteins [81].

12.4 Green Biorefinery Concept 12.4.1 Fundamentals and Status Quo

Green biorefineries are complex systems based on ecological technology for comprehensive (holistic), substantial, and energy utilization of renewable resources and natural materials in the form of green and waste biomass from focused sustainable regional land utilization. Such green biomass is, e.g., grass from cultivation of permanent grassland, fallow land cultivation, nature reserves, or green crops, for example alfalfa, clover, and immature cereals from extensive land cultivation. Thus, green plants are a natural chemical factory and food plant. Careful wet fractionation technology is used as a first step (primary refinery) to isolate the substances in their native form. Thus, the green crop (or humid organic waste goods) is separated into a fiber-rich press cake (PC) and a nutrient-rich green juice (GJ). Besides, cellulose and starch, the press cake contains valuable dyes and pigments, crude drugs, and other organic substances. The green juice contains proteins, free amino acids, organic acids, dyes, enzymes, hormones, minerals, high quality crude drugs, and other organic substances. By use of this biotechnology, ecotechnology, and “soft” and “green” chemistry, these valuable materials can be isolated in their natural form or, by mild careful conversion, can be utilized economically [7]. Activity in the green biorefinery field is increasing and developing into an independent aspect of the large field of biomass technology. Raw material and technological aspects of this system are particularly characterized by consideration of sustainability criteria and incorporation of technology from regional and rural living and business (sustainable economy, sustainable agriculture, and sustainable regional development). The term “green biorefinery” is, on the one hand, used for model processes but, on the other hand, also used for an entire program. To “refine” is originally French (raffiner) and means “to improve something, to purify”. A refinery is, by definition, a technical facility for purification, separation, and refining of materials and products. “Green” in the field of plants means, simultaneously, high concentrations of chlorophyll, nutrients, and water, “bio” is Greek (bios) and means “live”, something biological and natural. Programmatically, the green biorefinery stands for technology (refinery) formed by imitation of nature (biologically) with the target being soft and sustainable [7].

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Fig. 12.2 Green biorefinery combined with a green crop-drying plant. Concept of the Havelland biorefinery, Selbelang, State of Brandenburg, Germany [82, 83].

In principle, the primary conversion technology is set up on the basis of the water content of the different raw materials [84]. It is reasonable to use mechanical pressing for green nature-wet biomass in the first step. Traditionally green crop fractionation was regarded as providing edible protein for humans. Fractionation separates the crop into pressed matter, which can be used to feed ruminants, and leaf protein concentrate (LPC) that can be eaten directly by humans [15–17, 68]. Different choppers and shredders, e.g. hammer-mills and different presses, e.g. screw-presses, roller-presses, and piston-presses, are used as technical equipment for fractionation of green biomass [85–88]. The literature also mentions the separation of proteins [89, 90]. The company “France Luzerne” has realized a process for separation of proteins on an industrial scale. The resources, mainly alfalfa, are treated by four main process steps: · pressing, · heat coagulation, · centrifugation, and · drying. The technical specifications of the product and, especially, its high xanthophyll content make it very useful in poultry farming for egg yolk and broilers (Table 12.7) [91].

12.4 Green Biorefinery Concept Table 12.7 Industrial production of PX (protein–xanthophylls) and their specification [91]. Product

Production Quantity year

Specification

PX 1 PX 1 PX Super

1977 1980 1997

50% crude protein and 1.000 ppm of total xanthophylls 50% crude protein and 1.000 ppm of total xanthophylls 52% crude protein and 1.250 ppm of total xanthophylls

1300 T 6200 T 13000 T

12.4.2 Wet Fractionation and Primary Refinery

The special first feature of the green biorefinery is the wet fractionation of green biomass (Fig. 12.3 A). This is also called first fractionation step or primary refinery step. (It includes, for example, the harvest, fractionation, conservation, and storage of the primary fraction.) Here, fresh harvest and waste goods are treated. Thus, the plant compounds are mostly unadulterated; the green goods should, in any case, be treated immediately, however. This processing step, usually performed by means of an industrial press, produces a fiber-rich, water-insoluble, solid material, press cake (PC), and a nutrient-rich green juice (GJ) or brown juice (BJ). The wet fractionation is based on soft separation of water-soluble and water-insoluble components of the green biomass. The silage wet-fractionation is a form of the primary refinery technology (Fig. 12.3 C). The green goods are conserved by organic acids or fermentation processes before treatment by the procedure shown. Treatment of silage from green resources has many advantages (decentralized raw material preparation, simple and low price conservation and storage, reasonable whole year operation of the biorefinery, etc.) [92]. The end products of silage are different from that of the substances in the green juice, because silage fermentation degrades the cell walls and modifies or converts substances because of the biotechnological processes involved. The so called “decomposition” methods are the third category of primary refinery technology (Fig. 12.3 B). “Decomposition” methods are mainly applied to the humid or dry whole plant. The processes work with enzymatic, fermentative, hydrolytic, chemical, thermal, or combined thermal and fractionation methods. The strength (depth of operation) of the decomposition varies, and ranges from low (enzymatic, fermentative) to high intensity (chemical, hydrolytic). For every step classification is needed to check if it belongs to green biorefinery technology. A high single yield of products can be achieved if the complete plant decomposition occurs at the primary refinery step (e.g. saccharification, which increases the total amount of sugars in the raw charge). But these procedures reduce the level of native product diversity. Nevertheless decomposition methods have been regarded as feasible technologically and economically. This is also true for the secondary prod-

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Fig. 12.3 The green biorefinery – primary refinery. Methods for fractionation of green crops [7].

uct lines. Problems with the green biorefinery system may be solved by further development of new biotechnological decomposition methods. All primary fractionations after the secondary refinery steps contain processes for substantial and energy utilization of the fractionation products. The kind and number of the secondary fractionation steps are determined by the composition and energy potential of the input green biomass and waste biomass, the type of technology, and the marketability of potential products of the refinery. The company Avebe operates a pilot plant at Veendam (The Netherlands) and has formed a consortium with other partners to explore the potential of grass to supply a range of fiber, protein, and nutraceutical products. The current pilot plant processes 3 tons of fresh grass per hour. Depending on the outcome, a full-scale (200 tons per hour, 50 000 tons per year) factory may be commissioned. The intended full-scale plant requires 1000 ha per annum of mixed grass sources harvested from a radius of up to 50 km [93].

12.5 Processes and Products

The concept of the Havelland biorefinery, Selbelang, State of Brandenburg, Germany is the fractionation of 25 000 tons of fresh green biomass per year, in its first stage of expansion, and the production of proteins and fermentation medium from green juice. Production of feed, tech-paper, and chlorophyll from press cake [94] (Fig. 12.2) will also be established. The Austrian-Concept is based on a decentralized system to take into account small scale agriculture. The system is, however, built around grass silage fermentation and the production of lactic acid, amino acids (hydrolyzed proteins), and fiber [92]. The problem of storage of fresh green biomass must be solved, i.e. green biomass must be preserved such that it is available for longer periods of time to enable continuous year-round operation of the plant. The development of sustainable green biorefinery systems requires a combination of central and decentralized/localized units, i.e. large-scale conversion and processing plants that take advantage of economies of scale must be combined with smaller and localized units as close to biomass feedstock as possible, resulting in improving rural economies and reducing the environmental impact of transportation [95].


The kind and number of products from a green biorefinery is nearly unlimited, if the fractal character of the biosynthesis and biochemistry of green plant materials is considered [96]. Characterization of a plant by a new analytical method usually discovers, in addition to the main components, innumerable new side products. Not all substances have been discovered and technologically obtained from natural products even from plants with great trading importance, e.g. alfalfa [97]. For the green biorefinery the main products, side-products and impurities are of interest [98]. Economic aspects reduce the diversity of products of interest, however. Soft technology such as biotechnology is used to reduce the complex molecules of natural materials. Nevertheless, the scientific field of ecotechnology develops new methods, preferring a reduction of technological strength (depth of operation). This can be done by using, e.g., biodiversity before molecular modification or applying less intensive methods, etc. [99]. 12.5.1 The Juice Fraction 12.5.1.1 Green Juice In the (especially) freshly pressed (Fig. 12.3 A) green juice (GJ) we can find proteins, lipids, glycoproteins, lectins, sugars, free amino acids, dyes (carotenes), hormones, enzymes, minerals, and other materials. The GJ can be fractionated by heat, treatment with organic and inorganic acids, acid anaerobic fermenta-

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tion, centrifugation, and gel filtration into a leaf nutrient concentrate (LNC) and a brown juice (BJ). The LNC consists of a mixture of chloroplast and other organell membranes plus denatured soluble plant-cell proteins. The composition of a LNC is: true protein (60–70%); lipid (especially palmitic acid, linoleic acid, and linolenic acid) (20–30%), starch (5–10%), ash (1–10%), carotenoide/polyene dyes: b-carotene (1–2 g kg–1), and xanthophyll [6, 100]. The LNC is mainly used for nonruminant feed to enhance the color (red b-carotene, or lutein) of chicken skin or egg yolk. It also produces tender meat in chickens, ducks, and pigs. Feeding pigs with LNC results in pork with an increased content of healthy oleic and linoleic fatty acids in the fat [101]. Sugars (in Particular Glucose, Fructose, and Fructans) GJ and BJ contains valuable special sugars and have highly valuable and sometimes expensive applications. Other sugars in GJ are erythrose, rhamnose, xylose, galactose, mannose, mannitol, maltose, and derivatives, for example myoinositol and glycerin. Before these compounds are fermented they are studied with regard to their potential characterization, and isolation [98, 102]. Dyes and Vitamins Green leaf nutrient concentrate (GLNC) enriched in b-carotene may have anti-cancer effects. b-Carotene (provitamin A) and xanthophyll are used in cosmetic drugs and as food, textiles, and toy-coloring agents (see also chlorophyll) [28, 29, 31]. Green juice contains further vitamins, for example vitamin B1, vitamin B2, and vitamin E [103]. Fatty Acids GLNC is also rich in oleic and linoleic fatty acids, especially palmitic acid, linoleic acid, and linolenic acid. The lipids provide good health value. The lipids can be separated by steam distillation. They are also of interest to the cosmetic industry [100]. Crude Drugs/Ingredients Because the BJ contains specific secondary plant substances, for example saponins and nicotine, these can be separated from the juice for pharmacological or pesticide purposes (isolation is described elsewhere [97]). Proteins The proteins in the green juice can be fractionated by advanced technology, in a second step, into a green leaf nutrient concentrate GLNC. The main protein is the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, also known as fraction-I (F-I) protein (rubisco EC 4.1.1.39). In alfalfa leaves proteins account for 30 to 70% of total nitrogen, depending on the physiological stage or genotype [106]. Rubisco has a molecular weight (MW) between 500 000 and 600 000 Daltons and is composed of eight large and eight small subunits with MW of approximately 55 000 and 12 500, respectively. The sedimentation coefficient is close to 18.5 S. Rubisco has a compact, tightly folded three-dimensional structure typical of globular proteins. Because of its amino acid composition Rubisco is mildly acidic and is negatively charged at neutral pH (isoelectric

12.5 Processes and Products Table 12.8 Comparison of the amino acid composition of the different protein fractions of lucerne [113, 114], according to [124]. Protein

Rubisco White protein Green protein Soluble protein Oligomeric soluble protein Membranous protein

Amino acids (parts per thousand, by weight) Hydrophilic (H)

Charged

Apolar (A)

Small

H/A

414 421 432 491 451 427

289 303 286 333 310 288

285 272 268 239 275 299

180 183 180 143 166 169

1.45 1.55 1.61 2.10 1.70 1.40

Hydrophilic: Asp + Glu + Ser + Thr + Arg + Lys + His Charged: Asp + Glu + Arg + Lys Apolar: Val + Ile + Leu + Phe + Met Small: Gly + Ala

point pH 4.4–4.7) [107]. It also has a relatively high average hydrophobic value of 1275 cal/residue, calculated according to Bigelow [108]. Native Rubisco from alfalfa contains 90 sulfhydryl groups, of which eight are “free” (one per protomer), 36 are exposed after denaturation by SDS, and 46 are involved in the formation of disulfide bonds within the Rubisco subunits [109]. The denaturation temperature of alfalfa rubisco varies between 70 (pH 7.5) and 61 8C (pH 10.3) [110]. More details about Rubisco are available in reviews [106, 111]. Fraction-II protein consists of a mixture of proteins originating from the chloroplasts and cytoplasm with molecular weights from 10 000 to 300 000 Daltons and sedimentation constants from 4 S to 10 S [112]. On the basis of amino acid composition, Rubisco and the green and white fraction of leaf proteins are regarded as hydrophobic (Table 12.8). The F I protein can be used in medical diets to enhance recovery from brain damage, where a high calorie/high protein diet is needed. People with kidney problems can easily digest F I protein with no negative effects on body metabolism. Both F I and F II proteins are advocated for solid foods and drinks as a supplement. The nutritive value and functional properties of LNC and white leaf protein isolated for incorporation in human diets have been reviewed [68, 69, 115–118]. 12.5.2 GJ Drinks/Alternative Life

Young green cereal leaves are used for production of health food grass juices and cosmetics. Dried, finely ground, and resolved young leaves are used as “green tea” and added to health-food drinks [119, 120]. Those familiar with folk medicine also know much about the effects of wild mixed grasses, herbs, and herb teas [97].

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12.5.2.1 Silage Juice To facilitate continuous year-round operation of a green biorefinery it may be very reasonable to introduce common agricultural technology. For that reason the concept involves not only processing of directly cut grass, but also of silage, which can be prepared in the growing season and stored in a silo. Silage is the product formed when grass or other material of sufficiently high moisture content (grass optimum of 28 to 35% dry matter; maize 25–30% dry matter) liable to spoilage by aerobic microorganisms is stored anaerobically. It is formed by the process referred to as ensilage which occurs in a vessel or structure called a silo. Normally during ensilage the fodder undergoes acid fermentation in which bacteria produce lactic, acetic, and butyric acids from sugars present in the raw material. The net result is a reduction in pH (to approximately pH 4 to 4.5) which prevents the growth of spoilage microorganisms, most of which are intolerant of acid conditions [121].

Table 12.9 Physicochemical characteristics of grass silage juice [123]. Property

Value

Conductivity (20.5 8C) pH (20.5 8C) Dry matter Color

35.8 mS 4.04 13.6% Dark brown

Cations (g L–1) K+ Na+ NH4+ Ca2+ Mg2+

15.63 0.15 1.22 1.78 0.50

Anions (g L–1) Lactate Acetate Cl– NO–3 PO3– 4 SO2– 4

37.54 2.08 6.41 2.13 4.38 2.55

Sugars (g L–1) Glucose Fructose Saccharose Arabinose Xylose Galactose Mannitol Amino acid

8.88 14.99 5.36 1.72 1.44 2.86 3.09 26.13


The silage juice (Fig. 12.3 C) contains a relatively high concentration of lactic acid, amino acids, sugars, and inorganic salts. Protein and peptide degradation occurs during ensiling. In silage juices only 5 to 10% of the crude proteins (organic nitrogen compounds) are peptides > 1.2 kD (*15 amino acids). At least 18 amino acids are found in the juice with a total amino acid content of 26.13 g L–1. Among the most important are alanine, leucine, lysine, GABA (caminobutyric acid), aspartic acid, and isoleucine (all in the l form) [122, 123] (Table 12.9). 12.5.3 Ingredients and Specialities 12.5.3.1 Proteins/Polysaccharides The polysaccharide and protein components of Festuca spp cell walls have been transformed into emulsifiers by extraction and treatment with xylan-hydrolyzing enzyme preparations. The emulsifiers are useful, for example, for food, cosmetics, pharmaceuticals, and industrial chemicals applications [125].

12.5.3.2 Cholesterol Mediation It has been demonstrated that polysaccharide–lignin complexes from fodder grasses are active sorbents of cholic acid, a metabolite of cholesterol [126]. Equations have been derived for calculating the sorption of cholic acid by the grass material. The equations can be used to construct dietary fiber with the desired properties, for example for cholesterol metabolism in humans and animals.

12.5.3.3 Antifeedants Festuca arundinacea and Lolium perenne can become infected with fungal endophytes (Neotyphodium spp). The symbiosis between plant and fungus leads to the synthesis of alkaloids that have been shown to be either toxic or act as feeding detergents against insect pests. Alkaloid production/accumulation in Festuca arundinacea and Lolium perenne is enhanced by reduced mowing frequency [132]. Such alkaloids may have a role as insecticides for agrochemical use [104, 105] or in the clinic as a result of their pharmacology [79].

12.5.3.4 Silica A process has been described for manufacture of high-purity amorphous silica from biogenic materials [127]. Rice hulls are given as the example. The hulls are finely divided, screened, subjected to a surfactant wash, rinsed, and soaked in water to accelerate and enhance penetration of an oxidizing solution. The oxidizing solution removes organic compounds, and volatile impurities are removed by heated oxidation to leave silica. The remaining silica may be rinsed with water, acid solution, or other solution to remove even trace impurities. At

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the end of the process, a fine white amorphous silica of extremely high purity is produced.

12.5.3.5 Silicon Carbide Rye grass has been proposed as a raw material for the production of polytypically pure b-silicon carbide in an economically effective and ecologically compatible procedure. When the particle size of starting raw material is defined then the particle size of the developing silicon carbide is also controlled [128]. Silicon carbide has many industrial applications and is a valuable chemical used for cutting, grinding, and polishing applications. Silicon carbide is also used in the electronics industry in hostile environments where its ability to function in high-temperature, high-radiation conditions overcomes the limitations of conventional silicon-based systems. It is used as a component of blue and violet light-emitting diodes.

12.5.3.6 Filter Aids Highly purified biogenic silica has an intricate and diatomaceous SiO2 structure and a high-SiO2 specific volume. These products are extremely bright and can be used in filtration processes [129]. In one example the adsorbent has been used for the removal of proteins in chillproofing of beer [130].

12.5.3.7 Zeolites Artificial zeolites are manufactured by heating a mixture of grass husks and plants with aqueous alkali solutions to elute silicic components. These are mixed with aluminum-enriching agents and treated under heat and pressure. The zeolites have high cation-exchange properties and are be useful as fertilizers [131]. 12.5.4 The Press-Cake (Fiber) Fraction

The products and product groups described below are technologically possible after fractionation in accordance with Fig. 12.3 A and C. Use of the PC as feed (silage, bale press food, green pellets) is well known [133]. Furthermore, extraction of plant dyes (chlorophyll, carotenes, xanthophyll) [25, 28] and applications in the food and candle industry [31], in environmental analysis [134] or, after refining, in cosmetics, medicine, biochemistry [135], electronics (nematic liquid crystals) [136], and photovoltaics (organic dyes [32]) have also been described in the literature. Because of the structural similarity of chlorophyll and blood hemoglobin one can expect interesting developments in the field of plant dyes and colorants. The resulting fraction will, substantially, be thermally treated analogous to unex-


Fig. 12.4 Classification of the major components of grass press cake, hay, and late hay crop (by analogy with Ref. [143]).

tracted PC. The suitability (and applicability) as feed depends mainly on the corresponding extraction compounds and has to be tested. The PC fraction can be separated by analogy with wood raw materials into its main components (Fig. 12.4). On the one hand, this green plant fractionation does not seem to make much economic sense today (because of wood competition). There are, on the other hand, interesting applications for special vegetable celluloses, hemicelluloses, and lignin. The “green plant” polyoses (hemicelluloses) are nutrient-physiologically valuable [137]. Furthermore, they can be used (similarly to plant rubber) as protecting colloids, emulsifiers in cosmetics, thickeners in the food industry [138 a], adhesives, additives in the pulp and paper industry, stabilizers for environmentally friendly inks and dyes [138 b], or as thickeners for crude oil drilling [139]. Lignin is one component of press cake. Isolated lignin can be used as a dispersant in the food industry, as stabilizer for foams and bitumen, or as an environmental friendly adhesive [140–142].

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12.5.4.1 Fibers Non-wood fibers have been used to manufacture all kinds of paper, including printing, writing, and packaging. Such feedstock is expected to play an important role in improving the sustainability of the pulp and paper industry [144], by enabling more rational utilization of forest resources. Non-wood fiber pulps can be used effectively in combination with recycled papers, improving many of their attributes and enabling overall cost reduction because of a decrease in the use of starch [145]. Semi-chemical Pulping The process starts with atmospheric alkali cooking in a continuous digester [145]. The semi-chemical pulp obtained is washed, refined, screened, and sent to the paper mill for corrugated paper manufacture. From the black liquor obtained in the pulping process, lignin is initially recovered by precipitation then given a post-treatment to improve its filterability. Most of the silica remains with the filtrate and the resulting lignin cake is high in purity and contains less than 1% silica and less than 3.5% sugars. Lignin sales increase overall mill revenues and lead to a possible reduction of the minimum plant scale required for economic operation. The filtrate after lignin recovery can be processed in a biological treatment plant. Alternatively, oxygen-based wet oxidation of the filtrate can be used to generate energy and green liquor. From the latter, a precipitate that typically accounts for 70–90% of the silica in the wet-oxidation feed can be filtered, effectively purging silica from the cycle. The filtered green liquor can be made caustic to generate white liquor for re-use in pulping. A study of the pulping characteristics and mineral composition of 16 field crops grown in Finland showed that the most suitable species for alkali cooking were the grass and cereal crops, which gave the highest pulp yields and the lowest amounts of rejects. On the basis of the test results, Festuca arundinacea, Festuca pratensis, reed canary grass, and spring barley were selected for further study [146]. Further work selected Festuca arundinacea and reed canary grass as worthwhile candidates [147]. Steam Explosion Steam explosion of ryegrass straw has been reported in a patent application to yield separate portions of usable straw pulp and a usable aqueous by-product comprising lignins and hemicellulose. The pulp was blended with Kraft pulp and old corrugated containers to make linerboard [148]. Mechanical Pulping A recent Chinese patent describes the production of nonpolluting grass pulp and a method for reclaiming its by-product [149]. Grass is processed into refined grass chip and refined grass residue, the refined grass chips are treated by soaking with water, softening, washing, and pulping to produce high-quality grass pulp, and the refined grass residue is mixed with an additive containing functional preparation (organic selenium, organic calcium) and carrier (refined grass powder) to produce a high-quality fiber feed.


Downstream Processing of the Grass Fiber Fraction (PC) The biorefinery primary process generates a fibrous press cake (Fig. 12.3 A and C). This fraction can either be further processed while wet – by applying technology used in the pulp und paper industry – or it can be dried. After mechanical fractionation grass and silage press cake particles are typically less than 3 cm in length. The structure of grass stems and leaves is mostly eliminated and the bulk has a fibrous appearance. Basic Properties of Grass Fibers Sfiligoj et al. [150] evaluated the fundamental physical properties of press cake fibers (from Ryegrass (Lolium hybridum), wheat (Triticum aestivum L.), red clover (Trifolium pratense), and lucerne (Medicago sativa L.) after mechanical separation. Investigation involved isolation of elementary fibers or fiber bundles from the press cake fraction using chemical or biological retting. For the resulting samples density, size, and strain-stress-behavior were analyzed in wet conditions (Table 12.10). There are no significant differences between mechanical properties of fiber bundles of different origin (green or ensiled grass). For the press cake of trefoil, ryegrass and alfalfa tenacity values of stem fiber bundles were measured in the range 11.4 to 21.4 cN/tex, leaf fiber bundles reached 6.8 to 13.1 cN/tex [150]. The geometrical properties (length and diameter) of press cake fibers were similar to those of soft wood fibers and the mechanical properties (e.g. tenacity and elongation) of elementary fibers were comparable with those of bast fibers (jute, hemp). Grass fibers, especially the dry fibers, have poor bending strength and are characterized by brittleness. They are, therefore, used for non-woven textiles, preferably for technical applications. Chemical analysis of different press cake fractions gave the average results (% dry matter content): *5.8–7.6% cellulose, 14.7–28.7% hemicellulose, and 27.5–31.6% lignin. Crude fiber was 29.1–32.9% and crude ash 6.3–9.1%. These values are for the first cut of grass and may alter for later harvests.

Table 12.10 Basic properties of grass fibers from press cake – green and ensiled. Property

Unit

Measured value

Fiber content, stem Fiber content, leaves Fiber length, stem a) Fiber length, leaves a) Fiber diameter Linear density b) Tenacity Elongation

[%] [%] [mm] [mm] [lm] [dte x] [cN/tex ] [%]

20.2–39.5 6.9–10.2 0.8–3.2 0.6–1.3 15–18 12–105 6–21 c) 1–6

a) b) c)

Elementary fibers Fiber bundles (technical fiber) Coir 15 cN/tex; jute 23–31 cN/tex; hemp 29–47 cN/tex

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Products From Grass Fiber (PC) Generally speaking, the grass and silage fibers fraction of the green biorefinery may be used as raw material for: · insulation material (mats, boards, loose fill material) · building panels (fiber and chip boards) · products used in horticulture and landscaping (mulch fleece, erosion-control, peat substitution) · bio-composites · packaging material · pore forming additives (e.g. brick and tile industry) · gypsum boards · pulp and paper [151] · thermoplastics [156]. Paper and Cardboard Paper has been manufactured from PC of lucerne [152, 153] and from reed canary grass, wild mix grass, and Cock’s foot [7, 154]. It has also been shown that the quality of grass cardboards is the same or even better (for the paper re-working industry) than analog waste paper and that they are also less expensive [7, 155]. Thermoplastics Adhesive films have been produced from grass fiber by preparation of alkali cellulose and then film-forming. The adhesive film can be used as agricultural mulching film or packing material [156]. Grass fiber has been proposed as a component of a biodegradable protein/starch-based thermoplastic composition. The grass fibers function as reinforcement filler. The composition is processed by conventional methods, for example extrusion and injection molding, into packaging material or articles that are low density and have high compressive strength and tensile strength and good resilience [157].

12.5.4.2 Chemicals It is reasonable to combine the primary refining of green biomass with fermentation processes for production of chemicals. This assumption is based on the high water content of the raw material and the occurrence of many different substances in green biomass, important for biotechnological processes. Green juice, brown juice, and silage juice (Fig. 12.3 A and C) contain all the necessary macroelements (minerals, peptides, amino acids, sugar) for making fermentation products [103, 123, 158–160, 168, 170]. After enrichment with further sources of carbohydrates (sugars from lignocellulosic feedstock, for example press cake) it should be possible to produce a variety of biotechnologically basic chemicals [7]. Chemicals which provide two or three functional groups and can be integrated into the product trees of the chemical industry are of most interest [162, 163]. An assortment of industrial biotechnologically produced chemicals are: · (C2) chemicals such as ethanol [160, 161, 166, 167] and acetic acid, · (C3) chemicals such as lactic acid [48, 159, 164, 165], acetone [167], and 1,3propandiol [171],

12.6 Green Biorefinery – Economic and Ecological Aspects

· (C4) chemicals such as n-butanol [167], · (C5) chemicals such as itaconic acid [171], and · (C6) chemicals such as lysine [158, 168, 170]. Products from lactic acid include, e.g., polylactic acid and ethyl lactate [83, 164, 172]. The biotechnological production of polyhydroxybutyrate from switchgrass is currently in a stage of industrial development, and chemical or combined chemical/biotechnological decomposition of lignocellulosic press cake or switchgrass can be used to produce basic chemicals [169]. Basic chemicals which can be used as precursors in genealogical trees are furfural [174] and xylitol [175, 176] from the hemicellulose line and hydroxymethylfurfural [177] and levulinic acid [178] from the cellulose line. Esparto grass is a particularly important source of xylitol [179], and grasses with a high concentration of fructan, for example Lolium perenne are a source of hydroxymethylfurfural. The same is true of chicory roots [180].

12.5.4.3 Residue Utilization Green juice, brown juice, and silage juice can be used as bio-fertilizer (soil bioactivators) to return to the soil the macro and micro mineral nutrients which were removed by harvesting the green crop [101]. The low-molecular-mass substances in this juice are quickly transformed into methane in fermentation units [183, 184]. This has been developed a production process for biogas, heat, and electricity in a combined green biorefinery–animal-breeding complex [7, 94]. Silage residues have been used as sources of natural chelates to improve the ecological and economical balance of leaching techniques for remediation of metal-polluted soils. Silage effluent containing a variety of aliphatic carboxylic acids, sugar acids, and amino acids has been used to remove approximately 75% of the cadmium and more than 50% of the copper and zinc from contaminated soils [181]. A trial led to the conclusion that biomass residues have potential to serve as extractants in remediation techniques. Porous carbon fibers have been obtained from cut grass by baking in an oven in a roped form for formation of coiled carbonized fibers. The porous carbon fibers are useful for sound absorbers, adsorbents, purification materials, and radio wave absorbers [182]. The press cake has also been used as a medium for growing mushrooms, as a mulch/green crop enhancer, and as a fertilizer [34].

12.6 Green Biorefinery – Economic and Ecological Aspects

Plant biomass is the only foreseeable sustainable source of organic fuels, chemicals, and other materials. A variety of forms of biomass, notably many ligno-cellulosic feedstocks, are potentially available on a large scale and are cost-competitive with low-cost petroleum whether considered on a mass or energy basis, in

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terms of price defined on a purchase or net basis for both current and projected mature technologies, or on a transfer basis for mature technology [185]. Green plant biomass and lignocellulosic feedstocks are the dominant source of feedstocks for biotechnological processes for production of chemicals and materials [7, 158, 162, 163, 173]. The development of integrated technology for conversion of biomass is essential for the economic and ecological production of products. The biomass industry or bio-industry produces basis chemicals such as ethanol (15 Mio tons per year), amino acids (1.5 Mio tons a year of which l-lysine amounts to 500 000 tons per year [186]), and lactic acid (200 000 tons per year). The target of a biorefinery is to establish a combination of a biomass–feedstock mix with a process and product mix [162, 163]. A life cycle assessment (LCA) is available for production of polylactic acid (capacity 140 000 tons per year) [187]. In total assessment of the utilization of biomass one must consider that plant cultivation must fulfill economic and ecological criteria. Agriculture both creates pressure on the environment and plays an important role in maintaining many cultural landscapes and semi-natural habitats [188]. Green crops, especially, are available in large quantities. Additionally, grassland can be cultivated sustainably [92]. European grassland experiments have shown that species-rich grassland cultivation has both ecological and economic advantages. With plant diversity grassland is more productive and protects the soil against nitrate leaching. Seventy-one species have been examined, of which 29 had significant influence on productivity. In particular, Trifolium pratense has an important function with regard to productivity. On sites where this species occurs more than 50% of the total biomass has been used. Legumes, like clover and herbs, also play an important role, as do fast-growing grasses [189]. An initial assessment of the concept of a green biorefinery was performed by S. Schidler et al. for the Austrian system approach [190]. An Austria-wide concept for use of biomass and cultivable land for renewable resources has yet to be developed; the same is true for Europe [191]. The size of such plants depends on the rural structures of the different regions. Concepts with more decentralized units would have a size of about 35 000 tons raw material per year [192] and central plants could have sizes of approximately 300 000 to 600 000 tons per year [174]. The synthetic method used for modeling biorefinery systems [192] is based on combinatorial acceleration of separable concave programming developed by Nagy et al. [193].

Table 12.11 Cost calculation for production of grass in comparison to straw, including intermediate storage [195]. Raw material

Yield t DM/ha

Late cut Grass 5.0 Straw 2.1

Work Person-h ha–1

Person-h t–1 DM

Euro ha–1

Euro t–1 DM

4.46 1.58

0.89 0.75

297 77

60 36

References

Cost calculation for raw materials in the green biorefinery are based on the supply of agricultural products. For late-cut grass and straw the costs of cultivation, harvest, and intermediate storage have been calculated for Germany (Table 12.11 [194]). Currently, the costs are US $ 30 per ton for corn stover or straw [196]. The prices for green pellets are also available and range from 80 1 per ton in Germany to 160 1 per ton in Sweden [42]. Production of pellets from silage has been calculated to be approximately 110 to 155 Euro per ton in Austria [190].


Technology and research challenges associated with converting plant biomass into commodity products must be considered in relation to green biomass in combination with lignocellulosic biomass [196] (converting biomass into reactive intermediates) and product diversification (converting reactive intermediates into useful products). After isolation of the valuable products (chlorophyll, carotenoids) biomass precursors such as carbohydrates and proteins must be considered. Their isolation as functional products and their biotechnological or chemical conversion into derivatives such as O-chemicals and N-chemicals must be included in the development of the relevant technologies. Biotechnological conversion methods must be integrated into the concepts of utilization of nature – wet biomass. It is necessary to establish green biorefinery demonstration plants which are best suited for the different regional rural structures of grassland agriculture and the cultural landscape.

Acknowledgment

The authors thank Michael Mandl and Niv Graf, Joanneum Research, Graz, Austria, for investigations silage fibers, and Werner Koschuh, University of Natural Resources and Applied Life Sciences, Vienna, for investigations of silage juice.

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zo, L.; Miyafuji, H.; Thomasser, C.; Bvochora, J.; Bamusi, S.; Braun, R.; Lignocellulose Feedstocks for the production of lactic acid, Chem. Biochem. Eng. Q. (CABEQ) 18(1) (2004) 55–63 McGrath, D.; Italian ryegrass as a source of fermentable sugars and protein feedstuff. Luxembourg: Commission of the European Communities, (1990). Quereshi, N. and Blaschek, H. P.; ABE production from corn: a recent economic evaluation. Journal of Industrial Microbiology and Biotechnology, 27, (2001) 292–297. Starke, I.; Kamm, B.; Kleinpeter, E.; Separation, identification and quantification of amino acids in l-lysine fermentation potato juices by gas chromatography/ mass spectrometry, Fresenius J. Anal. Chem., 371 (2001) 380–384 Kamm, B.; Kamm, M.; Schmidt, M.; Starke, I.; Kleinpeter, E.; Chemical and biochemical generation of carbohydrates from lignocellulose-feedstock (Lupinus nootkatensis), Quantification of glucose, Chemosphere (in press) Kiel, P.; Thomsen, M. H.; Andersen, M.; Plant Juice in the Biorefinery – Use of plant juice as fermentation medium, In this book Vorlop, K. D.; Willke, Th.; Prüße, U.; Biocatalytic and catalytic routes for the production of bulk and fine chemicals from renewable resources, In this book Datta, R.; Tsai, S.-P.; Lactic acid Production and potential uses: A Technology and Economics Assessment. In: Fuels and chemicals from biomass, B. C. Sara, J. Woodward (ed.), ACS Symposium series, ISSN 00976156 666, ACS, Washington D. C., 1997, pp. 224–236 Tullo, A.; Renewable Materials, Two Pacts May Help Spur Biomass Plastics; Chemical and Engineering News, March 28, 2005, www.CENONLINE.org Van Dyne, D. L.; Blasé, M. G.; Clements, L. D.; A Strategy for returning agriculture and rural America to longterm full employment using biomass refineries. In: J. Janeck (ed.) Perspec-

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tives on new crops and new uses. ASHS Press, Alexandria, VA, USA, 1999, 114–123 Jiang, Z., Chen, R. and Wu, P.; Hydrogenation of xylose to xylitol in tricklebed reactor. Riyong Huaxue Gongye, 6, (1998) 6–8. Nigam, P. and Singh, D.; Processes for fermentative production of xylitol – a sugar substitute. Process Biochemistry, 30, (1995) 124–124. Bhatt, S. and Shukla, R. P. HMF – a new route to industrial chemicals from sugars. Taiwan Sugar, 48, (2001) 4–15. Elliott, D. C.; Fitzpatrick, S. W.; Bozell, J. J.; Jarnefeld, J. L.; Bilski, R. J.; Moens, L.; Frye, Jr. J. G.; Wang, Y.; Neuenschwander, G. G.; Production of levulinic acid and use as a platform chemical for derived products; In: Proceedings of the fourth biomass conference of Americas, Biomass, A Growths Opportunity in Green Energy and Value added Products, Vol. 1, Overend, R. P.; Chornet, E. (ed.); Elsevier Science Ltd., 1999, 911–914, ISBN 008 0430198 Rothschild, Z., Silva, H. C., Braga, G. L., Carlomagno, D. N., Prado, I. M. R., de Carvalho, R., Polizello, A. C. M. and Spadaro, A. C. C.; Xylitol a non-cariogenic sugar obtained from grasses of the genus Aristida and related species. Arquivos de Biologica e Tecnologia, 34, (1991) 61–71. de Baynast, R. and Renard, C.; Processes for extraction and transformation of products with high fructose content from chicory roots (Cichorium intybus). Comptes Rendus de l’Académie d’Agriculture de France, 80, (1994) 31– 46. Fischer, K., Bipp, H.-P., Riemschneider, P., Leidmann, P., Bienek, D. and Kettrup, A.; Utilization of biomass residues for the remediation of metal-polluted soils. Environmental Science and Technology, 32, (1998) 2154–2231. Kato, H.; Porous carbon fibers from plant waste materials manufactured by baking the waste materials in an oven in the roped form for formation of coiled carbonized fibers and reuse of plant waste materials therefor. Japanese Patent Application, 2000303266 (2000).

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12 The Green Biorefinery Concept – Fundamentals and Potential 183 Linke, B.; Schelle, H.; Anaerobe Ver-

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fahren zur Reststoffbehandlung, In: The Green biorefinery, Kamm B, Kamm M, Soyez K (eds) The Green Biorefinery, Concept of technology, 1st International Symposium Green Biorefinery, Oct. 1997, Neuruppin, Society of ecological technology and system analysis, Berlin, (1998) 196–207 Verstrate, W.; Debeer, D.; Pena, M.; Lettinga, G.; Lens, P.; Anaerobic Bioprocessing of Organic Wastes, World Journal of Microbiology and Biotechnology, 12 (1996) 221–223 Fowler, P. A.; McLaughlin, A. R.; Hall, L. M.; The potential industrial uses of forage grasses including miscanthus; BioComposites Centre, University of Wales, Bangor, Gwynedd 2003; www.nnfcc.co.uk/library/reports/ download.cfm?id=60 Elements, Degussa Science Newsletter 7 (2005) 35 Vink, E. T. H.; Rabago, K. R.; Glassner, D. A.; Gruber, P. R.; Applications of life cycle assessment to NatureWorks polylactide (PLA) production, Polymer degradation and stability 80 (2003) 403– 419 IEEP, Contribution to the background study agriculture, The Institute for European Environmental Policy, 2004. Hector, A.; et al.; Plant Diversity and Productivity Experiments in European Grasslands, Science, 286 (1999) 1123– 1127 (a) Schidler, S., Technikfolgenabschätzung der Grünen Bioraffinerie, Teil I: Endbericht, Institut für TechnikfolgenAbschätzung, Österreichische Akademie der Wissenschaften, 2003; Schidler, S., Adensam, H., Hofmann, R.,

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Kromus, S., Will, M., 2003, Technikfolgen-Abschätzung der Grünen Bioraffinerie, Teil II: Materialsammlung, Institut für Technikfolgen-Abschätzung, Österreichische Akademie der Wissenschaften, 2003 (German) Narodoslawsky, M.; Kromus, S.; Development of decentral green biorefinery in Austria; In: biorefinica 2004, Proceedings and Papers, October, 27–28, Osnabrück, Eds.: Kamm, B.; Hempel, M.; Kamm, M; biopos e.V., Teltow 2004, p. 24; ISBN 3-00-015166-4 Halasz L., G. Povoden, M. Narodoslawsky: Process Synthesis for Renewable Resources, presented at the PRES 03, Hamilton, Canada, 2003 Nagy, A. B., F. Friedler, and L. T. Fan, Combinatorial Acceleration of Separable Concave Programming for Process Synthesis, presented at the AIChE Annual Meeting, Miami Beach, FL, USA, November 15–20, 1998. Fechner, M.; Hertwig, F.; Nachwachsende Rohstoffe auch vom Grünland, Neue Landwirtschaft 11, (1994) 29–31 Dale, B.; Encyclopedia of physical sciences and technology, vol 2, 3rd edn. McGraw-Hill, New York, 2002, pp. 141–157 (a) Lynd, L. R., Wyman, C. E. and Gerngross, T. U.; Biocommodity engineering. Biotechnology Progress, 15, (1999) 777–793. (b) Lynd, L. R.; Biomass Processing in Response to sustainability and Security Challenges – A Vision for What is possible, In: biorefinica 2004, Proceedings and Papers, October, 27– 28, Osnabrück, Eds.: Kamm, B.; Hempel, M.; Kamm, M.; biopos e.V., Teltow 2004, p. 20; ISBN 3-00-015166-4


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13 Plant Juice in the Biorefinery – Use of Plant Juice as Fermentation Medium Mette Hedegaard Thomsen, Margrethe Andersen, and Pauli Kiel

13.1 Introduction

Biotechnological utilization of waste and residues from agriculture and the agricultural industry has been the common goal for AgroFerm A/S and University of Southern Denmark. The concept of the green biorefinery has been described elsewhere [8]. Additionally, much experimental work has been performed on realizing these ideas for industrial purposes. This paper describes the possibility of using brown juice from the green crop-drying industry and potato juice from the potato starch industry as raw materials in Danish lactic acid production, as a basis for the production of bio-based polylactate to be used as packaging materials for fruit and vegetables. One single green crop-drying factory producing 50 000 tons of fodder pellets a year has enough brown juice to supply a 6 000 ton lactic acid factory with fermentation medium, and a 50 000 ton potato starch factory produces enough potato juice to supply a 35 000 ton lactic acid factory.


Since 1997 work has been performed on a research project on the production and testing of bio-based packaging materials for food. The work has been performed as co-operation between The Technical University of Denmark, The Danish Technological Institute, The Royal Danish Veterinary and Agricultural University, Risø National Laboratory, The University of Aarhus, and The University of Southern Denmark. Bio-based materials are defined as materials originating from agricultural sources, i.e. produced from renewable and biological raw materials. The advantages of such materials are that they are CO2-neutral and biodegradable.


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13 Plant Juice in the Biorefinery – Use of Plant Juice as Fermentation Medium

13.3 Biobased Poly(lactic Acid)

Poly(lactic acid) (PLA) is a bio-based, biodegradable polymer with much potential as a material for food packaging because of its mechanical properties. Because it is a moisture and gas barrier and can be used to produce flexible waterresistant films, PLA is suitable for packaging of respiring fruit and vegetables and for liquid food applications, e.g. juice. PLA can be used [1, 2] as a pure product or it can be used in combination with other polymers. It may contain natural extracts/components e.g. lignin and waxes acting as preservatives or antioxidants preventing oxidation-sensitive products from deteriorating [3]. For PLA to compete with conventional packaging materials it must be produced from cheap raw materials and by feasible processes. Our objective in this collaborative research project was to find the cheapest and most suitable raw materials for Danish production of lactic acid for polylactate production. 13.3.1 Fermentation Processes

PLA is produced by polymerization of lactic acid. Lactic acid can be produced by chemical synthesis or by a fermentation process. Fermentation is a process whereby carbohydrates such as sucrose, glucose, fructose, or lactose are converted by microorganisms into a desired product. Most residues from the agricultural industry contain such carbohydrates either free or bound in long chains of polysaccharides. Molasses – a waste product from sugar beet production – contains saccharose; whey – a waste product from cheese making – contains lactose; potato waste – from the potato starch or French fries industry – contains starch, which can be metabolized by certain strains of microorganisms or hydrolyzed to glucose. Straw contains cellulose and hemicelluloses. Cellulose can be hydrolyzed to glucose and hemicelluloses can be hydrolyzed to xylose, which also can be metabolized by some strains. Green and brown juice from the green crop-drying industry contains several types of carbohydrate and other nutrients. Some of the waste products are available free, others can be purchased at very favorable prices and could be used as cheap raw materials in feasible production of PLA. 13.3.2 The Green Biorefinery

In the green biorefinery, jointly described by the University of Southern Denmark and AgroFerm A/S, crops are converted by means of mechanical and biotechnological methods into useful materials such as food and feed products and additives, and into materials and organic chemical compounds and bio-energy. The crops used in the green biorefinery are crops that give a high yield, fit in with normal crop rotation, absorb large amounts of organic fertilizers, are robust and require a minimum of pesticides.

13.3 Biobased Poly(lactic Acid)

Fig. 13.1 The principles of the green biorefinery.

The crops are separated in a liquid fraction – the juice containing the soluble compounds and the press cake containing particles and insoluble high-molecular-weight compounds. Vitamins, colors, enzymes, and other phytochemicals can be isolated directly from the juice or press cake. The press cake can be used as animal feed or, after drying, as solid fuel. After extraction of the high-value compounds from the juice, it can be used as a substrate for fermentation [4]. The fermentation products can be any organic compounds, for example enzymes, antibiotics, biodegradable plastics, organic acids, alcohols and amino acids.

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13.3.3 Lactic Acid Fermentation

Lactic acid fermentation is normally performed by lactic acid bacteria. Lactic acid bacteria comprise several genera with similar physiology, metabolism, and nutritional needs. A primary similarity is that they all produce lactic acid as a major or sole end product and that they do not use oxygen in their metabolism (anaerobes). Nutritionally, lactic acid bacteria are extremely fastidious. A medium that will support their growth must contain many growth factors such as amino acids, peptides, nucleic acid derivatives, vitamins, salts, and fatty acids or fatty acid esters. When all these nutrients are present in the fermentation medium, lactic acid bacteria will convert carbohydrates to lactic acid. 13.3.4 Brown Juice as a Fermentation Medium

The green crop-drying industry in Denmark uses Italian rye grass, clover, and alfalfa as raw materials for production of green pellets. Approximately 300 000 tons of green pellets are produced in Denmark each year. The green crop-drying industry solves its energy-economical problems by pressing the green crop before drying. The by-product produced, green juice, typically has a dry-matter content of 3 to 8%. At some factories the green biomass is heated to 80 8C by steam before pressing, this causes the plant cells to burst and the protein to coagulate. The by-product produced is called brown juice. Typically brown juice has a dry-matter content of 4 to 8%. Approximately 200 000 m3 of brown juice is produced each year in Denmark. Although the green and brown juice are spread on the fields as fertilizer, pollution of ground water, particularly with nitrate, in the late autumn has led to stringent regulations on the use of plant juice as a fertilizer. In Denmark plant juice can be spread on green fields only in the autumn and not in the period between October 1st and February 1st [5]. The problem with the plant juice can be solved by storing the juice in large lagoons from October 1st. Because storage can easily result in foul-smelling waste products, we have studied other possibilities. It is common knowledge that grass and almost all kinds of crop decay if stored inappropriately, but it can be conserved by ensiling either by a spontaneous process in which the microorganisms present in the crops convert the carbohydrates to acid, or by adding a culture of lactic acid bacteria. Therefore the obvious solution is to use the brown juice as a fermentation medium for lactic acid fermentation [6].

13.4 Materials and Methods

13.4 Materials and Methods 13.4.1 Analytical Methods 13.4.1.1 Sugar Analysis Sugar analysis was performed by HPLC using a Bio-Rad IG Carbo C pre-column and a Bio-Rad Aminex HPX-87C column. Before HPLC analysis samples were subjected to ion exchange through a cation-exchange column (Amberlite CG-120) and an anion-exchange column (Dowex 1(4)).

13.4.1.2 Analysis of Organic Acids Analysis of organic acids by HPLC was performed using a Bio-Rad Aminex HPX-87H column. Before analysis of organic acids by HPLC, samples were subjected to ion exchange through a cation-exchange column (Amberlite IR-120) and an anion-exchange column (DEAE Sephadex A-25).

13.4.1.3 Analysis of Minerals Cations, anions and trace minerals were kindly analyzed by the central laboratory of the Danish Co-operative Farm Supply, DLG, using EU-approved methods.

13.4.1.4 Analysis of Vitamins Vitamins are analyzed by use of Bacto Assey Methods (Difco Manual, 11th edn, 1998).

13.4.1.5 Analysis of Amino Acids Analysis of free amino acids was performed by EU-approved method 98/64 1EC.

13.4.1.6 Analysis of Protein Nitrogen (N) analysis was performed by the Kjeldahl method and crude protein was calculated as N ´ 6.25. 13.4.2 Fed Batch Fermentation of Brown Juice with Lb. salivarius BC 1001

A 2-L continuously stirred tank reactor containing 1.5 L fresh (non-heat treated) brown juice (2% DM) was used for the fed-batch experiment. The bioreactor was equipped with a Mettler Toledo pH meter, heat sensor (pT100, MJK auto-

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mation) and a heating element (50 W, 220 V), to keep pH and temperature constant. pH was controlled automatically by addition of 4 m NaOH by a peristaltic pump, the computer program used for control and regulation of pH and temperature was Genesis 4.2. Growth of the strain was followed by measurement of the dry cell mass. This was achieved by centrifuging samples and washing twice with saline water (0.9%) before drying the samples in an oven at 190 8C overnight. During fermentation 2% glucose (40% solution) was added successively. 13.4.3 Pilot Scale Continuous Fermentation with Lb. salivarius BC 1001

The pilot scale experiment with continuous lactic acid fermentation of brown juice was performed at the green crop-drying factory – Dangrønt Products, Ringkøbing, Denmark. The brown juice produced from the pressing of the crops was cooled to 30 to 40 8C and fed into an 8 m3 tank and the tank was inoculated with 4 m3 bacterial culture (Lb. salivarius BC 1001). Fermentation was initiated as batch fermentation, and after the pH in the tank had dropped to approximately 4.5, substrate flow was started. The initial flow rate was 3.5 m3 h–1. The acidified BJ was led to a sedimentation tank from where the supernatant was pumped to a storage/buffer tank before evaporation to approximately 40% DM. 13.4.4 Study of Potato Juice Quality During Aerobic and Anaerobic Storage

Experiments were performed to investigate the quality of potato juice stored under aerobic and anaerobic conditions. The investigations were carried out in plastic containers with a volume of 25 L. The containers were filled with 10 L fresh potato juice from the Karup, Denmark, potato starch factory. For anaerobic storage the surface was covered with 1 to 2 cm corn oil. Containers were kept at a temperature between 15 and 20 8C.

13.5 Brown Juice 13.5.1 Chemical Composition

The chemical composition of the green and brown juice was analyzed to investigate the suitability of the brown juice as fermentation medium. The average composition of evaporated fresh brown juice determined from 42 different batches collected from June 9th 1998 to November 11th 2000 is shown in Table 13.1.

13.5 Brown Juice Table 13.1 Average composition of evaporated fresh brown juice determined from 42 different batches of brown juice collected from June 9th 1998 to November 11th 2000, and the calculated composition of average fresh brown juice with a dry-matter content of 4%. Component

Average evaporated fresh brown juice Maximum Content (g kg–1 DM)

DM (%) Density (kg L–1) pH

Minimum St. dev. (%)

Content (g L–1)

Average fresh brown juice Content (g L–1) (Calculated)

32.4 1.2 5.2

52.6 1.3 5.9

14.7 1.1 3.9

28.0 4.0 9.0

Sugar Glucose Fructose Sucrose Free sugars Fructan 1-Ketose Total

75.7 104.5 50.1 229.7 92.2 9.1 325.7

149.7 168.1 123.1 355.7 224.7 29.0 573.4

27.7 60.2 4.4 96.4 6.8 0.0 169.1

34.4 25.6 57.6 26.7 58.5 67.5 31.6

28.3 39.0 18.7 85.8 34.4 3.4 121.7

3.5 4.8 2.3 10.6 4.3 0.4 15.0

Acids Citric acid Malic acid Malonic acid Succinic acid Lactic acid Acetic acid Total

17.5 24.0 13.1 13.2 33.1 17.8 118.8

27.4 50.2 24.8 22.9 103.6 42.9 271.8

6.5 0.0 0.0 1.7 0.0 0.0 65.1

28.0 41.5 40.3 37.5 65.8 62.9 48.9

6.5 9.0 4.9 4.9 12.4 6.7 44.4

0.8 1.1 0.6 0.6 1.5 0.8 5.5

Cations Calcium Magnesium Sodium Potassium Ammonium Total

10.6 5.2 9.5 73.5 5.9 98.7

17.2 8.3 16.8 112.5 17.9 149.9

5.4 3.0 4.6 27.3 1.4 28.3

29.6 25.5 40.4 35.4 54.6 31.1

4.0 1.9 3.5 27.4 2.2 36.9

0.5 0.2 0.4 3.4 0.3 4.6

Anions Phosphate Chloride Nitrate Total

29.4 52.8 5.2 83.1

39.4 83.1 14.7 115.2

17.7 28.5 0.0 49.7

18.1 24.8 88.2 19.8

11.0 19.7 2.0 31.1

1.4 2.4 0.2 3.8

Trace-minerals Cobber Zinc Manganese Iron Total

0.013 0.106 0.077 0.392 0.585

0.020 0.216 0.156 1.023 1.277

0.000 0.048 0.017 0.126 0.289

33.2 46.6 42.3 47.4 37.4

4.0

0.005 0.040 0.029 0.146 0.219

0.001 0.005 0.004 0.018 0.027

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13 Plant Juice in the Biorefinery – Use of Plant Juice as Fermentation Medium Table 13.1 (continued) Component

Average evaporated fresh brown juice Maximum Content (g kg–1 DM)

Vitamins Pantothenic acid Niacin Thiamin Total Amino acids Lysine Serine Glutamine Glycine Alanine Asparagine Methionine Cystine Threonine Valine iso-Leucine Leucine Tyrosine Phenylalanine Histidine Arginine Total Crude protein

0.060 0.133 0.015 0.209 5.0 6.3 17.2 6.2 13.1 20.9 1.2 1.3 6.0 7.3 4.6 7.0 2.3 4.4 2.9 4.0 124.0 203.0

0.101 0.190 0.028 0.276 7.2 12.1 27.2 16.4 21.8 51.7 1.8 1.9 7.9 10.4 6.6 9.6 5.9 6.8 5.0 8.8 19.1 279.1

Minimum St. dev. (%)

0.035 0.068 0.011 0.154 3.2 4.2 2.9 4.0 8.3 10.3 0.6 0.8 3.8 4.6 2.6 4.2 0.0 2.3 0.5 0.5 167.5 133.2

27.139 17.676 23.158 14.865 17.5 25.8 24.8 33.8 20.5 42.1 22.5 24.0 18.0 19.7 20.7 20.7 70.6 24.7 28.1 32.5 20.7 18.3

Content (g L–1)

0.023 0.050 0.006 0.078 1.9 2.4 6.4 2.3 4.9 7.8 0.4 0.5 2.2 2.7 1.7 2.6 0.9 1.6 1.1 1.5 46.3 75.9

Average fresh brown juice Content (g L–1) (Calculated)

0.003 0.006 0.001 0.010 0.2 0.3 0.8 0.3 0.6 1.0 0.1 0.1 0.3 0.3 0.2 0.3 0.1 0.2 0.1 0.2 5.7 9.4

Mono-, di-, and trisaccharides are called free carbohydrates because they can be metabolized by most lactic acid bacteria. Fructans (including 1-ketose) are polymeric carbohydrates consisting of variable numbers of fructose molecules and terminal sucrose. Fructans can be decomposed to free carbohydrates both by enzymes in the crops, fructan fructohydrolases, and by some strains of lactic acid bacteria, especially in the genera Lactobacillus plantarum and Lactobacillus paracasei subspecies paracasei [7]. The enzymes in the crops are activated after harvesting, carving, and pressing. Alfalfa does not contain fructans. 13.5.2 Seasonal Variations

Table 13.1 also shows the highest and the lowest value for a component found in the 42 different batches of brown juice and the standard deviation. For most compounds there are very high deviations between batches. The composition of

13.5 Brown Juice

Fig. 13.2 Seasonal variation in free sugars and fructan in different batches of brown juice harvested from June 1st to November 6th 2000.

green crops is known to be influenced by many factors. These include climatic influences such as light and night temperature, the fall of rain, management practices, nitrogen application level, genotype, and relative proportions of leaf and stem. The composition of the brown juice is further more influenced by the type of crops harvested. Figure 13.2 shows the seasonal variation in free sugars and fructan in different batches of brown juice harvested from June 1st to November 6th 2000. The amount of free sugars in BJ has been found to vary between 135 and 340 g kg–1 DM and fructans between zero and 200 g kg–1 DM. The highest total amount of carbohydrate was found at the beginning of the season in June and lowest total amount of carbohydrate was found short periods in the beginning of August and September when the fructan content, especially, drops dramatically. Because alfalfa contains no fructan and is harvested only in this period it is likely that the drop is caused by a considerable increase in juice derived from alfalfa in the mixed brown juice. A high content of organic acid in the BJ during this period indicates that some of the sugars are converted to organic acid before pressing (Fig. 13.3). Figures 13.4 and 13.5 shows the variation in minerals and amino acid/protein in the brown juice. The amounts of cations and anions in the brown juice seem to increase toward the end of the season whereas amounts of trace minerals are more stable. The amount of amino acids and crude protein in the juice fluctuate throughout the harvesting season, but no great seasonal variations are found.

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Fig. 13.3 Seasonal variation in organic acids in different batches of brown juice harvested from June 1st to November 6th 2000.

Fig. 13.4 Seasonal variation in minerals in different batches of brown juice harvested from June 1st to November 6th 2000.

13.5 Brown Juice

Fig. 13.5 Seasonal variation in amino acids and crude protein in different batches of brown juice harvested from June 1st to November 6th 2000.

13.5.3 Lactic Acid Fermentation of Brown Juice

Different lactic acid bacteria have been tested for their ability to utilize the most common carbohydrates. Several strains have been found suitable, but despite the fact that Lactobacillus salivarius is unable to metabolize fructan, it has been chosen for lactic acid fermentation of brown juice because of its high growth rate. This high growth rate makes L. salivarius BC 1001 robust and capable of competing with other microorganisms for an unsterile substrate [6]. By choosing a robust, fast-growing microorganism that can compete with unwanted microorganisms in the brown juice, sterilization of the substrate can be avoided. The brown juice contains both carbohydrates and amino acids, which in combination can form Maillard compounds during heat sterilization. This will cause a decrease in the nutrient-content and reduce the quality of the juice, because of the toxicity of some Maillard compounds. Fermentation experiments with fresh non-heat-sterilized brown juice have shown that fructan is used in fermentations with L. salivarius even though this strain is unable to produce fructan-degrading enzymes. This is probably because of the activity of plant enzymes in the brown juice. Under monoseptic fermentation conditions fructan is not metabolized because enzymes in the juice are destroyed by heat sterilization [8]. Fed batch fermentation of fresh brown juice (2% DM) has shown that brown juice with only 2% DM contains enough nutrients in the substrate to achieve high lactic acid production. In this experiment the specific growth rate of Lb.

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Fig. 13.6 Growth and titration curve for fed batch fermentation with L. salivarius BC 1001 in fresh non-heat-sterilized brown juice with 2% DM, to which glucose syrup, 40%, was added successively.

salivarius BC 1001 was 0.9 h–1 and a cell mass concentration of approximately 3 g L–1 was achieved (Fig. 13.6). The composition of nutrients in the brown juice causes the growth of cell mass to stop at a certain concentration, whereupon the energy in the substrate is used exclusively for maintenance and production of lactic acid. This characteristic makes the brown juice suitable as a substrate for continuous lactic acid fermentation. It has been shown that the growth rate of L. salivarius BC 1001 is higher in brown juice than in MRS-bouillon, which is a substrate commonly used for lactic acid fermentation [8]. 13.5.4 The Green Crop-drying Industry as a Lactic Acid Producer

The green crop-drying industry in Denmark is located in Jutland. There are five factories with a total production of approximately 200 000 m3 green and brown juice 5% DM. It is shown in Fig. 13.6 that 2% DM is enough to achieve good conversion of carbohydrates to lactic acid. A single green crop-drying factory produces enough juice for lactic acid production of approximately 8000 tons per year if a carbohydrate source is added to achieve a lactic acid concentration of 8% in the fermentation broth. Experiments with continuous lactic acid fermentation of brown juice have been performed at the green crop-drying factory: Dangrønt Products, Ringkøbing, Denmark. Figure 13.7 shows the process. This pilot scale continuous lactic acid fermentation of brown juice was successful. The pH of brown juice was reduced to 4.7 and much of the sugar in the brown juice was converted to lactic acid. Table 13.2 shows the dry-matter content, density, and pH of the brown juice after the process. Table 13.3 shows results from analysis of the lactic acid fermented brown juice.

13.5 Brown Juice ,

Fig. 13.7 The brown juice produced from the pressing of the crops was cooled to 35 8C ± 5 8C and fed into a 8 m3 tank. The tank was inoculated with 4 m3 bacterial culture (Lb. salivarius BC 1001). Fermentation was initiated as batch fermentation, and after pH in the tank had dropped to approxi-

mately 4.5, substrate flow was started. The initial flow rate was 3.5 m3 h–1. The acidified BJ was fed into a sedimentation tank, from where the supernatant was pumped to a storage/buffer tank before evaporation to approximately 40% DM.

Table 13.2 Dry matter, density, and pH of lactic acid fermented and evaporated brown juice in pilot scale continuous fermentation at Dangrønt Products, Ringkøbing 16/10 2001. Dry matter

Density (kg L–1)

pH

45.3

1.2

4.7

The analysis of the lactic acid fermented brown juice shows that efficient lactic acid fermentation has occurred. The amount of lactic acid produced is 280 g kg–1 DM, leaving only small amounts of sugar in the medium. The strain used for this fermentation (Lb. salivarius BC 1001) is a homofermentative strain, and only small amount of acetic acid and succinic acid is present in the fermen-

307

(g L–1)

(g L–1)

77.2

132.0

55.7

Anions

Cations

Crude protein (g L–1)

103.5

143.4

245.2

0.440

Trace minerals (g L–1)

0.818

3.4

6.8

(g L–1) 1.8

(g L–1)

Free sugars Fructan

12.5

33.5

8.6

(g L–1) 18.0

(g L–1)

Total sugars Succinic

16.0

151.2

(g L–1)

Lactic

280.8

12.7

(g L–1)

Acetic

23.5

181.9

Organic acids (g L–1)

337.8

63.2

(g L–1)

Amino acids

117.3

Crude Cations Anions Trace Free sugars Fructan Total sugars SuccinicLacticAceticTotal Amino acids protein minerals acid acid acid org. acids (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM) (g kg–1 DM)

Table 13.3 Composition of lactic acid fermented and evaporated brown juice (45.3% DM) produced in pilot scale continuous fermentation at Dangrønt Products, Ringkøbing 16/10 2001.

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13.6 Potato Juice

tation product. Substantial amounts of nutrients (minerals and amino acids) remain in the brown juice after lactic acid fermentation, indicating that adding more carbohydrate could increase the yield of lactic acid. This would be necessary in the production of PLA to make the process feasible. This will be discussed in Section 13.7.

13.6 Potato Juice 13.6.1 Potato Juice as Fermentation Medium

At potato starch factories the potatoes are sorted, washed, and grated. After grating, the potato pulp is centrifuged and the starch, pulp, and potato juice are separated. Two waste products are produced: potato pulp and potato juice. The pulp is sold for animal feed and the juice is spread on fields as fertilizer. The potato starch industry thus faces the same problem as the green crop-drying industry – a waste product that must be stored in large lagoons from October 1st or be used for other purposes. Experiments have been performed to investigate the quality of potato juice with regard to pH, lactic acid, and acetic acid formation, when stored under aerobic and anaerobic conditions. Figures 13.8 and 13.9 show the results of these experiments. The figures show that aerobic and anaerobic storage of the juice causes a conspicuous drop in the pH of the juice within the first 7 days. This is because of lactic acid and acetic acid fermentation of the carbohydrates and some organic

Fig. 13.8 pH, lactic acid and acetic acid during aerobic storage of potato juice at a temperature between 15 and 20 8C.

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Fig. 13.9 pH, lactic acid and acetic acid during anaerobic storage of potato juice at a temperature between 15 and 20 8C.

acids in the potato juice by naturally occurring microorganisms in the juice. Further storage of the juice induces the pH level in the aerobic container to increase to over 6 whereas the pH in the anaerobic container remains stable at approximately 4.5. Accompanying this, a drop in lactic acid concentration and an increase in acetic acid concentration can be observed. At Karup potato starch factory the potato juice is fermented by adding lactic acid bacteria and molasses to achieve a pH of approximately 4.5. Continuous fermentation is performed in a 6000 m3 tank before it is led to big lagoons (140 000 m3) where it is stored. During storage of the potato juice in the big lagoons it can be difficult to maintain a low pH for a long time, primarily because of access to air. A rise in pH provides good growth conditions for bacteria other than lactic acid bacteria, resulting in obnoxious smells. The results in Figs. 13.8 and 13.9 show that prevention of access to air by (anaerobically) covering of the lagoons could be a way of maintaining the quality of the potato juice and thus prevent obnoxious smells. 13.6.2 The Potato Starch Industry as Lactic Acid Producer

There are four potato starch factories in Denmark situated in Northern Jutland, Karup, Brande, and Toftlund, manufacturing a total of approximately 1 million tons of potatoes each year. This gives a total of approximately 200 000 tons of potato starch, 150 000 tons of potato pulp (12 to 13% DM) and between 1 and 1.5 million tons of potato juice (2 to 4% DM) depending on the production methods. At the factory in Karup 300 000 tons of potato juice (2% DM) are produced each year. On that basis Karup potato starch factory could produce approximately 35 000 tons of lactic acid a year if a sufficient amount of carbohydrate is added to the juice.

13.7 Carbohydrate Source

13.7 Carbohydrate Source

To profitably utilize brown juice from the green crop-drying industry or potato juice from the potato starch industry, a carbohydrate source must be added. The carbohydrate source could be another waste product from the agricultural industry such as molasses from beet sugar production, whey from the dairy industry, or soy meal extract from the production of soy protein. It could also be refined sugar (white sugar), hydrolyzed straw, or hydrolyzed cereal grains, e.g. wheat. Wheat has advantages over other carbohydrate sources. Cereals, being much lower in moisture than molasses, are more energy intensive and have the advantage that they can be stored and transported easily. The sugar is in the form of starch and in addition cereal grains contain nutrients which can be separated easily from the grain and sold as lucrative by-products – bran, gluten, and A-

Wet separation (pressing)

Fig. 13.10 A green biorefinery based on green crops will be able to produce products based on organic acids and amino acids made by fermentation [4].

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starch. Gluten – a protein used in the baking industry – is the most profitable of these by-products. Webb and Wang describe a process for utilization of wheat as a carbohydrate source for lactic acid fermentation in which gluten is separated from the grain before hydrolysis of the starch. The filamentous fungus Aspergillus awamori is capable of producing enzymes to break down the starch to glucose [9]. As shown in Fig. 13.10, there are many possibilities of producing fermentation products on the basis of on green crops and potatoes and thereby changing the green crop-drying factories and potato starch factories to green biorefineries.

13.8 Purification of Lactic Acid

The lactic acid must be recovered and purified from the fermentation broth. The classical method is the calcium lactate process in which the lactic acid is precipitated as calcium lactate and treated with sulfuric acid. The resulting calcium sulfate is separated from the lactic acid solution by filtration and the liquid is evaporated. The process gives residual amounts of gypsum, and waste gypsum disposal can be a problem. The latest developments in lactic acid purification have been towards membrane processes such as ultrafiltration and electrodialysis. Other methods such as ion-exchange and solvent extraction involve heavy initial costs and operating costs; they are, therefore, not very suitable for low-cost production of lactic acid. Electrodialysis is a membrane process in which the membranes allow passage only of either anions or cations (ion-exchange membranes), the driving force being potential difference. Because the membranes are easily fouled if the fermentation broth contains large impurities and particles, these are removed by ultrafiltration before electrodialysis. Problems with membrane processes arise if the fermentation broth contains large amounts of organic particles and inorganic ions such as calcium and magnesium, which damage the membrane. Birgit and Michael Kamm use ultrafiltration, nanofiltration, and electrodialysis for purification of lactic acid from fermentation broth. They have developed a process whereby lactic acid is neutralized with piperazine, an amine that combined with two molecules of lactic acid makes piperazinium dilactate. The piperazinium dilactate can be converted to dilactid (a building block in the production of polylactate) without the production of undesired by-products. Birgit and Michael Kamm did not experience problems with fouling of the ultrafiltration membrane but some optimization of the nanofiltration and electrodialysis processes is still needed [10]. At the Technical University of Denmark a process has been developed in which the lactic acid is continuously removed and purified from the fermentation broth using various membrane processes (Donnan dialysis, electrodialysis with bi-polar membranes, and electrodialysis). In this process problems with fouling of the membranes are minimized or avoided [11].

References

13.9 Conclusion and Outlook

Waste products from the green crop-drying industry and the potato starch industry, brown juice and potato juice, contain all the nutrients necessary for lactic acid bacteria to convert carbohydrates to lactic acid. Brown juice 2% DM in a substrate contains enough nutrients to achieve good lactic acid production in continuous fermentation by addition of a carbohydrate source. It is possible to use other cheap waste products from the agricultural industry, for example molasses or hydrolyzed straw, or purer products such as refined sugar or hydrolyzed starch, as a carbohydrate source. Hydrolyzed B-starch from wheat has several advantages compared with other carbohydrate sources. The most important is that the grain contains nutrients, for example bran, gluten and A-starch, which can be separated easily from the grain and sold as lucrative by-products. At the Technical University of Denmark a very promising process has been developed for purification of lactic acid produced from agricultural waste products. This process can substantially reduce the cost of lactic acid production. The experiments reported in this article show it is possible to produce lactic acid from brown juice or potato juice on an industrial scale using non-sterile brown juice or potato juice for fermentation. The fermented juice can be stored under anaerobic conditions and used as a substrate for all-year round production of lactic acid, lysine, and many other fermentation products.

Acknowledgments

This work was supported by the Danish Ministry of Food, Agriculture and Fisheries under the program “Increased Utilization of Renewable Resources for Industrial Non-food Purposes” (1997–2001). We thank Vagn Hundelbøll, AgroFerm A/S, and Jens Mikkelsen, the potato starch factory in Karup for the interesting co-operation.

References 1 Shogren, R., 1997, Water Vapour Perme-

3 Petersen, K. et al., 1999, Potential of Bio-

ability of Biodegradable Polymers, J. Environ. Polym. Degrad. 5(2). 2 Weber, C. J., Biobased Packaging Materials for the Food Industry Status and Perspectives – A European Concerted Action, KVL Department of Dairy and Food Science, Frederiksberg, Denmark. Trio Design, Copenhagen

based Materials for Food Packaging, Trends Food Sci. Tech. 10, 52–68. 4 Andersen, M., Kiel, P., 1999, Method for Treating Organic Waste Materials, European Patent Application, 19 March 1999, WO 00156912. 5 The Danish Ministry of Environment and Energy’s order no. 823 of 16 Sep-

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13 Plant Juice in the Biorefinery – Use of Plant Juice as Fermentation Medium tember 1996. Order of the use of waste products for agricultural purpose. 6 Thomsen, M. H., Bech, D., Kiel, P., 2004, Manufacturing of Stabilized Brown Juice for l-lysine production – from University Lab Scale over Pilot Scale to Industrial Production. Chem. Biochem. Eng. Q. 18, 37–46. 7 Müller, M., Steller, J., 1995, Comparative Studies of the Degradation of Grass Fructans and Inulin by Strains of Lactobacillus paracasei subsp. paracasei and Lactobacillus plantarum. J. Appl. Bacteriol. 78, 229–236. 8 Andersen, M., Kiel, P., 2000, Integrated Utilisation of Green Biomass in the Green Biorefinery, Ind. Crops Prod. 11, 129–137.

9 Webb, C., Wang, R., 1997, Development

of a Generic Fermentation Feedstock from Whole Wheat Flour. In: Campel, G. M., Webb, C., McKee, S. L. (Eds) Cereals: Novel Uses and Processes, Plenum Press, New York, pp. 205–218. 10 Kamm, B., Kamm, M., Richter, K., Reimann, W., Siebert, A., 2000, Formation of Aminium Lactates in Lactic Acid Fermentation. Acta Biotechnol. 20(3/4), 289– 304. 11 Garde, A., Rype, J. U., Jonsson, G., 2000, A Method and Apparatus for Isolation of Ionic Species from a Liquid. Not yet published patent. Application no. PA 2000 01862.


Part III Biomass Production and Primary Biorefineries


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14 Biomass Commercialization and Agriculture Residue Collection James Hettenhaus

14.1 Introduction

In the next ten year biorefineries may be processing 100 million metric dry tons (dt) biomass annually for production of fuels and chemicals if a stretch goal set by the US Department of Energy is met [1–3]. Initially, feedstocks with no collection cost like paper mill sludge, bagasse, and rice hulls may be used to validate the conversion technology. They have no associated transport cost. Their quantities may also serve niche markets for high-value chemicals [4]. For larger markets like transportation fuels, their conversion is too small and costly to compete. The biorefining industry’s growth is predicated mostly on corn stover, supplemented with straw and grasses with a delivered price of $ 30 to $ 35 dt–1 [5, 6]. The quantity and ready availability makes stover an early feedstock choice for initial biorefineries (Table 14.1), US Ag residue feedstock availability. When the feedstock market is established with stover, other biomass feedstock, prairie grasses and energy crops, from wide geographic areas will emerge to supply biorefineries. While great strides have been made in improving the conversion process, there remains much uncertainty regarding the feedstock supply. Economies of scale require a biorefinery size of 500 to 2000 dt feedstock per day. Supplying these large quantities raises major issues including:

Table 14.1 US Ag residue feedstock availability, metric dry tons (millions). Corn stover Cereal straw Energy crops Bagasse Corn fiber Rice hulls Total dry tons

200 70 74 6 4 1 355


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· · · ·

agronomic systems for sustainable removal – maintaining soil quality; economic and environmental benefits for the farmer and other stakeholders; methods for improved feedstock collection, storage and transport; and infrastructure required for reliable feedstock supply.

More information is needed for the farmer to assess the opportunity for supplying the emerging biorefinery market for biomass feedstock. What is the value of the biomass to the soil and to the farmer? What cropping practice is needed for sustainable removal, balancing environmental and economic requirements? Previous studies of crop residue removal on soil quality are limited to small research plots that often do not align with current best practice. Although existing models provide guidelines for residue management to limit soil erosion, robust models that address soil quality and residue removal are just emerging. Bulky crop residues are collected to meet small needs, mostly for local farm use–bales for animal bedding. There is negligible collection infrastructure to serve large markets like biorefineries. Sizable investment – $ 50 to $ 100 million – is probably needed in the supply systems for biomass harvesting, collection, storage and transport to supply a 1 million dt year–1 to a biorefinery [1]. Who will make these investments – the farmer, the potential processor, or a biomass supplier that aggregates individual growers? Current supply systems for grain like corn and wheat are highly evolved and efficient infrastructures and may provide valuable extensions. Existing grain elevators may serve as collection points for biomass. Local conditions determine the potential feedstock quantities. Areas capable of providing large supplies have been identified – but like commercializing an idea, much work remains between knowing the location of promising biorefinery sites and reliably supplying them with economic, sustainable feedstock.


Commercial ventures that require large quantities of crop residues have a mixed history of success. Collected material has found more use as co-products than those that remain in the field after harvest. For example, corn fiber is mixed with steepwater and sold as corn gluten feed with a nutrient value 1.1 times cracked corn for ruminant animals [8]. Bagasse has a long history and is discussed in Case Study 2.2. Small quantities like rice hulls offer little economic incentive to develop markets, therefore most remains to be disposed of, often by burning as a fuel or simply land applied [9]. For biomass left in the field – stover, straw and grasses – collection and transportation cost increases the economic hurdle. In areas of the world lacking trees, non wood fiber is pulped, producing quality papers [10]. They make a relatively poor animal feed because of low protein content [8] and their use as a fuel is limited by their composition, especially the low thermal content compared with coal and natural gas.


During the last decade the list of unsuccessful ventures using bagasse, grass, straw, and stover for particle board has grown by more than a dozen [11, 12]. This mixed record hinders enthusiasm among many growers and others to consider feedstock collection as a viable route for biomass commercialization. 14.2.1 Case Study: Harlan, Iowa Corn Stover Collection Project

The largest recent corn stover collection project was undertaken in Harlan, Iowa in 1996 by Great Lakes Chemical. Modern collection methods – self loading and unloading wagons and high speed, over the road tractors – were used to reduce feedstock cost for the production of furfural [13, 14]. The stover revenue to the farmer was $ 3 to $ 12 dt–1, depending on the hauling distance (Table 14.2). Total feedstock requirements were approximately 100 000 dt per year. A collection center for approximately 50% of the total was constructed in Harlan, Iowa, USA, in 1996. The facility sampled and weighed all deliveries, then stacked bales for storage. During the year bales were removed from storage, milled, pelletized, and loaded into trailers and trucked 90 km to the furfural plant. The first year was a learning experience for all. Meetings with local producers were held, and many showed interest in collecting stover for added income, and as a way to remove most of it so the soil would warm in the spring for seed germination without having to plow. The amount collected complied with soil erosion guidelines. But at the conclusion little stover was delivered. Great Lakes had left the collection with the farmers who mostly used their resources for harvesting the corn grain, not baling. The second year Great Lakes employed contract balers and haulers, matching them up with the farmers and the result was an overwhelming collection success. More than 400 farmers committed 20 000 ha of their corn fields. Thirty plus custom harvesters were contracted to perform the baling. Self loading and unloading wagons pulled by high speed tractors were used for bale transport (Fig. 14.1). The bales were collected in the field – 17 round bales, approximately 9.5 dt – in less than 20 min. The wagon traveled at highway speeds, up to 90 km h–1, en route to the collection center. At the collection center the load was weighed, sampled for moisture, and unloaded. In less than 10 min the driver was on the way to the next field.

Table 14.2 Corn stover pricing summary. Revenue payments, dollars per dry ton (mkg) Radius, km Producers revenue Baler’s revenue Hauler’s revenue Total, delivered price

0–25 $ 12.00 $ 16.06 $ 6.71 $ 34.76

26–49 $ 9.05 $ 16.06 $ 9.65 $ 34.76

50–80 $ 6.12 $ 16.06 $ 12.58 $ 34.76

81–164 $ 3.19 $ 16.06 $ 15.51 $ 34.76

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Fig. 14.1 Load-and-go wagon with high-speed tractor.

a)

b) Fig. 14. 2 Corn stover bale storage.


Start-up problems were mostly baler-related: working to achieve a dense bale, with a minimum 550 kg dry weight. Bales are often sold as a unit, not on a dry weight process. Low bale density made transporting a losing proposition. These difficulties were worked out and several weeks of productive baling occurred before the first blizzard on October 27. Afterward, the field conditions were too wet for baling. Approximately 12 000 ha were actually collected, because of the wet, unusually warm winter. The season ended with a waiting list of more than 40,000 ha, more than enough cropland to meet the total plant requirements IF weather permitted. Meanwhile, the price of imported furfural dropped below the plant manufacturing cost, and the operation was bought out by another firm to make hydromulch. Economic, process, and product-quality problems slowed sales. Finally a bale fire destroyed most of their inventory, forcing a liquidation of their assets in 2002. The major findings from the collection operation include the following [15]: · Farmers are willing to sell excess stover when the economics fit. · Collection practices have to meet farmers’ requirements for success. · Logistics are a major factor for successful collection. · Contract operators are essential to meet the extra workload. · Shortening the harvest window is essential to reduce collection risk due to weather. · Bale storage is costly and adds no value. · One-pass harvest, with the grain, can reduce cost and harvest risk. · Bale fires are a serious hazard.

14.2.2 Case Study: Bagasse Storage – Dry or Wet?

Bagasse, the biomass remaining from sugar cane after the sucrose-containing juice has been extracted, has been used by the pulp and paper industry for more than a century [10]. It incurs no collection cost and competes favorably with wood pulp in a global market. The bagasse exits the mill with about 50% moisture. For stable storage it must be below 20% or above 60%. In the early 1920s the sugar cane industry investigated ways to store bagasse for processing to particleboard and pulp. Particleboard processors preferred dry material. Pulp mills readily accepted wet material.

14.2.2.1 Dry Storage Celotex produced insulation board, a dry process, and pursued ways to improve dry feedstock storage. Their effort resulted in using the heat from microbial fermentation to dry bales from 50% to less than 20% moisture [16]. The bales were sized and stacked to dissipate heat and acid fumes. Sheltered from the weather, bales kept for several years without serious deterioration (Figs. 14.3 and 14.4).

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Fig. 14.3 Bale stacking, circa 1930.

Fig. 14.4 Bale storage, 1930–1960.

Although this dry storage method was used for more than 40 years [17], a change to wet storage occurred in the 1960s because of increasing recognition of its advantages: · The bales were relatively small, weighing 115 kg “as is”. · Mechanical handling was slow and costly. · The bales had to be precisely stacked to vent fumes and dissipate heat. · Procedures were labor-intensive. · Several months were required to dry bales from 50 to 20% moisture. · Fire loss and increasing fire insurance costs.


14.2.2.2 Wet Storage Whereas Celotex pursued dry storage, other companies in the pulp and paper industry continued investigating wet storage, because pulping is a wet process. The results were more successful than dry storage. Wet storage of non-wood fibers was first commercialized by E. A. Ritter in 1950. Wet storage of bagasse has been in widespread use on a commercial scale since 1960 for both wet and dry downstream processes [18]. Figure 14.5 shows a typical collection area with a pile under construction in the foreground. Major studies on a commercial scale showed pulping the stored bagasse was superior to pulping dry bagasse and fresh, wet bagasse. The wet feedstock contained less solubles, required less chemical treatment, produced higher yields and had better processing characteristics compared with dry feedstock and fresh, wet bagasse [19]. In addition, wet feedstock stored for 6 months was superior to “green” bagasse, the fresh material from the current harvest in processing characteristics, with 80% less solubles and higher holocellulose composition. As a result, green bagasse is held over, for processing the following year [20]. Section 14.5.2 discusses storage further.

Fig. 14.5 Wet storage pile construction.

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14.3 Biomass Value

Lacking large uses of crop residues, studies have not addressed the impact of removal of stover [21] and straw [22] on soil quality, although several implications can be drawn [23]. An environmental and economic balance is required for sufficient retention of residues to avoid erosion losses and maintain soil quality, while economically removing excess residue as biomass feedstocks. The impact of different levels of surface removal depends on local conditions and practices. Maintaining this balance is key for success. 14.3.1 Soil Quality

Agricultural residues provide a key role in maintaining soil quality. Surface cover is needed to prevent wind and water erosion, retain soil moisture, recycle nutrients from the plant back to the soil, and support assorted life. When residue is removed, reduced inputs from the residue to the soil can result in a negative flux from the soil and a loss of soil organic matter, SOM, and other nutrients leading to a breakdown of soil structure. The amount of excess is a complex question. It depends on local factors like soil type, cropping practice, weather, and topography. For example, in some dry areas surface cover is required to retain moisture, mostly in western areas of the grain belt [24]. Further east, surface cover is given as a major reason for tilling, because the cover prevents the cold, wet soils from warming in the spring, delaying planting and reducing yield [25]. Recycling nutrients from the plant, especially P and K, back to the soil reduces the need for replacement. Conversely, in areas where manure is used, the P contained in the soil may already be too high, resulting in excessive run-off that contributes to algae formation in ponds and streams [26]. Surface cover provides shelter and food for many organisms – microbial and larger. Their contribution to the humic pool is important, but they also shelter destructive weed seeds, pests, and toxins that can harm the next crop [27]. Models are under development to better measure soil quality. For example, agricultural ecosystem models like Century, DayCent, and Cstore show some beneficial effects of removing residue while still meeting constraints of soil and wind erosion. Namely, nitrate leaching decreases tenfold in some situations and nitrous oxide emissions (a potent greenhouse gas) are also significantly reduced. These models can be used with actual field measurements for guidance in selecting among alternatives that best balance economic and environmental benefits [28]. Using field measurements with the soil conditioning index will show if the crop practice is correctly managing soil carbon and is recommended [29].

14.3 Biomass Value

14.3.2 Farmer Value

Most farmers are forced to manage crop residues in place. Present markets are negligible for straw and corn stover. Less than 5% is used for animal bedding and feed, with the major portion used on the site and then recycled as soiled bedding or manure. Some growers have historically rented harvested corn fields for grazing, charging $ 12 to $ 25 ha–1. This practice is declining as combines have become more efficient, lodging is less with Bt corn, and cattle ranching has grown more “factory-like” with heavy dependence on large feedlots. If residue were removed, the phosphorus (P) and potassium (K) content in straw and stover will eventually need to be replaced. The composition is typically 0.1% P and 1% K, valued at $ 3.50 dt–1 [6]. The N fertilizer value is more complex, and depends on crop rotation and local conditions. Reduced field operations are estimated to reduce inputs $ 24 ha–1 for preparation of the seed bed [30]. Carbon credits are likely to add additional economic incentive for US farmers. Reducing tillage or no-till sequesters about 0.3 to 0.5 metric tons C equiv ha–1. The increased soil carbon improves yields, and this benefit continues with each crop year. Eventually, over decades, soil carbon equilibrium is achieved. In the European Union, carbon is currently trading for about $ 35 per ton C equiv. A small, voluntary greenhouse gas trading market has been established for agricultural carbon sequestration as part of the Chicago Board of Trade, the Chicago Climate Exchange [31]. Recent efforts to move US policy in this direction call for a $ 26 per ton of C-equivalent credit that would fund renewable fuels research and development [32]. Reducing N fertilizer use is also possible, depending on crop rotation. Microbes desire a 10 : 1 ratio of C/N for breaking down residue. Because the C/N ratio of straw and stover is 40 to 70 : 1 , 10 kg N fertilizer addition per ton of residue is typically recommended to avoid denitrification of the next crop. For 250 ha of 9 dt ha–1 corn (170 bu acre–1), 30 to 40 tons of N fertilizer may be avoided. In addition to the out-of-pocket costs, environmental benefits include reducing N run-off to streams and groundwater, and reducing greenhouse gas – 0.17 to 3.5 tons of N2O/100 tonnes applied – 5 to 100 tonnes C equiv ha–1 [33]. The economic benefit from selling stover to the farmer is summarized for three production yields – 6.9 dt ha–1, 9.9 dt ha–1, and 10.6 dt ha–1 (130, 170, and 200 bu acre–1) in Table 14.3. More details are presented in Section 14.5. The example uses the following values: · delivered sale price is $ 33 dt–1 · moisture is 15% to adjust to dry basis · harvest index of 0.5, a 1 : 1 ratio of grain to stover · surface cover of 2.2 dt ha–1 left in the field, · P and K fertilizer value of $ 3.50 dt–1 removed with the stover · reduced field operations, $ 24 ha–1 · no credit for carbon sequestration or other inputs like N fertilizer.

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14 Biomass Commercialization and Agriculture Residue Collection Table 14.3 Stover field value before transportation and collection cost, $ ha–1. Production case

1

2

3

Stover production, dt ha–1, 1 : 1 ratio Surface cover, 2.2 dt ha–1, left in field Stover sales, dt ha–1 Stover revenue, $ 33 dt–1 P & K nutrient credit ($ 3.50 dt–1) Reduced field operations, $ 24 ha–1 Net value, bulk in field, $ ha–1

6.9 (2.2) 4.7 $ 155 (16) 24 $ 163

9.1 (2.2) 6.9 $ 225 (24) 24 $ 226

10.7 (2.2) 8.5 $ 278 (29) 24 $ 273

The net value in the field is $ 163 to $ 273 ha–1 before collection and transportation cost. The net margin after Collection and transportation costs are determined is given for two examples – typical baled stover collection (Table 14.4), and onepass collection (Table 14.5). Other costs like shrinkage or change in properties of the feedstock in storage, storage investment, and operation, and relative quality of final feedstock processed are not included in the examples. There is a large variation in these factors depending on the local situation. Baled stover, Table 14.4, is trucked within a 50 km radius – an average 70 km round trip using high speed tractors and “load and go wagons” as in Harlan, IA. Baling and transport cost adds $ 25 dt–1, leaving the farmer a net margin of $ 41 to $ 54 ha–1. A minimum $ 50 ha–1 return is most often cited to raise grower interest [34, 35].

Table 14.4 Stover sale net to farmer, $ ha–1 W/custom bale and 50 km radius collection site. Case

3.1

3.2

3.3

Net margin, bulk in field, $ ha–1 Less custom bale, $ 16.00 dt–1 Hauling, 50 km radius, $ 9.9 dt–1 Net margin to farmer after delivery, $ ha–1

$ 163 (75) (47) $ 41

$ 226 (109) (68) $ 49

$ 273 (135) (83) $ 54

Table 14.5 Sale net to farmer, $ ha–1, W/one-pass harvest and 3–25 km radius collection sites. Case

3.1

3.2

3.3

Net margin, bulk in field, $ ha–1 Less one-pass harvest, $ 40 ha–1 Field to collection site, $ 6.00 dt–1 Hauling, 50 km radius, $ 9.9 dt–1 Net margin to farmer after delivery, $ ha–1

$ 163 (40) (28) (35) $ 59

$ 226 (40) (41) (51) $ 93

$ 273 (40) (51) (63) $ 119

14.3 Biomass Value

One pass collection of grain and stover, with stover trucked from field to one of three collection centers within a 25 km radius – 35 km average round trip, stored above 60% moisture, then transported via rail to the biorefinery – offers more opportunity to reduce cost and reduce harvest risk [36]. Even with higher transport cost, $ 13.50 vs $ 9.90 dt–1, the farmer’s net margin after delivery ranges from $ 59 to $ 119 ha–1 (Table 14.5). The difference is greatest for fields with high yields, with nearly twice the margin compared with baling. One-pass harvest prototypes are under development, and further discussed in Section 14.5. 14.3.3 Processor Value

Carbohydrate feedstock is being seriously considered as an alternative to petroleum and natural gas feedstocks by the chemical and plastic materials industry. The escalating cost of fossil feedstocks and their pricing instability have contributed significantly to the industries low margins. Added environmental concerns, especially emissions that contribute to global warming have generally shrunk their market value. Low cost fermentation sugars coupled with rapid advances in biotech tools offers these industries a potentially economic and sustainable feedstock for production of transportation fuels, chemicals, and materials. For example, E85 fuel, 85% ethanol in gasoline, reduces greenhouse gases by 64% compared with gasoline. The effective benefit is 170 kg C equiv mitigated per dt corn stover processed compared with regular gasoline [37]. Sourcing low-cost feedstock is a common critical success factor. Successful crop residue-processing plants wish to achieve an economy of scale well below the local feedstock supply limits to accommodate expansion. Most biorefineries plan to have a business model that offers the grower an option to participate in the value chain to help ensure a win–win business relationship. An example is corn growers who are also shareholders in dry mill plants now growing hybrids with traits that produce more liters of ethanol kg–1 corn. They have no grain yield drag and processing this corn adds value to their stake in the dry mill [35]. Sustainable supply is of prime importance to the processors. Environmental concerns are a major driver for the industry to move from fossil feedstocks. Assurances will be needed that the long-term effect on the soil quality is neutral or better to avoid the claim that removal of residues is depleting the soil of needed nutrients [35]. Feedstock pricing has a major effect. The National Renewable and Energy Laboratory uses $ 33 dt–1 delivered cost to the biorefinery in the base case of their process. The result is based on extensive experimentation, process modeling and industry peer reviews. A $ 5 dt–1 change in feedstock price affects ethanol 1 c/ to 1.5 c/ L–1, depending on process yield [7]. Wet or dry feedstock can be processed, but if dry, some prefer bulk delivery of milled product to avoid the multi-million dollar investment in equipment and dust-explosion-proof installation [38].

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Clean feedstock with consistent composition and properties are desired, but more information remains to be provided by the processors. Because dirt, inorganics and ash add to the processing cost, and higher cellulose and hemicellulose content can increase yields, feedstock pricing based on composition and ease of processing is expected to emerge, not paying just per dry ton. Rapid analytical methods for compositional analysis are under development and are expected to be validated and available for incorporating in a feedstock payment program [39].

14.4 Sustainable Removal

Removing residues from the soil depletes the amount available for replenishment for nutrients and increases the possibility of erosion. Compensating for this will probably require revising some present agronomic practices. For example, leaving anchored stubble, 20 cm or more above the crown may suffice in some areas to control erosion, especially with narrow planting rows, e.g. 40 cm compared with 80 cm, the most prevalent practice for corn rows. This and other contemplated needs are discussed in this section. With a biorefinery, the excess is removed, processed into fuels – and “digested” by the autos – the CO2 exits from the auto’s exhaust, instead of being exuded from microbes in the field. The benefits are huge, BUT only when this is conducted in a sustainable manner by controlling erosion and maintaining soil quality, especially soil organic material (SOM). 14.4.1 Soil Organic Material

Tilling causes loss of SOM, an important measure of soil quality. If too much stover is removed or a cover crop is not planted that adds to the biomass, SOM can be depleted. SOM is more strongly affected by below-ground residues (i.e. roots), with above-ground residue contributing less to SOM formation. Studies at the National Soil Tilth Laboratory shows 80% or more of the surface material is lost as CO2 within months, and three times the amount of SOM comes from roots compared with surface material [40]. The tillage effect on soil carbon loss after corn harvest is shown for various field operations in Fig. 14.6 [41]. The CO2 flux emitted from the soil is shown for various cases over time. The amount of loss depends on the amount of disturbance – more exposure, more oxidation of the organic material and more lost soil carbon. The bottom line in the figure shows normal soil respiration as microbes and other organisms in the soil and on the soil surface emit CO2 as they digest biomass carbohydrates and lignin. The top line shows the highest loss when plowing the soil – an initial burst of CO2 occurs as the plow rips open the soil and the anaerobic soil environment is exposed to oxygen in the air.

14.4 Sustainable Removal

Fig. 14.6 Fall tillage effect on soil carbon.

No-till or reduced-till practices are needed to maintain soil organic material when stover is removed – if not, the carbon loss from tillage will deplete the soils’ carbon pool, resulting in negative effects on soil quality, including aggregate instability, less water-holding capacity, and an associated drop in productivity. 14.4.2 Soil Erosion Control

Loss of topsoil remains a concern and continues to be a severe problem in many areas. Not only does much soil get lost in waterways, erosion also depletes soil fertility. The SOM is greatest in the topsoil, and up to 20% of the SOM is estimated to eventually be lost to the atmosphere as a result of cropping practices [42]. For adequate feedstock supply and efficient collection less tillage will be required to control erosion in addition to maintaining SOM. With present tillage practices all of the residue must remain in 60% the corn fields and 70% of the wheat fields to comply with USDA erosion control guidelines. The USDA guidelines are based on extensive studies that indicate all the residue should remain in fields with conventional till, i.e. less than 30% of the surface is covered. The guidelines are based on “tolerable” soil loss, a judgmental value [21]. Less tillage, referred to as mulch till, has more than 30% of the surface covered, allows some residue removal while no-till or ridge till permits most of the surface material to be removed. Ridge-till or strip till, involves tilling a strip through the field, clearing away the residue in a space adequate for planting and warming the soil in the seed area for early germination. The ridge is favored with furrow irrigation. Without the ridge, it is referred to as strip till.

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For example, using the USDA water-erosion model, revised universal loss equation (RUSLE), and wind erosion equation (WEQ), the variation in required erosion cover for selected counties in the top corn-producing states – Illinois, Iowa, and Nebraska – are shown in Table 14.6. Wheat is included, because irrigated wheat yields enough to warrant straw collection in Peoria County, IL, Jasper County, IA, and Rock County, NE [43]. Unless wheat is irrigated, much of the wheat yield on dry land is below 2.2 dt ha–1, negating any straw removal. Even with no till, economic quantities are difficult unless yields approach corn, 8 to 10 dt ha–1. The historic tillage practices for corn and wheat for the US are summarized in Tables 14.7 and 14.8. The values are based on a nationwide survey conducted by the Conservation Technology Information Center (CTIC) [44]. Table 14.7 shows about 20% of corn is no-tilled or ridge-tilled, enabling efficient collection. More than 80% of farmers employ some form of tillage to manage surface residues, with 60% of corn fields conventionally tilled. This has become a larger task as yields have increased. In 1960, corn belt states averaged 3.6 tons ha–1. In 2003 the yield had increased to 10.0 tons ha–1. Adapting to no-till corn will be easier in many areas when some of the stover is economically collected, especially in the northern parts of the corn belt where cold moist soils can delay germination in the spring. Each day delayed is 25 kg corn grain lost according to local lore. Most corn growers till for removing, i.e., burying, corn stover to induce spring soil warming.

Table 14.6 County-level stover and straw cover required for water and wind erosion. State

IL IL IA IA NE NE

County

McLean Peoria Grundy Jasper Rock Dawson

Corn, dt ha–1

Wheat, dt ha–1

No-till

Mulch till

No-till

Mulch till

1.1 2.1 1.6 2.8 2.4 1.6

2.2 4.6 2.7 6.7 4.0 2.4

NA NA NA NA 1.1 1.1

NA NA NA NA 2.3 1.7

Table 14.7 Corn tillage practice, % total. Year

1994

1996

1998

2000

2002

No till Mulch Ridge till Conventional

18 22 3 60

17 23 3 60

16 23 3 61

18 19 2 63

19 16 2 64

14.4 Sustainable Removal Table 14.8 Wheat tillage practice, % total. Year

1994

1996

1998

2000

2002

No till Mulch Conventional

5 25 69

7 24 69

9 23 68

10 20 70

11 16 73

For wheat, less than 10% of the acres are no-till (Table 14.8). Weed control of wheat is the primary reason for tillage. Changing to no-till wheat is slowly gaining favor, increasing from 5% in 1994 to 11% in 2002. But conventional till has increased 4% over the same period, despite higher fuel costs for field operations. Evidently, the new investment in no-till equipment, $ 70 000 to $ 150 000, coupled with low commodity prices is a significant obstacle to changing. 14.4.3 Cover Crops

Cover crops can offset the erosion effect from collecting residues by restoring the surface cover, reducing wind and water erosion. Cover crops can also improve soil quality: their additional biomass builds SOM, especially from their roots. Cover crops also choke out weeds and may retain N in its root system over the winter, thereby reducing N2O emissions and nitrate leaching [45]. Cover crops require a higher level of management. Important factors for consideration include: · Selecting appropriate cover crops to fit in the rotation to avoid allelopathic effects, inhibition of growth in one species of plants by chemicals produced by other species · Planning to ensure enough growth occurs to provide the above benefits. Broadcast seeding may be used for the current crop to give it a timely start, and care must be taken not to hinder its growth during that crop’s harvest · Cover crop harvesting or killing growth needs to consider soil moisture conditions. It can deplete needed soil moisture in a dry spring, or if left to grow longer in a wet spring, deplete excess soil moisture · Growth must be stopped for some cover crops like rye before it goes to seed or it will interfere with future cash crops. Establishing local, credible test plots to investigate the impact of residue removal for various cropping practices is important to provide information to both the grower and the processor.

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14.5 Innovative Methods for Collection, Storage and Transport

Most previous studies focus on collecting and baling dry material after the grain harvest. Grain is the cash crop and is at risk until safely collected and stored. Collecting, storing, and transporting bulky straw and stover is secondary. Additional field operations add cost and increase risk. Straw is normally dry enough to bale, but corn stover must dry from 30–50% moisture to less than 20%. Feedstock drying and densification methods can reduce collection delays and increase density. These approaches may be appropriate when a dry, compacted material is desired for co-firing or for thermo-chemical processes. These operations increase cost to $ 50 dt–1 or more, however [46]. Densification inhibits wet processing and pellets need to be “reconstituted” by soaking in water to shorten digestion time for hydrolysis. One-pass harvest of corn grain and stover, wet storage, and transport to the processor seem to be advantageous where wet feedstock is acceptable, as already shown in Tables 14.4 and 14.5. Custom bailing and transporting bales from the field to a processing point within a 50 km has a relative return of $ 41–$ 54 ha–1 depending on the production. One-pass harvest and transport to 3–25 km collection centers for storage and then supplying the processing plant from these sites is estimated to increase margins to $ 59–$119 ha–1, a 44 to 118% improvement. The relative difference between custom baling and one-pass harvest with wet storage and transport for the farmer is shown in Table 14.9. These cost comparisons are relative. Other costs like shrinkage or change in properties of the feedstock in storage, storage investment and operation, and relative quality of final feedstock processed are not included. There is a large variation in these factors depending on the local situation. These are addressed in the following sub-sections. 14.5.1 Collection

Collection choices are divided into two general categories, baling the residue following harvest or one-pass collection of both grain and residue.

Table 14.9 Farmers net margin, stover sale comparison. Case

1

2

3

Stover yield, dt ha–1 A. Custom bale net margin, $ ha–1 B. One-pass, bulk, net margin, $ ha–1 % Improvement

6.9 $ 41 $ 59 44%

9.0 $ 49 $ 93 92%

10.6 $ 54 $ 119 118%


14.5.1.1 Baling Many studies have been made of baling biomass on a large scale. For straw, baling is an option. Cereal grains, like winter wheat, are grown in dryer areas and mature earlier than corn, enabling a longer harvest window. When cereal grain is ready to harvest, straw moisture is usually suitable for collection. In contrast, stover is typically too high in moisture, 30 to 50%. It must remain in the field to dry and be collected later. To speed drying, some flail the stalk. Raking is then required before baling, adding more cost, increasing the foreign matter, especially dirt, in the bales and compacting the soil. A wet harvest season can prevent its collection entirely, because of wet residue [13]. For industrial feedstock, baling only adds cost, $ 15 dt–1 or more at the field [30]. Bales also add cost at the processor for additional equipment and disposal of twine, wrap and foreign contamination [38].

14.5.1.2 One-pass Collection Achieving the target of $ 33 dt–1 with adequate farmer margins will probably exclude baling. This can be achieved with many variations. There are more choices for separation, i.e. ear and stalk, grain-stalk-cob, remove leaves and husk, or just leaves. Prototypes for one-pass harvest of straw and stover are under development, adapting existing equipment and examining new designs. One-pass systems have been compared with multi-pass alternatives for wheat [47] and stover [36, 48–50]. Results indicate one-pass harvest can deliver $ 33 dt–1 target price with $ 70 ha–1 or more margin to the farmer when the following conditions are met: · 9.0 dt ha–1 (175 bu acre–1) or more yield when 2.2 dt ha–1 or less cover maintains soil quality · 50 km or less collection radius · bulk delivery · 400 hours (700 ha) or more utilization of stover collection equipment.

Table 14.10 summarizes the harvest cost as a function of harvester utilization and yield, adjusted for 4th Q 2004 fuel pricing [51]. The key stover collection equipment issue resolves around one question: “What does customer need?” For example: · Corn with ear and stalk? Or grain and stover? · Number of harvester take-offs: 1, 2 or 3? – Component separation? Which parts? – Size reduction? How much? – Other pre-processing? · On-farm storage or at a collection site? Local requirements will vary. In areas where soybeans and corn rotate, the needs are different from wheat and corn or other crop rotations. For wheat, stripper headers work well now, leaving a standing stalk that is clean and readily harvested.

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14 Biomass Commercialization and Agriculture Residue Collection Table 14.10 Collection cost for forage harvester. Annual collection

Harvest cost per ha

Harvest price per dt

ha

Hours 1.8 ha h–1

Fixed cost

Direct cost

Total cost Case 4.1, +15% 7 dt ha–1

Case 4.2, 9 t ha–1

Case 4.3, 11 dt ha–1

250 500 1000 1500

140 281 561 842

$ 70 $ 35 $ 18 $ 12

$ 28 $ 28 $ 28 $ 28

$ 113 $ 72 $ 52 $ 45

$ 7.03 $ 4.52 $ 3.26 $ 2.84

$ 5.86 $ 3.76 $ 2.71 $ 2.36

$ 9.60 $ 6.20 $ 4.50 $ 3.90

Some areas may be satisfied with one take off, separating the grain and other components of the field. Others may desire to use existing equipment, but at the same speed, separating the grain from the stover in the field. One design offers three field separation possibilities as cobs offer commercial value as a carrier for herbicides and other chemicals, cat litter, and metal-polishing applications. Development cost for a new design is $ 2 million or more for prototypes, with millions more required to bring to market. The definition of the customer’s needs, regional differences, market size uncertainty and the cyclical economic performance of this industry makes significant up-front development beyond paper studies difficult to justify until the market for machine sales exist. Without subsidizing development, a “chicken and egg” dilemma exists. 14.5.2 Storage

Maintaining quality of feedstock in storage is a major concern. Stable storage is possible only when the material is dry, less than 20% moisture, or wet, greater than 60% moisture. In the US most industrial experience is with dry storage, mostly bales, and the associated need to keep dry and protect from fire. Pest infestations have not been noted with corn stover, but some report straw is more susceptible. Wet storage of large quantities is also familiar for silage, fermented forage crops. Ensiling green sorghum and corn plants has been practiced for years as a means of preserving feedstock for ruminant animals. These crops are chopped and stored in bags, bunkers, or silos at 65% moisture. The storage life can extend to years, with losses less than 3%. In addition, in many tropical areas “Ritter” wet storage is practiced to supply pulp mills – 250 000 to 500 000 dt piles are built via circulating liquor, saturating the feedstock to 80% moisture. Residual sugar results in fermentation that drops the pH below 4, halting microbial activity. Less area is required; fire is eliminated when stored above 60% moisture. A comparison of dry bale and wet bulk storage is shown in Table 14.11.

14.5 Innovative Methods for Collection, Storage and Transport Table 14.11 Dry and wet storage comparison [36]. Property

Dry (bales)

Wet storage

Dry density, lb ft–3 Storage area Storage loss Foreign matter and soil nutrients Non-volatile solubles removal Weather risk Fire hazard Investment Storage quantity

7 to 10 10 ´ > 10% High Process residue Rain High Low to high Small, mostly farm use

12 to 14 1´ < 5% Low Storage liquor Extreme cold None Medium to high Large, bagasse for pulp

14.5.2.1 Density The dry density of bales is about half that of wet stored material, ranging between 112 kg m3 for round bales to 160 kg m–3 for square bales. Wet storage density depends on the stack height, with 40 meters achieving 200 kg m–3 average pile density [52].

14.5.2.2 Storage Area Square bales require about ten times the wet storage space because of bale stacking limits, access corridors, and a measure of fire protection. The total area required for 1 million dry tons is approximately 500 acres for square bales. With wet storage, a 333 000 dt pile requires 15 acres. For 1 million dt storage, just 3 piles or about 50 acres is needed. The equivalent land rent depends on the local situation, adding zero to $ 2 or more per dt.

14.5.2.3 Storage Loss Bales are adversely affected by wet weather and without shelter can decompose and break apart. For 6'' dia ´ 5' bales, 30% of the mass is in the outer 4 inches and 25% weight loss can easily occur in one season. Stored inside barns, both round and square bales had 14% weight loss over 10 months in eastern Canada [53]. For wet storage, the major losses are the 5 to 8% solubles removed during storage. Typical cellulose and hemicellulose losses reported by the pulp and paper industry are 1 to 3% [54]. Surface loss depends on the total surface exposed relative to the stored tons. The higher the pile, the smaller the exposed surface and the surface loss. While there may be aesthetic or zoning limits, wet storage piles height is limited by design considerations for pump head and recovery, varying between 30 and 40 m. The lignin, pentosans, and holocellulose increased in proportion to decreases of the solubles in water, alcohol–benzene, and 1% caustic soda. The absolute quantity of lignin, pentosans, and holocellulose remained constant during the

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Fig. 14.7 Solubles removal in wet storage.

storage period. The loss in solubles during storage is shown in Fig. 14.7, declining from 10 to 3% within weeks, and fresh bagasse having 7% less solubles for the five trials. The pentosans and holocellulose, which consists of the alpha-cellulose plus the hemicelluloses, continue to increase over a longer period (Figs. 14.8 and 14.9).

Fig. 14.8 Pentosan change in wet storage.

Fig. 14.9 Holocellulose change in wet storage.


14.5.2.4 Foreign Matter and Solubles Bales contain soluble soil nutrients along with foreign material that can be deleterious during storage and processing. Wet storage has proven to remove dirt, foreign matter and solubles over time. Removing the nutrients during storage, returning them to the fields is much preferable to processing the bales and disposing of the process ash. Fewer solubles in the wet feedstock, with the absolute values of holocellulose and pentosans unchanged, increases the plant capacity up to the distillation step: 7% removal opens up 7% more pretreatment, hydrolysis, and fermentation capacity.

14.5.2.5 Storage Investment Storage investment cost must be investigated and evaluated as part of the supply chain – from collection through the disposition cost of the residue and storage liquors. Investment can vary widely for both types of storage. An uncovered stack of round bales on the ground requires negligible investment. Sheltered bale storage investment can be high, depending on the degree of automation. Wet storage investment may consist of a conveyor for building the pile and a water spray system to raise the pile moisture to above 60%. A more elaborate system is shown in Fig. 14.5 can add $ 1 million or more. Bale-storage systems were recently estimated for rice straw [55]. Short term, tarps were favored at a cost of $ 6 to $ 11 dt–1. Longer term, pole barns are favored, costing 50% less with the upfront investment. For a 1 million dt plant, the pole barn investment would range between $ 6 and $ 11 million. At the plant, the bale unloading, interim storage and handling system designed for NREL’s model has a $ 12.9 million capital cost, $ 2.94 dt–1. The operating cost is $ 2.62 dt–1 based on twelve operators, twenty-four hours a day, seven days a week, to handle the bales, adding a total of $ 5.56 dt–1 to the feedstock cost [38]. Truck or rail car unloading of wet or dry bulk material can be automatic, with minimum additional plant labor. 14.5.3 Transport

Transport is divided into two components: · harvest period for the annual crop, typically 20 to 40 days · biorefinery supply requirements: 2 000 to 3 000 dt day–1. During harvest, existing resources are stretched just collecting and transporting the grain in the field while keeping the combines operating continuously. Increasing the resources to accommodate up to 2.5 to 3 times that quantity from the filed is a serious increase.

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14.5.3.1 Harvest Transport Transportation distance from the field to a storage site during harvest needs to be short for truck requirements to remain manageable. Leaving the feedstock at the edge of the field or at multiple collection sites 20 to 30 km in diameter can reduce the trucking requirements, potentially reduce cost by better utilization of equipment, and reduce traffic congestion. Delivering 1 million dt to one site during harvest cause much congestion. For a 20-h delivery day, 100 trucks per hour rumble past, in and out of the location. Sugar cane harvest approaches this, with deliveries to some mill sites reaching 22 000 dt day–1, 1 000 or more truck deliveries. In areas with high yields, three collection sites with a 25 km radius (200 000 ha) can supply 1.5 million dt assuming 30% of the land is harvested. The average round trip is 35 km for each site. Increasing to four sites further reduces congestion during harvest and shortens trips by 35%, to 23 km. The simplest and most efficient logistics occur when the ear and stover are transported together, Weight and volume limits are met using standard trailer dimensions with the resulting bulk density. Baled or bulk stover loads generally reach a maximum volume before reaching the weight limit. Milling the stover or straw increases the bulk density, more closely approaching the maximum weight.

14.5.3.2 Biorefinery Supply Because an annual feedstock supply, 1 million dt, is not expected to be stored on the biorefinery sites, reliable supply is required by the biorefinery throughout the year. Processors have indicated a minimum one to two week supply is desired on site, with deliveries made daily, 5 to 6 days per week. Truck transportation is most common. Other transportation modes previously considered include rail, pipelines, and dirigibles. The viable transport options are truck and rail, and wet or dry feedstock. Truck transportation is most common. Rail is most efficient when prompt service is available. Pipelines are possible but, because of the high absorption of water by the fiber (80%), slurries of more than 96% water are desired – huge volumes that may require a 2nd pipeline for water return [56]. Truck Transport Truck transport has long been favored for both wet and dry materials for short distances. Dry material is bulky and flammable. Flaming trailers in transit are a serious hazard, especially in populated areas. Depending on biomass density and local regulations, the dimensional limits for road transport may be exceeded before the weight limit is reached. Because trucking cost is based on distance, $ 1.00 to $ 1.50 km–1, less than a full weight load results in higher cost. For example, round bale transport using load-and-go wagons was just 9 dt per trailer load, a transport cost twice that of a full load, normally 18.2 tons. Wet material from storage can be passed through a dewatering press and reduced to 50% moisture before transporting. It is perishable with this moisture content and requires timely delivery. Full truck loads are readily achieved, but

14.6 Establishing Feedstock Supply

50% water results in the same pay load as the load-and-go wagon. The water volume requires management. Rail Transport Rail shipments for short distances have become feasible as a result of improved practices, especially when shipments remain on the same line of a regional railroad. With multiple collection points along the rail line, the collection area for the biorefinery can be more than doubled. Moving a rail car 100 to 300 miles is $ 150 to $ 250 with little regard to weight [57]. The cost of rail transit was estimated to be $ 3.00 to $ 5.00 dt–1 for transporting feedstock several hundred miles. Modeling shows conversion costs drop 30% for a 4 million dt plant. Rail shipment of dry material has the associated fire liability issue. The higher weight limits make wet feedstock shipments possible at lower cost. Maximum load per car is usually 114 tonnes, 91 tonnes net, to accommodate grain shipments. Truck traffic increases with increased plant size while rail car shipments are more manageable. Assuming 50 car unit trains traveling at 50 km h–1 (off the main line), approximately 1.5 min is required for the train to pass a road crossing. In contrast, when the truck delivery is limited to just 10 hours each day and 6 days per week, the truck traffic just from feedstock delivery is 50 to 60 trucks in and out every hour for a 1 million dt plant. Storing 1/3 of the feedstock on site reduces the value accordingly, but the disruption is still substantial. Trucks are assumed to carry maximum loads (Table 14.12).

14.6 Establishing Feedstock Supply

At the present time in the US, the dairy herd feed lots in Jerome County Idaho purchase about 1 million dt of straw from the surrounding irrigated barley and wheat fields with yields often exceeding 10 dt grain ha–1. Grower participation and infrastructure evolved over years. It is 20 times the largest corn stover collection effort in Harlan, IA. Establishing a reliable feedstock supply system requires substantial investment, planning and outreach to the growers and other stakeholders.

Table 14.12 Plant feedstock requirements. Rail and truck traffic volume, units day–1. Units day–1 (60 h week–1 truck delivery)

Plant, dt (000) Mode

Moisture

1000

2000

4000

6000

Rail cars Trucks Trucks

Up to 70% 50% 15%

70 308 240

143 608 396

275 1230 960

418 1850 1920

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14.6.1 Infrastructure

In addition to the biorefinery investment, farmers and biomass suppliers will be faced with investment decisions in the range of $ 70 million or more for equipment, excluding feedstock storage facilities. The storage facilities could add on more millions.

14.6.1.1 Infrastructure Investment For the participating grower, new cropping practices may require investment in seed drills for planting, modifying existing equipment for less tillage and possibly narrow rows, managing cover crops, all exclusive of harvesting. Based on CTIC tillage survey, at least 50% of farmers require new tillage equipment. The new equipment can range up to $ 150 000 per farmer. If half of conventional till farmers make a collection commitment, their investment exceeds $ 50 million. Assuming a net margin of $ 10 dt–1, the grower needs to sell 15 000 dt for a simple pay back, the equivalent of 1500 high-yielding ha. Even half this amount is a significant hurdle for the grower assuming they offer 200 to 400 ha each, 25 to 50% of their crop, the payback time on the new equipment is 4 to 8 years. Other benefits, such as improved yields, may shorten this time [41]. In addition another $ 20 million investment is required for approximately 100 collection units needed for the annual campaign based on Section 14.5.2 – estimating a unit and the peripheral cost is equivalent to silage harvesters, $ 200 000 each, operating 40 days, averaging 1.8 ha h–1 and 14 hours per day. Collection sites may require additional investment in land, material handling equipment and year-round facilities to transfer the material to the biorefinery. Trucking during the harvest nearly triples to remove the stover along with the grain for one-pass harvest. Because of feedstock transportation demands, additional investment may be required to improve roads and bridges, rail track, and crossing to meet the increased traffic – an additional 2.5 million km of truck traffic from field to the three collection sites, with more for delivery to the biorefinery, depending on the use of rail.

14.6.1.2 Organization Infrastructure For a corn processor, procuring grain is as simple as a phone call to purchase feedstock. Standards for grain quality are in place. Delivery schedules are routine to establish. Payment is usually made to local grain elevators that in turn pay the growers. If needed, grain is readily obtained from more distant suppliers. None of this is in place for biomass. To supply 1 million dt, commitments from 1000 to 2000 farmers totaling 200 000 ha is required to insure adequate feedstock. Enlisting them is a timeconsuming task. One likely business model is to mimic the “grain elevator”

14.7 Perspectives and Outlook

model. Local growers already deal with them for their grain business [14]. Accounts are in place, and their managers are skilled in logistics issues. Feedstock from other areas could also be sourced.

14.7 Perspectives and Outlook

Industry segments are beginning to move toward carbohydrate feedstocks as alternatives to fossil fuels. With improved biotech tools their processing cost are becoming competitive with present methods of chemical synthesis. Price instability and higher prices of petroleum and natural gas, global warming policies, and higher liability insurance costs are accelerating interest in a move to more sustainable feedstocks. Global warming is driving policies like the Kyoto agreement, adding economic incentives in the form of carbon credits. Liability insurance companies are considering the potential for claims from policy holder emissions as they set rates for corporate coverage – all positive activities for the rural economy. Feedstock supply must be given significantly more attention if it is to serve as a sustainable platform for this industrial shift to have significance. Sourcing the feedstock quantity has a high risk without improved methods for delivery to the processor from the field. There are many areas for economic and environmental improvement before it enters the processing plant. The target price, $ 33 dt–1, seems achievable with one-pass harvest and bulk delivery of clean feedstock. Farmers are the key determinate for supplying the feedstock. Improved agronomic systems with more crop removal can also maintain soil quality. A business model with an option for the grower to participate in the value chain is important, because the bulky biomass is inherently local. Partnering with the processor insures a win–win for both. Short term, two to three years, dry feedstock is most likely to be chosen to supply biorefineries. Straw collection will be favored, because of to reduced supply risk compared to stover. Mid term, four to seven years, the economic and environmental advantages of one-pass harvest and wet storage will be validated. Chemical, biochemical, and microbial treatments will emerge, improving the feedstock “processability” from the collection centers before delivery to biorefineries. Long term, 2014 and beyond, other feedstocks – especially energy crops grown on marginal croplands – will emerge as the processing technology is more proven. Plant science will enhance the feedstock value and co-products from the biorefinery will become more significant in their economic impact on the product mix.

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Number: 01-16075, 2001 ASAE Annual International Meeting, http://www.meadowoodindustries.com/asae_paper.htm D. Lengel, Ag-Fiber Dot Gone: A Litany of Failure. Panel World July 2001: 8–9, 34–38. D. Glassner, David, James Hettenhaus, Tom Schechinger, Corn Stover Collection Project, Bioenergy ’98, Expanding Bioenergy Partnerships, Madison, WI, 1998 J. Hettenhaus, T. Schechinger, Corn Stover Harvest: Grower, Custom Operator and Processor Issues and Answers, Oak Ridge National Laboratory Contract 4500008274, 1999. J. Hettenhaus, T. Schechinger, Improved Corn Stover Harvest & Collection Methods, 3rd Annual AgFiber Technology Showcase, Memphis, TN, October 2000 E. Lathrop, C. Elbert and B. Treadway, Ind. Eng. Chem. 26:594–598, 1934. H. Hay and Lathrop, E. C. Storage of Crop Fibers and Preservation of their Properties. TAPPI Technical Association Papers, Series XXIV. P 412–418, 1941. J. Salaber and Maza. Ritter Biological Treatment Process for Bagasse Bulk Storage. TAPPI Non-wood Plant Fiber Pulping Progress Report, No 2, October 1971. J. Moebius, The Storage and Preservation of Bagasse in Bulk Form, Without Baling. Pulp and Paper Development in Africa and Near East, United Nations, N.Y. Volume II, 1966. J. Bruin, Gonin, C., McMaster, L., and Morgan, R., Wet Bulk Storage of Bagasse. Proc. Intern, Soc. of Sugar Cane Tech. (ISSCT) XV Congress: 1793–1820, 1974. L. Mann, Tolbert, V. and Cushman, J. (2002) Potential environmental effects of corn (Zea mays L.) stover removal with emphasis on soil organic matter and erosion. Agriculture, Ecosystems and Environment 89:149–166. M. Stumborg, L. Townley-Smith, and E. Coxworth (1995) Crop residue exportissues and concerns. Innovations in Straw Utilization Symp, Oct 23–25. 1995, Winnipeg, Manitoba. Agriculture

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and Agri-Food Canada Research, Swift Current, Sask. W. Wilhelm, and Cushman, J. (2003) Implications of Using Corn Stalks as A Biofuel Source: A Joint ARS and Doe Project [abstract]. Eos. Trans. Agu. 84(46), Fall Meet. SUPPL., Abstract B51b-05. J. Doran, W. Wilhelm and J. Power, Crop residue removal and soil productivity with no-till corn, sorghum and soybean, Soil Sci. Soc. of Am. J., 48, 3, 1984. D. Linden, C. Clapp and R. Dowdy, Long term corn grain and stover yields as a function of tillage and residue removal in east central Minnesota. Soil Till. Res. 56, 167–174, 2000. V. Beri, Sidhu, B. S., Bahl, G. S. and Bhat, A. K. (1995) Nitrogen and phosphorus transformations as affected by crop residue management practices and their influence on crop yield. Soil Use and Management 11: 51–54. P. Unger, Ed., Managing Agricultural Residues, 1994. CRC Press K. Paustian, J. Brenner, K. Killian, J. Cipra, S. Williams, E. T. Elliott, M. D. Eve, T. Kautza, and G. Bluhm (2002) State level analyses of C sequestration in agricultural soils. Pp. 193–204 in: J. M. Kimble, R. Lal, and R. F. Follett (eds.), Agriculture Practices and Policies for Carbon Sequestration in Soil. Lewis Pub, CRC Press, Boca Raton, Fl. Soil quality web site: http://csltest.ait.iastate.edu/SoilQualityWebsite/home.htm Iowa Farm Custom Rate Survey, 2004, http://www.extension.iastate.edu/Publications/FM1698.pdf Agricultural carbon sequestration, Chicago Climate Exchange, 2004, http://www.chicagoclimatex.com National Commission on Energy Policy, “Ending the Energy Stalemate: A Bipartisan Strategy to Meet America’s Energy Challenges,” 2004, http://www.energycommission.org CAST 1992. Preparing US Agriculture for global climate change. Council for Agricultural Science and Technology, Task Force Report 199. J. Hettenhaus, R. Wooley, J. Ashworth, Sugar Platform Colloquies National Re-

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newable Energy Laboratory, NREL/ACO1-31051-01, January 2002. Roadmap for Agriculture Biomass Feedstock Supply in the United States, November, 2003, Document Number: DOE/ NE-ID-11129 http://devafdc.nrel.gov/ pdfs/8245.pdf J. Atchison, J. Hettenhaus, Innovative Methods for Corn Stover Collecting, Handling, Storing and Transporting, National Renewable Energy Laboratory Subcontract No. ACO-1-31042-01, March 2003, www.afdc.doe.gov/pdfs/7241.pdf Levelton Engineering Ltd. and (S&T)2 Consultants Inc., Assessment of Net Emissions of Greenhouse Gases From Ethanol-Blended Gasoline in Canada: Lignocellulosic Feedstocks, 2000. Harris Group Inc., Process Design and Cost Estimate of Critical Equipment in the Biomass to Ethanol Process, Report No. 99-10600/13, Baled Feedstock Handling System, Revision 1w, Subcontract No. aco-9-29067-0, October 11, 2000 B. Hames, S. Thomas, A. Slitter, C. Roth and D. Templeton, Rapid Biomass Analysis: New Tools for Compositional Analysis of Corn Stover Feedstocks and Process from Ethanol Production, 24th Biotech Symp for Fuels and Chemicals, 2002. W. Gale, C. Gambardella, and T. Bailey (2000) Surface residue- and root-derived carbon in stable and unstable aggregates. Soil Sci Soc Am J 64(1):196–201. D. Reicosky, Kemper, W. D., Langdale, G. W., Douglas, C. L., Jr. and Rasmussen, P. E. (1995) Soil organic matter changes resulting from tillage and biomass production. J. Soil and Water Conservation May–June: 253–261. R. Lal, J. Kimble, R. Follett and C. Cole, The potential of US Cropland to Sequester Carbon and Mitigate the Greenhouse Effect, 1998, Sleeping Bear Press. R. Nelson (2002) Resource assessment and removal analysis for corn stover and wheat straw in the Eastern and Midwestern United States – rainfall and windinduced soil erosion methodology. Biomass and Bioenergy 22: 349–363.

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15 The Corn Wet Milling and Corn Dry Milling Industry – A Base for Biorefinery Technology Developments Donald L. Johnson

15.1 Introduction 15.1.1 Corn – Wet and Dry Milling – Existing Biorefineries

Corn dry milling has existed for hundreds of years – maize was ground with stones into flour for food consumption by early American populations. Modern corn refining, however, began in the mid 1800s when Thomas Kingsford started up his corn refining plant in Oswego, New York [1]. Corn refining is distinguished from corn milling in that the refining process separates the corn grain into its components, starch, fiber, protein, and oil, and further processes the starch into a substantial number of products. Corn “wet milling” is the aqueous slurry process by which the corn grain is separated into its component parts. Corn “dry milling”, in contrast, physically alters moist corn granules into composite products such as flakes, grits, meal, flour, and hominy feed, although some operations do separate germ and recover oil. The dry milling process produces food and industrial products based on flakes, meal, and flour, and also fermentation ethanol. Specialty products such as white corn flour for food uses and yellow corn flour-based adhesives, produced by what are termed the flour millers, are a small part, less than ten percent, of the industrial market [2] and will not be discussed further here. Those interested in the topic are referred to texts available on the subject [3]. Corn refining has been the fastest growing market for US agriculture over the past 25 years. This is attributed to the burgeoning high-fructose corn sweetener market early in the period, followed by rapid growth in fuel alcohol, and, more recently, by fermentation products. Corn refiners now use over 14% of the annual corn crop [4], exceeding 39 million metric tons (MT) of corn refined each year.


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15 The Corn Wet Milling and Corn Dry Milling Industry

15.2 The Corn Refinery 15.2.1 Wet Mill Refinery

A corn refinery can be described succinctly in five process steps, but is substantially more complicated in operation. Corn grain that has been received by truck and/or rail is inspected for moisture content and debris, passed through cleaners to remove foreign material and steeped in large tanks. Steeped corn is coarsely ground to loosen and free the low-density germ which is removed by centrifugation from the starch and fiber slurry. A second grind releases starch and gluten from the fibrous hulls, the fiber is further washed to remove residual starch and sent to a feedhouse. The low-density gluten is removed by centrifugation and a battery of cyclone cleaners washes residual protein from the starch. The 99.5+ percent pure “refined” starch is ready for further processing. Germ, which was removed early in the process, is subjected to further processing to remove the oil, which can be refined or sold as crude corn oil. The spent germ is combined with the fiber from the second grind, dried, and sold as corn gluten feed. The gluten is dried and sold as a 60% protein-feed supplement. “Wet milling” is so named because the corn is steeped in slightly acidic warm water and is processed as an aqueous slurry until dried or solubilized in downstream processing. 15.2.2 Dry Mill Refinery

A dry mill, or “mash,” ethanol plant, is a simpler process. Corn is received and cleaned as in the wet mill, but the clean corn is tempered with steam, ground, and wetted to a free flowing “mash,” from which the name is derived. The mash is superheated in a continuous cooker, to which acid and/or enzymes are added to solubilize the starch in the corn meal. Additional water is added to adjust the solids and temperature, and saccharifying enzymes are added. This mash is added to a fermenter, adjusted to appropriate solids and temperature, and yeasts are added to convert the sugars to alcohol. When the fermentation is complete, alcohol is removed by distillation and the residual “still bottoms” are recovered for animal feed. In such a plant, the only two products are ethanol and distillers dried grains and solubles (DDGS). Some plants now separate the germ before “mashing” – the added cost is justified by the value of the oil recovered. A wet mill corn refinery and a dry mill ethanol plant are compared in a process flow schematic diagram of Fig. 15.1. As might be expected, the capital investment for a mash ethanol plant is significantly lower than that of a corn refinery. The operating profits from the multitude of products normally overshadow the investment cost differences, however; this will be discussed later.

15.2 The Corn Refinery

Fig. 15.1 Process flow diagrams comparing wet to dry milling.

15.2.3 Waste Water Treatment

Another common feature of wet and dry mills is, as indicated in Fig. 15.1, the waste water treatment required. Both operations, but especially wet milling, are water-intensive processes. A corn refinery may require two hundred to two hundred fifty gallons per bushel of corn (a bushel is defined as 56 pounds (25.45 kilograms) of corn at 15.5 percent moisture) processed, most of it as process water that is vaporized, condensed, heated, and cooled needing very little treatment before being returned to its source. But five to ten percent contains organic material which must be treated before discharge into the environment. Meeting local, state and federal regulations for liquid wastes may add more than 10% to the total plant investment [5]. The corn milling industry and its equipment suppliers work diligently to minimize water usage.

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15.3 The Modern Corn Refinery 15.3.1 Background and Definition

The modern corn refinery is a model for developing future biorefineries. Corn refining has been compared with petroleum refining wherein flexibility of downstream processing is used to maximize profitability [6]. Thus the focus of this chapter is on wet milling, with some reference for comparative purposes to mash ethanol plants. Corn refining produces several products in large volume. As already mentioned, wet milling refines the corn into four components, starch (carbohydrate), gluten (protein), hull (fiber), and germ (corn oil). Moreover, the carbohydrate fraction, which is nearly 70 percent of the corn composition, is further processed and refined into products such a native starch, modified starch, dextrose, high fructose corn sweetener (HFCS), ethanol, glucose syrup, and special hydrolyzates. 15.3.2 Technologies and Products

In the United States, the largest producer and miller of corn in the world, plant capacities are rated in bushels per day. Refineries in the US range in grind capacity from 55 000 bushels/day (14 000 MT/day) to more than 550 000 bushels/ day (14 000 MT/day) [6]. A metric ton of corn grain yields, on average, 684 kg starch, 237 kg corn gluten feed (CGF), 45 kg gluten meal (60% protein) and 34 kg corn oil in a typical wet milling operation. Thus the largest corn refinery produces more than 5 million metric tons of pure carbohydrate per year to be sold as such and further processed into a myriad of refined products. The wet milling process, as alluded to earlier, is a countercurrent aqueous slurry process. The aqueous stream, called mill water, begins as fresh water entering the final washing step of the pure granular starch stream, flows backward through several recycle loops in intermediate processes, and exits from the freshest corn steeping tank as heavy steep water. The solid phase, beginning as corn kernels, flows from a steep tank forward through the separation processes as components are removed and exits the final washing step as a pure granular starch slurry. Steeping is accomplished by conveying corn grain into large steep tanks where it is exposed to warm water (circa 122 8F, 50 8C) for 30 to 40 h. A small amount of sulfur dioxide is added to maintain approximately 0.2% concentration to control bacterial growth. The corn kernels soften, loosening the hulls and disrupting gluten–starch bonds as the slightly acidic water diffuses into the swelling kernels. Steeping is currently a semi-continuous countercurrent process. Process water to which sulfur dioxide is added enters the first of a train of

15.3 The Modern Corn Refinery

steep tanks, each holding as much as 3000 to 5000 bushels (76 to 127 MT) of corn kernels. The steepwater flows continuously through the train while tanks of steeped corn are sequentially removed from the front end and fresh corn tanks added at the back. Flow rates are adjusted to provide the appropriate steeping time. Water exiting the final tank has been exposed to the corn the longest whereas the corn in that tank has been exposed to the steepwater the shortest time. The fully steeped tanks are drained and the kernels are sluiced to the first, or coarse, grind. Steeping process improvements have been proposed with the objective of shortening steeping time, eliminating sulfur dioxide, and other cost reductions [7] but none has yet been incorporated to any extent. Steeping yields an additional product, corn-steep liquor (CSL). This nutrient rich product can be concentrated for sale as condensed fermented corn extractives, more commonly called concentrated steep liquor. Steep liquor that is not used internally (called thin steep liquor if it has not been concentrated) as a fermentation nutrient or sold to other users as CSL, is combined with corn germ meal (germ from which the oil has been extracted) and hulls, the composite constituting CGF. Hydrocyclones remove the low-density corn germ from the coarsely ground slurry in the germ separation process. The germ of each kernel contains about 85% of the kernels’ oil. Germ is thoroughly washed over bent screens to remove residual starch, then dried and subjected to mechanical and chemical processing to remove the oil. The oil is further processed into either crude or refined corn oil, and the spent germ combined with hulls and steep liquor as described above. The degermed slurry is subjected to more extensive grinding in attrition mills to free the starch and gluten from the fibrous hull of the kernels. Slurry from the grinding mills flows over concave “bent” screens. The slotted screens enable starch and gluten particles to flow through, but not the fiber, effectively separating the fiber from the starch–gluten slurry. The fiber is rewashed to optimize starch–gluten recovery and combined with spent germ and CSL as already described. The starch–gluten slurry is centrifuged to remove the gluten (light phase) from the starch water slurry. Gluten, containing 60% protein is dried, and marketed primarily as a premium animal feed ingredient. The starch is subjected to exhaustive countercurrent washing to remove protein to less than 0.5% in the starch, commonly approximately 0.3% range. The last stage, in which the starch is washed to 99.5% purity (some oil and trace minerals also remain), is the only point in the milling process where fresh water is added. This essentially pure carbohydrate stream is ready for further processing into starch products, syrups, or fermentation products.

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15.3.3 Refinery Economy 15.3.3.1 Refinery Economy of Scale and Location Considerations A large corn refinery produces a multitude of products from the pure carbohydrate stream and strives to minimize the cost per unit of production of the carbohydrate and subsequent products also. Unit production cost consists of the cost of the corn and costs related to plant investment, labor, and conversion. The first two costs decrease as capacity increases whereas conversion cost is usually insensitive to capacity, on a unit production cost basis. Investment-related costs include maintenance, insurance, property tax, and general plant costs, which are a direct function of plant size. Doubling the plant capacity usually increases the investment required by only approximately 50%. Thus, the investment-related cost per unit of production decreases with plant size. Labor cost per unit of production also decreases as production capacity increases. Doubling the capacity may require very little added labor. Unit conversion cost includes charges for utilities such as steam, electricity, and water and are sensitive to volume. That is, doubling the capacity usually doubles the steam, electrical, and water requirements. As plant capacity increases, the investment per unit production decreases and asymptotes at some minimum. The investment in dollars/annual gallon plotted against annual capacity for a mash alcohol plant, for example, has been shown to level off at about the 70 000 bushel/day grind capacity [8]. The generally accepted size of an “economy of scale” corn refinery is 60 000 to 80 000 bushels/ day grind (1525 to 2033 MT/day). The size and location of a scale plant is also influenced by availability of raw material corn, water, electricity, transportation (rail, water and highway), and labor. To put this in context, a 100 000 bushel/day plant uses more than a square mile of corn crop production per day (based upon national average yield of 142 bushels/acre) [9]. Each days grind requires 28 railcar loads of corn. If the grind were split evenly between two products, corn syrup and ethanol, 25 rail cars of products, including the CGF and the oil would be transported from the plant. Transportation considerations are not trivial. Most large corn refineries grew from one or two primary products, aside from the attendant co-products, by adding finishing capacity to support a new product with an attendant grind expansion to support the new capacity. For example, a burgeoning HFCS market supported new syrup refining capacity with the attendant grind expansions at existing syrup manufacturers. For HFCS, dramatic growth also spurred green-field plant construction dedicated solely to HFCS production on the scale mentioned above. Even those plants, however, have now been expanded to furnish additional products.

15.4 Carbohydrate Refining

15.4 Carbohydrate Refining

The flexibility in operating a corn refinery is in the downstream processing of the primary product, the carbohydrate. The granular starch slurry can be dried to produce native, or “pearl” corn starch. Alternatively, some or all of the granular starch can be processed using chemical and/or physical methods, into modified starch products. Although important, the over nine billion pounds (4.1 million MT) of corn starch forecasted to be used in the year 2004 [10] is still a distant third to the quantity used for producing corn sweeteners or fuel alcohol. The greatest portion of the wet milled cornstarch is converted to sweeteners or ethanol. In this process the granular corn starch slurry is solubilized and saccharified using a combination of heat, acid, and enzymes. Large thermal cookers introduce high-pressure steam into the slurry exposing the granules to heat and shear to disrupt the granular structure. Acid and enzymes begin depolymerizing the high-molecular-weight glucose polymer chains. Large vessels, called saccharification tanks, provide long residence time for the continuous flowing starch suspension. Starch-hydrolyzing enzymes are used to saccharify the glucose polymer, converting it to short-chain hydrolyzates. The extent of hydrolysis determines whether it is a corn syrup (20 to 70 dextrose equivalent, DE) or dextrose syrup (94 to 98 DE). (Dextrose equivalent, DE, is a measure of the total reducing sugars in the syrup calculated as dextrose and expressed as a percentage of the total dry substance of the solution. The higher the DE, the greater the extent of hydrolysis.) Corn sweeteners are sold as a wide range of glucose or dextrose syrups depending upon DE and purity. These hydrolyzates are mechanically clarified and refined with carbon and ion exchange to produce the desired syrup. Converting to 95 or higher DE glucose syrup provides the refiner with three options. It can be refined as dextrose or 95 DE corn syrup, fermented to ethanol, or isomerized to HFCS. Having the high DE glucose stream also provides flexibility in the choice of ethanol fermentation. Batch, semi-continuous, or continuous fermentation are options available with a 95 DE syrup stream. Large refiners producing both HFCS and ethanol provide a common stream which enables production swings, sometimes as much as 50%, from HFCS to ethanol and vice-versa as demand fluctuates. In such circumstances, some of either capacity is idle at times. That “idle capacity” investment is a small part of the overall plant investment, however, and considered a small price to pay for the flexibility. HFCS requires a 95 DE glucose or higher feed to the isomerization reactors, the higher the better (while avoiding isomaltose reversion, which is discussed later). Fixed isomerizing enzymes convert some of the glucose to fructose, a very sweet carbohydrate monomer. (Sucrose is a dimer of a molecule of glucose and a molecule of fructose). A 42% fructose stream issues from the isomerizing reactors, the balance being glucose and any higher sugars which were present in the feed stream. The product is refined with carbon and ion exchange and concentrated for shipment. Large chromatographic columns are also used in an enriching process in which the components fructose, glucose, and higher su-

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gars are separated. An 85 to 90% fructose product results, which is blended with 42% fructose syrup to produce a range of fructose compositions. The preponderance of product is 55% fructose syrup, an extensively used syrup in the huge soft drink industry. Corn refiners produce large volumes of glucose syrups. This covers a broad range of products ranging from 20 DE to 70 DE, as already mentioned. Such products are widely used in the baking, canning, and confectionary industries. If a hydrolysate is to be converted to ethanol, some refiners will provide a somewhat lower DE stream to the fermenter to avoid isomaltose, a non-fermentable reversion sugar of glucose which occurs as a result of an equilibrium reaction at high DE. The higher sugars (oligomers) are hydrolyzed, by enzymes added to the fermenter, to hydrolyzates that are used by the glucose-fermenting organism. The fermenting organism consumes the glucose and other fermentable sugars, reversion conditions are avoided in the saccharification process, and overall yields are improved. Having appropriate converter capacity also enables the option to produce other fermentation products. Some vitamins and amino acids may require a highly pure dextrose whereas a simultaneous saccharification–fermentation process can readily utilize a low-DE syrup. The range of choices is broad and, as new industrial organisms are introduced, even more options will become available.


The modern corn refinery is a model for the application of biotechnology to the production of fuels, chemicals, and materials from abundantly available natural resources in a sustainable, environmentally acceptable manner. New commodity chemicals and materials, capable of replacing non-renewable petroleum-derived products are being developed and manufactured now from corn-derived glucose in such refineries. Other biomass sources of glucose that are equally or more abundantly available and potentially less expensive than corn can readily be incorporated into such a process environment as the technology for such utilization is developed.

References 1 B. W. Peckham 2000, The First Hundred

3 See for example, P. White and L. A.

Years of Corn Refining in the United States, in Corn Annual 2000, Corn Refiners Association, Washington, DC. 2 United States Department of Agriculture 2003, Economic Research Service, Feed Outlook, January 2003

Johnson (eds.) 2004, Corn: Chemistry and Technology, 2nd edn, American Association of Cereal Chemists, Eagan Press. 4 Corn Refiners Association 2003, Corn Annual 2003, Washington, DC. 5 P. W. Madson, and J. E. Murtagh 1991, Fuel Ethanol in USA: Review of Reasons

References for 75% Failure Rate of Plants Built, International Symposium on Alcohol Fuels, Firenze, 1991, available from Katzen International, Cincinnati, Ohio. 6 L. R. Lynd 2002, Principal Investigator, Strategic Biorefinery Analysis, NREL Subcontract ADZ-2-31086-01. 7 J. Randall et al. 1978, USP 4,106,487. 8 R. Katzen et al. 1994, Ethanol from Corn – State of the Art Technology and Econom-

ics, National Corn Growers Association Corn Utilization Conference V, June 1994, unpublished. 9 US Department of Agriculture, NASS, Agricultural statistics board. 10 US Department of Agriculture, Economic Research Service.

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Part IV Biomass Conversion: Processes and Technologies


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16 Enzymes for Biorefineries Sarah A. Teter, Feng Xu, Glenn E. Nedwin, and Joel R. Cherry

16.1 Introduction

The total amount of carbon and nitrogen biologically processed by photosynthesis into polymeric organic material is referred to as biomass. Plants are the main source of biomass and it is estimated that the total amount of biomass worldwide is approximately 1012 tons. Biomass is a renewable source of carbon building blocks, and there is great potential for converting this resource into a diverse array of biobased products. Just over a hundred years ago, 90% of our energy needs were supplied by biomass, largely by combustion of wood. Most non-fuel industrial products, including dyes, inks, paints, medicines, chemicals, fibers, and plastics, were made from trees, vegetables, and crops. By the 1970s, 70% of US energy and 95% of industrial products were derived from petroleum rather than from renewable sources. The finite petroleum resources, a host of environmental issues, coupled with a growing interest in reducing the US dependence on foreign energy and industrial feedstock sources, have fueled research and development into alternative energy sources and the economic utilization of biomass as a source of biobased products [1]. The conversion of agricultural commodities such as corn into fuel-grade ethanol is one successful alternative energy initiative. Gasoline containing 10% ethanol was introduced as a fuel for automobiles. Today, ethanol in gasoline continues as an additive replacement for the oxygenate methyl tertiary butyl ether (MBTE). Fuel ethanol production from sugar/starch biomaterials, for example corn and sugar cane, has become an economically viable industry. In North America, approximately 11 billion liters of ethanol are currently produced annually, with a projected growth rate of *20% for the foreseeable future. Comparing with sugar and starch based agricultural products, biomass has a much larger potential to become the renewable energy source of the future. Biomass includes agro/forest byproducts such as corn stover (corn leaves and stalks) and wood pulp and paper residues. It is estimated that in the US alone, Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 1 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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biomass is capable of yielding *100 billion liters of ethanol annually. In the US corn production generates *100 000 dry metric tons of corn stover [2], an excellent raw biomaterial containing a huge amount of under-utilized, energyrich lignocellulosics. The concept of the biorefinery is analogous to the concept of the petroleum refinery in the sense that an abundant feedstock is converted into many different products. Unlike a petroleum refinery, a biorefinery utilizes renewable feedstocks, such as plant-based starch or residual biomass (cellulose, hemicellulose, and lignin) which can be harvested and re-planted year after year. In one scenario, the feedstock is harvested, delivered to the refinery, and pretreated to make the cellulose accessible for enzymatic conversion to fermentable sugars. The resulting sugars are then fermented into primary products such as ethanol, lactic acid, or several other materials for industrial use. Unused residual materials are burned as fuel to generate heat and electricity. Biorefineries exist today, to a small but growing extent, where, for example, corn starch feedstocks are converted to high-fructose syrups, animal feed, oil, and various organic acids (for example citric, gluconic, itaconic and lactic acids). Like the petroleum refinery, it is expected that the process and products from a biorefinery will evolve to meet society’s demand. The first refineries in the 1860s produced kerosene for oil lamps to replace a dwindling supply of whale oil, and the byproducts, naphtha and tar, had few low value uses. With the development of the combustion engine, gasoline and light oil became dominant products and spurred the development of industries utilizing chemical byproducts of fuel production as chemical feedstocks for plastics, synthetic fibers, elastomers, drugs, and synthetic rubber. Although the primary focus of the biorefinery concept today is on fuels and energy production, large-scale implementation will stimulate development of a feedstock industry similar to that seen from the petrochemical refinery. With recent developments in biotechnology, many petrochemical-derived products can be replaced with industrial materials made from renewable resources. Biotechnology has had a positive impact on the cost-efficiency of enzymatic conversion of biomass to fermentable sugars and has increased the range of products that can be produced by genetic engineering of fermentative organisms. Biotechnological improvements may soon enable us to produce plants with altered properties that make them more amenable to refining. Biobased products have the potential to dramatically alter our world. The availability of technology enabling the refining of biomass will reduce the release of petroleum-originated greenhouse gases into the atmosphere, will largely decentralize fuel and chemical production, with concomitant improvement of rural economies, and will positively impact US national security by reduction of its dependence on foreign oil imports. In this chapter we consider the biorefining of agricultural residue materials, primarily focusing on recent advances in enzymatic catalysis in the conversion of biomass to fermentable sugars. We conclude with a short discussion of the prospects for various biorefinery products.

16.2 Biomass as a Substrate

16.2 Biomass as a Substrate 16.2.1 Composition of Biomass

A vast carbon source for biobased products is locked up in plant matter, the most abundant source of biomass on earth. The principal components of biomass are cellulose (30–50%), hemicellulose (20–30%), and lignin (20–30%); with starch, protein, and oils as minor components. The exact composition of each biomass varies depending both on the plant and on the residue collected (Table 16.1). The composition, in turn, determines the ease with which the biomass can be converted to useful products and/or intermediates and affects the functionality of the final product. The complex polymeric structure of crystalline bundles of cellulose embedded in a covalently linked matrix of hemicellulose and lignin poses a formidable challenge for solubilization and conversion to monomeric sugars. As can be seen in Table 16.1, the relative lignin, cellulose, and hemicellulose content of a variety of potential feedstocks is quite similar, yet even a 5% increase in lignin or hemicellulose content can significantly alter accessibility to enzymatic attack. Thus, the variation in the composition of a given biomass requires some tailoring of the conversion method.

16.2.1.1 Cellulose Cellulose is abundant in plant cell walls and comprises a linear beta-(1 ? 4) anhydroglucopyranose polymer (six-carbon sugars). The molecular weights of different celluloses can range from 200–2 000 kDa where the number of glucose

Table 16.1 Composition of representative biomass samples. Samples

Variety

Monterey pine Pinus radiata Hybrid poplar DN-34 Sugarcane bagasse Gramineae saccharum var. 65-7052 Corn stover Zea mays Switchgrass Alamo Wheat straw Thunderbird Barley straw Hordeum vulgare sp. Rice straw Oryza sativa sp.

% Mass Total Lignin

Cellulose

Hemicellulose

25.9 24 24

41.7 40 43

20.5 22 25

18 18 17 14 10

35 31 33 40 39

22 24 23 19 15

Source: http://www.eere.energy.gov/biomass/feedstock_databases.html

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residues can exceed 15 000 per polymer molecule. Cellulose has such extensive hydrogen bonding, because of the formation of overlapping, staggered flat sheets, that it is a highly recalcitrant and water-insoluble crystalline material. Within a cellulose chain, each d-glucosyl residue is rotated by approximately 1808 relative to its nearest neighbor residue, making cellobiose the repeating unit present in cellulose. Only agents that can attack the glycosidic linkages between the glucose residues or which can disrupt the hydrogen bonding can solubilize cellulose.

16.2.1.2 Hemicellulose Hemicelluloses are plant cell wall heteropolymeric sugars and sugar acids with a backbone of 1,4-linked b-d-pyranosyls in which O4 is in the equatorial orientation. They are usually shorter than celluloses, typically containing fewer than 200 1,4 linkages, highly branched, and easily hydrolyzed by strong acid or base. Hemicellulose serves as an interface between cellulose and lignin in plant cell walls, and may form covalent and non-covalent linkages with other cell-wall constituents, for example pectin, glucans and proteins. One major hemicellulose is xyloglucan, a beta-(1 ? 4) linked polymer of xylose with mono-, di-, or triglycosyl side-chains, via O6, composed of a variety of substituents, for example acetyl, arabinosyl, or glucuronosyl units. Other hemicelluloses include xylan, glucuronoxylan, arabinoxylan, mannan, glucomannan, and galactoglucomannan.

16.2.1.3 Lignin Lignin is a highly complex, amorphous and heterogeneous complex of substituted phenolic compounds, often comprising syringyl, guaiacyl, and p-hydroxyphenol components. It binds to hemicellulose and cellulose. Lignin is highly resistant to enzymatic, chemical, and microbial hydrolysis because of its extensive cross linking. It can, however, be pyrolyzed to form oil for fuel and resins.

16.2.1.4 Starch Starch is composed of glucose, as a mixture of amylose and amylopectin in varying ratios. Amylose is a linear alpha-(1 ? 4) d-glucopyranose polymer whereas amylopectin has a similar structure but with additional side branches of more than 20 glucose residues with alpha-(1 ? 6) linkages. Currently, corn starch is the primary raw material of several major grain-based products, for example ethanol, polylactide, plastics, some packing materials, and adhesives.

16.2 Biomass as a Substrate

16.2.1.5 Protein Proteins are polymers of amino acids. In plants, proteins serve as structural, functional, and regulatory agents. Catalytic proteins, the enzymes, are essential for a variety of plant physiological activity.

16.2.1.6 Lipids and Other Extracts Lipids are esters of moderate to long-chain fatty acids, either saturated or unsaturated. Acidic or basic hydrolysis yields the component fatty acid and alcohol. Triglycerides, esters of fatty acids with glycerol, constitute fats and oils. Esters of fatty acids with monohydric alcohols, often mixed with hydrocarbons, constitute waxes. Phospholipids are important components of cell membranes. Another major plant extract is terpene, made from isoprene (isopentane) units. 16.2.2 Biomass Pretreatment

In a natural setting, lignocellulosic biomass is broken down over a period of years by the accumulated action of physical disruption from the forces of nature (wind, rain, snow, heat, sunlight) and by the action of microbes that chemically and enzymatically degrade it into compounds they can use for their growth. The components of the plant cell wall that give it strength and rigidity, namely the intertwined network of cellulose, hemicellulose, and lignin, also make it resistant to breakdown, whether on a forest floor or in a biorefinery reactor. In contrast with natural decomposition of lignocellulose in nature, breakdown of the feedstock in a biorefinery must occur in a matter of hours or days rather than years. To accomplish this requires coordinated steps of physical disruption, chemical modification, and enzymatic action. The recalcitrant cellulose is relatively resistant to breakdown by microbial hydrolytic enzymes in its natural form, with only approximately 20% of the cellulose present in untreated biomass being hydrolyzed to glucose after treatment with high doses of enzymes. In pretreatment, plant materials are physically disrupted, under the action of stress/tear, temperature, pressure, and/or pH. To convert biomass into fermentable sugars, the purpose of the pretreatment is to disorder or remove cellulose– hemicellulose–lignin interactions and thereby improve access of hydrolytic enzymes to sugar polymers in subsequent steps in the biorefinery. Physical disruption usually begins with a reduction in the size of the plant material by milling, crushing, and/or chopping. For example, in the processing of sugar cane, the cane is first cut into segments and fed by conveyor into consecutive roller presses that both extract the cane juice (rich in sucrose) and physically crush the cane, producing a fibrous bagasse that has the consistency of sawdust. In corn-stover processing, the stover is initially chopped with knives or ball-milled to increase the exposed surface area and improve wetability. After physical disruption, pretreatment may continue with a chemical extraction designed to maximize subsequent enzymatic hydrolysis of the cellulose.

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This usually means modifying or removing lignin, which not only acts as a block to enzyme action by coating cellulose microfibrils in untreated biomass, but also interferes with enzymatic hydrolysis by directly absorbing some cellulose-active enzymes. The result of pretreatment is a cellulose with both improved solvent accessibility and reduced lignin interference with enzyme action. Numerous methods of biomass pretreatment have been described in the literature and are summarized below.

16.2.2.1 Dilute Acid Pretreatment This process is perhaps the most thoroughly studied of the pretreatment methods and consists of mixing the biomass with a solution of dilute strong acid (e.g. 5% sulfuric) in a pressurized reactor at high temperature (e.g. 160–200 8C) for 1–10 min then rapidly releasing the pressure. This method effectively hydrolyses most (up to 95%) of the hemicellulose to its constitutive C5 sugars (xylose, arabinose, etc.) [3]. Little lignin is removed by the process, but it is thought to melt and be “redistributed”, enabling improved cellulose hydrolysis. Pretreatment conditions must be adjusted, depending on the source of the biomass, to maximize hemicellulose hydrolysis, while at the same time minimizing the formation of compounds such as furfural and hydroxymethyl furan that are toxic to fermenting yeast and probably also to other organisms of industrial interest (a review on this topic is given elsewhere [4]). In literature reports the solids remaining after pretreatment are often washed before enzyme digestion, but this may not be economically practical in a biorefinery.

16.2.2.2 Ammonia Fiber Explosion This pretreatment utilizes ammonia mixed with biomass in 1:1 ratio under high pressure (1.4–3 atm) at temperatures of 60–110 8C for 5–15 min, then explosive pressure release. Because of it volatility, ammonia can be recycled with near quantitative recovery. Little (10–20%) lignin is removed, but enzymatic cellulose hydrolysis is reported to proceed to as much as 98% of theoretical yield at relatively high cellulase loadings (15 FPU g–1 glucan) [5]. Hemicellulose depolymerization is highly variable and depends on the moisture content of the biomass, but is typically quite low. Enzymatic cellulolysis therefore requires the presence of at least some hemicellulase activity to increase cellulose accessibility during hydrolysis.

16.2.2.3 Hot-wash Pretreatment This method involves passing hot water through a heated stationary biomass bed and, like dilute acid, has been reported to result in solubilization of more than 90% of the hemicellulose fraction [6]. The hemicellulose is largely converted to pentose oligomers which must be enzymatically converted to monosaccharides before fermentation. The performance of the pretreatment depends on temperature and flow rate, and requires washing for ca. 8–16 min. At high flow

16.3 Enzymes Involved in Biomass Biodegradation

rates and temperatures, the lignin content is reduced by as much as 46% and the process produces no significant amounts of compounds inhibitory to fermentation [7, 8]. Although the hydrothermal process does not require the acidresistant reactor materials of acid pretreatment, this advantage may be offset by increased water use and recovery costs.

16.2.2.4 Wet Oxidation Here molecular oxygen and water are applied to the biomass at high temperature and pressure. In a series of experiments reported by Varga [9], 60 g L–1 biomass incubated at 195 8C for 15 min under 12 atm O2 and containing 2 g L–1 Na2CO3 solubilized 10% of the cellulose, 60% of the hemicellulose, and 30% of the lignin present in corn stover. Enzymatic hydrolysis of the remaining solids after hydrolysis at 50 8C for 24 h using 25 FPU enzymes per gram of dry biomass achieved an 85% conversion of cellulose to glucose. Other methods of pretreatment involve the use of sodium hydroxide, lime, organic solvent extraction, or lime steam explosion, but are, in general, less studied than the methods described above. The critical issues in selecting a pretreatment for lignocellulosic biomass are sugar yield and composition, the cost of the process both in terms of energy and chemical costs, and the capital cost of building the system. The pretreatment system selected has an impact on all the downstream processes in the biorefinery and must be evaluated carefully. As described above, increased enzyme digestibility may be accompanied by an increase in byproducts that inhibit downstream fermentation, whereas less severe conditions may produce a substrate requiring excessive enzyme loadings for cellulose hydrolysis. This interplay between pretreatment, enzymatic digestion, and fermentation is the crux of current research projects to develop an integrated biorefinery.


Biomass degradation is required for the survival of many organisms including bacteria, fungi, plants, protozoa, insects, and herbivores (through symbiotic microbes). Investigation into the action of these enzymes on biomass or its components has been active for over sixty years [10–16]. With the advent of large-scale genome sequencing, the complexity of biomass degradation has become more apparent. Bacteria such as Clostridium thermocellum, Cytophaga hutchinsonii, Microbulbifer degradans, Rubrobacter xylanophilus, and Thermobifida fusca, and the fungi Trichoderma reesei and Phanerochaete chrysosporium have all been sequenced to at least draft form, revealing a diverse array of enzymes involved in carbohydrate degradation. Organisms devote much of their resources to degrading plant material, with well over fifty genes targeting polysaccharide degradation even in relatively simple bacteria.

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16.3.1 Glucanases or Cellulases

On the basis of sequence homology, cellulases can be grouped in the glycoside hydrolase (GH) family classification system of Coutinho and Henrissat (Carbohydrate-Active Enzymes server at URL: http://afmb.cnrs-mrs.fr/*cazy/CAZY/ index.html). Cellulases fall into families GH5–9, 12, 44, 45, 48, 61, and 74 [17]. Cellulases are often modular, comprising a catalytic core, a linker, and one or more cellulose-binding modules (CBM, which can be grouped into *13 families, part of *39 families of carbohydrate-binding domains). Such modular organization is found with other enzymes active on other insoluble polysaccharides, for example amylases and chitinases. The CBM can be at the N or C-terminus of the catalytic domain and is attached via a linker domain rich in proline, serine, and threonine. The 3D structure of many catalytic domains has been solved [18]. In general, the active site in exoglucanases is enclosed by two surface loops forming a tunnel, believed to be vital for conferring directionality in enzyme action on linear cellulose microfibrils. In contrast, the active site in endoglucanases has an open groove, enabling it to act in the middle of a cellulose chain. The 3D structure of many CBM has also been solved [19]. In general, a CBM has a flat surface with exposed aromatic side-chains that mediate binding to the hydrophobic cellulose surface. Cellulase is a general term encompassing a diverse set of enzymes that participate in the hydrolysis of cellulose into glucose. Cellulases include exo-1,4-b-dglucanases or cellobiohydrolases (CBH, EC 3.2.1.91), endo-1,4-b-d-glucanases (EG, EC 3.2.1.4), and b-glucosidases (BG, EC 3.2.1.21). EG act internally within a cellulose chain at amorphous cellulose regions to cleave glycosidic bonds, thereby fragmenting the cellulose polymer. CBH are believed to “processively” degrade a cellulose chain from either the reducing or non-reducing ends, and can cleave glycosidic bonds within crystalline cellulose regions to release the disaccharide cellobiose. Because EG create new reducing and non-reducing ends within cellulose chains for CBH attack, these two enzyme classes act synergistically in cellulose degradation. At high concentrations cellobiose or other cellooligosaccharides can inhibit CBH activity. Thus BG, which hydrolyses these soluble sugars to glucose, is often required in a “complete”, effective cellulolytic system. In addition to cellulase, phosphorylase can also cleave b-glycosidic bonds, especially those in cellodextrin, by phosphorylating a glucosyl unit [20]. 16.3.2 Hemicellulases

Hemicellulases are involved in degrading hemicellulose. Some cellulases, for example GH7 and GH74 EG, have significant xylanase or xyloglucanase side-activity. The structural similarity enables many hemicellulases and cellulases to be grouped into the same GH family [17].


On the basis of the products they form xylanases can be classified as endoxylanases (EC 3.2.1.8) and b-xylosidases (EC 3.2.1.37). On the basis of sequence, endoxylanases belong to the GH10 and GH11 families whereas b-xylosidases belong to GH3. Galacto/glucomannan-active mannanases can also be classified as endomannanases (EC 3.2.1.78) and b-mannosidases (EC 3.2.1.25). Other hydrolases are active on other polysaccharides commonly found in biomass, for example pectinase/polygalacturonase, arabinofuranohydrolase, arabinase, galactanase, glucoronidase, and acetylesterase [21]. 16.3.3 Nonhydrolytic Biomass-active Enzymes

In addition to hydrolases, various proteins and enzymes are active on lignocellulosics. Expansin and swollenin are not cellulolytic, but seem able to disrupt the hydrogen bond between cellulose chains, leading to structurally weakened cellulose [10]. Polysaccharide lyases cleave glycosidic bond by b-elimination, resulting in a double bond at the newly formed non-reducing end. Laccase, lignin peroxidase, and Mn peroxidase can oxidize lignin, thus loosening lignin–polysaccharide interactions or relieving lignin inhibition of polysaccharide hydrolases [22, 23]. 16.3.4 Synergism of Biomass-degrading Enzymes

The selection pressure on organisms to feed efficiently on biomass has led to the evolution of biological cellulolytic systems integrating highly specialized yet synergistic enzymes. Two distinct, effective systems have developed – the multicomponent, secreted, and non-complexed fungal/bacterial (aerobic) cellulases and the multi-component, scaffold-assembled, complexed bacterial (anaerobic) cellulosomes. One representative fungal cellulolytic system is that of Trichoderma reesei (syn. Hypocrea jecorina) [24]. One of the most effective cellulose degraders known, T. reesei secretes an array of cellulases, including CBH I or Cel7A (*60%), CBH II or Cel6A (*15%), EG I or Cel7B, EG II or Cel5A (*20%), and other minor components (for example as EG III or Cel12A, EG IV or Cel61A, EG V or Cel45A, EG VI or XG74A, BG I or Cel3A, Xyn I or Xyn11A, and swollenin). CBH I and CBH II differ in their affinity for reducing and non-reducing ends. The subtle yet significant difference among the various EG can accommodate the need to degrade a heterogeneous substrate such as cellulose. Commercial T. reesei cellulase preparations, for example Novozymes’s Celluclast 1.5L and Genencor International’s Spezyme, are widely used in a variety of applications. One representative bacteria cellulosome is that of Clostridium thermocellum. This cellulase system is organized around a *200 000 molecular mass scaffolding, a protein equipped with a dockerin, a CBM, and many cohesin domains. The dockerin attaches the assembly to the cell surface, and the cohesins anchor a variety of enzyme molecules (CBH, EG, etc.) by interacting with their dockerin domains [10,

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12]. Cellulosomes range from 0.5 to 50 megadaltons, and many cellulosomes can further aggregate into polycellulosomes. The catalytic domains of cellulosomic enzymes are very similar to those of their non-complexed counterparts. Co-localization of enzymes in a cellulosome may improve synergism by bringing necessary components together within the same vicinity, but the organization gives the hydrolases limited mobility in comparison with the non-complexed cellulases. Nevertheless, the cellulolytic activity of the supramolecular cellulosome is comparable with that of the multi-component, non-complexed fungal cellulase system [10]. An effective industrial cellulase preparation should include enzymes that can “multi-task”, and the enzyme function should be collaborative and synergistic. Several fungal cellulase preparations are available commercially but no bacterial cellulase preparation has yet been produced industrially. Available cellulase products, developed for detergent, textile, and other industries, are too expensive for a viable biorefinery. The economic considerations of protein production led us to choose Novozymes’s Celluclast 1.5L, produced by large scale batch fermentation of T. reesei, as a starting point for developing the next generation of biomass-targeting cellulase product. We focused on improving the activity of the T. reesei cellulase system while maintaining its already high protein productivity during fermentation. All studies were performed using acid-pretreated corn stover (PCS) as the substrate.

16.4 Cellulase Development for Biomass Conversion 16.4.1 Optimization of the CBH-EG-BG System 16.4.1.1 BG Supplement A “complete” cellulase system requires BG to hydrolyze cellobiose, a potent inhibitor of CBH and a precursor of fermentable glucose. Balancing the ratio of CBH, EG, and BG is vital for improved cellulose hydrolysis. T. reesei secretes at least two enzymes with BG activity at a very low level during normal celluloseinduced growth. We observed that Celluclast hydrolyzed cellulose with improved performance when assayed in a diafiltration–saccharification device which enables continuous removal of small sugars by filtration, compared with a closed vessel. Accumulation of CBH-inhibiting cellobiose in a closed vessel resulted in a slowing down of the overall reaction [25]. We exogenously supplemented Celluclast with an Aspergillus oryzae BG (belonging to the GH3 family). Addition of small amounts of BG, present as a few percent of total protein, enabled us to achieve equivalent conversion of cellulose in PCS with half the enzyme dosage of the unsupplemented Cellulase mix (Fig. 16.1). By expressing the A. oryzae BG in the T. reesei strain used to produce Celluclast 1.5L we were able to eliminate the need to ferment BG separately.

16.4 Cellulase Development for Biomass Conversion

Fig. 16.1 Improvement of PCS-hydrolyzing cellulases by addition of BG.

16.4.1.2 Novel Cellulases with Better Thermal Properties One focus of our research has been to obtain a collection of CBH, EG, and BG from a taxonomically diverse group of mesophilic, thermotolerant, and thermophilic fungi. Extending the number of cloned and characterized cellulases beyond currently reported enzymes could lead to discovery of enzymes with novel

Fig. 16.2 Phylogenetic tree comparing the catalytic core sequences of the novel GH7 CBH I genes to the catalytic core sequences of published genes.

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properties. In addition to T. reesei we have isolated other cellulolytic fungi that effectively degrade complex lignocellulosic substrates such as PCS, and sought novel CBH I enzymes (from the GH7 family), particularly those from thermophilic filamentous fungi. Thermally stable enzymes could enable cellulose hydrolysis at elevated temperature, which in theory should result in thermal enhancement of the enzymatic rate. We attempted to find a CBH I superior to T. reesei CBH I, which has an unfolding temperature Tm of 62.5 8C [26] and is known to be inactivated in hydrolysis reactions above 50 8C [27]. Many genes with a large amount of sequence identity with T. reesei CBH I (Cel7A) have been deposited in public databases. We used homology search tools, for example the Smith–Waterman, FASTA, BLAST (gap penalties and scoring matrices), Clustal W (multiple alignment), MEME, and MAST (motif searching) algorithms, to discover numerous GH family 7 genes with wide phylogenetic diversity (Fig. 16.2). They provided a resource for understanding the functional diversity of cellobiohydrolases. Of these genes, 15 were expressed in fungal hosts, purified and characterized. In hydrolyzing various cellulose substrates, a few enzymes from thermophilic fungi had thermal stability/activity superior to that of T. reesei CBH I.

Fig. 16.3 Improved CBH I (Cel7A) variants with enhanced activity at high temperatures. Activity at moderate temperature (at which the wild-type enzyme is stable) is plotted against the ratio of activity at a thermally challenging temperatures (at which the wildtype is unstable) divided by activity at the moderate temperature. The performance of

the wild type (wt) is marked as a triangle (s). The open circle (*) marks the position of a variant obtained by protein design. Closed diamonds (^) denote variants obtained from primary screens whereas open squares (`) show variants obtained by rounds of shuffling from pools of primary variants.


Because T. reesei CBH I has high specific activity on PCS at moderate temperature, we tried to improve its thermal stability, using structure-based design and directed molecular evolution. For structure-based rational design, we compared the coding sequences of thermostable CBH with their less stable counterparts and modeled their structures, informed by the published structures of glycosyl hydrolase domains [28–31]. This comparison revealed specific residues that could be mutated to enhance thermostability, and several of these mutations were created by site-directed mutagenesis. In addition, we generated random mutations in the CBH I gene and identified variants with improved activity at elevated temperature. As a result of both approaches we identified several substitutions in the CBH I gene that led to enhanced performance in hydrolyzing a soluble cellulase substrate at high temperature. DNA shuffling, the process of using recombination between genes with partial sequence identity, was used to find favorable combinations of the identified substitutions and to eliminate detrimental mutations [32] (Fig. 16.3). We expressed several of our thermally improved CBH I variants in Trichoderma host strains that lacked the native CBH I gene. Expression of the improved variants was achieved at levels that approximated those found in the wild-type parent strain, and their expression did not noticeably alter the levels of other proteins in the T. reesei secretome. We assayed complete broths containing variant CBH I enzymes for hydrolysis of PCS at temperatures higher than the optimal temperature for T. reesei native cellulases. Figure 16.4 shows that the presence of the variant CBH I improved the high-temperature saccharification of

Fig. 16.4 High-temperature hydrolysis by cellulase mix including variant CBH I. Saccharification of pretreated corn stover by two Trichoderma broths, one expressing the wild type CBH I (“WT CBH I”) and one expressing a CBH I variant (“Variant CBH I”). Solid

lines, 55 8C hydrolysis; dotted lines, 60 8C hydrolysis. Cellulases were used at equivalent protein loadings. Cellulose conversion was determined by measurement of reducing sugars.

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Fig. 16.5 Thermostabilization of A. oryzae BG by directed evolution. Residual activity is the ratio of BG activity after heat treatment at 68 8C for 10 min compared with the activity of an untreated sample. Activity was measured using 4-nitrophenyl b-D-glucopyranoside at pH 5.

PCS over the wt strain. The selected variants failed to surpass wt T. reesei cellulase mix at its optimum temperature (50 8C), however (data not shown). In addition to CBH I, we also improved the thermal stability of A. oryzae BG by directed molecular evolution. We screened for stabilized variants by measuring enzyme residual activity after brief thermal denaturation at temperatures that partially denature the wt enzyme. Several improved variants had better thermal stability, as measured by assessing residual activity after a ten-minute thermal challenge at 68 8C (Fig. 16.5).

16.4.1.3 Structure–Function Relationship of EG For efficient cellulose conversion, it is crucial that CBH and EG act synergistically; thus, EG activity and substrate specificity is important for maximizing cellulose hydrolysis. Microbial EG are grouped into many families according to the extent of their sequence identity [17]. T. reesei secretes at least six EG, with EG I and EG II making up as much as 15% of the total secreted protein. Although the substrate specificity of several individual representative EG has been thoroughly studied, a systematic, comparative investigation of EG from different GH families has yet to be made [33]. We assayed *20 EG from GH5, 7, and 45 with a variety of representative substrates to further define their substrate preferences (Fig. 16.6). Cel5 EG had significant mannanase activity, in addition to their cellulase activity. Cel7 EG had significant xyloglucanase activity, in addition to their cellulase activity. In contrast, Cel45 EG were “strict” cellulases, acting almost exclusively on b-1,4 linked glucose polymers. Among the EG, only Cel7 were active on p-nitrophenyl-b-d-cellobioside, a commonly used chromogenic surrogate substrate for cellulases. These results suggest that Cel45 have an active site groove that is more defined, or specifically “tuned” to accommodate a b-1,4-d-cellodextrin unit, compared with the active sites found in Cel5 and Cel7.


Fig. 16.6 Substrate specificity of endoglucanases. Substrates: PNPC, p-nitrophenyl-b-D-cellobioside; PASC, phosphoric acidswollen cellulose. T. reesei Cel7B is marked with an arrowhead (Tr Cel7B).

Elucidation of this structure–function relationship could assist us in tailor-making cellulase systems specific toward biomass with different cellulose–hemicellulose compositions [34]. 16.4.2 Other Proteins Potentially Beneficial for Biomass Conversion 16.4.2.1 Secretome of Cellulolytic Fungi It is known that the number of cellulase-encoding genes in cellulolytic microbes can far exceed the number (3 to 4) of components in the simplest “complete” cellulase system. The need for a microbe to have an extensive cellulase array might be related to the diversity/heterogeneity of its carbon source, or the requirement of other proteins, beyond the “canonical” composition (reducing-end-preferred CBH I, nonreducing-end-preferred CBH II, EG and BG), for its cellulolytic function. Different cellulolytic fungi can secrete sets of proteins with significantly different 2D electrophoretic patterns, as exemplified in Fig. 16.7. The explanation for this difference might be twofold. Different fungi use at least one of the four

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Fig. 16.7 Two-dimensional gels of proteins secreted by T. reesei and three other cellulolytic fungi grown on PCS. The “canonical” cellulases are marked.

“canonical” cellulases, but the cellulase(s), although highly similar in terms of amino acid sequence identity, can differ by posttranslational processing (i.e. IEF isoforms). In addition to the major cellulase(s), different fungi can secrete various minor proteins under cellulose-induced growth. The presence of these minor components indicates that other proteins (in addition to the four canonical ones) may play complementary/beneficial roles in cellulosic degradation. Among the proteins secreted by T. reesei, Cel12A, Cel61A, Cel45A, XG74A, Cel3A, Xyn11A, swollenin, and other proteins have been identified in addition to Cel7A, Cel6A, Cel7B, and Cel5A. Microarray cDNA/genomic DNA analysis, Expressed-Signal-Tags (EST), and other molecular biology tools have unveiled many unknown genes whose expression were induced by PCS (Fig. 16.8). Given this evolutionary diversity, we can expect other cellulolytic fungi/bacteria to utilize different sets of proteins. Some organisms may utilize novel component(s) in the enzyme mix; others might be unique with regard to the stoichiometry of the cellulases present.

16.4.2.2 Hydrolases The two CBH, six EG, and one BG known to be secreted by T. reesei belong to eight GH families, representing approximately half of the GH families known to include cellulases. It is possible that cellulases from other GH families could be beneficial to T. reesei cellulolytic system, by providing either complementary specificity, stronger synergism, reduced inhibition, enhanced reactivity, or in-


Fig. 16.8 Schematic of fungal genes induced by growth on PCS. The action of gene products on lignocellulose is depicted.

creased stability. For example, cellulases derived from hyperthermophilic microbes are of particular interest because of their superior thermal profile. One example of the benefits of pairing the T. reesei cellulolytic system with another quite different fungal cellulolytic system is shown in Fig. 16.9. A 1 : 1 mixture of the two cellulase preparations performed as well as the individual system dosed at twice as much as the T. reesei cellulolytic system alone, indicating a significant synergism between the two systems. Identification and transfer of those enzymes responsible for this synergism into the T. reesei host should create a single organism with significantly improved cellulose-hydrolysis activity.

Fig. 16.9 Effect of adding fungus Z proteins to T. reesei broth in hydrolyzing PCS at 50 8C.

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Thus, studying non-canonical cellulases is of interest for improving enzymatic PCS hydrolysis.

16.4.2.3 Nonhydrolytic proteins As shown in Fig. 16.8, several non-hydrolytic proteins seem to be up-regulated under cellulose induction. Some of these can be attributed to cellular processes directly relevant to cellulose utilization, but others apparently cannot. The contributions of these additional components to microbial biomass conversion should be an indispensable part of “cellulolyteomics” – understanding the global network of all cellulosics-active biomolecules. Investigating the mechanism and extent of action of the components on different feedstocks will be an important part of improving biomass conversion. Swollenin and phenolic oxidases are currently attracting attention because of their ability to disrupt cellulose and lignin, respectively; the two reactions are thought to be beneficial to biomass hydrolysis [10].

16.5 Expression of Cellulases

Many individual enzymes acting synergistically form an effective cellulase mix for conversion of lignocellulosics to sugars. Production of cellulase components individually is not economically feasible; instead, all the proteins necessary should be expressed and secreted by a single fungal host. The protein composition must be well balanced to take advantage of the optimum mix for cellulase synergy. At the same time, the overall total protein yield must be high. Considering these factors, it becomes clear that an important aspect of biorefinery research is the technology of protein expression in fungi. Enhancement of a cellulase production strain can be performed on two different levels; first, classical strain mutagenesis can be used, as has been reported previously for T. reesei [35]. Second, genetic engineering can be used to modify levels of endogenous gene expression and to introduce genes for heterologous expression of novel cellulase components. The use of genetic engineering to introduce and manipulate specific gene expression in T. reesei has been indispensable to our cellulase research program. We used several selective genetic markers to follow gene integration into the host and developed a variety of promoter elements to enable variable levels of gene expression. Because we required the ability to introduce several novel genes into the host strain simultaneously, we investigated transformation efficiency, and developed procedures for simultaneous co-transformation of different transgenes. These technological improvements enabled us to investigate, rapidly and efficiently, the effect of introducing different enzymes into the T. reesei cellulase mix.

16.6 Range of Biobased Products

Fig. 16.10 Signal peptide effect on BG secretion in T. reesei. T. reesei strains were genetically modified to heterologously express A. oryzae BG, either behind the native A. oryzae BG signal peptide, or behind a signal peptide from the H. insolens Cel45A. A. Relative BG activity measured in the

secreted fraction, using 4-nitrophenyl b-Dglucopyranoside at pH 5. B. SDS–PAGE of secreted proteins from the two T. reesei strains. The positions of molecular weight markers are labeled; the position of A. oryzae BG is marked with an arrow. The gels were stained with Coomassie Brilliant Blue.

In addition to controlling gene expression transcriptionally, by using promoters of different strengths, we focused on enhancing individual protein yield by optimizing protein secretion. One example is replacement of the A. oryzae BG signal sequence with a signal peptide from Humicola insolens Cel45A EG, which improved the BG secretion in T. reesei (Fig. 16.10). The objective of the research discussed here is to produce a single fermentation product with improved capability for converting the cellulose in pretreated corn stover to glucose. The economic impact of these improvements on a cornstover-based biorefinery that produces fuel ethanol will be discussed below.


The goal of the biorefinery is to utilize renewable feedstocks in the production of power, a variety of fuels, and chemicals. Several biobased products are already on the market, including fuel, industrial and potable ethanol, sweeteners (highfructose syrups and sorbitol), organic acids (citric and lactic acids), MSG, lysine, enzymes, polymers (xanthan gums), food and feed products, and specialty chemicals, with annual multi-billion dollar sales. Biochemicals produced from today’s biorefineries find their utility in diverse products such as adhesives, wallboards, resins, paper coatings and additives, textile sizing agents, foam packaging materials, solvents, cosmetics, toiletries, paints, plastics, food, animal feed, and pharmaceuticals. In deriving products from raw biomaterials, biorefineries employ two fundamental technological platforms – the syngas platform based on thermochemical

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gasification and the sugar-platform based on biochemical conversion to simple sugars. Biotechnology is currently being applied to the sugar platform, with extensive focus on enzymatic saccharification and whole-cell microbial fermentation of the resulting sugars. Oxygenated biomass-derived products, for example ethanol and other fermented organic compounds, will be key precursors to many industrial chemicals traditionally dependent on petroleum feedstocks. The tractability of a sugarplatform for biosynthesis of products is apparent in comparison with a petroleum-based industry. Biorefinery-based products have an advantage in that oxygenated intermediates, for example adipic acid or ethylene glycol, can be produced more readily, because of the presence of oxygen in the sugar and starch backbone. Because the raw materials can vary depending on the local source of fermentable sugars, the fermentation of such products is more flexible. 16.6.1 Fuels

In the US, fuels make up approximately 70% of the carbon consumed annually (more than 1.8 billion tons). Biobased ethanol and biodiesel are currently produced at higher cost than gasoline, and these renewable fuels account for less than 2% of total liquid fuel consumption. Developing biobased liquid fuels will require production cost reduction, including use of low-cost carbon sources, for example agricultural/forestry byproducts and urban wastes. As a direct result of research and development programs worldwide, fuel ethanol production from sugarcane/beet (direct fermentation of material obtained by crushing) and corn/wheat starch (by starch saccharification) has become a viable industry. Yeast-fermentation of simple sugars, particularly sucrose and glucose, has been used economically to produce ethanol, in amounts of more than 20 million tons per year [36]. In Brazil, fermentable sugars are obtained from mechanically processed sugar canes. In the US the sugars come mainly from enzyme-degraded corn and wheat starch. To extend this industry by tapping into inexpensive, readily available biomass materials such as corn stover, wheat straw, and other agro/forestry byproducts as sources for fermentable sugars, intensive research is being conducted to develop enzymes which convert lignocellulosics and other polysaccharides to simple sugars. In addition, research efforts are being focused on obtaining and improving microbes for fermenting diverse sugars [37, 38]. Production of fermentable sugars from cellulose is currently more expensive than their production from amylose (starch). This can partly be explained by the relative recalcitrance of cellulose – amylase hydrolysis is intrinsically faster and the kinetics of cellulase action require relatively higher loadings of enzyme. As a result of significant funding by the US Department of Energy and collaboration between Novozymes and the US National Renewable Energy Laboratory (NREL), however, substantial progress has been made toward reducing enzyme cost for conversion of cellulose to fermentable sugars. After the research

EtOH)


Fig. 16.11 Progress in enzyme cost reduction for corn stoverbased ethanol production. Symbols denote milestones achieved in reducing enzyme cost for production of ethanol from pretreated corn stover, as validated by the National Renewable Energy Laboratory (NREL.) Costs are specific to the corn stover feedstock (PCS) and NREL cost models.

and development advances mentioned above, more than 20-fold cost reduction has been achieved (Fig. 16.11). Importantly, the reduction of enzyme cost achieved in the last few years has had a major affect on the estimated cost of producing fuel ethanol from corn stover in a biorefinery. In 1999, the total expected cost for producing bioethanol was dominated by enzyme cost; today enzyme cost is comparable with the estimated costs of biomass feedstock collection or depreciation of capital. Using a 2004 “state of the technology” process cost estimate supplied by NREL, and an enzyme cost of $0.50 per gallon ethanol produced, ethanol derived from biomass has a total cost of about $2.50 per gallon [39]. Comparing costs for producing ethanol from corn starch saccharification (by amylase) and fermentation to projected costs for lignocellulosic-rich biomass-based ethanol production indicates that the biomass based industry is becoming economically viable. In addition to fuel ethanol, biomass-derived sugars can be fermented into combustible “biogas.” Mainly methane, the fermentation is accomplished by anaerobic bacteria, a technology already developed on small to medium-scale. Fuel ethanol seems to be the future for a biomass-based energy industry, however. Future research and development effort will probably be focused on cellulases with improved reactivity and stability, and on microbes with expanded sugar specificity (e.g. novel yeasts or pathway-engineered microbes capable of fermenting a variety of pentoses, hexoses, or oligomeric sugars).

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16.6.2 Fine/Specialty Chemicals

Biomass is abundant in C6 sugars (hexoses), of which glucose is the most common, and C5 sugars (pentoses), of which xylose is predominant. Several organic acids are currently produced from these starting sugars via fermentation by microorganisms. As noted in Fig. 16.12, lactic acid and succinic acid fermentation platforms can lead to numerous value-added products. The C5 sugars can also be fermented into a variety of xylose derivatives including itaconic acid, furfural, furfuryl alcohol, and 2-hydroxymethyltetrahydrofuran (Table 16.2). Among various biomass-derived organic acids being studied, lactic acid and, to a lesser extent, 3-hydroxybutyric acid have attracted the most attention, because of their use in the manufacture of plastics. In addition to biodegradability, reduced CO2 emission is another benefit of making these biopolymer/plastics [40]. 16.6.3 Fuel Cells

A clean, efficient, and easily rechargeable energy source, fuel cells have been actively studied in the past few decades. In fuel cells, chemical energy is converted into electricity by electrochemistry rather than combustion. Biofuel cells, in which enzymes or entire microbial cells serve as electron-transfer catalysts, use

Fig. 16.12 Schematic diagram of biorefinery products based on lactic and succinic acid fermentation platforms.

16.6 Range of Biobased Products Table 16.2 Industrial bioproduct opportunities. Technology platform

Chemical

Applications

Sugar fermentation

Lactic acid (currently biobased) Acidulant (food, drink), electroplating bath additive, mordant, textile/leather auxiliary Polylactide (currently biobased) Film and thermoformed packaging, fiber, fiberfill Ethyl lactate (currently Solvent, chemical intermediate biobased) 1,3-Propanediol Apparel, upholstery, specialty resins, other applications Succinic acid Surfactants/detergents, ion chelators, food, pharmaceuticals, antibiotics, amino acids, vitamins Succinic acid derivatives Surfactants, adhesives, printing inks, magnetic tapes, coating resins, plasticizer/ emulsifiers, deicing compounds, herbicide ingredients, chemical and pharmaceutical intermediates Bionolle 4,4 polyester Thermoplastic polymer applications 3-Hydroxypropionic acid Acrylates, acrylic fibers, polymers, resins n-Butanol Solvent, plasticizers, polymers, resins Itaconic acid Aluminum anodizing reagent, methyl acryl

relatively inexpensive, safe, and available “feeds”, for example alcohol or sugar, instead of hydrogen gas or its volatile derivatives used in conventional fuel cells [41]. Because of the safety and cost issues of conventional fuel cells, biofuel cells are quickly emerging; in the near future, mini and micro-scale biofuel cells could replace conventional batteries that power a variety of consumer and medical-implant devices. In the more distant future, scaled-up biofuel cells could serve as a major industrial energy source. Current research on biofuel cells focuses on how to improve performance in terms of speed, output, reliability and durability. Enzyme-mediated electrontransfer between feeds and electrodes is a focus of research and development. For example, alcohol dehydrogenase and sugar oxidase are being studied as facilitators for electron-donation from alcohols or sugars (e.g. glucose) to an anode, and laccase is being studied to facilitate the electron-accepting of O2 (air) from a cathode. In the future, the focus of biofuel cell research may shift to cheaper, more readily available feedstocks. Because both ethanol and glucose can be generated from biorefineries, we may envisage a next-generation biofuel cell that is powered by biomass. In such a fuel cell, biomass would be enzymatically converted into glucose (e.g. by cellulase), which would then be enzymatically oxidized on an electrode (e.g. by glucose oxidase). The extracted electrons would run through a wire, perform electric work, and then be used to enzymatically reduce O2 (e.g. by laccase). Biofuel cells may be set up as part of a biore-

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finery plant that generates electric power from biomass, or be used for wilderness exploration or military operations where fuel transport is logistically costly.

16.7 Biorefineries: Outlook and Perspectives 16.7.1 Potential of Biomass-based Material/Energy Sources

The goal of biorefinery is to utilize renewable feedstocks in the production of power, a variety of fuels, and chemicals. One goal for the next generation of biorefineries will be to more fully integrate facilities that can process a variety of biomass feedstocks into a full range of biochemical products. This integration would enable us to take advantage of the different product streams that might emanate from renewable feedstocks by bioconversion of products (Fig. 16.13). More than 100 million metric tons of fine, specialty, intermediate, and commodity chemicals are produced annually in the US. Today, only 10% of these

Fig. 16.13 The next biorefineries: Integrating a variety of feedstocks for a multitude of end products.

16.7 Biorefineries: Outlook and Perspectives

are biobased. Thus, there is tremendous growth potential for biorefineries, if the economics are competitive, if the environmental impact is favorable, and/or if novel products are created. 16.7.2 Economic Drivers Toward Sustainability

The chemical industry currently consumes approximately 8% of total petroleum and natural gas output to produce approximately 2500 products worth approximately $215 billion [36]. An attempt to replace part of the petrochemical processes with biomass-based biotechnological processes is driven by the need to curb greenhouse gas emission, upgrade agro/forestry industry value production, and reduce reliance on fossil resources. For biobased production to be economically feasible, the cost of producing the biobased product must compete favorably with the comparable petroleum-derived product. An additional or alternative economic driver for the biorefinery could be synthesis of novel products that provide unique utility, and that are unavailable or uneconomical via petroleum-based chemistry. With regard to improving economy, there are several technologies where cost efficiency should be improved. These include: · harvesting, collection and pretreatment of the biomass, which serve to unlock the fermentable sugars and increase the carbon conversion to the desired products; · enzymatic conversion of the polysaccharides in the pretreated biomass stover into glucose and other fermentable sugar monomers; and · microbial fermentation of the sugars to the desired products. Efficient enzyme catalysis is one of the primary economic barriers in the challenge to design an overall cost-effective process for converting biomass into fermentable sugars. The research efforts described here, and research efforts in similar work performed at Genencor International, have specifically focused on improving the efficiency of enzymatic hydrolysis, and progress thus far looks quite promising. Further work on metabolic engineering of microbial production strains should continue, as should efforts to better integrate biomass pretreatment, enzyme hydrolysis, and fermentation to avoid complications resulting from isolated efforts. Several major problems must be solved to enable commercialization of various biorefineries. To satisfy the optimum operating conditions of enzymes and microbes, raw biomass materials collected from diverse regions under diverse climates must be examined to assess the impact of biomass feedstock variability on pretreatment, enzymatic conversion, and fermentation. Optimizing these steps and integrating them into a robust, low cost, efficient, sustainable, and value-generating material–energy cycle is highly challenging, yet offers great social, economic, and environmental promise.

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17 Biocatalytic and Catalytic Routes for the Production of Bulk and Fine Chemicals from Renewable Resources Thomas Willke, Ulf Prüße, and Klaus-Dieter Vorlop

17.1 Introduction 17.1.1 Renewable Resources

The most important sources of renewable resources for industry are oil plants, starch plants, sugar plants, energy plants, and wood, but also waste and residues from agriculture and industry. The corresponding substrates for conversion processes are manifold and belong to such heterogeneous substance classes as oils, fats, glycerol, lignocellulose, cellulose, starch, inulin, sugar, complex biomass, etc. Fats and oils are already being used as feedstock in industry at a level of 15 million t a–1. The corresponding products are mainly applied in plastics, paints, lacquers, biotensides, and energy (as biodiesel). The potential of carbohydrates (starch, sugar, cellulose) is far from being fully exploited. In the year 2002/2003, approximately 143 million tons of sugar were produced worldwide (Germany 4 million tons). Of these, only 70 000 t (1.7%) were industrially employed as renewable resources in the pharmaceutical and chemical sector in Germany [1]. Because of the substantial quantities of (ligno)cellulose available, utilization of the material of biomass (wood, straw, waste, and residues) has huge potential. Countries with large amounts of wood, for example Canada, the USA, Scandinavia, or Austria, invest much effort to utilize this potential. There is much need for research. If it were possible to establish highly efficient enzymatic pulping processes, numerous bulk products (ethanol, butanol, lactic acid, etc.) could be produced at competitive prices.


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17 Biocatalytic and Catalytic Routes for the Production of Bulk and Fine Chemicals

17.1.2 Products

In 2001 the organic chemicals produced worldwide were estimated to be worth almost 1,900 billion Euros. Asia leads, with 586 billion Euros, followed by the European Union (519 billion Euros) and North America (508 billion Euros). The German chemical industry, with approximately 25% of the total European amount, recorded sales in 2002 of 133 billion Euros. In respect of price and amount, the products can be roughly classified into two sectors: (1) bulk chemicals or commodities (> 1 million t a–1, < US$2–4 kg–1), and (2) fine and specialty chemicals (< 1 million t a–1, > US$2–4 kg–1). Worldwide the sales of fine and specialty chemicals is approximately 250 billion US$ (Europe 120 billion US$), which, for Europe, is approximately 20% of all the chemical products sold.

17.1.2.1 Bulk Chemicals and Intermediates In bulk chemical synthesis the use of renewable resources as feedstock is rather limited. Relevant future applications include biobased synthesis gas, and products thereof, and a few selected products, for example ethanol. The potential use of renewable resources for the production of intermediates, for example, monomers or plasticizers for polymers, is mainly a question of the price of crude oil. At a crude oil price (2004) of approximately 50 US$ per barrel (0.25 1 L–1), biotechnological processes can hardly compete. With rising crude oil prices, chemical and biotechnological processes based on renewables become increasingly competitive with petrochemical-derived products, especially for product prices > 2 US$ kg–1.

17.1.2.2 Fine Chemicals and Specialties For fine chemicals the price of the product is not so critical – product functionality and purity, and quality assurance, are much more important. Extremely high prices are charged for pharmaceuticals, followed by cosmetics and food ingredients and some intermediates for industry. If such high prices are accepted by the market, sustainability forces the chemical industry to use economical and environmentally friendly processes. Thus largest chances of broader application of renewable resources, either by biotechnological or chemical processes, are in fine chemicals. Often, biotechnology is the only way to obtain the required product.



Until 1930 many important bulk products, for example fuels (ethanol, butanol), organic acids (acetic acid, citric acid, lactic acid), and other basic chemicals, were mainly produced from renewable resources. Process engineering was limited to fermentation by use of fungi or bacteria. In the past some basic chemicals (for example butanol and acetone) were produced exclusively by fermentation. With the development of petroleum chemistry, however, they were replaced by chemical– technical products. In the production of other chemicals (ethanol, citric acid, lactic acid, and acetic acid), biotechnological techniques have always been predominant, because the chemical–technical alternatives are not economical. There are several prognoses of the amount and range of worldwide petroleum reserves. Most of the experts predict that maximum production will be achieved in the next few decades [2] (Fig. 17.1). Countries with high energy demands and limited resources, for example the USA, have already realized this and are investing much effort in appropriate research [3]. In the USA, for example, substitution of fossil fuel with renewable resources in the production of liquid fuels and organic chemicals is envisaged to be 50% and 90%, respectively, by 2090 [4]. More realistic prognoses expect an increase to 25% in the next 30 years [5]. Shell Oil, for example, intends to provide 30% of the world‘s chemical and energy needs by use of biomass by 2050, corresponding to nearly US$ 150 billion [6]. DuPont, one of the largest manufacturers of plastics, intends to produce 25% of its products from renewable resources by the year 2010 [7]. Figure 17.2 summarizes several prognoses for the USA.

Fig. 17.1 Prognoses of crude oil production and estimated ultimate recovery (EUR) [2].

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Fig. 17.2 Biotechnologically produced liquid fuels and organic chemicals in the USA as share of total production [3] (estimated data for 2090).

17.3 Processes

Large-scale chemical processes are usually very sophisticated, because environmentally friendly catalytic conversions are used almost exclusively. Although they are usually performed at high temperatures and pressures, and although substrates and products are often detrimental to health or are environmentally significant, they can be regarded as clean processes. Nevertheless, chemical processes with low annual production volumes and/or multi-step synthetic path-

Table 17.1 Typical problems of biotechnological processes and possible solutions. Problem

Solution

Appropriate biocatalyst not available

Cell screening Strain optimization, mutation/selection Metabolic pathway design (MPD)

Productivity too low

High-cell density fermentation Cell recycle Immobilization

Substrates too expensive Cheap substrates often not suitable Unwanted by-products Metabolic pathways not optimum Low product concentration (production inhibition)

Cell screening, substrate screening Genetic engineering, MPD Strain optimization, mutation/selection

Product recovery Process control

In situ processes On-line analysis

17.3 Processes

ways, especially in the fine and specialty segment, are less efficient and less environmental friendly. In this segment more classic organic chemistry is used than catalytic processes, resulting in significant waste generation, emissions, and energy consumption. Biotechnological processes usually occur under mild conditions. Biocatalysts, substrates, intermediates, and by-products, and the product itself, are biodegradable. Water is usually used as a solvent. There are also frequent disadvantages, however, including low product concentration, low productivity and, hence, high recovery costs. Table 17.1 lists some problems and possible solutions. Some products are only accessible chemically, whereas biotechnology is more appropriate for others (chiral substances, some vitamins and amino acids, highly selective transformations with polyfunctional substrates, such as sugars). Numerous syntheses are conducted exclusively using enzymes (lipases, amylases, proteases, and, also, increasing in the future, cellulases). To establish a large-scale process based on a biochemical reaction it is preferable to have means available to hold back the catalyst (i.e. enzyme) in the bioreaction vessel. By immobilizing catalysts, for example growing, resting, or dead cells or enzymes, it is possible to retard them. 17.3.1 Immobilization

Different types of immobilization procedure have been developed for this purpose, as is shown in Fig. 17.3 [8]. Besides the advantage of easy retention, immobilized catalysts are also often more stable with regard to, for example, pH and temperature. When entrapped the catalysts are, moreover, protected against other bacteria and thus processes can run under non-sterile conditions, because potential contamination is washed out while the favored catalyst is specifically protected. Encapsulation of catalysts also has disadvantages, however: during the immobilization process the catalyst may be inactivated by physicochemical or physiological effects. Even if this does not happen, the overall activity of the immobilized system could be lower than that of the free catalyst, because of diffusion

Fig. 17.3 Different ways of immobilizing biocatalysts [8].

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limitations. To minimize this effect the particles should be small and applicable for the later application. Examples of processes developed, investigated, and optimized at the Institute of Technology and Biosystems Engineering are discussed in the sections below. 17.3.2 Biocatalytic Routes from Renewable Resources to Solvents or Fuels 17.3.2.1 Ethanol Production with Bacteria or Yeasts? Introduction Ethanol can be used as a liquid energy carrier, fuel additive, and feedstock in the chemical industry. Because of the costs – except for use in foods or stimulants – biotechnological production in Europe has not yet been profitable. In May 2003 an EU directive on the use of biofuels set a minimum level of 5.75% bio-fuels (including bioethanol) for all transport fuels sold by 2010 [9]. Thus ethanol will be more highly in demand in the future. The biotechnological production of ethanol – at the beginning of the 20th century mainly for fuel – is currently experiencing a renaissance. In the year 2001, worldwide annual ethanol production amounted to more than 30 billion liters. The leading role played by Brazil, where bioethanol from sugar cane has been used for more than 25 years, has currently been taken over by the USA, which will probably continue to occupy first place. In the USA, mainly corn or wheat starch is used. In the EU, with a total of approximately 2 billion liters, France (approx. 0.8 billion liters) is the largest producer; Germany produces only approximately 0.3 billion liters [10]. The Process Ethanol is produced by fermentation with yeasts or bacteria (Fig. 17.4). The classic route by use of yeasts has been known for thousands of years and is one of the oldest biotechnological processes used by mankind. Substrates are sugar-based feedstocks (sucrose, glucose, molasses) and also starch hydrolyzates. A study recently promoted by the German Research Foundation (DFG) determined costs based on grain starch for the German market. According to this study, substrate costs account for up to 50% of total production costs [11]. To find cheaper sources of renewable raw materials, various routes are being followed: · A search for organisms which, in addition to glucose, can utilize other sugars, for example pentoses (xylose, arabinose). Such sugars are the products of hydrolysis of hemicellulose, the basic constituent of wood, straw, and a variety of other plant residues [12–14]. · Screening for enzymes which split the raw materials into usable sugars as efficiently as possible, either directly or in coupled processes [15]. · Alteration of known or new organisms by genetic engineering (genetically modified microorganism, GMO) to enlarge their substrate spectrum [16, 17]. In the USA a new technique is currently being implemented on a pilot scale

17.3 Processes

Fig. 17.4 Process scheme for the production of ethanol from renewable resources.

[18]. The process uses genetically modified Zymomonas mobilis, which was given the ability to use pentoses (xylose) in addition to hexoses (glucose, fructose). This enables inexpensive substances which could not previously be used, for example, rice straw, to be transformed into ethanol with high yields [19]. Reduction of the process costs can be achieved by several methods: · Screening or genetic engineering of microorganisms with the goal of increasing productivity, ethanol tolerance (and hence achievable final product concentration), and yield. For example, use of the bacterium Zymomonas mobilis instead of conventional yeasts (Saccharomyces spp.) also results in increased product yields, besides the fivefold higher productivity. In addition, immobilization boosts volumetric productivity and, again, product yield. Furthermore, product tolerance, and with it the final ethanol concentration, is enhanced. Essential data for the process are listed in Table 17.2.

Table 17.2 Ethanol production with yeast or bacteria. Biocatalyst

Yeast Saccharomyces cerevisiae Bacterium Zymomonas mobilis

Productivity in kg ethanol m–3 h–1 Suspended cells

Immobilized cells

0.5–2

10–30

4–5

50–80

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Fig. 17.5 Cell-Immobilization procedure for the production of lens-shaped particles (LentiKats).

· Development of new fermentation methods which, in particular, include the use of immobilized (entrapped) cells, so that the process can be run under non sterile conditions or the biocatalyst can be easily recycled. The latter is particularly important for high-performance strains or genetically engineered microorganisms.

Fig. 17.6 Pilot plant for the continuous ethanol production with immobilized cells.

17.3 Processes

Fig. 17.7 Process design of an ethanol production plant. Effect of type of biocatalyst on plant size. Source: BMA–Starcosa 2001, Brunswick, Germany, capacity 60 000 L ethanol day–1.

For example, new immobilization technology based on lens-shaped particles (Fig. 17.5) [20] was adapted for production of ethanol from molasses in cooperation with the company BMA–Starcosa (Braunschweig, Germany). Continuous and stable ethanol production from untreated molasses was achieved over several months, even under nonsterile conditions. A pilot plant (Fig. 17.6) has been running at BMA–Starcosa, under the described conditions, since 2003. Figure 17.7 shows clearly the extent to which plant size, and consequently investment costs, can be reduced solely by introduction of immobilized cell systems for the biotechnological ethanol production. In combination with the above-mentioned improvements ethanol could be produced economically in the future. 17.3.3 Biocatalytic Route from Glycerol to 1,3-Propanediol 17.3.3.1 Introduction One of the applications of 1,3-propanediol (PD) is its use as a diol component in the plastic polytrimethyleneterephthalate (PTT), a new polymer with properties comparable with those of Nylon. It is preferably used for carpets (Corterra by Shell) or special textile fibers (Sorona by DuPont). Further applications are appearing in polyester resins, mainly in the paint industry.

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17.3.3.2 The Process PD can be produced biotechnologically from glycerol with the aid of bacteria (Fig. 17.8). Glycerol is mainly a by-product of fat splitting and biodiesel production. Further growth of the biodiesel market would result in a fall in the price of glycerol. One of the largest biodiesel producers in Germany (Nevest, Schwarzheide) filed for insolvency in December 2003, partly because of the decline in the price of glycerol from 1000 1/t to nearly half [21]. Some companies even pay to dispose of their glycerol. Glycerol–water from RME production, in particular, would be an interesting raw material if it could be used in fermentation without further pretreatment. Another method would be the utilization of glucose instead of glycerol, which would provide independence from the fluctuating glycerol market. Because no microorganisms are known which directly convert glucose into 1,3-propanediol, however, this technique requires mixed cultures or microorganisms designed by genetic engineering. Both possibilities have been examined. The gene-technological variant is favored by DuPont in cooperation with Genencor and is on the verge of technical implementation [22]. Under some conditions, however, the classic technique based on glycerol can be quite interesting technically and economically. A concerted, extensive search for new microorganisms (screening) and improved process design (fed-batch with pH-controlled substrate dosage) enabled product concentrations, which were relatively low at a maximum of 70 to 80 g L–1 as a result of product inhibition (Fig. 17.9), to be increased to more than 100 g L–1 (Fig. 17.10). Another advantage of the new technique and the new isolated strains is the use of lowpriced crude glycerol or glycerol–water (Fig. 17.11). This is a factor which should not be underestimated and has a direct effect on product costs

Fig. 17.8 Process scheme for 1,3-propanediol production from plant oil via glycerol or glycerol–water as a byproduct of biodiesel production.

17.3 Processes

Fig. 17.9 1,3-Propanediol production from pharma glycerol by use of a commercially available strain (pH-controlled fed batch, mineral medium with YE, 328C, pH 7.2).

Fig. 17.10 1,3-Propanediol production from pharma glycerol with a new strain from screening (35 8C, pH 7.0, other conditions as for Fig. 17.9).

(Fig. 17.12). Further on, use of immobilized cells (LentiKats; Section 17.3.2.1, above) rather than freely suspended cells, enables productivity to be increase from approximately 2 to 30 gPD L–1 h–1. Comparison of current (chemical) techniques with the new biotechnical techniques based on different substrates and glycerol qualities (= raw glycerol costs)

395

396


Fig. 17.11 1,3-Propanediol production from raw glycerol–water with a new strain from screening (conditions as for Fig. 17.10).

Fig. 17.12 Cost comparison of industrial 1,3-propanediol production, effect of substrate costs, (*) glucose or glycerol, respectively (Data from PERP 1998, substituted).

17.3 Processes

shows that biotechnology might be competitive with chemical techniques if crude glycerol is used (PERP 1998). 17.3.4 Biocatalytic Route from Inulin to Difructose Anhydride 17.3.4.1 Introduction Inulin is a linear b-2,1-linked polyfructane terminated with a glucose residue. Large amounts of inulin are contained in the roots and tubers of crops like dahlia, chicory, and Jerusalem artichoke. Inulins have a very limited market thus far, mainly because of the high cost of expensive separation and purification steps. For 1.5 to 2 1 kg–1 it is approximately four times as costly as competing glucose, starch, or sucrose. This also explains why short oligofructoses are synthesized enzymatically from sucrose for probiotic products rather than by partial hydrolysis of inulins. Future use of inulin-derived products is thus either in high-value markets, for example the functional food segment, or by converting inulin into intermediates which can be separated and purified at lower cost. One promising compound derived from inulin for this purpose is difructose anhydride (DFA III, Fig. 17.13). DFA III can be the basis for plastics and tensides. It can be crystallized as easily as sucrose after an ion-exchange step and hence can be produced at a price well below that of inulin. So far DFA III has not been introduced to the market, because no efficient enzyme and biotechnical process was available for the necessary bioconversion of inulin. To produce DFA III on a technical and industrial scale, large amounts of enzyme are needed. The following sections introduce a strategy showing how this problem can be solved [23, 24].

Chicory

Chemical feedstock

Fig. 17.13 Process for production of DFA III from inulin.

397

398


17.3.4.2 Enzyme Screening From 65 tested samples from extremophile locations, approximately 400 bacterial strains were obtained and investigated further. Four strains were found to produce DFA III and one strain (Buo141) identified as an Arthrobacter sp. expressed an enzyme, stable for weeks at elevated temperatures of 60 8C whereas the activity of the other strains declined within a few hours (Fig. 17.14). The new strain grows aerobically at ambient temperatures and secreted inulase II extracellulary but only with low activity, too low for economical industrial process. To overcome these limits, genetic engineering was used.

17.3.4.3 Genetic Engineering To increase production of the enzyme the gene encoding for the inulase II (ift gene) should be transferred to and expressed in an E. coli host. To gain access to the bacterial gene suitable primers for a polymerase chain reaction (PCR) were needed. For primer design, only two highly divergent sequences of inulase proteins were known. A special primer design based on phylogenetic analysis substantially accelerated isolation of the ift gene. The complete ift gene was obtained by screening the genomic library with this probe. As a result a plasmid was constructed which expressed an enzyme of 477 amino acids when transferred to E. coli. A cell-free extract of such a culture had an activity of 3000 U L–1, whereas most of this activity was detected intracellularly. In Arthrobacter the inulase II enzyme is expressed as an extracellular enzyme. Transport through the cell membrane is accomplished by means of a specific signal-transfer peptide which is part of the ift gene. Because of phylogenetic differences between the species Arthrobacter and Escherichia the transfer-peptide does not work in E. coli. In E. coli the enzyme remains intracellular, as was shown by analysis of the activity of disrupted cells and the supernatant of cultivations. Exact removal of the complete transferpeptide resulted in a one-hundredfold increase in activity. A further increase in

Fig. 17.14 Screening of inulinase II producers: comparison of long-term stability.

17.3 Processes

Fig. 17.15 Increase of inulinase II activity by genetic engineering.

activity of approx. 35% was possible because of a point-mutation induced by error-prone PCR. In position 221 of the enzyme a glycine was exchanged with arginine. (Fig. 17.15). To obtain sufficient large quantities of the enzyme the genetically modified organism was fermented.

17.3.4.4 Fermentation of the Recombinant E. coli The recombinant E. coli pMSiftOptR was fermented using an inexpensive technical medium. During the fermentation inulase activity was monitored. A final biomass concentration of 11 g L–1 (dry weight) and an overall activity of 1 760 000 U L–1 was measured (Fig. 17.16). Because high-density fermentations of E. coli are known to reach biomass concentrations of approximately 100 g L–1 it seems possible that after optimizing the fermentation step activity of at least 15 ´ 106 U L–1 is possible.

Fig. 17.16 Fermentation of the redombinant E. coli pMSiftOptR for production of inulinase II.

399

400


17.3.4.5 Enzyme Immobilization and Scale-up For immobilization of inulinase II, the molecular weight of the enzyme must be increased, otherwise the enzyme will be lost by diffusion out of the particle. Inulase II was therefore flocculated from a cell-free extract by co-crosslinking with glutardialdehyde and chitosan (Fig. 17.17). Fortunately, the enzyme is not damaged by glutardialdehyde. The crosslinked enzyme was then immobilized in alginate beads of different diameter in the range 500 to 800 lm and analyzed to compare their activity with regard to bead diameter (Fig. 17.18.) Although beads 500 lm in diameter had 54% activity compared with the value when the same beads were dissolved, beads of 600 lm had only 42% of the activity. For 850-lm beads only one third of the original activity was observed. This shows once again the benefits of using sufficiently small particles when working with encapsulated systems.

Fig. 17.17 Enzyme-immobilization: cross-linking of inulase II for molecular-weight enhancement to prevent enzyme loss by diffusion.

Fig. 17.18 Effect of bead diameter on the activity of encapsulated inulase II. Dissolved beads means that the formerly immobilized enzyme is released to prevent diffusion limitation.

17.3 Processes

a)

b) Fig. 17.19 Principle of JetCutting, and high-speed-motion picture of the cutting process showing the effect of correct adjustment.

To accomplish the task of producing the desired small droplets from the very viscous alginate–enzyme solution, a novel JetCutter technology was used. In comparison with other techniques, for example blow-off devices, vibrating nozzles or electrostatic forces, the JetCutter uses mechanical cutting of a continuous jet of liquid to produce small droplets; this is shown in Fig. 17.19 [25].

17.3.4.6 Summary Complete process development has been shown, starting from screening for an enzyme with the desired properties – an inulase II converting inulin to DFA III at elevated temperatures (60 8C). After screening and successful optimization of the enzyme by genetic engineering and construction of a genetically modified organism which expresses the enzyme in very high numbers, this strain was fermented to furnish large amounts of enzyme for immobilization. After immobilization by entrapment of the enzyme in hydrogel particles, an industrially

401

402


applicable enzyme formulation with an activity of 196 U g–1 (wet matter) is obtained. 17.3.5 Chemical Route from Sugars to Sugar Acids 17.3.5.1 Introduction The increasing use of low-molecular-mass carbohydrates (sugars) for production of chemical building blocks is of great interest both economically and ecologically. Besides the biotechnological routes presented above, chemical catalysis has great potential for functionalization of sugars to furnish fine chemicals or building blocks for the chemical industry. Sugar oxidation in the presence of supported noble metal catalysts has always been an attractive subject. The products are biodegradable compounds and have many potential applications. Gluconic acid, for example, is of great industrial interest. Annual production amounts to nearly 80 000 t; this is used as a noncorrosive and biologically degradable complexing agent in industry, and in numerous applications in food, pharmaceuticals, and cosmetics. Lactobionic acid and maltobionic acids – the oxidation products of the corresponding sugar monomers lactose and maltose, respectively – can be used in the detergent and pharmaceutical and food industries. Gluconic acid is mainly produced biotechnologically, because chemical catalysts are inadequate. With gold catalysts, this could be changed in the future (Fig. 17.20). Table 17.3 gives an overview of some important milestones in chemical catalytic glucose oxidation. The work of Prati and Rossi describes an charcoal-supported gold catalyst for glucose oxidation, which exceeds the previous Pt- and

Fig. 17.20 Chemical catalytic conversion of carbohydrates from renewable resources.

17.3 Processes Table 17.3 Milestones in the chemical catalytic oxidation of carbohydrates. Topic

Description

Working group

Beginnings

“Mannitol acid” with platinum-Mohr Oxidation of carbohydrates on platinum interpreted as oxidative dehydration

Group-Besanz (1861) Wieland (1912)

Systematic

Reactivity sequence of the moieties in carbo- Heyns and Paulsen hydrates on platinum (1950s/60s)

Bimetal catalysts

Doping of platinum and palladium catalysts with bismuth or lead (problem: long-term stability, Bi/Pb leaching)

Kuster/Bekkum (1980s)

Gold catalysts

Au/C for oxidation of glucose to gluconic acid highly active and selective (problem: long-term stability) Enhancement of long-term stability by new preparations on TiO2

Prati and Rossi (2001)

Mirescu and Prüße (2003)

Pd-based catalysts in activity and selectivity, which is nearly 100% to gluconic acid [26]. Nevertheless long-term stability is still far too low for industrial application. A breakthrough could be achieved by the use of titania- or alumina-supported gold catalysts, which combine high activity and selectivity with long-term stability, which seems sufficient for industrial applications.

17.3.5.2 Gold Catalysts For glucose oxidation a comparative study of catalysts used industrially for the same reaction shows the high selectivity of the new gold catalysts (Fig. 17.21). This gold catalyst is highly active over a wide range of pH, temperature, and glucose concentrations. Except for formation of small amounts of fructose by isomerization at temperatures above 70 8C and pH > 10, selectivity to glucose always exceeds 99.5%. These results confirm the outstanding properties of the gold catalysts in this reaction. Whereas the charcoal-supported catalysts of Prati and Rossi lose 50% activity after four replicate batches, the TiO2-supported catalysts remain stable. After 17 replicate batches no activity loss was observed. The mean activity was 425 mmolglucose gmetal–1 min–1, corresponding to an overall productivity of 5 kggluconic acid ggold–1 h–1. In addition to glucose, several other carbohydrates were tested with both TiO2 and Al2O3-supported gold catalysts. Fast and complete reaction occurs with pentoses (arabinose, ribose, xylose, and lyxose), hexoses (mannose, rhamnose, galactose, and n-acetylglucosamine), and some di- and oligosaccharides (lactose, cellobiose, melibiose, maltose, maltotriose, maltotetrose) each with a selectivity of virtually 100% (Fig. 17.22, Table 17.4). No reaction occurred with ketoses or blocked aldoses (fructose, isomaltulose, methylglucose, sorbose, sucrose, and trehalose).

403

404


Fig. 17.21 Catalytic oxidation of glucose: screening of catalysts and selectivity proven by HPLC.

Fig. 17.22 Catalytic oxidation of other carbohydrates: galactose, xylose, maltobiose and maltotriose with Au/TiO2 catalyst.

References Table 17.4 Catalytic oxidation of carbohydrates (conditions: cat: 0.6% Au/Al2O3, 40 8C, pH 9, 100 mmol substrate, conversion > 99%). Type

Substrate

Pentose (C5)

Arabinose Lyxose Ribose Xylose Galactose Hexoses (C6) Glucose N-Acetylglucose Mannose Rhamnose Disaccharides (C12) Cellobiose Lactose Maltose Melibiose

a)

Activity (mmol min–1 gMe–1)

Activity (kgproduct* h–1 gMe–1) a)

Selectivity (%)

334 145 234 251 338 165 295 172 180 282 84 177 54

4.1 1.8 2.8 3.1 4.8 2.3 4.8 2.4 2.3 6.7 1.9 4.0 1.3

> 99.9 > 99.9 > 99.9 > 99.9 > 99.9 > 99.9 > 99.9 > 99.9 > 99.9 > 99.9 >99.9 > 99.9 > 99.9

Potassium salt of acid

17.3.5.3 Summary The newly developed Au/TiO2 catalysts have outstanding properties, including pronounced substrate selectivity and extremely high product selectivity. Combination of these with very high activity and excellent long-term stability results in catalysts that meet all the requirements demanded from a “Synzyme” (synthetic enzyme). In the future, gold catalysts may be an alternative to the biotechnological process for industrial production of gluconic acid.

References 1 WVZ, Wirtschaftliche Vereinigung Zu-

5 BRDTAC, Vision for Bioenergy and Bio-

cker, Informationen zum Zuckermarkt Stand 12/2003. 2003, http://www.zuckerwirtschaft.de 2 K. Hiller and P. Kehrer, Erdöl Erdgas Kohle, 2000, 116, 9, 427. 3 BRDTAC, Roadmap for Biomass Technologies in the United States. 2002, Biomass R&D Technical Advisory Committee. 4 NRC, Biobased industrial products: priorities for research and commercialization. In: National Research Council (ed.), 2000, Washington, DC.

based Products in the United States. 2002, Biomass R&D Technical Advisory Committee. 6 OECD, Biotechnology for clean industrial products and processes, p. 30. 1998, OECD Publications, Paris Cedex. 7 Dupont, Press release: Genencor International and Dupont expand R&D collaboration to make key biobased polymer. 2001, http://www1.dupont.com/NASApp/dupontglobal/corp/index.jsp?page=/content/US/en_US/news/product/ 2001/pn03_12_01.ht ml

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17 Biocatalytic and Catalytic Routes for the Production of Bulk and Fine Chemicals 8 J. Klein and K. D. Vorlop, Immobiliza-

9

10 11

12

13

14

15 16

17

tion techniques: Cells. In: M. Moo-Young (ed), Comprehensive Biotechnology, 1985, 203–224. Pergamon Press, Oxford. EU, Directive 2003/30/EC of the European Parliament and of the Council of 8 May 2003 on the promotion of the use of biofuels or other renewable fuels for transport. Official Journal of the European Union, 2003, 46, L123, 42–47. C. Berg, World ethanol production 2001. 2001. A. Rosenberger, H. P. Kaul, T. Senn, W. Aufhammer, Costs of bioethanol production from winter cereals: the effect of growing conditions and crop production intensity levels. Industrial Crops and Products, 2002, 15, 2, 91–102. J. Zaldivar, J. Nielsen, L. Olsson, Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration. Applied Microbiology and Biotechnology, 2001, 56, 1–2, 17–34. Q. A. Nguyen, J. H. Dickow, B. W. Duff, J. D. Farmer, D. A. Glassner, K. N. Ibsen, M. F. Ruth, D. J. Schell, I. B. Thompson, M. P. Tucker, NREL/DOE ethanol pilotplant: Current status and capabilities. Bioresource Technology, 1996, 58, 2, 189– 196. H. G. Lawford and J. D. Rousseau, Cellulosic fuel ethanol – Alternative fermentation process designs with wild-type and recombinant zymomonas mobilis. Applied Biochemistry and Biotechnology, 2003, 105, 457–469. Iogen, EcoEthanol. 2003, http://www.iogen.ca/ L. O. Ingram, P. F. Gomez, X. Lai, M. Moniruzzaman, B. E. Wood, L. P. Yomano, S. W. York, Metabolic engineering of bacteria for ethanol production. Biotechnology and Bioengineering, 2002, 58, 2–3, 204-214. J. Zaldivar, A. Borges, B. Johansson, H. P. Smits, S. G. Villas-Boas, J. Nielsen, L. Olsson, Fermentation performance and intracellular metabolite patterns in laboratory and industrial xylose-fermenting Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 2002, 59, 4–5, 436–442.

18 US-Department of Energy: The DOE

19

20

21 22

23

24

25

26

ethanol pilot plant – a tool for commercialization. 2000. http://www.ott.doe.gov/ biofuels/pdfs/28397.pdf D. de Jesus and N. P. Nghiem, Student Abstracts: Chemistry at ORNL – Abstract Ethanol Production from Rice-Straw Hydrolyzate Using Zymomonas mobilis in a Continuous Fluidized-Bed Reactor (FBR). 2002, http://www.scied.science. doe.gov/scied/abstracts2000/ ornlchem.htm P. Wittlich, E. Capan, M. Schlieker, K.-D. Vorlop, U. Jahnz, Entrapment in LentiKats. In: V. A. Nedovic and R. Willaert (ed.), Fundamentals of Cell Immobilisation Biotechnology, 2004, 53–63. Focus on Biotechnology. Hofman, M. and Anné, Jozef. Kluwer Academic Publishers, Dordrecht. VDI, Ökosprit mit Makel. VDI-Nachrichten, 2004, 9, 11. S. K. Ritter, Green Reward – Presidential honors recognize innovative syntheses, process improvements, and new products that promote pollution prevention. Chemical and Engineering News, Science and Technology, 2003, 81, 26, 30–35. U. Jahnz, M. Schubert, H. Baars-Hibbe, K. D. Vorlop, Process for producing the potential food ingredient DFA III from inulin: screening, genetic engineering, fermentation and immobilisaton of inulase II. International Journal of Pharmaceutics, 2003, 256, 199–206. U. Jahnz, M. Schubert, K. D. Vorlop, Effective development of a biotechnical process: Screening, genetic engineering, and immobilization for the enzymatic conversion of inulin to DFA III on industrial scale. Landbauforschung Völkenrode, 2001, 51, 3, 131–136. U. Prüße and K. D. Vorlop, The Jetcutter technology. In: V. A. Nedovic and R. Willaert (eds.), Fundamentals of Cell Immobilisation Biotechnology, 2004, 295–309. Focus on Biotechnology. Hofman, M. and Anné, J., Kluwer Academic Publishers, Dordrecht. S. Biella, L. Prati, M. Rossi, Selective oxidation of D-glucose on gold catalyst. Journal of Catalysis, 2002, 206, 2, 242–247.

Biorefineries – Industrial Processes and Products Edited by Birgit Kamm, Patrick R. Gruber, and Michael Kamm


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Biorefineries – Industrial Processes and Products Status Quo and Future Directions Volume 2

Edited by Birgit Kamm, Patrick R. Gruber, and Michael Kamm

The Editors Dr. Birgit Kamm Research Institute Bioactive Polymer Systems biopos e.V. Kantstr. 55 14513 Teltow Germany Dr. Patrick R. Gruber President and CEO Outlast Technologies Inc. 5480 Valmont Road Boulder, CO 80301 USA Michael Kamm Biorefinery.de GmbH Stiftstr. 2 14471 Potsdam Germany

n All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library. Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Typsetting K+V Fotosatz GmbH, Beerfelden Printing Betz-Druck GmbH, Darmstadt Binding Litges & Dopf Buchbinderei GmbH, Heppenheim Printed in the Federal Republic of Germany Printed on acid-free paper ISBN-13: 978-3-527-31027-2 ISBN-10: 3-527-31027-4

V

Contents Volume 1 Part I

Background and Outline – Principles and Fundamentals

1

Biorefinery Systems – An Overview 3 Birgit Kamm, Michael Kamm, Patrick R. Gruber, and Stefan Kromus

2

Biomass Refining Global Impact – The Biobased Economy of the 21st Century 41 Bruce E. Dale and Seungdo Kim

3

Development of Biorefineries – Technical and Economic Considerations 67 Bill Dean, Tim Dodge, Fernando Valle, and Gopal Chotani

4

Biorefineries for the Chemical Industry – A Dutch Point of View 85 Ed de Jong, René van Ree Rea, Robert van Tuil, and Wolter Elbersen

Part II

Biorefinery Systems Lignocellulose Feedstock Biorefinery

5

The Lignocellulosic Biorefinery – A Strategy for Returning to a Sustainable Source of Fuels and Industrial Organic Chemicals 115 L. Davis Clements and Donald L. Van Dyne

6

Lignocellulosic Feedstock Biorefinery: History and Plant Development for Biomass Hydrolysis Raphael Katzen and Daniel J. Schell


129

VI

Contents

7

The Biofine Process – Production of Levulinic Acid, Furfural, and Formic Acid from Lignocellulosic Feedstocks 139 Daniel J. Hayes, Steve Fitzpatrick, Michael H. B. Hayes, and Julian R. H. Ross Whole Crop Biorefinery

8

A Whole Crop Biorefinery System: A Closed System for the Manufacture of Non-food Products from Cereals 165 Apostolis A. Koutinas, Rouhang Wang, Grant M. Campbell, and Colin Webb Fuel-oriented Biorefineries

9

Iogen’s Demonstration Process for Producing Ethanol from Cellulosic Biomass 193 Jeffrey S. Tolan

10

Sugar-based Biorefinery – Technology for Integrated Production of Poly(3-hydroxybutyrate), Sugar, and Ethanol 209 Carlos Eduardo Vaz Rossell, Paulo E. Mantelatto, José A. M. Agnelli, and Jefter Nascimento Biorefineries Based on Thermochemical Processing

11

Biomass Refineries Based on Hybrid Thermochemical-Biological Processing – An Overview Robert C. Brown

227

Green Biorefineries 253

12

The Green Biorefiner Concept – Fundamentals and Potential Stefan Kromus, Birgit Kamm, Michael Kamm, Paul Fowler, and Michael Narodoslawsky

13

Plant Juice in the Biorefinery – Use of Plant Juice as Fermentation Medium 295 Margrethe Andersen, Pauli Kiel, and Mette Hedegaard Thomsen

Part III

Biomass Production and Primary Biorefineries

14

Biomass Commercialization and Agriculture Residue Collection James Hettenhaus

317

Contents

15

The Corn Wet Milling and Corn Dry Milling Industry – A Base for Biorefinery Technology Developments 345 Donald L. Johnson

Part IV

Biomass Conversion: Processes and Technologies

16

Enzymes for Biorefineries 357 Sarah A. Teter, Feng Xu, Glenn E. Nedwin, and Joel R. Cherry

17

Biocatalytic and Catalytic Routes for the Production of Bulk and Fine Chemicals from Renewable Resources 385 Thomas Willke, Ulf Prüße, and Klaus-Dieter Vorlop Subjcet Index

407

Volume 2 Editor’s Preface XXIII Foreword XXV Henning Hopf Foreword XXVII Paul T. Anastas List of Contributors Part I

XXIX

Biobased Product Family Trees Carbohydrate-based Product Lines

1

1.1 1.2 1.3 1.3.1 1.3.2 1.3.3 1.3.4 1.3.5 1.3.6 1.3.7

The Key Sugars of Biomass: Availability, Present Non-Food Uses and Potential Future Development Lines 3 Frieder W. Lichtenthaler Introduction 3 Availability of Mono- and Disaccharides 4 Current Non-Food Industrial Uses of Sugars 7 Ethanol 7 Furfural 8 D-Sorbitol (: D-Glucitol) 9 Lactic Acid ? PolylacticAcid (PLA) 10 Sugar-based Surfactants 11 ‘Sorbitan’ Esters 11 N-Methyl-N-acyl-glucamides (NMGA) 12

VII

VIII

Contents

1.3.8 1.3.9 1.3.10 1.4 1.4.1 1.4.1.1 1.4.1.2 1.4.1.3 1.4.2 1.4.3 1.4.1.4 1.4.1.5 1.4.1.6 1.4.1.7 1.4.1.8 1.4.4 1.4.5 1.4.5.1 1.4.5.2 1.4.6 1.4.7 1.4.7.1 1.4.7.2 1.4.7.3 1.4.7.4 1.5

2

2.1 2.1.1 2.1.2 2.1.3 2.2 2.3 2.3.1 2.3.2 2.3.3 2.3.4

Alkylpolyglucosides (APG) 12 Sucrose Fatty Acid Monoesters 13 Pharmaceuticals and Vitamins 14 Toward Further Sugar-based Chemicals: Potential Development Lines 14 Furan Compounds 16 5-Hydroxymethylfurfural (HMF) 16 5-(Glucosyloxymethyl)furfural (GMF) 17 Furans with a Tetrahydroxybutyl Side-chain 19 Pyrones and Dihydropyranones 20 Sugar-derived Unsaturated N-Heterocycles 24 Pyrroles 24 Pyrazoles 26 Imidazoles 27 3-Pyridinols 28 Quinoxalines 28 Toward Sugar-based Aromatic Chemicals 29 Microbial Conversion of Six-carbon Sugars into Simple Carboxylic Acids and Alcohols 32 Carboxylic Acids 34 Potential Sugar-based Alcohol Commodities Obtained by Microbial Conversions 36 Chemical Conversion of Sugars into Carboxylic Acids 37 Biopolymers from Polymerizable Sugar Derivatives 40 Synthetic Biopolyesters 41 Microbial Polyesters 44 Polyamides 45 Sugar-based Olefinic Polymers (“Polyvinylsaccharides”) 47 Conclusion 49 References 51 Industrial Starch Platform – Status quo of Production, Modification and Application 61 Dietmar R. Grüll, Franz Jetzinger, Martin Kozich, Marnik M. Wastyn, and Robert Wittenberger Introduction 61 History of Starch 61 History of Industrial Starch Production 62 History of Starch Modification 62 Raw Material for Starch Production 63 Industrial Production of Starch 65 Maize and Waxy Maize 66 Wheat 66 Potato 69 Tapioca 70

Contents

2.3.5 2.4 2.5 2.5.1 2.5.1.1 2.5.1.2 2.5.1.3 2.5.1.4 2.5.2 2.5.2.1 2.5.2.2 2.5.2.3 2.6 2.6.1 2.6.1.1 2.6.1.2 2.6.2 2.6.2.1 2.6.2.2 2.6.2.3 2.6.3 2.6.4 2.6.5 2.6.6 2.6.7 2.6.8 2.7 2.7.1 2.7.2 2.7.3

Other Starches 71 Properties of Commercial Starches 71 Modification of Starch Water 76 Modification Technology 76 Slurry Process (Heterogeneous Conditions) 76 Dry Reactions 77 Paste Reactions (Homogeneous Conditions) 77 Extrusion Cooking 77 Types of Starch Modification 78 Physical Modification 78 Degraded Starches 79 Chemical Modification 80 Application of Starch and Starch Derivatives 82 The Paper and Corrugating Industries 83 Use of Starch in the Paper Industry 83 Use of Starch in the Corrugating Industry 85 The Textile Industry 85 Sizing Agents 85 Textile-printing Thickeners 86 Finishing Agents 86 Adhesives 87 Building Chemistry 87 Pharmaceuticals and Cosmetics 88 Laundry Starches 89 Bioconversion of Starch 89 Other Applications of Starch 91 Future Trends and Developments 92 Tailor-made Starches by Use of Biotechnological Tools 92 New Modification Technologies for New Properties 93 New Fields of Application 94 Bibliography 95

3

Lignocellulose-based Chemical Products and Product Family Trees 97 Birgit Kamm, Michael Kamm, Matthias Schmidt, Thomas Hirth, and Margit Schulze Introduction 97 Historical Outline of Chemical and Technical Aspects of Utilization Lignocellulose in the 19th and 20th Century 98 From the Beginnings of Lignocellulose Chemistry Until 1800 98 Lignocellulose Chemistry in the Eighteenth Century 99 Cellulose Saccharification 99 Oxalic Acid 99 Xyloidin and Nitrocellulose 99 Cellulose 100

3.1 3.2 3.2.1 3.2.2 3.2.2.1 3.2.2.2 3.2.2.3 3.2.2.4

IX

X

Contents

3.2.2.5 3.2.2.6 3.2.2.7 3.2.2.8 3.2.3 3.3 3.3.1 3.3.2 3.3.2.1 3.3.2.2 3.3.2.3 3.4 3.4.1 3.4.1.1 3.4.1.2 3.4.2 3.4.3 3.4.3.1 3.4.3.2 3.4.3.3 3.5 3.5.1 3.5.2 3.6 3.6.1 3.6.2 3.6.2.1 3.6.2.2 3.6.3 3.6.3.1 3.6.3.2 3.7 3.7.1 3.7.2 3.7.2.1 3.7.2.2 3.7.2.3 3.7.2.4 3.7.2.5 3.7.2.6 3.7.2.7 3.7.3 3.8

Levulinic Acid 100 Lignin 101 Hemicellulose (Polyoses) and Furfural 101 Lignocellulose 102 Industrial Lignocellulose Utilization in the 19th and Beginning of the 20th Century 102 Lignocellulosic Raw Material 103 Definition 103 Sources and Composition 105 Sources 105 Chemical Composition of Lignocelluloses 106 Carbohydrates in Lignocelluloses 108 Lignocelluloses in Biorefineries 110 Background 110 Example 1 110 Example 2 110 LCF Biorefinery 111 LCF Conversion Methods 113 Pretreatment Methods 113 Chemical Pulping Methods 114 Enzymatic Methods 115 Lignin-based Product Lines 116 Isolation and Application Areas 116 A Lignin-based Product Family Tree 117 Hemicellulose-based Product Lines 119 Isolation and Application Areas 119 A Hemicellulose-based Product Family Tree 119 Mannan/Mannose Product Lines 119 Xylan/Xylose Product Line 120 Furfural and Furfural-based Products 122 Furfural 122 A Furfural-based Family Tree 127 Cellulose-based Product Lines 127 Isolation, Fractionation and Application Areas 127 Cellulose-based Key Chemicals 128 Glucose 128 Sorbitol 129 Glucosides 130 Fructose 131 Ethanol 132 Hydroxymethylfurfural 133 Levulinic Acid 134 An HMF and Levulinic Acid-based Family Tree 135 Outlook and Perspectives 138 References 139

Contents

Lignin Line and Lignin-based Product Family Trees 4 4.1 4.2 4.3 4.3.1 4.3.2 4.3.3 4.3.3.1 4.3.3.2 4.4 4.4.1 4.4.2 4.4.3 4.4.4

5 5.1 5.2 5.2.1 5.2.2 5.2.3 5.3 5.3.1 5.3.1.1 5.3.1.2 5.3.1.3 5.3.2 5.3.2.1 5.3.2.2 5.3.3 5.3.4 5.3.5 5.4 5.4.1 5.4.2 5.5 5.5.1 5.5.2

Lignin Chemistry and its Role in Biomass Conversion Gösta Brunow Introduction 151 Historical Overview 152 The Structure of Lignin 152 Definition 152 The Bonding of the Phenylpropane Units 153 Bonding Patterns and Functional Groups 156 General 156 Survey of Different Types of Lignin Unit 156 Role of Lignin in Biomass Conversion 159 Introduction 159 Low-molecular-weight Chemicals from Lignin 160 Polymeric Products 160 Biodegradation 160 References 160

151

Industrial Lignin Production and Applications 165 E. Kendall Pye Introduction 165 Historical Outline of Lignin Production and Applications 168 Lignosulfonates from the Sulfite Pulping Industry 168 Lignin from the Kraft Pulping Industry 169 Lignin from the Soda Pulping Industry 170 Existing Industrial Lignin Products 172 Lignosulfonates 172 Chemical Characteristics of Lignosulfonates 172 Lignosulfonate Producers 173 Markets for Lignosulfonates 174 Kraft Pulping and Kraft Lignin Recovery 175 Producers of Kraft Lignin 175 Markets for Kraft Lignin 175 Lignins Produced from the Soda Process 176 Lignin from Other Biomass Processing Operations 176 Comparisons of the Physical and Chemical Properties of Commercially Available Lignins 176 Lignin from Biorefineries 177 Advantages of Lignin and Hemicellulose Removal on Saccharification and Fermentation of Cellulose 177 Lignin from an Organosolv Biorefinery 179 Applications and Markets for Lignin 181 Phenol–Formaldehyde Resin Applications 181 The Potential Use of Biorefinery Lignin in Phenolic Resins 181

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5.5.3 5.5.4 5.5.5 5.5.6 5.5.7 5.5.8 5.5.9 5.5.10 5.5.11 5.6 5.6.1 5.6.2 5.6.3 5.7 5.7.1 5.7.2 5.7.3 5.7.4 5.7.5 5.8 5.8.1 5.8.2 5.8.3 5.8.4 5.9

Panelboard Adhesives 183 Thermoset Resins for Molded Products 184 Friction Materials 184 Foundry Resins 184 Insulation Materials 185 Decorative Laminates 185 Panel and Door Binders 185 Rubber Processing 186 The Opportunity for Lignin in Phenol–Formaldehyde Resin Markets 187 Lignin as an Antioxidant 187 Antioxidants in Animal Feed Supplements 188 Antioxidants in the Rubber Industry 188 Antioxidants in the Lubricants Industry 188 Applications for Water-soluble, Derivatized Lignins 189 Concrete Admixtures 189 Dye Dispersants 190 Asphalt Emulsifiers 192 Agricultural Applications 192 Dispersants for Herbicides, Pesticides and Fungicides 193 New and Emerging Markets for Lignin 194 Printed Circuit Board Resins 194 Animal Health Applications 195 Animal Feed Supplement 196 Carbon Fibers for Mass-produced Vehicles 196 Conclusions and Perspectives 198 References 199 Protein Line and Amino Acid-based Product Family Trees

6

6.1 6.2 6.2.1 6.2.2 6.2.3 6.3 6.4 6.4.1 6.4.2 6.4.3

Towards Integration of Biorefinery and Microbial Amino Acid Production 201 Achim Marx, Volker F. Wendisch, Ralf Kelle, and Stefan Buchholz Introduction 201 Present State of the Industry 202 Microbial Amino Acid Production 202 Biorefinery and the Building-block Concept 202 Metabolic Engineering and the Building-block Concept 204 Environmental and Commercial Consideration of Microbial Amino Acid Production Integrated in a Biorefinery 205 Technical Constraints for Integration of Microbial Amino Acid Fermentation into a Biorefinery 209 Mono-septic Operation 209 Carbon Sources 209 Nitrogen Source 211

Contents

6.4.4 6.4.5 6.4.6 6.4.7 6.5

Phosphorus Source 211 Mixing and Oxygen Supply 212 Toxicity 212 Cultivation Temperature 213 Outlook and Perspectives 213 Acknowledgment 214 References 215

7

Protein-based Polymers: Mechanistic Foundations for Bioproduction and Engineering 217 Dan W. Urry Introduction 217 Definitions 217 Proteins and Protein-based Polymers 217 Two Basic Principles for Protein-based Polymer Engineering 217 Proteins in Aqueous Media 218 Thermodynamics of Proteins in Water 218 Exothermic Hydration of Apolar Groups 218 The Change in Gibbs Free Energy of Hydrophobic Association 218 The Apolar–Polar Repulsive Free Energy of Hydration, DG8ap 218 The Inverse Temperature Transition for Hydrophobic Association 219 The Role of Elasticity in the Engineering of Protein-based Polymers 219 Near Ideal Elasticity Provides for Efficient Energy Conversion 219 Mechanism of Near Ideal Elasticity 220 Many of the Advantages of Protein-based Polymeric Materials 220 Historical Outline 221 Historical Beginnings of (Elastic) Protein-based Polymer Development 221 Mechanistic Foundations: Fundamental Engineering Principles 222 The Hydrophobic Consilient Mechanism 222 The Elastic Consilient Mechanism 223 Highlights of Bioproduction 223 Bioproduction 224 Gene Construction using Recombinant DNA Technology 225 Preparation of Monomer Genes and the PCR Technique 225 Transformation, Monomer Gene Production and Sequence Verification 226 Monomer Gene Concatenation Produces Multimer Genes of Monomer 226 E. coli Transformation for Protein-based Polymer Expression 227 Fermentation using Transformed E. coli 227 Purification of Protein-based Polymers 227

7.1 7.1.1 7.1.1.1 7.1.1.2 7.1.2 7.1.3 7.1.3.1 7.1.3.2 7.1.3.3 7.1.4 7.1.5 7.1.5.1 7.1.5.2 7.1.6 7.2 7.2.1 7.2.2 7.2.2.1 7.2.2.2 7.2.3 7.3 7.3.1 7.3.1.1 7.3.1.2 7.3.1.3 7.3.2 7.3.3 7.4

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Contents

7.4.1 7.4.1.1 7.4.1.2 7.4.2 7.4.2.1 7.4.2.2 7.4.2.3 7.4.3 7.4.3.1 7.4.3.2 7.4.3.3 7.4.3.4 7.4.3.5 7.4.3.6 7.5 7.5.1 7.5.2 7.5.2.1 7.5.2.2 7.5.2.3 7.5.2.4 7.5.2.5 7.5.3 7.6 7.6.1 7.6.1.1 7.6.1.2 7.6.1.3 7.6.2 7.6.2.1

Use of the Inverse Temperature Transition as a Method of Purification 228 Purification by Phase Separation as Demonstrated by SDS–PAGE 228 Purification by Phase Separation Shown by Carbon-14-labeled E. coli 228 Physical Characterization and Verification of Product Integrity 229 Gross Visualization of the Phase Separated Product 229 Sequence Integrity and Purity Evaluated by Nuclear Magnetic Resonance 229 Mass Spectra Reaffirm Size of Expressed Polymer 229 Biocompatibility 230 The Challenge of Using E. coli-produced Protein as a Biomaterial 230 Removal of Endotoxins and Determination of Levels 230 Western Immunoblot Technique to Demonstrate Level of Purity 230 Western Immunodotblot Technique to Demonstrate Medical Grade Purity 231 Subcutaneous Injection in the Guinea-pig 231 ASTM Tests 232 Mechanistic Foundations for Engineering Protein-based Polymers 232 Phenomenological Axioms 232 The Change in Gibbs Free Energy for Hydrophobic Association, DGHA 232 The Change in Gibbs Free Energy Attending a Phase Transition, dDGt(v) 234 The DGHA-based Hydrophobicity Scale for Amino Acid Residues 234 DG8HA-based Hydrophobicity Scale of Prosthetic Groups, etc. 235 Comprehensive Hydrophobic Effect: dGHA Responds to all Variables 237 The Apolar–Polar Repulsive Free Energy of Hydration, DGap 237 The Coupling of Hydrophobic and Elastic Mechanisms 237 Examples of Applications 238 Soft Tissue Restoration 238 Prevention of Post-surgical Adhesions 238 Soft Tissue Augmentation 238 Soft Tissue Reconstruction: The Concept of Temporary Functional Scaffoldings 239 Controlled Release Devices for Amphiphilic Drugs and Therapeutics 240 The Use of DGap in the Design of Controlled-release Devices 240

Contents

7.6.2.2 7.6.3 7.6.4 7.6.5 7.7 7.7.1 7.7.2 7.8 7.8.1 7.8.2

Prevention of Pressure Ulcers by Means of Elastic Patches for Drug Delivery 240 Fibers of Improved Elastic Moduli and Break Stresses and Strains 241 Programmably Biodegradable Thermoplastics 241 Acoustic Absorption 242 Outlook and Perspectives 242 List of Gene Constructions and Expressed Protein-based Polymers 242 Efforts Toward Low-cost Production in other Microbes and in Plants 242 Patents 245 Patents of D.W. Urry on Protein-based Polymers 245 Result of Ex Parte Patent Reexamination Request to the USPTO 245 Acknowledgment 249 References 249 Biobased Fats (Lipids) and Oils

8

8.1 8.2 8.2.1 8.2.1.1 8.2.1.2 8.2.1.3 8.2.2 8.2.3 8.2.4 8.2.5 8.2.5.1 8.2.5.2 8.2.5.3 8.2.6 8.2.7 8.3 8.3.1 8.3.1.1 8.3.1.2 8.3.2

New Syntheses with Oils and Fats as Renewable Raw Materials for the Chemical Industry 253 Ursula Biermann, Wolfgang Friedt, Siegmund Lang, Wilfried Lühs, Guido Machmüller, Jürgen O. Metzger, Mark Rüsch gen. Klaas, Hans J. Schäfer, Manfred P. Schneider Introduction 253 Reactions of Unsaturated Fatty Compounds 254 Oxidations 254 New Methods for the Epoxidation of Unsaturated Fatty Acids 254 Oxidation to vic-Dihydroxy Fatty Acids 257 Oxidative Cleavage 258 Transition Metal-Catalyzed Syntheses of Aromatic Compounds 259 Olefin Metathesis 259 Pericyclic Reactions 260 Radical Additions 261 Solvent-Free, Copper-Initiated Additions of 2-Halocarboxylates 262 Addition of Perfluoroalkyl Iodides 263 Thermal Addition of Alkanes 264 Lewis Acid-Induced Cationic Addition 264 Nucleophilic Addition to Reversed-Polarity Unsaturated Fatty Acids 265 Reactions of Saturated Fatty Compounds 266 Radical C–C Coupling 266 Oxidative Coupling of C2 Anions of Fatty Acids 266 Anodic Homo- and Heterocoupling of Fatty Acids (Kolbe Electrolysis) 267 Functionalization of C–H Bonds 269

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8.3.2.1 8.3.2.2 8.4 8.4.1 8.4.1.1 8.4.1.2 8.4.2 8.4.2.1 8.4.2.2 8.4.3 8.5 8.5.1 8.5.2 8.5.3 8.5.3.1 8.5.3.2 8.5.3.3 8.5.3.4 8.5.3.5 8.5.4 8.6

9

9.1 9.2 9.3 9.4 9.4.1 9.4.1.1 9.4.1.2 9.4.2 9.4.3 9.4.3.1 9.4.3.2 9.4.3.3 9.4.3.4 9.4.3.5 9.4.3.6

Oxidation of Nonactivated C–H Bonds 269 Oxidation of Allylic C–H Bonds 269 Enzymatic Reactions 270 Lipase Catalyzed Transformations 270 Lipase-Catalyzed Syntheses of Monoglycerides and Diglycerides 270 Lipase-Catalyzed Syntheses of Carbohydrate Esters 272 Microbial Transformations 272 Microbial Hydration of Unsaturated Fatty Acids 272 Microbial x- and b-Oxidation of Fatty Acids 273 Microbial Conversion of Oils/Fats and Glucose into Glycolipids 274 Improvement in Natural Oils and Fats by Plant Breeding 275 Gene Technology as an Extension of the Methodological Repertoire of Plant Breeding 275 New Oil Qualities by Oil Designed with Available Agricultural Varieties 276 Overview of Renewable Raw Materials Optimized by Breeding 277 Soybean 277 Rapeseed 277 Sunflower 280 Peanut 281 Linseed 281 Concluding Remarks on the Use of Gene Technology 281 Future Prospects 282 Acknowledgments 282 References 282 Industrial Development and Application of Biobased Oleochemicals 291 Karlheinz Hill Introduction 291 The Raw Materials 292 Ecological Compatibility 293 Examples of Products 294 Oleochemicals for Polymer Applications 295 Dimerdiols Based on Dimer Acid 297 Polyols Based on Epoxides 298 Biodegradable Fatty Acid Esters for Lubricants 299 Surfactants and Emulsifiers Derived from Vegetable Oil 301 Fatty Alcohol Sulfate (FAS) 303 Acylated Proteins and Amino Acids (Protein–Fatty Acid Condensates) 304 Carbohydrate-based Surfactants – Alkyl Polyglycosides 305 Alkyl Polyglycoside Carboxylate 307 Polyol Esters 307 Multifunctional Care Additives for Skin and Hair 309

Contents

9.4.4 9.4.4.1 9.4.4.2 9.4.4.3 9.5 9.6

Emollients 310 Introduction 310 Dialkyl Carbonate 311 Guerbet Alcohols 311 Perspectives 312 Trademarks 312 References 312 Special Ingredients and Subsequent Products

10 10.1 10.2 10.3 10.3.1 10.3.2 10.3.3 10.3.4 10.3.5 10.3.6 10.3.7 10.4

11

11.1 11.2 11.3 11.3.1 11.3.2 11.3.3 11.4 11.4.1 11.4.2 11.4.3 11.5 11.6 11.7

Phytochemicals, Dyes, and Pigments in the Biorefinery Context George A. Kraus Introduction 315 Historical Outline 316 Phytochemicals from Corn and Soybeans 317 Phytosterols 317 Lecithin 318 Tocopherols 319 Carotenoids 320 Phytoestrogens 321 Saponins 321 Protease Inhibitors 322 Outlook and Perspectives 323 References 323 Adding Color to Green Chemistry? An Overview of the Fundamentals and Potential of Chlorophylls Mathias O. Senge and Julia Richter Introduction 325 Historical Outline 325 Chlorophyll Fundamentals 326 Occurrence and Basic Structures 326 Principles of Chlorophyll Chemistry 327 Isolation of Chlorophylls 328 Chlorophyll Breakdown and Chemical Transformations 330 Biological Chlorophyll Catabolism 330 Geological Chlorophyll Degradation – Petroporphyrins 331 Chemical Degradation of Chlorophylls 333 Industrial Uses of Chlorophyll Derivatives 335 A Look at “Green” Chlorophyll Chemistry 337 Outlook and Perspectives 339 Acknowledgment 341 References and Notes 341

315

325

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Part II

Biobased Industrial Products, Materials and Consumer Products

12

Industrial Chemicals from Biomass – Industrial Concepts 347 Johan Thoen and Rainer Busch Introduction 347 Historical Outline 347 Basic Principles 349 Primary Conversion Technologies of Biomass 350 Gasification 350 Hydrothermolysis 351 Fermentation to Ethanol 351 Current Status 351 Europe 351 United States 352 Products 353 Industrial Concepts 354 Introduction 354 Biorefinery Concepts 355 Classes of Bioproduct 356 Opportunities for Industrial Bioproducts 357 Product Categories Based on C6-Carbon Sugars to Bioproducts 358 Product Categories Based on C5-Carbon Sugars to Bioproducts 358 Thermochemical Conversion of Sugars to Bioproducts 360 Thermochemical Conversion of Oils and Lipid Based Bioproducts 361 Bioproducts via Gasification 361 Bioproducts via Pyrolysis 362 Biocomposites 362 Outlook and Perspectives 362 References 364

12.1 12.2 12.3 12.3.1 12.3.1.1 12.3.1.2 12.3.1.3 12.4 12.4.1 12.4.2 12.4.3 12.5 12.5.1 12.5.2 12.5.3 12.5.4 12.5.5 12.5.6 12.5.7 12.5.8 12.5.9 12.5.10 12.5.11 12.6

13

13.1 13.2 13.3 13.4 13.5 13.5.1 13.6 13.6.1 13.6.2 13.7

Succinic Acid – A Model Building Block for Chemical Production from Renewable Resources 367 Todd Werpy, John Frye, and John Holladay Introduction 367 Economics of Feedstock Supply 368 Succinic Acid Fermentation 369 Succinic Acid Catalytic Transformations 372 Current Petrochemical Technology 373 1,4-BDO, THF, GBL, and NMP 373 Current Biobased Technology 375 1,4-BDO, GBL, and NMP 375 Derivatives of Diammonium Succinate 376 Conclusions 378 References 378

Contents

14 14.1 14.2 14.2.1 14.2.1.1 14.2.1.2 14.2.2 14.2.2.1 14.2.2.2 14.2.2.3 14.2.2.4 14.2.3 14.2.3.1 14.2.3.2 14.2.4 14.2.4.1 14.2.4.2 14.3 14.3.1 14.4 14.5 14.5.1 14.5.2 14.5.3 14.5.3.1 14.5.3.2 14.5.3.3 14.5.3.4 14.6 14.7 14.8 14.9 14.10

15 15.1 15.1.1 15.1.2

Polylactic Acid from Renewable Resources Patrick Gruber, David E. Henton, and Jack Starr 381 Introduction 381 Lactic Acid 382 Lactic Acid Production Routes 382 Chemical Synthesis 382 Fermentation 383 Production by Fermentation 384 Microorganisms 384 Sugar Feedstock 385 Nutrients 385 Neutralizing Agent 385 Acidification 386 Strong Acid Addition 386 Salt Splitting Technology 387 Purification 388 Cell Removal 388 Separation of Residual Sugars, Nutrients and Fermentation By-products 388 PLA Production 390 Polymerization of Lactide 392 Control of Crystalline Melting Point 394 Rheology Control by Molecular Weight and Branching 396 Melt Rheology of Linear PLA 397 Melt Rheology of Branched PLA 397 Branching Technology 398 Multi-functional Polymerization Initiators 398 Hydroxy Cyclic Ester and/or Carbonate Polymerization Initiators 398 Multi-cyclic Ester, Multi-cyclic Carbonate and/or Multi-cyclic Epoxy Comonomers 398 Free Radical Cross-linking 399 Melt Stability 399 Applications and Performance 400 PLA Stereocomplex 401 Fossil Resource Use and Green House Gases 402 Summary 402 Abbreviations 403 References 404 Biobased Consumer Products for Cosmetics Thomas C. Kripp Introduction and Historical Outline 409 Cosmetics Past and Present 409 Bionics: Learning from Nature 410

409

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15.2 15.2.1 15.2.2 15.2.3 15.2.4 15.2.5 15.2.6 15.3 15.3.1 15.3.2 15.3.3 15.3.3.1 15.3.3.2 15.3.4 15.3.5 15.4 15.4.1 15.4.2 15.4.3 15.4.4 15.4.4.1 15.4.4.2 15.4.5 15.5 15.5.1 15.5.2 15.5.3 15.5.4 15.5.5 15.5.6 15.5.6.1 15.5.6.2 15.5.7 15.5.8 15.6 15.6.1 15.6.2 15.6.3 15.6.3.1 15.6.3.2 15.6.3.3 15.6.4

Betaine, The Conditioner Made from Sugar Beet 410 Occurrence 410 Chemical Properties 411 Production 411 Use and Fields of Application 412 Innovation Through Combination: Betaine Esters 414 Summary and Prospects 415 Chitosan, Hair-setting Agent from the Ocean 415 Chitin, a Precursor of Chitosan 415 Occurrence of Chitin 415 Production 416 Purification of Chitin 416 Production of Chitosan 417 Chitosan in cosmetic products 419 Summary and Prospect 421 From Energy Reserve to Shampoo Bottle: Biopol 422 Biodegradable Packages 422 What is “Biopol”? 423 Biodegradability of Biopol 424 The Long Way to the Shampoo Bottle 426 Product Development 426 Market Launch 427 Quo vadis, Biopol? 428 Natural Apple-peel Wax: Protection for Hair and Skin 429 Raw Material Source 429 Apple-peel Wax 430 Observations 430 Production of Apple-peel Wax 432 Chemical Composition 433 Mode of Action and Uses 433 Skin Cosmetics 434 Hair Care 434 Market Launch 436 Summary and Prospects 436 Ilex Resin: From Shiny Leaves to Shiny Hair 437 Holly 437 Extraction of a Resin Fraction 438 Effects in Cosmetics 439 Skin Care 439 Hair Care 439 Styling 440 Summary and Prospects 440 References 441

Contents

Part III

Biobased Industry: Economy, Commercialization and Sustainability

16

Industrial Biotech – Setting Conditions to Capitalize on the Economic Potential 445 Rolf Bachmann and Jens Riese Introduction 445 Time to Exploit the Potential 446 How Far Can it Go? 446 Better Technology, Faster Results 447 Environmentally and Balance-sheet Friendly 448 Rekindling Chemicals Innovation 450 Increasing Corporate Action in all Segments 451 The Importance of Residual Biomass 452 Why Waste Biomass Works 452 Economic Benefits and Regulation 452 Still a Long Way to Go 454 Collaboration Will Push Biomass Conversion Forward 454 Overcoming the Challenges Ahead 455 Internal Obstacles 455 External Challenges 456 Overcoming Challenges 457 Case 1: Building a Biotech Strategy 457 Case 2: Identifying the Right Opportunities 458 Case 3: Managing Uncertainties 459 Case 4: Preparing the Launch and Market Development 460 Case 5: Building a Favorable External Environment 461 More Needs to be Done 461

16.1 16.2 16.2.1 16.2.2 16.2.3 16.2.4 16.2.5 16.3 16.3.1 16.3.2 16.3.3 16.3.4 16.4 16.4.1 16.4.2 16.5 16.5.1 16.5.2 16.5.3 16.5.4 16.5.5 16.6

Subject Index

463

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Editor’s Preface In the year 2003 when the idea for this set of books “Biorefineries, Biobased Industrial Processes, and Products” arose, the topic of biorefineries as means of processing industrial material and efficient utilization of renewable products had been primarily a side issue beyond the borders of the United States of America. This situation has changed dramatically over the last two years. Today in almost every developed and emerging nation much work is being conducted on biorefinery systems, driven by the rising cost of oil and the desire of to move away from petrochemical-based systems. In these books we do not claim to describe and discuss everything that belongs or even might belong to the topic of biorefineries – that would be impossible. There are many types of biorefinery, and the state of the technology is changing very rapidly as new and focused effort is directed toward making biorefineries a commercial reality. It is a very exciting time for those interested in biorefineries – technologies for bio-conversion have advanced to a state in which they are becoming practical on a large scale, economics are leaning more favourably to the direction of renewable feedstocks, and chemical process knowledge is being applied to biobased systems. As the editors of the first comprehensive biorefinery book we saw it as our duty to provide, first of all, a general framework for the subject – addressing the main issues associated with biorefineries, the principles and basics of biorefinery systems, the basic technology, industrial products which fall within the scope of biorefineries, and, finally, technology and products that will fall within the scope of biorefineries in the future. To provide a reliable description of the state of biorefinery research and development and of industrial implementations, strategies, and future developments we asked eighty-five experts from universities, research and development institutes, and industry and commerce to present their views, their results, their implementations, and their ideas on the topic. The results of their contributions are thirty-three articles organized into seven sections. Our very special thanks go to all the authors. We are especially indebted to Dr. Hubert Pelc from Wiley-VCH publishing, who worked with us on the concept and then, later, on the development and implementation of the book. Thanks go also to Dr. Bettina Bems from Wiley-


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Editor’s Preface

VCH publishing, who managed with admirable professionalism and very much patience, and to the three editors and eighty-five authors from three different continents. We are also indebted to Hans-Jochen Schmitt, also of Wiley-VCH publishing, who had the not always easy task of arranging the manuscripts in a form ready for publication. Maybe in 2030, when a biobased economy utilizing biorefinery technology has become a fundamental part of national and globally connected economies, someone will wonder what had been thought and written about the subject of biorefineries at the beginning of the 21st century. Hopefully this book will be highly representative. Until then we hope it will contribute to the promotion of international biorefinery developments. Teltow-Seehof (Germany) Boulder, CO (USA) Potsdam (Germany) November 2005

Birgit Kamm Patrick R. Gruber Michael Kamm

XXV

Foreword One-hundred-and-fifty years after the beginning of coal-based chemistry and 50 years after the beginning of petroleum-based chemistry industrial chemistry is now entering a new era. In the twenty-first century utilization of renewable raw materials will gain importance in the chemical conversion of substances in industry. Partial or even complete re-adjustment of whole economies to renewable raw materials will require completely new approaches in research, development, and production. Chemical and biological sciences will play a leading role in the building of future industries. New synergies between biological, physical, chemical, and technical sciences must be elaborated and established and special requirements will be placed on raw material and on product-line efficiency and sustainability. The necessary change from chemistry based on a fossil raw material to biology-based modern science and technology is an intellectual challenge for both researchers and engineers. Chemists should support this change and collaborate closely with their colleagues in adjoining disciplines, for example biotechnology, agriculture, forestry, and the material sciences. The German Chemical Society will help direct this necessary development by supporting within its structure new kinds of organization for chemists to work on this subject in universities, research institutes, and industry. This two-volume book is based on the approach developed by biorefinery-systems – transfer of the logic and efficiency of today’s petrochemical product lines and product family trees into manipulation of biomass. Raw biomass materials are mechanically separated into substances for chemical conversion into other products by different methods, which may be biotechnological, thermochemical, and thermal. Review of biomass processes and products developed in the past but widely forgotten in the petroleum age will be as important as the presentation of new methods, processes, and products that still require an enormous amount of research and development today. Henning Hopf President of the German Chemical Society Frankfurt (Germany) November 2005


XXVII

Foreword On October 5, 2005, the Nobel Prize Committee made an interesting and important statement with regard to the prize in chemistry. It said, “This represents a great step forward for ‘green chemistry’, reducing potentially hazardous waste through smarter production. [This research] is an example of how important basic science has been applied for the benefit of man, society and the environment.” By making this statement, the Nobel committee recognized what a new generation of scientists has known for quite some time, that by working at the most fundamental level – the molecular level – we are able to design our products, processes, and systems in ways that are sustainable. There is general recognition that the current system by which we produce the goods and services needed by society is not sustainable. This unsustainability takes many forms. It would be legitimate to note that in our current system of production we rely largely on finite feedstocks extracted from the Earth that are being depleted at a rate that cannot be sustained indefinitely. It is equally legitimate to recognize that our current production efficiency results in more than 90% of the material used in the production process ending up as waste, i.e. less than 10% of the material ends up in the desired product. Yet another condition of unsustainability is in our current energy use; this not only relies largely on finite energy sources but also results in degradation of the environment that cannot be continued as the growing population and demands of the developing world emerge over the course of the twenty-first century. Finally, the products and processes we have designed since the industrial revolution have accomplished their goals without full consideration of their impact and consequence on humans and the biosphere, with many examples of toxic and hazardous substances being distributed throughout the globe and into our bodies. If we are to change this unsustainable path, it will need the direct and committed engagement of our best scientists and engineers to design the future differently from the past. We will need to proceed with a broader perspective such that when we design for efficiency, effectiveness, and performance, we now must recognize that these terms include sustainability – a minimized impact on humans and the environment. An essential part of meeting the challenge of designing for sustainability will be based on the nature of the materials we use as starting materials and feedstocks. Any sustainable future must ensure that the materials on which we base Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 2 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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our economic infrastructure are renewable rather than depleting. The rate of renewability is also important because certainly one could argue that petroleum is renewable if you have a few million years to wait. Serious analysis would, however, necessitate that the rate of renewability is connected to the rate of use. There are options for how to approach this technological challenge, for example using waste products from one process as a feedstock for another, that are well thought through in industrial ecology models. There is, however, recognition that an essential part of a sustainable future will be based on appropriate and innovative uses of our biologically-based feedstocks. This book addresses the essential questions and challenges of moving toward a sustainable society in which bio-based feedstocks, processes, and products are fundamental pillars of the economy. The authors discuss not only the important scientific and technical issues surrounding this transition but also the necessary topics of economics, infrastructure, and policy. It is only by means of this type of holistic approach that movement toward genuine sustainability will be able to occur where the societal, economic, and environmental needs are met for the current generation while preserving the ability of future generations to meet their needs. While it will be clear to the reader that the topics presented in this book are important, it is at least as important that the reader understand that these topics – and the transition to a sustainable path that they address – are urgent. At this point in history it is necessary that all who are capable of advancing the transition to a more sustainable society, engage in doing so with the level of energy, innovation, and creativity that is required to meet the challenge. Paul T. Anastas Director of the Green Chemistry Institute Washington, D.C. November, 2005

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List of Contributors (Volume 1 and 2) José A. M. Agnelli Universidade Federal de São Carlos Departamento de Engenharia de Materiais Rodovia Washington Luis (SP-310) São Carlos, São Paulo Brazil

Robert C. Brown Center for Sustainable Environmental Technologies Iowa State University 286 Metals Development Building Ames, IO 50011 USA

Margrethe Andersen AgroFerm A/S Limfjordsvej 4 6715 Esbjerg N Denmark

Gösta Brunow Department of Chemistry University of Helsinki A. I. Virtasen aukio 1 00014 Helsinki Finland

Rolf Bachmann McKinsey and Company Inc Zurich Office Alpenstrasse 3 8065 Zürich Switzerland Ursula Biermann Fachbereich Chemie Carl von Ossietzky Universität Oldenburg Postfach 2603 26111 Oldenburg Germany

Stefan Buchholz Degussa AG Creavis Projecthouse ProFerm Rodenbacher Chaussee 4 63403 Hanau-Wolfgang Germany Rainer Busch Dow Deutschland GmbH & Co. OHG Industriestrasse 1 77836 Rheinmünster Germany


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Grant M. Campbell Satake Centre for Grain Process Engineering School of Chemical Engineering and Analytical Science The University of Manchester Sackville Street Manchester M60 1QD UK Joel R. Cherry Novozymes Biotech Inc 1445 Drew Ave Davis, CA 95616 USA Gopal Chotani Genencor International 925 Page Mill Road Palo Alto, CA 94304 USA L. Davis Clements Renewable Products Development Laboratories 3114 NE 45th Ave. Portland, OR 97213 USA Bruce E. Dale Department of Chemical Engineering and Materials Science Michigan State University East Lansing, MI 48824 USA Bill Dean Genencor International 925 Page Mill Road Palo Alto, CA 94304 USA

Tim Dodge Genencor International 925 Page Mill Road Palo Alto, CA 94304 USA Donald L. Van Dyne Agricultural Economics University of Missouri – Columbia 214c Mumford Hall Columbia, MO 65211 USA Wolter Elbersen Agrotechnology and Food Innovations B. V. P.O. Box 17 6700 AA Wageningen The Netherlands Steve Fitzpatrick Biofine 245 Winter Street Waltham, MA 02154 USA Paul Fowler The BioComposites Centre University of Wales Bangor Gwynedd LL57 2UW UK Wolfgang Friedt Institut für Pflanzenbau und Pflanzenzüchtung 1 Justus-Liebig-Universität Giessen Heinrich-Buff-Ring 26–32 35392 Giessen Germany


John Frye Pacific Northwest National Laboratory P.O. Box 999/K2-12 Richland, WA 99352 USA

James R. Hettenhaus CEA Inc 3211 Trefoil Drive Charlotte, NC 28226 USA

Patrick R. Gruber President and CEO Outlast Technologies Incorporated 5480 Valmont Road Suite 200 Boulder, CO 80301 USA

Karlheinz Hill Cognis Deutschland GmbH & Co. KG Paul-Thomas-Straße 56 40599 Düsseldorf Germany

Dietmar R. Grüll Südzucker Aktiengesellschaft Mannheim/Ochsenfurt Wormser Strasse 11 67283 Obrigheim/Pfalz Germany Daniel J. Hayes Department of Chemical & Environmental Sciences University of Limerick Limerick Ireland Michael H. B. Hayes Department of Chemical & Environmental Sciences University of Limerick Limerick Ireland David E. Henton Nature Works LLC (former Cargill Dow LLC) 15305 Minnetonka Blvd Minnetonka, MN 55345 USA

Thomas Hirth Fraunhofer-Institut Chemische Technologie Joseph-von-Fraunhoferstraße 7 76327 Pfinztal Germany John Holladay Pacific Northwest National Laboratory P.O. Box 999/K2-12 Richland, WA 99352 USA Franz Jetzinger Zuckerforschung Tulln Gesellschaft mbH Josef-Reither-Strasse 21–23 3430 Tulln Austria Donald L. Johnson Biobased Industrial Products Consulting 29 Cape Fear Drive Hertford, NC 27944 USA Ed de Jong Agrotechnology and Food Innovations B.V. P.O. Box 17 6700 AA Wageningen The Netherlands

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Birgit Kamm Research Institute Bioactive Polymer Systems (biopos e.V.) Research Centre Teltow-Seehof Kantstraße 55 14513 Teltow Germany Michael Kamm Biorefinery.de GmbH Stiftstraße 2 14471 Potsdam Germany and Laboratories Teltow Kantstraße 55 14513 Teltow Germany Raphael Katzen 9220 Bonita Beach Road Suite 2000 Bonita Springs, FL 34135 USA Ralf Kelle Degussa AG R & D Feed Additives Kantstrasse 2 33790 Halle/Westfalen Germany Pauli Kiel Biotest Aps Gl. Skolevej 47 6731 Tjæreborg Denmark Seungdo Kim Department of Chemical Engineering and Materials Science Michigan State University East Lansing, MI 48824 USA

Apostolis A. Koutinas Satake Centre for Grain Process Engineering School of Chemical Engineering and Analytical Science The University of Manchester Sackville Street Manchester M60 1QD UK Martin Kozich Zuckerforschung Tulln Gesellschaft mbH Josef-Reither-Strasse 21–23 3430 Tulln Austria George A. Kraus Department of Chemistry Iowa State University 1605 Gilman Hall Ames, IA 50011-3111 USA Thomas C. Kripp Wella AG Abt. FON Berliner Allee 65 64274 Darmstadt Germany Stefan Kromus BioRefSYS-BioRefinery Systems Innovationszentrum Ländlicher Raum Auersbach 130 8330 Feldbach Austria


Siegmund Lang Institut für Biochemie und Biotechnologie Technische Universität zu Braunschweig Spielmannstraße 7 38106 Braunschweig Germany

Achim Marx Degussa AG Creavis Projecthouse ProFerm Rodenbacher Chaussee 4 63403 Hanau-Wolfgang Germany

Frieder W. Lichtenthaler Institute of Organic Chemistry Darmstadt University of Technology Petersenstraße 22 64287 Darmstadt Germany

Jürgen O. Metzger Fachbereich Chemie Carl von Ossietzky Universität Oldenburg Postfach 2603 26111 Oldenburg Germany

Wilfried Lühs Institut für Pflanzenbau und Pflanzenzüchtung 1 Justus-Liebig-Universität Giessen Heinrich-Buff-Ring 26–32 35392 Giessen Germany

Michael Narodoslawsky Graz University of Technology Institute of Resource Efficient and Sustainable Systems (RNS) Inffeldgasse 21 B 8010 Graz Austria

Guido Machmüller FB 9 – Organische Chemie Bergische Universität GH Wuppertal Gaußstraße 20 42097 Wuppertal Germany

Jefter Nascimento PHB Industrial SA Fazenda da Pedra s/n – C. Postal 02 CEP 14150 Servana São Paulo Brazil

Paulo E. Mantelatto Centro de Tecnologia Canavieira (formerly Centro de Tecnologia Copersucar) Fazenda Santo Antonio CP 162 13400-970 Piracicaba Brazil

Glenn E. Nedwin Novozymes Biotech Inc 1445 Drew Ave Davis, CA 95616 USA

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Ulf Prüße Federal Agricultural Research Centre (FAL) Institute of Technology and Biosystems Engineering Bundesallee 50 38116 Braunschweig Germany

Carlos Eduardo Vaz Rossell Centro de Tecnologia Canavieira (formerly Centro de Tecnologia Copersucar) Fazenda Santo Antonio CP 162 13400-970 Piracicaba Brazil

E. Kendall Pye Lignol Innovations Corp. 3650 Westbrook Mall Vancouver, BC V6S 2L2 Canada

Mark Rüsch gen. Klaas Department Technology University of Applied Sciences Neubrandenburg Brodaer Straße 2 17033 Neubrandenburg Germany

René van Ree Rea Energy research Centre of the Netherlands (ECN) – Biomass Department P.O. Box 1 1755 ZG Petten The Netherlands Julia Richter Institut für Chemie Universität Potsdam Karl-Liebknecht-Str. 24–25 14476 Golm Germany Jens Riese McKinsey and Company Inc Munich Office Prinzregentenstraße 22 80538 München Germany Julian R. H. Ross University of Limerick Department of Chemical & Environmental Sciences Limerick Ireland

Hans J. Schäfer Organisch-Chemisches Institut Universität Münster Corrensstraße 40 48149 Münster Germany Daniel J. Schell National Bioenergy Center National Renewable Energy Laboratory 1617 Cole Blvd. Golden, CO 80401-3393 USA Matthias Schmidt Biorefinery.de GmbH Stiftstraße 2 14471 Potsdam Germany Manfred P. Schneider FB 9 – Organische Chemie Bergische Universität GH Wuppertal Gaußstraße 20 42097 Wuppertal Germany


Margit Schulze FB Angewandte Naturwissenschaften FH Bonn-Rhein-Sieg Grantham-Allee 20 53754 Sankt Augustin Germany

Robert van Tuil Agrotechnology and Food Innovations B.V. P.O. Box 17 6700 AA Wageningen The Netherlands

Mathias O. Senge SFI Tetrapyrrole Laboratory School of Chemistry Trinity College Dublin Dublin 2 Ireland

Dan W. Urry BioTechnology Institute University of Minnesota Twin Cities Campus 1479 Gortner Avenue Suite 240 St. Paul, MN 55108-6106 USA and Bioelastics Inc. 2423 Vestavia Drive Vestavia Hills, AL 35216-1333 USA

Jack Starr Cargill Dow LLC 15305 Minnetonka Blvd Minnetonka, MN 55345 USA Sarah A. Teter Novozymes Biotech Inc 1445 Drew Ave Davis, CA 95616 USA Johan Thoen Dow Europe GmbH Bachtobelstrasse 3 8810 Horgen Switzerland Mette Hedegaard Thomsen Risø National Laboratory Biosystems Department Frederiksbovgvej 399 4000 Roskilde Denmark Jeffrey S. Tolan Iogen Corporation 8 Colonnade Road Ottawa Ontario K2E 7M6 Canada

Fernando Valle Genencor International 925 Page Mill Road Palo Alto, CA 94304 USA Klaus-Dieter Vorlop Federal Agricultural Research Centre (FAL) Institute of Technology and Biosystems Engineering Bundesallee 50 38116 Braunschweig Germany Rouhang Wang Satake Centre for Grain Process Engineering School of Chemical Engineering and Analytical Science The University of Manchester Sackville Street Manchester M60 1QD UK

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Marnik M. Wastyn Zuckerforschung Tulln Gesellschaft mbH Josef-Reither-Strasse 21–23 3430 Tulln Austria Colin Webb Satake Centre for Grain Process Engineering School of Chemical Engineering and Analytical Science The University of Manchester Sackville Street Manchester M60 1QD UK Volker F. Wendisch Institute of Biotechnology 1 Research Center Juelich 52425 Juelich Germany Todd Werpy Pacific Northwest National Laboratory P.O. Box 999/K2-12 Richland, WA 99352 USA

Thomas Willke Federal Agricultural Research Centre (FAL) Institute of Technology and Biosystems Engineering Bundesallee 50 38116 Braunschweig Germany Robert Wittenberger Zuckerforschung Tulln Gesellschaft mbH Josef-Reither-Strasse 21–23 3430 Tulln Austria Feng Xu Novozymes Biotech Inc 1445 Drew Ave Davis, CA 95616 USA


Part I Biobased Product Family Trees


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Carbohydrate-based Product Lines 1 The Key Sugars of Biomass: Availability, Present Non-Food Uses and Potential Future Development Lines Frieder W. Lichtenthaler

1.1 Introduction

Because our fossil raw materials derived from prehistoric organic matter are irrevocably decreasing – the end of cheap oil is realistically predicted to occur in the next 2–3 decades, i.e. 2040 at the latest [1–3] – and because pressure on our environment is building up, the progressive change-over of chemical industry to renewable feedstocks emerges as an inevitable necessity [3–5]. The terrestrial biomass which Nature graciously provides us on an annual basis is considerably more complex than fossil raw materials, constituting a multifaceted accumulation of low- and high-molecular-weight products, exemplified by sugars, hydroxy and amino acids, lipids, and biopolymers such as cellulose, hemicelluloses, chitin, starch, lignin, and proteins. By far the most important class of organic material in terms of volume produced is carbohydrates, which represent approximately 75% of the annually renewable biomass of about 180 billion tons (Fig. 1.1). Of these, only a minor fraction (ca. 5%) is used by man, the rest decays and recycles along natural pathways. Thus, carbohydrates, a single class of natural products are – aside from their traditional uses for food, lumber, paper, and heat – the major biofeedstocks from which to develop industrially and economically viable organic chemicals and materials to replace those derived from petrochemical sources.


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1 The Key Sugars of Biomass

Fig. 1.1 Distribution of types of natural products in biomass.

1.2 Availability of Mono- and Disaccharides

The bulk of the annually renewable carbohydrate biomass is polysaccharides, yet their nonfood utilization is confined to the textile, paper, and coating industries, either as such or in the form of simple esters and ethers. Organic commodity chemicals, however, are of low-molecular-weight, hence they are more expediently acquired from low-molecular-weight carbohydrates than from polysaccharides. Accordingly, the constituent repeating units of these polysaccharides – glucose (cellulose, starch), fructose (inulin), xylose (hemicelluloses), etc., or disaccharide versions thereof – are the actual carbohydrate raw materials for organic chemicals with tailor-made industrial applications: they are inexpensive, ton-scale accessible and provide an ensuing chemistry, better worked out and more variable than that of their polymers. Table 1.1 lists the availability and bulk-quantity prices of the eight least expensive sugars, some sugar-alcohols and sugar-derived acids – all well below 1 10 kg–1 – compared with some basic chemicals and organic solvents from petrochemical sources. The result is stunning, because the five cheapest sugars, their alcohols, and some important sugar-derived acids are within the same price range as basic organic bulk chemicals such as naphtha, ethylene, acetaldehyde, or aniline. Actually, the first three of these sugars, sucrose, glucose, and lactose, are in the price range of some of the standard organic solvents. The uniqueness of this situation becomes even more imposing when looking at the availability of these sugars. Sucrose, “the royal carbohydrate”, has for centuries been the worlds most abundantly produced organic compound, current annual production being an impressive 140 million tons [6]. Similarly bulk scale-accessible are its component sugars d-glucose, produced by hydrolysis of starch [7], and d-fructose, generated either from glucose by base-induced isomerization or from inulin or sucrose by hydrolysis [8]. Isomaltulose, an a(1 ? 6) isomer of sucrose, has recently become accessible on an industrial scale through enzymatic transglucosylation [9], lactose and maltose are available in large quantities from whey [10] and starch [11], d-xylose, the cheapest pentose, from woodor straw-derived xylans. l-Sorbose is the cheapest, large-scale accessible l-sugar, because of its production from d-sorbitol (= d-glucitol) in the Vitamin C fabrication process [12]. The sugar alcohols d-sorbitol, erythritol [13], d-xylitol, and dmannitol [14], each of comparatively high yearly production via hydrogenation of

1.2 Availability of Mono- and Disaccharides Table 1.1 Annual production volume and prices of simple sugars, sugar-derived alcohols and acids compared with some petrochemically derived basic chemicals and solvents.

Sugars

Sugar alcohols

Sugar-derived acids

Amino acids Petrochemicals

Solvents

a)

b)

World production a) (metric t year–1)

Price b) (1 kg–1)

Sucrose d-Glucose Lactose d-Fructose Isomaltulose Maltose d-Xylose l-Sorbose d-Sorbitol Erythritol Xylitol d-Mannitol Citric acid d-Gluconic acid l-Lactic acid l-Tartaric acid l-Ascorbic acid l-Glutamic acid l-Lysine

140 000 000 30 000 000 295 000 60 000 70 000 3 000 25 000 60 000 650 000 30 000 30 000 30 000 1 500 000 100 000 150 000 35 000 80 000 1 500 000 740 000

0.20 0.30 0.60 1.00 2.00 3.00 4.50 7.50 1.80 2.25 5.00 8.00 1.00 1.40 1.75 6.00 8.00 1.20 2.00

Ethylene Propylene Benzene Terephthalic acid Aniline Acetaldehyde Adipic acid Methanol Toluene Acetone

90 000 000 45 000 000 23 000 000 12 000 000 1 300 000 900 000 1 500 000 25 000 000 6 500 000 3 200 000

0.40 0.35 0.40 0.70 0.95 1.10 1.70 0.15 0.25 0.55

Reliable data are available for world production of sucrose only, the figure given referring to the crop cycle 2004/2005 [6]. All other data are average values based on estimates from producers and/or suppliers as the production volume of many products is not publicly available. Prices given are those attainable in early 2005 for bulk delivery of crystalline material (where applicable) based on pricing information from sugar industry (sugars) and the Chemical Market Reporter 2005 (acids, basic chemicals, and solvents). The listings are intended as a benchmark rather than a basis for negotiations between producers and customers. Quotations for less pure products are, in part, sizeably lower, e.g. the commercial sweetener “high fructose syrup”, which contains up to 95% fructose, may readily be used for largescale preparative purposes.

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their parent aldoses, are mainly used as food ingredients, because of their sweetening properties, yet also have potential as inexpensive raw materials for broad-scale preparative purposes. The same holds for d-gluconic acid [15] and the other sugar-derived acids listed. Despite their large-scale accessibility at comparatively low cost, it must seem surprising that the chemical industry currently utilizes these mono- and disaccharides to a minor extent only as feedstock for organic chemicals – a state of affairs amply documented by the fact that of the 100 major organic chemicals manufactured in the US in 1995 [16], only seven were derived from biofeedstocks, and five of these – ethanol, sorbitol, citric acid, lysine and glutamic acid – used carbohydrates as the raw material source. Intense efforts within the last decade [17–26] to boost the acquisition of organic chemicals from the sugars in Table 1.1 have not basically changed this picture. There are a variety of reasons for this. Current use of fossil raw materials is more economic and, as important, the process technology for conversion of petrochemical raw materials into organic chemicals is exceedingly well developed and basically different from that required for transforming carbohydrates into products with industrial application profiles. This situation originates from the inherently different chemical structures of the two types of raw material, of which the essence is manifested in their structure-based names (Fig. 1.2). Our fossil resources are hydrocarbons, distinctly hydrophobic, oxygen-free and devoid of functionality, thus, organic functional groups such as hydroxyl, amino, aldehyde, acid, ester or halo functionalities have to be introduced – usually into olefinic hydrocarbons such as ethylene, propylene, and butane – to obtain the industrially important intermediate chemicals. In contrast, annually renewables are carbohydrates, overfunctionalized with hydroxyl groups and pronouncedly hydrophilic. Needless to say, that the methods required for converting carbohy-

Fig. 1.2 Hydrocarbons vs. carbohydrates: more than a play on words, because their names, taken literally, reveal the basic differences in their utilization as organic raw materials.

1.3 Current Non-Food Industrial Uses of Sugars

drates into viable industrial chemicals – reduction of oxygen content with introduction of C = C and C = O unsaturation – are diametrically opposed to those prevalent in petrochemical industry. As higher oil prices, environmental issues, and regulations begin to adversely affect the manufacture of chemicals from fossil raw materials, the transition to a biobased production system is unavoidable, strongly emphasizing the need for systematically elaborating appropriate chemical and microbial process methods to convert carbohydrates – they are the major biofeedstocks to fill the gap between dwindling oil supply and demand – into industrially useful products, be it bulk, intermediate, and fine chemicals, pharmaceuticals, agrochemicals, highvalue-added specialty chemicals, or simply enantiomerically pure building blocks for organic synthesis.


Current utilization of carbohydrates as a feedstock for the chemical industry – be it for bulk, commodity, intermediate, fine, or high-value-added specialty chemicals – is modest when considering their ready availability at low cost and the huge as yet unexploited potential. The examples presently realized on an industrial scale are outlined briefly. 1.3.1 Ethanol

With production of about 24 million tons in 2004 (300 mill hL [27]), fermentation ethanol (“bioethanol”) is the largest-volume biobased chemical today. The principal organism for fermentation is Saccharomyces cerevisiae, an ascomycetous yeast that can grow on a wide variety carbohydrate feedstocks – sugar crops, and sugar-containing by-products such as sugar cane, sugar beet, sorghum, molasses, and – after hydrolysis to glucose – starchy crops such as corn, potatoes, and grain, or cellulosic materials, e.g. wood pulping sludges from pulp and paper mills [28 a]. Recent developments [28 b] replace the conventional yeast by bacteria (Zymomonas nobilis) and/or genetically engineered organisms, which seems to improve productivity significantly. The manufacturing costs are said to be approximately the same as those for its production from ethylene in a plant of comparable size [28 c]. The large growth in production of industrial-grade fermentation ethanol within recent years is less because of its use as a solvent and starting material for follow-up chemicals such as acetaldehyde, ethyl esters (e.g. EtOAc) and ethers (Et2O) – these mostly result from ethylene-based processing lines – but because of its high potential as a fuel additive. It is either directly mixed with standard gasoline at a level of 5%, or indirectly in the form of ETBE (ethyl t-butyl ether) in proportions of up to 15%; a hefty government subsidy is, however, required (re-

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moval of gasoline tax) if it is to remain competitive. The growth opportunities for fuel-grade bioethanol are enormous and are predicted to increase substantially within the next five years. 1.3.2 Furfural

With an annual production of approximately 250 000 tons, furfural (2-furfuraldehyde) seems to be the only unsaturated large-volume organic chemical prepared from carbohydrate sources. The technical process involves exposure of agricultural or forestry waste – hemicellulose content up to 25% d-xylose polysaccharides (xylosans) – to aqueous acid and fairly high temperatures; the xylosans are first hydrolyzed then undergo acid-induced cyclo-dehydration [29]. The chemistry of furfural is well developed, providing a host of versatile industrial chemicals by simple straightforward operations (Scheme 1.1): furfuryl alcohol (2) and its tetrahydro derivative 1 (hydrogenation), furfurylamine 3 (reductive amination), furoic acid 4 (oxidation), and furanacrylic acid 5 (Perkin re-

Scheme 1.1 Furan commodity chemicals derived from pentosans in agricultural wastes (corn cobs, oat hulls, bagasse, wood chips).


action), or furylidene ketones 6 (aldol condensations). Furfural is also the key chemical for the commercial production of furan (by catalytic decarbonylation) and tetrahydrofuran (8) (hydrogenation), thereby providing a biomass-based alternative to its petrochemical production by dehydration of 1,4-butanediol [29]. Further importance of these furan chemicals stems from their ring-cleavage chemistry [30], which has led to a variety of other established chemicals, for example fumaric, maleic, and levulinic acids, the last a by-product of production of furfural [31]. The susceptibility of the furan ring in these compounds to electrophilic substitution at C-5 has been widely exploited. Mineral acid-promoted condensation with aldehydes or ketones converts 3 into difurfural diamine 9 [32], whereas esters of 2-furoic acid afford the respective difurfuryl dicarboxylate (10 ? 11). Both – the latter on saponification – are relevant monomer components for the generation of polyesters and polyamides [33].

Most of the furfural currently produced is used as a selective solvent in the refining of lubricating oil and, with furfuryl alcohol in condensations with formaldehyde, phenol, acetone, or urea, to yield resins with complex, ill-defined structures, yet excellent thermosetting properties, most notably high corrosion resistance, low fire hazard, and extreme physical strength [29]; they are extensively used in the foundry industry as cores for high-quality castings. 1.3.3 D-Sorbitol (: D-Glucitol)

Although the main consumer of its sizable annual production (Table 1.1) is the food industry, primarily as a non-calorific sweetening agent and as a key intermediate in the production of ascorbic acid (vitamin C) [12], it has important non-food applications, because of its moisture conditioning, softening, and plastifying properties. These result in its use in adhesives, paper, printing, textiles, cellulose-based foil, and pharmaceutical formulations. Other non-food applications of d-sorbitol result from etherification and polycondensation reactions which provide biodegradable polyetherpolyols used for soft polyurethane foams and melamine–formaldehyde or phenol resins [34]. Sizable amounts of d-sorbitol are also used for production of the sorbitan ester surfactants (cf. below).

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1.3.4 Lactic Acid ? Polylactic Acid (PLA)

Large amounts of d-glucose – in the crude form as obtainable from corn, potatoes, or molasses by acid hydrolysis – are used in industrial fermentation processes for production of lactic acid (Scheme 1.2), citric acid, and different amino acids, for example l-lysine or l-glutamic acid. Although the major use of these products is in food and related industries, recent non-food exploitation of lactic acid have made it a large-scale, organic commodity chemical. Most of it is subsequently polymerized via its cyclic dimer (lactide) to polylactic acid [36, 37], a high molecular weight polyester. Because of its high strength, PLA can be fabricated into fibers, films, and rods that are fully biodegradable (? lactic acid, CO2) and compostable, having degraded within 45–60 days. Accordingly, PLA and copolymers of lactic and glycolic acid are of particular significance for food packaging and in agricultural or gardening applications, but are highly suitable materials for surgical implants and sutures, because they are bioresorbable. Cargill, since 1989, has invested some $ 750 million to develop and commercialize polylactic acid (tradename “NatureWorks”), its Nebraska plant with an annual capacity of 140 000 metric tons opened in 2002 [36, 37]. Thus, polylactides, because they combine favorable economics with green sustainability, are poised to compete in large-volume markets that are now the domain of thermoplastic polymers derived from petrochemical sources. Another green development based on lactic acid is its ethyl ester (“Vertec”) that has been marketed for applications in specialty coatings, inks, and directly

Scheme 1.2 Production and uses of lactic acid.


for cleaning because of its high performance and versatility [38]. As a most benign solvent – green, readily biodegradable, and excellent toxicology – it has the potential to displace a variety of petrochemically based solvents such as acetone, DMF, toluene or N-methylpyrrolidone in industrial processes. 1.3.5 Sugar-based Surfactants

Utilization of cheap, bulk-scale accessible sugars as the hydrophilic component and fatty acids or fatty alcohol as the lipophilic component provides non-ionic surfactants which are nontoxic, non-skin-irritating and fully biodegradable. Typical examples of such industrially relevant surfactants are fatty acid esters of sorbitol (sorbitan esters) and of sucrose, fatty acid amides of 1-methylamino-1deoxy-d-glucitol (NMGA) and, most pronounced in terms of volume produced, fatty alcohol glucosides, the so-called alkyl polyglucosides (APGs) [39]. 1.3.6 ‘Sorbitan’ Esters

Bulk-scale accessible d-sorbitol (cf. Table 1.1) readily undergoes dehydration on exposure to mineral acid at fairly high temperatures to give anhydrosorbitol or sorbitan, de facto a mixture of sorbitol and its 1,4-anhydro and 1,4 : 3,6-dianhydro derivatives, the exact composition depending on the conditions employed [34] (Scheme 1.3). Esterification of this mixture with C16/C18 fatty acid chlorides/base or transesterification with their methyl esters leads to either sorbitan monoesters (SMS for sorbitan monostearate), or di- and tri-esters. Because of their favorable hydrophilic/hydrophobic balance (HLB) values, sorbitan esters find use as non-ionic surfactants and as solubilizers and emulsifiers in cosmetics, pharmaceuticals, textile processing, and a variety of other formula-

Scheme 1.3 Dehydration of D-sorbitol to “sorbitan”, giving the ‘sorbitan monoester’ surfactant on esterification with C16/C18 fatty acids.

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tions [34]. Having been commercially available since the 1940s, they de facto constitute one of the first fully green synthetic surfactants, presently produced at an estimated 20 000 t a–1. 1.3.7 N-Methyl-N-acyl-glucamides (NMGA)

Reductive amination of d-glucose with methylamine smoothly generates the aminoalditol, 1-methylamino-1-deoxy-d-glucitol, which, on amidation with fatty acids, gives the corresponding fatty acid amides, carrying a methyl group and a pentahydroxylated six-carbon chain on the amido nitrogen:

The NMGA have highly advantageous ecological and toxicological properties which brought about their use as surfactants, cleansing agents, and in cosmetics applications [39, 40]. 1.3.8 Alkylpolyglucosides (APG)

Commercially produced by several companies – most notably by Cognis with a capacity in the 50 000 t a–1 range, Kao, Seppic, and ICI – APG’s are by far the most important non-ionic surfactants. They are fatty alcohol glucosides with an alcohol chain length normally between C8 and C14. Their industrial synthesis entails either direct acid-catalyzed Fischer glycosidation of glucose (in the form of a syrupy starch hydrolysate) or starch itself. The alternative process consists of two stages, the first being Fischer glycosidation with n-butanol to butyl glycosides which are subsequently subjected to acid-promoted transacetalization [35] (see also Chapter 9 in this volume, Hill). The resulting product mixtures contain the a-d-glucosides predominantly, as designated in the formula (Scheme 1.4) and are marketed as such. APG are not skin-irritating, have good foaming properties, and are completely biodegradable, hence are widely used in manual dishwashing detergents and in the formulation of shampoos, hair conditioners, and other personal care products [35].


Scheme 1.4 Synthesis of alkyl polyglucosides (APG).

1.3.9 Sucrose Fatty Acid Monoesters

These are currently produced at an approximate 5000 t · a–1 only, and are mostly used in cosmetic and personal care formulations because of their attractive dermatological properties. Produced by transesterification of fatty acid methyl esters or fats, the resulting sucrose monoester (if 1 : 1 molar ratios have been used in the process) is not a defined product acylated exclusively at the primary glucose-6-OH, as indicated in the formula, but at the other primary and some secondary OH groups also [41]:

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1.3.10 Pharmaceuticals and Vitamins

Aside the enormous amount of sugars, mostly glucose and sucrose, that flows into the fermentative production of amino and hydroxy acids (cf. Table 1.1), of which a substantial part is for food use, a significant volume of these sugars is used in fermentation processes furnishing high-value-added products – antibiotics and vitamins, much too complex in their structures as to be generated by chemical synthesis. Figure 1.3 lists several representative examples – penicillins and cephalosporins with an estimated world production in the 70 000 t a–1 range, the aminoglycoside antibiotics of the kanamycin and spectinomycin type, or the recently optimized bioprocesses for bulk-scale production of vitamin C and B6. Some sugar-derived drugs obtained by chemical means have also reached some importance, e.g. ranitidine (Zantac), an inhibitor of gastric acid secretion – one of the top 30 drugs based on sales [42] – isosorbide dinitrate, a coronary vasodilatator [43], or topiramate, a fructose-derived anticonvulsant drug with high antiepileptic efficacy [44].

1.4 Toward Further Sugar-based Chemicals: Potential Development Lines

Considering the large-scale, low-cost availability of the basic biomass-sugars listed in Table 1.1, most notably sucrose, glucose and fructose, their present non-food utilization by chemical industry is modest indeed, i.e. the huge potential as the raw material for further viable industrial chemicals and materials is largely untapped. In view of the need for the chemical industry to somehow bring about the changeover from fossil raw materials to biofeedstocks, most notably carbohydrates because they are more readily accessible from agricultural crops and waste materials than any other natural products, their further exploitation to produce industrially viable products is one of the major “green” challenges. The attempt to trace those sugar-based development lines along which the further exploitation of the key sugars of biomass is likely to proceed, implies assessment of many imponderables, particularly with regard to current dynamics in exploiting genetically engineered enzymes and the products resulting from them. Hence, some important directions along which relevant developments are likely to proceed are surely to be missed. Nevertheless, an “inventory” based on the present status may be expedient for focusing efforts on those areas where useful methods leading to promising products already exist and wait to be further developed.


6)

Fig. 1.3 Sugar-derived high-value-added products – antibiotics, vitamins, and pharmaceuticals.

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1.4.1 Furan Compounds

In addition to furfural, an established sugar-based five-carbon commodity with versatile industrial applications (cf. above), several other furan compounds, readily prepared from sugars, hold high promise as industrial intermediate chemicals, albeit – for purely economic reasons – they are not (yet) produced on an industrial scale.

1.4.1.1

5-Hydroxymethylfurfural (HMF) Like many petroleum-derived basic chemicals, e.g. adipic acid or hexamethylenediamine, HMF is a six-carbon compound with broad application potential, inasmuch it is readily accessible from fructose or inulin hydrolysates by acid-induced elimination of three moles of water [45]. Even a pilot-plant-size process has been elaborated [46]. HMF has been used for the manufacture of special phenolic resins of type 12, because acid catalysis induces its aldehyde and hydroxymethyl group to react with phenol [47].

12

Of high industrial potential as intermediate chemicals are the various HMF-derived products for which well worked-out, large-scale-adaptable production procedures are available (Scheme 1.5). Of these, the 5-hydroxymethylfuroic acid 13, the 2,5-dicarboxylic acid (19), the 1,6-diamine (15), and the respective 1,6-diol (17) are most versatile intermediate chemicals of high industrial potential, because they are six-carbon monomers that could replace adipic acid, or alkyldiols, or hexamethylenediamine in the production of polyamides and polyesters. Indeed, an impressive series of furan polyesters and polyamides has been prepared [30] in which the furandicarboxylic acid 19 replaces terephthalic and isophthalic acids in current industrial products (cf. below). None has yet proved economically competitive with existing products, however. Thus, HMF is not yet produced on an industrial scale. Tentative assessment of its economics compared with those of petrochemical raw materials clearly reveals the underlying reasons – ton prices of naphtha and ethylene are in the 150–400 1 range; those of aniline (500 1 t–1) and fructose (*1000 1 t–1) , in particular, are substantially higher, entailing an HMF-marketing price of at least 2500 1 t–1 – too expensive at present for a bulk-scale industrial product. Accordingly, as long as the economic situation favors fossil raw materials, applications of HMF lie in high value-added products, for example pharmaceuticals or special niche materials.


Scheme 1.5 Versatile intermediate chemicals from HMF [48–52].

1.4.1.2 5-(Glucosyloxymethyl)furfural (GMF) The industrial production of isomaltulose from sucrose (Table 1.1) – for food reasons, because it is hydrogenated to an equimolar mixture of glucosyla(1 ? 6)-glucitol and -mannitol [9], the low calorie sweetener isomalt [53] – has made it a lucrative target for generating disaccharide intermediates of industrial potential. For example, air oxidation in strongly alkaline solution smoothly provides the glucosyl-a(1 ? 5)-d-arabinonic acid in the form of its lactone 20 in excellent yield [54]. Alternatively, acid-induced dehydration of the fructose portion gives a-d-glucosyloxymethylfurfural (a-GMF, 21) (Scheme 1.6). As this process is also feasible in a continuous flow reactor [55 a], the compound may be readily produced on large scale. As a glucosylated HMF, 20 provides a rich chemistry toward products with broad application profiles. Oxidation with chlorite gives the glucosylfuroic acid 21; when Pt/O2 is used in aqueous medium the dicarboxylic acid 22 is gener-

17

18


Scheme 1.6 Conversion of sucrose into products with industrial potential [54–56].

ated [56]. Both of these reactions proceed in excellent yield. Aldol-type condensations deliver derivatives with polymerizable double bonds, most notably the methylenation product 24, which polymerizes spontaneously, and the acrylic acid 25 [55], expected to yield novel, hydrophilic polymers with interesting performance profiles. Reductive amination provides GMF-amine; N-acylation of this with fatty acid chlorides gives compounds of type 26, non-ionic surface-active agents in which hydrophobic fat-alkyl residue and hydrophilic glucose part


are separated by a quasi-aromatic spacer. They also have useful liquid-crystalline properties [57], as do esters of glucosylfuroic acid 21 with long-chain alcohols (? 27), which combine high surface activity with biocompatibility, rendering them promising candidates for biomedical applications.

1.4.1.3 Furans with a Tetrahydroxybutyl Side-chain Another simple, one-step entry from hexoses to more highly substituted furans is their ZnCl2-mediated reaction with 1,3-dicarbonyl compounds such as 2,4pentanedione or ethyl acetoacetate. Because only the first two sugar carbons contribute to the formation of the furan, a distinctly hydrophilic tetrahydroxybutyl side-chain is produced. Thus, d-glucose smoothly provides furans 28 and 29 with the d-arabino configuration in the polyol fragment [58]; these can be shortened oxidatively to the dicarboxylic acid (29 ? 30) or a variety of other furan building blocks (Scheme 1.7). Of biological relevance seem to be ensuing products of type 32, i.e. amino acids with the carboxyl group in the hydrophobic portion of the molecule and the amino group in the hydrophilic portion, because they have been used to prepare hetarylene-carbopeptoid libraries by combinatorial techniques [59]. In contrast, under mildly basic conditions (aqueous bicarbonate at 85 8C), d-glucose reacts with pentane-2,4-dione in an entirely different manner, producing, via C-addition and subsequent retroaldo-type elimination of OAc–, the 2-Cglucosylpropanone 31 [60]. Because this conversion can be performed with the unprotected sugar and in aqueous solution with simple reagents, it may legitimately be referred to as a prototype of green and/or sustainable sugar transformations. The procedure is equally feasible with other aldohexoses and with d-fructose [61], thus, is one of the cleanest and most efficient preparative entries

19

20


Scheme 1.7 One-pot conversions of D-glucose into hydrophilic furans [58] or, alternatively, into C-glucosides by reaction with acetylacetone [60].

into the area of C-glycosides, which, as stable “mimics” of the usual O-glycosides, command major interest as glycosidase inhibitors [62]. 1.4.2 Pyrones and Dihydropyranones

The bulk-scale-accessible mono- and disaccharides of Table 1.1 invariably adopt pyran cyclohemiacetal forms, from which well-elaborated, efficient reaction channels lead to an unusually large variety of unsaturated pyran building blocks, for example pyrones, dihydropyrans, and dihydropyranones, of which the last two have the additional advantage of being enantiomerically pure. They are treated only cursorily in this context, because their potential as ideally functionalized six-carbon building blocks, particularly for the preparation of pharmaceutical targets, has not been utilized comprehensively. They may, however, be regarded as high-value-added specialty chemicals. Pyrones of type 33 (kojic acid) and 34 are readily obtained from d-glucose, the former either enzymatically by growing Aspergillus oxyzae on steamed rice [63] or chemically via pyran 3,2-enolones [64, 65], or the a-pyrone 34 by oxidation to d-gluconic acid [15] and acetylation [66]. Both, at present, are of little significance as six-carbon building blocks, despite a surprisingly effective route from 34 to cyclopentanoid products of type 35 [67] which is surmised to have industrial potential. Other, potentially useful derivatizations of d-glucose – entry reactions with which the tautomeric forms are fixed – were elaborated before the turn of the 19th century – thiation to the acyclic dithio acetals, isopropylidenation to furanoid systems, or generation of pyran structures, such as glucosides, glucals, and


Asp. oryzae

1. Oxid. 2. Ac2O/D

!

DMAP 90%

!

hydroxyglucalesters (Scheme 1.8) [68]. Of particular interest in the context of versatile sugar-based six-carbon building blocks are the pyranoid glycal and hydroxyglycal esters, because they not only have the oxygen content of d-glucose reduced – a precondition for elaboration of industrially viable products – but carry olefinic unsaturation in the pyran ring. Despite the ready accessibility of these glucal and hydroxyglucal esters, and their well-developed ensuing chemistry, their exploitation as industrial intermediates is exceedingly modest. Nevertheless, to emphasize their potential toward industrial intermediates, whether as enantiomerically pure building blocks

Scheme 1.8 Readily accessible, tautomerically fixed D-glucose derivatives which can be used to produce versatile building blocks [68].

21

22


for the synthesis of non-carbohydrate natural products [65, 69] or for agrochemicals and/or high-value-added pharmaceuticals, a particularly versatile array of six-carbon dihydropyranones is listed in Fig. 1.4; all are accessible from d-glucose (via the glucal and hydroxyglucal esters) in no more than three to five straightforward steps.

Fig. 1.4 Pyranoid six-carbon building blocks accessible from Dglucose via glucal (upper half) or hydroxyglucal esters (lower entries) intermediates. All products require no more than three to five straightforward steps from D-glucose [70–78].


A bicyclic dihydropyranone, levoglucosenone, is accessible even more directly by vacuum pyrolysis of waste paper [79]. Although the yield achievable is relatively low – levoglucosan is also formed, their proportions depending on the exact conditions (Scheme 1.9) – relatively large amounts can be amassed quickly; levoglucosenone has been used for synthesis of a diverse variety of natural products in the enantiomerically pure form [80].

Scheme 1.9 High-vacuum pyrolysis of cellulose [79].

Similarly convenient are the acquisition of the three dihydropyranones 36–38 (Fig. 1.5), requiring two and three steps from maltose and sucrose, respectively [81], their “left-over” glycosyl and fructosyl residues serving as acid-labile blocking groups, in contrast with the alkali-sensitive ester functions.

Fig. 1.5 Simple, disaccharide-derived dihydropyranones [81].

All these pyran building blocks are enantiomerically pure and have a unique, highly diverse array of functional groups to which the armory of preparative organic methodology can directly be applied. The enolone esters in Fig. 1.4, for example, have three differently functionalized carbonyl groups, one being free, the other two masked in enol ester and acetal form. In addition, the enolone structural unit is flanked by chiral centers, so any addition reaction to either carbonyl or enolic double bond proceeds with high stereoselectivity. As for the disaccharide-derived building blocks 36–38, they feature functionality in one of the

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24


units and – in the form of the intact glycosyl moiety – a cheap acid-sensitive protecting group in the other. In addition to their extensive use in the total synthesis of enantiomerically pure non-carbohydrate natural products [65, 69] these pyran building blocks have found little use as high-value-added chemicals. If suitable targets and appropriate preparative outlets are found, however, particularly toward pharmaceutically promising compound libraries via combinatorial techniques, these pyran building blocks are likely to become a plethora of attractive, industrially relevant specialty chemicals. 1.4.3 Sugar-derived Unsaturated N-Heterocycles

Although transformation of sugars into trace amounts of N-heterocycles occurs extensively on exposure of foodstuffs to heat (Maillard reaction [82]), and although a variety of nitrogen heterocycles have been generated from saccharide derivatives [83], procedures meeting preparative standards are exceedingly scarce. Recent improvements of existing procedures and the development of new methods has led to the more ready acquisition of various N-heterocycles from carbohydrates, e.g. imidazoles, pyrroles, pyrazoles, pyridines, and quinoxalines which, because of their sugar derivation, have hydrophilic side-chains – a favorable asset particularly in pharmaceutical applications. A brief overview on those N-heterocycles readily accessible from the basic sugars in practical, large-scale adaptable procedures should enhance their utilization as solid intermediate chemicals.

1.4.3.1 Pyrroles The generation of pyrroles by heating a glycerol solution of lactose-derived ammonium salt of galactaric acid over a free flame [84] seems to be the highestyielding acquisition (40%) from a carbohydrate source – a process that, in this or a modified form, does not seem to be utilized industrially.

2,5-Disubstituted pyrroles are accessible from carbohydrate sources via HMF in a preparatively straightforward reaction sequence involving photooxidative furan ring opening and cyclization of the saturated 2,5-diketones with ammonia or amines [85]:


These reaction sequences can directly be transferred to GMF, leading to pyrroles carrying an additional glucosyl residue [85]. Pyrroles with an equally hydrophilic tetrahydroxybutyl substituent, e.g. 39, are available from d-glucosamine by exposure to acetylacetone or ethyl acetoacetate under mildly basic conditions [86] or in a one-pot reaction from d-fructose by heating with acetylacetone and ammonium carbonate in DMSO [87].

The hydroxylated side-chain can, of course, be oxidatively shortened to give a variety of simple pyrrole building blocks, for example carboxylic acid 40, or cyclized to a furanoid ring (39 ? 41) [86], compounds that may be regarded as C-nucleosides.

25

26


1.4.3.2 Pyrazoles Expeditious four-step approaches to 1-phenylpyrazol-3-carboxaldehydes with 5hydroxymethyl, 5-dihydroxyethyl, or 5-glucosyloxymethyl substituents has been elaborated starting from d-xylose [88], d-glucose, and isomaltulose [89], respectively.

As illustrated for d-xylose, its osazone, nearly quantitatively formed on reaction with phenylhydrazine, readily gives the pyrazole when added to acetic anhydride under reflux. Subsequent removal of the N-acetylphenylhydrazone residue with formaldehyde/acetic acid and de-O-acetylation provides a pyrazole-aldehyde (57% overall yield from d-xylose), a versatile heterocyclic building block, useful for preparation of pharmaceuticals or monomers for the generation of polyamides and polyesters, e.g. the diamino and diol derivatives [88]:


1.4.3.3 Imidazoles A variety of imidazoles carrying hydrophilic substituents in the 4-position are readily accessible in one-pot procedures from standard monosaccharides. Of those, the formation of 4-hydroxymethylimidazole by Cu(II)-promoted reaction with formaldehyde and conc. ammonia [90] is rather unique, because obviously retroaldolization to glyceraldehyde and dihydroxyacetone is involved (Scheme 1.10). The retroaldol fission can be partially suppressed, however, on heating d-fructose with formamidinium acetate in liquid ammonia in a pressure vessel [91] or with formamidinium acetate in the presence of boric acid and hydrazine, obviously proceeding via a boric acid complex of the bishydrazone of d-glucosone [56].

Scheme 1.10 D-Fructose-derived hydrophilic imidazoles. A: CH2O, aq. NH3, CuCO3/Cu(OH)2, 2 h, 100 8C [90]; B: formamidine · HOAc/liq. NH3, 15 h, 75 8C [91]; C: N2H4/formamidine · HOAc, H3BO3, 3 h reflux [56].

These conditions can be readily applied to pentoses or disaccharides with acceptable yields, as exemplified with d-xylose [91] and isomaltulose [56] in onepot procedures:

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28


1.4.3.4 3-Pyridinols The conversion of pentosans or pentoses into 3-pyridinol can be effected in a practical three-step sequence, involving acid-induced dehydration to furfural, reductive amination to furfurylamine, and subsequent oxidation with hydrogen peroxide [51, 92], the last step conceivably proceeding through the stage of a 2,5-dihydroxy-2,5-dihydrofurfurylamine, which forms the pyridine nucleus by dehydration to a 5-aminopentenal intermediate and intramolecular aldimine formation. The pyridinol is a prominent intermediate chemical for the preparation of herbicides and insecticides [93], and cholinergic drugs of the pyridostigmine type.

For conversion of furfurylamines with readily oxidizable hydroxyl groups, e.g. those derived from fructose via HMF/bromine in water/methanol, the entire multistep process to the hydroxymethylpyridinol is effected in a one-pot procedure [94]:

1.4.3.5 Quinoxalines Useful one-pot procedures are also available for the conversion of various monosaccharides into tetrahydroxybutyl-substituted quinoxalines, the preparatively most favorable conditions seem to be reaction of fructose with hydrazine, o-phenylenediamine, and boric acid in dilute acetic acid by bubbling oxygen through the solution [95], the decisive intermediate being the bis-hydrazone of d-glucosone:


On briefly heating the quinoxaline under reflux in aqueous acid with excess hydrazine or phenylhydrazine, a surprising oxidative cyclization occurs with formation of the trihydroxypropyl-substituted flavazols [96]. 1.4.4 Toward Sugar-based Aromatic Chemicals

On the basis of a 1995 compilation [16], twenty of the 100 major organic chemicals in the US were aromatic compounds, invariably manufactured from fossil raw materials, mostly from the BTX (benzene–toluene–xylene) fraction derived from naphthas in refineries. There are very few alternatives. The direct thermochemical conversion of biomass to an equivalent BTX product is not realistically feasible, because only small amounts of monocyclic aromatic hydrocarbons – phenols of the catechol and pyrogallol series – are formed on pyrolysis or thermal cracking of woody feedstocks. The same is true for exposure of simple sugars, for example d-xylose, d-glucose, or d-fructose, to either basic or slightly acidic aqueous conditions at 100–160 8C [97]. Vanillin, however, is a by-product of the manufacture of cellulose pulp by the action of alkali on basic calcium lignosulfonate and may be isolated in yields of up to 25%.

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30


An entirely different, highly promising approach from sugars to industrially relevant aromatic compounds is based on microbial conversions along the shikimic acid pathway by using genetically modified biocatalysts. By incorporation of the genomic portion encoding the synthesis of 3-dehydroquinic and 3-dehydroshikimic acid into Escherichia coli constructs, the carbon flow is channeled into accumulation of large amounts of either quinic or shikimic acid [98] (Scheme 1.11), rendering their availability independent of often difficult isolation from plant sources. These improvements are likely to lead to pronounced expansion of the synthetic utilization of these enantiomerically pure carbocycles, not only in the pharmaceutical industry, where quinic acid is already used as the starting material for the synthesis of the anti-influenza drug Tamiflu (oseltamir phosphate [99]), but for production of bulk-scale commodity chemicals such as hydroquinone [100] or phenol [101] by application of simple chemical transformations. The powerful potential of metabolic engineering is similarly manifested in the E. coli biocatalyst-promoted conversion of d-glucose into protocatechuic acid, which can be readily decarboxylated to catechol [102]. This two-step process (cf. Scheme 1.11), feasible in a 24% overall yield, may replace the present process used to manufacture of this 25 000 t a–1 petrochemical commodity [103]. Of similar significance seems the genetically modified microbe-catalyzed conversion of d-glucose into gallic acid and pyrogallol [104], the accessibility of which currently relies on isolation from plant sources, despite a spectrum of uses, particularly as educts for pharmaceuticals [105]. The recent unraveling of the biosynthesis of phloroglucinol could also pave the way to its production from glucose. By expressing the enzyme from Pseudomonas fluorescens that assembles three molecules of malonyl coenzyme A into an activated diketoheptanediote which subsequently undergoes cyclization and conceivably spontaneous aromatization, in Escherichia coli, phloroglucinol can be generated from glucose in yields of up to 10 g L–1 [106].


Scheme 1.11 Metabolic engineering of the shikimic acid pathway intermediates toward aromatic chemicals: chemical transformations; ? bioconversions with biocatalysts. (a) E. coli QP1.1/pKD12.138 [98], (b) E. coli SP1.1PTS–/pSC6.090B [99],

(c) E. coli KL3/pWL2.46B [101], (d) E. coli KL7/pSK6.161 [102]. Abbreviations: PEP = phosphoenolpyruvic acid, EHP = erythrose-4phosphate, DAHP = 3-deoxy-D-arabino-heptulosonic acid 7-phosphate, DHQ = 3-dehydroquinic acid, DHS = 3-dehydroshikimic acid.

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32


Scheme 1.12 Potential generation of C7 plant acids from D-glucose [107].

Whereas protocatechuic and gallic acid are generated via the shikimic acid pathway, other C7 plant acids of c-pyrone structure are biosynthesized from d-glucose via 3-deoxy-manno-octulosonate 8-phosphate (KDO 8-P), as a result of aldol-type addition of PEP to d-arabino-5-phosphate (Scheme 1.12) [107]. Although widespread throughout the plant kingdom, daucic, chelidonic, and meconic acids are of limited availability because of their capricious isolation from the individual plants. Given their utility for industrial purposes when readily accessible, metabolic engineering of the genes involved in the respective enzymes is likely to be the proper approach to their large-scale acquisition. 1.4.5 Microbial Conversion of Six-carbon Sugars into Simple Carboxylic Acids and Alcohols

Some experts predict that biotechnology will produce up to 20% of the industrial chemicals by 2010 – from the current level of 5% [108, 109]. Undoubtedly, such an increase will receive its major thrust from the various genetically engineered bioprocesses currently under industrial investigation, most notably those that involve bioconversion of sugars into industrially relevant bio-alcohols other than ethanol and into simple C3–C5 carboxylic acids other than those already exploited (i.e. lactic, citric, and tartaric acids, and a variety of amino acids – cf. Table 1.1). Table 1.2 lists a variety of acids and alcohols that can be obtained by

1.4 Toward Further Sugar-based Chemicals: Potential Development Lines Table 1.2 C3–C5 carboxylic acids and alcohols producible by microbial fermentation a).

Acids

Alcohols

Product b)

Substrate

Microorganism

Propionic acid

Various sugars

Pyruvic acid 3-Hydroxypropionic acid Butyric acid 3-Hydroxybutyric acid Succinic acid

Glucose Glucose

Fumaric acid

Various sugars

l-Malic acid

Various sugars

Itaconic acid

Various sugars

2-Oxoglutaric acid

Glucose

Clostridium sp. Propionibacterium shermanii Pseudomonas aeruginora E. coli constructs Clostridium butyricum Alcaligenes eutrophus Actinobacillus succinogenes Mannheimia succiniciproducens Rhizopus nigricans Rhizopus arrhizus Parcolobactrum sp. Brevibacterium sp. Aspergillus terreus Aspergillus itaconicus Pseudomonas fluorescens

n-Propanol i-Propanol 1,2-Propandiol 1,3-Propanediol

Glucose Various sugars Glucose Glucose, glycerol

Glycerol n-Butanol 2,3-Butanediol 1,2,4-Butanetriol a) b)

Glucose Various sugars

Clostridium fallax Clostridium sp. E. coli constructs Clostridium pasteurianum E. coli mutants Glucose yeasts Various sugars Clostridium butylicum Glucose, xylose Klebsiella pneumoniae Bacillus polymyxa Xylose (xylonic acid) E. coli construct

Compiled from Ref. [110] Acids already exploited industrially, i.e. lactic, tartaric, and citric acid (cf. Table 1.1) are not listed here.

microbial production and substantial activity in this field is to be expected. Which of these products, however, are likely to enter industrial production will be determined by a variety of factors – demand, availability of a genetically engineered biocatalyst, and, not least, by economics, with rising oil prices increasingly providing more favorable conditions. The products with significant industrial potential marked in bold in Table 1.2 are briefly discussed below in respect of the current status of their microbial production and future prospects.

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1.4.5.1 Carboxylic Acids The US Department of Energy (DOE) recently published a list of twelve sugarderived chemicals worthy of industrial exploitation [111], of which five are simple carboxylic acids accessible by biotransformation of sugars, mostly glucose: · 3-hydroxypropionic acid (3-HPA) · four-carbon 1,4-diacids (malic, fumaric, and succinic acids) · itaconic acid (IA) · aspartic acid (Asp) · glutamic acid (Glu).

Each of these are considered to have high “building block potential”, either as monomers for the production of novel polyesters and polyamides [112] or as a starting material for a variety of commodity chemicals, currently produced petrochemically. Accordingly, these five carboxylic acids are to become economically viable products if low-cost fermentation routes can be developed and implemented on an industrial level. Even for glutamic acid – for which several fermentation processes have been industrially realized to meet the need for its sodium salt as a flavor enhancer – the productivity of the organism and the final fermentation titer must be improved if it is to become an attractive candidate for chemical and microbial follow-up transformations [111 a]. The same is true for aspartic acid, for which Krebs cycle pathway engineering of biocatalytic organisms to overproduce oxaloacetate (cf. Scheme 1.13) are required to provide a

Scheme 1.13 Glycolytic pathway leading to the L-malic, fumaric, and succinic acids [114].


product competitive with the (racemic) one obtained currently from petrochemical-derived fumaric acid by amination [111 b]. 3-Hydroxypropionic Acid (3-HPA) Structurally isomeric to lactic acid, 3-HPA represents a three-carbon building block with the potential to become a key intermediate for a variety of high-volume chemicals – malonic and acrylic acids, methacrylate, acrylonitrile, 1,3-propanediol, etc. [111 c]. Cargill is developing a low-cost fermentation route by metabolic engineering of the microbial biocatalyst that produces 3-HPA under anaerobic conditions [113 a], yet it will take another one or two years for the process to reach commercial viability [113 b].

Unlike a product such as lactic acid, one of the attractions of 3-HPA is that it is not manufactured commercially, either by chemical or biological means. Fumaric Acid Fumaric acid, a metabolite of many fungi, lichens, mosses and some plants, which is mainly used as the diacid component in alkyd resins [115], is produced commercially to some extent by fermentation of glucose with Rhizopus arrhizus [116], yet productivity improvements seem essential for the product to be an option for replacing its petrochemical production by catalytic isomerization of maleic acid. Malic Acid Most of the malic acid produced, approximately 10 000 t a–1, is racemic, because it is derived from petrochemically produced fumaric acid. The Lform can also be produced from fumaric acid by hydration with immobilized cells of Brevibacterium or Corynebacterium species. Succinic Acid Succinic acid is used to produce food and pharmaceutical products, surfactants and detergents, biodegradable solvents and plastics, and ingredients to stimulate animal and plant growth. Although it is a common metabolite formed by plants, animals, and microorganisms, its current commercial production of 15 000 tons per year is from petroleum, i.e. by hydrogenation of malic acid. The major technical hurdles for succinic acid as green, renewable bulk scale commodity chemical – 1,4-butanediol, THF, c-butyrolactone, or pyrrolidones are industrially relevant products – entail the development of very lowcost fermentation routes from sugar feedstocks. Currently available anaerobic fermentations of glucose include use of an organism genetically cloned from Aspergillus succinoproducens, an engineered E. coli strain developed by DOE laboratories [111 d], and several others [117]. The processes are currently under active

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development. Production costs must be at or below $ 0.25 lb–1 to match petrochemical production [111d]. Itaconic Acid (IA) Structurally an a-substituted methacrylic acid, IA is a C5 building block with significant market opportunities. It is currently produced by fungal fermentation at approximately 10 000 t a–1 [118] and mainly used as a specialty co-monomer in acrylic or methacrylic resins, e.g. incorporation of small amounts of IA into polyacrylonitrile significantly improves its dyeability.

To become a commodity chemical, productivity improvements with the currently used fungi Aspergillus terrous and Aspergillus itaconicus are required, and progress seems to be encouraging [119]. To be competitive to analogous commodities, the crucial production price of about 0.25 $ lb–1 possesses to be reached [111 e] – a significant technical challenge still to be solved.

1.4.5.2 Potential Sugar-based Alcohol Commodities by Microbial Conversions 1,3-Propanediol Both the diol and the dicarboxylic acid components of poly(trimethylene terephthalate), a high performance polyester fiber with extensive applications in clothing textiles and carpeting, are currently manufactured from petrochemical raw materials.

For the diol portion of the polyester, 1,3-propanediol, however, biobased alternatives have been developed which rely on microbial conversion of glycerol [122], a by-product of biodiesel production, or of corn-derived glucose [123]. For the latter conversion, DuPont has developed a biocatalyst, engineered by incorporating genes from bakers’ yeast and Klebsiella pneumoniae into E. coli, which efficiently converts corn-derived glucose in 1,3-propanediol [120, 123]. The bioprocess, currently being implemented on an industrial scale in a Tennessee manufacturing plant by a DuPont/Tate & Lyle joint venture will be providing bulk quantities from 2006 [124].


1,2-Propanediol In its racemic form, 1,2-propanediol is a petroleum-based high-volume chemical with an annual production of over 500 000 t, mostly used for manufacture of unsaturated polyester resins, yet also with excellent antifreeze properties. Enantiomerically pure (R)-1,2-propanediol accumulates along two different pathways via DAHP (cf. Scheme 1.11) and methylglyoxal, which is then reduced with either hydroxyacetone or lactaldehyde as intermediates. Both routes have been examined for microbial production from glucose by means of genetically engineered biocatalysts, obtained either by expressing glycerol dehydrogenase genes or by overexpressing the methylglyoxal synthase gene in E. coli [125]. This work provides a basis for further strain and process improvement. Another approach entails inoculating silos of chopped whole-crop maize with Lactobacillus buchneri. After storage for four months yields of 50 g · kg–1 were reported [126]. Thus prospects for elaborating an economically sound bioprocess look promising. 1,2,4-Butanetriol Used as an intermediate chemical for alkyd resins and rocket fuels, 1,2,4-butanetriol is presently prepared commercially from malic acid by high-pressure hydrogenation or hydride reduction of its methyl ester. In a novel’ environmentally benign route to this chemical, wood-derived d-xylose is microbially oxidized to d-xylonic acid, followed by multistep conversion to the product by use of a biocatalyst specially engineered by inserting Pseudomonas putida plasmids into E. coli [127]. Although further metabolic engineering is required to increase product concentration and yields, microbial generation of 1,2,4-butanetriol is a clear alternative to its acquisition by chemical procedures.

1.4.6 Chemical Conversion of Sugars into Carboxylic Acids

The sugar-derived carboxylic acids mentioned in Table 1.1, i.e. gluconic, citric, lactic, tartaric, and ascorbic acid, are accessible in bulk by fermentation processes and may be considered (and used as) commodity chemicals despite being mostly used for food purposes. In addition to these, however, there are several industrially attractive carboxylic acids obtainable from sugars by chemical means which have high potential as versatile building blocks.

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In the 2004 DOE report [111], four of these sugar-derived carboxylic acids have already been singled out as suitable candidates for further development: · furan-2,5-dicarboxylic acid · glucaric acid · levulinic acid, and · 3-hydroxybutyrolactone yet there are many others that equally merit the development and implementation of low-cost preparative procedures to become competitive products. They are addressed briefly here. The high industrial potential of furan-2,5-dicarboxylic acid (19) (cf. Scheme 1.5), accessible from fructose or fructosans (inulin) via HMF [45], has already been emphasized, because it could replace petroleum-derived diacids such as adipic or terephthalic acid in the production of polyesters and polyamides. The aldaric acids of the key hexoses and pentoses as highly hydrophilic diacids also have much potential, similar to that of the sugar platform – d-glucaric acid, a direct nitric acid oxidation product of glucose or starch [128], usually isolated as its 1,4-lactone, galactaric and xylaric acid, accessible from lactose [129] and from dxylose or hemicellulosic xylans. The technical barriers to their large-scale production are mainly development of efficient and selective oxidation technology for these sugars to eliminate the need for nitric acid as oxidant. Investment in Pt-catalyzed oxidation with oxygen seems to be a promising approach.

Because sucrose is cheaper than its component sugars d-glucose and d-fructose, and available in unprecedented quantities (cf. Table 1.1) and exceptional purity, carboxylic acids resulting from selective oxidation of its primary hydroxyl groups are likely of even higher industrial relevance. Because of the persistence of an


intersaccharidic water-bridge of the 2g-HO H2O HO-1–f type in aqueous solution [130], oxidation of sucrose with air in the presence of 0.5% Pt/C at 35 8C gives a 9 : 9 : 1 ratio of the 6g-, 6f- and 1f-saccharonic acids [131]. On further oxidation, particularly when using large amounts of the Pt catalyst and higher temperatures (80–100 8C), the formation of sucrose-6g,6f-dicarboxylic acid 42 has been observed [132], yet a preparatively useful procedure for its acquisition was developed only recently [133] by combining Pt/air-oxidation with continuous electrodialytic removal of 42, thereby protecting it from further oxidation. Otherwise, on letting the reaction proceed, the sucrose-tricarboxylate is obtained [134]. An useful alternative oxidant to the tricarboxylate 43 is the NaOCl/TEMPO system [135], particularly when applying high-frequency ultrasound; this results in yields in the 70% range [136] (Scheme 1.14). Levulinic acid (“LA”) and formic acid are end products of the acidic and thermal decomposition of lignocellulosic material, their multi-step formation from the hexoses contained therein proceeding through HMF as the key intermediate; the hemicellulose part, mostly xylans, furnishes furfural [31, 45 b]. A commercially viable fractionation technology for the specific, high-yield acquisition of LA has been developed (Chapter 7 in the first volume of this book, “The Biofine Process”), rendering it an attractive option as an important biorefinery platform chemical [111 g]. Levulinic acid is a starting material for a large number of higher-value products, because it can be converted by established procedures into products such as acrylic and succinic acids, pyrrolidines, diphenolic acid (44), which has the potential of

Scheme 1.14 Catalytic oxidation of sucrose [133, 134].

39

40


replacing bisphenol A in the manufacture of polycarbonate resins, or 5-aminolevulinic acid (45), applied in agriculture as a herbicide and as a growth-promoting factor for plants. Another asset is its convertibility into 2-methyltetrahydrofuran (46), which is used as a liquid fuel extender. The esters of LA, e.g. ethyl levulinate, have similar industrial potential as oxygenated additives to diesel fuels.

3-Hydroxybutyrolactone (3-HBL) is now a specialty chemical for fairly high value pharmaceuticals. The 2004 DOE report [111h] places it in the list of the top twelve sugar-based candidates worthy of industrial exploitation. Its generation from starch by oxidative (H2O2) degradation is regarded as “messy”, because it involves multiple steps and results in a variety of side-products. Thus, this process must be improved, or alternatives found; one such alternative is reduction and cyclization of microbially produced l-malic acid (cf. Section 1.4.5.1) – to fully utilize the potential of this four-carbon building block for the production of a variety of industrially important tetrahydrofuranoid derivatives.

1.4.7 Biopolymers from Polymerizable Sugar Derivatives

Today, biocompatibility and biodegradability are key functional requirements in the design of new polymeric materials, whether polyesters, polyamides, or polyurethanes [137]. If composed of sugar-derived monomer components, such poly-


mers are non-toxic and biodegradable and, hence, have minimal impact on waste management. They can be safely incinerated and, by composting, can be returned to the ecosystem harmlessly in a carbon dioxide-neutral process.

1.4.7.1 Synthetic Biopolyesters Polyester production worldwide is estimated to be approximately 20 million t a–1, of which only a small fraction is based on renewable monomers such as polyols, dicarboxylic acids, or hydroxyalkanoic acids, despite the fact that a huge variety of these building blocks are amenable to either chemical or biotechnological production. As amply illustrated by the large variety of sugar-derived di- and polyols, hydroxyacids, and dicarboxylic acids in Tables 1.3 and 1.4 – only those which are reasonably accessible are listed – the number of possible polyesters is immense, and not all conceivable combinations have been implemented and evaluated for their application profiles. The only one of industrial relevance today is Cargill’s polylactic acid (PLA), used as a benign, biodegradable material for packaging, for disposable single use items, and for medical devices (vide supra). On the verge though of becoming an industrial bioproduct is DuPont’s poly(trimethylene terephthalate) – its high-performance fiber Sorona – because one of its components, the presently petroleum-derived 1,3-propanediol, is being replaced by one obtained from glucose by microbial fermentation. Of the vast number of polyesters prepared from the monomers listed in the tables, those containing furan residues have attracted particular interest [30, 33, 138]. 5-Hydroxymethyl-2-furoic acid, for example, gave a mixture of linear (47) and cyclic products (48) on polycondensation [139, 140] whereas the 2-furoylacrylic acid analog afforded polyester 49; 2,5-furandicarboxylic acid has been polyesterified with a series of aliphatic diols (? 50 [140]), with dianhydrosorbitol (? 52 [141]), or with bisphenols (? 53 [142]) (cf. Fig. 1.6). Even the all-furan polyester 54 has been successfully prepared from its respective monomeric components [30] – like polyesters 47–50, 52, and 54, a “fully green”, naturally resourced product. The same applies to polyester 51, composed of 1,3-propanediol and the furan-2,5-diacid – in fact an analog of DuPont’s Sorona in which the terephthalic acid portion is replaced by a bio-counterpart. Given the same fiber properties it would rightfully deserve the “clothing from a cornfield” attribute [143]. Despite the versatile application profiles of these polyesters – and a vast number of others have been synthesized – they have been prepared only on the laboratory scale and used in experimental applications and so currently are “academic curiosities” rather than industrially promising products. They will remain that, however, only as long as the economics are in favor of the production of the monomeric components from fossil raw materials – a scenario that will change within the next 25–30 years [1].

41

42

1 The Key Sugars of Biomass Table 1.3 Sugar-based alcohols and diamines suitable as monomers for polyesters, polyamides, or polyurethanes. Compound

Derivation 1,2-Propanediol

D-Glucose

1,3-Propanediol

D-Glucose

Glycerol

Fats (D-Glucose)

2,5-Bis(hydroxymethyl)furan

D-Fructose

1,6-Dianhydro-D-sorbitol

D-Glucose

3,5-Bis(hydroxymethyl)pyrazole

D-Xylose

Xylitol

D-Xylose

D-Sorbitol

D-Glucose

1,6-Diamino-1,6-dideoxy-D-glucitol

D-Glucose

2,5-Diamino-1,4 : 3,6-dianhydrosorbitol

D-Glucose

2,5-Bis(aminomethyl)furan

D-Fructose

1.4 Toward Further Sugar-based Chemicals: Potential Development Lines Table 1.4 Sugar-derived carboxylic acids for potential use as monomers for polyesters and polyamides. Compound

Derivation Lactic acid

D-Glucose

3-Hydroxybutyric acid

D-Glucose

5-Hydroxymethylfuroic acid

D-Fructose

3-Hydroxyvaleric acid

D-Glucose

Xylaric acid

D-Xylose

D-Glucaric

D-Glucose

acid

D-Galactaric

acid

Lactose

Furan-2,5-dicarboxylic acid

D-Fructose

Pyrazol-3,5-dicarboxylic acid

D-Xylose

Sucrose-6,6'-dicarboxylic acid

Sucrose

GMF-6,6'-dicarboxylic acid

Sucrose

5-Aminomethylfuroic acid

D-Fructose

6-Amino-D-gluconic acid lactam (R = H, CH3)

D-Glucose

43

44


Fig. 1.6 Polyesters containing furan moieties.

1.4.7.2 Microbial Polyesters Microbial polyesters, or PHA for poly(hydroxyalkanoates) [144, 145], constitute a large and versatile family of polyesters produced by various bacteria in which they are deposited within the bacterial cell wall as a storage polymer. Industrial processes have been developed for poly(3-hydroxybutyrate) [poly(3HB)] and a polymer consisting of 3-hydroxybutyric and 3-hydroxyvalerianic acid [ ? poly(3HB)-co-3(HV), tradename Biopol]. Both polyesters have outstanding properties in respect of thermoplastic behavior, biocompatibility, and biodegradability, and hence have wide applications in cosmetics, hygiene, and agricultural materials, in drug delivery systems, and in medical surgery.

Other representative PHA’s synthesized by microorganisms contain 3-hydroxyhexanoic, 3-hydroxyoctanoic, and malic acid as repeating units. It is to be expected that improvement of fermentation strategies, e.g. by recombinant E. coli harboring the microbial PHA biosynthesis genes, will enhance the economic viability of PHA production, currently on an approximate 3000 t a–1 level, as they have the potential to replace numerous chemosynthetic polymers in many applications.


1.4.7.3 Polyamides More than 90% of the polyamides produced worldwide, amounting to approximately 5.8 million tons in 1998 [146], are based on six-carbon monomers, i.e. caprolactam (Nylon-6), and adipic acid/hexamethylenediamine (Nylon-66), the manufacture of which is based exclusively in petroleum-based pathways [146].

When considering the large variety of aminocarboxylic acids, dicarboxylic acids, and diamines reasonably well accessible from common six-carbon sugars [147, 148] – expedient examples are listed in Tables 1.3 and 1.4 – substitution of petroleum-based monomers of these polyamides by those derived from sugars seems particularly obvious and promising. Of the myriad of possible combinations of these sugar-derived monomers either with themselves or with the common, petrochemically-derived diamines and dicarboxylic acids, an immense number have been realized. Here only a few of these polyamides are covered as examples. Solution or interfacial polycondensation of galactaric acid dichloride in its acetylated form with a variety of aliphatic and aromatic diamines resulted in a series of polyamides [149], the one resulting from 1,6-diaminohexane resembling a Nylon-6,6 in which half of the methylene hydrogen atoms of the usual adipic acid are substituted by acetoxy groups (R = Ac). These can be deacylated with aqueous ammonia to give the tetrahydroxylated Nylon-66:

Use of the lactone monomethyl ester of d-glucaric acid proved advantageous in the generation of stereoregular polyglucaramides, effected with an impressive array of aliphatic and aromatic diamines [149]:

45

46


Of similar practical utility is the sucrose-6,6'-dicarboxylic acid (42), readily obtained from sucrose by Pt-catalyzed oxidation with oxygen in aqueous medium (cf. Scheme 1.14), which on amidation of its dimethyl ester with fat-amines provided surface-active diamines of type 55 with remarkable tensidometric properties [150], whereas reaction with hexamethylenediamine furnished the interesting, highly hydrophilic polyamide 56 [151].

Sugar-based “quasi-aromatic” monomers for polyamides, i.e. the furan-2,5-dicarboxylic acid, seem particularly relevant because they have the potential to replace petrochemically derived terephthalic or isophthalic acid in current industrial products. The furan-1,6-diamine has similar potential as a substitute for pphenylenediamine. Indeed, a series of such furan polyamides has been prepared [152] using the dicarboxylic acid and aliphatic and aromatic diamines. Of these, the polyamide resulting from condensation with p-phenylenediamine, which de facto is an analog of the commercially introduced Kevlar, has particularly promising decomposition and glass temperature properties [153], distinctly better than those of the all-furan polyamides:


Despite of the impressive array of highly useful products – their application profiles compare favorably with those of well known commercial polyamides – none of these sugar-derived polyamides is currently produced on an industrial scale; the reasons are purely economic, because the products derived from fossil raw material sources are still cheaper, on average by a factor of five. Eventually, however, with the end of cheap oil predicted for 2040 [1], and the pressure on our environment increasing, this untoward situation for products from carbohydrate feedstocks will change.

1.4.7.4 Sugar-based Olefinic Polymers (“Polyvinylsaccharides”) The synthesis of sugars carrying O-linked residues with polymerizable double bonds (“vinylsaccharides”) and their radical or cationic co-polymerization has been extensively pursued over the last 50 years, the first example apparently going back to Reppe in 1939 [154]. The following list (Fig. 1.7) gives relevant examples of the huge number of monosaccharide-derived “olefins” that have been prepared and subjected to polymerization as such, and to co-polymerizations with a variety of the petroleum-based standard monomers (methyl methacrylate, methyl acrylate, acrylonitrile, styrene, etc.). The products obtained invariably consist of a C–C backbone with pendant sugar residues:

Because the sugar groups along the carbon chain may be freed from their Oprotection by smooth simple reactions, highly hydrophilic, in most cases watersoluble polymers are obtained. These are not accessible from the respective monomers with free OH groups, because these usually give polymers of low molecular weight only, due to the increasing insolubility of oligomers.

47

48


Fig. 1.7 Selected examples of pyranoid, furanoid, and acyclic sugar derivatives carrying ether-, ester-, amide- or C–C-linked residues with polymerizable C = C double bonds. Their polymers and various copolymers with styrene, acrylonitrile, and/ or methacrylate have been characterized.

Sucrose, the most readily accessible bulk-scale sugar, has received particular attention in this context. A large variety of mono- and disubstituted ethers and esters have been prepared – mostly not clearly defined with respect to attachment of the residues to one or two of the eight OH groups – and subjected to radical or ionic homo- and co-polymerizations – acryloyl, and methacryloyl esters, styryl ethers, ethers with primary amino groups, etc. [162–165]. Some of the respective polymers are depicted schematically in Fig. 1.8.

1.5 Conclusion

Fig. 1.8 Idealized structures of a linear polymer resulting from radical polymerization of a mono-O-methacroylsucrose (left) and a 1 : 1 copolymerization product with styrene. Di-O-substituted “vinylsucroses” are deemed to lead to crosslinked polymers (right).

Despite the broad and versatile application profiles of these polymers, mainly because of their enhanced hydrophilicity (compared with their hydrophobic petroleum-derived counterparts), none seems to be produced commercially; one reason is that only the sugar portion of these polymers is biodegradable, leaving an extended carbon chain. Because biodegradability is currently a major issue [137], these polyvinylsaccharides are unlikely to become petrochemical substitution options in the near future.

1.5 Conclusion

The non-food utilization of inexpensive, bulk scale-accessible, low-molecularweight carbohydrates – sucrose, glucose, xylose and fructose being the most readily accessible – is at a rather modest level in terms of large-scale manufactured commodities currently on the market. The unusually diverse stock of readily accessible products described in this account, which cover a wide range of industrial application profiles, lies mostly unexploited – for economic reasons mainly, because equivalent products based on petrochemical raw materials are distinctly cheaper. Notwithstanding, a basic change in this scenario is clearly foreseeable. As depletion of our fossil raw materials progresses, chemical products derived from them will inevitably increase in price, such that biobased products will become competitive. Realistic predictions [1–3] expect this by 2040 at the latest. In the meantime, it is imperative that carbohydrates are systematically further exploited toward efficient, environmentally benign, and economical process methods for their large-scale conversion into industrially viable products, whether bulk, intermediate, or fine chemicals, pharmaceuticals, or polymeric organic materials. General conceptual formulations toward this goal are available [4, 5, 166], yet their straightforward implementation is currently exceedingly slow. To enhance this, it is essential that national and supranational funding institutions – in Europe the corresponding EU bodies (in the European Commission’s 7th framework program?) and/or the European Renewable Resources and

49

50


Materials Association [167] – play a substantially more dynamic role than hitherto, e.g. by the elaboration of a conclusive funding strategy for the material utilization of biorenewables rather than for biofuels and bioenergy only – a funding strategy capable of providing and maintaining a judicious balance between applied and basic research. Applied research ordinarily has predefined goals usually applying known scientific facts to the solution of practical problems, and has constraints that require results in a short time. It certainly should be funded and implemented on a broad basis, yet its short-term pressures, e.g. the expectation of obtaining marketable products within a three-year time frame, are often counter-innovative and tend to dodge research projects too complex to be solved in a few years. Basic (fundamental) research, in contrast, is driven by the interest and curiosity of an investigator usually following his intuition, be it an unresolved phenomenon, conflicting literature data, or the unconstrained exploration of a new inter-

Fig. 1.9 Ronald Searle’s notion [172] of miners searching for gold with conventional means along much-(re)searched main roads.

References

face between existing methods; it frequently creates its aims and specific targets along the way. Basic research of this sort inspires project proposals “for which it is infeasible or impractical to express a performance goal in an objective, quantifiable, and measurable form” [168]. Because Europe’s national granting agencies, e.g. the FNR [169] or AGRICE [170], do not have provisions enabling funding for such proposals, they have no chance of being supported [171]. Nevertheless, it is the author’s firm conviction that new, industrially viable, sugar-based chemicals and materials are likely to emerge as much from basic research as from mission-oriented applied investigations. Thus, there is an urgent need to create an instrument in our granting agency system that gives innovative basic research projects an equal chance of support. In this context, a pertinent cartoon (Fig. 1.9) seems to catch the essence of the present scenario – “biogold”-bearing seams are not only to be found along the common, much (re)searched main roads with conventional, generally applied means, but also – maybe even more likely – in remote, virgin, basic territory with unconventional, innovative methodology, and a healthy measure of intuition and serendipity. For full utilization of the “biogold” lying in carbohydrates – widely available from the plant kingdom and easily recovered at low cost – any promising, innovative research project, irrespective of involving mission-oriented, applied investigations or non-predefined basic explorations, should receive generous support either by funding institutions or by the chemical industry and/or both. Economically sound biobased alternatives to petrochemicals – various potential examples are contained in this review – will then become available as a matter of course.

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characterization of polymers containing linear sugar moieties as side groups”, Eur. Polymer J. 2001, 38, 273–280. V. Kollonitsch, Sucrose Chemicals, The Internat. Sugar Research Foundation, Washington, D. C., 1970. H. Gruber, G. Greber, “Reactive sucrose derivatives”, in Carbohydrates as Organic Raw Materials, F. W. Lichtenthaler, ed., VCH, Weinheim, 1991, pp 95–116. D. Jhurry, A. Deffieux, M. Fontanille, I. Betremieux, J. Mentech, G. Descotes, “Linear polymers with sucrose sidechains”, Makromol. Chem. 1992, 193, 2997–3007. (b) E. Fanton, C. Fayet, J. Gelas, A. Deffieux, M. Fontanille, D. Jhurry, “Synthesis of 4-O- and 6-Omonoacryloyl derivatives of sucrose. Polymerization and copolymerization with styrene”, Carbohydr. Res. 1993, 240, 143–152. D. Jhurry, A. Deffieux, “Sucrose-based polymers: polyurethanes with sucrose in the main chain”, Eur. Polym. J. 1997, 33, 1577–1582. M. Eissen, J. O. Metzger, E. Schmidt, U. Schneidewind, “Concepts on the contribution of chemistry to a sustainable development”, Angew. Chem. 2002, 114, 402–424; Angew. Chem. Int. Ed. 2002, 41, 414–436. Errma, http://www.errma.com US Government Results Act, 1993; http://www.whitehouse.gov/omb/ mgmt-gpra/gplaw2m.html Fachagentur Nachwachsende Rohstoffe; http:///www.fnr.de Agriculture pour la Chimie et l’Energie; http://www.ademe.fr/partenaires/ agrice/htdocs/present01.htm The sugar-based projects presently funded by the FNR qualify, without exception, for mission-oriented applied research. R. Searle, From frozen north to filthy lucre, Viking Press, New York, 1964.

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61

2 Industrial Starch Platform – Status quo of Production, Modification and Application Dietmar R. Grüll, Franz Jetzinger, Martin Kozich, Marnik M. Wastyn, and Robert Wittenberger

2.1 Introduction

Starch is the most abundant storage carbohydrate and is generated in many plants by photosynthesis. The plant assimilates carbon dioxide from the atmosphere and transforms it into glucose, the basic molecule for the synthesis of starch. These molecules are stored in tubers, roots, seeds, and fruits. Starch is composed of small granules which are insoluble in cold water. The sizes of these granules is characteristic of the origin of the starch and varies from 1 to more than 100 lm. 2.1.1 History of Starch

Although the early history of starch use is mainly unrecorded, some interesting examples of its industrial uses are known. Starch paste was used by the ancient Egyptians to cement strips of papyrus stem together for use as writing paper as early as 4000 B.C. In a treatise Cato described a process used by the Romans for separating starch from grain in 170 B.C., and 300 years later Celsus, a Greek physician, described starch as a wholesome dietary product. Pliny the Elder (23– 74 A.D.) stated that the Romans used wheat starch to make papyrus documents, to stiffen and whiten cloth and to powder their hair. In his Greek Herbal, Dioscorides (1st century A.D.) recommended the use of wheat starch for the treatment of ulcers, sores and eye inflammations. Around 312 A.D. starch was shown to provide resistance to ink penetration in Chinese paper, so starches from rice, wheat and barley came to be commonly used at that time. In 975 A.D. Abu Mansur, an Arab pharmacologist, described a method to treat wounds with an artificial honey made by mixing starch with saliva. Starch was used in northern Europe to stiffen linen, possibly as early as in the 13th to 14th centuries. Colored starches were used as cosmetics, and uncolored starches were priBiorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 2 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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2 Industrial Starch Platform – Status quo of Production, Modification and Application

marily used as hair powder. The Puritans used blue starch until it was banned by Queen Elizabeth in 1596. 2.1.2 History of Industrial Starch Production

The most common sources of starch are maize, potato, wheat, tapioca, and rice. Starches are isolated by wet grinding of the grain or tuber, followed by wet sifting, centrifugation, and drying. Wheat starch is an exception in that it may also be extracted from flour. These steps remove most of the other plant components, for example proteins, oil, and fiber. Because of their availability, wheat and barley were the first sources of starch to be commercially wet-milled. The first American wheat starch plant was founded in Utica, New York, in 1807. Wet milling of potato starch began in Hillsborough County, New Hampshire, in 1824, and in Europe at about the same time in Foxhol, The Netherlands, where Willem Albert Scholten founded his first potato-starch mill. Wet milling of corn began in 1844, when the Colgate wheat starch mill in Jersey City, New Jersey, was converted to processing corn using Thomas Kingford’s alkaline purification. 2.1.3 History of Starch Modification

Searching for substitutes for gum Arabic and tragacanth, Bouillon-Lagrange first reported the production of dextrin by roasting starch in 1804. The industrial use of dextrins, however, only became widespread after the accidental discovery of the torrefaction method for producing dextrins (now called British gums). In 1821 a fire broke out in an Irish textile factory where starch was used for sizing the yarns. After the fire was extinguished it was found that a part of the stored starch had turned brown due to the action of the heat and was soluble in cold water. So it could be used to produce a thick, adhesive paste. As access to cane sugar from the West Indies was limited during the Napoleonic wars, alternative technologies were investigated to produce sweeteners. In 1811, Kirchoff discovered that sugar could be produced from potato starch by hydrolysis with sulfuric acid. Concurrent with the opening of large potato starch mills, the use of sugar from starch in wines began in Germany in 1830. In 1874 Naegeli found that partial acid hydrolysis of starch granules produced a residue containing short linear molecules. His products and those of Lintner twelve years later formed the basis for today’s fluid starches. Bellmas in Germany and Duryea in the US both filed patents for acid-thinned starches at the beginning of the 20th century.

2.2 Raw Material for Starch Production

2.2 Raw Material for Starch Production

Virtually all plants contain carbohydrates. We are primarily interested in those plants that store these carbohydrates in the form of starch granules which can be extracted. Worldwide, starch is mainly derived from cereals, but it is also obtained in significant quantities from tubers and roots, especially in Europe and Asia. The main sources of starch are maize, wheat, potatoes, rice, and cassava, from which tapioca starch is derived (Fig. 2.1). Many other crops are also used for starch production, such as sorghum, sweet potato, arrowroot, taro, barley, oat, rye, peas, beans, lentils, and yam, but the quantities are small. Even exhaustive research on agronomic and phenotypic properties of tropical starch crops (e.g. arrowroot, sago, taro, sweet potato, and yam) has not made them competitive on an international scale. As a result, maize, wheat, potatoes, and tapioca dominate world markets for starches in both food and non-food industries. Worldwide, maize is the dominating crop for starch production, in particular in the US, where, apart from maize starch, only 1% of wheat starch and minor amounts of potato starch are produced. In 2000, the US starch production amounted to 24.6 million tons. The primary starch crops in the EU, including the new member states, are maize, wheat, and starch potatoes, so the European starch industry is more widely diversified in its raw material sources than other

Fig. 2.1 Starch production by raw material, worldwide, in 2000.

Fig. 2.2 Starch production by raw material in the EU in 2000.

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Fig. 2.3 Starch production worldwide in 2000.

Fig. 2.4 Development of EU starch production.

starch industries elsewhere. The different raw materials each supply a large share of total starch output, as shown in Fig. 2.2. In 2000, the world starch market was estimated to be approximately 48.5 million tons, of which the EU holds a share of approximately 17% or 8.5 million tons (Fig. 2.3). During the past 20 years the quantities of starch produced and the demand in food and non-food areas have increased continually. Whereas the increase worldwide was about threefold (Fig. 2.5) in the past 20 years, the increase in the EU was some 2.5-fold (Fig. 2.4); this was mainly based on an increase in maize and wheat-starch production capacity.

2.3 Industrial Production of Starch

Fig. 2.5 Development world starch production.


Starch production technologies depend on the type of raw material, because there are some basic differences between cereals, roots, and tubers. Due to its suitability for large-scale storage, cereal-starch may be produced throughout the year, whereas tuber and root-starch is produced only immediately after harvest. The composition of the most important raw materials used as sources of starch is summarized in Table 2.1.

Table 2.1 Composition of starch raw materials (median values).

Moisture (%) Starch (%) Protein (%) Minerals/Ash (%) Sugars (%) Fiber (%) Fat (%)

Maize

Potato

Tapioca

Wheat

Rice

16 62 8.2 1.2 2.2 2.2 4.2

75 19 2 1.2 1.1 1.6 0.1

70 24 1.5 2 0.5 0.7 0.5

13 60 13 1.7 8 1.3 3

14 77 7 0.5 0.3 0.3 0.4

65

66


2.3.1 Maize and Waxy Maize

As starch has been produced industrially from yellow maize for more than a century, the process is technically well developed. Typically maize starch factories have a daily processing capacity of 500–2000 tons of maize. The production of maize starch (Fig. 2.6) starts with steeping purified maize kernels. This means that maize is not just swelling in water, which has a temperature of about 50 8C and a pH of 3.8–4.2, but these conditions cause a controlled lactic acid fermentation and loosen the protein matrix. Steeping is an important step, because the maize kernels absorb up to 45% (wet basis) of water, release solubles, absorb sulfur dioxide (0.2–0.4 g per kg) and become soft enough to be suitable for separation of its components. After steeping, the grains are coarsely ground in an attrition mill, and then the germs are separated in special hydrocyclones. The grinding and separation processes must be performed twice for complete removal of the germs. This is followed by fine grinding in a pin mill or impact mill in order to completely disrupt the cells. At this stage the starch is freely in suspension and is led over bend screen cascades for separation from fiber and other maize components. The separated residues are dehydrated and dried for use as an animal feed component referred to as maize feed. The crude starch milk still contains all the dissolved proteins. This fraction is called gluten, and most of it is separated off by means of two successive nozzle type continuous centrifugal separators. The gluten is dehydrated by means of a rotary vacuum filter, then dried and used as a feed additive similar to maize feed. The starch milk, which still contains approximately 2% of protein after separation, is then refined in a multi-step cyclone plant. The refined starch milk, having a residual protein content of approximately 0.3%, is dehydrated in peeler centrifuges and dried by means of a flash dryer. The residual water content of the starch at this stage is less than 14%, and the product is storable. In some cases the starch production plant is connected with saccharification and ethanol production plants. Waxy maize is a special form of yellow maize. Its starch contains more than 99% amylopectin. The production of waxy maize starch is similar to normal yellow maize starch. 2.3.2 Wheat

Wheat starch can be produced either from wheat flour or by wet milling of wheat grain. Industrial production of wheat starch is almost exclusively from wheat flour. There are a large number of processes which are basically similar but differ in the means of producing and further processing the wheat dough. In the first step the wheat flour is stirred with water in special mixing machines to form a dough or a suspension (Fig. 2.7). Then the wheat paste is separated


Fig. 2.6 Maize starch production.

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68


Fig. 2.7 Wheat starch production.

in extractors, decanters, or in a hydro-cyclone plant. Fiber and gluten residues are subsequently removed by sieving through centrisieves. Wheat starch is characterized in that it is comprised of two fractions of different granule size; these are obtained separately while producing the starch. The bigger wheat starch granules of 20–50 lm in diameter are called prime starch and comprise 90% of the starch. This starch is refined by means of separators or a multi cyclone plant; normally the product obtained contains less than 0.3% of residual protein. The second-grade starch, which comprises the smaller granules of 2–15 lm in diameter, is purified by means of separators. It has a higher protein content and is contaminated with pentosans and hemicelluloses. Dehydration and drying of prime starch is similar to the process in the production of maize starch. The paste fraction is usually dried with a ring dryer.


2.3.3 Potato

Potato is the most important tuber used for industrial isolation of starch. It has the advantage that the extraction process is simpler than that used for cereals, because it is not necessary to swell or mill them or prepare a dough. The disadvantages are the lower starch content of tubers and the large quantity of fruit water produced, which is the main cause of sewage problems arising from potato starch plants. Processing (Fig. 2.8) starts after purification of the tubers by rasping by means of a rotary saw blade rasp, in which potatoes are converted into a mash. This process results in a nearly complete disruption of the potato cells, which therefore release the starch. Because of this disruption technique, starch yields in modern potato starch plants are in excess of 97%. Sulfur dioxide is added to the rasped potatoes to avoid discoloration, which is a result of the action of phenol oxidases.

Fig. 2.8 Potato starch production.

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70


In modern plants the high protein fruit water is separated off by means of decanters before the starch is extracted. This reduces the problem of sewage, and the valuable potato protein may be separated. The soluble proteins are coagulated by treatment with acid and heat and then separated in decanters. The starch in the rasped potatoes is isolated by means of centrisieves, yielding 99% of pure starch. The remaining pulp is drained or pressed off and used directly as feed while still damp or dried in flash dryers. The dilute starch milk had to be concentrated in separators or multi cyclone units prior to the next step of processing. Potato starch is refined in several steps in hydro cyclone plants. The starch is dewatered by rotary vacuum filtration and subsequently dried in a flash dryer. 2.3.4 Tapioca

Tapioca starch is a root starch which is produced from the roots of manioc or cassava plants. Its starch content is about as low as that of potato. One disad-

Fig. 2.9 Tapioca starch production.

2.4 Properties of Commercial Starches

vantage is that the roots are not storable. Therefore they have to be processed within 24 h of harvesting. Another problem is that the roots contain the glycoside linamarin, from which prussic acid may emerge during decomposition. Most of the steps of industrial tapioca starch production are similar to those of potato starch production (Fig. 2.9). After purification the roots are first cut into potato-size pieces with a crusher and then rubbed with a rotary saw blade rasp, which is the same as that used for potato rasping. The starch is extracted in centrisieves. The steps of concentration and refining correspond to those in potato-starch production. The only difference is that finer sieves are used because the starch granules are smaller in size. Like maize starch, tapioca starch is dewatered with peeler centrifuges and subsequently dried. 2.3.5 Other Starches

Apart from the main starch sources described above, there are a number of starch containing plants of less importance for industrial application. They are, however, worth mentioning because of their often very interesting properties. Sweet potatoes, for example, are used for starch production in Japan and are processed in the same way as normal potatoes. In the field of cereal starches, rice and millet are used regionally for industrial starch production, whereas rye and barley are rarely utilized because of their lower availability, their low starch content and their difficult production. Rice starch is usually produced from broken rice. Rice is first steeped under alkaline conditions and then ground and washed through a sieve. The separated fiber fraction is used as feed. The starch milk is separated from the paste by means of separators, dewatered in centrifuges and subsequently dried. Millet starch production is similar to the maize starch process. Therefore, it can be produced in maize starch plants, although an adaptation of the plant is necessary, because the millet starch granules are smaller and therefore require the use of smaller sieves and some changes in the milling process. The starch from different kinds of peas, beans and tropical plants such as banana, shoti roots and curcuma has been investigated because of its scientifically interesting properties. The production of so-called “tropical starch” is still in the experimental stage although it has been ascribed great potential in the future.


As starch is a polymer synthesized in plants, it has different physical and chemical properties depending on its origin. All starches are synthesized in the plant from glucose molecules and stored as water-insoluble granules in several different parts of the plant. During the starch synthesis two types of polysaccharide, namely amylose and amylopectin, are generated at a ratio of approximately 1 : 3

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Fig. 2.10 Molecular weight distribution of different starches (determined by SEC).

in most kinds of starches. Amylose is a largely linear molecule built from 1,4 linked a-D-glucopyranosyl units having an average molecular mass of 1 ´ 105 to 1 ´ 106. Amylopectin is different from amylose in that it has both a-1,4 and a1,6 bonds and a higher molecular mass of 1 ´ 107 to 1 ´ 108. The molecular mass varies substantially, depending on the method of isolation and determination. A method for the determination of the molecular mass of the polymers is a liquid chromatographic method called size-exclusion chromatography (SEC), in which the molecules are separated according to their hydrodynamic volume. Figure 2.10 shows the molecular mass distributions of six different types of starch. Because of these different structures, the two fractions of starch have different specific properties. Amylopectin is responsible for the partial crystallinity of starch, with the crystalline properties resulting from clusters of layers of the amylopectin molecules. The crystalline units are separated by amorphous layers. When studied by light microscopy with polarized light, these layers appear as Maltese crosses because of the optical properties of the starch granules (Fig. 2.11). These optical properties enable assessment of whether or not the starch granules are intact, because they are lost after e.g. thermal treatment. The shape and size of starch granules can be measured microscopically, which allows identification of the origin of the starch. The shape of the granules is difficult to describe, because it is never uniform. The shapes often range from round to oval, oblong, kidney-shaped, edged, polyhedral, and rosetteshaped. The size of the granules is between 1 lm and approximately 100 lm, and each type of starch usually contains a broad distribution (Table 2.2). Ama-


Fig. 2.11 Maltese crosses of potato starch in polarized light.

ranth and rice starch are examples of starches having small granules, potato starch is an example of a large granule starch. Diffraction patterns obtained by X-ray irradiation of starch allow identification of different types of starch and further classification of the material. Cereal starches have been assigned to type A, root and tuber starches to type B, and mixed forms (e.g. legume starch) to type C. The diffraction patterns result from different degrees of polymerization of the amylopectin side chains, resulting in double helix chains of different pack density. In type B, the double helices are less closely packed than in type A, in which more water can be stored, giving a different diffraction pattern. Commercially produced starch contains several contaminants, the amounts and types of which are generally dependent on the type of starch plant but also on the quality of the production process. Because the properties and quality of the starch can be severely affected by these substances, attempts are being made to reduce the total amount to less than 1%. This is usually achieved in a modern production plant. Typical accompanying substances are proteins, lipids, and salts. Phosphorus is especially important, because it is bonded to starch in the form of a salt of phosphoric acid or, in potato starch, an ester. This bonding to the hydroxyl groups of starch strongly affects the properties of the starch, particularly potato starch. Potato starch has greater swelling properties, stronger anionic character and forms a paste of clearly higher viscosity stability. The main minerals in starch are sodium, potassium, calcium, and magnesium.

73

a) b) c)

[8C]

[BU]

[%] d.s. [%] d.s. [%] d.s. [J/g]

[lm]

[%] [%] d.s. [%] d.s.

Bimodal distribution Resulting mainly from salt Resulting mainly from phospholipids

Swelling power

Brabender peak viscosity (concentration) Gelatinization temperature

Particle size range (median value) Protein content Phosphorus Ash DH (DSC)

Source Moisture Amylose Amylopectin Crystal structure Particle shape

24

870 (6.25%) 62–80

Maize 200 Extractives: alcohol–benzene extract

Table 3.3 presents the compositions of different types of straw. Straw species are more uniform in composition than wood species. Straw usually has a lower cellulose content than wood, but despite this it has a total carbohydrate fraction (holocellulose) approximately equal to that of wood. This is because of its high hemicellulose and low lignin content compared with wood. The ash content of straw is higher than that of wood [57]. Differences also arise as a result of different investigation and pulping methods [58, 59]; because of this complexity, it is still difficult to determine specific group composition. The data shown in Tables 3.3 to 3.6 are collected from different sources. Table 3.4 Chemical composition of different types of American straw a) [57]. Name (raw material)

Latin name of plant

Lignin (%)

Alphacellulose b) (%)

Hemicellulose pentosans only (%)

Extractives c) (%)

Ash (%)

Rice straw Barley straw Wheat straw Rye straw Oat straw Flax shives Soybean stalks

Oryza Hordeum vulgare Triticum aestivum Secale cereale Avena sativa Linum Glycine max

11.9 14.5 16.7 19.0 17.5 22.3 19.8

36.2 33.8 39.9 37.6 39.4 34.9 34.5

24.5 24.7 28.2 30.5 27.1 23.6 24.8

4.6 4.7 3.7 3.2 4.4 4.1 3.9

16.1 6.4 6.6 4.3 7.2 3.5 2.3

a) b) c)

As a percentage of dry matter Alpha-cellulose in 17.5% NaOH or 24% KOH solution, insoluble part with degree of polymerization > 200 Extractives: alcohol–benzene extract

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3 Lignocellulose-based Chemical Products and Product Family Trees Table 3.5 Ranges of variation of the chemical composition of different lignocellulosic feedstock [50]. Feedstock

Softwood Hardwood Cereal straw Maize straw Rape straw Recovered paper

Cellulose (%)

40–48 30–43 38–40 35–41 38–41 50–70

Hemicellulose (polyoses) Hexoses (%)

Pentoses (%)

12–15 2–5 2–5 2 – –

7–10 17–25 17–21 15–28 17–22 6–15

Lignin (%)

26–31 20–25 6–21 10–17 19–22 15–25

Table 3.6 Composition of selected biomass materials a) [60]. Feedstock

Cellulose (%)

Hemicellulose (%)

Lignin (%)

Tarnbark oak Corn stover Red clover hay Bagasse Oat hulls Newspaper Processed MSW b)

44.8 36.5 26.7 41.3 33.7 61.0 47.0

19.6 28.1 20.6 20.4 20.5 16.0 25.0

24.8 10.4 15.1 14.9 13.5 21.0 12.0

a) b)

As a percentage of dry matter Municipal solid wastes

3.3.2.3 Carbohydrates in Lignocelluloses Lignocellulosic feedstocks (LCF) consist of 65 to 85% carbohydrates, mainly polysaccharides. Linear and branched polymers of glucose are summarized as so-called glucan (polyglucan); cellulose also belongs to group of polysaccharides, as also do amylose, laminatin, and lichenin. Hemicelluloses mainly consist of polysaccharides such as mannan, xylan, and galactan. Arabinan, polymeric acetates, acetate derivatives of hemicellulose, and polyuronoic acid (polymeric aldehyde acids) are also included as carbohydrate building blocks (Fig. 3.9 [61, 62]). Mannan and xylan are the most common polysaccharides of polyoses. Mannanes are polyoses (hemicelluloses) built from mannose instead of glucose subunits. The chains consist of 1,4-linked mannose monomers forming a b-pyranose. Xylanes (C5H8O4) are polyoses (hemicelluloses) that are part of many trees, cereals, straw, glumes, bran, pectin, tragant, and, in common continental plants, mostly as heteroxylanes. Xylanases are special hemicellulases (enzymes) that can hydrolyze xylane into xylose and other pentoses. Such xylanases are generated by fungi. Genotechnologically modified Zymomonas mobilis bacteria can ferment xylane into ethanol [63]. Galactanes are polysaccharides (glycanes) that are mainly composed of galactoses. Arabinanes (C5H8O4)n are polyoses of

3.3 Lignocellulosic Raw Material

109

Fig. 3.9 Carbohydrates in lignocelluloses.

Table 3.7 Carbohydrate composition of different lignocellulosic feedstock a) [50]. Component plant name

Glucan (%)

Mannan (%)

White wood fir, Abies alba Douglas fir, Pseudotsuga menziesiicaesia Oak, Quercus Aspen, Populus tremula Maize cob Wheat straw

46.5

11.6

43.46

a)

Xylan (%)

Galactan (%)

Arabinan (%)

Total carbo- Lignin hydrates (%) (%)

6.8

1.2

1.6

67.70

26.7

10.76

2.77

4.66

2.67

64.32

31.30

40.63 45.97

1.97 2.10

19.19 17.74

1.22 7.9

0.36 1.23

63.37 67.83

23.91 20.30

34.0 37.0

0.5 0.3

14.0 18.9

1.0 6.5

1.7 5.6

51.20 62.30

13.1 13.6

As a percentage of dry matter

linear a-(1,5)-glycosidic linked l-arabinofuranoses with single l-arabinose units connected via a-(1,3)-glycosidic bonds to the main chain [62]. The amount of the different carbohydrates in lignocelluloses varies very much depending on wood type and age (one-year-old plants compared with older trees) (Fig. 3.8 and Table 3.7).

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3 Lignocellulose-based Chemical Products and Product Family Trees

3.4 Lignocelluloses in Biorefineries 3.4.1 Background

An important aspect of utilization of biomass as a chemical raw material is the cost of corresponding raw materials. In 2002, approximate raw material prices were: US $ 30 t–1 for corn stover or straw (LCF); US $ 3 bushel–1 or US $ 110 t–1 for corn or grain [64]. The objective of all hitherto introduced processing methods based on lignocellulosic feedstocks is to produce fibers, paper, pulp, or sugar. Because currently only the fraction is cellulose used, however, most of the valuable biomass raw material is lost. The following two examples should help explain this in more detail.

3.4.1.1 Example 1 Is an entire tree (from roots to treetop) as biomass is processed into pulp a significant weight loss is observed. After pre-treatment and pulping more than 70% of the original mass is lost. Before arrival at the plant the “biomass tree” will already have lost 40% of its original mass; a further 30% will be lost during the pulping process [65].

3.4.1.2 Example 2 Table 3.3 shows the composition of wheat straw [52]; none of the three main components cellulose, hemicellulose, or lignin is dominant. So, economically all three components must be processed to be efficient. The following conclusions could therefore be drawn. · Every plant, every feedstock, is a raw material that must be converted entirely. · All components of such a raw material are of special value and therefore must be treated within a complex integrated process.

There are still many problems to be solved, e.g. with regard to conversion of LCF into precursors. It is still the problem that one or more components may be destroyed or changed in structure during conversion. Another unsolved problem is that lignin still is not really used but regarded as waste. To solve the problem completely it will be necessary to learn from more than 100 years experience with and efficiency of petroleum-based chemistry. Petroleum refineries deliver well-defined, chemically-pure basic chemicals (building blocks) that are easily handled. On the basis of these simple building blocks, more complex intermediates can be generated in a controlled manner by chemical reactions. Because of an almost countless number of combinations, those intermediates then can be converted into a broad variety of even more complex intermediates and/or final products. The principles of the highly efficient fossil-based chemical industry – specific product lines, building blocks, product family trees, commodity chemicals, so-

3.4 Lignocelluloses in Biorefineries

called coupling production processes and backwards integrated production – should be transferred and adapted appropriately to bio-based chemistry. By following this approach one ends up with the concept of biorefineries [7, 66–73], integrated biobased “platforms” and building block structures [74, 76]. The term biorefinery may, in general, be defined as follows [72]: A biorefinery is an entire integrated complex concept of a processing plant in which biomass feedstocks are extracted and converted into a broad spectrum of valuable products analogous to petrochemical refineries. 3.4.2 LCF Biorefinery

A lignocellulosic biorefinery (LCF biorefinery) converts lignocelluloses into fuels, chemicals, polymers, and many other materials. A general scheme of a biorefinery is shown in Fig. 3.10. Here, all thermo-chemical processes, for example combustion, gasification, and pyrolysis (syngas platform) are considered solely as utilization of waste or residual materials. An LCF biorefinery is, analogous to most biorefineries, a rather complex and integrated system of conventionally used technology and more recent, modern processes. Complex technology resources are (with others): · the processing of cereal waste into furfural [39, 40], e.g. the well-known production of flakes and furfural from oats [English Patent (E.P.) 203,691 (1923), French Patent (F.P.) 570 531 (1923)] an industrial-scale process of Quaker Oats, Illinois, USA [77, 78] · commercial levulinic acid production using hexoses of low-cost cellulose products; in 1956 levulinic acid was regarded for the first time as a “platform chemical substance” of high synthetic potential [79–81]; and

Fig. 3.10 General scheme of a lignocellulosic biorefinery.

111

112


Fig. 3.11 The general equation of the conversion in the LCF-biorefinery.

· the complex technological approaches of wood processing according to Timell 1961 [82], James 1969 [83], or Prink and Pohlmann 1972 [84], the straw processing methods according to Puls and Dietrich 1980 [85], or the studies of Shen 1982–88 in the field of wood grass processing [86]. Lignocellulose materials consist of three primary chemical fractions or precursors: 1. hemicellulose/polyoses, sugar polymer of predominantly pentoses, 2. cellulose, a glucose polymer, and 3. lignin, a polymer based on phenol subunits. A general equation for the conversion of LCF precursors into “intermediate platforms” sugar (xylose, glucose) and lignin is given in Fig. 3.11 [7, 68, 71, 87] and an overview of the potential products of an LCF biorefinery is shown in Fig. 3.12.

Fig. 3.12 Products of lignocellulosic feedstock biorefinery (LCF-Biorefinery, Phase III) [7, 71, 87].


Furfural and hydroxymethylfurfural are particularly interesting products. Furfural is the starting material for the production of Nylon 6,6 and Nylon 6. The original process for production of Nylon 6,6 was based on furfural. The last of these production plants was closed in 1961 in the United States for economic reasons (low price of petroleum). Nevertheless, the market for Nylon 6 is huge. There are, however, still some problems to be solved in the LCF process, e.g. utilization of lignin as a fuel, adhesive, or binder. This still is unsatisfactory, because the lignin scaffold contains substantial amounts of monoaromatic hydrocarbons which, if isolated economically and efficiently, could add significant value to the primary process. It should be noted that no natural enzymes have yet been found that can split naturally formed lignin into basic monomers as easily as can occur for carbohydrates or proteins. 3.4.3 LCF Conversion Methods 3.4.3.1 Pretreatment Methods Several physical and/or chemical methods can be used for pre-treatment of lignocelluloses, especially to separate cellulose from its protective sheath of lignin and increase the surface area of the cellulose crystallite by size reduction and swelling [88]: 1. Physical pre-treatment [89] a) Milling and grinding b)High-pressure steaming and steam explosion c) Extrusion and expansion d)High-energy radiation e) Pyrolysis 2. Chemical methods [57, 90, 93] a) Alkali treatment (e.g. NH3, NH4SO3, NaOH) b)Acid treatment (e.g. H2SO4, HCl, H3PO4) c) Gas treatment (e.g. ClO2, NO2, SO2) d)Oxidizing agents (e.g. H2O2, O3) [92] e) Cellulose solvents (e.g. Cadoxen, CMCS) [93] f) Solvent extraction of lignin (e.g. ethanol–water, benzene–ethanol, ethylene glycol, butanol–water) g) Swelling agents [94]. 3. Biological methods [90 b, 95–97] a) Lignin-consuming microorganisms (fungi, e.g. Phanerochaete chrysosporium; bacteria, e.g. Nocardica sp.) b)Cellulose-attacking microorganisms (fungi, e.g. brown rot) c) Lignin and cellulose-attacking microorganisms (fungi, e.g. white and red rot) d)Lignin and/or cellulose attacking insects (e.g. termites).

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3.4.3.2 Chemical Pulping Methods Chemical and chemical–thermal pulping processes of lignocelluloses can be divided into two main groups [50, 57 b, 90 c, 91]: 1. Processes with lignin and cellolignin as residues a) Steam/pressure processes (autohydrolysis) b)Hydrolysis with weak acids c) Hydrolysis with concentrated acids or water-free acids 2. Processes with cellulose and cellulose/hemicellulose as residues a) Alkaline pulping b)Sulfate pulping c) Sulfite pulping d)Organosolv process e) Phenol process (Battelle) f) Extraction with H2O (pressure, high temperature).

Figure 3.13 shows combined steam alkali pulping of lignocellulose. All lignocellulose feedstock contains the three components lignin, hemicellulose, and cellulose. There are, however, differences between the way and the extent these compounds are chemically and physically connected. There are also differences between the reactivity of the components, depending on the pulping reagents used. There is, nevertheless, a demand for enhanced selectivity of the process which, in the end, means reducing yields and often also changing the characteristics of the target product (e.g. degree of polymerization, mechanical

Fig. 3.13 Combined steam-alkaline pulping of lignocelluloses.


strength, etc.). Therefore, all processes are a compromise focused on the final product.

3.4.3.3 Enzymatic Methods Biodegradation of untreated native lignocellulose is a very slow process, giving rise to little degradation, often below 20% [85, 98–100]. Thus, the low rate and extent of conversion inhibit the development of an economically feasible biotechnological process for hydrolysis of lignocelluloses. To increase the susceptibility of cellulosic material, structural modification by means of various pretreatment processes is essential (see above, “Pretreatment methods”). Enzymatic hydrolysis of pre-treated lignin-free cellulose (obtained from lignocellulose) is technically feasible. Enzymatic hydrolysis of cellulose accomplishes degradation of cellulose to glucose. Glucose is an important sugar building block for chemicals and fuels [8]. Enzymatic hydrolysis as a heterogeneous catalytic reaction is typically characterized by an insoluble reactant (cellulose) and a soluble catalyst (enzymes) [57 b]. Several comprehensive reviews have been published on the mode of attack by cellulases on crystalline cellulose. The rate of this reaction is affected by both structural features of the cellulose and the mode of enzyme action [90 e, 101–105]. Enzymatic hydrolysis processes are of interest because enzymes exclusively catalyze specific reactions (stereospecific and/or stereoselective reactions). Therefore, different from acidic hydrolysis, there are no side reactions or by-products and the hydrolysis can potentially be performed with yields approaching 100%. All enzymatic hydrolysis processes consist of four major steps that may be combined in a different ways – pretreatment, enzyme production, hydrolysis, and fermentation. During the last few years enzymatic hydrolysis processes have gained importance, particularly for ethanol production based on lignocellulose [87, 90, 91, 106–108] (Fig. 14).

Fig. 3.14 Enzymatic transformation of lignocelluloses into ethanol.

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3.5 Lignin-based Product Lines 3.5.1 Isolation and Application Areas

Current and future application of lignin are a broad field of increasing importance, as demonstrated during the last 20 years by an increasing number of worldwide scientific publications and patents on basic scientific knowledge and technological aspects. One reason for this development is an increasing appreciation of lignocelluloses as a renewable resource. Lignin as a raw material is still very far from intensive utilization, however, despite its high potential for several applications with regard to its chemistry, properties, and the large amounts available from pulp production or LCF biorefinery processes. During lignin isolation, there are mainly two problems to be solved – maintaining the natural lignin structure and achieving high yields. Usually, however, the chemical structure of the isolated lignin is changed to some extent. The principal isolation methods are shown Fig. 3.15 [34, 48, 54, 109 , 110–114]. Today, approximately 50 Mio tons alkalignin- and lignosulfonic acids are produced per year during pulp production, although only a very few percent of these acids are used. Combustion combined with recycling of chemicals is still the most efficient process (economically). Both sulfate and (increasingly) sulfite pulping processes include combustion (in combination with recycling). The heat generated covers 80 to 100% of the energy demand of the pulping factories (except bleaching processes). Although, there are several application areas for lignin

Fig. 3.15 Processes for isolation of lignin [109 a].

3.5 Lignin-based Product Lines

Fig. 3.16 Areas of application of different insulated and modified lignins [48].

and modified lignins; their market potentials would significantly increase in a biobased economy [34, 48, 50, 54, 110–118] (Fig. 3.16). 3.5.2 A Lignin-based Product Family Tree

Processes for substantial utilization of lignin can be divided into four groups: · utilization in polymeric forms: e.g. as adhesives for wood materials, cement additive for enhanced low-temperature durability and low-temperature resistance etc.; · utilization as polymer component: co-reactant for polymers and resins; · fractionation into low-molecular-mass particles and monomers: e.g. generation of vanillin from softwood lignosulfonates; production of dimethyl sulfoxide (an important solvent); and · complete degradation to gas, oil and coal by pyrolysis. Figure 3.17 shows a potential chemical lignin-based product family tree. Alkaline hydrolysis/oxidation is already technically established, e.g. vanillin production; residues are used as dispersants. Alkaline demethylation via sulfide is used for DMS production, which later could be used for DMSO solvent production [50, 119]. Up to 33% phenolic substances can be obtained by addition of NaS and NaOH at temperatures of 250 to 290 8C [120].

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Fig. 3.17 A lignin-based product family tree [48].

An interesting recently developed process is the catalytic pulping of wood in sodium hydroxide and water. By this method syringaldehyde, 3,4,5-trimethoxybenzaldehyde, vanillin, and levulinic acid are synthesized within one integrated biorefinery regime [121, 122]. It is also possible to obtain well-defined products such as phenols and cresols by catalytic hydrogenation/hydrocracking. Best known is the so-called Noguchi process [123]. Reports of investigations in the US reveal production of up to 38% monophenols and 7–8% higher phenols [50]. This demonstrates the potential of lignin as raw material particularly for synthesis of cresols more efficiently and less expensive than from coal or crude oil. Pyrolytic reactions and gasification, respectively, are analogous for lignin and coal processing. A major part of lignin hydrolysis and gasification is comparable with the corresponding woodprocessing steps. In addition, partial oxidation with oxygen can be performed to produce syngas (here, it must be considered that this process might lead to large amounts of ash). In very fast oxidation at 1200 8C with subsequent quenching up to 23% w/w acetylene is obtained [124]. In addition, lignin has an important potential as a co-reactant for synthesis of polymers and resins (phenolic compounds, furans, epoxides, urethanes, urea– formaldehydes, and others). Although well-known, many new developments and novel potential applications have been discussed for lignin-based polymers and resins [114, 116, 118, 125].

3.6 Hemicellulose-based Product Lines

3.6 Hemicellulose-based Product Lines 3.6.1 Isolation and Application Areas

The building blocks of hemicellulose (polyoses) are glucose, xylose, mannose, galactose, arabinose, and rhamnose. Polymeric polyoses consist of a specific combination of these building blocks. Hardwood and straw, for example, contain more xylanes (pentosans, degradation to pentoses); soft wood contains mainly glucomannanes [hexosans, degradation to hexoses] (see also Tables 3.5 and 3.7). Industrial or technical supply of pure hemicellulose fractions is still a very laborious process. Current wood pulping (sulfite pulping and corresponding pre-hydrolyzates) generate fractions of hemicellulose that are still contaminated with cellulose and/or lignin residues. Therefore, many applications are limited to a so-called “low-tech” sector, such as energy regeneration, production of feed yeast, adhesives for chipboards or fermentation to alcohol [50]. The latter is becoming increasingly important because improved processes for fermentation of hemicellulose pentoses have been developed in which ethanol yields are 30% higher than for conventional fermentation [104, 126]. Potential sources for almost pure hemicellulose fractions are: · pre-hydrolysis (acid hydrolysis), · precipitation component/solution component (Organosolv process, Batelle process), · steam-/pressure-processes in water, · aqueous extraction at higher temperature. To use the rich chemical and biotechnological potential of hemicelluloses, the hydrolysis of hemicellulose to xylose (pentose) and mannose (hexose) is preferentially used (Fig. 3.9). The resulting xylose, isolated and purified, could be used as a building block for subsequent reactions, for example oxidation, reduction, or esterification, to generate a wide array of technical products (Section 3.6.2, Fig. 3.18). 3.6.2 A Hemicellulose-based Product Family Tree 3.6.2.1 Mannan/Mannose Product Lines Figure 3.18 shows a technically feasible chemical/biotechnical hemicellulosebased product family tree in which chemical reactions are preferred. In the following text the hexoses of polyoses mannane/glucomannane are discussed only briefly because the LCF concepts are mainly based on utilization of pentose-rich plants (annual plants, grass, straw, seed vessels, grain etc.). In addition, it must be considered that chemical conversions of mannose always compete with those

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3 Lignocellulose-based Chemical Products and Product Family Trees Table 3.8 Potentially pure chemicals derived from glucomannan [127]. Product

Source

Process

Possible application

Mannose Mannitol (Manna sugar, Mannit)

Softwood a) Mannose b)

Acid hydrolysis Reduction

Methyl mannoside

Mannose b)

Methanolysis

Sodium mannose bisulfide

Mannose b)

Bisulfite addition

Mannoheptonic acid

Mannose b)

Kiliani synthesis

Food products, chemicals Sweetening agents, carrier for medicine, resins, drying oils, plasticizers, emulsifiers Polyurethane, polyester, polyethers Synthesis of medicinals, germicides, dyes, carrier for sulfite reagents Additives in alkaline washing compounds, detergent scale inhibitors, additive to cement

a) b)

Spent liquors from pulping softwood Residues from softwoods or softwood pulping liquors

of glucose; however, glucose is available from lignocellulose (cellulose fraction) in a more efficient way. Furthermore, the reaction of mannitol (C6 sugar alcohol) to arabitol (C5 sugar alcohol) is of growing interest. Production of arabitol from sugar beet pulp via reduction of arabinose is, anyway, economically more efficient. LCF-mannan will gain a special importance as a “hexose supplier” for fermentation to ethanol or yeast products, because of waste liquor can be used without mannose isolation. Table 3.8 shows some technically feasible conversion processes for glucomannan [127]. If mannose could be produced from polyoses of lignocelluloses in an economically advantageous way, however [128], its importance would be focused on hydrogenation to the sugar alcohol mannitol and subsequent catalytic hydrogenation via C–C bond cleavage to glycols and polyalcohols for polymer synthesis (e.g. polyurethane) [129] or the use of mannitol derivatives as simple or even chiral building blocks [130–132].

3.6.2.2 Xylan/Xylose Product Line Figure 3.18 shows a product line preferentially focused on xylane. Isolation of xylan (e.g. by precipitation from alkaline lignocellulose extract using alcohol) and subsequent hydrolysis to xylose is currently not profitable, however. More efficient is isolation of xylose from the hydrolyzate. So, different technical hydrolysis processes have been developed for production of xylose from lignocelluloses, e.g. acid processes with dilute sulfuric acid at higher temperatures [109 b, 133], alkaline processes and mixed alkaline–acid processes [134, 135], processes based on ion exchange [136, 137], and the use of nitrogen oxides as pretreatment agents [138, 139].


Fig. 3.18 A hemicellulose-based chemical product family tree.

Xylose (d-xylose) is the representative pentose of lignocelluloses. Pentoses are reducing sugars, built up of five carbon atoms. Xylose is also the cheapest pentose, readily accessible from wood- or straw-derived xylans. In 2004 approximately 25 000 tons of d-xylose were produced worldwide [128]. Xylose crystallizes much more rapidly than glucose and forms crystals of uniform grain size. The crystals can easily be separated from the mother liquid. Xylose can be used as a sweetener in the form of crystalline powder or large crystals. Table 3.9 summarizes the most important processes and applications of xylan and xylose (see also Fig. 3.18). The principal derivatives of xylose are xylitol and furfural. Xylitol is obtained by catalytic-high pressure hydrogenation of xylose and was first synthesized in 1891 by Fisher and Strobel by reduction of xylose using sodium amalgam. The reaction conditions of the hydrogenation are the same as those for sorbitol manufacture (below). Reduction catalysts are nickel fixed to different supports or Raney Nickel. Xylitol, as sorbitol, is sweet and used for food sweetening and as a moisture-retaining agent in cosmetics (e.g. in tooth pastes). Use of xylitol as a raw material in the chemical industry is analogous to sorbitol. In the 1960s large amounts of xylitol produced in the former USSR were used as starting material for production of vitamin C [50 b, 127]; today, sorbitol is preferentially used for this process. Xylitol can also be used as raw material for alkyd resins, surfactants, and plasticizers [109 b, 128]. Furfural is produced when three molecules of water are removed from xylose under ring-closure. Furfural chemistry is discussed more in detail in Section 3.6.3.

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3 Lignocellulose-based Chemical Products and Product Family Trees Table 3.9 Potentially pure chemicals derived from xylan [140]. Product

Source

Process

Possible application

d-Xylose

Xylan

Acid hydrolysis

Xylitol

Xylose a, b)

Reduction

Xylonic acid, tartaric acid, triox glutaric acid Furfural

Xylose

Oxidation of spent sulfite liquors

Secondary products, food additives, detergents after esterification, polyurethane from methyl ether Sweetener, humectant, plastic plasticizer Binders, sequestering agents

a) b) c)

Xylan c), Xylose Acidic dehydration

Plastics, solvents, chemical products

Wood and agricultural wastes Spent pulping liquors Vegetable materials

3.6.3 Furfural and Furfural-based Products 3.6.3.1 Furfural Furfural (the structural formula is given in Fig. 3.18) is the oldest and most important substance industrially produced from hemicellulose [141, 142]. In 1998, approximately 142,000 t a1 furfural were been produced worldwide, mainly in China, the Dominican Republic, South Africa, and the USA [41]. Industrially used raw materials are: corn cobs (23.4%), oat hulls (22.3%), cottonseed hull bran (18.6%), cane trash (17.4%), and rice hulls (11.4%) – the numbers given in parentheses are potential furfural yields [143]. Synthesis of furfural on the basis of fossil raw materials (e.g. catalytic oxidation of 1,3-dienes) is economically not competitive. Because of the acidic hydrolysis, the biobased synthesis of furfural can be combined with alcohol production based on lignocellulose. Furfural is a liquid with a boiling point of 161.7 8C that is almost infinitely miscible with all solvents except saturated aliphatic compounds. Freshly distilled, furfural is a colorless, temperature-stable liquid with a very high durability under anaerobic conditions. All technically established processes start with pentosan-containing raw materials. The primary reaction is acidic hydrolysis (1). Kinetically, this reaction depends on proton concentration and temperature [144]. The second step is a dehydration of pentoses to furfural (2) (Fig. 3.19). Finally, furfural is obtained by steam distillation, another time-determining reaction step [145]. The following processes are known for preparation of furfural; except for (9) and (10) all are used industrially applied [147] (1–4, 6, 8, 12 [41]; 5, 7, 9, [50 c]; 10 [146, 147]; 11 [143]).


Fig. 3.19 Furfural formation starting with pentosan.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Quaker Oats batch process (dilute H2SO4, T 153 8C) Chinese process (dilute H2SO4, T 160 8C) Agrifurane process (1% H2SO4, rector cascade, 177 8C to 161 8C) Continuous Quaker Oats process (dilute H2SO4) Roni and Sebara process (dilute and conc. H2SO4) Rosenlew process (catalytic hydrolysis with acetic and formic acid as catalyst) Duipopetrovski process (dilute HCl) Escher Wyss process (cat. hydrolysis with H2SO4, acetic and formic acid) Steug-Sevo process (steam 18 bar) Supratherm process/Staketech process (high temperature, 200 8C) Natta process (HCl, normal pressure) Process based on spent sulfite

Fig. 3.20 Furfural-based Nylon process.

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The main applications of furfural can be summarized as [41 128, 148]: 1. Starting material for the production of derivatives such as furfuryl alcohol, furan, methylfuran, tetrahydrofuran, and furoic acid. Approximately 60% of all furfural produced is used to make furfuryl alcohol. Even adiponitrile for Nylon was produced from furfural from 1946 to 1961 (Fig. 3.20, see also Fig. 3.21). 2. As an extractant in the refining of lubricating oils, diesel fuels, and vegetable oils. 3. As a fungicide. 4. As a nematocide. " Fig. 3.21 A furfural-based chemical product family tree: (1) furfural from biomass and/or hemicellulose: acid hydrolysis [40]. (2) furan from furfural: (a) cat. decarbonylation, -CO, cat: Zn-, Fe(II)-, or Cr2O3/ZnO or KOH/ NaOH [149–152]; (b) thermal decarbonylation with steam over Ca(OH)2 and slaked lime at 350 8C, y: 90% [153]. (3) furoic acid from furfural: (a) by Cannizarro reaction; (b) by oxidation with O2 [150, 154] (b) by oxidation with O2 [155, 156]. (4) furan from furoic acid: -CO2, decarboxylation [154]. (5) tetrahydrofuranyl alcohol from furfural: cat. vaporphase Hydrogenation, Ni-, or Ru(IV)-oxide, or CuCrO3 cat. [157–159]. (6) furfuryl alcohol from furfural: red., Ni- and CuCrO-cat. [160, 161]. (7) methyl furan from furfural: (a) cat. red., H2 + Ni-cat. (+ furfuryl alcohol) [162, 163] (b) cat. hydrogenation, CU-Cr-cat. [164]. (8) fufuryl amine from furfural: reductive amination [165]. (9) 5-nitro furfural from furfural: nitration with HNO3 in (CH3CO)2O [109 c, 166]. (10) tetrahydrofuranyl alcohol from furfuryl alcohol: cat. hydrogenation [152, 167–169]. (11) levulinic acid from furfuryl alcohol: by heating in hydrochloric acidic ethyl methyl ketone, y: 90–93% [170]. (12) methyl furan from furfuryl alcohol: cat. hydrogenation, Cu-Fe-cat., y: 80% [162]. (13) methyltetrahydrofuran (MTHF) from methylfuran: Cat. hydrogenation, cat: Raney Ni, 200 8C [161]. (14) methyltetrahydrofuran (MTHF) from tetrahydrofuranyl alcohol: cat. hydrogenation [109 c]. (15) succinic acid from furfural: (a) electrolytic oxidation (by product maleic acid) [109 c]; (b) cat. oxidation with O2 [109 c, 171]. (16) maleic

anhydride from furfural: oxidation [172]. (17) maleic anhydride from furan: oxidation in gas phase [173]. (18) maleic acid from furfural: cat oxidation, modif. V2O5-cat, Ni/Al-Fe-Tubes [172]. (19) fumaric acid from furfural: cat. oxidat. with NaClO3 in pres. of V2O5-cat. [174]. (20) furyliden acrolein from furfural: + acetaldehyde (cond.) [40]. (21) furyliden ketone from furfural: aldol condensation [175, 176]. (22) 2-furanacrylic acid from furfural: (a) + malonic acid in pres. of pyridine (Perkin reaction) [177, 178]; (b) + acetic anhydride, by 150 8C in the presence of potassium acetate [179]. (23) furanacrylonitrile from furfural: + cyanoacetic acid in pres. of ammonium acetate and pyridine [180]. (24) 2-furyl-2-nitroethylene from furfural: aldol cond./nitroacetic acid or esters and base [109 c, 179, 181]. (25) tetrahydrofuran (THF) from furan: cat. hydrogenation, cat: paladous oxide [182–185]. (26) 1,4-butanediol from furan: cat. hydrogenation by-product of THF-process [186]. (27) thiofuran from furan: reaction of H2S in presence of alumina by heating [187, 188]. (28) pyrrole from furan: reaction with NH3 over Al2O3-cat by heating [189, 190]. (29) 1,4-butanediol from succinic acid: reduction of succinic diethyl ester with sodium in ethanol [191]. (30) 1,4-butanediol from THF: hydrogenation and hydration [192 a, 193]. (31) tetrahydrofuran (THF) from 1,4-butanediol: cat. dehydration [193, 194]. (32) succinic acid from THF: oxidation with HNO3 or NO3 [195]. (33) maleic anhydride from succinic acid: dehydration [196]. (34) maleic


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3 Lignocellulose-based Chemical Products and Product Family Trees Legend Fig. 3.21 (continued) anhydride from THF: cat. airborne oxidation [196]. (35) maleic acid from THF: cat. airborne oxidation in gas phase [197]. (36) maleic acid from maleic anhydride: [198]. (37) fumaric acid from maleic acid: (a) cis-trans isomerism by heating with HCl solution [199], (b) cis-trans isomerism with H2O2, thiourea, and ammonium persulfate by 100 8C [198]. (38) DL-malic acid from fumaric acid: cat. or thermal hydration [54, 200]. (39) DL-malic acid from maleic acid: thermal hydration [201]. (40) DL-tartaric acid from maleic acid: Oxidation with H2O2 or KClO4 [202]. (41) maleic acid from furoic acid: oxidation with ClO–2 (by-product 2-furancarboic acid) [203, 204]. (42) tetrahydrofuroic acid from furoic acid: cat. Hydrogenation [150]. (43) 2,5-furandicarboxylic acid (FDCA) from furoic acid: react. of potassium salt of furoic acid over CdI2-cat [205]. (44) 5-nitrofurancarboxylic acid from furoic acid: Nitration with HNO3 [109 c, 166]. (45) a-ketoglutaric acid from furoic acid: oxidation under esterification and ether forming, then oxid. ring open./ hydrolysis [109 c, 206]. (46) 1,2,5-trihydroxybutane from tetrahydrofuranyl alcohol: with (CH3CO)2O/ZnCl2 to 1,2,5-triacetyl-butane and then KOH/CH3OH [192 a]. (47) 2,3-dihydropyran (DHP) from tetrahydrofuranyl alcohol: cat, (alumina-cont. cat.) dehydration and ring expansion at 200–500 8C [152 b, 207]. (48) acrolein + ethylene from DHP: by thermolysis [41, 192 b]. (49) 2-hydroxytetrahydropyran (R=H) and tetrahydropyran (THP) ethers from DHP: ethers by addition of ROH [41, 208]. (50) 2-cyano-THP from DHP: addition of cyanide [208]. (51) tetrahydropyran (THP) from DHP: cat. hydrogenation [152 b, 207]. (52) 1,5-pentanediol] from DHP: cat. Hydrogenation in presence of Raney-Ni [209, 210]. (53) d-valerolactone from DHP: cat. Hydrogenation [41, 208]. (54) d-valerolactone from 1,5-pentanediol: select. oxidation and lactonization [211]. (55) glutaric acid from d-valerolactone: cat. oxidation [212–214].

(56) c-valerolactone from d-valerolactone: isomerization, a) H2SO4-cat. in H2O [215], b) with J2 [216]. (57) d-cyanovaleric acid from d-valerolactone: NaCN, H2O [192 c, 215]. (58) e-caprolactam from d-cyanovaleric acid: hydrogenation, (+ 4H, – H2O) [192 c, 215]. (59) 1,5-dichloropentane from THP: acidolysis with 2 HCl [208]. (60) 1,5-dicyanopentane from 1,5-dichloropentane: + KCN in Methanol, 130 8C [217]. (61) 1,7-diaminoheptane from 1,5-dicyanopentane: cat. hydrogenation, Ni-cat. [218]. (62) 1,5-dicyanopentane from 2-cyano-THP: acidolysis [192 b]. (63) pimelic acid from 1,5-dicyanopentane: hydration [219, 220]. (64) butanol from THF: cat. or acid hydration [221, 222 a]. (65) d-valerolactone from THF: + CO, cat. carbonylation [223, 224]. (66) c-valerolactone from THF: cat. methylation [216, 225]. (67) 1,3-butadiene from THF: -H2O, cat. Dehydration [222 b]. (68) c-butyrolactone from THF: a) oxidation with O2 [223]; b) oxidation by bromate and lactonization [226]. (69) c-valerolactone from c-butyrolactone: cat. oxidation and methylation [227]. (70) pyrrolidone from c-butyrolactone: reaction with NH3 [228]. (71) N-vinylpyrrolidone from pyrrolidone: N-alkylation [228]. (72) adipic acid from THF: 2 CO + 2 H2O, cat. (Ni(CO)4 and NiCl2 [229]. (73) 1,4-dichlorobutane from THF: (a) with HCl and higher pressure [152 a, 222 c] (b) in presence of water removal agents H2SO4/ZnCl2/SOCl2 [230]. (74) poly(THF) from THF: polymeris. in pres. of oxonium salts, chlorosulfonic acid, or phosphor(V)-chloride [231]. (75) 4,4'-dichlorodibutyl ether from THF: + HCl, cat. [222 b]. (76) poly(THF) from 4,4'-dichlorodibutyl ether: + KOH, 1,4-butanediol [222 b]. (77) adiponitrile from 4,4-dichlorodibutyl ether: KCN and normal pressure, or NaCN and higher pressure [222 d, 232]. (78) 1,6diaminohexane from adiponitrile: hydrogenation under higher pressure over Ni- or Cocat. [222 d, 233]. (79) adipic acid from adiponitrile: + 4 H2O, hydrolysis [234].

3.7 Cellulose-based Product Lines

3.6.3.2 A Furfural-based Family Tree Furfural is a very interesting chemical compound. Fig. 3.21 shows a furfuralbased family tree with technically important products. Some are commercial products, e.g. furfuryl alcohol, others, for example furan, tetrahydrofuran, adiponitrile, were of economic interest until they were replaced by fossil-based products. Products such as methylfuran and methyltetrahydrofuran will gain in importance in the future, for example as fuel additives. In general, in a biobased economy, all products of a furfural family tree will gain more importance because of improved economy and a modified market demand.

3.7 Cellulose-based Product Lines 3.7.1 Isolation, Fractionation and Application Areas

Cellulose is by far the most frequent organic compound. Cotton and other cellulose-rich fibers (ramie, flax, cannabis) release their cellulose rather easily; in cellulose production from lignocellulose (wood, reed, straw, cane strash, cornstalks, or sunflower stalks, and others) special pulping processes must be performed to separate lignin and other polyoses and to obtain cellulose of uniform molecular weight. The most important chemical pulping processes are discussed in Section 3.4.3 (see also Fig. 3.13), the generated cellulose is called pulp. The corresponding pulping process can often be identified by the prefix of the pulp, e.g. sulfite-pulp, sulfate-pulp, sodium hydroxide-pulp etc. Cellulose is one of the most important raw materials for a variety of branches of industry. By far the largest amounts of cellulose are used in the paper and textile industries. In addition, cellulose is used in medicine and pharmacy. Cellulose is also a raw material for many plastics, fibers, rayon (viscose silk), vulcanized fiber, and cellophane. Further cellulose derivatives are cellulose wadding (batting), gun cotton, cellulose-based varnishes, and adsorbent materials for chromatographic use and other applications. Several cellulose derivatives are used in ion-exchange chromatography. Microcrystalline cellulose and cellulose powder are used as fillers or binders in tablets, as sedimentation delayers in tooth pastes and creams, in cigarettes as tobacco substitutes, and as filter materials, emulsifiers, dispersants, and filtration additives in the food industry, etc. Another application is bacterial transformation of cellulose into so-called single-cell protein (SCP) [54]. Use of cellulose while maintaining its original structure is discussed in detail in Ref. [48]. In the following text “cellulose chemistry” describes the chemistry of the corresponding cellulose conversion products, e.g. glucose, the major hexose of lignocellulose sugars, and its derivatives, for example fructose, polyalcohol, methylglucoside, hydroxymethylfurfural (HMF), and levulinic acid. When using cellulose prepared from lignocellulose feedstocks for conversion processes, it should always be checked in advance if the pure isolated and thus ex-

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pensive cellulose pulp is necessary as raw material for further conversion, or if it is possible to convert the desired hexoses by appropriate processes using the lignocellulose itself (instead of isolated cellulose pulp). Thus, occasionally, e.g. ethanol production, even hexoses from hemicellulose can be used. Looking for appropriate conversion methods, it is also reasonable to consider biorefinery principles [45]. 3.7.2 Cellulose-based Key Chemicals 3.7.2.1 Glucose Glucose, a hexose (d-glucose, dextrose, grape-sugar, IUPAC/IUB abbreviation dGlc, acyclic aldose, monosaccharide) is the most important cellulose derivative of lignocellulose. The importance of glucose is primarily a result of the frequency of the molecule (Section 3.3.1). Glucose also plays an outstanding role as so-called “intermediate platform” or “key chemical” within the concept of biobased products [75]. For biotechnical conversion, in particular, glucose is of special significance [128]. d-Glucose or dextrose occurs as a pure crystalline material; for medical applications, glucose is available as 5 to 50% aqueous solution. Most glucose is currently produced by hydrolysis of starch [235]. Most is used as glucose syrup for candy production; for this application an increasing amount of enzymatically isomerized glucose syrup, so-called “isosyrup”, is used. In the chemical industry, large amounts of glucose are used for chemical synthesis, e.g. of sorbit, gluconic acid, ascorbinic acid, glutaminic acid, and glutamates or methylglycoside and for technical application, for example fermentation into ethanol, acetic acid and lactic acid [236]. In recent years production of d-glucose and glucose syrups, respectively, starting from cellulose and lignocellulose, has attrached increasing interest, especially ethanol production from lignocellulose [90 d]. Industrial chemical processes of lignocellulose saccharification are mainly based on acid hydrolysis in so-called wood saccharification by the Bergius (Bergius-Rheinau process) [237] and Scholler (Scholler-Tornisch process) [238, 239], processes that were industrially used between 1930 and 1960 [240]. After World War II crystalline glucose was also successfully manufactured in the so-called New Rheinau process [241]. In recent times, enzymatic methods have been increasingly used for extraction of cellulose degradation products, e.g. saccharification of cellulose (see also Fig. 3.14). Although this degradation works successfully when applied to pure cellulose, it is still a problem to use lignocelluloses, particularly non-uniform lignocelluloses. Here, pretreatment and prepulping of lignocellulose are necessary to end up with economically efficient saccharification. Nevertheless, the first success has been obtained in enzymatic saccharification [90 e, 91]. Fig. 3.22 shows a chemical industrial cellulose-derived product family tree based on glucose as so-called “intermediate platform”. This does not mean, however, that crystalline cellulose or cellulose syrup is necessary as starting material for all the processes and product lines listed.


Fig. 3.22 Chemically industrial cellulose-based product family tree.

Remarkable recent publications have described glucose oxidation via heterogeneous catalysis, enzymes, or fermentation to products such as glucosone, 6-aldehyde-d-glucose, gluconic acid, glucuronic acid, glucaric acid, 2-keto-d-gluconic acid, 5-keto-d-gluconic acid, 2,5-di-keto-gluconic acid, l-ascorbic acid (vitamin C, by fermentation), and Koji acid [242].

3.7.2.2 Sorbitol d-Sorbitol (d-glucitol according to IUPAC/IUB; glucit, C6H14O6) is a crystalline sugar with approximately 50% of the sweetening efficiency of saccharose. d-Sorbitol belongs to the hexite group; it is a hexa-alcohol (sugar alcohol, polyalcohol, polyol) which can intramolecularly split off one or two molecules water and can form cyclic ethers (e.g. sorbitan and sorbit) (Fig. 3.23) [243]. Sorbitol is obtained when a glucose

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Fig. 3.23 Synthesis of cyclic ether based on sorbitol and glycose [243].

solution, e.g. the hydrolysis solution is hydrogenated. Manufacture of sorbitol from glucose has already been industrialized and the technique is firmly established. The first technical production of d-sorbitol was electrolytic reduction of glucose solution in caustic soda and sodium sulfate as electrolyte, with amalgamated lead as the cathode and an anode chamber filled with dilute sulfuric acid [244, 245]. Today, catalytic hydrogenation, especially high-pressure hydrogenation (Raney Ni catalyst, hydrogen pressure 100–150 atm, temperature 100– 150 8C) of glucose or starch and saccharose is preferred [54]. The chemical relationship of sorbitol and glycerin with the glycols on the one hand side and the carbohydrates on the other is decisive for its multi-purpose application. Sorbitol is widely used as an additive for paper, fiber, tobacco, cosmetics, leather, and pharmaceuticals, because it is suitably hygroscopic and maintains a fixed moisture content despite changes in atmospheric humidity. Crystalline sorbitol can be used as a sweetener in the form of a powder or as large crystals (rock sugar), and as dietary sugar. Sorbitol is also widely used as a raw material in the chemical industry. d-Sorbitol is used commercially as a raw material for vitamin C (l-ascorbic acid) production and was used as raw material for explosives during the war. Its most important current application is its use as a raw material for alkyd resins and surfactants [245–247] (Fig. 3.22).

3.7.2.3 Glucosides Glucosides are the glycosides of glucose. “Glycosides” is a collective term for an extensive group of plant substances and synthetic compounds that – by boiling in water or dilute acids or by treatment with glycosidases – can be fractionated into one or more carbohydrates (mono- or oligosaccharides) or into one or more aglycones (or non-sugars). The chemistry and biochemistry of glycosides is therefore very extensive [248]. In the following text glucosides are discussed as compounds in which the reducing group of sugar is condensed with an alcohol or phenol to form acetals. Among different types of glucosides the methyglucosides (formula in Fig. 3.24) are commercially important. A simple methylgluco-


Fig. 3.24 Synthesis of methylglucoside.

side is synthesized by treatment of glucose with methanol in presence of 1% hydrochloric acid as catalyst (Fischer reaction; Fig. 24). A variety of acids have been proposed as catalyst [249–252], but cation exchange resins of the sulfate type are most commonly used. The yield is 88% of the theoretical value [253]. Methylglucoside is used to manufacture alkyd resins. The alkyd resin obtained by esterification of linseed oil and soybean oil has excellent properties. Drying oil obtained from fatty acid and methylglucoside forms a strong membrane, with satisfactory waterproof and adhesive properties, and is used as raw material for paints [109 c]. Application is also being developed in the surfactant field [249, 250].

3.7.2.4 Fructose d-Fructose (levulose, fruit sugar) is the sweetest of the sugars. On a relative scale with sucrose taken as 100 the sweetness of fructose is 173. The molecular weight and the elemental composition of glucose and fructose are exactly the same. The only difference is that glucose is of the aldehyde type whereas fructose is of the keto type (ketohexoses). The most important natural sources are saccharose and inulin. A variety of methods have been proposed for converting glucose into fructose by isomerization. In general, these can be divided into chemical and enzymatic methods. Until 1970 chemical isomerization in alkaline solution with alkaline catalysts such as ammonia, slaked lime, lime water, sodium carbonate, potassium carbonate, or caustic soda via d-glucose-1,2-enediol was preferred [254–256]; today enzymatic and mixed enzymatic/chemical catalysis methods are of advantage. Under optimum conditions the enzyme glucose-2-oxidase converts > 99% of the glucose into glucosone, which can subsequently be transformed into d-fructose by use of a metal catalyst, e.g. Pd or Raney Ni, and hydrogen at elevated temperature and pressure, or by homogenous NaBH4 reduction [242, 257] (Fig. 3.25). d-Fructose is used as sugar substitute in the pharmaceutical and dietetics industries, particularly in cases of diabetes. For the biobased chemical industry, fructose is an outstanding starting material for manufacture of 5-hydroxymethylfurfural (HMF) and levulinic acid [175]. Nevertheless, both these important building blocks are also available from non-specific hexoses (Section 3.7.2). Utilization of natural fructose raw material, for example inulin (polyfructosan) from special cultures such as artichoke and topinambur (Jerusalem artichoke) or saccharose (sucrose) from sugar cane and sugar beet, and utilization of semi-

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Fig. 3.25 Synthesis of d-fructose from d-glucose [242, 257].

synthetic d-fructose for HMF and levulinic acid, will both be exposed to significant economic pressure.

3.7.2.5 Ethanol The significance of ethanol as an important bulk product from lignocellulose conversion and from the different processes of utilization of lignocellulose-based hexoses and pentoses has already been discussed. In this section the importance of ethanol among biobased products as a C2 building-block chemical is reviewed. Most currently produced ethanol is used in beverage industry. Technically, ethanol is a valuable solvent for fats, oils, and resins, particularly in lacquer and varnish manufacture and also for production of essences. Ethanol is the most important solvent for scents and perfumes and cosmetics (aftershaves, hair tonic, etc.). Because of its germicidal effect, ethanol is used as a preservative and a disinfectant.

Fig. 3.26 An ethanol-based chemical product family tree.


As substrate for protein generation (SCP), ethanol can replace petroleum. Because of its high calorific value (29 kJ g–1 or 7 kcal g–1), ethanol in the form of denatured alcohol or so-called solid alcohol is used for combustion or (mixed with gasoline) as a fuel (gasohol, E10- or E85-biofuel etc.). Use of ethanol as raw material for synthesis of important industrial chemicals is most promising for those countries that decided to use ethanol for fuel production. Changing the raw material basis from cost-intensive agricultural products (e.g. grain, corn) to lignocelluloses (e.g. straw) and introducing energy-saving biotechnological processing steps could further improve the competitiveness of bio-based versus fossil-based ethanol. Ethanol is the starting material for many chemicals, for example diethyl ether, chloroform, ethyl chloride, dyes, and pharmaceutical compounds [258]. Fig. 3.26 shows an industrial chemical product family tree. The product lines for ethyl lactate and ethylene, and the corresponding derivatives are of particular interest in chemical industry [259].

3.7.2.6 Hydroxymethylfurfural Two very important biobased building blocks, 5-hydroxymethylfurfural (HMF) and levulinic acid, are available via acid-catalyzed dehydration of hexoses. Formally, elimination of one molecule of water leads to a mixture of levulinic acid and formic acid. It is assumed that during degradation HMF is formed as an intermediate which is not stable in acids and degrades into levulinic and formic acids. This means that three molecules water are eliminated to form HMF, which then reacts with two molecules of water in a kind of “re-hydration” to form levulinic acid and formic acid [260, 261] (Fig. 3.3). To achieve high yields in the isolation of HMF, it is essential to suppress HMF hydrolysis, which is most successful in non-aqueous systems [20]. 5-Hydroxymethylfurfural (HMF) is a versatile sugar derivative which can be regarded as a key intermediate between bio-based carbohydrate chemistry and mineral oil-based industrial organic chemistry. HMF is a common dehydration product of all hexoses and thus is an aldehyde, an alcohol, an aromatic compound, a cis diene, and a difunctional diene. HMF is available from biomass in a simple dehydration reaction and is convertible into di- and tetrahydrofuran derivatives and also benzene, pyridazine, pyridine, and other derivatives [262. 263]. Some possible reactions and applications are shown in Figs. 3.27 and 3.29. Starting with HMF, furan-2,5-dicarboxylic acid (FDCA) is available in an onestep reaction [264, 265]. Terephthalic acid or isophthalic acid are bulk products which could be replaced by furan-2,5-dicarboxylic acid (FDCA) (Fig. 3.27). Currently, fossil-based terephthalic acid is the most important dicarbonic acid, produced on the million ton scale and mainly used for polyester production, e.g. poly(ethylene terephthalate) (PET) and polycondensation products of terephthalic acid (TPA) and ethylene glycol (EG) for packaging, beverage bottles, foil, etc. [266]. Polymers, especially polyesters and polyamides have been prepared using FDCA and other HMF derivatives instead of terephthalic acid [267, 268].

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Fig. 3.27 Comparison of the syntheses of TPA and FDCA [269].

HMF can be produced by three different acid catalyzed dehydration processes in which the ketohexose fructose is mainly used as starting material [262, 269, 270]: 1. reaction in aqueous solution with H2SO4 as catalyst, a simple process, but HMF yields are low (50–60%) (Fig. 3.27); 2. elimination of water in DMSO as solvent leads to high HMF yields, but subsequent isolation of HMF is rather difficult; and 3. elimination of water in multi-phase reaction mixtures, thereby continuously collecting the generated HMF by use of an appropriate solvent.

3.7.2.7 Levulinic Acid Levulinic acid, or 4-oxopentanoic acid (m.p. 33.5 8C, b.p. 245 8C), is the simplest member of the comparatively rare class of organic compounds known as c-keto acids. Having both a ketone carbonyl group and an acidic carboxyl group it reacts as a ketone and as a fatty acid. The chemical structure of levulinic acid can be represented as CH3COCH2CH2COOH [271]. Levulinic acid is formed by acid hydrolysis of hexoses and hexosic materials (Fig. 3.3, Section 3.7.2). Levulinic acid is easily converted into chemical derivatives [80, 81, 272] (Fig. 3.29) and, because of its high stability, can be used as a liquid fuel extender [273]. Fig. 3.28 gives a summary of possible applications. Levulinic acid is a starting product for preparation of organic chemicals, dyestuffs, polymers, pharmaceutically active compounds, and flavor substances. Levulinic acid is also an inhibitor of chlorophyll synthesis. If levulinic acid is used in food products, rigorous purity, color, and stability requirements must be met. Esters of levulinic acid are known to be useful as plasticizers and solvents, and have been suggested as fuel additives. Levulinic acid is useful as a solvent, as a food-flavoring agent, and as a starting material for preparation of a variety of industrial and pharmaceutical compounds such as diphenolic acid (useful as a component of protective and decorative finishes) and calcium levulinate (a particularly suitable form of calcium for intravenous injection used for calcium replenishment and to treat hypocalcemic states). Levulinic acid is also useful for preparing a glass-like synthetic resin, as a constituent of hydraulic brake fluids,


Fig. 3.28 Levulinic acid – overview of production and application.

and in the manufacture of nylon and rubber. Use of the sodium salt of levulinic acid as a replacement for ethylene glycols as an antifreeze has also been proposed [81]. 3.7.3 An HMF and Levulinic Acid-based Family Tree

HMF and levulinic acid both are multifunctional compounds with a very broad reaction and application potential. Figure 3.29 shows a chemical family tree with HMF- and levulinic-based products that already have or might achieve technical importance. Today, some of these products are as economically efficient as levulinic acid itself, others were of economic significance years ago and were then replaced by fossil-based products, e.g. several hydroxymethylfuranyl derivatives. Several others could gain importance in the future, for example 2,5furandicarboxylic acid (FDCA) as a monomer for polymer synthesis and methyltetrahydrofuran (MTHF) as a fuel additive. In a biobased economy, market demand and economic efficiency of all the products discussed will develop extremely positively.

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3.7 Cellulose-based Product Lines 3 Fig. 3.29 An HMF and levulinic acid-based chemical product family tree: (1) 5-hydroxymethylfurfural (HMF) from hexosic material: acid hydrolysis [20, 274]. (2) levulinic acid (LEVA) direct from biomass or over HMF: acid hydrolysis [81, 272, 275, 276]. (3) levulinic acid (LEVA) from HMF: cleavage in acidic medium [277]. (4) levulinate esters from cellulose: acid cat. + alcohol, + higher temp. [278]. (5) levulinate esters from HMF: acid. cat. + ROH [278]. (6) levulinate esters from LEVA: + ROH [279, 280]. (7) aldaric acids from hexoses: oxidation of glucose (or hexoses): (a) acid oxid. HNO3; (b) cat. oxid. O2, Pt on C; (c) biotechn. by Aspergillus niger [242]. (8) 2,5-furandicarboxylic acid (FDCA) from aldaric acids: (a) with dehydrating agents, e.g. HBr [281, 282]; (b) by cyclodehydration of mucic acid with p-TsOH at 140 8C [283]; (c) esters, from d-glucaric acid, alcs. and acids by using microwave radiation [284]. (9) 2,5-furandicarboxylic acid (FDCA) from hydroxymethyl furoic acid: by cat. oxyd. with charcoal-on-Pt-cat. [285]. (10) 2,5-furandicarboxylic acid (FDCA) from HMF: cat. oxydat. different cat. methods [286–289]. (11) 2,5-bis(hydroxymethyl)furan (BHMF) from HMF: (a) by cat. hydrogenation [290]; (b) by Cannizzaro [291]. (12) 2,5-bis(hydroxymethyl)furan (BHMF) from FDCA: cat. hydrogenation [292]. (13) 2,5-furandicarbaldehyde (FDC) from HMF: cat. oxyd. BaMnO4, 93% [289, 293]. (14) 2,5-furandicarbaldehyde (FDC) from BHMF: cat. hydrogenation [292]. (15) 2,5-furandicarbaldehyde (FDC) from FDCA: cat. hydrogenation [292]. (16) 2,5-furandicarboxylic acid (FDCA) from FDC: cat. oxid. AgO2, 80% [289]. (17) 5-hydroxymethylfuroic acid from HMF: (a) cat. with Ag2O, 100 8C, 75% [289] ; (b) by Cannizzaro [291]. (18) methyl malonate from HMF: electro-oxidation on platinum anode in methanol, LiClO4 electrolyte [294]. (19) bis(5-methylfurfuryl)ether from HMF: TsOH, 89% [295, 296]. (20) 2-aminomethyl-5-hydroxymethylfuran from HMF: reductive amination, Ni/H2, NH3, 72% [297]. (21) 2,5-bis(aminomethyl)furan from HMF: + NH2OH, then Ni/H2 [289]. (22) 5-hydroxymethyl-furylideneacetic acid from HMF: + malonic acid, cat. react in pyridine [298]. (23) 5-carboxy-2-furylideneacetic acid from 5-hydroxmethyl-furylidene-

acetic acid: electrochemical oxidation at a Ni-oxide-hydroxide anode [298]. (24) 2,5bis(hydroxymethyl)tetrahydrofuran from HMF: cat. hydrogenation, Raney Ni, 90% [283, 290, 292]. (25) 1,2,5-trihydroxyhexane from HMF: cat. hydrogenation, cat: Ru/C, 96% [290]. (26) 1,2,5-trihydroxyhex-3-ene from HMF: cat. hydrogenation, cat: Pt or Ru [290]. (27) 2,5-bis(hydroxymethyl)tetrahydrofuran from FDCA: cat. reduction, cat: RaneyNi [292]. (28) 2,5-bis(hydroxymethyl)tetrahydrofuran from BHMF: cat. hydrogenation, cat: Ru/C, T. neutral med. [283, 290]. (29) 2,5-bis(aminomethyl) tetrahydrofuran from FDCA: cat. hydrogen, NH3 [290]. (30) succinic acid from FDCA: (a) Potassium Salt of FDCA and Brom to dibromosuccinic acid and then hydrogenation [299 a]; (b) FDCA and Brom in heat water to fumaric acid and then hydrogenation [299 b]. (31) succinic acid from LEVA: (a) cat. oxidation, O2/V2O5 [272, 300]; (b) H2O2 on Cu-cat. [81]. (32) 2-oxoglutaric acid from LEVA: cat. oxidation (Riley reaction) SeO2 [272, 301]. (33) 5-methyl-2-pyrrolidone deriv. from LEVA: + R-NH2, reductive amination over Co-, Raney Ni, Pt or Pd-catalysts [302–305]. (34) 5-furfurylidenelevulinic acid from LEVA: + furfural [306–308]. (35) dilevulinic acid from 5-furfurylidenelevulinic acid: acids treatment [272, 309]. (36) sebacic acid from dilevulinic acid: cat. hydrogenation, H2/Ni [272, 309]. (37) 4,4-diaryl subst. valeric acids (diphenolic acid) from LEVA: acid-cat. condensation with phenols or naphthols [310–312]. (38) 1-keto-non-6-en from LEVA, + hex-3enoic acid [313]. (39) acrylic acid from LEVA: condensat. with aldehydes and ketone splitting [80]. (40) b-acetylacrylic acid from LEVAesters: oxidation of levulinic acid esters with SeO2 [314]. (41) a-angelica lactone from LEVA, cat. dehydration, cat e.g. phosphoric acid, [81, 315]. (42) b-angelica lactone from a-angelica lactone: base cat. (cat. e.g. tert. Amine) isomerization [81]. (43) c-valerolactone from a-angelica lactone: reduction [316]. (44) c-subst.-b-acetyl-c-butyrolactones from a-angelica lactone: rect. with aldehydes in presence of BF3-O(C2H5) [317]. (45) 4-hydroxypentanoic acid from LEVA: cat. hydrogenation or reduction with diluted HCl [318].

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3 Lignocellulose-based Chemical Products and Product Family Trees Legend 3.29 (continued) (46) c-valerolactone from hydroxypentanoic acid: acidic dehydration [318, 319]. (47) c-valerolactone from LEVA: (a) cat. hydrogenation, cat: Raney-Ni on Pt [273, 320]; (b) Platinum-Group metal catalyst (e.g. Rh/C) in the presence of hydrogen [321]. (48) 1,4-pentanediol from c-valerolactone: cat. hydrogenation [316]. (49) 1,4-pentanediol from LEVA: cat. hydrogenation, cat: Raney-Ni on Pt [320]. (50) 2-methyl-THF (MTHF) from 1,4-pentanediol: dehydration [316]. (51) 1,4-pentadiene from 1,4-pentanediol: cat. dehydration [322]. (52) 2-methyl-THF (MTHF) from LEVA: cat. hydrogenation, 240 8C, 100 atm (ca. 101 bar) [47, 316, 323]. (53) 5-cyano-4methylpent-4-enoic acid from LEVA: Knoevenagel condensation with cyanoacetic acid [324]. (54) 3-methyladipic acid from 5-cyano4-methylpent-4-enoic acid: reduction of double bond and acid hydrolysis [324]. (55) thio-

tenol (oxymethyl thiophene) from LEVA, heating with P4S10 [80, 324]. (56) [5-methyl5-fluoro-c-butyrolactone] from LEVA, fluorinating with SF4 (over-5-hydroxy-c-valerolactone) [326]. (57) dihydropyridazinones from LEVA: + hydrazine or derivatives [327, 328]. (58) pyridazinones from dihydropyridazinones: oxidation with Br2 or SeO2 [327, 328]. (59) dihydropyridazinone-3-carboxylic acid from dihydropyridazinones: oxid. w. 10% HNO3 [329]. (60) glutamic acid from dihydropyridazinone-3-carboxylic acid: cat. hydrogenation, H2/Ni [329]. (61) brominating levulinic acids from LEVA: + Br2 in different solv. [272, 330]. (62) d-aminolevulinic acid, (5-aminolevulinic acid) from LEVA or LEVAesters: (a) regioselect. Bromination to 5-bromolevulinic acid and then react. w. alkali metal diformylamide [331]; (b) fermentation by photosynthetic bacterium (Rhodobacter sphaeroides IFO 12203) [332].


In a biobased economy, lignocelluloses will most probably be the main source of raw materials. First, there are a variety of sources of lignocellulose (e.g. wood, straw, reeds, grass, etc.) and lignocellulose is the abundant continental biomass. There are also other sources, for example cellulose-containing waste materials from public life (recovered paper, hospital waste, municipal waste etc.) and industrial waste products (e.g. pulp and paper industry). Second, lignocelluloses, with their main components cellulose, hemicellulose, and lignin, contain organic structures that serve as source for a variety of derivatives and conversion products. There are almost inexhaustible possibilities in chemistry and biotechnology to use lignocellulose and corresponding derivatives. In addition, industrially established processes and products have already been developed in the past (e.g. saccharification, furfural-based nylon production); unfortunately they could not compete with extremely inexpensive petroleum. Those processes and corresponding experience can be used for further development. Third, lignocelluloses are, to a large extent, independent of economic policy (in contrast with agricultural products such as corn, grain, sugar beet, availability of hemicellulose raw materials is not state-controlled); this, together with their reasonable raw material prices, makes lignocelluloses very interesting for industrial use. (Prices for lignocellulose corn stover or straw are approximately 30% of those of corn and grain.)

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Fourth, lignocelluloses can be produced even in environmentally sound less intensive agriculture and forestry which is another positive effect, in addition to the general CO2-neutrality of biomass. The main requirement for economic success of lignocelluloses, its technologies and its products seems to be an integrated approach of lignocellulose processing and utilization. By analogy with the extremely successful petrochemistry, it is absolutely essential to improve biorefinery technologies and to develop sustainable and marketable product lines, multiproduct systems, and competitive biobased products.

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Lignin Line and Lignin-based Product Family Trees 4 Lignin Chemistry and its Role in Biomass Conversion Gösta Brunow

4.1 Introduction

Lignin is among the most abundant biopolymers on earth and, being renewable, it has attracted much effort to make use of lignin as feedstock in biomass conversion processes. Lignins are an essential component of the woody stems of arborescent gymnosperms and angiosperms in which amounts range from 15 to 36%. Lignins are not restricted to arborescent plants, they are found as integral cell wall constituents in all vascular plants including the herbaceous varieties. The lignin in the cell walls is intimately mixed with the carbohydrate components. The structure of the polymer is complex and irregular and isolation of lignin from other plant constituents is not easy. Lignin is an essential component of higher plants, giving them rigidity, water-impermeability, and resistance against microbial decay. In the pulp and paper industry, lignin is removed chemically and residual lignin in pulp is removed or degraded using bleaching agents, e.g. chlorine dioxide, oxygen, or ozone. In mechanical pulping much energy is needed to eliminate the cementing effect of lignin. Vast amounts of lignin derivatives from pulp and paper industry are created and these compounds are a threat to the environment if not detoxified or otherwise treated in effluent treatment plants. White-rot fungi are the only organisms able to mineralize lignin efficiently to carbon dioxide and water by processes initially catalyzed by extracellular enzymes. Biotechnological applications of these fungi and their lignin-modifying enzymes are being developed as alternative methods for pulping and bleaching and for bioremediation, i.e. removal of toxic pollutants from soil, groundwater, and effluents.


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4 Lignin Chemistry and its Role in Biomass Conversion

4.2 Historical Overview

Lignin is encountered industrially during the process of papermaking from wood. This involves the chemical and mechanical separation of the cellulosic fibers from woody or other lignified plant material. The chemical separation of lignin from cellulose has been termed “delignification” and it is one of the complex processes of the pulp and paper industry. The lignin products resulting from the delignification processes may vary widely in properties depending on which delignification process has been employed and at which stage of delignification the lignin was isolated. The sulfate or Kraft process produces the largest amount of pulp. The sulfate or kraft lignin has consequently been the most plentiful lignin available in spent pulping liquors. The kraft lignin can be recovered in reasonably high yields by acidifying and filtering the precipitated lignin. Very few kraft mills do process kraft lignin for sale; the bulk of the spent liquor lignin is burned for the production of energy. The fuel value of the organic matter in the black liquor from the kraft process is substantial. The value of any chemical to be produced from black liquor must be high enough to compensate for the cost of the capital installation and of the additional fuel used to replace the lignin removed.

4.3 The Structure of Lignin 4.3.1 Definition

The term “lignin” is often loosely used both for the lignin in the intact wood and for preparations obtained from diverse procedures with the objective of separating the lignin from other cell-wall constituents. To be precise, the lignin in the cell wall should be termed “protolignin”, and all other preparations “lignin products” with a clear declaration of what procedure was used to obtain the lignin. A published description (Brunow et al. 1998) gives an useful overview of our present knowledge about the chemical composition of protolignin: This description is not a chemical definition of protolignins, but summarizes the main structural features based on knowledge available today. Protolignins are biopolymers consisting of phenylpropane units with an oxygen atom at the p-position (as OH or O–C) and with none, one, or two methoxy groups in the positions ortho to this oxygen atom. These ortho positions may alternatively be C-substituted or O-substituted with substituents other than methoxy. Only a few of the aromatic units are substituted in other ring positions. A few percent of the building blocks in protolignins are not phenylpropane units. The side chain is missing or shortened, or the unit is replaced by a quinoid group. The phenylpropane units are attached to one another by a series of characteristic linkages

4.3 The Structure of Lignin

(b-O-4, b-5, b-b, etc.) or, alternatively, exist as members of a series of characteristic end groups (e.g. cinnamaldehyde units). Practically all the types of structural element detected in protolignins have been demonstrated to be formed on oxidation of the p-hydroxycinnamyl alcohols in vitro (Freudenberg 1968; Adler 1977). The structural elements in protolignins are not linked to one another in any particular order. Protolignins are not optically active. The polymer is branched and cross-linking occurs. In addition, the following facts should be noted: (1) there are strong indications of the occurrence of linkages between protolignin and carbohydrates, (2) some types of protolignin are esterified with phenolic acids (grass lignins with p-coumaric acid and other lignins, for example aspen lignin, with p-hydroxybenzoic acid), and (3) scattered observations suggest that there are some units, for example, dihydroconiferyl alcohol units, that cannot be thought to have been produced by oxidation of p-hydroxycinnamyl alcohols. 4.3.2 The Bonding of the Phenylpropane Units

Phenylpropane units of types 1 (guaiacylpropane), 2 (syringylpropane), and 3 (phydroxyphenylpropane) are the main building blocks in lignins. The proportions of 1–3 differ with the botanical origin of the lignin. The biosynthesis of lignins is regarded as proceeding via oxidative polymerization of three primary precursors – the p-hydroxycinnamyl alcohols 4–6. It has been shown that practically all the types of structural element detected in lignins are formed by in vitro enzymic oxidation of the p-hydroxycinnamyl alcohols 4–6 and phenolic lignin model compounds.

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The growth of the lignin polymer is visualized as an oxidative coupling between a phenoxy radical formed from a monolignol (4–6) with a radical formed from a phenolic center in the growing polymer. The isolation of numerous dimeric lignin degradation products derived from lignin structures consisting of different types of unit show that cross-coupling of units of types 1–3 occurs during the biosynthesis. It follows from the types of reaction that the structural elements in lignins are not linked to each other in any particular order and that lignins do not have optical activity. That this is the case has recently been demonstrated (Ralph et al. 1999; Matsumoto et al. 1999). The reaction sequences do not rule out the possibility that certain sequences of units are more probable than others. There are strong indications of the occurrence of linkages between lignin and carbohydrates. Certain types of lignin are esterified with phenolic acids (grass lignins with p-coumaric acid and other lignins (e.g. aspen lignin) with p-hydroxybenzoic acid). It has been found that there are some types of units in lignins, for example dihydroconiferyl alcohol units, that cannot be thought to be produced by oxidation of p-hydroxycinnamyl alcohols. The proportions of structural units of types 1–3 provides a basis for the classification of lignins. The composition of some important classes of lignins based on this criterion are listed in Table 4.1. According to tracer studies (Tomimura et al. 1980) roughly 50% of the 5-positions and a small percentage of the 2- and 6-positions in the guaiacyl units (1) in softwood and hardwood lignins carry a C or O-substituent (“condensed units”). The so called nucleus exchange method gives similar results (Funaoka et al. 1992). As judged from a recent reassessment of the nucleus exchange method, however, the number of condensed units determined by this method can be expected to be too low (Chan et al. 1995). Results based on studies of degradation products obtained on permanganate oxidation of lignins point to a smaller proportion of condensed guaiacyl units (condensed guaiacyl units/total number of guaiacyl units * 0.4) (Erickson et al. 1973; Larsson and Miksche 1971). A combination of degradation (thioacidolysis) and subsequent examination by gel per-

Table 4.1 Approximate composition (%) of some important classes of lignins.

Softwood lignin Hardwood lignin a) Grass lignin b) Compression wood lignin

1

2

3

95% 50% 70% 70%

1% 50% 25% 0%

4% 2% 5% 30%

a) Most hardwood lignins are composed of approximately equal amounts of 1 and 2 but quite a few exceptions are known. Some hardwood lignins are esterified with p-hydroxybenzoic acid. b) p-Coumaric acid attached by ester linkages not included.


meation chromatography is an alternative procedure for determination of the extent of condensation (Suckling et al. 1994). The lignin polymer is expected to be branched and cross-linking may occur to some extent. If the lignin were a linear polymer the number of interconnections per unit in a molecule consisting of n units would be (n–1)/n and cannot exceed 1. This is true even if branching occurs. Cross-linking leads to the formation of rings of units. A consequence of this is that the number of interconnections per unit increases (values larger than 1 are possible). Available lignin data suggest that the number of rings is small and it can therefore be assumed that the number of interconnections per unit is close to 1. In this chapter we have tried to amalgamate the results from studies of lignin in situ with those obtained in studies of isolated lignins. Most of the data given emerge from examinations of milled wood lignin (MWL), however (Björkman 1956; Lundquist 1992). Comparative studies of wood and MWL (Erickson et al. 1973; Lapierre et al. 1991; Rolando et al. 1992; Lapierre and Lundquist 1999) suggest that MWL is, in most respects, representative of the lignin in wood. The nature of the functional groups and bonding patterns in lignins are nowadays well established, although many results regarding the quantitative contribution of different types of structural element are contradictory and controversial. This is not surprising, because it is very difficult to obtain reliable quantita-

Scheme 4.1 Important structural features in lignins.

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tive lignin data. An example of the types of difficulty encountered in quantitative lignin analysis occurs in calculation of the abundance of condensed units in lignins on the basis of 1H NMR spectroscopy (Ludwig et al. 1964; Lundquist 1980). Calculation of condensed units is based on integration of the signal from aromatic protons (corrected for the contribution of some vinyl protons) together with determination of the number of phenylpropane units and consideration of the distribution of units 1–3 in the sample. A rather optimistic estimate of the experimental error in a determination of the number of aromatic protons/unit by this method would be ± 5%. If, for instance, the number of aromatic protons/unit is found to be 2.5 in a softwood lignin sample (composition, see Table 4.1) a 5% error makes determination of the extent of condensation uncertain (50% ± 12.5%). In this and in many other instances quantification of functional groups and bonding patterns in lignins leads to uncertain results despite basically sound approaches. The problems related to lignin analysis are outlined in Brunow et al. (1998). 4.3.3 Bonding Patterns and Functional Groups 4.3.3.1 General Biosynthetic considerations are often used among the arguments for the existence of particular structural units in lignins. Biosynthetic arguments are only pointed out occasionally in the discussion below. Each one of the types of unit discussed below can be expected to be present in all the different classes of lignin listed in Table 4.1. For simplicity the lignin units are usually depicted as noncondensed guaiacylpropane units (1) but when applicable the formulas also represent the other types of phenylpropane unit. Data given for hardwood lignins refer to such lignins composed of similar amounts of units of types 1 and 2.

4.3.3.2 Survey of Different Types of Lignin Unit Arylglycerol Units Attached to an Adjacent Unit by a b-O-4 Linkage Evidence for the occurrence of arylglycerol units attached to an adjacent unit by a b-O-4 linkage (linkages between units A and B, and D and E, see Scheme 4.1) as an important lignin structure was obtained by synthesis of appropriate model compounds and comparison of their reactions with lignin reactions. Both models and lignins gave, for instance, so called Hibbert ketones on acid degradation (Adler et al. 1957). The existence of lignin structures of this type was later confirmed in numerous studies. It has been shown that all the three types of phenylpropane unit (1–3) participate in structures of the arylglycerol b-aryl ether type. Degradation products obtained from the three types of unit are, for example, obtained on acidolysis (Lundquist 1992), thioacidolysis (Rolando et al. 1992) and DRFC degradation (Lu and Ralph 1998). Distribution of the diastereomeric forms in lignins has been studied by NMR spectral methods. The results are in


agreement to the extent that approximately equal amounts of the diastereomeric forms are found in softwood lignins and there is a predominance of the erythro form in hardwood lignins (Lundquist 1980; Nimz et al. 1984; Hauteville et al. 1986; Ede and Ralph 1996; Saake et al. 1996). Studies of the stereochemistry based on the formation of threonic acid and erythronic acid on ozonation (Matsumoto et al. 1986; Taneda et al. 1989; Sarkanen et al. 1992) are largely in accordance with the NMR spectroscopic results. The distribution of diastereomeric forms of arylglycerol units attached to guaiacylpropane (1) units and syringylpropane (2) units in a hardwood lignin has been studied using the 2D INADEQUATE experiment (Bardet et al. 1998). The results show that the predominance of erythro forms is because of the presence of large amounts of erythro forms attached to syringylpropane units. NMR studies suggest there are 30– 40% units of this type in softwood lignins (Robert and Brunow 1984; Lundquist 1991; Jiang and Argyropoulos 1994) and 40–50% such units in hardwood lignins (Lundquist 1991; Argyropoulos 1994). Most of the b-O-4 units in hardwoods are linked to syringylpropane units (Bardet et al. 1998). Phenylpropane Units Attached to an Adjacent Unit by a b-5 Linkage Units linked to an adjacent unit by a b-5 linkage are present in phenylcoumaran structures (B–C in Scheme 4.1). Freudenberg and co-workers (1968) have shown that permanganate oxidation of methylated lignin gives isohemipinic acid. This acid can be expected to originate from b-5-linked units, and labeling studies have shown that this is true to some extent (Freudenberg 1968). Acidolysis studies provided evidence of the occurrence of a significant number of b-5 units in softwood lignin (Adler and Lundquist 1963). Ozonolysis similarly confirms the occurrence of units of this type and indicates primarily the trans stereochemistry (Habu et al. 1990). Attempts to detect the cis form in spruce lignin by 1H NMR spectroscopy based on model compound data (Iliefski et al. 1997) have failed (Lundquist 1999). The occurrence of b-5 units in lignins has been confirmed in a large number of studies of degradation products (Lai and Sarkanen 1971) and in several 1H NMR and 13C NMR spectroscopic studies. On the basis of UV spectroscopic acidolysis studies (Li and Lundquist 1997) it was concluded there are approximately 10% b-5 units in spruce lignin. This figure should be adjusted somewhat downwards because of interference of diguaiacylstilbene formed from b-1 structures on acid treatment (Lundquist 1992; Lai and Sarkanen 1971). Recent acidolysis studies point to a frequency of 6–9% units in spruce lignin (Li and Lundquist 1999). Permanganate studies suggest 12% b-5linked units (Erickson et al. 1973) and this agrees fairly well with results obtained from ozonolysis studies (Habu et al. 1990). The number of units of this type in hardwood lignins is comparatively small (Larsson and Miksche 1971; Landucci et al. 1992). Phenylpropane Units Attached to an Adjacent Unit by a b–b Linkage Units linked to an adjacent unit by a b-b linkage are present in pinoresinol structures and in analogous structures. Solvolytic (Lapierre et al. 1991; Wallis 1971) and reductive

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(Lapierre et al. 1991; Nimz 1974; Sakakibara 1992) degradation studies provide evidence for the occurrence of different types of pinoresinol structure in lignins. Both pinoresinol and syringaresinol structures have been detected in lignins by a variety of NMR spectroscopic techniques (Lundquist 1991; Kilpeläinen et al. 1994). Ogiyama and Kondo determined the abundance of b-b units in a softwood lignin to be 5–10% (Ogiyama and Kondo 1968). NMR spectral studies suggest somewhat lower values (Lundquist and Stomberg 1988). The formation of 3,4-divanillyltetrahydrofuran on acidolysis (Lundquist and Stomberg 1988; see also references cited therein dealing with the formation of 2,3-divanillyl-1,4-butanediol from lignin) or thioacidolysis of softwood lignin (Lapierre and Lundquist 1999) suggests the occurrence of b-b-linked units with reduced side-chains. These structures cannot be formed by oxidation of coniferyl alcohol. Biphenyl Units, Dibenzodioxocin Structures and Diaryl Ether Structures The occurrence of biphenyl units in lignins has been demonstrated in studies of lignin degradation products. Methylation then permanganate oxidation gives dehydrodiveratric acid (Erickson et al. 1973). Degradation by reductive methods gives dehydrodicoerulignol (Lapierre et al. 1991; Nimz 1974; Sakakibara 1992). This provides unambiguous proof of the occurrence of phenylpropane units in lignins attached to each other by a biphenyl linkage. Pew (1963) attempted to estimate the number of biphenyl units in softwood lignin by a UV spectrometric method. He concluded that “coniferous lignin may well contain 25% or more biphenyl-linked units.” Permanganate oxidation studies suggested that 19% of the lignin units are of the biphenyl type in softwood lignin (Erickson et al. 1973). This estimate is based on results obtained by permanganate oxidation of lignin pre-treated by cupric oxide oxidation. Model compound studies (Pearl and Beyer 1954; Bose et al. 1998) show that biphenyl coupling occurs to some extent on cupric oxide oxidation. This indicates that the value derived from permanganate oxidation studies is slightly too high. On the basis 13C NMR examinations Drumond et al. (1989) arrived at a higher value for the frequency of biphenyl units (24–26%). The accuracy of this estimate can be questioned, because no separate signals from biphenyl units can be discerned in the spectra. In view of the many sources of error the results obtained by different methods are not directly incompatible. Estimation of biphenyl units is complicated by the recent finding (Karhunen et al. 1995) that there are significant amounts of dibenzodioxocin structures in lignins. Conclusive evidence of the occurrence of units of this type (Scheme 4.1, units C–D) has been obtained by NMR spectral studies (Karhunen et al. 1995). Tentative estimates suggest there are approximately 6% such units in softwood lignin (Sipilä and Brunow, unpublished results). This implies that approximately 12% biphenyl units may be present in dibenzodioxocin structures. From model compound studies (Karhunen et al. 1999) it can be concluded that the biphenyl units in dibenzodioxocin structures are included in the estimates of biphenyl units by NMR spectroscopy and permanganate oxidation. The number of biphenyl units is smaller in hardwood lig-

4.4 Role of Lignin in Biomass Conversion

nins. Permanganate oxidation suggests there are 9% such units in birch lignin (Larsson and Miksche 1971). The occurrence of diaryl ether structures in lignin was concluded on the basis of permanganate oxidation (Freudenberg 1968) and was later confirmed in studies of lignin degradation by reductive methods (Lapierre et al. 1991; Nimz 1974; Sakakibara 1992). Permanganate oxidation studies suggest 3.5% of such units in softwood lignin (Erickson et al. 1973) and significantly larger amounts (6.5%) in hardwood lignin (Larsson and Miksche 1971). Phenylpropane Units Attached to an Adjacent Unit by a b-1 Linkage The occurrence of b-1 structures of the 1,2-diaryl-1,3-propanediol type in lignins was discovered by studying degradation products (Lundquist and Miksche 1965; Nimz 1965). Almost all types of solvolytic and reductive degradation of lignins yield comparatively large amounts of dimeric products that can be envisaged as originating from structures of this type. Nevertheless, it has not, until recently, been possible to obtain spectroscopic evidence of the occurrence of b-1 structures in lignins (Kilpeläinen et al. 1994; Lundquist 1987). Degradation studies suggest, however, much larger amounts of such structures than do the spectroscopic studies. Two possible explanations of the discrepancy have been suggested – uneven distribution of the b-1 structures in lignins (Lapierre et al. 1991; Ede et al. 1996) or formation of b-1 structures during lignin degradation from cyclohexadienone precursors (Brunow and Lundquist 1991; Lundquist 1987). 1H NMR spectroscopy suggest some 1–2% of such units in softwood lignins and perhaps as much as 5% in hardwood lignins (Lundquist 1987). The number of cyclohexadienone units has been estimated to about 1% in softwood lignin in a recent study (Zhang and Gellerstedt 1999).

4.4 Role of Lignin in Biomass Conversion 4.4.1 Introduction

Because of the very nature of this complex organic polymer and its derivatives, there has been rather slow development towards an increasing number of uses and products. Improved understanding of the structure of lignins and their derivatives will help researchers to relate their experimentation to a fundamentally sound framework of ideas.

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4.4.2 Low-molecular-weight Chemicals from Lignin

At present only a few simple chemicals (such as vanillin, dimethylsulfoxide) are produced in commercial quantities from lignin. The main reason is the absence of competitive production methods. An important factor is the cost of separating the lignin from large volumes of aqueous industrial pulping liquors. Even after drying the product is not pure lignin but contains the ash, carbohydrates, and extraneous material of the spent liquor. In the long run lignin is a renewable resource but it may well be that it will become important only when petroleum becomes an expensive raw material. 4.4.3 Polymeric Products

The uses to which polymeric lignin products can be put may be broadly separated into three classes: (1) combustion, (2) utilization of the surface active properties of salts of a lignin derivative, and (3) condensation of lignin so that it becomes an integral part of the product. A useful review of the utilization of polymeric lignin products is found in Hoyt and Goheen (1971). 4.4.4 Biodegradation

White-rot fungi have been shown to degrade lignin in an essentially oxidative process. Degradation of the lignin polymer occurs in the side chains, which are oxidized with formation of carbonyl and carboxyl groups, and in the aromatic nuclei, which are oxidatively cleaved after demethylation and introduction of hydroxyl groups in phenolic units to give 2,3- and/or 3,4-dihydroxyphenyl moieties. It is assumed (Higuchi 1990) that various lignin structural units are degraded by the mediation of extracellular enzymes which attack both low-molecular-weight and polymeric substrates via many intermediate products along several different pathways. In practice it has proved difficult to imitate with individual enzymes the degradation of lignin achieved by basidiomycetes. Oxidative enzymes (peroxidases and laccases) tend to polymerize the lignin via coupling of radical intermediates. Further research is needed to clarify the mechanism that prevents the polymerization when lignin is degraded in vivo.

References Adler, E., JM Pepper, E Eriksoo. (1957) Action of mineral acid on lignin and model substances of guaiacylglycerol-beta-aryl ether type. Ind Eng Chem 49:1391–1392, 1957.

Adler, E., K Lundquist. (1963) Spectrochemical estimation of phenylcoumaran elements in lignin. Acta Chem Scand 17:13– 26, 1963.

References Adler, E. (1977) Lignin chemistry – past, present and future. Wood Sci Technol 11:169–218, 1977. Argyropoulos, DS. (1994) Quantitative phosphorus-31 NMR analysis of six soluble lignins. J Wood Chem Technol 14:65–82, 1994. Bardet, M., D Robert, K Lundquist, S von Unge. (1998) Distribution of erythro and threo forms of different types of b-O-4 structures in aspen lignin by 13C NMR using the 2D INADEQUATE experiment. Magn Reson Chem 36:597–600, 1998. Björkman, A. (1956) Studies on finely divided wood. Part I. Extraction of lignin with neutral solvents. Sven Papperstidn 59:477–485, 1956. Bose, SK., KL Wilson, RC Francis, M Aoyama. (1998) Lignin analysis by permanganate oxidation. I. Native spruce lignin. Holzforschung 52:297–303, 1998. Brunow, G., K Lundquist, G Gellerstedt. (1998) Lignin. In: E Sjöström, R Alén, eds. Analytical Methods in Wood Chemistry, Pulping and Papermaking. Berlin: Springer, 1998, pp 77–124. Brunow, G., K Lundquist. (1991) On the acid-catalysed alkylation of lignins. Holzforschung 45:37–40. Chan, FD., KL Nguyen, AFA Wallis. (1995) Estimation of the aromatic units in lignin by nucleus exchange – a reassessment of the method. J Wood Chem Technol 15:473–491, 1995. Drumond, M., M Aoyama, C-L Chen, D Robert. (1989) Substituent effects on C-13 chemical shifts of aromatic carbons in biphenyl type lignin model compounds. J Wood Chem Technol 9:421–441, 1989. Ede, RM., J Ralph, KM Torr, BSW Dawson. (1996) A 2D NMR Investigation of the heterogeneity of distribution of diarylpropane structures in extracted Pinus radiata lignins. Holzforschung 50:161–164. Ede, RM., J Ralph. (1996) Assignment of 2D TOCSY spectra of lignins: the role of lignin model compounds. Magn Reson Chem 34:261–268, 1996. Erickson, M., S Larsson, GE Miksche. (1973) Gaschromatographische Analyse von Ligninoxydationsprodukten. VIII. Zur Struktur des Lignins der Fichte. Acta Chem Scand 27:903–914, 1973.

Freudenberg, K. (1968) The constitution and biosynthesis of lignin. In: K Freudenberg, AC Neish, eds. Constitution and Biosynthesis of Lignin. Berlin–Heidelberg: Springer, 1968, pp 47–122. Funaoka, M., I Abe, VI Chiang. (1992) Nucleus exchange reaction. In: SY Lin, CW Dence, eds. Methods in Lignin Chemistry. Berlin: Springer, 1992, pp 369–386. Habu, N., Y Matsumoto, A Ishizu, J Nakano. (1990) The role of the diarylpropane structure as a minor constituent in spruce lignin. Holzforschung 44:67–71, 1990. Hauteville, M., K Lundquist, S von Unge. (1986) NMR studies of lignins. 7. 1H NMR spectroscopic investigation of the distribution of erythro and threo forms of b-O-4 structures in lignins. Acta Chem Scand B40:31–35, 1986. Higuchi, T. (1990) Lignin biochemistry: Biosynthesis and biodegradation, Wood Sci. and Technol. 24, 23–63. Hoyt, CH., DW Goheen. (1971) Polymeric Products. In: KV Sarkanen, CH Ludwig, eds. Lignins – Occurrence, Formation, Structure and Reactions. New York: WileyInterscience, 1971, pp 833–865. Jiang, Z-H., DS Argyropoulos. (1994) The stereoselective degradation of arylglycerolbeta-aryl ethers during kraft pulping. J Pulp Pap Sci 20:J183–J188, 1994. Karhunen, P., J Mikkola, A Pajunen, G Brunow. (1999) The behaviour of dibenzodioxocin structures in lignin during alkaline pulping processes. Nord Pulp Pap Res J 14:123–128, 1999. Karhunen, P., P Rummakko, A Pajunen, G Brunow. (1996) Synthesis and crystal structure determination of model compounds for the dibenzodioxocine structure occurring in wood lignins. J Chem Soc, Perkin Trans 1 1996:2303–2308, 1996. Karhunen, P., P Rummakko, J Sipilä, Brunow G. (1995) Dibenzodioxocins; a novel type of linkage in softwood lignins. Tetrahedron Lett 36:169–170, 1995. Kilpeläinen, I., E Ämmälahti, G Brunow, D Robert. (1994) Application of three-dimensional HMQC–HOHAHA NMR spectroscopy to wood lignin, a natural polymer. Tetrahedron Lett 35:9267–9270, 1994. Kilpeläinen, I., J Sipilä, G Brunow, K Lundquist, RM Ede. (1994) Application of two-

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4 Lignin Chemistry and its Role in Biomass Conversion dimensional NMR spectroscopy to wood lignin structure determination and identification of some minor structural units of hard- and softwood lignins. J Agric Food Chem 42:2790–2794, 1994. Lai, YZ., KV Sarkanen. (1971) Isolation and structural studies. In: KV Sarkanen, CH Ludwig, eds. Lignins – Occurrence, Formation, Structure and Reactions. New York: Wiley-Interscience, 1971, pp 165– 240. Landucci, LL., GC Deka, DN Roy. (1992) A 13 C NMR study of milled wood lignins from hybrid Salix clones. Holzforschung 46:505–511, 1992. Lapierre, C., B Pollet, B Monties, C Rolando. (1991) Thioacidolysis of spruce lignin: GC–MS analysis of the main dimers recovered after Raney nickel desulphuration. Holzforschung 45:61–68, 1991. Lapierre, C., B Pollet, B Monties. (1991) Heterogeneous distribution of diarylpropane structures in spruce lignin. Phytochemistry 30:659–662. Lapierre, C., K Lundquist. (1999) Investigations of low molecular weight and high molecular weight lignin fractions. Nord Pulp Pap Res J 14:158–162, 170, 1999. Larsson, S., GE Miksche. (1971) Gaschromatographische Analyse von Ligninoxydationsprodukten. IV. Zur Struktur des Lignins der Birke. Acta Chem Scand 25:647–662, 1971. Li, S., K Lundquist. (1997) Synthesis of lignin models of b-5 type. Acta Chem Scand 51:1224–1228, 1997. Li, S., K Lundquist. (1999) Acid reactions of lignin models of b-5 type. Holzforschung 53:39–42, 1999. Li, S., T Iliefski, K Lundquist, AFA Wallis. (1997) Reassignment of relative stereochemistry at C-7 and C-8 in arylcoumaran neolignans. Phytochemistry 46:929–934, 1997. Lu, F., J Ralph. (1998) The DFRC method for lignin analysis. 2. Monomers from isolated lignins. J Agric Food Chem 46:547– 552, 1998. Ludwig, CH., BJ Nist, JL McCarthy. (1964) Lignin. XIII. The high resolution nuclear magnetic resonance spectroscopy of protons in acetylated lignins. J Am Chem Soc 86:1196–1202, 1964.

Lundquist, K., GE Miksche. (1965) Nachweis eines neuen Verknüpfungsprinzips von Guaiacylpropaneinheiten im Fichtenlignin. Tetrahedron Lett: 2131–2136, 1965. Lundquist, K., R Stomberg. (1988) On the occurrence of structural elements of the lignan type (b–b structures) in lignins. The crystal structures of (+)-pinoresinol and (±)-trans-divanillyltetrahydrofuran. Holzforschung 42:375–384, 1988. Lundquist, K., S Li. (1999) Structural analysis of lignin and lignin degradation products. Proceedings of 10th International Symposium on Wood and Pulping Chemistry Vol 1, Yokohama, 1999, pp 2–10. Lundquist, K. (1980) NMR studies of lignins. 4. Investigation of spruce lignin by 1 H NMR spectroscopy. Acta Chem Scand B34:21–26, 1980. Lundquist, K. (1987) On the occurrence of b-1 structures in lignins. J Wood Chem Technol 7:179–185. Lundquist, K. (1991) 1H NMR spectral studies of lignins. Quantitative estimates of some types of structural elements. Nord Pulp Pap Res J 6:140–146, 1991. Lundquist, K. (1992) Acidolysis. In: SY Lin, CW Dence, eds. Methods in Lignin Chemistry. Berlin: Springer, 1992, pp 289–300. Lundquist, K. (1992) Wood. In: SY Lin, CW Dence, eds. Methods in Lignin Chemistry. Berlin: Springer, 1992, pp 65–70. Matsumoto, Y., A Ishizu, J Nakano. (1986) Studies on chemical structure of lignin by ozonation. Holzforschung 40 (Suppl):81– 85, 1986. Matsumoto, Y., T Akiyama, A Ishizu, G Meshitsuka, K Lundquist. (1999) Proof of the presence of racemic forms of arylglycerolb-aryl ether structures in lignin – studies on stereo structure of lignin by ozonation. Proceedings of 10th International Symposium on Wood and Pulping Chemistry Vol 1, Yokohama, 1999, pp 126–129. Nimz, H. (1965) Über die milde Hydrolyse des Buchenlignins, II. Isolierung eines 1.2-Diaryl-propan-Derivates und seine Überführung in ein Hydroxystilben. Chem Ber 98:3160–3164, 1965. Nimz, H. (1974) Beech lignin – proposal of a constitutional scheme. Angew Chem Int Ed 13:313–321, 1974.

References Nimz, H. H., U Tschirner, M Stähle, R Lehmann, M Schlosser. (1984) Carbon-13 NMR spectra of lignins, 10. Comparison of structural units in spruce and beech lignin. J Wood Chem Technol 4:265–284, 1984. Ogiyama, K., T Kondo. (1968) On the pinoresinol type of structural units in lignin molecule. IV. The changes of dilactone-yield during enzymic dehydrogenation. Mokuzai Gakkaishi 14:416–420, 1968. Pearl, IA., DL Beyer. (1954) Studies on lignin and related products. IX. Cupric oxide oxidation of lignin model substances. J Am Chem Soc 76:2224–2226, 1954. Pew, JC. (1963) Evidence of a biphenyl group in lignin. J Org Chem 28:1048– 1054, 1963. Ralph, J., J Peng, F Lu, RD Hatfield, RF Helm. (1999) Are lignins optically active? J Agric Food Chem 47:2991–2996, 1999. Robert, DR., G Brunow. (1984) Quantitative estimation of hydroxyl groups in milled wood lignin from spruce and in a dehydrogenation polymer from coniferyl alcohol using 13C NMR spectroscopy. Holzforschung 38:85–90, 1984. Rolando, C., B Monties, C Lapierre. (1992) Thioacidolysis. In: SY Lin, CW Dence, eds. Methods in Lignin Chemistry. Berlin: Springer, 1992, pp 334–349. Saake, B., DS Argyropoulos, O Beinhoff, O Faix. (1996) A comparison of lignin polymer models (DHPs) and lignins by 31P

NMR spectroscopy. Phytochemistry 43:499–507, 1996. Sakakibara, A. (1992) Hydrogenolysis. In: SY Lin, CW Dence, eds. Methods in Lignin Chemistry. Berlin: Springer, 1992, pp 350–368. Sarkanen, KV., A Islam, CD Anderson. (1992) Ozonation. In: SY Lin, C.W. Dence, eds. Methods in Lignin Chemistry. Berlin: Springer, 1992, pp 387–406. Suckling, ID., MF Pasco, B Hortling, J Sundquist. (1994) Assessment of lignin condensation by GPC analysis of lignin thioacidolysis products. Holzforschung 48:501–503, 1994. Taneda, H., N Habu, J Nakano. (1989) Characterization of the side chain steric structures in the various lignins. Holzforschung 43:187–190, 1989. Tomimura, Y., T Yokoi, N Terashima. (1980) Heterogeneity in formation of lignin. V. Degree of condensation in guaiacyl nucleus. Mokuzai Gakkaishi 26:37–42, 1980. Wallis, AFA. (1971) Solvolysis by acids and bases. In: KV Sarkanen, CH Ludwig, eds. Lignins – Occurrence, Formation, Structure and Reactions. New York: Wiley-Interscience, 1971, pp 345–372. Zhang, L., G Gellerstedt. (1999) Detection and determination of carbonyls and quinones by modern NMR techniques. Proceedings of 10th International Symposium on Wood and Pulping Chemistry Vol 2, Yokohama, pp 164–170.

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5 Industrial Lignin Production and Applications E. Kendall Pye

5.1 Introduction

As one of the three major polymers always present in woody biomass, lignin represents a considerable proportion of the structural components of plants. It can account for approximately 10–12% of the aerial portion of some short annual plants and up to 30% or more for some coniferous trees [1]. As such it is claimed to be the second most abundant organic chemical on earth [2] and is therefore a major renewable chemical that should not be overlooked, either in volume or value. Significant commercial markets, totaling over one million tons per year, already exist for lignins that have been recovered from chemical pulp mills [3]. The value of lignin in these markets is generally an order of magnitude higher than its fuel value. With the anticipated future construction of biorefineries processing lignocellulosic feedstocks, the amount of lignin potentially available for marketing for its chemical value, rather than its fuel value, is likely to be enormous. Furthermore, lignin from some types of biorefineries will most probably have better performance characteristics and be of greater commercial value for its chemical properties than lignins from existing chemical pulping operations. The availability of these “new” lignins with enhanced physical and chemical properties will no doubt stimulate major new markets for this renewable material. However, it is likely that these materials will find their initial markets in the same sectors as the current lignin products. A review of the manufacturing, properties and present markets for lignins from pulping operations is therefore instructive in assessing the potential economic value of lignin that might be recovered from biorefineries processing lignocellulosic materials. Presently lignin is separated on an industrial scale from wood and from some annual plants, such as straw, bagasse and flax, by the chemical pulping industry, using primarily the kraft, sulfite and soda pulping processes [4]. The vast majority of this lignin is not isolated and recovered but is burned in chemical recovery boilers as components of the concentrated black liquor feed. As such it provides low value fuel to the pulp mill for the production of steam and power Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 2 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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[4]. A small number of companies have, on the other hand, introduced systems for the partial recovery and purification of lignin and have developed significant businesses that market these materials to a large number of different industries for diverse applications [3]. In future, biorefineries processing lignocellulosic materials with the primary purpose of producing fermentable sugars from cellulose and hemicellulose, will generate lignin in very substantial quantities. The lignin produced by these operations will almost certainly be chemically different from the presently available industrial lignins and they will most probably be closer to the chemical structure of the native material. Consequently, new applications will arise for these more versatile products, but they will also be capable of competing in the existing lignin markets, representing strong competition to the presently available lignin products. The lignins that are currently produced and marketed today are recovered from the cooking liquors of the chemical pulping industry. They are, however, chemically modified to a greater or lesser extent by the chemistry of the specific chemical pulping process from which they are derived [1]. In the sulfite pulping process, for example, sulfonic acid groups are introduced into partially hydrolyzed lignin in the wood converting it into a fully water-soluble lignosulfonate. This becomes a major component, together with the hemicellulose sugars, of the spent sulfite liquors. In the kraft pulping process the native lignin is converted into lower molecular weight fragments of thiolignin by the introduction of thiol groups through the action of the sodium sulfide employed in the process. This thiolignin is soluble in the strong alkali present in the cooking liquor, but can be precipitated and recovered by acidification of the kraft black liquor [1]. Soda pulping causes a hydrolytic cleavage of the native lignin into smaller fragments that are soluble in the strongly alkaline cooking liquors, but the resultant lignin is otherwise relatively unmodified chemically. The markets and the applications of the various forms of lignin presently recovered from commercial chemical pulping operations are therefore determined by the nature of the lignin that is produced by each process. As mentioned previously, when compared with the total amounts that are separated from wood and woody fibers by the chemical pulping industry, the amounts of the various forms of lignin that are presently recovered and sold commercially are very small, representing a few percent of all that is extracted by that industry. Worldwide, almost all of the lignin separated from woody feedstocks by the pulping industry is burned in chemical recovery furnaces because of the need to recover and recycle the inorganic cooking chemicals. An exception to this is in some lesser developed countries where many small soda pulp mills that process annual fibers cannot afford chemical recovery boilers and therefore dispose of their cook liquors into the environment, either directly or following minimal treatment. This latter activity is now being actively discouraged by almost all governments, with one result being the closure over the last decade of many small chemical pulp mills, especially in China [5]. In the larger chemical pulp mills, where chemical recovery boilers are an economic necessity, the lignin is part of a low value fuel that provides steam and power to the pulp mill.

5.1 Introduction

Several lignocellulosic biorefinery technologies that are now under development produce lignin as a wet, solid residue that remains after the saccharification and/or fermentation stages, often in a form contaminated with residual cellulose. As such, these particular biorefinery processes find it most convenient to use this lignin residue as a low value boiler fuel to provide power and steam for the process [6]. On the other hand, pure lignin that can be readily produced by some biorefinery technologies, such as organosolv technology, could be an excellent source of industrial specialty and commodity chemicals, displacing materials now produced from crude oil and natural gas [7]. Lignin, with its dominant aromatic chemical structure, represents a potential new source of aromatic chemicals and other products that are used extensively in industry. Simply using this versatile material as a fuel would represent its lowest value application. In principle, using known technology, higher value, purified lignin can be recovered equally effectively from either the original woody biomass feedstock prior to saccharification, or from the residues of the saccharification and/or fermentation stages. However, it is now becoming well recognized that the presence of lignin and also hemicellulose to some extent, can significantly inhibit the velocity and efficiency of cellulose saccharification by cellulolytic enzymes, and even saccharification by acid hydrolysis [8]. Feedstock pretreatment by either mechanical or chemical methods, which partially removes or modifies these two materials, is therefore a universal feature of almost all presently proposed biorefinery processes. Most of these pretreatments, such as steam explosion [9], dilute acid hydrolysis [10], and ammonia fiber explosion (AFEX) [11], serve to disrupt the physical and possibly the chemical relationships between the cellulose, hemicellulose and lignin, which in native woody biomass is highly integrated. Such pretreatments can influence the chemical structure and the physical properties of the lignin making it more or less valuable in potential commercial applications. Most of the proposed biorefinery pretreatments will produce a lignin that has been partially depolymerized by hydrolysis, mostly at the aryl–alkyl ether linkages [12], creating a lignin product that, unlike the majority of presently available lignins, has no sulfur-containing substituents. Provided that the conditions of the pretreatment and the lignin recovery do not introduce substantial recondensation of the lignin fragments the number average and the weight average molecular weight of these lignin products should be small (1000–2000) and the polydispersity should be low [7], leading to a very uniform product. This is different from the situation that exists for currently available commercial lignins. Lignosulfonates from the sulfite pulping process, for example, have a very high degree of sulfonic acid substitution and a very high polydispersity [13]. Over the past fifty years or so, numerous research papers and conferences have been devoted to the development of specialty and commodity applications for pure, unmodified lignin [3, 14, 21], but today few of these exciting applications have been commercialized. The reason is clear. Until very recently, the only lignins available commercially and in large quantities were thiolignins

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from the kraft pulping process and lignosulfonates from the sulfite pulping process. These chemically-modified lignins are presently being marketed primarily by two major suppliers with roots in the pulp and paper industry, the Specialty Chemicals division of MeadWestvaco Corporation and Borregaard Lignotech division of Borregaard Industries [3]. Chemically nonderivatized lignins, of the type used in many investigative studies and of the type likely to be produced by biorefineries, have not been available in the quantities required to support a significant commercial market. With the anticipated commercial introduction of biorefineries processing lignocellulosics, this situation is about to change. These biorefineries could produce large quantities of lignin in a relatively uniform and purified form. Such lignins can either be used for low value fuel, or they can be used for much higher value industrial chemical applications, which would substantially improve the economics of the biorefineries producing them. The lignin output from these future biorefineries could become the raw material for a major new industry making products that either substitute for chemicals presently made from crude oil or natural gas, or products that are new to the market place. Some current and future market and product opportunities are presented in this chapter.

5.2 Historical Outline of Lignin Production and Applications

An excellent and detailed account of the discovery and history of lignin, as well as the history of the chemical pulping industry, has recently been provided by J. L. McCarthy and A. Islam [15]. Starting with the discovery of lignin in 1838 by Payen in France [16], there has been a substantial continuing interest in lignin both at the industrial and the chemical research levels. This interest accelerated with the introduction of a process in 1866 to produce pulp for papermaking from woody materials by chemical pulping (in which the bulk of the lignin is removed in order to release intact individual fibers) using sulfurous acid [17]. Before this time high quality printing and writing paper had been produced almost exclusively from old rags. 5.2.1 Lignosulfonates from the Sulfite Pulping Industry

The first chemical wood pulp mill, using a calcium based sulfite liquor, was built in Sweden in 1874 followed by a mill in the US in 1882 and a mill in Canada in 1888 [15]. From that time on the sulfite pulping process became the dominant chemical pulping process for wood until the kraft process started to expand in the 1930s following the invention of the Tomlinson recovery furnace. Prior to this time, the lignin-containing “spent liquors” from the sulfite pulp mills were discharged into the environment, usually into the nearest waterway.


As this practice became more socially and environmentally unacceptable alternative disposal mechanisms were introduced including the spraying of these materials onto road surfaces for dust suppression and their inclusion into animal feeds. These spent sulfite liquors from the sulfite pulping industry were erroneously referred to as “lignin” in the trade, a misnomer that still is problematic for many attempts to convince individuals in the pulp and paper industry of the high value of purified lignin. In fact, the solids composition of this sulfite spent liquor, known as “lignin”, includes a substantial quantity of inorganic materials and even the organic fraction contains approximately 50% sugars, which accounts for its value as an animal feed additive. Lignosulfonate therefore represents considerably less than 50% by weight of the total solids of the so-called “lignin” from a sulfite mill. In the early part of the twentieth century a small number of sulfite pulp mills began to consider the potential commercial value of purified lignosulfonates and initiated investigations on applications and commercial development. Perhaps the earliest of these, in 1909, was the Marathon Corporation at their Rothschild mill near Wausau, Wisconsin, in the US [18]. This led, starting in 1927, to the development of commercial products from the organic components of the spent sulfite liquor and was a milestone in the commercial development of lignin-based products. Full-scale commercial production of lignosulfonate products started at Marathon with the construction of a production facility in 1936. Marathon, after passing through several corporate hands that included American Can, Reed Lignin and Daishowa Chemicals, is now the North American flagship site of Borregaard LignoTech, Inc., the major supplier of specialized products from lignosulfonates that are produced by the sulfite pulping industry [18]. With the successful introduction of chemical recovery furnaces for the kraft process in the 1930s, many sulfite mills changed from the classical calciumbased sulfite liquors to using pulping liquors containing cations such as sodium, ammonium and magnesium [15]. These systems provided not only technical advantages for the pulping operations, but also allowed the use of recovery furnace systems that recovered both the cation and the SO2. In these systems the lignosulfonate is burned and it is therefore no longer available for industrial applications. Today, purified lignosulfonates are produced from those sulfite pulp mills that do not practice chemical recovery, or that have spent liquor in excess of their capacity to burn it. 5.2.2 Lignin from the Kraft Pulping Industry

With the invention of the Tomlinson chemical recovery furnace in the early 1930s the kraft process started to displace the sulfite process as the primary chemical wood pulping technology, except for the production of some specialty pulps such as dissolving pulps. Even though a number of specialty sulfite pulp mills have been constructed in the intervening period, the sulfite pulping indus-

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try has been a steadily declining fraction of the overall chemical pulping industry since that time; a trend that continues today. In 1942, at Charleston, South Carolina, the Westvaco Company started to produce lignin products from the black liquors obtained from the kraft pulping of softwoods and hardwoods. That company, now MeadWestvaco Corporation, through its Specialty Chemicals division, continues as the dominant supplier of lignin products obtained from kraft black liquor. While this business appears to be quite profitable, MeadWestvaco has attracted few competitors for its kraft lignin business, probably because the recovery of kraft lignin from black liquors is neither simple nor inexpensive. Furthermore, the manufacture and marketing of lignin products appears as a complicating divergence from their principle business for most pulp and paper companies. Today, vast quantities of lignin are produced in the kraft industry worldwide, probably greater than 70 million tonnes per year, but of this amount more than 99% is burned in chemical recovery furnaces and is not recovered for industrial applications. Separation and recovery of significant quantities of lignin from kraft black liquors changes the organic to inorganic ratio in the recovery furnace feed and will potentially disrupt operations of those critical pieces of equipment. With the huge scale of present day chemical pulp mills, any possibility for an expensive disruption of normal operations is carefully avoided by most company managers. It is likely that no more than 100 000 tonnes per year of kraft lignin is marketed for its chemical value, worldwide. However, a significant fraction of this amount is chemically modified to convert it into a water-soluble sulfonated lignin that competes in some applications against lignosulfonates from the sulfite pulping industry [19]. 5.2.3 Lignin from the Soda Pulping Industry

While it is the oldest of the chemical pulping processes, the soda process [4], in which chemical pulp is produced by the delignifying action of sodium hydroxide, is now almost exclusively used for the production of chemical pulps from annual plants, such as sugar cane bagasse, flax and cereal straws. Soda pulping is not as effective a pulping process as kraft or sulfite when applied to either hardwoods or softwoods. Because of the relatively high ash content of the raw materials, annual fiber pulp mills usually face the serious problem of a high inorganic content, particularly silica, in the soda black liquors, which restricts their ability to use chemical recovery methods similar to the kraft process. Furthermore, because of the seasonal nature of the availability of the annual fiber feedstock most of these mills are very small and are mostly located in developing countries. In the past, the standard treatment of the black liquors from these pulp mills has been to discharge them directly into the environment. Lignin has not been recovered from such sources in the past primarily because of the relatively unsophisticated nature of the industry in the regions that practice soda pulping.


However, the lignin produced in the soda pulping of annual fibers is potentially a very useful material. Unlike kraft and sulfite lignins, soda lignins have not had sulfur introduced into their chemical structure. In addition, they have interesting thermal and solubility properties that make them especially valuable in certain commercial applications. One company, Granit S.A., of Lausanne, Switzerland, is now selling sulfur-free lignin from the soda pulping of annual fibers for higher value applications [20]. As noted above, most annual fiber pulp mills are small and located in developing countries, so the availability of this lignin is a concern as the markets for it expand. Granit has addressed this problem in an innovative way. Recognizing that small annual fiber soda pulp mills have severe difficulties dealing with their silica-containing pulping liquors, Granit has developed a technology that allows these mills to solve their liquor disposal problems while at the same time providing Granit with the lignin product it requires for its lignin marketing business [22]. The lignin recovery process developed by Granit, called the Lignin Precipitation System (the LPS process), recovers lignin from the soda black liquor [23]. In this process, the black liquor is first filtered to remove any contaminating pulp fibers. The filtered liquor is then acidified to create a lignin slurry, which is conditioned in a maturation step and then filtered to remove the lignin solids. Following washing, the lignin cake is dried to a powder of high purity lignin containing about 5% moisture. The removal of lignin reduces the COD of the original black liquor by about 50%, which can then be treated either by anaerobic digestion or by wet oxidation. The capital cost of the combined system would appear to much lower than that of a typical alkali recovery system used in conventional pulp mills, which in any case would be difficult to scale down to a size appropriate for the small annual fiber mills. The interesting feature of this business arrangement is that Granit will purchase the high purity dried lignin powder from the mill [24]. Thus, installation of the LPS system increases revenues to the mill, solves an environment problem and generates lignin product that is required by the lignin marketing arm of Granit. The first commercial installation of the LPS technology was undertaken at the flax pulping mill of Papeteries du Léman in Thonon, France, in 2000 [20]. In this operation the entire black liquor generated from the soda pulping of flax is fed into the relatively small LPS lignin recovery unit. After the lignin is filtered from the slurry and dried, the filtrate is sent presently to a municipal treatment plant, but soon it will be treated in a Granit wet oxidation reactor that will be part of a new on-site effluent treatment plant. A second LPS system is under construction at a mill in India and is anticipated to be operational in 2005. The potential lignin production is over 10 000 tonne per year of dried high purity lignin powder ready for the market. Interesting alternative uses of the LPS technology are for the debottlenecking of an existing conventional alkali recovery system, or for the expansion of pulping capacity in an existing mill.

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5.3 Existing Industrial Lignin Products 5.3.1 Lignosulfonates

As previously stated, the sulfite pulping industry, in its earlier days, simply discharged its spent liquor into the environment. As this practice became less acceptable and also for economic reasons, some in the industry started to look for alternative uses for the spent sulfite liquor. Initially, the simplest approach was to partially evaporate the liquor to reduce its volume and its transportation cost and then to use the concentrated material as road dust suppressant, or animal feed supplement [3]. One of the processes used to upgrade the spent liquors involves the fermentation of the sugars in the liquor, which represents close to 50% of the organic material, to produce food and fodder torula yeasts. The remaining partially purified lignosulfonates can then be concentrated and modified to produce a series of industrial products suitable for commercial application. Such a plant is operated by Wausau Paper Mills at the Rhinelander sulfite mill in Wisconsin, but other similar plants have been constructed in the western US but are not presently operating. As the sulfite pulp mills that supplied the spent liquors were closed the fermentation and lignosulfonate plants were also mostly closed.

5.3.1.1 Chemical Characteristics of Lignosulfonates The sulfite pulping process can be operated either at acid, neutral or alkaline pH. In all cases the native lignin in the wood is cleaved into smaller fragments and the bulky sulfonic acid group is introduced at the a and/or the c carbons of the phenylpropane unit of the lignin. The reactions responsible for this modification include sulfonation (the introduction of the sulfonic acid group mostly at the a-position), hydrolysis (the hydrolytic cleavage of the aryl–alkyl ether bonds between the phenylpropane groups, mostly the b-O-4 linkage), sulfitolysis (similar to hydrolysis, but leads to the formation of lignosulfonic acid, and condensation (reactions that produce new linkages between the lignin fragments, resulting in higher molecular weight components). The resulting lignosulfonate can have a broad range of molecular weights that might range from several hundred up to ninety thousand daltons, or greater. Unless deliberately fractionated, the same lignosulfonate product will span this entire molecular weight range, leading to a relatively nonuniformly functioning product in some applications. Because of the introduction of the bulky, polar sulfonic acid groups into the molecule, lignosulfonates are water soluble over essentially the entire useful pH range. Consequently, they find their greatest utility in applications that exploit their surfactant and dispersant properties. The sulfonic acid group cannot be readily or economically removed from the molecule. Therefore lignosulfonates, especially with their high polydispersity, do not

5.3 Existing Industrial Lignin Products

represent a useful source of underivatized sulfur-free lignin. Lignosulfonates are considered to be non-toxic at typical usage levels [3] and are regularly used in animal feeds and agricultural and horticultural applications.

5.3.1.2 Lignosulfonate Producers With the steady decline in the number of operating sulfite pulp mills worldwide, the number of lignosulfonate producers has declined and these have been consolidated into just a few suppliers [3]. Over the last thirty years or so, previously wellknown suppliers of lignosulfonates, such as Crown Zellerbach, Rayonier Silvichemicals, American Can, Georgia-Pacific and Reed Lignin have gone out of the business. Almost always this was because of the sale, or more frequently the permanent closure, of the sulfite pulp mill that supplied the raw material for the business. A classic example is that of Georgia-Pacific which had a major lignosulfonates business, producing almost 200 000 tonnes per year of solids, until the company closed its Bellingham, Washington, sulfite mill in March 2001 for reasons unrelated to the ligosulfonate business. At this point, without a supply of raw material Georgia-Pacific exited the lignosulfonates business. Today the worldwide lignosulfonates business is dominated by Borregaard LignoTech, which makes a number of products and grades. LignoTech has operations worldwide and markets about 400 000 tonnes of lignosulfonate solids per year [3]. There are several smaller sellers of lignosulfonate products, including Tembec, Fraser Papers and Nippon Papers in Japan that have total sales just less than those of Borregaard. Even as recently as twenty years ago it could be calculated that the worldwide production of commercial lignosulfonate chemicals was well in excess of 1.2 million tonnes per year. But, with the precipitous closure in 2001 of the Bellingham sulfite mill that was a major production source, the available supply of lignosulfonates dropped to below 800 000 tonnes per year, which caused a considerable short term market disruption. Earlier, in 1997, Borregaard assured itself of an additional significant supply of lignosulfonates when it signed an agreement with Sappi/Saiccor to construct a 55 000 ton per year lignosulfonates plant at its Umkomass sulfite mill in Natal, S. Africa that started up in 1998. This plant was expanded to 155 000 tonnes per year in 2003 partly in response to the capacity shortfall caused by the closure of the Georgia-Pacific Bellingham mill [18]. The Umkomass mill has two pulping lines: one a calcium sulfite line of approximately 450 tonnes per day of pulp, while the other is a magnesium sulfite line of approximately 550 tonnes per day of pulp that has its own recovery boiler. At the time, this calcium sulfite line represented one of the larger non-utilized lignosulfonate sources available for lignosulfonate recovery anywhere in the world. The project also benefited Sappi by removing one of its environmental problems, since in the past the mill had discharged the spent liquors into the ocean [18]. An example of a typical smaller lignosulfonate producer is Fraser Papers Inc. that produces about 170 tons of liquid lignosulfonate products daily at its Park Falls, Wisconsin, mill. This pulp mill produces 170 tons of calcium bisulfite

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birch hardwood pulp per day. These lignosulfonate products are sold in liquid form having a solids content of between 52% and 62% and a pH as low as 2–3 and as high as pH 7–8, depending on the grade. The more purified grades, which are used primarily as binders and dispersants, have approximately 5% sugars, 4–5% calcium, and 6% sulfur as a percent of dry matter, with up to 80% of the solids being lignin. The less pure grades would have between 30% and 35% of the solids as sugars and about 55% as lignin.

5.3.1.3 Markets for Lignosulfonates Many smaller lignosulfonate producers still rely on the traditional markets for spent sulfite liquors such as road dust suppression, feed pellet binders and animal feed additives. But the larger producers exploit newer, more specialized markets with products that are relatively more purified and refined. A partial list of applications and markets for lignosulfonates is given in Table 5.1. In many of these applications lignosulfonates act as dispersants, emulsifiers and surfactants. For example, in concrete admixtures, the lignosulfonate performs as a plasticizer, reducing the amount of water required to produce a workable concrete mixture and thereby creating a final concrete having greater compressive strength, durability, density and better uniformity. They act in a similar manner in the production of bricks, ceramics and refractories by dispersing the clay particles and reducing the amount of water required in the early stages of manufacture. This reduces drying costs and increases the “green” strength of the products, which holds the shape better before the formed bodies are sent to the kiln for firing. As binders in feed pellet production and for agricultural fertilizers and minerals, the lignosulfonates coat and bind the small particles together providing greater granule strength, less dusting and better handling. Because of the water solubility of the lignosulfonates, the products break down easier in the environ-

Table 5.1 Major applications of lignosulfonate products. Applications of crude spent liquor lignosulfonates

Applications of refined lignosulfonates

Feed and pellet binders Feed molasses extenders Dust suppression and road stabilization Granulation and agglomeration Plant micronutrients and horticulture Agricultural dispersants and emulsifiers Grinding aids Metal ore processing

Oil well drilling fluids Dye and pigment dispersants Protein precipitants Tanning agent Gypsum board manufacture Cement manufacture Concrete admixtures Refractory clays and ceramics Carbon black Phenolic resins Lead acid storage battery plates

5.3 Existing Industrial Lignin Products

ment or, in the case of feed pellets, in the animals digestive tract. An added advantage in pelletizing operations is that the lignosulfonates provide lubrication for the die of the pellet forming machine, which reduces the energy consumption and increases productivity of the pelletizing equipment. 5.3.2 Kraft Pulping and Kraft Lignin Recovery

The kraft pulping process has now become the dominant chemical pulping process for making pulp from wood. It uses a combination of sodium hydroxide and sodium sulfide to delignify the wood by causing thiolysis mostly of the aryl–alkyl ether linkages, which occur predominantly at the b carbon of the phenylpropane unit. This thiolysis introduces a thiol group into the partially depolymerized lignin, which can account for about 2–3% by weight of the lignin product. This lignin becomes a component of the kraft black liquor, which in almost all kraft pulp mills is evaporated to greater than 60% by weight of solids and then burned in an on-site kraft chemical recovery boiler [4]. The purpose of the recovery boiler is to allow the recover and recycle of the inorganic cooking chemicals. The burning of the concentrated black liquor produces steam, which can be used for power production and also process steam. To recover kraft lignin it is necessary to remove some lignin from the black liquor prior to its entry into the recovery boiler. This can be done by acidifying the black liquor, initially with carbon dioxide from flue gas, and then by addition of sulfuric acid. A precipitate of sodium lignate is formed, which is coagulated and filtered and in most circumstances dried. Only a small portion of the lignin in the black liquor can be recovered otherwise the organic content of the concentrated liquor becomes too low to support combustion in the recovery boiler.

5.3.2.1 Producers of Kraft Lignin At this time the one major producer of kraft lignin is the MeadWestvaco Corporation at Charleston Heights, South Carolina, USA. Kraft lignin is also sold by Borregaard LignoTech. MeadWestvaco uses some of its production of kraft lignin internally to make sulfonated derivatives for many of the lignosulfonate markets and other products.

5.3.2.2 Markets for Kraft Lignin Kraft lignins find applications [19] mostly as: · Rubber reinforcers · Activated carbon · Carbon black substitutes · Phenolic resin components · Raw materials for production of methylsulfonates, which find applications in mostly the same markets as lignosulfonates, but in some cases with superior performance.

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5.3.3 Lignins Produced from the Soda Process

As stated previously, the Swiss company Granit is now producing and marketing annual fiber lignin from the soda pulping process [20]. The markets for this product are developing rapidly and it is finding applications in many of the markets currently being exploited by kraft lignin producers. However, with its particular advantageous properties it is likely to find new markets, especially in animal nutrition and health, which are not presently being served by other forms of lignin. 5.3.4 Lignin from Other Biomass Processing Operations

Furfural is made by treatment of pentose-rich biomass, such as bagasse and oat hulls, with strong mineral acid at high temperature. These conditions favor the hydrolysis of hemicellulose and the dehydration of the resultant pentose sugars to furfural. Following recovery of the furfural a solid residue consisting mostly of lignin and cellulose remains in the digester. This residue can be extracted with solvent or alkali to dissolve and recover a substantial fraction of the lignin. One commercial form of this lignin, derived from bagasse residues from a furfural plant, is known as Sucrolin. Because it is derived from an annual fiber, the methoxy content of this lignin is extremely low, which could imply greater reactivity than lignins from wood. The solubility characteristics of this lignin, however, are similar to kraft lignin and it might be anticipated that the lignin is more condensed than lignins produced under less intense conditions, such as the soda process. 5.3.5 Comparisons of the Physical and Chemical Properties of Commercially Available Lignins

A general comparison of the chemical and physical properties of the three commercially available lignin is given in Table 5.2. It can be seen that lignin from the soda process is distinguished from the other two since it contains no measurable sulfur and has a very low water solubility.

5.4 Lignin from Biorefineries Table 5.2 Comparative properties of commercial lignins. Property

Softwood kraft lignin

Softwood lignosulfonate

Soda lignin from straw

Carbon, % Hydrogen, % Methoxy, % Ash, % Wood sugars, % Sulfur, % Water solubility Tg, 8C Softening pt. 8C Mol. wt., MN

66 5.9 14 3 Low 1.6 Low 140 Not detected 2000

53 5.4 12.5 2.5 Up to 50% 6–7.9 Very high Not detected Not detected 400–150 000

56 7.5 N/A < 2.5 2.5–3.5 N/A Very low 150 N/A 2300–2900

5.4 Lignin from Biorefineries

5.4.1 Advantages of Lignin and Hemicellulose Removal on Saccharification and Fermentation of Cellulose

Strong mineral acid can be employed to saccharify cellulose, as was done in the Scholler process and other processes reviewed by Wenzl [25], to produce ethanol from wood. However, acid hydrolysis creates a number of significant difficulties. Strong mineral acid will readily penetrate the walls of the woody material and quickly initiate both cellulose and hemicellulose saccharification but, being a rather indiscriminate catalyst, it also induces other less desirable reactions in wood hydrolysis. These side reactions include the dehydration of pentose and hexose sugars to form furfural and 5-hydroxymethylfurfural, two potent inhibitors of fermentation organisms. This not only makes it difficult to ferment the resultant sugars directly, but it also leads to a significant reduction of glucose yield below the theoretical maximum. Additionally, strong mineral acid, at the elevated temperatures required for cellulose hydrolysis, induces potentially undesirable condensation reactions in the lignin, thus reducing its versatility and value as a chemical intermediate and product. Consequently, lignin recovered from acid hydrolysis processes is usually utilized only as a boiler fuel. Cellulase enzymes are much more specific catalysts that operate closer to ambient temperature than do acid hydrolysis systems. They do not catalyze reactions in lignin, although they may have secondary hydrolytic activity on hemicellulose. Neither do they create fermentation inhibitors. As a consequence, they have the ability to carry out almost complete saccharification of relatively clean cellulose, when the enzyme profile is appropriately adjusted. The glucose yields

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from cellulose using enzymatic hydrolysis can be greater than 90% of theoretical [26], compared with acid-catalyzed hydrolysis which may be little better than 70% of theoretical. For these and other reasons the technology focus for future lignocellulosic biorefineries has now mostly shifted towards enzymatic saccharification of cellulose, especially with the recent major reductions in the price of cellulase enzymes [27]. Starch saccharification has moved almost exclusively towards enzymatic systems, for similar reasons. However, enzymes are large bulky catalysts that cannot readily diffuse through the encrusting hemicellulose and lignin that surrounds the cellulose in woody materials. Cellulase enzymes react very slowly with native lignocellulosic materials. For this and other practical reasons biorefinery technology for lignocellulosic conversion has now found it essential to include some type of feedstock pretreatment stage. Such pretreatments must accomplish the physical disruption of the native lignocellulosic biomass sufficiently to allow the diffusion and penetration of the enzymes into the biomass structure and give ready access to the cellulose molecules in the primary and secondary walls of the woody fiber. Woody biomass pretreatments that are under investigation include (1) one stage steam explosion, (2) two stage steam explosion in the presence of SO2 or sulfuric acid, (3) ammonia fiber explosion (AFEX), (4) dilute mineral acid, and (5) organosolv delignification. More exotic pretreatments, such as treatment with super critical water, are also being investigated. The various fiber explosion processes and dilute mineral acid pretreatments have the effect of hydrolyzing and solubilizing hemicellulose and also of opening up the tight structure of the fiber structure to expose cellulose more for enzymatic attack. They do not remove lignin from the pretreated material unless specific extraction steps, usually with hot ethanol or hot alkali, are included after the initial explosion stage. Recent studies have indicated that the presence of lignin and even hemicellulose in the pretreated biomass can reduce the speed and possibly the extent of enzymatic saccharification of the cellulose [28]. Partly this is because of nonspecific, and possibly irreversible, binding of the cellulase enzymes to the lignin. The effect of this phenomenon, which can be overcome by adding excess enzymes, is to increase the enzyme requirement and consequently the operating costs of the biorefinery. Organosolv pretreatment, which involves treating the raw lignocellulosic material with an aqueous organic solvent (frequently ethanol) at temperatures in the range of 180–200 8C, specifically hydrolyzes most of the hemicellulose and lignin and causes them to dissolve in the liquor [29]. Following washing of the remaining solid fiber, the residual lignin is a minor part of the finely disrupted material. Studies have shown that organosolv-pretreated woody biomass is highly susceptible to cellulase hydrolysis, with the extent of cellulose saccharification being greater than 90% of theoretical [26]. Even though dehydration products of the sugars are formed in organosolv processes, they do not contaminate the saccharification and fermentation stages because the solid fiber is washed extensively to remove and recover lignin that is dissolved in the liquor retained in the fibers following the pretreatment. The other advantage of organosolv pretreatment is that a relatively pure, partially hydrolyzed lignin product is easily

5.4 Lignin from Biorefineries

recovered from the liquor as well as various components of the hydrolyzed hemicellulose [29]. This lignin has excellent properties for use in various commercial applications [7]. It should also be possible to recover a useful lignin from the solid residuals that remain following saccharification and fermentation of woody biomass that has been pretreated by steam explosion and possibly dilute acid hydrolysis. This could be accomplished by subjecting these lignin-rich residuals, which would be contaminated with undissolved cellulose and hemicellulose to an organosolv extraction. However, this approach would lose the advantage of removing the lignin prior to enzymatic saccharification. Alternatively, the lignin-rich residuals of the saccharification stage, despite their contamination with polysaccharides, might find a valuable use in industry. 5.4.2 Lignin from an Organosolv Biorefinery

Recent interest in environmentally friendly chemical pulping has encouraged the investigation and development of organosolv pulping. Various organosolv processes have been proposed and investigated over the last twenty-five years or so. These include the Alcell process [29], using ethanol; Acetosolv, using acetic acid; Formacell, using formic acid; pulping with phenol; and the Organocell process using methanol. The Alcell process, developed by Repap Enterprises Inc, during 1987–1997, is perhaps the most commercially advanced of the ethanol-based organosolv pulping processes. This technology employs an aqueous solution of ethanol as the pulping liquor. Cooking at relatively high temperatures, approximately 1958C (and consequently relatively high pressures, about 28 bars) allows the production of a bleachable pulp from hardwoods and from most annual fibers. A key characteristic of this process is the required recovery of a natural “organosolv” lignin as one of a series of co-products that also include bleachable pulp, furfural, acetic acid and xylose [29]. The Alcell pulping process was operated in a pre-commercial demonstration plant at Repap’s Miramichi pulp and paper mill in New Brunswick, Canada. This demonstration plant is shown in Fig. 5.1. This plant, with a design capacity of 30 tonnes of pulp per day, operated intermittently from 1989 to 1996 and produced more than 3700 tonnes of organosolv lignin. This lignin was in the form of a dry powder and was sold in supersacks containing approximately 800 kg each. All of the lignin produced at the plant was sold commercially during the time the plant operated, except for some minor portion that was used for internal study and development purposes. Unfortunately, with the financial collapse and consequent breakup of Repap in 1997, this plant was closed, but the technology has since been acquired by Lignol Innovations Corporation of Vancouver, Canada, and is now being commercialized as a biorefinery technology with the cellulose fraction being used for ethanol production instead of pulp [30]. The availability of tonnage quantities of this new form of lignin ignited a veritable explosion of interest in identifying new applications for this product. Large

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Fig. 5.1 View of the organosolv-based Alcell Demonstration Plant at Miramichi, New Brunswick, Canada, showing the distillation column for ethanol recovery (left) and the lignin dryer building (right, with loading door).

samples were distributed to universities and to companies for applications testing in many different market areas. Commercial sales started as early as 1990. In 1995, when Repap was preparing to construct a 450 tonne per day Alcell pulp mill at Atholville, New Brunswick, several studies were commissioned to identify marketing strategies for the 56 000 tonnes of organosolv lignin output from this mill. These studies clearly showed that several hundred thousand tonnes of lignin per year could be easily sold at that time for an average price close to the price of PF resins. A different form of organosolv pulping technology was commercialized in Germany in 1992 under the name of Organocell. Initially, it was the intention of Organocell to recover lignin and market it as a co-product. Before mill construction a significant amount of successful development work was undertaken to identify markets in Europe for this lignin product. Unfortunately, process changes introduced during mill construction eliminated the ability to recover lignin [31].

5.5 Applications and Markets for Lignin

5.5 Applications and Markets for Lignin 5.5.1 Phenol–Formaldehyde Resin Applications

One of the earliest types of thermoset synthetic resins is phenol–formaldehyde (PF) resin produced by reacting phenol and formaldehyde in the presence of an acid or alkaline catalyst. PF resins now have a wide range of industrial applications [32]. These resins display a useful range of hardness, physical strength, glossy finish, electrical properties, heat resistance and chemical stability. They are one of the most widely used groups of plastics in the world, with total annual market size that can now be estimated to be greater than 2.5 million tonnes. The estimated worldwide capacities, markets and major applications are excellently reviewed by others [33]. Phenolic resins are normally divided into resols or novolacs. Resols are socalled one-stage resins that are most usually used as binders in the production of exterior grade wood panels (such as plywood, particleboard and OSB), glass fiber insulation, friction materials, foundry sand molds and resin-saturated laminated products. Novolacs are so-called two-stage phenolic resins that require the presence of a crosslinking agent to cure. They are used principally in abrasives, protective coatings, friction materials, heat-resistant and general purpose molding compounds, foundry sand mold binders and as specialized binders. Since they contain a higher proportion of phenol to formaldehyde, novolacs usually are more costly than resols. The worldwide demand for phenolic resins is generally increasing in line with world economic activity, since it is used in many broad-based industrial activities and in housing. The highest prices are for specialized phenolic resins used in the manufacture of heat-resistant and high electrical resistant molded products, as well as in the manufacture of abrasives. Since phenol is now produced almost exclusively from benzene instead of from coking operations, sharp increases in crude oil prices that have occurred in 2004 have caused a significant rise in the cost of raw materials and a consequent increase in the prices for phenolic resins. 5.5.2 The Potential Use of Biorefinery Lignin in Phenolic Resins

A simple examination of the chemical structure of native lignin [15] shows some interesting similarities to the structure of phenol–formaldehyde resins. Lignin consists of a three-dimensional amorphous matrix of crosslinked phenylpropanoid units. These residues, depending on their botanical source, may have, to a greater or lesser extent, substitution of methoxy groups on the ortho positions to the hydroxyl group on the aromatic ring. Lignin from annual plants, with a high proportion of p-hydroxy phenylpropane residues, will have a

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significantly lesser degree of ortho substitution, while lignins from hardwoods, with a high content of syringyl moieties, will have a high proportion of ortho substituents on the aromatic ring. Softwood lignin with a high content of guaiacyl groups (more than 90%) and some p-hydroxy phenylpropane moieties will have an intermediate degree of methoxy substitution. Both native lignin and phenol–formaldehyde resins are polyphenolic materials consisting of phenolic rings linked together by short aliphatic chains. However, the form of these linkages in lignin are much more diverse and different from the linkages in PF resins. In lignin many of the crosslinking groups are aryl– alkyl ethers, but there are numerous carbon–carbon links also. Furthermore, lignin has a significant number of functional groups, such as aliphatic hydroxyls, on the propane residues, a feature that is not shared with phenolic resins. Nevertheless, the structural similarities are close enough to suggest a strong compatibility between these two materials and even opportunities for chemical interaction between them, with or without added crosslinking agents. Lignosulfonates and thiolignins have been marketed for incorporation into phenolic resins for some time, but they do not appear to have met with significant acceptance by the resin industry. Some of the problems experienced by current lignins in this market may be overcome with biorefinery lignins, but not by all forms of biorefinery lignins. Organosolv processing of woody biomass can readily yield a nonderivatized lignin having high purity, significant chemical reactivity, low polydispersity and low molecular weight. Organosolv lignins from hardwood produced in tonnage quantities by the Alcell process in Miramichi, Canada, in the 1990s has been readily substituted into phenolic resins [7] and used successfully on a commercial basis. Commercial applications in which it was used included in phenolic resins used in OSB manufacture and the production of friction materials. Numerous other applications where organosolv lignin was being substituted in part or in whole for phenolic resins were also under development at that time, such as in saturating resins for laminates production, stiffening agents for container boards and in rubber processing additives. Figure 5.2 shows samples of several commercial and pre-commercial products manufactured using Alcell organosolv lignin. The manner of substituting lignin for phenol–formaldehyde resins can vary from a simple blending of dry powder lignin with dry powder phenolic resin to the use of organosolv lignin as a primary phenolic component during the manufacture of the resin. In both cases the lignin represents the substitution of a renewable material for nonrenewable chemicals produced from fossil carbon. The advantages to the users and suppliers of phenolic resins of partial or complete substitution of biorefinery lignin are numerous. They include the potential of lower cost raw material and the opportunity to receive valuable carbon credits since phenolic resins are produced from phenol, now almost totally produced from crude oil, and formaldehyde, which is a product of natural gas. In addition, recent studies have shown that the incorporation of organosolv lignin into PF resins that are used in the manufacture of OSB will reduce the emis-


Fig. 5.2 Some commercial and pre-commercial products made with organosolv lignin from hardwoods. On the left is an automobile brake pad, next to a pot handle made from a molding compound. To the rear are

sections of oriented strand board (OSB) and other panel boards, while on the right is a section of rubber belting. In the middle is a sample of the original organosolv lignin powder, with a granulated version to its left.

sions of formaldehyde from the press during manufacture [34]. This is a major advantage to manufacturers because these emissions of a known carcinogen are being closely monitored and regulated by various governments. An added benefit to OSB manufacturers is that it has been shown that the substitution of up to 35% of PF resin with an equal weight of organosolv lignin will provide substantial improvements to the final board properties, especially in reduced swell of the wet board and a higher modulus of rupture following the boil test [7]. 5.5.3 Panelboard Adhesives

Resin binders for exterior grade wood panel production is the largest part of the market for PF resins, representing over half of the PF resin used today. The market is divided mostly between plywood (a declining market), waferboard and oriented strandboard (OSB), and exterior grade granulated wood panels, such as particleboard. Plywood uses only liquid resins, while waferboard and OSB resins are divided between dry powder and liquid forms. For a number of technical and production reasons, isocyanate resins, despite their higher cost, are becoming the favored resins for the core layer of OSB, while PF resins remain the resins of choice for the two face layers. The estimated North American demand for this market segment is over 550 000 tonnes annually on a solids basis, while total world demand for this purpose is probably more than 1.1 million tonnes annually.

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In work conducted in the 1970s it was shown that lignin could replace up to 50% or more of the expensive MDI on an equal weight basis in an isocyanate resin used in wood binder applications [35]. The properties of the finished board were in many cases superior to the control board with 100% isocyanate resins. Isocyanate resins sell for about double the price of PF resins on a solids basis. 5.5.4 Thermoset Resins for Molded Products

One of the oldest applications for PF resins is in the production of phenolic molding compounds that are used to make a variety of products in the automotive, electrical and appliance fields, such as electrical circuit boxes and circuit breaker components, heat resistant pot holders and handles (Fig. 5.2). They normally consist of 50% PF resin and 50% fillers, but their exact composition is determined by their application. In the US the phenolic molding compound market is approximately 100 000 tonnes per year, which equates to more than 55 000 tonnes per year of PF resin, while the total world usage of PF resin in this application is about 200 000 tonnes per year. 5.5.5 Friction Materials

During the manufacture of friction products, such as brake pads, brake shoes and clutch facings, PF resins are added to the initial mix of components in the range of 2–5% by weight. They serve as green strength binders to hold the shape of the final product during molding and while in the bake oven. In the bake cycle the PF resins are carbonized and form a matrix that supports the other components. Approximately 27 000 tonnes per year of PF resins are used in the manufacture of friction materials in the US, while world consumption is probably in excess of 120 000 tonnes per year. Lignin can substitute for a significant amount of this PF resin in friction materials [36]. Before the closing of the Alcell plant in 1996, Repap was regularly selling organosolv lignin to a commercial brake lining manufacturer who was substituting lignin for PF resin at a 20% level, but a higher level of substitution was anticipated following these successful introductory levels (Fig. 5.2). Organosolv lignin was found to provide some technical advantages to the final product, in addition to cost advantages. 5.5.6 Foundry Resins

PF resins are used to bind the sand in metal casting molds and core. Mostly the molds are made of green sand without binders, but the more delicate sand cores are bonded with PF and other types of resins, such as furan resins. These


resins have the advantage that they are burned out of the sand, which can then be recycled. The incorporation of lignin into these resins would have the same advantage. Phenolics account for slightly more than 50% of the resins used in the foundry market. In North America approximately 55 000 to 60 000 tonnes of phenolic resins are used in the foundry mold binder market each year. Worldwide the usage is estimated to be about 150 000 tonnes per year. Alcell organosolv lignin was found to effectively displace more than 20% of furan and phenolic foundry resins without loss of performance. 5.5.7 Insulation Materials

Glass fiber in insulation batting is bonded together using mostly liquid phenolic resins but some powder resins, about 15–20% of the total, are used in certain specialty applications. PF resins are used because they are temperature stable and flame resistant. They are also used in phenolic foams, mineral wool and waste fiber insulation. In the latter case, waste fiber such as cotton, polyester and wool is shredded and bonded together with powder resin and molded. Much of this material is used for acoustical insulation and applications. In North America over 110 000 tonnes of phenolic resins are used by the insulation materials market sector each year. The prices are similar to those for resins used in other applications. 5.5.8 Decorative Laminates

A major market for PF resins is in the manufacture of decorative laminates. Decorative laminates are made up of numerous layers of kraft paper that are saturated generally with alcohol-based liquid PF resin. It is then partly cured and dried before a photographic melamine surface layer is attached and the entire system is placed in a press to be bonded. The amount of PF resin usage in the US is approximately 100 000 tonnes per year, while the total world demand for this application is probably close to 300 000 tonnes per year. Prices in this application are similar to those in other PF resin applications. 5.5.9 Panel and Door Binders

There is a growing market for PF resins in the production of laminated and veneered doors and certain types of molded fibrous structural panels. While this is a fast growing business it is relatively small compared to the production of structural wood panels for roofing, siding and underlaying, such as OSB and plywood.

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5.5.10 Rubber Processing

Several applications for lignin have been identified in the rubber industry. These are: · phenolic tackifiers · antioxidants · reinforcers. Tack is the term given to the resistance to separation of two materials after they have been brought in contact with each other, under light pressure, for a short time. One type of tack is classified as autoadhesive tack, where the two materials have the same chemical composition. The other type, adhesive tack, is used where the two materials are of different composition. In the production of multi-layered rubber products, such as tires and belting, tack is needed to hold the components together during manufacture. Various additives, known as tackifiers, are added to rubber formulations to increase tack in the compound. Three types of tackifying resins are mostly used – hydrocarbon resins, rosin and its derivatives, and phenol–formaldehyde resins. Interestingly, the molecular weight of the typical tackifying resins is around 2000 or less, a number consistent with the number average weight of several soda and organosolv lignins. As a result of recognizing these similarities Repap entered into a collaborative development program in the early 1990s with two major rubber products manufacturers for the evaluation of Alcell lignin as a tackifier in rubber processing. This program demonstrated the capacity of dry powder Alcell lignin to function as an effective tackifier in SBR compounds. Initially, lignin was used as a 50% replacement of PF resin. Because tackifiers also affect the cured physical properties of rubber, it was necessary to not only test for tack improvement, but also to test the hardness and tensile strength of the cured rubber as well as other properties such as cut growth resistance and resistance to oils and solvents. A patent covering this application was awarded in the US and several other countries [37]. Reinforcers are additives employed to obtain certain performance properties in rubber products, such as tires, where abrasion resistance, in particular, is important. Reinforcing agents are normally carbon blacks, inorganic fillers, or reinforcing resins. Kraft lignin powders are also used in this application and it has been suggested that, because of the specific gravity of organosolv lignin powders, they should also be effective reinforcers. A major advantage in this application was that the lignin also acted as an antioxidant. In the manufacture of rubber products, antioxidants are also added to the compound. Therefore, lignin acted as a multi-functional additive; a major advantage, since a single additive could be used in place of the two additives normally employed.

5.6 Lignin as an Antioxidant

5.5.11 The Opportunity for Lignin in Phenol–Formaldehyde Resin Markets

As phenolic resin substitutes, natural lignins have the following characteristics and advantages, which should provide inducements for their acceptance by existing users. · They are highly compatible with phenolic resins, which allows for effective partial substitution and resin mixing. This accommodates applications where blending to achieve specific performance properties is necessary. · Lignin condenses under heat without the need for inorganic acid or base catalysts. This, together with its very low ash content, is useful for resin use in the electronics industry. · It is a natural product that does not release formaldehyde during condensation. This is important in the wood panel industry, which is under tight regulation of formaldehyde release during the hot pressing stage of panel manufacture. · Lignin is a renewable natural product that should easily qualify for carbon credits when it is used to displace phenol–formaldehyde resins. · Lignin also brings additional properties, such as being a polymeric hindered phenol antioxidant, which makes it multifunctional in many applications.

5.6 Lignin as an Antioxidant

Antioxidants are a group of chemicals that inhibit atmospheric oxidation and its degradative effects on polymer systems, lubricants, foodstuffs and animal feed additives. They minimize degradation during fabrication, storage and use. In polymers and long chain molecules, such as fatty acids and polyalkanes, chemical bonds are broken under the influence of heat, ionizing radiation, mechanical stress and chemical reactions to form free radicals. Oxygenation of these free radicals forms peroxy radicals that initiate a chain reaction, which will eventually badly degrade the polymer or long chain molecule, frequently introducing colored compounds into the product. In order to inhibit oxidation in this chain reaction and to slow polymer degradation, free radical scavengers are introduced into the polymer or foodstuff. These are known as primary antioxidants, which include hindered phenols and secondary arylamines. Lignin is a polymeric hindered phenol, a class of compounds known to be effective primary antioxidants. Alcell organosolv lignin was tested for its antioxidant properties and found to be an effective antioxidant in grease, rubber and animal vitamin supplements. Presently, the major antioxidants used in industry include butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate (PG), natural tocopherols and polymeric hindered phenols. These are all expensive materials ($ 3.00 per kg) and many of them are petroleum-based. Particularly in food and

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feed applications, they are highly regulated. Some synthetic antioxidants continue to be under suspicion for adverse health effects. Lignin is a natural product, which is ingested daily by both humans and animals. It might therefore be expected to be a preferred form of natural antioxidant especially in food and feed since there is a high likelihood that lignin is physiologically benign in humans. 5.6.1 Antioxidants in Animal Feed Supplements

In 1996 a leading feed vitamin supplier tested Alcell lignin to assess its potential as a natural antioxidant able to protect the potency of the antioxidant vitamins, such as vitamin E in animal feed vitamin supplements. It was reported that the lignin functioned well in this application. Interestingly, the lignin also served the added function as a pellet binder. 5.6.2 Antioxidants in the Rubber Industry

Antioxidants are also used in the rubber industry to suppress deterioration in rubber products subject to long term exposure of air and sunlight, such as the sidewalls of automobile tires. A number of different antioxidants are used by the industry that can cost upwards of $ 3.00 per kg. Since lignin is a polymeric hindered phenol, with hardwood lignin being especially hindered because of its dominant syringyl structure, lignin will compete in the middle price range of antioxidants. 5.6.3 Antioxidants in the Lubricants Industry

An area that is very attractive for lignin as an antioxidant is in the field of lubricating greases. In this application the dark color of lignin is not usually important and it is not necessary for it to be soluble in the grease. Addition levels to greases is between 1% and 10% by weight, depending on product and application. Alcell organosolv lignin was marketed in 1996 as a multifunctional grease additive (antioxidant and extreme pressure/antiwear additive) through a specialty lubricants additives company for a price competitive with existing antioxidants. Lubricating greases represent only 2% of all lubricant sales. Sales of antioxidant/antiwear additives for greases in the US in the late 1990s were approximately 3.0 million kg with a value in excess of $10 million. Many customers are looking for “green additives” that are nonmetallic, nonchlorinated, nonsulfur additives and also are looking for natural products with low cost relative to the conventional additives.

5.7 Applications for Water-soluble, Derivatized Lignins

Approximately 300 000 tonnes of antioxidant/antiwear additives for lubricating oils are being sold annually in the US, with a total value of approximately $ 520 million. Globally, the market was twice this size, with a value of over $ 1 billion per year.


As discussed earlier, lignosulfonates from the sulfite pulping industry and deliberately sulfonated kraft lignins represent the largest single form of lignin used in industrial applications. Together these products account for approximately 1 million tonnes per year, worldwide. They function mostly as dispersants, emulsifiers and surfactants in numerous commercial applications. Hydrolysis lignins obtained from future biorefineries will also be capable of being modified to produce equivalent or, because of their greater purity, improved materials for these markets. As the sulfite pulping process has moved from being used to produce commodity papermaking pulps to being the source for the manufacture of specialty dissolving pulps, more sulfite mills have closed over the last fifty years or so, and continue to close. Most of the recovered lignosulfonates from the remaining mills is going into the surfactants, dispersants and emulsifiers markets. With the increase in demand for these products and the need for improved performance, some market share has been lost to synthetic products. Sulfonated kraft lignins are also sold into this market by MeadWestvaco. Methylsulfonated biorefinery lignins should easily enter this market. 5.7.1 Concrete Admixtures

Water-reducing concrete admixtures are surface-active chemicals that function to break down cement particle agglomerates, thereby enhancing the fluidity and workability of the fresh concrete. The less water that is present in the fresh concrete mix, the higher the strength of the final set concrete. Water reducing admixtures can therefore be used to reduce the total amount of concrete and therefore the weight of concrete needed to achieve a desired strength. Ideally, water-reducing admixtures should have no negative effects on the strength and durability of the hardened concrete. Also, they should have no detrimental side effects such as excessive air entrainment or retardation of set time. The addition rate and therefore the effectiveness of many admixtures currently in use is limited by these undesirable side effects. Current commercial water-reducing admixtures fall into four broad categories: 1. modified and unmodified lignosulfonates and sulfonated kraft lignins 2. sulfonated melamine formaldehyde condensates 3. sulfonated naphthalene formaldehyde condensates

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Fig. 5.3 Concrete test cylinder made with methyl-sulfonated organosolv lignin, shown next to a brake pad for size comparison.

4. others, including polycarboxylic acids, and mixtures of acids, amines and polysaccharides. The first three categories rely upon sulfonic acid groups to impart a negative charge on the cement particle. The positioning of sulfonic acid groups on lignosulfonates is a direct result of the pulping process chemistry. This may not be ideal for this application and deliberately sulfonated natural lignins may yield higher performing products than lignosulfonates. Methyl-sulfonated Alcell lignin was tested as a water reducer in concrete and found to be quite effective. Figure 5.3 shows a concrete cylinder produced with methyl-sulfonated Alcell lignin that is used for testing of compressive strength. 5.7.2 Dye Dispersants

Before the advent of synthetic fibers, fabrics were made from naturally occurring materials such as wool, cotton, linen etc. These natural fibers were dyed from solution, in a dye bath. The dissolved, dyestuff molecule chemically bonded to reactive sites on the fiber surface. Solutions exhibited uniform concentration so that the fabric was evenly-colored. Synthetic fibers have now become equally as important as natural materials in clothing, furniture coverings, drapes, and carpeting. Since many of the newly discovered fibers were synthetic polymers, they were chemically unreactive.


Often it proved impossible to color them using traditional dyestuffs or dyeing techniques because they would not react with the dyestuff molecule. A new technique became necessary and, by the 1960s, one had been developed, known as disperse dyeing. In this process, the dye is applied to the fabric in the form of a fine, uniform suspension. The insoluble particles of dye, uniformly dispersed, are physically entrapped within the polymer structure. In effect, they are in a solid solution. Disperse dyes account for about 100 000 tonnes per year. Of the different classes of dyestuff, only disperse dyes (by far the most important), sulfur dyes in some applications and, to a lesser extent, vat dyes, are in the insoluble form during the dyeing process. All types of dye must be applied in a manner that produces uniformly colored fabric, free of specks or color shadings. In the case of disperse dyes, this means that the dispersion must be extremely uniform and contain particles of dyestuff in the 1-lm diameter size range. The only way to meet these criteria is to use dispersing agents. Dispersing agents are incorporated into the dyestuff during its manufacture (both before and after the milling process) and may amount to as much as 50% of the finished product. In addition to providing excellent dispersion, the dispersing agent must also offer other properties, including: · low staining · ability to maintain stable dispersions at high temperatures (above 100 8C) · nonfoaming · contain no sludge (fabric is an excellent filter) · nonreactive with the dye molecule · be readily spray-dried · assist in grinding. It is not easy to meet these demanding requirements. Over the past 30 years, the dyestuffs industry has found only three dispersants that will perform adequately: 1. Sulfonated kraft lignin 2. Lignosulfonate 3. Condensation products of naphthalene sulfonate. Sulfonated kraft lignin and lignosulfonates together account for about 90% of the dispersant market. While they are the best products available, it is generally admitted that they are far from ideal. The third class of products, the naphthalene sulfonates, account for 5 to 10% of the dispersant market. They exhibit questionable high-temperature stability – a vital property – and are expensive. They are seldom used in formulations in North America, but they are often used in vat dyes for cotton in other countries. The total amount of lignin-based dye dispersants used worldwide is about 25 000 tonnes annually.

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5.7.3 Asphalt Emulsifiers

The strong move to water-based asphalt emulsions and away from organic solvent-based systems has been driven by environmental concerns about organic solvent releases into the atmosphere. Asphalt emulsions are now used extensively in a variety of applications including road building, road and driveway sealing, soil stabilization, surface coating of asphalt pavement and built-up roofing. In these applications the emulsions are formed and stabilized with emulsifiers that are generally used at a level of about 1 to 2% by weight of the emulsion. Pinova, a division of Hercules, of Wilmington, DE, markets a series of asphalt emulsifiers based on polymerized rosin, Vinsol resin (a “bottoms” fraction from the refining of rosin) and rosin acids. These materials are obtained from rosins extracted from Southern Pine stumpwood. For a number of reasons the availability of these rosins is declining and Hercules has been searching for new raw materials to support its asphalt emulsifier business. During discussions with Repap in the mid-1990s Hercules became aware of the new organosolv lignin product from the Alcell process and tested its performance in asphalt emulsifiers. These trials were successful and two US patents [38, 39] was awarded and assigned to Hercules covering the formulation of lignin-containing asphalt emulsifiers and the asphalt emulsions containing such emulsifiers. These new emulsifiers were prepared by combining and melting lignin and polymerized rosin to obtain a homogeneous molten blend, then cooling the blend until it solidified. In a preferred embodiment, the ingredients further comprise added rosin acids and Vinsol resin, and the lignin is an organosolv lignin. The total world output of asphalt is well in excess of one billion tons. It is stated that 90% of all road networks are asphalt. Lignin-based asphalt emulsifiers should be well received by the industry. There is a continuing public concern about leaching of asphalt components into the surrounding soils and into ground and surface water. The leaching of lignin would have no negative environmental consequences, and may even have positive effects. 5.7.4 Agricultural Applications

Lignin products from conventional chemical pulping operations have for a long time been used in agriculture. Lignosulfonates from the sulfite pulping industry have found applications as feed pellet binders, dispersants for insecticides, fungicides and herbicides, and even as dust suppressants on farm roads. Initially these applications were driven by a need of the sulfite pulping industry to find uses for its spent sulfite pulping liquors. In these applications there was little need to purify the spent liquors and so the product was frequently a dried powder of the spent liquor, or even the liquor itself.


Worldwide, agriculture has several emerging needs that can be satisfied by hydrolysis lignins, such as organosolv and biorefinery lignins. Among these needs are · improved environmental performance · improved animal health without using human applicable antibiotics · greater efficiency in use of resources · lower costs of operation · improved productivity · insulation from rising energy costs · lower usage of fossil carbon-based chemicals. Natural hydrolysis lignins have been shown to be multifunctional and have properties that help satisfy these needs of agriculture. They include: · biodegradability, with the degradation products having a positive environmental effect · photodegradability · physiologically active · specific antimicrobial effects · natural antioxidant · relatively immunity to rising energy prices · natural product not derived from fossil carbon sources. These properties have been exploited recently in several new applications in agriculture. They include: · enhanced feed efficiency in calves · antidiarrheic in cattle · antioxidant component in vitamin supplement formulas for animals · slow release matrix for fertilizer and pesticide applications, with positive environmental effects · beneficial effects on rumen metabolism · reduced ammonia concentrations in broiler operations. 5.7.5 Dispersants for Herbicides, Pesticides and Fungicides

The active ingredient in herbicide formulations is generally an organic chemical that is insoluble or immiscible in water. In this regard, herbicide formulations are closely related to disperse dyes. Many of the manufacturers are the same, particularly the giant Swiss and German chemical companies. With the advent of micro-emulsion technology, post-emergent herbicides, and the greater use of dry granular compositions, herbicide manufacturers and formulators now require that the dispersant system do more than merely emulsify or disperse the active ingredient in water. Now, the additive must also be compatible with fertilizers and other pesticides, insecticides or fungicides, to allow combined-field application. In addition, the additive should increase the efficiency of the application by improving penetration of the leaf or insect.

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A range of surfactants provide these properties to some degree or another. In the United States, the annual surfactant market in herbicide applications totals about 45 000 tonnes and is divided between several product classes, principally lignin-based surfactants and synthetic surfactants, such as alkylphenol ethoxylates. The total market in the US for herbicide surfactants is about 50 000 tonnes annually, of which only about 5% is currently lignin based. The total world market for lignin-based herbicide surfactants is approximately 18 000 to 20 000 tonnes per year.

5.8 New and Emerging Markets for Lignin

As can be readily seen from the above descriptions of the present markets for lignin and its water-soluble derivatives, there is an existing major market opportunity for hydrolysis lignins that might be obtained from new biorefinery operations. Not only will the new lignins promise superior performance in many applications that currently are filled by the lignosulfonates and kraft lignins, but, because of their anticipated superior purity and functionality, they will open major new markets that presently do not exist. Already there are indications of such future market opportunities that were being developed from the very limited supply of organosolv lignins generated from Repap and from the limited work undertaken by Organocell. Some of these exciting opportunities are presented below. 5.8.1 Printed Circuit Board Resins

A report from the IBM company in 1996 [40] identified a novel application for lignin in the manufacture of printed circuit boards (PCBs) for the electronics industry. In the manufacture of PCBs the glass fabric, which is the largest part of the PCB, is dip coated with a resin. This is usually an epoxy resin, dissolved in a low boiling solvent, such as methyl ethyl ketone or acetone. The release of these solvents during resin curing is an undesirable health and environmental issue. At that time, IBM was in the process of examining its various production processes and operations in order to substitute, where possible, “green” technology. One area of interest was the use of renewable biopolymers to replace the oilbased epoxy resins typically used in the industry. Lignin became of interest because its molecular structure could provide the thermal stability and chemical resistance that was required for PCBs. It was reported that in initial research, PCBs produced with an epoxy resin containing 50% lignin showed better thermal and electrical performance than current high volume PCBs and that the resin cost was significantly lower than standard resins. Furthermore, this material reduced the dependence on fossil fuels.


5.8.2 Animal Health Applications

In the mid 1980s, Organocell, of Munich, developed a methanol-based organosolv pulping process for wood from which they obtained an organosolv lignin. This lignin was manufactured in sub-tonnage quantities in their pilot plant and was distributed to various academic institutions in Germany for applications research. Some of this lignin was sent to Professor J. Gropp, of the Veterinary Department of the Ludwig-Maximilians University of Munich. He and his students tested various applications in animal feeds and several doctoral theses were produced from this work [41–43]. The conclusions from this work can be summarized as follows. Prophylactic use of 1.5 to 4.5% organosolv lignin in the feed of piglets inoculated with pathogenic E. coli led to a significant decrease in morbidity and intensity of diarrhea. · The reduction of feed efficiency caused by diarrhea could be avoided by feeding 1.5% lignin. · The therapeutic administration of 3.0% and 6.0% lignin after appearance of the diarrhea resulted in an improved fecal consistency and a reduced water excretion. · Nevertheless, the beneficial effects of feed efficiency and average daily weight gain showed a maximum at 4.5%, with 6.0% lignin proving to be deleterious to these criteria. · The egg laying performance of hens was negatively affected by the presence of lignin in the diet. The antidiarrheic effects of dietary lignin in piglets were also demonstrated in calves. The addition level having optimal effects was very similar for both animal models, with both showing negative effects at 6% and above. Support for these observations comes from US Patent #4, 473 556 awarded in 1984 that describes the use of a feed formulation containing acid-hydrolysis lignin (Scholler lignin), used in the treatment of gastrointestinal disturbances in farm animals. The significance of these observations is that there is often substantial morbidity, especially in young farm animals, as a direct result of acute gastrointestinal disturbances. In the past sub-therapeutic levels of antibiotics have been used to reduce the incidence of this problem and the economic losses associated with it. The same antibiotics are used to treat human infections and there is widespread concern over the appearance of antibiotic-resistant bacterial strains that result from this practice. Many European countries have now banned the broad use of human therapeutic antibiotics in farm animal husbandry. An alternative, nonantibiotic treatment for this problem would be of great importance.

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5.8.3 Animal Feed Supplement

As a result of the above observations on the positive effects of lignin on animal gastrointestinal disturbances, a major well controlled feeding trial and related research program at the experimental farm of McGill University was undertaken in 1995–96. The feeding trials involved groups of veal calves that were fed rations containing no or different levels of organosolv lignin from the Alcell process. The microbial studies involved in vitro and in vivo experiments on the responses of various bacteria to lignin. Some of the results of these studies have been published and reported at scientific conferences [44, 45]. Among the most interesting results was the observation that 1.25% of added organosolv lignin to the diet of calves increased weight gain by 12% and reduced ammonia concentrations in the feces. In addition it was noted that enteric bacteria were found to adhere to the lignin particles and that at certain levels lignin destroyed Grampositive and Gram-negative bacteria. It was concluded that lignin has the potential to alter feed utilization by animals through its effect on the physiology and microbiology of the gut. The importance of the finding on weight gain and feed efficiency cannot be overstated. If these observations are carried through to the feeding and fattening of mature cattle, lignin could significantly enhance the economics of feedlot operations, together with a resultant benefit to the environment. This one application could become a major market for natural lignins. 5.8.4 Carbon Fibers for Mass-produced Vehicles

A fascinating and potentially very large new market for lignin is for the production of low cost carbon fibers for use in automobile and light truck body components. It is well recognized that major transportation fuel savings, and an equivalent reduction in the demand for oil, would be achieved if the weight of vehicles could be reduced. There would also be a beneficial concomitant reduction in emissions. Today, despite a rising use of lower weight materials, between half and two thirds of the weight of most vehicles is contributed by ferrous metals. Much of these could be replaced by components made from lighter weight carbon fiber composites, which could reduce the weight of the vehicle by about one third. Such carbon fiber composite materials are used extensively in the aerospace industry where they have proven their value and performance. Carbon fibers for these applications are made mostly from oil-derived pitch and polyacrylonitrile (PAN) and in their final form cost in excess of $ 25 per kg. In order for auto body parts made from carbon fiber composites to be pricecompetitive in today’s mass-produced vehicles, it has been determined that the carbon fibers would need to cost less than half this amount. This price cannot be achieved using existing feedstocks, especially as oil prices continue to climb.


What is needed to achieve this goal is a high carbon content, relatively low priced, preferably renewable feedstock that can be produced on a large scale, the price of which would be relatively independent of the price of oil. Researchers at the Oak Ridge National Laboratory in Tennessee, USA, are investigating the use of lignin for this application [46, 47]. Most of their work has been with kraft lignin, but they have also successfully used organosolv lignin in this application. This work has produced industrial grade carbon fibers suitable for use in vehicles from blends of lignin and post-consumer recycled polyester and other polymers. The commercial kraft lignin that was used had to be washed free of contaminants and desalted prior to use to avoid the presence of voids in the fibers, which would significantly reduce their strength and value. Purer lignins produced from biorefinery processes could eliminate the need for this processing step. Figure 5.4 shows spools of the melt spun lignin/polymer fibers containing different amounts of lignin. Figure 5.5 displays microscopic images of lignin/polyethylene oxide fibers at different stages in the manufacture of carbon fibers [47]. Once this application becomes fully developed it has been estimated that if each newly manufactured vehicle in the US used only 10 kg of carbon fiber, there would be an annual demand for 125 000 tonnes of carbon fiber. With a lignin carbon content of 0.68 and an expected carbon yield of 0.45–0.55, just this limited consumption could require up to approximately 250 000 tonnes of lignin annually and could support a lignin price greater than US $ 1.20 per kg. Each new vehicle could ultimately use many times the 10 kg suggested in this illustration. In fact, it is conceivable that each vehicle could ultimately use up to 500 kg of carbon fiber making the carbon fiber demand for US manufactured vehicles alone at least 4 million tonnes per year, which in turn would require up to 8 million tonnes of lignin per year. Today the worldwide demand for carbon fiber is approximately 28 000 tonnes per year. Therefore, capacity would have to increase by close to five times just to satisfy this application at a level of 10 kg per new vehicle in the US alone and a further fifty times to meet the ultimate potential demand.

Fig. 5.4 Spools of melt spun fibers made from blends of kraft lignin and polymer.

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Fig. 5.5 Microscopic images of fibers made from blends of kraft lignin and polyethylene oxide during the manufacture of carbon fibers.

5.9 Conclusions and Perspectives

Lignin that is now being recovered from conventional chemical pulping operations has established a significant commercial market in numerous industries and applications. These markets basically employ two different forms of lignin. The first of these are the water-soluble lignosulfonates recovered from the sulfite pulping process and the deliberately sulfonated lignins derived from the kraft process. The second form of commercial lignin is the thiolignin obtained from the kraft process. Because of their markedly different physical and chemical properties, each of these has established its own range of applications, with very little overlap between them. The combined worldwide market that presently exists for these two product forms appears to be about one million tonnes per year. With the increasing cost of crude oil and natural gas and the developing trend and incentives towards the use of renewable chemicals to replace fossil-carbon-derived materials it can only be anticipated that these traditional markets for lignin products will expand. At the same time, the number of pulp mills that practice the sulfite process are slowly declining, which raises the question as to where the supply to meet this increased demand will come from. An additional trend in the pulping industry is the ever increasing scale of new kraft mills and the frequent closure of small kraft mills now regarded as too small to be competitive. It is unlikely that recovery of lignin will be an attractive option for the very large kraft mills because of the process integration with large recovery boilers. This situation can benefit future biorefineries that will be processing lignocellulosic feedstocks. Certain biorefinery pretreatment technologies, such as organosolv processes, will have the capability to produce a superior form of pure lignin ideal for chemical applications. The marketing of this lignin as a co-product will add substantial revenues and vastly improve the economics of the biorefinery. The new biorefineries will more than likely be on a considerably smaller scale than world-scale kraft pulp mills that are now processing 3 000 to 4 000 tonnes of wood on a dry weight basis per day. Consequently biorefineries will be more flexible and better suited to produce and market some of the specialized lignins that will emerge from them.

References

The past experience from the production of large volumes of Alcell lignin shows that the availability of commercial quantities of these improved lignins will stimulate sizeable new market opportunities that do not presently exist for lignins from the chemical pulping industry. Some of these new markets are already clearly visible. What is needed now is the commercial-scale production of the lignin to service these opportunities.

References 1 R. Alén, Forest Products Chemistry, Fapet 2

3

4

5

6 7

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Oy, Helsinki, Finland, 2000. W. G. Glasser, R. A. Northey, T. P. Schultz, Lignin: Historical, Biological and Materials Perspectives, ACS Symposium Series 742, American Chemical Society, Washington, DC, 2000. J. D. Gargulak, S. E. Lebo. In Lignin: Historical, Biological and Materials Perspectives, ACS Symposium Series 742, American Chemical Society, Washington, DC, 2000, p. 304. G. A. Smook, Handbook for Pulp and Paper Technologists, 2nd edn., Joint Textbook Committee of the Paper Industry, TAPPI, Atlanta, USA, and CPPA, Montreal, Canada. X.-J. Zhong, A. Gan, Profit Through Innovation, Pira International, London, UK, 1997, p. 222–224. F. Zimbardi, E. Ricci, G. Braccio. Applied Biochem. Biotechnol. 2002, 18/20, 89–99. J. H. Lora, C. F. Wu, E. K. Pye, and J. J. Balatinecz, In Lignin: Properties and Materials. W.G. Glasser and S. Sarkanen (Eds.). American Chemical Society Symposium Series 397. Washington, DC, USA 1989, p. 312–324. X. Pan, X. Zhang, D. J. Gregg, J. N. Saddler. Applied Biochem. Biotechnol. 2004, 113/116, 1103–1114. A. Boussaid, A. R. Esteghlalian, D. J. Gregg, K. H. Lee, J. N. Saddler, Applied Biochem. Biotechnol. 2000, 84/86, 693– 705. G. P. van Walsum, Applied Biochem. Biotechnol. 2001, 91/93, 317–328. F. Teymouri, L. Laureano-Perez, H. Alizadeh, B. E. Dale, Applied Biochem. Biotechnol. 2004, 113/116, 951–963.

12 R. Alén, Forest Products Chemistry, Fapet

Oy, Helsinki, Finland, 2000, p. 66. 13 W. G. Glasser, Forest Prod. J. 1981, 31,

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W. G. Glasser, S. Sarkanen, American Chemical Society Symposium Series 397. Washington, DC, USA, 1989. J. L. McCarthy and A. Islam, Lignin: Historical, Biological and Materials Perspectives, ACS Symposium Series 742, American Chemical society, Washington, DC, 2000, p. 2–99. A. Payen, Compt. Rend. 1838, 7, 1052. B. C. Tilghman, British Patent No. 2924, 1866. Borregaard LignoTech website. http:// www.ltus.com/about us/history.html. Meadwestvaco website, http:// www.meadwestvaco.com/specialtychemicalshome.nsf Granit S. A. website, http://www.granit.net Chemical modification, Properties, and Usage of Lignin, 2002, ed. T. Q. Hu, Kluwer Academic/Plenum Publishers, New York. J. H. Lora, A. Abächerli, F. Doppenberg, 2000, Tappi Pulping Conference Proceedings, Tappi Press, Atlanta, USA. J. H. Lora, Proceedings of the 6th International Conference of Pulp and Paper Industry – Paerex, December 5–7, 2003, New Delhi, India. J. H. Lora, personal communication, 2004. H. F. J. Wenzl, The Chemical Technology of Wood, 1970, Academic Press, New York. X. Pan, C. Arato, N. Gilkes, D. Gregg, W. Mabee, K. Pye, Z. Xiao, X. Zhang, J. Saddler, “Biorefining of Softwoods Using Ethanol Organosolv Pulping – Prelimin-

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1997, “Asphalt emulsion with lignin-containing emulsifiers”. R. R. Suchanec, US Patent #5 656 733, 1997, “Lignin-containing resinous compositions”. J. M. Shaw, S. L. Buchwalter, J. C. Hedrick, S. K. Kang, L. L. Kosbar, J. D. Gelorme, D. A. Lewis, S. Purushothaman, R. Saraf and A. Viehbeck, Printed Circuit Fabrication, 1996, 19, 38–44. R. Stich, Doctoral Thesis, Ludwig-Maximilians University of Munich, 1984, “Tests for Different Lignin Effects in Veal Calves, Rats, Laying Hens and Quails”. A. J. Kloner, Doctoral Thesis, Ludwig-Maximilians University of Munich, 1985, “Reduction in Infectious Piglet Diarrhea by Lignin”. A. Ott, Doctoral Thesis, Ludwig-Maximilians University of Munich, 1983, “Effect of Dietary Lignin on Nutrient Utilization of Piglets”. L. E. Phillip, E. S. Idziak, Proc. of the XVIII International Grassland Congress, June 1997, Winnipeg, Canada. L. Phillip, E. Idziak, S. Kubow, Proc. of the Eastern Nutrition Conference, Animal Nutrition Conference of Canada, May 25, 2000. J. F. Kadla, S. Kubo, R. A. Venditti, R. D. Gilbert, A. L. Compere, W. Griffith. 2002. Carbon, 40, 2913. W. L. Griffith, A. L. Compere, J. T. Shaffer, C. F. Leitten, R. D. Gilbert, J. F. Kadia, S. Kubo, R. A. Venditti, 2000, Proc. Midwest Advanced Materials and Processing Conference, Sept 12–24, Dearborn, MI, USA. Society for the Advancement of Material and Process Engineering.


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Protein Line and Amino Acid-based Product Family Trees 6 Towards Integration of Biorefinery and Microbial Amino Acid Production Achim Marx, Volker F. Wendisch, Ralf Kelle, and Stefan Buchholz

6.1 Introduction

In the chemical industry the utilization of feed stocks produced in biorefineries is an emerging field of interest, because a wide range of low cost raw materials is potentially available. On the other hand amino acid production is a large-volume, steadily growing business already with several decades of experience with the utilization of renewable resources such as sucrose, molasses and starch hydrolyzates. Integrating both concepts opens up further opportunities for a sustainable production of amino acids. In our contribution we discuss environmental, commercial and technical aspects of microbial amino acid production (MAAP) integrated in a biorefinery. The optimization of MAAP has made tremendous progress during the last four decades. In this article we restrict our review to biotechnologically produced amino acids with a world-wide yearly production capacity of more than thousand tons per year. Because excellent reviews are already available [1–4] we do not cover details of metabolic engineering, strain development, alternative fermentation processes, and downstream processing. Instead the major focus of our contribution is to illustrate synergies of developments at the interface between emerging biorefinery and the well established amino acid industry and to outline how to integrate both.


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6 Towards Integration of Biorefinery and Microbial Amino Acid Production

6.2 Present State of the Industry 6.2.1 Microbial Amino Acid Production

Amino acids are produced by extraction from protein-hydrolyzates, by chemical synthesis, by enzymatic synthesis, or by fermentation. Because of tremendous progress in strain development during the last two decades, the fermentation industry has grown rapidly with a steady increase of process efficiency. Estimated world-wide production by fermentation in 1996 was 1000, 250, 4, 1.3, and 1.2 thousand tons per year for mono sodium glutamate, l-lysine HCl, lthreonine, l-glutamine, and l-arginine, respectively [2]. The fermentative production of l-tryptophan, l-valine, l-leucine, l-isoleucine, l-histidine, l-proline, and l-serine accounts for world-wide production capacities below 600 tons per year. For the bulk amino acids used for food and feed applications, the carbon source is the major variable cost followed by variable energy cost and the cost of the nitrogen source. Other raw material costs are less than 5% of the variable cost. Fix costs have been significantly reduced by economy of scale, so that variable cost dominates the overall manufacturing cost. Rational strain development, the application of genome information, and information about metabolic fluxes, metabolite concentrations, the transcriptome, and the proteome have led to the development of high-performance production strains with high productivity and high conversion of the carbon source. Recently, success stories for application of metabolic engineering for improving lysine production by Corynebacterium glutamicum have been reviewed [3]. 6.2.2 Biorefinery and the Building-block Concept

Biorefineries are defined as processing facilities that extract carbohydrates, oils, lignin, and other materials from biomass and convert them into multiple products including fuels and high-value chemicals and materials [5]. Many technical concepts are discussed, mainly focusing on the pretreatment and hydrolysis of lignocellulose-based feedstocks [6]. Processing of green leaf material toward high amino acid-containing grass juice as a raw material for fermentation has also been used for commercial amino acid production [7]. The biorefinery definition is derived from the petrochemical definition but biorefineries can potentially yield many high-value organic products that oil refineries cannot. Recently, a comprehensive overview on the variety of biobased products which are already produced in biorefineries or may originate from biorefineries in the future has been presented [5]. Biobased products fall into three categories: commodity chemicals (including fuels), specialty chemicals, and materials. Either direct physical or chemical processing of biomass such as cellulose, starch, oils,

6.2 Present State of the Industry

protein, or lignin results in biobased products or indirect processing from carbohydrates by biotechnological methods such as microbial (e.g., fermentation) and enzymatic processing. Competitive microbial fermentation of large-volume amino acids is already closely integrated with large-scale starch-based (corn milling) or sucrose-based (sugar cane) biorefinery operations. But the question arises if there are more favorable process options, if MAAP is integrated in a lignocellulose-based operation. The fermentation of amino acids might benefit from experience of lignocellulose-based ethanol production. Requirements of feedstock quality are, however, substantially higher, because the MAAP process is more sophisticated than the ethanol process and a consistent fermentation performance is of high importance. The downstream process for feed-grade amino acids will, on the other hand, tolerate the use of more impure carbon sources, compared with the very sensitive downstream processes for biobased polymers. Production of high volume, high value feed additives like lysine and threonine will therefore have lower technological hurdles to overcome for successful integration into a lignocellulose-based biorefinery concept than other large-scale fermentation processes (e.g. poly(lactic acid), 1,3-propanediol). The motivation for the integration of fermentation processes into a lignocellulose-based biorefinery has been the availability of low-cost carbon sources and the reduction of energy cost, because of the utilization of waste material for generation of steam and electricity. Despite the 20- to 30-fold reduction of enzyme costs for cellulose hydrolysis [6, 8], however, significant disadvantages of the lignocellulose-base biorefinery still exist: · the use of dilute, acidic carbohydrate solution from hydrolysis and the slower hydrolysis of cellulose at higher temperatures compared with hydrolysis of starch increase the specific capital investment [9] · the lower protein content of lignocellulose material compared with corn or wheat and the absence of an established commercial application for co-products results in no additional benefit from co-products [9] · despite progress in improving the utilization of xylose (C5 sugar from hemicellulose) by yeast and other industrial important production organisms, utilization of xylose is still not as efficient as the use of dextrose or sucrose. Therefore, despite recent technical improvements, cellulose-based ethanol still has significantly higher manufacturing cost compared to starch-based ethanol. This cost disadvantage will only increase when cellulose-based production of fermentation products with a greater need for consistent raw material quality and more stringent downstream requirements are compared with the starch-based or sucrose-based technologies. For a starch-based operation the state-of-the-art technology converts 97% of the starch into rather pure and well-defined fermentable sugars with 95% dextrose content. For the sucrose-based operation from sugar cane, no hydrolysis is necessary in the first place, to yield a well defined carbon source. Lignocellulose-based technology has a long way to go to achieve this with the same level of process stability.

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6 Towards Integration of Biorefinery and Microbial Amino Acid Production Table 6.1 Building-block concept in biorefinery. Twelve sugarderived building blocks which serve as a platform for the synthesis of derivatives or secondary chemicals [5]. 1,4-Succinic, fumaric and malic acids 2,5-Furandicarboxylic acid 3-Hydroxypropionic acid Aspartic acid Glucaric acid Glutamic acid Itaconic acid Levulinic acid 3-Hydroxybutyrolactone Glycerol Sorbitol Xylitol/arabinitol

For all renewable resources processed in a biorefinery, irrespective of the main carbon source, the so called building block concept, which has been illustrated in detail [5], enables potential generation of additional value from co-products. The building block concept introduces twelve building blocks that can be produced from sugar by biological or chemical conversion and can be subsequently converted to a number of high-value biobased chemicals or materials. Building blocks, as considered in the biorefinery concept, are chemicals such as succinic acid with multiple functional groups with the potential to be transferred into new families of useful molecules (Table 6.1). This will eventually also benefit MAAP if it actually leads to reduced cost for carbon sources and energy supply. For example, it has recently been reported that succinic acid can be produced economically and efficiently by fermentation with Mannheimia succiniciproducens at a product yield of 55% and productivity of 3.19 g L–1 h–1 [10] from an inexpensive biomass-based wood hydrolyzate. In an integrated biorefinery it should be also possible to use a variety of coproducts and intermediates for MAAP; there will, however, also be limitations to this concept that will be discussed in more detail in Section 6.3. 6.2.3 Metabolic Engineering and the Building-block Concept

Because, in multi-product operations, flexibility is mandatory to adapt to everchanging market situations for raw materials and products, it will be important that the production organisms and technical constraints of fermentation processes integrated in a biorefinery enable the effective conversion of a wide variety of substrates. This can be achieved by application of metabolic engineering, and with intelligent design of bioreactors and flexible utility operation. Metabolic engineering has been defined as the improvement of enzymatic, transport, and regulatory functions of the cell with the application of recombinant DNA

6.3 Environmental and Commercial Consideration of Microbial Amino Acid

technologies [11]. Developments in metabolic engineering have been reviewed extensively [12–14]. Analytical tools have been developed to investigate intracellular metabolic fluxes [3, 14, 15]. For metabolic flux analysis the building block concept introduced by Neidhardt et al. [16] is of central importance. According to this concept in microbial intermediary metabolism twelve building blocks are derived from carbon sources which subsequently are required for biosynthesis of biomass and products formed by a microorganism. Interestingly, a similar concept has been defined in biorefinery (Table 6.1). In Table 6.2 the building block concept of cellular metabolism is illustrated for C. glutamicum. Here only eleven central intermediates of metabolism were considered to calculate the molecule demand to generate one gram of dry cell weight [17]. For the predominant amino acid production species C. glutamicum this concept has been extensively applied [17–19] so that nowadays the metabolic network is well understood. With this knowledge of metabolic flux distributions for a large number of physiological states it is feasible to determine the complex readjustment of metabolic networks in response to changes in environmental conditions or to changes in the metabolic network structure. Large differences in intracellular building block demand and metabolic fluxes were observed in C. glutamicum when the production of glutamate and lysine were compared with physiological state of growth [20]. Another example for alterations in metabolic network structure is the modification of cofactor dependency of important enzymes. In a lysine-producing C. glutamicum strain a glutamate dehydrogenase was introduced which is dependent on NADH instead of NADPH. The building block demand and metabolic fluxes changed dramatically [21]. Similar redirections of metabolic fluxes might occur with use of alternative carbon sources from biorefinery side streams which differ in their degree of reduction. During the last two decades a platform was established which enables us to quantify metabolic effects rather than just to evaluate them qualitatively. This way it will be possible to quantify the effects of new carbon source input from emerging biorefinery. Subsequently, metabolic engineering strategies can be designed for rational metabolic engineering or for traditional strain breeding. Also, models and control schemes for a cost-effective operation and control of a biorefinery considering metabolic constraints and substrate supply of the different fermentation operations can be developed.

6.3 Environmental and Commercial Consideration of Microbial Amino Acid Production Integrated in a Biorefinery

The role of amino acids in world-wide ecology and in relation to the future development of the world population has been evaluated recently [22]. Feeding animals in a well balanced manner leads to reduced release of ammonia into the environment. When methionine instead of soybean meal is given to poultry the amount of ammonia released to the environment is reduced more than tenfold

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Table 6.2 Building block concept in metabolic engineering of microbial amino acid production. Eleven building blocks are produced from carbon sources in microbial metabolism and subsequently are used as intermediates for biosynthetic pathways. The typical demand in lmol to build one gram of dry cell weight of C. glutamicum is shown. These data are relevant for a leucine auxotroph strain. For a leucine prototroph strain the demand for pyr and accoa is 2685 and 2939 lmol per gram of dry cell weight, respectively. The three letter code for amino acids is indicated. Precursor

Amount

Stoichiometry g6p

Ala Arg Asx Cys Glx Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val Dmp Protein RNA DNA Lipids LPS Peptidoglycan Glycogen CI units Polyamines Precursor Total

f6p

606 189 399 87 806 361 71 202 0 202 146 133 170 225 275 54 81 284 146

ri5p

e4p

gap

pga

pep

pyr

accoa

oaa

akg

1 1 1 1 1 1 1 1 2 1 1

1 1 1 1

2 1 1 1

1

0

0

125 630 100

51

16 55

24

1 1

268

1 2

0

129

673 368 50 129 24

482

24 28

pep 534

2 1 1724

0

1 1370 262 50

1165

83

2116 329 55

28

28

pyr 1807

accoa 2500

oaa 1710

59 akg 1252

154 49 g6p 205

f6p 71

ri5p 879

e4p 268

gap 129

pga 1293

Abbreviations: Dmp, diaminopimelate; g6p, glucose 6-phosphate; f6p, fructose 6phosphate; ri5p, ribose 5-phosphate; e4p, erythrose 4-phosphate; gap, glyceraldehyde 3-phosphate; pga, 3-phosphoglycerate; pep, phosphoenolpyruvate; accoa, acetyl coenzyme A; oaa, oxaloacetate; akg, a-ketoglutarate [17]

6.3 Environmental and Commercial Consideration of Microbial Amino Acid

[22]. In this way surplus nitrogen and especially nitrate can be avoided so that ground-water quality is improved. The same is true for substitution of soybean meal by lysine and threonine in low-protein diets. An ecological balance for the fermentative production of polyhydroxyalkanoates revealed that the major burden on the environment was caused upstream of the fermentation in the supply chain which provides the substrate for the fermentation [23]. Similar results were obtained for the fermentative production of lysine and threonine [24]. This means that a re-engineering in the sugar-providing industry could further improve the ecological impact of MAAP. Because the lignocellulose-based process is significantly less efficient for the preparation of the monomer carbon source, however, compared with the well established starch or sucrose-based process there might not be a significant improvement. Considering the ecological balance the most efficient process will be the preparation of a sucrose stream from sugar cane and utilization of sugar cane bagasse for energy generation. The utilization of bagasse for energy supply or supply of lignocellulose-based carbon sources has a significant advantage over corn stover, because it is already available at the processing site and no costs arise for collection and transport. The markets for the amino acids glutamate, lysine, and threonine are characterized by dynamic growth and they are highly competitive. Monosodium glutamate is used in food as a taste enhancer which is added in prepared food at 0.1 to 0.8% or even more in East Asian dishes. Lysine is used as a feed additive and the addition of 1 kg lysine · HCl increases poultry feed quality to the same extent as addition of 35 kg soybean meal [22]. Also threonine is used as feed additive especially in diets for pig and poultry. Addition of up to 0.75% of threonine to sorghum-peanut meal increases breast meat deposition by more than 15%. Lysine and monosodium glutamate are the largest products in the category of MAAP, and the total market value for amino acids including threonine and tryptophan was estimated to be in the range of 2.5 billion 1 in 2004. A publication by Ajinomoto illustrates the decrease of manufacturing cost for lysine and threonine during the last two decades [25]. It becomes obvious that in this highly competitive market the product price stabilizes on a quite narrow low price level. Innovative concepts for reduction of production cost are of tremendous importance to stay competitive in MAAP. The size and location of a MAAP site is affected by the following considerations: · availability and price of carbon sources as major single cost · availability and price of electricity, gas, and steam supply · availability and price of nitrogen source · equipment prices and prices for engineering and construction driving capital investment · proximity to the market and large-scale customers to achieve low transport and distribution cost · currency effects in a global market.

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6 Towards Integration of Biorefinery and Microbial Amino Acid Production Table 6.3 Commercially available lysine preparations for feed use based on different downstream technologies [3]. Product preparation

Lysine-base Downstreaming content (%) steps

Lysine HCl [26]

78.8

Liquid lysine Approx. 50 [27] Granulated 40–50 lysine sulfate [28]

Wastes

Biomass separation, Biomass, organic ion exchange, drying acids, salts, ammonia, water Biomass separation, Biomass, salts evaporation, filtration Evaporation, Almost none spray drying

Advantages

No crystallization

No drying step No wastes, no separation step

The same considerations are valid if MAAP is evaluated in the context of any biorefinery operation. MAAP processes consist of four major steps: receiving, storage and sterile preparation of carbon sources and other raw materials; cultivation of the production strain in an aerobic process; downstream of fermentation broth; and purification of amino acids and waste treatment. Table 6.3 illustrates, as an example, that depending on the downstream process the overall process yield and thus the quantity of waste materials differs significantly [3, 4]. Even though the process flow in the downstream part of the production of a special lysine sulfate product which contains the whole culture broth was simplified to a minimum number of unit operations, great challenges had to be overcome to enable good handling properties and variations of lysine content in the final product. Because, in the feed-additive market, customers require only a guaranteed minimum content of the active substance and handling properties suitable for large scale operations (low dustiness, low caking tendency, good flow ability, high bulk density) there is room for the development of further low-purity product forms if the savings in manufacturing cost are substantially higher than the costs of product registration and marketing efforts for a new product form. Even if a feed mill is integrated into a biorefinery operation and a liquid, amino acid-enriched product [29] is used directly for the feed preparation, large variation of the amino acid content will be very problematic for highquality feed preparation. This puts significant higher constraints on process control of MAAP within a biorefinery operation compared with production of ethanol.

6.4 Technical Constraints for Integration of Microbial Amino Acid Fermentation into a Biorefinery

6.4 Technical Constraints for Integration of Microbial Amino Acid Fermentation into a Biorefinery 6.4.1 Mono-septic Operation

Because all amino acid fermentation processes operate between 30 8C and 40 8C at approximately neutral pH conditions they are highly susceptible to contamination. Contaminants usually have higher growth rates than the production organisms. They also might degrade amino acids during the cultivation or add enzymatic activities that lead to increased degradation during further processing of the fermentation broth. Contaminants also may express toxins that are not desired for food and feed applications. This means that much effort must devoted to ensuring sufficient sterilization of all incoming raw materials, monoseptic operation of the fermentation process, and sufficient sanitation of all downstream equipment. Sterilization of the raw material is especially difficult if large amounts of suspended solids or precipitation result in fouling of heat-exchanger surfaces. Increased viscosity also negatively affects turbulent flow in continuous sterilizers and thus reduces sterilization efficiency. This means that flexible utilization of different raw material streams for fermentation will most probably result in increased investment and an increased maintenance effort to ensure sterile operation. This must be taken into account if the benefit to overall production cost is evaluated. 6.4.2 Carbon Sources

The broadening of substrate utilization spectrum for MAAP will be of central importance for a competitive production process. During the last two decades metabolic engineering strategies have mainly focused on the final terminal biosynthetic pathways. For lysine biosynthesis this resulted in extremely high product yields of up to 0.5 g lysine · HCl g–1 sucrose or glucose [3]. Threonine production strains which are able to grow on ethanol [30] or methanol [31] are available. Production of lysine [32, 33] and glutamate [34] from methanol has also been described. A future challenge will be the broadening of the substrate-utilization spectrum of production strains for MAAP. Because Saccharomyces cerevisiae is of great industrial importance and is thoroughly characterized in its genetics and in its physiology, different substrate utilization systems have been established for this yeast [35]. Similar strategies must be used to broaden the substrate utilization spectrum of production strains in MAAP. In particular, to confer polymer-degrading capabilities on hosts, access to the substrate must be ensured by efficient secretion of the enzymes from the host. Additional requirements arise for sugar transport and for a new set of metabolic activities. The design of these

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new sets of metabolic activities is a challenge which must be addressed by a rational design of the metabolic network coping with the optimization of a large variety of cellular regulation systems. Lignocellulose is the most abundant biomass in the biosphere and enzymes for utilization of lignocellulose must attack in a sequential concerted and synergistic manner. Hydrolyzates of lignocellulose contain compounds that are inhibitory to most microorganisms, so strain development is required to facilitate the development of robust “platform” biocatalysts that can ferment biomass sugars into either ethanol or other desired biobased products with economically viable rates and yields on industrial scales. Tolerance to harsh environments, including elevated temperatures, high salt, and low pH, will be essential. Currently, available strains are severely limited in pentose utilization and exhibit poor hydrolyzate tolerance. Zymomonas mobilis has been successfully engineered to utilize xylose for ethanol production by introducing a xylose assimilation pathway and two genes encoding transketolase and transaldolase resulting in a product yield which is 85% of the theoretical maximum [36]. Acetate has not been considered as a building block in the biorefinery concept because of its lower potential compared with acetone, which is already a petrochemical byproduct. Nevertheless, it is worthwhile discussing acetate as an alternative or additional carbon source to sucrose or glucose because acetate metabolism of C. glutamicum has been investigated in detail by using modern analytical tools of metabolic engineering [37–39]. Metabolic flux analysis revealed how the carbon flow inside the bacterial cell is readjusted and how intermediate building block metabolism is affected. For example, in vivo citric acid cycle activity was increased almost twofold when acetate was fed compared with growth on glucose plus acetate [37]. Furthermore, the synthesis of building blocks was superior during growth on glucose plus acetate. Interestingly, a recent publication [38] has revealed that even small amounts of acetate, injected with a glucose acetate pulse into a steady-state continuous culture, significantly increased the experimental lysine yield from C. glutamicum. The use of carbon sources such as palmitate and propionate has been evaluated [39]. On palmitate and propionate very low growth rates and biomass yields have been observed. This kind of information might help in the design of optimum carbon source cocktails at the interface between biorefinery and MAAP. A similar study has been performed for other carbon sources [40]. This kind of information will, in the future, help to redesign metabolism of C. glutamicum for the utilization of new carbon sources from the biorefinery. In this context it is important to know where redox equivalents from carbon sources can be channeled into the electron transport chain and how efficient energy generation will be for different carbon sources. For C. glutamicum this topic has been reviewed by Bott and Niebisch [41]. Although lactose is very dilute in whey it has been addressed as carbon source for C. glutamicum. The introduction of the lac operon of Escherichia coli resulted in growth of C. glutamicum on lactose and with the use of heterologous expression of the lac and gal genes from Lactococcus lactus, and Lactobacillus del-

6.4 Technical Constraints for Integration of Microbial Amino Acid Fermentation into a Biorefinery

brueckii lysine was formed at a concentration of up to 2 g L–1 [42, 43] which is far from target values which would be regarded as economical. So, there are ways of utilizing a wide range of carbon sources for MAAP, but for a biorefinery operation they must all be available at consistent qualities and quantities at a price significantly lower than starch hydrolyzate or sucrose. The use of MAAP as a sink for undesired co-product stream with a highly variable composition will not be competitive. 6.4.3 Nitrogen Source

Complex nitrogen sources such as corn steep liquor or yeast extract are already added to MAAP processes to increase productivity and stability of the microbial fermentations. They do not contribute significantly to the supply of nitrogen sources. This is achieved with low-cost alternatives, for example ammonium sulfate or liquid ammonia. Corn steep liquor is a by-product in the production of corn starch and contains amino acids, nucleic acids, vitamins, minerals, and a significant amount of phosphorus. The natural fluctuation in quality of complex nitrogen sources results in fluctuations of MAAP process performance. Intensive quality monitoring for the raw materials is required. This must be considered when new nitrogen sources from a biorefinery are to be introduced in MAAP. The use of high-value amino acid or peptide fractions as nitrogen source in amino acid fermentation could be an outlet for these components, however, only if other applications of those amino acids are not successful. This application would force a biorefinery operation to price those “high-value” nitrogen sources at the same level as “low-value” ammonium sulfate or ammonia. 6.4.4 Phosphorus Source

Sufficient supply of phosphorus is essential for oxidative metabolism. Fermentation media contain synthetic and/or complex phosphorus sources. Interestingly, corn steep liquor as a complex source of phosphorus is a traditional product of corn processing and, depending on the steeping process, the major fraction of phosphorus is available in the inorganic form or bound as inositol phosphate. Media must be carefully designed with regard to phosphorus input because excess input leads to extensive oxidative metabolism and low biomass and product yields. On the other hand, low input results in slow biomass formation, weak intermediate energy supply, weak export capacity, and low productivity. Phosphate limitation elicits a specific gene expression response in C. glutamicum [44]. By the introduction of simultaneous and concerted carbon and phosphate limitation continuous fermentation for lysine production has been optimized [45]. All mass flows in a biorefinery must certainly be evaluated for their phosphorus content before they can be integrated in MAAP.

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6.4.5 Mixing and Oxygen Supply

Depending on the extent of reduction of the carbon source and on the energetic efficiency of MAAP processes more or less oxygen is required to produce biomass and amino acids. The fermentation equipment for mixing, oxygen supply, and heat removal will have to handle a rather large range of requirements, especially when flexible utilization of carbon sources with different physical properties (i.e. viscosity) and different reduction state of the carbon atoms are used. If the carbon sources are less oxidized than sugars, the fermentation will use significantly more oxygen and produce more CO2. For E. coli this effect seems to be uncritical up to a concentration of 30% of carbon dioxide in the exhaust gas [46]. 6.4.6 Toxicity

A major problem of the usage of raw materials from biorefinery in MAAP may be the toxicity of components which are contained in the raw material. This topic has already been addressed in Section 6.4.2 when the use of lignocellulose was discussed. Advanced monitoring technologies such as cytometry [47, 48] and DNA microarray analysis of gene expression [49, 50] might be used to monitor toxicity effects, to reveal the nature of toxicity, and to search for metabolic engineering or strain breeding strategies to overcome the hurdles caused by toxic compounds. Recently, metabolic flux analysis has been applied to acidtolerant yeast Candida milleri which would be ideal to convert industrial pulp mill xylose containing waste streams into xylitol [51]. This kind of investigation is a first step in the direction of a comprehensive understanding of metabolic network responses to stimuli from substrate streams which originate from biorefineries. It is crucial to select robust host strains such as, in this case, C. milleri in advance and afterwards to optimize the metabolic pathways for maximum product formation. In the field of MAAP this approach is in its infancy, because metabolic engineering efforts have been focused on well established hosts. Alternatively, before proceeding with further optimization of terminal pathways for amino acid biosynthesis, metabolic engineering and classical strain breeding should be initiated for improvement of resistance against toxic effects. A recently described example is the development of a raffinate (ammonium sulfate rich eluent from ion exchange)-resistant lysine production strain that enables re-utilization of the ammonium sulfate generated as a waste stream during purification [52].


6.4.7 Cultivation Temperature

A significant part of production costs is caused by cooling demand. To reduce costs of cooling utilities C. glutamicum strains have been selected which are characterized by a growth optimum at 40 8C instead of 30 8C [53]. This strategy might be useful to integrate mass flows from biorefinery into MAAP processes so that costly cooling operations are avoided.


Biorefineries are defined as processing facilities that extract carbohydrates, oils, lignin, and other materials from biomass, and convert them into multiple products including fuels and high-value chemicals and materials [5]. In our contribution we have illustrated the major hurdles which must be overcome to generate value in industrial production of amino acids. Sucrose, molasses, and starch hydrolyzates have been used as renewable carbon sources for decades. Existing starch processing already has the advantage that co-product markets as for gluten and corn oil contribute to overall plant economics. In industrial amino acid production experience in handling these product streams is available. Based on this experience it is obvious that potential new product streams which might enter MAAP must be of low cost and characterized by low concentrations of impurities and by low fluctuations in raw material composition. It will be a prerequisite for the success of the biorefinery concept to yield new well defined carbon sources of high purity and lower cost compared with starch or sugar-based feedstock. If these characteristics can be ensured in future the integration of biorefineries and MAAP will be successful. Especially, with regard to downstream processing and product formulation, stable product characteristics and guaranteed purity must be delivered by a MAAP process which is integrated into a biorefinery. Not only will the large part of variable cost attributable to the carbon source in MAAP be reduced substantially, but also the fixed cost will be reduced when different co-products can be produced in one biorefinery. We have illustrated the example of generation of energy or even an additional carbon source from sugar cane bagasse. The processing of natural compounds requires extensive equipment size and energy consumption for water handling. This problem must be addressed to improve cost structure. The availability and price of carbon and nitrogen sources, electricity, gas, and steam supply, and equipment prices and prices for engineering and construction must be considered together, to define the optimum for economic operation of MAAP as part of a biorefinery. Proximity to the market and to large-scale customers guarantees low transport and distribution cost which means that sophisticated optimization of the supply chain must be

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performed. An additional option might be to combine biorefinery locations with animal feed-production facilities, to make use of synergies in logistic planning and transportation. If all prerequisites for the establishment of this kind of biorefinery concept are fulfilled, lignocellulose-based processes will be beneficial for the economics and ecology of MAAP. Taken together, the biorefinery concept promises to provide cheap carbon sources with unlimited availability, environmentally friendly processes, and valuable co-products. Glycerol and ethanol might in future be inexpensive and might be used for the biotechnological MAAP as an alternative to sugar substrates, depending on public regulation. Even for chemical synthesis of amino acids and other specialty chemicals glycerol and ethanol might be of importance in the future. Until now we have considered topics related to the business model of MAAP. The basis for a successful operation of this business process is a well elaborated technology platform. To establish this platform a systematic approach must be designed which guarantees that future product streams are compatible within a biorefinery so that they can be combined and integrated. The so called building block concept in biorefineries, which has been illustrated in detail [5], has been defined to support the systematic design of biorefineries. If technical solutions for this concept become available, large opportunities will arise for MAAP. We have illustrated in our contribution that also in metabolic engineering a similar building block concept has been defined. Using this concept metabolic engineering has contributed to tremendous progress in MAAP during the last two decades. Now it will be the challenge to redirect metabolic fluxes in microorganisms depending on changes in carbon sources, phosphorus source, mixing and oxygen supply, toxicity, and cultivation temperature. The building block concept will help to systematically develop platform microbes with the use of metabolic engineering. The question arises whether to build technology platforms in homologous systems or to intensify the establishment of heterologous pathways for broadening of the substrate utilization spectrum for MAAP in selected host organisms. The answer must be found individually, because we just have started the journey toward developing robust high-performance strains which can efficiently use new carbon sources and can resist harmful effects in their microenvironment. If the major hurdles described in our contribution are overcome the concept of biorefinery is beneficial for MAAP. The major benefit will originate from the additional value of co-products for which a clear marketing and sales concept must be demonstrated.

Acknowledgment

We acknowledge the support of Dr Klaus Huthmacher, Professor Dr Wolfgang Leuchtenberger, and Dr Michael Binder, Degussa AG, Hanau, Germany.

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7 Protein-based Polymers: Mechanistic Foundations for Bioproduction and Engineering Dan W. Urry

7.1 Introduction 7.1.1 Definitions 7.1.1.1 Proteins and Protein-based Polymers A protein is a polypeptide, (–NH–CHR–CO–)n, where R is the side chain of any of the twenty different amino acid residues available to biological protein synthesis, or in practice the R group can also be a chemical or enzymatic modification thereof. In the living organism the molecular weight can range from a few thousand Daltons, for example, some tens of residues, to two million Daltons, that is, twenty thousand residues. Protein-based polymers are polymers of repeating peptide sequences, The repeating unit may be as small as a dipeptide, (–NH–CHR–CO–NH–CHR'–CO–)n, or as large as hundreds of peptide residues and n can be as large as several hundred, or more. Protein-based polymers hold promise of low-cost production by means of recombinant DNA technology. By this process genes are constructed that encode for the desired peptide sequence, and a biological organism is transformed to produce (express) the designed protein-based polymer sequence that could even be a thermoplastic. Thus, proteins and protein-based polymers constitute renewable resources with the potential, at least in part, to replace petroleum-based polymers.

7.1.1.2 Two Basic Principles for Protein-based Polymer Engineering As introduced here, two basic principles, referred to as the hydrophobic and elastic consilient mechanisms, for protein-based polymer engineering bear on controlling hydrophobic association and utilizing near ideal elasticity [1]. Moreover, an understanding of hydrophobic association is key to bioproduction, purification and function, and the nature of elasticity relates to efficient function and to an enabling of a remarkable biocompatibility for medical applications. Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 2 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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7 Protein-based Polymers: Mechanistic Foundations for Bioproduction and Engineering

7.1.2 Proteins in Aqueous Media

Proteins in an aqueous milieu provide the ultimate in amphiphilic polymers. There is absolute control of sequence where in each position can occur any one of twenty disparate amino acid residues, ranging from very hydrophobic to very polar (for example, charged), and this occurs with strict maintenance of stereochemistry. The result is a structural and functional diversity that is unmatched by any other polymer. 7.1.3 Thermodynamics of Proteins in Water 7.1.3.1 Exothermic Hydration of Apolar Groups In 1937 Butler reported the fundamental finding that dissolution of CH2 groups of an alcohol series in water is an exothermic reaction [2]. Thinking within the framework of the Gibbs free energy for solubility, DG(solubility) = DH–TDS. For the methanol to n-pentanol series, the heat released (DH) on hydration is 1.3 kcal mol–1 CH2 with the (–TDS) entropic term limiting solubility by a +1.7 kcal mol–1 CH2. This requires that there be structured (low entropy) water surrounding dissolved hydrophobic groups, and that too much hydrophobic hydration means insolubility (Section 7.1.4 below).

7.1.3.2 The Change in Gibbs Free Energy of Hydrophobic Association The most fundamental property deciding aqueous protein-based polymer structure formation and function resides in DG8HA, the change in Gibbs free energy for hydrophobic association, that is, for loss of solubility of hydrophobic groups [3]. Insight into the primary operative component of DG8HA gains from the old adage, “oil and vinegar do not mix.” In proteins and protein-based polymers, however, oil-like and vinegar-like groups are forced by sequence to coexist along a chain molecule; they are forced to interact. As one might expect from the old adage, they display their reluctance to intermix by a water-mediated repulsion. Oil-like (apolar) and vinegar-like (polar) groups of proteins, forced by primary, secondary, tertiary and quaternary structure to coexist, repulse each other as each seeks hydration unperturbed by the other. Apolar and polar groups compete for hydration!

7.1.3.3 The Apolar–Polar Repulsive Free Energy of Hydration, DG8ap The operative component of DG8HA is the apolar–polar repulsive free energy of hydration, DGap; it measures the competition for hydration [4]. These fundamental thermodynamic quantities, DG8HA and DGap, are quantifiable by a number of experimental methods. The value of DGap can be determined using differential scanning calorimetry as the change in DG8HA attending ionization of a functional R group, and it can also be measured using acid–base titrations from

7.1 Introduction

the shift that occurs in pKa of an ionizable side-chain on changing the hydrophobicity of the protein-based polymer. From the standpoint of bioproduction, ionization can give solubility and neutralization can effect selective phase separation for purification. 7.1.4 The Inverse Temperature Transition for Hydrophobic Association

By the Second Law of Thermodynamics, an increase in temperature results in an overall decrease in order of the total system, which in our case is water plus protein-based polymer. With the correct balance of oil-like and vinegar-like groups, raising the temperature through the transition range results in a phase separation in which the protein-based polymer goes from being randomly dispersed in solution to being more ordered, and even crystalline, on separation [4]. This is called an inverse temperature transition. Structured water surrounding the dissolved polymer, which becomes less ordered bulk water as the polymer associates, makes the inverse temperature transition congruent with the Second Law. Such water does exist surrounding oil-like (hydrophobic) groups and is called hydrophobic hydration. Thus, the inverse temperature transition represents a transition from a state of hydrophobic groups being hydrated to the loss of hydrophobic hydration as the hydrophobic groups associate. Development of too much hydrophobic hydration for a given temperature results in the insolubility due to hydrophobic association. On the other hand, in the process of achieving their own hydration, charged species disrupt hydrophobic hydration, even the hydrophobic hydration that forms during a transient hydrophobic dissociation. In this way charged species decrease the amount of hydrophobic hydration, and thereby disrupt hydrophobic association. As considered above, the driving force for loss of solubility (hydrophobic association) derives from the development of too much hydrophobic hydration for a given temperature [2]. Since DG(solubility) = DH–TDS, insolubility occurs as the term (–TDS) becomes too positive and dominates the inherently negative DH term. 7.1.5 The Role of Elasticity in the Engineering of Protein-based Polymers 7.1.5.1 Near Ideal Elasticity Provides for Efficient Energy Conversion In general, an energy input changes hydrophobic association, and as the means of achieving function, the input energy is transiently stored in chain deformation. Efficient energy conversion requires that most or all of the energy expended during elastic deformation be recovered on relaxation, as can only occur with near ideal elasticity. This is most apparent in the performance of mechanical work. During an isometric contraction, for example, the input energy converts to an increase in elastic force. If the force–relaxation curve falls below the force–extension curve, the result is called hysteresis, and the result is that only a part of the input energy spent on extension is recovered on relaxation. There-

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fore, without near ideal elasticity, too little energy of deformation becomes available for the function of producing motion

7.1.5.2 Mechanism of Near Ideal Elasticity A mechanism of elasticity that allows for near complete recovery of the deformation energy on relaxation is central to the design of an efficient protein-based machine that is to perform mechanical work. The structure and mechanism should be such as to yield the experimental result of a near perfectly reversible stress–strain curve. In our view, such near ideal elasticity arises, in general, from a decrease in chain mobility within the extended chain segment. Specifically, the decrease in chain mobility on extension is well described as a decrease in amplitude of torsion angle oscillations within the load-bearing chain. This allows that very little energy is lost from the chain to its environment, such as to associated chains that bear no load. 7.1.6 Many of the Advantages of Protein-based Polymeric Materials

One of the most important advantages of protein-based polymers is that they constitute perhaps the most favorable model proteins with which to determine the sought-after engineering principles. This, of course, becomes possible due to the absolute control of amino acid sequence and due to the availability of a diverse set of residues from which to choose. In addition to the model proteins on which they are based, these principles provide a consilience, “a common groundwork of explanation” [5] for the hydrophobic mechanism that is relevant to all amphiphilic polymers and for an entropic elastic mechanism for all polymers of whatever composition so long as the polymer exhibits a chain mobility that becomes damped on deformation. For these reasons the derived engineering principles are referred to as the hydrophobic and elastic consilient mechanisms. Table 7.1 lists eighteen advantages of protein-based polymers for their utilization as advanced materials for the future. Each item on the list warrants a substantial paragraph, but length limitations require that the listing suffice. A number of the advantages, however, become apparent in the following sections more specifically focused on the mechanistic foundations of bioproduction and engineering.

7.2 Historical Outline Table 7.1 Many of the advantages of protein-based polymeric materials. 1 2 3 4 5 6 7 8 9 10 11 12

13 14 15 16 17 18

Two modes of synthesis: chemical and recombinant DNA technology Diversity of monomers Precise control of the sequence of amino acid residues Exact control of stereochemistry Precise chain lengths Capacity to introduce natural bioactive peptide sequences Circumstances of protein function to guide analyses and approach Properties and uses beyond those of known proteins, e.g. thermoplastics (polymers that melt) Low cost of bioproduction Produced from renewable resources Axioms for protein-based polymer engineering Perform pair-wise energy conversions using intensive variables of mechanical force, pressure, chemical potential, temperature, electrochemical potential, and electromagnetic radiation Quantitative principles available for effective design of protein-based polymer machines Allows for most efficient mechanism for function in an aqueous environment Remarkable biocompatibility of elastic protein-based polymers In vivo breakdown products simply amino acids A near infinite number of polymer compositions Permits de novo design of specific composition for each intended application

7.2 Historical Outline 7.2.1 Historical Beginnings of (Elastic) Protein-based Polymer Development

As the result of a collaboration in 1969 with S. M. Partridge of Bristol, England, we reported that a temperature-induced phase separation of a-elastin, the Partridge 70 kDa chemical fragmentation product of purified ligament elastic fiber, resulted in increased intramolecular order of this protein fragment [6]. Subsequent observation, by means of transmission electron microscopy with negative staining, showed that on phase separation a-elastin increased its intermolecular order as represented by formation of filaments and fibrils. These heat-induced increases in intramolecular and intermolecular order gave rise to the term inverse temperature transition [7]. In 1971, based on amino acid composition and on in vivo properties of the elastic fiber, the proposal was made that glycine containing repeating tetra-, penta-, and hexapeptide sequences would be probable in the elastic fiber [8]. Shortly thereafter, L. B. Sandberg, then at the University of Utah, called by telephone to report that his research group had found a repeating tetrapeptide in elastin [9]. Subsequently, they found that the dominant repeating peptide sequence was a pentapeptide [10]. Our response was to synthesize repeating tetra-, penta-, hexa- and nonapeptide sequences, to carry out many physical characterizations [11], to crosslinked high mo-

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lecular weight polypentapeptides, and to find the latter material to be highly elastic [12]. Then followed the proposal of a new mechanism of elasticity in 1982 [13], which was supported by test syntheses, by many more physical characterizations [14, 15], and by calculations using the ECEPP molecular mechanics approach of Scheraga [13] and the CHARMM molecular dynamics program of Karplus [16]. The development of elastic-contractile model proteins capable of diverse energy conversions, based on inverse temperature transitions, rapidly ensued formation of crosslinked elastic matrices that contracted either on raising the temperature above Tt, the onset of the transition, or on lowering Tt from above to below the operating temperature. Five phenomenological Axioms summarize the use of Tt for design of Tt-type polymers capable of eighteen classes of pairwise energy conversions [4]. Development of the mechanistic foundations followed enumeration of the Axioms. 7.2.2 Mechanistic Foundations: Fundamental Engineering Principles

Development of the mechanistic foundations proceeded on two distinct tracks that then couple to achieve function. The two tracks, referred to as the hydrophobic and elastic consilient mechanisms, couple hydrophobic association or apolar–polar repulsion to elastic deformation to produce motion or even chemical work.

7.2.2.1 The Hydrophobic Consilient Mechanism The change in the Gibbs free energy of hydrophobic association, DGHA, undergirds the hydrophobic consilient mechanism [1, 3, 4]. Initial insights began with expectation (based on the Second Law of Thermodynamics) that the increase in order (structure formation) of elastin peptides on raising the temperature was due to hydrophobic association [7]. Again many physical characterizations were utilized, including direct observation of hydrophobic association and the accompanying decrease in water structure as ordered hydrophobic hydration became disordered bulk water [4, 17]. The apolar–polar repulsive free energy of hydration, DGap, represents the operative component of DGHA. DGap derives most effectively from changes in the presence of charged states associated with protein, such as the ionization of carboxyls and the binding of phosphates. Stretch-induced [18] and hydrophobic-induced [19] pKa shifts demonstrate competition for hydration and allow evaluation of DGap by the entirely independent experimental means of acid–base titrations. The identification of hydrophobic hydration and the direct observation that charge formation destroys hydrophobic hydration provide confirmation of the competition for hydration [20]. The change in DGHA resulting from charge formation, as measured from differential scanning calorimetry, gave the same value of DGap as obtained from acid–base titration data of hydrophobic-induced pKa shifts [1, 3, 21]. Such are these highlights in the historical development of the hydrophobic consilient mechanism.


7.2.2.2 The Elastic Consilient Mechanism The history of the development of the elastic consilient mechanism has been one of controversy. Our view noted above that the damping of internal chain dynamics on extension of even a single chain gives rise to an entropic elastic force [16] has been decried by adherents to the established classical (random chain network) theory of rubber elasticity [22, 23] and even by those currently subscribing to the view that solvent entropy changes attending changes in hydrophobic association give rise to an entropic elastic force [24]. In your author’s view, the issue is clear. First, the proposed contribution of solvent entropy changes resolves on consideration of the development of elastic force under isometric conditions. Elastic force development at fixed extension results in the development of entropic elastic force due to a negative change in entropy, whether due to thermally or chemically driven hydrophobic association [1, 21, 25]. Under these conditions for hydrophobic association, the entropy change is positive for solvent, but negative for developed elastic force. Second, in AFM single-chain force–extension studies a single chain, fixed at both ends, develops entropic elastic force [26, 27]. It would appear that extension of a random chain network comprised of a Gaussian distribution of end-to-end chain lengths is not required for entropic elasticity. Having eliminated the solventbased mechanism and also invoking an elementary statistical mechanics analysis [26, 27], entropic elastic force derives from decreased amplitude of chain torsional angle oscillations on extension [15, 16]. Analyses of crystal data for the Rieske Iron Protein support this understanding of elastic force development. Hydrophobic association stretches a single chain that on relaxation moves a protein domain to transfer an electron in Complex III of the electron transport chain. Also our crystal data analyses for the myosin II motor of muscle contraction argues that the power stroke due to ATP hydrolysis and loss of phosphate causes two hydrophobic associations that stretch interconnecting single chains to produce elastic force under isometric conditions of a near rigor state [1, 21].

7.2.3 Highlights of Bioproduction

The history of protein-based polymer bioproduction essentially began in 1986 with an Accelerated Research Initiative of the Office of Naval Research (ONR) with the purpose of developing biotechnology, specifically recombinant DNA technology, for the production of protein-based adhesives and elastomers. This five-year initiative, administered by Michael Marron, rightly receives the credit as the critical starting point for the production of the hundreds of transformed E. coli of Table 7.5 (below) and the approximately one hundred and fifty (150) expressed protein-based polymers. Of the many highlights of this striking development, several may be illustrated (Figs. 7.1–7.3).

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Fig. 7.1 A. E. coli transformed to express the phase-separated protein-based polymer, (GVGVP)121, with the expression product seen as large phase-separated inclusion bodies which comprise approximately 80%

of the volume of the transformed E. coli. B. The same strain of E. coli before transformation. Reproduced with permission from Ref. [28].

Fig. 7.2 Two-hundred and thirty-seven-gram yield of a single fermentation in 1997 of transformed E. coli that produced (GVGIP)260. Now the yields can be several times greater.

7.3 Bioproduction

Bioproduction of protein-based polymers may be considered in three stages: (1) gene construction using recombinant DNA technology, (2) transformation of the cell to be used for expression, and (3) fermentation of the transformed microbial cell. This process has been reported in detail for the production of (GVGVP)10 ´ n, where the monomer gene encodes for ten pentameric repeats without DNA repeats [29].

7.3 Bioproduction

Fig. 7.3 Three thermoplastics (polymer melts pulled into plastic fibers). Two are plastics of our daily experience (B, polypropylene; C, polystyrene) whereas the third, A, is a protein-based plastic that melts at 160 8C and does not decompose until 250 8C. Thus, using living organisms, the potential is to prepare protein-based polymeric materials with properties inaccessible to living organ-

isms but of growing interest to society. Furthermore, protein-based polymer thermoplastics can be programmed to biodegrade in a sufficiently wet environment with halflives that could be varied from days to decades. It might be said that these proteinbased thermoplastics portend food rather than death for marine life. From Ref. [1].

7.3.1 Gene Construction using Recombinant DNA Technology 7.3.1.1 Preparation of Monomer Genes and the PCR Technique [29] A protein sequence of no more than about 50 residues is chosen. Such a length makes possible chemical synthesis of two nucleotide sequences, each of about 80 to 90 bases, one from the 3' end and the other from the 5' end. These two base sequences associate by base pairing with an overlap of some 10 to 20 base pairs. The polymerase chain reaction (PCR) is then used to complete the double stranded DNA sequence. The appropriate restriction enzyme is used to cut the ends of the double stranded DNA to produce the monomer gene for insertion into the chosen plasmid. Commonly, the BamH 1 restriction site is used for insertion into pUC-118.

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7.3.1.2 Transformation, Monomer Gene Production and Sequence Verification The prepared plasmid, in our case pUC-118 containing the monomer gene, is introduced into the chosen strain of E. coli, which is grown up to prepare many copies of the monomer gene. The purified large-scale monomer gene preparation is sequenced to verify that the chemical synthesis did not introduce sequence errors, and prepared for concatenation (polymerization) into multimer genes.

7.3.1.3 Monomer Gene Concatenation Produces Multimer Genes of Monomer [30] After excision exposes the PflM 1 restriction site, which allows only head-to-tail alignment, the ligase reaction polymerizes the monomer gene into multimer genes. In Fig. 7.4 the gene ladder of lane 2 shows each multimer gene. Lane 1 is the monomeric gene fragment. Lanes 3, 4, and 5 contain pUC-118 and genes encoding (GVGVP)10 ´ n+1 released on treatment with restriction endonuclease, Bam H 1. Lane 3 gives (GVGVP)10 ´ 4+1, which is (GVGVP)41. Lane 4 gives (GVGVP)121, and Lane 5 gives (GVGVP)251. As seen in Lane 5, the capacity to make genes is so effective, that the gene is larger than the plasmid into which it is placed. In fact, it has been possible to prepare and to express genes that encode for the equivalent of more than 400 repeats of the pentamer. In terms of

Fig. 7.4 Gene-ladder. From Ref. [30].

7.4 Purification of Protein-based Polymers

molecular weights that would be some 200 kD, whereas the original maximum size was thought to be 50 kD in E. coli. 7.3.2 E. coli Transformation for Protein-based Polymer Expression

The multimer gene, prepared in pUC-118, is excised and inserted into an expression vector, such as pET-23d, which is specialized for expression of the designed protein-based polymer and inserted into a strain of E. coli, selected for the expression [29]. Figure 7.1 B, above, shows the non-transformed E. coli, whereas Fig. 7.1A shows the strain of E. coli, transformed to produce (GVGVP)121. This demonstrates the high production capacity of the chosen expression system, for example, the BL21(DE3) strain of E. coli, to produce the elastic protein-based polymer. 7.3.3 Fermentation using Transformed E. coli

A portion of a glycerol stock of the transformed E. coli is utilized to grow an inoculum for addition to a large fermenter. In addition to the choice of carbon source, nitrogen source, salts, and cofactors, the medium contains an antibiotic to which the chosen expression vector confers resistance and an inducer that turns on the expression vector after the cell density has reached an optimal level. In the example given above the antibiotic was antimycin and the induction system was designed for IPTG. Figure 7.2, above, is an example of an early single fermentation using a working volume of 400 L. The yield of a single fermentation for this product, (GVGIP)260, was 237 grams after purification, by phase separation, to less than 1 ppm impurities.


The purification of protein-based polymers that exhibit inverse temperature (phase) transitions is addressed in three sections: (1) The essential properties of the inverse temperature transition are discussed as a very effective method of purification. It should be realized that all of the means of changing Tt and DGHA in Tables 7.3 and 7.4, below, can be useful means for selective phase separation of the designed protein-based polymer from the cell lysate. (2) A number of physical methods, such as multi-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry are shown to be particularly effective in the verification of product. And (3) after achieving adequate purification, subcutaneous injection in the guinea-pig is shown to provide effective demonstration of the remarkable biocompatibility of elastic protein-based polymers, where large quantities of biomaterial disperse within days without eliciting any evidence of having been present.

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7.4.1 Use of the Inverse Temperature Transition as a Method of Purification 7.4.1.1 Purification by Phase Separation as Demonstrated by SDS–PAGE [30] Figure 7.5, below, uses sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) to demonstrate the usefulness of the inverse temperature transition exhibited by the elastic protein-based polymer, (GVGVP)141 as a method of purification. Using the negative CuCl2 stain, the expressed product is seen as the bulge in Lane 1. Lane 4 shows how the effective utilization of the inverse temperature transition purifies these protein-based polymers.

7.4.1.2 Purification by Phase Separation Shown by Carbon-14-labeled E. coli [31] Without showing the data, the effectiveness of the phase separation was followed by C-14 labeling of all of the components of E. coli except the proteinbased polymer [1]. With each phase separation the purification improved by a factor of ten or more.

Fig. 7.5 SDS–PAGE with CuCl2 stain of expressed (GVGVP)141 demonstrates use of phase transition to separate from E. coli proteins. Lane 1: Crude lysate of transformed E. coli. Lane 2: Precipitate of cold centrifuga-

tion leaving polymer in solution. Lane 3: Supernatant of cold spin after heating to phase separate (GVGVP)141. Lane 4: Phase separated elastic protein-based polymer on heating. From [30].


7.4.2 Physical Characterization and Verification of Product Integrity 7.4.2.1 Gross Visualization of the Phase Separated Product In Fig. 7.6 direct visualization of the phase separated product provides graphic demonstration of both the physical nature of the product and the effectiveness of the inverse temperature transition as a means of purification.

7.4.2.2 Sequence Integrity and Purity Evaluated by Nuclear Magnetic Resonance Routinely, one-dimensional nuclear magnetic resonance (NMR) verifies the absence of impurities and other molecular groups. Two-dimensional NMR, by proton–proton through bond couplings, gives proton assignments, and, using proton–proton through space nuclear Overhauser interactions, allows determination of the sequence integrity of the repeating unit [32]. In this way the complete sequence of the repeat monomer can be verified. Now, verification of number of repeats follows below.

7.4.2.3 Mass Spectra Reaffirm Size of Expressed Polymer [32] The agarose gel gene ladder allows determination of gene size, as seen in Fig. 7.4, where the integral number of monomer repeats of the designed gene can be discerned from the number of monomer gene repeats plus one pentamer. Thus, one has an expected molecular weight for the expressed proteinbased polymer. Using matrix assisted laser desorption time-of-flight (MALDI–

Fig. 7.6 A. Phase separated, taffy-like mass of (GVGVP)251 produced by a single fermentation. The mass readily pulls into long, fine viscoelastic strands [1]. B. A small amount of the protein-based polymer, (GVGIP)320, phase separated into the bottom of a 250 mL centrifugation bottle to form a pan-

cake, which in C. is pulled into a tough band of a meter in length. The addition of a single CH2 group per pentamer causes this dramatic change in physical property. Crosslinked (GVGVP)251 exhibits reversible stress– strain curves, whereas (GVGIP)320 exhibits a marked hysteresis. From Ref. [1].

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TOF) mass spectrometry, the whole-molecule (polymer) peak can be determined plus or minus a number of sodium ions, if neutral or negatively charged, or the number of chloride ions if positively charged. Such efforts allow confirmation of polymer size [32]. Accordingly, a combination of physical methods achieves verification of structure. 7.4.3 Biocompatibility 7.4.3.1 The Challenge of Using E. coli-produced Protein as a Biomaterial Required purification levels pose a challenge for protein biomaterials produced by E. coli fermentation. The target level for purification of protein pharmaceuticals has been to achieve impurities of less than 10 ppm (parts per million). Since biomaterials may be used in amounts one thousand to one million times greater than a pharmaceutical, purification requires an equivalent increase in level of purity. The most challenging test situation, that is, a gold standard, would be for the introduced biomaterial to have released all of its impurities in a relatively short time, say in a few days, without an adverse reaction from the host tissues. This challenge has been met. When injected subcutaneously in the guinea-pig, purified (GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP)36(GVGVP) does disperse in a short time without a trace of ever having been present (Fig. 7.7 A below).

7.4.3.2 Removal of Endotoxins and Determination of Levels After phase separation, a pre-filtration step followed by an ultrafiltration approach is used to remove endotoxins. Determination of endotoxin levels utilizes a 20 mg mL–1 protein-based polymer solution in the Pyrotell Limulus Amebocyte Lysate test (Associates of Cape Cod, Inc). The values obtained for the endotoxin units per mg (EU mg–1) can be routinely less than 0.01 and often less than 0.003 [32].

7.4.3.3 Western Immunoblot Technique to Demonstrate Level of Purity In the standard Western blotting technique, 5 lg per well of positive control (lyzed E. coli supernatant) and of test E. coli-produced protein-based polymer(s) are run in parallel lanes of a 12.5% SDS–PAGE gel. The separated protein bands, as in Lane 3 of Fig. 7.5, are then transferred, by contact, to a nitrocellulose membrane. After suitable treatment, the membranes are incubated with polyclonal rabbit anti-E. coli immunoglobulin G (IgG) as the primary antibody. The membranes are then incubated with biotinylated anti-rabbit IgG. The detection procedure utilizes an alkaline phosphatase procedure to detect antigens to E. coli proteins with a limit of 5 pg. When no bands are detected from an application of 5 lg, the E. coli protein impurity is considered to be below 1 ppm (one part per million). Phase separations by means of elicited inverse temperature transitions are sufficient to achieve this level of purification using any of the


means of Tables 7.3 and 7.4 and Section 7.5 below. It is important to realize, however, that this level of purification is insufficient when using the proteinbased polymer in vivo, as a biomaterial that can release all of its impurities in a short time without leaving any trace of its ever having been present.

7.4.3.4 Western Immunodotblot Technique to Demonstrate Medical Grade Purity Based on the subcutaneous injection test, discussed below, an impurity level of less than 1 ppm is not satisfactory. Accordingly, a more sensitive technique is required to assess impurity level. The next step is to use the Western Immunodotblot technique, where 1 mg of test sample is placed in a single spot and the above technique is again employed. With a detection limit of 5 pg, the absence of detection (absence of a spot) indicates an impurity level of less than 5 ppb (parts per billion). This level of purification is required for protein-based polymer to be a candidate for medical use. The ultimate test uses subcutaneous injection, as discussed immediately below.

7.4.3.5 Subcutaneous Injection in the Guinea-pig Protein-based polymer (30 mg) at phase separation concentration in normal saline is injected subcutaneously in the guinea-pig into the region of the fatty layer. The injection site is followed externally as a bump that results from the volume of injected sample. In the case of the protein-based polymer, (GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP)36(GVGVP), the bump lasted for no more than several days. At two weeks the injection site was examined histologically. As seen in Fig. 7.7A, no evidence is found of the polymer’s ever having been injected. When (GVGVP)251 is injected, the bump is retained, and the in-

Fig. 7.7 Histological sections of subcutaneous injections in the guinea-pig using three different protein-based polymer compositions. The injected polymers are in A. (GVGVP GVGVP GEGVP GVGVP GVGVP

GVGVP)36(GVGVP), in B. (GVGVP)251 and in C. {(GVGVP)10–GVGVPGRGDSP– (GVGVP)10}16(GVGVP). The results of A and C will be discussed below in Section 7.6.1. From Ref. [32].

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jected polymer is seen in Fig. 7.7 B to be intact and surrounded by a fine fibrous capsule [32].

7.4.3.6 ASTM Tests It should be noted in addition to the tests routinely run above, three compositions, (GVGVP)n [33], (GGAP)n [30], and (AVGVP)n [34], have been examined using the complete set of eleven tests recommended by the American Society for the Testing of Materials (ASTM) for materials to be in contact with tissues, tissue fluids and blood. As this set of tests had been run on chemically synthesized polymers, they were continued with microbially synthesized polymers to achieve an adequate understanding of the impurities derived from E. coli. Elastic protein-based polymers, (GVGVP)n and (GGAP)n, exhibit truly remarkable biocompatibility. The plastic protein-based polymer, (AVGVP)n, exhibits good biocompatibility, but being inelastic and an unnatural sequence, its biocompatibility is not as extraordinary.

7.5 Mechanistic Foundations for Engineering Protein-based Polymers

The mechanistic foundations derive from determination of the physical basis for a set of Axioms that enumerate observations arising out of elastic–contractile model proteins designed de novo for energy conversions. As yet unexplained by popular descriptions of protein function, the Tt-based Axioms argue that new mechanistic understandings be sought. Therefore, this section begins with a table that succinctly states these practical Axioms [4] in terms of changes in Tt that function as simple on–off switches for contraction/relaxation by hydrophobic association/dissociation. 7.5.1 Phenomenological Axioms (see Table 7.2) 7.5.2 The Change in Gibbs Free Energy for Hydrophobic Association, DGHA

The experimentally developed phenomenological Axioms, listed below in Table 7.2, do not derive from present treatments of protein structure and function. Therefore, the Axioms require that physical bases, i.e., mechanistic foundations, be sought for these observations. Over the last two decades, our focus has been to design and characterize a particular class of protein-based polymers called elastic–contractile model proteins with which to develop the mechanistic foundations. In short, virtually every experimental variable that effects a change in protein structure and function changes the Gibbs free energy of hydrophobic

7.5 Mechanistic Foundations for Engineering Protein-based Polymers Table 7.2 Phenomenological AXIOMS for protein-based polymer engineering. AXIOM 1: The change in temperature interval, over which occurs the hydrophobic association transition of a host protein-based polymer on introduction of different guest substituents, becomes a practical measure of relative hydrophobic character of the substituents, and it approximates the change in free energy of the resulting hydrophobically associated state. This provides the phenomenological data-base for design of protein-based machines. AXIOM 2: Heating to raise the temperature from below to above the temperature interval for hydrophobic association of crosslinked elastic protein-based polymers drives contraction with the performance of mechanical work. This is thermally driven contraction inherent in inverse temperature transitions. Example:

Thermo « mechanical transduction

AXIOM 3: At constant temperature, any energy input that changes the temperature interval for hydrophobic association in a protein-based polymer can drive contraction with the performance of mechanical work Examples:

Chemo « mechanical transduction Baro « mechanical transduction

Electro « mechanical transduction Photo « mechanical transduction

AXIOM 4: Two or more different functional groups of an amphiphilic macromolecule, each of which can be acted upon by a different energy input that changes the temperature interval for hydrophobic association, become coupled one to the other by acting upon the same hydrophobic association domain. In other words, an energy input acts on one functional constituent to change its hydrophobicity and that change in hydrophobicity becomes an energy output that changes the other functional constituent due to its dependency on hydrophobicity. This axiom is the fundamental energy-coupling axiom; it utilizes the hydrophobic association transition common to all energy conversions by the consilient mechanism. Importantly, in doing so, it involves the performance of kinds of work in addition to mechanical work. Examples:

Electro « chemical transduction Electro « thermal transduction Baro « electrical transduction Photo « voltaic transduction Thermo « chemical transduction Photo « thermal transduction Baro « thermal transduction Baro « chemical transduction Photo « baric transduction Photo « chemical transduction Chemo « chemical transduction Electro « electrical transduction Electromagnetic radiation (1) « electromagnetic radiation (2) transduction

AXIOM 5: More hydrophobic domains make more efficient the energy conversions involving polar constituents undergoing conversion between more and less hydrophobic functional states. This is the efficiency axiom. (Poising or Biasing)

association, DGHA. Because of this and all that has been demonstrated by simply controlling hydrophobic association, your author believes DGHA to be the underlying thermodynamic quantity that dictates protein function. Accordingly, a proper derivation of the quantity, an analysis of its component parts, and reali-

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zation of the range of experimental variables contributing to DGHA are central to understanding bioproduction, purification and engineering of protein-based polymers. By way of example, every change in DGHA of Tables 7.3 and 7.4, below, provides a unique opportunity for purification and engineering. The treatment begins with the derived statement of the change in Gibbs free energy attending a phase transition, rewrites the statement with respect to inverse temperature transitions, and uses differential scanning calorimetry to obtain the necessary heats and temperatures of the transition for calculation of DGHA. The first useful result of the treatment is the DG8HA-based hydrophobicity scale for the amino acid residues and where relevant in each of their uncharged and ionized states. The next step brings in prosthetic groups and chemical modifications of side chains relevant to biological systems. Finally, the effects of solvent-based variables on DGHA are noted. All of these results guide bioproduction and engineering.

7.5.2.1 The Change in Gibbs Free Energy Attending a Phase Transition, dDGt(v) [3] The relationship that the chemical potential for molecules undergoing a phase transition is the same in solution as in the phase separated state means that the heat of the transition, DHt, equals TtDSt, which is then written for both a reference state, ref, and a state resulting from the change in a variable, v. Suitable identification of the change in the heat, dDHt, and entropy, dDSt, of the transition as the result of v allows the statement [3] that,

dDGt v dDHt v

Tt vdDSt v

1

7.5.2.2 The DGHA-based Hydrophobicity Scale for Amino Acid Residues [3] Rewriting Eq. (1) for the endothermic inverse temperature transition allows that dDGt(v) = DGHA(v). When the variable is the amino acid residue, X, within the pentamer, dDGt(v) = DGHA(GXGVP) where DGHA is written per mole GXGVP. If the glycine residue (Gly, G) is taken as the zero reference, DG8HA(GGGVP) = 0. Thus, at the temperature of the inverse temperature transition, Tt(GXGVP), we can write:

DGHA GXGVP DH t GGGVP

DH t GXGVP

2

Using the basis set of protein-based polymers, poly[ fX(GXGVP),fV(GVGVP)], where fX and fV are mole fractions with fX + fV = 1, the experimental values of Tt and DHt are determined for values of fX and linearly extrapolated to fX = 1. At fX = 1, these reference values are indicated by the bold faced quantities, Tt and DHt. The resulting DG8HA-based hydrophobicity scale for the naturally occurring amino acid residues is given in Table 7.3 for the experimental conditions of 40 mg mL–1, mw & 100 kDa in 0.15 m NaCl, 0.01 m phosphate.

7.5 Mechanistic Foundations for Engineering Protein-based Polymers Table 7.3 Hydrophobicity Scale in terms of DG8HA , the change in Gibbs free energy of hydrophobic association for amino acid residues. b

Residue X

Tt (8C)

DG8HA(GXGVP) kcal mol–1 pentamer

W F Y H8 L I V M H+ C E8 P A T D8 K8 N G S R Q Y D K+ E S-Pi

Trp Phe Tyr His8 Leu Ile Val Met His+ Cys Glu(COOH) Pro Ala Thr Asp(COOH) Lys(NH2) Asn Gly Ser Arg Gln Tyr(}-O–) Asp(COO–) Lys(NH+3 ) Glu(COO–) Ser(PO2– 4 )

–105 –45 –75 –10 (Tt) 5 10 26 15 30 (Tt) 30 (Tt) 20 (2) 40 50 60 40 40 (38) 50 55 60 60 (Tt) 70 140 170 (Tt) (104) (218) 860 (Tt)

–7.00 –6.15 –5.85 –4.80 (from graph) –4.05 –3.65 –2.50 –1.50 –1.90 (from graph) –1.90 (from graph) –1.30 (–1.50) –1.10 –0.75 –0.60 –0.40 –0.05 (–0.60) –0.05 0.00 +0.55 +0.80 (from graph) +0.75 +1.95 *+3.4 (from graph) (+2.94) (+3.72) *+8.0 (from graph)

Data in parentheses utilized microbial preparations of poly(30-mers), e.g. (GVGVP GVGVP GXGVP GVGVP GVGVP GVGVP)n, with n & 40. The notation (from graph) indicates that the value of Tt was used to obtain DG8HA(GXGVP) from the experimental sigmoid curve of Tt versus DG8HA of Ref. [3]. Table adapted from Ref. [3].

7.5.2.3 DG8HA-based Hydrophobicity Scale of Prosthetic Groups, etc. For this set of comparative data, the modification is carried out by addition of the new chemical group to the side chain of a functional amino acid residue and indicated by enclosure in brackets. Using the example of a functional glutamic acid residue (Glu, E), DG8HA may be defined for addition of reduced nicotinamide adenine dinucleotide (NADH) by amide linkage as DG8HA(GK{NADH}GVP). In this way the basis is exactly the same as for the DG8HA-based hydrophobicity scale for amino acid residues of Table 7.3.

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Included in Table 7.4 are reduced and different oxidative states of N-methylnicotinamide (NMeN), oxidized and reduced nicotinamide adenine dinucleotide (NAD and NADH), oxidized and reduced flavin adenine dinucleotide (FAD and FADH2), adenosine monophosphate (AMP), sulfated serine, threonine, and tyrosine (Ser{–O–SO3H}, Thr{–O–SO3H}, and Tyr{–O–SO3H}, respectively), nitrated tyrosine (Tyr{–O–NO–2}), and finally phosphorylated serine (Ser{PO2– 4 }). Table 7.4 shows large effects of changing the oxidative states of the most common redox groups of biology and of changing the state of phosphorylation. The DGHA on reduction of FAD is about two-thirds that of NAD as might be expected for their roles in oxidative phosphorylation. On inclusion of the effects of salt, solvent and ligands on DGHA , one gains a comprehensive understandTable 7.4 DG8HA -based Hydrophobicity Scale (preliminary Tt and DG8HA values) to include chemical modifications and prosthetic groups of proteinsa). Residue X

DGHA (kcal mol1)

Lys{dihydro NmeN}b, d) Glu{NADH}c) Lys{6-OH tetrahydro NMeN}b, d) Glu{FADH2} Glu{AMP} Ser{–O–SO3H} Thr{–O–SO3H} Glu{NAD}c) Lys{NMeN,oxidized}b, d) Glu{FAD} Tyr{–O– SO3H}e) Tyr{–O–NO2–}f) g) Ser{PO2– 4 }

7.0 5.5 3.0 2.5 +1.0 +1.5 +2.0 +2.0 +2.0 +2.0 +2.5 +3.5 +8.0

a) b)

c)

d)

e) f) g)

The usual conditions are for 40 mg mL–1 polymer, 0.15 m NaCl and 0.01 m phosphate at pH 7.4 NMeN is for N-methylnicotinamide pendant on a lysyl side chain, i.e. N-methylnicotinate attached by amide linkage to the –NH2 of Lys and the most hydrophobic reduced state is N-methyl-1,6-dihydronicotinamide (dihydro NMeN), and the second reduced state is N-methyl-6-OH-1,4,5,6-tetrahydronicotinamide or (6-OH tetrahydro-NMeN) For the oxidized and reduced nicotinamide adenine dinucleotides, the conditions were 2.5 mg mL–1 polymer, 0.2 m sodium bicarbonate buffer at pH 9.2 For the oxidized and reduced N-methylnicotinamide, the conditions were 5.0 mg mL–1 polymer, 0.1 m potassium bicarbonate buffer at pH 9.5, 0.1 m potassium chloride The pKa of polymer bound –O–SO3H is 8.2 The pKa of Tyr{–O–NO2} is 7.2 Gross estimates of DG8HA using the Tt values in the right column in combination with the Tt versus DG8HA values from sigmoid curve of Tt versus DG8HA. Adapted from Ref. [1].

[g

Tt at fX = 1 (8C) 130 30 15 25 70 80 100 120 120 120 140 220 860

7.5 Mechanistic Foundations for Engineering Protein-based Polymers

ing of the hydrophobic effect in biology, a universal foundation for protein and protein-based polymer function.

7.5.2.4 Comprehensive Hydrophobic Effect: DGHA Responds to all Variables Tables of the effect of salts, solvents and additional ligands on DGHA are available [1], but space limitations do not permit their inclusion here. Even so, it is important to make the fundamental point here. The significance and general relevance of DGHA gains from consideration of the following perspective of Gregorio Weber [35]: “A complete description of the energetics of hemoglobin, or of any other oligomeric protein of similar size and complexity, is well nigh impossible. It would involve not only the determination of the energetic couplings of any number of ligands with each other and with the subunit interactions but also the variations of these quantities with pH, temperature, and pressure.” Indeed, the unifying result of the comprehensive hydrophobic effect comes from finding that virtually every variable of interest in protein and protein-based polymer function brings about, and is responsive to, a change in DGHA [1]. Thus, the solution to the problem is no longer “well nigh impossible,” but rather the solution simplifies due to the common denominator of the hydrophobic consilient mechanism.

7.5.2.5 The Apolar–Polar Repulsive Free Energy of Hydration, DGap The apolar–polar repulsive free energy of hydration can be seen in Table 7.3 in terms of the change in DGHA on ionization of the Glu carboxylate of 5.22 kcal mol–1 (GEGVP). The DGap repulsion is also measured from the shift in pKa on increasing hydrophobicity where DGap = 2.3RTDpKa [4]. Whether determined from the heat and temperature of the transition or from a titration curve, DGap is the same. The repulsion that arises out of a competition for hydration between apolar and polar groups provides the basis for function. 7.5.3 The Coupling of Hydrophobic and Elastic Mechanisms

A brief description of the elastic mechanism occurs above in Sections 7.5.1 and 7.5.2. The coupling of hydrophobic and elastic mechanisms can occur in several ways. In the elastic contractile model proteins for energy conversion and in the myosin II motor of muscle contraction an energy input causes hydrophobic associations that stretch interconnecting chain segments [1, 21]. The stretched chains retract (i.e., contract) to perform mechanical work in a smooth, efficient conversion of the input energy into the work output of producing motion. In the ATP synthase example, proton chemical energy is transduced into the chemical energy of ADP phosphorylation, but it occurs in two steps. Again, chemically driven hydrophobic association produces (rotary) motion with elastic deformation that in turn uses apolar–polar repulsion to produce ATP. Thus, motion involving

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elastic deformation is central to this energy conversion even though motion is neither the initial input energy nor the final output energy [1].

7.6 Examples of Applications

Examples of applications are literally endless. Many are addressed in Chapter 9 of Ref. [1]. Here, mention will be made of applications in each of the wide-ranging medical areas of soft tissue restoration (3) and drug delivery (2). In areas with both medical and nonmedical utility are biodegradable thermoplastics that can be programmed for rate of degradation, fibers with improved elastic moduli and strength and sound absorption. 7.6.1 Soft Tissue Restoration

Soft tissue restoration can be considered in terms of three categories: (1) prevention of post-surgical adhesions to restore the tissues to a near normal state, (2) soft tissue augmentation, for example, to support the urinary bladder and improve body contours, and (3) soft tissue reconstruction where elastic–contractile materials function as temporary functional scaffoldings that induce normal cells of the tissue to remodel into a natural tissue.

7.6.1.1 Prevention of Post-surgical Adhesions The application for which the most work has been completed is the prevention of post-surgical adhesions. This work has been performed in three separate animal model studies: (1) the bloodied and feces-contaminated abdominal cavity of the rat [36], (2) the strabismus surgery model of the rabbit [37], and (3) the spinal surgery (post-laminectomy) model in the rabbit [38] and goat. In each case emplacement of crosslinked matrices and gels of (GVGVP)251 or of (GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP)36(GVGVP) prevented adhesions, and when purified to the highest level, the host exhibited no reaction whatever to the protein-based polymers. Once large-scale purification of medical grade polymers is in place, this application should move quickly to commercialization.

7.6.1.2 Soft Tissue Augmentation One approach to soft tissue augmentation is to introduce an inert material that would occupy the desired volume and stay in place. This could be achieved as shown above in Fig. 7.7 B. An approach uniquely possible with protein-based polymers is to include in the sequence a bioactive peptide such as GRGDSP. This peptide acts both as a chemoattractant [39] and a cell attachment sequence


[40]. When injected subcutaneously in the guinea-pig, it causes a generation of natural tissue with angiogenesis, as shown in Fig. 7.7 C [32]. This shows the diversity of responses to simple changes in protein-based polymer composition. With just one Glu residue in six GVGVPs, the injected sample disperses in a few days without a trace (Fig. 7.7 A). In the absence of the Glu residue, (GVGVP)251 forms a large inclusion body the size of the injected bolus (Fig. 7.7 B). On replacing the Glu used in Fig. 7.7 A with GRGDSP, the generation of natural tissue of Fig. 7.7 C occurs. These polymers exhibit remarkable versatility.

7.6.1.3 Soft Tissue Reconstruction: The Concept of Temporary Functional Scaffoldings The concept of a temporary functional scaffolding derives from two principal factors. The initial enabling feature is the capacity to prepare biocompatible elastic matrices of protein-based polymers that match the compliance of the tissue to be replaced and contain attachment sequences for the natural cells of the tissue. Second is the important feature that tissue cells function as mechanochemical transducers. They attach to the extracellular matrix, sense the changing tensional forces, and remodel the tissue continually in order to sustain the sensed forces. Important tissues that have been considered in this context are vascular wall, intervertebral discs and urinary bladder. Consideration of the latter is given in Fig. 7.8 where the dynamic changes chosen to simulate the filling and emptying of the urinary bladder did indeed stimulate cellular proliferation and extracellular matrix elaboration. Elastic protein-based polymers make a temporary functional scaffolding approach possible.

Fig. 7.8 Outgrowth of human uroepithelial cells from a ureteral explant (dark area) onto a crosslinked elastic matrix of {(GVGIP)10– GVGVPGRGDSP–(GVGVP)10}12(GVGVP). The elastic matrix, with an elastic modulus that closely matches that of the human urinary bladder, is placed within a chamber

designed to simulate bladder filling and emptying. A. The static case where good outgrowth is seen. B. The dynamic case of filling in three hours and emptying in 25 s shows enhanced cellular proliferation and much greater development of extracellular matrix. Reproduced from reference [41].

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7.6.2 Controlled Release Devices for Amphiphilic Drugs and Therapeutics

The capacity to use elastic protein-based polymers as controlled release devices is as diverse as the energy conversions of Table 7.2 and as expansive as the many advantages of Table 7.1. The extraordinary biocompatibility of elastic protein-based polymers amplifies the opportunity, for example, due to the possibility of no fibrous capsule formation in vivo that can defeat the competence of the drug release device. Here, we note two examples of the uniqueness of elastic protein-based polymers in light of the mechanistic foundations. One is the utilization of the apolar–polar repulsive free energy, DGap, in the design of families of polymers that can give a predetermined set of near constant release rates. Second is the advantage of an elastic cushion for controlled release patches to prevent pressure ulcer formation.

7.6.2.1 The Use of DGap in the Design of Controlled-release Devices Protein-based polymer designs utilize charged side chains with methodical increases in hydrophobicity, commonly by replacing, in each six pentamer repeat, 2, 3, 4, and then 5 valine (Val, V) residues by much more hydrophobic phenylalanine (Phe, F) residues. The result is a systematic shift in pKa values that are quantifiable by means of DGap = 2.3RTDpKa. Generally, the polymer, with one ionized functional group per 30-mer (5 ´ 6 pentamers), is soluble in solution. Addition of an amphiphilic pharmaceutical of the opposite charge lowers the value of Tt below the operating temperature, and the pharmaceutical effects phase separation to form the loaded drug-delivery vehicle. The level of pharmaceutical release depends on the DGap value of the polymer’s functional group. For a constant surface area zero order release occurs, and the drug delivery vehicle disperses at the same rate as the drug. The loading and zero-order release of positively charged drugs and therapeutics, using negatively charged protein-based polymers, has been demonstrated for Leu-enkephalin amide [41] and Naltrexone (a narcotic antagonist) [1]. All cationic anesthetics, analgesics, and protein could be released in this remarkably controlled manner using the DGap approach. Analogously, using positively charged protein-based polymers, the loading and controlled release of negatively charged drugs and therapeutics, such as dexamethasone phosphate and betamethasone phosphate (anti-inflammatory glucocorticoids) [42], anti-sense oligonucleotides [43], including genes and proteins with net negative charge could be released in this manner [1].

7.6.2.2 Prevention of Pressure Ulcers by Means of Elastic Patches for Drug Delivery Pressure ulcers appear over bony prominences where the pressure leads to skin necrosis and ulcers. In the Swaim coaptation cast model for the dog model, even the elastic patch (Fig. 7.9) made of crosslinked (GVGIP)260 placed over the bony


Fig. 7.9 A. Disk-shaped patches of crosslinked (GVGIP)260 contain Dazmegrel from left to right of 0, 0.1, 1.0, and 10 mg cm–2. B. The two polymers, (GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP)36–(GVGVP) and (GVGVP GVGVP GKGVP GVGVP GVGVP GVGVP)22(GVGVP), were crosslinked by amide linkage to form a fiber with an elastic modulus of 3.86 ´ 106 Pa, a break stress of 1.3 ´ 106 Pa and a break strain of

350%. C. The two polymers, (GVGVPGVG FPGEGFPGVGV PGVGVPGVGV P)40– (GVGVP) and (GVGVP GVGFP GKGFP GVGVP GVGVP GVGVP)22(GVGVP), were similarly treated to obtain fibers with an elastic modulus of 6.1 ´ 106 Pa, a break stress of 6.9 ´ 106 Pa and a break strain of 280%. The bar indicates 100 lm. B and C reproduced from Ref. [45].

prominence pressure point without drug, appeared to have efficacy in preventing pressure ulcer formation. When loaded with dazmegrel (a thromboxane synthetase inhibitor), efficacy appeared to be complete as long as the patch did not split or tear [44]. The patches split 25% of the time at shear pressure points. Tougher patches have since been made exhibiting twice the essential work of fracture [45]. 7.6.3 Fibers of Improved Elastic Moduli and Break Stresses and Strains

The carboxyl and amine functions in the polymers of Fig. 7.9 exhibit hydrophobic-induced pKa shifts. Formation of the –COO–···+H3N ion pairs on mixing of the polymers reduces Tt and DGHA from the values of the individual polymers. This aligns the polymers for chemical crosslinking and results in improved elastic moduli and break stress values by one to two orders of magnitude (see legend of Fig. 7.9). 7.6.4 Programmably Biodegradable Thermoplastics

Thermoplastics derive from the designed repeat pentamer, (AVGVP)n, that exhibits an inverse temperature transition, but to a plastic rather than elastic-like state [46]. When the polymer is made more hydrophobic, the dry polymer melts and can be pulled into the fibers of Fig. 7.3 A. Also, introduction of asparagine residues allows for timed breakdown to the carboxylate of aspartic acid. This raises the value of Tt, and in water the plastic surface swells to become biodegradable. Since life cannot exist at the 160 8C required to melt the plastic, we have designed protein-based polymers for functions that go beyond what nature has had reason to evolve. Nonetheless, society can find uses for such environmentally friendly plastics.

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7.6.5 Acoustic Absorption

To the best of our knowledge, elastic protein-based polymer (GVGIP)320 has an intense mechanical resonance in the acoustic absorption range with an absorption intensity that is an order of magnitude greater than the usually considered sound-absorbing petroleum-based elastomers. Once the relevant “idol of the past” [47] abates, this material can be expected in numerous sound absorption applications.

7.7 Outlook and Perspectives 7.7.1 List of Gene Constructions and Expressed Protein-based Polymers

Appropriate in considering the list of Table 7.5 is a quotation due to Jacob Bronowski “In effect, the modern problem is no longer to design a structure from the materials (available) but to design materials for a structure” [48]. Each of the gene constructs of Table 7.5 was made for a specific purpose, either for elucidating an understanding of mechanism or for a specific application or for both. Indeed, no longer is it required to coerce, by physical manipulation, many structure/function uses out of a single composition of matter. Now, one can design a specific polymer sequence to be near optimal for the particular application and then use physical manipulation in the role of fine-tuning the product for its designed application. This is the power of protein-based polymeric materials. Each transformed E. coli, below, becomes the factory for producing, with extraordinary fidelity, the specific designed protein-based polymer. 7.7.2 Efforts Toward Low-cost Production in other Microbes and in Plants

Bioproduction of protein-based polymers has been under consideration using tobacco [49, 50], mushrooms [51], yeast [52], and the seeds of arabidopsis, canola, and soy [53]. Early optimism that cost of production by E. coli could reach levels of less than $ 10.00 kg–1 has yet to be realized. Production by yeast, as proposed by Casal [52], gains from the capacity for protein-based polymer to be excreted from the yeast cell, where harvesting could proceed without cell destruction, and purification would not be as demanding as described above for E. coli [52]. The promise of truly low-cost production appears to reside with plants. One attractive approach, considered by Somers [53], would be to express proteinbased polymer in seed as a value-added product under circumstances where the cost of production would be the cost of purification. For example, production in the canola seed would occur with retention of the canola oil product, and the

7.7 Outlook and Perspectives Table 7.5 List of gene constructions and expressed proteinbased polymers of Bioelastics Research. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

{(GVGVP)10}n(GVGVP): n = 25*, 14*, 12*, 4* (GVGVP GVGFP GHGFP GVGVP GVGFP GFGFP)n(GVGVP): n = 25*, 15, 12, 9, 8, 7, 5, 3 (GVGVP GVGFP GKGFP GVGVP GVGFP GFGFP)n(GVGVP): n = 75*, 41*, 33*, 22*, 15, 12, 6*, 5, 2 (GVGVP GVGFP GEGFP GVGVP GVGFP GFGFP)n(GVGVP): n = 42*, 32*, 17*, 14*, 8*, 6*, 4*, 3*, 2* (GVGVP GVGFP GDGFP GVGVP GVGFP GFGFP)n(GVGVP): n = 12*, 8*, 7, 6, 4 (GVGVP GVGFP GEGFP GVGVP GVGFP GKGVP)n(GVGVP): n = 40, 21*, 20, 11*, 10, 8, 7 (GVGVP GVGKP GEGFP GVGVP GVGFP GFGVP)n(GVGVP): n = 48, 39*, 26, 22*, 21, 20, 17, 15, 12, 9, 7 (GVGVP GVGFP GEGFP GVGVP GVGVP GKGVP)n(GVGVP): n = 22*, 20, 18, 15, 13, 11, 10, 9, 8, 7, 6, 5, 4 (GVGVP GVGKP GEGFP GVGVP GVGVP GFGVP)n(GVGVP): n = 38, 22*, 15, 6 {(AVGVP)10}n(GVGVP): n = 29*, 7*, 4*, 2 {(GVGIP)10}n(GVGVP): n = 26*, 15*, 13*, 7*, 4* (GVGIP GFGEP GEGFP GVGVP GFGFP GFGIP GVGIP GFGEP GEGFP GVGVP GFGFP GFGIP)n(GVGVP): n = 30*, 24, 20*, 13*, 11, 10, 6, 5 (GVGIP GFGEP GEGFP GVGVP GFGFP GFGIP GVGIP GFGEP GEGFP GVGVP GFGFP GFGIP GVGVP GVGRGYSLG VP)n(GVGVP): n = 20* (GVGVP GVGFP GEGFP GVGVP GVGFP GVGFP)n(GVGVP): n = 41*, 29, 15*, 12, 9, 7, 6, 3 (GVGVP GVGVP GEGVP GVGVP GVGFP GFGFP)n(GVGVP): n = 60*, 39*, 24*, 15 (GVGVP GVGFP GEGFP GVGVP GVGVP GVGVP)n(GVGVP): n = 40*, 25, 16, 14, 10, 8, 7, 6, 5, 4, 3, 2 (GVGVP GVGFP GKGFP GVGVP GVGFP GVGFP)n(GVGVP): n = 21*, 12, 6 (GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)n(GVGVP): n = 45*, 22*, 20, 15, 14*, 12*, 7 (GVGVP GVGFP GKGFP GVGVP GVGVP GVGVP)n(GVGVP): n = 26*, 22*, 17*, 16, 14*, 13, 11, 10, 9 {(GVGIP)10–GVGVPGRGDSP–(GVGIP)10}n(GVGVP): n = 21, 18*, 11* {(GVGVP)10–GVGVPGRGDSP–(GVGVP)10}n(GVGVP): n = 18*, 15*, 8* {(GVGIP)10–GVGVPGRGDSP–(GVGVP)10}n(GVGVP): n = 12*, 8* {(GVGIP)10–GVGRGYSLGIP–(GVGIP)10}n: n = 10*, 4* [{(GGVP)3(GGFP)}3]n: n = 27, 22 GKGKAPGK–{(GVGVP)10}n: n = 10* GKGKAPGK–{(AVGVP)10}n: n = 20*, 5* {(GVGVP)10(GVGVAP)8(GVGVP)10}n: n = 8*, 5*, 2, 1 {(AFGFPAEGFP)5}n(GVGVP): n = 53, 22, 18, 16*, 14, 6 {(GVGFPGEGFPGFGVP)3}n(GVGVP): n = 30*, 14*, 12 {(GVGVP)10–GVGVPGNGVP(GVGVP)10}n(GVGVP): n = 10*, 5* {(GVGIP)10–GVGIPGNGIP}n(GVGVP): n = 30*, 40* {GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP}n(GVGVP)YGSEFELRRQACGRTRAPP–PPPLPSGC: n = 44*, 36* {GVGVP GVGVP GKGVP GVGVP GVGVP GVGVP}n(GVGVP): n = 55*, 36*, 22* {(FEGFPAEGFP)5}n(GVGVP): n = 19* {(GVGVP)10–GVGRGYSLGIP–(GVGVP)10)}n: n = 12*, 7*

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7 Protein-based Polymers: Mechanistic Foundations for Bioproduction and Engineering Table 7.5 (continued) 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78

{(GVGVP)10–GVGRGYSLGIP–(GVGIP)10)}n: n = 7* {(GVGIP)10–FPGVGQKRPSKRSKYLIP–(GVGIP)10)}n: n = 11*, 6* {(GVGVP)10–FPGVGQKRPSKRSKYLIP–(GVGVP)10}n(GVGVP): n = 9*, 6* {(GVGVP)10–FPGVGQKRPSKRSKYLIP–(GVGIP)10}n(GVGVP): n = 9*, 7*, 5* GKGKAPGK–(GVGVP GVGVP GEGVP GVGVPGVGVP GVGVP}n(GVGVP): n = 1 {(FEGVP)10}n: n = 28*, 15*, 11, 4 {(GVGVP)10–GVGVPGRGDSP–(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2}n(GVGVP): n = 8*, 5 GKGKAPGK–{(GVGVP)10–GVGVPGRGDSP–(GVGVP)10}n: n = 7* (FEGFP AFGFP)5: n = 33, 29, 26, 25, 23, 19, 18, 16, 14, 11, 6 {(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2–GVGVPGRGDSP–(GVGVP)10 (GVGVAP)8}n: n = 11*, 8*, 4*, 3* {(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2– (GVGVP)10(GVGVAP)8}n: n = 12*, 8*, 4* {(GVGIP)11–GYGIP–(GVGIP)10}2(GVGIP):Rea): n = 11*, 10, 9, 8, 7, 6*, 5 {(FVGEP FVGFP)5}n: n = 18, 17, 12, 2, 1 (GIGVP GAGVP GIGVP GIGVP GAGVP GIGVP)n: n =22*, 11* {(GVGIP)11–GYGIP}n(GVGIP):Rea): n = 18*, 17, 16, 15, 14, 13, 12, 11*, 6, 3 {(GVGVP)10}n(GVGVP): Rea): n = 28, 26, 18, 17*, 16, 15, 14,13*, 12, 11, 6, 4 {(GVGIP)10}n(GVGVP): Rea): n = 32*, 25*, 21*, 15*, 9*, 6*, 4*, 2* [{(GVGIP GEGIP GVGIP)2}2]n: n = 20*, 9* [{GVGIP GEGIP (GVGIP)4}2]n: n = 23*, 10* [(GVGIP GEGIP GVGIP GEGIP GVGIP GVGIP)2]n: n = 26*, 17*, 15* [{(GVGIP GKGIP GVGIP)2}2]n: n = 19, 18, 17, 12 [{GVGIP GKGIP (GVGIP)4}2]n: n = 21*, 12* [(GVGIP GKGIP GVGIP GKGIP GVGIP GVGIP)2]n: n = 73, 41, 21, 17, 14, 11 [(GVGIP)8(GYGIP]n: n = 32*, 24*, 18* [{(GVGIP)5GYGIP}2]n: n = 20*, 12*, 11* [{(GVGIP)2GYGIP}3]n: n = 26, 15*, 14 {(GVGIP)10}n1–c)Fn3–{(GVGIP)10}n2: n1 = 16 and n2 = 6 {GVGVP GVGVP GEGVP GVGVP GVGVP GVGVP}n(GVGVP): n = 42*, 23*, 22*, 18*, 16* AKKKKKKG–{(GVGIP)10}n–VCCC: n = 15* AKKKKKKG–{(GVGIP)10}n–VCCC: n = 18* GKGKAPGK–{(GGAP)12}n(GVGVP): n = 1 { (GGAP)12}n: n = 1 {(FVGVP FEGVP)5}n: n = 1 {[(GVGVP)2–GFGVP]3}n: n = 1 {(GVGIP)11–GSGIP–(GVGIP)10}n(GVGIP):Rea): n = 1 {(GVGIP)11–GTGIP–(GVGIP)10}n(GVGIP):Rea): n = 1 {(FFGEP)10}n: n = 1 {(GVGIP)11–GSGIP}n(GVGIP):Rea): n = 1 {(GVGIP)11–GTGIP}n(GVGIP):Rea): n = 1 {(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2–GVGVPGRGDSP–(GVGVP GVGVP GKGVP GVGVP GVGFP GFGFP)2}n: n = 1 (GIGVP GAGVP GKGVP GIGVP GAGVP GIGVP)n: n = 1 (GIGVP GAGFP GKGFP GIGVP GAGVP GIGVP)n: n = 1 (GIGVP GAGFP GKGFP GIGVP GAGFP GVGFP)n: n = 1

7.8 Patents Table 7.5 (continued) 79

*) a)

b)

c)

{ACPGCGGVGIPCPGCG–[(GVGIP)26–b)Fn3(RGYSLG)–(GVGIP)6] CPGCGGVGIPCPGCG}n: n = 1 Indicates that the protein-based polymer was expressed Re: Indicates that the gene was made explicitly using the same codon for a repeating residue in the sequence and then a different codon was used in the next repeat Fn3(RGYSLG) stands for the tenth type III domain of fibronectin in which the cell attachment sequence, GRGDSP, was replaced by the kinase site, RGYSLG, as a site for phosphorylation Fn3 stands for the tenth type III domain of fibronectin. Adapted from Ref. [1]

protein-based polymer would be in the protein meal byproduct, currently used for cattle feed at costs of less than a dollar per pound. Water-based purification, using the inverse temperature transition, would allow selected removal of the protein-based polymer and return the remaining protein for cattle feed. In short, the time can be anticipated when the cost of production of protein-based polymers will compete favorably with that of petroleum-based polymers. Accordingly, the outlook is promising for protein-based polymers to make a significant impact in the marketplace of the near future.

7.8 Patents 7.8.1 Patents of D. W. Urry on Protein-based Polymers

A list of your author’s patents exclusively on uses of protein-based polymers is given in Table 7.6. There are many additional patent drafts and concepts for patents that have yet to be drafted. These will be pursued as finances allow. In addition, there are enabling trade secrets that have been maintained. 7.8.2 Result of Ex Parte Patent Reexamination Request to the USPTO

Our routine utilization of GVGVP, or any of the five permutations of this sequence, in development of the mechanistic foundations and the applications considered above is apparent. As noted above in Section 7.2, the efforts, specifically using GVGVP and VPGVG, date back more than three decades. Nonetheless, a patent issued in 1993 that claimed the use of recombinant DNA technology to produce GVGVP and VPGVG. Once the patent appeared collaboration/license agreements between Bioelastics Research (BRL), the entity that held the

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7 Protein-based Polymers: Mechanistic Foundations for Bioproduction and Engineering Table 7.6 D. W. Urry patents on protein-based polymers. Country

Patent no.

Date filed

Date issued

1 Inventors: D. W. Urry and K. Okamoto Title: Synthetic Elastomeric Insoluble Crosslinked Polypentapeptide USA 4,132,746 07/09/76 01/02/79 Canada 1,103,238 02/26/79 06/16/81 2 Inventors: D. W. Urry and K. Okamoto Title: Synthetic Elastomeric Insoluble Crosslinked Polypentapeptide USA 4,187,852 08/14/78 02/12/80 3 Inventor: D. W. Urry Title: Elastomeric Composite Material Comprising a Polypeptide USA 4,474,851 10/02/81 10/02/84 4 Inventor: D. W. Urry Title: Elastomeric Material Comprising a Polypentapeptide of Opposite Chirality in Position Three USA 4,500,700 12/23/82 02/19/85 5 Inventor: D. W. Urry Title: Enzymatically Crosslinked Bioelastomers USA 4,589,882 09/19/83 05/20/86 6 Inventors: D. W. Urry and R. M. Senior Title: Stimulation of Chemotaxis by Chemotactic Peptides (Hexapeptide) USA 4,605,413 09/19/83 08/12/86 7 Inventors: D. W. Urry and M. M. Long Title: Stimulation of Chemotaxis by Chemotactic Peptides (Nonapeptide) USA 4,693,718 10/31/85 09/15/87 8 Inventors: D. W. Urry and K. U. M. Prasad Title: Temperature Correlated Force and Structure Development of Elastin Polytetra and penta USA 4,783,523 08/27/86 11/08/88 Europe EP 0,321,496 03/30/94 Japan 2,726,420 08/27/87 12/05/97 9 Inventors: D. W. Urry and K. U. M. Prasad Title: Segmented Polypeptide Bioelastomers to Modulate Elastic Modulus USA 4,870,055 04/08/88 09/26/89 Japan 2,085,090 04/17/87 08/23/96 10 Inventor: D. W. Urry Title: Bioelastomer Containing Tetra/Pentapeptide Units USA 4,898,926 06/15/87 02/06/90 Europe 06/13/88 11 Inventor: D. W. Urry Title: Reversible Mechanochemical Engines Comprised of Bioelastomers Capable of Interconversion of Chemical and Mechanical Work USA 5,032,271 06/15/87 07/16/91 Europe EP 0,425,491 06/13/88 07/20/94 12 Inventor: D. W. Urry Title: Reversible Mechanochemical Engines Comprised of Bioelastomers Capable of Interconversion of Chemical and Mechanical Work USA 5,085,055 04/30/91 02/04/92

7.8 Patents Table 7.6 (continued). Country

Patent no.

Date filed

Date issued

13 Inventor: D. W. Urry Title: Reversible Mechanochemical Engines Comprised of Bioelastomers Capable of Interconversion of Chemical and Mechanical Work USA 5,255,518 12/24/91 10/26/93 14 Inventors: D. W. Urry and M. M. Long Title: Stimulation of Chemotaxis by Chemotactic Peptides USA 4,976,734 09/19/87 12/11/90 Europe EP 0,366,777 06/13/88 07/20/94 15 Inventor: D. W. Urry Title: Bioelastomeric Materials Suitable for burn areas or the Protection from Adhesions USA 5,250,516 04/21/88 10/05/93 Europe EP 0,365,655 09/21/94 Japan 2,820,750 04/14/89 08/28/98 16 Inventor: D. W. Urry Title: Elastomeric Polypeptides as Vascular Prosthetic Materials USA 5,336,256 04/22/88 08/09/94 Europe EP 0,365,654 01/12/94 Japan 2,115,962 04/14/89 12/06/96 17 Inventor: D. W. Urry Title: Polynonapeptide Bioelastomers Having an Increased Elastic Modulus USA 5,064,430 02/23/89 11/12/91 18 Inventor: D. W. Urry Title: Polymers Capable of Baromechanical and Barochemical Transduction USA 5,226,292 04/22/91 07/13/93 19 Inventor: D. W. Urry Title: Superabsorbent Materials and Uses Thereof USA 5,393,602 04/19/91 02/28/95 Europe EP 0,580,811 03/10/93 08/04/99 Japan 4,510,189 03/10/92 07/26/02 20 Inventor: D. W. Urry Title: Superabsorbent Materials and Uses Thereof USA 5,520,672 02/06/95 05/28/96 21 Inventor: D. W. Urry Title: Elastomeric Polypeptide Matrices for Preventing Adhesion of Biological Materials USA 5,527,610 05/20/94 06/18/96 Japan 05/20/95 22 Inventor: D. W. Urry Title: Elastomeric Polypeptide Matrices for Preventing Adhesion of Biological Materials USA 5,519,004 06/07/95 05/21/96 23 Inventor: D. W. Urry Title: Bioelastomeric Drug Delivery System USA 6,328,996 B1 10/03/94 12/11/01 Europe EP 0,449,592 11/30/94 Japan 1,994,740 11/22/95

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7 Protein-based Polymers: Mechanistic Foundations for Bioproduction and Engineering Table 7.6 (continued). Country

Patent no.

Date filed

Date issued

24 Inventors: D. W. Urry, Peter Shewry and U. Prasad Kari Title: Bioelastomers Suitable as Food Product Additives USA 5,972,406 10/13/95 10/26/99 Europe 96,912,815.6 04/15/96 Pending Japan 8-531,270 04/23/2002 Pending 25 Inventors: D. W. Urry, H. Daniell, D. McPherson and J. Xu Title: Hyperexpression of Bioelastomeric Polypeptides USA 6,004,782 10/13/95 12/21/99 26 Inventor: D. W. Urry Title: Bioelastomers Suitable as Food Product Additives USA 5,900,405 06/07/95 05/04/99 Europe 0,830,509 06/07/96 03/25/98 Japan 9-519,202 08/06/97 Pending 27 Inventors: D. W. Urry, D. McPherson and J. Xu Title: A Simple Method for the Purification of a Bioelastic Polymer USA 5,854,387 10/13/95 12/29/98 Europe 96,912,768.7 04/15/96 Pending Japan 8-531261 04/16/2002 Pending 28 Inventor: D. W. Urry Title: Acoustic Absorption Polymers and Their Methods of Use USA 09,746,371 12/20/00 Pending Europe PCT/US 00/ 7/22/02 Pending 34,658 29 Inventor: D. W. Urry Title: Bioelastomer Nanomachines and Biosensors (NEMS) USA 09/888,260 06/21/01 Pending Europe PCT/US01/20045 06/21/01 Pending 30 Inventor: D. W. Urry Title: Injectable Implants for Tissue Augmentation And Generation USA 09/837,969 04/18/01 In Allowance Canada 2,319,558 02/26/99 Pending 31 Inventor: D. W. Urry Title: Injectable Implants for Tissue Augmentation And Generation USA 09/841,321 04/23/01 Pending Europe 99,908,590.5 02/26/99 Pending Canada 2,319,558 02/26/99 Pending Japan 533,072/2000 02/26/99 Pending 32 Inventors: D. W. Urry, P. Glazer and T. M. Parker Title: Injectable Implants for Tissue Augmentation And Generation (Disk Repair) USA 6,533,819 B1 04/23/01 03/18/03 Table adapted from Ref. [1].

Note added in Proof: Since the completing of the manuscript for this book, I have learned, following my resignation as General Partner of Bioeleaxtec Research Ltd. that the prosecution and maintenance of this patent portfolio did not continue.

References

rights to the patents of Table 6, and major US companies as well as an important Option Agreement with a major foreign entity terminated. In my view, this was largely due to the perceived lack of BRL’s access to bioproduction of this pivotal repeating unit. It was thought that only an interested party, prepared to initiate litigation, would allow BRL access to GVGVP and VPGVG for commercialization. Fortunately, the legal firm for BRL suggested that an Ex Parte Patent Reexamination Request to the US Patent and Trademark Office (USPTO) would provide the opportunity, at relatively low cost, to remove the impairing claims. The submitted Request, which presented the BRL prior art, resulted in the relatively recent rejection of the claims by the Examiner, and the entity that owned the patent containing the offending claims made no effort to overcome the rejections. Accordingly, this major impediment for moving forward with commercialization has been removed. Note added in Proof: On August 2005, just prior to receiving these page proofs quite inexplicably, your author has learned that the Examiner has allowed amended claims to read on the other three permutation of the polypentapeptide, namely VGVPG, GVPGV, and PGVGV. These should be rejected on the same basis as were GVGVP and VPGVG. For example, one can group the pentameric repeats of figures 7.8 and 7.9 to reach as repeats of any of this five permutations.

Acknowledgment

The author gratefully acknowledges support of the Office of Naval Research (ONR) and Program Officers, Michael Marron and Keith Ward, by means of Grant No. N00014-98-1-0656 and Contract No. N00014-00-C-0178.

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C. F. Hendrix and M.M. Long, Biochemistry, 1976, 15, 4083–4089. 13 D. W. Urry, C. M. Venkatachalam, M. M. Long and K. U. Prasad, In Conformation in Biology, (R. Srinivasan and R H. Sarma, Eds.) Adenine Press, USA, 1982, pp. 11–27. 14 D. W. Urry, Methods in Enzymology, 1982, 82, 673–716, (L.W. Cunningham and

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29 D. T. McPherson, J. Xu, and D. W. Urry,

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Protein Expression and Purification, 1996, 7, 51–57. D. W. Urry, A. Nicol, D. T. McPherson, C. M. Harris, T. M. Parker, J. Xu, D. C. Gowda and P. R. Shewry, In Encyclopedic Handbook of Biomaterials and Bioengineering – Part A – Materials, Vol. 2, (D. L. Wise, D. J. Trantolo, D. E. Altobelli, M. J. Yazemski, J. D. Gresser and E. R. Schwartz, Eds.) Marcel Dekker, Inc., New York, 1995, pp. 1619–1673, A. Pattanaik, T. M. Parker and D. W. Urry, unpublished results. D. W. Urry, A. Pattanaik, J. Xu, T. C. Woods, D. T. McPherson and T. M. Parker, J. Biomater. Sci. Polymer Edn., 1998, 9, 1015–1048; Reproduced in Polymers for Tissue Engineering, (M. S. Shoichet and J. A. Hubbell, Eds,) VSP BV, The Netherlands, 1998, pp. 19–52. D. W. Urry, T. M. Parker, M. C. Reid, and D. C. Gowda, J. Bioactive Compatible Polym., 1991, 6, 263–282. D. W. Urry, D. T. McPherson, J. Xu, D. C. Gowda, and T. M. Parker, In Industrial Biotechnological Polymers, (C. Gebelein and C. E. Carraher, Jr., Eds,), Technomic, Lancaster, PA, 1995, pp. 259–281. G. Weber, Protein Interactions, Chapman & Hall, Inc. 1992, p. 104. L. D. Hoban, M. Pierce, J. Quance, I. Hayward, A. McKee, D. C. Gowda, D. W. Urry and T. Williams, J. Surgical Res., 1994, 56, 179–183. F. J. Elsas, D. C. Gowda and D. W. Urry, J. Pediatr. Ophthalmol. Strabismus, 1992, 29, 284–286. R. N. Alkalay, D. H. Kim, D. W. Urry, J. Xu, T. M. Parker, and P. A. Glazer, Spine, 2003, 28, 1659–1665. M. M. Long, V. J. King, K. U. Prasad and D. W. Urry, Biochim. Biophys. Acta, 1987, 928, 114–118. M. D. Pierschbacher and E. Ruoslahti, Nature, 1984, 309, 30–33. D. W. Urry, A. Pattanaik, M. A. Accavitti, C.-X. Luan, D. T. McPherson, J. Xu, D. C. Gowda, T. M. Parker, C. M. Harris, and N. Jing, In Handbook of Biodegradable Polymers, (A. J. Domb, J. Kost and D. M. Wiseman, Eds.) Harwood Aca-

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demic Publishers, Chur, Switzerland, 1997, pp. 367–386. D. W. Urry, T. C. Woods, L. C. Hayes, J. Xu, D. T. McPherson, M. Iwama, M. Furuta, T. Hayashi, M. Murata, and S. M. Parker, In: Tissue Engineering and Novel Delivery Systems, Marcel Dekker, Inc., New York, 2004, pp. 31–54. T. C. Woods, Ph.D. Dissertation of The University of Alabama at Birmingham, 1998. B. Kemppainen, N.-Z. Wang, S. Swaim, D. W. Urry, C.-X. Luan, J. Xu, E. Sartin, R. Gillette, S. Hinkle and S. Coolman, Wound Repair and Regeneration, 2004, 12, 453–460. D. W. Urry, J. Xu, W. Wang, L. Hayes, F. Prochazka, and T. M. Parker, Mat. Res. Soc. Symp. Proc.: Materials Inspired by Biology, 2003, 774, 81–92. D. W. Urry, J. Jaggard, K. U. Prasad, T. Parker, and R. D. Harris, “Poly(Val1–

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Pro2–Ala3–Val4–Gly5): A Reversible, Inverse Thermoplastic,” In Biotechnology and Polymers, (C. G. Gebelein, Ed.), Plenum Press, New York, 1991, 265–274. P. Zagorin, Francis Bacon, 1998, p. 82. J. Bronowski, “The Ascent of Man,” Little, Brown and Company, Boston, 1973, p. 110. X. Zhang, C. Guda, R. Datta, R. Dute, D. W. Urry, and H. Daniell, Letters, 1995, 17, 1279–1284. X. Zhang, D. W. Urry, and H. Daniell, Plant Cell Reports, 1996, 16, 174–179. R. W. Herzog, N. K. Singh, D. W. Urry and H. Daniell, Applied Microbiol. & Biotech., 1997, 47, 368–372. Collaboration with Prof. Margarida Casal, Dept. Biology, University of Minho, Braga, Portugal. Collaboration with Prof. D. A. Somers, Dept. Agronomy and Plant Genetics, University of Minnesota.

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Biobased Fats (Lipids) and Oils

8 New Syntheses with Oils and Fats as Renewable Raw Materials for the Chemical Industry Ursula Biermann, Wolfgang Friedt, Siegmund Lang, Wilfried Lühs, Guido Machmüller, Jürgen O. Metzger, Mark Rüsch gen. Klaas, Hans J. Schäfer, Manfred P. Schneider

This work is dedicated to Professor Siegfried Warwel, Münster, for his important contributions to the development of oleochemistry.

8.1 Introduction

Sustainable development had become the key ideal of the 20th century [1]. In the search for sustainable chemistry, considerable importance is being attached to renewable raw materials which exploit the synthetic capabilities of nature [2, 3]. Oils and fats of vegetable and animal origin make up the greatest proportion of the current consumption of renewable raw materials in the chemical industry, since they offer to chemistry a large number of possibilities for applications which can be rarely met by petrochemistry. The extent of the use of natural oils and fats in chemistry was summarized in 1988 [4]. It stated that “more than 90% of oleochemical reactions have been those occurring at the fatty acid carboxy group, while less than 10% have involved transformations of the alkyl chain. However, future progress will be along the lines of these latter types of reactions with their potential for considerably extending the range of compounds obtainable from oils and fats. Such progress is essential for a growth in the use of oils and fats as renewable raw materials”. For the future, this means that “oils and fats of vegetable and animal origin offer possibilities for providing chemistry with a wealth of reaction products which will be of great value in the future. The chemical possibilities of renewable oils and fats are still very far from being fully exploited. Interdisciplinary collaboration involving chemistry, biochemistry, plant breeding, and agriculture is necessary to extend the successful applications of this technology.” A good example of this is the alkyl polyglycosides, the use and properties of which have been recently reviewed [5]. Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 2 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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8 New Syntheses with Oils and Fats as Renewable Raw Materials for the Chemical Industry

We report here the advances made in the chemistry and biotechnology of fatty materials over the last ten years and include the improvements in natural oils and fats by plant breeding.

8.2 Reactions of Unsaturated Fatty Compounds

By means of simple industrial reactions, fatty materials are available from vegetable oils in such purity that they may be used for further chemical conversions and for the synthesis of chemically pure compounds. Predominantly, oleic acid 1 a and elaidic acid (E)-1 a, petroselinic acid 2 a, erucic acid 3 a, linoleic acid 4 a, and linolenic acid 5 a have been used in the syntheses described below (Fig. 8.1). Ricinoleic acid 6 a carries an additional hydroxyl group which is useful in stereo- and regioselective syntheses. By pyrolysis of 6 b and subsequent hydrolysis, 10-undecenoic acid 7 a, a x-unsaturated carboxylic acid, is obtained [4], which is very useful for selective reactions. Both 9-decenoic acid 8 a and 13-tetradecenoic acid 9 a are accessible by the metathesis reaction of ethylene with oleic acid 1 a and erucic acid 3 a, respectively, thus extending the range of x-unsaturated fatty compounds available (Section 8.2.3). Conjuenic acid 10 a, with conjugated double bonds, is obtained as a regio- and stereoisomeric mixture by the isomerization of linoleic acid 4 a [4]. The alkyne fatty compounds 11–13, with internal or terminal triple bonds, are readily available on a laboratory scale [6]. The epoxides 14–16, the synthesis of which has been greatly improved recently, are also available as reactive fatty compounds (see Section 8.2.1). Methyl ricinoleate 6 b may be oxidized to methyl 12-oxooleate 17 which, in turn, may be readily isomerized to the enone 18 [7]. Similarly, the allyl alcohol 19, obtained by selenium oxidation of methyl 10-undecenoate 7 b (see Section 8.3.2.2), can be dehydrogenated to the x-unsaturated enone 20 [7 b]. The fatty compounds 17–20 are suitable substrates for interesting follow-up reactions. 8.2.1 Oxidations 8.2.1.1 New Methods for the Epoxidation of Unsaturated Fatty Acids Unsaturated fatty compounds are preferably epoxidized on an industrial scale by the in situ performic acid procedure [4]. Numerous new methods have been used, particularly with oleic acid, such as epoxidation with aldehydes and molecular oxygen [8], dioxiranes [9–11], H2O2/tungsten heteropolyacids [12, 13], and H2O2/ methyl trioxorhenium [14–17]. Epoxidation by the Halcon process [18]. with alkyl hydroperoxides also succeeds with unsaturated fatty compounds [19, 20]. As yet, however, none of these methods has achieved industrial significance. Chemo-enzymatic epoxidation is of considerable interest because this method totally suppresses undesirable ring opening of the epoxide. Initially, the unsaturated fatty acid [21] or ester [22] is converted into an unsaturated percarboxylic


Fig. 8.1 Starting materials for the synthesis of novel fatty acids: Oleic acid 1 a, elaidic acid (E)-1 a, petroselinic acid 2 a, erucic acid 3 a, linoleic acid 4 a, linolenic acid 5 a, ricinoleic acid 6 a, 10-undecenoic acid 7 a, 9-decenoic acid 8 a, 13-tetradecenoic acid 9 a, conjuenoic acid 10 a (regio- and stereoisomeric mixture), stearoleic acid 11 a, 17-octadecynoic acid 12 a, 10-undecynoic acid 13 a, cis-

9,10-epoxyoctadecanoic acid 14 a, cis-9,10;cis12,13-bisepoxyoctadecanoic acid 15 a, cis9,10;cis-12,13;cis-15,16-trisepoxyoctadecanoic acid 16 a, 12-oxooleic acid 17 a, 12-oxooctadec-10-enoic acid 18 a, 9-hydroxy-10-undecenoic acid 19 a, 9-oxo-10-undecenoic acid 20 a, and the respective methyl esters 1 b–20 b and alcohols 1 c–20 c.

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Scheme 8.1 Reaction principle of the chemo-enzymatic “self” epoxidation of unsaturated fatty acids: intermediate enzymatic formation of peroxyoleic acid 21 from oleic acid 1 a [23].

acid, such as 21, by a lipase-catalyzed reaction with H2O2 and is then self-epoxidized in an essentially intermolecular reaction (Scheme 8.1) [23]. The second reaction step occurs without involvement of the enzyme, following the rules of the Prileshajev epoxidation. Excellent stability and activity is shown by Novozym 435, a Candida antarctica lipase B immobilized on polyacryl. This remarkable, readily separable, heterogeneous biocatalyst can be used several times without loss of activity; a turnover of more than 200 000 moles of product per mole of catalyst has been achieved. If vegetable oils are subjected to perhydrolysis, they are likewise epoxidized by the peroxy fatty acid formed (Scheme 8.2) [24]. The formation of mono- and diglycerides can be fully suppressed by the addition of 5 mol% free fatty acid. Soybean and other vegetable oils have been oxidized by these methods with conversions and selectivities of 90% and above. Even with the highly unsaturated linseed oil, the selectivity of this reaction is maintained. Industrially, vegetable oil epoxides are currently used mainly as PVC stabilizers. New applications have been opened by the possibility of photochemically initiated cationic curing [25]. Follow-Up Reactions of Epoxides to Aziridines and Episulfides Epoxidized fats, such as 14–16, are reactive reactants for a number of interesting follow-up pro-

Scheme 8.2 Chemo-enzymatic epoxidation of vegetable oils [24].


Scheme 8.3 Synthesis of methyl epiminooctadecanoate 22 a from methyl epoxyoctadecanoate 14 b: a) NaN3, NH4Cl, EtOH, H2O; b) Ph3P, THF [26]. Methyl di- and triepiminooctadecanoates 22 b and 22 c can be synthesized in a similar manner from the methyl di- and triepoxyoctadecanoates 15 b and 16 b [27].

Scheme 8.4 Synthesis of methyl epithiooctadecanoate 23 a from methyl epoxyoctadecanoate 14 b: a) HC(=S)N(CH3)2, CF3COOH, ClCH2CH2Cl. Methyl diepithiooctadecanoate 23 b can be synthesized in a similar manner from methyl diepoxyoctadecanoate 15 b [28].

cesses [4]. The corresponding methyl epiminooctadecanoates 22 a–c have been synthesized as potentially bioactive compounds (Scheme 8.3) [26, 27]. The aziridines 22 and the episulfides 23 [28], the latter being accessible from the epoxides 14 b and 15 b (Scheme 8.4), are interesting intermediates in the synthesis of heterocyclic and highly functionalized fatty compounds.

8.2.1.2 Oxidation to vic-Dihydroxy Fatty Acids Vicinal diols of unsaturated fatty compounds – polyols for polyurethanes based on renewable raw materials – may be prepared by epoxidation and subsequent nucleophilic ring opening of the epoxide. Since harsh reaction conditions are technically necessary for ring opening of fatty epoxides [29], the direct synthesis of vicinal dihydroxy fatty acids is an interesting alternative.

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Scheme 8.5 Enantioselective oxidation of methyl elaidate (E)1 b with AD-mix-a to methyl (–)-(9S,10S)- and with AD-mix-b to methyl (+)-(9R,10R)-dihydroxyoctadecanoate 24 [35a]. a) AD-mix, MeSO2NH2, H2O, tBuOH, 0 8C.

The hydroxylation of oleic acid 1 a by H2O2 and a molybdenum [30], tungsten [31], or rhenium-based catalysts [32, 33]. affords syn diols via the epoxide intermediate. The enantioselective oxidation of methyl elaidate (E)-1 b to the chiral syn-dihydroxyoctadecanoate (S,S)-24 and its enantiomer with AD-mix-a and AD-mix-b [34], respectively, in a yield of 97% and an enantiomeric excess of 95% ee is especially noteworthy (Scheme 8.5) [35 a]. With methyl oleate 1 b, the enantiomeric excess was very low using this method. The enantiomerically enriched carboxylates derived from 24 associate to gels in carbon tetrachloride. Like the corresponding gels from enantiomerically pure ricinoleic acid salts [36], they form helical fibers which can be visualized with atomic force microscopy [35 b].

8.2.1.3 Oxidative Cleavage The cleavage of oleic acid 1 a to pelargonic 25 and azelaic acids 26 a with ozone as oxidant is the most important industrial application of ozonolysis [4, 37]. A catalytic alternative, which uses a more suitable and safe oxidant is of considerable interest [4]. The direct oxidative cleavage of internal C–C double bonds with peracetic acid and ruthenium catalysts or with H2O2 and Mo, W, or Re-based catalysts leads to yields of only 50–60% (Scheme 8.6) [38]. In contrast, terminal C–C double bonds can be cleaved in yields of 80% with Ru(acac)3/CH3CO3H [39] or Re2O7/ H2O2 [40] (acac = acetylacetonato). This gives rise to the possibility of initially converting natural, internally unsaturated fatty acids into x-unsaturated fatty acid methyl esters such as 8 b and 9 b by means of metathesis, followed by oxidative cleavage. The advantage here is that the production of azelaic acid 26 and pelargonic acid 25 can be uncoupled, independent of the oxidation method.

Scheme 8.6 Transition metal-catalyzed oxidative cleavage of methyl oleate 1 b to pelargonic acid 25 and azelaic acid half ester 26 with peracetic acid [39] or hydrogen peroxide [40]. a) [Ru(acac)3]/CH3CO3H or Re2O7/H2O2.


8.2.2 Transition Metal-Catalyzed Syntheses of Aromatic Compounds

The route to aromatic compounds from renewable raw materials is of importance [4]. The transition metal catalyzed trimerization of the alkyne fatty compounds 11 and 13 gives the highly functionalized aromatic species 27 and 28, respectively (Scheme 8.7), and co-trimerization with nitrile moieties affords the highly varied and functionalized pyridine derivatives 29 (Scheme 8.8) [42].

Scheme 8.7 Cyclotrimerization of the internal alkyne 11 c and the terminal alkyne 13 b to the regioisomeric benzene derivatives 27 and 28 [42]. TMS=trimethylsilyl, Cp=cyclopentadienyl, cod = cycloocta-1,5-diene.

Scheme 8.8 Cyclization of methyl 17-octadecynoate 12 b with nitrile species to the pyridine derivatives 29 [42]

8.2.3 Olefin Metathesis

Transition metal metathesis of olefins, which is used in the industrial petrochemistry and polymer chemistry for the production of special olefins and unsaturated polymers, is also applicable to unsaturated fatty acid esters. However, the low loading of the expensive catalysts has, until now, stood in the way of the technical utilization of this interesting reaction in oleochemistry [4]. Over the past few years, Warwel et al. have developed significantly more active catalysts in the form of Re2O7 · B2O3/Al2O3 · SiO2 + SnBu4 and CH3ReO3

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Scheme 8.9 Co-metathesis of methyl oleate 1 b and ethylene to methyl 9-decenoate 8 b and 1-decene. The ester 1 b used (new sunflower) was 87% pure, the conversions and selectivities each > 90%, and the yields of 8 b were > 80% [43].

+ B2O3 · Al2O3 · SiO2 and successfully tested them in a series of metathesis transformations [43]. The industrial application of olefin metathesis to unsaturated fatty compounds thus moves realistically nearer. Scheme 8.9 illustrates the co-metathesis of methyl oleate 1 b and ethylene to form methyl 9-decenoate 8 b and 1-decene. Similarly, methyl 13-tetradecenoate 9 b and 1-decene are obtained from methyl erucate 3 b and ethylene [43]. Methyltrioxorhenium is also a suitable catalyst for the metathesis of unsaturated fatty compounds [44]. 8.2.4 Pericyclic Reactions

The thermal Diels-Alder reaction of methyl conjuenate 10 b with electrondeficient dienophiles has been thoroughly investigated [4]. and is carried out industrially with maleic anhydride. With a Lewis acid, such as boron trichloride or tin tetrachloride, and catalytic amounts of iodine it was possible to obtain the cycloadducts 31 with the dienophiles 30, even at room temperature (Scheme 8.10) [45]. Building on the cycloaddition of dimethyl acetylenedicarboxylate 32 to 10 b, a further aromatic synthesis has been developed. Adduct 33 was dehydrogenated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to the dimethyl phthalate 34 into which the fatty acid side chain is incorporated (Scheme 8.11).

Scheme 8.10 Diels-Alder reactions of methyl conjuenates 10 b with the dienophiles 30 a–d to the regioisomeric addition products 31 [45].


Scheme 8.11 Diels-Alder addition reactions of methyl conjuenates 10 b and dimethyl acetylenedicarboxylate 32 to the cycloaddition product 33 which was dehydrogenated to the dimethyl phthalates 34 with DDQ [45]. BHT = 4-methyl-2,6bis[(1,1-dimethyl)ethyl]phenol.

In addition, Diels-Alder reactions with the enones 18 and 20 as dienophiles [45, 46], ene reactions [47, 48], [2+2] cycloadditions of ketenes [49], and isocyanates [50], to unsaturated fatty compounds, as well as sigmatropic [3,3] rearrangements of allylvinyl ethers derived from fats [49], have been investigated, which lead to a number of interesting and novel fatty compounds. 8.2.5 Radical Additions

With the development of modern preparative radical chemistry, radical additions to unsaturated fatty compounds with the formation of new C–C bonds have been investigated systematically. Normal tin hydride radical chemistry [51] cannot be applied to the sterically constrained, internal, and electron-rich double bonds, as in 1 b. In contrast, enolizable compounds such as acetic acid, malonic acid, monomethyl malonate, and cyanoacetic acid were added to the fatty acid

Scheme 8.12 Manganese(III) acetateinduced radical addition of malonic acid to methyl oleate 1 b with formation of the regioisomeric c-lactones 35 [53–55].

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esters 1 b and 7 b by initiation with manganese(III) acetate [52] to give the respective c-lactones such as 35 (Scheme 8.12) [53–55]. Unfortunately, higher carboxylic acids cannot be oxidized to radicals with manganese(III) acetate and added to alkenes [54, 55]. For this purpose, a new method was developed.

8.2.5.1 Solvent-Free, Copper-Initiated Additions of 2-Halocarboxylates Higher carboxylic acids can be added to alkenes, such as unsaturated fatty compounds, as their a-haloesters, a process initiated by electron transfer from copper [56–58]. The addition of 2-iodocarboxylates, for example, methyl 2-iodopropanoate 36 a, to 7 b gave the c-lactone 37 in high yields (Scheme 8.13). The reaction procedure is very simple: The unsaturated fatty compound, the 2-halocarboxylate, and commercial copper powder are mixed without further pretreatment and heated at 100–130 8C under an inert atmosphere. After a simple work-up, analytically pure products are obtained in good yields. Esters of 2-iodocarboxylic acids can be obtained in situ from the readily available bromo compounds by the addition of a stoichiometric amount of sodium iodide. Methyl 2-bromopropanoate 36 b was also added to methyl oleate 1 b with copper by the addition of stoichiometric amounts of sodium iodide. The regioisomeric addition products 38 a were isolated in 58% yield. The addition–elimination product 38 b was isolated as a byproduct (Scheme 8.14). Comparable results were achieved with methyl petroselinate 2 b and methyl erucate 3 b [57, 58]. This generally applicable addition reaction was also carried out with bromomalo-

Scheme 8.13 Copper-initiated addition of methyl 2-iodopropanoate 36 a to methyl 10-undecenoate 7 b [56–58].

Scheme 8.14 Copper-initiated addition of methyl 2-bromopropanoate 36 b to methyl oleate 1 b in the presence of sodium iodide yields the regioisomeric c-lactones 38 a and the addition–elimination product 38 b [56–58].


Scheme 8.15 Radical cyclization of methyl 2-iodopetroselinate 39 induced by AgOAc/SnCl2 (a-40 : b-40 : trans-40 = 35 : 31 : 34) [59].

nates, 2-bromo-3-alkylsuccinates, and a,a'-diiododicarboxylates, among others, in good to very good yields [57, 58]. In an analogous manner, 2-haloalkane nitriles have also undergone addition [56–58]. The reaction can also be used for intramolecular cyclization. The cyclization of methyl 2-iodopetroselinate 39 to the cyclopentane derivatives 40 was best carried out with the initiator system AgOAc/SnCl2 (Scheme 8.15) [59].

8.2.5.2 Addition of Perfluoroalkyl Iodides Radical additions of perfluoroalkyl iodides to terminally unsaturated carboxylic acids such as 10-undecenoic acid 7 a with 2,2'-azobisisobutylnitrile (AIBN) as initiator give perfluoroalkylated products in good yields [60]. In contrast, for radical additions to alkenes with internal double bonds, such as methyl oleate 1 b, the addition products are only obtained in very low yields by this method [61].

Scheme 8.16 Synthesis of 9- and 10-perfluoroalkyloctadecanoic acids 42 as a regioisomeric mixture: Addition of perfluoroalkyl iodides to 1 b give the regioisomeric perfluoroalkylated iodoesters 41, which were then reduced to iodine-free esters and hydrolyzed to free perfluoroalkylated fatty acids 42 [61, 62].

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Perfluoroalkyl iodides 41 can be added to both methyl 10-undecenoate 7 b and methyl oleate 1 b with good to very good yields if the reaction is initiated by electron transfer from metals such as finely divided silver [61], copper powder [62], or lead with a catalytic amount of copper(II) acetate [62] (Scheme 8.16). The best yields of addition product 41 are obtained with copper powder or with lead/Cu(OAc)2 [62]. Perfluoroalkylated fatty compounds such as 42 are of interest because of their surfactant properties [63].

8.2.5.3 Thermal Addition of Alkanes Alkylated fatty acids have interesting properties [64] and an effective synthesis of these products is important [4]. The ane reaction is the thermally initiated radical addition of alkanes to alkenes at elevated temperatures (200–450 8C) and pressures (200–250 bar) [65]. The addition of cyclohexane to methyl 10-undecenoate 7 b gave methyl 11-cyclohexylundecanoate 43 (Scheme 8.17) [66]; 11-cyclohexylundecanoic acid is the main lipid of thermophilic archaebacteria [67].

Scheme 8.17 Thermally initiated addition of cyclohexane to methyl 10-undecenoate 7 b. The reaction was carried out in a high pressure–high temperature flow reactor [66].

8.2.6 Lewis Acid-Induced Cationic Addition

x-Hydroxycarboxylic acids, including alkyl-branched acids such as 44, which are of interest as polyester components, are obtained with high selectivity by the ene addition of formaldehyde to unsaturated fatty acids (Scheme 8.18) [68]. However, stoichiometric amounts of dimethylaluminum chloride or ethylaluminum dichloride are used as reagents [69, 70]. A catalytic variant would be highly significant. The acid (Z)-45 (Scheme 8.18), obtained by the addition of formaldehyde to 10-undecenoic acid 7 a, induces wound healing of tissue damage in soybeans by stimulation of callus formation at the damaged site [71]. Ene additions of formaldehyde to natural oils proceed with formation of the respective di- and trifunctionalized triglycerides [72], and jojoba oil gives mixtures of 1 : 1 and 1 : 2 adducts [73]. Homoallyl ethers are obtained in an analogous reaction with acetals [74]. Formaldehyde and higher aldehydes react with unsaturated fatty compounds in the presence of aluminum chloride to form the corresponding alkyl-substituted 4-chlorotetrahydropyrans in good yields and with high selectivity [75]. The reaction of two equivalents of formaldehyde with, for example, methyl oleate


Scheme 8.18 Me2AlCl-induced addition of paraformaldehyde to oleic acid 1 a to give the regioisomeric homoallyl alcohols 44. The corresponding addition to 10-undecenoic acid 7 a gives the homoallyl alcohol 45 [(E):(Z) = 4 : 1] [68].

Scheme 8.19 AlCl3-induced addition of two equivalents of paraformaldehyde to methyl oleate 1 b to give the 4-chlorotetrahydropyrans 46 (mixture of two regioisomers) [75].

1 b, gave the 3,5-dialkyl-substituted 4-chlorotetrahydropyran 46 (Scheme 8.19). Variation of the alkene, on the one hand, and the carbonyl component, on the other, leads to a broad range of alkyl-substituted chlorotetrahydropyrans. The Friedel-Crafts acylation is an interesting and versatile method for the functionalization of unsaturated fatty compounds [76]. The EtAlCl2-induced acylation of oleic acid 1 a, among others, with acyl chlorides 47 gave the (E)-configured b,c-unsaturated oxocarboxylic acids 48 with high selectivity (Scheme 8.20). Cyclic anhydrides, such as succinic anhydride, gave oxo diacids in a similar manner [76]. The acylation products 48 are substrates for a number of interesting follow-up reactions, for example, 48 g for Nazarov cyclizations [77]. 8.2.7 Nucleophilic Addition to Reversed-Polarity Unsaturated Fatty Acids

Addition to the double bond of unsaturated fatty acids mainly occurs with electrophiles (Section 8.2.6), radicals (Section 8.2.5), or in pericyclic reactions (Section 8.2.4). Totally new coupling possibilities arise when the polarity of the electron-

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Scheme 8.20 EtAlCl2-induced Friedel-Crafts acylations of oleic acid 1 a with the acyl chlorides 47 a–g give the unsaturated regioisomeric oxocarboxylic acids 48 a–g [76].

rich double bond is reversed, as in the enone fatty acids 18 and 20. In this way, a number of nucleophiles may be coupled to the double bond by Michael additions [78]. Interesting and novel fatty compounds have also been obtained from the enones 18 and 20 in Stetter [7, 78] and Mukaiyama additions [79]. Similarly, methyl conjuenate 10 b has been treated anodically with numerous alcohols to afford methyl dialkoxyoctadecanoates, some of which had interesting surfactant properties [80]. Numerous carbon, oxygen, and nitrogen-based nucleophiles may be inserted into fatty allyl carbonates by palladium catalysis (from the corresponding allyl alcohols; Section 8.3.2.2) in very good yields [79 b, 79 d].

8.3 Reactions of Saturated Fatty Compounds 8.3.1 Radical C–C Coupling 8.3.1.1 Oxidative Coupling of C2 Anions of Fatty Acids The C–C coupling, with the concurrent formation of symmetrical products, may be achieved by the dimerization of two radicals. Radicals may be formed selectively under mild conditions in high concentrations by the oxidation of anionic precursors. Unsaturated fatty acids possess several sites with comparably high C–H acidities, which are suitable for anionization and subsequent reactions,

8.3 Reactions of Saturated Fatty Compounds Scheme 8.21 Radical a,a' dimerization of the fatty acid methyl esters 49. Ozonolysis of the dimer 50 c gives the dimethyl tetracarboxylate 51 [81]. LDA = lithium diisopropylamide.

particularly the a-positioned C–H bond of the ester group. The fatty acid methyl esters 49 were anionized and treated oxidatively with 0.9 equivalents CuBr2. In this way, the dimers 50 were formed with a (d,l) : meso ratio of about 1.2 : 1 (Scheme 8.21) [81 a]. The dimethyl ester of the tetracarboxylic acid 51 was obtained from 50 c by ozonolysis in 90% yield.

8.3.1.2 Anodic Homo- and Heterocoupling of Fatty Acids (Kolbe Electrolysis) The anodic decarboxylation of aliphatic carboxylic acids gives a rapid, and also technically useful, path to radicals for dimerization and coupling (Kolbe electrolysis) [82]. This efficient synthetic method was used extensively in homocouplings with natural and modified fatty acids, such as in the preparation of specifically functionalized alkanes [83] or in the preparation of long chain diesters. Isostearic acid may be dimerized in 63% yield to a methyl-branched C34 hydrocarbon whose cosmetic property profile resembles that of squalane. As the half ester with the currently greatest number of carbon atoms, the methyl ester of the C36 dimeric fatty acid was coupled in 38% yield to a C70 dimethyl dicarboxylate [83, 84]. In addition, the dimeric fatty acids 53 may be obtained from Diels-Alder adducts of fatty acids such as 52 (Section 8.2.4) by homocoupling (Scheme 8.22 a) [47], or dicarboxylic acids with four alkyl chains, such as 55, are obtained from 2,2'-coupled diacids like 54 (Section 8.3.1.1, Scheme 8.22 b) [78 b]. Through heterocoupling, that is, the electrolysis of two different carboxylic acids, new unsaturated fatty acids are formed [82 a, 84, 85 a], such as methyl 17-octadecenoate 56 (Scheme 8.23 a) [84], partially perfluoronated fatty acids like 57 (Sche-

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Scheme 8.22 Homocoupling of the half esters 52 and 54 by Kolbe electrolysis to give the dimers 53 [45] and 55 [78 b] respectively.

Scheme 8.23 Kolbe electrolysis of two different fatty acids: Synthesis a) of new x-unsaturated fatty acids such as 56 [84], b) of perfluoroalkylated fatty acids such as 57 [84], and c) of C-glycosides such as 59 [86 a].

8.3 Reactions of Saturated Fatty Compounds

me 8.23 b) [84], pheromones [85 b], C-glycosides 59 formed by co-electrolysis with carbohydrate carboxylic acids 58 (Scheme 8.23 c) [86], or long chain diesters [85 b]. 8.3.2 Functionalization of C–H Bonds

Selective reactions at the alkyl chain of fatty acids are still rare but are of great interest [4].

8.3.2.1 Oxidation of Nonactivated C–H Bonds Nonactivated C–H bonds may be functionalized chemically [87] or enzymatically [88] (Section 8.4.2.5). Particularly important, but yet to be solved satisfactorily, is the regioselectivity of C–H functionalization. Notable advances have been achieved by photochemical gas phase chlorination of fatty acids that are adsorbed onto aluminum oxide with chlorine or tBuOCl. For this reaction, selectivities increase with increasing chain length of the fatty acid. Stearic acid 60 reacts with chlorine or tBuOCl at –35 8C in the x-(x-2) position to form the chlorostearic acids 61 in yields of 96% and 93%, respectively (Scheme 8.24) [89]. The selectivity is significantly better than with the more established radical chlorination with dialkylchloramines in an acidic medium [90, 91]. The methyl esters of shorter chain fatty acids and fatty alcohols may be hydroxylated with amine oxides with good conversions and (x-1)-(x-2) selectivities [7 a].

Scheme 8.24 Photochemical gas-phase chlorination of stearic acid 60 adsorbed on aluminum oxide to chlorostearic acids 61 with chlorine (relative product distribution (rpd): x-2 14.2%; x-1 38.4%; x 43.7%) and tBuOCl (rpd: x-2 11.6%; x-1 51.5%;x 30.1%) [89].

8.3.2.2 Oxidation of Allylic C–H Bonds The allylic C–H bonds of unsaturated fatty acids are activated C–H bonds, which, in principle, may be functionalized with a number of oxidizing agents. For the allylic oxidation of methyl 10-undecenoate 7 b and methyl oleate 1 b, SeO2/tBuOOH has been shown to be suitable [7]. The reaction with singlet oxygen has proved to be considerably more suitable for the preparation of the allyl alcohol 62. For this purpose, 1 b was photooxygenated with oxygen by means of a high pressure sodium-vapor lamp and tetraphenylporphin as sensitizer, and the resulting hydroperoxide 63 was reduced

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Scheme 8.25 Photooxygenation of methyl oleate 1 b with singlet oxygen and tetraphenylporphin as sensitizer to give a) allyl alcohols 62 and b) a,b-unsaturated ketones 18 (both regioisomeric mixtures form) [79 b, 79 c].

with triphenylphosphine (Scheme 8.25) [79 b, 79 c]. In the presence of acetic anhydride, pyridine, and catalytic amounts of 4-dimethylaminopyridine (DMAP), the hydroperoxide may converted directly into the regioisomeric mixture of the enone fatty acids 18 [79 b, 79 c]. Since the photooxidation occurs even in sunlight, unsaturated fatty acids oxidized in the allylic position are available by a route which is particularly favorable from both an economical and an ecological viewpoint.

8.4 Enzymatic Reactions 8.4.1 Lipase-Catalyzed Transformations

Lipases and their applications have been reviewed in this journal in 1998 [92]. We will limit ourselves therefore to a few examples of selective syntheses of fatty compounds.

8.4.1.1 Lipase-Catalyzed Syntheses of Monoglycerides and Diglycerides Mono- and diglycerides (partial glycerides) are among the most important nonionic surfactants emerging from oleochemistry. They are used widely as emulsifiers in the preparation of foodstuff. “Monoglycerides” are prepared on a large scale by the glycerolysis of natural fats and oils in the presence of inorganic catalysts and must be subsequently purified by molecular distillation [92]. Biocatalytic transformations offer a more gentle alternative to this process [4]. Consider-

8.4 Enzymatic Reactions

able advances have been made here in the last few years but no breakthrough could yet be considered economic. Glycerol, protected as the isopropylidene [93] or phenylboroester [94, 95] derivatives, can be converted into pure monoglycerides 68 with interesting surfactant properties in the presence of a lipase from Rhizomucor miehei (lipozyme) and free fatty acids as acyl donors (Scheme 8.26). If glycerol is immobilized on silica gel, the esterification runs surprisingly smoothly in aprotic solvents such as n-hexane or tert-butylmethyl ether in the presence of different lipases and acyl donors (free fatty acids, fatty acid methyl esters, vinyl esters, triglycerides, and so forth) [96–98]. Thus, for example, isomerically pure (> 98%) 1,3-sn-dilaurin 70 is readily obtained with vinyl laurate 69 in the presence of a “1,3-selective” lipase (Scheme 8.27) [96, 99]. Because of their ready accessibility, up to the kilogram scale, and their high purity and stability, these 1,3-sn-diglycerides represent interesting building blocks for further surfactant compounds (for example, by coupling with amino acids) and for the preparation of reagents for lipid modification of natural products [100]. If the 1(3)-sn-monoglycerides 68 are prepared by this method, they must be selectively separated from the reaction mixture, since they are very readily esterified by the lipases to 1,3-sn-diglycerides. This separation is achieved by exploiting the poor solubility of the monoglycerides 68 at low temperatures in appropriate solvent mixtures [97, 98]. The method has proved to be exceptionally suit-

Scheme 8.26 Lipase-catalyzed synthesis of 1(3)-sn-monoglycerides 68 by acylation of glyceryl phenylborates 66 with fatty acids to give 67 once the protecting group is cleaved [94, 95].

Scheme 8.27 Lipase-catalyzed synthesis of 1,3-sn-diglycerides, such as 1,3-sn-dilaurin 70, by the vinyl lauroate (69) acylation of glycerol immobilized on silica gel [96, 99].

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able for the conversion of natural fats and oils from palm kernels, coconuts, soybeans, sunflowers, and rapeseeds into the respective monoglyceride mixtures. Such highest-quality products are particularly well suited for uses in the cosmetic and pharmaceutical industries.

8.4.1.2 Lipase-Catalyzed Syntheses of Carbohydrate Esters Alkylpolyglucosides (APGs) and (polyethoxylated) sorbitan esters (Span, Tween) – both directly or indirectly derivatives of glucose – are already used extensively as nonionic surfactants or emulsifiers [5]. The lipase-catalyzed synthesis of carbohydrate esters has been recently reviewed [92]. It is particularly noteworthy, that in aprotic solvents such as tetrahydrofuran, dioxan, monoglyme, or diglyme, monosaccharides such as d-glucose, d-galactose, d-mannose, and d-fructose can be transformed regioselectively and in high yields into the respective 6-O-acyl derivatives in the presence of the lipase from Candida antarctica B (Novozym SP 435). Thus, for example 6-O-lauroyld-glucose 72 was obtained directly from glucose 71 and lauric acid 49 d (Scheme 8.28). The method may also be applied to the esterification of l-ascorbic acid and even, if only to a limited extent, to saccharose. The fatty acids caprylic, capric, lauric, myristic, palmitic, stearic, oleic, 12-hydroxystearic, and erucic acids have been used as acyl donors [101].

Scheme 8.28 Lipase-catalyzed selective esterification of glucose 71 with lauric acid 49 d in aprotic solvents to give 6-Olauroyl-D-glucose 72 [101].

8.4.2 Microbial Transformations 8.4.2.1 Microbial Hydration of Unsaturated Fatty Acids The chemical addition of water to unsaturated fatty compounds such as 1 a is neither regioselective nor stereoselective [102]. In contrast, microbial hydration is frequently both regio- and stereoselective. Microbial water attachment to an unsaturated fatty acid was first reported by Wallen et al. in 1962 [103]. The authors observed that a Pseudomonas species, isolated from fat-containing materials, hydrated oleic acid 1 a to (R)-10-hydroxystearic acid 73 in 14% yield (Scheme 8.29 a). This water attachment was also observed with the bacterial genera Nocardia, Rhodococcus, Corynebacterium, and Micrococcus [104–106]. Compound 73 was obtained in 45% yield with the yeast

8.4 Enzymatic Reactions

Scheme 8.29 Microbial enantioselective and regioselective addition of water to a) oleic acid 1 a [103–106]; b) linoleic acid 4 a [108]; and c) linolenic acid 5 a [108]. to give the 10-hydroxy fatty acids 73, 75, and 76; further, d) 73 can be enzymatically dehydrogenated to 10-oxooctadecanoic acid 74 [107].

Saccharomyces cerevisiae [107]. The hydration product 73 can be oxidized to 10oxooctadecanoic acid 74 in a subsequent enzymatic dehydrogenation reaction (Scheme 8.29 d) [107]. Microbes Lactobacillus plantarum and Nocardia cholesteriolicum assisted the formation of (Z)-10-hydroxyoctadec-12-enoic acid 75 in 71% yield from linoleic acid 4 a (Scheme 8.29 b) [108]. a-Linolenic acid 5 a was converted into (12Z,15Z)-10hydroxyoctadeca-12,15-dienoic acid 76 in 77% yield (Scheme 8.29 c) [108]. The hydratase was not active towards trans-unsaturated fatty acids, such as elaidic acid (E)-1 a, and unsaturated fatty acids without a double bond in the 9 position, such as erucic acid 3 a. Enzymatic hydration activity occurred less readily with a greater number of double bonds in the substrate. Since hydratases from a number of bacteria and yeasts convert (9Z)-fatty acids into 10-hydroxy fatty acids, it can be assumed that the C10 specificity is universal in nature [109]. In the future, it is expected that protein structure elucidation in algae, higher plants, and marine lifeforms will advance and these enzymes will cloned in microorganisms so that biocatalysts will be available for synthetic use in ever larger amounts [110].

8.4.2.2 Microbial x- and b-Oxidation of Fatty Acids Microbial x-oxidation of fatty acids, which leads to dicarboxylic acids, is of great interest [4]. Advances have been made here in recent years: Yi and Rehm [111] were able to convert oleic acid 1 a into the corresponding unsaturated dicarboxylic acids 77 with yeast of the genus Candida tropicalis (Scheme 8.30 a). With alkaline fermentation procedures, it was possible to increase the yields of 77 from 23 to 50% and also to oxidize solid fatty acids, such as palmitic acid, stearic acid, and erucic acid, to the respective dicarboxylic acids [112 a]. In a batch

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Scheme 8.30 Microbial x-oxidation of oleic acid 1 a to a) (Z)9-octadecendioic acid 77 with Candida tropicalis [111, 112 a] and b) (Z)-3-hydroxy-9-octadecendioic acid 78 with the yeast mutant Candida tropicalis M25 [114].

fermentation with 70 g L–1 palmitic acid, hexadecanedioic acid was obtained in a yield of 36% and a concentration of 28.1 g L–1. In comparison with reported yields [113], this value is among the highest values that have been obtained with genetically unaltered microorganisms. This reaction is therefore not subject to the gene technology regulations and is therefore of additional industrial interest. The yields are significantly lower with linoleic acid 4 a and ricinoleic acid 6 a [112 b]. Oleic acid 1 a can be transformed into the unsaturated hydroxy diacid 78 with 76% ee with the yeast mutant Candida tropicalis DSM 3152. The productivity of the strain is improved by N-methyl-N'-nitro-N-nitrosoguanidine (NMG) mutagenesis, and in a Fed batch fermentation with the mutant Candida tropicalis M25 19.4 g L–1 78 were obtained from oleic acid (Scheme 8.30 b) [114]. Under similar conditions, 6.1 g L–1 hydroxydiene diacid were obtained with 30 mL L–1 linoleic acid [115]. 8.4.3 Microbial Conversion of Oils/Fats and Glucose into Glycolipids

The broad structural palette of biosurfactants, along with their numerous applications and biosynthetic pathways, were comprehensively reviewed by Kosaric [116], Ratledge [117], Desai and Banat [118], Banat [119], and Lang and Wagner [120]. Of particular interest, because of the remarkably high yield, 300–400 g L– 1 , is the sophorose lipid formation with Candida bombolica from glucose and rapeseed oil as substrates [121, 122]. Moreover, natural oils, fatty acid methyl esters, and free fatty acids have been converted into glycolipids in the presence of Ustilago maydis DSM 4500. In comparison with natural oils, the use of free fatty acids has brought about an increase in yield to 30 g L–1 glycolipid containing 90 wt% of mannosyl erythritolipids [123].

8.5 Improvement in Natural Oils and Fats by Plant Breeding


In recent years, knowledge of the biochemical relationships of plant metabolism – in particular, of the biosynthesis of the storage fats in commercial use – has increased considerably [124]. Breeding has always been aimed mainly at an improvement in the yield performance of useful plants. Efforts are also now being made to meet the demands of industry for tailor-made oils and fats. The potential for future growth in this area is mainly expected where suitability for chemical processing already exists due to the natural structure or purity of the vegetable raw material. Possibilities for the genetic modification of oil plants exist primarily in respect of the composition of the storage lipids, since, where chain length, number, and position of double bonds and functional groups are concerned, nature has already generated an enormous variety of fatty acids. Even drastic variations in the fatty acid pattern of seeds are tolerated by the plants and the seedlings. 8.5.1 Gene Technology as an Extension of the Methodological Repertoire of Plant Breeding

Concurrent with the increasing demand for renewable raw materials, modern biotechniques, including gene technology, have made enormous steps in extending the methodological repertoire of plant breeding, so that today’s breeding procedures are even more efficient and selective. Although classical plant breeding, when combined with experimental mutagenesis (“mutation breeding”) and modern in vitro cell- and tissue-culture methods, has frequently proved to be successful in oil plants such as, for example, soybean, rapeseed, sunflower, or linseed [125, 126], gene technology offers an additional, universal approach for changing the amount and composition of the stored oil [127, 128]. This development was recently made possible by a series of methodological improvements. This is illustrated by continued progress both in natural vectortransformation systems based upon natural infection by the soil-borne bacterium Agrobacterium tumefaciens (or A. rhizogenes) and in a series of vector-free transformation systems where, for example, the foreign recombinant gene is integrated into cells lacking a cell wall (protoplasts) or by bombardment of regenerable meristems with DNA-loaded particles [129, 130]. In molecular-biological experiment, the necessary regulatory “gene switches” (promoters) as expression signals are normally combined (cloned) with structural parts of previously isolated genes (structural genes) to form a new functional unit, a chimeric gene [131]. Moreover, selection markers, namely, genes which confer antibiotic or herbicide resistance and thus permit a selection of successfully transformed plant cells, are also frequently inserted [129]. A widely used method for the genetic modification of useful plants, which are used particularly for the prevention of the undesired expression of speciesspecific genes, is the “antisense RNA” approach. Although the mode of action

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in transgenic plants is not yet fully understood, the simplest explanation is that the species-specific sense RNA binds to the transferred, complementary antisense RNA and, thus, the translation and biosynthesis of the relevant protein is inhibited. The resulting double-stranded RNA hybrid appears to be degraded very rapidly by nucleic-acid digesting enzymes (the nucleases, in this case RNAse H) [132]. The extent of the gene expression achieved is dependent to a significant extent upon the transcriptionally active promoter used. Thus, in the preparation of the gene constructs that are to be transferred, bacterial or viral promoters are frequently used. Meanwhile, however, a series of seed-specific promoter sequences such as those of napin, phaseolin, or oleosin genes have also been successfully used [131, 133]. Since at present, almost exclusive reliance is placed upon cell cultures or embryonic tissue in vitro for genetic transformation, further improvements are also indispensable for the regeneration to intact, transgenic plants; this has also been achieved [129]. 8.5.2 New Oil Qualities by Oil Designed with Available Agricultural Varieties

New genetic variation is fundamental to every commercial breeding activity, that is, selection is only successful if the characteristic to be changed varies in the starting material. Because of the intensive breeding to which they have been subject, the variability for new, desirable quality properties in agriculturally cultivated plants is, in practise, highly restricted. In contrast, a very rich reservoir of genetic resources for industrially interesting raw material for fatty compound in high purity is present in wild plants. These are, for example, medium to very long chain fatty acids as well as fatty acids with unusual functionality resulting from the number and position of double bonds or the presence of hydroxyl, oxo, or epoxy groups [134, 135]. Plant breeding efforts to domesticate wild plants such as species of the genera Cuphea, Calendula, Euphorbia, Vernonia, Lesquerella, Crambe, or Limnanthes, in order to develop useful plants that may be more productive, are not lacking [136]. Where the genetic distance between the wild and the cultivated species is not too large, it is possible, in principle, to transfer the desired, quality improving property into the cultivated form by conventional methods of interspecific and intergeneric hybridization or with the support of biotechniques (for example, “embryo rescue”). Such breeding programs are, among others, very laborious, since many adverse properties of the “gene donor”, such as the low yield capability, late ripening, or low shattering resistance of a wild species, is also transferred. These unfavorable properties must once more be eliminated with difficulty through repeated backcrossing and subsequent selection (often even without success). This clarifies why transferring a novel oil quality into a high yielding, agronomically adapted plant species by conventional methods is indeed possible, but is fraught with difficulty. In this scenario, gene technology is well suited to accelerate breeding progress or, in many cases, to make it even possible. In practice, this grants the ability to


implant a specific and desired quality property from distantly related plant species, from microorganisms (such as bacterial or yeasts), or even from mammals without detrimental effects to the genetic background or yield capabilities of the productive species. Numerous genes (cDNA clones) exist for the biosynthesis of unusual fatty acids such as ricinoleic, petroselinic, linoleic, vernolic, or crepenynic acids, which may be cloned and transformed in cultivated plants. With the help of this material, it should become more possible to optimize genotypes (such as species) for the production of oleochemical raw materials [127, 137–139]. 8.5.3 Overview of Renewable Raw Materials Optimized by Breeding

A series of oil plants of world-wide significance is suitable for the production of renewable raw materials, namely, for the extraction of oils and fats with a specific fatty acid composition. Thus, commercially exploited oil seeds such as soybean (Glycine max), rapeseed (Brassica napus), sunflower (Helianthus annuus), peanut (Arachis hypogaea), or linseed (Linum usitatissimum) now exhibit a considerable variation in their fatty acid pattern, both in nature and as modified by breeding (Table 8.1) [126, 140–143]. Where “nonfood” uses are concerned, genetic engineering approaches can make a special contribution to the expansion in the wealth of raw materials available to oleochemistry, such as increasing the content of individual fatty acids or drastically changing the oil quality by the introduction of a new fatty acid. Within this context, variants of important oil seeds, which have become available by plant breeding with different methods, will be discussed in the following on the basis of selected examples (Table 8.2).

8.5.3.1 Soybean As a result of intensive quality breeding, the fatty acid pattern of the soya bean is remarkably variable. In addition to the low linolenic acid varieties, which should contribute considerably to the improvement in oxidative stability (mainly in the food oil area), there are further varieties with modified proportions of individual saturated fatty acids (Table 8.2) [144–146]. “High oleic” (HO) soybeans have been produced by routes based on genetic engineering. It has been estimated that 40 000 ha of this variety was planted in the USA in 1998 [138, 147].

8.5.3.2 Rapeseed For a number of reasons, the intensive, ongoing work inducing alterations in the oil quality of cultivated plants is currently concentrated on rapeseed (B. napus). Since both summer and winter forms of this species are available, they can be planted as oil plant in climatically different regions of the world. A further advantage of rapeseed over other cultivated species is in its accessibility to biotechnological methods and, in particular, in its capability for transformation and regeneration [141, 148].

277

conventional high erucic acid 0 or 00 (canola) low linolenic acid high lauric acid conventional high oleic acid (HO) conventional high oleic acid (HO) conventional low linolenic acid (linola)

soybean rapeseed

a)

– – – – 37 – –

12 : 0 a)

mutagenesis

– –

– natural mutation –

mutagenesis

conventional natural mutation mutagenesis gene technology

Origin

– –

– –

– – – – 4 – –

14 : 0

6 6

12 6

11 3 4 4 3 7 3

16 : 0

4 3

4 2

4 1 2 2 1 5 4

18 : 0

18 15

47 81

23 11 60 61 33 19 83

18 : 1

14 73

31 3

54 12 21 28 12 68 10

18 : 2

58 3

– –

8 9 10 3 7 – –

18 : 3

– –

– –

– 8 1 1 – – –

20 : 1

12 : 0 = lauric acid, 14 : 0 = myristic acid, 16 : 0 = palmitic acid, 18 : 0 = stearic acid 60, 18 : 1 = oleic acid 1 a, 18 : 2 = linoleic acid 4 a, 18 : 3 = linolenic acid 5 a, 20 : 1 = eicosenoic acid, 22 : 1 = erucic acid 3 a.

linseed

peanut

sunflower

Variant

Type

Table 8.1 Commercially available fatty acid variants of important oil seeds.

– –

– –

– 52 1 – – – –

22 : 1

– –

6 8

– 4 1 1 3 1 –

Others

[126] [143]

[142] [142]

[126] [126] [126] [140] [141] [126] [126]

Source

278


low linolenic acid low palmitic acid high palmitic acid high stearic acid high oleic acid (HO) high myristic acid high stearin high oleic acid (HO) high oleic acid (HO) low linolen high palmitin high stearin high oleic acid combined with low saturated fatty acids high palmitin

soybean

b) c) d)

a)

– – – –

gene technology mutagenesis mutagenesis mutagenesis

–

–

gene technology

mutagenesis

17.7 – –

– – – – –

14 : 0 a)

gene technology gene technology mutagenesis

mutagenesis mutagenesis mutagenesis mutagenesis gene technology

Method

27.8

3.8 25.2 5.1 3.2

4.3

23.1 4 4.2

10.5 3.7 17.3 8.4 6.6

16 : 0

1.8

1.5 3.5 26.0 2.4

1.4

2.4 29 2.2

4.6 3.7 2.9 28.1 3.6

18 : 0

17.5

68.5 11.4 13.8 92.1

84.1

33.7 15 80.2

23.2 24.1 16.8 19.8 84.9

18 : 1

6.0

22.1 55.1 55.1 2.3

5.2

14.8 19 4.5

59.6 58.9 54.5 35.5 0.6

18 : 2

42.0

1.2 – – –

2.9

3.8 22 5.2

2.0 8.9 8.3 6.6 1.9

18 : 3

–

1.1 – – –

0.9

– 1 1.8

– – – – –

20 : 1

14 : 0 = myristic acid, 16 : 0 = palmitic acid, 18 : 0 = stearic acid 60, 18 : 1 = oleic acid 1 a, 18 : 2 = linoleic acid 4 a, 18 : 3 = linolenic acid 5 a, 20 : 1 = eicosenoic acid, 22 : 1 = erucic acid 3 a; includes 6% arachidic acid (20 : 0), 2% behenic acid (22 : 0), 1% lignoceric acid (24 : 0); includes 3.7% palmitoleic acid (16 : 1); palmitoleic acid (16 : 1).

linseed

sunflower

rapeseed

Variant

Type

Table 8.2 Extreme fatty acid variants in breeding material from important oil seeds.

–

– – – –

–

– – –

– – – – –

22 : 1

[133] [158] [158] [144]

[164]

4.8 d)

[133]

[155] [153] [151]

[144] [145] [146] [146] [147]

Source

1.8 4.8 c) – –

1.2

4.5 10 b) 1.9

– 0.7 0.2 1.6 2.4

Others

8.5 Improvement in Natural Oils and Fats by Plant Breeding 279

280


Rapeseed oil is very rich in erucic acid (3 a), a widely sought raw material for many nonfood uses [149, 150]. In the context of improvement in nutritional oil quality, the low erucic acid varieties – named zero and double zero or canola types, which exhibit about 60% oleic acid (1 a) – were developed by classical breeding methods. The breeding of linolenic acid deficient (< 3% 5 a) or high oleic acid (> 80% 1 a) rapeseed forms has been achieved both by induced mutation [151, 152] and genetically by inhibition of the inherent 12- or 15-desaturase genes [133] (Table 8.2). Anti-sense inhibition of the desaturation step in Brassica rapa (a close relative of B. napus) yields up to 40% stearic acid 60 in the seed oil and has been already tested under field conditions [153]. In approaches which promise success, special fatty acid variants, which previously could not be realized in rapeseed, have been developed by gene technology. In this way, success was obtained in establishing the synthesis of short- and medium-chain saturated fatty acids (chain lengths of 8–14 carbon atoms), which are of special interest for oleochemistry and which could only be obtained previously from imported tropical fats (coconut, palm kernels) [154, 155]. Most advanced is the development of “high lauric acid rapeseed” with about 40–50% lauric acid by Calgene (CA, USA), which depends on the transfer of a thioesterase gene from the Californian bay (Umbellularia californica) and which has been already commercially planted [141, 154]. There is a constant demand for high erucic acid rapeseed oil for industrial use. Here, breeding is devoted to increasing the fraction of this very long chain fatty acid well above the current maximum of 55–60% 3 a. It has been known for a long time that, because of the nonoccupation of the middle of the three triacylglycerol positions by erucic acid 3 a, a (theoretical) maximum of 67% cannot be exceeded [149]. However, partial success was achieved recently when transgenic rape forms were developed with varying contents of trierucin (trierucoylglycerol) in the seed oil by transfer of the gene for sn-2-acyltransferase (lysophosphatidic acid acyltransferase, LPAAT) from different Limnanthes species (meadowfoam) and by inhibition of the inherent LPAAT of rapeseed [141, 156].

8.5.3.3 Sunflower In addition to the conventional sunflower oils, which exhibit a high content of linoleic acid (4 a), HO types were developed experimentally some time ago by mutagenesis [126, 157]. Furthermore, forms with increased proportions of saturated fatty acids, which could provide advantages for margarine production (Table 8.2), have been produced by mutagenic treatment [158]. However, the industrial use of HO sunflower oil requires that the content of saturated fatty acids should be as low as possible. Breeding has already reduced the content of stearic acid 60 to 1.5%, which adversely affects the solidification temperature and the cloud point. Under favorable climatic and cultivation conditions, a stable proportion of 90% 1 a with a concurrently reduced content of stearic acid 60 could be achieved from current HO sunflower lines or hybrids developed in this way [144]. In contrast for nonfood purposes in Germany, economic reasons de-


mand at least 83% oleic acid 1 a in the product in order to avoid additional purification steps and thus increase the advantage to this production, against 1 a derived from the competing raw material, beef tallow [159].

8.5.3.4 Peanut In the case of the peanut, an HO mutant (breeding line F435 from the University of Florida) was found in the available varieties which was then used to breed varieties which provide an oil with high oxidative stability [142, 160].

8.5.3.5 Linseed If the possibilities for the use of linseed oil in the nonfood areas are considered, its main uses are in the production of dyes, coatings, and linoleum [135]. On the other hand, in the oleochemical area, the high reactivity of the polyene structure of linseed fatty acids results in a pronounced sensitivity towards autoxidation of products based on linseed oil as well as complex reaction pathways which lead to poorly defined products [161]. From this point of view, breeding efforts are being made to improve the variability of linseed oil in respect of its fatty acid and triglyceride composition in order to provide new, specific oil qualities [162]. Here too, the classical approach of mutagenesis has also been used to breed new linseed varieties with a linolenic acid 5 a content of less than 5% (linola quality) [143, 163] or with an increased palmitic acid content in the oil [164]. 8.5.4 Concluding Remarks on the Use of Gene Technology

The preceding presentation clearly illustrates that gene technology is a very suitable breeding instrument in order to induce new genetic variation. In the ideal situation, it allows the breeder to introduce totally new qualities into cultivated plant varieties with greater precision without impairing the performance capabilities of the respective genotype. There are certain barriers to the realization and use of gene technology because of the poor acceptance by some end users; this applies especially to the nutritional and feed area (novel food, novel feed). However, since the novel products do not enter the human food chain directly, it is not surprising that the initial applications of modern bio- and gene technology methods are found in the technical and chemical area, where they provide vegetable raw materials of improved quality and yield. In this way, important but limited raw material resources can be saved for future generations. To what extent these “new” plant types achieve practical relevance depends on economic factors. Thus, from the viewpoint of industry, demand will only be generated if new raw materials of vegetable origin are available in sufficient quantities at competitive prices, economically viable isolation of the relevant components is possible, and they are held in a higher esteem and preference to alternatives which come, for example, from the petrochemical industry.

281

282


8.6 Future Prospects

After years of relative stagnation, the synthesis of novel fatty compounds based on oils and fats has made important advances. With the breeding of new oil plants – including the use of gene technology – numerous fatty compounds of adequate purity are now available which makes them attractive for synthesis. The use of modern synthetic methods together with enzymatic and microbiological methods has lead to an extraordinary expansion in the potential for the synthesis of novel fatty compounds, which are selectively modified in the alkyl chain. These are now being investigated for their action, properties, and possibilities for new applications. However, numerous synthetic problems remain unsolved and solutions must be found in the coming years. Chemists, biotechnologists, and plant breeders are all challenged to continue development of the advances made in recent years in an integrated, interdisciplinary approach and thus prepare the way for oils and fats to be increasingly used as renewable raw materials in the chemical industry.

Acknowledgments

We thank the Departments of Bildung und Forschung, and Ernährung, Landwirtschaft und Forsten of the German government, as well as the companies Bayer AG, Henkel KGaA, Harburger Fettchemie, Brinckmann & Mergel GmbH, Hoechst, BASF AG, Condea Chemie GmbH, Süd-Chemie AG, and Wella AG for inspirational and material support of our work.

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9 Industrial Development and Application of Biobased Oleochemicals Karlheinz Hill

9.1 Introduction

Vegetable oils and fats are important constituents of human and animal foodstuffs. Some grades are used industrially and, together with carbohydrates and proteins, are numbered among renewable resources compared with fossil and mineral raw materials, whose occurrence is limited and finite. For new products price, performance, and product safety are equally important criteria and have a correspondingly high importance at the start of product development. To ensure high product safety for consumers and the environment renewable resources have often been shown to have advantages compared with petrochemical raw materials and can, therefore, be regarded as being the ideal raw material. Results from oleochemistry show that the use of vegetable fats and oils enables the development of competitive, powerful products which are both consumerfriendly and environmentally-friendly. Products from recent developments fit this requirement profile. In polymer applications derivatives of oils and fats, for example epoxides, polyols, and dimerization products based on unsaturated fatty acids, are used as plastic additives or components for composites or for polymers such as polyamides and polyurethanes. In the lubricant sector oleochemically-based fatty acid esters have proved to be powerful alternatives to conventional mineral oil products. For home and personal care applications a wide range of products, for example surfactants, emulsifiers, emollients, and waxes based on vegetable oil derivatives has proved to provide extraordinary performance benefits to the endcustomer. Selected products, for example the anionic surfactant fatty alcohol sulfate, have been investigated thoroughly with regard to their environmental impact compared with petrochemical based products, by life-cycle-analysis. Other product examples include carbohydrate-based surfactants and oleochemical based emulsifiers, waxes, and emollients.


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9 Industrial Development and Application of Biobased Oleochemicals

9.2 The Raw Materials [1–3]

The sources of oils and fats are a variety of vegetable and animal raw materials (e.g. tallow, lard), with the vegetable raw materials soybean, palm, rapeseed, and sunflower oil being the most important in terms of the amounts involved (Fig. 9.1). Of approximately 125 million tonnes of fats and oils produced worldwide in 2003, by far the largest share was used in human foodstuffs. For oleochemistry 17.4 million tonnes were available. The composition of the fatty acids contained in the oil (fatty acid spectrum) determines the further use of the oils. Special attention must be given to coconut oil and palm kernel oil (lauric oils) because they contain a high proportion of fatty acids of short or medium chain length (mainly 12 and 14 carbon atoms – C12, C14). For example, these are particularly suitable for further processing to surfactants, for washing and cleansing agents, and to cosmetics. Palm, soybean, rapeseed, and sunflower, and animal fats such as tallow contain mainly long-chain fatty acids (C18, saturated and unsaturated) and are used as raw materials for polymer applications, surfactants, and lubricants. Figure 9.2 shows the composition of a typical lauric oil (coconut oil) compared with that of sunflower oil. Fats and oils are available in large amounts. In recent years the amounts produced have increased continuously by approximately 3% per year. Price development – similar to crude oil – has not been constant, but has sometimes undergone drastic changes. In parallel with the price increases in crude oil in the seventies caused by shortage of supply (oil crisis), price jumps in the fats and oils sector have also occurred; this has been particularly noticeable for coconut oil which in those days was the most important source of short-chain fatty acids. In this market palm kernel oil, a second source of short-chain fatty acids,

Fig. 9.1 World production of oils and fats in 2003 (in million tonnes) and main uses (source: Oil World).

9.3 Ecological Compatibility

Fig. 9.2 Composition of coconut and sunflower oil.

has had a stabilizing effect on raw material availability and price. In the medium and long term it must be assumed that fats and oils will be offered at competitive prices. The increasing demand will be met by the amounts produced, which are also increasing [3, 4].

9.3 Ecological Compatibility

On the basis of on results from cycle analyses (see Chapter 9.4.3.1) and selected ecological and toxicological studies one can assume that products based on renewable resources are usually more ecologically compatible than petrochemical-based substances – an important criterion in the development of a new product, just as price and performance are [5]. This general assumption must be proven for each new product, however, so ecological compatibility plays a decisive role in all research and development projects. Basically, it covers two different aspects – remaining in the environment and the effects on the environment (Table 9.1). A variety of criteria are used to evaluate these two aspects. By exposure analysis the expected environmental concentration of a particular substance (in wastewater, in exhaust gases, or in a sewage treatment plant) is estimated, taking into consideration the amount of the substance produced and its biodegradation behavior. The effect on the environment, e.g. toxicity to organisms such as fish, algae, or microorganisms, is determined by a series of standardized testing methods. The two results are compared with each other. If the expected environmental concentration of a particular substance is less than the amount at which negative effects can no longer be determined then the product

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Table 9.1 Evaluation of environmental compatibility of chemicals. Method Basic information – Environmental fate – Environmental effect Criteria – Environmental concentration – Ecotoxicological concentration Evaluation

Result

Biodegradation tests Ecotoxicity tests Exposure analysis, Biodegradation Analysis of effects Comparison PEC vs. PNEC

PEC: Predicted environmental concentration PNEC: Predicted no effect concentration PEC < PNEC: environmentally compatible

is ecologically compatible [6]. Apart from ecological investigations, toxicological tests and microbiological and dermatological investigations are also carried out. In the framework of a successful marketing strategy all data relevant to product safety for the environment and the consumer are evaluated at each stage of product development (selection of raw materials, development of test formulations and application tests, process development, development of packaging, testing consumer satisfaction in test markets).

9.4 Examples of Products

Oils and fats are triglycerides which typically consist of glycerin and saturated and unsaturated fatty acids. There are a few exceptions to this rule, for example castor oil, a glycerol triester of 12-hydroxyoleic acid (ricinoleic acid). Triglycerides have two chemically reactive sites – the double bond in the unsaturated fatty acid chain and the acid group of the fatty acid chain. In product development based on triglycerides most derivatization reactions are performed out at the carboxyl group (> 90%) whereas oleochemical reactions involving the alkyl chain or double bond amount to less than 10% (Fig. 9.3). For most other uses oils and fats must be split into the so-called oleochemical base materials – fatty acid methyl esters, fatty acids, glycerol, and, as hydrogenation products of the fatty acid methyl esters, fatty alcohols (Fig. 9.4) [1]. In the

Fig. 9.3 Reactive sites in triglycerides.


Fig. 9.4 Industrial processing of natural oils and fats and selected product derivatives.

text below innovative products derived from glycerides, fatty acids, or fatty alcohols are discussed – oleochemicals for polymers, esters for lubricants, and surfactants, emulsifiers, and emollients for home and personal care applications. 9.4.1 Oleochemicals for Polymer Applications

The use of oleochemicals in polymers has a long tradition. One can differentiate between their use as polymer materials, for example linseed oil and soybean oil as drying oils, polymer stabilizers and additives, for example epoxidized soybean oil as plasticizer, and building blocks for polymers, for example dicarboxylic acids for polyesters or polyamides (Table 9.2) [7]. Considering the total market for polymers of approximately 150 million tonnes in 1997 the share of oleochemical based products is relatively small – or, in other terms, the potential for these products is very high. Without doubt there is still a trend in the use of naturally derived materials for polymer applications, especially in niche markets. As an example, the demand for linseed oil for production of linoleum has increased from 10,000 tonnes in 1975 to 50,000 tonnes in 1998 (coming from 120,000 tonnes in 1960!) [8 a]. Epoxidized soybean oil (ESO) as a plastic additive has a relatively stable market of approximately 100,000 tonnes/year [8 b]. A few years ago research was undertaken to use oleochemicals to build up matrices for natural fiber reinforced plastics [9]. The use of natural fibers, for example flax, hemp, sisal, and yuca is of increasing interest for a variety of applications, among them the automotive and public transportation industries, in which the composites could be used in door pockets, covers, and instrument panels, and for sound insulation [9 a]. Other applications could be in the manu-

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9 Industrial Development and Application of Biobased Oleochemicals Table 9.2 Oleochemicals for polymers – selected examples [7].

Polymer materials – polymerized soybean oil, castor oil – polymerized linseed oil Polymer additives – epoxides – soaps (Ba/Cd, Ca/Zn) – fatty acid esters, – amides, waxes Building blocks for polymers – dicarboxylic acids – ether-/ester polyols

Product/use

Source

Drying oils Linoleum

Soybean oil, castor oil Linseed oil

Stabilizers, plasticizers Stabilizers Lubricants

Soybean oil Stearic acid Rapeseed oil

Polyamides, polyesters, alkyd resins, polyurethanes

Tall oil, soybean oil, castor oil, sun-flower Oil, linseed oil, oleic acid

facture of furniture. In this field Cognis was coordinating a research project, funded by the Federal Ministry of Food, Agriculture and Forestry (BML) and the National Agency for Renewable Resources (FNR) – project partners being the German Aerospace Center (DLR) and Wilkhahn. The objective was the development of a matrix-system with a high content of renewable raw materials (70– 75%) and comparable or better performance compared with purely petrochemical based matrices. Various oleochemical based monomers, for example epoxidized oils, maleinated oils, polyols and amidated fats were investigated and tested, including manufacture of prototype products based on epoxidized oil (Tribest) [9 b]. In the meantime Alstom has used this technology in the manufacture of urban public transport vehicles (Hamburger Hochbahn) [9 c]. Oleochemical-based dicarboxylic acids – azelaic, sebacic, and dimer acids (Figs. 9.5 and 9.6) – amount to approximately 100,000 tonnes/year as compo-

Fig. 9.5 Building blocks for polymers based on natural oils.


Fig. 9.6 Dimerization of unsaturated fatty acids.

nents for polymers. This is approximately 0.5% of the total dicarboxylic acid market for this application, in which phthalic and terephthalic acids represent 87%. The chemical nature of these oleochemical derived dicarboxylic acids can alter or modify condensation polymers, and therefore will remain a special niche market area. Some of these special properties are elasticity, flexibility, high impact strength, hydrolytic stability, hydrophobicity, lower glass transition temperatures, and flexibility [10]. The crucial reactions in the development of building blocks for polymers based on oils and fats are caustic oxidation, ozonolysis, dimerization, (aut)oxidation, epoxidation, and epoxy ring opening. All of these reactions except biooxidation [10 b] are performed on the double bond of an unsaturated fatty acid or glyceride. Figure 9.5 summarizes the end products and areas of application derived from these reactions. In the following text recent developments in the field of diols and polyols for polyurethanes will be presented in more detail [11].

9.4.1.1 Dimerdiols Based on Dimer Acid [11, 12] Dimerization of vegetable oleic acid or tall oil fatty acid (TOFA) yields dimer acids, originally introduced in the 1950s by General Mills Chemicals and Emery (both now Cognis Corporation). The reaction is very complex resulting in a mixture of aliphatic branched and cyclic C36-diacids (dimer acid) as the main products, besides trimer acids and higher condensed polymer acids and a mixture of isostearic acid and unreacted oleic and stearic acid. Hydrogenation of dimer acid methyl ester or dimerization of oleyl alcohol leads to dimer alcohols (dimer diols) (Fig. 9.6). Oligomers based on dimer diol are industrially manufactured by acid-catalyzed dehydration of dimer diol. The reaction can be easily monitored by determination of the amount of water produced. Oligomers in the molecular weight range 1000–2000 are commercially available by this route

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Table 9.3 Specificaitons of high molecular weight aliphatic diols [11].

Outer appearance Hydroxyl value Viscosity (25 8C, mPas) Melting Point (8C) Composition – monomer (%) – dimer (%) – trimer (%) Trademark a)

Dimer diol a)

Dimer diol a)

12-Hydroxystearyl alcohol

1,10-Decane diol

Yellow liquid 180–200 3500–4300 –

Colorless liquid 180–210 1800–2800 –

White flakes 345–360 Solid 61–65

White flakes 625–645 Solid 68–73

13 68 19 Sovermol 650NS

2 >96 2 Sovermol 908

– – – Sovermol 912

– >98 – Sovermol 110

Molecular weight = 1000–2000 by oligomerization of dimer diol

(Table 9.3). Another method used to produce oligomers is the transesterification of dimer diol with dimethyl carbonate. The resulting dimer diol polycarbonate has a average molecular weight of 2000. Both types of oligomer, ethers and carbonates, are more chemically stable than dimer diol polyesters. Because of their improved stability toward hydrolysis and oxidation dimer diol polyethers (and dimer diol polycarbonates) are used as soft segments in the preparation of thermoplastic polyurethanes. Polyurethanes prepared from these oleochemical building blocks are very hydrophobic and have the expected stability. The products were almost unaffected when stored either in 60% sulfuric acid or 20% sodium hydroxide solution at 60 8C for 7 weeks. There was no significant change in the weight of the testing sticks. For comparison, ester-based polyurethanes used as standards were destroyed completely under these testing conditions after one week (sulfuric acid) and two weeks (sodium hydroxide), respectively. Soft segments based on dimer diol ethers are used to prepare saponification-resistant TPU-seals, which withstand contact with aggressive aqueous media at elevated temperatures. A typical field of application is in nutrition technology [13].

9.4.1.2 Polyols Based on Epoxides [13, 14] Low-molecular-weight liquid epoxy polyol esters or ethers which can be used as polyols for polyurethane systems are obtained by reaction of epoxidized oils with low-molecular-weight mono- or polyfunctional alcohols or acids. Depending on the reaction conditions either polyols with high OH-functionality (complete reaction) or epoxy polyol esters with remaining epoxy groups (partial conversion) are obtained (Fig. 9.7). Although hydroxyl-functional triglyceride oils have found applications in casting resins and adhesives the carboxyl groups of the triglyceride backbone are not fully resistant to hydrolytic attack, particularly by alkali. To overcome this specific behavior fatty acids are used as starting oleo-


Fig. 9.7 Oleochemical polyols for polyurethanes (brand name: Sovermol).

chemicals in the preparation of (epoxy) polyols. In principal three categories of product are obtained by this route – fatty acid derivatives with reactive OHgroups in the ester position only, e.g. glycerol monostearate, fatty acid derivatives with reactive OH-groups in the ester position and in the hydrocarbon chain, e.g. glycerol monoricinoleate, and fatty acid derivatives with reactive OHgroups in the hydrocarbon chain only. These new polyols are of low molecular weight and relatively low viscosity. They have outstanding hydrolytic stability against both alkali and acids and very high chemical resistance toward corrosive solvents such as super fuel. In addition they have significantly improved mechanical properties compared with hydroxyl functional oils after reaction with aliphatic isocyanates [11]. Oleochemical polyols have an average molecular weight of 250 to 2500. Because of their relatively low viscosity and compatibility with methylene di(phenylisocyanate) (MDI) they are particularly suitable for solvent-free, two pack, full solids polyurethane systems, to be applied as thin decorative or protective coatings by brush, roller or spraying. They can also be applied in thick coatings, bearing even high filler loads. In industrial flooring applications, self leveling polyurethane or epoxy/polyurethane multilayer systems have good chemical and mechanical properties and benefits such as minimum shrinkage, high mechanical strength and durability, and favorable cost of installation. They are widely used for wear and crack-resistant floorings on parking decks, for concrete protection in assembly areas, and in large kitchens, slaughterhouses, and grocers, because of the ease of cleaning. Oleochemical polyols can also be used to bind porous filler materials, for example perlite, and rubber particles for applications of composites in construction, soil protection, sport tracks, and playing fields [8, 11]. 9.4.2 Biodegradable Fatty Acid Esters for Lubricants [15]

Apart from being used as “bio-diesel”, fatty acid esters, which are obtained from fatty acids and alcohols, are becoming increasingly interesting as biodegradable replacements for mineral oils. In some application areas, for example chain saw

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oil, gearbox oils, hydraulic oils, and lubricants for crude oil production these oleochemical products have already proved themselves. Esters for lubricant applications are divided into five groups: monocarboxylic acid esters (monoesters), dicarboxylic acid esters (diesters), glycerol esters, polyol esters, and complex esters. Monoesters are obtained by reacting carboxylic acids with alkyl chain lengths from C8 to C22 and branched or linear alcohols. Typical diesters are obtained, for example, from adipic, sebacic, azelaic, or dimer fatty acids by reaction with butanol, ethyl hexanol, isodecanol, isotridecanol, or Guerbet alcohol (see Chapter 9.4.4.3). Using renewable resources as a base, sebacic acid is obtained from castor oil by oxidation with lead(II) oxide as catalyst. Azelaic acid and dimer fatty acids are obtained from oleic acid by technical processes – the first by ozone cleavage, with pelargonic acid being formed as a coupling product, the latter by thermal dimerization (see also previous section). Although the diesters already have excellent lubricating properties, their thermal stability is surpassed by the polyol esters. These products are based on polyols with a quaternary carbon atom – for this reason glycerol esters form a separate class of products (Fig. 9.8). Complex esters are formed by esterification of polyols with mixtures of mono, di, and tricarboxylic acids and are oligomer mixtures; from a technical application viewpoint they are characterized by their high shear stability [15 a]. The decisive factor is that the specially designed fatty acid esters which are used as replacements for mineral oil products not only have ecologically compatible properties but also comparable or even better performance than that of conventional products. That this is possible can be demonstrated very clearly by an example from crude oil production. In coastal drilling (e.g. in the North Sea) the demands placed on the lubricants (drilling fluids) are particularly high. The drilling fluid is pumped to the surface together with the drill cuttings and after coarse separation disposed of directly into the sea. In addition to the good lubricating effect the biodegradability assumes particular importance in this application. A specially developed fatty acid ester (Petrofree) not only fulfils the re-

Fig. 9.8 Polyols used for the manufacture of complex esters.

9.4 Examples of Products Table 9.4 European potential market for biodegradable lubricants (1000 tonnes year–1) a). Application

Total

Biodegradable lubricants

Automotive oils Hydraulic oils Turbine oils Compressor oils Industrial gear oils Metal working oils Demolding oils Chainsaw oils Process oils Lubricating greases

2305 750 200 65 200 500 110 60 600 100

250 200 20 25 10 10 110 60 200 100

a)

According to Reference [15b].

quirement of biodegradability but also has a better lubricating effect than products based on mineral oils [16]. Current developments include the use of specially designed fatty acid esters in a wide range of applications as biodegradable lubricants, and environmentally friendly alternatives are available for almost all mineral oil-based products. In Europe, the long-term potential is estimated to be 10–20% of the total market (500,000–1,000,000 tonnes/year; Table 9.4) [15 c]. In 1997 40,000 tonnes of biodegradable lubricants were sold in Germany alone (4.5% of the total market) [15 c]. An increase of this share is the objective of a variety of measures taken by government and other authorities. The success of these efforts will finally also depend on statutory regulations which should govern the use of environmentally-friendly products [17]. 9.4.3 Surfactants and Emulsifiers Derived from Vegetable Oil

The basic manner in which surfactants act is determined by their structure. With their hydrophilic head and hydrophobic tail, surfactant molecules interpose themselves between water and water-insoluble substances. By enriching themselves at the boundaries which water forms with air or oil they lower its surface tension; as ingredients in soaps and washing agents they make contact with soiled material in this way. When dissolved in water at high concentrations these molecules group themselves together to form spherical structures (micelles); their inwards-pointing hydrophobic groups surround soil particles and keep these in solution. Surfactants are usually classified as anionic, cationic, nonionic, or amphoteric, depending on the type and charge of the hydrophilic groups (Fig. 9.9) [18 a].

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Fig. 9.9 Production of surfactants and examples of products.

Surfactants are used in a wide range of fields. By far the most important fields of application are the washing and cleansing sectors and in textile treatment and cosmetics; these use more than 50% of the total amount of surfactants. Surfactants are also used in the food sector, in crop protection, in mining, and in the production of paints, coatings, inks, and adhesives. The basic manufacturing routes to important surfactants are listed out in Fig. 9.9. It is true that the most important surfactant on the basis of the amount produced, apart from soap, is still the petrochemical-based alkyl benzene sulfonate; in recent years, however, a continuous trend toward surfactants based on renewable resources has become apparent. The total worldwide market amounts to approximately 19.2 million tonnes (2000, including soap). The amounts involved, broken down Table 9.5 Global surfactant consumption in 2000 a). Surfactant class

1000 tonnes

Soap Anionic surfactants – Alkyl benzene sulfonates – Fatty alcohol ether sulfates – Fatty alcohol sulfate Nonionic surfactants – Alcohol ethoxylates – Alkylphenol ethoxylates Cationic surfactants Amphoteric surfactants Others (including carbohydrate-based surfactants)

8800

a)

Soap, Perf., Cosmet., November 2000, p. 51; Colin A. Houston and Associates

3400 1000 500 800 700 810 180 2900


Fig. 9.10 Worldwide surfactant market.

into the individual surfactant classes, are summarized in Table 9.5; Fig. 9.10 shows the regional distribution [18 b].

9.4.3.1 Fatty Alcohol Sulfate (FAS) [5 a, b] Fatty alcohol sulfate has been known for a long time and has already been used as surfactant in a variety of products, for example detergents. It is produced directly from fatty alcohol by reaction with sulfur trioxide (SO3) gas (1–8% v/v in air or nitrogen) in a falling-film reactor. The crude product is then neutralized with aqueous sodium hydroxide, using a buffer if necessary (Fig. 9.11). Some forms of the product, for example aqueous solutions or granulates, are typically produced according to the requirements of the market. Fatty alcohol sulfates are readily biodegradable (no metabolites from the degradation) under both aerobic and anaerobic conditions. Because FAS can be produced either from vegetable oil-based or petrochemical-based fatty alcohol (Fig. 9.9), both types have been evaluated in a life-cycle analysis with a positive overall result for the natural product. For vegetable

Fig. 9.11 Synthesis of fatty alcohol sulfate (FAS).

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based fatty alcohol sulfate the analysis starts with harvesting of the oil fruits (palm kernels or coconuts) and their processing to isolate the desired plant oil. Subsequent transesterification and hydrogenation of the methyl ester intermediates leads to the fatty alcohols, which are finally sulfated to produce the desired product. On the basis of on this analysis the environmental impact of vegetable oil-based fatty alcohol sulfate compared with the petrochemical based product is: · 70% less use of fossil resources · 50% less emission to the atmosphere · 15% less waste · 50% more emission to water (low toxic waste water from small, decentralized oil plants)

9.4.3.2 Acylated Proteins and Amino Acids (Protein–Fatty Acid Condensates) [19] In the development of protein–fatty acid condensates it was possible to combine the renewable resources fatty acids (from vegetable oil) and protein, which can be obtained both from animal waste (leather) and from many plants, to construct a surfactant structure with a hydrophobic (fatty acid) and a hydrophilic (protein) part (Fig. 9.12). This was achieved by reacting protein hydrolyzate with fatty acid chloride under Schotten–Baumann conditions using water as solvent. The products obtained have an excellent skin compatibility and also a good cleaning effect (particularly on the skin) and, in combination with other surfactants, lead to an increase in performance. For instance, even small additions of the acylated protein hydrolyzate improve the skin compatibility of other products. This protective effect could be because of the amphoteric nature of the product. There is an interaction between the protein–fatty acid condensate and skin collagen. This could lead to the formation of a protective layer, which reduces excessive attack of surfactants on the upper layers of the skin, their strong degreasing effect, and the direct interaction of anionic surfactants with the skin. Comparable reaction conditions have been used to develop acylated amino acids, and acyl glutamates, in particular, have found broad uses in recent years (Fig. 9.13). In the personal care market, fatty acid derivatives of proteins and amino acids (glutamic acid) are mainly used in mild shower and bath products, mild shampoos, surfactant-based face cleansers, cold-wave preparations and fixatives, baby wash formulations, and special emulsifiers for “leave-on” products.

Fig. 9.12 Structure of protein hydrolyzate fatty acid condensates.


Fig. 9.13 Synthesis of acyl glutamate.

9.4.3.3 Carbohydrate-based Surfactants – Alkyl Polyglycosides [20] The development of surfactants based on carbohydrates and oils is the result of a product concept which is based on the exclusive use of renewable resources. In industry saccharose, glucose, and sorbitol, which are available in large amounts and at attractive prices, are used as the preferred carbohydrate raw materials. The selective functionalization of saccharose and sorbitol with fatty acids for construction of a perfect amphiphilic structure cannot be realized by simple technical processes, because of the polyfunctionality of the molecule. This is why the products offered on the market contain different amounts of mono-, di-, and tri-esters and are, therefore, only suitable for particular applications, e.g. as emulsifiers for foodstuffs and cosmetics or, for the sorbitan esters, also in technical branches such as explosives and in emulsion polymerization. The ideal raw material for selective derivatization is glucose. Reaction with fatty alcohol produces alkyl glucosides; N-methylglucamides are prepared by reductive amination with methylamine and subsequent acylation. Both products have proved to be highly effective surfactants in washing and cleansing agents. The alkyl glucosides have also established themselves in the cosmetic products sector, as auxiliaries in crop protection formulations, and as surfactants in industrial cleansing agents. On the basis of yearly production they can already be regarded as the most important sugar surfactants. Alkyl polyglycosides have been known for a long time but only in the 1990s, after several years’ research work, reaction conditions were developed, which enabled manufacture on a commercial scale. The structure on which these compounds are based corresponds exactly to the surfactant model described above. The hydrophobic (or lipophilic) hydrocarbon chain is formed by a fatty alcohol (dodecanol/tetradecanol) obtained from palm kernel oil or coconut oil. The hydrophilic part of the molecule is based on glucose (dextrose) obtained from starch (Fig. 9.14).

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Fig. 9.14 Synthesis of alkyl polyglycosides.

The chemical challenge to process technology was to find reaction conditions which enabled the fatty alcohol to react directly with glucose on a commercial scale and at acceptable cost. To develop a method that was as environmentally friendly as possible the use of solvents was rejected. Method development was successfully completed and Cognis was the first company to offer alkyl polyglycosides on an industrial scale of the required quality. Currently Cognis has an annual capacity of approx. 50,000 tonnes available for manufacture of this class of compound (other manufacturers include Kao, Seppic, ICI, and LG). By combining vegetable oil and sugar as raw materials it has for the first time become possible to offer commercially important amounts of nonionic surfactants which are completely based on renewable resources (Fig. 9.15).

Fig. 9.15 Manufacturing processes for alkyl polyglycosides.


Unique properties had previously been observed for alkyl polyglycosides, particularly in combination with other surfactants. For example, use of alkyl polyglycosides in light-duty detergent or shampoo formulation means that the total amount of surfactant can be reduced without sacrificing performance. In other combinations a particularly stable and fine foam can be produced which protects sensitive textiles during the washing process. Toxicological and ecological laboratory investigations have also produced favorable results. Alkyl polyglycosides have a good compatibility with the eyes, skin, and mucous membranes and even reduce the irritant effects of surfactant combinations. On top of this they are completely biodegradable, both aerobically and anaerobically. The relatively favorable classification (for surfactants) as class I under the German water hazard classification (WGK I) is a consequence of this.

9.4.3.4 Alkyl Polyglycoside Carboxylate [21] Alkyl polyglucoside carboxylate (INCI name sodium lauryl glucose carboxylate (and) lauryl glucoside, Plantapon LGC Sorb), is a new anionic surfactant with an excellent performance in personal care cleansing applications. In shampoo and shower bath formulations the anionic surfactant has good foaming behavior. In body wash applications it improves sensorial effects. These properties make Plantapon LGC Sorb suitable for several cosmetic applications, e.g. mild facial wash gel, mild baby shampoo, mild body wash for sensitive skin, wet wipes, and special sulfate-free shampoo applications. A new industrial process based on reaction of sodium monochloroacetate with aqueous alkyl polyglycoside (without additional solvents) enables economical and ecologically favorable manufacture of this product (Fig. 9.16).

9.4.3.5 Polyol Esters [22] Polyglycerol Esters Emulsifiers based on glycerol or polyglycerol are a class of product well known commercially and used particularly in products for personal care and in food production. Further development will focus on the design and

Fig. 9.16 Synthesis of alkyl polyglycoside carboxylate.

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Table 9.6 Properties of selected polyglycerol esters as emulsifiers for personal care and food technology. INCI name

Trade name

Properties

Polyglyceryl-3-diisostearate Polyglyceryl-2-dipolyhydroxystearate PEG-4-polyglyceryl-2-stearate Pentaerythritol distearate Polyglycerol stearate (E 475) Polyglycerol polyricinoleate (E 476)

Lameform TGI Dehymuls PGPH Lamecreme DGE 18 Cutina PES Polymuls 4G Polymuls PGPR

W/O – emulsifier MW = 725; pale yellow liquid W/O – emulsifier for lotions and creams MW >3000; yellow, cloudy, viscous (ca. 15 000 mPas, Brookfield, 23 8C) O/W – emulsifier Consistency wax with sensorial benefits Food – emulsifier Food – emulsifier

optimization of specific emulsifier formulations. The combination of different types of emulsifier can lead to new uses for mono- and diglycerides. Polyglycerol esters are obtained by esterification of polyglycerol, which is produced by oligomerization of glycerol under basic conditions, with fatty acids. The properties of the different products can be adjusted by selection of the type of polyglycerol used and the chain lengths and chain type of the fatty acids used. Selected types of product of different molecular weight are shown as an example in Table 9.6. For example, Dehymuls PGPH is recommended for use in body lotions. It leaves the skin with a smooth, non greasy, and well cared for feeling, spreads easily, and is absorbed quickly. Pentaerythritol Esters As for glycerol esters, these esters are produced by esterification of pentaerythritol with the desired fatty acids. For example, under defined reaction conditions and use of stearic acid at a defined concentration, pentaerythritol distearate has been recently developed as an off-white wax with a very weak odor (Cutina PES). This type of product is offered as co-emulsifier and consistency factor for cosmetic products with high sensorial elegance and can be applied in a variety of formulations (Fig. 9.17). Emulsifier compound based on polyglycerol ester and alkyl polyglycoside Requirements for modern emulsifiers not only include outstanding performance

Fig. 9.17 Chemical structure of pentaerythritol distearate (main component in Cutina PES).


Fig. 9.18 Capacity increase in cold emulsification processes using Eumulgin VL 75 as emulsifier.

but also compatibility with modern emulsification techniques and balanced sensory feeling. One product which fulfils these requirements is a compound based on glycerin, alkyl polyglycoside and polyglyceryl-2-dipolyhydroxystearate (Eumulgin VL 75). In combination with selected emollients it enables the preparation of O/W emulsions of high quality and stability (small droplet sizes). In addition, because of the liquid appearance of the product, and, as a consequence, the possibility of cold processing, manufacturing time and cost of preparation of emulsions are significantly reduced (Fig. 9.18).

9.4.3.6 Multifunctional Care Additives for Skin and Hair [23] Intelligent combination of alkyl polyglycoside and glyceryl oleate resulted in a new product which combines emulsifying and cleansing properties with outstanding care effects, such as enhancing of the skin lipid layer. This effect is proven in a standardized test by washing the forearm, rinsing, drying, and extracting the lipid layer on the skin with ethanol pads. The lipid content is measured by quantitative analysis of glycerol oleate in the extract (Fig. 9.19). Technical data for the product (Lamesoft PO 65) are summarized in Table 9.7. For application in hair-care products it was found that a compound based on dioctyl ether from coconut or palm kernel oil-based octanol (Cetiol LDO) enables the formulation to be silicone oil-free. Cetiol LDO is a highly efficient hair-care additive and is particularly suitable for the use in hair-cleansing preparations to improve the tactile hair feel and hair gloss. Cetiol LDO, in combina-

Table 9.7 Technical data for Lamesoft PO 65. Application Composition Dry residue Lipid pH value (5%) Viscosity (Brookfield, 23 8C)

Multifunctional care additive for clear and pearlescent cleansing preparations Glyceryl Oleate (lipid), Coco Glucoside (surfactant) 65–70% ca. 30% 3.0–3.5 max. 12 000 mPas

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Fig. 9.19 Evaluation of lipid layer enhancing effect.

tion with wax esters and cationic polymers also has the benefits of silicon-free body-cleansing preparations with regard to improved sensorial skin properties. 9.4.4 Emollients [24] 9.4.4.1 Introduction The physicochemical nature of the oil-phase components in a cosmetic emulsion, the emollients, determines their skin-care effects, such as smoothing, spreading, sensorial appearance. Test methods have been developed to characterize and classify the numerous emollients available on the market, for example

Table 9.8 List of selected emollients. Structure

Emollient

INCI name

Ester

Cetiol A Cetiol LC Eutanol G16 S Cetiol V Cetiol J 600 Myritol 318 Eutanol G 16 Eutanol G Cetiol S Cetiol OE Cetiol CC

Hexyl laurate Coco-caprylate Hexyldecyl stearate Decyl oleate Oleyl erucate Caprylic/capric triglyceride Hexyl decanol Octyl dodecanol Diethylhexyl-cyclohexane Dicaprylyl ether Dicaprylyl carbonate

Guerbet Alcohols Hydrocarbons Ethers Carbonates


silicones, paraffins, and oleochemical-based products. The latter include glycerides, esters, alcohols, ethers, and carbonates with tailor-made structures depending on the performance needed (Table 9.8). With regard to additional effects, however, there is still a demand for new products with unique performance properties.

9.4.4.2 Dialkyl Carbonate One example of a new class of compound in this field is dioctyl carbonate. The product is synthesized by transesterification of octanol and dimethyl carbonate in the presence of alkali catalyst (Fig. 9.20). Dioctyl carbonate (Cetiol CC) is a dry emollient with excellent dermatological compatibility and a comprehensive and convincing performance profile for various applications in the personal care segment. Properties reported are good emulsifiability, outstanding behavior in Deo/AP formulations, solubilizing and dispersing properties for sun care, and, last but not least, the special sensorial feeling given to the final formulation (Table 9.9).

9.4.4.3 Guerbet Alcohols [25] R. C. Guerbet in 1899 discovered the self condensation reaction of alcohols, which, via the aldehyde as an intermediate, lead to branched structures (2-alkyl alcohols) as shown in Fig. 9.21. Starting from fatty alcohols from vegetable sources, for example octanol and decanol, the corresponding C16 and C20 alcohols are produced, 2-hexyldecanol and 2-octyldecanol. The reaction is conducted under alkali catalysis and at high temperatures (>200 8C). Over the years, both

Fig. 9.20 Synthesis of dialkyl carbonates.

Table 9.9 Main properties of dioctyl carbonate. Chemical structure

INCI Spreading value Sensory feeling Skin compatibility Stability Origin Biodegrability

Dicaprylyl carbonate 1600 mm2/10 min Dry, volatile silicone-oil like Excellent Hydrolysis stable Vegetable oil (fatty alcohol) Yes

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Fig. 9.21 Synthesis of Guerbet alcohol 2-hexyldecanol (Eutanol G 16).

products have proven to be efficient emollients, but are also used for other applications, for example as plasticizers or as components of lubricants (Fig. 9.21). 9.5 Perspectives

Examples of recent product innovations from oleochemistry illustrate the successful sustainable development of environmentally compatible and powerful products. It can be assumed that in the future further possibilities for using renewable resources will be intensively investigated. Here the combination of a variety of vegetable raw materials to form new products and intelligent product concepts in order to meet market and consumer needs will be a challenge for research and development. However, this will depend on the scope of implementation of the Bioenergy and Biofuel Strategy as part of the Kyoto protocol, too. The subsidized use of vegetable oils for bioenergy and biofuel production is completely contradictory to their established use in nutrition and to future industrial developments of high value added, oleochemical-based products. Therefore edible oils and fats should not be part of this biomass regulation and subsidies for biofuel and bioenergy in general should be more flexible [26]. 9.6 Trademarks

APG, Cetiol, Cutina, Dehymuls, Emulgade, Eumulgin, Eutanol, Lamecreme, Lameform, Lamesoft, Myritol, Polymuls, Sovermol, and Tribest are registered trademarks of the Cognis group. Petrofree is a registered trademark of Baroid Drilling.

References 1 (a) H. Baumann, M. Bühler, H. Fochem,

F. Hirsinger, H. Zoebelein, J. Falbe, Angew. Chem. Int. Ed. Engl. 1988, 27, 41; (b) A. Behler, M. Biermann, N. Huebner, L. Zander, A. Westfechtel, 95th AOCS Meeting, Cincinnati, USA, 2004.

2 H. Eierdanz (Ed.), Perspektiven nachwach-

sender Rohstoffe in der Chemie, VCH, Weinheim, 1996. 3 (a) B. Brackmann, C. Hager, The statistical world of raw materials, fatty alcohols and surfactants, Proceedings 6th World

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Surfactant Congress, Cesio 2004, Berlin, June 20–23, 2004; (b) P. Renaud, Natural-based fatty alcohols: Completely in line with the future?, Proceedings 6th World Surfactant Congress, Cesio 2004, Berlin, June 20–23, 2004; (c) G. Kreienfeld, G. Stoll, Surfactants in Consumer Products and Raw Material Situation – A Brief Survey. In: K. Hill, W. von Rybinski, G. Stoll (Eds.), Alkyl Polyglycosides – Technology, Properties and Applications, VCH, Weinheim, 1997, p. 225. (a) G. Röbbelen, Pflanzliche Öle als Rohstoffbasis – Potential und Veränderungen in der Verfügbarkeit. In: Tagungsband 3. Symposium Nachwachsende Rohstoffe – Perspektiven für die Chemie, Schriftenreihe des Bundesministeriums für Ernährung, Landwitschaft und Forsten, Landwirtschaftsverlag, Münster, 1994, p. 115; (b) K. Hill, Agro–Food Industry Hi-Tech, 1998, 9[5], 9. (a) F. Hirsinger, F. Bunzel, Ökobilanz von Fettalkoholsulfat – Petrochemische versus oleochemische Rohstoffe. In: H. Eierdanz (Ed.), Perspektiven nachwachsender Rohstoffe in der Chemie, VCH, Weinheim, 1996, p. 228; (b) M. Stalmans, M. et al., Tenside Surf. Det., 1995, 32, 84; (c) M. K. Patel, A. Theiß, E. Worrell, E., Resources, Conservation and Recycling, 1999, 25, 61. J. Steber, Textilveredlung 1991, 26, 348. R. Höfer, Anwendungstechnische Aspekte der Verwendung natürlicher Öle und ihrer Derivate in der Polymer – Synthese und – Verarbeitung, In: H. Eierdanz (Ed.), Perspektiven nachwachsender Rohstoffe in der Chemie, VCH, Weinheim, 1996, pp. 91– 106. (a) B. Schulte, B. Schneider, Linoleum: Traditionelle und moderne Problemlösung für den Fußboden auf Basis nachwachsender Rohstoffe, In: H. Eierdanz (Ed.), Perspektiven nachwachsender Rohstoffe in der Chemie, VCH, Weinheim, 1996, p. 338– 344; (b) M. W. Formo, Industrial Use of Soybean Oil, in Proceedings of the 21st World Congress of the International Society of Fat Research (ISF), The Hague, 1995, P. J. Barnes & Associates (Eds.), pp. 519–527. (a) D. Schäfer, Einsatz und Potential naturfaserverstärkter Kunststoffe in der

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Automobilindustrie, in Gülzower Fachgespräche: Nachwachsende Rohstoffe – Von der Forschung zum Markt, Workshop 25./26. 05. 1998, Fachagentur Nachwachsende Rohstoffe e.V. (Ed.), 1998, pp. 27–47; (b) B. Dahlke, H. Larbig, H. D. Scherzer, R. Poltrock, J. Cell. Plastics, 1998, 34, 361; (c) J. Neubauer, Der Schritt in die Industrialisierung – Nachwachsende Rohstoffe im Schienenfahrzeugbau, Conference presentation at Konstruktionswerkstoffe aus nachwachsenden Rohstoffen 2002, September 2002, Braunschweig. (a) R. Fayter, Technical Reactions for Production of Oleochemical Monomers, In: H. Eierdanz (Ed.), Perspektiven nachwachsender Rohstoffe in der Chemie, VCH, Weinheim, 1996, p. 107–117; (b) D. L. Craft, R. C. Wilson, D. Eirich, Y. Zhang, Candida CYP52A2A promoter and uses in increasing gene expression in yeast for dicarboxylic acid production, PCT Int. Appl. WO2002008412, 2002, (Cognis). R. Höfer, Oleochemical polyols – New Raw Materials for Polyurethane Applications, Proceedings European Coatings Conference, Berlin, 1999. A. Heidbreder, R. Höfer, R. Grützmacher, A. Westfechtel, C. W. Blewett, Fett/Lipid, 1999, 101, 418. J. Möschel, Thermoplastische Polyurethane sowie ihre Verwendung, DE-PS 19512310, 1995, (Parker-Prädifa). (a) B. Gruber, R. Höfer, H. Kluth, A. Meffert, Fat Sci. Technol., 1987, 89, 147; (b) P. Daute, R. Gruetzmacher, R. Höfer, A. Westfechtel, Fat Sci. Technol., 1993, 95, 91. (a) F. Bongardt, A. Willing, J. Synthetic Lubrication, 2003, 20-1 (April), 53; (b) J. Legrand, K. Dürr, Agro–Food Industry Hi-Tech, 1998, 9[5], 16; (c) Th. Mang, Fett/Lipid, 1998, 100, 524. C.-P. Herold (Cognis Deutschland GmbH & Co.KG), personal communication. Bundesministerium für Ernährung, Landwirtschaft und Forsten (Ed.), Bericht über den Einsatz biologisch schnell abbaubarer Schmierstoffe und Hydraulikflüssigkeiten und Maßnahmen der Bundesregierung, 1996, Germany.

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Products: Theory, Technology, Applications, Springer, Heidelberg, 1987; (b) W. Dolkemeyer, Surfactants on the Eve of the Third Millennium, 5th World Surfactant Congress, Cesio 2000, Florence May 29–June 02, 2000. (a) A. Sander, E. Eilers, A. Heilemann, E. von Kries, Fett/Lipid, 1997, 99, 115. (a) M. Biermann, K. Schmid, P. Schulz, Starch/Stärke, 1993, 45, 281; (b) J. Knaut, G. Kreienfeld, Chimica Oggi 1993, 41; (c) K. Hill, W. von Rybinski, G. Stoll (Eds.), Alkyl Polyglycosides – Technology, Properties and Applications, VCH, Weinheim, 1997; (d) W. von Rybinski, K. Hill, Angew. Chem. Int. Ed., 1998, 37, 1328. A. Behler, H. Hensen, W. Seipel, Alkyl Polyglycoside Carboxylate – A New Anionic Surfactant, Proceedings 6th World Surfactant Congress, Cesio 2004, Berlin, June 20–23, 2004. C. Mitchell, A. Ansmann, S. Bruening, U. Issberner, S. Nefkens, Formulation Design by Texture Modifications, in Conference Proceedings 51st SEPAWA Kongress, Würzburg, 2004, pp. 283.

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C. Nieendick, Cosmetics & Toiletries, 2004, 119 (5), 79; (b) W. Seipel, N. Boyxen, Moderne Formulierungskonzepte mit CareEffekten, in Conference Proceedings 51st SEPAWA Kongress, Würzburg, 2004, pp. 70. 24 (a) H. Tesmann, Nachwachsende Rohstoffe in der Kosmetik, in Ref. [2], pp. 31–39; (b) R. Kawa, A. Ansmann, B. Jackwerth, M. Leonard, Parfüm. Kosmet., 1999, 80, 17; (c) Th. Förster, U. Issberner, H. Hensen, J. Surfactants and Detergents, 2000, 3, 345; (d) B. Jackwerth, Cetiol CC – The New Benchmark for Dry Emollients, In-Cosmetics 2000, Barcelona, April 2000. 25 (a) R. C. Guerbet, C. R. Hebd, Seances Acad. Sci., 1899, 128, 5118; (b) K. S. Markley, Fatty Acids (Vol. 2), Interscience Publishers, 1961, New York, p. 1353. 26 H. Sauthoff, Bioenergy and Biofuels – Opportunity or Threat for Oils and Fats Trading?, Handout and Lecture, FOSFA “Contact Day”, London, September 8, 2005.


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Special Ingredients and Subsequent Products 10 Phytochemicals, Dyes, and Pigments in the Biorefinery Context George A. Kraus

10.1 Introduction

The idea of a biorefinery is modeled after the highly successful oil refinery wherein petroleum is converted into gasoline, oil, and monomers such as ethylene and propylene. As a result of several decades of improvements, these petroleum refineries are flexible and efficient [1–3]. They produce high-volume chemicals plus a large number of low volume, high-value materials. For the biorefinery to be successful, it must produce high-volume fuels such as ethanol or biodiesel plus monomers and a portfolio of high-value chemicals for niche markets. An outline of a biorefinery is depicted in Fig. 10.1. The monomers produced by the biorefinery will probably be diols and acids, because the feedstocks are more highly oxygenated than petroleum. Among the high-value products produced by the biorefinery will be phytochemicals, dyes, and pigments. Occasionally these phytochemicals are foods or drugs. Often (e.g.

Fig. 10.1 The biorefinery. Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 2 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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the saponins, phytoestrogens, and the protease inhibitors) the pharmacological potential has only recently begun to be realized.


The fundamentals of a biorefinery already exist in the corn and soybean industries. The corn wet milling industry converts approximately two billion bushels of corn per year into corn oil, starch, corn gluten feed, corn gluten meal, and corn germ meal plus a variety of sugars and other chemicals [4–6]. The basic outline of a corn wet milling plant is shown in Fig. 10.2. In the wet milling process, corn is first soaked in water containing sulfur dioxide. It is then ground and the components are physically separated using a series of centrifuges, screens, and washes. Some of the separated fractions then undergo additional refining steps. In a soybean-processing plant, the soy protein is separated from the oil [7–9] and the crude soybean oil is then purified by degumming to remove lecithin, by refining to remove fatty acids, by bleaching with clay to remove chlorophylls, and by deodorization (steam distillation) to remove phytosterols and tocopherols. This process is outlined in Fig. 10.3.

Fig. 10.2 Corn wet milling.

Fig. 10.3 Soybean processing.

10.3 Phytochemicals from Corn and Soybeans

The deodorizer distillate contains phytochemicals such as phytosterols and tocopherols. The soy protein contains phytochemicals such as saponins and protease inhibitors. Some cooperatives then convert the soybean oil into biodiesel by treating the oil with methanol or ethanol and a catalyst [10, 11]. Biodiesel is formed by a transesterification reaction which is catalyzed by acid or, more commonly, by base. It is the methyl or ethyl ester of a fatty acid. The byproduct of this reaction is glycerin, a hygroscopic triol. Glycerin can be converted into higher-value products by catalysis or biocatalysis [12, 13]. Glycerin can also be converted into 1,3-propanediol which is used in the synthesis of Sorona [14].


Corn and soybeans are, at least initially, the most likely feedstocks for a biorefinery. There is an enormous knowledge base about the fundamental plant science of corn and soybeans [15]. Additionally, the means of production and harvesting of these two crops on a million-acre scale is well documented and is improved each year. I have therefore focused this chapter on describing the phytochemicals, dyes, and pigments from these two crops. Phytochemicals are chemicals from plants. We will focus on chemicals that are reported to have beneficial health effects but are not essential for life. 10.3.1 Phytosterols

Phytosterols are present in both corn and soybeans. They are separated from soybean oil by a steam distillation process and are isolated in the deodorizer distillate with other organic chemicals. The structures of the major phytosterols found in corn and soybeans are depicted in Fig. 10.4. The mixture obtained from soybeans consists of sitosterol (60%), sigmasterol (20%), and campesterol (20%). These sterols are converted into commercially important steroids by microbial fermentation [16]. Biotransformation procedures for the efficient production of androst-4-ene-3,17-dione, androsta-1,4-diene-3,17dione, and testosterone have been reported [17, 18]. Sitosterol has recently been shown to reduce plasma VLDL and LDL (“bad”) cholesterol and increases plasma HDL (“good”) cholesterol [19, 20]. Because approximately 25% of American adults have high cholesterol, the market for this high-value phytochemical may soon increase dramatically. Corn contained dimethyl sterols and monomethyl sterols which amount to approximately 6% of the total sterol content. In corn and soybeans, sterols are also present as esters and as glycosides. In Illinois high oil corn, 53% of the sterols are free sterols, 46% of the sterols are acylated, and 1% of the sterols are present as glycosides [21]. Most of the acylated sterols are esters of linoleic acid.

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Fig. 10.4 Phytosterols from corn and soybeans.

10.3.2 Lecithin

Lecithin, also known as phosphatidylcholine, is available from both corn and soybeans. In soybean oil processing it is separated from crude soybean oil at the degumming step. The structure of lecithin is depicted in Fig. 10.5. Although the structure in Fig. 10.5 shows the phosphatidylcholine unit, commercial lecithin is a mixture of phospholipids in which R1CO2 represents a mixture of saturated fatty acids and R2CO2 represents a mixture of, mostly, unsaturated fatty acids. The major soy phospholipids have been separated by means of liquid chromatography with use of a flame ionization detector [22]. Lecithin is used commercially as an emulsifier. Lecithin also has several beneficial effects at the cellular level. It is involved in cell-membrane function, fat transport, and cholinergic neurotransmission [23]. Lecithin reduces plasma cholesterol levels [24] and provides a source of choline which is beneficial for cognitive development [25].

Fig. 10.5 The structure of lecithin.


10.3.3 Tocopherols

Tocopherols are antioxidants that are present in both corn and soybeans. They are separated from soybean oil in the deodorizer distillate fraction. The structures of the tocopherols are shown in Fig. 10.6. In soybeans the major constituent is c-tocopherol (62%), followed by d-tocopherol (25%), a-tocopherol (12%), and b-tocopherol (1%) [26]. In corn the major component is c-tocopherol, with a-tocopherol second. The total tocopherol content of corn varies as a function of genetics and geography, with a range of 500–1000 mg tocopherols per kilogram of corn oil [27]. The tocopherols are a source of vitamin E. Natural vitamin E is a-tocopherol in which each of the three stereogenic centers has the R configuration. The b, c,

Fig. 10.6 Structures of the tocopherols.

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and d isomers can be converted into vitamin E or used as antioxidants in the animal feed industry [28]. Vitamin E has cardioprotective activity [29]. 10.3.4 Carotenoids

The main pigments in yellow corn are carotenes and xanthophylls. Depending on the growing conditions, corn contains from 20–80 mg kg–1 carotenes and xanthophylls [30]. The main carotenes are b-carotene and b-cryptoxanthin. Their structures are shown in Fig. 10.7. Because of the conjugated polyene structure of carotenes, they are not as stable as other phytochemicals present in corn. They can be readily degraded by oxygen in the air, by a combination of oxygen and sunlight, and also by heat. The time of storage and storage conditions are, therefore, factors in the yield of carotenoids. Carotenoids are a significant dietary source of vitamin A for animals. The efficiency of conversion of carotenes into vitamin A varies with the type of animal. Carotenes are also good antioxidants [31].

Fig. 10.7 Structures of the carotenoids.


Xanthophylls are pigments responsible for color but do not function as precursors to vitamin A. The principal xanthophylls in corn are lutein and zeaxanthin. Their structures also are illustrated in Fig. 10.7. Like carotenes, the xanthophylls are labile in light, heat, and air. They are also good antioxidants. Chromatography-based detection methods have been developed for the carotenes and xanthophylls [32]. Lutein has recently been recognized as an effective drug for treatment of macular degeneration, a disease that affects eyesight [33]. Although marigolds are currently the primary source of lutein, genetic modification of corn may lead to enhanced levels of the compound. 10.3.5 Phytoestrogens

Phytoestrogens are found in soybean protein. Levels of these phytochemicals are approximately 400 mg kg–1 soy protein [34]. Soybeans, particularly tofu, are the major dietary source of isoflavanoids. Isoflavone levels can vary as a function of genetics, growing conditions, and processing techniques. The main phytoestrogens in soybeans are isoflavanoids such as genistein and daidzein. In non-fermented foods these compounds are present mainly as the b-glycosides [35]. The structures of genistein and daidzein are depicted in Fig. 10.8. These phytochemicals are associated with reduced incidence of breast cancer and prostate cancer [36]. Genistein is reported to reduce levels of LDL (“bad”) cholesterol [37]. 10.3.6 Saponins

Saponins are glycosylated terpenes found in several crops, including soybeans. Structures of representative soybean saponins aglycones are illustrated in Fig. 10.9. Soybeans have a saponin content of approximately 6% by weight in whole soybeans. A procedure for producing a purified saponin fraction on an industrial scale was recently patented [38]. Soy saponins have several health benefits. Soy saponins may reduce cholesterol levels in humans by binding to cholesterol, thus preventing its re-adsorption into the blood stream [39]. Saponins have been reported to inhibit accumulation

Fig. 10.8 Structures of genistein and daidzein.

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Fig. 10.9 Structures of saponin aglycones.

of visceral fat, providing a mechanism for controlling obesity [40]. Saponins also bind tightly to bile acids, thereby reducing the risk of colon cancer [41]. Saponins have also been reported to remove toxins and hardened matter from the intestinal tract [42]. Researchers have recently reported an in-vitro effect of saponins on the infectivity of the human immunodeficiency virus [43]. Saponins have also been reported to protect against aflatoxin-induced mutagenicity. They were ranked as more effective than tocopherol or l-ascorbic acid [44]. 10.3.7 Protease Inhibitors

Over the past decade, protease inhibitors, particularly those derived from soybeans, have emerged as potential cancer chemopreventive agents. Soybeans contain significant amounts of protease inhibitors – approximately 6% by weight of total soybean protein is protease inhibitors [45]. Protease inhibitors have also been found in crops such as corn and rice. The inhibitor known as the Bowman–Birk inhibitor, more commonly referred to as BBI, has received the most attention [46]. This inhibitor makes up a significant fraction of the inhibitors in soy protein and is readily available in commercial tofu. The molecular structure of BBI, a polypeptide of molecular weight 8000, has recently been determined [47]. Extensive experimentation has shown that BBI is a potent chymotrypsin and trypsin inhibitor. Although BBI has been effective in animal trials, results of human trials are not yet known. On the basis of public health records in Asia and the United States, there certainly seems to be a connection between soybean consumption and the incidence of breast, colon, and prostate cancers [48]. BBI has been recommended as a radioprotector for normal tissue to enhance radiotherapy [49]. The soybean protease inhibitor SBTI has also been characterized.

References

Corn gluten contains inhibitors with powerful angiotensin I enzyme-converting enzyme inhibitory activity [50]. A 12 kD protease/a-amylase inhibitor was recently isolated from corn seed. CI-4a, a cysteine protease inhibitor in corn endosperm has been purified and characterized [51]. Inhibitors of corn endosperm sulfhydryl protease P-Ia, the main factor for zein degradation during germination, have been isolated and characterized [52].


Phytochemicals are high-value chemicals for niche markets. As methods for their isolation become better defined and as their biological activity in humans is better understood, demand for these chemicals is very likely to increase substantially. There are, moreover, other, less well understood, phytochemicals such as conjugated linoleic acid and other protease inhibitors that could eventually add more value to the biorefinery.

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11 Adding Color to Green Chemistry? An Overview of the Fundamentals and Potential of Chlorophylls Mathias O. Senge and Julia Richter

11.1 Introduction

Chlorophylls, often termed the “pigments of life” are green colored macrocyclic pigments which are the primary photosynthetic pigments. The term chlorophyll, coined by Berzelius in 1838, is derived from Greek and indicates the green of leaves [1]. In fact, as green pigments they are responsible for primary biochemical energy generation in nature and are the only indication of life on earth visible from outer space. Current efforts to switch chemistry from a system based on the use of nonrenewable resources (geological organic carbon deposits) to one based on the utilization of renewable bioresources are one of the cornerstones of efforts often labeled “green chemistry” [2]. Although “green” is taken to imply an environmentally friendly approach, chlorophylls in a literal sense are the green chemical components responsible for beauty in our natural environment (vegetation). If so, is green chemistry really green? At present, no. Even a cursory inspection reveals that all practical efforts aimed at the use of biological materials in chemistry rely solely on the use of nongreen materials. This article aims to describe some of the fundamentals and highlights of chlorophyll chemistry and gives a preliminary assessment whether chlorophylls might be a useful bioresource.


Chlorophylls have been studied for almost 200 years [1] and, although much of their basic chemistry was established almost 100 years ago [3], real breakthroughs in their exact biochemical function were only made in recent decades as a result of X-ray crystallographic analysis both of the reaction center [4] and of light harvesting complexes [5]. Until WW II most studies were concerned Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 2 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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with their basic structural chemistry. This was followed by elucidation of the basic anabolic pathways of chlorophyll in the higher plants, photophysical studies of their function in photosynthesis, and most recently their catabolism. Parallel to studies on chlorophylls a and b from higher plants, recent decades have seen many efforts to elucidate the structure, function, and biosynthesis of photosynthetic pigments from unicellular organisms and photosynthetic bacteria [6]. Chlorophylls have never been used as bulk chemicals for transformation into other compounds on a technical scale. As outlined below, since the middle of the last century phytochlorin and rhodochlorin derivatives have found some use as food additives and antiodor compounds, often in the form of simple plant extracts. During the last twenty years pheophorbide derivatives have been used as starting materials for the synthesis of novel photosensitizers (vide infra) and are under active investigation as compounds for solar energy conversion and hydrogen production. These efforts are still in a developmental stage, however, and require only gram-scale amounts of material [7].

11.3 Chlorophyll Fundamentals 11.3.1 Occurrence and Basic Structures

Chlorophylls and the related bacteriochlorophylls are the ubiquitous pigments of photosynthetic organisms. As such they share common structural principles and functions. They are involved either in light harvesting (exciton transfer) as antenna pigments or in charge separation (electron transfer) as reaction-center pigments. The best-known pigment is chlorophyll a, 1, which occurs in all organisms with oxygenic photosynthesis (Fig. 11.1). In higher plants it is accompanied in a 3 : 1 ratio by chlorophyll b, 3, in which the 7-methyl group has been oxidized to a formyl group. Both compounds typically consist of the tetrapyrrole moiety and a C-20 terpenoid alcohol, phytol. Most compounds are magnesium chelates, but the free base of chlorophyll a, pheophorbide a, 2, is also active in electron transfer. Chlorophylls a and b can be obtained easily from plants or algae and are the focus of this article. Many other, similar photosynthetic pigments occur in nature, however [8]. Approximately one hundred related pigments have now been isolated and all share either a phytochlorin 4 or 7,8-dihydrophytochlorin framework. For example, such compounds include chlorophyll d, 5, from Rhodophytes, bacteriochlorophylls c, 6, d, and e (which are chlorins 9 and have significant variability in their peripheral groups) [6, 9] from Chlorobiaceae and Chloroflexaceae, and bacteriochlorophylls a, 7, and b (true bacteriochlorins 10) found in Rhodospirillales. Other natural pigments are chlorophyll c, bacteriochlorophyll g, and many related compounds with different esterified isoprenoid alcohols. Chemically re-

11.3 Chlorophyll Fundamentals

Fig. 11.1 Naturally occurring chlorophylls and related systems. Small numbers in the chlorophyll a formula indicate the IUPAC numbering scheme. R groups may vary in different organisms or at different stages of development.

lated chlorins have also been found in many oxidoreductases, marine sponges, tunicates, and in Bonella viridis. The deep-sea dragon fish Malacosteus niger even uses a chlorophyll derivative as a visual pigment [10]. Most of these are believed to be derived from chlorophyll which is processed by the plant or animal. 11.3.2 Principles of Chlorophyll Chemistry

Chlorophylls are heteroaromatic compounds and the aromatic character of the underlying tetrapyrrole moiety and the reactivity of the functional groups in the side chains govern their chemistry. Three different classes of tetrapyrrole, differentiated by their oxidation level, occur in nature (Fig. 11.2): porphyrins (8, e.g., hemes), chlorins (9, e.g. chlorophylls), and bacteriochlorins (10, e.g., bacteriochlorophylls). The overall reactivity of chlorophyll, a cyclic tetrapyrrole with a fused five-membered ring, is that of a standard phytochlorin, 4. Such compounds are capable of coordinating almost any known metal with the core nitrogen atoms. Together with the conformational flexibility of the macrocycle and the variability of its side chains, this accounts for their unique role in photosynthesis [11, 12].

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Fig. 11.2 Basic tetrapyrrole structures and reactivity of chlorophyll.

The basic aromatic system is susceptible mainly to electrophilic reactions, e.g. halogenations, with a preference for reaction at C20, the meso position closest to the reduced pyrrole ring. The C7–C8 double bond in phytochlorin (next to R1) is not part of the aromatic delocalization pathway and is thus a convenient point for modification of the chlorophyll macrocycle by addition and oxidation reactions. The side-chain substituents undergo standard transformations, e.g. the C3 vinyl group undergoes addition and oxidation reactions, the ester groups at C173 and C132 can be saponified or transesterified, and the reactivity of ring E is usually governed by enolization and follow-up chemistry of the b-keto ester system. The latter includes the so-called allomerization reactions, i.e. oxidative degradation reactions involving oxidations, hydroxylation, ring-opening or decarboxylations of the isocyclic pentanone ring. The book by Scheer [13] remains the most comprehensive treatise on chlorophyll chemistry and function and a comprehensive review on side-chain transformation has recently been published by Pavlov and Ponomarev [14]. 11.3.3 Isolation of Chlorophylls

Because of their prominent biological role, many different methods have been developed for the qualitative and quantitative extraction of chlorophyll a and b from plants and algae [15]. All methods require extraction of the pigments with an organic solvent or boiling water and, despite all efforts, no single solvent or solvent mixture has been found that will extract the pigments quantitatively and unaltered. The natural chlorophylls are exceptionally labile compounds that readily undergo reactions in the macrocycle core, at C20, and at the doubly activated 132-position [16]. Thus, they rapidly become demetalated and/or lose the phytyl side-chain to yield pheophorbides 12, or undergo other chemical alterations (Fig. 11.3).

11.3 Chlorophyll Fundamentals

Fig. 11.3 Structures of selected chlorophyll a derivatives formed during extraction.

Often, this is not only the result of the chemicals added but is promoted by the plant material itself. For example, plants may have an acidic cytoplasm resulting in the formation of pheophytins, or contain oxidative enzymes that promote oxidation reactions. The most prominent and most easily formed byproducts of the extraction process are the allomerization products, notably epimerized 13 or hydroxylated derivatives 14 of chlorophyll [16, 17]. Likewise, chlorophyllase often remains active during extraction resulting in the formation of acidic chlorophyllides 15 or re-esterified derivatives thereof when using alcohols for extraction. Efforts to denature the enzymes before extraction (e.g. by scalding) will result in the formation of isomerization products. All the different reactions described, and many others, can occur concomitantly at different stages, giving rise to a broad range of extraction artifacts. Thus it is almost impossible to extract plant materials without concurrent formation of a variety of chlorophyll derivatives. Exceptional precautions must be taken (inert gas, cell break up by liquid nitrogen, etc.) to isolate chlorophylls quantitatively and unaltered. This precludes any large-scale technical use of chlorophyll and any chemical or technical use of chlorophyll must be based on chemically more stable derivatives thereof. In practical terms, small-scale extractions (1–5 g plant material) may be performed with almost any “green” organism, usually with chilled acetone as extraction solvent. A typical procedure involves disrupting the material in a blender in the presence of acetone, filtration, re-extraction of plant material, and concentration of the combined extracts by rotary evaporation. Medium-scale extractions (up to 1 kg plant material) are best performed using fresh spinach. The material is first heated in boiling water (1–2 min), filtration followed by extraction with methanol–petroleum ether mixtures then typically yields 2 to 4 g crude pigment extract. For large-scale extractions (1–5 kg) dried alfalfa meal or air-dried blue–green algae, notably Spirulina, are the best sources. In our hands the best results were obtained by freezing Spirulina with liquid nitrogen and extracting the pigments with acetone to yield approximately 2–5 g chlorophyll a kg–1 dried algae. Spirulina contains only chlorophyll a, a major benefit which circumvents the a/b separation problem. Although a variety of chromatographic

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methods have been developed to separate the “a” and “b” series, the best “largescale” method utilizes chemical modification of the formyl group in pheophytin b with Girard’s reagent T [18]. Dried blue-green algae are still the best material for the “large-scale” preparation of pure chlorophyll derivatives and have served for decades as the source of starting materials for research groups involved in chlorophyll chemistry. Except for production of “chlorophyllin” (see below) no attempts have been made to extract chlorophyll or derivatives thereof on an industrial scale. A “dye fraction” is, nevertheless, one of the fractions routinely obtained when green materials (grass, lucerne, alfalfa, etc.) are processed in a green biorefinery [19] and could serve as a source of chlorophyll derivatives.

11.4 Chlorophyll Breakdown and Chemical Transformations

Any consideration of the potential of chlorophylls as a bioresource for fine chemicals is critically dependent on the types of compound that can be derived from it. Thus, we must take a look at chemical transformations occurring either in nature or in the laboratory. 11.4.1 Biological Chlorophyll Catabolism

Although more than 109 tons of chlorophyll are degraded each year on earth (and account for the natural beauty of fall vegetation), the biological breakdown of chlorophyll had remained an enigma until about a decade ago [20]. The only chemical reactions known to be involved in senescence and natural breakdown of chlorophyll were similar to those encountered during pigment extraction. Loss of Mg, dephytylation, and some changes at the isocyclic pentanone ring seemed to be early steps but nothing was known about the fate of the macrocycle and the putative ring-opening reaction. Work by the groups of Kräutler, Matile, and Gossauer showed that the central step is a ring-opening reaction at the 5-position [21, 22]. This is in contrast with the situation encountered for heme, which is oxidatively cleaved at the 20-position. As shown in Fig. 11.4, the crucial steps of chlorophyll degradation begin by conversion of chlorophyll a into pheophorbide a, 12, followed by enzymatic transformation into the bilinone, 16. During this step the macrocycle undergoes oxidative C5 ring-opening, incorporates two oxygen atoms (the CHO one from O2) and is saturated at the 10-position. This reaction is catalyzed by a monooxygenase and the red compound 16 is further converted to the still fluorescing compound 17, and finally into the nonfluorescing derivative 18, along with some changes in the side chains which increase the hydrophilicity of the breakdown products. Chlorophyll b is first converted into chlorophyll a and then subjected to the same reactions.


Fig. 11.4 Outline of chlorophyll degradation in senescent plants [22].

There is currently no evidence that chlorophyll is degraded further to smaller nontetrapyrrolic compounds. The primary function of chlorophyll catabolism during senescence is one of detoxification. Downsizing of the photosynthetic machinery during low light conditions and degradation of the photosystems during senescence releases chlorophylls and the carotenoids necessary for photoprotection in intact chlorophyll–protein complexes. Unbound chlorophylls are potent photosensitizers and produce highly toxic singlet oxygen (see Fig. 11.8). Prevention of this reaction, rather than recycling of nutrients, is the primary rationale behind chlorophyll degradation. Likewise, phytol is not recycled. Its intracellular level remains largely constant during senescence and it is transesterified to phytyl acetate and found in gerontoplasts [22]. 11.4.2 Geological Chlorophyll Degradation – Petroporphyrins

It is known that chlorophyll is not only degraded in living systems but also undergoes degradation in sediments [23]. Up to a few percent of all annual primary organic production is accumulated in sediments and under appropriate conditions (low thermal or oxidative stress) significant amounts of organic compounds will remain identifiable in sediments as fossil molecules [24]. These compounds can serve as geochemical biomarkers as long as they retain enough structural resemblance to biochemical compounds. Indeed, porphyrins are found in many deposits and their concentration in sediments can vary from 1 to 3000 ppm. They have been termed geoporphyrins, petroporphyrins, or sedimentary porphyrins. Their concentration in coal is usually below that in crude oils, and even one porphyrin-based mineral, abelsonite, has been identified [25]. Tetrapyrroles have been isolated with the organic matter from almost all sediments, and are found at geological ages ranging from the Precambrian to recent. Interest in these pigments is related to two areas of research. In practical terms, different deposits have different porphyrin compositions, both with regard to type and relative content of pigments. Thus, the petroporphyrins can be used as a fingerprint for different oil shales. Indeed, HPLC analysis of the pet-

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roporphyrin fraction often serves as an important analytical tool in the oil industry. Second, the close structural resemblance between some geoporphyrins and biochemical pigments (e.g. chlorophyll a) might enable development of a diagenetic scheme for their formation. Indeed, Treibs’s basic hypothesis [23] that petroporphyrins are derived from chlorophylls (and porphyrins) has more than stood the test of time. By now more than 90 different petroporphyrins have been identified, some with very unusual structures. The classic example is desoxophylloerythroporphyrin, 20 (a derivative of phytoporphyrin in modern nomenclature), which clearly bears a close resemblance to chlorophyll a (Fig. 11.5). Even etioporphyrin, 19, another well-known geoporphyrin, may be envisaged as being derived from 20 by cleavage of ring V, or from heme-type pigments [26]. Geoporphyrins are derived from almost all different types of chlorophyll (e.g., 21, 22). Indeed, even some “petrochlorins” (e.g. phytochlorins 4 and 23) have been found. Many of these compounds have been prepared by total synthesis, and for many a chemical rational can be given for their chemical formation. Their natural formation involves extreme diagenetic conditions (geological timescale, heat, pressure, catalysts, . . .). Most of the reactions involving geoporphyrin formation are easily identified – loss of magnesium, remetalation with VO, Ni, or other metals, aromatization of ring IV, vinyl and keto group reduction, cleavage of the methyl ester, decarboxylation – and are standard reactions of the porphyrin chemist [24]. Fossils of phytol 26, e.g. phytane 27 and pristine 28, are prominent constituents of sediments also [27]. Finally, elucidation of the structure of geoporphyrins with yet unknown diagenetic origin (e.g. 24) might lead to the identification of yet unknown biochemical pigments (Fig. 11.6). In the context of this chapter, such pigments can serve as typical examples of exceptionally stable compounds which can be derived from natural pigments. Besides their importance in palaeobiology, geochemistry, and as an analytical tool, however, they are of no industrial importance.

Fig. 11.5 Treibs’s scheme for petroporphyrin formation [23].


Fig. 11.6 Selected petroporphyrins and fossil phytol derivatives [24].

11.4.3 Chemical Degradation of Chlorophylls

As outlined in Section 11.3.2, chlorophylls contain many reactive positions that can serve as an entry for degradation reactions. A survey of all possible reactions is beyond the scope of this article and the reader is referred to relevant reviews [3, 11, 14, 28]. Broadly, chlorophyll chemistry is divided into functional group transformations of the side chains, manipulations involving ring V, and reactions involving the macrocyclic ring system. The first two will be of either no importance or must be prevented during utilization of chlorophyll-derived compounds from biomass (Section 11.6). The third is another possible obstacle to the use of chlorophylls. For our purpose, three types of macrocycle reaction are important. First, like any other porphyrin, the meso positions or chlorophyll derivatives are highly susceptible to electrophilic substitution. In chlorophylls the 20 position is especially reactive and readily undergoes reactions with electrophiles [29], sometimes even during chromatography on silica gel (e.g. to yield 29) [30] (Fig. 11.7). A variety of substitution reactions can also be used to disrupt the chlorophyll system at the 20 position [22]. The second reaction is the chlorin to porphyrin conversion. Any chlorin that has hydrogen atoms on the sp3-hybridized centers of the reduced ring can be oxidized to the respective porphyrin. Oxidation can be achieved by use of a variety of oxidants including oxygen [28]. Likewise, reductions to hydroporphyrins and other reactions of the macrocycle are possible. Most of these are of interest

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Fig. 11.7 Selected reactions of chlorophyll involving the macrocycle.

Fig. 11.8 Chlorophyll as a photosensitizer.

only to the specialist, however. Under appropriate conditions photochemical reductions, notably the Krasnovskii reduction to 30, can occur [31]. The most important and potentially most troublesome reaction is photooxygenation [32]. Chlorophylls are potent photosensitizers and will produce singlet oxygen in the presence of air or triplet oxygen [33]. Thus, chlorophylls can undergo self-destruction (Fig. 11.8). The chemistry of this photooxygenation is very involved and differs somewhat for individual types of chlorophyll [28, 34]. While being partially responsible for the low stability of chlorophylls in solution [35], and for unwanted side reactions in foodstuffs [36], the same reaction has great potential for future applications. Chlorophyll and its derivatives may be used as photosensitizers to affect desired chemical transformations and they have been used for applications in photodynamic therapy (PDT, below) [37].

11.5 Industrial Uses of Chlorophyll Derivatives

11.5 Industrial Uses of Chlorophyll Derivatives

A historical look at the industrial use of chlorophyll (and derivatives) in the 20th century shows that it has been mainly used as a green pigment in the nutrition industry (in Europe food additive E140 for chlorophyll and E 141 for chlorophyllin in cakes, beverages, sweets, and ice-cream, etc.) and in pharmaceutical and cosmetic products (color no. 125 in toothpaste, as a soap pigment, or in shampoos). The older literature also describes its use in candles [38] and as a lipophilic oil-bleaching additive (to neutralize the yellow color of oils in foodstuffs or to give them a greener touch) [39]. Chlorophyll is usually used in the form of chlorophyllin and its metal complexes. Chlorophyllin is an inhomogeneous water-soluble material. It is prepared by saponification of the phytyl side chain with NaOH and exchange of the central magnesium atom for copper (or other metals). The harsh reaction conditions (and the use of the natural chlorophyll a/b mixture) results in the formation of a mixture of compounds (Fig. 11.9). Most prominent constituents are the derivatives 31,32-didehydrorhodochlorin (notably the chlorin e6, 31, and e4, 32, derivatives in the old nomenclature), pheophorbide salts (e.g. 33) and the typical allomerization products [40]. Chlorophyllin is a stable pigment with an intense light green to dark blue–green color. Related formulations are sodium zinc chlorophyllin, chlorophyll paste, oil-soluble chlorophyll, and sodium magnesium chlorophyllin [41].

Fig. 11.9 “Chlorophyllin”.

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Chlorophyllin production also yields large amounts of phytol 26, a colorless oil by-product [42]. This compound has found industrial uses in the synthesis of vitamin K1 (phylloquinone) [43–46] and as the lipophilic part of vitamin E [47]. Besides the traditional use of chlorophyll and its derivatives as pigments, early investigations on the medicinal use of chlorophyllin in the 1940s led to a first boom in chlorophyll use and initiated more serious investigation of its medicinal properties [48]. During those times it was used in bathroom tissue, diapers, chewing gum, bed sheets, shoe liners, toothpaste [49], and other daily products, mostly as an antiodorant [50]. Chlorophyll preparations are still available as OTC medicine to reduce fecal odor because of incontinence or to reduce odor from a colostomy or ileostomy. Other applications involved use in wound healing, germ killing, and treatment of infections and inflammations (use of bandages, antiseptic ointments, surgical dressings) [48]. A late revival of chlorophyll use started in the 1980s when consumer demand for natural pigments in food and other products increased [51]. Although chlorophyllin is currently used as a food coloring agent in Europe and in Asia, chlorophyll(in) is not approved by the FDA in the USA as a food additive. It is permitted in drugs or cosmetics at concentrations < 1% [52, 53]. Annual chlorophyllin production is estimated to be several thousand tons worldwide. An exact assessment of the total amount of “industrial chlorophyll” is difficult with many companies being located in the USA, India, China, and Japan. Since the 1980s use of chlorophyllin has not been limited to pigmentation but the list of putative applications is growing daily – wound healing, treatment of ulcers, treatment of acute and chronic hepatitis, treatment and prevention of cancer [54], recovery of liver function [41], antiodorant in the digestive system, treatment of iron deficiency anemia, etc. [55]. As a result, there is a growing market for chlorophyll formulations as dietary supplements. Some of these are based on actual research, most are rather dubious [56]. There is, nevertheless, growing evidence for medicinal use of chlorophylls. Antimutagenic effects, both in vitro and in animal models, have been proven, notably against aflatoxins [57]. Likewise, there are indications of an anticarcinogenic role [58, 59]. For example, an animal study revealed inhibition of dioxin absorption and increased fecal excretion of dioxin [60]. At the very least, these results indicate the need for further research and offer the promise of future applications of chlorophyll. Photodynamic therapy is the one clearly established medicinal application of chlorophyll derivatives to date [37, 61, 62]. This method relies on selective accumulation of a photosensitizer in target tissue where it can be activated with light to produce toxic singlet oxygen resulting in, e.g., tumor necrosis, as outlined in Fig. 11.8. Although developed in the early seventies, this concept is only now making its potential felt in oncology, as antiviral and antibacterial PDT, and in the treatment of diseases such as age-related macular generation and psoriasis. Several porphyrin-based compounds have been approved for medicinal applications and others are in phase-2 trials. Amongst other tetrapyrroles,

11.6 A Look at “Green” Chlorophyll Chemistry

Fig. 11.10 Visible light-induced hydrogen production system [64, 65].

chlorophyll derivatives are currently under active investigation and show great promise. The current status of this field has been reviewed [37]. The use of chlorophyll derivatives in technical applications is still in the early developmental stage. Topics of current interest are both solar energy conversion and hydrogen production [63]. A representative investigation of biohydrogen evolution uses Zn-chlorophyll a. This is prepared from chlorophyll a derived from Spirulina and its photostability and photosensitizing activity are superior to those of the natural magnesium complex. The use of chlorophyll derivatives is based on their absorption in the visible region. Besides the photosensitizer the photo-induced hydrogen generation-systems also require an electron donor (for example nicotinamide adenine dinucleotide (NADH) regenerated by glucose dehydrogenase (GDH)), an electron relay (Fig. 11.10, MV+/MV2+) and a catalyst (for example colloidal platinum) [64, 65]. Another modern example is the use of chlorin e6, 36, also derived from Spirulina in bio-photovoltaics [66]. Here, the conversion device uses chlorin e6 in a dye-sensitized solar cell (DSSC) [67] with a nanocrystalline TiO2 film. The green pigment absorbs visible light (to yield the chromophores in an excited singlet state). In the next step its emission is quenched by TiO2 with effective electron injection from 1(chlorin e6)* into the TiO2 conduction band. The final aim is the production of low-cost solid-state photovoltaic cells. Organometallic compounds such as ruthenium(II) polypyridyl complexes have been used frequently in this field [68, 69]. Chlorophyll derivatives, however, have a better near-infrared response and the use of environmentally undesirable heavy metals can be avoided.

11.6 A Look at “Green” Chlorophyll Chemistry

Currently, the inherent lability of the natural pigments precludes any direct commercial use. Exceptions are dried algae or plant materials or freeze dried extracts of these. These are hardly uses in the context of a biorefinery, however [70], and even if a suitable application would be found, regulatory problems might arise with regard to GMP standards. Thus, any rational use of the pig-

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ments, i.e. the macrocyclic part of the chlorophylls, must involve a simple and facile extraction process and some simple chemical transformations that produce chemically defined materials of high purity and stability. These requirements are counteracted by the need to produce chemicals that are still sufficiently reactive (e.g. carry functional groups) that enable transformations into desirable and marketable target compounds. A variety of extraction methods have been developed for small-scale extraction, and it should be possible to transfer these to large-scale systems. Ignoring the chlorophyll a/b problem for now, there are several possibilities of utilizing chlorophyll derivatives as starting materials for other fine chemicals. Almost all start with conversion of the chlorophylls to the respective pheophorbides, i.e. demetalation and hydrolysis of the 172-ester. This reaction is preferably performed in the presence of an alcohol to yield a pheophorbide ester, 34, which can be handled in organic solvents. Often it is beneficial to hydrogenate the 3-vinyl group to yield “meso” compounds (35). There are two means of circumventing allomerization problems. Either the 132 ester can be removed by demethoxycarbonylation [18] to yield the “pyro” compounds 37, or ring V can be opened by a variety of nucleophiles to yield chlorin e6 36 and/or various other derivatives of rhodochlorin. Naturally, at all stages it is possible to oxidize the chlorins to the respective porphyrins (e.g. 38). Fig. 11.11 shows a few selected reactions that can be applied to chlorophylls in different sequences to yield relatively stable derivatives. Rhodochlorin and phytochlorin derivatives, in particular, serve as very important intermediates in contemporary chlorophyll chemistry [14, 28]. Any use of chlorophylls as fine chemicals will start with these or closely related compounds [71]. As a result of ongoing studies in PDT (vide supra) and the photobiological relevance of these pigments, the chemistry of these compounds is well developed and most of the reactions can be performed on a multigram scale. Note that oxidation of chlorins to the porphyrins typically results in “red” chemistry, because of a blue shift of the absorption maxima. All of the compounds outlined in Fig. 11.11 can be prepared from crude chlorophyll a in a few steps with standard chemicals. Porphyrin chemists are continually adding new and exciting compounds to this field. Current efforts concentrate on modeling the bacteriochlorophylls c–e [72], developing new electrontransfer compounds [73], modeling compounds for photosynthesis [74], and applications in biomedicine [37]. Thus, no significant efforts in synthetic chlorophyll chemistry are necessary in respect of their use from biorefineries. Rather, the biotechnological side and the technical side of the extraction processes must be resolved. One of the most overlooked areas of chlorophyll chemistry is their use as new materials. For example, significant enhancement of their photochemical stability is possible by adsorbing them on solid phases (silica or polymer supports or smectite conjugates) [75–79]. Such silica-adsorbed materials have been shown to be capable of generating hydrogen gas when coupled with appropriate electron carriers [76], whereas PEG-coupled chlide could function in the coupled generation of glutamate [80]. Despite these promising results, very few groups


Fig. 11.11 Some chlorophyll transformations and key intermediates of chlorophyll chemistry.

are active in this area. The situation is similar with regard to applications in solar energy conversion. Although this has been the catchword in almost any modern publication dealing with chlorophyll chemistry, very few recent papers actually deal with the use of isolated chlorophyll derivatives in photovoltaic cells [66, 81]. Both areas require major research efforts to yield useful applications. If successful, however, these would provide the impetus necessary to further stimulate this whole line of research.


There are currently no large-scale commercial uses of chemically pure chlorophyll-derived materials. The few specialized applications there are very narrow in focus and require only small amounts of material (multi-gram to kg scale), which is conveniently prepared from a dedicated source, e.g. Spirulina. On the

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other hand, well-established major industrial uses of tetrapyrroles, for example use as coloring agents in inks, paints, or plastics and rubber, are exclusively based on nonnatural technical pigments. For example the annual production of phthalocyanines is about 70 000 metric tons. In fact, the technically most important green pigment is “Phthalo green”, a chlorinated copper phthalocyanine. These compounds are derived from simple and cheap starting materials in a few steps. It is therefore doubtful if any bioresource-derived pigments can compete with bulk organic pigments from established synthetic chemistry [82]. We currently have a situation in which people interested in chlorophylls extract chlorophyll from bioresources and discard everything else, whereas people interested in biorefinery systems utilize most materials except chlorophylls, despite listing chlorophylls and pigments as one of the target fractions [83]. There are still several misconceptions in the latter area. For example, a green “pigment/dye” fraction is taken to imply that the content can be used as an industrial pigment. Lack of knowledge about the heterogeneity of the relevant biomass fraction and about the chemical instability of natural tetrapyrrole pigments persists. Also, scientific progress in mimicking nature using artificially generated systems (e.g., artificial photosynthesis or solar energy conversion) is often taken as an argument for the use of natural biorenewable systems [84]. From a tetrapyrrole chemist’s viewpoint a biorefinery has three limitations that currently negate any use of the “green fraction”. First, any mass-produced large-scale crop that may be used as raw material will contain chlorophylls a and b, necessitating chromatographic or chemical separation of the two pigment series. Similarly, the green dye fraction contains many other pigments that cannot be removed by a simple step (e.g. filtration) but will always involve chromatography (with associated costs and environmental impact because of solvent use) if pure compounds are required. Third, compared with carbohydrates, the green fraction is one of the smallest in terms of mass [85] but one of the most complex in terms of content. This prevents its use as a source of pigments as bulk chemicals. In conclusion, no case can currently be made for industrial-scale use of the green material from biorefineries. This is, however, partially a result of the absence of any relevant studies in the subject, the lack of appropriate extraction methods to yield pigments in high purity, a nonexistent industrial chlorophyll chemistry, and the lack of large-scale industrial uses for chlorophyll-derived pigments. What is needed? In the short term, first, a quantitative assessment of the content and composition of the dye fractions derived from green biorefineries. Next, such analyses must be correlated with different extraction methods and processing conditions. When these data are available, it might be possible to optimize the work-up conditions for the “green fraction” with regard to optimum use of the tetrapyrroles. This would also open the possibility of using the green fraction for industrial scale production of chlorophyllin, the only chlorophyll-derived material immediately marketable. In parallel with this, chemists must develop simple, cost-effective, and environmentally benign reactions that can be

References and Notes

applied to the crude chlorophyll fraction to yield homogenous and pure materials for use as fine chemicals. Similarly, use of the “green biomass fraction” must be compared economically and environmentally with dedicated use of, perhaps, bioengineered blue green algae as a biological source of tetrapyrroles [85]. Last, but not least, more industrial applications must be found for natural tetrapyrrole pigments and developed to provide an economic stimulus for this area. Only then will “green” be truly a part of green chemistry.

Acknowledgment

Writing of this article and our own research was made possible by generous funding from the Science Foundation Ireland.

References and Notes 1 Krasnovsky, A. A. Jr. Photosynth. Res. 2

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2003, 76, 389–403. Besides efforts to develop nonhazardous chemical procedures, see: http://www. epa.gov/greenchemistry/ Willstätter, R.; Stoll, A. Untersuchungen über Chlorophyll. Methoden und Ergebnisse; J. Springer: Berlin, 1913. Deisenhofer, J.; Michel, H. Angew. Chem., Int. Ed. Engl. 1989, 28, 829–847. McDermott, G.; Prince, S. M.; Freer, A. A.; Hawthornthwaite-Lawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Nature 1998, 374, 517–521. Senge, M. O.; Smith, K. M. In Anoxygenic Photosynthetic Bacteria; Blankenship, R. E., Madigan, M. T., Bauer, C. E., Eds.; Kluwer Academic Publ.: Dordrecht, 1995; pp. 137–151. Even after 23 years in chlorophyll chemistry, the largest amount of a chemically pure chlorophyll derivative I have ever seen, was about 300 g. Scheer, H. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, 1991, pp. 3–30. Senge, M. O. Chem. Unserer Zeit 1992, 26, 86–93. Douglas, R. H.; Partridge, J. C.; Dulai, K.; Hunt, D.; Mullineaux, C. W.; Tauber, A. Y.; Hynninen, P. H. Nature 1998, 393, 423–424.

11 Senge, M. O.; Wiehe, A.; Ryppa, C. In

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Chlorophylls and Bacteriochlorophylls: Biochemistry, Biophysics and Biological Functions; Grimm, B., Porra, R. J., Rüdiger, W., Scheer, H., Eds.; Kluwer Academic Press: Dordrecht, 2004; in press. Senge, M. O. J. Photochem. Photobiol. B: Biol. 1992, 16, 3–36. Scheer, H. (Ed.) Chlorophylls; CRC Press: Boca Raton, 1991. Pavlov, V. Y.; Ponomarev, G. V. Khim. Geterosikl. Soed. 2004, 483–519; Chem. Heterocycl. Chem. 2004, 40, 393–425. Svec, W. A. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, 1991, pp. 89–143. Grese, R. P.; Cerny, R. C.; Gross, M. O.; Senge, M. J. Am. Soc. Mass Spectrom. 1990, 1, 72–84. Hynninen, P. H.; Hyvarinen, K. J. Org. Chem. 2002, 67, 4055–4061. Kenner, G. W.; McCombie, S. W.; Smith, K. M. J. Chem. Soc., Perkin Trans. 1 1973, 2517–2523. Kamm, B.; Kamm, M. Chem. Biochem. Eng. Q. 2004, 18, 1–6. Hendry, G. A.; Houghton, J. D.; Brown, S. B. New Phytol. 1987, 107, 25–302. Gossauer, A.; Engel, N. J. Photochem. Photobiol. B: Biol. 1996, 32, 141–151. Kräutler, B.; Matile, P. Acc. Chem. Res. 1999, 32, 35–43. Treibs, A. Liebigs Ann. 1935, 510, 42.

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Ed.; CRC Press: Boca Raton, 1991, pp. 339–364. Storm, C. B.; Krane, J.; Skjetne, T.; Telnaes, N.; Branthaver, J.; Baker, E. W. Science 1984, 233, 1075–1076. However, the ratio of “green” to “red” organic material contributing to sediments is about 100000 : 1 [24]. Rontani, J. F.; Grossi, V. Org. Geochem. 1995, 23, 355–366. Hynninen, P. H. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, 1991, pp. 145–209. Woodward, R. B.; Skaric, V. J. Am. Chem. Soc. 1961, 83, 4676–4678. Senge, M.; Senger, H. Photochem. Photobiol. 1988, 48, 711–717. Scheer, H.; Katz, J. J. Proc. Natl. Acad. Sci. USA 1974, 71, 1626–1629. Wasser, P. K.; Fuhrhop, J.-H. Ann. N.Y. Acad. Sci. 1973, 206, 533–548. Inhoffen, H. H. Pure Appl. Chem. 1968, 17, 443. Llewellyn, C. A.; Mantoura, R. F. C.; Brereton, R. G. Photochem. Photobiol. 1990, 52, 1037–1041; 1043–1047. Peled, A.; Dror, Y.; Baal-Zedaka, I.; Porat, A.; Michrin, N.; Lapsker, I. Syn. Metals 2000, 115, 167–171. Min, D. B.; Boff, J. M. Compr. Rev. Food Sci. Food Saf. 2002, 1, 58–72. Nyman, E. S.; Hynninen, P. H. J. Photochem. Photobiol. B.: Biol. 2004, 73, 1–23. Arpe, H.-J., Ed. Ullmann. Industrielle organische Chemie; 4th ed.; Wiley–VCH: Weinheim, 1988, 11, pp. 127–129. Kephart, J. C. Econ. Bot. 1955, 9, 3–38. Strell, M.; Kalojanoff, A.; Zuther, F. Arzneimittel-Forsch. 1955, 5, 640–642; 1956, 6, 8–11. See http://www.fengming.com for several products and use. Fugmann, B., Ed. Römpp Lexikon Naturstoffe. Thieme: Stuttgart, 1997, pp. 491. Almquist, H. J.; Klose, A. A. J Am. Chem. Soc. 1939, 61, 2557. Binkley, S. B.; Cheney, L. C. ; Holcomb, W. H.; McKee, R. W.; Thayer, S. A.; MacCorquodale, D. W.; Doisy, E. A. J. Am. Chem. Soc. 1939, 61, 2558. Fieser, L. F. J. Am. Chem. Soc. 1939, 61, 2559, 2661, 3467.

46 Isler, O. Amer. Patent 1939, 2325681. 47 Schmid, M.; Gerber, F.; Hirth, G. Helv.

Chim. Acta 1982, 65, 684–702. 48 See the review by R. H. Dashwood,

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Linus Pauling Information Center 2004 at http://lpi.oregonstate.edu/infocenter/ phytochemicals/chlorophylls/. Hein, J. W. J. Am. Dent. Assoc. 1954, 48, 14–19. See the online-book by R. L. Seibold 1990, chapter 4 at www.wheatgrass.com, USA, Kansas. Legally, dyes derived directly from biomass may not be termed a “natural color”, although they may not require certification by the FDA. Gilman, V. Chem. Eng. News 2003, 81, 34. In fact, the only FDA certified green food color (Green No. 3, Fast Green) is a triphenylmethane dye. Imai, K.; Aimoto, T.; Sato, M.; Watanabe, K.; Kimura, R.; Murata, T. Chem. Pharm. Bull. 1986, 34, 4287–4293. A critical analysis of the literature will be published elsewhere. A few excerpts from web pages: “Chlorophyll is one of the greatest food substances for cleansing the bowel and other elimination systems, such as the liver and the blood. – Chlorophyll is effective against anemia and stimulates the production of red blood cells in the body. It also helps carry oxygen around the body and to the brain. – Brain Food – . . .it essentially has the same effects in the body as iron, so it builds our own blood naturally. – It is ... a whole food ... – wonder tonic.” Sudakin, D. L. J. Toxicol. Clin. Toxicol. 2003, 41, 195–204. Waladkhani, A. R.; Clemens, M. R. Int. J. Mol. Med. 1998, 1, 747–753. Chernomorsky, S.; Segelmann, A.; Poretz, R. D. Teratogen. Carcin. Mut. 1999, 19, 313–322. Morita, K.; Ogata M.; Hasegawa, T. Environ. Health Persp. 2001, 109, 289–294. Spikes, J. D.; Bommer, J. C. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, 1991, pp. 1181–1204.

References and Notes 62 Bonnett, R. Chemical Aspects of Photody-

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namic Therapy; Gordon and Breach Sci. Publ.: Amsterdam, 2000. Himeshima, N.; Amao, Y. Energ. Fuel. 2003, 1641–1644. Woodward, J.; Orr, M.; Cordray, K.; Greenbaum, E. Nature 2000, 405, 1014– 1015. Inoue, T.; Kumar, S. N.; Karnachi, T.; Okura, I. Chem. Lett. 1999, 147–148. Amao, Y.; Komori, T. Biosens. Bioelectron. 2004, 19, 843–847. O’Regann, B.; Grätzel, M. Nature 1991, 353, 737–740. Nazeeruddin, M. K.; Pechy, P.; Renouard, T.; Zakeeruddin, S. M.; Humphry-Baker, R.; Compte, P.; Liska, P.; Cevey, L.; Coast, E.; Shklover, V.; Spiccia, L.; Deacon, G. B.; Bignozzi, C. A.; Grätzel, M. J. Amer. Chem. Soc. 2001, 123, 1613– 1624. Smestad, G.; Bignozzi, C.; Argazzi, R. Sol. Energ. Mat. Sol. C. 1994, 32, 259– 288. Kamm, B.; Kamm, M. Appl. Microbiol. Biotechnol. 2004, 64, 137–145. The preparation of chlorophyllin uses similar principles: exchange of the metal to increase stability, ester saponification to yield a water soluble compound. However, due to the drastic reaction conditions allomerization reactions take place to some extent; in addition often the a/b mix is used. Thus, as described, chlorophyllin is not really a chemically defined pure compound. Tamiaki, H.; Omodo, M.; Saga Y.; Morishita, H. Tetrahedron 2003, 59, 4337– 4350. Helaja J.; Tauber A. Y.; Tkachenko, N. V.; Lemmetyinen, H.; Kilpelainen, I.; Hyn-

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ninen, P. H. J. Chem. Soc., Perkin Trans. 1 1999, 2403–2408. Fajer, J. Photosynth. Res. 2004, 80, 175– 182. Furukawa, H.; Kuroda, K.; Watanabe, T. Chem. Lett. 2000, 1256–1257. Itoh, T.; Yano, K.; Inada, Y.; Fukushima, Y. J. Am. Chem. Soc. 2002, 124, 13437– 13441. Itoh, T.; Ishij, A.; Kodera, Y.; Matsushima, A.; Hiroto, M.; Nishimura, H.; Tsuzuki, T.; Kamachi, T.; Okura, I.; Inada, Y. Bioconj. Chem. 1998, 9, 409–412. Ishii, A.; Itoh, T.; Kodera, Y.; Matushima, A.; Hiroto, M.; Nishimura, H.; Inada, Y. Res. Chem. Interm. 1997, 23, 683–689. Ishii, A.; Itoh, T.; Kageyama, H.; Mizoguchi, T.; Kodera, Y.; Matsushima, A.; Torii, K.; Inada, Y. Dyes Pigments 1995, 28, 77–82. Asada, H.; Itoh, T.; Kodera, Y.; Matsushima, A.; Hiroto, M.; Nishimura, H.; Inada, Y. Biotechn. Bioeng. 2001, 76, 86– 90. Komori, T.; Amao, Y. Electrochemistry 2003, 71, 174–176. Herbst, W.; Hunger, K. Industrial Organic Pigments: Production, Properties, Applications; 3rd ed.; Wiley-VCH: Weinheim, 2004. Kromus, S.; Wachter, B.; Koschuh, W.; Mandl, M.; Krotscheck, C.; Naradoslawsky, M. Chem. Biochem. Eng. Q. 2004, 18, 7–12. Moser, A. Chem. Biochem. Eng. Q. 2001, 15, 33–41. The chlorophyll content of green feedstocks is approximately 0.2% compared with 2–3% for blue–green algae.

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Part II Biobased Industrial Products, Materials and Consumer Products


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12 Industrial Chemicals from Biomass – Industrial Concepts Johan Thoen and Rainer Busch

12.1 Introduction

The era of a chemical industry based on mineral oil, gas and coal has lasted almost 150 years, but will gradually come to an end in the course of the next 50 to 75 years. The two main reasons for this development are: · The stocks of fossil resources are finite. This has generally been accepted since the Club of Rome published its report “Limits to Growth” in 1972. First, mineral oil stocks will be exhausted around 2050 if we continue our present way of life. Times during which more oil was found each year than was consumed are definitely over. Natural gas will last slightly longer (some 75 years), and coal will last longest (> 200 years). · Environmental considerations. All kinds of pollution from global warming to acid rain, from smog to ground water pollution, can be linked to the use of fossil fuels. Hence we are really living on the threshold of a new era in chemistry – and unfortunately in most chemical research and education this has not yet been sufficiently recognized. The hypothesis has been formulated that from 2040 onwards we will no longer use fossil organic raw materials, as a consequence of exhaustion and environmental considerations. The fundamental question, however, is: “Is this technologically feasible while maintaining our present way of life in terms of food, organic materials and energy consumption?”


The use of renewable resources as raw materials for technical applications is certainly not new. Humanity already used natural materials from the first civilizations onward to meet their basic needs. The first industrial activity was also Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 2 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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largely based on the use of renewable resources and this continued until the industrial revolution. This means that until 1850, all organic consumer products and industrial raw materials were plant-based. Within the relatively short period of 150 years society changed from a mainly plant-based economy to an economy based on fossil fuels (coal until the end of the 19th century, until 1950 mineral oil, and now, increasingly, natural gas). Wood supplied 70% of the fuel demand in 1870, in 1920 70% came from coal, in 1970 70% from mineral oil. The use of renewable materials declined substantially with time, mainly as a consequence of the extremely low prices of petrochemical resources. Currently, approximately 96% of all organic chemical substances are based on fossil resources. Nevertheless, several important industries are still based on renewable raw materials. Half of the fiber used in the textile industry is natural material (cotton, wool, flax), the oleo-chemical industry supplies society’s daily hygienic needs for soaps and detergents that are based on vegetable oils. The building industry continues to use natural fiber for thermal insulation purposes. Petrochemistry does not always offer a realistic alternative to the use of renewable materials. Classic examples are, for example, in the production of antibiotics and of drugs where fermentation processes play an important role. Moreover, industrial biotechnology frequently has further significant performance benefits compared with conventional chemical technology, for example higher reaction rates, increased conversion efficiency, improved product purity, reduced energy consumption, and significantly reduced chemical waste generation. This branch of biotechnology has recently been named “White Biotechnology”. The penetration of bio-based product today is estimated at 5% with growth potential up to 10–20% by the year 2010. The Vision for Bioenergy and Biobased Products in the United States and the related Roadmap have established far reaching goals for increasing the role of biobased energy and products in this country. The oil crisis of the 1970s gave renewed impetus to the use of renewable resources. The worlds increasing dependence on fossil resources, and the finite availability of these, created serious concern. The concern was, however, largely channeled in the direction of energy security. Renewable materials were less of a concern and all attention disappeared when the oil price dropped. With increasing awareness and concern in the 1990s about industrial and consumer waste, and its effect on the environment, the need arose for better biodegradable intermediates and final end products. Those products can naturally degrade into components that are absorbed back into the natural cycle. Biodegradability was the new key property of many new products and they were often based on renewable resources, in view of their intrinsic biodegradability. With regard to fossil reserves, the world is now faced with the dilemma that while crude oil is being consumed faster than ever, “proven oil reserves” have remained mostly unchanged. The cost of exploration and exploitation of crude oil increases, which is reflected in increasing oil prices. In contrast with this, agricultural raw materials such as wheat, corn, sugar, and oil crops are becoming cheaper as a fundamental consequence of increasing agricultural efficiency and yield. This

12.3 Basic Principles

trend will most probably continue for some time to come. This long-term trend may be perturbed by the transitory effects of market imbalances and politics but for a growing number of applications the economic balance is tipping toward the use of renewable resources; this is also true of bulk chemicals. Process and catalysis technology revolutionized the chemical industry in the 20th Century. Now the same thing is happening for the production of industrial chemicals from biomass. A wave of project initiatives is under way globally; the objective of these is to convert renewable resources into industrial chemicals. Industrial biotechnology uses biological systems in conjunction with existing and new thermochemistry, for production of useful chemical entities. Biotechnology is mainly based on biocatalysis and bioprocessing (the use of enzymes and cells to catalyze chemical reactions) and fermentation technology (directed use of microorganisms), in combination with recent breakthroughs in the forefront of molecular genetics and metabolic engineering.

12.3 Basic Principles

Biomass can be used in different ways to provide us with organic compounds and materials: 1. Nature already produces the desired structures, and isolation of these components usually requires only physical methods. Examples include polysaccharides (cellulose, starch, alginate, pectin, agar, chitin, inulin), disaccharides (sucrose and lactose) and triglycerides, lecithin, natural rubber, gelatin, flavors and fragrances, etc. Some current production volumes are: sucrose 115 ´ 106 t a–1, triglycerides 85 ´ 106 t a–1, natural rubber 5.5 ´ 106 t a–1. Cotton, the natural cellulose fiber, is produced in a volume of 20 ´ 106 t a–1, an amount which equals the sum of all synthetic fibers (volume order: polyester > polyamide > acrylic). The possibilities of producing organic chemicals directly by and from plants by means of plant biotechnology will increase dramatically. The plant is the “plant” of the future. 2. One step (bio)chemical modification of naturally produced structures included in (1), above. Examples include cellulose and starch derivatives, glucose and fructose, glycerol, fatty acids; ethanol, citric acid, glutamic acid and lactic acid by fermentation. Lactulose, lactitol, and lactobionic acid by isomerization, hydrogenation, and oxidation, respectively, from lactose. Nature provides a variety of fine starting materials for pharmaceuticals. 3. By several-step modifications of naturally produced structures included in (1), above, organic chemicals and organic materials are obtained from natural products. For example: ethanol can be converted to today’s no. 1 organic chemical, ethylene; sorbitol and mannitol can be produced by hydrogenation of glucose and sucrose, respectively; vitamin C can be obtained in several steps from glucose; fatty alcohols and amines can be obtained from triglycerides; and succinic acid can be obtained from glucose and CO2.

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4. “Back to C1-Chemistry” by using biomass as the carbon and hydrogen source, converting it into small fragments (synthesis gas) and building it up again to the desired structures. In the above the focus has been on chemical structures and less on the product areas. For some large product groups it can be stated that the green label (renewables-based) is accepted as a selling advantage.. 12.3.1 Primary Conversion Technologies of Biomass

An overview of technically feasible technology for conversion of biomass, ranked according to water content, is given in Fig. 12.1. The three most important technologies will be dealt with in some detail.

12.3.1.1 Gasification Biomass can be converted into power plant fuel by gasification with high yield and in an environmentally friendly fashion. Also, in the longer term, the economics of this process look good, especially for energy crops. Gasification takes place with air, at temperatures of approximately 850 8C. The gas consists of 13% H2, 17% CO, 4% CH4, 12% CO2, 13% H2O, and 40% N2, which has a calorific value of 6 MJ. In the 2040 scenario, 80 EJ a–1 could be produced from waste streams and 200 EJ a–1 from energy crops, on a global scale. The removal of sulfur-containing components, tar, char, and ash from the gas is critical for use in gas turbines and for methanol production. The technology is promising. Many pilot plants are in operation, large installations are in the planning phase. The gas could presumably also be used in Fischer–Tropsch synthesis.

Fig. 12.1 Biomass conversion technology.

12.4 Current Status

12.3.1.2 Hydrothermolysis During the period 1982–1993, the Royal Dutch Shell Laboratory developed a process to convert biomass into liquid fuel, so-called bio-crude. This process is called HTU (hydro-thermal upgrading). First, biomass is treated in an aqueous slurry at 200 8C and 30 bar, followed by a treatment at 330 8C and 200 bar. This process results in a bio-crude, an oil with a low oxygen content which can be further upgraded by catalytic hydrodeoxygenation to a high-quality naphtha or diesel oil with very low oxygen, nitrogen, and sulfur content. The oil yield is approximately 40%, on the basis of the biomass feed stock. Wood, agricultural, and domestic (green) waste streams have been successfully used as feedstocks. According to Shell, this HTU process is the cheapest route to liquid biofuels. Its cost price would be approximately $20–40 per barrel, compared with fossil crude oil today at approximately $12 per barrel. Hence, the process is not yet economical under the current tax regime. This HTU process and many variants of this process lead directly to biocrude, from which known transport fuels and petrochemicals can be manufactured, without the extra sulfur-removing steps, etc., which are necessary with fossil fuel.

12.3.1.3 Fermentation to Ethanol By fermentation of biomass (sugars, grain, cellulose, etc.) with yeast a 6.5–11% solution of ethanol in water is formed, from which 95 or 100% ethanol can be obtained by distillation (or membrane-filtration, or distillation-adsorption). Depending on the feedstock, chemical or enzymatic hydrolysis is sometimes required first, to convert the biomass into monosaccharides. Alcohol is a raw material for many organic chemicals among which, as was already mentioned, is today’s no. 1 organic chemical, ethylene. In India over 400 000 t a–1 of alcohol is used to make “alcochemicals” with acetic acid and ethylene glycol as numbers 1 and 2. In both India and China, Moreover, aqueous alcohol is used directly for aromatic ethylation (with ethylbenzene, 1,4-diethylbenzene, and 4-ethyltoluene). Ethanol can also be used directly as a liquid fuel. The technology is well developed and applied on a large scale in the USA (corn-based) and in Brazil (sugar cane-based). It is expected that, as a result of better enzymatic hydrolysis and ethanol processing, with increasing fossil fuel prices, bioethanol prices will become competitive with gasoline in 2010.

12.4 Current Status 12.4.1 Europe

Renewable raw materials as industrial feedstock for the manufacture of chemical substances and products have recently received attention from policy makers in some European Union Member States. It has been recognized that use of renewable raw materials as industrial feedstock is already well established in the

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energy and transport sectors. In contrast, manufacture of chemicals and other products from renewable resources, for example oils from oilseed crops, starch from cereals and potatoes, and cellulose from straw and wood has only recently received attention from policy makers in some member states. By employing physical, chemical and biochemical processes, these materials can be converted into polymers, lubricants, solvents, surfactants and specialty chemicals for which fossil fuels have traditionally been used. At the EU level crop-derived raw materials entered the political agenda in 2000 with the establishment of a working group “Renewable Raw Materials” within the European Climate Change Program. The objective of this working group was to quantify the possible reduction in greenhouse gas emissions arising from wider use of RRM-based materials in manufacturing. Although its current direct contribution to the reduction of green house gas emissions has been shown to be rather modest (about 8 million tonnes of CO2 equivalent by 2010), so called indirect reductions associated with product performance improvements could be substantially higher (more than 30 Mt CO2 equivalent). Moreover, the potential for significant reduction in GHG emissions is expected to arise in the medium to long term as a result of use of RRM in connection with application of biotechnology. Beside environmental benefits there will, in the future, be other advantages to be derived from the wider use of RRM in industry by: · improving the economic competitiveness of EU industry and agriculture by giving incentives to use the most advanced technologies, including bio-technology; · providing social benefits by rejuvenating rural communities through establishment of local industries and by providing farmers with additional sources of income, thereby securing their jobs; and · further enhancing environmental protection by improving soil and water quality. Hence, by saving fossil resources the use of RRM in industry directly contributes to sustainable development, recently endorsed by heads of States and Governments at their summit in Gothenburg as one of the Community’s main political aims for the future. It is felt that this industrial sector needs a more detailed presentation of its situation and requirements, with a view to assessing what kind of action could be taken at a Community level to increase its prospects. 12.4.2 United States

In the United States, strong motivation has developed in the past decade to reduce the nation’s dependence on imported oil and to increase its own energy supplies by using a more diverse mix of domestic resources. In 1999 a presidential order triggered a series of initiatives for promotion of the use of renewable

12.4 Current Status

materials. The Biomass Research and Development Act of 2000 led to the establishment of the Biomass Research and Development Technical Advisory Committee, which issued the “Vision for Bioenergy and Biobased Products in the United States” and the “Roadmap for Biomass Technologies in the United States”. Challenging long-term goals have been established that will dramatically transform the role of biomass in the everyday lives of Americans. For production of chemicals and materials from biobased products, these goals predict a substantial increase from five percent of the current production of target US chemical commodities in 2001 to 12 percent in 2010, 18 percent in 2020, and 25 percent in 2030. By 2030, a well-established, economically viable, bioenergy and biobased products industry will create new economic opportunities for rural America, protect and enhance the environment, strengthen US energy-independence, provide economic security, and deliver improved products to consumers. Biobased products are a major new market opportunity for domestically grown biomass resources. It will be a new source of revenue not only for those who produce the feedstocks but also for the farmers and others who are involved in the production of biobased products. Continued research can significantly increase opportunities for biobased products, expand existing markets, and open entirely new markets. Current production of biobased textile fibers, polymers, adhesives, lubricants, soy-based inks, and other products is estimated at 12.4 billion pounds per year. Total production (biobased and non-biobased) is, however, in the hundreds of billions of pounds. The growth opportunities for biobased products are, therefore, enormous. As a result of advanced research, new concepts in industrial biorefinery could become a reality. In the industrial biorefinery, any combination of biofuels, electric power, materials, chemicals, and other products could be produced from local biomass resources. 12.4.3 Products

Renewable biobased products are products created from plant or crop-based resources such as agricultural crops and crop residues, forestry, pastures, and rangelands. Many of the products that could be made from renewable bioproducts are now made from petroleum (e.g. petrochemicals). Plant resources, mostly for paper products and chemical feedstocks, now provide approximately 5% of manufacturing inputs. Plant-based chemical products include paints, adhesives, lubricants, inks, polymers, and resins. Use of hydrocarbon resources is often much less expensive. For some chemical products plant inputs are already cost-competitive, and they are a significant feedstock. Plant-based systems are the major sources for ethanol, sorbitol, cellulose, citric acid, natural rubber, most amino acids, and all proteins. Companies from the chemical, life sciences, forestry, and agricultural communities are involved in establishing the renewable bioproducts industry. Their activities range from genetic engineering of new plant species to development of

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new technologies and processes for converting plants into useful industrial inputs. For example, DuPont recently developed a biobased method that uses corn instead of petroleum-based processes to produce a polymer platform for use in clothing, carpets, and automobile interiors. Similarly, Cargill Dow’s biorefinery in Blair, Nebraska is producing polylactide (PLA) polymers from corn sugar.

12.5 Industrial Concepts 12.5.1 Introduction

Total annual biomass production on our planet is estimated at 170 billion tons and consists of approximately 75% carbohydrates (sugars), 20% lignin, and 5% of other substances, for example oils and fats, proteins, terpenes, alkaloids, etc. Of this biomass production, 6 billion tons (3.5%) are presently being used for human needs, distributed as: · 3.7 billion tons (62%) for human food use, possibly via animal breeding as an intermediate step; · 2 billion tons of wood (33%) for energy use, paper, and construction needs; and · 300 million tons (5%) to meet the human needs for technical (non food) raw materials (clothing, detergents, chemicals, . . .). Other biomass is used in natural ecosystems or is lost by burning or natural mineralization processes. Use of bio-based materials has significant ecological advantages. Agricultural crops are the preferred starting raw materials, instead of using fossil resources such as crude oil and gas (Table 12.1). This technology consequently has a beneficial effect on greenhouse gas emissions and at the same time supports the agricultural sector producing the raw materials. The OECD has collected and analyzed case studies of the application of biotechnology in such diverse sectors as chemical, plastics, food processing, textiles, pulp and paper, mining, metal

Table 12.1 Average world market prices for raw materials. Raw material Petroleum Coal Ethylene Corn Straw Sugar

Average world market prices (2000–2003) 175 1 35 1 400 1 80 1 20 1 180 1

t–1 t–1 t–1 t–1 t–1 t–1

12.5 Industrial Concepts

refining, and energy. The case studies show that biotechnology not only reduces costs but also reduces the environmental footprint for a given level of production. Capital and operating costs are sometimes reduced by 10–50%. In others, energy and water use were reduced by 10–80% and use of petrochemical solvents was reduced by 90% or eliminated completely. There are several example in which biotechnology enabled development of new products whose properties, cost, and environmental performance could not be achieved by use of conventional chemical processes or petroleum as a feedstock. 12.5.2 Biorefinery Concepts

In biorefineries, biomass is used for production of high added-value chemicals, materials, intermediates, and fine chemicals together with the production of energy carriers, preferably in liquid phase, for its higher energy content and easier transport. In that respect, biorefineries can be compared with existing and fully integrated petrochemical refineries. A parallel approach is the concept of “BioCascade”, using dedicated crops in such a way that all the constituents a plant offers (oils, proteins, fibers, cellulose, lignin, etc. . .) result in a total product mix with the highest economic value (Fig. 12.2). A range of different technologies can be used industrially to convert the available biomass into renewable materials or energy carriers. Industrial activity in renewable materials is very often linked to the food sector. Many of the renewable raw materials that can be considered for technical industrial applications can be made in food processing plants. For example, sugar or glucose or natural oils for human food use are also important raw materials for industrial processes. The industrial sectors supplying the most important renewable raw materials are (Table 12.2): · the sugar and starch sector, which produces carbohydrates such as sugar, glucose, starch and molasses from plant materials like sugar beet, sugar cane, potatoes, wheat, corn, etc.; · the oil and fat processing sector which produces numerous oleo-chemical intermediates such as triglycerides, fatty acids, fatty alcohols and glycerol from plant materials like rape seeds, soybeans, palm oil, coconut; and · the wood processing sector, in particular the cellulose and the paper industry, produce mainly cellulose, cellulose derivatives, and lignin from wood. Biorefineries are large industrial factory complexes in which agricultural feedstocks are processed and fractionated into intermediate basic products that are then partially converted into final products. In several instances the intermediate basic products find their own applicability. The products often have little in common with the nature of the original plant feedstock. Biorefineries use physical, chemical, and biotechnological processes; fermentation technology and biocatalysis, are particularly important. This technology uses microorganisms and their enzymes to convert basic renewable raw materials. Thermochemical conversion pro-

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12 Industrial Chemicals from Biomass – Industrial Concepts Table 12.2 Estimated world production figures and indicative world market prices of several renewable and petrochemical raw materials.

Renewable raw materials Cellulose Sugar Starch Glucose Bio-ethanol Glutamic acid Petrochemicals Ethylene Propylene Benzene Terephthalic acid Isopropanol Caprolactam

Estimated world production (million tons per year)

Indicative world market price (1 per ton)

320 140 55 30 26 1

500 180 250 300 400 2000

85 45 23 12 2 3

400 350 400 700 700 2000

cesses, although having much potential in the future, have been insufficiently considered. Thermochemical thermolysis, pyrolysis, and gasification are well known technology for production of char, oil, and gases. Catalytic thermochemical conversions technology is mostly undiscovered for production of complex molecules from agricultural feedstocks. Fractionation technology is a preparation process intended to separate agricultural materials into separate families using chemical and physical methods. Industrial biotechnology, enzymatic technology, and thermochemical conversion technology are complementary with each other for full valorization of renewable feedstocks into a portfolio of value-added chemicals and of energy. The whole chain of different process steps, each using very different technology can occur within an industrial complex. These are then referred to as “biorefineries” analogous to petrochemical crude oil refineries. 12.5.3 Classes of Bioproduct

The many different industrial bioproducts produced today can be divided into four major categories. · Sugar and starch bioproducts derived by fermentation and thermochemical processes include alcohols, acids, starch, xanthum gum, and other products derived from biomass sugars. Primary feedstocks include sugarcane, sugar beet, corn, wheat, rice, potatoes, barley, grain. and wood. · Oil and lipid-based bioproducts including fatty acids, oils, glycerin, and a variety of vegetable oils derived from soy, canola, sunflower, or other oil seeds.


Fig. 12.2 Integrated biorefinery.

· Wood chemicals include tall oil, alkyd resins, rosins, pitch, fatty acids, lignin, turpentine, and other chemicals derived from trees. · Cellulose derivatives, fibers, and plastics, including products derived from cellulose, including cellulose acetates (cellophane) and other cellulose derivatives. 12.5.4 Opportunities for Industrial Bioproducts

For bioproducts including polymers, lubricants, solvents, adhesives, herbicides, and pharmaceuticals potential markets are wide ranging. Although bioproducts have already penetrated most of these markets to some extent (Table 12.5), new products and technology are emerging with the potential to further enhance performance, cost competitiveness, and market share (Table 12.4). Organic chemicals are the most direct and largest target for bioproducts. Using novel chemistry, the carbon and hydrogen moieties present in biomass can be rearranged to yields products equivalent to or better than the products that are produced from fossil feedstocks. Many common organic building blocks serve as monomers for the production of plastics, which is the largest opportunity for renewable materials (Table 12.3). Several advances in biotechnology, chemicals processing, engineering, chemistry, and separation technology are opening up new avenues for biobased polymers such as poly(lactic acid).

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12 Industrial Chemicals from Biomass – Industrial Concepts Table 12.3 Primary global markets for polymers. Material

Volume (million tons)

Polyethylene Polypropylene Poly(vinyl chloride) Poly(ethylene terephthalate) Polystyrene Butadiene/co-polymers Phenolic resins Polyamides

48 23 26 13 14 8 5.5 4

The other major organic chemicals markets for bioproducts include organic acids, alcohols, and solvents. Biomass-based ethanol is an established industry, with other emerging markets for other alcohols and bio-derived acids. Lubricants and greases were originally plant based but are now mostly petroleum-based. Increasing energy prices and growing environmental concerns over the impact of petroleum-based products are supporting the entrance back in the market of vegetable oil-based lubricants and greases. 12.5.5 Product Categories Based on C6-Carbon Sugars to Bioproducts

Biomass sugars are the most abundant renewable resource available. Many of the products used today, for example citric acid, ethanol, and lactic acid are produced by fermentation. With a vast range of microorganisms available and being discovered and exploited, the fermentation of sugars has great potential for the future. Two types of sugar are present in biomass – six-carbon sugars or hexoses, of which glucose is the most common, and five-carbon sugars or pentoses, of which xylose is most common. The most promising glucose derivatives include lactic acid, succinic acid, butanol, 3-hydroxypropionic acid, 1,3-propanediol, and polyhydroxyalkanoates. Lactic acidderivatives include poly(lactic acid) polymer, ethyl lactate solvent, acrylic acid, propylene glycol, and pyruvic acid. Succinic acid derivatives include tetrahydrofuran (THF), 1,4-butanediol (BDO), c-butyrolactone (GBL), and N-methylpyrrolidone (NMP). 3-Hydroxypropionic acid derivatives include acrylic acid, acrylonitrile, and acrylamide. 12.5.6 Product Categories Based on C5-Carbon Sugars to Bioproducts

Pentose sugars such as xylose have thus far been an untapped resource. The microorganisms currently available prefer glucose to pentose. By developing microorganisms that convert pentose sugars alone or in combination with glucose, the overall economics of biobased products can be improved by enabling full

12.5 Industrial Concepts Table 12.4 Industrial biobased product opportunities. Technology Platform

Chemical

Application

Sugar Fermentation

Lactic acid

Acidulant, electroplating additive, textile/leather auxiliary Thermoplastic polymer Solvent, intermediate Specialty resins Surfactant, food, pharma, antibiotics, amino acid and vitamin production Solvents, adhesives, paints, printing inks, tapes, plasticizer, emulsifier, de-icing compound, herbicide

Poly(lactic acid) Ethyl lactate 1,3-Propanediol Succinic acid

Succinic acid derivatives Tetrahydrofuran 1,4-Butanediol c-Butyrolactone N-Methylpyrrolidone 3-Hydroxypropionic acid and derivatives Acrylic acid Acrylonitrile Acrylamide n-Butanol

Sugar fermentation and thermochemical Sugar thermochemical

Oils and lipids

Biomass gasification

Biomass pyrolysis

Acrylates, acrylic polymers, fibers and resins

Solvent, plasticizer, polymer, resin Itaconic acid Aluminum anodizing agent, reactive comonomer Propylene glycol Solvent, chain extender in PU, antifreeze, plasticizer Levulinic acid and derivatives Oxygenates for fuels, biodeMethyltetrahydrofuran gradable herbicide, bisphenol d-Aminolevulinic acid A alternative, comonomer, Diphenolic acid solvent THF, 1,4-butanediol Lubricants and hydraulic Polyurethanes fluids Solvents Polymers Fischer–Tropsch and Fuels, solvent, aerosol, oxygengas-to-liquid products ate, intermediate for monoMethanol mers and polymers Higher alcohols Phenol–formaldehyde resins Plywood, oriented strandboard

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12 Industrial Chemicals from Biomass – Industrial Concepts Table 12.5 Current industrial bioproducts from biomass. Category

Technology

Feedstock

Chemical

Application

Starch and sugars

Biochemical

Carbohydrate biomass

Lactic acid, citric acid, ethanol, starch, sorbitol, levulinic acid, itaconic acid

Oil/lipids

Thermochemical Oils/lipids derived from soybean, canola, sunflower

Polymers, solvent, pharma, cleaners, coating, inks, surfactants, paints, adhesives, cosmetics Pharmaceuticals, resins, lubricants, paints, printing inks, foams, elastomers, coatings, personal care, surfactants Solvents, soaps, detergents, personal care, adhesives, inks, phenolic resins, plastics, textiles

Glycerin, alkyd resins, polyurethane, epoxidized oils, fatty alcohol, fatty acid/ester

Forest derivatives Thermochemical Pine, black Turpentine, oil, liquor, soft wood rosin, tall oil, lignin, cellulose

utilization of all carbohydrates present in biomass. Potentially important xylose derivatives are itanonic acid, furfural, furfuryl alcohol, and 2-hydroxymethyl tetrahydrofuran. 12.5.7 Thermochemical Conversion of Sugars to Bioproducts

Biomass sugars can be upgraded to value-added products by use of thermochemical conversion routes in addition to fermentation routes. Some thermochemical conversion routes have been used for many years, such as the production of sorbitol from glucose. In recent years, production of high value-added products from biomass has been advancing rapidly, because of the development of new and improved catalysis and process technologies. Much focus on sugar thermochemistry has been on sorbitol, levulinic acid, and their derivatives. In that respect, the conversion of sorbitol to glycols is creating new opportunities for bioproducts. Current production of propylene glycol and ethylene glycol is petroleum-based. Glycols are largely used in numerous polyester resins, copolymers, polyethers, and alkyd resins, and in personal care products, coatings, printing inks, heat-transfer fluids, and antifreeze. Levulinic acid has been well explored as a platform intermediate. The current production cost of fossil feedstock-based levulinic acid ($ 4.00–6.00


per pound) is prohibitive for large-scale applications. Levulinic acid can be produced from lignocellulose material such as paper mill sludge, paper and wood solid waste, and agricultural residues applying high temperature dilute-acid hydrolysis. The manufacturing cost for levulinic acid using such technology has the potential to be reduced to below $ 0.50 per pound. Important chemicals derived from levulinic acid include methyltetrahydrofuran (MTHF), d-aminolevulinic acid (DALA), and diphenolic acid (DPA). MTHF can be blended with gasoline up to 70% by volume without adverse engine performance or reduced mileage. MTHF will have to complete with bioethanol to penetrate the transportation fuel market. DALA is the active chemical in a new group of herbicides and pesticides. DPA is an alternative to bisphenol A which is used in polymers such as polycarbonates and as a comonomer in phenolic resins. Other potential derivatives of levulinic acid include tetrahydrofuran, 1,4-butanediol, c-butyrolactone and N-methylpyrrolidone. Thermochemical xylose derivatives include ethylene glycol, propylene glycol, and glycerol. High hemicellulose content agricultural fiber waste can be hydrolyzed to xylose and other C5 sugars. 12.5.8 Thermochemical Conversion of Oils and Lipid Based Bioproducts

The fatty acid methyl ester of soybean oil is an excellent biobased solvent. It is produced by transesterification of soy oil with methanol, resulting in a mixture of soy fatty acid methyl esters. Methyl soyate-based biobase solvents have been introduced commercially in recent years. They match or even surpass the performance of some conventional solvents while being cost-competitive. Methyl soyate has superior solvent properties, is readily biodegradable, and has a low toxicity compared with other common chemicals. The diversity of structure and inherent functionality of vegetable oils make them prime candidates for use in polymers and resins. The fatty acid chain in vegetable oils, which is hydrocarbon in nature, can be transformed with a spectrum of traditional and breakthrough chemistries to yield high-performance products with desirable properties. The chemistry being considered to modify and functionalize vegetable oils includes transesterification, epoxidation, hydroformylation, and metathesis. Transesterification and epoxidation are already being used to modify soy oil for use in industrial products. Hydroformylation and metathesis are well developed for use on petroleum feedstocks. The challenge will be to develop similar catalyst systems that are effective and efficient on vegetable oils. 12.5.9 Bioproducts via Gasification

The gas mixture produced by gasification is synthetic gas (syn-gas), a mixture of carbon monoxide, carbon dioxide, hydrogen, and methane. Syn-gas can serve as a fuel to produce power and/or may serve as one source of hydrogen for hydro-

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gen fuel cells. It can also be converted to valuable chemicals and liquid fuels. Methanol can be produced from syn-gas. Methanol is a versatile and key starting compound for production of several chemicals, for example acetic acid, formic acid, higher alcohols, MTBE, methyl chloride, methylamines, formaldehyde, or dimethyl ether. The methanol to olefins route (MTO) seems an interesting future route for production of propylene and ethylene which, in turn, are used to produce polyethylene, polypropylene, and other bulk components, for example propylene oxide and its derivatives. Ethanol can also be produced from syngas and this might become a cost-effective alternative to the sugar fermentation route. Fischer–Tropsch chemistry is another approach for converting syn-gas to valuable chemicals and fuels. The chemicals that can be produced include paraffins, monoolefins, aromatics, alcohols, aldehydes, ketones, and fatty acids. 12.5.10 Bioproducts via Pyrolysis

The pyrolysis technology that is closest to commercialization is the pyrolysis of high lignin-containing lignocelluloses; this yields a replacement for phenol in phenol–formaldehyde resins. Phenol–formaldehyde resins are used in plywood, oriented strandboard, and many other applications that serve the building and construction industry. 12.5.11 Biocomposites

Over the past few decades, high performance composites have replaced metals in some applications, and wood in many applications. Biobased materials have the potential to replace one or both parts of composite systems thereby maintaining or improving performance. Plant fibers are very ductile and do not splinter, producing panels that are more shatter-resistant than traditional composites made with wood flour or sawdust. Fibers that could be used include kenaf, jute, sisal, coir, flax, and straw.


Sustained economic growth depends on a secure supply of raw material inputs. With rapid world growth and continuing changes in consumer demands, there is a need to find additional, preferably renewable, resources for industrial production and energy needs. The need is growing to explore the developing technology front to capture opportunities that are provided by renewable resources. Technology advances are beginning to make an impact on reducing the cost of production of industrial products and fuels from biomass, making them more competitive with their equivalents produced from petroleum-based hydrocar-


bons. Developments in pyrolysis, gasification technology, separation technologies via centrifuges or membranes, and the use of enzymes and microbes as biological factories are enabling the extraction of value-added chemicals and intermediates from plant-based materials at competitive cost. As a consequence, industry is investing in the development of new bioproducts that are steadily gaining market share. In the chemicals and food-processing industries, companies are developing new technology that will enable more cost-effective production of industrial products from biomass. Approximately 5% of chemical sales currently depend on biotechnology, but that figure could jump to 10–20% by 2010. The Vision for Bioenergy and Biobased Products in the United States has put forward an ambitious goal for bioproducts. The share of target chemicals that are biobased is set at 25% by 2030. Ethanol is by far the largest-volume chemical made by bioprocessing, but several other categories, including citric and lactic acids, amino acids, and pharmaceutical intermediates are being investigated. The big shift, however, will be the growing importance of biotechnology processes to make bulk chemicals, polymers, and specialty chemicals. In the chemicals and food-processing industries, companies are developing new technology that will enable more cost-effective production of all kinds of industrial product from renewable resources. One example is a plastic polymer derived from corn that is being produced at a 300 million pound per year plant in Nebraska, a joint venture between one of the world’s largest grain merchants, Cargill, and The Dow Chemical Company, the largest chemical producer. Other chemical producers are exploring the use of lost-cost biomass processes to produce chemicals and plastics that are now made from more expensive petrochemical processes. Strategic partnerships between the chemical industry, food, textiles, and agricultural sectors are expected to become the mainstay of the emerging bio-products industry and foster its growth over the coming decades. Although we look forward and point to future potential, it is also recognized that change must start today. Change itself is often continuous, whereas breakthroughs occur at infrequent intervals. Successful progress in this field of new technology will be achieved by integrated and multidisciplinary research in a phase approach. Many current limitations of the use of renewable materials arise from attempts to fit carbohydrate chemistry into a hydrocarbon based chemistry pattern. This is often difficult. Use of renewable materials requires the development of concepts around alternative processing. In the short term, modified processes will enable economic use of renewable resources, and longer-term opportunities exist via the smart combination of chemistry with advanced engineering and with recent biotechnology advances. Performing the research and development sequentially will be slow and take many years. The optimum approach is to ensure coordinated, parallel processing of research results and key target area. Such an approach should also encourage partnership between the public and private sectors. Plant based renewable resources are strategic options to meet the growing needs for industrial building blocks. There will be economic, environmental

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and societal advantages from the development of this new resource base. The appropriate mix of research and development and technology development, in combination with market and public policies, can support the development and the demonstration of viable chemicals, intermediates, and materials, in combination with fuels, heat, and power supply. The impetus for new bio-products will continue to come from favorable government policies, the implementation of bio-refineries, and the desire to reduce the need for and dependence on imported oil. Perhaps the greatest factor driving the growth of bio-products will be acceptance by the public, business enterprises, and governments that biomass can provide a solution to some of the most pressing global resource problems. The impact of the bio-industry on rural development and economics has not yet been quantified, but could be impressive. Development of a bio-industry will require increased production and processing of bio-mass and could provide a boost to rural areas. It could create new income for farmers and foresters. Development of a larger bio-industry would require new processing, distribution, and logistics, and new service industries. This could potentially result in positive economic impacts on rural economic growth in many parts of the world. Using plants as feedstock instead of petroleum or natural gas can potentially reduce the amount of carbon dioxide emitted to the atmosphere. Globally, approximately 62 Gigatons of carbon are taken-up by plants annually in photosynthesis. Producing chemicals and industrial products from biomass directly reduces the associated carbon released during the production of fossil-based products.

References Ashford, Robert D., Ashford’s Dictionary of Industrial Chemicals, 2nd Edition, (Wavelength Publications Ltd., London, 2001) “Affordable Resins and Adhesives from Optimised Soybean Varieties”, US Department of Energy, Industrial Technologies Program Fact Sheet, May 2002 ACS Symposium Series 742: Lignin: Historical, Biological, and Materials Perspectives, W. G. Glasser, R. A. Northey and T. P. Schultz, 1999, ISBN 0-8412-3611-9 Barger, Paul. Methanol to olefins (MTO) and beyond. Catalytic Science Series 2002, 3 (Zeolites for Cleaner Technologies), 239– 260. Biomass Research and Development Act of 2000, http://www.bioproducts-bioenergy.gov/bio_act.html Bioprocessing, Reaping the Benefits of Renewable Resources, Chemical Week, February 11, 2004

“Current situation and future prospects of EU industry using renewable raw materials”, European Renewable Resources and Materials Association (EERMA), Brussels, 2002 Deamin, A. L., Small bugs, big business: The economic power of the microbe, Biotechnology Advances 18 (2000) 499–514 “Developing and Promoting Biobased Products and Bioenergy”, ordered by William J. Clinton, U.S. President, August 1999 (White House, 1999) M. Sasaki, Z. Fang, Y. Fukushima, T. Adschiri, K. Arai, Dissolution and Hydrolysis of Cellulose in Subcritical and Supercritical Water, Ind. Eng. Chem. Res. 2000, 39, 2883–2890 Eggersdorfer, M.; Meyer, J. Eckes, P.; Use of renewable resources for non-food materials; FEMS Microbiology Reviews 103 (1992) 355–364.

References EU FP6 Expression of Interest, http://eoi.cordis.lu/dsp_details.cfm?ID=25887, 2002 “Functionalized Vegetable Oils for Utilization as Polymer Building Blocks”, US Department of Energy, Industrial Technologies Program Fact Sheet, May 2001, http:// www.oit.doe.gov/agriculture Dale, B. E.; Greening the chemical industry: research and development priorities for biobased industrial products, J Chem. Technol. Biotechnol. 78: 1093–1103 (2003) Industrial Biotech and Sustainable Chemistry, European Biotechnology News, No 1–2, Volume 3, 2004, pp. 28–29 Industrial Biotechnology and Sustainable Chemistry, Royal Belgian Academy Council of Applied Sciences, January 2004 Johnston, S., “Emissions and Reduction of Greenhouse Gases from Agriculture and Food Manufacturing”, S.C. Johnson Associates, Inc., for the U.S. Department of Energy, December 1999 Industrial Bioproducts; Today and Tomorrow, US Department of Energy, July 2003 Matsumura, Y.; Minowa, T., International Journal of Hydrogen Energy 29, 2004, 701– 707 Mangold, E. C., Munradaz, M .A., Quellette, R. P., Farah, O. G., and Cheremisinoff, P. N., Coal Liquification and Gasification Technologies, (Ann Arbor Science Publishers, Inc., Michigan) 1982 NACHRICHTEN – Forschungszentrum Karlsruhe Jahrg. 33 1/2001, p. 59–70, A. Kruse, ITC NACHRICHTEN – Forschungszentrum Karlsruhe Jahrg. 35 – 3/2003

NSF Workshop on “Catalysis for Biorenewables Conversion”, April 13–14, 2004 www.egr.msu.edu/apps/nsfworkshop OECD2001 The Application of Biotechnology to Industrial Sustainability (www.oecd.org/ sti/biotechnology) Okkerse, H; Van Bekkum, H. From fossil to green. Green Chemistry, April 1999, 107– 114 Oleochemical Manufacture and Applications, Edited by F. D. Gunstone and R. J. Hamilton, CRC-Press 2001, ISBN 1-84127-219-1 Steps towards a sustainable development, A White Book for R&D of energy-efficient technologies, Eberhard Jochem, March 2004, A Project of Novatlantis – Sustainability at the ETH-Domain (Zurich-CH) D. H. Meadows, D. L. Meadows, Jorgen Randers, William W. Behrens III, The Limits to Growth, A Report to The Club of Rome (1972), Universe Books, New York, 1972 The Refining Process, Corn Refiners Association, Washington DC, August 2002 Transforming biomass to hydrocarbon mixtures in near-critical or supercritical water: USP 6,180,845 B1 “Trash to Treasure”, DOE Pulse, No 50, Feb. 28 2000, http://www.ornl.gov/news/pulse Vision and Roadmap for Bioenergy and Biobased Products in the United States, DOE, October 2002 http://www.bioproductsbioenergy.gov/board.html White Biotechnology: Gateway to a More Sustainable Future, www.europabio.org, 2003 H. Wiedenroth, Zuckerrüben-Magazin, Nr. 32, September 2002

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13 Succinic Acid – A Model Building Block for Chemical Production from Renewable Resources Todd Werpy, John Frye, and John Holladay

13.1 Introduction

The U.S. currently produces about 10 billion bushels of corn annually, with about 20%, or 2 billion bushels, processed by either wet or dry milling. Approximately 550–600 million bushels of the processed corn are used to produce ethanol. The remainder is processed to produce food and non-food products. While corn wet milling has been practiced since the mid 1800s, significant technology advancements have continued to improve processing efficiency and bring about a substantial reduction in water usage and energy requirements. The corn wet milling process consists of seven major unit operations: 1) corn cleaning and inspection, 2) steeping, 3) grinding, 4) germ separation, 5) fiber separation, 6) starch and protein separation, and 7) downstream processing. The major components from corn wet milling include corn oil (1.5 pounds per bushel), corn gluten meal (2.6 pounds per bushel 60% protein), corn gluten feed (13.5 pounds per bushel – 20% protein), and starch (32 pounds per bushel). Corn oil is extracted from the germ, refined and used to produce finished oil. Corn gluten feed is produced from combining the fiber fraction with the steep water followed by drying, and is sold primarily as animal feed. Corn gluten meal is derived from the protein fraction, and is also sold as animal feed. The starch is recovered and converted by several processes to produce various starch products. The starch is also enzymatically hydrolyzed and used as a feedstock to produce products such as high fructose corn syrup, ethanol, lactic acid, lysine, citric acid, and a variety of other fermentation products. However, through further technology advancements, additional valuable products can be derived from corn processing operations. Several new technologies are under development for the production of value added chemicals from starch. This is driven by the desire to expand the capacity at existing corn processing facilities and for improving the overall economics of the integrated biorefinery. The integrated biorefinery is defined here as a facility producing both fuels and chemicals. Specific products currently under developBiorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 2 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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13 Succinic Acid – A Model Building Block for Chemical Production from Renewable Resources

Fig. 13.1 Starburst diagram of succinic acid as a building block chemical.

ment or in various stages of commercialization include lactic acid for the production of polylactic acid, direct hydrogenolysis of sorbitol for the production of propylene glycol and ethylene glycol and the production of polyols for polyurethanes. The product targets have been established because of the potential economic attractiveness and synergistic fit with the existing corn wet milling infrastructure. One of the major considerations for the development of new technologies that can be utilized in a corn wet mill for the production of new chemical products is the concept of platform building blocks. This concept is based on the fact that a single building block has the potential to create a significant number of final products. Succinic acid represents a building block that can be used as a starting material for producing a large number of commodity and specialty chemicals. Succinic acid itself is derived via the fermentation of both C6 and C5 sugars. The derivatives of succinic acid can be obtained via a variety of catalytic processes, primarily hydrogenation. Succinic acid and potential derivatives of succinic acid are represented in the “starburst” diagram below (Fig. 13.1).

13.2 Economics of Feedstock Supply

The major feedstock from corn wet milling to be utilized for the production of fuels and chemicals is starch. Starch costs are estimated to be in the range of $0.05–0.06 per pound. The cost of starch is based on the overall corn wet milling process and the co-product value associated with the oil, corn gluten feed, and corn gluten meal. The actual starch cost depends on both the specific corn wet mill and the size of the mill. Corn pricing has remained essentially constant over the past several decades. The average annual corn price over the past 10 years has been $2.37/bushel.

13.3 Succinic Acid Fermentation

The fiber fraction of corn (5.5 pounds per bushel) is another feedstock opportunity that represents a low cost source of five carbon sugars, xylose and arabinose, that could be used as a fermentation feedstock. Corn fiber is currently sold as part of corn gluten feed, and the value of the feed is on the order of $0.05 per pound. The real value in corn gluten feed is the protein, and the fiber is primarily used as a carrier for the protein. The economics of the fermentation of glucose to succinic acid are driven by a number of key factors including sugar costs, yield of succinic acid from sugar, media costs and productivity. Within these technical challenges the two major issues are the development of an organism that can use a minimal media and improving the overall productivity. The minimal media requirement is that a low cost media such as corn steep liquor can be the sole source of nutrients. It is also essential that the productivity be high enough that the capital costs do not become dominant in the economics. Initial economic analysis has shown that the productivity requirements be on the order of 2–3 g L–1 h–1. The challenge in developing a competitive process for the production of succinic acid and its derivatives is developing a fermentation process that can produce succinic acid at a cost structure comparable to the cost of maleic anhydride from butane. This cost structure must allow for purification costs of succinic acid as well as the overall cost of the fermentation. The economics of the catalytic transformations are competitive with the catalytic conversion of maleic anhydride.

13.3 Succinic Acid Fermentation

In the last decade numerous patents have issued on anaerobic production of succinic acid, microorganisms for such fermentations, and methods for purification, including both separations from cellular biomass and salt splitting. Two US organizations that have worked extensively in this area are Michigan Biotechnology Institute (MBI) [1] and Argonne National Laboratory [2]. Other notable work has taken place at University of Georgia [3] and outside the US includes laboratories in Japan [4] and Korea [5]. Succinate fermentation faces several challenges that must be overcome before an industrial process can be realized. A few illustrative examples are given below. The first challenge is robustness of the organism. Microorganisms that naturally produce succinic acid are often not able to tolerate large concentrations of the acid or its salt and either die or cease growth and active metabo-

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lism, making such organisms unsuitable for industrial use [1 a, 2 a]. This results in low final titers which adds to production and separation costs. A second challenge is media cost. Often expensive nutrients found in yeast extract are required. These might include tryptophan, cysteine, methionine biotin, folic acid, thiamine, riboflavin, and various others [1c]. Niacin may be the important yeast extract component as the organism must regenerate NAD rapidly to achieve the required productivity levels. The cost of fermentation nutrients can have substantial effect on production costs. A third challenge is production of co-products such as acetic acid or pyruvic acid [1 c]. These co-products result in lower titers of desired products and must be removed if additional catalytic upgrading of succinate is to be done. A fourth challenge is the narrow pH range under which bacterial microorganisms can operate. The required neutral conditions for maintaining succinate production (usually between pH 5.8–7.2) [1 a], necessitates base neutralization. Common bases include ammonia or sodium or potassium hydroxide. Thus the actual fermentation product is not succinic acid but rather the ammonium, sodium or potassium salt. Acidification can represent of to 30% of the total cost of organic acid production. The patent literature provides examples of relatively high succinate concentrations produced at neutral pH. Examples of three microbial variants developed by MBI are given in Table 13.1. The data was obtained from shake flask experiments using a peptone, yeast extract, glucose medium [1 b]. The final titer for succinate approached and even surpassed 90 g L–1. However, substantial amounts of either acetate or pyruvate were co-produced. Furthermore, the nutrient concentrations, even for the “low nutrient” conditions were substantial. It is interesting to note that the organism that gave low acetate had an increased production of pyruvate. Organism FZ 6 was selected for further evaluation using various nutrient conditions. Results are shown in Table 13.2. Increasing the corn steep liquor concentration had a dramatic effect on the final titer, whereas increasing the yeast extract concentration had only a moderate effect. Examples with lower yeast extract were not provided.

Table 13.1 Succinate fermentation described by MBI (US Patent 5,573,931). Vial fermentation with “low nutrient“ conditions (corn steep 10 g L–1, yeast extract 5 g L–1). Strain

130Z FZ 6 FZ 21 a)

Concentration (g L–1)

Yield a)

Succinate

Acetate

Formate

Pyruvate

Propionate

68.5 86.9 96.4

15.1 4.9 16.5

3.9 0 0

9.8 12.5 4.4

0.9 0.7 3.6

Yield = (grams succinic acid/grams dextrose) ´ 100

85 94 89

13.3 Succinic Acid Fermentation Table 13.2 Effect of nutrients on FZ 6 succinate yield (g L–1). (Reproduced from US Patent 5,573,931). Nutrient level

CSL a)

Yeast extract

Ave succinate

Yield (wt%)

“Low” “Medium” “High”

10 15 10

5 5 15

84.9 93.4 90.4

92 94 82

a)

Corn steep liquor

The high producing succinate strain FZ 21, was also examined in a 1 L fermenter using 15 g L–1 yeast extract, corn steep liquor, MgCO3 (for pH control) and glucose. The succinate yield was 78%. Results are shown in Fig. 13.2. Other groups have also been involved in strain development. Strains developed at Argonne have been demonstrated on more complex carbohydrate streams. For example they have used industrially produced rice straw hydrolysate (Arkenol, Inc.) with Difco yeast extract (5 g L–1), tryptone (10 g L–1) as well as various buffers. Figure 13.3 shows data for the consumption of the two major

Fig. 13.2 Succinate production with time (Reproduced from US Patent 5,573,931).

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Fig. 13.3 Glucose and xylose uptake during fermentation (Reproduced from US Patent 6,743,610).

sugars7glucose and xylose7and production of major products7succinate, acetate, and ethanol. In this example the final succinate titer reached 63 g L–1 in 192 h. The amount of acetate co-produced was 4.9 g L–1. In summary, there has been substantial strain development for the purpose of commercial succinate production. The efforts described have concentrated on improvements in final titer, organism robustness, and to a lesser extent selectivity (less acetate). The Argonne group has also demonstrated the use of more complex carbohydrate hydrolysate streams. In all examples, fermentations were done at neutral pH. However, it is believed that several groups are working on low pH fermentations.

13.4 Succinic Acid Catalytic Transformations

The chemical conversion of succinic to produce both commodity chemicals and specialty chemicals is shown in Fig. 13.4. The major products include 1,4-butanediol (1,4-BDO), tetrahydrofuran (THF) and c-butyrolactone (GBL). These products can all be produced via catalytic hydrogenation and are analogous to the products produced from maleic anhydride via hydrogenation. One of the potential specialty

13.5 Current Petrochemical Technology

Fig. 13.4 Derivatives of succinic acid.

products that can be produced from succinic acid, or more appropriately from diammonium succinate (DAS) is N-methylpyrrolidinone (NMP).

13.5 Current Petrochemical Technology

13.5.1 1,4-BDO, THF, GBL, and NMP

Several catalytic technologies have been developed in the past for the production of 1,4-BDO from petroleum based resources. Early technology development included a two step process based on Reppe chemistry. The first step in the process is the conversion of acetylene and formaldehyde to produce primarily 1,4butynediol. The second step is the catalytic hydrogenation of 1,4-butynediol to 1,4-BDO. The 1,4-BDO produced can be dehydrated to produce THF.

A second well know technology is the Lyondell propylene oxide based process. This process utilizes propylene oxide as the starting material and converts PO to allyl alcohol via isomerization. The allyl alcohol is converted to 4-hydroxybutyraldehyde via hydroformylation with CO and H2. The final step is the hydrogenation of the aldehyde to produce 1,4-BDO.

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A third process utilizes butadiene as the starting material. In this process, butadiene is reacted with acetic acid and oxygen to produce the intermediate 1,4-diacetoxy-2-butene and water. The 1,4-diacetoxy-2-butene is then hydrogenated over a catalyst to form the saturated intermediate which is then hydrolyzed to produce 1,4-BDO.

The process developed by Huntsman employs a catalyst for the direct oxidation of butane to maleic anhydride. Maleic anhydride is then reacted with methanol to produce dimethyl maleate. The dimethyl maleate is hydrogenated to produce 1,4-BDO.

The final process is the process developed by BP Amoco and Lurgi. The process is also based on the direct oxidation of butane to maleic anhydride. This process differs from the Huntsman process in that the maleic anhydride is directly reduced over a catalyst to produce THF.

It is currently believed that this is the low cost technology for THF production. All of these technologies are described in more detail in the open literature and the patent literature [6]. The production of THF can be achieved from any of the technologies that produce 1,4-BDO. In one form or another BDO or one of its precursors is dehydrated to yield THF. The overall yield is generally greater than 90%. GBL is produced by the cyclic dehydrogenation of 1,4-BDO. The majority of GBL produced is used for the production of N-methylpyrrolidinone (NMP). In

13.6 Current Biobased Technology

the process to produce NMP, GBL is converted in a three stage liquid phase process. The reaction is carried out by reacting GBL with methylamine and water. After reaction the final NMP produce is recovered via distillation with methylamine and water being recycled back to the process.

13.6 Current Biobased Technology 13.6.1 1,4-BDO, GBL, and NMP

There is extensive patent literature regarding the conversion of maleic acid and succinic acid to produce 1,4-BDO, THF, GBL and NMP, the bulk of this work has been developed utilizing petrochemical feedstocks as the starting material. There has been a relatively low level of effort on the direct aqueous phase catalytic conversion of bio-derived succinic acid to value added products. The remainder of this chapter will describe work at Pacific Northwest National Laboratory on the catalysis of converting succinic acid and diammonium succinate and describe some of the unique challenges associated with fermentation derived starting materials. Pacific Northwest National Laboratory has developed a series of supported metal catalysts for the highly selective, aqueous phase hydrogenation of succinic acid to GBL. The basic catalysts materials consist of an active form of palladium on a specific carbon support. Selection of the carbon support with the appropriate surface chemistry and porosity is critical in achieving highly selective catalysts. Pacific Northwest National Laboratory has demonstrated that an active palladium on carbon can afford yields of GBL greater than 95%. The reaction is carried out in either a batch mode or in a continuous mode. Operating temperatures are on the order of 150–175 8C with pressures ranging from 800–2000 psig. The weight percent of succinic acid can range from 5–25%. An important aspect of this technology development is that during the fermentation of glucose to produce succinic acid, acetic acid is often seen as one of the co-products. The technology developed at Pacific Northwest National Laboratory is selective in the hydrogenation of succinic acid in the presence of acetic acid, to the point that none of the acetic acid is hydrogenated to ethanol. This has important economic implications in that acetic acid could be recovered as a co-product and that selective reduction of succinic acid without the reduction of acetic acid reduces the total consumption of hydrogen. A second element of this work relates to the production of 1,4-BDO. In an attempt to maximize the yields of 1,4-BDO, a process was developed in which GBL produced by hydrogenation of succinic acid is separated via distillation. The purified GBL is then catalytically hydrogenated over bimetallic catalysts supported on carbon. The bimetallic include nickel and palladium coupled with rhenium. These catalysts have afford selectivity greater than 95% to 1,4-BDO

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from GBL. Operating temperatures range from 150–200 8C with hydrogen pressures ranging from 1000 to 2000 psig. 13.6.2 Derivatives of Diammonium Succinate

As has been described previously succinic acid from the fermentation is an excellent building block for the production of industrially important chemical intermediates. One of the challenges with the fermentation is that neutralization is required which leads to significant downstream processing to convert the succinic acid salt back to the free acid. One strategy for deriving products from succinic acid fermentations is to identify products that can be obtained directly from the salts. One route that affords the economical production of chemicals from diammonium succinate is the formation of 2-pyrrolidone (2P) and Nmethylpyrrolidone (NMP). In addition to 2P and NMP, derivatives such as N-vinyl-2-pyrrolidone can also be produced. The reaction chemistry for these products is shown in Fig. 13.5. 2-Pyrrolidone can be produced by the direct hydrogenation of aqueous diammonium succinate with hydrogen in the presence of an active metal catalyst [7]. A mixed product of NMP and 2P can be produced from the conversion of DAS in the presence of methanol and an active metal catalyst. Figure 13.6 shows the results of converting aqueous DAS in the presence of methanol and hydrogen over a supported rhodium catalyst. Conversion for this reaction is near 100% with a yield of NMP of about 50% and a yield of 2P of about 30%. The remainder of the product is a polymer of 2P. It is interesting to note the formation of N-methylsuccinimide in the early part of the reaction, which converts to NMP as the reaction proceeds. Once 2P is formed it essentially remains during the course of the reaction as shown in Fig. 13.6.

Fig. 13.5 Conversion pathways of diammonium succinate.

13.6 Current Biobased Technology

Fig. 13.6 NMP and 2P production over a rhodium catalyst.

As was described above NMP can be produced in a single-stage reaction with DAS, hydrogen and methanol. The drawback of the single-stage process is that a mixture of both NMP and 2P is formed. This mixture would require substantial separation costs to obtain a pure NMP product. Employing a two-stage process in which N-methylsuccinimide is formed first, prior to reduction, leads to near-complete conversion and selectivity to NMP. Results of the hydrogenation of N-methylsuccinimide are given in Table 13.3. The reaction was carried out at 1900 psig hydrogen for the catalyst and temperature described. It is evident from the results that pre-forming NMS is preferred to obtain high levels of NMP. Using the appropriate catalyst and reaction conditions, virtually 100% NMP can be obtained when starting with NMS [8–11].

Table 13.3 Production of NMP from NMS. Catalyst composition

Feedstock

Reaction temperature

NMP +2P Yield

NMP:2P Ratio

2.5%Rh/2.5%Re/C 2.5%Rh/2.5%Re/C 2.5%Rh/2.5%Zr/C

NMS NMS NMS

265 8C 200 8C 200 8C

54% (5 h) 89% (8 h) 81%

5.3 67.3 38.1

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13.7 Conclusions

The fermentation of glucose and other sugars derived from biomass for the production of succinic acid provides a valuable building block for the production of industrially important chemicals. There are still several challenges before commercialization will be realized. The first major hurdle is the need for further cost reductions in the fermentation of glucose to succinic acid. The specific cost barriers include reducing the media requirements so that a low cost nutrient source such as corn steep liquor could be used, improving the volumetric productivity of the organism and ultimately a low pH fermentation. From a process perspective, reducing the cost of separation/purification would have a significant overall economic impact. With respect to catalysis, the overall yields for the conversion of succinic acid and diammonium succinate to commercially important chemical intermediates needs to be demonstrated on a longer term continuous basis which will prove that the catalyst lifetime is commercially viable. In general, the potential for biobased products to replace or displace existing petrochemical routes to value added products continues to move closer to commercialization. Overcoming a broad range of technical barriers in fermentation, separations and catalysis will be critical to the realization of economically viable biobased products from renewals.

References 1 (a) Guettler, M. V.; Jain, M. K.; Soni, B. K.

US Patent 5,723,322; March 3, 1998 (Michigan Biotechnology Institute). “Process for making succinic acid, microorganisms for use in the process and methods of obtaining the microorganisms.” (b) Guettler, M. V.; Jain, M. K.; Rumler, D. US Patent 5,573,931; Nov 12, 1996 (Michigan Biotechnology Institute). “Method for making succinic acid bacterial variants for use in the process, and methods for obtaining variants.” (c) Guettler, M. V.; Jain, M. K. US Patent 5,521,075; May 28, 1996 (Michigan Biotechnology Institute). “Method for making succinic acid, Anaerobiospirillum succiniciprodens variants for use in process and methods for obtaining variants.” (d) Guettler, M. V.; Jain, M. K.; Soni, B. K. US Patent 5,504,004; April 2, 1996 (Michigan Biotechnology Institute). “Process for making succinic acid, microorganisms for use in the process and

methods of obtaining the microorganisms.” (e) Ponnampalam E. US Patent US Patent 6,284,904; Sep 4, 2001 (Michigan Biotechnology Institute). “Purification of organic acids using anion exchange chromatography.” (f) Berglund, K. A.; Elankovan P.; Glassner, D. A. US Patent 5,034,105; July 23, 1991 (Michigan Biotechnology Institute). “Carboxylic acid purification and crystallization process.” 2 (a) Donnelly, M. I.; Sanville-Millard, C. Y.; Nghiem, N. P. US Patent 6,743,610; June 1, 2004 (The University of Chicago). “Method to produce succinic acid from raw hydrolysate.” (b) Donnelly, M. I.; Millard, C. S.; Stols, L. US Patent RE37,393; Sep. 25, 2001 (The University of Chicago). “Mutant E. Coli strain with increased succinic acid production.” (c) Donnelly, M. I.; Sanville-Millard, C.; Chatterjee, R. US Patent 6,159,738; Dec. 12, 2000 (The University of Chicago). “Method for construction of bacterial

References

3

4

5

6

strains with increased succinic acid production.” (d) Nghiem, N. P.; Donnelly, M.; Millard, C. S.; Stols, L. US Patent 5,869,301; Feb 9, 1999 (Lockhead Martin Energy Research Corporation). “Method for the production of dicarboxylic acids.” Gorkan, R. R.; Eiteman, E. US Patent 6,455,284, Sep. 24, 2002. (The University of Georgia Research Foundation). “Metabolically engineered E. Coli for enhanced production of oxaloacetate derived biochemicals.” Okino, S.; Inui, M.; Yukawa, H. Poster at the Biotechnology for Fuels and Chemicals Conference, 2003. Chang, H. N.; Chang, Y. K.; Kwon, S. H.; Lee, W. G.; Lee, P. C.; Yoo, I. K.; Lim, S. J. US Patent 6,596,521; July 22, 2003 (Korea Advanced Institute of Science and Technology). “Method for manufacturing organic acid by high efficiency continuous fermentation.” See for example Kirk–Othmer Encyclopedia of Chemical Technology, 2005 on-line edition.

7 Frye Jr., J. G.; Zacher, A. H.; Werpy, T. A.;

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9

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11

Wang, Y. “Catalytic Preparation of Pyrrolidones from Renewable Resources” in Catalysis of Organic Reactions, Sowa Jr., J. R. Ed; Taylor and Frances, Boca Raton Werpy, T. A.; Frye, Jr, J. G.; Wang, Y.; Zacher, A. H. U.S. Patent 6,706,893; March 16, 2004 (Battelle Memorial Institute) “Methods of Making Pyrrolidones” Werpy, T. A.; Frye, Jr. J. G,; Wang, Y.; Zacher, A. H. U.S. Patent 6,670,483; December 30, 2003 (Battelle Memorial Institute) “Methods of Making Pyrrolidones” Werpy, T. A.; Frye, Jr, J. G.; Wang, Y.; Zacher, A. H. U.S. Patent 6,632,951; October 14, 2003 (Battelle Memorial Institute) “Methods of Making Pyrrolidones” Werpy, T. A.; Frye, Jr, J. G.; Wang, Y.; Zacher, A. H. U.S. Patent 6,603,021; August 15, 2004 (Battelle Memorial Institute) “Methods of Making Pyrrolidones”

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14 Polylactic Acid from Renewable Resources Patrick Gruber, David E. Henton, and Jack Starr

14.1 Introduction

In today’s world of green chemistry and concern for the environment, polylactic acid (PLA) is interesting. One hundred percent of the carbon in PLA originates from carbon dioxide in the atmosphere. PLA has very versatile properties, giving it the ability to compete in a wide variety of markets, and it is economical. The types of lactic acid used to make the polymer combined with the length of polymer chain, and the extent of branching control the versatility of PLA. The stereoisomers of lactic acid required for PLA can only be produced economically by bioprocesses. PLA also rapidly degrades in the environment under composting conditions and the byproducts are of very low toxicity, eventually being converted to carbon dioxide and water. PLA is an interesting combination, it performs well, is economical, and it is “green”. Polylactic acid (PLA) is a rigid thermoplastic polymer that can be semi-crystalline or totally amorphous, depending on the stereochemical purity of the polymer backbone. l-(–)-Lactic acid (2-hydroxypropionic acid) is the natural and most common form of the acid, but d-(+)-lactic acid can also be produced by microorganisms or by racemization and this “impurity” acts much like comonomers in other polymers such as poly(ethylene terephthalate) (PET) or polyethylene (PE). In PET, diethylene glycol or isophthalic acid is copolymerized into the backbone at low levels (1–10%) to control the rate of crystallization. In the same way, d-lactic acid units are incorporated into l-PLA to optimize the crystallization kinetics for specific fabrication processes and applications. PLA is a unique polymer that in many ways looks like PET, also a polyester, but also performs much like polypropylene (PP), a polyolefin. It may eventually be the polymer with the broadest range of applications because of its ability to be stress crystallized, thermally crystallized, impact modified, filled, co-polymerized, and processed in most polymer processing equipment. It can be formed into transparent films or injection molded into blow-moldable preforms for bottles, similar to PET. PLA also has excellent organoleptic characteristics and is excellent for food contact and related packaging applications. Biorefineries – Industrial Processes and Products. Status Quo and Future Directions. Vol. 2 Edited by Birgit Kamm, Patrick R. Gruber, Michael Kamm Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-31027-4

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14 Polylactic Acid from Renewable Resources

Although PLA has a unique combination of characteristics, commercial growth has been limited by high production costs (greater than $ 2/lb), limited availability of both the monomer and polymer, and limited performance in applications. Until now PLA has enjoyed little success in replacing petroleumbased plastics in commodity applications, with most initial uses limited to biomedical applications such as sutures [1]. PLA is not a new to the world polymer industry. Carothers [2] investigated the production of PLA from the cyclic dimer (lactide) of lactic acid as early as 1932. Even before that, low-molecular-weight dimers and oligomers were detected when water was removed from an aqueous solution of lactic acid [3]. The announcement of the formation of a new company, Cargill Dow LLC, in 1997 brought two large companies together to focus on the production and marketing of PLA with the intention of significantly reducing the cost of production and broadening the performance of PLA, and developing the markets [4].

14.2 Lactic Acid

Lactic acid (2-hydroxypropionic acid) is an a-hydroxy organic acid. It is commercially available as a colorless to slightly yellow solution at concentrations of typically 50%, 80% and 88%. A variety of lactate salts, for example calcium lactate and sodium lactate, and lactate esters are also available commercially. Lactic acid is an important food ingredient used for its acidity, flavoring, and ability to control microorganisms. Lactic acid is also used in a variety of industrial and pharmaceutical applications. 14.2.1 Lactic Acid Production Routes 14.2.1.1 Chemical Synthesis Lactic acid can be made as a racemic mixture using lactonitrile as starting material. This route has diminished because many commercial applications require the l-lactic acid isomer and various manufacturers have left the business. Musashino Chemical Laboratories in Japan remains one of the few producers of racemic lactic acid. A chemical synthesis route may prove to be economical if combined with either a low-cost chiral separation or a stereoselective synthesis. These unit operations are commercial for the production of low volume, high cost chemicals such as pharmaceuticals, but have not routinely been applied into high volume, low cost commodity chemicals.

14.2 Lactic Acid

14.2.1.2 Fermentation The fermentation of lactic acid by humans is very old, as shown in the production of yogurt and other foods. In the last hundred years, people have mastered the fermentation and purification of lactic acid for food and industrial use. The anaerobic fermentation of sugars to produce lactic acid is the main commercial route. Fermentation can provide either l-lactic acid or d-lactic acid with a chiral purity near 100%. The exact chiral purity if the lactic acid formed depends on the characteristics of the microorganism used for fermentation. Current lactic acid production by fermentation is limited to the small number of companies listed in Table 14.1. There are numerous other smaller lactic acid manufacturers, particularly in China, that are not listed here. Virtually all lactic acid manufacturers have some affiliation with a company that provides the sugar feedstock for the lactic acid fermentation. There has been a large increase in the worldwide lactic acid capacity in the last decade as the PGLA-1, B&G, and Cargill Dow plants have all started up production between 1998 and 2004. All three of these plants have been the result of joint ventures. Another joint venture between Dupont and ConAgra, named EcoChem, was established in the early 1990s to produce lactic acid. EcoChem closed down the manufacturing plant in 1994 because of production problems. Cargill Dow has the largest single plant at a declared capacity of 400 million lbs per year. Virtually all the lactic acid produced at the Cargill Dow facility is used internally for the production of lactic acid-based polymers. Cargill Dow does sell lactide, a six-membered ring consisting of two lactic acid moieties that can be easily converted back to lactic acid, by addition of water, or to other chemical derivatives. For the other seven manufacturing plants listed in Table 14.1, for which the main applications are the food and industrial markets, the declared capacities range from 20 million to 100 million lbs per year.

Table 14.1 Lactic acid manufacturers. Manufacturing sites

Main application

Affiliations

Food and Industrial

Subsidiary of CSM

PGLA-1

The Netherlands, Spain, Brazil Nebraska, USA

Food and Industrial

Galactic B&G

Belgium China

Food and Industrial Food and Industrial

Archer Daniels Midland (ADM) Cargill Dow LLC

Illinois, USA

Food and Industrial

Joint venture between Purac and Cargill Owned by Finasucre Joint venture between Galactic and BBCA Biochemicals –

Nebraska, USA

Polymers

Purac

Joint venture between Cargill and Dow Chemical

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14.2.2 Production by Fermentation

To make a commercial grade lactic acid product by fermentation, there must be four main processing areas. These processing areas and their purpose are listed in Table 14.2. A single-unit operation may actually be accomplishing two purposes, such as the removal of volatile impurities during a concentration step. Successful lactic acid fermentation needs the right combination of a microorganism, sugars, nutrients, and neutralizing agent to meet the productivity, lactic acid concentration, and low impurities levels to economically produce lactic acid of sufficient purity for PLA production.

14.2.2.1 Microorganisms Several microorganisms are able to produce lactic acid in an economically viable process. There is probably no perfect microorganism because each company and geographic location has different sugar sources, nutrient availability, and separation technology. For instance, a Lactobacillus microorganism needs lowcost nutrients like corn steep liquor, but this system may result in higher separation costs to produce a high purity product. Alternatively, the use of Rhizopus with a defined medium may produce lower yields, but the lower separation costs of a relatively clean broth could make it viable. Although there are numerous microorganisms that can make lactic acid, Table 14.3 lists the genus of three microorganisms that have the characteristics of a commercially viable lactic acid-producing microorganism. The characteristics listed for each genus are generalizations from the literature. Given the diversity within each genus, it is likely that a strain exists that is contrary to the generalized characterization listed here. Producers regard their specific production strain as part of their competitive advantage.

Table 14.2 Lactic acid processing areas. Area

Purpose

Fermentation

Microbial conversion of sugars to lactic acid, addition of neutralizing agent to maintain pH in fermentor and make lactate salts Convert lactate salts to lactic acid Separation of cells, nutrients, and residual sugars to obtain lactic acid solution that meets needed purity requirement. Removal of water from lactic acid solution to achieve required concentration

Acidification Purification Concentration

14.2 Lactic Acid Table 14.3 Commercially viable lactic acid-producing microorganisms.

Lactobacillus Bacillus Rhizopus

Positive Characteristics

Negative Characteristics

High productivity Low by-products High operating temperatures High chiral purity Low requirement for complex nutrients Naturally produces enzymes for starch saccharification

Susceptible to phage Requires complex media Higher by-products Lower yields Need to control morphology to enable easy cell removal

14.2.2.2 Sugar Feedstock Depending on location, the sugar source is either sucrose from sugar cane or sugar beet or dextrose corn syrups made from corn (maize). Because the cost of the sugar feedstock is a large portion of the production cost of lactic acid, virtually all lactic acid production is integrated with a manufacturing plant that produces the sugar feedstock. Other carbohydrate sources have been tried. The EcoChem plant was located in Wisconsin, USA, and used lactose from cheese whey as a sugar source. Production facilities integrated with corn milling could possibly use corn starch as a cheaper sugar source. The use of sugars from lignocellulosics for lactic acid fermentation may be the next major technology advance to lower the cost of sugar feedstocks for fermentation. Because pentose sugars are also derived from lignocellulosics, biocatalysts with the ability to convert pentoses to lactic acid are needed.

14.2.2.3 Nutrients The choice of nutrients depends on the production microorganism. Complex nutrients that are typically used would be corn-steep liquor and yeast extract paste. These are available commercially from several suppliers. Most microorganisms require various salts, particularly ammonium phosphate, and vitamins. If a complex nutrient is used, it may provide much of the microorganism’s salt and vitamin needs. Salt and vitamins may need to be supplemented into the media as the amount of complex nutrient decreases.

14.2.2.4 Neutralizing Agent Because the microorganisms actually produce lactic acid, without addition of neutralizing agent or base to form a lactate salt the pH of the fermentation would drop quickly and lactic acid production would decrease significantly. Lime (Ca(OH)2) and chalk (Ca(CO3) are used industrially as neutralizing agents to control the pH of the fermentor between 5.0 and 6.8, depending on the exact

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production microorganism. Other neutralizing agents have been proposed, for example ammonia (ammonium hydroxide), sodium hydroxide, and potassium hydroxide. EcoChem reportedly formed ammonium lactate in their process. The choice of the neutralizing agent has important implications for the acidulation area of the plant. When a base is added to control pH, the cation of the base then forms a lactate salt. The cation of the lactate salt must be bio-compatible with the microorganism and easily removed during purification. If a microorganism could produce lactic acid in a low-pH (< 3.8) environment, most of the lactic acid produced in the fermenter would be in the protonated form. This fermentation would have significant cost advantage, because it would use less neutralizing agent and less acidifying agent, and produce less by-product salt. For instance, Cargill Incorporated. has developed a microorganism that is able to produce lactic acid at a final pH of less than 4.0 [5]. 14.2.3 Acidification

The fermentation area produces a lactate salt, because the fermenters operate at near neutral pH. The acidulation area of the plant needs to convert the lactate salt to lactic acid. There are two main options for the acidification – strong acid addition and salt-splitting.

14.2.3.1 Strong Acid Addition By directly adding sulfuric acid to the lactate salt broth, lactic acid is formed and a by-product salt is formed. Most industrial processes form calcium lactate in the fermenters and add sulfuric acid to the broth to crystallize out calcium sulfate dihydrate (gypsum). The resulting solids are then removed by filtration. As the lactic acid market grows, the cost and ability to dispose of gypsum will be a concern. Lactic acid manufacturers have worked to develop alternatives to land filling gypsum, for example placing the material in the drywall, cement, or agriculture industries. Gypsum is a relatively low-value by-product salt but this route has been used commercially because of the low cost of the calcium base and sulfuric acid, and the potential of putting the gypsum into industrial use. Other combinations of bases and acids have been proposed, such as ammonia for pH control and sulfuric acid to acidify thus producing ammonium sulfate as the by-product salt. Ammonium sulfate can be sold as a fertilizer. Because ammonia is more expensive than the calcium bases, the sale of the ammonium sulfate has to more than make up for the higher cost of raw materials. Ammonium sulfate is also more soluble in water than calcium sulfate, which complicates the separation process.

14.2 Lactic Acid

14.2.3.2 Salt Splitting Technology Considerable research effort has been devoted to reforming the neutralizing agent from the lactate salt in a process called salt splitting. A “black box” flow sheet of the salt splitting process for sodium lactate is shown in Fig. 14.1. Many processes have been proposed to accomplish salt splitting. At least three salt-splitting options have been piloted in attempts to understand the process and economics. They are water-splitting electrodialysis, thermal cracking of ammonium lactate, and carbon dioxide/amine extraction. In water-splitting electrodialysis, water is split using electric force in a bipolar membrane. The lactate anion passes through an anion-selective membrane to combine with the proton to form lactic acid. The cation passes through a cation selective membrane to combine with the hydroxyl anion to form a cation hydroxide, such as sodium hydroxide. This water-splitting electrodialysis option has been investigated by both ADM and Cargill in conjunction with Michigan Biotechnology Institute. Because ammonia is volatile, ammonium lactate can be salt-split by vaporizing ammonia from the broth. As ammonia is removed from solution, the solution becomes acidic, thus making it more difficult to remove more ammonia. To drive the salt-splitting reaction toward high conversion, the acidity associated with the lactic acid must be addressed. One method to do this would be to add an alcohol, for example ethanol (EtOH), to the ammonium lactate solution. The alcohol reacts with the lactic acid to form a lactate ester. The lactate ester is then vaporized to remove the lactic acid and enable more ammonia to be vaporized. The overall chemistry is shown below.

NH4 Lac EtOH ! NH3 " EtLac " Carbon dioxide is a weak acid when it dissolves into water as carbonic acid. The carbonic acid can provide a proton to acidify a small fraction of the lactate salt. This sequence of chemical reactions is shown below. CO2 H2 O NaLac ! H2 CO3 NaLac ! H HCO3 NaLac ! NaHCO3 H Lac

Fig. 14.1 Salt splitting of sodium lactate.

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As the solution becomes acidic, the system reaches an equilibrium. The lactic acid must be removed from the aqueous phase to achieve high conversion to lactic acid. Cargill solved this problem by extracting the lactic acid into an amine-based solvent [6]. 14.2.4 Purification

Several different purification processes have been commercialized on an industrial scale. The choice of a purification process for lactic acid depends the amount of residual sugars, residual nutrients, and fermentation by-products in the broth and the purity required for the application. In all cases the microorganisms or cells must be removed from the broth.

14.2.4.1 Cell Removal The options for cell removal depend on the choice of the production microorganism. For a Lactobacillus or Bacillus-based fermentation, a simple filtration step would not work well because the cells are too small and would leak by the filter cloth. These small cells must be flocculated before filtration to achieve a clarified broth. A clarified liquid can be obtained by decantation from the flocculated cells. Crossflow filtration using a 0.1 to 1.0 lm membrane could also provide an economical method for removing the microorganisms. For a Rhizopus-based fermentation, the cell morphology must be controlled as a pellet to enable easy filtration. Methods are known for controlling this aspect of the fermentation. A belt filter or pressure filter can provide adequate broth clarity for Rhizopus fermentation, with good pellet morphology.

14.2.4.2 Separation of Residual Sugars, Nutrients and Fermentation By-products Three separation schemes are used on a commercial scale – solvent extraction, direct distillation, and distillation of lactate ester. After these operations, the lactic acid solution may need to be passed through activated carbon and cation and anion-exchange resin to produce a clear to slightly yellow, deionized product. Solvent Extraction Solvent extraction is performed using a solvent comprising, typically, three components – a long chain tertiary amine, a water-immiscible alcohol, and an aliphatic hydrocarbon. The tertiary amine, for example trioctylamine, acts as a base to form an ion pair with the lactic acid in the organic phase. The water-immiscible alcohol, for example octanol, is designed to increase the distribution of lactic acid in the organic phase. Other functional groups such as ketones and phosphates can be used to replace the alcohol. The aliphatic hydrocarbon is used to control the viscosity and phase separation characteristics. The basic nature of the amine enables good selectivity for lactic acid

14.2 Lactic Acid

Fig. 14.2 Amine solvent extraction process flow diagram.

over residual sugars and nutrients, but does not provide good separation from acidic impurities. The forward extraction of the lactic acid into the solvent phase is performed at relatively low temperatures to help increase the distribution of the lactic acid into the solvent. The lactic acid is released from the amine and the solvent phase by back extracting the lactic acid into an aqueous solution at high temperatures. A process flowsheet for this solvent extraction process is shown in Fig. 14.2. Solvent exchange can be integrated with salt-splitting operations such as those described above with carbon dioxide. The solvent extraction acts as a second phase to help drive the salt splitting reaction to high conversion. Solvent extraction could also be used to sequester the lactic acid when vaporizing the ammonia from ammonium lactate. Direct Distillation Lactic acid can be purified by distillation. The distillation scheme can be a single stage or multi-stage depending on the purification required. This can be an efficient method for separating low volatility compounds like sugars and amino acids from lactic acid. A single stage distillation is less efficient for separating fermentation by-products that have a vapor pressure near the vapor pressure of lactic acid. In a multi-stage distillation, care has to be taken to minimize the formation of lactic acid oligomers, such as lactyllactate, that have significantly lower vapor pressures and may result in loss of yield. Distillation of Lactate Ester The distillation of lactic acid can be difficult, because it can form lactic acid oligomers, require high temperatures, and low pressures. By reacting lactic acid with an alcohol, such as ethanol, to form a lactate ester, many of the issues with the distillation of lactic acid can be avoided. High-purity lactic acid can be obtained by this process as multi-stage distillation has the potential to separate other carboxylic acids. A simplified process flowsheet with ethanol as the alcohol is shown in Fig. 14.3. The alcohol must be recycled in the process which adds complexity. The water and the alcohol have higher vapor pressures than ethyl lactate, so they preferentially go into the vapor phase compared with the lactate ester. Thus ethanol must be recycled back to the esterification reactor and water must be removed from the system to drive the esterification reaction to high conversions. Re-

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Fig. 14.3 Ethyl lactate purification process flow diagram.

searchers at Argonne National Laboratories have developed a pervaporation process that has the potential to minimize the cycle streams around the esterification reactor by using a selective membrane [7]. This route may have benefits for companies like Archer Daniels Midland and Cargill, that also have ethanol production, compared with other routes. This purification scheme may also favor companies that have significant sales of lactate esters, for example ethyl lactate, because they would not have to hydrolyze the esters to produce a final product. Concentration Finally the lactic acid is concentrated using standard technology and then transferred via pipeline to the PLA plant. Typically the lactic acid would have an optical purity of >98% at about a 60–70% concentration.

14.3 PLA Production

Production of commercially useful PLA requires control of the optical composition of the polymer, control of rheology, and stabilization of the polymer, all with high yield under simple processing conditions. Control of the optical composition affects both the melt behavior in polymer-converting equipment and the end use properties. Control of the rheology enables PLA to be processed on a wide variety of polymer processing equipment, and melt stabilization makes the PLA stable to conditions typically seen in polymer-fabrication operations. PLA can be prepared by both direct condensation of lactic acid and by the ring opening polymerization of the cyclic lactide dimer, as shown in Fig. 14.4.

14.3 PLA Production

Because the direct condensation route is an equilibrium reaction, difficulties removing trace amounts of water in the late stages of polymerization usually limit the ultimate molecular weight achievable by this approach. Most work has focused on the ring opening polymerization of lactide, although other approaches, for example azeotropic distillation to drive the removal of water in the direct esterification process, have been evaluated [8]. Cargill Dow LLC has developed a patented, low cost, continuous process for the production of lactic acid-based polymers [9]. The process combines the substantial environmental and economic benefits of synthesizing both lactide and PLA in the melt rather than in solution and, for the first time, provides a commercially viable biodegradable commodity polymer made from renewable resources. The process starts with lactic acid produced by fermentation of dextrose, followed by a continuous condensation reaction of aqueous lactic acid to produce low molecular weight PLA pre-polymer (Fig. 14.5). Next, the low-molecular-weight oligomers are converted into a mixture of lactide stereoisomers using tin catalysis to enhance the rate and selectivity of the intramolecular cyclization reaction. The molten lactide mixture is then purified by vacuum distillation. Finally, PLA high polymer is produced using a tin-catalyzed, ring-opening lactide polymerization in the melt, completely eliminating the use of costly and environmentally unfriendly solvents. When polymerization is complete any remaining monomer is removed under vacuum and recycled to the beginning of the process (Fig. 14.6). This process is currently in operation in a recently constructed 300 million lb/yr commercial-scale PLA plant in Blair, Nebraska, USA. As we move into the 21st century, increased utilization of renewable resources will be one of the strong drivers for sustainable products. Reduced energy consumption, waste generation, and emission of greenhouse gases will take on greater emphasis. Polylactic acid is the first commodity plastic to incorporate these principles and Cargill Dow LLC was awarded the 2002 Presidential Green Chemistry Award for their process to produce NatureWorks PLA. The published literature on PLA is extensive and has been reviewed in detail in several recent publications [10].

Fig. 14.4 Polymerization routes to polylactic acid.

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Fig. 14.5 Schematic of PLA production via prepolymer and lactide.

Fig. 14.6 Non-solvent process to prepare polylactic acid.

14.3.1 Polymerization of Lactide

Many catalyst systems have been evaluated for polymerization of lactide, including complexes of aluminum, zinc, tin, and lanthanides. Even strong bases such as metal alkoxides have been used with some success. Depending on the catalyst system and reaction conditions, almost all conceivable mechanisms (cation-

14.3 PLA Production

ic [11], anionic [12], coordination [13], etc.) have been proposed to explain the kinetics, side reactions, and nature of the end groups observed in lactide polymerization. Tin compounds, especially tin(II) bis-2-ethylhexanoic acid (tin octoate), are preferred for bulk polymerization of lactide, because of their solubility in molten lactide, high catalytic activity, and low rate of racemization of the polymer. Conversions > 90% with less than 1% racemization can be achieved while providing polymer with high molecular weight. The polymerization of lactide using tin octoate is thought to occur via a coordination–insertion mechanism [13] with ring opening of the lactide to add two lactyl units (a single lactide unit) to the growing end of the polymer chain, as shown schematically in Fig. 14.7. The tin catalyst facilitates the polymerization, but hydroxyl or other nucleophilic species are the actual initiators. There are usually several hundred ppm of hydroxyl impurities in the lactide from water, lactic acid, and linear dimers and trimers. High molecular weight polymer, good reaction rate, and low levels of racemization are obtained with tin octoate catalyzed polymerization of lactide. Typical conditions for polymerization are 180–210 8C, tin octoate concentrations of 100– 1000 ppm, and 2–5 h to reach 95% conversion. The polymerization is first-order in both catalyst and lactide. Hydroxyl-containing initiators such as 1-octanol are frequently used to both control molecular weight and accelerate the reaction. In addition to the work performed on catalysts and comonomers, a significant amount of work has been devoted to designing the optimum polymerization process from a cost and versatility perspective. Most of this work has used lactide dimer as the starting point. Batch polymerization and continuous processes can be used. Continuous processes (Fig. 14.6) have a cost and productivity advantage and thus are the focus of most work. Stirred tank or pipe reactors have been evaluated alone and in combination. Because of the low energy of the ring-opening polymerization and the potential to obtain high rates of polymerization, melt extruders have been extensively evaluated as reactors to produce PLA. Several groups have reported or patented specific aspects of the use of extruders or combinations of several reactor concepts to produce PLA [14–27]. Dupont [15] recognized the importance of high rates of lactide polymerization on the economic viability of using extruders. They investigated the issues surrounding hydroxyl initiators, catalysts, and acidic impurities. Dupont’s patent

Fig. 14.7 Coordination-insertion chain growth mechanism of lactide to PLA; R = growing polymer chain.

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[15] (US 5,310,599) provides examples showing the dramatic (negative) effect of acidic impurities, such as the linear dimer (DP2), etc., on the rate of lactide polymerization. The acid impurities coordinate with the tin catalyst and render it ineffective as a ring-opening site of polymerization. As an example, 389,000 MW PLA can be made with a 5-min residence time (6000 : 1 monomer:Sn octoate) at 180 8C in the absence of acidic impurities and 98% conversion is obtained. The conversion in 5 min drops to 50% when the impurty:catalyst ratio is 2:1 and to 12% in 5 min if the ratio is 6:1. The rate of polymerization of lactide can be accelerated by use of hydroxyl initiators such as butanol or by higher catalyst loading. Increased initiator levels will, however, reduce the molecular weight while more catalyst will reduce the thermal stability and increase the color of the PLA.

14.4 Control of Crystalline Melting Point

PLA is a hard, brittle plastic which can exist in either the amorphous or semicrystalline state, depending on the stereochemistry and thermal history. Common physical properties such as density, heat capacity, and mechanical and rheological properties are very dependent on its transition temperatures. For amorphous PLA, the glass transition (Tg) determines the upper use temperature for most commercial applications. For semi-crystalline PLA, both the Tg (*58 8C) and melting point (Tm), 130–230 8C, (depending on structure) are important for determining the usage temperatures for different applications. Both of these transitions, Tg and Tm, are highly affected by overall optical composition, primary structure, thermal history, and molecular weight. Several excellent reviews have been written which include details of the properties and characteristics of PLA [28–32]. Having two optical arrangements for lactic acid (l-lactic acid, d-lactic acid), and three optical arrangements for lactide (l-lactide, d-lactide, meso-lactide), the variety of primary structures available for PLA is substantial. In addition, many other monomers have been copolymerized with lactic acid and/or lactide, a topic which will not be covered in this chapter. Techniques for measuring PLA stereo-sequences by NMR have been published by Thakur et al. [33]. The most common commercial polymers of PLA are optical copolymers of predominantly l-lactide, with small amounts of d- and meso-lactides, made by bulk polymerization with tin octoate catalyst by ring-opening polymerization (ROP). Although these copolymers are usually described as random, there is evidence of some stereo selectivity. The selectivity of tin octoate is discussed by Thakur et al. [34]. Condensation polymerization of, mostly, l-lactic acid with small amounts of d-lactic acid, polymerized in solution, has also been used. The optical comonomers introduce “kinks” in PLA’s natural helical conformation and “defects” in the crystal arrangement, which results in depression of the melting point, reduction in the level of attainable crystallinity, and reduction in

14.4 Control of Crystalline Melting Point

the rate of crystallization. Optical purity (OP) is common nomenclature for describing polymers of this variety. As OP decreases, crystallization eventually becomes impossible and the polymer is amorphous (occurring about when OP 10%). It can be used for setting mousses, hair sprays, gels, and waxes and for hair tip repair fluids, i.e. formulations preventing split ends, and for leave-on conditioners. 15.6.4 Summary and Prospects

Ilex resin is a new natural material with many useful cosmetic properties. As an active ingredient it enhances the performance of numerous product groups in hair and skin care and, because it is derived from a very decorative ornamental and useful plant and has an obvious active principle working in its natural habitat, its efficacy is readily substantiated. Ilex resin is extracted from a plant cultivated in many countries as an agricultural crop. There are no supply problems, and being renewable the substance is available in virtually unlimited amounts. The resin is obtained by a purely physical solvent-free process with and without undergoing chemical change, simply by extraction of maté leaves with supercritical carbon dioxide. Because it is nontoxic, this process has recently largely replaced conventional solvent extraction for foodstuffs (e.g. decaffeination), spices, and drugs. Biodegradability, climate

Fig. 15.29 Increase of hair luster by application of a product containing an ilex resin.

References

safety, and sparing of fossil resources are additional benefits of all chemically unaltered natural substances. Ample toxicological testing has shown that ilex resin is not an irritant to the skin and the eye mucosa, that it is not mutagenic, and has no sensitization potential. The first hair conditioners and shampoos containing ilex resin were launched under Wella’s “Lifetex” brand in 1998 and hair-care products containing this natural material are now marketed nearly everywhere in the world. The trade name is “Ilex Gloss”, and – according to EU legislation – the INCI term is the botanical name of the raw material source, that is “Ilex paraguariensis”.

References 1 Lang, G., Clausen, T., Chitosan and Be-

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4 5

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taine, Wella AG Darmstadt, company brochure, revised edition (2004). Kripp, T. C., Von der Naturstoffisolierung bis zur Trendformulierung. SOeFW-Journal 123, 20–27 (1997). Lang, G., Clausen, T., Chitosan and Betaine, Wella AG Darmstadt, company brochure, revised edition (2004). Imhoff, J. F.; Rodriguez-Valera, F.: Journal of Bacteriology (1984), pp 478–479. Bagnasco, S.; Balaban, R.; Fales, H. M.; Yang, Y.-M.; Burg, M.: The Journal of Biological Chemistry, Vol. 261, No. 13 (1986) pp 5872–5877. Grattan, S. R.; Grieve, C. M.: Plant and Soil 85, pp 3–9 (1985). Coughlan, S. J.; Heber, U.: Planta (1982), 156: pp 62– 69. Semmler, F.: Ther. d. Gegenw. 116 (1977), pp 2113–2124. Babucke, G.; Sarre, H.: Med. Klin. 68 (1973), pp 1109–1113. Schroeder F., Konrad, E., Lang, G.: Wella AG DE 3527974 (1987). Bimczok, R., Racky, E. D., Lang, G.: Wella AG EP 747468 A1 (1996). Bimczok, R., Lang, G.: Wella AG EP 0750904 A1 (1997). Racky, E.-D.: Wella AG DE19640086 C2 (1998). Lang, G., Bimczok, R., Czigler, T., Kripp, T.: Wella AG EP 826661 A2 (1999). Lang, G., Clausen, T., Chitosan and Betaine, Wella AG Darmstadt, company brochure, revised edition (2004).

16 Römpp Chemie Lexikon, Thieme Verlag

17 18 19 20

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Stuttgart, Online DE Version (2002– 2004). Zechmeister, L.; Toth, G.: Fortschritte Chem. Org. Naturstoffe II (1939) p 212. Muzzarelli, R.A.A.: Chitin. Pergamom Press, New York (1977), p 5 ff. “Panzerknacker”, High Tech 7 (1988), p 29 ff. Zikakis, J. P. (Ed.): Chitin, Chitosan and Related Enzymes. Academic Press Inc. (1984), S. XVIII. Sandford, P. A.; Hutchings, G. P.: Prog. Biotechnol. 3 (1987) p 363. Groß, P.; Konrad, E.; Mager, H.: DE PS 26 27 419 (1976). Bernadet, M.: FR 1552076 (1969). Gleckler, G. C.; Goebel, J. C.: US 4035267 (1977). Yanagida, T.: JP 61/210014 A2 [86/ 210014] (1986). Konrad, E.; Gross, P.; Mager, H.: EP PS 0 002 506 (1987). Waki, M.; Tsuruta, E.: Jpn. Kokai Tokkyo Koho, JP 62/223108 A2 [87/223108] (1986). Kripp, T.: Industrielle Erfahrungen mit Verpackungen aus Nachwachsenden Rohstoffen. Vortrag und Tagungsband des Zentrums für Europäische Wirtschaftsförderung und der IHK Karlsruhe, (1994) pp 44–53. Schlegel, H. G.: Allgemeine Mikrobiologie, Thieme Stuttgart, New York, 5. Auflage (1981), p 65. Römpp Chemie Lexikon, Thieme Verlag Stuttgart, Online DE Version (2002– 2004).

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lungswasserbau, Wassergüte und Abfallwirtschaft. Nachr. Chem. Techn. Lab. 39 (1991) Nr. 10 p 1112. www.umweltfibel.de/lexikon/pq/lex_p_ polymilchsaeure.htm. Danone, personal communication 5 July 2004. Financial Times 15 February 1994. Kripp, T. C., Von der Naturstoffisolierung bis zur Trendformulierung. SOeFW-Journal 123/1 (1997) pp 20–27. Kolattukudy, P.E., Cutin, suberin and waxes. Comprehensive biochemistry of plants, IV, 571–645, Academic Press, New York (1980 b). Herbstreith & Fox, Neuenbürg, Firmenprospekt “Von der Apfelernte zum Pektin”. Sando, C. E., Constituents of the wax-like coating on the surface of the apple, J. Biol. Chem., 56, 457–468 (1923). Chibnall, A. C., Piper, S. H., Pollard, A.., Smith, J. A. B., Williams, E. F., The wax constituents of the apple cuticle, Biochem. J. 25, 2095–2110 (1931). Fernandes, A.., Baker, E., Martin, J., Studies on plant cuticle, VI. The isola-

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tion and fractionation of cuticular waxes, Ann. Appl. Biol., 53, 43–58 (1964). Kaufmann, R., Höcker, H., XPS, eine Methode zur Charakterisierung von Faserveränderungen in der Oberfläche., Fachz. Lab. 4, 348–352 GIT-Verlag, Darmstadt (1994). Lang, G., Hoch, D., Konrad, E., Geibel, W., Wendel, H., Kripp, T.. EP 0 583 ;438 B1 “Verfahren zur Gewinnung von Apfelwachs, durch diese Verfahren erhältliches Apfelwachs sowie apfelwachshaltige kosmetische Mittel” (1993). Kripp, T. C., Ilex Resin, a Novel Renewable Raw Material for Cosmetics. SOeFW-Journal 126, 18-21 (2000). Kripp, T. C., Von der Naturstoffisolierung bis zur Trendformulierung. SOeFW-Journal 123, 20-27 (1997). Kripp, T. C., Bormuth, H., Franzke, M., Baecker, S., Schröder, F. and Kischka, K. H., Cosmetic Agent with an Ilex resin content, process for extraction of Ilex resin and Ilex resin obtained thereby. WO 97/30680. Wella AG, Berliner Allee 65, D-64274 Darmstadt (DE).


Part III Biobased Industry: Economy, Commercialization and Sustainability


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16 Industrial Biotech – Setting Conditions to Capitalize on the Economic Potential Rolf Bachmann and Jens Riese

16.1 Introduction

Today’s pharmaceutical industry would be almost unthinkable without biotechnology. Practically every new drug has relied on biotech tools for discovery and development and an increasing number of drugs themselves are biological in nature. This is a growing trend. Biotechnology has also increased in importance in agriculture. In the United States, for example, approximately 80% of soy and corn plants grown today are genetically modified. The use of biotechnology in food production is not without controversy, however, and these products face consumer skepticism especially in Europe. After these inroads made by “red” and “green” biotechnology, a third wave is beginning to spread – “white” or industrial biotechnology. It enables companies to manufacture new, innovative products or existing products more effectively and efficiently than with conventional processes. Biotechnology mostly uses renewable materials and copies tried-and-tested natural processes to produce industrial goods. This often saves resources, reducing the burden on the environment. Biotechnology lays a foundation for sustainable development in which social, ecological, and economic concerns can all be reconciled. The potential for industrial biotech is broadly recognized, and chemical and biotech companies are starting to move into this space and increase their presence. The success of biotechnological processes for vitamin production, the growth of the enzyme industry, and the introduction of cost-competitive biopolymers, for example, all hint at the possibilities. Major challenges still lie ahead in transforming this potential into economic value, however, and here we set out some of these challenges and discuss ways to tackle them.


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16.2 Time to Exploit the Potential

For those who remain unconvinced that biotech is more than just a passing fad, approximately 5% of the estimated US$ 1.2 trillion total chemical sales already depend on biotech. (Here, “dependency” is defined as at least one production step using biotechnology; 100% of the sales of a chemical product were accounted for in a multi-step production process.) The global market for bio-based ethanol is worth US$ 15 billion alone; other basic organic molecules, for example citric acid (US$ 2 billion) and lactic acid, are produced by fermentation, and so are all but three amino acids (approx. US$ 4 billion). Various basic, advanced, and active pharmaceutical ingredients produced by the fine chemical industry are worth US$ 7.5 billion; the attractive enzyme market has reached US$ 2 billion in sales and is growing by more than 5% per year, and specialty chemicals for flavors, fragrances, and other applications add several more billion dollars. Biotech is changing industrial production in three specific ways. First, sugars, vegetable oils, and ultimately waste biomass are replacing fossil fuel feedstock (oil and natural gas). Second, bioprocesses such as fermentation, biocatalysis, and, in the future, plant- and animal-based production may replay chemical syntheses. Last, new bioproducts are emerging including bio-based polymers, enzymes for use in textiles or feed, and innovative nutritional ingredients. 16.2.1 How Far Can it Go?

McKinsey & Company has analyzed the market potential by looking at the technological and market trends in the chemical industry, taking an inventory of existing commercial and research and development biotech activities, estimating the likely penetration in different chemical market segments, and finally by conducting interviews and discussions with more than 100 industry executives (Fig. 16.1). The results suggest that biotech could have an impact on 10% of chemical sales by 2010, double what it is today. Initial analyses at the beginning of the decade indicated that 20% might have been achievable in the same timescale. Today this looks highly unlikely, and even the 10% figure will not be reached without effort. One of the most important factors affecting this development is the price difference between fossil fuel feedstocks and biological carbohydrate feedstocks, especially for those products with a high relative feedstock cost. Consumer acceptance is also vital – as we have seen in agricultural biotech with the widespread rejection in Europe of genetically modified foods, despite their acceptance in the US. Thus far there is no significant consumer movement against industrial biotech – indeed, as we show later, it actually has strong environmental benefits. Consumer and environmental organizations have not actually publicly endorsed industrial biotech, however.


Fig. 16.1 Biotech adoption could more than double by 2010.

The regulatory framework is another important factor. Supportive regulatory procedures, active government support (e.g. grants, and renewable purchasing policies) and the extent to which less sustainable production methods need to compensate for their environmental impact will all be a fundamental prop if the industry is to grow. This sets the context for the final success factor – investment by the chemical companies. Without this industrial biotech will remain no more than a dream. The promise of industrial biotech has been around for decades, and failures far outnumber success stories. So it is not surprising that chemical companies are hesitant to move forward. The prospects look brighter than ever, however, because of three key drivers – advances in technology, environmental and economic benefits, and the need for innovation in the chemical industry. 16.2.2 Better Technology, Faster Results

A broad spectrum of enzymes and fermentation systems are already available to the market, and the number is increasing all the time. The pace of biotech development has picked up to the extent that it can now take a matter of weeks rather than years to develop new, highly specific, and efficient enzymes. Until recently the slow pace of development has hindered the use of enzymes in pharmaceutical production, where drugs need to be developed quickly. Now DSM’s Pharmaceutical Product Unit, for example, is exploiting them systematically as

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a competitive advantage. Enzymes are also becoming more resistant to harsh environments such as heat and acidity, and are cheaper to produce, opening inroads into other industrial production processes, for example pulp and paper, oil exploration, and textile processing. 16.2.3 Environmentally and Balance-sheet Friendly

The increased pressure for sustainable production is also helping to spur the industry’s prospects. Two reports – one authored by the OECD (OECD Report “The Application of Biotechnology to Industrial Sustainability” (2001), which includes 21 case studies on the impact of biotechnology on the environment), the other by a consortium of companies, industry associations, the Öko-Institut, and McKinsey – clearly demonstrated that industrial biotech can help create jobs, boost profits, and benefit the environment (Fig. 16.2).

Fig. 16.2 Case studies demonstrate sustainability.

Green Economics

Several studies of the environmental impact of replacing chemical synthesis with biotech routes have demonstrated the benefits of industrial biotech. The German chemical company BASF was able to adopt biotech processes to transform production of Vitamin B2. Traditionally, this requires a complicated eight-step chemical process, but biotech reduces that to just one step. Soy oil is fed to a mould and Vitamin B2 is recovered as yellow crystals directly from this fermentation process. This has cut production costs by 40% and reduced CO2 emissions by 30% and waste by 95%.


The antibiotic Cephalexin has been produced on an industrial scale by the Dutch chemicals firm DSM for several years. Metabolic pathway engineering helped establish a bio-route that reduced substantially the number of steps needed in the process. The biotech process uses 65% less energy, 65% less input chemicals, is water-based, and generates less waste. In total, it has halved the variable costs of the process. Novozymes, a Danish biotech company, produces enzymes for the scouring process in the textiles industry. Scouring, which removes the brown, non-cellulose parts of cotton, traditionally requires a harsh alkaline chemical solution. Use of enzymes not only reduces discharges into the water by 60%, but reduces energy costs by a quarter. Environmental and economic benefits go hand in hand: the new process is also 20% cheaper than the chemical treatment. Cargill Dow’s bio-polymer PLA made from corn requires 25 to 55% fewer fossil resources than the conventional polymers against which it competes. With the help of biomass and potentially other forms of renewable energy for processing, the joint venture between Cargill and Dow Chemical believes PLA could even become a net carbon sink. In the near future DuPont’s Sorona® polymer will be based on propanediol (PDO) produced by fermentation, in collaboration with Tate and Lyle. This is estimated to reduce greenhouse gas emissions by approximately 40%.

When oil and gas are used as production feedstock, carbon is removed from the earth and bound into the chemicals that constitute plastics and other materials. Eventually, the plastic is discarded and burned and CO2 is released into the atmosphere – a one-way cycle that produces significant pollution. When biomass is used, the CO2 released helps provide the raw material for further production, because it can be absorbed by plants, which can in turn be used as new feedstock. This closed cycle theoretically results in a neutral carbon balance, thereby reducing greenhouse gas emissions. The increased energy efficiency that comes from replacing fossil fuels has led McKinsey to estimate that 7% of the Kyoto target on emissions could be achieved (based on the full range of case studies in the reports mentioned above, and the figure of 10% of chemical product sales relying on biotech). When we reach the stage where 20% of chemical products are produced using biotechnology, the contribution will increase from 7 to 20%. The economic benefits alone are also driving the adoption of biotechnology. The case studies for Vitamin B2 and Cephalexin (see box), and dozens of pharmaceutical intermediates suggest that cost savings of 50% and more are not unlikely. The savings can come directly from lower variable costs, but also from reduced capital expenditure for simpler production assets, or from reduced scale and therefore lower risk, transportation costs, and/or overcapacity.

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16.2.4 Rekindling Chemicals Innovation

Finally, interest in biotech has increased recently because of its role in product innovation. At a time of increasing competition from Asia in established products and the subsequent commoditization and strong price decline, chemical companies are once again looking at innovation as a key source of differentiation. At the 2004 World Economic Forum in Davos, the leaders of the world’s largest chemical companies generally shared the view that biotechnology will be the predominant driver of innovation for their organizations, and placed it among the key drivers of change for the decade ahead. The importance of stimulating innovation can be seen by looking at the introduction of new polymers. Over the course of the 20th century the development of fossil-fuel-based polymers increased steadily through to the post-war period, stimulated by the abundance and low cost of basic petrochemicals. It has, however, declined dramatically since 1960. Innovation in the traditional polymer industry today is mainly related to the application and blending of these polymers, rather than the invention of new ones (Fig. 16.3). Just as low-cost petrochemical building blocks such as ethylene, propylene, and butadiene became available with the introduction of crackers in the 1930s, we are seeing the emergence of new bio-based building blocks today. These include lactic acid, which can be polymerized to the biopolymer PLA. PLA has started to replace polyester because of its competitive cost and new applications. Lactic acid can also be processed into chiral drugs, acrylic acid, propylene glycol, food additives, and more (Fig. 16.4).

Fig. 16.3 The innovation potential for fossil-based building blocks appears to be exhausted.


Fig. 16.4 Bioethanol: an early beneficiary of low-cost biomass.

Other examples of innovation abound – Cargill is exploring the potential of 3hydroxyproprionic acid as a new building block; BASF is looking into new chemistry around the simple organic molecule succinic acid; and DuPont will use cheap PDO as a monomer for its Sorona® polymer. Overall, we are seeing the emergence of a green chemistry that complements the traditional product trees and that gives the industry more innovation headroom. 16.2.5 Increasing Corporate Action in all Segments

All these economic, regulatory, and environmental factors are encouraging companies in all major chemical market segments to make more definite moves In fine chemicals, half of all life science chemicals at DSM are based on biotech – a wolume which is worth US$ 1.5 billion. BASF has committed more than 1 500 million to explore the potential of plant-based biotechnology. DuPont has invested more than US$ 500 million in the development of Sorona®, and Cargill Dow’s investments for PLA are of a similar scale. Ciba Specially Chemicals has introduced an enzymatic process for acrylic acid, BP is exploring industrial biotech for oil exploration and basic chemicals, Degussa has recently opened a second project house dedicated to biotechnology, Givaudan is using biotech extensively for new flavors and aromas, and Novozymes has an annual research and development budget of more than 1 100 million for new and enhanced enzymes. Finally, dedicated industrial biotechnology companies such as Codexis, Diversa, and Genencor have established themselves in the market and have encouraged a new generation of start-ups to emerge.

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16.3 The Importance of Residual Biomass

Early introductions of bio-based products have shown that the willingness to pay a high “green” premium is limited to a small portion of the customer base. For broadbased adoption, new products must be competitive with existing offerings. In this context, the use of alternative low-cost feedstock could give industrial biotech another boost (as, of course, will the recent increases in the price of natural oil and gas). 16.3.1 Why Waste Biomass Works

The most promising alternative feedstock is waste biomass. It comes primarily from agricultural sources such as straw and corn stover. This material is abundant, cheap, and largely serves no other purpose. It is also obviously renewable and contains three useful raw materials: · cellulose and hemicellulose, which can be turned into sugars; · proteins that can be used in animal feed and for industrial products such as hydrolysates; and · lignin that may be used as a combustible fuel; indeed it can power the very biorefineries that process biomass, making them virtually self-sufficient. Bioethanol is one of the first and the largest markets to profit from cheap biomass feedstock. Ethanol is usually produced from dextrose, which in the US tends to be derived from corn. The first ethanol biorefinery based on waste biomass is already online. It is a Canadian venture operated by Iogen and receiving investment from Shell, Petro Canada, and the Canadian government. With an annual capacity of 700,000 liters it is semi-commercial in scale and too inefficient to compete on cost with conventional ethanol refineries. The technology is expected to improve quickly, however. The process sounds relatively simple. Straw is delivered to the refinery where it is chemically and/or physically pretreated. Enzymes then decouple the cellulose and hemicellulose chains, breaking them down into individual sugars. Most of the sugar yielded will be used as feedstock for fermentation. As bacteria or yeast cells break down the sugar it is fermented into ethanol. The organisms can also be modified to produce vitamins, organic acids, and other substances. The primary products that emerge from the biorefinery are often subject to further downstream processing. 16.3.2 Economic Benefits and Regulation

Biomass-based biorefineries have the potential to reduce sugar costs from about 8 to 9 cents per pound in the US today to approximately 4 cents per pound in three to four years, and lower still as the biorefineries become more integrated. It is even possible to see a case where the net cost of producing sugar in an in-

16.3 The Importance of Residual Biomass

Fig. 16.5 Bio-based building blocks emerge as a source of new products.

tegrated biorefinery is zero because by-products such as lignin and proteins generate the value. It is important to realize that the price of conventionally produced sugar will also fall as genetic modification makes a variety of productivity improvements possible. The technology for the cost-competitive, large-scale application of ethanol could be ready in the next few years, and the value-creation potential for shifting from corn to biomass for ethanol is estimated to be US$ 10 to 16 billion by 2010 (Fig. 16.5). (This figure is derived from industry estimates for the increasing use of biomass as feedstock. After calculating marginal and overall cost savings, we multiplied the result by typical profit multiples for the industry. We assumed that the price of ethanol remains stable. The resulting change in market capitalization is the value creation.) Legislation is already in place to support production of fuel ethanol in countries such as Canada and Brazil, and the EU has committed to ensuring that 5.75% of all transportation fuel in the EU will be bio-based – a target that cannot be achieved by biodiesel alone. Several European companies, for example Abengoa and Südzucker, have therefore announced aggressive plans for expansion in ethanol in the coming years. Estimates for the US market suggest that 15 billion gallons (57 billion liters) of bioethanol will be produced from a combination of biomass and corn-based feedstock by 2020; and the cost of ethanol could come down from approximately $ 1.20 per gallon (32 cents per liter) today to as low as 40 cents per gallon (11 cents per liter) by 2010.

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16.3.3 Still a Long Way to Go

For this to happen, all steps of the process need to undergo major efficiency improvements. The cost of enzymes must fall by 90% from its 2003 level. This might sound unrealistic, but it is not unusual to increase the effectiveness of enzyme production by factors of 10, 100 or even 1000. By modifying the amino acid sequences of the cellulase and hemicellulase enzymes, biochemists from Genencor and Novozymes have been able to make them dramatically more effective, and recent advances suggest the cost target for the enzymes will be exceeded in due course. The US Department of Energy predicts that 2 billion gallons (7.5 billion liters) of ethanol will be derived from biomass by 2010. This figure, depends, however, on whether companies are willing to invest. Despite major government support, especially in the US, no major companies have yet aggressively pursued this. Cargill, DuPont, Deere, Shell, and others are putting in moderate investments of US$ 10 to 15 million often related to matching government grants, thereby reserving an option to play in the future. The competitive threat is still perceived as low, and companies are afraid to risk being the first mover. Only the technology companies, in particular Novozymes and Genencor, and the Spanish pioneer Abengoa have publicly announced aggressive growth plans and commitments to biomass-based ethanol. It is not just the chemical or the biotech companies that need to make the investments; it goes all the way down the value chain. Farmers need the right sort of equipment, storage facilities for corn stover must be built, pretreatment needs investment, etc. No one owns the full value chain, so collaborating with partners is essential. In fact, the value chain does not yet exist, and collaboration between different parties is required if it is to be built. 16.3.4 Collaboration Will Push Biomass Conversion Forward

There are three collaboration models. The first, which prevails today, is where multiple players are involved in diverse activities with very little explicit collaboration and without enough investment to launch a new value chain. In the second, entrepreneurial pioneers invest substantially to bundle research and development efforts and build the new value chain. This is a high-risk approach, but has potentially high rewards. It also enables the company to assume a clear leadership role. The final model is a structured network in which multiple players from different parts of the value chain cooperate closely. The risks – and therefore the returns – are shared out and each company can focus on its core competency. Such networks have yet to emerge. When they do, they will have much to accomplish, including collecting, warehousing, and treating biomass, and constructing biorefineries. One possible drawback of this model is the introduction of governance and interface inefficiency.

16.4 Overcoming the Challenges Ahead

It will be interesting to see how governments and the various players along the value chain will act in the coming years, and whether a real breakthrough in the broad commercial use of waste biomass will happen. Next to the advantages for the environment, political stability, and jobs in rural areas, it could give a welcome boost to the broader adoption of industrial biotech.

16.4 Overcoming the Challenges Ahead

The path from awareness to profit for companies engaged in biotech is long and arduous (Fig. 16.6). Most companies have already taken the first step and are aware of the opportunities and the threats of biotech. Some have even made the commitment to invest; but from there onward is a long staircase with many internal and external challenges to manage. 16.4.1 Internal Obstacles

The first challenge for chemical companies in turning the biotech promise into reality is to create the awareness of its economic potential and benefits for sustainability. We have already come a long way in achieving this. The next challenge is to convert the awareness into a commitment to act. Investors and senior managers have recently demonstrated their willingness to act if they are

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Fig. 16.6 Staircase of challenges from awareness to profit.

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presented with good opportunities. This has held true particularly for incremental investments. Those high-risk/high-return areas that require major investments, for example biomass conversion, still face great skepticism. Furthermore, very few companies have progressed beyond an opportunistic use of biotech and have formulated a true biotech strategy, with a clear focus on where and how to compete. To execute a strategy, a company needs the right assets, capabilities, and networks, and no one has all the requirements in-house. Identifying where the gaps lie and understanding how best to fill them is not easy, and feedback on partnerships in this field has been mixed. The next step is to identify and select the right opportunities. There is usually no shortage of ideas, as illustrated, for example, by the impressive theoretical product trees rooted in the new bio-based building blocks. The difficulty is selecting the right ones; as mentioned earlier, the list of failed biotech investments is much longer than the list of successes. Failure cannot be avoided. The trick – as in the world of venture capital – is to manage the uncertainty and to build a portfolio of good prospects. And, as with venture capital, the starting point is a solid business case for the opportunities deemed right for investment. The most common mistakes are overestimating the addressable market size and market uptake (mostly by defining it too broadly), and underestimating the investments, in particular for ongoing market development and application development after the initial research and development investment. With a portfolio of projects in hand, there remains the challenge of separating the biotech wheat from the biotech chaff. Companies often hang on to secondtier projects far too long, rather than focusing their scarce resources on the few most promising. Investors are also no longer willing to tie their money up in projects that might take a decade to break even. Research and development work in biopolymers, for example, began in the 1980s, so we need to find ways to accelerate the whole process. The launch and market development might be the last steps, but they are also the ones that can make all the difference. The art is to focus on the right market segments and customers with the right value proposition, to align the value chain to adopt your innovation, and to use partners appropriately. Indeed, managing partnerships plays a role in several steps of this staircase of success. Surrounding this set of internal challenges are external pressures that also need close attention, but which can be met. 16.4.2 External Challenges

There are four distinct external challenges. · Consumer acceptance is not yet a big issue, but is one to monitor. Supermarket chains in the UK have refused a biopolymer because it was derived from genetically modified plants, even though it is an eco-friendly material. There is even a discussion as to whether vitamins produced by fermentation should

16.5 Overcoming Challenges

be labeled GM. The environmental NGOs and pressure groups have been relatively quiet on industrial biotech so far, but it would only take one of the more influential groups putting it on the agenda for lengthy delays to be possible. The industry therefore needs to invest resources in educating people on the benefits. · The cost differential between hydrocarbon (oil, natural gas) and carbohydrate (sugar, biomass) feedstock is clearly a moving target. In a climate of rising oil prices, carbohydrates look even more appealing but such a situation cannot be relied upon permanently. Companies need to consider potential price changes, and analyze the sensitivity of biotech investment cases against various assumptions on future feedstock costs. · The regulatory situation is certainly liable to change but is also subject to the influence of various interest groups. The proponents of industrial biotechnology, represented, for example, by the BIO and EuropaBio industry associations, need to maintain or even increase the level of activity on this front. This, in turn, requires commitment from their members. · The success of company strategies and investment decisions ultimately depends on the moves of competitors. Products must be distinctive; intellectual property must be in place to introduce a new biotech process; and an eye must be kept out for opportunities to align interests and join forces. It is therefore essential to watch and anticipate competitor moves as well as possible.


A growing number of case studies demonstrate that all these challenges can be overcome. Usually, the trick is to tailor well-established good management practices and problem-solving approaches from comparable situations to the specific needs of industrial biotech. These approaches do not have to come from the chemical industry itself. For example, chemical companies have looked at risk management tools in financial services to learn how to deal with uncertainty. Here, we set out a number of cases with which McKinsey has been involved, and in which companies have managed to capture value from industrial biotech. 16.5.1 Case 1: Building a Biotech Strategy

After years of internal discussions, and smaller investments with promising results, the board of a chemical company decided to take a major step in industrial biotech. It looked at a number of different growth fields and biotech emerged as the one with the highest innovation potential and a good fit with the company’s broader capabilities and position in the value chain. There were

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Fig. 16.7 Building the biotech strategy for a chemical company.

many potential entry points and business opportunities, some of which had internal champions and others that did not seem worth pursuing, but none had a clear rationale. The company approached the challenge from two sides. It determined its distinctive skills and assets through a benchmarking process. At the same time, it assessed the relevant opportunities and threats – both competitors’ recent or announced moves and the potential for competitors if the company chose not to enter a specific area. This produced a list of strategic options – a combination of areas that seemed most attractive for the client to enter and different business models and value chain positions that would match these areas. The next step was to assess these different strategic options along a set of criteria that included economic value, feasibility, risk, investments, fit with overall strategy, and portfolio of initiatives (Fig. 16.7). The final decision was made after extensive discussions and interviews both within and outside the company. It is too early to determine the financial success, but the company has already achieved alignment between the overall strategy, the level of investment and the organizational setup. 16.5.2 Case 2: Identifying the Right Opportunities

Another chemical company already had a clear biotech strategy in place and had built the capabilities, assets, and networks required to implement it. Execution was already successfully underway in several business units and new biobased products and processes had started to generate healthy profits. The company was, however, wondering how best to apply biotechnology to a recently ac-


quired business. In particular, it was seeking ways to change the old chemical production processes to new, more competitive synthesis routes. The project scope expanded to include a complete review of the company’s core product strategies, which included a detailed assessment of competitor moves, market trends, etc. This was important because a new biotech process can easily take five years or more to develop, so it is critical to understand whether it will result in a distinctive cost position after that time. Regulations and customer sensitivities also change – would there be a “bio-based” premium or a “genetically modified” discount for a product produced by fermentation? The scope was also extended on the technology side. While one team investigated the potential for new biotech routes, a competing team tried to optimize the existing process, including analyzing different locations, and a third team searched for the best alternative chemical routes. In the end, biotech was just one of the solutions. Each potential solution was assessed, and the final one was chosen based on the basis of the best risk/reward ratio. This led to an implementation roadmap and a decision tree, as further technical feasibility studies were required to prove the concept and assist in making the final choice. Of the fifteen products under investigation, biotech was the best solution for five of them; in four cases a new chemical process was found; incremental improvements to the existing chemical process were made in three further cases; two products were moved to China, one was stopped completely and bought instead from a low-cost producer. So even though the impetus for the project was to see how biotech might have an impact on the company’s processes, the outcome was an improvement of every process in a variety of ways. By 2010, it is predicted that costs will have fallen by 60% on average for these fifteen processes. 16.5.3 Case 3: Managing Uncertainties

This company was building a portfolio of industrial biotech projects but it was difficult to predict the future cost and revenues of each one and to determine which ran a relatively high risk of failure. The company wanted to make the risks more transparent and to improve its decision making against a background of high uncertainty. The solution was inspired by riskmanagement practices used in the venture capital and pharmaceutical industries. The basis of many of these tools is a Monte Carlo simulation, which randomly combines potential parameters to assess the probability of certain resulting values. It was used to estimate the process economics of a new biotech process. Instead of using a best guess for yield, cycle times and other key process characteristics, a best-, base-, and worst-case scenario were each assigned a certain probability of occurrence. The Monte Carlo simulation combined these randomly and generated a graph that showed the potential total production costs against its probability of occurrence. For example, it turned out that there was a 60% chance that a new process was going to

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be at least 30% cheaper than the old one. Based on the investment required, this was still considered to be too risky and the project was terminated. In another case, the analysis revealed a spread of potential outcomes that did not enable any conclusive recommendation. A more detailed assessment showed that the range of outcomes was extremely broad for one process characteristic, and the company decided to invest in another six months of laboratory work to understand this better and then repeat the assessment with a better defined range of potential outcomes. This is just one example demonstrating that uncertainty and risk are not reasons to refrain from rational decision making. 16.5.4 Case 4: Preparing the Launch and Market Development

This company was on the very last step of the staircase towards profitability in industrial biotech. It had a strategy, a highly capable organization, a great product, a new production facility, and some funding. Its problem was that the potential market applications for its new bio-product were so numerous that it was impossible to pursue them all simultaneously with its limited resources. It was also becoming clear that the product was not equally suited to all applications and that each market required a different product positioning. The company was looking for a go-to-market strategy for its new blockbuster. The full list of potential applications and addressable market segments were assessed against: 1. the relative strength of the new product’s value proposition to the customer in terms of price and performance compared with existing offerings; 2. the size and attractiveness of the addressable market; and 3. the ease of capturing the value, i.e. the time and effort it would take to develop the product applications and markets and the hurdles for adoption of the product along the value chain. The last point required much attention because consumers had already expressed considerable interest in some of the applications and the economics looked attractive. Interviews with companies along the value chain and supporting analyses showed, however, that the investment of intermediaries in the value chain made adoption very unlikely. In other market segments, retailers were concerned about the brand risk and were not willing to proceed without further demonstration of fitness-for-use and safety. These segments were therefore put on the back burner, but will be reignited when results from other segments support this market case. In the end, a handful of segments were chosen as top-priority targets for immediate focus and specific targets, marketing strategies, and implementation plans were put in place. In some other market segments and geographies partnering was the preferred strategy, mostly because partners had better customer access or application technologies than the company. The remaining segments were put on hold. It is too early to measure the success in financial terms, but

16.6 More Needs to be Done

the company now feels it has a clear focus and funding has recently been renewed by investors, so the new go-to-market strategy can be implemented. 16.5.5 Case 5: Building a Favorable External Environment

The previous case referred to overcoming internal hurdles, but the final case study illustrates how companies and industry associations can work together to tackle external challenges: to educate opinion leaders and decision makers and avoid a negative atmosphere that could prevent the economic and environmental potential from being realized. In 2003, EuropaBio decided to launch a project together with BIO (its American big sister) and some of its most prominent industrial members. BASF, Cargill Dow, DSM, DuPont, Genencor, and Novozymes all committed funds, data, and resources to put together case-studies on recent industrial biotech innovations. The German Öko-Institut, which has a good reputation and credibility with environmental interest groups, was asked to ensure that the environmental impact assessment was sound, and McKinsey was brought in to manage the process and perform the economic analyses. The resulting report included case studies and high-level recommendations for policy makers. It was presented at several prestigious conferences, and is still cited regularly in the trade and general press. Moreover, it has opened the door for EuropaBio to enter a dialogue with the key policy makers in the European Commission. One of the outcomes was to establish round-table discussions with the key stakeholders, including industry, academia, governments, and consumers. Several consumer interest groups are meanwhile supporting industrial biotech, and, more importantly, none is campaigning against it!

16.6 More Needs to be Done

The potential of industrial biotech to benefit the triple P – “profits, planet, and people” – has been broadly recognized and companies are starting to turn it into reality. There are significant challenges in making this happen, but the cases shown here encourage us to think that they can and will be overcome. Nevertheless, the effort required from chemical companies in terms of making and managing investments is significant. The speed and extent to which the potential of industrial biotech is realized will largely depend on how determined and successful chemical companies are in forging ahead. Another part of the equation is government support. Various US institutions, especially the Department of Energy, have supported industrial biotech significantly in the past. It is important that this level of investment is sustained or even increased. There are rumors that part of this funding will be redirected towards other technologies,

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but this would send a negative signal that could have repercussions far beyond the direct loss of investment. In contrast to the situation in the US, the EU has yet to implement any major initiatives to support industrial biotechnology. Such support would be more than desirable and should concentrate on three specific areas. First, the EU should formulate a vision and a long-term strategy for developing the biotechnology industry and all the relevant groups should be included in drafting such a strategy. Second, legislators should be challenged to create a favorable environment, which can include temporary support for lower prices for carbohydrate feedstock that Europe is not producing competitively. Third, the EU should promote research to intensify technological development in Europe. It feels as if we are teetering on the edge of an economic breakthrough in industrial biotech. If governments help create a conducive environment and companies understand how to move forward without taking unnecessary risks, this could be the beginning of a genuine revolution in the application of science. It is an exciting time to be involved in this industry, but there are still challenges ahead that we must all rise to meet if industrial biotech is to fulfill its sizeable potential.


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Subject Index Numbers in front of the page numbers refer to Volume 1 and 2: e.g., 2: 282 refers to page 282 in volume 2 a A. niger CBX-209, levoglucosan fermentation 1: 229 A. rhizogenes 2: 275 absorption, acoustic 2: 242 acetaldehyde, glucose product family 1: 21 acetate, biorefinery concept 2: 210 acetic acid, glucose product family 1: 21 Acetobacterium woodii 1: 235 acetogenic bacteria, metabolic pathways 1: 234 acetogens 1: 233 acetoin, biomass building blocks 1: 22 acetone, glucose product family 1: 21 acetyl-CoA 1: 233, 236 acetylated starches 2: 80 acid addition, lactic acid 2: 386 acid-catalyzed dehydration 1: 91 acid-catalyzed hydrolysis, cellulose 1: 133 acid-catalyzed stages, biofine process 1: 144 acid conversion, cellulose 1: 129 acid cooking, straw 1: 193 acid-forming anaerobes 1: 235 acid hydrolysis, carbohydrate polysaccharides 1: 144 acid hydrolysis of polysaccharides 1: 140 acid hydrolysis process 1: 130–133, 199 acid-insoluble ligneous components, feedstock 1: 146 acid prehydrolysis 1: 200 acidification, lactic acid 2: 386 acidogenic anaerobes 1: 235 acids – dilute 1: 362 – sugar-derived 2: 5 aconitic acid, glucose product family 1: 21 acoustic absorption, elastic protein-based polymer 2: 242 acrylic acid, glucose product family 1: 21

acyclic sugar derivatives 2: 48 acyl glutamate, synthesis 2: 305 acylated proteins 2: 304 addition – cationic 2: 264–265 – copper-initiated 2: 262–263 – perfluoroalkyl iodides 2: 263–264 additive chemicals 1: 91 additive replacement, ethanol 1: 357 adhesions prevention, post-surgical 2: 238 adhesive films 1: 282 adhesive tack 2: 186 adhesives 2: 86 advanced materials, protein-based polymeric materials 2: 220 aerobic storage, potato juice 1: 300, 309 agarose gel gene ladder 2: 229 age of sustainability, modeling tools 1: 57– 60 agribusiness, integrated production 1: 8 agricultural applications, lignin 2: 192 agricultural crop residues 1: 117 agricultural ecosystem modeling 1: 57–60 agricultural land, net requirement 1: 54–55 agricultural varieties, oil qualities 2: 276 agricultural waste 1: 68 agriculture residue collection 1: 317–344 agrification 1: 96 Agrobacterium tumefaciens 2: 275 agroindustry, sugar 1: 209–211 agrosector, dutch 1: 96 air-blown gasification 1: 232 air classification 1: 176 – oat grain 1: 183 Alcaligenes eutrophus, PHB accumulation 2: 424 alcell demonstration plant 2: 180 alcell process 2: 179 alcohol commodities, sugar-based 2: 36–37


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Subject Index alcohols – microbial conversion 2: 32–34 – microbial fermentation 2: 33 – production 1: 235–236 – sugar-based 2: 42 – sugar-derived 2: 5 alfalfa 1: 254 – chlorophyll extraction 2: 329 – cultivation 1: 260 Alfalfa New Products Initiative see ANPI alfaprox procedure 1: 257 algal fungi, chitin occurrence 2: 415 aliphatic diols, high molecular weight 2: 298 alkali pretreatments 1: 200 alkaloids 1: 268 alkanes 1: 268 – thermal addition 2: 264 alkyl polyglycoside carboxylate 2: 307 alkyl polyglycosides 2: 272, 305 – emulsifiers 2: 308 – manufacturing processes 2: 306 – synthesis 2: 306 – see also APG allylic C–H Bonds, oxidation 2: 269–270 alternative life, GJ Drinks 1: 275–277 American Society for the Testing of Materials see ASTM american straw, chemical composition 2: 107 amino acid-based product family trees 2: 201–216 amino acid composition, lucerne 1: 275 amino acid production, microbial 2: 201–216 amino acid residues, hydrophobicity scale 2: 234–235 amino acid units, proteins 1: 122 amino acids 1: 267, 2: 304 – analysis 1: 299 – brown juice content 1: 305 – fermentation 2: 203 – markets 2: 207 d-aminolevulinic acid see DALA ammonia fiber explosion, pretreatment 1: 362 ammonium lactate 2: 387 amphiphilic drugs, controlled release devices 2: 240 amylases 1: 197 amylopectin – physical structure 1: 140 – starch synthesis 2: 71

amylose – physical structure 1: 140 – starch synthesis 2: 71 anaerobe bacteria, lactic acid fermentation 1: 298 anaerobes, acidogenic 1: 235 anaerobic fermentations, succinic acid 2: 35 anaerobic production, succinic acid 2: 369 anaerobic storage, potato juice 1: 300, 310 analytical assays, antioxidant activities 1: 186 analytical methods, lactic acid fermentation 1: 299 1,6-anhydro-b-d-glucose, chemical structure 1: 245 d-anhydroglucopyranose units 1: 140 anhydrosugar 1: 229, 243, 245 animal bedding, stover 1: 325 animal feed, nutrient-enriched 1: 170 animal feed supplements – antioxidants 2: 188 – lignin 2: 196 animal health 2: 195–196 anions of fatty acids, oxidative coupling 2: 266 anodic coupling, fatty acids 2: 267–269 ANPI 1: 260 anthracenes 1: 118 antibiotic-resistant bacterial strains 2: 195 antibiotics 2: 15 antidiarrheic effects, dietary lignin 2: 195 antifeedants 1: 277 antifreeze protein 1: 269 antimutagenic effects, chlorophyll 2: 336 antioxidant activity, oat bran-rich fractions 1: 186 antioxidants 1: 186 – animal feed supplements 2: 188 – ferulic acid 1: 179 – lignin 2: 187–189 – lubricants industry 2: 188 – rubber industry 2: 188 – synthetic 2: 188 antisense RNA approach 2: 275 APG 2: 11–12, 272 – synthesis 2: 13 apolar groups, exothermic hydration 2: 218 apolar–polar repulsive free energy of hydration 2: 218 apple-peel wax 2: 430–432 – components 2: 434

Subject Index – market launch 2: 436–437 – natural 2: 429–437 – production 2: 432–433 – skin protection 2: 434 aquatic biomass 1: 91 aqueous media, proteins 2: 218 aqueous phase hydrogenation 2: 375 arabinanes 2: 109 arabinose, reaction 1: 199 arabinoxylans 1: 178 Arachis hypogaea 2: 277 aromatic chemicals, sugar-based 2: 29 aromatic compounds 1: 118 – renewable raw materials 2: 259 – transition metal-catalyzed syntheses 2: 259 aromatic functionality, char 1: 156 Arthrobacter 1: 398 arthropods, chitin occurrence 2: 416 arylglycerol units 2: 156 ascorbic acid, d-sorbitol 2: 9 ash components, feedstock 1: 146 asparagine residues, biodegradable thermoplastics 2: 241 aspartic acid (ASP) 2: 34 – basic biobased chemicals 1: 22 Aspergilli 1: 181 Aspergillus 1: 202 Aspergillus itaconicus, IA 2: 36 Aspergillus oryzae 2: 20 – cellulase development 1: 366 – thermostabilization 1: 370 Aspergillus succinoproducens 2: 35 Aspergillus terrous, IA 2: 36 asphalt emulsifiers, lignin-based 2: 192–193 ASTM, tests 2: 232 austria-wide concept, biomass usage 1: 284 austrian-concept, biorefinery 1: 273 autoadhesive tack 2: 186 autotrophic acetogens, syngas fermentation 1: 233 autotrophic bacterium AVGVP, biocompatibility 2: 232 axioms, phenomenological 2: 232–234

b B-starch 2: 68 Bacillus megaterium 2: 424 bacteria, biodegradation 1: 363 bacteria cellulosome 1: 365 bacteria destruction, lignin 2: 196 bacteriochlorophylls 2: 326

bagasse 1: 91 – brazil production 1: 210 – energy source 1: 222 – world production 1: 51 bagasse storage, case study 1: 321 bale storage 1: 322 – corn stover 1: 320 bale transport, Iowa Corn Stover Collection Project 1: 319 baling 1: 333 – dry material 1: 332 BAS 1: 268 basic chemicals – cellulose 1: 17 – glucose 1: 17 – starch 1: 17 basic principles, biotechnology 2: 349–351 basic substances, biorefinery 1: 18 batch fermentation, brown juice 1: 299–300 BBI 2: 322 14-BDO 2: 373, 375 beer streams 1: 134 benzene 1: 87 benzene derivatives 1: 118 – cyclotrimerization 2: 259 benzene–toluene–xylene see BTX Bergius, F. 1: 5 Berzelius, J. J. 1: 5 Beta vulgaris 2: 410 betaine 2: 410–415 – chemical properties 2: 411 – chemical structure 2: 411 – usage 2: 412–414 betaine esters 2: 414–415 BG 1: 203, 364 BG Supplement 1: 366–367 binder, starch 2: 84 bio-alcohols, sugar conversion 2: 32 bio-based building blocks 2: 453 – emergence 2: 450 biobased consumer products, cosmetics 2: 409–442 biobased economy 2: 138 – 3-pillar model 1: 3 – existing 1: 43 – growth 1: 44–45 – historical outline 1: 42–45 Biobased Industrial Products, initiative group 1: 16 bio-based industry, transition 1: 93–96 bio-based materials 2: 354 biobased oleochemicals, industrial development 2: 291–314

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Subject Index biobased poly(lactic acid) 1: 296 biobased production, integrated 1: 8–12 biobased products – market opportunity 2: 353 – markets 2: 348 biobased technology, current 2: 375–377 bio-cascade, biorefinery concepts 2: 355 biocatalysts 1: 68 – development 1: 108 – genetically engineered 2: 37 – improvement, 3-HPA 2: 35 biocatalytic routes – chemicals production 1: 385–406 – ethanol production 1: 390–393 biochemical refinery, secondary 1: 104–106 biochemicals 1: 13 biocompatibility 2: 230–232 biocomposites 2: 362 bioconversion – biomass processing 1: 98 – fermentation 1: 104 – starch 2: 89–91 bio-counterpart, petroleum-derived polymers 2: 41 biocrude 1: 98, 2: 351 biodegradability – general aspects 1: 213 – intrinsic 2: 348 biodegradable films, gluten 1: 181 biodegradable lubricants, european potential market 2: 301 biodegradable packages 2: 422–423 biodegradable plastics 1: 182 – sugar cane 1: 212–216 biodegradable polymer 1: 91 biodegradable thermoplastics, programmable 2: 241 biodegradation – definition 1: 213 – white-rot fungi 2: 160 biodiesel 1: 116, 152 – production 1: 126 bio-ethanol 2: 451–452 bio-fertilizer, grasses 1: 283 biofine char 1: 155–158 – insoluble components 1: 146 biofine plants – byproducts 1: 145 – costs 1: 159 biofine process 1: 139–164 – advantages 1: 146 – economics 1: 158–161 – yields 1: 145–146

biofuel cells 1: 379 biofuels 1: 182 – directive 1: 15 – promotion 1: 94 biogas 1: 30, 377 biogenic amorphous silica see BAS biological inhibitors, fast pyrolysis 1: 249 biological raw materials, product classes 1: 13 biomass – availability 1: 99–101 – commercialization 1: 317–344 – components 1: 22 – composition 1: 16, 119, 359, 2: 108 – compositional variety 1: 45 – conversion 2: 151–163, 350, 455–456 – definition 1: 12–14 – depolymerization 1: 123 – diversification 1: 54–55 – hydrolysis 1: 129–138 – hydrolyzate 1: 78 – industrial 1: 13 – industrial chemicals 2: 347–365 – key sugars 2: 3–59 – lignocellulose 2: 97 – local 1: 56–57 – multi-quality 1: 92 – policy targets 1: 85 – polysaccharide-containing 1: 105 – pretreatment 1: 107, 361–363 – recycling 1: 117 – refining 1: 41–66, 107, 227–252 – sustainability 1: 93–97, 106 – technology 1: 14–16, 93–97 – thermochemical processing 1: 249 biomass-based industrial products 1: 87 biomass-based products, estimated EU potential 1: 89 biomass carbon resources 1: 116 biomass chemistry, comparison with petroleum 1: 118–122 biomass content, classes 2: 4 biomass feedstocks 1: 45 – costs 1: 48–50 – required properties 1: 50 biomass flux, The Netherlands 1: 99 biomass fuels 1: 103 biomass gasifiers, types 1: 231 biomass industry, chemical production numbers 1: 284 biomass-nylon-process 1: 26 Biomass Research and Development Technical Advisory Committee 1: 135

Subject Index biomass streams 1: 100 biomass substitution volume 1: 85 biomass suppliers 1: 118 Biomass Technical Advisory Committee see BTAC biomass value 1: 324–328 biomaterials 1: 13 bionics 2: 410 bio-oil – characteristics 1: 243 – fermentation 1: 229, 244 – yield 1: 241 Biopol 2: 44, 422–424 – biodegradability 2: 424 – future 2: 428–429 biopolyesters, synthetic 2: 41 biopolymers 2: 40–47 – cellulose 2: 104 bioprocessing, consolidated 1: 56 bioproduct opportunities, industrial 1: 379 bioproduction – highlights 2: 223 – mechanistic foundations 2: 217–251 – protein-based polymers 2: 223–227 bioproducts, classification 2: 356–357 bioreactor engineering 1: 108 biorefineries – basic principles 1: 17 – bio-oil based 1: 229 – Brazil 1: 71 – building-block concept 2: 202–204 – cellulosic 1: 55–56 – chlorophyll disregard 2: 338 – conceptual schematic diagram 1: 239 – definition 1: 19–22, 116, 227, 358 – development 1: 67–83 – disadvantage 1: 46 – fuel-oriented 1: 193 – future integration 1: 380 – generations 1: 19–20 – green see green biorefinery – integration 2: 201–216 – lignin 2: 177 – lignocelluloses 2: 110–115 – lignocellulosic 1: 115–128 – lignocellulosic feedstock 1: 24–26, 129–138 – MAAP 2: 209 – near future production 1: 317 – oats based 1: 183–187 – phase III 1: 19 – plant juice 1: 295–314 – possible products 1: 45–47

– primary research areas 1: 101–103 – principles 1: 16 – raw material 1: 45–47, 253 – sugar-based 1: 209 – supply 1: 45–52 – technological development 1: 53–56 – wet mill-based 1: 28 – wheat based 1: 167–183 – whole-crop 1: 24, 26–29 biorefinery complex, cost estimates 1: 118– 122 biorefinery concepts 1: 98–99, 2: 355–356 – definition 1: 166 – elements 1: 81 biorefinery context 2: 315–324 biorefinery evolution 1: 69 biorefinery I, sucrose-based 1: 68 biorefinery II, starch-based 1: 69 biorefinery III 1: 69 biorefinery lignin, substitution 2: 182 biorefinery model 1: 68 biorefinery process, integrated 1: 102 biorefinery products 1: 11 biorefinery research, current 1: 11 biorefinery supply, transport options 1: 338 biorefinery systems 1: 3–40, 23 – history 1: 4–16 – sustainability 1: 56–65, 60–65 – whole crop 1: 165–191 biorefinery technology developments, milling industries 1: 345–353 biorefinery two platforms concept 1: 24 biorefinery wastes 1: 56 biosyngas 1: 98 biosynthesis, poly(3-hydroxybutyric acid) 1: 224 biosynthesis genes, Escherichia coli 2: 44 bio-synthetics, car production 1: 9 biotech, industrial 2: 445–462 biotech adoption 2: 447 biotech development, pace 2: 447 biotech strategy 2: 457 biotechnological processes, typical problems 1: 388–389 biotechnology, predictions 2: 32 biphenyl units, lignin 2: 158 1,5-biphosphatecarboxylase/oxygenase 1: 255 bisphenol A see BPA black liquors – Kraft pulping 2: 170 – soda pulping 2: 171

467

468

Subject Index blood plasma substitutes, starch 2: 89 blue starch 2: 62 Boehringer, A. 1: 7 bonding patterns 2: 156–159 Boudouard reaction, syngas production 1: 230 Bowman–Birk inhibitor see BBI BPA 1: 148 Brabender viskograph 2: 75 Braconnot, H. 1: 5 bran, wheat milling 1: 170 Branched PLA, melt rheology 2: 397 branching, rheology control 2: 396 branching technology 2: 398 Brassica napus 2: 277 Brazil, agroindustry 1: 209–211 breeding material, fatty acid variants 2: 279 Brevibacterium 2: 35 British gums 2: 62 bromination, LA 1: 150 Brookfield viscometer 2: 75 brown juice (BJ) 1: 271, 274, 300–308 – average composition 1: 301–302 – batch fermentation 1: 299–300 – carbohydrate addition 1: 311 – composition of nutrients 1: 305 – fermentation medium 1: 298 – lactic acid fermentation 1: 305–306 – lactic acid source 1: 295 – quality variations 1: 302–305 – storage alternatives 1: 298 BTAC 1: 14 BTX 2: 29 building-block concept 2: 204 – biorefinery 2: 202 – metabolic engineering 2: 204, 206 building blocks 1: 22–23, 98 – biobased 2: 450, 453 – heterocyclic 2: 26 – protolignins 2: 152 – succinic acid 2: 367–379 – sugar derived chemicals 2: 34 building chemistry 2: 87 bulk chemicals 1: 386 – production routes 1: 385–406 Burkholderia spp, poly(3-hydroxybutyric acid) biosynthesis 1: 224 business structure 1: 117–118 butadiene – 1,4-BDO 2: 373 – glucose product family 1: 21 1,4-butanediol 1: 149 2,3-butanediol, glucose product family 1: 21

1,2,4-butanetriol 2: 37 n-butanol, glucose product family 1: 21 Butyribacterium methylotrophicum 1: 228 – representatives 1: 235 c-butyrolactone see GBL by-products – animal feed 1: 100 – biorefinery 1: 23 – brown juice 1: 311

c C1 compounds 1: 233 – syngas fermentation 1: 228 C5-carbon sugars, product categories 2: 358–360 C6-carbon sugars, product categories 2: 358 C–C coupling, radical 2: 266–269 C–C double bonds, oxidative cleavage 2: 258 C. glutamicum 2: 205, 210 – phosphorus supply 2: 211 C–H bonds, functionalization 2: 269–270 C. milleri 2: 212 C-nucleosides 2: 25 (C-x)-chemicals 1: 21 C2 anions, oxidative coupling 2: 266 C2 building-block chemical, ethanol 2: 132 C3–C5 carboxylic acids, microbial fermentation 2: 33 C3 plants – protein yield 1: 253 – yield 1: 258 C4 plants – protein yield 1: 253 – yield 1: 258 C7 plant acids, potential generation 2: 32 CAFI 1: 136 calcium lactate 1: 106 cancer chemopreventive agents 2: 322 cancer therapies, DALA 1: 150 Candida antarctica, lipase B 2: 256 Candida bombolica 2: 274 Candida tropicalis 2: 273 Candida tropicalis DSM 3152 2: 274 Candida tropicalis M 25 2: 274 capital costs, biorefinery 1: 240 carbohydrate-based product lines 2: 3–59 carbohydrate-based surfactants 2: 305 carbohydrate composition, lignocellulosic feedstock 2: 109 carbohydrate content, changes 1: 266 carbohydrate esters, lipase-catalyzed syntheses 2: 272 carbohydrate homopolysaccharides 1: 139

Subject Index carbohydrate polymers, cellulose 1: 55 carbohydrate polysaccharides, acid hydrolysis 1: 144 carbohydrate refining 1: 351 carbohydrate source, addition to brown juice 1: 311–312 carbohydrate stream, corn refinery 1: 349 carbohydrates 1: 89–90, 2: 108 – annually renewable 2: 6 – biorefinery 1: 18 – catalytic oxidation 1: 403–404 – chemical catalytic conversion 1: 402 – contained in biomass 2: 3 – heating 2: 24 carbon – recycling 1: 116 – renewable 1: 43 carbon-14-labeled Escherichia coli, purification 2: 228–229 carbon-based plant material, yearly amount 1: 43 carbon dioxide – glucose product family 1: 21 – recycling 1: 117 carbon dioxide sink, PLA 2: 402 carbon fibers – annual demand 2: 197 – porous 1: 283 – vehicle production 2: 196–197 carbon-oxygen reaction, syngas production 1: 230 carbon-processing industries 1: 42–44 carbon sequestration 1: 62 carbon sources 2: 209 – fermentable 1: 78 – industrial 1: 67 – reduced cost 2: 204 carbon sugars 2: 358 carbon-water reaction, syngas production 1: 230 carbonate polymerization 2: 398 carboxylic acids 2: 34–36 – addition 2: 262 – chemical conversion 2: 37–40 – microbial conversion 2: 32–34 – sugar-based 2: 43 carboxymethylation 2: 77 cardboard, from press cake fibers 1: 282 care additives, multifunctional 2: 309 carotene 1: 257 – industrial production 1: 9 carotenoids 2: 320 Carothers, W. H. 1: 8

carton production 2: 84 case studies, sustainable production 2: 448 catabolism, chlorophyll 2: 330–331 catalysis technology 2: 349 catalysts – bio-oil production 1: 244 – carbohydrates conversion 1: 403 – metal-based 1: 228, 233 catalytic decarbonylation, furan 2: 9 catalytic hydrogenation – LA 1: 151 – sorbitol 2: 130 catalytic pulping, wood 2: 118 catalytic routes, chemicals production 1: 385–406 catalytic transformations 2: 270–272 – succinic acid 2: 372–375 catechol 2: 30 cationic addition, Lewis acid-induced 2: 264–265 cationic polymers, hair 2: 419 cationic surfactants 2: 412 – structure 2: 414 CBH 1: 203, 364 CBH-EG-BG System, optimization 1: 366–371 CBH I 1: 76 CBH I (Cel7A) variants, thermal activity 1: 368 CBM, cellulase families 1: 364 CC 1: 61 cell contents 1: 265–269 cell-immobilization 1: 392 cell removal 2: 388 cell wall, structural constituents 1: 260–265 cellobiohydrolase see CBH cellobiohydrolase I see CBH I cellulase development 1: 366–375 cellulase enzyme performance 1: 74 – improved 1: 76–77 cellulase enzyme production 1: 194 cellulase enzymes 1: 74, 2: 177–178 – costs 1: 72–73 – production 1: 201–202 – superior 1: 205 – thermal stability 1: 76 cellulase expression inducers, disaccharide sophorose 1: 76 cellulase mix, lignocellulosic conversion 1: 374 cellulase production economics, improved 1: 74–77

469

470

Subject Index cellulase production strain, enhancement 1: 374 cellulase saccharification, plant development 1: 134 cellulases – biodegradation 1: 364 – commercial status 1: 202 – expression 1: 374–375 – improvements 1: 367 – novel 1: 367–370 cellulolyteomics 1: 374 cellulolytic fungi – protein gels 1: 372 – secretome 1: 371–373 cellulose 1: 6, 71–73, 90, 121 – accessibility 1: 55 – acetate 1: 243 – acid conversion 1: 129 – biosynthesis 1: 90 – chemical composition 1: 359 – chemical conversion to LA 1: 144 – chemical structure 2: 418 – conversion rates 1: 130 – digestibility 1: 261 – enzymatic hydrolysis 2: 115 – fermentation 2: 177–179 – glucan source 1: 139 – high-vacuum pyrolysis 2: 23 – history 2: 100 – hydrolysis 1: 26, 194, 199, 202–205 – isolation 2: 127 – plant content 1: 261 cellulose-based biorefinery III 1: 69 cellulose-based product family tree, industrial 2: 129 cellulose-based product lines 2: 127 cellulose-binding modules see CBM cellulose derivatives 2: 357 – principal 1: 90 cellulose fiber, pretreatment 1: 72 cellulose-hydrolyzing enzymes 1: 17 cellulose saccharification 2: 99 cellulosic biomass 1: 68, 197 – conversion to fuel 1: 52 – ethanol production 1: 193 – recalcitrance 1: 56 cellulosic biomass conversion 1: 71 cellulosic biorefineries, process development 1: 55–56 cellulosic feedstocks, hydrolysis 1: 140 cement 2: 87 CENTURY model 1: 60–65 cereal fractionation, advanced 1: 173

cereal fractionation plants, categories 1: 167 cereal fractionation processes see CFP cereal grains, baling 1: 333 cereal waste, LCF biorefinery 2: 111 cereals 1: 26, 165–191 – starch sources 2: 63 cetiol CC 2: 311 CFP 1: 166 chain length, cellulose 1: 195 char 1: 145 – biofine process residual 1: 155 chemical composition, apple-peel wax 2: 433 chemical conversion, sugars 2: 37–40 chemical degradation, chlorophyll 2: 333 chemical digestion, intracellular poly(hydroxyalkanoates) 1: 218 chemical fractions, lignocellulose 1: 24 chemical industry 1: 97 – biorefineries 1: 85–111 – renewable raw materials 2: 253–289 chemical modification, naturally produced structures 2: 349 chemical pulping 2: 166 – environmentally friendly 2: 179 – LCF 2: 114 chemical sources, grasses 1: 282–283 chemical transformation steps, petrochemical industry 1: 88 chemicals 1: 22–23 – basic 2: 5 – biobased 1: 22 – biomass compounds 1: 119 – fossil sources 1: 120 – from biomass 1: 108 – from renewable resources 2: 367–379 – glucomannan derived 2: 120 – lignocellulose-based 2: 97–150 – low-molecular-weight 2: 160 – organic 1: 124 – product family tree 2: 124–126, 132 – production 1: 386 – production routes 1: 385–406 – special 1: 378 chemo-enzymatic epoxidation 2: 254 chemo-enzymatic self epoxidation, reaction principle 2: 256 chemoattractant, peptides 2: 238 chemopreventive agents, cancer 2: 322 chemurgy 1: 9 chiral purity, lactic acid 2: 383 chitin 1: 182 – chemical structure 2: 418

Subject Index – chitosan precursor 2: 415 – deacetylation 2: 417 – occurrence 2: 415–419 – purification 2: 416–417 chitosan 2: 415–422 – chemical structure 2: 418 – production 2: 417 chitosan derivatives 2: 421 chitosonium salts, water vapor sorption 2: 421 chlorophyll 2: 325–343 – biological catabolism 2: 330–334 – breakdown 2: 330 – chemistry 2: 327 – commercial production 1: 257 – degradation 2: 331, 333 – derivatives 2: 335–339 – fundamentals 2: 326 – historical outline 2: 325 – industrial production 1: 9 – isolation 2: 328 – new materials 2: 338 – reactivity 2: 328 – structure 2: 327 chlorophyllin 2: 335 cholesterol level, decrease 1: 180 cholesterol mediation 1: 277 cholesterol reduction, b-glucan 1: 185 chopping, pretreatment 1: 361 chrisgas-project 1: 103 Chromatium okenii 2: 424 circuit board resins 2: 194–195 citrates 1: 91 citric acid, glucose product family 1: 21 Clostridium ljungdahlii 1: 228, 235 Clostridium methoxybenzovorans SR3 1: 179 Clostridium thermoaceticum 1: 235 Clostridium thermocellum 1: 365 clothing, synthetic fibers 2: 190 CO2 flux 1: 328 CO2 production, MAAP 2: 212 CO2 sequestration 1: 173 co-polymerization, starch 1: 27 co-products 2: 370 – sugar fermentation 2: 375 coconut oil 2: 292 collection, baling dry material 1: 332 collection cost, forage harvester 1: 334 commercial consideration, MAAP 2: 205–209 comonomers, multi-cyclic 2: 398 company closures, lignosulfonate producers 2: 173

competitive prices, biobased products 2: 49 competitors, external challenges 2: 457 components, cereals 1: 166 composite materials, carbon fiber 2: 196 compositional variety, biomass 1: 45 concentration, lactic acid 2: 390 concept, all biomass is local 1: 57 concrete, self-leveling 2: 88 concrete admixtures 2: 189–190 conditioner, sugar beet 2: 410–415 conditioning agent, natural 2: 436 coniferyl alcohol, oxidation 2: 158 consolidated bioprocessing 1: 56 Consortium for Advanced Fundamentals and Innovation see CAFI consumer acceptance, external challenges 2: 457 consumer products 2: 409–442 continuous cultivation see CC continuous fermentation 1: 300 controlled-release devices, design 2: 240 conversion, chlorin 2: 333 conversion efficiency 1: 196 conversion steps – biorefinery 1: 23 – lignocellulosic biorefinery 1: 24 conversion technologies 1: 108 – primary 1: 270, 2: 350 cooking liquors 2: 166 coordination–insertion mechanism, lactide polymerisation 2: 393 copolyesters, PHB 1: 215 copolymer, PHV-PHB 2: 426 copper-initiated additions 2: 262–263 corn 1: 26 – phytochemicals 2: 317 – wet milling 1: 28 corn continuous cultivation 1: 61 corn dry milling, biorefinery example 1: 70 corn dry milling industry 1: 345–353 corn grain, export reduction 1: 43 corn oil, corn refinery products 1: 348 corn pricing 2: 368 corn refinery, modern 1: 348 corn refining 1: 346–347 corn–soybean rotation 1: 61 corn starch, pearl 1: 351 corn-steep liquor 1: 349 corn stover, world production 1: 51 corn stover bale storage 1: 320 corn stover pricing 1: 319 corn stover structure 1: 74 corn syrup, carbohydrate refining 1: 351

471

472

Subject Index corn tillage practice 1: 330 corn wet milling industry 1: 345–353 corn wet milling process 2: 367 corncobs 1: 91 corporate action, increasing 2: 451 corrugating industry, starch usage 2: 83–84 Corynebacterium efficiens 1: 80 Corynebacterium 2: 35 cosmetic emulsion, oil-phase components 2: 310 cosmetic lipids, occlusion testing 2: 434 cosmetics – chitosan 2: 419 – consumer products 2: 409–442 – history 2: 409–410 – ilex resin 2: 439 – starch usage 2: 88–89 cost components, ethanol production 1: 73 cost disadvantage, cellulose-based ethanol 2: 203 cost efficiency 1: 381 cost estimates, biorefinery complex 1: 118 cost generators, waste 1: 96 cost savings, biotechnology 2: 450–451 costs – antioxidants 2: 188 – biomass vs. petroleum 1: 48–50 – feedstock 1: 196 – MAAP 2: 202 – processing systems 1: 53 cotton 1: 90 coupling, oxidative 2: 266 cover crops 1: 331 crop-drying industry, grass usage 1: 298 crop residues 1: 45 – commercial 1: 318 – world production 1: 51 cropping system 1: 61 crops, starch sources 2: 63 cross-linking – free radical 2: 399 – starch modifications 2: 81 cross-reactions 1: 145 crotonaldehyde, glucose product family 1: 21 crude drugs, juice fraction 1: 274 crude fiber, plant content 1: 261 crude oil 2: 292 – high prices 1: 115 crude petroleum, separation 1: 119 crude starch milk 2: 66 crushing, pretreatment 1: 361

crystalline cellulose 1: 195 crystalline melting point, control 2: 394 crystallinity, starch 2: 72 CSL 1: 349 cultivation temperature 2: 213 curl-retention test 2: 420 curled hair, swatches relaxation 2: 419 cycle times, PHB 1: 216 cyclization, methyl 17-octadecanoate 2: 259 cyclodextrins 2: 90 cyclotrimerization, benzene derivatives 2: 259 Cyprus papyrus 2: 98

d Dactylis glomerata 1: 261 – alkanes 1: 268 – amino acid composition 1: 267 – sugar 1: 265 DALA 1: 149–150 DDGS 1: 71 debranning apparatus 1: 174 decomposition methods, primary refinery 1: 271 decorative laminates 2: 185 deformation energy, recovery 2: 220 degradation, chlorophyll 2: 331, 333 – definition 1: 213 degradation resistance, cellulose fibrils 1: 140 degree of polymerization, cellulose 1: 195 demonstration process, iogen’s 1: 193 density, bales 1: 335 department of energy (DOE) 1: 19, 74 depolymerization, biomass 1: 123 designer proteins 1: 122 development lines, sugar-based chemicals 2: 14 development trap, underdeveloped countries 1: 52 dextrins 2: 79 dextrose 2: 128 – production 1: 44 – starch hydrolysis 1: 5 dextrose syrup, carbohydrate refining 1: 351 DFA III, production 1: 397 diacids, replacement 2: 38 1,4-diacids 2: 34 – basic biobased chemicals 1: 22 dialkyl carbonates 2: 311 – synthesis 2: 311 diamines, sugar-based 2: 42

Subject Index diammonium succinate 2: 376 diastereomeric forms, lignin 2: 157 dibenzodioxocin structures, lignin 2: 158 Diels-Alder reaction, methyl conjugenate 2: 260 diesel 1: 119 – low-smoke formulation 1: 153 dietary lignin, antidiarrheic effects 2: 195 diethyl ether, glucose product family 1: 21 diffraction patterns, starch 2: 73 difructose anhydride 1: 397–402 digestibility, cellulose 1: 261 diglycerides, lipase-catalyzed syntheses 2: 270–272 dihydropyranones 2: 20 – disaccharide-derived 2: 24 dilactide, glucose product family 1: 21 dilute acid hydrolysis 1: 200 dilute acids – pretreatment 1: 362 – starch treatment 2: 76 dilute sulfuric acid, biofine process 1: 142 dilute-sulfuric-acid hydrolysis, cellulose 1: 132 dimer acid 2: 297–298 dimerdiols, dimer acid based 2: 297–298 dimerization, radical 2: 267 dimethyltetrahydrofuran see DMTHF diphenolic acid 1: 148 direct distillation 2: 389 disaccharide sophorose 1: 76 disaccharides, availability 2: 4–7 disposal problems, Biopol 2: 428 dissociation, of industries from petrochemical 1: 94 distillation of lactate ester 2: 389 distillers dried grains and solubles see DDGS DM 1: 261 DMTHF 1: 152 DOE see department of energy door binders 2: 185–186 downdraft gasifiers 1: 231 downstream processing – grass fiber fraction 1: 281 – poly(3-hydroxybutyric acid) 1: 218–220 drilling fluids, starch derivatives 2: 91 drugs, sugar derived 2: 14 dry fractionation, wheat 1: 176–183 dry matter 1: 261 dry mill refinery 1: 346–347 dry milling 1: 27, 70 – operations 1: 166

dry reactions, starch modifications 2: 77 dry storage, bagasse 1: 321 DSM, transition process 1: 93 Duales System, biodegradable bottle 2: 427 Dutch Energy Research Strategy 1: 109 dye dispersants 2: 190–192 dyes – biorefinery context 2: 315–324 – juice fraction 1: 274 dyestuff 2: 191

e E. coli see Escherichia coli E10-Fuel 1: 9 ECN 1: 109 ecological aspects, green biorefinery 1: 283–285 ecological balance, fermentative production 2: 207 ecological compatibility, biobased oleochemicals 2: 293 economic aspects, green biorefinery 1: 283–285 economic barriers, biotechnology 1: 381 economic benefits 2: 452–454 economic clusters, new synthesis 1: 95 economic forces 1: 41 economic potential – biotechnology 2: 446–451 – industrial biotech 2: 445–462 economics, biofine process 1: 158–161 economies of scale 1: 159 – biodiesel plant 1: 127 economy, biobased 1: 41–66 economy growth 1: 67 economy of scale – biorefineries 1: 350 – furfural 1: 125 ecosystem modeling 1: 57–60 edible films, gluten 1: 181 efficiencies, biofine process 1: 145–146 efficiency improvements, biotechnology 2: 454 efficient energy conversion, elasticity provides 2: 219 EG (endoglucanase) 1: 203, 364, 2: 133 – structure–function relationship 1: 370– 371 Ekman, C. D. 1: 6 EL (ethyl levulinate) 1: 152–153 elastic consilient mechanism 2: 223 – protein-based polymer engineering 2: 217

473

474

Subject Index elastic mechanisms, coupling to hydrophobic mechanism 2: 237–238 elastic moduli, fibers 2: 241 elastic protein-based polymers, temporary functional scaffoldings 2: 239 elasticity, protein-based polymers 2: 219– 220 Elbow washing test, betaine 2: 413 electricity 1: 46 – biomass share 1: 14, 16 electricity generation, biomass 1: 44 electrodialysis, lactic acid purification 1: 312 electrophilic substitution, furan 2: 9 emollients 2: 310 emulsifiers – lecithin 2: 318 – polyglycerol esters 2: 308 – vegetable oil 2: 301 emulsion(co)polymerization process, starch derivatives 2: 90 endoglucanase see EG endotoxins, removal 2: 230 enediol, dehydration 1: 142 Energie Onderzoeks Strategie see EOS energy and protein coproduction 1: 55 energy balance, simultaneous processing of sugars 1: 221 energy conversion, efficient 2: 219 energy costs, impacts 1: 115 energy crops, renewable carbon 1: 44 energy efficiency, fossil fuels replacement 2: 449 Energy Research Center of the Netherlands see ECN energy sources, biomass-based 1: 380 engine efficiency loss, diesel 1: 153 engineered organisms 1: 68 engineering – mechanistic foundations 2: 217–251 – protein-based polymers 2: 219–220, 232–238 engineering principles – fundamental 2: 222 – protein-based polymers 2: 220 entire barrel of biomass 1: 54–55 entropic elastic force, proteins 2: 223 environmental aspects, biodegradable plastics 1: 212 environmental benefits 1: 117 environmental consideration, MAAP 2: 205–209 environmental impact, production process 1: 92

environmental improvements 1: 64 environmentally friendly, biotech 2: 448– 450 enzymatic conversion 1: 130 enzymatic digestion, poly(3-hydroxybutyrateco-valerate) 1: 219–221 enzymatic hydrolysis 1: 79, 147 – cellulose 2: 115 – improvements 1: 205 – reactions 1: 203 enzymatic hydrolysis process 1: 134 enzymatic methods, LCF 2: 115 enzymatic oxidizing systems 1: 178 enzymatic processes, starch degradation 2: 79 enzymatic reactions 2: 270–274 enzymatic synthesis, MAAP 2: 202 enzymatic transport, improvements 2: 204 enzyme-based plant development 1: 134 enzyme broth 1: 201 enzyme catalysis, economic barriers 1: 381 enzyme cost reduction, ethanol production 1: 377 enzyme dosage 1: 366 enzyme immobilization 1: 399–402 enzyme performance, cellulase 1: 76–77 enzyme production 1: 202 – bran 1: 172 enzyme recovery 1: 74 enzyme requirement, increase 2: 178 enzyme screening 1: 398 enzyme system, optimisation 1: 74 enzymes 1: 357–383, 2: 90 – biodegradation 1: 363 – biomass conversion 1: 68 – cellulase 1: 201–202 – cost reduction 1: 56 – markets 2: 446 – nonhydrolytic 1: 365 – oxidative 1: 213 – recycling 1: 205 – superior 1: 205 – synergism 1: 365–366 – thermally stable 1: 368 EOS 1: 109 epoxidation – chemo-enzymatic 2: 254 – new methods 2: 254–257 epoxides 2: 254 – polyols 2: 298–299 – PVC stabilizers 2: 256 equilibrium concentration, protonated glycoside 1: 141

Subject Index erosion 1: 327 – cover crops 1: 331 – prevention 1: 61 ERRMA 1: 89 erucic acid, high 2: 280 erythro form, lignin 2: 157 Escherichia coli 1: 206, 2: 30 – bioengineered 2: 35–37, 44 – carbon-14-labeled 2: 228–229 – cost of production 2: 242 – fermentation 2: 230 – inulin production 1: 398 – recombinant 1: 399 – transformation 2: 227–230 esparto grass, xylitol source 1: 283 esterification, starch 2: 80 esters, lubricant applications 2: 300 ETBE 2: 7 ethanol 1: 89, 104, 146, 209, 2: 7–8, 132 – additive replacement 1: 357 – fermentation 1: 11, 2: 120, 351 – global market 2: 446 – glucose product family 1: 21 – lignocellulose transformation 2: 115 – predictions 2: 454 – vapor pressure 1: 151 – wood hydrolyses 1: 5 ethanol production 1: 125, 130, 193, 209–210, 389 – advantages 1: 197 – cellulase 1: 201 – costs 1: 72, 246 – enzyme cost reduction 1: 377 – sucrose 1: 70 ethanol production plant, process design 1: 393 ethanol recovery 1: 206–207 ether structures, lignin 2: 158 etherification, starch 1: 27, 2: 80 ethyl t-butyl ether 2: 7 ethyl ester, LA derivatives 2: 10 2-ethyl hexanol, glucose product family 1: 21 ethyl lactate, glucose product family 1: 21 ethyl levulinate (EL), properties 1: 151 ethylene, glucose product family 1: 21 EU directives 1: 94 Eubacterium limosum 1: 235 Europe, biomass conversion 2: 351 European grassland, yield 1: 259 European Renewable Resources and Materials Association see ERRMA excess water, removal 1: 219

exothermic hydration, apolar groups 2: 218 expansin, enzymatic hydrolysis 1: 365 expressed protein-based polymers 2: 242–245 expression vector, gene 2: 227 external challenges 2: 456 external environment, biotechnology 2: 461 extraction, chlorophyll 2: 328 extraction methods, chlorophyll 2: 337 extraction processes, PHB 1: 218 extrusion cooking, starch modifications 2: 77 extrusion processes, PHB 1: 216

f fabric coloring 2: 191 FAME 1: 152 farmer value 1: 325–327 FAS 2: 303 – synthesis 2: 303 fast pyrolysis 1: 229, 241 – biorefinery 1: 246–248 – products 1: 242 – reaction pathways 1: 243 fast-pyrolysis plant, schematic diagram 1: 244 fat hardening 1: 7 fats – microbial conversion 2: 274 – new syntheses 2: 253–289 fatty acid esters, biodegradable 2: 299–301 fatty acid methyl esters see FAME fatty acid oil seeds variants, commercially available 2: 278 fatty acids 1: 90–92 – anodic coupling 2: 267–269 – apple-peel wax components 2: 433 – chain length 2: 292 – epoxidation 2: 254–257 – juice fraction 1: 274 – microbial oxidation 2: 273–274 – nucleophilic addition 2: 265 – oxidative coupling 2: 266 – triglycerides 1: 122 – unsaturated 2: 272–273 – vic-dihydroxy 2: 257–258 fatty alcohol sulfate 2: 303 fatty alcohols 2: 294 fatty compounds – reactions 2: 266–270 – unsaturated 2: 254–266

475

476

Subject Index FDCA (furan-2,5-dicarboxylic acid) 2: 38, 133 – basic biobased chemicals 1: 22 – synthesis 2: 134 feedstocks 1: 100, 105 – alternative 2: 452 – composition 1: 195 – conversion 1: 68–73 – fibrous 1: 239 – insoluble components 1: 146 – pretreatment 2: 167, 178 – pricing 1: 327 – production 1: 181 – quality 1: 196, 334 – selection 1: 194–198 – sucrose-based 1: 197 – supply 1: 317, 2: 368–369 – thermogravimetric analyses 1: 156 fermentable-carbon-cost 1: 68 fermentable carbon source 1: 78 fermentable sugars 1: 67–73 fermentation 1: 74, 85, 123, 146, 2: 356 – E. coli 1: 399 – Escherichia coli transformation 2: 227 – amino acids 2: 203 – biomass processing 1: 98 – bio-oils 1: 229 – cellulose 2: 177–179 – commercial lactic acid production 2: 384 – continuous 1: 300 – economics 2: 369 – ethanol 1: 390–391, 2: 351 – fungal 1: 181 – glucose 1: 31 – guidelines 1: 21–22 – lactic acid 1: 298, 2: 383 – microbial 1: 181 – PHB production 1: 217 – rhizopus-based 2: 388 – succinic acid 2: 369 – syngas 1: 233–239 fermentation broth, no processing 1: 75 fermentation by-products 2: 388 fermentation ethanol 2: 7 fermentation industry, starch 2: 89 fermentation inhibitors, bio-oil 1: 245 fermentation medium – brown juice 1: 298 – pearled grain flour 1: 176 – plant juice 1: 295–314 – potato juice 1: 309–310 fermentation organisms 1: 77–81 fermentation process 1: 104–106

– inhibitors 1: 78 – performance 1: 78 – PLA 1: 296–297 fermenters, ethanol production 1: 201 fertilizer – ash 1: 161 – nitrogen 1: 325 ferulic acid 1: 178–179 Festuca arundinacea 1: 261 – alkaloid production 1: 277 – fructans 1: 267 – sugars 1: 266 Festuca pratensis 1: 261 – amino acid composition 1: 267 – sugar 1: 265 Festuca spp, proteins 1: 277 fiber fraction 1: 278–285 – corn 2: 369 – grass 1: 281 fibers – biodegradable 2: 10 – corn refinery products 1: 348 – high-performance 2: 41 – improved 2: 241 – Kraft lignin 2: 197–198 – paper 2: 84 – press-cake components 1: 280–282 fibrous biomass, fast pyrolysis 1: 246 film-forming agents, chitosan 2: 419 films, water-retentive properties 2: 420 filter aids, purified biogenic silica 1: 278 fine chemicals 1: 386 – production routes 1: 385–406 finishing agents 2: 86 Fischer glycosidation, APG synthesis 2: 12 Fischer–Tropsch process 1: 158 flocculants, starch derivatives 2: 91 flow dynamics, agricultural ecosystems 1: 60 fluidized bed gasifiers 1: 231 foams, production 2: 9 follow-up chemicals, ethanol 2: 7 follow-up products, biorefinery 1: 24 food 1: 13 food preservative, ferulic acid 1: 179 forage crops 1: 45 forestry ecosystem modeling 1: 57–60 forestry waste, furfural hydrolysis 2: 8 formic acid 1: 153–154, 2: 39 – production 1: 139–164 formulation 1: 74 forward extraction, lactic acid 2: 389 fossil-based raw material substitution 1: 85

Subject Index fossil carbon-processing industries 1: 42–44 fossil fuel substitution 1: 85 fossil fuels replacement, energy efficiency 2: 449 fossil organic raw materials 2: 347 fossil resources, dependence 1: 92 foundry, starch derivatives 2: 91 foundry resins 2: 184–185 Fownes, G. 1: 6 fraction-I protein 1: 274 – economic interest 1: 255 fraction-II protein 1: 275 fractionation, green crops 1: 272 fractionation process, oats based 1: 183–187 free energy of hydration, repulsive 2: 218 free radical cross-linking 2: 399 friction materials 2: 184 Friedel-Crafts acylation 2: 265 fructans 1: 267 – enzymatic decomposition 1: 302 fructose 2: 131 d-fructose, synthesis 2: 132 frying oil, byproducts 1: 100 fuel additives 1: 150 – ethanol 1: 26 fuel alcohol, production 1: 193 fuel cells 1: 378 fuel ethanol, legislative support 2: 453 fuel ethanol program, Brazil 1: 210 fuel gas 1: 102 fuel-oriented biorefineries 1: 193–208 fuel production 1: 53 – starch 1: 181 fuel source, sustainable 1: 115–128 fuels 1: 13 – biobased products 1: 376 – biofine char 1: 155 fumaric acid 2: 35 functional foods 1: 180 functional group transformations, side chains 2: 333 functional groups 2: 156–159 – addition to hydrocarbons 1: 119 – LA 1: 147 functionalization, C–H bonds 2: 269–270 fungal cellulolytic system 1: 365 fungal fermentations 1: 181 fungal genes, schematic representation 1: 373 fungi, cellulolytic 1: 371–373 fungicides, lignin-based dispersants 2: 193

fungus, wood-rotting 1: 201 fungus Z proteins 1: 373 furan 2: 19 – hydrophilic 2: 20 – polyesters 2: 44 furan commodity chemicals 2: 8 furan compounds 2: 16 furan derivatives 2: 98 furan-2,5-dicarboxylic acid (FDCA) 2: 38, 133 – basic biobased chemicals 1: 22 – synthesis 2: 134 furan polyamides 2: 46 furan resins 1: 154 furanoid sugar derivatives 2: 48 furfural 1: 6, 78, 91, 124–125, 154, 199, 2: 8, 121–127 – biomass building blocks 1: 22 – chemical structure 1: 143 – formation 2: 123 – history 2: 101 – lignocellulosic products 1: 25 – mass yield 1: 145 – production 1: 142 – yields 2: 102 furfural production 1: 125, 133, 139–164 furfuryl alcohol 1: 154 furfurylamines, conversion 2: 28 future biorefineries, lignocellulosic materials processing 2: 166 future development lines, sugars 2: 3–59

g galactanes 2: 108 gas-phase chlorination, photochemical 2: 269 gas to liquids see GTL gasification 1: 31, 85, 101, 123, 227, 2: 350 – air-blown 1: 232 – bioproducts 2: 361 – bran 1: 172 – coal 1: 157 – fundamentals 1: 230 – seperation of value components 1: 103 gasification-based systems, hybrid processing 1: 230–241 gasifier temperatures 1: 232 gasoline 1: 119 – replacement 1: 49 gasoline market, USA 1: 71 GBL 1: 149, 2: 373, 375 GEGVP, repulsive free energy 2: 237 gelatinization, starch 2: 78

477

478

Subject Index gelatinization temperature 2: 75 gene constructions 2: 225–227 – expressed protein-based polymers 2: 242–245 gene ladder 2: 229 – GVGVP 2: 226 gene technology, plant breeding 2: 275, 281 Genencor International 1: 74, 77 generation-I biorefinery 1: 19 generation-II biorefinery 1: 19 generation-III biorefinery 1: 19–20 genetic engineering 1: 374, 398 – ethanol production 1: 391 genetically engineered organisms, fermentation 2: 7 genetically modified crops 1: 96 geographical distribution, refineries 1: 57 geoporphyrins 2: 331 germ, corn refinery products 1: 348 GGAP, biocompatibility 2: 232 GH 1: 364 GH families, Trichoderma reesei 1: 373 Gibbs free energy, change 2: 218, 222, 232 – phase transition 2: 234 GJ see green juice glass fiber, resins 2: 185 GLNC, juice fraction 1: 274 global warming 1: 94 GLU see glutamic acid (GLU) glucan 1: 139, 2: 108 b-glucan 1: 175–176, 185 glucanases, biodegradation 1: 364 glucaric acid 2: 38 – basic biobased chemicals 1: 22 d-glucitol 2: 9 gluconic acid 1: 402 – biomass building blocks 1: 22 Gluconobacter oxydans 1: 80 glucosamine 1: 182 d-glucosamine, pyrroles synthesis 2: 25 glucose 1: 17, 2: 128–128 – electrolytic reduction 2: 130 – microbial conversion 2: 274 – nitric acid oxidation product 2: 38 – wood saccharification 1: 5 d-glucose – derivatives 2: 22 – one-pot conversions 2: 20 – reductive amination 2: 12 glucose fermentation, plant development 1: 134 glucose-product family tree 1: 21–22

d-glucose residues, Lolium perenne 1: 264 glucose yields 2: 177 b-glucosidase see BG glucosides 2: 130–133 5-(glucosyloxymethyl)furfural 2: 16 glutamate, markets 2: 207 glutamic acid (GLU) 2: 34, 304 – basic biobased chemicals 1: 22 gluten, corn refinery products 1: 348 glycanes 2: 108 glycerol 2: 271 – basic biobased chemicals 1: 22 – biocatalytic route 1: 393–397 glycine, chemical structure 2: 411 Glycine max 2: 277 glycolipids, microbial conversion 2: 274 glycoside hydrolase family classification system see GH glycosidic bonds 1: 140 GMF 2: 17, 25 gold catalysts 1: 403 grain 1: 45 grain wet-milling, biorefinery example 1: 70 grass – composition 1: 262–263 – key components 1: 260–269 – production costs 1: 284 grass fibers – basic properties 1: 281 – downstream processing 1: 281 – products 1: 282 grass press cake, major components 1: 279 grass silage juice, physicochemical characteristics 1: 276 grassland feedstocks, availability 1: 259– 260 gravity separator, acid hydrolysis 1: 145 green biorefiner concept 1: 253–294 green biorefineries 1: 19, 24, 29–31 – concept 1: 269–273, 296–297 – ecological aspects 1: 283–285 – economic aspects 1: 283–285 – products 1: 31 – raw materials 1: 258–269 green chemistry, chlorophyll 2: 325–343 green crop-drying plant 1: 270 green crops, industrial use 1: 9 green cycle, sugar cane industry 1: 210–211 green harvesting residue material 1: 258 green house gases 2: 402 green juice (GJ) 1: 30, 269, 271, 273–277 green leaf nutrient concentrate 1: 274

Subject Index green natural gas 1: 103 green pellets, production amount 1: 260 green plant material, composition 1: 258 green plant parts, fractionation 1: 256 greenhouse gas reduction 1: 62 greenhouse gases, emission 1: 197 gross visualization, phase separated product 2: 229 ground water pollution, brown juice 1: 298 growth, biorefining industry’s 1: 317 growth phase, fermentation 1: 218 GTL technology 1: 158 guerbet alcohols 2: 311 – synthesis 2: 312 guinea-pig, protein-based polymer injection 2: 231 gum arabic, substitute 2: 62 GVGIP 2: 240 – patches 2: 241 GVGVP 2: 223 – adhesions prevention 2: 238 – biocompatibility 2: 232 GVL 1: 151 gypsum 1: 106, 2: 87, 386

h Haarmann, W. 1: 7 hair, protection 2: 429–437 hair care 2: 434–436 – ilex resin 2: 439–440 hair conditioners, betaine derivatives 2: 414 hair-setting agent 2: 415–422 hair surface, cationic compound deposition 2: 413 hair swatches, standardized 2: 435 Hale, W. J. 1: 9 half esters, homocoupling 2: 268 Halobacterium sp NRC-1 1: 80 2-halocarboxylates, copper-initiated additions 2: 262–263 hammer mill 1: 176 hardwood, composition 2: 106 hardwood lignins 2: 154, 157 harvest cost 1: 333 harvest transport 1: 338–339 3-HBL see 3-hydroxybutyrolactone heating value, biofine char 1: 155 Helianthus annuus 2: 277 heliogerme 1: 179 hemicellulases 1: 364 hemicellulose 1: 72, 121, 198, 2: 104 – accessibility 1: 55

– chemical composition 1: 360 – feedstock content 1: 359 – history 2: 101 – hydrolysis 1: 362 – isolation 2: 119 – plant content 1: 261 – quantities 1: 264 – removal 1: 199 hemicellulose removal, advantages 2: 177– 179 hemicellulose-based product lines 2: 119 hemicellulose concentrations, forage grasses 1: 264 hemicellulose content, stem tissue 1: 265 hemicellulose polysaccharides, hydrolysis 1: 141 herbaceous species 1: 50 herbicidal treatment, highly selective 1: 150 herbicides 1: 149 – lignin-based dispersants 2: 193–194 heterocoupling, fatty acids 2: 267–269 heteropolymeric sugars, hemicellulose 1: 360 hexadecanedioic acid, yield 2: 274 hexose sugars 1: 78 HFCS 1: 351 HFRR 1: 153 high frequency reciprocating ring test see HFRR high-performance fiber 2: 41 high-value-added products, sugar-derived 2: 14–15 high value pharmaceuticals 2: 40 higher-value products, levulinic acid 2: 39 HMF (hydroxymethylfurfural) 1: 130, 133, 142, 2: 16, 133 – levulinic acid process 2: 100 – manufacture 2: 131 HMF based family tree 2: 135 Holly 2: 437–438 holocellulose change, wet storage 1: 336 homocoupling, fatty acids 2: 267–269 homofermentative strain, Lactobacillus salivarius 1: 307 hot-wash, pretreatment 1: 362 3-HPA 2: 34–35 HTU process, liquid biofuels 2: 351 Humicola grisea var thermoidea 1: 77 Humicola insolens 1: 375 Humicola 1: 202 hybrid processing, biomass 1: 227 hybrid thermochemical-biological processing 1: 227–252

479

480

Subject Index hydration – microbial 2: 272–273 – repulsive free energy 2: 237–238 hydrocarbons – apple-peel wax components 2: 433 – fossil 2: 6 – linear 1: 118 hydrochloric acid, carbon hydrolysis 1: 131 hydrogen bonding, cellulose 1: 360 hydrogenation reaction, syngas production 1: 230 hydrolases 1: 373–375 hydrolysability, biopol 2: 425 hydrolysis – biomass 1: 129–138 – furfural 2: 8 – hemicellulose 1: 362 – starch 1: 5 hydrolysis reactors, novel 1: 205 hydrolytic enzymes, costs 1: 105 hydrolytic liquefaction 1: 123 hydrolyzate, biomass 1: 78 hydrophilic imidazoles, d-fructose-derived 2: 27 hydrophilic side-chains 2: 24 hydrophilic/hydrophobic balance, sorbitan esters 2: 11 hydrophobic association, Gibbs free energy 2: 218, 232 – input energy 2: 219 – inverse temperature transition 2: 219 hydrophobic coating 2: 434 hydrophobic consilient mechanism 2: 222 hydrophobic effect, comprehensive 2: 237 hydrophobic hydration 2: 218 hydrophobic mechanism, protein-based polymer engineering 2: 217 hydrophobicity scale – Gibbs free energy 2: 234–235 – prosthetic groups 2: 235–236 hydrothermal conditioning, granular starch 2: 78 hydrothermolysis 2: 351 hydroxy cyclic ester 2: 398 hydroxyalkylation, starch 2: 80 3-hydroxybutyrolactone (3-HBL) 2: 38, 40 – basic biobased chemicals 1: 22 hydroxymethylfurfural (HMF) 1: 78, 2: 16, 133 – formation 1: 143 – hydration 1: 143 – lignocellulosic products 1: 25 3-hydroxypropionic acid (3-HPA) 2: 34–35

– basic biobased chemicals 1: 22 hydroxypropylation 2: 77 3-hydroxyvalerate see PHV

i IA see itaconic acid ideal elasticity 2: 219 – mechanism 2: 220 ift gene 1: 398 IgG 2: 230 Ilex aquifolium 2: 437–438 Ilex paraguariensis 2: 438 Ilex resin 2: 437 Ilex species 2: 437 imidazoles 2: 27 – hydrophilic 2: 27 immobilization 1: 389 – enzyme 1: 399–402 – lipase B 2: 256 immobilization technology 1: 392 immunoblot technique, western 2: 230–231 immunoglobulin G 2: 230 income generator 1: 96 inducing sugar 1: 201 industrial biobased products 2: 359 industrial biomass 1: 13 industrial bioproducts, opportunities 2: 357 industrial biotech 2: 445–462 industrial chemicals – biomass-derived 1: 124, 2: 347–365 – fossil sources 1: 120 – sustainable 1: 115–128 industrial concepts – biobased materials 2: 354–362 – biomass 2: 347–365 industrial feedstock, baling 1: 333 industrial product family, development 1: 18 industrial products, biomass-based 1: 87 industrial resources, historical 1: 4–8 industrial starch platform 2: 61–95 industrial uses, sugars 2: 7–14 industries, fossil carbon-processing 1: 42–44 infection, natural 2: 275 infrastructure investment 1: 340 inhibitors, enzyme activity 2: 322–323 initiators 2: 398 injection processes, PHB 1: 216 injections, histological sections 2: 231 innovation potential, fossil-based building blocks 2: 450

Subject Index insulation materials, resins 2: 185 integrated biorefinery applications 1: 125 integrated biorefinery process, detailed view 1: 102 integrated biorefining systems, sustainability 1: 56–65, 60–65 integrated process chain approach, biomass processing 1: 98 integrated processing facility 1: 45 integrated production, sugar 1: 209 intergeneric hybridization, plant breeding 2: 276 intermediate chemicals, HMF derived 2: 16 intermediate products, biorefineries 1: 46 intermediates 1: 386 – biofine process 1: 145 intermolecular order, proteins 2: 221 internal obstacles 2: 456 intracellular poly(3-hydroxybutyric acid) 1: 218–219 intracellular reserve material, PHB 2: 424 intramolecular cyclization, addition 2: 263 inulase II gene 1: 398 inulin 1: 267 – biocatalytic route 1: 397–402 – fructose source 2: 131 inulinase II, immobilization 1: 399 inverse temperature transition – hydrophobic association 2: 219 – protein-based polymers 2: 227 – purification 2: 228–229 investment costs, poly(3-hydroxybutyric acid) 1: 222–223 investments, biomass supplies 1: 318 Iogen’s demonstration process 1: 193 – schematic 1: 194 Iowa corn stover collection project 1: 319– 321 isoamyl alcohol, extraction of PHB 1: 219 isoascorbinic acid, glucose product family 1: 21 isolation, lignin 2: 116 isomaltulose, industrial production 2: 17 isosorbide dinitrate 2: 14 itaconic acid (IA) 2: 34, 36 – basic biobased chemicals 1: 22 – glucose product family 1: 21

j jet milling 1: 176 jetcutting 1: 401 juice fraction 1: 273–285

k Kevlar 2: 46 key chemicals, cellulose-based 2: 128 key intermediates – chlorophyll chemistry 2: 338 – HMF 2: 133 key sugars 2: 3–59 – exploitation 2: 14 Kirchhoff, G. S. C. 1: 5 Kirchoff 2: 62 Klebsiella pneumoniae 2: 36 Klebsiella 1: 206 kojic acid 2: 20 – glucose product family 1: 21 Kolbe electrolysis 2: 267–269 Kraft black liquor 2: 175 Kraft lignin, producers 2: 175 Kraft lignin recovery 2: 175 Kraft pulping 2: 175 Kraft pulping industry, lignin 2: 169–170 Kyoto objectives, dutch 1: 85

l LA see lactic acid, levulinic acid lactate ester, distillation 2: 389 lactic acid (LA) 1: 7, 11, 91, 2: 10–11, 382 – biomass building blocks 1: 22 – composition 1: 308 – fermentation 1: 11, 298, 303–305, 305– 306, 378 – glucose product family 1: 21 – manufacturers 2: 383 – production 1: 306, 312, 2: 382 – sources 1: 296 – usage 2: 10 lactide, polymerization 2: 392–396 Lactobacillus buchneri, 1,2-propanediol 2: 37 Lactobacillus delbrueckii 2: 210 Lactobacillus paracasei subspecies paracasei 1: 302 Lactobacillus plantarum 1: 302, 2: 273 Lactobacillus salivarius 1: 305 Lactococcus lactus 2: 210 laundry starches 2: 89–91 lauric oils 2: 292 Lb salivarius BC 1001, fermentation 1: 299–300 LCA, polylactic acid 1: 284 LCF see lignocellulosic feedstock LCF-mannan 2: 120 LCI, PLA 2: 402 leaf dyes, first production 1: 257–258

481

482

Subject Index leaf nutrient concentrate 1: 274 leaf protein concentrate 1: 254 learning from nature, bionics 2: 410 leaves, Ilex resin 2: 437 Leblanc, N. 1: 7 lecithin 2: 318 levoglucosan 1: 229, 243, 247 levoglucosan hydrolysis, alternative 1: 245 levoglucosenone 2: 21 levuglucosan, biomass building blocks 1: 22 levulinate esters 1: 153 levulinic acid (LA) 1: 6–7, 143–144, 2: 38–39, 134, 361 – basic biobased chemicals 1: 22 – bromination 1: 150 – catalytic hydrogenation 1: 151 – history 2: 100 – maximum theoretical yield 1: 145 – oxidation 1: 149 – production 1: 139–164, 2: 111 – reaction to diphenolic acid 1: 148 levulinic acid-based family tree 2: 135 Lewis acid-induced cationic addition 2: 264–265 life cycle analysis 1: 57 life cycle assessment see LCA life-cycle inventory 2: 402 lignin 1: 7, 72, 121, 195, 2: 104 – antioxidant 2: 187–189 – approximate composition 2: 154 – biodegradation 2: 160 – cell wall constituents 2: 151 – chemical composition 1: 360 – chemical linkages 2: 182 – commercially available 2: 176 – emerging markets 2: 194–198 – feedstock content 1: 359 – gasification 2: 118 – history 2: 101 – hydrolysis 2: 118 – Kraft pulping industry 2: 169–170 – markets 2: 166, 175, 181, 198 – organosolv biorefinery 2: 179–181 – plant content 1: 261 – press cake component 1: 279 – purified 2: 167 – pyrolytic 1: 241 – soda pulping industry 2: 170–172 – structural units 2: 105 – structure 2: 152–159 – utilization 2: 117 – water-soluble 2: 189–194

lignin-based product lines 2: 116–118 lignin chemistry, biomass conversion 2: 151–163 lignin content 1: 265 lignin isolation 2: 116 lignin polymer 2: 155 – growth 2: 154 lignin precipitation system 2: 171 lignin processing 1: 194, 205–206 lignin production – historical outline 2: 168–172 – industrial 2: 165–200 lignin products 2: 152 – existing 2: 172–177 lignin recovery process 2: 171 lignin removal, advantages 2: 177–179 lignin unit, different types 2: 156–159 lignocellulose 1: 74 – biorefinery 2: 111 – enzymatic sequence 2: 210 – history 2: 102 lignocellulose-based chemical products 2: 97–150 lignocellulose chemistry, historical outline 2: 98–99 lignocellulose structure 1: 121 lignocellulose utilization – industrial 2: 102 – technical aspects 2: 98 lignocelluloses 1: 10 – carbohydrates 2: 108 lignocellulosic, raw material 2: 103 lignocellulosic biomass, pretreatment 1: 361 lignocellulosic biorefineries, PLA 2: 403 lignocellulosic biorefinery 1: 115–128 – chemistry 1: 122–125 lignocellulosic feedstock (LCF) 1: 24, 125, 139–164 – biorefinery 1: 24–26, 129–138, 2: 111–113 – chemical composition 2: 106–108 – conversion methods 2: 113–115 – definition 2: 103 – major groups 2: 103 – sources 2: 105 lignocellulosic fractionation 1: 139–144 lignocellulosic materials 1: 45, 105 lignocellulosic technology, conventional 1: 146–147 lignosulfonate, dye dispersants 2: 191 lignosulfonate producers 2: 173–174 lignosulfonates 2: 168–169, 172–175 – markets 2: 174

Subject Index lignosulfonic acid, vanillin production 1: 7 linear hydrocarbons 1: 118 linear PLA, melt rheology 2: 397 linear polymer, idealized structure 2: 49 b-1 linkage, phenylpropane units 2: 159 b-b linkage, phenylpropane units 2: 157 b-O-4 linkage, arylglycerol units 2: 156 linseed 2: 281 Linum usitatissimum 2: 277 lipase-catalyzed syntheses 2: 270–272 – carbohydrate esters 2: 272 lipase-catalyzed transformations 2: 270– 272 lipid based bioproducts 2: 361 lipid layer enhancing effect, evaluation 2: 310 lipids 1: 7 – chemical composition 1: 361 lipotropic factor, betaine 2: 411 liquefaction 1: 123, 126 liquid biofuels 1: 49 liquid epoxy polyol esters 2: 298 liquid fuel production, missing part 1: 55 liquid transportation fuels 1: 46 LNC, composition 1: 274 load-and-go wagon 1: 320 local ownership, biorefinery 1: 56 Lolium hybridum, press cake fibers 1: 281 Lolium multiflorum – alkanes 1: 268 – silica 1: 268 Lolium perenne 1: 261, 264 – alkaloid production 1: 277 – alkanes 1: 268 – amino acid composition 1: 267 – antifreeze protein 1: 269 – fructans 1: 267 – minerals 1: 268 – silica 1: 268 – sugar 1: 265 – water-soluble carbohydrate 1: 266 low-cost production 2: 242 low nutrient conditions, succinate fermentation 2: 370 LPC 1: 268 – first industrial process 1: 256 – first production 1: 254–257 – quality 1: 258 LPS process 2: 171 lubricants, fatty acid esters 2: 299–301 lubricants industry, antioxidants 2: 188 lucerne, protein fractions 1: 275 lyocell 1: 90

lyondell propylene oxide, 1,4-BDO 2: 373 lysine – biomass building blocks 1: 22 – markets 2: 207 lysine fermentation 1: 11 lysine preparations, commercially available 2: 208 lysine yield, C. glutamicum 2: 210

m MAAP 2: 201–202 – ecological impact 2: 207 – environmental and commercial consideration 2: 205–209 – technical constraints 2: 209 MAAP processes – cultivation temperature 2: 213 – major steps 2: 208 – nitrogen source 2: 211 macrocycle 2: 334 macrocyclic ring system, reactions 2: 333 Madison-Scholler process 1: 132 Maillard reaction 2: 24 maize starch production 2: 66, 67 MALDI-TOF, polymer size 2: 229 maleic acid, conversion 2: 375 malic acid 2: 35 – glucose product family 1: 21 malonic acid, biomass building blocks 1: 22 maltol, glucose product family 1: 21 managing uncertainties, biotechnology 2: 459 mannan 2: 108 mannan/mannose product lines 2: 119 d-mannitol 1: 267 margarine 1: 7 Marggraf, A. S. 1: 5 market development, biotechnology 2: 460 market launch – apple-peel wax 2: 436–437 – biodegradable bottle 2: 427–428 market potential 2: 446 – LA 1: 147 – succinic acid 1: 149 market price, furfural 1: 154 markets, lignin 2: 198 mass spectra, polymer size 2: 229–230 material sources – biomass-based 1: 380 – renewable 2: 355 materials design 1: 108 matrices, natural fibers 2: 295

483

484

Subject Index matrix assisted laser desorption time-offlight spectrometry 2: 229 MBTE 1: 357 MDI 2: 299 mechanical pulping 1: 280 mechanical separation, cereals 1: 26 mechanistic foundations 2: 217–251 media cost 2: 370 Medicago sativa L 1: 255 – press cake fibers 1: 281 medical grade purity 2: 230 Mellier, M. A. C. 1: 6 Melsens, G. F. 1: 5 melt, polylactic acid synthesis 2: 391 melt rheology – branched PLA 2: 397 – linear PLA 2: 397 melt stability, PLA 2: 399 melting enthalpy, PLA 2: 401 membrane electrodialysis 1: 106 membrane process, lactic acid purification 1: 312 metabolic engineering 2: 204 metabolic flux distributions, C. glutamicum 2: 205 metabolic pathways – optimize 2: 212 – PHB synthesis 1: 238 – syngas fermentation 1: 234 metal-based catalysts 1: 228, 233 metal catalysts, aqueous phase hydrogenation 2: 375 metal complexes, chlorophyllin 2: 335 methanation, syngas production 1: 231 methane, glucose product family 1: 21 methanol, glucose product family 1: 21 methanol synthesis, syngas 1: 158 methyl 17-octadecanoate, cyclization 2: 259 methyl 2-iodopetroselinate, radical cyclization 2: 263 methyl conjugate, Diels-Alder reaction 2: 260–261 methyl elaidate, enantioselective oxidation 2: 258 methyl epiminooctadecanoate, synthesis 2: 257 methyl oleate – co-metathesis 2: 260 – oxidative cleavage 2: 258 methyl tertiary butyl ether 1: 357 1-methylamino-1-deoxy-d-glucitol 2: 11 methylene di(phenylisocyanate) 2: 299 methylglucoside, synthesis 2: 131

methyltetrahydrofuran see MTHF Michael addition 2: 81 Michaelis-Menten constant 1: 77 Michaelis-Menten kinetics, enzymatic hydrolysis 1: 204 microbial activity, wet storage 1: 334 microbial amino acid production see MAAP microbial bioconversions, milling byproducts 1: 172 microbial biomass 1: 106 microbial biosynthesis 1: 104 microbial conversions – oils/fats and glucose 2: 274 – six-carbon sugars 2: 32–34 – sugar 2: 30 – sugar-based 2: 36–37 microbial fermentation, drying bales 1: 321 microbial oxidation, fatty acids 2: 273–274 microbial polyesters 2: 44–45 microbial transformations 2: 272–274 microfibril 1: 195 microorganisms – acetyl-coa forming 1: 233 – commercial lactic acid production 2: 384–385 – important 1: 80 – usage 1: 146 middlings 1: 170 mill water 1: 348 milled wood lignin 2: 155 milling – industries 1: 345–353 – pretreatment 1: 361 – process flow diagrams 1: 347 milling byproducts, wheat flour 1: 169–173 milling efficiency, increase 1: 173 milling operations 1: 166 minerals 1: 268 – analysis 1: 299 – brown juice content 1: 304 Mitscherlich, A. 1: 8 mix-polymerization, starch 1: 27 mixed sugars 1: 98 model building block, succinic acid 2: 367–379 modeling, ecosystem 1: 57–60 modern corn refinery 1: 348–350 molasses 1: 222 molasses fermentation 1: 131 mold temperature, PHB 1: 216 molecular weight, rheology control 2: 396

Subject Index monitoring technologies, toxicity 2: 212 mono-septic operation 2: 209 monocyclic aromatic hydrocarbons, biorefinery by-product 2: 29 monoglycerides, lipase-catalyzed syntheses 2: 270–272 monomer genes – concatenation 2: 226 – preparation 2: 225 – production 2: 226 monomers 1: 46 – biorefinery 2: 315 – quasi-aromatic 2: 46 monosaccharide production 1: 180 monosaccharides – availability 2: 4–7 – conversion 2: 28 MSW Management, coupling with fuel production 1: 126 MTBE 1: 71 MTHF 1: 150, 2: 135 – formation from LA 1: 152 mulch till 1: 329 Mulder, G. J. 1: 6 multi-cyclic carbonate comonomers 2: 398 multi-cyclic epoxy comonomers 2: 398 multi-cyclic ester comonomers 2: 398 multi-functional polymerization initiators, branching 2: 398 multi-quality biomass 1: 92 multifunctional care additives 2: 309 multifunctional compounds 2: 135 multimer genes 2: 226 multiple feedstock capability 1: 68 municipal solid waste 1: 116–117 – management 1: 125 mutagenesis 1: 75–77 mutated spores, fermentation 1: 75 MWL 2: 155

n N-heterocycles, sugar-derived 2: 24 Naegeli 2: 62 naltrexone, controlled-release devices 2: 240 naphtha 1: 86 naphthalene sulfonate, dye dispersants 2: 191 naphthenic compounds 1: 118 National Farm Chemurgic Council 1: 9 National Renewable Energy Laboratory process see NREL natural lignin, recovery 2: 179

natural fibers 1: 90 natural oils – formaldehyde additions 2: 264 – improvements 2: 275–281 – industrial processing 2: 295 – polymer building blocks 2: 296 natural substance, definition 2: 422 natural vector transformation systems 2: 275 NatureWorks 2: 10 near ideal elasticity 2: 219 – mechanism 2: 220 net corn cost 1: 50 network polymer, lignin 1: 121 neutralizing agent, lactic acid 2: 385 nitrocellulose, history 2: 99 nitrogen leaching 1: 63 nitrogen source 2: 211 NMGA 2: 11–12 NMP 2: 373, 375–376 – rhodium catalytical production 2: 377 NMR, purity 2: 229 Nocardia cholesteriolicum 2: 273 non-carbohydrate natural products, synthesis 2: 20 non-food products – manufacture 1: 165–191 – renewable resources 1: 11 non-food uses, sugars 2: 3–59 non-recyclable organic solid waste materials see NROSW non-starch polysaccharides 1: 175 non-wood fibers 1: 280 nonactivated C–H bonds, oxidation 2: 269 nonhydrolytic proteins 1: 374 nontraditional microorganisms 1: 80 Normann, W. 1: 7 novel fatty acids synthesis, starting materials 2: 255 novel plastics, 1,3-propanediol 1: 182 novolacs 2: 181 novozym 435 2: 256 novozymes 1: 77 NREL 1: 19, 22, 72, 74, 150 NROSW 1: 126 NSP 1: 175 nuclear magnetic resonance 2: 229 nucleophilic addition, unsaturated fatty acids 2: 265 nucleus exchange method, lignin 2: 154 nutrient replacement 1: 324–325 nutrients 2: 388 – lactic acid 2: 385

485

486

Subject Index – oat 1: 183 – replenishment 1: 327 – wheat 1: 168 nutritional value, re-growth 1: 261 nylon 6, markets 2: 113 nylon-6,6 2: 45 nylon process, furfural-based 2: 123

o oat based biorefinery, schematic 1: 184 oat bran-rich fractions, value-added byproducts 1: 185–187 oat composition 1: 183 oat gum 1: 185 occurrence, betaine 2: 410–411 OFP 1: 151 oil 1: 98 – microbial conversion 2: 274 – new syntheses 2: 253–289 – thermochemical conversion 2: 361 oil and lipid-based bioproducts 2: 356 oil-based surfactants 1: 90 oil crisis 2: 348 oil fruits, FAS 2: 304 oil industry, sections 1: 86 oil-like proteins, repulsion 2: 218 oil production, permanent decline 1: 42 oil qualities 2: 276–277 oils and fats, world production 2: 292 oilseeds 1: 45 olefin, metathesis 2: 259–260 olefinic polymers, sugar-based 2: 47 olefins, monosaccharide-derived 2: 47 oleic acid 2: 254 oleochemical base materials 2: 294 oleochemical-based dicarboxylic acids 2: 296 oleochemical industry 1: 122 oleochemicals – biobased 2: 291–314 – polymer applications 2: 295 one-pass collection 1: 333–335 one-pass harvest 1: 332 one step biochemical modification, naturally produced structures 2: 349 one-way cycle, oil and gas feedstock 2: 449 OP 2: 395 – PLA 2: 401 operating costs – biofine plants 1: 160 – biorefinery 1: 240 optical purity 2: 395 organic acids 1: 78

– analysis 1: 299 – brown juice content 1: 304 – commercially important 1: 79 – production 1: 234–235 organic chemicals – bioproduction 1: 182 – fossil sources 1: 120 – industrial 1: 115–128, 124 – levulinic acid 2: 134 – renewable carbon 1: 44 organisms, engineered 1: 68 organization infrastructure 1: 340 organosolv biorefinery, lignin 2: 179–181 organosolv lignin, products 2: 183 organosolv pretreatment, lignin 2: 178 oseltamir phosphate, synthesis 2: 30 oxalic acid 2: 99 oxidation 2: 254–258 – enantioselective 2: 258 – fatty compounds 2: 257–258 – selective 2: 38 – starch 2: 79 b-oxidation, fatty acids 2: 273–274 x-oxidation, fatty acids 2: 273–274 oxidation technology, development 2: 38 oxidative cleavage 2: 258 – transition metal-catalyzed 2: 258 oxidative coupling 2: 266 oxidative enzymes, biodegradable plastics 1: 213 oxidative metabolism, phosphorus supply 2: 211 oxidative polymerization, lignin polymerization 2: 153 oxidative states, changes 2: 236 4-oxopentanoic acid 1: 6 oxygen supply 2: 212 ozone, cleavage of fatty compounds 2: 258 ozone-forming potential, P-Series fuels see OFP

p P-Series fuels 1: 151 Pachysolen tannophilus 1: 147 Pacific Northwest Laboratory see PNL Pacific Northwest National Laboratory see PNNL palm kernel oil 2: 292 panel binders 2: 185 panelboard adhesives 2: 183–184 paper – adhesion 2: 87 – from press cake fibers 1: 282

Subject Index paper industries, starch usage 2: 83 paper mill waste 1: 134 parasorbic acid, glucose product family 1: 21 partial glycerides 2: 270 particle size, feedstock materials 1: 144 paste reactions, starch modifications 2: 77 pasture lands 1: 52 patents – protein-based polymers 2: 245–249 – reexamination request 2: 245–249 Payen, A. 1: 6 PC 1: 30, 269, 271 – downstream processing 1: 281 PCB, lignin containing 2: 194 PCR technique 2: 225 PCS-hydrolyzing cellulases, improvements 1: 367 PD 1: 393–397 PDLA 2: 395 PDO 1: 11 peanut 2: 281 pearl corn starch, carbohydrate refining 1: 351 pearling 1: 173–176 – oat grain 1: 183 pectin substances 1: 265 Penicillium 1: 202 pentaerythritol esters 2: 308 2,3-pentane dione, glucose product family 1: 21 “pentanes-plus” 1: 151 pentosan change, wet storage 1: 336 pentosans, conversion 2: 28 pentose fermentation 1: 206 pentose sugars 1: 78 pentoses 1: 91 – conversion 2: 28 peptide sequences, repeating 2: 217 Peptostreptococcus productus 1: 235 perfluoroalkyl iodides, addition 2: 263–264 perfluoroalkylated products, synthesis 2: 263 performic acid procedure 2: 254 pericarp 1: 183 – wheat 1: 167 pericyclic reactions 2: 260–261 pesticides, lignin-based dispersants 2: 193 PET 2: 133 petrochemical industry 1: 86 – transformation steps 1: 88 petrochemical technology 2: 373 petroleum

– dependence 1: 115 – structural shift 1: 116 petroleum-based pathways, polyamides 2: 45 petroleum chemistry, comparison with biomass 1: 118–122 petroleum costs 1: 48–50 petroleum dependence, reduction 1: 71 petroleum feedstocks 1: 45 petroleum refineries 1: 16 petroleum refining industry, development 1: 41 petroleum reserves, prognoses 1: 387 petroporphyrin formation 2: 332 petroporphyrins 2: 331–332 PF 2: 181 PF resins, markets 2: 183 pH adjustment 1: 79 PHA 1: 182, 214, 236, 239, 2: 44 – accumulation 1: 236 pharmaceuticals 1: 13, 2: 14–15 – intermediate 2: 40 – preparation 2: 26 – purification target level 2: 230 – starch usage 2: 88–89 phase III-biorefineries 1: 19–20 phase separated product, gross visualization 2: 229 phase separation, purification 2: 228 phase transition, Gibbs free energy 2: 234 PHB 1: 209, 238 – chemical structure 1: 214 – copolyesters 1: 215 – intracellular reserve material 2: 423 – lifetime of products 1: 214 – synthesis 1: 237–238 – yield determination 1: 238 PHB-PHV copolymer, brittleness 2: 426 phenol–formaldehyde resin 2: 181 phenol–formaldehyde resin markets, lignin 2: 187 phenolic acids 1: 178 phenolic–carbohydrate complexes, Lolium perenne 1: 264 phenolic molding compound market 2: 184 phenolic resins 2: 16, 181, 185 – biorefinery lignin 2: 181–183 phenomenological axioms, engineering protein-based polymers 2: 232–234 phenyl-propanoid units, crosslinked 2: 181 phenylpropane units 2: 157, 159 – bonding 2: 153–156

487

488

Subject Index phloroglucinol, biosynthesis 2: 30 phospholipids 1: 361 phosphorus source 2: 211 phosphorylation, changes 2: 236 photochemical gas-phase chlorination 2: 269 photodynamic therapy, chlorophyll derivatives 2: 336 photosensitizer, chlorophyll 2: 334 photosynthesis 1: 12, 42 photosynthesis enzyme, plant content 1: 255 photosynthetic bacteria 1: 229 photosynthetic pigments 1: 257, 2: 326 phthalo green 2: 338 PHV 1: 237, 2: 426 phylogenetic tree, gene sequences 1: 367 phytic acid 1: 170 phytochemicals, biorefinery context 2: 315–324 phytoestrogens 2: 321–322 phytosterols 2: 317–318 Pichia yeast 1: 206 Picrophilus torridus 1: 80 pigments – biorefinery context 2: 315–324 – carotenoids 2: 320 – chlorophyll 2: 336 pilot plants, biomass fermentation 1: 135 Pirie, N. W. 1: 9 PLA 2: 10–11, 41, 381 – biobased 1: 296–299 – high polymer 2: 391 – melt rheology 2: 397 – production 2: 390–396 – properties 2: 400 – resins 2: 400 – semi-crystalline 2: 394 – stereocomplex 2: 401 plant breeding, oil improvement 2: 275–281 plant cuticle, schematic 2: 431 plant development, biomass hydrolysis 1: 129–138 plant infection, fungal endophytes 1: 277 plant material – usage 1: 90 – yearly amount 1: 43 plant resources 2: 353 plant usage, historical 1: 254 plasma cholesterol, reduction 2: 317–318, 321 plasticization, starch 1: 27

plasticizer, biodegradable bottle 2: 427 plastics – biodegradable 1: 182, 212–216 – novel 1: 182 platform chemical 1: 147 platform molecules 1: 182 PLLA 2: 395 plug-flow reactor 1: 144 PNL process 1: 151–152 PNNL 1: 22 poly(3-hydroxybutyric acid) polymer 1: 214–216 poly(hydroxyalkanoate) production, future milestone 1: 224 poly(hydroxyalkanoates) 1: 238, 2: 44 poly(3-hydroxybutyrate-co-valerate), enzymatic digestion 1: 219 poly(hydroxybutyrate) – monomer 1: 237 – processing 1: 215–216 – switchgrass 1: 283 poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) 1: 214–215 poly(3-hydroxybutyric acid) 1: 212–213 – biosynthesis 1: 224 – downstream processing 1: 218–219 – production process 1: 217–223 – sugar fermentation 1: 217 poly-b-hydroxy butyric acid 2: 423 poly(lactic acid) 1: 8, 296 poly(tetramethylene ether glycol) see PTMEG poly(vinyl chloride), cements see PVC polyamides 1: 122, 2: 45–47 polyesters – fiber 2: 36 – furan containing 2: 44 – microbial 2: 44–45 – production 1: 236–239 polyetherpolyols, biodegradable 2: 9 polyglucaramides, stereoregular 2: 46 polyglycerol ester, emulsifier 2: 308 polylactic acid 2: 10, 41 – non-solvent process 2: 392 – polymerization routes 2: 391 – production capacity 1: 284 – renewable resources 2: 381–407 polymer building blocks, natural oils 2: 296 polymer development, protein-based 2: 221–222 polymer industry, starch 1: 28 polymer size, mass spectra 2: 229–230

Subject Index polymerase chain reaction 2: 225 polymeric materials, protein-based 2: 220–221 polymeric products, polymeric lignin 2: 160 polymerizable sugar derivatives 2: 40–47 polymerization initiators, multi-functional 2: 398 polymers 1: 46 – biobased 1: 11 – oils and fats 2: 291 – oleochemicals 2: 295 – PHB 1: 209 – protein-based 2: 217–251 polyol esters 2: 307 polyols, epoxides based 2: 298–299 polyoses, history 2: 101 polypeptide, protein definition 2: 217 polysaccharides 1: 89, 121, 277 – acid hydrolysis 1: 141–142 – repeating units 2: 4 polytrimethyleneterephthalate see PTT polyurethane foams, production 2: 9 polyurethane stretch fibers 1: 149 polyurethanes, oleochemical building blocks 2: 298 polyvinylsaccharides 2: 47 pomace 2: 432 porous carbon fibers 1: 283 potato 2: 69–70 potato juice 1: 309–310 – lactic acid source 1: 295 – quality 1: 300 potato starch crystals 2: 73 potato starch industry, lactic acid producer 1: 310 potato starch production 2: 69 potential future market, formic acid 1: 154 potential screening 1: 22–23 power technologies, renewable 1: 139 precursors, biomass targets 1: 17 preprocessing, biomass 1: 46 press cake 1: 257, 269 press cake fibers, basic properties 1: 281 press-cake fraction 1: 278–285 press juice 1: 257 pressure ulcers, prevention 2: 240 pretreatment 1: 135, 198–200 – biomass 1: 107 – cornstover 1: 245 – dilute acid 1: 246 – LCF 2: 113 – lignocellulosic biomass 1: 361

– solvent-based 1: 200 – straw 1: 193 price changes, external challenges 2: 457 price difference, fossil fuel feedstocks 2: 446 price swings, oil 1: 48, 52 prices, renewable carbon feedstock 1: 50 primary antioxidants 2: 187 primary conversion technologies 1: 270, 2: 350 primary refinery 1: 269, 272 – wet fractionation 1: 271–273 primary starch 2: 85 primary streams, raw materials 1: 92 prime starch 2: 68 printed circuit boards 2: 194 process economics 1: 53 process optimization, lignocellulose-based operation 2: 203 process scheme, integrated 1: 127 processing technologies, neccessary for biorefineries 1: 46 processor value, stover 1: 327 product development, biodegradable bottle 2: 426–427 product diversification, biomass 1: 54–55 product family tree 2: 97–150 – amino acid-based 2: 201–216 – glucose 1: 21–22 – hemicellulose-based 2: 121 – HMF and levulinic acid-based 2: 136–138 – lignin-based 2: 117–118 – syngas 1: 33 product flow-chart, biobased 1: 23 product innovation, biotechnology 2: 450 product integrity, verification 2: 229–230 product lines – biobased 1: 375 – carbohydrate-based 2: 3–59 – hemicellulose-based 2: 119 product spectrum, chemical compounds 1: 105–106 product yield 1: 53–54 prognoses 1: 387 propanediol, glucose product family 1: 21 1,2-propanediol, racemic form 2: 37 1,3-propanediol 2: 36 propionic acid, biomass building blocks 1: 22 propylene, glucose product family 1: 21 prosthetic groups, hydrophobicity scale 2: 235 protease inhibitors 2: 322

489

490

Subject Index protective action, hair keratin 2: 436 protective film, skin care 2: 439 protein-based polymers 2: 217–251 – charged side chains 2: 240 – development 2: 221–222 – elasticity 2: 219–220 – engineering 2: 217, 232–238 – expression 2: 227–230 – gene constructions 2: 242–245 – materials 2: 220–221 – order 2: 219 – patents 2: 245–249 – purification 2: 227–230 protein content, comparison between plants and animals 1: 255 protein–fatty acid condensates 2: 304 protein gels, cellulolytic fungi 1: 372 protein generation, ethanol 2: 133 protein-hydrolyzates, extraction 2: 202 protein line 2: 201–216 protein repulsion 2: 218 protein–xanthophylls 1: 271 proteins 1: 46, 122, 268, 277, 2: 217 – acylated 2: 304 – analysis 1: 299 – aqueous media 2: 218 – biomass conversion 1: 371–375 – brown juice content 1: 305 – chemical composition 1: 361 – crude starch milk 2: 66 – juice fraction 1: 274 – potato starch production 2: 70 – thermodynamics 2: 218 protolignin 2: 152 protonated glycoside, cellulose hydrolysis 1: 141 Pseudomonas fluorescens 2: 30 Pseudomonas putida 1: 245, 2: 37 PTMEG 1: 149 PTT production 1: 393–396 pulp 1: 6 pulping – environmentally friendly 2: 179 – mechanical 1: 280 – semi-chemical 1: 280 pure chemicals, xylan derived 2: 122 purification 1: 218–219, 2: 388 – chitin 2: 416–417 – inverse temperature transition 2: 228– 229 – lactic acid 1: 312 – phase separation 2: 228 – PHB 1: 218

– protein-based polymers 2: 227–230 purity – evaluation 2: 229 – medical grade 2: 231 PVC 1: 149 PVC stabilizers, vegetable oil epoxides 2: 256 PX (protein-xanthophylls), production numbers 1: 271 pyran, building blocks 2: 21 pyranoid, building blocks 2: 23 pyranoid sugar derivatives 2: 48 pyrazoles 2: 26 3-pyridinols 2: 28 pyrogallol 2: 30 pyrolysis 1: 123, 230 – bioproducts 2: 362 pyrolysis oil 1: 98 – production 1: 244 pyrolysis products, cornstover 1: 246 pyrolytic char 1: 247 pyrolytic lignin 1: 241 pyrolytic liquid, yield 1: 241 pyrones 2: 20 pyrroles 2: 24 pyrrolidone solvents, manufacture 1: 149

q quasi-aromatic monomers, sugar-based 2: 46 quinoxalines 2: 28 – sugar derivative 2: 24

r racemic mixture, lactic acid 2: 382 radical additions 2: 261 – malonic acid 2: 261 – perfluoroalkyl iodides 2: 263 radical C–C coupling 2: 266–269 radical cyclization, methyl 2-iodopetroselinate 2: 263 rail transport, feedstock 1: 339 Ralstonia eutropha 1: 217, 237 ranitidine 2: 14 rapeseed 2: 277 rapeseed oil, industrial use 2: 280 rapid pyrolysis 1: 227 – cellulose 1: 243 rate of biodegradation 1: 213 raw material costs 1: 53 raw materials – appropriate 1: 165 – aromatic compounds 2: 259

Subject Index – biomass 1: 12–14 – biorefineries 1: 45–47 – costs 2: 110 – oleochemicals 2: 292–293 – renewable 2: 253–289 – sterilization 2: 209 – world market prices 2: 356 RBAEF Project 1: 43, 52 reaction system costs 1: 53 reactive sites, triglycerides 2: 294 recombinant DNA technologies – application 2: 204 – gene construction 2: 225–227 – protein-based polymers 2: 217 recovery 1: 218–219 recycling 1: 106 – plastics 1: 212 refined biomass 1: 98 refineries – hybrid biomass processing 1: 227–252 – thermochemical 1: 101–103 refinery economy 1: 350 refining, biomass 1: 41–66, 107 reformulated gasoline see RFG regioselective syntheses, ricinoleic acid 2: 254 re-growth, nutritional value 1: 261 regulatory framework, biotechnology 2: 447 regulatory situation, external challenges 2: 457 reinforcers, lignin 2: 186 renewable carbon feedstock prices 1: 50 renewable energy law 1: 15 renewable material, definition 2: 422 renewable-power technologies 1: 139 renewable raw materials 2: 355 – oils and fats 2: 253–289 – optimized by breeding 2: 277–281 renewable resources 2: 347 – industrial conversion 1: 5 – integrated utilization 1: 10–11 – non-food products 1: 11 – polylactic acid 2: 381–407 – prognoses 1: 387 – sources 1: 385 repulsive free energy 2: 222 – apolar–polar 2: 218 – hydration 2: 237 residual biomass, importance 2: 452–457 residual sugars, separation 2: 388 residue utilization 1: 283–285 resin binders 2: 183

resin fraction, holly 2: 438–439 resin industry 2: 182 resins – PCB 2: 194 – thermoset 2: 184 resols 2: 181 retroaldolization, imidazoles 2: 27 retrogradation 2: 75 reversed-polarity unsaturated fatty acids, nucleophilic addition 2: 265 RFG 1: 151 rheological properties – b-glucan 1: 185 – NSP 1: 175 rheology control 2: 396 Rhizomucor miehei 2: 271 Rhizopus arrhizus 2: 35 rhizopus-based fermentation 2: 388 rhodium catalyst, NMP production 2: 377 Rhodopseudomonas gelatinosa 1: 237 Rhodospirillum rubrum 1: 237, 239 Rhodospirillus rubrum 1: 229 ribulose 1: 255 rice, starch production 2: 71 rice straw, world production 1: 51 ricinoleic acid 2: 254 ridge-till 1: 329 right opportunities, biotechnology 2: 458 ring-opening – chlorophyll 2: 330 – nucleophilic 2: 257 ring-opening polymerization 2: 394 ring structures, saccharides 1: 121 rings, lignin polymer 2: 155 Ritter, E. A. 1: 323 rocket fuels 2: 37 Role of Biomass in America’s Energy Future see RBAEF Project ROP 2: 394 Rothamsted process 1: 256 Roulle, H. M. 1: 254 rubber industry, antioxidants 2: 188 rubber processing, lignin 2: 186 rubisco 1: 274 – plant content 1: 255 Rubrivivax gelatinosus 1: 236–237 rye grasses, digestibility 1: 261 ryegrass, fiber properties 1: 281

s saccharides 1: 121 saccharification 1: 32, 2: 128, 177–179 – cellulose 2: 99

491

492

Subject Index – wood 1: 5–6 Saccharomyces 1: 194 Saccharomyces cerevisiae 1: 7, 146, 2: 209 Saccharomyces yeast 1: 206 saccharose, fructose source 2: 131 Saccharum officinarum 1: 210 salt splitting technology, lactic acid 2: 387 saponins 2: 321–322 satake pearling system 1: 174 saturated fatty compounds, reactions 2: 266–270 SAXS, lamellar thickness 2: 395 scaffoldings, temporary functional 2: 239 scenarios, intgeration of industries 1: 94 Scholler process 1: 131 Scholten, W. A. 2: 62 screening 1: 22–23, 77 screening methods 1: 75–76 scutellum, wheat 1: 169 SDS–PAGE 2: 228 – purification 2: 228 SEC 2: 72 second-grade starch 2: 68 secondary biochemical refinery 1: 104–106 secondary biorefining processes, thermochemical 1: 103 secondary starch secondary streams, raw materials 1: 92 secretome, cellulolytic fungi 1: 371–373 sectoral integration, bio-based industry 1: 93–96 seed, wheat 1: 167 selective oxidation, carboxylic acids 2: 38 self-leveling concrete 2: 87 semi-chemical pulping, lignin extraction 1: 280 separation of biomass, technically feasible 1: 17 separation system costs 1: 53 sequence integrity, evaluation 2: 229 sequence verification 2: 226 sequenced genomes, microorganisms 1: 80 sequestration, carbon 1: 62 serine, biomass building blocks 1: 22 shampoo bottle – Biopol 2: 422 – degradation 2: 425 – market launch 2: 427 Shell, transition process 1: 93 shellfish industry, chitin source 2: 416 shikimic acid, metabolic engineering 2: 31 side-chain, oxidativ shortening 2: 25 side-streams, fermentation 1: 106

signal peptide effect 1: 375 silage additive, formic acid 1: 153 silage juice 1: 276–277 silage residues, reusage 1: 283 silage wet-fractionation, primary refinery 1: 271 silica 1: 268, 277 silicon carbide, rye grass 1: 278 simultaneous saccharification and fermentation 1: 134 sitosterol 2: 317 six-carbon sugars, microbial conversion 2: 32–34 sixth framework program, EU 1: 103 size-exclusion chromatography 2: 72 sizing agents 2: 85 skin, protection 2: 429–437 skin care, ilex resin 2: 439 skin cosmetics 2: 434 slaughterhouse wastes, byproducts 1: 100 slurry process, starch modifications 2: 76– 78 small-angle X-ray scattering 2: 395 small-scale extractions, chlorophyll 2: 329 smell, obnoxious 1: 310 soap 2: 409 soap production, history 1: 7 soda process, lignin 2: 176 soda pulping industry, lignin 2: 170–172 sodium dodecyl sulfate polyacrylamide gel electrophoresis 2: 228 sodium lactate, salt splitting 2: 387 soft tissue augmentation 2: 238 soft tissue reconstruction 2: 239 soft tissue restoration 2: 238 softwood, composition 2: 106 softwood lignins 2: 157 soil bioactivators, grass juices 1: 283 soil carbon equilibrium 1: 325 soil carbon loss 1: 328 soil coverage, stover 1: 329 soil erosion control 1: 329 soil organic material 1: 328–329 soil organic matter see SOM soil quality 1: 324 – models 1: 324 solid state bioprocessing see SSB solubilization, cellulose 1: 359 solubles removal, wet storage 1: 336 solvent, selective 2: 9 solvent extraction 1: 219–221, 2: 388 SOM, loss 1: 324, 328 sorbic acid, glucose product family 1: 21

Subject Index sorbitan esters 2: 11, 272 sorbitol 2: 129–130 – basic biobased chemicals 1: 22 d-sorbitol 2: 9 – dehydration 2: 11 Sorghum dochna 1: 255 sorona 2: 41 sovermol 2: 299 soybean 2: 277 – phytochemicals 2: 317 – processing 2: 316 – saponins 2: 321 spandex 1: 149 special ingredients 2: 315–324 special sugars, juice fraction 1: 274 specialties 1: 386 specialty chemicals, bio-based 1: 91 Spirulina, chlorophyll extraction 2: 329 spores, mutated 1: 75 SSB, fungal 1: 172 SSF 1: 71, 146, 203 – process 1: 79 starch 1: 27, 67, 70–71, 121, 181, 268 – acetylated 2: 80 – bioconversion 2: 89–91 – chemical composition 1: 360 – chemical source 1: 50 – commercial 2: 71–76 – common sources 2: 62 – composition 2: 74 – corn refinery products 1: 348 – degraded 2: 79 – ethanol raw material 1: 197–198 – glucan source 1: 139 – history 2: 61 – industrial production 2: 65 – modification 2: 61–95 – nitric acid oxidation product 2: 38 – production 2: 61–95 – properties changes 2: 78 – quality 2: 73 – raw materials composition 2: 65 – syrups 2: 89 – tailor-made 2: 92–93 – total consumption 2: 82 – world market 2: 64 – yield 2: 69 starch-based biorefinery II 1: 69 starch derivatives 2: 82–91 starch ethers, building chemistry 2: 87 starch–gluten slurry, corn refinery 1: 349 starch granules, reshaping 2: 75 starch hydrolysis 1: 5

starch modification, types 2: 81 starch modification technology 2: 76 starch platform, industrial 2: 61–95 starch saccharification 2: 178 starch water, modification 2: 76–81 steam 1: 46 steam-alkaline pulping, lignocelluloses 2: 114 steam explosion 1: 280 – pretreatment 1: 198 steam gasification 1: 156 stearic acid, photochemical gas-phase chlorination 2: 269 steep liquor 1: 349 steeping, corn 1: 348 steepwater 1: 349 stereoregular polyglucaramides 2: 46 stereoselective syntheses, ricinoleic acid 2: 254 steric hindrance, enzymatic hydrolysis 1: 147 sterigel 1: 178 sterols, soybeans 2: 317 storage 1: 334 – bagasse 1: 321 – baling dry material 1: 332 – brown juice 1: 298 – potato juice 1: storage area, square bales 1: 335 storage investment cost 1: 337 storage loss 1: 335 storage polymer, PHA 1: 239 stover, economic benefit 1: 325 stover field value 1: 326 stover revenue, farmers income 1: 319 strain development 2: 371 straw – baling 1: 333 – LCF biorefinery 1: 26 – world production 1: 51 straw species 2: 107 straw waste, wood saccharification 1: 10 Streptomyces setonii 1: 179, 245 strip till 1: 329 strong acid addition, lactic acid 2: 386 structural features, lignin 2: 155 structure-based design, enzyme improvement 1: 369 structure–function relationship, EG 1: 370 styling, ilex resin 2: 440 substance classes, apple-peel wax 2: 433 substrate recalcitrance 1: 204 succinate fermentation 2: 369

493

494

Subject Index succinate strain FZ 21 2: 371 succinic acid 1: 149, 2: 35 – catalytic transformations 2: 372 – conversion 2: 375 – derivatives 2: 373 – fermentation 1: 378, 2: 369–372 – model building block 2: 367–379 – wheat flour milling byproducts 1: 172 sucrose 1: 70 – catalytic oxidation 2: 39 – conversion 2: 18 – ethanol raw material 1: 197–198 sucrose-6,6'-dicarboxylic acid 2: 46 sucrose-based biorefinery I 1: 68 sucrose fatty acid monoesters 2: 13 sugar 1: 209 – analysis 1: 299 – bulk-quantity prices 2: 4 – chemical conversion 2: 37–40 – contained in biomass 2: 3–59 – fermentable 1: 68 – fermentation problems 1: 146 – increasing demand 1: 67 – juice fraction 1: 274 – mixed 1: 98 – non-food industrial uses 2: 7–14 – nonionic surfactants raw materials 2: 306 – plant contents 1: 265 – simple 2: 5 – thermochemical conversion 2: 360 – yield 1: 130 sugar acids, chemical route 1: 402–405 sugar and starch bioproducts 2: 356 sugar-based biorefinery 1: 209 sugar-based chemicals 2: 14 sugar-based olefinic polymers 2: 47 sugar-based surfactants 2: 11–12 sugar beet 2: 410 sugar biorefinery 1: 70 sugar cane industry, Brazil 1: 209–211 sugar cane processing 1: 211 – steps 1: 222 sugar composition, dependence on harvesting time 1: 265 sugar content, brown juice 1: 303 sugar conversion, efficiency 1: 136 sugar crops 1: 45 sugar derivatives 2: 48 – polymerizable 2: 40–47 sugar feedstock, carbohydrate sources 2: 385 sugar fermentation 1: 194, 206–207 – poly(3-hydroxybutyric acid) 1: 217

– sulfite pulp process 1: 8 sugar mill – Brazil 1: 210 – poly(3-hydroxybutyric acid) production 1: 221 sugar platform 1: 32 – intermediate 1: 31 sugar production 1: 5 sugar residues, Lolium perenne 1: 264 sugar syrup 1: 222 sugar transformations, prototype 2: 19 sugarcane bagasse 1: 74 sulfite pulp process, historical improvement 1: 8 sulfite pulping industry 2: 189 – lignosulfonates 2: 168–169 sulfite pulping process 2: 172 Sulfolobus sulfataricus P2 1: 80 Sulfolobus tokodaii strain 7 1: 80 sulfonated Kraft lignin, dye dispersants 2: 191 sulfur, lignin structure 2: 171 sulfur-bearing gases, catalyst poisoning 1: 234 sulfur-containing components, removal 2: 350 sulfur emissions, diesel 1: 153 sulfuric acid, hydrolysis of cellulose 1: 130 sunflower 2: 280 surface cover, fields 1: 324 surfactants – carbohydrate-based 2: 305 – cationic 2: 412 – classification 2: 301 – nonionic 2: 272 – oil-based 1: 90 – production 2: 302 – sugar-based 2: 11–12 – vegetable oil 2: 301 – worldwide market 2: 303 sustainability 1: 92, 96 – biomass 1: 106 – biorefining systems 1: 56–65 – economic drivers 1: 381 – integrated biorefining systems 1: 60–65 sustainable development 2: 253 sustainable production 2: 448 sweet potato, starch production 2: 71 sweeteners, alternative 2: 62 switchgrass, polyhydroxybutyrate source 1: 283 swollenin, enzymatic hydrolysis 1: 365

Subject Index syngas 1: 26, 30–31, 46, 98, 126, 157, 2: 361 – composition 1: 232 – fermentation 1: 228–229, 233–239, 239– 241 – platform 1: 31–32 – product family tree 1: 33 – technology 1: 240–241 syntheses 1: 123 – lactic acid 2: 382 – lipase-catalyzed 2: 270–272 – petroleum compounds 1: 119 – with oils and fats 2: 253–289 synthesis gas 1: 182 synthesis of structure, petrochemistry 1: 123 synthetic biofuels 1: 30 synthetic biopolyesters 2: 41 synthetic rubber 1: 131

t tablet coatings 2: 88 tack 2: 186 tall oil fatty acid 2: 297 Tamiflu, synthesis 2: 30 tapioca 2: 70–71 tapioca starch production 2: 70 tar-formation, acid hydrolysis 1: 144 target chemicals, biobased 1: 14, 16 target crops, feedstock production 1: 15 tars, undesirable 1: 231 technical constraints, MAAP 2: 209 technical prerequisite, cellulosic biorefineries 1: 55–56 technoeconomic factor, dominant 1: 53–54 technological outline, biorefinery systems 1: 4–8 technological pathways, transformation process 1: 87 temperature adjustment 1: 79 temperature transition, inverse 2: 219 temporary functional scaffoldings 2: 239 terpenes 1: 91 terrestrial biomass, content 2: 3 tetrahydrofuran see THF tetrahydroxybutyl side-chain, furans 2: 19 tetrapyrrole structures 2: 328 TEWL 2: 434 textile-glass-fiber-industry, starch usage 2: 91 textile industry, starch usage 2: 85 The Netherlands, bio-based industry 1: 93–96 thermal addition of alkanes 2: 264

Thermatoga maritima 1: 80 thermochemical biorefinery concept, ECN 1: 104 thermochemical conversion 1: 31 – biomass processing 1: 98 – catalytic 2: 356 – oils 2: 361 – optimization 1: 108 – sugars 2: 360 thermochemical liquefaction 1: 123 thermochemical processing, biomass 1: 249 thermochemical refinery 1: 101–103 thermogravimetric analyses, feedstock 1: 156 Thermoplasma acidophilum, microrganisms 1: 80 thermoplastic polymer, PLA 2: 381 thermoplastics 2: 225 – adhesive film 1: 282 – thermoplastic 1: 215 thermoset resins 2: 184 THF 1: 149, 2: 373 thickeners, textile-printing 2: 86 threonine – biomass building blocks 1: 22 – markets 2: 207 Tiemann, F. 1: 7 Tilgham, B. C. 1: 6 tillage effect, soil carbon loss 1: 328 tillage practice 1: 330–331 tin-catalyzed lactide polymerization 2: 391 tin hydride radical chemistry 2: 261 tin octoate catalyzed polymerization, lactide 2: 393 TNPP, melt stability improvement 2: 399 tocopherols 2: 319–320 toxicity 2: 212 TPA, synthesis 2: 134 traffic congestion 1: 338 traffic problems, feedstock 1: 338–339 tragacanth, substitute 2: 62 transepidermal water loss 2: 434 transesterification, sucrose fatty acid monoesters production 2: 13 transition metal-catalyzed syntheses, aromatic compounds 2: 259 transition metal metathesis, olefins 2: 259 transport – baling dry material 1: 332 – crops 1: 337 transportation fuels, biomass share 1: 14, 16 Treibs’s scheme, petroporphyrin formation 2: 332

495

496

Subject Index triacylglycerides 1: 121–122 Trichoderma 1: 201–202 Trichoderma cellulase 1: 75 – enzymes 1: 204 Trichoderma reesei 1: 77, 130, 134, 365, 375 – cellulase development 1: 366 – enzyme improvement 1: 369 – GH families 1: 373 – protein secretion 1: 372 Trichoderma viride 1: 134 trichomes 1: 183 Trifolium pratense – economic importance 1: 284 – press cake fibers 1: 281 triglyceride oils, hydroxyl-functional 2: 298 triglycerides 1: 121–122, 2: 294 tris(nonylphenyl) phosphite 2: 399 truck transport, feedstock 1: 338 tunneling, dry-jet process 2: 88 turpentine, crude 1: 91 two platforms concept 1: 31 – biorefinery 1: 24 two-use ethic 1: 116 two-uses ethics, MSW 1: 126

u Udic–Rheinau process 1: 131 Umbellularia californica 2: 280 unicarbonotroph 1: 239 unicarbonotrophic acetogens, syngas fermentation 1: 233 United States, biomass conversion 2: 352– 353 unsaturated fatty acids – dimerization 2: 297 – epoxidation 2: 254–257 – microbial hydration 2: 272–273 – nucleophilic addition 2: 265 unsaturated fatty compounds, reactions 2: 254–266 unsaturated N-heterocycles, sugar-derived 2: 24 updraft gasifiers 1: 231 US patent and trademark office 2: 245– 249 USPTO 2: 245–249 Ustilago maydis DSM 4500 2: 274

v c-valerolactone see GVL value-added byproducts – bran-rich wheat fractions 1: 178 – oat bran-rich fractions 1: 185–187

value-added components, bran-rich wheat fractions 1: 175 value-added products 2: 360 value chain approach, biomass processing 1: 97 vanillin 1: 7, 179, 2: 30 vegetable oil – emulsifiers 2: 301 – nonionic surfactants raw materials 2: 306 vegetable oil epoxides, PVC stabilizers 2: 256 vegetable oils 1: 121, 2: 254, 291 – chemo-enzymatic epoxidation 2: 256 vehicle production, lignin 2: 196–197 Vertec 2: 10 vic-dihydroxy fatty acids 2: 257–258 vigorous mixing, starch modifications 2: 77 vinegar-like proteins, repulsion 2: 218 vinyl acetate, glucose product family 1: 21 vinylsaccharides 2: 47 viscosity, starch 2: 73 viskose process, history 2: 102 vital wheat gluten 1: 180 vitamin A source, carotenoids 2: 320 vitamin E source 2: 319 vitamins 2: 14–15 – analysis 1: 299 – juice fraction 1: 274 – wheat 1: 169 vitriol oil 2: 99 von Walden, P. 1: 8

w wagons, self loading and unloading 1: 319 waste biomass 1: 259, 2: 452 – biorefinery products 1: 11 waste-products, processing 1: 8 waste streams, cost generator 1: 96 waste treatment costs 1: 53 waste water treatment 1: 347 wastes, biorefinery 1: 56 water, enantioselective addition 2: 273 water-gas shift reaction, syngas production 1: 230 water-retentive properties, chitosonium salts 2: 420 water-solubility, chitosan 2: 417 water-splitting electrodialysis 2: 387 watersoluble carbohydrates see WSC wax, isolating 2: 432 wax coating, apple 2: 431 wax esters, apple-peel wax components 2: 433

Subject Index waxy maize, starch production 2: 66 western immunoblot technique, purity 2: 230–231 wet fractionation 1: 257, 271–273 – green biomass 1: 29–30 wet mill-based biorefinery – products 1: 29–31 – whole crop 1: 28 wet mill refinery 1: 346–347 wet-milling 1: 48, 70 – corn 2: 367 wet oxidation, pretreatment 1: 363 wet storage – bagasse 1: 323 – silage 1: 334 wheat 2: 66 – chemical composition 1: 169 – composition 1: 167 wheat-based biorefinery, schematic 1: 177 wheat flour, secondary processing 1: 169– 173 wheat flour milling byproducts – annual amount 1: 173 – biorefinery 1: 171 wheat germ 1: 179 wheat germ oil, purified 1: 179 wheat kernel – exploitation 1: 180–183 – morphology 1: 168 wheat milling efficiency, increase 1: 173 wheat separation processes, advanced 1: 173–176 wheat starch production 2: 68 wheat straw – ethanol production 1: 193 – world production 1: 51 wheat tillage practice 1: 331 wheatfeed 1: 170 whey 1: 296 white biotechnology 2: 445 white-rot fungi 2: 151 whole-crop biorefinery 1: 24, 26–29, 165– 191 – products 1: 27

window of processibility, PHB 1: 216 winter cover crop 1: 61 wood chemicals 2: 357 wood chemistry, origin 1: 10 wood hydrolyses 1: 5 wood-hydrolysis pilot plant, US 1: 131 wood processing, LCF biorefinery 2: 112 wood saccharification 1: 5–6 woody biomass 1: 245 woody crops 1: 45 WSC 1: 146 – press cake 1: 257

x xanthophylls 1: 257, 2: 321 XPS 2: 435 X-ray photoelectron spectroscopy 2: 435 xylan 1: 129, 2: 108 xylan/xylose product line 2: 120 xylitol 2: 121 xylitol/arabinitol, basic biobased chemicals 1: 22 xylitol source, esparto grass 1: 283 xyloglucan 1: 360 xyloidin, history 2: 99 xylonic acid, biomass building blocks 1: 22 xylose – crystals 2: 121 – fermentation problems 1: 146 – monomeric 1: 198 d-xylose, pyrazole synthesis 2: 26

y yeast – ascomycetous 2: 7 – ethanol production 1: 194, 206 yeast extract, MAAP processes 2: 211

z Z. mobilis see Zymomonas mobilis zeolites 1: 278 zwitterion, betaine 2: 411 Zymomonas 1: 206 Zymomonas mobilis 1: 133, 2: 7, 108, 210

497

Biorefineries - Industrial Processes and Products: Status Quo and Future Directions

Status Quo

Status Quo

Status Quo

Status Quo

Status Quo

Microbial Processes and Products

Reactive Distillation Status and Future Directions Kai Sundmacher Achim Kienle

Neutrino oscillations: present status and future plans

Orbital Disease: Present Status and Future Challenges

Neutrino Oscillations: Present Status and Future Plans

Bailey's Industrial Oil And Fat Products

Biobased Industrial Products: Research and Commercialization Priorities

Wto and Global Governance The: Future Directions

Submarine slope systems: processes and products

Energy Technology and Directions for the Future

Travel Survey Methods: Quality and Future Directions

Future Directions in Biocatalysis

Industrial catalysis: optimizing catalysts and processes

Clinical Aphasiology: Future Directions

Future Directions in Biocatalysis

Food Constituents and Oral Health: Current Status and Future Prospects

Deformation Mechanisms, Rheology and Tectonics: Current Status and Future Perspectives

Management of Industrial Cleaning Technology and Processes

Industrial Catalysis: Optimizing Catalysts and Processes

Solar and Space Weather Radiophysics: Current Status and Future Developments

Industrial Archaeology: Future Directions (Contributions To Global Historical Archaeology)

Science and Technology in Kazakhstan: Current Status and Future Prospects

Combining Targeted Biological Agents With Radiotherapy: Current Status and Future Directions

Safety and Reliability of Industrial Products, Systems and Structures

Universal and Accessible Design for Products, Services, and Processes

Biorefineries - Industrial Processes and Products: Status Quo and Future Directions

Status Quo

Status Quo

Status Quo

Status Quo

Status Quo

Microbial Processes and Products

Reactive Distillation Status and Future Directions Kai Sundmacher Achim Kienle

Neutrino oscillations: present status and future plans

Orbital Disease: Present Status and Future Challenges

Neutrino Oscillations: Present Status and Future Plans

Bailey's Industrial Oil And Fat Products

Biobased Industrial Products: Research and Commercialization Priorities

Wto and Global Governance The: Future Directions

Submarine slope systems: processes and products

Energy Technology and Directions for the Future

Travel Survey Methods: Quality and Future Directions

Future Directions in Biocatalysis

Industrial catalysis: optimizing catalysts and processes

Clinical Aphasiology: Future Directions

Future Directions in Biocatalysis

Food Constituents and Oral Health: Current Status and Future Prospects

Deformation Mechanisms, Rheology and Tectonics: Current Status and Future Perspectives

Management of Industrial Cleaning Technology and Processes

Industrial Catalysis: Optimizing Catalysts and Processes

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