Current Developments in Solid-state Fermentation

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Author: Pandey A. | Soccol C.R. | Larroche C.

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Current Developments in Solid-state Fermentation i

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Current Developments in Solid-state Fermentation !

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Editors

Ashok Pandey Carlos Ricardo 8occol Christian Larroche

Springer ASIATECH PUBLISHERS, INC. New Delhi

ISBN: 978-0-387-75212-9

e-ISBN: 978-0-387-75213-6

Library of Congress Control Number: 2007938881

2008 Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary fights. 987654321 springer.com

Published by N.K. Muraleedharan for Asiatech Publishers, Inc. E-20, Arya Nagar, CGHS, Plot No. 91, I.P. Extension, Patparganj, Delhi 110092 Ph.: +91-11-22724601

9 Fax: +91-11-22724601

e-mail: [email protected] ~ www.asiatechpublishersinc.com Printed in India

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There could not be any better view than the above about the application of solid-state fermentation for developing bioprocesses involving micro-organisms, especially filamentous fungi. Over the period of last two decades, there has been significant resurgent in solid-state fermentation due to numerous benefits it offers, especially on engineering and environmental aspects. SSF has shown much promise in the development of several bioprocesses and products, which include high volumetric productivity, relatively higher concentration of the products, less effluent generation, and simple fermentation equipments, etc. This resurgent gained further momentum during the last 5-10 years with the development of knowledge-base in the fundamental and applied aspects. A good deal of information has been generated in the published literature and patented information. Several commercial ventures have come up based on SSF in different parts of the world. It was, thus, thought crucial to publish a document allowing to get state-of-the art information in this area, in order to demonstrate that the wellestablished liquid stirred tank is not always the best technical solution. During 2001, a book was published by Asiatech Publishers, Inc. on SSE The book was well received globally. The present book is based on the previous work, although that was an authored volume while this one is an edited work. Also, the coverage in the present volume is much wider and comprehensive. The book covers a wide range of topics in the field of solid-state fermentation. The contents of the book have been distributed in four parts. The Part 1 deals with the General and Fundamentals aspects of SSF and comprises eight chapters. The Chapter 1 is Introductory and describes the history, development and scientific elaboration of SSE The Chapter 2 discusses various general issues

Preface vi

related with SSF. The Chapter 3 describes the factors that influence SSF, which include physio-chemical and biological factors. The Chapters 4 and 5 are on kinetics and water relations in SSF, respectively. The Chapter 6 describes the designs of different bioreactors (fermenters) developed and used for carrying out SSF in laboratory, or at commercial scale. The Chapter 7 is on instrumentation and controls in SSF and the chapter 8 describes the use of informatics in SSF. The Part 2 of the book comprises four chapters, which are on the production of bulk chemicals and products in SSE These include industrial enzymes, organic acids, spores and mushrooms. The Part 3 of the book has also four chapters, which are on the use of SSF for the specialty chemicals such as gibberellic acid, antibiotics and other commercially valuable secondary metabolites, pigments and aroma compounds. The fourth and last part of the book (Part 4) deals with the use of SSF for miscellaneous application such as SSF for the food and feed applications, agro-industrial residues as substrates in the SSF and the production of silage and vermicompost. All the chapters have incorporated the most significant developments taken place during the last 5-10 years with state-of-art information. We are hopeful that the book would be useful to the students, teachers, researchers and professionals interested in the area of the industrial biotechnology and microbiology.

Ashok Pandey Carlos Ricardo Soccol Christian Larroche

Contents

1. Introduction ........................................................................................ 3

Ashok Pandey, Carlos Ricardo Soccol & Christian Larroche 2. General Considerations about Solid-state Fermentation Processes .......... 13

Ashok Pandey, Christian Larroche & Carlos Ricardo Soccol 3. Factors Affecting Solid-state Fermentation .............. :. ........................... 26

Jose A Rodriguez-Leon, Carlos R Soccol, Ashok Pandey & Daniel E. Rodriguez 4. Kinetics in Solid-state Fermentation .................................................... 48

Jose A Rodriguez-Leon, Carlos R Soccol, Ashok Pandey & Daniel E Rodriguez 5. Water Relations in Solid-state Fermentation ......................................... 74

Patrick Gervais 6. Aspects o f D e s i g n o f Bioreactors in SSF ......................................... 117

G Giovannozzi Sermanni & Nicola Tiso 7. Instrumentation and Controls in SSF ................................................ 145

Wilerson Sturm, Dario Eduardo Amaral Dergint & Carlos Ricardo Soccol 8. Informatics in SSF .......................................................................... 168 .

Wilerson Sturm, Carlos R Soccol, Dario Eduardo Amaral Dergint, Jose A Rodriguez-Le6n & Deiva Canali Navarro Vieira Magalhaes

viii Contents

9. Production of Enzymes .................................................................... 181

Sudheer Kumar Singh, George Szakacs, Carlos Ricardo Soccol & Ashok Pandey 10. Production of Organic Acids ............................................................ 205

Carlos R Soccol, Luciana PS Vandenberghe, Cristine Rodrigues, Adriane Bianchi Pedroni Medeiros, Christian Larroche & Ashok Pandey 11. Production of Spores ....................................................................... 230

Sumitra Ramachandran, Christian Larroche & Ashok Pandey 12. Mushroom Production ..................................................................... 253

Leifa Fan, Carlos Ricardo Soccol & Ashok Pandey

13. Gibberellic Acid Production .............................................................. 275

Cristina Maria Monteiro Machado & Carlos R Soccol 14. Production of Antibiotics and Other Commercially Valuable Secondary Metabolites ..................................................................... 302

Javier Barrios-Gonzdlez & Armando Mejia 15. Production of Pigments ............ ......................................................... 337

J~lio Cesar de Carvalho, Carlos Ricardo Soccol, Sumathy Babitha, Ashok Pandey & Adenise Lorenci Woiciechowski 16. Production of Aroma Compounds ..................................................... 356

Carlos R Soccol, Adriane BP Medeiros, Luciana PS Vandenberghe, Marlene Soares & Ashok Pandey

17. Solid-state Fermentation for Food and Feed Applications ................... 379

Maria A. Longo, Francisco J Deive, Alberto Dominguez & M ~ Angeles Sanromdn 18. Agro-industrial Residues as Substrates in SSF ................................... 412

Reeta Rani Singhania, Carlos Ricardo Soccol & Ashok Pandey 19. Preservation of Forage Crops by Solid-state Lactic Acid ................... 443 Fermentation-Ensiling: Zwi G Weinberg 20. Potential of Vermicomposting Technology in Solid Waste Management ................................................................. 468

Vinod K Garg, Renuka Gupta & Anoop Yadav

Contributors

Sumathy Babitha

Leifa Fan

3iotechnology Division National Institute for Interdisciplinary Science & Technology, ~formerly Regional Research Laboratory) Trivandrum-695 019, India

Institute of Horticulture Zhejiang Academy of Agricultural Sciences 198 Shiqiao Road 310021, Hangzhou-ZJ, P. R. China

Javier Barrios-Gonz~ilez

Department of Environmental Science and Engineering, Guru Jambheshwar University of Science and Technology, Hisar 125 001, India

Departamento de Biotecnologia Universidad Aut6noma Metropolitana Iztapalapa, San Rafael Atlixco 186, Col. Vicentina 39340 M6xico D.F., M6xico

Vinod Kumar Garg

Patrick Gervais

Bioprocess Engineering and Biotechnology Division, Federal University of Paran/t CEP 81530-970 Curitiba-PR, Brazil

Laboratoire de G6nie des Proc6d6s Alimentaires et Biotechnologiques ENSBANA 1, Esplanade Erasme 21000 Dijon, France

Francisco J. Deive

Renuka Gupta

Department of Chemical Engineering Isaac Newton Building University of Vigo Lagoas Marcosende 36310 Vigo, Spain


Jfilio Cesar de Carvalho

Dario Eduardo Amaral Dergint

Christian Larroche

Departamento de Eletr6nica Universidade Tecnol6gica Federal do Paranfi CEP 80230-901, Curitiba- PR, Brazil

Laboratoire de G6nie Chimique et Biochimique (LGCB), CUST - Universit6 Blaise Pascal, 24, avenue des Landais, B.P. 206, F- 63174 Aubi~re Cedex, France

Alberto Dominguez

Department of Chemical Engineering Isaac Newton Building University of Vigo Lagoas Marcosende 36310 Vi~o, Spain

x

Contributors

Maria A Longo

Daniel E Rodriguez

Department of Chemical Engineering Isaac Newton Building University of Vigo Lagoas Marcosende 36310 Vigo, Spain

Bioprocess Engineering and Biotechnology Division, Federal University of Paran~i CEP 81530-970 Curitiba-PR, Brazil

Cristina Maria Monteiro Machado

Embrapa Vegetables BR 060 km 09 70359-970 Brasilia-DF Brazil Deiva Canali Navarro Vieira Magalhies

Departamento de Eletrrnica Universidade Tecnol6gica Federal do Paranfi CEP 80230-901, Curitiba- PR, Brazil Adriane BP Medeiros

Bioprocess Engineering and Biotechnology Division, Federal University of Paranfi CEP 81530-970 Curitiba-PR, Brazil AMejia

Departamento de Biotecnologia Universidad Aut6noma Metropolitana, Iztapalapa, San Rafael Atlixco 186, Col. Vicentina, 09340 Mrxico D. F., Mrxico Ashok Pandey

Biotechnology Division National Institute for Interdisciplinary Science & Technology, (formerly Regional Research Laboratory) Trivandrum-695 019, India Sumitra Ramachandran

Laboratoire de Grnie Chimique et Biochimique (LGCB), CUST- Universit6 Blaise Pascal, 24, avenue des Landais, B.P. 206, F- 63174 Aubi&e Cedex, France C Rodrigues

Bioprocess Engineering and Biotechnology Division, Federal University of Paran~ CEP 81530-970 Curitiba-PR, _Brazil

Jose A Rodriguez-Le6n

Bioprocess Engineering and Biotechnology Division, Federal University of Paran~i CEP 81530-970 Curitiba-PR, Brazil M" Angeles Sanrom~in

Department of Chemical Engineering Isaac Newton Building University of Vigo Lagoas Marcosende 36310 Vigo, Spain G Giovannozzi Sermanni

Agrobiology and Agrochemistry Departmen Tuscia University Viterbo, Italy Sudheer Kumar Singh

Biotechnology Division National Institute for Interdisciplinary Science & Technology, (formerly Regional Research Laboratory) Trivandrum-695 019, India Reeta Rani Singhania

Biotechnology Division National Institute for Interdisciplinary Science & Technology, (formerly Regional Research Laboratory) Trivandrum-695 019, India Maarlene Soares

Bioprocess Engineering and Biotechnology Division, Federal University of Paran~i CEP 81530-970 Curitiba-PR, Brazil Carlos Ricardo Soccol

Bioprocess Engineering and Biotechnology Divi~on, Federal University of Paran~ CEP 81530-970 Curitiba-PR, Brazil

Oontributors xi George Szakacs

Department of Agricultural Chemical Technology, Technical University of Budapest 1111 Budapest, Gellert ter 4 Hungary Nicola Tiso

Agrobiology and Agrochemistry Department Tuscia University Viterbo, Italy Wilerson Sturm

Bioprocess Engineering and Biotechnology Division, Federal University of Parand CEP 81530-970, Curitiba-PR, Brazil Lueiana PS Vandenberghe

Bioprocess Engineering and Biotechnology Division, Federal University of Paran~i CEP 81530-970 Curitiba-PR, Brazil

Zwi G Weinberg Forage Preservation and By-Products Research Unit, Department of Food Science Agricultural Research Organization The Volcani Center Bet Dagan 50250, Israel Adenise Lorenci Woiciechowski

Bioprocess Engineering and Biotechnology Division, Federal University of Parand CEP 81530-970 Curitiba-PR, Brazil Anoop Yadav


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1.1 BIOPROCESS DEVELOPMENT Solid-state (substrate) fermentation (SSF) is generally defined as the growth of the micro-organisms on (moist) solid material in the absence or near-absence of free water. In recent years, SSF has shown much promise in the development of several bioprocesses and products. It seems that two terms, solid-state fermentation and solid substrate fermentation have often been ambiguously used. It would be logical only to distinguish these two terms. Solid substrate fermentation should be used to define only those processes in which the substrate itself acts as carbon/energy source, occurring in the absence or near-absence of free water; solid-state fermentation should define any fermentation process occurring in the absence or near-absence of free water, employing a natural substrate as above, or an inert substrate used as solid support. The history, development and scientific elaboration of SSF have been reviewed by several authors from time to time. Evidently, food fermentation and production of enzymes were the areas where SSF originated. The recorded history of SSF was described in Asia before the birth of Christ on cheese making by Penicillium rouquefortii. Egyptians were reported to make bread using SSF process in 2000 BC. The use of soy sauce koji in China was reported in the years 1000 BP and probably in the years 3000 BP (koji process involves fermentation of steamed rice as solid substrate by the fungal strain of Aspergillus oryzae). The koji process migrated from China to Japan by the Buddhist priests in seventh century. During this period, there were mentions of several fermented foods such as tempeh, miso etc., in many South-East Asian countries. Tempeh and miso use steamed and cracked legume seeds as solid substrate and fermentation is carded out using non-pathogenic fungal strains such as Rhizopus sp.

During the 18th century, for the first time, SSF was used for the production of vinegar from the apple pomace. The period also saw the development of leather tanning process using gallic acid. The period of late 19th century saw the development of composting and solid waste treatment using SSF processes. The beginning of 20th century witnessed for the first time the production of primary metabolites such as enzymes and organic acids using micro-organisms in SSE In these processes, mostly fungal cultures were used as producing organisms. It was this period exactly when maiden concepts appeared to develop fermenters (bioreactors) suitable for SSF processes, which led to the development of drum-type of fermenters. The period of 1940's has been termed as the 'Golden Era' of fermentation industry, which saw the dramatic discovery and development of the wonder drug, penicillin. Penicillin was produced using liquid as well as solid culturing techniques. However, this was the period, when, for the reasons best known to the researchers of that period, much attention was paid on the development of liquid fermentation processes and some how or other, knowingly or unknowingly, SSF got totally neglected. This was typically the case with the penicillin, which continued for all other fermentation products. Consequently, there were no serious efforts by the researchers to develop SSF systems, except a few isolated studies, which still continued focusing SSE Even with such low profiles, during the period of 1950-60, reports were published describing steroid transformations in SSF using fungal cultures, which was yet another milestone achievement in the history of SSE This was again followed by the period of 1970's when fungal cultures were successfully cultivated in SSF for the production of mycotoxins, which resulted a significant impact on cancer research. During this period, yet another important application oriented finding of SSF research was on the production of protein enriched cattle feed (singlecell protein). Enormous work, since then has been carried out on this process using a large number of substrates and micro-organisms, and various processes with techno-economic feasibility have been successfully developed. Thus, though historically known since centuries, SSF gained a fresh attention from researchers and industries all over the world since recent few years, mainly due to few major advantages which it offers over liquid (submerged) fermentation (SmF), particularly in the areas of solid waste management, biomass energy conservation and its application to produce high v a l u e - low volume products such as biologically active secondary metabolites, etc., apart from the production of food, feed, fuel and traditional bulk chemicals. _

Attempts were made to trace the history of SSF describing the general features and also the aspects of fermenter design in SSF (Pandey 1991, 1992, 2003, Pandey et al., 2001, Durand 2003). These reviews discussed various developments since historical time. During 1991-2006, more than 1400 publications have

appeared in various joumals, proceedings and books, apart from several important publications in book form, or special issue of joumals. A few reviews have also been presented discussing some particular features of SSF from time to time. A special review by CW Hesseltine was on the Thom Award Address, which reprinted his work on SSE This very well signified the biotechnological potential of SSF globally. Significantly, it has been CW Hesseltine who first consolidated the scientific information on SSF in 1977 (Pandey 2003, Pandey et al., 2000, 2001, Hoelker 2004, Robinson et al., 2001, Mitchell et al., 2002, Tengerdy and Szakacs 2003, Weinberg and Ashbell 2003, Gervais and Molin 2003, Pandey and Ramachandran 2005). Current decade has witnessed an unprecedented spurt in SSF for the development of bioprocesses such as bioremediation and biodegradation of hazardous compounds, biological detoxification of agro-industrial residues, biotransformation of crops and crop-residues for nutritional enrichment, biopulping, and production of value-added products such as biologically active secondary metabolites, including antibiotics, alkaloids, plant growth factors, etc., enzymes, organic acids, biopesticides, including mycopesticides and bioherbicides, biosurfactants, biofuel, aroma compounds, etc. in SSF system. During the past time, most of such processes were eventually termed as 'low-technology' systems but presently seen to be a promising one for the production of value-added 'low volume-high cost' products such as biopharmaceuticals. SSF processes offer potential advantages in bioremediation and biological detoxification of hazardous and toxic compounds. With the advent of biotechnological innovations, mainly in the area of enzyme and fermentation technology, many new avenues have opened for the application of SSE Over the past few years, the increasing demand for the natural products in the food industry has encouraged remarkable efforts towards the development of biotechnological processes for the production of flavour compounds. The use of SSF as a means to improve economical feasibility of these processes would be of potential benefit (Longo and Sanroman 2006). Holker et al., (2004) opined that despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF), it was very difficult to draw general conclusion from the data presented. The authors remarked that this was due to the lack of proper standardisation that would allow objective comparison with other processes. Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported. They further commented that on a gram bench-scale SSF appeared to be superior to submerged fermentation technology (SmF) in several aspects. However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, 02, substrate and moisture gradients. Recently, Viniegra-Gonzalez and Favela-Torres (2006)

made a critical review of the phenomenon of resistance to catabolite repression of enzyme synthesis by SSE commenting the practical and theoretical importance of such phenomenon, together with the current ideas to explain it. Mathematical models have been considered as important tools for optimizing the design and operation of SSF bioreactors. Such models must describe the transport phenomena within the substrate bed and mass and energy exchanges between the bed and the other subsystems of the bioreactor, such as the bioreactor wall and headspace gases. The sophistication with which this has been done for SSF, has improved markedly over the last decade, or so (Mitchell et al., 2003). Mathematical models also must describe the kinetics of microbial growth, how this is affected by the environmental conditions and how this growth affects the environmental conditions. This is done at two levels of sophistication. In many bioreactor models the kinetics are described by the simple empirical equations. However, other models that address the interaction of growth with intraparticle diffusion of enzymes, hydrolysis products and 02 with the use of mechanistic equations have also been proposed, and give insights into how these microscale processes can potentially limit the overall performance of a bioreactor (Mitchell et al., 2004). An important development has been in developing sensors and measurements in SSF processes. In a review, Bellon-Maurel et al., (2003) discussed current on-line methods and innovative applications of methods with a potential to measure parameters in SSE Given the complexity and heterogeneity of the solid medium, process variables are not easily accessible and measurable. Direct measurements of temperature, pH, and water content are considered employing classical sensors, and indirect measurements of the biomass by respirometry or pressure drop (PD). More recent methods such as: aroma sensing, infrared spectrometry, artificial vision, and tomographic techniques (X-rays, Magnetic Resonance Imaging or MRI) should be explored.

1.2 ASPECTS OF DESIGN OF FERMENTER FOR SSF Design of fermenter for SSF processes is an important aspect. However, in spite of strong resurgent of SSF in last ten years, bioreactor design aspects have not been given enough attention by the researchers, although there are certainly path-breaking developments. Present knowledge, however, does not provide state-of-art information about an ideal fermenter for SSF processes. Table 2 gives an overview of different types of bioreactors used in solid-state fermentation as fermenters. As is evident, most of the designs are based on two models: tray type or drum type with or without mixing devices and modifications. Koji process for soy sauce is considered as a representative of SSF processes. Traditionally this has been carried out in wooden trays. Attempts were also made to operate koji process in drum type of bioreactors. However,

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Table 1. History and development of solid-state fermentation

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Table 2. Developments in design of fermenter for solid-state fermentation

Source: Pandey 1991, Pandey et al., 2001, Meien et al., 2004, Chen et al., 2005,Ye et al., 2006,

Fem~indez-Fem~indez & P6rez-Correa, 2006, Rodrfguez-Couto et al., 2006.

not much successes were achieved. As early as in 1964, a 5-gallons rotating drum type of bioreactor was used for the production of fungal enzymes. Some researchers proposed some modifications such as dividing the drum by baffles (three- or four-chambered) to achieve improved mixing and better product formation. While initially drum-type of bioreactors were thought to be useful for processes such as protein enrichment of cattle feed, several attempts were made to use them for production of other products such as enzymes and mycotoxins. For the ease of handing and cost-effectiveness, cement mixers of various sizes have also been tried as bioreactor for SSF processes. A breadmaking blender was modified to suit the process requirements of cattle feed production using agricultural residues. Drum-type of bioreactors have often been used for the bioprocesses where mixing of the substrate during fermentation was recommended useful, which could be achieved by the rotation. There has not been any clear principle behind the extent of mixing of the substrate. While most of the researchers have used a low rotation speed (1-15 rpm), some have used high rates of rotation. One disadvantage, in particular with high rates of rotation, was the damage to fungal mycelia. However, there are contradicting reports concerning the damage per purporated to be cast by mixing. For example, in some reports the mixing (hand or mechanical) of a 3-day duration tray Koji for soya sauce production was considered to be essential while other reports stressed the serious check to enzyme production and heat generation due to the mixing. In case of the composting processes, mixing in long drums mounted on a gentle incline position on continuous mode has been considered to produce excellent effect. During the last 20 years, the concept of column and deep trough type of bioreactor design emerged. These were reported to be useful for the product developments with efficient process controls, particularly for heat removal. However, much is still to be done to realize an optimally suitable size of column for multiple purposes. Moreover, scale-up of column bioreactors poses difficulties. Durand (2003) reviewed the various reactor designs for SSF, focusing on the differences between lab-scale and industrial-scale designs. He highlighted the main designs that have emerged over the last 10 years and the potential for scaling-up for each category of reactor. Robinson and Nigam (2003) reviewed the bioreactor designs and their use for protein production under SSF conditions using various agricultural by-products. The advantages and disadvantages of various bioreactors and their potential for scale-up are described. SSF was proposed as a suitable low-tech strategy for protein enrichment for animal feed by converting a previously low value substance into a more nutritionally valuable one. Recently, Khanahmadi et al., (2006) described the performance of continuous solid-state bioreactors having two different solid substrates flow

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patterns, namely plug flow and completely mixed flow. The performance was quantified for both steady-state and transient operation using a simple mathematical model.

References Bellon-Maurel V, Orliac O & Christen P, 2003, Sensors and measurements in solidstate fermentation: A review, Process Biochemistry, 38, 881-896. Chen I-IZ, Xu J & Li ZI-I, 2005, Temperature control at different bed depths in a novel solid-state fermentation system with two dynamic changes of air, Biochemical Engineering Journal, 23, 117-122. Couto SR & Sanrom~in MA, 2006, Application of solid-state fermentation to food industryA review, Journal of Food Engineering, 76, 291-302. Durand A, 2003, Bioreactor designs for solid-state fermentation, Biochemical Engineering Journal, 13, 113-125. Fermindez-Fermindez M & P6rez-Correa RJ, 2006, Realistic model of a solid substrate fermentation packed-bed pilot bioreactor, Process Biochemistry, 42 (2), 224-234. Gervais P & Molin P, 2003, The role of water in solid-state fermentation, Biochemical Engineering Journal, 13, 85-101. I-I61ker U, H6fer M & Lenz J, 2004, Biotechnological advantages of laboratoryscale solid-state fermentation with fungi, Applied Microbiology and Biotechnology, 64,175-186. Khanahmadi M, Mitchell DA, Beheshti M & Roostaazad R, 2006, Continuous solidstate fermentation as affected by substrate flow pattern, Chemical Engineering Science, 61,2675-2687. Longo MA, Sanromffn M.A, 2006, Production of food aroma compounds: Microbial and enzymatic methodologies, Food Technology and Biotechnology, 44, 335-353. Mitchell DA, Von Meien OF, Krieger N & Dalsenter FDH, 2004, A review of recent developments in modeling of microbial growth kinetics and intraparticle phenomena in solid-state fermentation, Biochemical Engineering Journal, 14, 15-26. Mitchell DA, Von Meien OF & Krieger N, 2003, Recent developments in modeling of solid-state fermentation: Heat and mass transfer in bioreactors, Biochemical Engineering Journal, 13, 137-147. Mitchell DA, Berovic M & Krieger N, 2002, Overview of solid-state bioprocessing, Biotechnology Annual Review, 8, 183-225. Meien OF, Luz Jr LFL, Mitchell DA, P6rez-Correa RJ, Agosin E, Fermindez-Fermindez M & Arcas JA, 2004, Control strategies for intermittently mixed, forcefully aerated solidstate fermentation bioreactors based on the analysis of a distributed parameter model, Chemical Engineering Science, 59, 4493-4504.

/m Pandey A, 2003, Solid-state fermentation, Biochemical Engineering Journal, 13, 81-84. Pandey A, 1992, Recent process developments in solid-state fermentation. Process Biochemistry, 27 (2), 109-117. Pandey A, 1991, Aspects for design of fermenter in solid-state fermentation. Process Biochemistry, 26 (3), 355-361. Pandey A, Soccol CR, Rodriquez-Leon JA & Nigam P, 2001, Solid-statefermentation in Biotechnology, Asiatech Publishers, New Delhi, India. Pandey A, Soccol CR & Mitchell D, 2000, New developments in solid-state fermentation: I bioprocesses and products. Process Biochemistry, 35 (10), 1153-1169. Pandey A & Ramachandran S, 2005, Process developments in solid-state fermentation for food applications, In- Food Biotechnology, 2nd edition, K Shetty, G Paliyath, A Pometto & RE Levin (eds), Taylor & Francis, New York, pp 87-110.

Robinson T & Nigam P, 2003, Bioreactor design for protein enrichment of agricultural residues by solid-state fermentation, Biochemical Engineering Journal, 13, 113-125 Robinson T, Singh D & Nigam P, 2001, Solid-state fermentation: A promising microbial technology for secondary metabolite production, Applied Microbiology and Biotechnology, 55, 284-289. Rodriguez-Couto S, L6pez E & Angeles-Sanrom~in M, 2006, Utilisation of grape seeds for laccase production in solid-state fermentors, Journal of Food Engineering, 74, 263-267. Tengerdy RP & Szakacs G, 2003, Bioconversion of lignocellulose in solid substrate fermentation, Biochemical Engineering Journal 13, 169-179. Viniegra-Gonzaqez G & Favela-Torres E, 2006, Why solid-state fermentation seems to be resistant to catabolite repression?, Food Technology and Biotechnology, 44, 397-406.

Weinberg ZG & Ashbell G, 2003, Engineering aspects of ensiling, Biochemical Engineering Journal, 13, 181-188. Ye SD, Ying SH, Chert C & Feng MG, 2006, New solid-state fermentation chamber for bulk production of aerial conidia of fungal biocontrol agents on rice, Biotechnology Letters, 28,799-804.

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2.1 INTRODUCTION SSF offers numerous advantages over submerged fermentation (SmF). These include high volumetric productivity, relatively higher concentration of the products, less effluent generation, and simple fermentation equipments, etc. The major factors, which affect microbial growth and activity in SSF include the selection of a suitable micro-organism and substrate, pre-treatment of the substrate, particle size (inter-particle space and surface area) of the substrate, moisture content and water activity (aw) of the substrate, relative humidity, type and size of inoculum, temperature of fermenting matter, removal of metabolic heat generated during respiration, period of cultivation, maintenance of uniformity in environment of SSF, and the gaseous atmosphere, i.e. oxygen consumption rate and carbon dioxide evolution rate. Based upon the type of micro-organisms involved, SSF processes can be classified into two main groups, natural (indigenous) SSF, and pure culture SSF using individual strains, or a mixed-culture. Composting and ensiling are the two best examples of SSF processes, which involve natural micro-flora. Bioprocesses involving pure culture SSF are also known since long. One such unique example is Koji process utilizing a culture of Aspergillus oryzae. In industrial SSF processes, however, generally pure cultures are used as it helps in optimum substrate utilization for the targeted product. Examples are the production of enzymes, organic acids, bioactive secondary metabolites, etc. Examples of SSF processes involving mixed (pure) cultures are bioconversion of agro-industrial residues such as wheat straw for protein enrichment in which a cellulolytic organism (e.g. a fungal strain of Chaetomium cellulolyticum) and a yeast strain (e.g. Candida utilis) are used. In nature, SSF is mostly carried out by the mixed cultures in which several micro-organisms show development and symbiotic co-operation.

Selection of a suitable strain is one of the most important criteria in SSE The vast majority of wild type micro-organisms are incapable of producing commercially acceptable yields of the desired strains product. The importance of the micro-organism can be seen from the fact that culture of Aspergillus niger can produce as many as 21 types of enzymes (Table 1) while one enzyme alone can be produced by several microorganisms in varying quantities. For example, alpha amylase can be produced by as many as 64 different micro-organisms (Table 2). It is also important to ensure that the selected microbial strain is best suited for SSF as some times the same strain's potential could be not best exploited in SSF but in other systems. Such an example is illustrated in Table 3. As is evident, the strain of Rhizopus formosaensis produced glucoamylase enzyme in varying concentrations when grown as surface culture in submerged, or solid-state fermentation, or as submerged culture. Yet another crucial aspect involves the screening of the strain for its efficiency to produce the desired end-product as the sole product, although there could be other associated activities but these should be in trace amounts only. This could be exemplified from the results described in Table 4, which showed a case study involving the production of alpha amylase by a strain of Trichoderma viride. Indeed this study involved screening of various carbons sources as well. With all the substrates, there was production of another enzyme, namely protease in varying concentrations. These results showed that although corn starch gave the best yields of alpha amylase, the organism also produced simultaneously protease in significant quantities, which, in fact was more than the targeted end-product, i.e. alpha amylase. Thus, corn starch was not a suitable substrate for this process using the selected strain. When lactose was used as the substrate, alpha amylase production was almost negligible and protease production was dominant. However, yield of protease in this case was almost half to that produced by corn starch, which showed that although strain produced only protease as major end-product, this was not suitable for that (protease production) also, as with other substrates, enzyme yields were higher than this case. The results demonstrated that the strain was neither a suitable choice for the production of alpha amylase, nor for protease from the substrates used in the experiment. From these examples, it could be concluded that the selection of the strain for any process development, and so for SSF as well, is an extremely significant step and must be studied with utmost care. While doing so, it would be necessary to keep in mind all the necessary associated factors such as process technique, substrate, associated products formation, etc. The selection of a substrate for SSF process depends upon several factors, mainly related with the cost and availability and, thus, may involve their screening. Agro-industrial residues, particularly of tropical origin offer potential advantages for their application as substrates. In SSF process, the solid substrate not only

Table 1. Spectrum of enzymes obtainable from the strains of

Aspergillus niger

Table 2. Micro-organisms reported to produce alpha amylase

m Table 3. Glucoamylase production by a strain of Rhizopus formosaensis

Table 4. Screening of alpha amylase production by a strain of Trichoderma viride

supplies the nutrients to the microbial culture growing in it, but also serves as an anchorage for the cells. The substrate that provides all the needed nutrients to the micro-organisms growing in it should be considered as the ideal substrate. However, some of the nutrients may be available in sub-optimal concentrations, or even not present in the substrates. In such cases, it would be necessary to supplement them externally. It has also been a practice to pre-treat (chemically, or mechanically) some substrates before use in SSF processes (e.g. lignocellulosics), which makes them more easily accessible for microbial growth. Among the several other factors, which are important for the microbial growth and activity in a particular substrate, particle size and moisture level/water activity are the most critical. Generally, smaller substrate particles would provide larger surface area for the microbial attack and, thus, should be considered as a desirable factor. However, too small substrate particles may result substrate agglomeration in most of the cases, which may interfere with microbial respiration/ aeration, and, thus, may result poor cellular growth. At the same time, larger particles provide better respiration/aeration efficiency (due to increased interparticle space) but provide limited surface for the microbial attack. Thus, it would be necessary to arrive at a compromised particle size for a particular process. Table 5 shows a typical example of particle size distribution in three different commercial samples of wheat bran, which is the most frequently used substrate in SSF processes. Sample A and B were obtained from a local market in India while sample C was prepared by mixing A and B in 1:1 ratio (w/w). Sample A contained 76% particle smaller than 500 microns while sample B has only 11% particles smaller than 500 microns. Sample C showed a wide

Table 5. Particle size distribution in a commercial samples of wheat bran

Source:

Pandey A, 1991, Bioresource Technololgy, 37, 169-172

distribution of particles of various sizes containing approximately 44% smaller and 56% large than 500 microns. When SSF was carried out using these samples by taking individual sized particles prepared from these samples by sieving and individual samples as well, best results on enzyme production were obtained by the substrate containing particles of 425-500 microns, followed by 500-600 microns. Substrates with lower particles resulted in lower enzyme activity and the same was true for bigger particles substrates. The lowest enzyme activity was obtained with the substrate containing particles bigger than 1.4 mm. The specific enzyme activity of sample A was much better than sample B but this was best in sample C, which showed a unique distribution of particles of different sizes. Although, enzyme titres obtained from sample C was not the same as from the best individual sized particles substrate, from the techno-economic feasibility point of view, it was the most appropriate to use the mixed substrate. Substrate moisture and water activity play very important role in SSE The water activity of the medium is considered as a fundamental parameter for the mass transfer of the water and solutes across the cell membrane. In fact, the control of this parameter could be used to control and modify the metabolic activity of the micro-organism. In general the types of micro-organisms that can grow in SSF system are determined by the water activity factor, aw. Water activity is defined as the relative humidity of the gaseous atmosphere in equilibrium with the substrate. Pure water has aw = 1.00 and aw decreases with the addition of solutes. Bacteria generally grow at higher aw values while filamentous fungi and some yeast can grow relatively at lower aw values. Due to this fact, filamentous fungi have often been preferred over the other groups

of micro-organisms for SSF processes. In case of fungal growth in SSF, higher water activity generally results sporulation, while low water activity with suitable range favours spore germination and mycelial growth. Research on the selection of a suitable substrate has mainly centred around tropical agro-industrial crops and residues. These include crops such as cassava, soybean, sugar beet, sweet potato, potato, and sweet sorghum, crop residues such as bran and straw of wheat and rice, hull of soy, corn and rice, bagasse of sugarcane and cassava, residues of coffee processing industry such as coffee pulp, coffee husk, coffee spent-ground, residues of fruit-processing industries such as pomace of apple and grape, wastes of pine-apple and carrot processing, banana waste, waste of oil-processing mills such as coconut cake, soybean cake, peanut cake, canola meal and palm oil mill waste, and others such as saw-dust, corn cobs, carob pods, tea waste, chicory roots, etc. Wheat bran has been the prime among all. Many processes have been developed that utilize these as raw material for the production of bulk chemicals and valueadded fine products. We would not go into details of these aspects here, as there is a separate chapter exclusively devoted to this in the next section. In relation to SSF processes using inert substrates, two approaches have been adopted; one, in which synthetic materials such as Amberlite, or polyurethane are used, and other which utilises natural materials such as sugarcane bagasse as the inert solid support. Since natural substrates create problems in the fermentation kinetics studies (due to their heterogeneous nature), such studies could better be performed using synthetic inert solid substrates.

2.2 ESTIMATION OF GROWTH IN SSF In SSF, estimation of biomass, which is essential for the kinetic studies, poses difficulties as generally the microbial cells remain attached with the substrate and it is difficult to separate them. In case of the fungal cultures, it is more difficult because fungal mycelia penetrate into the substrate particles. Generally the estimation of growth of microorganism in SSF is carried out by the indirect methods, although for model studies, some methods are used, which provide direct growth estimation. For example, gelatin could be used to grow microbial cells, e.g. Saccharomyces cerevisae in solid cultures and after the growth, gelatin could be melted at about 37~ and subsequently the cells could be recovered from this melted medium by centrifugation. For yeast cells, one another method has been applied which involved the washing of fermented matter with water and then counting the cells in the washed medium. However, the efficiency of method largely depends upon the efficiency of washing out the cells from the fermented matter.

m ,,,,,~,,~,,!~,,,~,~,,,~~i~~,,,,,,,,,!,~,~,,,~,,,~i~i~,,,~ ~~~i~~~~~!~!~i!~ Among the indirect or the other methods used for the estimation of microbial growth in SSF, assay of biomass components such as DNA, glucosamine, ergosterol and protein (Kjeldahl) or assay of metabolic activity (respirometry) have been considered useful. Estimation of glucosamine for fungal biomass assay has been considered as the most suitable and frequently used method. In recent times, particularly while using column type of bioreactors, estimation of oxygen utilization rate (OUR) and carbon dioxide evolution rate (CER) are considered to be most accurate for the determination of growth of the microorganisms in SSE Some more details about growth estimation methods have been provided in another chapter in the next section.

2.3 CONCEPT OF STERILITY Sterility is very often required in any SmF process because many contaminants could out-compete the process organism under the conditions of high water availability provided. Often SSF processes involve an organism, which grows quite rapidly under the low water conditions, and if an active inoculum is added to a (cooked) substrate, the process organism is able to out-compete the contaminating organisms. Thus, strict aseptic operation of the bioreactor may not be essential in SSF, although, of course, operation should be carried out in as clean a manner as possible. The less stringent design requirements for such bioreactors, and correspondingly lower costs could be considered as favourable point for the SSF process, providing an economic advantage over the SmF process. However, there are a number of products for which SSF has good potential but for which the process organisms grow generally relatively slowly, which could be overtaken by contaminants. For example, this is the case in the production of gibberellic acid by Giberella sp. In such cases, it would be essential to use a bioreactor, which can be operated under aseptic conditions. In such a case, the bioreactor costs would be expected to be similar to those for SmF bioreactors, and SSF would only be chosen if it could provide any specific advantage, such as higher product titres, or lower downstream processing costs, which finally could lead to better economic performance.

2.4 ACADEMIC RESEARCH VS. INDUSTRIAL APPLICATIONS In order to understand this, let's look into some fundamental questions. There is a lot of research going on into SSF - why?There are relatively few commercial applications (at least compared to SmF) - why? A general opinion about the choice of fermentation method for the production of any microbial product would normally be SmF, unless there appears a particular reason why SSF should be chosen. There is no question that SmF is intrinsically less problematic-

heat transfer is better, and homogeneity is much-much better. SSF would be chosen if (a) particular economic conditions favored it (therefore, in some parts of the world some enzymes are produced by SSF, whereas in other parts, the same enzymes are produced by SmF), (b) the product is only produced in SSF - or if produced in both systems, the SSF product is far superior (for example, fungal spores for use as biopesticides tend to be much more robust when they are produced in SSF compared to when they are produced in SmF, and some fungi simply don't sporulate well in SLF), (c) use of solids becomes an imperative (government regulations in response to environmental pressures caused by dumping of organic solids). Thus, although SSF has potential mainly in specific areas, under the conditions that it is "off the main track", it has received relatively little attention. Its potential to operate reliably at large-scale simply has not been investigated to the same degree as the SmF method. It is possible that SSF processes could be routinely operated at large-scale, following rational design rules - but we currently don't know enough to really be sure if this is the case or not. Certainly, there are a few quite successful SSF processes - such as enzyme production processes, various biopesticide production processes, etc, even if many of these are at relatively small scales. There is also the case with the koji industry. SSF should not be seen as a technology, which can simply replace SmF. In fact, SmF has many features, which would make it the preferred method in a case where SmF and SSF had similar economic performances. The greater homogeneity in a SmF system simply makes the process less problematic. The relatively few commercial SSF processes compared to the range of products, which have been investigated in the laboratory, could probably be a confirmation of this. However, there are a number of products for which SSF is the superior production technology, and a number of large-scale SSF processes have begun operating commercially. There is a continued need to develop SSF technology to allow such processes to operate at their full potential.

2.5 COMPARISON OF CHARACTERISTICS OF SSF AND SUBMERGED FERMENTATION Table 6 highlights the major differences in the characteristics of solid and submerged fermentation. As is evident, due to static nature of substrate and microorganism, the system poses difficulties for heat and mass transfer effects. Due to respiration, carbon dioxide evolves and being heavier than oxygen, remains accumulated in the substrate bed, which in turn results in increase in the temperature of the fermenting bed. This needs to be controlled. Two other characteristics of SSF, mainly low physical energy requirements and high human

Table 6. Comparison of characteristics of SSF and submerged fermentation

energy requirement led to an untrue assumption that SSF was a low technology. In fact, poor knowledge, or understanding of fundamental aspects of SSF is the main reasons behind these two aspects. Since last 10-15 years, scenario is changing fast and it is hoped that generation of data based knowledge on fundamental aspects of SSF would lead to the development of modem fermenters with automation as is the case of SmF.

2.6 ECONOMICALLY AND INDUSTRIALLY IMPORTANT ADVANTAGES OF SSF As has been pointed out in the beginning of this chapter, there are several industrially and economically important advantages, which SSF offers over SmF and essentially due to these reasons, there is strong resurgence of SSE A glimpse of Table 7 provides an insight about these advantages. Higher product titres in most o f the SSF process, less water need in up-stream processing resulting lesser wastewater generation in down stream processing (which heavily reduces the cost of down-stream processing) are the major advantageous point of SSF processes. Since fermenters used for SSF have no complicated design and have not much automated control systems (the points, which need to be studied and developed), SSF systems require lower capital investment. Moreover, since the substrates used are generally agro-industrial residues, fermentation media are generally simpler without requiring complex nutrients such as vitamins, etc. This also leads to economical advantage and lower recurring expenditure. In the absence of any rigorous mixing, there is no foam formation, which normally is faced in SmF and is controlled by addition of anti-foaming agents, which leads to increase in pollution load. One another advantage, which SSF offers is the application of fermented solids directly without isolating the product. An example is the production of glucoamylase enzyme using wheat bran admixed with corn flour in SSF by A. niger. After fermentation, wet fermented matter

Table 7. Economically and industrially important advantages of SSF

containing glucoamylase could be directly used for the hydrolysis of cassava flour to produce fermentable sugars. Hydrolysis efficiencies were as good as obtained with the purified enzyme. Fermented matter could be dried and stored at room temperature without any significant loss in the activity. However, this aspect should be evaluated according to the need of user.

References Aidoo K. E, Hendry R & Wood BJB, 1982, Solid substrate fermentations, Advances in Applied Microbiology, 28, 201-237.

Docile HW, Mitchell DA & Rolz CE, (Eds), 1992, Solid-state Fermentation, Elsevier Applied Science, London. Durand A, 1998, Solid-state fermentation, Biofutur, 181, 41-43. Hesseltine CW, 1999, A microbe's view of fermentation -Thom Award Address (Reprinted from Developments in Industrial Microbiology, 1981; 22, 1-18), Journal of Industrial Microbiology and Biotechnology, 22, 482-499. Jonas R, Pandey A & Tharun G (Eds), 2004, BiotechnologicalAdvances and Applications in Bioconversion of Renewable Raw Materials, Doehring Druck, Braunschweig, Germany Joshi VK & Pandey A, (Eds), 1999, Biotechnology: Food Fermentation. Educational Publishers & Distributors, New Delhi, Vols I & II.

Lonsane BK, Ghildyal NP, Budiatman S & Ramakrishna SV, 1985, Engineering aspects of solid-state fermentation. Enzyme and Microbial Technology, 7, 258-265.

!iiiiiiiiiii!FiiiFiiFiiiii!iiii! ............hi................!........!ii!! ............ii.... .... Mitchell DA, Krieger N, Stuart DM & Pandey A, 2000, New developments in solid-state fermentation: II Rational approaches for bioreactor design and operation, Process Biochemistry, 35, 1211-1225. Mitchell DA, Pandey A, Sangsurasak & Krieger N, 2000, Scale-up strategies for packedbed bioreactors for solid-state fermentation, Process Biochemistry, 35, 167-178 Pandey A (Ed), 2004, Concise Encyclopedia of Bioresource Technology, Haworth Press, New York, USA.

Pandey A (Guest Ed), 2003, Special Issue on solid-state fermentation, Biochemical Engineering Journal, 13, 79-218. Pandey A (Ed), 1998, Advances in Biotechnology, Educational Publishers & Distributors, New Delhi.

Pandey A (Ed), 1996, Solid-state Fermentation, Special Issue of Journal of Scientific and Industrial Research, 55 (10-11), 311-482. Pandey A (Ed), 1994, Solid-state Fermentation, Wiley Eastern Publishers, New Delhi. Pandey A, 1992, Receent developments in solid-state fermentation, Process Biochemistry, 27, 109-117.

Pandey A, 1991, Aspects for design of fermenter in solid-state fermentation, Process Biochemistry, 26 (3), 355-361. Pandey A & Soccol CR, 2000, Economic utilization of crop residues for value addition A futuristic approach, Journal of Scientific and Industrial Research, 59 (1), 12-22. Pandey A & Soccol CR, 1998, Potential applications of cellulosic residues for the production of bulk chemicals and value added products. In- Trends in Carbohydrate Chemistry, (Eds. P. L. Soni & V Kumar), Vol. 5, Surya International Publications, Dehradun, India, pp 83-88.

Pandey A, Webb C, Soccol CR & Larroche C (Eds), 2006, Enzyme Technology, Springer Science, USA, p 740. Pandey A, Soccoi CR & Mitchell DA, 2000, New developments in solid-state fermentation, Process Biochemistry, 35, 1153-1169. Pandey A, Nigam P, Soccol CR, Singh D, Soccoi VT & Mohan R, 2000, Advances in microbial amylases. Biotechnology and Applied Biochemistry, 31, 135-152.

Pandey A, Soccol CR, Nigam P, Soccol VT, Vandenbereghe & Mohan R, 2000, Biotechnological potential of agro-industrial residues: II Cassava bagasse. Bioresource Technology, 74 (1), 81-87 Pandey A, Soccol CR, Nigam P & Soccol VT, 2000, Biotechnological potential of agroindustrial residues: I Sugarcane bagasse, Bioresource Technology, 74 (1), 69-80. Pandey A, Benjamin S, Nigam P, Krieger N & Soccol VT, 1999, The Realm of microbial lipases in biotechnology, Biotechnology and Applied Biochemistry, 29 (2), 119-131.

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Pandey A, Selvakumar P, Soccoi CR, Soccol VT, Krieger N & Fontana JD, 1999, Recent developments in microbial inulinases - Its production, properties and industrial applications, Applied Biochemistry and Biotechnology, 81 (1), 35-52. Pandey A, Selvakumar P, Soccol CR & Nigam P, 1999, Solid-state fermentation for the production of industrial enzymes, Current Science, 77 (1), 149-162. Pandey A, Azmi W, Singh J & Banerjee UC, 1999, Fermentation types and factors affecting it. In - Biotechnology: Food Fermentation, (Eds VK Joshi & A Pandey), Educational Publishers & Distributors, New Delhi, Vol. I, pp. 383-426. Raimbault M, Soccol CR & Chuzel G (Eds), 1998, Intemational Training Course on Solid-state Fermentation, Oct 6-10, 1997, Curitiba, Brazil, published by ORSTOM, Montpellier. Raimbault M (Ed), 1988, Solid-state fermentation in bioconversion of agro-industrial raw materials, Proceedings of the Seminar on SSE July 25-27, 1988, ORSTOM, Montpellier, published by ORSTOM, Montpellier. Roussos S, Soccol CR, Pandey A & Augur C (Eds), 2003, New Horizons in Biotechnology, Kluwer Academic Publishers, Dordrecht, The Netherlands, p 449. Roussos S, Lonsane BK, Raimbault M & Viniegra-Gonzalez G (Eds), 1995, Advances in Solid Substrate Fermentation, Kluwer Academic Publishers, Dordrecht. Roussos S, Raimbault M, Prebois JP & Lonsane BK, 1993, Zymotis, a large scale solid state fermenter- design and evaluation, Applied Biochemistry and Biotechnology, 42, 37-52.

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3.1 INTRODUCTION To analyze the factors that may influence in solid-state fermentation, it is necessary to take into account a consideration which kind of factor we are considering. Generally, two types of factors exist:

Biological factors: These factors are related with the biology, metabolic process and reproduction of a living species or organism. These determine the behaviour of the particular species in a specific way.

Physico-chemical factors: These factors are related with the physico-chemical phenomena occurring in the system. These factors affect such mechanisms of the system that could be related with the transport phenomena of momentum, energy and mass with all the thermodynamics considerations that it could imply. Of course, all these factors are intimately related and cannot be considered as independent one from another. It is necessary to distinguish when a particular factor is affecting a particular response and when it is affected. In a specific SSF process, all the factors covered will be present at the same time. The duality that a factor could have as biological and as physico-chemical factor need to be considered. For example, in SSF the viscosity variation of air flowing though the solid matrix with temperature increase due to the heat evolved in the process is meaningless in comparison to the biological response to this increase. It is also very important to determine which factors are independent, or dependent. In SSF, factors are generally considered as independent, although this might not be the case. A factor can be independently related to the biological behaviour

but dependent when it is related to the physico-chemical process and v i c e versa. Consider, for example, temperature. It is well established that microorganisms grow better at a particular optimum temperature and have a relative narrow range of temperature for living. Here, the temperature is an independent factor. However, in the system temperature, will rise due to the effects of heat generation and transfer. In this sense, temperature is a dependent factor. It is very important to keep in mind above considerations. While analyzing the real factors that decide the development of a SSF process (as we shall see in future chapters from their interactions), these will have impact on: 9 strains selection, 9 design and characteristics of a particular medium for fermentation, 9 substrate conditioning, 9 importance of control of specific factors, 9 control criteria, 9 design of reactors.

3.2 CARBON SOURCE AND CARBON/NITROGEN RELATIONSHIP (C/N) Type, source and nature of carbon and nitrogen are among the most important factors for any fermentation process. Carbon source represents the energetic source that will be available for the growth of the micro-organism. It could be as simple as a pure monosaccharide compound such as glucose, or as complex as polymeric molecules such as cellulose, or starch. As a matter of fact in any fermentation process when selecting a proper energy, or carbon source, there could be two points of view: a)

Select the carbon source as a function of the micro-organism to be employed and the products to obtain. This means in the widest sense to formulate the proper medium.

b)

Select the adequate micro-organism in order to employ a particular substrate component. This point of view is related with the use of resources such as industrial residues, use of eventually harmful wastes to the environment or raw materials that could improve the economics of established technology. This, of course, means too that the media need to be formulated.

While dealing with media formulations, it is necessary to take into account the biomass composition. Cellular biomass presents as average 40-50% carbon, 30-50% oxygen, 6-8% hydrogen and 3-12% nitrogen. Other elements such as phosphorus, sulphur and metals are important as part of the cell and its metabolism but are present in such small quantities that need to be considered for their particular significance.

From all the principal components of cellular biomass, carbon source is the most important one and should be given significant consideration. The yield of a product in kinetic studies i s based on the consumption of carbon source ((Yx/s) Theoretically, this parameter has a value of 0.5 when the carbon sources are sugars. Nitrogen is the next most crucial factor that determines the growth of micro-organisms and needs to be calculated or determined to obtain maximum biomass. This is based on the yield (Yx/s) and the biomass composition. The other two components, oxygen and hydrogen are obtained through the organic carbon source. Generally, it is believed that the ratio between the mass of carbon and nitrogen (C/N) is most crucial for a particular process to obtain a specified product. This statement is valid when the metabolite to produce is non-growth associate or even partially associated. In the case when the metabolite is associated to growth it is better from the beginning of the process to bring the balanced medium. Besides, when dealing with fungi, it is necessary to take into account how the C/N ratio could induce or delay sporulation. Exercise 3.1 Determine the ratio of C/N that is needed to obtain biomass from a strain of Aspergillus niger. The empirical formula for this strain is CH1.77 00.72 No.085. Consider a 50% yield of biomass based on sugar consumption (g biomass produced/g sugar consumed), b) In a solid state fermentation process with an initial concentration of total reduced sugars of 30% dried basis, which is the amount of (NH4)2SO4 that must be added to the medium for obtain the maximum biomass and which is the ratio C/N in the medium. Solution: a)

Basis of calculation: 1 mol biomass

Molecular weight of biomass: 12.011 + 1.77 + 0.72(16) + 0.085 (14.008) = 26.5 g Carbon weight needed to produce 1 mol biomass: 12.011 g Nitrogen weight required to produce 1 mol biomass: 0.085 (14.008) = 1.19 g Ratio C/N weight in the biomass: 12.011/1.19 = 10.09 b) Basis of calculation: 100 g of medium dry weight basis Biomass to produce (g): 30(0.5) = 15 g of biomass Biomass to produce (mol): 15/26.5 = 0.566 mol biomass Nitrogen required: 1.19(0.566) = 0.67 g Weight ratio carbon/sugar considering total sugar as glucose" 12/(12 + 2 + 16) = 0.4 g Carbon present in the substrate: 0.4(30) = 12 g

Ratio C/N weight in the substrate: 12/0.67 = 17.91 Ratio N/(NH4)2SO4: 2(14) / 2(14) + 8 +32 + 4(16) = 0.212 (NH4)2SO4 required in the medium: 0.67/0.212 = 3.16 g Reader must take into account that the ratios of C/N in the biomass and in the medium are related by the yield and the biomass composition. Normally, a C/ N ratio of 16 is frequently found suitable for fermentation processes. From the Exercise 3.1, we can see that when a medium is formulated, it is very important to know not only the sources of carbon and nitrogen but also the chemical composition of biomass and the yield expected in the process. These considerations shall allow designing the medium in such a way that there will not an excess of any of the components. This means that the materials required would be used adequately and not in excess with the consequent waste of materials and increase in the production cost. As it has been pointed out before, it must be kept in mind that when we are dealing with the production partially, or not associated product with the growth, C/N could vary. Then a particular research about which is the best C/N ratio must be done.

3.3 TEMPERATURE Biological processes are characterized by the fact that they are developed in relatively very narrow range of temperature. High temperature limits for development could be found in many cases at values not higher than 60-80~ and with particular strains at utmost at 120~ The significance of temperature in the development of a biological process is such that it could determine effects such important as protein denaturization, enzymatic inhibition, promotion, or inhibition on the production of a particular metabolite, cells death etc. Importance of temperature in the growth of micro-organism brings to a classification considering the temperature, or the temperature range in which the microorganism grows.

3.3.1. Extremo-thermophiles There are some micro-organisms, which survive at temperatures as high as 400~ (for example those found on sea-beds in sulphur-chimneys). These may have optimum activity between 100-250~

3.3.2 Thermophiles These micro-organisms present capacity of growth at such temperatures as 80-95~ with optimum growth temperature in a range between 45-70~

3.3.3. Mesophiles The capacity of growth for these micro-organisms does not exceed a temperature of 50~ Optimum temperatures for growth lay between 30-45~

3.3.4. Psychrophiles These micro-organisms can grow at temperatures below 0~ for growth between 10-20~

with optimum

The above classification is somewhat arbitrary and no strict adherence should be given to the temperature ranges as these may vary according to different authors. If we know which kind of micro-organism we have, we can determine its adequacy for a particular process. Normally, the fermentation processes (SSF as well as SmF) develop with mesophilic microbial strains. However, the trend is to look for thermophilic ones that in some sense could allow solving the problems of heat evolution during the process. However, mesophilic strains are by far the most existent. Temperature rise due to exothermic characteristics of fermentation processes is one of the main difficulties faced in SSE Temperature control is more difficult in SSF processes when compared with SmF processes. In the case of SSF, the solid characteristics of the substrate lacks the homogeneity in the reactor and, therefore, results the existence of temperature gradients. Such facts make the heat exchange difficult in the system. A typical example of the temperature effect in SSF can be observed in the process of composting. This process is developed spontaneously through the growth of the natural micro-flora present in it. As temperature arises due heat evolution, the activity of micro-organisms belonging to mesophilic temperature range growth at environmental conditions trends to diminish and start to impose higher temperature range (thermophilic) micro-organisms. Undoubtedly the temperature control is one of the factors that decide the good development of SSF and must be taken into account when any SSF process is design.

3.4 MOISTURE AND WATER ACTIVITY (a.) Moisture is a factor that is intimately related with the definition SSF and with the characteristics of the biological material. The importance of water in the system is due to the fact that the great majority of viable cells are characterized by moisture content of 70-80%. From this simple observation, it is easy to arrive to the conclusion about the necessity to have a certain quantity of water available for new cell synthesis. Importance of moisture for SSF processes has been well documented.

In a general sense, it has been established that in the case of bacteria the moisture of the solid matrix must be higher than 70%. For yeast, the moisture range can be a little wide as 72-60% and in the case of fungi the range could be as wide as 70-20%. This certainty by itself could signify a great advantage for the development of a specific SSF process. In a case where the optimum moisture level for growth for employed micro-organism is relatively low, this could be a natural barrier to possible contaminants. In this case the need of a sterilization treatment could be less rigorous or even absent. In relation to water interchange in the system, a good measure is provided by the interchange between the substrate and airflow. The mass transfer mechanism between the different components of the substrate and the airflow is studied by Thermodynamics, based on the free energy concept. Any substance (A) present in a mixture has a vapour pressure that will correspond to the equilibrium between the concentration of the substance (A) in the mixture and the concentration of the substance (A) in the gaseous phase. This vapour pressure is evaluated from the Raoult's Law as: ea = P'AXA

where: Pa: vapor pressure of substance A in the gaseous mixture at a particular temperature.

P'A: vapor pressure of pure substance A in the gaseous mixture at a particular temperature. XA: molar fraction of A in the mixture. Considering that the gaseous phase is in equilibrium with the mixture (in our case with the solid substrate) and assuming that this phase is an ideal gas, the molar fraction of the substance (YA) in the gaseous mixture can be calculated as:

YA PA I'--AXA 1'1 et =

Where: Pt: total pressure of the gas in the system However assuming that Raoult's Law is applied only for ideal mixtures, therefore,

I i i iii!

iii i ii!

ii. . . .

iiii]iilJiiiiiiiiiiiiiiiiiiii!iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii!iiiiii ...........ilii!i_ii....

when we are dealing with non-ideal mixtures the former equation is modify by the following expression: Pa = ptAXA'YA

Where: ~/A: activity coefficient of substance A From the preceding equation, water activity (aw) is defined already as: P)v aw

=

.......

=

XA~

Pt

It must be noted that the concept of water activity (aw) was introduced as a definition in which the gaseous phase is considered as an ideal gas. This assumption at normal work pressures and temperatures are fair. Note that for the definition it was not necessary to consider the mixtures as ideal. The water activity (aw) is measured using different methods and equipments, which basically imply to measure the respective pressure between the mixture and the gaseous phase in equilibrium. At the same time it must be take into account that in the equation in which the water activity (aw) is defined as the ratio pw/Pt is the definition of the relative humidity of the gaseous phase. From this regard, values for the water activity (aw) were determined in which micro-organisms could grow or not. In the case of bacteria normally they can grow at water activities higher than 0.95 but in the case of fungi there are some that can grow even at such low water activities as 0.6. The importance of moisture and water activity (aw) imply that while preparing a substrate, it is necessary to consider the exact quantities to be added to the substrate in order to have the proper required for the system. Exercise 3.2 A solid-state fermentation will be developed to obtain protein enrichment of cassava flour. A strain of Rhizopus oryzae will be employed. This strain has 32% protein content on weight dry basis. The flour has moisture content of 12% and a starch content of 83% humid basis. As nitrogen source urea will be used. In addition, KH2PO 4 would also be used for the phosphorus requirements of 0.2% in dried biomass. Fermentation starts with a 10% w/p of a liquid inoculum of threedays old. The starting medium must have a 62% of initial humidity.

Calculate the quantity of the different components of the substrate that must be added. Make all the pertinent assumptions. Basis of calculation: 100 g humid substrate Total solid balance: a) Initial total solid in the substrate: 100 (1 - 0.62) = 38 g b) Initial cassava flour at to employ (assumed): 42 g c) Initial dry weight of cassava flour: 42(1 - 0.12) = 37 g d) % starch fermented from the cassava flour (assumed): 50 e) Fermented cassava flour: 37 (0.5)= 18.5 g f) Yield biomass/substrate (Yx/s) (assumed): 50% g) Biomass produced: 18.5 (0.5) = 9.25 g h) Protein produced: 9.25(0.32) = 2.96 g i) Nitrogen required in the process: 2.96/6.25 = 0.47 g j) Urea required in the process: 0.47/0.466 = 1.01 g k) KH2PO 4 required: 9.25 (0.002)/0.23 = 0.08 g Checking the total solid to add: 37 + 1.01 + 0.08 = 38.09

(see a)

Water balance: a) Total water required in the substrate: 100(0.62) = 62 ml b) Water added with the cassava flour: 42(0.12) = 5 ml c) Water added with the inoculum: 100(0.1) = 10 ml d) water to be added: 62 - 5 - 10 = 47 ml Substrate formulation with inoculum included: 42 g cassava flour 1 g urea, 0.08 g de KH2PO 4 47 ml water From the Exercise 3.2, it is observed that the media formulation in SSF is a little more complicated that when dealing with submerged fermentation conditioned for the initial moisture or the water activity that must be observed.

3.5 pH pH is one another among the other most important factors for any fermentation process. Each micro-organism possesses a pH range for its growth and activity with a optimum value in between the range. A very noticeable example about how pH influences in the microbial development is offered by the use of Lactobacillus sp. as preserving of storage fibers. The growth of the micro-

organism results in lowering the pH to level of ca. 3. This low pH avoids the growth of parasite micro-organism that could spoil the material, pH control is one of the problems not yet solved in SSF processes. This problem is due to the lack of proper equipment and electrodes to determine the pH in solid materials and the existence of pH gradients due to the heterogeneous characteristics of the process. An attempt to overcome the problem of pH variability during the SSF processes is obtained by the substrate formulation considering the buffeting capacity of the different components employed or by the use of buffer formulation with components that have no deleterious influence on the biological activity. In general, it can be observed that fungi have pH ranges for growth between 3.5-6.0, yeast between 4.5-7.0 and bacteria a little higher than the last ones. However, this should not be taken as a rule. It should be kept in mind that some Lactobacillus sp. and other bacteria can grow at such low pH as 2.

3.6 AERATION AND AGITATION These two factors have generally a determinant influence in SSF due to two fundamental aspects: demand in the aerobic processes, 9 heat and mass transport phenomena in a heterogeneous system.

9

0 2

02 demand does not present the difficulties from the physiological point of view observed in the submerged fermentation. It is not necessary to have mechanisms, physical or chemical that allow that the 02 be dissolved previously in a liquid phase before it could be employed for the metabolic process. In this sense, 02 demand in SSF can be satisfied with relatively low aeration levels. Considering the aeration levels, it is very important that they can be expressed through intensive and not extensive values in order to make independent levels from the scale in which the process is developing. A criterion to express the aeration in a particular system is obtained by the airflow intensity or VKgM. This value is the airflow expressed by unit substrate weight, commonly as 1 air Kg-lm -1. It must be made clear that the weight considered for the calculation was on dry or wet basis. Once the level of aeration is found as an intensive factor, this value could serve as aeration criteria for higher scales of the process. This indeed does not mean that the value can be taken as scale-up criteria, but could be used as a starting point at the new levels. Exercise 3.3 To develop the SSF of 100 g of substrate from the medium reported in Exercise 3.2 with an airflow of 2.5 l/h. Determine the

airflow that must be used to ferment 1 t of this substrate with a humidity of 60%. Solution:

Base for calculation 100 g substrate. VKgM: 2.5/(0.038)(60) = 1.1 1 air Kg-1

m -1

Airflow required for 1 t substrate: 1.1(400)(60) = 26400 1/h R. Other point to take into account with the aeration is the air quality at the entrance to the fermenter, in particular, the air moisture. Use of saturated air is a common practice to avoid substrate drying or change in water activity. This procedure is not always advisable. Airflow, as we will see in other chapter, is the main tool to transfer the heat evolved during the process. Water evaporation and heat transfer with the surrounding are the processes to keep the process temperature in proper limits. Obviously, a contradiction exists between the heat to eliminate and the moisture in the system, but as we will see, a variable flow of air and its quality could improve temperature control. As has been pointed out earlier, in heat transfer there occurs the existence of gradients owing to the heterogeneous nature of the system. Substrate heterogeneity causes too possible air canalisation. When air canalisation occurs, there will be non-aerated zones that could produce non-desired cell metabolism changes. A possible solution to solve heterogeneity problems is agitation. Agitation could improve the homogeneity and disrupt gradients. However, if it is fight that agitation improves homogeneity, there may exist problems related to the integrity of the microbial cell. This may be particularly true related to fungi mycelium. Some authors have reclaimed mycelium damage when agitation was used. Others have employed intermittent agitation, often with good results. In case of bacteria or yeast, agitation could favour the biomass drag related to airflow mainly when cells are not tight enough on substrate surface. It is, therefore, necessary to study agitation and analyse its influence particularly for a determinate process.

3.7 PARTICLE SIZE Particle size of the substrate has a particular importance related to substrate characterization and system capacity to interchange with microbial growth and heat and mass transfer during SSF process. It determines several parameters through which a heterogeneous system is characterized. One of these parameters is the void fraction (8), which is defined as:

t3-

V V0

where: (e): void fraction of the system. (Dimensionless) V : volume empty space with particle. (m 3) Vo: void volume. (m 3) In relation to particle size, however, it must be kept in mind that in SSF processes it does not remain constant and tends to diminish. Mycelium formation is the main cause for void fraction variation. This is particularly true in fermentation in which fungi are used. This variation could even provoke canalisation during the process. Matrix, or solid substrate characteristic is closely related with SSF systems. One of these characteristics is the surface on which the process develops. Several types of particle were employed. Synthetic materials such as Amberlite pearls or natural materials such as agro-industrial residues (e.g. sugarcane bagasse) can also be used as matrix for SSF processes. When dealing with agro-industrial natural materials, generally it is a practise to pre-treat them to achieve improved microbial attack. Reduction in particle size through milling is one of such pretreatment, which provides larger surface area for microbial growth. However, it must be underlined that particle size could show an optimum range. Very tiny particle could produce matrix contraction or compaction, enhance channelling problems, increasing mass and heat transfer problems etc. Considering first synthetic materials as matrix for SSF process and presuming that all the particles are spherical and equal, superficial area is defined as"

Sp = gD 5 Where: Sp 9 superficial area (m 2) Dp: sphere diameter (m) The superficial volume of the particle (sphere) is defined as: Vp =

1

6

rtD3p

Where: Vp: superficial sphere volume (m 3) From the above relations, the ratio surface/volume for a sphere can be obtained

as" Sp

6

When dealing with non-spherical particles, superficial surface and superficial volume are defined as" Sp = 6b D 2p

and

Vp -9

6a D 2p

respectively. In the last expressions, a and b are constants that express in some way the form of the particles and are determined experimentally. More useful is the ratio between the superficial surface/superficial volume obtained for any irregular particle from these expressions as:

Sp

6q)s

Vp

Dp

Where Cps = b/a 9s is called as the form factor. The form factor is estimated from measurements of particles in their possible dimensions, as for example, width and length. The reader could look for these methods in any book dealing with Chemical Engineering Process. As it is obvious, the form factor for a sphere is 1. What is important to distinguish in this section is that the above definitions and other complementary ones not only characterize the solid substrate but must be taken into account when dealing with such problems as mass and heat transfer in SSF processes. As a general conclusion from this chapter, the reader must keep in mind that all those factors that may affect the good

development, or a particular activity of any micro-organism. Such factors as the influence of growth factors, inhibition substances, growth promoters, etc., need to be considered as particular demands of a particular process. When considering these factors, it must take into account the particular characteristics of the solid matrix in SSF processes and its heterogeneous form.

ENERGY BALANCE IN A SOLID-STATE FERMENTATION PROCESS

3.8 INTRODUCTION One of the principal problems faced in solid-state fermentation is to describe the way in which the energy evolved in the process could be evacuated. Undoubtedly, this is a problem that determines not only the development of the process but its economic possibility too. As it could be expected, the energy produced during the fermentation is responsible for factors such as the temperature rise in the system, air demand, specific growth rate of the micro-organism employed and even the type of reactor to be used. In this sense, it is extremely important to perform an overall energy balance that may allow establishing these relationships among the factors already postulated.

3.9 OVERALL ENERGY BALANCE When an energy balance is attempted in a solid-state fermentation process, it must be taken into account the following mechanisms: -

Heat evolved that dissipates as heat transfer from the solid substrate to the air that flows through the bed. Heat transfer from the wall of the reactor to the surroundings. Metabolic heat produced during the fermentation.

In the first mechanism, it is very important to consider the air conditions such as air humidity, air temperature and air flow, while the second one is directed by the heat transfer coefficient through the fermenter wall and the temperature gradient between the system and the surroundings. In a macroscopic energy balance, these conditions are considered but it requires the estimation of the heat evolved during the process. The macroscopic balance in the fermenter at steady state is as follows" Ein + Eme t = Eou t + Esurr

Where:

Ein: energy in the inlet air (kcal/h) Emet: energy released by microbial metabolism (kcal/h)

3.1

Eout: energy in the outlet air (kcal/h)

Esurr: energy loss to surroundings through the wall of the fermentor (kcal~)

The energy in the air (Ein and Eout) may be estimated from the enthalpy of air. Air enthalpy is generally defined by: i=

3.2

(0.24 + 0 . 4 5 H ) T + ~, H

Where: i

~

H"

enthalpy of humid air (kcal/kg dry air). absolute humidity of air (kg water/kg dry air). latent heat of vaporization of water (kcal/kg water).

In the Equation 3.1, the energy loss to the surroundings is expressed as: 3.3

Esurr'- hA (Tou t - Tsurr)

Where: h A

9 heat transfer coefficient of the fermenter wall (kcal/m2/h/~ 9 wall surface area of fermenter (mZ). Tout " temperature of the outlet air (~ T~urr" emperature of the surroundings (~

Substituting Eq 3.2 and 3.3 in 3.1, rearranging and considering Emet as Qm it may be deduced that: W g

Qm - hA ( T~ =

- Tin )

0 . 2 4 ( T o u t - Ti~ ) + 0 . 4 5 ( ToutHou, - Ti, n ~ ) + ~( H out- H ~, )

3.4

In Eq. 3.4, it was considered that the temperature of inlet air was the same as the temperature of the surroundings and that Wg was the mass velocity of dry air (kg dry air/h). Furthermore in Eq 3.4, the t er m 0.45(Toutnout-Tinnin) corresponding to metabolic water production could be overlooked, therefore, Eq. 3.4 can be simplified to: Qm - h A ( Tout - T~. ) Wg =

0.24( Tou, - T~, )+ /q,( nou~ " H ~ , )

3.5

In Eq. 3.5, the term Qm may be estimated from the Eq. 3.1 already deduced which is related to the biomass synthesis rate (Rb). In relation to the biomass

synthesis appraisal for a particular process, it is required the model that describes such synthesis. Several models are reported for characterizing this behaviour in solid-state fermentation and among them the archetypal model that describes the exponential growth phase was also reported. Thus, if the model for the exponential growth could be applied, it may be postulated that: 3.6

X = Xo e~t

Where: X Xo kt t

9 9 9 9

cell mass produced at a particular time (kg) initial cell mass (kg) specific growth rate at the exponential phase (h-1) time (h)

Hence, from Eq. 3.6:

Rb-

dX dt

3.7

- t-tX oe ut

Relating the Eq. 3.5, 3.7 and 3.1, following is obtained:

Q ~ (1-rlT1)tYb 7 b l't

o e I.tt_ hA(Tout- Tin)

3.8

Wg= ~(nout

-

H in) @ 0.24(Tout- Tin)

It must be kept in mind that if other model is used to represent the growth of the micro-organism and not the exponential one, the procedure would be the same requiring solely that the Eq. 3.6 and 3.7 be defined for the new assumption. The inferences that could be done from Eq. 3.8 are remarkable. First of all this expression allows to consider the specific growth rate (it) of the micro-organism employed in the process as a function of the air demand and air quality, both of them acting as regulating factors. Accordingly, air demand and air quality must be considered variable components of the process. In this sense, they could be a medium for attempting the control of solid state fermentation processes. It must be denoted that normally the solid state fermentations reported anywhere consider the air demand of the process and its quality as constants.

not differ very much, this value was assumed as fair. The absolute humidity (H) and the latent heat of vaporization of water ()~) was taken from the psychrometric chart and the thermodynamic table as 0.036 kg water/kg dry air and 560 kcal/kg water, respectively, assuming saturated conditions and an average value for the latent heat of vaporization of water. Substituting the required values in Eq. 3.8 is obtained the expected expression for the heat evolved in this system as:

AQ Rq = ~

At

= 0.39

t.t e ~t

3.9

The general expression that considers the whole system for this particular process may be obtained from Eq. 3.8 and 3.9 and from the values already discussed:

0.39 t.te ut- 0.58(Tou~- 35) Wg =

0.24(Tou, - 35) + 560(H out-O.O36)

3.10

The Eq. 3.9 showed an exponential pattern like it was expected considering that it was assumed an exponential model for the growth of the micro-organism. On the other hand, in Eq. 3.10, the dry air demand (Wg) is expressed as a function of outlet temperature (Tout), the maximum specific growth rate deduced from the exponential phase (g) and the absolute humidity of the exhaust air (Hour). For resolving this equation, the variability of the absolute humidity (Hour) as a function of the exhausted air temperature from the fermenter must be calculated. From the definition of the absolute humidity and considering that the exhaust air that leaves the reactor is saturated, following expression is depicted:

H=

0.62 Ps 1-Ps

3.11

where" H

Ps:

~

absolute humidity (kg water/kg dry air) vapor pressure of saturated air (atm)

An empirical expression was reported for the partial pressure of saturated air (Ps) as a function of temperature from the modeling of the psychometric chart, so:

.

ax+bx2+Cx 4

p= = 2 1 8 x 10t(!.,.d~(a47-x~ ~

3.12

Where the coefficient values in the empirical equation are: a: b: c: d:

3.2438 5.8683 x 10-3 1.1702 x 10-8 2.1878 x 10 -3

and: x = 3 4 2 - Ts

3.13

Where: Ts: temperature of saturated air (~

It must be noted that this last expression relates the partial pressure of saturated air with temperature through Eq. 3.12, and hence the absolute humidity with temperature through Eq. 3.11. Other parameter that must be calculated is the dry air mass velocity (Wg), which was considered constant during the whole process and was estimated from the constant airflow employed (4 l/h) as already reported. Its value was 0.036 kg dry air/h. For this estimation it was considered that the inlet air was saturated at 35 ~ There are two possibilities for solving the equation system offered by Eq. 3.10 to 3.13. Specific growth rate (kt) could be considered as an independent variable or as a constant value during the process. In both the conditions, the system was solved for the outlet temperature. In latter case when we assumed the specific growth rate as constant, the value selected was 0.30 h -1. The results for the predicted temperatures are shown in Table 1. As it may be seen from the table, specific growth rate showed a tendency of decrease during the fermentation and after the first eight hours, it was 50% of the initial value. It could be quite possible that this pattem responded to the evolved heat in the process. As we may see, for a theoretical value for the specific growth rate of the order of 0.3 h-1 after 10 h of fermentation, the temperature would be of the order of 40~ This meant that it could be supposed that the process was auto regulated as has suggested by Eq. 3.10. One important conclusion from this behaviour is the need of change in the air demand, or the air quality during the process according the Eq. 3.10 in order

to preserve high specific growth rates and not to develop the process at constant air flow rates, or constant air quality as is often done. Considering the variation of the air flow, or the quality of air in the process, it is very important not to overlook the effect that the substrate humidity produces over the microbial growth, because the air quality determines the water mass transfer between the substrate and the flowing air and therefore, it could produce a drying in the system at a level that may not be allowed. Another conclusion not to be overlooked is to consider the design of reactor, or fermenter that allows a better heat transfer in order to diminish air flow requirements. As a general conclusion, it may be stated that the method here reported for the modeling of solid-state fermentation process seems to be fairly good and could bring a basis for the development of a mechanism of control in such processes.

Table 1. Comparison between the values reported in solid state fermentation system of cassava and the values estimated by the macrscopic heat balance based in the process electron balance,

*values estimated considering p at a value constant of 0.30 h-~

.................................................

...............

................

NOMENCLATURE A

Ein greet Eout surr

H h i 1

Ms N N Nb

No Ns nc nil ni

nn no

P Ps

Q Qo Qm

q

Ro

Rq Tin

Tout

Ts t X

Xo Ys

wall surface area of fermentor (m 2) energy in the inlet air (kcal/h) energy released by microbial growth (kcal/h) energy in the outlet air (kcal/h) energy exchanged with the surroundings (kcal/h) absolute humidity of air (kg water/kg air) heat transfer coefficient of the fermented wall (kcal/m2/h/oC) enthalpy of humid air (kcal/kg dry air) atomic ratio of oxygen to carbon in a substance (dimensionless) biomass produced in a period of time (kg) consumed oxygen in a period of time (kg) consumed substrate in a period of time (kg) available electrons in a molecule/atom carbon (equiv. available electrons/g atom carbon) available electrons in a molecule equiv, available electrons transfered to biomass equiv, available electrons transfered to oxygen equiv, available electrons transfered from substrate number of atom carbon in a molecule number of atom hydrogen in a molecule number of a particular atom in a molecule number of atom nitrogen in a molecule number of atom oxygen in a molecule atomic ratio of hydrogen to carbon in a molecule (dimensionless) vapor pressure of saturated air (atm) heat evolved during a period of time (kcal) heat evolution per equivalent of consumed oxygen (kcal/g equiv.) rate of metabolic heat (kcal/h) atomic ratio of nitrogen to carbon in a substance (dimensionless) rate of biomass synthesis (kg/h) rate of oxygen consumption or respiration rate (kg/h) rate of heat evolved in the process (kcal/h) rate of substrate consumption (kg/h) temperature of inlet air (~ temperature of outlet air (~ temperature of saturated air (~ time (h) mass velocity of air (kg dry air/h) cell mass produced in a particular time (kg) initial cell mass (kg) yield based on oxygen consumption (dimensionless) yield based on substrate consumption (dimensionless)

I[ll % 1] ~t t~b O"s

i~~

....

~

ii!J~ii~ii~

!!}j

~~iik!iiF .....ii.......!i..!.Jl..i...Jjii........Ji..!.iH...ii.i!.i.!i.!.i!....ii!i ........iii}i ...........

reductance degree of dry biomass (equiv available electrons/g atom carbon) reductance degree of substrate (equiv. available electrons/g atom carbon) biomass energetic yield or fraction of energy in the substrate which is converted to biomass (dimensionless) specific growth rate in the exponential phase (h-1) latent heat of vaporization of water (kcal/kg of water) weight fraction carbon in biomass (dimensionless) weight fraction hydrogen in substrate (dimensionless)

References Auria R, Palacios J & Revah S, 1992, Determination of the interparticular effective diffusion coefficient for CO2 and 02 in solid state fermentation. Biotechnology and Bioengineering, 39, 898-902. Carrizalez V, Rodriguez H & Sardifias I, 1981, Biotechnology and Bioengineering, 23, 321. Dale BE, Beba A, M'Nasria H, Alvarez-Martinez R, Nebot E, Nazmul Kerim M & Tengerdy RP, 1987, Memorias Simposium Latin Biotecnology, Antilla, Guatemala, p. 89. Desfargues C, Larroche C & Gross JB, 1987, Biotechnology and Bioengineering, 29, 1050. Erickson LE, Minkievich IG & Eroshin VK, 1978, Biotechnology and Bioengineering, 20, 1575. Erickson LE, Minkievich IG & Eroshin VK, 1979, Biotechnology and Bioengineering, 21,575. Minkievich IG & Eroshin VK, 1973, Folia Microbiologica, 18, 376. Minkievich IG, 1983, Biotechnology and Bioengineering, 25, 1267. Narahara N, Koyama Y, Yoshida T, Atthasapunna P & Tagushi H, 1984, Journal of Fermentation Technology, 62 (5), 453. Okasaki N, Sugama S & Tanaka T, 1980, Journal of Fermentation Technology, 58 (5), 471. Rairnbault M, 1981, Travaux et Documents de l' O.R.S.T.O.M. no. 27. Ramana Murthy MVR, Karanth NG & Raghava Rao KSMS, 1993, Biochemical engineering aspects of solid-state fermentation. Advances in Applied Microbiology, 38: 99-147. Rathbun BL & Shuler ML, 1983, Heat and mass transfer effects in static solid-substrate fermentations: Design of fermentors chambers. Biotechnology and Bioengineering, 25, 929-938.

Rodriguez Le6n JA, Bechstedt W, Echevarria J, Sierra N, Delgado G, Daniel A & Martinez O, 1986, Optimization of solid state fermentation of citrus dried peel by Aspergillus niger in a packed bed column. Acta Biotechnologica, 6 (3), 253-258. Rodriguez Le6n JA, Echevarria J, Sierra N, Daniel A & Martinez O, 1985, Solid-state fermentation of dried citrus peel. Biotechnology Letters, 7 (8), 577-580. Rodriguez Le6n JA, Garcia I & Sierra N, 1983, Thermal methods to study transformations of citrus wastes by solid fermentations, Boletfn Informaci6n Cientffico T~cnica del Instituto de Qufmica y Biologfa Experimental de la Academia de Ciencias de Cuba. 3 (1), 19-21.

Rodriguez Le6n JA, Sastre L, Echevarria J, Delgado G & Bechstedt W, 1988, A mathematical approach for the estimation of biomass production rate in solid state fermentation. Acta Biotechnologica, 8 (4), 299-302.

Rodriguez Le6n JA, Sastre L, Echevarria J, Delgado G & Bechstedt W, 1988, Acta Biotechnologica, 8 (4), 299-302. Rodriguez-Leon JA, Domenech F, Leon M, Mendez DE & Pandey A, 1999, Production of spores of Trichoderma harzianum on sugar cane molasses and bagasse pith in solidstate fermentation. Brazilian Archives of Biology and Technology, 41 (1), 69-76.

Rodriguez-Leon JA, Torres JA, Echevarria J &Saura G, 1991, Acta Biotechnologica, 11,9-14.

Rodriguez-Leon JA, Torres JA, Echevarria J & Saura G, 1991, Energy balance in solid state fermentation processes. Acta Biotechnologica, 11, 9-14. Sarrette M, Nout MJR, Gervais P & Rombouts FM, 1992, Effect of water activity on production and activity of Rhizopus oligosporus polysaccharides, Applied Microbiology and Biotechnology, 37 (4), 420-425. Sato K, Nagatani M, Nakamura K & Sato S, 1983, Growth estimation of Candida lipolitica from oxygen uptake in solid state culture with forced aeration. Journal of Fermentation Technology, 61 (6), 623-629. Saucedo-Castafieda G, Guti~rrez-Rojas M, Bacquet G, Raimbault M & ViniegraGonz~ilez G, 1990, Heat transfer simulation in solid substrate fermentation. Biotechnology and Bioengineering, 25, 802-808. Saucedo-Castafieda G, Lonsane BK, Navarro JM, Roussos S & Raimbault M, 1992, Importance of medium pH in solid state fermentation for growth of Schwanniomyces castellii. Letters in Applied Microbiology, 15, 164-167. Smits JP, Rinzema A, Tramper J, van Sonsbeek HM, Hage JC, Kaynak A & Knol W, 1998, The influence of temperature on kinetics in solid-state fermentation. Enzyme and Microbial Technology, 22, 50-57.

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of S o l i d - s t a t e

Fermentation

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4.1 INTRODUCTION Kinetic studies in SSF are generally considered difficult as the heterogeneous nature of the SSF processes poses difficulty in biomass estimation. This is because the biomass cannot be separated from the fermenting substrate, a fact, which does not allow to obtain a homogeneous sample during the fermentation. These problems are particularly acute in the case of fungal growth and mycelium production. In this chapter we shall study different alternatives that allow kinetic performing in SSF in an objective and satisfactory manner. Undoubtedly, kinetic process is the procedure that describes the development of the process. Even with the difficulties that are encountered in SSF processes, kinetic procedure cannot be substituted by goodwill, subjectivism, or even by the simple and overall process description. Although the reader can consult several books related with submerged fermentation in which particular concepts of kinetics in fermentation are dealing, we prefer to introduce these concepts in a simple and gradual manner. The fundamental objective is that the reader could distinguish between the validity of a concept and the possible difficulty in its implementation. The importance of kinetic resides in the fact that these studies imply the determination of important parameters such as: 9 9 9 9 9

specific growth rate process yield process productivity heat evolved process control criteria

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9 strategy for the production of a particular product scale-up considerations

9

4.2 SPECIFIC GROWTH RATE A kinetic study or the kinetics of a process is determined by the variation of one or several variables during the fermentation time. From this point of view, any available (measured) variable can be chosen to determine kinetic characteristics. The selected variable will be related with other factors in the process. It must take into account how these factors are related and which is dependent or independent factor. Factors identification and their relationships eventually will permit the establishment of appropriate models describing the process or part of it, postulate studies for process optimization, determine control criteria etc. It must be underlined that kinetic means necessarily the determination of at least one variable with the time bearing in mind that time; at the same time, could be explicit or implicit in the models, which represent the process kinetic. In a fermentative process, the variable used to establish the process kinetic has been usually the variation of biomass (synthesis) in the system, variation of substrate content, variation of a product (metabolite), determination of 02 consumed or CO2 evolved, heat evolved, etc. Among these variables, biomass synthesis during the process and substrate consumption determination has to be conducted following the procedures that represent kinetically any fermentation process. Considering the biomass variation during running time in the process, in a general manner it can be said that this variation depends on several factors, hence, dx

~ = f ( x , s , T , etc.) dt

4.1

Where: X

~

t

9

S

"

T"

biomass concentration (g biomass/1) time (h) substrate (g substrate/1) temperature (~

First term in the equation 3.1 is a velocity or kinetic term. It represents a unit change (in our case biomass concentration) by time unit. Figure 4.1 shows a characteristic curve showing biomass synthesis in a process.

Figure 4.1 Variation: (a) cell concentration, and b) substrate content during a batch submerged process employing a strain of Lactobacillus acidophilus

From Figure 4.1, the process development can be described in a very simple manner and one can determine parameters such as: 9 9 9 9

Maximum cell concentration obtained in the fermentation Time delay in the rise of biomass content in the reactor Substrate consumed in the process at any time Yield biomass/substrate obtained in the process

However, although kinetic is implicit in the data, we cannot say anything about parameters that characterize it in a proper way. Monod (1942) was one of the first scientists that described mathematically a fermentation process. He proposed a model that related biomass synthesis with substrate consumed. The model is:

S

~--~max gsq-S

4.2

Where: specific growth rate (h-1) maximum specific growth rate (h -1) ktrnax : S : substrate concentration (g/l) Ks : ffinity constant biomassa/substrate (g/l) In the model proposed by Monod (4.2), time was not explicit but included in a term called specific growth rate (kt). This variable (as the name indicates) is related with velocity or rate (dx/dt) as well as with the intensity relating the velocity or rate term with the quantity of biomass present at a particular time

in the fermenter. The model proposed by Monod is, of course, process dependent. The parameters, It, ~tmax and Ks determine values for a particular process. At the same time it must be pointed out that specific growth rate is a definition and hence, is independent of the process. This definition is expressed as:

1 dX

//=~~

X dt

4.3

Where: X:

t :

biomass concentration at a particular time t (g/l) time (h) specific growth rate (h-1)

Specific growth rate (It) defined t~ough Eq. 4.3 permits the representation of kinetic pattern of microbial growth through different phases, known as growth phases and are expressed as: ,~

Lag phase: In this phase, the specific growth rate is practically null (~t = 0) and represents a stage in which microbial biomass do not start to multiply. The time this phase last is one of the characteristics to determine in the fermentation process. The period till which this phase lasts depends up on factors such as physiology of the micro-organism, environment, type of inoculum, etc.

Accelerated growth phase: This phase is characterized for the initiation of growth by the synthesis of new cells. During this phase m is >0. The lasting time of this phase is relatively short considering the whole process.

Logarithmic or exponential growth phase: This phase is characterized by the fact that the specific growth rate is constant during the whole phase. Logarithmic specific growth rate is at the same time the highest specific growth rate that can be attained in a fermentation process. This is the parameter that first characterizes fermentation from a kinetic point of view.

Desacelerated growth phase: In this phase, the biomass synthesis rate starts to demit in the process and it can be postulated that ~Lmax > ~ > 0.

Stationary growth phase: In this phase, a equilibrium is established between the quantity of cells that are synthesized and the quantity of cells that are no longer viable or loose their capacity to reproduce. In this phase it can be postulated that ~t = 0.

Negative growth rate phase: In this phase, more number of cells loose their capacity of reproduction or viability than the cells that can reproduce. In this phase the growth can be represented as kt < O. The study of each of the microbial growth phases can be done through the application of Eq. 4.3 using different available mathematical tools. A specific mathematical procedure can be found in the works of Kono and Asai (1969). Anyway, readers should keep in mind that the definitions given for the different phases could change according to different authors but their significance and proper mathematical definitions not.

4.3 MICROBIAL LOGARIHTMIC OR EXPONENTIAL GROWTH PHASE The microbial logarithmic or exponential growth phase is, as said before, the most important phase that distinguishes fermentation. It is characterize by the specific growth rate (kt) as constant and reaches the maximum value that can be obtained in the process. Is this value (kt = ktmax) a parameter that identifies immediately the significance of the process? Customarily when it refers about specific growth rate with anymore specification, it refers to the specific growth rate that belongs to the logarithmic phase that is the maximum specific growth which can be attained due to particular conditions. All through this book this appraisal will be considered except when other indications are given. Certainly the specific growth rate (kt) is a function of several variables or factors such as: temperature, pH, substrate and in solid state fermentation of substrate moisture and water activity (aw). Applying to Eq. 4.3, the statement that in the logarithmic or exponential phase kt = k, where k is a constant, can be stated that:

f

x dx

t

4.4

A from Equation (3.4):

In X-- =~tt Xo

4.5

Where: biomass concentration at time t of the logarithmic or exponential growth (g/l) Xo: biomass initial concentration at time t = to (g/l) ~t: specific growth rate at the logarithmic or exponential phase (h-i) t : time that corresponds to the biomass concentration X (h) X"

Regarding the initial biomass at time t = to, it must be recalled that in strictly sense, this corresponds to the biomass concentration at the initial exponential or logarithmic growth. Basically, the initial concentration at the beginning of the process is practically unchanged and therefore, initial biomass content can be taken as the value for the calculations. From Eq. 4.5, it is easy to obtain the doubling time value. This is other parameter that characterizes fermentation and it is the time that takes biomass concentration to achieve its double value. For the calculation, we considered that X = 2 Xo y to = 0 and integrating Equation 3.5 gave: In 2

0.693

t d = ~ = ~ ~[,/ max ~,/max

4.6

Where:

td: doubling time (h) Also, a very important parameter, the biomass productivity (P) related to the biomass synthesis, can be obtained considering the value of the specific growth rate (g). Biomass productivity is defined as: P = pX

4.7

Where: P: biomass productivity (g biomass/1 h) Other parameter directly related to the specific growth rate is substrate concentration although in its definition the specific growth rate is not explicit, the yield is based on substrate consumption (Yx/s) and is defined as"

So Yx/~ =

-

St

~ Xs-Xo

Where: Yras : yield based on substrate consumption (dimensionless) So : initial substrate concentration (g/l) St : final substrate concentration (g/l) X0 9 initial biomass concentration (g/l) Xt : final biomass concentration (g/l)

4.8

From the definitions, it could be concluded that the concepts and corresponding mathematical models or expressions were defined not considering the type of fermentation. In this sense, all these statement are equally valid for submerged fermentation as well as for solid- state fermentation. The only problem to overcome in case of solid-state process is the adequacy of proper measurements that allows the estimation of these parameters. Exercise 4.1 Determine from data reported in Figure 4.1: a) the beginning of the logarithmic phase, b) time at fermentation should be stopped case biomass synthesis is the only purpose of the fermentation, c) the yield based on substrate consumption, d) the initial of the stationary phase, e) the maximum specific growth rate that was attained in the process and f) time in which biomass duplicates. Solution:

To solve the present problem the following table is made from the date reported in Figure 4.1.

Note that negatives values for In X simple correspond to the natural logarithmic of decimal value and obviously do not alter the values to determine for the corresponding parameters, v. gr. ktmax. A graphic representation of In X vs. t allows a much easier estimate of the different questions asked in the problem and a more clearly picture of the process. Reader is suggested to compare this figure with Figure 4.1. Calculations: a) Beginning of the logarithmic phase: From the constructed graphic, it is observed that the exponential phase begins four hours after the beginning of the fermentation.

Fig 4.2 graphic representation of In X vs. t

b) Time at fermentation should be stopped because biomass synthesis is the only purpose of the fermentation: It is the corresponding time at the end of the exponential phase. From the figure it can be seen that the logarithmic phase ended at 10 h. This is the time when fermentation must be stopped. Going further is unproductive as we are concerned with biomass production only. c) Yield based on substrate consumption Yx/s" zSX

Yx/x=~=

2.45 - 0.08

10.0-5.1 overall, dimensionless)

=0.4837g de biomass/g de substrate consumed (as

Expressed as % Yx/s = 48.37 % It must be observed that to determine the yield, the final values in the yield expression correspond to the end of the logarithmic or exponential phase (in our case at 10 h). If we would consider all the time the fermentation ran, the estimate for the yield would be 30.54 %. This fact is very important to take into account. It'indicates that fermentation should be stopped at 10 h. After this time, endogenous process starts to became more important and consumed sugars are employed in major quantity than in exponential phase for this purpose. The reader must consider that in case a metabolite is the principle aim of the process, the analysis would be different mainly in case when secondary metabolites are produced. d) The initial stationary phase" From the graphic, it is taken at 14 h. e) Maximum specific growth rate (~)" The maximum specific growth rate that is reached in this process corresponds to the specific growth rate in the

logarithmic or exponential phase. From Eq. 3.5, it is obvious that the best way to estimate this value is making a lineal regression with minimum squared adjustment for In X vs t in time interval between 4 and 10. The slope corresponding to line fixing is the value of the specific growth rate, This value is constant during the whole phase and maximum (~max ---- ~). In our case: ~trnax = 0.540 h -1 r 2 = 0.9945 (It should be noted that the estimate was made considering only four points. Undoubtedly a better estimate could be done if there were more points to consider). f) Time in which biomass duplicates: This time is already calculated from Eq. 4.7, hence, 0.693 td

--

--

0.540

1.28h

4.4 KINETICS MEASUREMENT IN SOLID-STATE FERMENTATION As we have seen in the previous section, the concepts developed considering the parameters that characterize the development of a fermentation process do not have to take into account the type of the process considered. However, the way the original data need to be handled to estimate the different parameters, the corresponding mathematical expressions and the representative statistics that must be considered are strictly related with the kind of process. The principal kinetic pattern that is observed in a biochemical process is viewing the biomass synthesis during the time. In a submerged fermentation process this is normally done through measurement of biomass at a particular time by the so-called direct methods. Such methods include direct cell counting, dry biomass determination or optical density determination. However, these methods cannot be considered in a solid-state fermentation process due to the fact that biomass is partially, or completely attached, or imbedded to the solid surface. This causes difficulty in the measurement of whole biomass. Besides, the heterogeneous character of the system demands a more precisely acquirement of the samples, a fact that is not faced in submerged, or homogeneous fermentation. Considering all the above facts, several methods have been developed for biomass determination that we could divide as direct or indirect measurements. Direct

measurements are based on direct separation of biomass followed by normal standard procedures as established for submerged biomass determination (cell counting, etc.). The main problem with this method is the necessity of a whole extraction of biomass from the remained solid substrate. The employment of innocuous detergent in order to guarantee the whole extraction of the biomass from the sample was attempted. However, in the case of mycelium production, the method is not feasible due to the impossibility of a complete separation of biomass. Indirect measurements are based on the determination of a particular component of the cell, or the mycelium that is not present in the solid substrate. It includes: a) Glucosamine content determination: The method is based on the fact that glucosamine is a monomer component as acetylglucosamine of chitin. Chitin is an insoluble polymer present in the mycelium. The process consists in the depolymerisation of chitin, followed by the liberated glucosamine determination. Principal difficulties with this method are the lengthy analytical procedure, which takes about 24 hours and the sample adequacy as statistically representative. b) Ergosterol method: The method is based as the former one due to the presence of ergosterol in the biomass but presents same difficulties as glucosamine content determination. c) DNA determination: The basis of this method is the precise increase of the DNA content in the medium as the biomass develops due to the fact that DNA is a constant cellular component. The principal errors that could be made are related with sample adequacy and the possible consumption of cell DNA containing during the process. Another difficulty encountered is related with the DNA isolation and procedures with determination, which takes a long time. d) Protein determination: This may be the most intended method for direct estimation of biomass. The principal problems of this determination are how exactly protein content is determined and which part of the protein present in the substrate are not consumed, or transformed. It seems that when the solid matrix has no or high protein content, the method could work reliable. For protein estimation, it is usual to determine the N content by the Kjeldahl determination, previous precipitation of the present protein, but a more accurate procedure could be obtained by using an amino acid analyzer. As has been pointed out previously, all these methods are subject to an appropriate sampling. This is more significant at the initial stages of the process, or when

the problems with gradients are not solved. Besides, in particular processes biomass, or mycelium could be damaged by sample acquirement. This fact does not allow a response that could be taken into account to modify the process or to control it.

e) Metabolic Gas Balance Method: This method overcomes the sample adequacy, damage of biomass, or mycelium and is an on-line and fast delivery method. As a matter of fact, this method could be considered a direct measurement of the process's kinetics. 02 consumption and/or CO2 evolved during the process are linearly related with biomass synthesis in an aerobic system. But it is also an indirect method for biomass estimation. In an anaerobic process, CO2 evolved is a direct indication of biomass synthesis and associated product formation as it occurs in the alcoholic fermentation. The method considers the determination of exhaust gas (exhaust air) composition determination from the fermenter during fermentation. The procedure implies a balance for 02 and CO2 considering the airflow through the fermenter. A general scheme about volumetric composition of entrance and exhaust air in the fermenter is provided in Figure 4.3. The balance for 02 consumption considering this system is: 0 2

consumed

= 0 2

in

-

02 out

4.9

Assuming that in the reactor only pure air enters whose composition in % volumetric basis is 20.9 and 79.1 for O2 and N2, respectively. The volumetric flows for the entrance are obtained as: Vo2e "- (20.9/100) F e

4.10

VN2e -~ (79.1/100)F e

4.11

Where:

Vo2e: volumetric Oxygen flow at fermentor entrance (l/h) Vnze: volumetric Nitrogen flow at fermentor entrance (l/h) Fe " air flow at fermentor entrance (l/h) Volumetric flow at the exit for :each component of the air are estimated based on the volumetric composition of exit air and considering that this exhaust air is composed only by 02, CO2 and N2. Thus, we can postulate that: go2 s = (% O2s/100) Fs

4.12

VCO2sm~(% CO2s/100) Fs

4.13

!!!m % O2s

Fs % C02s %N2=100-%02-

%C02

% 02 = 20.9

Fe ~ N2 = 79.1

Figure 4.3. Volumetric composition of air in the flow at entrance and exit from a solid-state fermenter

VN2~= (100- % O2s - % CO2s)/100) Fs

4.14

Where: volumetric VCO2s" volumetric VN2s : volumetric Fs 9 volumetric Wo2 s

9

Oxygen flow at fermenter exit (l/h) Carbon Dioxide flow at fermenter exit (l/h) Nitrogen flow at fermenter exit (l&) airflow at fermenter exit (l/h)

The volumetric Oxygen consumed (VO2cons.) flow is obtained relating Eq. 4.9, 4.12 and 4.10 and reordering so: VO2~ons. = (20.9/100) F~ - (% O2s/100) F s

4.15

It must be noticed that air is a compressible fluid. This means that entrance and exit flow cannot be assumed to be the same because air density changes with air composition. The relation between F e and Fs is obtained considering that N2 is not consumed during the process, hence it can be stated that: gNzentra = VN2sal e

4.16

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Relating Eq. 4.11, 4.14 and 4.16, we obtain the relation between airflow at the entrance and exit of the reactor: 79.1Fe Fs =

4.17

%C02)

(1O0- ~

From Eq. 4.15 and 4.17, we obtain the volumetric oxygen consumed flow (VO2cons) considering the airflow measured at fermenter entrance as:

VOzcons = (0.209 -

0.791%02

(100- %02- %C02)

) Fe

4.18

The corresponding expression for the CO2 (WfO2evold.) evolved during the process is obtained in the same way. Assuming that there is no CO2 in the air at the entrance, the volumetric VCO2evold. is expressed as function of entrance airflow as: VCO2prod. =

0.791%CO2

Fe

(lO0-%Oz-%COa)

4.19

Molar, or gravimetric flow for respective gases in the air is calculated from the respective volumetric flows. Considering the volumetric 02 consumed and CO2 evolved and assuming that air at normal fermentation conditions can be assumed as a perfect gas, the molar or volumetric flows are readily achieved. From the ideal gas law it can be postulated that 1 mol of any component at normal conditions of temperature (273 ~ and pressure (1 atm) occupied 22.4 1. In this consideration, corrections for real temperature must be taken but for pressure can be neglected. Exercise 4.2 In a solid-state fermentation process at 30~ the entrance airflow and composition of exhausted air were measured as shown in Table 1. Represent in a graphic the gravimetric flow for oxygen consumed and carbon dioxide evolved during the process. From this data, it is very convenient to complement Table 1 as is reported in Table 2. Values reported in Table 2 for corrected entrance airflow (Fcorr.) correspond to correction made for temperature at entrance (30~ In this case, pressure correction was overlooked. Applying ideal gas law in this data and considering ideal gas constant as 0.082 atm 1/mol ~ the corrected entrance air flow (Fcorr.) is: Fcorr. = Fair(303/273) = 1.11 Fair 1/h

Table 1, Experimental data

Table 2. Results obtained from original data processing

VO2cons = (0.209-

V f O2evolv. -~

0.791%O2

(100- %02- %CO2)

0.791%CO2

(100- % 0 2 - %CO2)

Fcorr.

)Fcorr. 1 of O2/h

1 of CO2/h

moles O2/h = VO2cons./0.082 (303) moles CO2/h = VCO2prod./0.082 (303) g de O2/h = 32 VO2cons./0.082 (303) g de CO2/h = 44 VCO2cons./0.082 (303) From the data as above, the obtained graphic would be as shown in Figure 4.4:

Figure 4.4. 0 2 consumed and CO2produced 4.5 OXYGEN BALANCE DURING MICROBIAL GROWTH In every fermentative process, it is important totake into account, which part of the substrate, or energy obtained by biodegradation (redox processes) from the substrate is employed in the_endogenous process, which part is used for biomass reproduction and which part is employed in synthesizing non-constitutive metabolites of biomass. It is pointed out that the endogenous process is characterized by the synthesis of the material of each cell not considering cell duplication. The pattern of consumed oxygen during the whole fermentation in an aerobic process is an overall pattern that considers the oxygen consumption for growth and maintenance. Cell maintenance represents the endogenous metabolism and their significance implies that a balance for oxygen consumption must be done.

!!!i!!!! ,i i!, In general terms and considering a process in which only biomass and not extra-cellular metabolites are produced, it can be stated that: 02 consumed = 02 employed in + O2 employed in biomass endogenous synthesis process

4.20

The consumed oxygen can be calculated by the procedure already shown above, which considers the composition of air at the entrance and exit of the solid-state fermentation reactor and the particular flow for each component. The Oxygen Uptake Rate (OUR) in the process is defined as:

RO2cons =

AO2 ~

4.21

At

Where:

RO2 cons" 0 2 consumed at time interval Dt (g 02 employed in biomass synthesis (02

RO2 sin t

AO2 sint. -- ~

1

sint.)

O 2 / t l ).

is obtained by the expression:

AX

4.22

-"

At

Yx / o A t

Where: RO2sint. : 0 2

employed for biomass synthesis (g

O2/11 ).

Yrao

9 yield based on consumed O2 for biomass synthesis at time interval considered (g biomass/g O2)

DX

: biomass synthesized during time interval considered (g)

At

: time interval considered (h)

In Eq. 4.22, the 02 consumption rate (OUR) is related with the coefficient that defines the yield based on oxygen consumption (Yx/o). This yield can be defined through Eq. 4.8 considering 02 as the substrate in that expression. The oxygen employed for the endogenous process is estimated from the expression defined by Monod in which is employed the concept of maintenance coefficient as:

m-~ 1 X

eno

dt

4.23

Then considering in the former expression the 0 2 as substrate it can be postulated that:

RO2endog. =

mO2endogeno At

= mX

4.24

Where:

endog" O2 employed in endogenous process (g O2/h).

RO 2

m

9 maintenance coefficient (g O2/g biomas h).

X

9 biomass at a particular time (g).

Relating Eq. 4.20, 4.22 and 4.24, we get:

AO2 At

1 AX Yx / o At

+ mX

4.25

Equation 4.25 allows to estimate the quantity of biomass that exists at a particular time of the fermentation from the data of consumed 02 by finite determination of this consumption at particular time intervals From Eq. 4.25, it can asserted that:

-0

RO .... dt =

dt + mxdt -0 Yx i o -0

4.26

Term ~ R O 2 cons in Eq. 4.26 indicates that consumed 0 2 in time t=n can be evaluated from the relation between the oxygen consumption rate (OUR) and time. In Exercise 4.1,1this value is the area under the curve reported in Figure 4.1 in which it represents the OUR vs. t. A first explicit solution of Eq. 4.26 could be obtained if the function, which represents the curve reported in Figure 4.1 would be available. However, the solution of the differential Eq. 4.26 was postulated by numerical methods. Applying the Trapeze Rule to the numerical integration of the first term in the expression, we get:

;,=o dOzdtdt '="

(

-2At ( dO2)' =~

+ 2((dO2dt

(dO2)2 + ' " + - Z(dO2)n - 1)

~d02 "~

+(-;t ).)

4.27

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iiii/

Where:

dO2)

--'~-.t = i "

02

consumed at the time interval i (g O2]h ).

At: time interval considered for the estimation (h). It can be postulated that considering the yield based on consumed 0 2 as constant (Yx/o = k) is obtained as:

i ;-,, y~~odX = 1 (X,-Xo) -o Yx l o

4.28

Where: X 0 : biomass at the beginning of the time interval At (g). Xn : biomass at the end of the time interval At t (g). The value of last term in Eq. 4.27 is achieved employing the same procedure already used for the determination of Eq 4.28:

S,

=o

mxdt = m

,',( 2

Xo +

+ X

+ ...X~ -1) + X~

)

4.29

It must be underlined that in the deduction of Eq. 4.29 the maintenance coefficient was considered also as constant (m = k) during the time interval. Relating Eq. 4.26, 4.27, 4.28 and 4.29 and rearranging, the following equation is attained:

=

2

at

=0+

=

i--1 'T-

--

-

-

i--1 4.30

Where to simplify the Eq. 4.30, it was considered that: a = mYx/oAt

4.31

The reader must keep in mind that the yields based on oxygen consumption as the maintenance coefficient are considered constant during the time interval evaluated. Eq. 4.30 allows to estimate the biomass value at a determined time t = tn if the preceding values of biomass are already known during the period. As it is noticeable, if previous values for biomass are unknown and the only value available is the initial biomass and OUR pattern during the whole period, corresponding biomass can be determined step by step by successive iterations.

Once the biomass values are estimated through Eq. 4.30 for the different time intervals in which OUR are measured, the specific growth rate value at the logarithmic or exponential phase can be achieved. As has been postulated before, this is one of the most important parameters to determine in any fermentation process and is not the only but the main parameter to define the kinetic of fermentation.

Exercise 4.4. Determine the 02 consumed in the process reported in Exercise 4.1. Solution: 0 2 consumed in the whole process, or in a particular time interval is obtained estimating ht and area under the surface from a figure that relates OUR vs. t as in Exercise 4.1. To solve the present exercise is better to construct the following Table.

As is observed, Dt values are constant except for interval between 20 and 24 h. In the preceding deductions, time interval was considered constant so when evaluating the area under the curve, this must be taken into account. Applying Eq. 4.27 in time interval from 0-20 h, we get: 2/2 [ 0 + 3.136 + 2( 0 + 0.064 + 0.064 + 0.064 + 0.384 + 0.8 + 0.672 + 0.832 + 1.664)] = 12.224 g de 0 2

O2consume d --

Through same equation (4.27) for time interval between 22-24 h, we obtained: O2consumed =

4/2 (3.136 + 1.664) = 12.16 g de 0 2

And, at last for time interval 24-30 h we get: O2consumed =

2/2 [2.944 + 0 + 2 (2.656 + 0.672 + 0.16)] = 9.92 g of 0 2

Total 0 2 consumed = 12.224 + 12.16 + 9.92 = 34.304 g

0 2

R.

4.6 RESPIRATION QUOTIENT AND SOME CONSIDERATIONS ABOUT KINETIC DETERMINATION IN SOLID-STATE FERMENTATION As it has been already discussed in the section 1.4, the kinetic of a solid-state fermentation can be studied considering the pattern of consumed 02 or oxygen uptake rate (OUR), achieving in an explicit manner the biomass value and pattern through Eq. 4.30. The constant term "a" in Eq. 4.30 is defined by Eq. 4.31. This determines the validity how to employ Eq. 4.30. The cases in which yield based on oxygen consumption (Yx/o), or maintenance coefficient (~t) after a particular interval are no longer constant must be checked. An element that provides an insight about the statement of constant coefficient for yield and maintenance is provided by the respiration quotient (Q), which is defined as: C 0 2produced

Q =

4.32

0 2consumed

Where: Q: respiration quotient (dimensionless) Considering the metabolic overall balance of an aerobic fermentation as sugars +

n 0 2

,,

I~

biomass +

n CO 2

4.33

where n is the number of the moles of 0 2 consumed and of C O 2 evolved during the process, is a straight forward determination that the value for the respiration quotient (Q) in such a process is 1. Take into account that the value for the respiration quotient could vary as a function of the stequiometry for other substrate. The following figure contains the values for the respiration quotient that correspond to the data from Exercise 4.1. In this, an acceptable pattern is obtained considering some values distortion due to measurement problems. In other word, there is

no evidence for another pattern, which does not stand with postulated in Eq. 4.33. The respiration quotient pattern can also indicate which kind of kinetics we are dealing with in a particular fermentation according to Gaden classification as function of microbial growth. The classification proposed by Gaden is: a) Associated growth: In this kinetic pattern, the product to achieve is obtained at the same time biomass is produced. An example of this kind of fermentation is given by those processes in which biomass, or a particular intra-cellular constituent, or a direct metabolite as result of normal growth is the principal goal. This pattern can be found in single cell protein and vitamins production. To this type of fermentation corresponds a respiration quotient constant and a value close to 1.

b) Partially associated growth: This pattern is mainly related with a process in which an extra-cellular metabolite is produced after growth in the exponential, or logarithmic phase starts. This type of fermentation represents very fairly the production of induced metabolites like in induced enzymes production. In such induced enzyme production processes, the enzyme synthesis is repressed by initial inhibitors, which in many cases are simple sugars. Substrate contains sugar and enzymes promoters. Initial sugar content allows an initial growth without enzyme production. When sugar level falls in the medium, the induced enzyme production starts to attack the so-called promoter, v. gr. a polysaccharide related with the particular enzyme to produce. In this case, it is normal to assume that after the start of induced metabolite production, the yield in the process changes and is not any more constant due to new energy requirements by the cells to synthesize the induced metabolite. Also, the maintenance coefficient is no longer constant. As a matter of fact, a fall could be expected in the yield and a augment in the maintenance coefficient values. These facts imply that:

It is necessary to estimate the values for yields and maintenance coefficient after the period a non-associated or a partially associated metabolite is produced due to variation in energy and oxygen demand for maintenance and microbial growth. The respiration quotient is no more constant after the non-associated metabolite production begins. The value of the quotient should be lower than 1 (in case substrates are polysaccharides from simple sugars units) because it would be a consumption of 02, not only related to biomass synthesis but to synthesis of a new product as well.

c) Non-associated growth: The kinetic pattern of this kind of process corresponds to a process in which a metabolite is produced under cellular stress or not normal conditions for microbial growth. Usually the metabolite synthesis initiates after the exponential growth phase is terminated or at its end. The maximum biomass can practically be achieved. In this process, it is valid also to assume that the respiration quotient has a value lower than 1 in case sugar or related molecules are the substrate. Thus, the values must be determined for particular processes and conditions. Based on this reasoning, one can postulate the following strategy when dealing with solid state fermentation 02 and CO2 pattern from these fermentations. If it is observed that in a time interval the respiration quotient is approximately 1 in case substrates are simple sugars or related substances, it is fair to assume that the yield based on oxygen consumption (Yx/o) and the maintenance coefficient (m) can be presumed to be constant in the whole time interval. If data shows a defined tendency in the sense that the respiration quotient is Q < 1, it is valid to assume a possible production of one or several non-constitutive metabolites that till then were not produced. From this period, it is indispensable to calculate new values for Yx/o Y m. When the value for the respiration quotient is bigger than 1, it must be considered, if we are dealing with an aerated fermentation, that the higher production of CO2 related to 02 corresponds to a bad functioning of aeration and the existence of anaerobic zones in the system that must be resolved at once. It must be kept in mind that all these considerations are based on simple sugar, or related carbohydrates, or polysaccharides. When dealing with other source of substrate, the particulars and the overall metabolic pathway must be taken into consideration in order to sustain a proper value for the respiration

. . . . . .!iiiiiiiiiiiiiii!iiiiii!!iii!!!!iii! ..... !!!ii i!!!!ii i!i!i!i!iii!iiii!iiiiii!iiiiiiiiiiii!!iiiiiiiii iiii!!!

quotient. Undoubtedly, it is not only the consumed 02, or the CO2 evolved during a solid-state fermentation, the ones that represent the kinetic pattern. Closely associated with these is the respiratory quotient. By consideration of all these patterns one can solve Eq. 4.30 and have proper kinetic parameters.

4.7 PROCEDURES TO DETERMINE THE SPECIFIC GROWTH RATE IN SOLID-STATE FERMENTATION PROCESSES As we have seen in preceding sections, the problem to determine if it is feasible or not to consider the yield based on oxygen consumption (Yx/o) and the maintenance coefficient (It) constant in the whole time interval considered when dealing with Eq. 4.30 reduces. At the same, time it is necessary to know, or obtain the proper coefficient values for Yx/o and m. In that sense, following decisions could be considered: Take values for the coefficients Yx/o and m from former experiences. This decision is valid only when it is completely sure that there is no doubt about their values. Statistic procedures help in this assumption. However, it dose not seem very proper to assume coefficient values outside the experiment. Mainly, it may be loose information of the actual experience for which the values were assumed and their variation related to former experiences. Whatever the case be, the reader must have in mind how to deal with the assumptions and their validity. Another procedure consists in adjusting by trial and error process the parameters that coincide with values estimated by Eq. 3.30. This method implies to have the real biomass content for a particular time determined analytically and the corresponding patterns for consumed 02 and CO2. Certainly there is no need to have more than two or three values for real biomass in the fermentation. Biomass determination should be made at particular times when change in respiration quotient is expected. When it is certain that respiration quotient does not vary, only two determination must be made, that is at the beginning and the end of the fermentation, and then proceed to the trial and error estimation comparing values of biomass determined at chosen points and the ones predicted by equation 4.30. No need to mention that after the parameters Yx/o and rn estimated, biomass value for any point or at any time of the fermentation is already there. Obviously, this procedure could be burden and require for computer calculation but undoubtedly it is more proper than the former procedure of considering values outside the system. It is interesting to note that doing all these considerations, there has not been any need to distinguish if the fermentation is submerged, or solid and even

more the analysis made considering the 0 2 balance can be made with substrate balance. The only statement is that for solid-state fermentation the above procedure seems to be very proper in order to have on-line information about process kinetic and how is the process developing.

Exercise 4.6 The pattern for 02 consumption in a solid-state fermentation is reported in the two first column of the table reported in this problem. It was determined analytically that initial biomass was 0.2 g and at the end of the process 18.6 g. Determine the biomass kinetic pattern and show in a graphic form and calculate the maximum specific growth rate. To estimate the biomass synthesized in each time interval, we applied the Eq. 4.30. To do so, it is necessary to know, or estimate the values for Yrao and m. From Eq. 4.29, the consumed oxygen (O2cons.) will be: O2cons. -" 2 2(0+0.0027+2(0+0.0488+0.0461+0.0693+0.3418+0.7335+0.6163+

0.7553+1.4881+2.8140+2.6584+2.3798+0.6031+0.1410)) = 0.0027 + 2(12.6955) = 25.3937 g of 02 A first estimate could be done considering that the maintenance coefficient was negligible and therefore the consumed oxygen was used in biomass synthesis. From above considerations we can assume and calculate that: m = 0.0001 g O2/g biomass h Yx/o =

18.6-0.2 25.3938

(assumed).

= 0.725 g biomass/g 02 cons.

With these values the constant (a) of Eq. 4.31 is determined as" a = (0.0001)(0.725)(2)

= 0.000145

Calculation of X2: X2 = (1.45 (0.5 (0 + 0) + 0) + 0.2 - 0.000145 (0.2)) / 1.00007246 = 0.2

Calculation of X41 X4 = (1.45 (0.5 (0 + 0.0488) + 0) + 0.2 - 0.000145 (0.4)) / 1.00007246 = 0.2353 and so on successively As observed, an estimate for biomass at 30 h gave a value of 18.586. Comparing this result with the biomass content determined analytically, it could be well concluded that the initial assumptions seemed valid. The kinetic pattern of biomass synthesis is obtained plotting In Xn vs t as is showen in the figure as below:

The maximum specific growth rate corresponds to the specific growth rate in the exponential, or logarithmic phase. This value is estimated from the slope of the line observed in the figure through lineal regression. From figure it can be assumed that the exponential phase began at hour 4 and lasted till 26 hours. This was the time interval for estimating the specific growth rate and results give a kt = 0. 217 h -1 with a regression coefficient r = 0.9922.

References Aiba S, Humphrey AE & Millis N, 1973, Biochemical Engineering. Academic Press. New York. Brand D, Pandey h~ Leon JR, Brand I & Soccol CR, 2002 Relationship beteween coffee husk caffeine degradation and respiration of aspergillus sp. in solid-state fermentation. Applied Biochemistry and Biotechnology, 102, 169-178. Carvalho JC, Oishi BO, Brand D, Rodriguez-Leon JA, Pandey A & Soccoi, CR, 2006, Relation between growth, respirometric analysis and biopigments production from monascus by solid substrate fermentation. Biochemical Engineering Journal, 29 (3), 262-269. Leveau JY and Bouix M, Cinftiques microbiennes. In Biotechnologie, 4th edition. R. Schneider coordenateur. Tec Doc. Paris. Matchman SE, Jordan BR and Wood DA, Estimation of fungal biomass by!three different methods. Applied Microbial Biotechnology, 21, 108. 1985. Okasaki N, Sugama S and y Tanaka T, Mathematical model of surface Culture of koji mold. Journal of Fermentation Technology, 58, (5), 471. 1980. Rodriguez Leon JA, Echevarrfa J, Sierra N, Daniel A & Martfnez O, 1985, Solid-state fermentation of dried citrus peel. Biotechnology Letters, 7 (8), 577-580. Rodriguez Leon JA, Bechstedt W, Echevarria J, Sierra N, Delgado G, Daniel A & Martfnez O, 1986, Optimization of solid state fermentation of citrus dried peel by Aspergillus niger in a packed bed column. Acta Biotechnologica, 6 (3), 253-258. Rodriguez Leon JA, Sastre L, Echevarria J, Delgado G & Bechstedt W, 1988, A mathematical approach for the estimation of biomass production rate in solid state fermentation. Acta Biotechnologica, 8 (4), 299-302. Rodriguez-Leon JA, Torres JA, Echevarria J & Saura G, 1991, Energy balance in solid state fermentation processes. Acta Biotechnologica, 11, 9-14. Rodriguez-Leon JA, Domenech F, Leon M, Mendez DE & Pandey A, 1999, Production of spores of Trichoderma harzianum on sugar cane molasses and bagasse pith in solid state fermentation. Brazilian Archives of Biology and Technology, 41, (1), 69-76. Rodriguez-Leon JA, Bueno Gloria, Rodriguez DE & Delgado Grizei, True and apparent yields and maintenance coefficient and their significance on fermentation kinetics. New Horizons in Biotechnology, Editorial Kluver, 1,201-212, 2003. Sato K, Nagatani M, Nakamura K & Sato S, 1983, Growth estimation of Candida lipolitica from oxygen uptake in solid state culture with forced aeration. Journal of Fermentation Technology, 61 (6), 623-629.

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ilii

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i

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!

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5.1 INTRODUCTION For the majority of filamentous fungal species, solid-state media are the natural life media. Growth can occur on the surface, or within the whole substrate, depending on the porosity. Industrial solid-state fermentations have been developed largely in traditional food industries such as cheese, Oriental fermentations, fermented vegetables, meat, and other products, and in biotechnological industries such as antibiotics and enzymes. The use of water-insoluble materials in microbial metabolism has been characterized in solid-state fermentation (Moo-Young et al., 1983). Since substrate utilization could also occur in a suspension, the definition of solid-state fermentation requires the absence, or near absence of solution in the interparticule volume (Hesseltine 1972; Lonsane et al., 1985). Such an absence has been related to the water holding capacity (WHC) of the support which could reach up to 10 g water/g of dry matter in the case of cellulose fibers (Grigelmo-Miguel & Martin-Belloso 1999). The empty or void space must then be mainly occupied by a gaseous phase (Durand et al., 1983). It is well recognized that the growth and metabolism of microorganisms always or almost always occur in the liquid phase. Solute as well as dissolved gas transfers take place in the aqueous film surrounding the microorganisms. So, even in solid-state fermentations, the microorganisms are in a liquid medium. Solute diffusion in the substrate must occur in the liquid phase, and gaseous diffusion in the substrate can occur in the liquid as well as in the gaseous phase. Thus, the replacement of the water phase by the gaseous phase increases oxygen transfer but prevents solute diffusion in most of the substrates (Gervais & Bensoussan 1994). Mostly, the three phases (gas, solid, and liquid) are

present in the liquid as well as in the solid media, but not in the same proportions, and so kinetic, diffusional, rheological, and thermodynamic (aw, osmotic pressure) properties are modified. It is difficult to give an exact definition of solid-state fermentations based on the proportion of each phase because the biophysical properties of the mixture depend on the nature of the liquid and solid components. Solid-state fermentations are generally characterized by a reduced liquid phase, (approximately the water-holding capacity of the substrate in the case of the water-liquid phase, i.e. no presence of free water) (Pandey, 2003) and present a high viscosity. Thus, mixing such media leads to great shear forces that drastically injure the microbial cells. Solid-state media could then be defined by high viscosity and no continuous possibility of mixing and could also be considered as heterogeneous media in terms of microbial population and solute concentration. Indeed, the gradients of water and solutes cannot be prevented in SSF as shown by works of Nagel et al., (2002). The major difference between liquid fermentations and solid-state fermentations reside in the mixing ability. Liquid fermentations are generally perfectly mixed reactions, and so each part of the reactor contains at any time the same quantity of microorganisms, nutrients, and metabolites. Perfect mixing of liquid fermentations prevents the formation of large boundary layers around the microorganisms. Such layers drastically limit the diffusion phenomena of nutrients and metabolites and prevent the removal of the heat of the fermentation process, which could represent quite high values, about 500 W/m 3 in moist solid substrate (Oostra et al., 2000) and so limit the metabolism of the microorganisms. In solid-state fermentations, four alternatives could be used in order to improve the homogeneity of the cell population and corresponding substrate concentration: -

Efficient mixing during, or just after the inoculation. Sequential mixing.

--

The use of mobile microorganisms, most often filamentous fungi; the forward growth of such microorganisms allows the occupation of new substrate with high levels of nutritive components (if solutes diffusion rate is limited and smaller than growth rate).

--

Forced aeration through the solid substrate, which allows the control of water content and therefore water activity; the removal of volatile compounds and CO 2 together with heat produced during the fermentation; and supply of 0 2 for aerobic metabolism.

Growth of filamentous fungi has been shown to be dependent on physicochemical parameters such as thermodynamics (aw, temperature) (Scott 1957; Gervais et al., 1988d) and chemicals (glucose, 02 and CO2, nutrient concentrations)

(Trinci & Collinge 1973; Trinci 1969). Numerous workers have shown the evidence of such dependence but there are many results that are sometimes difficult to synthesize. This chapter intends to identify and model the influence of the conditions of the medium hydration on the microbial development. First, some physical parameters, relating to the water state of the medium and implicated in the variations of metabolism, will be examined for solid-state media. Then, the mechanisms of the action of water at the cellular level will be discussed, particularly in the light of the works of our laboratory. The roles of water in biological systems are numerous and the mechanisms of action and interaction of this molecule with organic molecules are not totally elucidated. Two fundamental functions can be distinguished (Gervais et al., 1996): a solvent function on the level of the organism as well as of the cell, provides nutrients and scavenges wastes, or metabolites, under the dissolved form. a structural function which is implicated in the stability and the function of the biological structures organized at the molecular and cellular levels. At the molecular level, the role of water in the stabilisation of the structure of the biopolymers, such as proteins, nucleotides and carbohydrates, was recognized aboutthirty years ago (Schwan 1965; Kuntz 1971). At cellular level, the role of water molecules in the stabilisation of the lamellar structure of the plasma membranes and thus in the preservation of membrane permeability has been shown (Crowe et al., 1982; Wolfe & Steponkus 1983; Quinn 1985). In the intracellular medium, the molecules of water linked with other molecules, such as polyols, sugars or enzymes, contribute to the maintenance of the cellular volume, especially when the cell is placed in a hypertonic medium and particularly during desiccation or freezing conditions (Mazur et al., 1981). From these different functions, we can understand why water is essential to fungal metabolism in solid state fermentation: *If the quantity of the water becomes insufficient and does not allow a good diffusion of solutes and gas, the cell metabolism slows, or can stop, because of a lack of substrates or through too high concentration of inhibitive metabolites in or near the cell. *If the intracellular or extracellular quantity of water does not allow the maintenance of the functional properties of some enzymes (Todd 1972), their inactivity creates disequilibrium in the metabolic chain of the cells. *In the same way, if the transfer of water induced by water stress leads to a denaturation of the mechanical structure of the plasmic membrane, all the properties of permeability and transport through the membrane are affected

and the cell is then perturbed (Wolfe & Steponkus 1983; DeLoecker et al., 1978).

5.2. WATER CHARACTERISATION OF A SOLID-STATE MEDIUM In a heterogeneous system such as a solid-state medium, numerous parameters can describe the state of water. Two types of parameters can be distinguished:

Thermodynamic parameters, relative to steady state systems describe the energy state of water, the mainspring of water transfer between two systems such as cells and substrates. Evolution of biological systems is very low compared with that of physical systems, so such systems can be frequently considered at steady state.

Dynamic or kinetic parameters define the resistance of the medium to the water transfer. Solute diffusion as well as cellular absorption occurs in aqueous medium. The diffusional properties of water interact on these transfers, and the dynamic properties of water molecules are modified when they are near other molecules. The diffusion laws are deduced from the atomic theory of Brownian movement established by Einstein. The relation between the flow and the concentration gradient is given by Fick's law and the coefficient of proportionality is called diffusion coefficient or diffusivity; its value is closely dependent on water concentration in the medium. In fact, thermodynamic parameters and kinetic parameters, although fundamentally different, are always related to the water content of the medium and therefore dependent. Nevertheless, this degree of dependence is extremely variable and the limiting factors for the transfer of water in a system can have a kinetic or thermodynamic origin.

5.2.1 Thermodynamic parameters The parameters generally used to characterize the thermodynamic state of water in a system may be defined from the two fundamental laws of thermodynamics.

5.2.1.1 Chemical water potential The chemical water potential of a system is equivalent to the partial free energy of water:

-G-w

5.1

...........

iUigiii!iiiliii!ii~

where T is temperature, P is pressure, w is the water suffix, n is the number of moles, j is the solute suffix, G is free energy, G w is the partial molar free energy of water, and ~tw is the water chemical potential in the solution. The potential difference can be estimated in relation to a standard reference state. For a solution:

l.tw _po

_ ~-w( p _ jr )

5.2

with

7r-RT(CM+A2C2+A3C3+...)

5.3

For a porous solid: //w _ / t o _ ~-w( p _ , t . )

5.4

hpg

5.5

with ~ r -

For a heterogeneoussystem: as a solid

state medium (solutes and porous solids)"

/2 w - / 2 ~ - Vw ( P - / r - ' t ' )

5.6

where pw~ is the chemical potential of pure water, r~ is the osmotic pressure, C is the solute concentration, M is the solute molar weight, Ai is the ith viriel osmotic coefficient, R is the perfect gas constant, volume, x is the suction pressure.

Vw

is the water molar

5.2.1.2 Water potential of a system The water potential (W) of a system is defined by:

~ Vw

(Pa)

5.7

The water potential W can be written as the sum of terms of different origins: kI/ - kI/p "1- kIJ s -I" kI'/m

5.8

where Wp = P, water potential pressure (hydrostatic pressure), Ws = -r~, osmotic

iiii

i iiii

water potential (solute effect: osmotic pressure), and (solid matrix effect: adsorption, capillarity).

!iii kI; m

=-re, matrix potential

Other parameters such as temperature or gravity can affect the water energy state of a system. Nevertheless, in biological systems, most of the time, only two components W= and ~I'tm c a n describe the systems. The distinction between 9 = and ltlJm is especially valid at the macroscopic level; at the molecular level, the same physicochemical phenomena such as adsorption and size effects are implicated.

5.2.1.3 Water activity The water activity concept is used to describe the equilibrium between a heterogeneous system and the surrounding water vapour phase (considered as a perfect gas.). The water activity of a solution is given by:

aw

- p ew o --

5.9

~'w " X w

w

where Pw is the partial pressure of water vapor equilibrated with the solution, Pw ~ is the pressure of water vapor above pure water in the same conditions, Xw is the water molecular fraction, and aw is the water activity coefficient (dependent on the concentration). Guggenheim (1967) demonstrated the relation between the chemical potential and water activity" l.tw - l.t ~ - R T l n a

w

5.10

and so, according to equation 5.6: RTlna

w

V-w

5.11

5.2.1.4 Thermodynamic properties of water: Molecular basis Case o f s o l u t i o n s

The thermodynamic water properties of a solution are related to three types of molecular interactions: solute-solute, solute-solvent and solvent-solvent. The hydration phenomenon corresponds to water-solute interactions and can be studied alone at solute concentrations corresponding to an infinite dilution, where the solute-solute interactions therefore become negligible. For electrolyte solutions,

this dilution threshold can be estimated using the Debye-Htickel's law; for nonelectrolytes, an objective method to estimate this threshold does not yet exist. The thermodynamic reference for the hydration term is composed by the ideal solution which presents no solute-water interaction. For this ideal solution, the aw value is exactly equal to the water molar fraction (aw = Xw), i.e. the water activity coefficient )'w is equal to 1. From this model, some deviations are observed which can be attributed to three types of phenomena: ~

Solute molecular size vs. solvent molecular size. Flory (1953), using the theory of the "liquid network", showed that the binary solution entropy and consequently its eventual deviation from an ideal solution depends on the respective size of the solvent and solute molecules.

2.

Solute molecule hydration or solvation.

3.

Other molecular interactions (solute-solute).

Case of Insoluble Substances The previous types of interactions can occur depending on the chemical nature of the polymers, hydrophilic or hydrophobic. But, in addition, the energy state of water is closely dependent on the thermodynamic level of the geometry of the medium and especially on the dimensions of the pores. Fisher & Israelachvili (1979) showed that the thermodynamic properties of liquids trapped in microscopic pores can be described by Kelvin's equation, the basis of the nucleation theory, for pore sizes as small as a few nanometres. The Kelvin equation can be written as:

lnPw=po

PwRT Mw [-a--~--(Pw-P~

5.12

where rm is the radius of the curve of the water meniscus in the pore, t~ is the surface tension at temperature T, and Pw is the water volumetric weight.

Pw - pO is negligible for RTln a w

ty

Mw

Pwrm

t7 rm

-~,

so:

5.13

5.14

and then RTlna

w

a

=

Vw

rm

=W

5.15

In this case, the energy state of water is dependent only on the curve radius of the meniscus and then on the particles or pore sizes of the material. The porosity of a heterogeneous system, such as a solid substrate, is modified during drying or hydration (bulking), as the curve radius of water meniscus decreases. Nevertheless, for definite water content the matrix water potential provides an estimation of liquid-gas and solid-liquid interactions, generated by very thin pore size (capillarity), which can characterize biological structures such as membranes or macromolecules.

5.2.2. Dynamic parameters These parameters allow the characterization of the water resistance to transfer in a medium. The parameters that are used are the coefficients of the rotational and translational diffusion of water. These coefficients are related to the friction coefficient by Einstein's equation: kT D

~

m ~

5.16

where D is the diffusion coefficient of a particle in a liquid, ~ is the friction coefficient, k is Boltzmann's constant, T is the temperature. The hydrodynamic model allows the calculation of ~ in the case of large molecules with a radius a, for the translational diffusion { = 6 rtarl (11= liquid viscosity) and for the rotational diffusion { = 8rca3rl . Water content is a parameter that represents the diffusional properties of a slightly hydrated medium. In fact, in the case of the study of the rotational diffusivity of the solute molecules (Le Meste & Voilley, 1988), as in the case of the translational diffusivity of water molecules (Simatos & Karel 1988), the relation proposed between diffusivity and water content is established for definite scales of water content. The thermodynamic and kinetic parameters of a medium are linked by the water content. Nevertheless, this link, represented by the sorption isotherm of the medium, can be formalized by equations only for simple model systems. In most cases, this link can only be established experimentally. Previous studies (Bruin & Luyben 1980) have confmned that the water diffusivity in polymer solutions is essentially dependent on the water content of the solutions.

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Le Meste & Voilley (1988) even showed an antagonism between the kinetic and thermodynamic parameters. A unique factor representing both the energy state of water and its solvent properties in a heterogeneous system does not exist. Nevertheless, it seems possible to draw general conclusions. For dilute solutions, the solute diffusion is not a limiting factor, and the solute nature has only a small influence. If the solvent and the solute molecules are of compatible sizes, there is little deviation from Raoult's law. In this case, thermodynamic factors allow the prediction of the water transfer in the system. For more concentrated solutions, homogeneous or heterogeneous, as found in solid-state media, the previous hypotheses are no longer true; the diffusion phenomena can become limiting according to the sorption properties of the solutes or solids. The physicochemical nature of the molecules can then interfere. For certain enzymatic activities, which can be developed over almost the total scale of water content (5-100% wt./wt.) (Bouanda 1983) or of the water activity (Goldberg et al., 1988), the previous considerations are particularly important to the understanding of the systems. For microbial activity there may be a hierarchy in the action of kinetic or thermodynamic parameters on the cellular physiology. In submerged cultivations, limitation would essentially be of a thermodynamic origin, but in solid state cultivation, limitation should be thermodynamic as well as dynamic in origin.

5.2.3 Water sensors For many years, several authors reviewed the principles and engineering aspects of SSF (Bellon-Morel et a1.,2003; Raghavarao et al, 2003). Because of the heterogeneity and complexity of the media and the variety of reactors, monitoring and controlling environmental factors like water activity pose problems in SSE As described below, water acts in numerous reactions: biomass development, metabolic reactions, enzyme activities, transport of nutrient, extra-cellular metabolite and gas. Water content is considered optimum when reaching the Water Holding Capacity (WHC) of the substrate (Durand et al, 1993), which varies between 30 and 85% (w/w) depending on the substrate. As water content is evolving during the fermentation (evaporation due to metabolic heat and substrate hydrolysis or metabolic water production) such variation must be taken in account in order to prevent any productivity decrease.

As water content "off-line" sensors process by dry weight measurement, KarlFisher titration or Proximity Equilibration Cell Method (Xavier & Karanth, 1992), they are difficult to integrate into control schemes due to the typically long

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delays in samples analysis; so recent works tend to optimize "on-line" rapid measurements to study water dynamics during SSF. In this way, various tomographic techniques, adapted from soil and food areas, like microwaves (Narayanan & Vu 2000), Magnetic Resonance Imaging (Maniette et al., 1999), X-rays and y-rays (Mohsenin 1984) are studied. Interest of "on-line" measurement of water activity during SSF was developed by several authors (Durand & Chdreau 1988; Barstow et al., 1988; Ramesh et al., 1990; De Reu et al., 1993; Ghildyal et al., 1994). The most common on-line system to measure water activity is the capacitive sensor, designed to regulate the circulating air humidity was improved by Gervais (1989) by separating the capacitive element from the medium by a thin ethylenepolytetrafluoride membrane which allows on-line and accurate measurements of water activity. Other sensors which are based on changes in electrical behaviour of an e l e m e n t - usually resistance- after water adsorption are described (Labuza et al., 1976). As these sensors have slow response times, an other approach is based on the modification of the optical absorbance of a mirror where dew condenses, resulting from the the atmosphere in equilibrium with sample (Bramorski et al., 1998; Aunia et al., 1995; Engel 1995).

5.3 WATER PROPERTIES RELATED TO FUNGAL DEVELOPMENT AND PHYSIOLOGY The introduction of the water activity concept (and then of the water potential) in microbiology dates from 1953 (Scott 1953). This parameter has been and still is widely used as a predictive criterion of the physiological functions of microorganisms and thus, of food preservation (although physicochemical factors like non-enzymatic oxidations also intervene). Ben-Amotz & Avron (1978) have used the influence of water activity or osmotic pressure to increase the glycerol production by unicellular algae. It is thus a statement to show the water activity (aw) effects on the physiology of microorganisms. Nevertheless, if there are numerous observations, the explanation is different. In recent years, the systematic utilization of this parameter has been debated by specialists to qualify the water situation of a medium (Franks 1995). For these authors, the thermodynamic considerations are insufficient to explain the water effect in a medium. Kinetic parameters should also be included. The respective influence of kinetic and thermodynamic parameters in fermentation media should be investigated.

5.3.1. Influence of water thermodynamic parameters 5.3.1.1 Cellular mechanisms It is recognized that there is no active water transport in cells and that water moves freely according to the gradient of the water potential. Thermodynamic

models relating the energy equilibrium of the intra- and extracellular media have been developed by many authors (Griffin 1981; Steudle et al., 1983). At equilibrium, the water potential of the protoplasm wP is equal to the water potential of the medium WE: W e = wP

5.17

The variation scale of ~Fe according to WE is dependent on the species. For xerotolerant species, WP can decrease to -40 MPa without damaging the cells. The protoplasmic water potential we can be restated in terms of its constituent elements as follows: LI/P

P

P

P

--lkI/p -+-kI-/s -.~-kI-/m

5.18

where ~P = P - n -x, with P the hydrostatic pressure, n the osmotic pressure, P P and1: the suction pressure. ~ p and ~ p are the water osmotic and matrix potentials P of the protoplasm, and ~p the turgor potential of the protoplasm created by the plasmic membrane and the cellular walls. This latter potential corresponds to the pressure permitting the initiation of cellular plasmolysis. For most P filamentous fungi, ~ p = - 1 MPa, a value that can rise t o - 3 MPa for xerotolerant species.

5.3.1.2 Fungal growth Growth of microorganisms is very sensitive to environmental parameters such as temperature (Suutari et al., 1990), water potential or water activity (Scott 1957). In the cell, there is no active water transport and water moves according to the water potential gradient between the intra- and extracellular medium. In heterogeneous media, water potential or water activity is related to the solute concentration, capillary forces and absorption properties of the insoluble solid substrate. Numerous workers have demonstrated the sensitivity of hyphal extension (Robertson & Rivzi 1968; Wilson & Griffin 1979; Robertson 1958) fungal spores germination and aroma production (Gervais et al., 1988b; Gervais et al., 1988c) to water stress. A variety of experiments have shown that a turgor pressure exists in hyphal tips (Money 1997). First of all, a theoretical approach (Gervais et al., 1999) could explicit the great importance of turgor pressure on the fungal growth.

5.3.1.2.1 Turgor pressure as a driving force" theoretical approach .'. Equilibrium state and cell wall tension: Cell turgor pressure is defined as the hydrostatic pressure due to the difference between the intracellular and extracellular osmotic pressures at a steady state characterized by a steady cell volume. Turgor pressure exists in all types of cells: microorganism, plant and animal cells. In all theses cases, cell turgor pressure corresponds to an overpressure which allows the cell morphology, elongation, division and hence the increase in biomass:

Pt = I-Ii -lie with Pt the turgor pressure, osmotic pressure.

5.19 11 i

the intracellular osmotic pressure,

II e

the external

The turgor pressure value is balanced by the cell wall resistance of the organisms (Pr), the higher the wall resistance the higher is the turgor pressure (Trinci 1978). This can be explained as follows: since the cell wall must be in equilibrium, it has to be under tension so that each surface element can balance the intra and extracellular osmotic pressures. This translates mechanically into a compensating pressure, given by the ratio of a surface tension over a curvature radius that is exactly as in the case of a soap bubble. It follows that for a given organism: et = er

5.20

where Pr will be a constant value unless wall geometry or properties are modified. The growth of individual cells is discontinuous and can be separated into two phases: a slow evolution phase and a rapid division phase corresponding to mitosis or budding. The turgor pressure was found to be constant for a cell at a specific physiological stage and the budding phenomena in yeasts was found to be related to a specific and localized hydrolysis of the cell wall which induces an important decrease in wall resistance and leads to a volumetric expansion of the cell wall through a passive flux of water into the cell.

.'. Non equilibrium state: fungal cell growth: For fungal species, it is largely admitted that the growth occurs continuously at the apex of numerous hyphae of a colony and at constant growth rate under fixed conditions of the medium (i.e. pH, temperature and water activity) through the continuous deformation induced by a constant overpressure exerted on the renewed thin membrane of the apex (Scott 1957; Gervais et al., 1988d), and the maintenance of the turgor pressure could not be sufficient to explain

the growth phenomena (Zhu & Boyer; Kaminky et al., 1992; Money & Harold 1993). Nevertheless, Trinci (1974) has observed oscillations of the mycelium growth rate just after spore germination. During hyphal elongation, synthesis of cell materials such as cytoplasmic organelles or membrane components must continuously occur in order to keep the cell wall constant in mechanical resistance in spite of the continuous deformation induced by the turgor pressure (Bartnicki-Garcia 1973). The most numerous components in the apical zone are the apical vesicles that originate in the Spitzenk6rper (or Vesicle Supply Centre): a central and dense component of the apex consisting of accumulated vesicles and presumably anchored to the apex walls (Howard 1981). Hence, cell growth is the result of a dynamic balance between the maintenance of an intracellular turgor pressure via solute synthesis and water input, and a synthesis of macromolecules which causes the membrane to provide a constant resistance despite elongation phenomenon (Wessels 1993). We give further details below. In the case of continuously growing cells such as filamentous fungal tips, the steady state conditions mentioned in the preceding section (see equation 20) can no longer be applied. So the evaluation of intracellular turgor pressure must take into account the type of system under consideration: it will be different between non-extending cells (such as bacteria, yeasts, or plant cells) for which both cell volume V and intracellular osmotic pressure ~i could be considered as constant, and extending non equilibrium cells such as fungi. In order to study more precisely the case of fungi, let us consider the example of fungal hyphae growth during a time interval dr. During this interval, dn~ moles of a solute j are synthesized or incorporated in the internal medium which induces a dI-I i increase in osmotic pressure, a s l-I i could be considered for diluted media as equal to:

Hi = RT 2_~Cj

5.21

j=l

with R = ideal gas constant; Cj = molar concentration of intracellular solute j; and T = temperature. Such an osmotic pressure increase, added to a continuous apex wall elongation (through vesicle incorporation), will simultaneously provoke a thermodynamic water flow into the cell which will decrease the Cj concentration and so will simultaneously reduce the osmotic pressure level of-dl-Ii as shown in the previous sentence. All these previous phenomena occur simultaneously, which leads to a continuous hyphal extension. Cell growth could then be attributed to a constant increase in the osmolytes concentration near the apical part which is continuously balanced through a water flow input therein. So in the case of filamentous fungi, growth regarded as volume variation (essentially due to water

input) should occur under a constant intracellular osmotic pressure (Hi). The increase in the cell volume is in turn limited by the cell wall's mechanical properties, which were previously discussed in this section. Assuming that the tension developed by the cell wall is constant in the apical part during hyphal growth (i.e. the continuously synthesized wall exhibits steady mechanical properties), it could be possible to calculate the cell pressure balance for extending fungi. In this case the sum of the exerted pressures is no longer equal to zero (as previously shown for non-continuously extending cells in equation 20) but equal to a pressure loss value (AP) which is the origin of the continuous apical extension, (that is, water inflow to the cell). At a time t, it could be written: (I'Ii-lie)

or

- Pr = ~

Pt -- Pr

= z~~

5.22

This very simple equation allows us to describe fungal growth; thus turgor pressure in extending cells is no longer counterbalanced only by the membrane pressure resistance (Pr) but also by the apical deformation (due to AP). Previous works (Money & Harold 1993; Lockhart 1965) have not considered the parameter AP in the turgor pressure evaluation or measurement in continuously extending cells. Now it is possible to relate the volume variation dV during the time dt to this constant pressure loss AP over time (as Hi, lie and Pr are constant) and to physical characteristics of the fungal apex through the application of an equation (Kedem & Katchalsky 1958) describing laminar water flow driven by pressure gradients between intra and extracellular media: 1 dV A dt = LpAP

or

dV ~dt -

5.23

ALpAP

with A = exchange surface where the water flow takes place (m2),

dV dt

m

water flow between intra and extracellular media (m3.sq), Lp = hydraulic permeability of the apex wall (m.s-l.pa-1), = ( I I i - I ' I e ) - Pr (Pa). Integration of previous equation 5.23 yields: V = (ALpz~)t + Vo

5.24

In obtaining (5.24) it is assumed that the surface A is constant versus time. Indeed the parameter A is relative to the surface of the apex where the water flow takes place, i.e. where the pressure gradient occurs. This apex wall surface corresponds to the cell's active volume which can be defined as the hyphal apex volume which is osmotically balanced or equilibrated through both continuous

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incorporation or synthesis of solutes and water input. Indeed it was shown by 14C glucose labelling (Trinci 1971; F~vre 1979; F~vre & Rougier 1982) that glucose incorporation was about ten times higher at the apex than in the subapical zone of Saprolegnia monorca. Such a local increase in solute incorporation (mainly sugars and ions) would immediately trigger a fast water input to the fungus, this is known to be many times faster than solute diffusion towards the rear of the apex (Mauro 1957). This confirms the view that the rate of hyphal growth will be only controlled by the water input in the apical part, as equation (5.24) estimates. With these assumptions, the fungal growth expansion is represented by equation 5.24. As the hyphal growth rate is unidirectional (x axis), the volume variation dV during the time dt could be written dV = dx. S and where dx is the axial hyphal extension and S is the section of the hyphal tube. Consequently, equation 5.23 becomes:

dx dt

--=

ALp zSaP S

or

Ap=

S dx Lpta--7-'d ----;

5.25

dx These equations imply that the hyphal growth rate ~ is constant for defined medium conditions (A, Lp, AP,, S are constant), a well known fact since Scott (1957). The expression of the turgor pressure could be easily obtained from the previous equation 5.25. Substituting AP by its value (Pt" Pr) given by equation 5.22 in equation 5.25 gives:

Pt --Pr +K --~

s with K=A---~p

5.26

Comparison of this equation with equation (20) allows us to conclude that in the case of growing tips the turgor pressure, considered as a potential pressure, is greater than the equilibrium value Pt " Pr (case of non-extending cells). Equation 5.26 trivially reduces to 5.20 when cell extension stops. This difference between non extending cells and growing tips is easily understandable, because in the case of continuous hyphal elongation, the part IK-~t j of the turgor pressure continuously drives the volume expansion and so cannot be measured just through the membrane pressure resistance Pr.

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Turgor pressure as a driving force" experimental approach An experimental approach of this theory has been proposed in a previous work (Gervais et al., 1999) where the hyphal growth rate of A. orizae has been shown to be related to the pressure AP as proposed in equation 5.25 (see Table 1). The measured AP part of the turgor pressure is plotted in Fig. 1 against hyphal extension rate. It can be seen that the higher the AP, the higher the apical rate. The variability of the AP measurements has not clearly allowed showing a linear relation between the two parameters as expected from equation 5.25. Nevertheless, the relationship between the two parameters is clearly an increasing function. Further experiments will allow us to explore this relation. Table 1: Evaluation of AP values for Aspergillus orizae cultivated at different levels of water potential.

*Values into brackets represent confidence intervals at 95%.

Figure 1: Representation of the hyphal extension rate as a function of the pressure •P.

The theory developed here allowed us to better understand the role of turgor pressure in fungi and mobile microorganisms. For such organisms a main part of the turgor pressure (AP) was shown to be continuously used for cell volume expansion.

5.3.1.3 Radial growth rate and hyphal orientation An increase in the osmotic pressure (or decrease in the water activity for a heterogeneous medium) of the extracellular medium leads to a two-phases cell response (Zimmermann 1978): (1) A very fast but passive exit of cell water, for a few seconds, with a corresponding decrease in cell volume for protoplasts or decrease in turgor pressure for cells with walls. For such cells, the variation of the turgor potential is related to cellular volume modifications through the elastic properties of the cellular walls, defined by the elastic volumetric coefficient ~ according to the equation: AV AP = e - V

5.27

This coefficient is related to turgor pressure and cellular volume; it can vary from 5 bars to 600 bars according to species and pressure conditions. This first phase, can lead, in the case of large gradients of osmotic pressure, to either an exit of most of the free water from the cell, and thus to a metabolically inert cell that can survive over long periods, or to cellular death essentially due to the alteration of membrane properties (Cudd & Steponkus 1987; Gervais et al., 1996b). (2) A second compensation phase during which the cell allows the input of permeable solutes or the biosynthesis of intracellular metabolites. This accumulation in response to a hydric stress has been identified in microorganisms, plant and animal cells (Gilles 1975; Hellebust 1976; Luard 1982) and allows the cell to recover its initial volume and to grow again at lower aw value (depending on the fungal strain, generally above 0.90). For a given aw value the turgor pressure maintenance or more specifically the AP value has been shown (see previous section) to be the driving force of the fungal growth rate. Numerous experiments have demonstrated the influence of the water activity of the culture medium on the survival, growth, and metabolism of microorganisms cultivated on different types of medium (Mossel 1975; Troller 1980; Richard-Molard et al., 1982; Jackobsen 1985). In all cases, optimum growth and metabolic production occurred at media aw values of less than 1.

Macroscopic considerations Numerous authors (Gervais et al., 1988d; Bull & Trinci 1977; Raimbault 1981) have described the macroscopic development of a fungal colony. From a point inoculum, the growth is isotropic and then circular. After a lag phase due to the germination of the spores and after a short exponential phase, colony radial

extension rate reaches a constant value (Scott 1957; Grajek & Gervais 1987a). Water activity of the medium, which is a thermodynamic parameter, was found to have an effect on radial growth and biomass production (Gervais et al., 1988d; Raimbault 1981; Trinci & Bandury 1967) . There is an optimal water activity value for radial extension rate which depends on the fungal species. Nevertheless Inch & Trinci (1987) pointed out that colony radial growth did not always provide a relevant variable for estimating the effect of environmental factors on mould growth. Previous results about fungal radial extension rate as a function of the water activity of the medium (Gervais et al., 1988b) have confirmed that the colony diameter was linearly time-dependent (Trinci 1969). For each water activity, a mean extension rate could be approached using the slope of the corresponding radial growth. The radial extension rate values were then related to the water activity value as shown in Fig. 2. The shape of the curves obtained was characteristic of the answer of a microorganism growth to a variation of water potential as proposed previously (Scott, 1957; Griffin 1981). The optimum growth always occurred in a medium in which the water activity was clearly less than 1, corresponding to a water osmotic potential scale between -0.1 MPa and -10 MPa. From Fig. 2, the optimal water activity value for growth was found to be 0.99 for Trichoderma viride TS; below 0.90 no development occurred. These values allowed this fungus to be classified in a group constituted of moderately water-sensitive fungi. Moreover, these results have been confirmed 1100

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by a study of the growth of this fungus led on sugar-beet pulp (Grajek & Gervais 1987a). For Penicillium roqueforti, the optimal water activity value was found to be 0.97 and development occurred over the whole range studied. However, at 0.90 the time lag was greater than five days. This fungus could be classified as a xerotolerant species. Otherwise, for the whole range of water activities studied, growth occurred faster in Trichoderma viride TS than in Penicillium roqueforti. The maximal radial extension rate values were 390 and 140 mm/h, respectively. This difference could be attributed to the nature of the microorganisms while the culture parameters, such as temperature and pH, were optimized.

Microscopic considerations Microscopic observations of colony growth on solid media reveal that hyphae tend to grow away from each other, and this has been explained by chemotropism (Robinson & Griffith, 1977); that is, each growing tip depletes substrates around itself and thus causes neighbouring tips to grow away toward more favourable conditions. Moreover, hyphae are subjected to steric interactions with other hyphae and tips as the colony growths. At short distances, binary interactions (tip-hyphae) involve a local spatial rearrangement, resulting in a slowing down of the tip extension rate. This leads to a maximum biomass surface density for given environmental conditions, whereas hyphal extension provides an alternative to motility, enabling hyphae to extend toward new food sources, branching provides a means to optimize colonization and substrate utilization. Growth of new tips is a process driven by complex intracellular mechanisms characterized by cytoplasm polarization (Prosser, 1990), so there is uncertainty about when and where a new tip will branch out of a hypha. One way of describing this mechanism is to assume that tips are generated by a stochastic process, characterized by a branching probability. Then, growth of new tips is also subjected to the same steric interactions just mentioned. That is, tips near colony center are likely to stop growing prematurely, so a maximum of (arrested) tip surface density arises therein. At the colony edge, tips are initially free to grow and extend. Therefore, tip growth is modulated by the local biomass surface density, incorporating a bias to the local growth rate. Therefore, the visual aspect of a growth colony can be explained by microscopic events: hyphal growth rate, branching frequency and direction of hyphal growth. Molin et al., (1992) have tried to describe the microscopic characteristics of hyphal growth and to study the effect of water activity on these parameters. The study was performed using image analysis, as previous authors (Adams & Thomas 1988) did for submerged cultures. This work was also intended to

link microscopic events with the macroscopic behaviour of a filamentous fungal colony.

Comparison between apical and radial growth rate allows to evaluate the hyphal orientation The relationship between radial growth rate and hyphal growth rate is not necessarily an equality: Vr may be different from Vh. The observation of a colony front shows that hyphae grow in all directions and the contribution of each apex to colony expansion is the projection of apex elongation upon the reference radial axis. The distribution of tip orientations could be measured by the angular deviation from the radial orientation (~h). Hyphal growth rate (Vh) and radial growth rate (Vr) measurements were made on two filamentous fungi solid cultivations: Trichoderma viride TS, Rhizopus oligosporus CBS 338.62. Five water activities were tested: the optimal aw of each fungus (0.995 for R. oligosporus and 0.990 for T. viride), two lower values (0.970 and 0.950) and another value close to 1 (0.998). The microscopic and macroscopic results of growth versus aw are presented in Table 2 and Figure 3.

Table 2: Dispersion in tip axes (V r /Vh) and tips orientation ( ~ ) versus water activity

*Optimal awfor R. oligosporus radial extension rate. **Optimal % for T. viride radial extension rate.

The optimal aw for radial growth rate was also optimal for the hyphal growth rate. Hyphal growth rate was always greater than radial growth rate, showing that the hyphae do not simply extend in the radial direction. The value of the ratio Vr/Vh given in Table 2 was always greater than 0.64, implying that the dispersion in tip axes was not evenly distributed. It means that the angles Oth, representative of the tips orientations, were not uniformly distributed onto 2 n radians.

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Hyphal Orientation Measurements as a function of the medium aw The means of the angles (O~h) were always found to be close to 7z as shown in Table 2, so that the tip orientations were centred around the radial direction of the colony for each fungus species and each water activity value. Angles distribution was not uniform. The angles were distributed according to a normal distribution with a mean of ~ radians and a standard deviation (a) which varied with water activity. The normality of these distributions was tested and Lhe tests showed that the distributions were not different from normal distributions. The more the water activity deviated from the optimal growth value, the higher

was the standard deviation. Orientation towards the colony margin, which signifies good spreading orientation, decreased when the medium became unfavourable. Apices of T viride were found to grow more radially than those of R. oligosporus. The previous described work (Molin et al., 1992) has shown that hyphae which were in the front of the colony had a favoured direction of growth which was the radial axis of the colony. The distribution of tip orientations was not uniform, but followed normal distribution and the standard deviation depended on the species and on aw values. Hence, the adjustment of filamentous fungi to the changing water activity environment would imply modifications in hyphal growth rate and in orientation of this growth. Furthermore, the use of the colony radial extension rate as the only growth criterion could induce some bias for the interpretation of experimental results about environmental parameter effects on fungal growth, since the hyphal growth rate is not always linearly linked to the radial extension rate.

5.3.1.4 Biomass evolution of a fungal SSF In a recent work (Ferret et al., 1999), a quantitative model predicting biomass growth on solid media has been developed. The model takes into account steric interactions between hyphae and tips at the microscopic level (competition for substrate and tips-hypha collisions). These interactions induce a slowing down of the hyphal, population-average extension rate and are responsible, at the microscopic level, for the distribution of tip orientations observed at the colony border. At the macroscopic level, a limiting value of the colony radial extension rate is attained. A mathematical model that combines hyphal branching, tip diffusion, and biomass growth was proposed to explain such behaviour. The main role of the substrate aw was particularly studied and integrated in this model. Experiments using Gibberella fujikuroi at different water activity levels were performed to validate the model; good agreement between experiments and simulations was achieved. Most parameters can be measured by simple image analysis on the peripheral growth zone, and they have clear physical meaning; that is they correspond to properties of single, leading hyphae. The model can be used to describe two-dimensional (2D) solid media fermentation experiments under varying culture conditions. The model was also extended by same authors to a three-dimensonal (3D) SSF of Gibberella fujikuroi grown on stacked amberlite spheres in a glass column. Steric growth limitations were found to be closely linked to the way of stacking the spheres which is related to the very old Kepler problem. The use of the rhombic dodecahedron surrounding each amberlite sphere has allowed developing an efficient model (Ferret et al., 2004).

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Others complex models that address the interactions of microbial growth with intraparticule diffusion of enzymes, hydrolysis products and 02 with the use of mechanistic equations have also been proposed and give insights into how this microscale processes can potentially limit the overall performance of a bioreactor. Models of this type have been recently reviewed by Mitchell et al., 2004. For many bioreactor models, the SSF kinetics are described by simple empirical equations. For instance, a recent kinetic model was applied to obtain noise free CO2 production rate values which in turn was used in water and energy balances. Finally, the evolution of other relevant variables including total and active biomass, 02 consumption rate, secondary metabolite production and dry mass degradation were also obtained (Lekanda & Perez-Correa 2004). An other empirical model for maximum biomass and maximum specific growth rate of A. niger as a function of the moisture content of the medium (steamed wheat bran) has been proposed by Hamidi-Esfahani et al., (2004).

5.3.1.5 Sporulation Solid-state fermentation is largely used for fungal spore production (Dorta et al., 1996) and it has been shown that spore size is different from that obtained from submerged cultivations (Feng et al., 2000). Water is a main parameter inducing sporulation and recent work (Dorta & Arcas 1998) has shown that sporulation pattern for Metarhizium anisopliae is directly related to water content even if others parameters as light frequency (Masangkay et al., 2000) and oxygen concentration are known to influence conidiation. Results obtained in our laboratory on the sporulation of two species are presented in Figure 4. A final numeration of the spores was performed after 10 days of culture. For the two fungi, sporulation did not occur in the youngest mycelial filaments, particularly in the peripheral zone. The intensity of sporulation was weaker for Trichoderma viride TS than for Penicillium roqueforti, certainly due to the pH conditions. The sporulation of Trichoderma viride TS attained a maximum for aw = 0.98 and the values decreased above as well as below this value. Maximum sporulation values of Penicillium roqueforti were obtained at aw of 0.96 as shown in Figure 4. For both fungi, optimal aw values for sporulation were slightly lower than optimal values for growth. Nevertheless for Trichoderma viride the minimal aw value allowing sporulation (c. 0.96) was greater than for growth (c. 0.92).

5.3.1.6 Germination The germination of a mould spore is a physiological reaction of a resting cell to modifications of the environmental conditions. The resting state of a spore

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is controlled by the internal presence of metabolic blocks, nutrient penetration barriers, self-inhibitors, and low water content (Smith 1978). After a certain time lag, three successive structural changes are observed during germination (Gottlieb 1978): the swelling of the mould spore, the emergence of a germ tube, and the elongation. Hydration of the medium is an important parameter of germination (Snow 1949). Germination has been observed in pure water for Alternaria and Cladosporium (Dickinson & Bottomley 1980). Penicillium frequentans and Trichoderma viride have been identified as strains with self-feeding spores (Sheridan & Sheeman 1980). However, other authors have shown that the conidia of P. notatum and T. lignorum needed carbon and nitrogen to germinate (Martin & Nicolas 1970). When the water availability was limited, the water activity (aw) of the medium strongly influenced the mould development (Gervais et al., 1988b). This parameter is widely used for quantifying the availability and the energy state of water. Nevertheless, aw alone cannot explain the biological variations; when the medium aw was adjusted by solutes the nature of the solute has an effect (Beuchat 1983). Only a few authors have tried to describe germination of conidia as a function of time. An empirical model for spore germination of Alternaria solani has been described, and further extended in order to develop a complex box model of germination (Waggoner & Parlinge 1975). A model proposed for conidial germination in Colletotrichum graminicola (Lapp & Skoropad 1976) fitted the

data well, but the parameters had little biological significance. The model proposed in a previous work (Gervais et al., 1988c) was based on a first order system application to germination which has been tested on T. viride and P. roqueforti. Water activity values from 0.94 to 0.99 were favourable to the germination of P. roqueforti; and the most favourable values for T. viride TS were 0.94 to c. 1. P. roqueforti germinated at minimum aw value of 0.86, which was higher than 0.83 found previously (Magnan & Lacey 1984) with glycerol-agar medium. But the absence of nutrients reduced the range of aw values where germination and growth were possible (Snow 1949). The influence of the low water activity values on germination is not well understood (Charlang & Horowitz 1974). The loss of an essential substance for germination for Neurospora crassa, Aspergillus nidulans and Penicillium ch~ysogenum at low water activity has been suggested (Charlang & Horowitz 1971; Charlang & Horowitz 1974). According to Gottlieb 1978 water allows the diffusion of self-inhibitors out of the spore. In all cases, the membrane permeability is important. Dehydration of the membrane would produce a molecular rearrangement of phospholipids leading to a rigidification and a loss of permeability of the membrane (Quinn 1985). The rearrangement would occur at an aw value which corresponds to the minimum water activity value observed for germination.

5.3.1.7 Metabolite production The production of secondary metabolites is also dependent in numerous cases on the water energy state in the medium which can be evaluated by thermodynamic parameters. Thus, the toxin production by filamentous fungi cultivated on solid support is related to the medium aw; the ochratoxin A production by Penicillium viridicatum is optimal for an aw of 0.87 (Northolt et al., 1984) and for an initial substrate water content of 31% (Lindenfelser & Ciegler 1975). In the same way, the water activity value of the substrate during giberellic acid production by G. fujikuroi was found to drastically decreased during SSE although optimal value was evaluated at 0.99 (Corona et al., 2005). Some hypotheses have been made on the influence of a decreased water activity on the metabolism of microorganisms. A new metabolic cycle of glycerol initiated by high concentrations in glycerol was discovered by Ben-Amotz and Avron (1981) in the halophilic alga Dunaliella. The result of this cycle is to transfer redox equivalents from NADH to NADPH at the expense of one ATE The enzyme of this cycle was found to be specific and had a very high Km to glycerol.

Since an increased synthesis of [3-carotene was found to follow the decrease in aw, it could he postulated that the intracellular accumulation of glycerol started the glycerol cycle which resulted in a transfer of redox equivalents from NADH to NADPH which favoured biosynthesis pathways and particularly ~-carotene synthesis. The synthesized solutes responsible for osmoregulation, called compatible solutes, are essentially polyols, amino acids and ions. The organic solutes thus defined interfere with enzymatic activities and are generally very water-soluble. With regard to the rate of some syntheses in response to a water stress (3 minutes for the isofluoride synthesis by Ochramonas malhamensis) (Krauss 1974), it seems that these syntheses could be initiated by the membrane deformation.

Water activity and enzymatic reactions Water activity can act through the osmoregulation process of the cell or through a direct enzymatic action, as previously described by numerous authors (Beuchat 1983; Nishio et al., 1979). We have confirmed such a direct action by a study on enzymatic activity related to the mass of enzyme produced versus water activity. The influence of water activity (aw) on the biosynthesis of polygalacturonase, D-xylanase and 13-glucosidase in a solid culture system of Trichoderma viride was previously studied (Grajek & Gervais 1987b). It was found that the production of enzymes was strongly affected by the water activity of the substrate and slightly by the nature of the aw depressor used. The maximum polygalacturonase and D-xylanase production occurred at aw 0.99 whatever the depressor used whereas ~-glucosidase formation was favoured between aw 0.96 and 0.98 depending upon the solute (0.96 for sorbitol, 0.97 for glycerol, 0.98 for NaC1). The influence of water activity on the catalytic effect of enzymes using sodium chloride, glycerol and sorbitol as aw depressors was also investigated and shown to be drastically influent as proposed in Figure 5. It should be noticed that the optimal value for enzymatic synthesis was found to be different from the optimal value found for catalytic activity. Recent work proposed an optimal 55% moisture content value for a maximum xylanase production by Trichoderma longibrachiatum (Ridder at al 1999). Nevertheless, in the case of xylanase production by Aspergillus tereus, the optimal water content was found to be 75%. A water content of 71% corresponding to a water activity of 0.921 was found to increase the production of cyclodepsipeptides in solid state fermentation by Metarhizium anisopliae (Liu & Tzeng 1999). The Doehlert experimental design was applied by Taragano and Pilosof 1999 to optimize water activity, pH, and fermentation time conditions for Aspergillus niger 148 pectinolytic activities production in solid-state (SSF) and submerged (SmF) fermentation. The

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Figure 5: Effect of water activity on the product release in hydrolir reaction using enzyme solution from T. viride TS; aw depressors: NaCl (D), glycerol (A), sorbitol (O) for A: Polygalacturonir acid => Galacturonir acid; B: Xylan => Xylose and C." Cellobiose ~ Glucose.

fermentation technique had a great influence on the composition of pectinases produced by the fungus. Production of polygalacturonase was 5-fold higher in SmF than in SSE However, pectin lyase production was 3 times higher in SSF than in SmF. Pectinesterase was only a 30% higher in SSF than in SmF. Optimization of the process was based on minimum pectinesterase production and maximal pectin lyase production. The optimal conditions to obtain this composition by a SSF process were: initial pH 6.5-7; initial aw 0.93-0.94, and 2 days of fermentation. For the SmF process they were initial pH 5.5-6.2, initial aw above 0.99, and 3 days of fermentation. The results suggest that A. niger 148 overproduces pectin lyase under the optimized SSF culture conditions, as the concentration in the fermentation extract was around 0.25 g/l, representing --- 65% of total extracellular proteins produced by the fungus. In the same way, comparison of exopectinase production by Aspergillus niger was studied between solid state and submerged fermentation (Diaz-Godinez et al., 2001). In this case an enhancement of enzyme activity was observed in SSF for high water activity (0.99) with sucrose addition (40g/L) linked to high level of biomass production. In order to increase production of different metabolites by Aspergillus strains, parasexual crosses between selected strains may be considered and recent works have shown the drastic effect of water activity on invertase production in SSF by improved diploide strains of Aspergillus niger (Montiel-Gonzalez et al., 2004). Lipase production by Candida rugosa

was enhanced by mixed solid substrate fermentation proposed by Benjamin and Pandey (1998) with an initial aw value of 0.92.

Water activity and fungal aroma production The 2-heptanone bioconversion of the fungus Trichoderma viride TS from octanoic acid has been studied in Roux flasks. A continuous aeration (50 ml/h) allowed the heptanone produced, which is a highly volatile compound (Tallu 1986) to be analysed. In this case, sequential headspace chromatographic analysis was used (Gervais et al., 1988c). Aroma production versus aw of the liquid or solid media is shown in Figure 6. Production value was dramatically affected by the water activity variation of the media. In the solid medium the 2-heptanone production increased as the aw value of the medium decreased. Such results were obtained by the analysis of the extracellular medium and resulted from the excretion availability of the cell which combines intracellular production and permeability properties. Variation of water activity in solid fermentation media could lead to great modifications in intracellular accumulation and in extracellular excretion of aroma produced by moulds (Gervais et al., 1990). Enzyme inhibition and cell membrane permeability variation are involved in such mechanisms. This conclusion may be of great interest in the attempt to optimize metabolite production such as aroma, antibiotics, enzymes by means of aw control. A sensor that allows the continuous measurement of the osmotic pressure of a liquid medium could be used with this aim (Gervais 1989). In a previous work (Gervais & Sarrette 1990) it was shown that the aroma production by microorganisms is dependent not only on physicochemical factor like pH, 18 16 = ,~, 14 ._o =

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temperature (Latrasse et al., 1985) or water activity (Gervais et ai., 1988c), but also on the physiological state of the fungus. Maximum aroma production always occurred around 4.5 days which corresponded to a particular physiological state of the mycelium. In a recent work, Larroche et al., 1999 have studied the production of 2,5-Dimethylpyrazine (2,5-DMP) and tetramethylpyrazine (TFMP) using Bacillus subtilis IFO 3013 grown in solid substrate conditions. The optimal cultivation temperature was 40~ pH had to be monitored at 7.5, aeration rate had to be higher than 0.1 VVM and ground soybeans used as substrate were suspended in water. Optimization studies showed that the best way to increase the two above aroma compounds (until 2 g/L) involved massive enrichment of the medium with L-threonine and acetoin. Medeiros et al., 2000 have also determined the optimal initial moisture of the solid-substrate allowing optimizing the aroma production by Kluyveromyces marxianus.

5.3.2 Influence of Water Dynamic Parameters In the case of solid-state fermentations, the presence of an insoluble matrix generates a matrix potential (Wm) through the intermediary of water-solid reactions. The presence of solutes in the liquid medium gives another kind of potential linked with solute-liquid interaction, the osmotic potential (qJs)- These two potentials make up the total water potential of a fermentation medium if temperature, gravity, and pressure are constant. By measuring the respective effects of the matrix potential and the osmotic potential, Griffin (1981) has determined that these two potentials have a different effect on the growth of microorganisms at the same pressure. For the same value of aw, the growth measured on media with only solid-liquid interactions, i.e. the matrix potential, is always less than the growth measured on media with only solute-liquid interactions, i.e. the osmotic potential. However, these two types of potential should always have the same effect on the growth of microorganisms at a thermodynamic level. Some authors have suggested that this variation is due to the interference of diffusion phenomena with the growth of micro-organisms (Adebayo & Harris 1971; Griffin 1972). In unpublished work, we have demonstrated that variations in kinetic properties of the culture medium in relation to hydration can explain the physiological modifications of micro-organisms. The hydration of the solid substrate was related to the growth of the filamentous fungus Trichoderma viride (Gervais et al., 1988a). Experimental design was intended to discriminate the respective influence on the fungal growth of thermodynamic and kinetic (or dynamic) parameters related to water. For the same thermodynamic level of water (aw) and by adding silica gel to the medium, it was found that the decrease in water content could generate diffusional limitation of the fungal growth.

The results shown in Figure 7 show that for the same aw value, the increase in glucose concentration significantly increases the radial extension rate for very low water content value (Medium C: 2.22 g water/g insoluble dry matter), but has no action for high water content values (Medium A: 63.4 g water/g insoluble dry matter). In order to confirm the assumption of a diffusion limitation, the diffusion of glucose in previous media (A, B and C) was evaluated. The translational diffusion coefficient proposed (see Table 3) was determinated following Lyrett's method. Glucose diffusivity was found to decrease sharply when silica gel was added to the media. These results confirm the hypothesis that diffusion could be limited when the water content of the fermentation was decreasing. The rotational diffusivity (Drot) measurements determinated by using electronic spin resonance are also presented in Table 3. Similar results described previously showed a decrease in diffusivity of solutes and water molecules when there was a decrease in water content (Karel 1976; Biquet & Guilbert 1986). The values for the Drot[Otrans ratio in Table 3 show that this ratio stays almost constant whatever the water content is, implying that the tortuosity of the media used had little or no influence on solute diffusion in these media. The limiting factor on the growth of microorganisms was the diffusion rate of solutes in the gel and not the tortuosity of the medium. When there was a large amount of polymer, the water content was reduced and solute and water diffusion decreased for the same aw. The higher the proportion of the matrix potential, the lower the water content became. In fact, analysis of the sorption curves showed that insoluble polymers required a smaller quantity of water in order to reach the same aw as most solutes. MacDonald & Duniway 1978 carried out experiments on the same kind of soils as those used by Griffin & Luard (1979) and Griffin (1981). It can be deduced that in order to obtain a matrix potential level that corresponds to water activities 0.98 and 0.99 in their culture medium, extremely low water contents were required. Thus, diffusional factors combined with low water content in the culture can limit the growth of microorganisms in such solid media.

Table 3: Diffusion coefficients of glucose (and their ratio) in different silica gel media of different water content (A, B, C)

Figure 7: Radial growth rate of T. virideTS grown on gels with water activity 0.99. [ ( ~ ) Medium A (63.4 g water/g insoluble dry matter); ( ~ ) Medium B (5.47 g water/g insoluble dry matter); ( ~ ) Medium C (2.22 g water/g insoluble dry matter)]. 5.4 CONCLUSIONS In solid-state fermentation, water mass transfers are strongly related to other physico-chemical control parameters and especially to aeration and heat, and it is necessary to have a global approach for optimization.

5.4.1. Water and aeration Aeration has essentially two functions (Chahal 1983; Tabak & Cook 1968): (1) oxygen supply for aerobic metabolism and (2) removal of CO2, heat, water vapour, and volatile components produced during the metabolism. The aeration rate must also depend on the thickness of the layer and the porosity of the medium. The effects of pO2 and pCO2 in the gas environment are important variables and must be optimized for each medium, microorganism, and process (Raimbault & Alazard 1980; Lonsane et al., 1985; Bajracharya & Mudgett 1985; Huang et al., 1985; Levonen-Mufloz & Bone 1985). Aeration also has very important effects on hydration properties and heat regulation in solid-state fermentations. As shown previously, the water potential could be monitored through the relative humidity of the air, and most of the heat evolved would be transferred to the air latent heat of water vaporization. Sato et al., 1982 estimated this latent heat to account for about 80% of the total heat evolved. Such a ratio has been confirmed more recently by Sangsurasak & Mitchell (1998). The aeration in solid-state fermentations which controls air distribution in the medium can be characterized by means of residence time distribution determinations (Gervais et al., 1986).

As defined by Moo-Young et al., (1983), the most important O2 transfer mechanism is interparticles mass transfer. The dew point of the air used for aeration must be defined as a function of the air inlet temperature in order to control the relative humidity of the solid substrate. Modification of the air status occurs when air is passing through the solid bed. The relative humidity of the air generally increases due to evaporation; such phenomena can be localized and lead to heterogeneity in the medium if the flow rate is too slow. To prevent this type of problem, the temperature and water content of the substrate bed could be controlled by saturating the air at the air inlet but manipulating its temperature, coupled with a strategy of water replenishment when the water content of the bed falls below a threshold. Recent paper (Von Meien et al., 2004) has tested different control strategies based on classic proportional integral derivative (PID) and advanced dynamic matrix control (DMC) algorithms for an intermittently stirred, forcefully aerated SSF bioreactor. Pressure drop could also vary as the chemical composition of the solid state evolved. An air flow rate control loop could prevent these variations (Aunia et al., 1995). However, from a general point of view, gas diffusion (O2, CO2) occurs at very high rates with regard to the solute diffusion rates in liquid media and even at higher rates in the gas phase of solid state media. Therefore such diffusion will not generally constitute a limiting factor in SSF.

5.4.2. Water and heat Temperature regulation is directly related to water potential and aeration. Temperature is a factor that strongly influences culture growth, and in aerobic fermentations, a large amount of heat is produced during microbial growth, as shown by Gonzalez-Blanco and colleagues (Gonzales-Blanco et al., 1990) for A. terreus. A great limitation of solid-state fermentations is the difficulty of removing excess heat due essentially to the low thermal conductivity of the solid substrate. For instance, Oostra et al., (2000) have measured a thermal conductivity value of the bed of 0.1W.m-I.K-1. Saucedo-Castanada and colleagues (Saucedo-Castanada et al., 1990) have estimated the dimensionless B iot and Peclet numbers corresponding to the heat transfer during cassava solid-substrate fermentation by A. niger. It was shown that conduction through the l 1-fixed bed was the main heat transfer resistance. To maintain the optimal culture conditions, it was necessary to immediately eliminate the heat generated. Convective heat transfer from the reactor wall was not effective due to low mixing and internal heat transfer limitations. The method of evaporative cooling that inserts partially saturated air at lower temperature is the most interesting, as proposed by Durand et al., (1983) and Grajek (1987).

In practice, only air is used for temperature control in solid-state fermentations. The heat capacity of the air at maximal water saturation is less than the heat capacity of water for cooling in submerged fermentations. For these reasons, solid-state fermentations need a large quantity of air, exceeding the amount necessary for microbial respiration. At a semi-industrial scale, the limitation in cooling technique in solid-state fermentations enhances the use of thermophilic strains, as realized by numerous workers (Moo-Young et al., 1977; Viesturs et al., 1981; Moo-Young et al., 1983; Chahal 1983). From the previous considerations, the physicochemical requirements of solidstate fermentations can be summarized as follows: It is generally beneficial to keep the level of water content just below the water holding capacity of the solid substrate in order to optimize 02 requirements without limiting the liquid diffusion of nutrients. It is important to adjust the forced aeration with air with controlled relative humidity to optimize heat, CO2 and volatile evacuation, and 02 requirements. Since respiration produces water, endothermic evaporation phenomena will be the major energetic factor of heat evacuation. It is necessary to take into account the depth of the fermentation bed and to calculate the air temperature and the air flow rate in order to prevent rapid water saturation of forced air and condensation in the upper part of the bed. Indeed, residence time of air must be sufficiently short in order to not be saturated at the exit of the bed. It is necessary to take into account the evolution of the water sorption properties of the solid substrate during the fermentation to optimize water activity requirements, which are of major importance for growth. Indeed, the division of high-molecular-weight polymers by extracellular enzymes such as cellulases could lead to drastic decreases in water activity for the same water content, as shown previously. Therefore, the relative humidity of the forced air must be adjusted during the fermentation.

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6.1 INTRODUCTION It is well known that the solid-state fermentation (SSF) is a new technology with which antibiotics, enzymes, fine chemicals can be obtained by new bioprocesses with improved efficiency (Giovannozzi-Sermanni & Porri 1989; Pandey et al., 2000) if compared to the classical submerged fermentations. SSF processes can be defined as "the growth of microorganisms, mainly fungi, on moist solid materials in the absence of free-flowing water"(Cannel & MooYoung 1980). In the last few decades SSF has grown quickly in interest and importance and has been used for the production of antibiotics, alkaloids, aroma compounds, plant growth factors, enzymes, biofuel, and also for the bioremediation of polluting compounds. To understand tiae importance of the SSF, following must be kept in mind: 9 the biological sustainability is associated to the atmospheric content of carbon dioxide. 9 the content of carbon dioxide in the atmosphere increases continuously making the chemical energy obtained by the photosynthesis in sufficient to control its rise to which a quote coming from fossil carbon utilization is added (Fig. 1). 9 the needs of chemical energy requested by human societies are increasing rapidly. 9 the availability of water is dependent on environmental conditions and it is decreasing in many countries. 9 celluloses, hemicelluloses, lignines, starch and oils could be utilized by new technologies to avoid their loss through wild uncontrolled biological processes.

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From the above considerations, it seems that the better controlled biological utilization of lignocelluloses by environmentally friendly applications can deeply contribute to improve the sustainability. The degradation of water insoluble copolymers has as first steps hydrolytic and oxyreductive reactions, occurring in three-phases (gaseous, liquid and solid phases) systems and can help to efficiently solve problems through SSF, since in recent years studies have demonstrated their superior product yields and simplified downstream processes (Pandey et al., 1999). These factors increase to the possibility of producing more stable products, with less energy requirements, and make SSF a very attractive alternative to the conventional liquid fermentation, especially in the developing countries. The use of solid matter has serious implications in the engineering aspect of bioreactor design and operation. It is interesting to note that few enzymes, necessary for the biosynthesis and biodegradation of the lignocelluloses have been working in nature at least since 350 millions of years, but until now they are poorly utilized by means of controlled biological processes. These enzymes belong to hydrolytic enzymes (cellulases, hemicellulases, polyphenolases, pectinases, etc.) and oxidative ones (polyphenoloxydases, peroxydases, etc.).

6.2 CHEMICAL, BIOCHEMICAL AND MICROBIOLOGICAL SHORT OUTLINES Lignocelluloses are copolymers, characterized by highly complex chemical structure. Celluloses, lignins and hemicelluloses, chemically linked each other,

contain large quantities of chemical energy. The agricultural and forestry resources provide immense quantities of such materials but the amount of unutilized lignocellulose can reach easily the 50%. Keeping in mind that more than 3000 million tons of cereal straws roughly are produced per year worldwide and that these residues are mostly disposed of by burning and humification, it results a terrific loss of energy. As a consequence, the chemical energy of unutilized lignocellulosic matter constitute a huge source of energy which could be used for many applications beside the buming and humification. Cellulose, homopolymer of 1,4-glucose units, hemicellulose, heterogeneous carbohydrate polymer, and lignin, polymer of phenylpropanoidic units interconnected by a great variety of linkages, contain energy which can be utilized by a series of biochemical breakdown reactions, fundamental aspect for efficient applications. Therefore, first of all it is necessary the depolymerization of the water insoluble lignocellulose takes place to produce soluble compounds, and this degradation can start by few enzymes which allow the formation of lower molecular weight water soluble copolymers (Crestini et al., 1998) (Fig.2). It is interesting to note that simple phenolic compounds can have also effects on redox enzymes activity (Giovannozzi-Sermanni 1981).

6.3 ROLE OF MAIN PARAMETERS IN SSF 6.3.1 General considerations Targeting a SSF production for a possible industrial interest, two are the ways to approach the problem:

Fig. 2. Pathways of biodegradation of lignocelluloses by enzymes.

the characteristics of the wanted products determine the choice of the starting matter, generally a low-cost agricultural resource simply available. the possibility to use huge amount of available residues encourages the development of new processes to produce value-added goods, by improving also the biological sustainability. Therefore, the choice of microorganisms is of great importance, which are dependent on parameters such as pH, temperature, moisture content, etc. Consequently the development of devoted bioreactors useful to utilize the set of parameters chosen for the process is of paramount importance. Also, some physical and engineering specifics not directly dependent on the biochemistry of the process must be considered, such as employed materials, analytical controls, economical factors, maintenance, pretreatment and downstream treatments, etc.

6.3.2 Parameters affecting the bioprocess The most important parameters characterizing an SSF process are the following"

water activity and moisture content of the substrate. This variable has influence on biodegradation processes, biosynthesis and secretion of metabolites (Perez-Guerra et al., 2003). temperature and heat transfer. This affects directly bioprocesses characteristics, spores germination, cells growth and the efficiency of the process. pH. It determines the enzyme activities, given that each of them can be dependent on different optimal pH values. aeration. It determines the 02 and CO2 concentration inside the bioreactor, regulating the removal of CO2 and volatile compounds (such as ethylene), the relative humidity, and it improves the heat transfer. nutrient diffusion. It affects the nutrient concentration and regulates the actions of enzymes over the solid substrate (normally water insoluble). mixing. It helps heat removal, gas exchange, water content, uniformity and influences the process conditions. septic conditions. It is required to avoid the release of the process organism in the environment and the entry of polluting microorganisms.

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particle size (density). It regulates the utilization of the substrate by the microorganism at a molecular level, the gas-liquid interfacial area and the thickness of the wet fungal layer. It makes the oxygen utilization dependent on this parameter (a too small particle size may interfere with microbial respiration, causing poor growth; a large particle size provides only a limited surface for microbial attack) (Pandey et a1.,1999).

physico-mechanical properties of the solid matrix. The matrix should have wide surface area and should stand gentle stirring or compression if required by the chosen fermentation to avoid sticking behaviour during the process.

microbiological inoculum. Its type, relative humidity, amount, etc. influence the growth rate.

biomass uniformity. It is of fundamental importance to maintain uniform biochemical reactions all over the biomass. morphology of the microorganism. The most important characteristics are to penetrate into a solid substrate with a low water activity, assimilating complex and variable mixtures of nutrients, the capacity of adherence and i.e. the presence or not of septum in the hyphae for mechanical resistance. Most of the above parameters are strictly interconnected, but few of them appear to be of major importance in the development of new processes.

6.3.2.1 Temperature and heat transfer Respiration during the growth, which is dependent to the oxygen consumption and CO2 formation, is highly exothermic and heat generation is directly related to the level of metabolic activities of the microorganisms. The removal of metabolic heat during large-scale SSF is one of the most critical issues since microbial growth is particularly sensitive to the rise of temperature, given that the heat generation produces thermal gradients (Schutyser et al., 2003; SaucedoCastafieda et al., 1990). Many approaches are available using different cooling techniques to enhance the heat transfer and to control the microbial growth, such as mixing, forced aeration, evaporative cooling, utilization of cooling jackets and additional cooling surfaces, that are just the most used systems to achieve the best thermal conditions. These approaches and their role in designing will be considered in detail in par. 6.4.

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6.3.2.2 Water activity and moisture content Water has a solvent function providing nutrients and scavenges wastes, and a structural function which, is involved in the stability and the function of the biological structures (Gervais & Molin 2003). Water in SSF systems is present in a complexed form within the solid matrix or as a thin layer either absorbed on the surface of the particles, or in the capillary region of the solid. The excessive lack of free water does not allow good diffusion of the nutrients and gas exchange, and can cause a loss of the functional properties of some enzymes and a disequilibrium on the metabolic chain of the cells. On the other hand, an excess of water can perturb the transport phenomena at cellular and macroscopical level. Water requirements should be defined in terms of water activity (aw), a thermodynamic parameter related to the chemical potential of water. Aw is well correlated to the relative humidity (RH) as follows: aw = RH/100 = P/P0 where p = vapour pressure of water in the substrate and P0 = vapour pressure of pure water at the corresponding temperature (Raimbault 1998). A reduction in aw normally extends the lag phase of the microbial growth, causing a low biomass production.

6.3.2.3 pH The substrate and medium acidity has great importance in the bioprocess, not only because the pH partly screens the microbial growth from a large number of competitors but also because partly gives a fight habitat for the microorganism. In fact the metabolic regulation routes, among other factors, may be controlled by pH (Fig. 3) (Giovannozzi Sermanni et al., 1978).

6.3.2.4 Aeration The rate limiting step in an aerobic submerged liquid fermentation (SmF) is usually the transfer of 02 from the gas phase to the liquid phase. Therefore the single most important consideration in the design and scale up of bioreactors for submerged liquid fermentations is to provide a sufficiently high rate of O2 transfer into the liquid medium. Regarding to the SSF processes, especially in packed bed bioreactors, both heat and mass transfer are important, and SSF in packed bed bioreactors, overheating is a major problem (Saucedo-Castaneda et al., 1992; Gowthaman et al., 1993), due to unidirectional flow of air. As the air passes through the column and removes heat from the substrate, the air warms up, loosing its cooling efficiency through the column. Consequently, a

Fig. 3. Influence of pH on the possible regulation of cellulose and lignin degradation by mycelial and microbial metabolism (pH stat) (modified from Giovannozzi et al., 1978). temperature gradient is established along the column with the highest temperature at the outlet end of the bed. In a rotating drum bioreactor, the gas exchange is obtained by passing the air through the biomass. In this way, it is possible to maintain a constant chosen 02 and CO2 value as shown in Fig. 4, and the total fluxed air during the fermentation and the gas exchange can be recorded continuously to maintain the reproducibility of the process. For example, after few days of fermentation the CO2 and the 02 levels can be modified and maintained at a chosen value. In an earlier study, Fung and Mitchell (1995) showed that maximum 02 uptake rates were higher in a baffled rotating drum than in an unbaffled one, so that 02 limitation could occur in the unbaffled drum too (Marsh et al., 1998).

6.4 GENERAL ASPECTS OF SSF BIOREACTOR DESIGN The use of solid matrix, either as an inert support or substrate support, has relevant implications on the engineering aspect of bioreactor design and operation (Robinson & Nigam 2003). Maximization of the rate of formation and the yield of product within the bioreactor are a key of the optimizing the bioprocess. Nevertheless, SSF bioreactor systems have not yet reached a high degree of

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development, mainly due to the problems associated to the solid beds like poor mixing, heat transfer and material handling. Some of the desired features of a solid-state bioreactor system are the following (Raghavarao et al., 2003): the constitutive material has to be cheap, inert and resistant to the corrosion and abrasion. the system has to be preferably microbiological contamination-free to prevent accidental hazardous due to biological pollution. the control and regulation of the operational parameters must be efficient. the uniformity of biomass must be provided. the bioreactor design has to simplify the maintenance, the loading-unloading and the product recovery (according to the labour cost of the countries where the SSF process is developed). It is possible to classify the categories of bioreactor for SSF by their size and by the quantity of dry solid substrate involved, so that a lab-scale would process from few grams to few kilograms, a prepilot-scale from few to several kilograms, a pilot-scale several kilograms and an industrial-scale up to several tons. During the scaling-up of the process, the availability of bioreactors designs decreases considerably due to the reasons shown in 6.4.3 and 6.4.4.

6.4.1 Lab-scale SSF at laboratory-scale shows several biotechnological advantages, if compared to SmF, such as high fermentation productivity, high end-concentration of products, high product stability, low catabolic repression, cultivation of microorganisms able to grow on water-insoluble substrates, and often the demand on sterility is not requested (H61ker et al., 2004). It is also possible at lab-scale to remove properly the metabolic heat generated by the fermentation, keeping the culture vessel in a temperature-controlled environment (water bath, cooling jacket, etc.).

Approximately 90% of all industrial enzymes are produced by SmF, frequently using specifically optimized, genetically manipulated microorganisms. However, almost all these enzymes could also be produced in SSF using wild-type microorganisms (Filer 2001; Pandey et al., 2001). It has been shown that enzyme production in solid state is higher than in submerged fermentation (Viniegra-Gonz~ilez et al., 2003). It is very interesting also that the stability of the produced enzymes at high temperature or extreme pH, are better in SSF (Deschamps and Huet 1985; Acuna-Arguelles et al., 1995). Catabolite repression or protein degradation by proteases, severe problems in SmF, were often reduced or absent in SSF (Solis-Pereira et al., 1993; Aguilar et al., 2001). Since 1959, when the first example of laboratory-scale fermentor was described (Giovannozzi, 1959) (Fig. 5), studies on laboratory-scale system represent a powerful aid to develop SSF bioprocesses and a useful front of data for modelling the process.

The solid-state bioreactor f contains a metal net basket a which is filled with lignocellulosic matter. The funnel e may contain substrate useful for the growth of microorganisms. All the apparatus can be sterilized in a steam pressure oven. The substrate of funnel can be inoculated with microorganisms chosen for the trial. After few days the liquid culture is transferred into the bottom part of the bioreactor so that the matter contained in the basket is inoculated with a single chosen microorganism. Then the liquid phase is pumped away through the r tap. The bottom b part of the bioreactor can be utilized to contain different kinds of liquids useful for the trial. Through d and c taps equipped with sterile filters it is possible to control the gaseous phase composition of the bioreactor, particularly oxygen and carbon dioxide. Hence this solid state bioreactor, the first described in the scientific literature, permits to control and to follow the bioconversions of lignocellulosic matter and to test the effects of different microorganisms on the biodegradation of them.

Fig. 5. Lab-scale fermenter (Giovannozzi 1959).

It is significant to note that recent equipments organized in batteries and computer controlled (Fig. 6), useful for a systematic investigation, utilizes single units very similar to the first one already shown. Laboratory equipments such as Petri dishes, jars, wide mouth Erlenmeyer flasks, Roux bottles, roller bottles are of some help for their simplicity and easiness in handling. In the last few decades, more complex lab-scale units have been developed, such as the one patented by an OSTROM team between 1975-1980. It consists of a small column filled of inoculated medium, humidified, and thermoregulated by a waterbath. It is useful for screening studies and for the first step of parameters optimization. The small quantity of used medium (few grams), and the geometry of the glass column guarantee an easy maintenance of the temperature in the reactors. The design of this reactor, however, does not permit sampling during the fermentation and hence, during the process, one entire column has to be utilized for each analysis. A new generation of small reactors was developed by an INRA-team in France. Such reactors have a working volume of about 1 1, a relative humidity probe, a cooling coil on the air circuit and a heating cover for the vessel (Fig. 7). These changes enhanced the regulation of the water content during the process. Each reactor is automatically controlled by a computer. Several teams have designed bioreactors based on continuous mixing of the solid substrate/support. Such characteristic is suggested by the aptitude of some filamentous fungi to agglomerate. Therefore, to avoid such behaviour which alter deeply the biological efficiency the mixing can be useful. The bioreactors can be perforated drums (Fig. 8-1), like the one described by Kalogeris et al., (1999), rotating drums (Fig. 8-2), or horizontal paddle mixers (Fig. 9). All those drum bioreactors are designed to allow adequate mixing of the medium and good aeration.

Fig. 6. Schematic packed column bioreactor system. 1) air pump, 2) air filter, 3) humidifier, 4) air distributor, 5) battery of columns in water bath, 6) silica gel columns, 7) gas sampler, 8) chromatograph, 9) PC unit (redrawn from Medeiros et al., 2001).

Fig. 7. Schematic and photography of a lab-scale sterile fermenter: 1) heating cover, 2) insulating jacket, 3) biomass temperature probe, 4) stainless steel sieve, 5) air inlet temperature probe, 6) relative humidity probe, 7) heaters, 8) level probe, 9) water temperature probe, 10) massic flow meter (redrawn from Durand 2003).

Fig. 8. Schematic of a rocking drum bioreactor (1), a rotating drum bioreactor (2), and photography of a lab-scale battery of drums controlled by a PC unit device (3).

Fig. 9. Photography of a paddle mixer. Schematic of a stirred horizontal bioreactor: 1) air inlet, 2) air outlet, 3) cooling jacket, 4) paddles, 5) motor, 6) temperature probe (redrawn from Durand 2003).

With or without a water-jacket, this type of reactors is required for the continuous mixing to increase the contact between the reactor wall and the solid medium and also to provide oxygen to the microorganisms. For rotating drum bioreactors, as an horizontal cylinder, the mixing is obtained by the tumbling motion of the solid medium which may be aided by baffles on the inner wall of the rotating drum (perforated or not). With these bioreactors, agglomeration of substrate particles during the growth of the mycelium can happen.

6.4.2 Pre-pilot and pilot plants Only a few designs of bioreactors have been studied and applied to pilot-scale, or industrial level. The limited application of SSF for large-scale is due to some further important parameters present for the scaling-up, such as: the mycelium hyphae, which can be damaged by heavy mechanical stirring, particularly if they have no septa, allowing only few possible designs to respect the aeration and heat removal needs. the solid medium, which can agglomerate during the process causing shrinkage, air channelling and problems during the operations. That makes difficult the heat removal and the gas transfer and severely restricts the design strategies available. the procedures of inoculation, control and the difficulties associated to the sterilization or pasteurization of large volumes as well. Particularly beyond three days of mycelial growth, it could be prohibitive working in non-sterile conditions (Fig. 10). 9 the versatility of the reactor for a flexible usage in different conditions.

Fig. 10. Scheme of an SSF pilot plant and related controls.

the maintenance and the procedures of filling, emptying, cleaning of the bioreactor. 9 the maintenance of uniformity, difficult for large volumes of biomass. Few of the previous described bioreactors have been used to develop the industrial and pilot plants that can be divided in categories based on the mixing and aeration strategies adopted (Table l) (Mitchell et al., 2000): 9 static, pulsed mixed, continuosly mixed. 9 unforced or forced aeration (trough the biomass). Table 1. Summary of most used bioreactor typologies.

6.4.2.1 Static beds without forced aeration (tray bioreactors) This category is the oldest and the simplest in design, typified by the tray fermenters used in the Koji process. The solid substrate is loaded on trays (made of wood, plastic or metal, perforated or not) in thin layers (typically 5 -

15 cm) and placed one above the other with a gap of few centimetres into a thermostated room as shown in Fig. 11. There is fully empirical and experimental evidence of poor heat and mass transfer in trays, limiting the thickness of the substrate bed, that must be not more than a few centimetres. Hence, such reactors are restrictive in the volume of the solid matrix used: only thin layer can be used to avoid overheating and to guarantee aerobic conditions. Temperature and relative humidity are the only external parameters that can be controlled. The scale-up in this case is easy (increasing the number of trays used), but requires large operational area, intensive labour and it is difficult to apply for sterile processes.

Fig. 11. Schematic of a koji-type reactor: (1) water inlet, (2) UV tube, (3) (4) (5) air blowers, (6) (7) air filters, (8)air outlet, (9) humidifier, (10) heater, (11) Air inlet, (12) Trays, (redrawn from Durand 2003).

6.4.2.2 Static beds with forced aeration (packed beds) In a packed-bed bioreactor, conditioned air is usually blown through a sieve which supports the substrate. Experimental work demonstrates that the temperature gradients limit considerably the bioreactor performance. These bioreactors have been reported being interesting for their efficient process controls, especially for heat removal, which is due to an ample exchange surface. Strategies to prevent overheating problems have been examined (Ashley et al., 1999), finding more useful the mixing if tolerated by the microorganism than the periodic air reversal. Packed-bed bioreactors show some limitations such as irregular growth and poor heat removal if scaled up (Robinson & Nigam 2003). Targeting a particularly efficient heat removal, Roussos et a1.,(1993) developed the Zymotis bioreactor equipped with vertical rectangular cooling plates closelyspaced and inserted directly into the bed (Fig. 12). The geometry of such type

of bioreactor is particularly promising, but the loading-unloading operations can be uneasy and appears difficult to obtain septic conditions when large volumes are utilized.

Fig.12. Zymotis bioreactor; photography(Durand 2003) and schematic.

6.4.2.3 Pulsed mixing without forced aeration (discontinuously rotating drum) The drum bioreactor intermittently rotates operating like a tray bioreactor during the static periods.The pulsed stirring prevents the agglomeration of the mycelium and the shear is less effective on the mycelium than in a continuous rotating drum. This design has been demonstrated scalable up to 4 m 3 (Fig. 13). This rotating drum can work intermittently or continuously and is equipped by an internal sterile aeration system that allows air circulation trough the substrate if required, a regulation of internal gas phase composition (automatically or manually controlled), a cooling/heating jacket and a sterile inoculation system. By the way it shows some limitations in heat transfer control, that is the greatest barrier in scaling up.

6.4.2.4 Pulsed mixing with forced aeration (intermittently stirred beds) It is possible to enhance the homogeneity of the cell population and the substrate concentration in the SSF systems adopting sequential mixing and by imposing forced aeration through the solid substrate it is possible to allow the control of water content, water activity, the removal of volatile compounds, CO2 and

Fig. 13. Rotating drum bioreactor (4m 3) at University of Tuscia; inside view of the bioreactor with complete mycelial colonization at the end of a fermentation before unloading (Giovannozzi-Sermanni et a11994).

heat generated during the fermentation. The geometry is similar to the one used for packed bed, but it includes a mixing system for the biomass like a screw or an other kind of agitation device (Fig. 14). The capacity of such kind of bioreactor has been scaled up to several tons. The intermittently stirred bed bioreactors are promising for non-sterile processes, but their design can be complex and not simply scalable, if sterile conditions are needed.

6.4.2.5 Continuously mixed without forced aeration (continuously rotating drums) This type of bioreactor consists of a continuously rotating horizontal drum that holds the substrate bed. It can be equipped with baffles and within the air is not forcefully blown through the bed. The effect of the rotational speed and the other mixing parameters on the fermentation was largely studied by several

Fig. 14. Schematic of a cylindrical (1), conical (2), screw mixers and an helical blade mixer (3).

teams (Schutyser 2003). To increase the performance of such bioreactor, it is possible to use a baffled geometry and/or use high rotational rates (up to 1050 rev. min 1).

6.4.2.6 Continuously mixed with forced aeration The most important types of this category of bioreactor are: 9 the rocking drum bioreactor. 9 the gas-solid fluidized bed. 9 the continuously stirred aerated bed. Frequently, water is replenished, by spraying it as a fine mist on the biomass or dripping it into the substrate trough perforated tubes. This allows high evaporative cooling. It has been reported that the continuous mixing improves the heat transport to the fermenter wall reducing the need of evaporative cooling and, consequently, the problems related to the substrate drying. The evaporative cooling retains its importance for large scale mixed bioreactors (Nagel et al., 2001).

6.4.2. 7 Other designs In the last few years, some interesting unusual novel designs for SSF bioreactor have been proposed. These designs are based on unusual features and for this reason, they differ visibly from the previous typologies. A patented solid-state fermentation based on periodic air-forced pressure oscillation (SFPAPO) has been described by Chen & Sun (Fig 15). An immersion bioreactor, based on intermittently immersion in a liquid medium has been described (Rivela et al., 2000; Rodriguez-Couto & Sanrom~in 2006). It consists of a jacketed cylindrical

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glass vessel with a round bottom, inside which several wire mesh baskets filled with colonized support are placed. They can move upwards and downwards by means of a pneumatic system, remaining more time outside than inside the medium (Fig. 16).

Fig. 16 Scheme of an immersion bioreactor (mechanical agitation; humidified air): (1) medium, (2) sampling port, (3) air inlet, (4) water cooling, (5) compressor, (6) pneumatic system, (7) gas exit, (8) time controller. (redrawn from Rivela et al., 2000).

6.4.3 Scaling-up SSF scaling-up, essential for uses at industrial scale, raises severe engineering problems due to the increase of temperature, pH, 02, substrate and moisture gradients. Hence, most published reviews also focus on progress towards industrial engineering. The scale-up of bioreactors is usually based on empirical criteria, related to transport processes (Hsu & Wu 2002), but the application of mathematical modelling techniques to describe the phenomena within the system is the basis of the most significant improvements in designing and scaling-up SSE Lenz et al., (2004) and Pandey (2003) have shown to these be a powerful aid for the designing and the defining of large-scale bioreactors (Raghavarao et al., 2003). A large-scale prototype of an intermittently stirred bed ( l t o n capacity) designed at INRA has been described by Durand & Chereau in 1988 (Fig. 17). An important concept in the design a bioreactor is the possibility to let take place in one single vessel as many major operations as possible. The pilot plant described by Grant et al., in 1978 is a good example in this respect. In this bioreactor, it was possible to hydrolize (121~ 30min, 0.5N H2SO 4, 7:3 liquid:solid) the substrate (100kg of ryegrass straw per batch), to treat it with ammonia to raise the pH (to 5.0), to inoculate it (with Candida utilis) and to conduct the fermentation holding the material stationary with air blowing up through it, as shown in Fig. 18.

Fig.17 A. Schematic of a large-scale mixed reactor: (1)inoculated biomass, (2) agitation device, (3) probes, (4) air inlet, (5) cooling water, (6) steam, (7) air conditioner, (8) blower, (9) regulator, (10) (11) valves, (12) screw conveyer, (13) outlet (redraw from Xue et al., 1992) B. photographies of a large scale bioreactor (INRA-Dijon, France) (Durand 2003, Durand et al., 1996),

On the same concept, a novel efficient design of integrated matrix bioreactor, called the PLAFRACTOR TM, consisting of a computer controlled device, using complex fermentation control algorithms, has been patented (Suryanarayan & Mazumdar 1999; Suryanarayan 2001). All the operation, such as sterilization, cooling, inoculation, control of fermentation condition, extraction of the product and post-sterilization of the substrate, are all done in one single equipment, maintaining all the advantages of SSF (Fig. 19). A particularly interesting novel design was scaled-up and reported by Hongzhang et al., (2002). It consists of a cylindrical steel container with the capacity of 70 m 3, used as the fermentation vessel, loaded by trays made up of stainless steel mesh placed horizontally, or vertically. In order to enhance the biological

Fig. 18. Pilot plant (2,270 I): operations and photography (Grant et al., 1978). activity by external stimulation, thermal and mass transfers, the fermentor is supplied by two dynamic changes of air consisting of internal/external circulation and periodic air pressure pulsation (Fig.20). This system provides sufficient gas exchanges, more room for fungal propagation and efficient heat removal without disrupting the mycelia. On these basis, the reactor design was scaledup from 8 1 to 50 1, 25 m 3 and 70 m 3.

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Fig. 19 Plafractor TM bioreactor: schematic with; (1) fluids into communicating channels, (2) fluids out from non-communicating channels, (3) air/steam/ inoculum in, (4) vent, (5) extraction fluid in, (6) extract (Suryanarayan 2003).

Fig. 20 Photography of of the 70 m3 industrial bioreactor (a); schematic diagram of external (b); and internal (c); circulation of (d): air; schematic of the reactor: 1. door, 2. trays, 3. vessel, 4. fan system; (e); diagram of the periodic pressure changes (drawn from Hongzhang et al., 2002).

6.5 APPLICATION One interesting example of the application of SSF enzymes production is given by the preparation of cellulose for paper mills, where the opening of the fibres needs large amounts of thermal, mechanical and chemical energy. Enzymes produced by SSF of wheat straw have been used to prepare paper pulps from annual plant residues. By the rotating drum bioreactor after seven days of fermentation with a Lentinus edodes strain, a well colonized biomass is obtained (Fig. 14). With the aid of a press, it is squeezed and the liquid phase is used as a source of enzyme, particularly redox enzymes. Enzymatic mixture applied to annual plant residues have excellent technical properties of high yield paper obtained with kenaf bast strands, cereal straws. With corn stalks (Giovannozzi Sermanni et al., 1994) remarkable improvements of freeness and strength properties with respect to the control are also obtained, as well as a higher lignin extractability. Previous investigations, carried out on wheat straw and corn stalks, showed that the enzymatic incubations produced chemical modifications, which were not evident after the pre-treatment only. In fact, positive effects, in terms of lignin extractability, were recorded after alkaline cooking. This phenomenon suggested the possibility of a lignin functionalization, which rendered it more extractable under alkalineconditions and/or a physical modification of the biotreated material, such as an enhanced porosity, which facilitated the accessibility of the cooking liquor, thereby improving its effectiveness. The cellulose content of enzyme treated-samples (ET) did not differ from the corresponding controls, confirming the poor cellulolytic capability of the Lentinus edodes strain. By incubating recycled fibers with enzymatic cocktails containing cellulases and hemicellulases, the results suggest that the enzymes could act on the surface of the fibers (peeling effect), removing those components with a great affinity for water, poorly contributing to the hydrogen-bonding potential of fibers (Pommier 1989). The biotreatments have poor, or no effects on the opacity and brightness, whereas the latter parameter was reduced by 20 and 15% in wheat straw and corn stalks samples, respectively. The phenomenon increases the strength properties accompanying reduced optical properties during biomechanical pulping of loblolly pine chips (Akhtar et al., 1992; 1993). The enzyme treatment show a significant energy savings during fiberization and refining. The results obtained by using hexoenzymes allows to obtain the following main results: the amounts of alkali employed in the cooking process for enzymaticallytreated samples is reduced by 40 % with respect to the untreated controls

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(4% vs 7% NaOH on o.d. weight basis). All comparisons are made at the same freeness values of pulps (30-35 S.R. degrees). significant energy savings during fiberization and refining operation (50%) are observed in enzyme treated samples. no significant differences in the optical properties are observed between the controls and the enzymatically-treated samples, with the exception of kenaf and wheat, being the brighmess values of ET samples lower than the corresponding CT samples.

6.6 CONTROLS Monitoring these processes requires the measurement of environmental parameters (temperature, pH, water content and activity) and the carbon cycle (biomass, substrate concentration, CO2). However, given the complexity and heterogeneity of the solid medium, these variables are not easily accessible and measurable (Bellon-Maurel et al., 2003). Temperature sensors are generally inserted radially at various distances from the centre of the fermentor and linked to control systems. At the industrial scale, temperature regulation is linked to the moisture content of the solid phase. Direct measurements of temperature, pH, and water content are considered employing classical sensors, and indirect measurements of the biomass by respirometry or pressure drop (PD). More recent methods have been tested and successfully tested, such as: aroma sensing, infrared spectrometry, artificial vision, and tomographic techniques (X-rays, Magnetic Resonance Imaging or MRI). Therefore, the study and development of novel on-line methods and innovative applications of methods with a potential to measure parameters in SSF are an important task to improve the performance of the bioreactors.

6.7 CONCLUDING REMARKS The abundance of scientific literature on the solid-state bioreactors makes difficult to organize a comprehensive treatment of the issues given the complexity of the matter. Many solid materials can be utilized for SSF such as soils, animal residues, hides, but without any doubt the most attractive solid-state substrates for new processes are the lignocelluloses, present all over the Planet. By keeping in mind that the amounts of agriculture residues can reach up to the 50% of totally produced biomass, the paramount importance of the SSF is obvious for utilizing the enormous amount of available chemical energy improving at the same time the biological sustainability.

The practical approach for the production of metabolites and enzymes can be related to different areas (paper deinking, paper recycling, agricultural residues utilization, pesticides biodegradation, fodders, olive and seeds oils residues, pruning, fuels, paper pulp production, etc.) and each of them can require some different sets of biotechnological conditions. As it results from the data of the scientific literature, for obtaining profitable results, it is necessary to have dedicated bioreactors, which can take into account same particular characteristics of the new processes such as hyphen fragility if filamentous fungi are used and/or dissipation of heat due to the metabolic respiration. Hence, the CO2/O2 ratios of gas phase inside the bioreactor are important to judge and to define the more useful bioconversion conditions. In fact, these ratios can describe the bioreactor like a living cell, where the biochemical activity can determine different respiratory quotients. Nevertheless, the description in the literature of many different solid state bioreactors suggests that a bioreactor suitable for all the bioconversion conditions seems still under progress, being perhaps the working flexibility the best aspect to achieve.

Acknowledgements Giovanni Giovannozzi shows appreciation and thanks to N. Tiso for the hard work on the literature references.

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Pandey A, Selvakumar P, Soccol CR & Nigam P, 1999, Solid-state fermentation for the production of industrial enzymes, Current Science, 77 (1), 149-162. Prrez-Guerra N, Torrado-Agrasar A, L6pez-Macias C & Pastrana L, 2003, Main characteristics and applications of solid substrate fermentation, Electronic Journal of Environmental, Agricultural and Food Chemistry, 2 (3), 343-350. Pommier J-C, Fuentes J-L & Goma G, 1989, Tappi Journal, 72 (6), 187-191. Raghavarao KSMS, Ranganatham TV & Karanth NG, 2003, Some engineering aspects of solid-state fermentation, Biochemical Engineering Journal, 13, 127-135. Raimbault M, 1998, General and microbiological aspects of solid substrate fermentation, Electronic Journal of Biotechnology, 1 (3), 15. Rivela I, Rodrigez Couto S & Sanrom~n A, 2000, Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor, Biotechnology Letters, 22, 1443-1447.

Robinson T & Nigam P, 2003, Bioreactor design for protein enrichment of agricultural residues by solid-state fermentation, Biochemical Engineering Journal, 13, 197-203. Rodriguez Couto S & Sanrom~n M A, 2006, Application of solid-state fermentation to food industry - A review, Journal of Food Engineering, 76 (33), 291-302 Roussos S, RaimbaultM, Prebois J-P & Lonsane BK, 1993, Zymotis, a large scale solidstate fermenter, Applied Biochemistry and Biotechnology, 42 (1), 37-52. Saucedo-Castafieda G, Gutirrrez-Rojas M, Bacquet G, Raimbault M & ViniegraGonz~lez, 1990, Hest transfer simulation in solid substrate fermentation, Biotechnology and Bioengineering, 35 (5), 802-808. Saucedo-Castafieda G, Lonsane BK, Navarro JM & Roussos S, 1992, Applied Microbiology and Biotechnology, 37, 580-582. Schutyser MAI, 2003, Mixed solid-state fermentation: numerical modelling and experimental validation. Schutyser MAI, Weber FJ, Briels WJ, Rinzema A & Boom RM, 2002, Heat and Water Transfer in a Rotating Drum Containing Solid Substrate Particles, Biotechnology and Bioengineering, 82 (5), 552-563. Solis-Pereira S, Favela-Torres E, Viniegra-Gonz~lez G & Gutierrez-Rojas M, 1993, Effect of different carbon sources on the synthesis of pectinases in Aspergillus niger in submerged and solid-state fermentation, Applied Microbiology and Biotechnology, 39, 36-41.

Suryanarayan S, 2001, In: Proceeding of the International Conference on New Horizons in Biotechnology, Trivandrum, April 18-21. Suryanarayan S, 2003, Current industrial practice in solid state fermentations for secondary metabolite production: the Biocon India experience, Biochemical Engineering Journal, 13, 189-195.

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i i i!i!!

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Instrumentation and Control in SSF

,, iiiiii

Wilerson Sturm, Dario Eduardo Amaral Dergint & Carlos Ricardo Soccol

i i Di' 7.1 INTRODUCTION This chapter has the purpose of explaining some of the Instrumentation and Control concepts and their application in solid-state fermentation. When dealing with instrumentation, it is important to keep in mind that it could be divided in four parts: Sensors, Actuators, Control, and Communication Network. Some of the newest technologies appear first in industrial sensors and controlling systems, indeed some of them are specific necessities developed. Industrial automation and control demands large amounts of investment, thus these devices suppliers do not have doubts about using the more updated technology, even if costs are high. However, the usual sensors and control devices are more and more cost effective due to electronic industry development and the demand and supply behavior. Nowadays, it is possible to choose between the centralized or distributed control systems. According to Rana and Taneja (1988), in the centralized control system, there is only one control device, but with high velocity and processing power, which is responsible for analyzing all the process signals and making decisions. The distributed control system normally uses some industrial network to interconnect the sensors, controllers, operator terminals, and actuators, which embraces the number of parts of a controlled process. Smith (2004) asserts that in the future, the computer will be an integral part of most bioreactor systems.

7.2 SENSORS This section discusses about the usual sensors and some specific ones, but the approach is only for the industrial processes or pilot plants, because the sensor could mean a large scope of devices. Thus, here we will focus only at industrial variables.

According to Yacynych and Twork (1990), improved sensor technology could be the driving force for new control strategies development, which could result more productive fermentations. However, there is a lack of good sensor probes that allow on-line analysis on the chemical components of the fermentation process, what means difficulties in real time control(Smith 2004). Yacynych and Twork (1990) have described the challenges to develop sensors, which could be readily implemented for more effective process control. Other feature to take into account is the necessity of sterilization when working with a bioreactor, hence, any sensor must be capable of sterilization conditions in situ, during steam sterilization for instance, or remotely, before being applied to the process (Bryce and Mansi 1999).

7.2.1 Temperature sensors Temperature variable control or, at least, monitoring is present in almost every industrial plant in (Sturm 2004). It has been stated that any SSF control system should include temperature measurements, for instance monitoring the temperature distribution and measuring inlet and outlet air temperature so that the energy and water balances could be kept under control (Pandey 2004). There are many types and protection constructions for temperature probes, but two of them are more usual in industrial applications: thermocouples and Ptl00 (Pandey 2004). Normally, these devices are externally protected with a stainless steel tube, or even ceramic materials tubes depending on the application conditions. Thus, an external protection with stainless steel could be appropriate for SSF process, taking into account the chemical components and sterilization. 7.2.1.1 Thermocouple A thermocouple construction is very simple. Two different metallic, or alloy, wires are welded at one edge, called hot junction and the other edge, called as the cold junction, is kept opened. Discovered in 1821 by T.J. Seebeck, the socalled Seebeck Effect could be explained as the appearance of a low electrical voltage signal when there is some difference between the temperature at the hot junction and the cold one, which is connected to the instrument. Because of the proportionality with the difference, it is necessary to measure the temperature, close to the instrument or cold junction, and add to the final result. Its installation must be made under strict technical conditions to use this sensor, because an inversion, bad union, or different materials used as conductors could result in error. Low cost, wide measuring range and its fast and linear response are responsible for their popularity among the process engineers. The more usual are the types called T, copper and constantan, type J, iron and constantan, and K, chromel and alumel alloys, depending on the temperature range and the costs. Nowadays, the low signal generated in their terminals is not quite a

problem because of the new high input impedance circuits and the use of programmable microcontrollers, instead of circuitry based equipments (Sturm 2004).

7.2.1.2 PtlO0 or RTD The Ptl00 or RTD, Resistance Thermometer Detector, construction is based on a kind of insulating ceramic material with a Platinum wire winding around it. The signal present in its terminals is an electrical resistance directly proportional to the temperature. Ptl00 is a standard; Pt comes from Platinum and the number 100 is 100 ohms at 0~ with a variation rate of 0.4 ohm per degree Celsius. Ptl00 are stable, precise, with fast response, and do not need periodic calibration, although they are more expensive and fragile compared to thermocouples. This fragility is due to the insulating base, which could disrupt when under strong vibration or mechanical impact. Normally, this kind of temperature sensor is installed using three wires, so that the error caused by the line resistance can be minimized in the internal measurement circuit. The following figure shows the usual protection for both Thermocouples and Ptl00; however, there are many other possibilities, including cheaper ones.

7.2.1.3 Thermistor The thermistor is a thermal resistor, constructed with semiconductor materials to present more sensibility (McComb 1999). This is one of the most cheap solutions in measuring the temperature. However, it's low linearity and narrow range of temperature demands more electronics, less accuracy, and more specific application uses. It can provide a negative temperature coefficient, known as NTC or positive temperature coefficient, the PTC. Using technologies such as microcontrollers and instrument amplifiers, it is possible to enlarge the scope of its applications, but keeping in mind their limitations.

7.2.1.4 Integrated sensor Nowadays, many integrated sensors are available. These devices are semiconductor based, but have an internal circuit able to turn the output more linear and

accurate. The range of temperature could be about -55 to 150~ One usual sensor is the National Semiconductor, LM35 as described in National 2000, with small packages and very stable signal. This chip integrates internal circuits responsible for the linearization and calibration, thus it needs less external circuits to work.

7.2.2 Flow s e n s o r When facing a flow sensor application, it is necessary to pay attention in many specification and performance data, because the variety of choices is vast (Liptak, 1993). It is necessary to know the material in which the flow has to be measured, liquid or gas, and its range for a specific bioreactor. Other characteristic to keep in mind is the losses in the energy flow, translated in losses caused by the turbulence and fluid friction into the sensor or transducer. These losses impact indirectly in costs (Sturm 2004).The air flow is often recorded on-line by a flow meter fitted at the exit of the reactor, including rotameters, which is only a monitoring device and not a sensor, thermal mass flow meters, anemometers, and differential pressure sensors (Christen et al., 2003). It is very important to measure the flow at the entrance and exit of the bioreactor to make sure about volumetric gas evaluation (Pandey 2004).

7.2.2.1 Flow s e n s o r b y differential pressure The following figure shows an example of differential pressure application, using two tubes, Pitot and Venturi. The difference between these two pressures, indicated as h or the differential high between two liquid columns, is proportional to the fluid velocity so that the flow can be calculated (Sturm 2004).

7.2.2.2 Flow sensor type venturi tube This sensor is a differential pressure device. Giovanni B. Venturi in 1797 postulated that fluids under pressure when pass through convergent tubes increased their velocity and loose pressure but the opposite occurred in the divergent tubes. Using pressure sensors, the flow can be measured (Liptak, 1993). For instance, in the following figure, velocity indicated as V2 would be higher than V1, due

to the different areas indicated as S1 and $2, although the pressure in S1 would be higher than $2.

This sensor does not add much resistance to the fluid flow as an advantage, but due to the pressure measurement needs, it is not low cost choice in much of the cases.

7.2.2.3 Flow sensor by thermal dispersion When fluids pass through the heated sensors, it causes loss of temperature due to the convective cooling effects at a rate proportional to the fluid velocity (Soloman 1998). The heated sensor must be at a constant temperature, above the temperature of the gas flow being measured. The electrical energy required to maintain a constant temperature in the velocity sensor is proportional to the cooling effects on the measured gas flow, so that the velocity could be inferred. This kind of sensor is, in present time, very accurate and has a large scale of application ranges, though it is not an inexpensive technology yet. The resolution, no moving parts, and accuracy together with a real time measure possibility could be advantageous, hence a good analysis about the costs and benefits must be made.

7.2.2.4 Orifice plate This differential pressure flow sensor consists of a plate installed in the tube, which measures the flow with a central or eccentric orifice (Saleh, 2002). When the fluid faces the orifice in the line, it causes a pressure change before and after the hole. The difference between the pressure before and after the orifice is proportional to the fluid's velocity, hence, the flow can be estimated with a quite accuracy. This kind of solution is very simple to install and at low cost, but has the disadvantage of adding resistance to the flow crossing the orifice. Thus, when there are many sensors in the same line, it could be prohibitive.

7.2.2.5 Turbine sensor The turbine flow sensor may be the low cost model because of its simplicity. This sensor consists of a kind of turbine blade inserted inside the tube, by

which the flow is measured (Herman and Alerich 1998). The moving fluid causes the turbine to turn. The speed of blade's turning is proportional to the flow. The electrical signal is determined by this speed.

7.2.2.6 Magnetic sensor The magnetic flow sensor works based on the Faraday's Law, which states that when there is a conductor moving through a magnetic field, a voltage will be induced into the conductor, depending on the strength of the magnetic field and the conductor velocity (Cheremisinoff 1988). In this case, the conductor is a moving liquid. This sensor works only with the conductive liquids, thus could be useful in measuring the water flow or any other liquid addictives addition to the fermentation process. There are many advantages in using this kind of sensor, such as accuracy and less resistance to the fluid flow, but it is not a low cost solution. The following figure is a schematic view of a magnetic flow sensor, where E1 and E2 indicate the electrodes of output electrical signal.

7.2.3 Humidity sensor Depending on the bioreactor type, humidity control is as critical as temperature control concerning to the bioreactor performance because of its influence on oxygen transfer, hence causes a reduction in the growth rate (Pandey, 2004).There are mainly two types of humidity sensors, and their construction is based on the sensitive polymers coating of an electrode base-plate (Soloman, 1998). One of these is a capacitive sensor, whose output signal causes a capacitance change depending on the humidity. The other type is a resistive output sensor that changes electrical resistance according to the humidity changes. In comparison with the capacitive, this sensor has a slower output response, but for most

processes, it is enough. The capacitive humidity sensor is faster than the resistive but is more expensive.

7.2.3.1 Moisture and water activity In solid-state fermentation, it is very crucial that exact quantity of water is added to the substrate so as to achieve a suitable water activity (aw) (Pandey et al., 2001). In SSF process, the water activity is variable because of the evaporation and metabolism (Dorta et al., 1994). Water activity could be more important factor than the water content for microbial growth and metabolism, (Tsao and Brainard 1999). It is important to keep in mind that water activity is closely related, but not equal to the water content. Water content is often measured off-line by dry weight determination, but it is not the same of water available for the activity of the microorganism. One of the most common methods to measure water activity is the capacitive sensor, designed to regulate the circulating air humidity (Christen et al., 2003). It is possible to measure the relative gas humidity in a cell separated from the solid phase by a hydrophobic membrane (Gervais 1989). In a chamber, a capacitive element absorbs the water vapour in equilibrium with the surrounding environment, which makes the dielectric constant of the capacitor to change. This change can generate an electrical signal to be measured proportionally to the water activity. However, there is still very slow response for most bioreactors process.

7.2.4 pH s e n s o r Variations in pH result are indicators of changes in the metabolic activity in SSF (Christen et al., 2003). Flat-ended electrodes are normally used to measure pH directly on the surfaces of solid substrates, but off-line and usually can be made by measuring the pH of aqueous suspensions or extracts of the solid sample (Scheper and Berovic 2000).There are new pH probes models, without the necessity of temperature compensation and filling of solution, and can be connected to interfaces which are able to output standard signals and industrial communication networks protocols (Myrvoll 2003). But in SSF, due to the lack of free water, it is difficult to control pH accurately.

7.2.5 0 2 and C O 2 s e n s o r s

In SSF, the 02 consumption and CO2 produced allow to estimate the growth kinetics of the microorganism (Pandey et al., 2001). This also helps in determining the respiratory quotient, defined as the ratio of CO2 produced and 02 consumed. Oxygen has a strong affinity for a magnetic field and this property is not shared by many other gases (Liptak 1994). In SSF, this is the only gas which has paramagnetic property, thus, it is one of the most common characteristics

to use in measuring oxygen consumption direct from the process. In deflection type sensor, the paramagnetic behavior of oxygen is compared to another gas, both inside a magnetic field. The more concentrate is the oxygen sample, the more unbalanced is the system, which measures how deflected is some mechanical device. However, this kind of sensor is quite expensive, although very accurate. The thermal type sensor is based on the paramagnetic property of the oxygen and the flow of the oxygen changes the thermal balance of a temperature sensor, proportionally to gas concentration, generating an electrical output signal. This sensor has the disadvantages of its sensitivity to heat and low precision (Christen et al., 2003). There is a type of oxygen sensor which operates like a metal/air battery (Jardine and McCallum 1994). Oxygen in contact with the cathode is reduced to hydroxyl ions, with a balancing reaction of lead oxidation at the anode, thus an electrical current is generated proportionally to the rate of oxygen consumption, following the Faraday's Law (mentioned in the section about magnetic flow sensor). This kind of sensor's lifetime depends on the availability of lead, which stops working when all the lead is oxidized. For continuous monitoring applications an absolute, non-dispersive infrared (NDIR) gas analyzer based upon a single path, dual wavelength, infrared detection system could be a low cost, low maintenance CO2 analyzer (Liptak 1994).Infrared spectrometry is commonly used to record CO2, allowing to evaluate microbial growth, to validate gas diffusion models, and to monitor fermentation processes (Christen et al., 2003). Accordingly to Brown et al., (1989), using proper columns balances, it is possible to resolve 02 and CO2 simultaneously performing a gas chromatography with two concentric columns. Christen et al., (2003) stated that one column must be filled with a porous polymer or silica gel to retain CO2 from the air and the other with a molecular sieve, which was able to separate 02. Each compound can be quantified by a heat conductivity detector, or catharometer. This solution could be used on-line to store and analyze data.

7.3 ACTUATORS Sensors and actuators are the components of the control system, which come into contact with the fermentation process, thus need to be designed to facilitate sterilization, asepsis, insertion, and removal (Omstead 1990). In the specific case of SSF, some actuators could be outside of the process itself. Actuators such as micro switches, limit switches and contactors are electrically very simple, because of their mechanics and well-known function. This section will discuss about newest and more complex actuators.

7.3.1 Motor To perform many kind of movement it is necessary to apply an electric motor. The ventilation is indirectly or directly accomplished by a motor attached to an air pump or compressor as well as the rotating parts to achieve the proper mixing of the fermenting substrate. Motors are often supplied by Alternating Current (AC), or Direct Current (DC). In industrial application, the AC supplied motors are most common (Valkenburgh 1993). Using a Frequency Inverter, an electronic device, it is possible to change the speed of three phase motors. Day by day, these devices are turning more cost effective, using thyristors such as SCR. It is common nowadays to use the insulated gate bipolar transistors, IGBT, with high frequency and updated technologies (Bradley 1995). Hence, it is possible to control some variables by changing a motor velocity. The use of servomotor is normally prohibitive because it is often expensive; however, it has advantages in controlling speed and position with high accurate rates.

7.3.2 Valve The correct use of valves is indispensable for instrumentation design. There are many types of the valves. Mechanical and manually activated models are of lower cost, but these are not very useful in automated control loop. Motorized and Proportional Valves as explained by Janocha (2004), could be the correct alternative for controlled processes. These kinds of valves are able to vary from completely closed until completely open, depending on the input signal, often used 4 to 20 mA as control signal for proportional types. Hence, to accomplish with the automatic control requirements such as accuracy and resolution, these could be used to control the air inlet flow, pressure component, other substances volume to be added to the process, etc. 7.3.3 Heater power control There are two main ways to control the heater power in the industrial applications by Phase Angle and Pulse Width Modulation (PWM) (Ibrahim 2002). Phase Angle is one of the most common methods to control the heater power, where the start of each mains power supply half-cycle, applied to resistance heater, is delayed by an angle. In electricity it means some amount of time, but it is important to keep in mind that each full-cycle lasts just around 16.6 miliseconds when taking account the frequency of 60 Hz. Electronic components called thyristors are used in order to change the triggering angle, so that the power is delivered to the heater element. The other method PWM, or Duty Cycle is able to turn on and off very fast in order to cut a number of full-cycles and leaving the other cycles applied to the heater element. Both of them are useful in temperature control with electrical heaters because they are able to apply from zero to the full available power to process heating depending on the

controller signal. Accordingly to Rexford and Giuliani (2004), it turns the control loop more accurate and reliable, but generates electrical noise which can cause some equipments malfunction.

7.4 CONTROL When dealing with control strategies, some terms appear very often such as Setpoint, SP, which is the desired value of some parameter under control, Manipulated Variable, MV, which is the variable that is changed by the control strategy to act directly for the process, and Present Value, PV, which is the value at the analyzed or real time. A controlled parameter must maintain its value, i.e. should stay at stable value, or as close as possible considering the tolerances, and when faced with a disturbance, must return to the previous state as soon as possible.

7.4.1 On-Off control One of the most simple control methods or strategies is the so-called On/Off control (Montague 1997). Its action is shown in the following figure. Here, a temperature control is used as an example. The heating power is either fully switched on when the temperature is below the Setpoint or fully turned off when it is above. As a result, the temperature oscillates around the Setpoint, the amplitude and the time period of the oscillation is a specific function of the process, or the thermal lag between the heating energy source and the temperature sensor. To minimize the output "bouncing" as the measured temperature crosses the Setpoint, the controller does not turn on and off at precisely the same point. Instead, a small differential known as hysteresis is used, whose value depends on the process behavior to maintain the Setpoint at the average value of total oscillating curve. On/Off control is satisfactory only for the non-critical process applications where some oscillation in the present value is permissible.

7.4.2 PID control Where there is a more critical process application, On/Off control would not reach the results needed. In this case, the PID, Proportional Integral and Derivative method could be the correct choice, and is widely used in the industrial plants. To achieve the strategic comprehension, this method will be analyzed in details.

7.4.2.1 Proportional control A proportional controller takes place to perform better than the On/Off control type by applying power to the heater, for example, to a temperature control application, proportionally to the difference in temperature between the process and the Setpoint. This difference is usually called error. P is known as the proportional gain of the controller. When this gain is increased, the system tends to respond faster to the changes in Setpoint value, or facing a disturbance, but becomes progressively faded and eventually unstable. The final process temperature is used to stabilize below the set-point with this system because of a kind of equilibrium between the energy supplied to the system and the resistance from it. In other words, the proportional amount of energy is too weak to reach the set point, taking into account that the proportional action will be linked to the error and close to Setpoint this value tends to be little (Montague (1997). This error is normally called steady-state error.

7.4.2.2 Proportional and derivative control When only proportional controller is used at high gain, the amount of PV may reach above the Setpoint, and problems could appear. In order to minimize these problems, a term proportional to the time-derivative of the error signal can be added to the output signal or control signal (Omstead 1990). This term is the variation rate of error in relation to the time. Normally, it is called derivative time and expressed in seconds, depending on the controller manufacturer. This technique is known as PD control. Its response is faster than the proportional control.

7.4.2.3 Proportional integral and derivative control- PID The PD control described above can deal properly with the overshoot and stability problems associated with the proportional control but it does not solve the problem with the steady-state error. This error can be eliminated, at least minimized, by the use of relatively low gain, achieved by adding an integral term to the control function which becomes: Ger de S c - P.e + ~ l e.dt + T d ~ + B Ti Jo dt

Where: Sc is the output signal of control; P is the proportional band; G is a constant; Ti is the integral Time; e is the error or (SP- PV); Td is the derivative time; and B is a kind of Bias (Bryce and Mansi 1999). The following figure shows the usual behavior of PID control.

The mathematical difficulties with the Integral and Differential Calculus eventually encountered in the previous function can be more easily understood with a time sampled function, generating a table of values and the equation changes to:

DMVN = r e

EN

At

EN ~ + - - E N TI

"~d

--:-:(CVN 2CVN ,+CVN 2)

)

/~t

MV N = MVN , + AMV N Where Kc represents the proportional gain, TI the integral time, Td the derivative time, CVN is the controlled variable, SPN is the Setpoint and MVN is the controller output at the current sample N. The current error is calculated as EN=SPN-CVN (Birol et al., 2002).

7.4.2.4 Advanced PID control In many years of continuous development of new control application techniques, some algorithms have been added to the usual PID. Some manufacturers offer more specialized control devices. One of these advances is the Fuzzy Logic algorithm working together with the traditional PID (Shaw 1998), which is able to recognize situations when fuzzy logic would offer certain advantages

over the traditional control strategies, particularly in controlling the highly nonlinear and time-variant plants and processes.

7.4.3 MMI and SCADA A MMI system (Man Machine Interface), or HMI (Human Machine Interface) could be explained as the connecting point between the operator and the machine. Most of the cases, it is an operator panel or a Personal Computer (PC), or a workstation (Evrendilek et al., 1997). Nevertheless, in the both cases, the transmitting and receiving data are from the sensors and actuators in the process. Widely used in industry plants processes, Supervisory Control and Data Acquisition (SCADA) systems could be used in the biotechnological processes also. The pioneer SCADA masters were normally strongly manufacturer-based software, as well as hardware, with no interchangeability between the different manufacturers products and at that time the investment were high (Strauss 2003). The development and cost effective personal computer, nowadays present in every industrial plant, actually forced suppliers of SCADA software to develop their products to be compatible with PC use, with low cost devices. Using a PC as a SCADA-MMI system, the following items will be necessary: SCADA Software: Software program able to work together with the PC like a SCADA system; Programmable Logical Controller (PLC) Communication card, or some communication standard protocol for the whole devices; Communication driver: It is a kind of translator between the SCADAMMI system and the process devices. The bioreactor can be connected, or in communication, with the SCADA system, whose function is to collect all the data and store it for later retrieval and supposed to be analyzed. It ought to be kept in mind that the SCADA is not a database, and usually cannot give answers to database type queries but using resources to present the operator with comprehensive information on plant status (Bryce and Mansi 1999). The control function distributed in the process, performed by the controller loops or PLC. Most of the applications are proprietary in nature using proprietary hardware and commercial software pack, because there is almost no open source SCADA software available (Gorodeckij et al., 2003). However, there are many efforts to create and to tum into available open software tools, with its own advantages and disadvantages of course (Goodman 2001).These solution methods have a strong and close relation with communication networks, which are discussed in the next section.

7.5 COMMUNICATION NETWORK For the bioreactor automation, it is necessary to plan for the communication system. This planning is necessary to certify all the devices such as sensors, actuators, interfaces, and management systems, to be interchangeable and operational. The correct choice can guarantee maintenance, evolutionary and flexible control requirements also. With this point of view, this section presents the technologies still in use, and the trends for the communication devices.

7 . 5 . 1 . 4 to 20mA The 4 to 20mA communication method had spread when the electronic elements in control devices and sensors took place. It was developed in the sixties of last century (TEI 2006). This communication system has the following features: connection by metallic twisted pair wires, normally shielded to avoid electrical noise, which accomplished the communication and power supply; polarization of electrical current of 4mA through the communication wire; range of data transmission from 4 to 20mA; high noise immunity; easy circuit design and maintenance; system low cost; fast identification of system fault by loop current monitoring such as wire disruption, bad contact, and circuit malfunction; high connection distances (Dergint 1990). This kind of devices could present some inconveniences due to the increasing process control complexity. For each control signal, it is necessary to have a different twisted pair of wires, hence, with many control nodes, the maintenance, identification, installation or position changes could turn into a serious problem (Dergint 1990). 7.5.2 Serial communication Due to electronics evolution, new and more sophisticated communication protocols take place, allowing the bidirectional data transfer between the two equipments. Two patterns can be pointed out, parallel and serial communication. The parallel communication is almost obsolete due to the short interconnection distance between the equipments. For instance, GPIB an 8 bit parallel communication developed for workbench instruments connection is still in use today (GPIB tutorial 2005). This system's advantage is the automatic bit rate speed operation, which allows working with different equipments from different manufacturer. It has been reported that with the component integration and increasing velocity of electronics, the serial communication is the trend for the interconnection devices nowadays (Wong 2006). These included the RS485 and communication protocols such as Fieldbus (Thomesse 1998).

7.5.2.1 RS 485/RS 232 The most well-known serial communication is the RS232, useful to devices connect printers, mouse to Personal Computers, but today almost disused due

to connection distance available (30 meters maximum), and peer to peer connectivity (Dergint 1990). However, the electrical protocol RS485 is quite different from the RS232 (Wong 2006). It uses differential transmission, allowing distances in kilometers. Using the multipoint connection, it is possible to use until 32 elements per bus, and some manufacturers actually offer up to 128 devices. Combining the low cost, it is one of the most usual options for the industrial environment complex processes. The following figure shows the RS485 devices bus connection, where D - Driver; R - Receiver; T - Transceiver; and T R - Termination Resistor.

7.5.2.2 USB/FireWire The USB connection uses a serial communication protocol also. It has replaced RS232 system and is useful to interconnect office's devices such as printers, scanners, web cam, photograph cameras, etc. Due to the low cost, many other devices are now available with this interface. The basic features are: connection up to 127 devices, 5 meters distance and transmission velocity from 12 Mbps (Mega bits per second) for USB 1.0 to up to 480Mbps for USB 2.0 (Axelson 2005). FireWire is an USB protocol competitor and is useful for the similar applications (Axelson 2005). Technically, it shows more reliable performance than the old USB versions; however, this protocol is not so well-known among the PC framework users. Its features are 127 devices connection, distance of 5 meters and transmission speed of 400 Mbps (1394a) and 800 Mbps (1394b) (Wong 2006).

7.5.3 FieldBus Fieldbus is a network developed for the industrial automation environments. It took place in the 1980s but began popularizing in the beginning of twenty first

century. This delay occurred because of many proprietary and non-proprietary systems competition and sophisticated framework levels and sub-levels requirements such as memory availability and low cost (Smar 2001). Today, the field buses can be divided in SensorBus, DeviceBus and FieldBus. The following figure shows this distinction.

Nevertheless, with the technological evolution and its consequent production cost reduction, the trend is the FieldBus application in the whole process. In general terms, DeviceBus and SensorBus could be considered Fieldbus network simplifications (LeBlanc 2000). The FieldBus framework complies with a three layers simplification from the OSI (Open System Interconnection) at ISO (International Standard Organization), which has totally seven layers (Betlem and Roffel, 2004). However, as can be observed in the following figure, the framework is organized in three layers OSI plus one, Physical, MAC (medium access control), LLC (logical link control), Application and User (Wright et al., 2003).

The physical layer makes the interconnection with the medium, which has two types, RS485 based and IEC 1158-2 standard. The latter is more expensive and possesses some advantages compared to RS485, such as data and power supply at the same bus, communication protocol normally using the diphase

Manchester method, which is superior in comparison with the RS serial types, galvanic insulation between the devices and the bus to (Lewis 2001).

Actually, every FieldBus standard supports the physical medium described above, especially IEC 1158-2 (61158), with differences in data link layer, subdivided in MAC and LLC. MAC has the task of managing the medium access and LLC to certify the reliable messages exchanging. Over these layers two basic mechanisms are implemented which are master-slave and producer-consumer. Profibus uses the multi master framework with the sequential monitoring over the slave nodes, and FIP (Factory Instrumentation Protocol) applies producerconsumer (Dergint 1990). It also implements cyclic and event communication mechanisms. They are important for the process control algorithms application. The application layer has tasks, such as writing and reading devices variables and has a directory for process configuration also. The User layer was introduced in FieldBus to assure the devices inter operation. Hence, this as well as the usual control PID 4 to 20mA, could be interchanged with different manufacturers devices without new configuration (Lewis 2001). The User layer defines three types of the Function Blocks: Resource Block, Function Blocks and Transducer Blocks. Resource Block describes the Fieldbus device characteristics, such as

device number, manufacturer, and series number. Function Block defines the control system behavior. The input and output parameters can be connected through the Fiseldbus. Each Function Block execution is precisely scheduled. There can be many Function Blocks in one User Application. Fieldbus Foundation defined 10 standard Function Blocks for the basic control specified by the FF-891 standard part 2 specifications.

The initial set of 10 function blocks includes from the analog and digital inputs until PID algorithm control, as pointed out by Appendix D 2002 and Override and Split Range Control. Advanced function blocks intends to cover 20 (19 in Foundation) operations including ramp generators and dynamic compensators, FF-891 standard part 3 specifications. The function blocks can be inside a field device providing required function; for instance, a flow meter can have a function block inside with the analog input, and a proportional valve can have a PID control function block together with an analog output, hence, these two devices could make a complete flow loop control. Finally, the Transducer Blocks turn unattached the Function Block from the local input and output functions required for the sensors and output command of hardware, containing information such as calibration, date, and sensor type. Usually, each input and output Function Block has one Transducer Block. The following objects are defined in the User Application: Link Objects, Trend Objects, Alarm Objects, View Objects which can be useful for Man Machine Interfaces.

7.5.4 Profibus (Process Field Bus) Profibus was developed to support the manufacturers need and became the standard DIN V 19245 in 1989/1995. In 1997, it was defined in the international standard EN 50170, 1996 and 1999 it was included in IEC 61158/IEC 61784

standards (Wright et al., 2003). Profibus is the widely employed model, and is an open field network, which is not dependant of Manufacturers and allows communication between different brand devices without any specific adjustment (Betlem and Roffel 2004). The framework complies with OSI reference model and ISO 7498 standard, using 1, 2, and 7 layers (Dergint 1990). Profibus allows real time and high velocity applications and complex communication tasks. Its standard form can be separated in three subdivisions: Decentralized Periphery- DE master-slave, low cost, high velocity and manufactory oriented; Process Automation- PA, intrinsically safe, adopt User Layer and process oriented; Fieldbus Message S y s t e m - FMS, multi-master/peer to peer, IEEE 802.4 simplification, presented in IEEE 1984 for heavy communication tasks, without critical time requirements. Basic Features are: maximum of 127 stations, making use of repeaters; Non Return to Zero-NRZ coded (DP) and diphase Manchester L (PA); physical medium is twisted pair or fiber optics; electrical standard RS485 or IEC 1158-2; bus velocity using RS485 (DP) from 9600 till 12Mbps and using IEC 1158-2 (PA) of 31,25 kbps; maximum cable length of 1200 meters, using 9600 bps bit rate (DP) and 1900 meters per bus at (PA).

7.5.5 WorldFip It was developed foremost to support industrial process requirements. In 1989, it was defined in French standard NFC 46-600 and nowadays accepted in international standard EN 50170/3 and EN 50254/3 as well as included in IEC 61158. Its framework is very flexibl6, especially because it adopts producerdistributor-consumer as exposed in EN 50170, 1996. It is also an open field network, using OSI reference model in conformity with ISO 7498, using 1, 2, and 7 layers (Betlem and Roffel, 2004). Its basic features are: identifiers are coded using 16 bit integers; up to 65535 variables can be named; producerdistributor-consumer to simplify distributed system projects; maximum of 256 nodes with repeaters; diphase Manchester L coded; physical medium is twisted pair or fiber optics; electrical standard IEC 1158-2; bus velocity using IEC 1158-2 of 31,25 kbps or 1Mbps or 2,5 Mbps; maximum cable length of 40 km (Wright et al., 2003).

7.5.6 Zigbee ZigBee implements a low cost and low power consumption wireless network, based in IEEE 802.15.4 standard (Wheeler 2006). Its specification was ratified on 14th December 2004 and 13th June 2005 and the ZigBee Alliance became public available specification. The cost estimation is only U$ 1.00 per node. The frequency band work is distributed among the countries as: 915 MHz in the USA, 868 MHz in Europe and 2.4 GHz in most countries.

The ZigBee protocol is supposed to be on-board applications working with low rate of data transmission and low power consumption (Yacoub and Yang 2006). It can be suitable for industrial control, on-board sensors, medical data logging, and so forth. Due to low consumption, a ZigBee system could work more than one year with the same battery. The Basic Features are: three device types, ZigBee coordinator(ZC), ZigBee Router (ZR) and ZigBee End Device (ZED); cable length from 10 to 100 meters depending on the specific environment conditions; the physical medium access protocol uses "carrier sense, multiple access/collision avoidance" (CSMA/CA), transmission based in fixed timing schedule, and for real time requirements uses Guaranteed Time Slots (GTS); Addressing space of up to 64 bit address devices with 65535 networks; topologiesstar, peer-to-peer and mesh.

7.6 CONCLUSIONS This chapter had the purpose of exposing and discussing about the Instrumentation and Control for a specific area, solid-state fermentation. Attempts were made to focus here only the current devices, equipments and communication methods for the process considered as well as the new strategies and trends. Depending on the process size and behavior, it would be necessary to analyze what kind of method would be useful. There are many variables to be taken into account, such as control type and accuracy needs, stability, number of control loops, distance between the process and the control system, response time and tolerances, specific sensors availability, control and communication network interfaces necessity, and at last but not the least important, the budget. Because of vast quantifies of manufacturers and suppliers, as well as the different technologies, it is necessary to know how to identify not exactly the very best solution, but the most adequate one.

Acknowledgments The authors acknowledge Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq and CAPES for financial support.

9. . . .

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References Appendix D, 2002, PID Function Block, Rosemount Model 3244MV MultiVariable Temperature Transmitter with Foundation Fieldbus, FIELDBUS-FBUS_34A, Rosemount, www. emers onproce s s. com/ro semount/document/manual s/3 244 ff/3 244f_pid. pdf, accessed on 20/11/2006. Axelsom, 2005, Jan. Usb Complete, pp 196-198, Madison: Lakeview Research LLC. Betlem BB & Roffel B, 2004, Advanced Practical Process Control, p 309, Springer: New York. Birol G, Undey C & Cinar A, 2002, A Modular Simulation Package for Fed-Batch Fermentation: Penicillin Production, Computers and Chemical Engineering, 26, 15531565.

Bradley DA, 1995, Power Electronics. pp 39-42, CRC Press: London. Brown PR, Giddings JC & Gruchka E, 1989, Advances in Chromatography, p 73, Marcel Dekker: New York.

Bryce CFA & Mansi EM, 1999, Fermentation Microbiology and Biotechnology, pp 203-236, Taylor & Francis: London.

Cheremisinoff NP, 1988, Flow Measurement for Engineers and Scientists, pp 311-312, Marcel Dekker: New York. Christen P, Veronique BM & Orliac O, 2003, Sensors and measurements in solid-state fermentation: a review, Process Biochemistry, 38, 881-896. Dergint DEA, 1990, Um Barramento de Campo para Automa~ao da Garantia da Qualidade da Produ~o; 1990, Dissertation; CEFET-PR, Curitiba, Brasil. Dorta B, Bosch A, Arcas I & Ertola R, 1994, Water balance in solid-state fermentation without forced aeration, Enzyme and Microbial Technology, 16, 562-565.

EN 50170, 1996, General Purpose Field Communication System, European Standard, ~.l

.FC.

Evrendilek C, Wilhelmsen D, Gezer E & Ince AN, 1997, Planning and Architectural Design of Modern Command Control Communications and Information Systems, pp 106-107, Springer: Massachusetts.

Gervais P, 1989, A new sensor allowing continuous water activity measurement of submerged or solid substrate fermentations, Biotechnology and Bioengineering, 33, 266-271.

Goodman G, 2006, Puffin Projects Lead Open Source Parade, 23 April 2001. www.isa.org, accessed on 17/11/2006.

Gorodeckij VI & Popyack L (eds), 2003, Computer Network Security, p 171, Springer: New York.

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GPIB Tutorial, 2006, TransEra Corporation's, http://www.transera.com/htbasic/ tutgpib.html, accessed on 13/11/2006. Herman SL & Alerich WN, 1998, Industrial Motor Control. p 143, Thomson Delmar Learning: New York. Ibrahim D, 2002, Microcontroller-Based Temperature Monitoring and Control, pp 203204, Newnes: Oxford. I E E E - 1984, International Standard 802.4: IEEE standards for local area networks: tokenpassing buss access method and physical layer specifications.

Janocha H, 2004, Actuators: Basics and Applications. pp 167-170, Springer: New York. Jardine FM & McCallum RI, 1994, Engineering and Health in Compressed Air Work, pp 334-335, Spon Press: London. Leblanc C, 2000, The Future of Industrial Networking and Connectivity;Dedicated Systems; 2000 Q1, http://www.dedicated-systems.com, accessed on 11/11/2006. Lewis RW, 2001, Modelling Control Systems, IET: London. Liptak BG, 1993, Flow Measurement, CRC Press: Pennsylvania. Liptak BG, 1994, Analytical Instrumentation, CRC Press: Pennsylvania. McCombs DM, 1999, Detecting the World, CMP Books: Berkeley. Montague G (ed), 1997, Monitoring & Control of Fermenters, pp 89-92, Institution of Chemical Engineers (IChemE): London. Myrvoll F (ed), 2003, Field Measurements in Geomechanics, Taylor Francis: Lisse (UK). National Semiconductors Corporation, 2006,. LM35 Data Sheet, www.national.com, accessed 10/11/2006. Omstead DR, 1990, Computer Control of Fermentation Processes,CRC Press" Boca Raton. Pandey A, Soccol CR, Rodriguez-Leon JA & Nigam P, 2001, Solid-state Fermentation in Biotechnology: Fundamentals and Applications, Asiatech Publishers, Inc. New Delhi, India. Pandey A (ed), 2004, Concise Encyclopedia of Bioresource Technology, Haworth Press: New York. Rana SP & Taneja SK, 1988, A Distributed Architecture for Automated Manufacturing, International Journal of Advanced Manufacturing Technology, 3, 5. Rexford KB & Giuliani PR, 2004, Electrical Control for Machines, Thomson Delmar Learning: New York. Saleh JM, 2002, Fluid Flow Handbook, McGraw-Hill Professional: New York. Scheper T & Berovic M, 2000, New Products and New Areas of Bioprocess Engineering, Springer: Berlin.

Shaw IS, 1998, Fuzzy Control of Industrial Systems, Springer: Massachusetts. SMAR, 2001, Fieldbus Tutorial a Foundation: fieldbus technology overview, www.smar.com.br, accessed on 15/11/2006. Smith JE, 2004, Biotechnology. Cambridge University Press: Cambridge. Soloman S, 1998, Sensors Handbook, McGraw-Hill Professional: New York. Strauss C, 2003, Practical Electrical Network Automation and Communication Systems, Newnes: Burlington. Sturm W, 2004, Sensores Industriais: Conceitos Teoricos e Aplicaq6es Praticas, Papel Virtual Editora: Rio de Janeiro. TEI Controls Inc. "4-20 mA Description", E.U.A, www.teicontrols.com/4-20.html, accessed on 20/11/2006. Thomesse JP, 1998, A Review of the Fieldbuses, Annual Reviews in Control, 22, 35-45 Tsao GT & Brainard AP, 1999, Recent Progress in Bioconversion of Lignocellulosics, Springer: New York.

USB 2.0 Specification; Universal ~Serial Bus, http://www.usb.org/developers/docs/, accessed on 13/11/2006. Valkenburgh AV, 1993, Basic Electricity, Thomson Delmar Learning: Indianapolis. Wheeler A, 2006, ZigBee Wireless Networks for Industrial Systems - White Paper, Jun2006. www.microcontroller.com, accessed on 13/11/2006. Wong H, 2006, Overview of Serial Communications; Web Presentation, National Semiconductor, www.national.com/AU/design/0,4706,0_34_,00/html, accessed on 13/ 11/2006. Wright E, Park J & Mackay S, 2003, Practical Data Communications for Instrumentation and Control, Newnes: Oxford. Yacoub M & Yang GZ, 2006, Body Sensor Networks, Springer: London.

Yacynych AM (ed), 1990, Sensors in Bioprocess Control, Marcel Dekker: New York.

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!iiiiiiiiiWilerson ii Sturm, Carlos R Soccol iiii!iii!!iDario ii! Eduardo Amaral Dergint, iii!i!iii!iiJose ii~ A Rodrfguez-Le6n & i!iiiiiii!i!Deiva ii!ilil Canali Navarro Vieira Magalh~es 8.1 INTRODUCTION The technological progress in informatics and computation sciences has been increasing faster than most could imagine or even understand, not only because of its velocity but essentially because of its large scope of applications. With constant improvements of performance features and relative drop of prices, these equipments are more and more cost effective. One of the areas where the demand for more computing power is most notorious is in biotechnology, especially in DNA researches, where it could turn into the driving force of development because of the need to store and analyze the enormous amount of genomic and proteomic data. However, almost the same has been taken place in all other biotechnology areas. Actually the advances are not only in hardware specifications. Indeed there are new programming techniques, new and powerful languages, and even new entire programming frameworks. Together with the hardware performance these software developments allow more complex problems solution. This chapter will discuss some of the new techniques, solutions and challenges concerning the computing uses in biotechnology and show some examples of implementation like Fersol2 software, for instance.

8.2 PROGRAMMING LANGUAGES A major problem to design simulation system is the task of choosing an adequate programming language. Normally, it is necessary to make equilibrium between the following criteria that are considered more important in this situation: rapid implementation, normally to analyze non complex problem; independence of operational system (OS), normally Windows or Linux; velocity, when the algorithms are CPU intensive; flexibility, simulating and controlling a real-time system; evolutive, developed blocks are easily re-utilizable by objects; diffused, easy to find skilled people to use a language. Here the languages analyzed are C++, C#, Java and VB (Visual Basic) which fit with the criteria presented.

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Obs.: More positive is better: - single negative, + single positive, ++ double positive. In the context of biological simulations, VB are most utilized to make simple analysis. However, if the objective is to make evolutive platform, languages like C++, C# and Java must be considered. But, with the above criteria, the best language is C#. This language is new, but has some interesting characteristics: JAVA like, C++ like, and open standard. This hybrid nature makes C# flexible and powerful to develop virtually any application.

8.2.1 Agent and multi-agent system Conjugated with the language, it is important to adopt a flexible and evolutionary methodology to develop the biotechnological simulating / controlling system. According to Jennings et al., (1998), the agents autonomous and the multiagent systems represent a new manner of analyzing, projecting, and implementing complex systems by software. This approach is made up of a very powerful whole of tools, techniques and metaphors which are very interesting for the development of software. According to this approach "An agent is a computer system, situated in some environment, which is capable of flexible autonomous action in order to meet its design objectives". This work intends to check this approach potentiality for the study of biological problems. The importance of the multiagent system lies in its capacity of complex problems resolution, that use to be difficult to identify and describe perfectly. The multiagent system is based on a certain number of agents which try to give a good response for a specific situation. To achieve that, they can exchange information and also be able to learn "how to move". The solution is a system evolution function, which is "itself' the result of a process of training. The training can be seen in two different levels: individual or by the system. The best manner of solving a complex, large and/or unforeseeable problem is to develop a number of specific functionalities and components modular which are specialized in the resolution of a particular aspect of the problem. Here, the modular or almost modular components are the agents. Another characteristic

of real problems is that they are open and distributed systems. In such a situation, it is impossible to know its components in advance and moreover, those behavior changes with time. Sycara (1998) defined a Multi-Agent System (MAS) as a loosely coupled network of problem solvers that worked together to solve problems that were beyond the individual knowledge or capabilities of each problem solver. Nowadays, the most problems solution is based on single agent. However, the MAS has a great difference; it is not necessary to know exactly what the system needs to do before its implementation. The system can be improved with a little effort when it turns necessary or when a solution is reached. The multi-agent system allows simulations closer to the real world, because it is possible to take into account a great diversity of variables and to articulate them to check the results. The constraints of this method are related to the system project and the results comprehension. Accordance to Dergint (1999), due to the system complex nature, it is still not easy to know or determine analytically the conclusion or the results obtained automatically. For instance, biological control and monitoring systems with MAS can use a heterogeneous strategy to find a solution, it gives characteristics as robustness and flexibility.

8.3 MODELING AND SIMULATION Accordingly to Harrington and Tumay (2000), computer simulation modeling allows to see the results of a process without investing time and resources to test the real one. Most of simulations are able to show results with a kind of time acceleration. It, thus, becomes clear that it brings the possibility of saving resources and time. However, it demands more complete mathematical models describing the process behavior. Instead of committing resources to implement a new real process, simulation allows to test some theories previously such as changing process parameters, process scale, and initial conditions, without the real costs and specially the whole kind of risks. In addition, there is the possibility of turning the simulation model into a control solution or, at least, part of that.

8.3.1 Examples of techniques There are many strategies for modeling and simulating industrial processes. Bioprocesses specifically have some peculiarities such as the uncertainties in the model parameters, normally described by ordinary differential equations, taking into account kinetic coefficients, for instance, and some not totally described relations as the yield and specific growth rate, depending on the product and microorganism involved, besides the processes behavior usually contain non-linearities. These characteristics lead to discuss about the importance

of making use of modem mathematical modeling techniques in accordance to Edelstein-Keshet (2004). In some cases it is unfeasible to take into consideration all the variables and its behavior, or even know all of them, hence new approaches are indispensable to face these kind of problem. The following sections show several examples of useful methods.

8. 3.1.1 Fuzzy logic The concept of Fuzzy Logic, accordingly to Zadeh (1965), was presented at first time not as a control methodology, but as a way of processing data, considering partial set membership rather than crisp set membership or nonmembership, as the so-called Classical Logic. As presented in Zadeh (1990), Professor Lotfi A. Zadeh explained that people do not require precise numerical information input, and yet were capable of highly adaptive control. Human beings often consider imprecise expressions like "tall", "expensive", or "not so far" (Sturm, 2005). However, the processing mode of a computer is limited to a one-or-zero, everything-or-nothing, or true-false mode of thinking. Gupta (1977) asserts that Fuzzy Logic is able to remove barriers between human being communication mode and computer processing mode. Professor Zadeh proposed the concept of linguistic or "fuzzy" variables, which invited to think as linguistic objects or words instead of numbers. Fuzzy Logic presents a rule-based approach to describe the process behavior rather than attempting to model the system mathematically or the modeling task is empirically-based, depending on human experience and not exactly technical understanding of the whole process. This approach allows to handle poor defined problems, where there are variables not exactly known, concerning to its behavior or importance to some specific process, which frequently happens in bioprocesses. Thus, it is not necessary to consider all the variables; in most cases it could be prohibitive and the nonlinearities are often absorbed by the proper Fuzzy Logic manner of describing problems and working with them.

8.3.1.2 Neural networks According to Norgaard et al., (2000), most of human abilities were learned by examples. That is how emerged the interest of transferring this didactic model into a computer's program. Neural Networks are a kind of approach designed to mimic the real human brain neural connections. Anthony and Bartlett (1999) mentioned that an Artificial Neural Network needs a training process, where examples were presented and were supposed to be learned. A neural network consists of a large number of simple neurons, which is often richly interconnected and each one is a part of program with normally very simple function to execute. The so-called synaptic weight associated with the connections between

neurons will determine the characteristics of the network. During the training process, the network automatically adjusts the values of synaptic weights. Actually, only one neuron has no function but the pattern of synaptic weight values in the whole network that carries information. The patterns are generated by the network and, because of that, it is not possible to describe analytically the whole process used to solve the problem. Norgaard et al., (2000) explains that the interest in using this approach is because of the ability of working with non-linear systems and the learning capacity. Sometimes researchers know the behavior of a specific Bioprocess, but are not able to describe each variable influence itself. Thus a Neural Network could be able to learn from the practical examples showed, even if the mathematical model is not understandable as an equation or something like that, but presenting the same results as the real process or manipulating the control parameters, leading to an adequate and stable work value.

8.3.1.3 Genetic algorithms (GA) According to the theory suggested by Charles Darwin in 1859, the biological alive species evolution would be due to the natural selection resulting from the competition for food and the reproduction ("struggle for life"). Holland et al., (1986) asserts that this mechanism of creation/selection is very simple, therefore, its application as software algorithms seems to be natural. The introduction of these mechanisms on computer solutions took place in early seventies and received the name of Genetic Algorithms known by the initials GA. The way that genetic algorithms treat a problem is very simple. Firstly, GA works in prospecting on a group of objects, not on an object. The group of objects is a whole population of different objects. Each element of the group receives a fitness value. This value allows the object to know which is the best solution to a given problem and the fitness is calculated in an objective way by the means of an evaluation function. This evaluation function is the process part which is guided by a known field. However, the process of prospecting is not guided in a specific knowledge field. The individuals or objects are coded by sequences of bits so that it is possible to make an analogy with the chromosomes. As in the biological creatures, the genetic operations on these sequences of bits are not found on a specific field of knowledge. The characteristics of the individuals are coded by chromosomes neutral sequences and the genetic operations are made on these chromosomes. Thanks to this characteristic of specific science field independence, the genetic algorithms are universal and of special interest to prospect in areas whose the results possibilities are not known. With a population and their fitness a new generation is created. The process of creation analogically follows the natural process of evolution where the most powerful individuals have more chance to reproduce. Rising generation,

thus, will replace, with a certain probability, the old individuals less adapted to their medium. GA turns possible to prospect in most of the target medium and in a short lapse of time. Normally the prospecting must finish when an optimum is found in target space. The probability of having found a good optimum increases accordingly to the number of individuals used in simulation.

8.4 THE FERSOL2 EXAMPLE The new software Fersol2 is based upon the so-called Fersol. This software was developed in 1987, running only on DOS operational system, using the methods described by in Rodriguez Leon et al., (1988) to manipulate solidstate fermentation (SSF) parameters. In SSE instead of what happens in submerged fermentation, the kinetic process behavior is very difficult to analyze. It is due to the impossible separation of biomass from the solid substrate and because of its heterogeneous characteristic (Pandey et al., 2001). The interest in studying kinetic process behavior is because of the fact that it allows many important parameters determination such as specific growth rate, process yield and productivity, heat evolved, process control criteria, strategy for the production of a particular product, and scale-up considerations (Pandey et al., 2001). The biomass synthesis is one of the most important patterns present in a fermentation bioprocess. In submerged fermentation process, this parameter can be normally measured through direct methods such as cell counting, dry biomass or optical density determination. Nevertheless, in SSF the proper characteristic is that the biomass will be attached to solid surface, hence, it is not so easy to measure directly. Thus, some indirect methods can be used as the determination of protein content or another cell component (Raimbault 1981; Okasaki et al., 1980; Matchman et al., 1985), or through the oxygen consumption and production of CO2 in (Nishio et al., 1979; Carrizalez et al., 1981; Koba et al., 1986). Regarding the software solution, the 02 consumed and/or CO2 evolved seems to be more adequate because of on-line measurement possibility and fast results (Pandey et al., 2001) and this method could be considered as a direct measurement of process kinetics, although in true sense it is still an indirect measure of biomass synthesis. To develop the Fersol2 software, it was decided that it should run on Microsoft Windows operational system because of the advantages and resources offered by the system. It was decided to use DotNet platform and C Sharp (C#) programming language, both with new concepts of programming, because of the performance, resources, and longevity concerning the time to turn obsolete. The platform allows to develop solutions which runs under Windows and Linux operational systems, adding more versatility.

8.4.1 Farsol2 calculations As pointed out in Pandey et al., (2001) and observing all the considerations, using some computing power it is possible to estimate the biomass (Xn) through an appropriate software solution. Making use of a mathematical set of method called Numerical Calculus, Fersol2 is able to calculate the integral and differential equations. As an example to evaluate the total oxygen consumed, it is useful the integral equation described by Pandey et al., (2001):

ftt

dt

From the Numerical Calculus, the numerical integration is applied by the Trapeze Rule, which leads to a discrimination of the theoretical equation, but in this specific case the values are taken from a table of oxygen quantity consumed, hence it is not quite a problem. The result is equivalent to the area under the oxygen consumption curve, determined by some periodic measures. On the other hand, some problems need an iterative method to solve. To determinate the biomass (Xn), making use of Metabolic Gas Balance Method described in Pandey et al., (2001), it is necessary to make an adjust in the biomass curve using the following equation:

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Where a = m.Yx/o.At. Taking into account some real measures of the biomass at a determined time and the initial process conditions, a kind of trial and error method is used to adjust the intermediary values. Numerical Calculus presents many iterative methods to solve this kind of problem because the trials should not be chosen randomly, it is necessary to maximize the convergence and it allows using some stop criteria to the number of iterations. In the present time the available computers, concerning to performance features, and the software development tools allow this kind of calculation without any additional care. Instead, some years ago the programmers should keep in mind the limitations of computing power, thus the algorithms were developed to adequate the problem complexity to the time to solve it, in other words there were some problems which could be unfeasible since would take much long to solve.

8.4.2 Fersol2 results As has been pointed out in the previous section, the Fersol2 software uses updated techniques to manipulate all the parameters. After adjusting the biomass values, it estimates many other process indicators such as, process yield based on consumed oxygen for biomass synthesis, specific growth rate at the logarithmic or exponential phase, and maintenance coefficient. It is possible to show virtually in only one window all the parameters, it turn easier to visualize the process behavior. The software interface is shown in the following figure.

It is possible to plot the curves of oxygen, biomass, and logarithmic biomass. The following figure 1 and figure 2 show the oxygen and the biomass curves. Using the Windows or Linux operational system and additional software as text editors or spreadsheet applications software as Microsoft Office's Excel and Word or the contents of Open Office, for instance, Fersol2 allows to export or import all the values and graphics; thus, it turns easier to analyze and produce documentation.

8.5 THE DATA ACQUISITION AND CONTROL SOFTWARE EXAMPLE Some of considerations pointed out in the previous section take into account the necessity of having on-line information about the fermentation process behavior, concerning to temperature, air flow, oxygen consuming, and CO2

Fig. 1.

Fig. 2. production, for instance. Sometimes to control or monitor a process it is necessary to consider real time analysis, which is all situation where there is a time window to execute some operation and/or fix some system fault, after this the error will be considered irreversible. Normally critical time applications are real time applications also. Nowadays it is possible to use the communication ports such as RS232 or, today more commonly, USB ports to make the acquisition of external sensor signals accordingly to Kelly and Bai, (2005). In addition, some protocols are getting more usual as Ethernet and Fieldbus in industrial

plants and a strong trend in wireless communication as BlueTooth will certainly be present in a great number of applications. The data transfer rate has been increasing and communication protocols became more friendly specially when using the updated Windows versions and appropriate programming platform. Thus, this kind of solution turned more cost effective. The controlling devices available in industrial commerce are normally open communication protocol and the development frameworks allow making the communication drivers more friendly by the programmers. Using adequate sensors and controlling devices, accordingly to Wang and Tan (2006), an acquisition or control software can read any parameter value and transfer it into the computer, besides the distributed control possibility allows to keep these parameters monitored even if the communication between process and computer is interrupted. In this case with eventual loss of data, of course, but keeping the advantage of maintaining the system under local control. Acquisition or control software can also display values on the screen such as tables and graphics in real time and making some calculations such as unit conversion and values statistics evaluation in accordance to Wang and Tan (2006).

8.6 CONCLUSIONS The Solid State Fermentation process carries many peculiarities that lead to the necessity of investigating new approaches to solve several kinds of complex problems. The technological advance, in informatics and computation sciences, allows the researchers and developers to use updated techniques with even more increased performance and resources availability. These characteristics permit using some sophisticated methods with cost effective performance. An evolutive solution software could be adequate because of its flexibility and adaptation capacity. Some of the solutions discussed demonstrate that the proper indirect measures necessity in SSF is, itself, a strong argument to make use of more complete mathematical models, with the consequent need to apply more accurate analysis, which is only possible using the informatics features available nowadays.

Acknowledgment The authors acknowledge Conselho Nacional de Desenvolvimento Cientifico e Tecnologico -CNPq for financial support and Universidade Federal do Parana - UFPR.

References Anthony, Martin, Bartlett & Peter L, 1999, Neural Network Learning: Theoretical Foundations. 403p. p 1,5. Cambridge University Press: Cambridge. Carrizalez V, Rodriguez H & Sardifia I, 1981, Determination of the specific growth rate of molds on semi-solid cultures. Biotechnology and Bioengineering, 23, 321.

Dergint Dario EA, 1999, Apprentissage Collectif et Milieux Innovateurs 9l~tude de Cas Grenoble et Simulations Multi-Agents - Thesis. p 567. UTC, Compi~gne. Edelstein-Keshet L, 2004, Mathematical Models in Biology. p 586. Siam: Philadelphia. Gupta MM, 1977, Fuzzy Automata and Decision Processes. New York: Elsevier.

Harrington H. James, & Tumay Kerim, 2000, Simulation Modeling Methods: To Reduce Risks and Increase Performance. p 1-3. p 379. McGraw-Hill: New York. Holland John H, Keith J Nisbett, Richard E Thagard & Paul R, 1986, Induction: Processes of Inference, Learning and Discovery. Cambridge, Mass: MIT Press.

Jennings Nicholas R, Sycara Katia & Wooldridge Michael, 1998, A Roadmap of Agent Research and Development- Autonomous Agents and Multi-Agent Systems. pp. 7-38. Boston: Kluwer Academic Publishers. Kelly Laurie & Bai Ying, 2005, The Windows Serial Port Programming Handbook. p 4, 5. CRC Press LLC: Charlotte.

Koba Y, Feroza B, Fujio Y & Ueda S, 1986, Journal of Fermentation Technology, 62, 2, p. 175. Matchman SE, Jordan BR & Wood DA, 1985, Estimation of fungal biomass by three different methods. Applied Microbiology and Biotechnology, 21, p. 108. Nishio N, Tai Tai & K Nagai S, 1979, Hydrolase production by Aspergillus niger in solid state cultivation. European Jounal of Applied Microbiology and Biotechnology, 8, p. 263. Norgaard M, Ravn O, Poulsen NK & Hansen LK, 2000, Neural Networks for Modelling and Control of Dynamic Systems: A Practitioner's Handbook. 246 p. Springer-Verlag London Limited: Great Britain. Okasald N, Sugama S & Tanaka T, 1980, Mathematical model of surface culture of koji mold, Journal of Fermentation Technology, 58, 5, p. 471.

Pandey A, Soccol CR, Rodriguez-le6n JA & Nigam P, 2001, Solid State Fermentation in Biotechnology: Fundamentals and Applications. Asiatech Publishers, Inc. New Dehli. India. Raimbault M, 1981, Travaux et Documents de 1'ORSTOM, No. 27, Paris. Rodriguez Le6n J A, Sastre L, Echevarria J, Delgado G & Bechstedt W, 1988, A Mathematical Approach for the estimation of biomass production rate in solid-state fermentation, Acta Biotechnologica, 8 (4), 299-302,

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Sturm Wilerson, 2005, Avalia~o do Potencial de Uso da L6gica Fuzzy para a Identifica~o

de Indicadores de CompetSncias no Currfculo Lattes. Dissertation. p 104. April-25-2005. Centro Federal de Educa~ao Tecnol6gica do Parami. Curitiba. Syeara Katia P, 1998, Multiagent Systems, AI Magazine, 79-92. Wang Lingfeng, Tan & Kay Chen, 2006, Industrial Automation Software Design, p 158,159 John Wiley and Sons: New Jersey. Zadeh LA, 1965, Fuzzy Sets. Information and Control, 8, 338-353. Zadeh LA, 1990, The birth and evolution of fuzzy logic. In: TURKSEN, I.B. Proceedings of NAFIP'90.

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iiii!iiiii iiiiiiiiiiiiiii!! !ii 9.1 INTRODUCTION The process of solid-state fermentation for food applications is one of the oldest knowledges available to humans. To many communities it is a part of their traditional knowledge to prepare the fermented food (Hesseltine, 1977). The expanded form of Japanese word for Koji relates to growth of mold. The traditional fermentations such as Koji, Tofu, miso, sauceges, pickles, ensilling are the extension of the traditional fermentation knowledge exploiting the GRAS strains of fungi and bacteria to carry out the fermentation. The processes provide extracellular fungal enzymes and have been the basis to initiate the microbial enzymes production by SSF in industrial environment. The koji process undoubtedly influenced the first production of microbial enzymes by SSF on industrial scale. The emergence of modem SSF based enzyme industry may be attributed to the entrepreneurship of Jokichi Takamine and later by Underkofler's efforts in producing mold bran enzymes for saccharification of grain (Bennett, 2001). The discovery of Penicillin in early thirtee's and streptomycin, chloramphenicol and tetracyclin's in early fifty's took the sheen of the emerging SSF process and emphasized on submerged fermentation. The SSF process development was still in its infancy and was considered not suitable for many of the potential large-scale applications. However, recently with lot of effort being put into value addition of agroindustrial residues and publications emphasizing the possibilities in SSF (Pandey, 1992, 1994, 1999, 2000, 2001; Pandey and Soccol, 2000; Lonsane & Ghildyal, 1992; Babu & Satyanarayana, 1996; Haltrich et al., 1996; Mitra et al., 1996; Selvakumar et al., 1996; Tengerdy, 1998) commercial operations using SSF process were developed in countries such as Japan, India, USA, and France. The successful operation of the companies utilizing the SSF process ensured that the process gets its due attention and apart from enzymes the pharmaceutical products and other secondary metabolites were also manufactured. The major

applications of solid-state fermentation involve processes exploiting fungal systems and to a lesser extent bacterial systems, however, recently some actinomycetes were also reported especially for production of bioactive compounds using SSF (Kota and Sridhar, 1998; Sircar et al., 1998; Saudagar et al., 2006). The fungal systems are an obvious choice, as these systems require solid support matrices for their growth under natural circumstances. In natural habitats fungi grow on wood, bark, leaves of plant, decaying organic matter and other moist surfaces. Given this tendency for adhered growth under natural environments fungi have been exploited for maximum extent. The enzymes being produced by SSF include cellulases, hemicellulases, pectinase, amylases, ct- and ~galactosidases, caffeinase, tannase, proteases etc. The major support matrices include bran's (wheat, rice, barly), oil cakes (sesame, soy, olive, coconut, mustard), bagasse (sugarcane, cassava, orange), coffee pulp and husk (Pandey and Soccol, 2000) and also substrates such as Jackfruit seed powder, tamarind seed powder. In the present chapter the authors are presenting a brief account of developments in SSF process during last five years as the earlier developments had already been discussed in various reviews and b-bok chapters.

9.2 MiCRO-ORGANISMS AND METHODS OF IMPROVING SSF PRODUCTIVITY The potential candidates for enzyme production using SSF process are bacteria and fungi. The bacteria from diverse habitat have been isolated and studied for fermentative applications however mesophilic and themophilic fungi have been more successfully utilized. A number of commercial enzymes such as alphaamylase and glucoamylases, pectinases, hemicellulases, phytases, xylanases and proteases are produced by fungi. The fungal systems used for koji preparations such as A. sojae, A. oryzae and other like A. niger are considered as GRAS strains (Generally Regarded as Safe). The fungal strains such as A. terreus, A. tamarii, A. ustus, Rhizopus oryzae, Rhizopus oligosporus and Penicillium sp. strains are also being frequently explored for production of enzymes. Apart from the commonly used fungal strains, bacterial strains such as Bacillus spp. are being increasingly exploited (Table 1). The yield enhancement of the SSF process has been mostly studied by traditional approach of screening for new culture with improved production and their process optimisation. However, now recombinant DNA approach using genetically engineered systems is also being explored. The approach is of potential importance given the ability to modify the host genome by introducing novel genes responsible for improved traits. The recombinant DNA approach has been successfully applied for studying the phytase, amylase, protease and biochemicals such as diacetal, folate, riboflavin, mannitol and lactic acid. The recombinant DNA approach

has been successfully exploited for improvement of lactic acid bacteria especialy for applications in dairy industry. The applications included flavour enhancement, resistance to bacteriophages, addition of nutritional components and stability and structure of end products. However, the recombinant approach requires the cultural conditions of the modified organisms to be examined to provide conditions that would fully exploit the increased potential of the culture. Also, the process of strain improvement involves the continual genetic modification of the culture, followed by reappraisals of its cultural requirements (Stanbury et al., 1995) and needs special training to cater to these requirements. The initial efforts in genetic modifications were concentrated using traditional approaches such as exposing to mutagenic agents such as ethyl methyl sulphonate, NNG, NTG and UV radiations. However, to exploit enzyme production fight metabolic regulatory elements need to be modified using recombinant DNA approaches. This approach of modifying the entire production pathways is called as metabolic pathway engineering. This requires complete knowledge of the pathway and various control elements working in it.

9.3 SUBSTRATES USED FOR THE PRODUCTION OF ENZYMES IN SSF The recent SSF process studies have explored a variety of substrates varying from agro-residues to wastes of industries such as potato chips, spent brewing grain, paper and wood processing industries. The wastes such as saw dust and wood chippings have a huge potential for cellulases and hemicellulases production. Substrates such as sugar cane bagasse, wheat bran, rice bran, maize bran, gram bran, wheat straw, rice straw, rice husk, soyhull, sago hampas, grapevine trimmings dust, saw dust, corncobs, coconut coir pith, banana waste, tea waste, cassava waste, palm oil mill waste, aspen pulp, sugar beet pulp, sweet sorghum pulp, apple pomace, peanut meal, rapeseed cake, coconut oil cake, mustard oil cake, cassava flour, wheat flour, corn flour, steamed rice, steam pre-treated willow, starch, etc. have been studied for SSF applications. Recently green gram husk was used as a good support for alkaline protease production (Prakasham et al., 2006). Also oil cakes and their combinations have been explored recently with much success for phytase, amylase and protease production. The other developments include the solid-state fermentation process using inert support material supplemented with chemically defined liquid media for production of enzymes, metabolites and biological control agents (Ooijkaas et al., 2000). The SSF process may have a major application in production of bio-control agents for large-scale field applications given the requirement of biocontrol agents at an economical price and ease of directly using the fermentation media. The several studies have already reported effect of substrate, its particle size and moisture level/water activity in the media.

Table 1. Microorganisms for enzyme production using SSF

9.4 SSF REACTOR DEVELOPMENT The delay in SSF being the major mode of fermentation can be partly attributed to the bioreactors initially available. During the initial phases mostly tray type of fermenters were in use with poor instrumentation support and also, heat generated during the process was poorly dissipated. Although recently research was directed on SSF reactor development however, simultaneous instrumentation development has so far been lagging. Initially, automated koji (tray) cultivation was most frequently applied (Pandey et al., 1999). Later on different bioreactor configurations such as periodic pressure solid state fermenter (Tao et al., 1999), immersion, expanded bed and tray type reactor (Couto et al., 2002), intermittent agitation rotating drum type (Kalogeris et al., 2003) and a new bioreactor "PlaFractor" (Suryanarayan, 2003) were developed for the production of enzymes, biocontrol agents and pharmaceuticals. The traditional tray type reactors consist stacked trays contained in a container with sterile humidified air being circulated for aeration and moisture maintenence. Although, the column bioreactors have been used for enzyme production studies (Pandey et al., 1996; Mitchell et al., 1999) however, these reactors suffering from differences in growth, enzyme synthesis and sporulation pattern in different segments of the column (Pandey et al., 1996) could not be used for industrial scale fermentation. Recently some efforts were made to improve upon the tray bioreactors by using spouted bed bioreactor. The studies suggested that intermittent spouting of tray reactor with air achieved high production levels of (~-amylase with yields comparable to packed bed bioreactor (Silva and Yang, 1998). Also, periodically dynamic changes of air have been found effective in controlling temperature instead of agitation and rotation that damage or disrupt fungal mycelia and reduce the porosity of the substrates (Chen et al., 2005).

9.5 FACTORS AFFECTING ENZYME PRODUCTION IN SSF SYSTEMS The various factor which need to be studied for successful SSF process development include: a suitable substrate and microorganism, the treatment requirement for easy substrate availability to micro-organism, substrate particle size, water content and water activity of the substrate, relative humidity, nature of inoculum, temperature stability, heat dissipation and maintenance of uniformity, duration of fermentation (Pandey et al., 1999). Although, the SSF systems have been known for their simplicity and relatively low level of instrumentation requirements but for process automation purposes more instrumentation support is necessary. Although, the PlaFractor fermenter developed at B iocon India, has been developed keeping the process automation requirements in mind. However, various aspects of SSF processes such as development of probes mainly for moisture measurement, control and regulation of process variables,

analytical procedures to specific media and measurement of the gaseous environment still need to be explored (Durand, 2003). Also, various problems of scale like mass compaction, shrinkage, and reduction of the heat transfer (Durand, 2003) etc. require careful studies. The factors affecting the fermentation scale-up in SSF are mainly poor heat removal (Mitchell et al., 2003) leading to problems in temperature control and oxygen limitation (dos Santos et al., 2004). The air being poor conductor of heat is not suitable for conducting the heat and also it requires maintaining certain level of moisture, which can only be done by providing humidified air. However, the disadvantage of poor heat removal can be used advantageously using thermophilic organisms which require elevated temperatures (Kalogeris et al., 2003)'

9.6 ENZYMES PRODUCED BY SSF Although much work has been carried out on SSF for the production of enzymes of industrial importance such as proteases, cellulases, ligninases, xylanase, pectinase, amylase, glucoamylase, etc., attempts are also being made to study SSF processes for the production of inulinases, phytases, tannase, phenolic acid esterase, microbial rennet, aryl-alcohol oxidase, oligosaccharide oxidase, tannin acyl hydrolase, a-L arabinofuranosidase, etc. using SSF systems. In the following sections, a brief account on production of selected enzymes in SSF systems would be discussed. 9.6.1 Cellulases and hemicellulases Cellulose is an insoluble molecule consisting of glucose units ranging from 2000-10000 residues while some chains may be still bigger in size. The glucose units are arranged linearly as ~-(1-4)-D-glucopyranose and form crystals. The intra-molecular and intra-strand hydrogen bonds hold the network. The cellulase has many uses as an anticake agent, emulsifier, stabilizer, dispersing agent, thickener, and gelling agent and also has water-holding capacity. The cellulases represent a group of enzymes capable in breaking cellulose. They comprise of endoglucanases (1,4-[3-D-glucan glucanohydrolases), exoglucanases or cellobiohydrolases (1,4-~-D-glucan cellobiohydrolases) and ~-glucosidases or cellobiases ([3-D-glucoside glucohydrolases). The cellulases have various applications ranging from extraction and/or clarification of fruit and vegetable juices, to baking, brewing, biostoning of denim and deinking of the recycled pulp and paper.

Trichoderma reesei is the most studied mesophilic fungus and five endoglucanases, two cellobiohydrolases and one ~-glucosidase have been already identified from this source. The other organisms studied include Aspergillus niger, Melanocarpus sp., Scymlidium thermophilum and Thermoascus aurantiacus. The cellulase

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production studies in Melanocarpus sp. and Scytalidium thermophilum suggested that expression profile of different components of cellulase complex in thermophilic fungi, Melanocarpus sp. MTCC 3922 is independently regulated while in Scytalidium thermophilum MTCC 4520 it is co-regulated (Kaur et al., 2006). The cellulose rich agricultural crop wastes such as wheat straw, rice straw, corncob, corn stover, wheat bran, etc. have been used for cellulase production. Similarly, the SSF has been extensively utilized for production of hemicellulases. The hemicelluloses are a group of complex carbohydrates containing xylans, uronic acid and arabinose. The main chain of xylans is built from 13-1, 4-1inked xylopyranosyl residues. The backbone is usually substituted to various degrees by residues of 4-O-methyl-D-glucuronic acid, D-glucuronic acid, or L-arabinofuranose, and in some cases is also esterified by acetyl groups (Biely, 2003). Due to the structural heterogeneity of the xylans, xylan-degrading enzyme systems include several hydrolytic enzymes. The best known of these are endo-~-l,4-xylanases, which attack the main chain of xylans, and 13-xylosidases which hydrolyze xylo-oligosaccharides to D-xylose (Haltrich et al., 1996). Xylanases have various applications including production of oligosaccharides, baking, starch recovery from wheat flours and aid in the extraction and clarification of fruit juices. The hemicellulases also find application in feed industry to improve the digestibility of feed and in paper and pulp industry for biopulping and help in reducing the consumption of chlorides for developing an ecofriendly process The xylanase production has recently been reported from lot of fungal systems such as Thermomyces lanuginosus, Thermoascus aurantiacus, Aspergillus awamori, A. niger, A. oryzae, Penicillium canescens, Ceriporiopsis subvermispora, Melanocarpus albomyces, P. thermophila J18 and Trichoderma reesei. The substrates used mostly for xylanase production include wheat bran, corn cobs, sugarcane bagasse, bagasse pulp, spent sulphite liquor, rice straw, wheat straw, sorghum flour and eucalyptus pulp. However, lignocellulosic materials especially wheat bran has been more successful in production with higher titers being attributed to its hemicellulose nature, favorable degradability and the presence of some nutrients in the carbon source (Sonia et al., 2005). Also, wheat straw is reported to be ideally suitable for xylanase production in T. aurantiacus and Penicillium canescens cultures (Kalogeris et al., 1999; Bakri et al., 2003).

9.6.2 Ligninases Lignins are three-dimensional phenylpropanoid polymers, considerably resistant to microbial degradation in comparison with polysaccharides and other naturally occurring biopolymers. Ligninases have applications in delignification of lignocellulosic materials, which can be used as the feedstock for the production of biofuels, paper pulp and animal feedstuff. These may also be used in pulp

bleaching, paper mill wastewater detoxification, pollutant degradation, or conversion of lignin into valuable chemicals. Lignin itself is good asphalt binder with applications in road constructions and also has fuel applications. The Ligninases comprise a group of enzymes represented by Lignin peroxidase (LIP, EC- 1.11.1.7), manganese peroxidase (MnP, EC- 1.11.1.13) and laccase (EC-1.10.3.2). LiP and MnP are heme-containing glycoprotein which require hydrogen peroxide as an oxidant. LiP oxidizes non-phenolic lignin structures by abstracting one electron and generating cation radicals, while, MnP oxidizes Mn(II) to Mn(III), which then oxidizes phenolic compounds to phenoxy radicals. This leads to the decomposition of lignin substructure. Laccases are multicopper phenol oxidases, which reduces oxygen to water and simultaneously catalyze the oxidation of aromatic pollutants like anilines and phenols. Several methods using laccase, immobilized laccase and laccase/mediator system have been developed for the treatment of the textile effluents. This enzyme decolorizes some azo dyes without direct cleavage of the azo bond through a highly nonspecific free radical mechanism, thereby avoiding the formation of toxic aromatic amines. Recently, laccases produced from various organisms such as whiterot fungus Daedalea quercina, Stereum hirsutum and Peniophora sp and Streptomyces cyaneus were studied for decolorization of synthetic dyes (Baldrian, 2004) and biopulping of softwood chips in SSF (Wolfaardt et al., 2004; Berrocal et al., 2004). The biological pretreatment studies of wheat straw resulted in better quality cellulose pulps.

9.6.3 Proteases Proteases constitute a very large and complex group of enzymes, which differ in properties such as substrate specificity, active site and catalytic mechanism, pH and temperature activity and stability profiles. Milk clotting enzymes have been in use to transform milk into products such as cheese. Use of proteases ,/ 4n baking is another important food application. The inability of the plant and animal proteases to meet the demands led to an increased interest in microbial proteases. Proteases from microbial sources are preferred to the enzymes from plant and animal sources because of their ease of manipulation for biotechnological applications. Microbial proteases are classified based on the pH range of their activity as acidic, neutral and alkaline proteases and based on the functional group at their active site as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases. Neutral proteases are active within a narrow pH range, have pH optima near neutral, have relatively low thermal tolerance and have applications in preparation of food hydrolysates, baking, protein modification, in leather, animal feeds and pharmaceutical industries. Aspergillus oryzae is the predominant source of the

enzyme. Its affinity for hydrophobic amino acids is an advantage in minimizing the bitterness in protein hydrolysates. While, acid proteases may be classified into two groups based on weather their catalytic activity resembles rennin or pepsin. Rennins like enzymes are characterized by their ability to clot milk and have major commercial importance in cheese manufacture. The group of pepsin like acid proteases includes bovine and porcine pepsin and microbial acid proteases mainly from Aspergilli. The microbial acid proteases have applications in the hydrolysis of soybean protein in soy sauce manufacture, improvement of baking properties and digestive aids. Microbial pepsin-like acid proteases mainly produced by strains of Aspergillus and Rhizopus spp. are active in acidic pH and have moderate temperature tolerance. Solid-state fermentation for proteases has been a preferred mode of fermentation with fungal systems, because of the presence of extracellular enzymes making their recovery easier. The SSF processes usually being simpler can use cheaper agro-industrial residues such as wheat bran, rice bran, sesame oil cake, soybean oil cake, coconut oil cake etc. for production. The recovery also is easier because, the enzyme is produced in concentrated form. The neutral and acidic proteases are widely used in food industry for cheese ripening, meat tenderization, in brewing industry, and for production of protein hydrolysates, as a digestive aid and in bread making.

9.6.4 Lipases Lipases catalyse the hydrolysis of ester bonds at the interface between water insoluble fatty acid ester or glyceride phase and the enzyme-containing aqueous phase. Lipases find their application in hydrolysis of fats and oils, in transesterification, production of biosurfactants, digestive aid formulation, detergents, enantiomeric separations and steriospecific transformations. Lipases are being increasingly explored for tolerance to organic solvents with applications in organic synthesis for chiral selective reactions The chirality is a key factor in the efficacy of many drug products and agrochemicals, and thus the production of single enantiomers of chiral intermediates is becoming increasingly important in the pharmaceutical industry. Although, lipases from various microorganisms have been produced but commercially available lipases are mostly from Aspergillus niger and Rhizopus oryzae. The R. oryzae lipase has found application in bio-diesel production (Ban et al., 2001). Various agro-industfial residues such as coconut oil cake extract, cotton cake, soy cake, gingelly oil cake, olive oil cake, sugarcane bagasse, barley bran, wheat bran, babassu oil cake have been evaluated singly as well as in various combinations, for lipase production. Apart from fungal systems such as Aspergillus niger, Rhizopus oryzae, Penicillium restrictum

Penicillium simplicissimum, Trichoderma versicolour, and T. Hirsuta, bacterial systems such as Bacillus megaterium have also been studied recently for lipase production under SSF conditions.

9.6.5 Galactosidases and pectinases There has been considerable interest to produce t~- galactosidase (EC- 3.2.1.22), ~-galactosidase (EC- 3.2.1.23) and polygalacturoanase (PG, EC- 3.2.1.15) in SSF processes. These enzymes have application in the pharmaceutical and food industries. ~-Galactosidase (t~-D-galactopyranoside galactohydrolase EC 3.2.1.22) finds applications in industries ranging from beet sugar production to hydrolysis of raffinose and stachyose present in soybean, cowpea and other leguminous crops. The presence of these oligosaccharides is the reason of diarrhoea and flatulence caused by consumption of soy products and pretreatment with ct- galactosidase may help their enhanced applications in food and feed industries. Aspergillus oryzae has been mostly used as a source of tx-galactosidase. Although, recently actinomycetes have also been studied for ct- galactosidase production but the relative advantage of Aspergillus oryzae as a GRAS strain makes its enzyme acceptable for food and feed applications. ~i-Galactosidase (or lactase) hydrolyzes the milk sugar, lactose, to its components glucose and galactose and thus finds applications in milk and milk products meant for lactose intolerant peoples, for prevention of lactose crystallization in frozen and condensed milk products, for the reduction of water pollution caused by whey and also for increasing the sweetening properties of lactose. Although, production of ~-galactosidase has been studied with various orgaisms such as Trichoderma reesei, Aspergillus niger, Bifidobacteria, Lactobacillus acidophilus, Kluyveromyces fragilis, kluyveromyces lactis, Kluyveromyces marxianus. However, only Trichoderma reesei and Aspergillus niger have been studied for SSF production. The various substrates used for fermentation include arabinoxylan, wheat bran and lactose etc. and further exploitation using SSF is desired. The pectinases, a complex of pectin-degrading enzymes, consist mainly of pectin methylesterases, endo- and exo-polygalacturonases, pectin lyases and causes deesterification, chain splitting and glycoside-bond cleavage. They have been used extensively by fruit, wine and vegetable industries for various functions such as maceration, extraction, liquefaction, clarification and valorization. AspergiUus niger, A. carbonarius, A. sojae and Rhizopus sp. are the most frequently used fungi for pectinase production in commercial scale. A variety of substrates such as soy and wheat flour, sugar beet pulp, deseeded sunflower head, orange pomace, lemon pulp, orange bagasse, sugarcane bagasse have been studied singly and in combination for pectinase production.

9.6.6 Glutaminase L- glutaminase (L-glutamine amidohydrolase - E.C. 3.5.1.2) is the enzyme deamidating L-glutamine and plays a major role in the cellular nitrogen metabolism of both prokaryotes and eukaryotes. L-glutaminase is useful in the food industry as it increases the glutamic acid content of the fermented food thereby imparting a unique flavour. Besides its food value, ability of this enzyme to bring about degradation of glutamine poses it as a possible candidate for enzyme therapy, which may replace or combine with L-asparaginase in the treatment of acute lymphocytic leukaemia. However, not much is reported recently on L-glutaminase production. The recent reports on L-glutaminase production reported the exploitation of Zygosaccharomyces rouxii NRRL-Y 2547, Cryptococcus nodaensis, Beauveria sp and Streptomyces rimosus but only Zygosaccharomyces rouxii NRRL-Y 2547 and Beauveria sp. were studied for SSF applications. The substrates such as wheat bran and sesamum oil cake have been studied apart from inert supports such as polystyrene supplemented with glucose and L-glutamine.

9.6.7 Amylases The amylases can be broadly classified into two major classes of alpha-amylase and glucoamylase. The a-amylase (endo-1, 4-t~-D-glucan glucohydrolase, EC3.2.1.1) randomly cleaves the 1,4-a-D- glucosidic linkages between adjacent glucose units in linear amylose chain, and glucoamylase or amyloglucosidase (exo-1, 4-o~-D-glucan glucanohydrolase, EC- 3.2.1.3) hydrolyses single glucose units from the non-reducing ends of amylose and amylopectin in a stepwise manner. The glucoamylases are capable of hydrolysing both t~-1,4 and a-1,6 linkages. The enzymes have applications in the starch processing, food, fermentation, textile, detergent and paper industries. The amylases are used in baking industry to improve the quality of dough, in fermentation industry for glucose production for fermentative applications, in pharmaceutical for preparation of glucose syrups and in feed industry to act as a digestive aid. The Aspergillus oryzae has been a preferred organism for fungal alpha-amylases for food and pharmaceutical applications while both Apergillus and Rhizopus spp. have been used for glucoamylase production. The recent studies suggested that oil cakes/ meal such as coconut oil cake, soy meal may also be used efficiently in mix substrate fermentation for amylase production. The other in expensive substrates such as spent brewing grain, rice husk, rice flakes, cassava starch, sugar cane bagasse, orange bagasse, molasses, rice bran, maize meal, millet cereal, wheat flakes, barley bran, crushed maize, corncobs and crushed wheat have also been studied in solid state fermentation (SSF). However, in most of the studies wheat bran has been found to be the best substrate for amylase production in SSE Given the requirement of starch processing industry to have thermotolerant amylase, the efforts were directed in identifying thermostable amylases. The bacterial and actinomycetes amylases were found to be having thermostability

properties. However, for food related applications amylases from actinomycetes may not found much favour. The bacterial alpha-amylase from B. stearothermophilus GRE1, Bacillus subtilis, Bacillus licheniformis, Geobacillus thermoleovorans have been found to be moderate to highly thermostable while alpha-amylase from other sources such as Bacillus cereus MTCC 1305, Bacillus amyloliquefaciens and fungal systems such as Thermomyces lanuginosus have also been studied. The fungal glucoamylase production has already been reported using inexpensive nutrient sources such as wheat bran, tea waste and coconut oil cake. However, recently rice-processing waste (coarse, medium and fine waste) along with rice powder was also studied for glucoamylase production. The various isolates studied include Aspergillus sp. HA-2, AspergiUus niger, Scytalidium thermophilum,

Aspergillus awamori, Rhizopus oligosporus and Thermomucor indicae-seudaticae. The thermophilic mold Thermomucor indicae-seudaticae was reported for production of a thermostable and neutral glucoamylase optimally active at 40~ and pH=7, when grown in wheat bran moistened with a salt solution in 1:2.5 (w/v) ratio supplemented with 2% cotton oil seed cake (Kumar and Satyanarayana, 2004).

9.6.8 Phytase Phytases (myo-inositol hexakisphosphate phosphohydrolase, EC 3.1.3.8) catalyses the release of phosphate from phytate (myso-inositol hexaksiphosphate). Several cereal grains, legumes and oilseeds, etc. have their phosphorus stored in the form of phytate. The phytate phosphorus is not easily accessible for monogastric animals due to low inherent phytase activity and passes in manure. The areas with high density of cattle and swine farming suffer from ground water contamination due to the high-phytate phosphorus manure. The pollution of water bodies due to high phosphate manure has raised serious problems of water blooms and growth of toxin producers. Phytases appear of significant value in effectively controlling phosphate pollution. Although, phytase can be produced from a host of sources including plants, animals and micro-organisms. Microbial sources, however, are promising for their commercial exploitations. Strains of Aspergillus spp., chiefly A. ficuum and A. niger have most commonly been employed for industrial purposes. Apart from them Rhizopus oligosporus, Rhizopus oryzae, Mucor racemosus and Aspergillus ficuum have also been studied for phytase production. Given the desirability of thermostable phytase to survive the feed pelleting process search has now began for thermotolerant phytase and attempts have already been made to use genetic engineering to manipulate the phytase for thermostability. The traditional approach using thermophilic molds such as Sporotrichum thermophile has also been evaluated for thermostable phytase production. Recent studies suggest agro-industrial residues such as

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sesame oil cake, coconut oil cake, groundnut oil cake and soy oil cake are suitable as a single substrate as well as in combination for phytase production under SSF conditions.

9.6.9 Inulinase Inulin is a naturally occurring polyfructan in plants consisting of linear chains of 13 (2,1)-linked fructose residues attached to a terminal sucrose molecule. The polysaccharide is widely distributed in plants such as Jerusalem artichoke, Chicory, Dahlia and Asparagus. Microbial inulinases (2,1-13-D fructan fructanohydrolase (EC 3.2.1.7) are usually inducible and exo-acting enzymes, which catalyse the hydrolysis of inulin by releasing the end fructose molecule. Different Aspergillus, Staphylococcus sp. and Kluyveromyces strains have been studied for the production of inulinases. The solid-state fermentation for the production of inulinase using coconut oil cake and ssugarcane bagasse has been studied as support and carbon source for production of inulinase. The nitrogen supplementation in the form of corn steep liquor has also been studied. The optimum fermentation conditions for inulinase production were found to be: 36~ and 20 wt.% of corn steep liquor using Kluyveromycesmarxiana (Mazutti et al., 2006). However not much work has been reported of late in inulinase production using SSF and most of the work still remains in SmF conditions. Although, studies were conducted by immobilizing the conidia of Aspergillus niger 20 Osm producing extracellular inulinase on pumice stones and polyurethane sponge for use in repeated-batch processes, only some factors affecting inulinase biosynthesis by the mycelium were studied. It was observed that immobilization enabled repeated-batch enzyme production and as many as six subsequent 24 h batches could be fermented by using the same carrier (Skowronek and Fiedurek, 2006). However given the importance of inulinase in production of mannitol, inulo-oligosaccharides~low caloric oligosaccharides, bio-ethanol and also high fructose syrup for sweetening applications, improved inulinase production remains to be explored by SSE 9.6.10. Miscellaneous e n z y m e s The various other enzymes reportedly being explored using SSF process include tannase, chitinase, invertase, and alpha L-arabinofuranosidase etc. 9.7. RECOVERY OF THE ENZYMES Recovery process for enzymes depends upon the nature of product and its intended application. The higher end applications may require more purified enzymes while the industries such as textile, paper and pulp and feed industry may require the crude enzyme preparations. While the enzyme required for

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pharmaceutical and organic synthesis may be the purified one to avoid any side reactions leading to undesired by products. Since most of the enzymes are secreted in the medium hence, they may be simply extracted with water or buffer solutions after mixing for 30 min- 1 hr and removing the mycelia by centrifugation. Since most of the industries desire to have a cost effective downstream process and most of the enzyme preparations have been found useful even in the crude form, hence the enzyme extraction is the preferred mode of recovery. Extraction temperature and pH of the extractant may be sometimes important for enzyme stability. Further purification of enzyme preparations may be brought by ammonium sulphate or acid precipitation followed by ultrafiltration and column chromatography. However, this may lead to overall cost increase of final product.

9.8 CONCLUSIONS The SSF process has been found to be cost effective for many enzyme production processes vis a vis SmF. The increasing cost of feed for SmF, value addition to agro-industrial residues and ease of operation makes the SSF process more favourable. Also, SSF process requires less initial capital and incurs low operating cost and is suitable for agriculture-based economies. The production of industrial enzymes by SSF will have an important role in future biotechnologies. The focus in SSF application is required to study for enzyme specific SSF process development, development of host specific environments, SSF targeted fungi and bacteria and on their genetic improvement for desired tasks. The emerging trends also suggest development of IPR for SSF related processes. The SSF offers suitable conditions for microorganisms capable of growing at elevated temperatures and future developments may lead to their better exploitation of thermotolerant organisms and products by SSE

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Nampoothiri KM, Baiju TV, Sandhya C, Sabu A, Szakacs G & Pandey A, 2004, Process optimization for antifungal chitinase production by Trichoderma harzianum, Process Biochemistry, 39, 1583-1590. Ooijkaas LP, Weber F J, Buitelaar RM, Tramper J & Rinzema A, 2000, Defined media and inert supports: their potential as solid-state fermentation production systems, Trends in Biotechnology, 18, 356-360. Pandey A & Soccol CR, 1998, Bioconversion ofbiomass: A case study of lignocellulosics bioconversions in solid state fermentation, Brazilian Archives of Biology and Technology, 41,379-390.

Pandey A & Soccol CR, 2000, Economic Utilization of Crop Residues for Value Addition: A Futuristic Approach, Journal of Scientific ad Industrial Research, 59 (1), 12-22. Pandey A, 1992, Recent process developments in solid state fermentation, Process Biochemistry, 27, 109-117. Pandey A, 1994, Solid state fermentation - An overview, Solid State Fermentation, A Pandey (ed), Wiley Eastern Ltd, New Delhi, India, pp 3-10. Pandey A, Selvakumar P & Ashakumary L, 1996, Performance of a column bioreactor for glucoamylase synthesis by Aspergillus niger in SSF, Process Biochemistry, 31, 43-46.

Pandey A, Selvakumar P & Ashakumary L, 1996, Performance of a column bioreactor for glucoamylase synthesis by Aspergillus niger in SSF, Process Biochemistry, 31, 43-46. Pandey A, Selvakumar P, Soccol CR & Nigam P, 1999, Solid-state fermentation for the production of industrial enzymes, Current Science, 77, 149-162. Pandey A, Soccol CR & Mitchell D, 2000, New developments in solid-state fermentation. I. Bioprocesses and products. Process Biochemistry, 35, 1153-1169. Pandey A, Soccol CR, Rodriguez-Leon JA & Nigam P, 2001, Solid-state fermentation in biotechnology: fundamentals and applications. Asiatech Publishers Inc, New Delhi. Park YS, Kang SW, Lee JS, Hong SI & Kim SW, 2002, Xylanase production in solidstate fermentation by Aspergillus niger mutant using statistical experimental designs, Applied Microbiology Biotechnology, 58, 761-766.

Patidar P, Agrawal D, Banerjee T & Patil S, 2005, Optimisation of process parameters for chitinase production by soil isolates of Penicillium chrysogenum under solid substrate fermentation, Process Biochemistry, 40, 2962-2967.

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Suryanarayan S, 2003, Current industrial practice in solid state fermentations for secondary metabolite production: the Biocon India experience, Biochemical Engineering Journal, 13, 189-195. Szendefy J, Szakacs G, Christopher L, 2006, Potential of solid-state fermentation enzymes of Aspergillus oryzae in biobleaching of paper pulp, Enzyme & Microbial Technology, 39, 1354-1360. Tao S, Beihui L, Zuohu L & Deming L, 1999, Effects of air pressure amplitude on cellulase productivity by Trichoderma viride SL-1 in periodic pressure solid-state fermenter, Process Biochemistry, 34, 25-29.

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Virupakshi S, Gireesh Babu K, Gaikwad SR & Naik GR, 2005, Production of a xylanolytic enzyme by a thermoalkaliphilic Bacillus sp. JB-99 in solid-state fermentation, Process Biochemistry, 40, 431-435. Wolfaardt F, Taljaard JL, Jacobs A, Male JR & Rabie C J, 2004, Assessment of woodinhabiting basidiomycetes for biokraft pulping of softwood chips, Bioresource Technology, 95, 25-30.

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i:i,{:~,ii:!,i:~i{.:i,:.,Christian i:,.:i,~.ii,:,:, Larroche & Ashok Pandey 10.1 INTRODUCTION The global market for fermentation products was estimated as $14.1 billion in 2004 and was expected to rise at an average annual growth rate (AAGR) of 4.7% to $17.8 billion in 2009. In this context, organic acids represent the third largest category among the other products. Total market value of organic acid is expected to rise to $3 million in 2009 (Marz 2005). Organic acids are among the most versatile ingredients in food and beverage industries. Citric acid, acetic acid, lactic acid, tartaric acid, malic acid, gluconic acid, propionic acid and fumaric acid are some of the organic acids used widely in various industries. They are obtained as the end-products or sometimes as the intermediate components of a particular biochemical cycle. Generally, organic acids are produced commercially either by chemical synthesis or fermentation. However, fermentation processes are the most commonly used method. All organic acids of tricarboxylic acid cycle can be produced in high yields in microbiological processes. Among fermentation processes, the production of organic acids is dominated by submerged fermentation. Table 1 presents some facts about global production of organic acids. Global production of citric acid has reached 1.4 million tons with annual growths of 3.5-4.0% in demand/consumption rate of citric acid. In terms of volume, citric acid and acetic acid together account for about three-quarters of food acidulants. The production of organic acids by solid-state fermentation (SSF) is associated with the historical development of SSF. Citric acid has been known to be produced in SSF for many years while other organic acids such as lactic acid, fumaric acid, oxalic acid and gluconic acid have been reported to be produced in SSF only during the last few years. Many by-products and residues of the agro-industries can be utilized in their production by solid-state fermentation, which minimizes environmental problems and also serves as economical alternative production method.

Table 1. Global production of organic acids

10.2 MICROBIAL PRODUCTION OF ORGANIC ACIDS A large number of microorganisms including bacteria such as Arthrobacter paraffinens, Bacillus licheniformis, Corynebacterium sp., Lactobacillus casei, L. helveticus, L. paracasei, Streptococcus thermophilus; fungi such as Aspergillus niger, A. aculeatus, A. carbonarius, A. awamori, A. foetidus, A. fonsecaeus, A. phoenicis, Rhizopus oryzae, Penicillium janthinellum; and yeasts such as Candida tropicalis, C. oleophila, C. guilliermondii, C. citroformans, Hansenula anamola, Yarrowia lipolytica, etc. have been employed for the production of organic acids (Ikeno et al., 1975, Kubicek and Rohr, 1986, Yokoya, 1992, Grewal & Kalra, 1995, Vandenberghe et al., 1999). In the case of citric acid production, some of them are not able to produce commercially acceptable yields due to the fact that citric acid is a metabolite of energy metabolism and its accumulation rises in appreciable amounts only under conditions of drastic imbalances. Among the strains, A. niger has remained the organism of choice for commercial production because it produces more levels of citric acid per time unit. The main advantages of using A. niger are its ease of handling, its ability to ferment a variety of cheap raw materials, and high yields. The problem associated with the production of citric acid by the yeasts is the simultaneous formation of isocitric acid. Efforts have been made to develop microbial strains which could produce high yields of organic acids. The most commonly employed technique has been by inducing mutations in parental strains using mutagens (Yokoya, 1992, Grewal & Kalra, 1995, Vandenberghe et al., 1999, Pandey et al., 2001, Ikram-ul-Haq et al., 2001). Among mutagens, y-radiation, UV-radiation and chemical mutagens

are often used. Among different approaches, one has been to combine the UV treatment with some chemical mutagens. The "single-spore technique" and the "passage method" are generally the principal methods of selecting strains. The first one has the disadvantage that mineral acid and organic acids (gluconic and oxalic acids) simulate the presence of citric acid (Kubicek and Rohr, 1986, Vandenberghe et al., 1999, Pandey et al., 2001, Rohr et al., 1983).

10.3 SUBSTRATES FOR ORGANIC ACIDS PRODUCTION Various agro-industrial residues such as cassava bagasse, coffee husk, apple pomace, soybean, potato residues, pineapple waste, wheat bran, kiwi fruit peel, kumara, okara, carob-pods, corn-cobs, sugarcane press-mud, citric pulp, etc. have been used in SSF for the production of organic acids. There has been an increasing trend towards efficient utilization and value-addition of these residues as they are generally very well adapted to solid-state cultures due to their cellulosic and starchy nature. Different organic acids have been produced using such residues as substrates, e.g. L (+) lactic acid (Soccol et al., 1994a,b, Shojaosati & Baboeipur, 2002), citric acid (Hang & Woodams, 1984, 1985, Pintado et al., 1998, Soccol et al., 1999, Soccol and Vandenberghe, 2003, Vandenberghe, 2000, Vandenberghe et al., 1999, 2000, 2004), oxalic acid (Leagon et al., 1999) and gluconic acid (Roukas et al., 2000, Singh et al., 2003). Table 2 summarizes the production of some organic acids produced by solid-state fermentation using different agro-industrial residues. 10.4 ORGANIC ACIDS PRODUCED BY SOLID-STATE FERMENTATION 10.4.1 Citric acid Citric acid (C6H807, 2-hydroxy-1,2,3-propane-tricarboxylic acid), a natural constituent and common metabolite of plants and animals, is the most versatile and widely used organic acid. Its global production of about 1.4 million tons was estimated by Business Communications Co. (BCC) in a recent study of fermentation chemical markets in 2004. The report showed that China accounted for 35-40 % of worldwide citric acid production (Graf, 2005, Food Production Daily, 2004). Citric acid is widely used to impart a pleasant, tart flavor to foods and beverages. It also finds application as a function of additive, detergents, pharmaceuticals, cosmetics and toiletries. 10. 4.1.1 Production Citric acid production by fermentation is the most economical and widely used method. More than 90 % of the citric acid produced in the world is obtained by fermentation which has its own advantages: operations are simple and stable; the plant is generally less complicated and needs less sophisticated control

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systems; technical skills required are lower; energy consumption is lower and frequent power failures do not critically affect the functioning of the plant. Citric acid production by fermentation can be divided into three phases, which include preparation and inoculation of the raw material, fermentation and recovery of the product. The industrial citric acid production can be carried out by three different ways: by submerged fermentation, surface fermentation and solid-state fermentation or "Koji" process (Yokoya, 1992, Grewal & Kalra, 1995, Vandenberghe et al., 1999, Soccol et al., 2006). In industrial citric acid fermentation, the large-scale spore production is made by using appropriate means and conditions such as direct inoculation in the production fermenter. Sometimes it is necessary to remove the remainder mineral of the raw material and add other nutrients such as phosphorous, magnesium and nitrogen for development of the mycelium and a good production of the citric acid. Several types of systems have been used for citric acid production in solidstate fermentation such as Erlenmeyer conical flasks, glass incubators, trays, rotating and horizontal drum bioreactors, packed-bed column bioreactor, singlelayer packed-bed, multi-layer packed-bed, e t c (Grewal & Kalra, 1995, Pandey et al., (2001), Vandenberghe et a1.,1999, 2004, Lu et al., 1997, Pintado et al., 1998, Prado et al., (2004). Classically, the solid-sate process has been carded out in trays with aeration. Higher yields (347 g/kg dry cassava bagasse) were obtained in flasks without any aeration, and very little sporulation was observed (Vandenberghe et al., 2004). Equivalent yields (309 g/kg of dry cassava bagasse) were obtained in column reactors only with variable aeration. This showed good perspective in using solid culture process for citric acid production in simple tray type fermenter. In fact, the scale-up study of production of citric acid showed that in tray bioreactors with 4-cm bed thickness 263 g/kg of dry cassava bagasse were attained (Vandenberghe et al., 2004). Several raw materials such as hydrocarbons, starchy materials including agroindustrial residues and molasses have been employed as substrates for citric acid production (Table 2) (Vandenberghe et al., 1999, 2000, Soccol et al., 2004, Yokoya, 1992, Hang & Woodams, 1984, 1985, 1998, Kolicheski, 1995, Vandenberghe, 2000, Soccol et al., 2004). To perform SSE the solid substrate is moistened to about 70%, depending on the substrate absorption capacity. The initial pH of the material is normally adjusted to 4.5-6.0 and the temperature of incubation is about 28-30 ~ depending on the microorganism used (Hossain et al., 1984, Xu et al., 1989, Vandenberghe et al., 1999, 2004, Pintado et al., 1998, Prado et al., 2004). The solid culture process is generally completed within 96 h under optimal conditions (Kubicek & Rohr, 1986, Vandenberghe et al., 1999). SSF processes also normally do not require complex nutritional

supplementation such as trace elements and also their presence may not be harmful (as it does in submerged fermentation). This is one of the important advantages of the solid culture (Grewal and Kalra, 1995, Vandenberghe et al., 1999, Soccol et al., 2004).

10.4.1.2 Chemical factors affecting the production Citric acid accumulation is strongly influenced by the type and concentration of carbon source (Soccol et al., 2004, Roukas & Kotzekidou, 1987). The presence of carbohydrates which are rapidly taken up by microorganisms has been found essential for a good production of citric acid (Yokoya, 1992). Among the easily metabolized carbohydrates, sucrose is the most favourable carbon source followed by glucose, fructose and galactose (Yokoya, 1992, Vandenberghe et al., 1999, Soccol et al., 2004). Table 3 shows the main factors that affect citric acid production.

Phosphorous: The presence of phosphate in the medium has a great effect on the yield of citric acid. Low levels of phosphate have positive effect on citric Table 3. Chemical factors affecting citric acid production

acid production. This effect acts at the level of enzyme activity and not at the level of gene expression. On the other hand, the presence of excess of phosphate leads to a decrease in the fixation of carbon dioxide, which in turn increases the formation of certain sugar acids, and the stimulation of growth (Kubicek & Rohr, 1986, Grewal & Kalra, 1995, Vandenberghe et al., 1999). Nitrogen: Citric acid production is directly influenced by the concentration and nature of the nitrogen source. Physiologically, ammonium salts are preferred such as urea, ammonium nitrate and sulphate, peptone, malt extract, etc. (Soccol et al., 2004). Acid anamonium compounds are preferred because their consumption leads to pH decrease, which is essential for the citric fermentation. However, it is necessary to maintain pH values in the first day of fermentation prior to a certain quantity biomass production. The concentration of nitrogen source required for citric acid fermentation is 0.1 to 0.4 g N/L (Yokoya, 1992, Kubicek & Rohr, 1986). High nitrogen concentrations increase fungal growth and sugar consumption but decrease the amount of citric acid produced (Vandenberghe et al., 1999). Trace elements: Trace metal ions have a significant impact on citric acid accumulation by A. niger (Roukas & Kotzekidou, 1987). Divalent metal ions such as zinc, manganese, iron, copper and magnesium affect citric acid production. It is crucial to take into account the interdependence of medium constituents. There is elevated production of citric acid only if a rigorous control of the trace elements availability is accomplished, but mainly in the submerged process. Lower alcohols: Lower alcohols in pure form inhibit citric acid production but if added as crude carbohydrates, enhance citric acid production. Methanol, ethanol, n-propanol, isopropanol or methylacetate neutralize the negative effect of the metals in citric acid production generally in amounts about 1 to 5 % (Ingram & Buttke, 1984, Dasgupta et al., 1994). Even so, optimal amount of methanol and ethanol depends upon the strain and the composition of the medium. Alcohols have been shown to act principally on membrane permeability in microorganisms by affecting phospholipid composition. Other studies showed that alcohols stimulate citric acid production by affecting growth and sporulation on space organization of the membrane or changes in lipid composition of the cell wall (Ingram & Buttke, 1984). Other compounds: Oils and fats are used in citric acid production to control the foam formation. In addition, some oils stimulate productivity and maintenance of fermentation processes. This lipids act as carbon sources and they are consumed during the fermentation, which is necessary to maintain its level above 0.05-0.3 % (Yokoya, 1992, Grewal & Kalra, 1995).

10.4.1.3 Physical factors affecting the production pH: The pH of a culture may change in response to microbial metabolic activities. The most obvious reason is the secretion of organic acids, such as citric acid, which causes the pH decrease. Changes in pH kinetics also depend highly on the microorganism. It is known that growth of Aspergillus sp., Penicillium sp. and Rhizopus sp. can results in decrease in pH very quickly to less than 3.0. For other groups of fungi such as Trichoderma, Sporotrichum, Pleurotus sp., pH is more stable between 4.0 and 5.0. The nature of the substrate and production technique also influences pH kinetics (Yokoya, 1992). It is desirable that initial pH must be very well defined and optimized depending on the microorganism, substrate and production technique.

Aeration: Since citric acid production is an aerobic process; oxygen supply has a determinant effect on its production. An interruption of aeration during batch fermentation could be quite harmful (Grewal & Kalra, 1995). Dissolved oxygen concentration influences the citric acid formation directly. It is important to maintain the oxygen concentration above 25 % saturation (Vandenberghe et al., 1999). Critical dissolved oxygen tension is 9-12 % of air saturation for growth phase and 12-13 % of air saturation for the production phase (Yokoya, 1992, Grewal & Kalra, 1995). The high demand of oxygen is reached by constructing appropriate aeration devices, which is also dependent on the viscosity of the fermentation broth. This is an additional reason why small compact pellets are the preferred mycelial forms of A. niger during the production. When the organism turns into developments of filaments, the dissolved oxygen tension rapidly falls to less than 50 % of its previous value, even if the dry weight has not increased by more than 5 %. Aeration should be performed through the medium during the whole process with the same intensity, although due to economic reasons, it is usually preferred to start with low aeration rates. The incorporation of the oxygen together with air in submerged process results in increment of citric acid production, but it is economically unviable (Yokoya, 1992). However, it is possible to circulate the oxygen in the fermenter since the carbonic gas is removed from the process. High aeration rates lead to high amounts of foam, especially during the growth phase, so the addition of antifoaming agents and the construction of mechanical "defoamers" are required to tackle this problem (Vandenberghe et al., 1999, Soccol et al., 2004). Forced aeration at the beginning of the process in solid-state fermentation packed-bed reactor affects metabolic rates, and thus, citric acid productivity. The study of citric acid production by A. niger in SSF revealed the importance of a CO2 rich atmosphere. It is known that an environment with high concentrations

of CO2 has a positive effect on citric acid synthesis. The high partial pressure of CO2 probably retards spore liberation of the filamentous fungi and favours citric acid synthesis with cassava bagasse as support/substrate. In fact, low oxygen environment is directly involved in the growth limitation, which is crucial for citric acid production. Low aeration rates (0.18 m3/kg dry CB/h) are supposed to limit the respiration activity of A. niger and, consequently, to turn the metabolism to citric acid synthesis and not to biomass production. It was also observed that strongly aerated cultures (0.3 m3/kg dry CB/h) increased sporulation. As the sporulation increased, the accumulation of citric acid decreased (Vandenberghe, 2000, Vandenberghe et al., 2004).

Production in semi-pilot scale: The influence of different percentages of gelatinized starch present in cassava bagasse on citric acid production by SSF was studied in horizontal drum and tray-type bioreactors. Gelatinization of starch of cassava bagasse was conducted in order to make starch structure more susceptible to its consumption by the fungus Aspergillus niger. Figure 1 shows the citric acid production in tray-type (TT) bioreactor with different percentages of TCB. The best result was 26.3 g/100g of dry CB with 80% of TCB and 4 cm of bed thickness. Citric acid values for 100 % of raw CB were negligible in TT bioreactors. Figure 2 presents citric acid production in horizontal drum (HD) bioreactor with different percentages of TCB. In this case, the best result was obtained with 100% of TCB yielding 26.9 g of citric acid/100g of dry CB. These results show the importance of the starch gelatinization, which surely made starch structure more accessible to fungus attacks. The influence of the TCB percentage on citric acid production was clearly observed in HD bioreactor where high degrees of gelatinization were favorable. On the other hand, in TT bioreactor, it was not necessary to gelatinize CB

Figure 1. Citric acid production in tray-type bioreactors with O, 40, 60, 80 and 100% of treated cassava bagasse in 2, 4 and 6 cm of bed thickness, Vandenverghe et at., (2004).

Figure 2. Citric acid production in horizontal drum bioreactor with O, 40, 60, 80 and 100% of treated cassava bagasse, Vandenverghe et at., (2004).

starch at high levels. This fact could be explained by the excessive material compaction, due to starch gelatinization, which limited the air insertion through out the medium. In HD bioreactor there was not high substrate compaction because it was revolved 3 or 4 times a day and its initial moisture was lower than in the process carried out in TT bioreactors (Table 4). There was no significant difference between the best results obtained in HD bioreactor (26.9 g of citric acid/100g of dry CB) and in TT bioreactors (26.3 g of citric acid/100g of dry CB).This proved that aeration was not a determinant factor for citric acid production in SSF, as it is in submerged citric acid fermentation. The study of the citric acid production for A. niger cultivated by SSF in trays also revealed the importance of the rich atmosphere in COe. The high partial pressure in CO2 probably delays the liberation of spores by filamentous fungus favoring the citric acid synthesis. The limitation of the growth is an important factor in the production of the citric acid. This fact was checked in previous studies in Erlenmeyer flasks where the limitation of Table 4. Citric acid production in bioreactors*

*Vandenverghe et at., (2004)

the growth provoked by a higher concentration of CO2 resulted higher citric acid accumulation (Vandenberghe, 2000). In trays higher bed thickness became more compact during fermentation than lower bed thickness. This was due to the fact that in more compact substrate layers there was more CO2 accumulation and, consequently, more citric acid production. The values for 80% of treated CB have not followed this trend, but in this case citric acid produced in 4 and 6 cm of bed thickness did not present significant difference as presented in Table 3. However, citric acid production for 100% of treated CB in all bed thickness was lower than 80% of treated CB due the excessive accumulation of CO2 caused by high compaction of the substrate. Another important factor to be considered was the fermentation time, which for trays (120h) was shortest than for the HD bioreactor (144 h). Moreover, energy consumption in trays was also lower considering the need of intermittent agitation of the substrate in HD bioreactor. Hence, the cost of the process conduced in TT bioreactors would be lower.

10.4.1.4 Recovery from SSF medium Citric acid from the fermented solid material is generally leached out using water. Recovery from the extracts is generally accomplished in three processes, precipitation, extraction and adsorption and absorption using ion-exchange resins. Precipitation is the classical method and it is performed by the addition of calcium oxide hydrate (milk of lime) to form the slightly soluble tri-calcium citrate tetrahydrate. The precipitated tri-calcium citrate is removed by filtration and washed several times with water. The precipitate is then treated with sulphuric acid to form calcium sulphate, which is removed by filtration. Mother liquor containing citric acid is treated with active carbon and passed through cation and anion exchangers. Several anion-exchange resins are commercially available. Finally, the liquor is concentrated in vacuum crystallisers at 20-25~ forming citric acid monohydrate. Anhydrous citric acid is prepared following the process of crystallisation at higher temperature than 25~

10. 4.1.5 Applications of citric acid As stated above, citric acid has got sevral industrial applications. Table 5 some of its significant used for different porposes.

10.4.2 Fumaric acid Fumaric acid is a useful intermediate in several chemical synthesis including esterification and polymerization, and in the manufacture of sizing resins for the paper industry (Petruccioli et al., 1996). It can easily be produced from

Table 5. Citric acid applications

Rhizopus arrhizus on an industrial scale, but fermentative production has long been abandoned for economic reasons and replaced by the cheaper acid-catalysed isomerization of maleic acid obtained by oxidation of benzene, or phtalic anhydride (Ueno, 2003). Biotechnological processes could possibly be developed using cheap and readily available substrates (Kosseva et al., 1998; Idris et al., 2003; Yan et al., 2001; Klinkenberg et al., 2001). Fumaric acid was produced in repeated batch processes using Rhizopus arrhizus NRRL 1526 immobilised in polyurethane sponge particles and cultivated in a fluidised-bed reactor. Acid production was optimized (12.3 g/l, 35.65% and 0.256 g/1 h of fumaric acid accumulation yield and volumetric productivity of the whole process, respectively) under the following conditions: carrier-particle concentration, 20% (v/v) of the working volume; glucose molasses, 50 g/l; ammonium sulphate, alternation of cycles with 0.0 and 0.5 g/1. 10.4.3 Lactic acid Lactic acid (C3H603: 2-hydroxypropionic acid) has an important position in the family of the carboxylic acids because of is applications in food and non-food industries. It is produced by fermentation (50%)and by chemical synthesis (50%). Lactic acid is present in plants and micro-organisms. Lactic acid has two enantiomers, L-(+) and D-(-)-lactic acid. The L-(+) isomer is used by human metabolism and is preferred for food use because D-(-) isomer is slightly toxic. Nevertheless, synthetic racemic (DL) lactic acid is the primary commercial form. Lactic acid is widely used in the food industry as tasteenhancing additive. Ferrous salts and the various L(+)-lactic acid salts are used in the pharmaceutical industry for their therapeutic qualities.

10. 4.3.1 Uses Lactic acid is used in foods, pharmaceutical, cosmetic and textile industries (Datta et al., 1995). The free acid is used as an acidulant and preservative in several food products such as cheese, meat, beer and jellies. Ammonium lactate is used as a source of non-protein nitrogen in feeds; sodium and calcium

stearoyl lactylates are used as emulsifiers and dough conditioners. Only the L(+) form of the lactic acid is metabolised in animal and human cells, because these cells can synthesize only L(+)-lactate dehydrogenase enzyme for the metabolism. Therefore, consumption of large quantity of D(-)-lactic acid results in its accumulation in the blood as unmetabolised. As a result of D(-)lactic acid accumulation, hyper-acidity of urine and decalcification may occur. In pharmaceutical industry, lactic acid is used for the adjustment of pH of pharmaceutical preparations and topical wart preparations, it dissolves wart tissues. Other applications are as a blood coagulant and dietary calcium source. Ethyl lactate is used in the preparation of anti-inflammatory drugs. Lactic acid is a good solvent, provides acidity in foods and beverages. Ethyl and butyl lactates are used as flavour ingredients. Lactic acid has some important applications in food industry such as for the production of fermented foods, in the pickling-process of gherkins, dill, olives, sauerkrauts, carrot and some leafy vegetables, and in the processing of oriental-foods. Lactic acid finds its application for the production of dairy products such as yoghurt, buttermilk, acidophilus milk, cottage cheese, creamy-cheese and fermented cheese etc. Recently, there has been increased interest in the lactic acid because of its application in the synthesis of biodegradable, biocompatible plastics and coatings. L(+)-lactic acid can be polymerised to form polylactic acid (PLA). This polymer can be used in the manufacture of new biodegradable plastics. The plastics prepared in such way are increasingly used in surgery for self-dissolving suture thread. Biodegradable plastics could play an increasing role in the industry by replacing or minimizing the use of non-degradable ordinary plastics to solve environmental pollution problems. These biodegradable plastics synthesized from lactic acid could be considered as a substitute for plastics manufactured from the petroleum products. Lactic acid has been used in manufacture of cellophane, resins, some herbicides and pesticides. Another important application of lactic acid is in textile and tanning industries.

10.4.3.2 Production The production of lactic acid by bacterial fermentation results into a racemic mixture of L(+) and D(-) isomers, although some strain can produce optically pure D(-)-lactic acid under specific conditions (Benthin and Villadsen, 1995). The fungal fermentation leads to the exclusive production of the L(+)isomer (Martak et al., 2003). The large-scale production of lactic acid is mainly obstructed by the cost of raw material. Application of agro-industrial waste residues in bioprocesses provides an alternative way to replace the costly raw materials (John et al., 2007). Notably improved productivity has been reached

with the employment of immobilized cells (Ogbonna et al., 1996, Ganguly et al., 2006). Recently, Singh et al., (2006) have discussed the metabolic engineering approaches for lactic acid fermentation. The initial efforts in LAB genetic modifications were concentrated mostly to develop LAB with enhanced qualities for food grade applications, using traditional approaches. The spontaneous mutations were also attempted by using insertion sequence (IS) elements. The LAB subjected to genetic improvement had been used in dairy industry for flavour enhancement, resistance to bacteriophages, addition of nutritional components and stability and structure of end products. Also various attempts have been made to develop efficient vector systems for metabolic engineering of lactic acid bacteria and expression systems for industrial Gram positive bacteria with low G+C content, controlled gene expression systems with the food grade inducer nicin etc. have been reported. However, with the recognition of polylactide as a biodegradable polymer, attempts were directed to reduce the cost of lactic acid production by genetically modifying the organism and also by using various cheaply available agro-industrial residues and process modifications to remove the lactic acid produced during the course of fermentation. Lactic acid production in SSF has been carried out using fungal as well as bacterial cultures. Strains of Rhizopus sp. have been common among the fungal cultures and that of Lactobacillus sp. among the bacterial cultures. Different crops such as cassa:~a and sweet sorghum and various crop-residues such as sugarcane bagasse, sugar cane press-mud, and carrot-processing waste can be used as substrates in fermentation for lactic acid production. A strain of R oryzae was used to evaluate the lactic acid production in different fermentation systems, using sugarcane bagasse impregnated with a nutrient solution. Both, the production level and productivity of lactic acid were higher in SSF with a yield of 137.0 g/1 and the productivity of 1.43 g/1 per h. One bacterial strain, L paracasei produced lactic acid in SSE Lactate concentration and yield were 90 g/kg and 91-95 %, respectively. The time required to complete SSF for lactic acid production is usually 120-200 h. Sugar cane press-mud has been used as substrate in SSF employing three bacterial cultures. L casei produced a higher concentration of lactic acid in comparison to L helveticus and Streptococcus thermophilus (Soccol et al., 1994a,b). A comparison in liquid and solid-state fermentation showed 93.8 and 137.0 g/1 of L(+)-lactic acid, and the productivity was 1.38 and 1.43 g/1/h in liquid- and solid-fermentations, respectively. John et al., (2006) exploited two agro-industrial wastes, cassava bagasse and sugarcane bagasse as raw material and inert support for L-lactic acid by solidstate fermentation. Gelatinised cassava bagasse was enzymatically hydroysed

and starch hydrolysate containing reducing sugars was used to impregnate the sugarcane bagasse. A maximum production of 249 mg/gds L-lactic acid was attained after 5 days with a conversion efficiency of 99%.

10.4.3.4 Extraction and recovery The traditional process on extraction and recovery of lactic acid after fermentation involves precipitation of calcium lactate and regeneration of lactic acid by addition of sulphuric acid followed by further purification steps, which are ion exchange and decolourisation. The extract is treated with hydrated lime for four purposes; this lime-treatment kills lactic acid bacteria, coagulates proteins, degrades residual sugars and solubilizes calcium lactate precipitated due to the reaction of lactic acid with calcium hydroxide and calcium carbonate present in fermenter. The solution is then filtered and the filtrate containing soluble calcium lactate is treated with sulphuric acid to precipitate calcium sulphate liberating lactic acid into the solution. The lactic acid solution is filtered to remove calcium sulphate precipitates and then bleached. The bleaching is perfon~aed by treatment with activated carbon. The bleached lactic acid solution is then concentrated to 3540% acidity in evaporator. The process of bleaching and evaporation is repeated 2-3 times to obtain lactic acid of different grades, such as technical-grade and food-grade lactic acid. Alternative processes are the extraction by liquid membranes, electrodialysis and ion exchange.

10.4.3.5 Factors affecting lactic acid SSF As mentioned above, in solid-state fermentation, aeration of the moistened medium is important. It controls the humidity of the solid support and simultaneously the heat-release of the fermentation metabolism. Aeration also provides the oxygen required for the fungal colonisation in solid medium. Therefore, the optimisation of aeration rate is very important in SSF and every SSF-process is different due to the use of different substrate and a different micro-organism. In one example of lactic acid production as described above, aeration rates of 0 to 100 ml/min per column reactor were tried; the maximal L(+)- lactic acid yield was obtained with the aeration rate of 20 ml/min. A decrease of 35% was caused in lactic acid yield at the aeration rate of 100ml/min. A higher aeration may cause a significant reduction in the lactic acid yield, which is directly related to an increase in the aerobic respiration rate of the fungus. Similarly, at very low or with a poor aeration, a significant amount of glucose may not be consumed in the fermentation that may result in decreased biosynthesis of lactic acid (Soccol et al., 1994a). Another important factor influencing the SSF is the inoculation rate of fungal spores. Various sizes of spore-inoculum using 105 to 108 spores in a suspension

form have been used to produce inoculum of A oryzae. The optimal seedinoculum has been found to be 10 6 spores per g glucose used. This inoculum size produced 120 g/1 lactic acid with complete glucose uptake and 75% yield of lactic acid. Inoculation rate and the lactic acid production are parallel. A similar variation in lactic acid yield has been observed as any variation occurred in inoculation rate (Ueno 2003). Lactic acid synthesis has been found to be affected by the glucose concentration in the fermentation medium. SSF using 12 to 24% glucose was performed to optimize sugar concentration for an enhanced yield of lactic acid. The optimum glucose concentration as the initial intake in the medium was 18%, producing 137g/1 lactic acid with a 76% yield. A higher concentration of glucose as 24% led to a decrease in lactic acid biosynthesis. Because of a partial consumption of glucose in the fermentation medium, a glucose concentration of about 16% produced 117 g/1 of lactic acid with a fermentation yield of 74%. If the glucose concentration used was lower as 12%, lactic acid production was reduced to 75 g/l, with a lower fermentation yield of 63% (Soccol et al., 1994b). The type of fermenter used is another factor affecting the product yield. A comparative study of SSF in column bioreactor and culture in Erlenmeyer flasks showed 77 and 74% fermentation yields, respectively. High quantities of fumaric acid production (20 g/l) were also noted, which in liquid fermentation was in small quantity (2.1 g/l). The culture conditions in SSF caused the synthesis of fumaric acid (Petruccioli et at., 1996).

10.4.4. Gluconic acid Gluconic acid is used in the food, beverages and pharmaceutical industries. It imparts a refreshing sour taste in many foods items such as wine, fruit juices etc. Sodium gluconate has a high sequestering power. It is a good chelator at alkaline pH; its action is comparatively better than EDTA, NTA and other chelators. Aqueous solutions of sodium gluconate are resistant to oxidation and reduction at high temperatures. It is an efficient plasticizer and a highly efficient set retarder. It is easily biodegradable (98 % at 48 h). It has an interesting property of inhibiting bitterness in foodstuffs. Concentrated gluconic acid solution contains certain lactone structures (neutral cyclic ester) showing antiseptic property. In the European Parliament and Council Directix~e No. 95/2/EC, gluconic acid is listed as a generally permitted food additive (E 574). The US FDA (Food and Drug Administration) has assigned sodium gluconate a GRAS (generally recognized as safe) status and its use in foodstuff is permitted without limitation (Ramachandran et al., 2006).

10. 4.4.1 U s e s Gluconic acid is a mild organic acid, which finds applications in the food industry. As stated above, it is a natural constituent in fruit juices and honey and is used in the pickling of foods. Its inner ester, Glucono-8-1actone imparts an initially sweet taste, which becomes later slightly acid. It is used in meat and in dairy products, particularly in baked goods as a component of leavening agent for preleavened products. It is used as a flavouring agent (for example, in sherbets) and also finds application to reduce fat absorption in doughnuts and cones. Foodstuffs containing D-glucono-8- lactone include bean curd, yoghurt, cottage, cheese, bread, confectionery and meat (Ramachandran et al., 2006). Generally speaking, gluconic acid and its salts are used in the formulation of food, pharmaceutical and hygienic products (Table 6). They are also used as mineral supplements to prevent the deficiency of calcium, iron, etc, and as buffer salts. Different salts of gluconic acid find various applications based on their properties. Sodium salt of gluconic acid has the outstanding property to chelate calcium and other di- and trivalent metal ions. It is used in the bottle washing preparations, where it helps in the prevention of scale formation and its removal from glass. It is well suited for removing calcareous deposits from metals and other surfaces, including milk or beer scale on galvanised iron or Table 6. Applications of gluconic acid and its derivatives Ramachandran et al,, (2006)

stainless steel. Its property of sequestering iron over a wide range of pH is exploited in the textile industry, where it prevents the deposition of iron and for desizing polyester and polyamide fabrics. It is also used in metallurgy for alkaline derusting. It is used in the washing of painted walls and removal of metal carbonate precipitates without causing corrosion. It also finds application as an additive to cement, controlling the setting time and increasing the strength and water resistance of the cement. It helps in the manufacture of frost and crack resistant concretes. It is also used in the household cleaning compounds such as mouthwashes. Calcium gluconate is used in pharmaceutical industry as a source of calcium for treating calcium deficiency by oral or intravenous administration. It also finds a place in animal nutrition. Iron gluconate and iron phospho gluconate are used in iron therapy. Zinc gluconate is used as an ingredient to treat common cold, wound healing and various disease caused by zinc deficiencies such as delayed sexual maturation, mental lethargy, skin changes, and susceptibility to infections.

10.4.4.2 Production Gluconic acid is a mild organic acid derived from glucose by a simple oxidation reaction. The reaction is facilitated by the enzyme glucose oxidase (fungus such as Aspergillus niger) and glucose dehydrogenase (bacteria such as Gluconobacter). Microbial production of gluconic acid is the preferred method and it dates back to several decades. The most studied and widely used fermentation process involves the fungus Aspergillus niger. Current commercial production of sodium gluconate uses submerged fermentation with A. niger It involves fed-batch cultivation with intermittent glucose feedings and the use of sodium hydroxide as neutralising agent, pH is held at 6.0-6.5 and the temperature at about 34~ The productivity of this process is very high, since glucose is converted at a rate of 15 g/L.h. It can be produced by synthetic medium by surface and submerged fermentation. Agricultural and industrial residues such as molasses (Rao & Panda 1994), grape must (Buzzini et al., 1993), whey, jerusalem artichoke (Kim & Kim 1992) were used as source of glucose. As regards SSF, there are only a few reports on the production of gluconic acid. Roukas (2005) reported the production of gluconic acid by solid-state fermentation on figs. The maximal gluconic acid concentration of 490 g/kg of dry fig with 63 % yield was obtained at initial moisture of 75% and at pH 7.0 after 9 days of fermentation. The addition of 6 % methanol into the substrate helped to increase the production of gluconic acid from 490 to 685 g/kg. Singh et al., (2003) performed SSF by using HC1 pretreated sugarcane bagasse and the highest level of gluconic acid (107 g/L) with 95 % yield was obtained. In comparison with the submerged culture, the degree of conversion was higher in SSF. The increased rate of product formation was due to the variations

of osmotic pressure, water content and dissolved oxygen. SSF was carried out for the production of A. niger spores, which was further used as a biocatalyst in the conversion of glucose to gluconic acid. Average rate of reaction was 1.5 g.L-1 h-1 with 102 g L-1 of gluconic acid produced out of 100 g L-1 glucose consumed after approximately 100 h reaction, which corresponded to a molar yield close to 93 % (Ramachandran et al., 2007).

10.5 CONCLUSIONS Production of organic acids using agro-industrial residues in solid-state fermentation could be significant in reducing the cost of the production. Also, such processes would be eco-friendly. Evidently production in SSF in comparison to liquid fermentation generally leads higher production.

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Hang YD & Woodams EE, 1985, Grape pomace: A novel substrate for microbial production of citric acid, Biotechnology Letters, 7, 253-254. Hang YD & Woodams EE, 1998, Production of citric acid from corncobs by Aspergillus niger. Bioresource Technology, 65, 251-253.

Hossain M, Brooks JD & Maddox IS, 1984, The effect of the sugar source on citric acid production by Aspergillus niger. Applied Microbiology and Biotechnology, 19, 393-397. Ikeno Y, Masuda YM, Tanno K, Oomori I & Takahashi N, 1975, Citric acid production from various raw materials by yeasts, Journal of Fermentation Technology, 53, 752-756. Ikram-ul-Haq S, Khurshid S, Ali H, Ashraf MA, Qadeer MI & Rajoka, 2001, Mutation of Aspergillus niger for hyperproduction of citric acid from black strap molasses, World Journal of Microbiology and Biotechnology, 17, 35-37. Ingram LO, & Buttke TM, 1984, Effects of alcohols on microorganisms, Advances in Microbial Physiology, 25, 253. John RP, Nampoothiri KM & Pandey A, 2007, Fermentative production of lactic acid from biomass: An overview on process developments and future perspectives, Applied Microbiology and Biotechnology, in press. John RP, Nampoothiri KM & Pandey A, 2006, Solid-state fermentation for L(+)-lactic acid production from agro wastes using Lactobacillus delbrueckii, Process Biochemistry, 41,759-763. Kim DM & Kim HS, 1992, Continuous production of gluconic acid and sorbitol from Jerusalim artichoke and glucose using an oxidoreductase of gymomonas mibilis and inulinase, Biotechnology and Bioengineering, 39, 336-342. Kim JW, Barrignton S, Sheppard J & Lee B, 2006, Nutrient optimization for the production of citric acid by Aspergillus niger NRRL 567 grown on peat moss enriched with glucose, Process Biochemistry, 41, 1253-1260.

Klinkerberg G, Lystad KQ, Levine DW & Dyrset N, 2001, Cell release from alginate immobilized Lactococcus lactic ssp. lactis in chitosan and alginate coated beds, Journal of Dairy Science, 84, 1118-1127. Kolicheski MB, 1995, Production of Citric Acid By Solid-State Fermentation using cassava Bagasse as Substrate, MScThesis, Curitiba, Brazil, 137 P. Kosseva M, Beschkov V, Kennedy JF, & Lloyd LL, 1998, Malolactic fermentation in Chardonnay wine by immobilised Lactobacillus casei cells, Process Biochemistry, 33, 793-797. Kubicek CP & Rohr M, 1986, Citric acid fermentation, CriticalReviews in Biotechnology, 3,331-373. Kumar D, Jain VK, Shanker G & Srivastava A, 2003, Citric acid production by solidstate fermentation using sugarcane bagasse, Process Biochemistry.

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Prado FC, Vandenberghe LPS, Lisboa C, Paca J, Pandey A & Soccol CR, 2004, Relation between citric acid production and respiration rate of Aspergillus niger in solid-state fermentation, Engineering of Life Sciences, 4 (2), 1-8. Rao S & Panda T, 1994, Critical analysis of the metal ions on gluconic acid production by Aspergillus niger using a treated Indian cane molasses, Bioprocess Engineering. 10,99-107. Ramachandran S, Fontanille P, Pandey A & Larroche C, 2006, Gluconic acid: Properties, application and microbial production, Food Technology and Biotechnology, 44 (2), 185-196. Ramachandran S, Fontanille P, Pandey A & Larroche C, 2007, Spores of Aspergillus niger as reservoir of glucose oxidase synthesizes during solid-state fermentation and their use as catalyst in gluconic acid production, Letters in Applied Microbiology, 44, 155-160.

Riihr M, Kubicek CP & Kominek J, 1983, Citric Acid, In- Biotechnology, Vol. 3, G. Reed, H. J. Rehm (Eds), Verlag Chemie, Weiheim, Germany, pp. 419-454. Roukas T & Kotzekidou P, 1987, Influence of some trace metals and stimulants on citric acid production from brewery wastes by Aspergillus niger, Enyme and Microbial Technology, 9, 291-294. Roukas T, 2005, Citric acid and gluconic acid production from fig by Aspergillus niger using solid-state fermentation, Joiurnal of Industrial Microbiology and Biotechnology, 25,298-304. Roukas, T, 1998, Carob pod: A new substrate for citric acid production by Aspergillus niger, Applied Biochemistry and Biotechnology, 74, 43-53. Shojaosadati SA & Babaeipour V, 2002, Citric acid production from apple pomace in multi-layer packed bed solid-state bioreactor, Process Biochemistry, 37, 909-914. Singh OV, Jain RK & Singh RP, 2003, Gluconic acid production under varying fermentation conditions by Aspergillus niger, Journal of Chemical Technology and Biotechnology, 78, 208-212. Singh SK, Ahmed SU & Pandey A, 2006, Metabolic engineering approaches for lactic acid production, Process Biochemistry, 41 (5), 991-1000. Soccol CR, Matin B, Raimbault M & Lebeault JM, 1994a, Potential of solid-state fermentation for production of L (+) lactic acid by Rhizopus oryzae, Applied Microbiology and Biotechnology, 41, 286-290. Soccol CR, Stonoga VL & Raimbault M, 1994b, Production of L (+) latic acid by Rhizopus species. World Journal of Microbiology and Biotechnology, 10 (4), 433--435. Soccol CR, Vandenberghe PLS, Rodriguez C & Pandey A, 2006, New perspectives for citric acid production and application, Food Technology and Biotechnology, 44 (2), 141-150. Soccol CR, Prado FC, Vandenberghe, LPS & Pandey A, 2004, General aspects in citric acid production by submerged and solid-state fermentation, In- Concise Encyclopedia of Bioresource Technology, A, Pandey (ed), New York: Haworth Press, pp. 652-664. Soccol CR, & Vandenberghe, LPS, 2003, Overview of applied solid-state fermentation in Brazil, Biochemical Engineering Journal, 13, 205-218. Soccol CR, Vandenberghe LPS & Lebeault JM, 1999, Production of citric acid by solidstate fermentation using A. niger. Pt. Br. DEINPI/PR 00175. Tran CT, Sly LI & Mitchell DA, 1998, Selection of a strain of Aspergillus for the production of citric acid from pineapple waste in solid-state fermentation, World Journal of Microbiology and Biotechnology, 14, 399-404. Ueno T, 2003, Lactic acid production using two food processing wastes, canned pineapple syrup and grape invertase as substrate and enzyme, Biotechnology Letters, 25, 573-577. Vandenberghe LPS, 2000, Development of process for citric acid production by solidstate fermentation using cassava agro-industrial residues, PhD Thesis (Doctorate). Universit6 de Technologie de Compi~gne, Compi6gne, France, p. 205.

Vandenberghe LPS, Soccol CR, Pandey A & Lebeault J-M, 1999, Review: Microbial production of citric acid, Brazilian Archives of Biology and Technology, 42, 263-276 Yandenberghe LPS, Soccol CR, Pandey A & Lebeault, J-M, 2000, Solid-state fermentation for the synthesis of citric acid by Aspergillus niger, Bioresource Technology, 74, 175-178. Yandenberghe LPS, Soccol CR, Prado FC & Pandey A, 2004, Comparison of citric acid production by solid-state fermentation in flask, column, tray and drum bioreactor, Applied

Biochemistry Biotechnology, 118, 1-10. Xu DB, Madrid CP, Rohr M & Kubieek CP, 1989, The influence of type and concentration of the carbon source on production of citric acid by Aspergillus niger. Applied Microbiology and Biotechnology, 30, 553-558.

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11.1. INTRODUCTION Spores are generally defined as small reproductive bodies that get detached from the microbial species to produce a new offspring. In the process they do not fuse with other reproductive bodies, but get germinated sooner or later. They widely vary in origin, size, shape, pigmentation, biological functions, etc. Microbial spores have a diameter ranging between 1-50 ~tm. However, the largest spores are the fungal ascospores, for eg. Varicellariamicrosticta, measuring 340 • 115 pm; the smallest spores are bacterial endospores measuring about 0.25 ~tm in diameter. Bacterial endospores remain dormant in the same place of origin and help the species to pass through an unfavourable period; such spores are termed as resting spores. Also, there are dispersal spores, those of Ascomycetes, whose explosive asci bursts with a hydrostatic pressure getting scattered even upto distance of 40 cm (Wolken et al., 2003). Spores play an important role in the lifecycle of fungi and they produce both sexual and/or asexual spores. The main classes of fungi producing sexual spores are Zygomycetes (Rhizopus), Ascomycetes (Neurospora, Talaromyces), Basidiomycetes (Puccinia, Agaricus) and their spores are called zygospores, ascospores, basidiospores respectively. There is another class of fungi, Deuteromycetes (Penicillium, Aspergillus) which produce only asexual spores called conidia. There are many fungi which are able to produce more than one kind of spores, e.g., Neurospora crassa produces two kinds of spores such as microconidia and macroconidia. Coming to bacteria, all bacteria do not produce spore. A few species of bacteria such as Bacillus and Clostridium have the ability to produce highly resistant structures known as endospores which resist a range of hazardous environments. Solid-state fermentation (SSF) has been widely exploited for the production of many value-added microbial products and bulk chemicals (Pandey 1992, Pandey 1994). SSF offers various advantages when compared to SmF which is widely

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

discussed elsewhere in the book. This is partly because solid-state processes have lower energy requirements, produce lesser wastewater and are environmentalfriendly as they resolve the problem of solid wastes disposal (Pandey 2003). There has been much development in the application of SSF in various areas such as bioremediation and biodegradation of hazardous compounds, biological detoxification of toxic agro-industrial residues, biotransformation of crops and crop residues for nutritional enrichment, biopulping, and production of valueadded products such as biologically active secondary metabolites, etc. (Pandey et al., 2000). One among the various applications of SSF is the production of spores. With regard to this aspect, SSF is the role model technology and dominates SmF as it yields high quantity and quality of spores. Growth and proliferation of microorganisms cultured in SSF highly resemble their natural environments (i.e. growth on a solid support in the presence of limited free water) enabling in producing spores with better properties. SSF being a cheaper and well adapted process meets the requirements of spore production with higher productivity, higher end concentration, higher product stability, etc. SSF presents several advantages over SmF as it gives better yields of homogenous and pure spores (Larroche & Gros 1992). Due to these and many reasons, discussed under section 11.4 in this chapter, SSF is the feasible and promising technology for the production of spores. Figure 1 shows some of the microbial spores commonly produced by SSF. This chapter

Fig. 1. Fungal and bacterial spores. From left to right Trichoderma harzianum (www.ngi-net.de), Aspergillus niger (www.moldbacteria.com ), Beauveria bassiana (www.vertigo.uqam.ca), Verticillium lecanii (www.invasive.org), Metarhizium anisopliae (www.invasive.org), Penicillium roquefortii (www.biocentrum.dtu.dk), Bacillus thuringiensis (www.gre.ac.uk)

will focus on the research work carried out on the process parameters governing SSF in spore production, production of bacterial and fungal spores by SSF, properties and applications of spores produced by SSF, advantages of the spores produced by SSF over SmF, etc.

11.2. WHY TO PRODUCE SPORES? Spores are generally regarded as agents for disease, contamination and biological warfare. However, they possess several advantages when compared to their corresponding vegetative cells. Spores have interesting properties such as resistance to high temperature, toxic compounds, desiccation, radiation, etc. Some yeast spore survives heat treatment up to 350 times compared to their vegetative cells (Dijksterhuis Samson 2002). Spores of Metarhizium flavoviride, known as green muscle | the commercial biocontrol agent is stable for more than 42 months at 4~ Spores of various Bacillus species are generally 10 to 100fold more resistant to UV than the corresponding vegetative cells (Setlow 2001). Apart from these, spores have the ability to germinate in response to external triggers such as plant hormones, host surface wax, extreme heat or pressure. Due to these significant properties spores find applications in biocontrol, bionsensing and tumor control (Wolken et al., 2003). Applications of spores produced by SSF are discussed in detail under section 11.5. Some spores are also known to have the same biocatalytic activity as that of the vegetative cells (Leuenberger 1984). In fermentation processes or biotransformation process, use of spore instead of mycelium results in less viscous medium, further leading to easier aeration and product recovery (Moskowitz, 1979). Moreover, spores are generally easier to store, and the lack of pellet formation results in greater homogeneity of the biocatalyst (Larroche & Gros 1997). In some cases, biotransformation reactions are carried out exclusively by spores; in others, vegetative cells perform the biotransformation less efficiently and for a considerably longer period of time compared to spores. Another significant advantage of using spores is that they show higher resistance towards toxic effects of bioconversion substrates and products. Spores of Penicillium digitatum were found to be 2.5 times more resistant towards various toxic compounds (geraniol, citral, etc.) when compared to their mycelium (Wolken et al., 2002). These are some of the special properties of the spores which add advantage in using them in various bioprocesses when compared to the use of their vegetative cells.

11.3. PRODUCTION OF SPORES BY SSF Spores produced by SSF are mostly that of fungal species; however some bacterial spores are also produced. Spores of entomopathogenic fungi such as Beauveria bassiana (Santa et al., 2005), Cordyceps spp, (Hussey & Tinsley 1981), Verticillium lecanii (Feng et al., 2000), Metarhizium anisopliae (Dorta

& Arcas 1998), Colletotrichum truncatum (Silman et al., 1993), Trichoderma harzianum (Munoz et al., 1995), Penicillium oxalicum (Pascual et al., 2000), Coniothyrium minitans (Oostra et al., 2000), Clonostachys rosea (Viccini et al., 2006) etc., have been widely produced in SSE Spores of bacteria such Bacillus thuringiensis (Capalbo et al., 2001, Wang 1988), B. sphaericus (Foda et al., 2003) are also produced by SSF for the use as insecticide. Fungal spores for applications in the food and other industries have also been been predominantly produced by SSF such as Penicillium roquefortii (Larroche & Gros 1992, Larroche & Gros 1989), P. nalgoviensis (Holker 2000), Aspergillus niger (Moksia et al., 1996), Mucor bacilliformis (Lareo et al., 2006), etc. Table 1 shows various lists of microbial spores produced by SSE Most of the fungi are maintained and preserved in the spore form in solid culture. Spores of the microorganisms are generally used as inoculum in many fermentation processes. These spores are developed in agar plates or slants containing nutrient medium. However for large-scale spore production, SSF using cheaper substrate is carried out. Though spore production by SSF process is simple, many environmental factors such as moisture content, water activity, temperature, oxygen levels and concentration of nutrients are taken into consideration as they significantly affect the microbial growth and sporulation. Selection of substrate is one of the most critical steps in spore production process in solid substrate fermentation as it provides both nutrition and support. Nutritional requirement of the microorganism is a crucial parameter for its growth and sporulation. Sporulation is associated with the synthesis of macromolecules which are needed to produce the sporulation-specific structures and storage molecules needed for germination (Dahlberg and van Etten 1982). Spore formation occurs without overall protein synthesis, but with the formation of wall. Thus chitin content at the end of growth is considered as a good indicator of spore formation in fungi (Desfarges et al., 1987). Supplementation of starchy material and trace elements are considered to increase the spore production. However, it is believed that certain nutrient limitation must be maintained to achieve a good sporulation yield (Larroche et al., 1988). Research showed that free reducing sugars accumulation (Moo Young et al., 1983) or nitrogen supplementation such as NH4 and urea (Raimbault 1981) does not induce sporulation. Cellulosic substrates are the best suited for spore production as it provides better aeration, less compaction problems and greater growth surface area (Lonsane et al., 1985). Starchy substrates are also reported as ideal for spore production. For e.g., buckwheat seed renders excellent mechanical properties (retention of structure, lack of agglomeration) along with high external sporulation (Larroche & Gros 1989). Wheat, wheat bran, rice, rice husk, rice bran, buckwheat seeds, oats, refused potatoes, soy bean flour, cassava flour,

Table 1. Various spores of microorganism produced by SSF

sesame seeds, linseed meal, cotton seed meal, etc. are some of the substrates widely used for spore production. Certain substrates (Oats, Weber et al., 1999) shrink due to the evaporation of water, resulting inadequate heat removal from the bed, subsequently yielding low spore. Hence, apart from the nutritional aspect, physical properties of the substrate such as porosity, water uptake capacity, mechanical strength, etc, should be taken into consideration, such that it provides higher spore yield and facilitates easy extraction of spores. Many inert supports have been used for the spore production such as hemp, perlite, bagasse (Weber et al., 1999), polyurethane foam (Lareo et al., 2006), pozzolano particles, glass beads, wood shavings, Biogrog N (Larroche & Gros 1989), etc. Inert support is preferred for its physical nature as it possesses uniform structure throughout the process, providing easy recovery of spores. Porosity studies of the pozzolano particles (inert support) were carried out and it was found suitable for spore production of P. roquefortii, as 90.5% of the spore accumulation was located at the periphery of the particles, resulting in easy extraction. Roussos et al., (1991) reported that bagasse absorbed conidiospores and imparted protection during vacuum concentration. Hemp provided the best spore yields due to its high water uptake capacity among the other inert supports (bagasse, perlite) tested (Weber et al., 1999). The colonization of the microorganism in the susbtrate generally leads to both internal and external spore production. It is preferable that the spores are produced in the external surface of the substrate as it is easier in the extraction process. It is also possible to modify this internal/external ratio of spore content by varying substrate pre-treatment (Larroche & Gros 1989). Temperature is considered as an independent process depending on the microorganism used; however it is one of the key parameters which should be controlled in spore production process. In SSF process, heat rise up is commonly observed as the system is usually a packed bed and sporulation response gets affected as consequence of the thermal gradients. Oostra et al., (2000) observed that metabolic heat production rate in SSF for spore production of C. minitans carried out in Erlenmeyer flask was 500 watt/m 3 bed. Various type of cooling mechanisms are used such as conductive cooling (wall cooling), convective (evaporative cooling) to control the temperature build up. However, conductive cooling in packed bed is not sufficient if large reactor volumes greater than 0.2 m radius is used, as radial temperature profiles becomes too steep because of the poor thermal conductivity. This results in un-uniform growth and hinders the sporulation. Convective cooling is comparatively more effective in controlling the heat build up; however problem of evaporative water losses cannot be ruled out. This further affects the water activity and shrinkage of substrate, hampering spore production. Mixing of the medium also could improve heat removal via the reactor wall because it increases the heat transfer coefficient

from solids to the wall (De Reu et al.,1995). Mixed bioreactor was found to be superior to non-mixed bioreactor for the spore production of Coniothyrium minitans (Oostra et al., 2000). There was no detrimental effect of mixing on spore production, but resulted in high spore yields. However, this is not universally applicable, depends on the process and microorgansim used as mixing significantly inhibits the sporulation of the microorganism. The substrate bed must remain static in fungal spore production because agitation causes damage to the reproductive hyphae, greatly reducing spore yields (Silman, 1980). Thus various parameters should be considered for maintaining the controlled temperature through out the bed, according to the microorganism and technique for largescale spore production by SSF. Moisture content and water activity (aw) of the substrate play crucial roles in spore production. Moisture content does not remain constant through out the cultivation as aeration, starch hydrolysis, metabolic water production, etc occurs causing variation in the moisture content. Also, in static cultivation without mixing, there occurs water loss due to evaporative cooling. Thus it is necessary to maintain the optimum moisture content throughout the cultivation. Sporulation increases linearly with the initial moisture content. By increasing the water content, cultivation time becomes shorter and results in better spore yields. Sporulation of Mucor bacilliformis on polyurethane foam were studied at different initial moisture content of the substrate and maximum spore production (6x108 spores/g inert support) was obtained with the highest moisture content (90% w/w) (Lareo et al., 2006). However, it is also possible to increase the spore content by maintaining low moisture content by adding an organic nitrogen source (Larroche et al., 1988). Spores of Metarhizium anisopliae on rice bran and rice husk with an initial moisture content of 47% (w/w). Water activity is highly dependent upon the water binding properties of substrate and aw close to one is optimal for spore production. Water activity remains high over a broad range of moisture content, so significant amounts of water can be evaporated before the water activities declines to critical values. Reduction in aw results in reduction of oxygen consumption rate and directly affects the spore number produced Reduced water activity has tremendous effect on spore production, for e.g., Coniothyrium minitans was very sensitive to reduced water activity; spore formation was completely inhibited at aw 0.97 (Weber et al., 1999). 'Free water release' phenomenon occurs during static cultivation of fungus for spore production. Due to this, aeration and homogenization of the substrate becomes difficult. Hence it is necessary to remove this free water release to achieve high spore yield. Semi-continuous cultivation with sequential emptying and filling was carried out for the production of spores (Larroche & Gros 1986). This eliminated the metabolic liquor released and was found to be the best way to get maximum spore. However this was not tried in large scale.

.................................... !!! il.......... !!!ii .............!i.................................................................................. !! ................................

Medium packing density also govems the spore production. In densely packed bed, non-homogenous growth and sporulation occur if aeration and temperature are not maintained uniform through out. The effect of packing density was studied and was found that when apparent density was increased upto 0.496 g/ml, sporulation was drastically affected. On top of the solid bed, spore yields were 35 times higher than those estimated at the bottom of the column. Also, spore yields were observed maximum at the periphery regardless of bed heights (Dorta & Arcas 1998). Sporulation response gets affected as a consequence of thermal and humidity gradients.

11.3.1. Fungal spores produced by SSF Conditions and parameters for spore production often differ based on the microorganism used. Production parameters governing spore production of some commonly used fungi are discussed here. Penicillium roquefortti was cultivated on buckwheat seeds with initial moisture content of 1.5 gHz0/gIDM which resulted in spore yield of 1.5xl01~ with productivity of 107/h/gIDM in 100 ml Erlenmeyer flask (Larroche et al., 1992). Maximal average productivity of these spores were 4• 4.4x106, 5.08• 6xl06external spores/gIDM/h when cultivation was carried out in drum fermentor, column fermentor, non agitated 1-L bottle and semi-continuous 1-L bottle respectively. External spores are considered as useful spores as it is easy to extract as grinding the substrate is required to recover the spores formed inside the substrate (Larroche & Gros 1986). Conidia of Coniothyrium minitans was produced in mixed scraped drum reactor (0.2 rpm) resulting in spore yield of 5• spores/kg dry oats after 450 h (Oostra et al., 2000). Spore yield of 9x1014 conidia/m 3 packed bed of C. minitans was achieved in 18 days, using hemp impregnated with a solution containing 100 g/dm 3 glucose and 20 g/dm 3 potato extract (Weber et al., 1999). McQuilken & Whipps (1995) carried out cultivation of C. minitans in spawn bags (22.5x56 cm with microporus strip for aeration) containing oats at 18-20 ~ and reported spore yield of 7.5x1011 spores/kg air dried material after 28 days of cultivation. Response surface methodology was performed to optimize the medium components for spore production of C. minitans (Chen et al., 2005). They used wheat bran as substrate and carried out cultivation in 250 ml Erlenmeyer flask for 8 days, with the supplementation of nutrient solution and produce 1.04x101~ conidia/glDM. Beauveria bassiana spores were produced using refused potatoes, coffee husks and sugarcane bagasse as substrates (Santa et al., 2005). They reported that inoculum concentration of 107 spores/g dry weight, initial moisture of 75% and temperature of 26~ allowed best spore production, which resulted in the

spore yield of 1.07• l~ spores/g dry weight in column fermentor in 10 days. Response surface methodology was carried out to optimise conditions for the maximal production of spores of B. bassiana. Conidial concentration of 2x1012 was obtained using parboiled rice (70 g) in autoclavable bags (20x30 cm) at pH 5-6 and aw 0.999 (Tarocco et al., 2005). Alves & Pereira (1989) obtained a yield of 2x1011 spores of B. bassiana/g powdered preparation using rice as a basic growth substrate. Each 100 kg rice generated 3 kg conidial powder, i.e. 6• conidia/kg rice. Spores of Trichoderma harzianum were produced in disc fermenter, column fermenter and Zymotis (a large scale SSF fermenter designed at ORSTOM lab) by SSF using bagasse and cassava flour as substrates. Five times higher production of conidiospores (5x101~ spores/g cassava flour) was achieved in Zymotis (with a load of 21 kg moist medium), as compared to the agar medium in Erlenmeyer flasks. They also reported that inclusion of feather meal to the medium resulted in 5 times increase in the spore production and use of bagasse absorbs the conidiospores and imparts protection during vacuum concentration (Roussos et al., 1991). SSF of V. lecanii on wheat bran and beet pulp mixture resulted in spore yield of 3.2x109 spores/g dry matter (Grajek 1994). Similar spore yields (1.5x109 spores/g) were obtained when SSF was carried on 100 g cooked rice in autoclavable bag cultivated at 24~ for 2 weeks (Feng et al., 2002). Spores of Metarhizium anisopliae was produced using rice bran and rice husk (1"1 on dry weight basis) as substrate with a initial moisture content of 47% in glass column bioreactors at a flow rate of 0.34/1/g for two weeks at 27~ (Dorta & Arcas 1998). Spores of Aspergillus niger (2xl09spores/dry substrate) was produced in packed bed column fermentor of buckwheat seed supplied with continuous aeration (0.075/1/min) for 216 h at 30~ with 50% initial moisture (Ramachandran et al., 2007b). Production of Penicillium nalgoviensis conidia on bread took 18 days to reach a spore count of lxl09 /g solid substrate (Holker 2000). Spores of Mucor bacilliformis was produced on polyurethane foam containing nutrient solution (C/N = 5) at an intial moisture of 90% with spore yield of 6x108 spores/g inert support (Lareo et al., 2006).

11.3.2. Bacterial spores produced by SSF Spores of B. thuringiensis were produced in polypropylene bags containing sterile moist rice with aw of 0.92 and incubated at 30~ for 4 days. Adams et al., (2002) used several varieties of broiler litter with initial moisture content of 60% as substrates for the spore production of B. thuringinesis. The bacteria was found to grow in methanol extracted litter to produce 5x 1010 cell forming units (CFU)/g litter (dry weight) and a spore count of lxl01~ CFU/g dry litter

was produced. Spores of B. thuringiensis was produced on ground soybean seeds as a substrate in the presence of talcum powder and wheat bran as carrier materials and the highest growth and sporulation were obtained at 10% (w/w) of ground soybean (Foda et al., 2002). Wang et al., 1988 reported that stable high quality B. thuringiensis spores were obtained using wheat bran, rice husk and lime powder as substrates. The authors claimed that this process as power saving with low cost. Agro-industrial residues such as wastes from pulp and paper industry, residual malt from beer industry, meal from residual cookies and biscuits from bakery industry, meal from chicken slaughter house were used for the production of spores of B. thuringiensis and high sporulation titres were obtained (Capalbo & Moraes, 1988). Capalbo et al., 1994 devised two column bioreactors such as fluidized bed and aerated fixed beds for SSF of B. thuringiensis and claimed that these two reactors could be used to solve and address the problems such as heat and mass transfer, aeration extent, sterility level, productivity, etc, encountered in SSF methodology. Foda et al., 2003 produced spores Bacillus sphaericus using cotton seed meal, sesame seed meal, fodder yeast and linen seed meal in the presence of wheat bran as a carrier material. They found that a final spore count of 25.2•176 CFU/g substrate using cottonseed meal.

11.3.3. Bioreactors used for spore production Several types of equipment are used for spore production in SSF such as petri dishes, bottles, wide-mouth Erlenmeyer flasks, etc which are simple without forced aeration and agitation, with temperature regulation by placing them in incubators. They are used for screening of substrates or micro-organisms in the preliminary stage. Spore production in large bioreactors have been carried out mostly in non-mixed reactors such as packed-bed column, tray bioreactors and mixed reactors such as drum, stirred aerated bed reactors (Oostra et al., 2000, Larroche & Gros 1989). Figure 2 shows non-mixed and mixed reactors used for spore production. Packed-bed column reactors provide adequate temperature control (wall cooling) and aeration (forced aeration from bottom). However, scale-up of spore production in packed bed reactors is difficult. One of the severe problems encountered in large scale SSF spore production is the heat accumulation; high temperatures adversely affect the microbial growth and sporulation. Although forced aeration acts also as a convective cooling water loss occur due to evaporation. This makes the substrate dry and critically affects the water activity, which in turn affects the sporulation process. Conductive cooling (wall cooling) is not efficient if the diameter of the reactor is large. Therefore, mixing is done to improve the heat removal. However, intensive mixing impedes the fungal growth and

Fig. 2A. Non mixed reactor- Zymotis used for production of spores of Trichoderma harzianum (Courtesy: Mitchell et al., 2002).

Fig. 2B. Mixed reactor - Scraped drum used for the production of spores of Coniothyrium minitans (Courtesy: Oostra et al., 2000). Fig. 2 Bioreactors used for spore production

affects the spore formation as mycelium is damaged and spore content is drastically reduced. There are reports which use mixed system bioreactors which give high yield of spores. Scraped drum reactor was used for the production of C. minitans spores. A hollow scraper was situated over the length of the reactor near the wall for mixing at a rate of 0.2 rpm. Relatively high sporulation yield was observed, and the authors claimed that mixing improved the sporulation and spore production of C. minitans in large scale could be effectively scaled up using mixed bioreactor. A comparative study of mixed and non mixed packed bed reactors was carried out by Durand et al., 1994 for the production of C. minitans spores. In packed bed reactor (15 L) without mixing spore yield of 9x1014 conidia/m 3 was produced in 18 days. Reactor was well insulated; it mimicked a large-scale packed bed reactor concerning the absence of conductive cooling. The spore number was comparable with that of spores produced in mixed packed bed reactor of 50 L (6x1014 conidia/m 3) produced in 13 days (Durand et al., 1994). This is because C. minitans tolerated the shear and collision forces due to mixing. This is not true with all the microorganisms.., for e.g. mixing of Penicillium roquefortii had a negative effect on the sporulation (Larroche & Gros 1986). A large scale solid-state fermentor was designed called Zymotis, in which the substrate bed remains static. This system is aerated and has an internal heat transfer plates for cooling. Productivity of conidiospores of Trichoderma harzianum using this bioreactor was five times higher as compared to the spores produced in Erlenmeyer flask containing agar medium (Roussos et al., 1993). Besides these types of bioreactor, a reactor consisting of hollow metallic tubes coated with solidified medium containing extract of nutrient solution (wheat bran, corn flour) at both outer and inner surface was designed exclusively for spore production (Pandey et al., 1996). Temperature and aeration was controlled and it was incubated at 28~ for 12-60 h. Aspergillus niger spores thus produced were found to produce 18% more glucoamylase enzyme than the spores produced in agar slants. Table 2 gives a summary of various reactors used in spore production and spore yield.

11.4. ADVANTAGES OF SSF OVER SMF PROCESS IN SPORE PRODUCTION It is a well known fact that the properties of some products produced by SSF are found to be superior to those produced by SmF processes. For e.g. fungal glucosidase produced by SSF is more thermotolerant than the enzyme produced

Table 2. Various bioreactor used for spore production.

by SmF (Deshchamps & Huet 1984). Similarly, with respect to the production of spore, SSF dominates the fermentation processes, this attraction prevails as the SSF system truly resembles the natural way of microbial life (especially that of fungi). This factor is responsible for the differences in morphological, functional and biochemical properties of spores produced by SSF and SmF, enabling the spores produced by former fermentation process to survive longer at natural and drastic environmental conditions. In SmF, some fungi are unable to sporulate (Penicillium spp), most fungi imperfecti do not produce conidia, but produce thin-walled mycelial fragments called blastospores, which are short lived in adverse conditions (Ferron 1978, Mc Coy et a1.,1984); some fungi produce only low amounts of spores e.g.C, minitans (McQuilken et al., 1997).

Fig. 3. Comparison of the spores produced by SSF and SmF - Scanning electron micrographs of spores of V. lecanii grown on rice (SSF) and submerged spores. Top - left to right spore produced by SSF submerged spore, low magnification (1A 7000., bar 1/a 2 Im). Bottom - left to right spore produced by SSF, submerged spore where; high magnification (~/a35,000., bar ~/a0.5 Im). [Courtesy Feng et al., 2002].

On the contrast, SSF has been reported to yield not only high quantity of spores, but better qualities such as greater resistance to UV-radiation and dessication during recovery process. Further more, they are highly viable (Munoz et al., 1995) and was found to retain enzyme activity even after longer storage period as that of fresh spores e.g., glucose oxidase activity of A. niger (Ramachandran et al., 2007b). Morphology of the V. lecanii spores produced by SSF was compared with the spores produced by SmF (Feng et al., 2002). Figure 3 shows the SEM images of the spores produced by SSF and SmE Spores produced by SSF were cylindrical, ellipsoid and relatively uniform in size with rough brittle surface, while spores from SmF were of irregular in shape and size (oval to oblong) and had smooth surface. It is considered that the warty, brittle outer layer of aerial conidia renders strong protection and resistance to adverse environmental conditions (Gooday 1981). Spores produced by SSF are hydrophobic and are

I..

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......................................................................................................................................................................

resistant to stress and are highly durable when compared to submerged spores which are hydrophilic (Munoz et al., 1995). They reported that the increased concentration of a large (14 kDa) hydrophobin-like protein excreted to the surface of aerial spores is responsible for the hydrophobicity of spores produced using solid culture. Pascual et al., 2000 found that spores of Penicillium oxalicum obtained by aerial conditions had a higher surface hydrophobicity, better rate of survial after 27 weeks of storage, were less damaged by freeze-drying, had higher biocontrol activity when compared to submerged conditions and thus concluded that SSF is the most suited system for the mass production of spores. There are some studies which use non agitated SmF process for spore production (McQuilken et al., 1997), however this technique is unsuitable for scale-up as it requires large surface area. Thus, SSF stands as the appropriate spore production technique (Oostra et al., 2000). Spores of bacteria produced by SSF also are comparatively superior to those produced by SmF. Devi et al., 2005 claimed that Bt spores produced by SSF was not only effective against larvae of castor semilooper, but the material cost for production was cheaper too (one kg of Bt would be US$ 0.70). Apart from being simple and economical, spores production by SSF when compared to SmF does not require higher cost for down stream processing, as it can be used directly. Spores of B. thuringiensis tolworthii were used directly in maize fields to control fall armyworm (Capalbo et al., 2001).

11.5. APPLICATIONS OF SPORES PRODUCED BY SSF Spores possess some interesting properties which make them a candidate member for various applications in the bioprocess technology. Spores of fungi are used as inoculum for most of the fermentation process including small scale and large scale level. Apart from these, mainly spores are used as biocontrol agents (insecticide, fungicide, pesticide), biocatalysts in various bioconversion reactions. Spores of Bacillus thuringiensis produced by SSF is applied as control agent against plant pests such as caterpillars of Lepidoptera, mosquito larvae, locusts, grasshoppers, etc. (Devi et al., 2005, Capalbo et al., 2001). Spores of C. minitans produced by SSF are used against the control of fungal pathogen Sclerotinia sclerotiorum that reduces yields of many crops. Spores of Colletotrichum truncatum protects soybean crop against Sesbania exalta (Silman et al., 1993). Spores of V. lecanii control white fly invasion of various crops (Barranco-Florido et al., 2002). Spores of Beauveria bassiana is used to control sugarcane borer and European corn borer, invading maize crop (Arcas et al., 1999, Desgranges et al., 1993). A list of microbial spore produced by SSF applied as biocontrol agents is illustrated in Table 3A.

. . . . . . . . . . . . . . .i.i. .i..~.~!~..!i . . . . . . . . .!.i.i.~!i~l.i i.....i.i!.!ii!......i .........!i.i!................................. !....i!!~i..i'.!ii ...i. . .i . . .!. ............ .....

........................ i..i~i~i.ili.i..................... i i i !iii i!i~~i.~i~.....~~.

Table 3. Application of spores produced by SSF A. In Biological control

B. As biocatalysts

Spores are also known to possess high biocatalytic activity because they generally exhibit high catalytic activity in terms of dry matter. Thus they are used in various biotransformation reactions. Spores of Penicillium roquefortii carded out transformation octanoic acid ~ 2-heptanone and produced blue cheese flavour (Larroche & Gros 1989). Spores of Aspergillus niger produced by SSF showed stable glucose oxidase activity even after long storage time and carried out biotransformation reaction of glucose to gluconic (Ramachandran et al., 2007b). Table 3B shows some of the reactions catalysed by spores of fungi produced by SSE Spores are also applied in various other fields; however spores produced by SSF were not tried for these applications. Spores carry out steroid and antibiotic transformation. The first reaction discovered to be catalysed by the spore is the hydroxylation. Spores of Aspergillus ochraceus produced l lt~-hydroxyprogesterone from progesterone. Spores of A. niger convert several sugars to citric acid, spores of Mucor rouxii reduce [3-ketoesters, spores of Aspergillus sydowi convert soluble starch to glucose. There are also various other potential applications of the spores. Spores of Bacillus are used as bioindicators in the validation of thermal sterilization processes (Spicher et al., 2002), also as biosensors (Rotman 2001). Spores also play an important role in the field of tumour detection and treatment (Mose 1970, Minton et al., 1995), etc. Spore suspensions are also administered as probiotics (Casula & Cutting 2002). In agricultural industry, spores are receiving increasing attention as potential alternatives to antibiotics for use as growth promoters (Wolken et al., 2003).

11.6. CONCLUSIONS SmF is currently carried out for the industrial production of various chemicals, although SSF renders various advantages such as high productivity, lower energy, etc. However, spore production is the only process where SSF dominates over SmF in all aspects and the latter cannot replace or compete with the SSF process. This is due to the yield of spores with preferred morphology, stability and various other significant properties. Not only spores of filamentous fungi, but spores of bacteria have also been successfully produced by SSE Thus, SSF meets the worldwide demand for spore production for its application in various fields. However, clear focus on designing the bioreactor for spore production is still to be optimised and adjusted to clarity. Although many bioreactors and systems have been adopted, which successfully meet the requirements of some fungi, scaling up of spore production in large-scale is yet to be met completely by ruling out heat build up.

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Lareo C, Sposito AF & Bossio AL, 2006 Volpe DC, Characterization of growth and sporulation of Mucor bacilliformis in solid state fermentation on an inert support, Enzyme and Microbial Technology, 38, 391-399. Larroche C & Gros JB, 1989, Strategies for spore production by Penicillium roquefortii using solid state fermentation techniques, Process Biochemistry, 24 (2), 97-103. Larroche C & Gros JB, 1992, Characterization of the growth and sporulation behaviour of Penicillium roquefortii in solid substrate fermentation by material and bioenergetic balances, Biotechnology and Bioengineering, 39, 815-827. Larroche C & Gros JB, 1997, Special transformation processes using fungal spores and immobilized cells, Advances in Biochemical Engineering and Biotechnology, 55, 179-220.

Larroche C, Desfarges C & Gros JB, 1986, Spore production of Penicillium roquefortii in simulated solid state fermentation, Biotechnology Letters, 8 (6), 453-456 Larroche C, Desfarges C & Gros JB, 1988, Optimization of the spore production of Penicillium roquefortii in solid substrate fermentation on buckwheat seeds, Applied Microbiology Biotechnology, 28, 85-92 Leuenberger HGW, 1984, Methodology. In; Biotechnology, Vol. 6a, Kieslick (eds), VErlag Chemie, Weinheim, Deerfield Beach, Basel, pp 5-9.

Lonsane BK, Ghildayal NP, Budiatman S & Ramakrishna SV, 1985, Engineering aspects of solid state fermentation, Enzyme and Microbial Technology, 7, 258-265 McCoy CW, Stamper DH & Tuveson RW, 1984, Conidiogenous cell difference among mutant and wild type pathotypes of Hirsutella thompsonii var. thompsonii, Journal of Invertebrate Pathology, 43, 414-421. McQuiken MP & Whipps JM, 1995, Production, survival and evaluation of solidsubstrate inocula of Coniothyrium minitans against Sclerotinia sclerotiorum, European Journal of Plant Pathology, 101, 101-110 McQuilken MP, Budge SP & Whipps JM, 1997, Production, survival and evaluation of liquid culture produced inocula of Coniothyrium minitans against Sclerotinina sclerotiorum, Biocontrol Science and Technology, 7, 23-26. Minton NP, Mauchline ML, Lemmon MJ, Brehm JK, Fox M, Michael NP, Giaccia A & Brown JM, 1995, Chemotherapeutic tumor targeting using clostridial spores, FEMS Microbial Reviews, 17, 357-364. Mitchell DA, von Meien OF, Luz Jr LFL & Krieger N, 2002, Evaluation of productivity of zymotis solid-state fermentation bioreactor based on total reactor volume, Food Technology & Biotechnology, 40 (2), 135-144.

Moksia, J, Larroche, C & Gros JB, 1996, Gluconate production by spores of Aspergillus niger. Biotechnology Letters 18, 1025-1030. Moo-Young M, Moreira AR & Tengerdy RP, 1983, Principles of solid-substrate fermentation, In; JE Smith, DR Berry, B Kristiansen (ed), The filamentous fungi, vol 4 (Fungal Technology), Edward Arnold, London, pp 117-144. Mose JR, 1970, The use of spores of bacteria for a serological tumor diagnosis (I), Z Krebsforsch, 72, 329-341. Moskowitz GJ, 1979, Inocula for blue-veined chesses and blue cheese flavour, pp 201210, In: Peppier AJ, PErlman D (eds), Microbial Technology, Vol. 2, 2nd edition, Academic, New York & London. Munoz GA, Agosin E, Cotoras M, San Martin R & Volpe D, 1995, Comparison of aerial and submerged spore properties for Trichoderma harzianum, FEMS Microbiology Letters, 125, 63-70. Oostra J, Tramper J & Rinzema A, 2000, Model-based bioreactor selection for largescale solidstate cultivation of Coniothyrium minitans spores on oats, Enzyme and Microbial Technology, 27, 652-663. Pandey A, 1992. Recent developments in solid-state fermentation, Process Biochemistry, 27, 109-117. Pandey A, 1994. Solid-state fermentation: An overview. In: Pandey, A. (Ed.), Solid-State Fermentation, Wiley Eastern, New Delhi, pp. 3-10. Pandey A, 2003, Solid-state fermentation, Biochemical Engineering Journal, 13 (2-3), 81-84. Pandey A, Selvakumar P, Ashakumary L & Damodaran AD, 1996, A novel process for the production of fungal spores in solid-state fermentation.. Indian patent IPA no 379/ DEL/96, Dated 23.2.96. Pandey A, Soccol CR & Mitchell D, 2000, New developments in solid-state fermentation: I-bioprocesses and products, Process Biochemistry, 35 (10), 1153-1169. Pascual S, De Cal A, Magan N & Melgarejo P, 2000, Surface hydrophobicity, viability and efficacy in biological control of Penicillium oxalicum spores produced in aerial and submerged culture, Journal of Applied Microbiology, 89, 847-853. Raimbault M, 1981, Solid-state fermentation: growth of filamentous fungi in starchy substrate. ORSTOM 127. Ramachandran S, Fontanille P, Pandey A & Larroche C, 2007a, Spores of Aspergillus niger as reservoir of glucose oxidase synthesized during solid-state fermentation and their use as catalyst in gluconic acid production, Letters in Applied Microbiology, 44, 155-160.

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Rotamn B, 2001, Using living spores for real-time biosensing, Genetic Engineering News, 21, 21. Roussos S, Olmas A, Raimbault M, Saucedo-Castaneda G & Lonsane B K, 1991, Startegies for large scale inoculum development for solid state fermentation system: Conidiospores of Trichoderma harzianum, Biotechnology Techniques, 5 (6), 415-420. Roussos S, Raimbault M, Prebois JP & Lonsane BK, 1993, Zymotis, a large scale solid state fermenter: design and evaluation, Applied Biochemistry and Biotechnology, 42, 37-52. Santa HSD, Santa ORD, Brand D, Vandenberghe LPS & Soccol CR, 2005, Spore production of Beauveria bassiana from agro-industrial residues, Brazilian Archives of Biology and Technology, 48, 51-60. Setlow P, 2001, Resistance of bacterial spores to ultraviolet light, Environmental Molecular Mutagenesis, 38, 97-104.

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W e b e r F J, Tramper J & Rinzema A, 1999, A simplified material and energy balance approach for process development and scale-up of Coniothyrium minitans conidia production by solid-state fermentation cultivation in a packed bed reactor, Biotechnology and Bioengineering, 65 (4), 447-458.

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12.1. INTRODUCTION Mushroom cultivation presents an economically important biotechnological industry that has markedly developed all over the world. It is estimated that more than 10 million metric ton of edible and medicinal mushrooms were produced in 2004 in various countries (Royse 2005), expanded by magnitudes over just the past decade, following strain and technical improvements, coupled with better acceptance of mushrooms and derivative products (Chang 1999, 2005). Mushroom production can convert the huge lignocellulosic waste materials into a wide diversity of products (edible or medicinal food, feed and fertilizers), protecting and regenerating the environment. In addition, mushroom production can generate equitable economic growth that has already had an impact at national and regional levels. This impact is expected to continue increasing and expanding in the future, because more than 70 % of agricultural and forest materials are nonproductive and have been wasted in the agro-industrial processing or even consuming period. The mushroom conversion has been named the "non-green revolution" (Chang 1999). However, the mushroom science is a relatively new applied science and the mushroom industry is still small compared to many plant crops, with the limited investment in the mushroom researches. As a consequence, scientific research on mushroom generally lags behind that of plant and animal (Sonnenberg et al., 2005). However, there are some achievements needed to be pointed out in the mushroom production by solid state fermentation. This article deals with the advances of mushroom production by solid state fermentation in the last decade.

12.2. SPECIES EXPLORED IN MUSHROOM PRODUCTION BY SSF There are about 10,000 known mushroom species, fleshy macrofungi, including some ascomycetes species. Half of them in principle are edible and many are ecotomycorrhizal, i.e. associated with tree roots (Chang 1993). Precious fruiting bodies of mycorrhizal fungi such as Cantharellus species (Chanterelles) and

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Boletus edulis (king boletus), favourites in Western counties, and Tricholoma matsutake (Matsutake), a favourite in Japan, still have to be collected from the wild with variable and unpredictable success form year to year (Redhead et al., 1997; Yamanaka 1997). Transplanting studies on the mycelium of C. lutescens in Switzerland showed that it is possible to inoculate forest grounds with a desired fungus and obtain fruiting bodies in the following year (Ayer and Egli 1997). A recent report of a single C. cibarius fruiting body obtained in culture (Danell and Camacho 1997) and the occasional success in producing a fruiting body of T. matsutake under artificial conditions (Iwase 1997) raise hopes that mushroom cultivation of ectomycorrhizal species might also be improved in the future to a commercial scale. It is a solid-state culture of mushroom inoculated in the nature by human kind.

Saprophytic species are cultivated for mushroom production on lignin and cellulosecontaining substrates such as wood logs, saw dust, straw and cotton waste, besides many other agricultural and industrial wastes, which can easily be transformed into mushroom fruit body and other products. The proficiency of fungi in converting substrate to protein is far superior to that of several plants and even animals (Yang 1986). Mushroom fruit body is low in calories, fat and cholesterol, while rich in protein, carbohydrate, fibers, vitamins and minerals. These nutritional properties make mushrooms a very good dietary food. In addition, consumption of mushrooms has positive effects on the general human health because of a number of species substances, referred to as nutraceuticals (Chang and Buswell 1996). The commercial potential of an edible species for any grower depends on a number of factors including edibility and shelf life, local and regional culinary customs, and the existence of a distribution network, among others. Some edible species are cultivated and consumed internationally while other edibles have not yet achieved worldwide culinary appeal and are, therefore, cultivated only in countries where a local market has developed. There are a few species that are cultivated strictly for their medicinal properties while a few are cultivated because they are good edibles and also have medicinal properties. The following species are currently cultivated on a commercial scale for specific marketing areas. Table 1 shows the mushroom species produced at a large scale in the region (Stamets 2000). However, in China, there are more than 50 species cultivated by solid-state fermentation (Table 2) (Huang 2000).

12.3. SUBSTRATES EXPLORED FOR MUSHROOM PRODUCTION BY SSF

Mushrooms are the reproductive bodies formed by mycelium, a strand like mass of white cells found in the growing substrate, which may be a dead tree

Table 1. Production of mushroom species at a large scale in the world or some countries

stump, a live tree, or organic material in the soil (Fan et al., 1990). Mushrooms are classified into three types according to their growing characteristics. Parasitic mushrooms attack a living host plant, usually a tree, and eventually kill it. They may also be found growing on dead trees, but they probably started growing while the tree was alive and contributed to its demise (Yang 1986). An example of a parasitic mushroom is the Honey mushroom (Armillaria mellea). This type of mushroom can be cultivated but will require a living host. Some parasitic mushrooms also function as saprophytes. Mycorrhizal mushrooms form a symbiotic (mutually beneficial) relationship with the roots of trees or bushes. In fact, the root tips of all plants are coated with a fungus, which breaks down the organic matter in the soil and makes it available to the plant. The plant in turn produces sugars and exchanges these with the fungus. Some of these symbiotic fungi produce mushroom (Chang 1993). Examples include the Boletes, Truffles, Chanterelles and Amanitas. Cultivation of this type of mushroom requires the other half of the symbiotic relationship, which is the live tree.

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However, the majority of cultivated mushroom is saprophytic mushrooms, which live on dead organic matter such as dead trees, stumps, old roots, grass, straw, compost, etc. Mushrooms in this group are those that are successfully cultivated, including Shiitake, Oyster, Champignons or White Button mushrooms (Agaricus spp.), Portobello, Enokitake, Reishi, Maitake, Paddy Straw mushroom, and many others. The principal substrates used in the production are rice straw, rice bran, wheat straw, wheat bran, sawdust, corncobs, corn stems, cotton

seed hulls, cotton straw, cotton waste from textile industry, coffee pulp, horse and chicken manure, bean straw, cocoa shell waste, banana leaves, oil palm pericarp waste, sugar-cane bagasse, cassava bagasse (Chang 1993, 1999). Table 3 shows the principal media and species used for mushroom production by solid-state cultivation (Fan et al., 1990). The other substrate explored in the recent years was the coffee husk, leaves and spent-ground, which are more important in the coffee producing and consuming countries. Just in Brazil, they are generated in more than two millions tons quantity yearly (Soccol, 1995; Fan et al., 1999a, b; Pandey and Soccol, 2000). Coffee husk is different from coffee pulp, in which coffee cherries are generally processed by the dry method in Brazil (ICO, 1998). Coffee husk is rich in organic nature and nutrients but also contains compounds such as caffeine, tannins, and polyphenols, which result in toxic nature of the husk. Coffee leaves are mostly collected during harvesting in large volume. Their disposal also difficulties in crop management as they could facilitates epidemic of pathogens and pests. Coffee spent-ground is obtained during the processing of raw coffee powder to prepare 'instant coffee'. It also shows toxic nature as contains caffeine and tannins, although in less quantities when compared with husk. Presence of caffeine, tannins and polyphenols has affected utilization of these residues beneficially and their disposal in the environment poses serious pollution concerns (Fan et al., 1999a, b, c; Pandey and Soccol, 2000). The oyster mushroom was produced by solid-state cultivation using coffee husk through selection of strains in the coffee extract medium. The some strains P. ostreatus showed better growth, in general, than P. sajor-caju (Fan et al., 2000a). Results demonstrated the biological efficiency of P. ostreatus LPB 09 on the coffee husk, spent-ground and the mixture of spent ground plus leaves (60:40) as 96.5, 90.4 and 76.7%, respectively in 60 days. When coffee leaf was used as the substrate, it took five days for full occupation of mycelia under the experimented conditions, but no fruit body was obtained. As was the case with Pleurotus, all the strains of Lentinus sp. screened grew well on coffee husk extract medium. The strain L. edodes LPB 02 showed best growth in general with high density mycelial growth (9.68 mm.day -1) and biomass (43.4 mg.plate -1) in 12 days. When coffee husk was used as the substrate, it took 20 days for full occupation of mycelia but with the time of growing the mycelia regressed and secreted water so no fruit body was obtained (Fan et al., 2000b). Beaux and Soccol (1996) utilised the coffee husk for growing of Lentinus and reported poor mycelial growth in comparison to other substrates. When coffee husk was subjected to a hot water treatment by boiling one-hour in the water, filtering and using the solid residue for cultivating Lentinus, the growth was very vigorous. After full occupation of mycelia, the transformation

Table 3 Mushroom cultivation media used in the production

of colour began. First fructification occurred after 60 days of inoculation and the biological efficiency reached at the 85.8%. When coffee spent ground was used as the substrate, it also took 20 days for full occupation of mycelia; first fructification occurred after 56 days of inoculation; the biological efficiency reached at the 88.7%. With the mixed substrate, it took 25 days for full occupation of mycelia; first fructification occurred after 65 days of inoculation and the biological efficiency reached at the 78.4% (Fan et al., 2000b).

F. velutipes grew well on coffee husk extract medium. The mycelial growth velocity was 7.87 mm.day -1 and biomass 45.78 mg.plate -1 in 10 days. When coffee husk was used as the substrate, it took 15 days for full occupation of mycelia and the primodia occurred after 25 days of inoculation. The biological efficiency reached at 55.8% (Fan et al., 2000c). With spent-ground as substrate,

it took 12 days for full occupation of mycelia; first fructification occurred after 21 days and the biological efficiency reached 78.3% at two flush in 45 days. Sanchez et al., (2002) used vineyard pruning and grape pomace for Pleurotus production using a solid-state fermentation (SSF). Biological efficiency and bioconversion ranged from 37.2 to 78.7% and from 16.7 to 38.8%, respectively. The best substrates for mycelial growth and mushroom yield were the mixtures with higher vineyard pruning content. Mahmoud (2006) used Pleurotus species to ferment water hyacinth (Eichhornia crassipes) plants without roots. The mushroom delignified the lignocellulose of water hyacinth 26.2 - 30.4% of the plant. Whey permeate was utilized as a growth substrate for the cultivation of mycelia from the edible mushroom L. edodes (Inglet et al., 2006).

12.4. PHYSIOLOGICAL CONTROL FOR MUSHROOM PRODUCTION BY SSF Fruiting body formation is very much influenced by the physiological condition and nutritional state of the mycelium. Substrates and nutritional supplements satisfy the needs of the fungi for growth and fruiting and, most importantly help to withstand microbial competitors (Scrase and Elliott 1998), although not necessarily optimal, since they are low in readily accessible nutrients. Supplementing meal increases fruiting body yields of Agaricus by 25-50% (De Groot et al., 1998). In the laboratory, the dung fungus C. cinereus and the wood-rotter S. commune fruit on rich, artificial media based on glucose and malt extract (Walser et al., 2000). For fruiting body induction, it is important to keep a balance between C and N sources - hyphal knots in C. cinereus will easily form into primordia when the C/N ratio becomes too high (Moore 1998a). The optimal ratio for A. bisporus has been determined to lie between 80:1 and 10:1 (Scrase and Elliott 1998). C/N metabolism and the proteins involved have mainly been studied in A. bisporus and C. cinereus (De Groot et al., 1998; Moore 1998a; Stoop and Mooibroek 1999). Since the C sources utilized by mushrooms are usually of a lignocellulosic character, the fungi during vegetative growth produce a wide range of enzymes to degrade the lignocellulosic substrates: peroxidases and laccases for lignin degradation and various types of glucanases, cellulases and xylanases for cellulose and hemicellulose degradation (De Groot et al., 1998; Stoop and Mooibroek 1999). Considerable changes in enzyme activities occur during fruiting, indicating a connection to the regulation of fruiting body development. For example in A. bisporus and L. edodes, laccase activities are highest just before fruiting initiation and decline rapidly with aggregate formation. Cellulose activities are highest when fruting bodies develop (De Groot et al., 1998; Ohga et al., 1999). With

the periodic fruiting of Agaricus in commercial cultures, enzyme activities therefore fluctuate in approximately weekly cycles (De Groot et al., 1998; Ohga et al., 1999). Substrates offered for mushroom production normally contain organic N sources and are low in free ammonium, since excess can inhibit growth or fruiting of mushroom. Study in A. bisporus gives evidence for the presence of several high-affinity transport systems for amino acids and NH4§ The intracellular pools of NH4§ largely regulate uptake (Kersten et al., 1999). When A. bisporus, C. cinereus and Volvariella volvacea are grown on protein as C source, excess N gained from protein degradation was found to be excreted as ammonium into the growth medium (De Groot et al., 1998; Moore 1998a). At least in C. cinereus, ammonium release is thought to play a positive role for fruiting body initiation as it inhibits the competing process of primordium formation from hyphal knots (Moore 1998a). Onset of fruiting body development correlates with nutritional exhaustion of the growth substrates. Fruiting body development for commercial mushroom production is thus often induced by coveting compost colonized by vegetative mycelium with a layer of moist peat and chalk, which only limited nutrients (Scrase and Elliott 1998). Typically, mycelia of mushroom are not uniformly competent to differentiate; and only young hyphae can be induced to initiate fruiting body development. Mechanical injury of established mycelium locally stimulates fruiting body development, because wounding causes outgrowth of fresh hyphae (Granado et al., 1997; Scrase and Elliott 1998). The molecular principles triggering differentiation are not known. Various substances with fruiting including activity in specific or several mushrooms have been described: cerebrosides (Kues 2000), sucrose esters of fatty acids and other surfactants (Magae and Itoh 1998; Magae 1999), cAMP and AMP (Wessels 1993; Kues 2000), anthranilic acid and indole (Wessels 1993; Samadder et al., 1997) and other substances of yet unknown nature present in fungal extracts (Butler and Pearce 1999). The variety of these activating substances probably reflects a very complex physiological control of fruiting body initiation. So far, signaling by cAMP has found most attention. In C. cinereus, cAMP levels were shown to rise with light-induced initial formation due to increased adenylate cyclase activity; and a high cAMP protein kinase activity is associated with the fruiting response. The levels of cAMP decrease again with proceeding primordial development (Moore 1998a; Kues 2000). Transcript analysis of the cloned adenylate cyclase gene cac suggests that regulation of protein activity for cAMP production occurs at the post-translational level (Bottoli et al., 1999). At the competent stage upon environmental induction, numerous hyphal knots, initials and primordial appear on a colony but only a few of these come to

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maturation. Relocation of nutrients is thought to occur throughout the mycelium to favour one specific or a few selected primordia. In C. cinereus, during the transition from vegetative growth to fruiting body development, a breakdown of storage polysaccharides (glycogen) in the substrate mycelium is observed. Instead, glycogen accumulates first in the base of the stem in the young primordium, from which it subsequently disappears and is translocated into the gill tissues during further development. Accumulation of glycogen in the gill tissues is highest upon karyogamy in the early stages of fruiting body maturation, but deposits are absent from the mature fruiting bodies. Glycogen translocation is accompanied by increased glycogen synthase and glycogen phosphorylase activities. It obviously provides the metabolic energy for mushroom development. Per fruiting body, this metabolic energy is estimated at 25 J (Moore 1998a). Glycogen concentrations also fluctuate in the vegetable mycelium of A. bisporus with the periods of fruiting. Moreover, synthesis of thehalose increases before hyphal aggregates arise. Upon translocation into emerging aggregates, thehalose is degraded by the developmentally regulated thehalose phosphorylase to glucose and glucose1-phosphate for energy and the C supply. Unlike C. cinereus, mannitol is accumulated in the developing fruiting bodies of A. bisporus and probably acts as an osmoticum during growth of the mushroom. It is synthesized form fructose by mannitol dehydrogenase, using NADPH as cofactor. This enzyme is also developmentally regulated, together with a specific glucose 6-phosphate dehydrogenase responsible for increased NADPH production (De Groot et al., 1998; Wannet et al., 1999).

12.5. ENVIRONMENTAL CONTROL FOR MUSHROOM PRODUCTION BY SSF Environmental conditions play a crucial role in the decision whether a fruiting body will be formed. The optimal environmental situation for mycelial growth and the subsequent fruiting is usually very distinct- fruiting body development is often induced after drastically altering the environmental circumstances. There is no universal set of the conditions that leads to fructification in all mushrooms. The conditions for growing and producing fruiting bodies of a given species in culture have to be empirically established; but the natural environment of the fungus may give valuable indications as how to proceed in this determination. Once the situation has been optimized for a certain fungus, this might help when establishing the cultural conditions for related species (Scrase and Elliott 1998) Production of the vegetative mycelium usually occurs over a wide range of temperatures. Likewise, the formation of hyphal knots and the steps leading to primordial formation might not be restricted to a specific temperature, although such development is usually favored at certain temperatures. Fruiting is typically

induced, after vegetative growth, by reducing the temperature by at least 5 ~ e.g. in A. bisporus to 16-18~ in C. cinereus to 25 - 28~ and in Flammulina velutipes to 18~ (Flegg and Wood 1985; Williams et al., 1985; Scrase and Elliott 1998; Wues 2000). Other parameters of fruiting body initiation and maturation include CO2 concentration, humidity, salinity and pH. Higher CO2 concentrations may stimulate mycelial growth and inhibit fruiting completely. At lower CO2 concentrations fruiting body might be malformed, with elongated stipes and reduced pilei (Flegg and Wood 1985; Kinugawa 1993; Wessels 1993; Scrase and Elliott 1998). High humidity (90-95%) is favourable for pining and fruiting (Flegg and Wood 1985; Kinugawa 1993; Wues 2000), but the moisture content of the substrate might be even more critical. The optimal water content for wooden substrate is 35-60% and for other substrate 60-80%. The values reflect the oxygen demand of the fungi in the substratum, balanced against their requirement for water (Flegg and Wood 1985; Scrase and Elliott 1998; Ohga 1999a). Mushrooms tolerate relatively high levels of salts for growth, but fruiting body development can be more sensitive. Likewise, mycelial growth is less affected by pH, but fruiting body development of several species occurs best at neutral or slightly acidic pH values around 6-7 (Flegg and Wood 1985; Kinugawa 1993) or, in Lentinula edodes at a pH of 4.0 (Ohga 1999b). Light has been implicated in the fruiting of several Coprinus species (Ellis et al., 1999; Kues 2000), Favolus arcularius (Kitamoto et al., 1972), Pleurotus ostreatus (Richartz and MacLellan 1987), F. velutipes (Kinugawa 1993), S. commune (Yli-Mattila 1991) and some others. However, light is not needed for the fruiting of A. bisporus (Wessels 1993). Usually, light positively influences hyphal aggregation and fruiting body maturation. In contrast, light can repress hyphal knot formation. When illumination is too strong or too long, further steps in fruiting body development is also blocked. A short light pulse of low energy is typically enough to induced hyphal aggregation. Fruiting body maturation needs longer exposures of higher energy, but strains of the same species can differ in their light requirements (Richartz and MacLellan 1987; Wessels 1993; Ellis et al., 1999; Kues 2000). Whenever tested, the active wavelengths in controlling fruiting body initiation and maturation were found to be in the blue light~V range (Ellis et al., 1999; Kues 2000). However, the fruiting of some species can also depend on the presence of other organisms. Many Agaricus strains only fruit when associated with microorganisms, e.g. pseudomonas. Experiments with charcoal suggest that the microorganisms do not influence fruiting by producing induction substances, but by eliminating inhibitory compounds (De Groot et al., 1998). Cho et al., (2003) isolated bacteria from the mycelial surface of Pleurotus ostreatus and investigated their role in fruiting body induction (fructification)

of the edible mushroom P. ostreatus. Analysis of the bacterial community that colonized the mycelium showed that the species composition and numbers of culturable bacteria differed according to the developmental stage of P. ostreatus. In particular, the population size of fluorescent pseudomonads increased during fruiting body induction. An experiment showed that inoculation of pure cultures of the mycelium with strains of fluorescent Pseudomonas spp. isolated from the mycelial plane of commercially produced mushrooms promoted the formation of primordia and enhanced the development of the basidiome of P. ostreatus. Results of this research strongly suggest that inoculation of the mycelium with specific bacteria may have beneficial applications for mushroom production.

12.6. ENZYMES INVOLVED IN MUSHROOM PRODUCTION BY SSF Mushrooms degrade the lignocellulosic substrate by means of hydrolytic extracellular enzymes like cellulases, pectinases and xylanases, along with oxidases and peroxidases to degrade the lignin. Lignin is a polymer of phenylpropane units connected by different C-C and C-CO-C linkages. This molecule is oxidized and degraded by a ligninase system, which is composed at least by three enzyme activities: lignin peroxidase (LIP), manganese dependent peroxidase (MnP) and laccase (Master et al., 2001). Cellulose is a linear polymer of glucose units, which can be hydrolyzed by the action of endoglucanases, cellobiohydrolases and bglucosidases; these enzymes are typically induced by their substrates (Magnelli and Forchiassin 1999). Hemicellulose is a heterogeneous, branched polymer. The backbone of the polymer is built of sugar monomers like xylose, in this case, the enzymes involved in its degradation are named xylanases. Similar to cellulases, the xylanases can act synergistically to achieve hydrolysis. Mushroom produces the extracellular enzymes necessary to degrade the substrate to soluble molecules that are transported to the intracellular space and stored as glycogen; later, the glucosamine content increased to the final of incubation period, reflecting, probably, the associate preparation for fruiting (Leatham 1985). Mansur et al., (1997) showed that the use of fructose instead of glucose resulted in a 100-fold increase in the specific Lac activity of Basidiomycetes. According to Master and Field (1998) and Hammel (1997), the ligninolytic enzymes are produced during the secondary metabolism under conditions of limited nitrogen. However, in Pleurotus ostreatus a high concentration of nitrogen in the medium (34 mM, N as glutamate) did not repress but rather slightly stimulated mineralization of lignin compared to the N-limited medium (Kaal et al., 1995). Martinez et al., (1994) showed that P. eryngii and related species have a limited attack on cellulose, less than 15% of the initial content, while xylan degradation was parallel to lignin degradation. However, contrary to results of Martinez et al., (1994) and Munoz et al., (1997), where Lac production in P.eryngii was strongly stimulated by alkali lignin that is present in straw. Martinez

et al., (1996) found MnP production in P. eryngii, P. ostreatus, P. pulmonarius and P. sajor-caju in peptone medium, but it was not detected in media with other investigated nitrogen organic sources (corn-step liquor, malt extract and ammonium tartrate). Peptone was also the best nitrogen source for MnP production in Cerrena unicolor IBB 62 under SSF conditions of grapevine sawdust (Elisashvili et al., 2001). Likewise, peptone supplementation stimulated MnP production in Bjerkandera adusta, Bjerkandera sp. strain BOS55, L. edodes, P. ostreatus and P. chrysosporium(Kaal et al., 1995). Ligninolytic and cellulolytic enzyme pattern and activities were influenced by composition of the substrate (Papinutti et al., 2003; D'Souza et al., 1999). Thus, the capacity of a particular substrate to induce or increase production of lignocellulases is another factor that indirectly confers ability to grow and fruiting. In addition, not only the enzyme profiles accounts for the capacity of degradation but also their physicochemical characteristics when they are secreted; more stable enzymes at their optimum pH activity are capable to produce a more extensive degradation of the substrate. It was observed that L. edodes showed a rapid decrease in laccase activity at the start of fruiting. Other reports from known edible fungi such as Agaricus bisporus (Wood and Goodenough 1977; Manning and Wood 1983) and L. edodes (Ohga 1992) showed similar changes in laccase and cellulase activities; during development laccase activity declined before fruiting (Lechner and Papinutti 2006).

12.7. ENZYME PRODUCTION USING MUSHROOM STRAINS BY SSF Polygalacturonase is widely used in food processing industries as a processing aid, in maceration, liquefication, extraction, clarification, and filtration of fruit or vegetable juices and wines (Cannel and Moo-Young 1980; Dosanjh and Hoondal 1996). For most common industrial uses, fungal PGs prove to be most useful because of higher enzyme activity and optimum activity at a lower pH range suited for most fruit and vegetable processing applications (Dziezak 1991). Lentinus edodes has the advantage of possessing GRAS (generally regarded as safe) status, which permits the use of its metabolites including its extracellular enzymes in the food processing industry (Pariza and Foster 1983). It was found that a high level of polygalacturonase activity produced by L. edodes during solid-state fermentation on strawberry pomace. Zheng and Shetty (1999) showed that it is feasible to use fruit processing wastes such as strawberry pomace as a raw material for the production of polygalacturonase by a foodgrade fungus L. edodes. This fungus is able to produce high levels of extracellular polygalacturonase during solid-state fermentation on strawberry pomace.

Polygalacturonase produced from L. edodes has a relatively higher temperature optimum (50~ and a lower pH optimum (pH 5.0), with good thermal stability (up to 50~ and a high tolerance to low pH (3.0-6.5). Considering the natural acidic pH of most fruit and vegetable tissues and juices, the acid tolerant property of L. edodes polygalacturonase makes the enzyme an ideal candidate for tissue maceration, juice extraction and clarification in the fruit and vegetable processing industry. This research also provided another value-added strategy for better utilization of food processing wastes (Zheng and Shetty 1999).

12.8. RATION RESEARCHES FOR ANIMALS USING MUSHROOM STRAINS BY SSF Some authors suggest that straw compost degraded by enzymes during mushroom production can be more easily digested by ruminants (Yang et al., 1993; Adamovic et al., 1995a). It contains more free sugars, more protein, less cellulose and lignin, with an increased content of ash compared with beginning material (Rajatham and Bano 1989). Bisaria et al., (1997) analyzed the bioconversion of agroresidues, rice straw, and wheat straw by P. sajor-caju in an attempt to increase its nutritional value for animal feed. Bioconversion of unsupplemented rice straw after 20 days of incubation showed a decrease of cellulose from 35.8 to 17.9% and a decrease of lignin from 17.2 to 9.5%. The in vitro dry matter digestibility (IVDMD) increased from 19.7 to 29.8%. A similar tendency was observed for wheat straw after SSE and the IVDMD augmented from 27.2 to 36.8%. In the case of rice straw, supplementation with ammonium nitrate resulted in the maximum loss of organic matter (34.7%). For wheat straw, urea supplementation caused the highest loss of organic matter. As with rice straw, the nitrogen supplementation increased the degradation of cellulose and hemicellulose but decreased the degradation of lignin. The highest IVDMD values in supplemented rice and wheat straw after 20 days of SSF were 31.8 and 34.8%, respectively. Karunanandaa and Varga (1996) found that hemicellulose in rice leaf was selectively and extensively consumed when evaluated in SSF of rice straw by P. sajorcaju. Fungus improved the IVDMD of rice leaf, primarily because of increased digestion of cellulose. The improvement of rice straw quality was dependent on fungal species, botanical fractions, and preparation of substrate prior to fungal decay. Nchez et al., (2002) establishes the potential use of biotransformation of winery byproducts through solid state fermentation by Pleurotus species to produce food of good quality for human consumption. Moreover, after mushrooms are harvested, it is possible to use the fermented viticulture wastes to feed ruminants. The nutritional value of these agroresidues might be increased by some strains of Pleurotus, whereas some antinutritional factors such as phenolic components are diminished (Nchez et al., 2002). However, it was suggested

that the spent mushroom waste after SSF were formulated on an apparent digestible organic matter basis so that it can be efficiently utilized by the animals (Bakshi et al., 1985; Adamovic et al., 1998). The spent straws may be incorporated in the feeds of ruminants (cattle and buffaloes) by replacing upto 50% of the plain straws (Dhanda et al., 2005) Although there was increase in the protein content of the fermented matter with the increase in spawn rate from 10-25%, there was not much difference between the results obtained with 10 and 25% (Fan et al., 2000a). During SSF, there was increase in the protein content of coffee husk, spent-ground and mixed substrate with the increase in cultivation period. The fibre content decreased simultaneously (Fan et al., 2000a). In contrary, the protein contents was increased almost similar to that with Pleurotus, Lentinus sp and F. velutipes (Fan et al., 2000b,c). Inclusion of pruning content had higher phenolic components and total sugars, better C/N ratio, and lower crude fat and total nitrogen than pomace. On the contrary, mycelium grew more slowly and scarcely in all treatments with l00% grape pomace. Moisture, protein, fat, and lignin contents were generally higher in mixtures with higher pomace proportion, whereas neutral detergent fiber, hemicellulose, and cellulose contents were higher with pruning content. Pleurotus strains may act depending on the availability of fiber fractions of substrate, and dynamic changes in digestion might occur as these fractions change during fungal growth. The recycling of viticulture residues through SSF by Pleurotus has great potential to produce human food and yields an available high-fiber feed for limited use inruminants (Sanchez et al., 2002). Mukherjee and Nandi (2005) worked on biological delignification of lignocellulosic biomass of water hyacinth following SSF by two Pleurotus spp., as well as suitability of using the highly digestible, protein-enriched, spent biomass as ruminant feed. Loss of organic matter was higher in all replicates inoculated with P. citrinopileatus than that with P. florida after all three degradation periods during both solid state fermentation (SSF) and under mushroom growing conditions, being at a maximum, 34.4 +/- 0.6% dry weight (d.w.) after third flush of mushrooms. Under both conditions, an extensive removal of hemicellulose during the initial growth period (22 d) and a delayed Iigninolysis were observed. Loss of hemicellulose and cellulose was also higher (45.6% and 37.1% d.w., respectively, after the third flush) in biomass incubated with P. citrinopileatus than that with P. florida in all sets. Delignification was, however, higher (30.2% after third flush) by P. florida. The amount of reducing sugar present in the degraded biomass increased gradually during incubation and the highest value (3.1%) was obtained by P. citrinopileatus after 48 d under mushroom growing conditions. Crude protein of the bioconverted biomass increased up to 32 d in

all replicates (maximum value 10.8% by P. florida in SSF), but decreased thereafter. Spent biomass enriched with mycelial protein of all replicates after 32 d, resulted improvement of dry matter digestibility, more so by P. florida (30.7%) than by P. citrinopileatus (27.1%) during SSF. The results showed that P. florida was more effective than P. citrinopileatus in delignification and is a biologically efficient species for production of highly digestible mycoproteinrich ruminant feed. Ghosh et al., (1998) worked on banana pseudostem biomass using Pleurotus and showed the efficient degrading capacity of the test fungi and demonstrated their potential use in the conversion of banana pseudostem biomass into mycelial protein-rich fermented animal feed.

12.9. BIOREMEDIATION AND DETOXIFICATION OF MUSHROOM STRAINS BY SSF Saprophytic mushrooms are natural decomposers because they secrete enzymes and acids that degrade organic polymers into simpler moieties. The dominion of mushroom mycelium is enormous since each colony extends long chains of cells forming a network that can occupy many square meters. Even the most popular edible mushroom (Agaricus bisporus) is capable of accumulating silver as demonstrated by Falandysz et al., (1994), who investigated the uptake of this element from artificially enriched substrates. Accumulations of up to 150 mg/kg (on a dry weight basis) of silver were found in the fruit bodies (caps and stalks) when the level of the metal in the substrate was more than 12 times lower, showing that A. bisporus is not only an extractor but is also an efficient concentrator for this element. Silver (as silver nitrate) at this concentration did not affect mycelia growth or the emergence of fruiting bodies. Weber et al., (1997) showed that the Basidiomycete Boletus badius is particularly efficient in accumulating gold and arsenic, which are stored in different parts of the mushrooms. Thus, gold is accumulated in caps and stalks, while arsenic accumulates in the hymenium. The amount of gold accumulated in the mushroom can be as high as 0.23 mg/kg. The ability of mushrooms to concentrate toxic metals such as arsenic and cadmium is clearly taxonomically related. Vetter (1994) investigated 88 species of the class Basidiomycetes. Significant accumulation of arsenic was found in species of Agaricus and in family related fungi, such as Macrolepiota rhacodes, Flammulina velutipes, Lepista nebularis, and Clitocybe inversa. The highest concentration of cadmium (35 mg/kg) was also found within the genus Agaricus. Although the accumulation of arsenic is rarely observed in fungi (most accumulating only a few mg/kg), some species appear to be particularly adept at both storing and transforming this element. Stijve (1999) investigated 300 species of mushroom and found that only six species showed abnormally high arsenic levels, but such levels ranged from 10 mg/kg up to 2.4 g/kg. In some

of the investigated fungal accumulators (e.g. Entoloma lividum) arsenic was stored as the toxic arsenite and arsenate, but in others, inorganic arsenic was converted into organic derivatives of significantly lower toxicity, such as monomethylarsonic acid (Arsenicumcoronaria), dimethylarsinic acid (Laccaria amethystina), and arsenobetaine (Sarcodom imbricatus and two Agaricus species), the last compound being a harmless metabolite that also occurs in sea fish and crustaceans. Some strains of the highly appreciated gourmet mushroom Lentinus edodes (Shiitake) have been shown to possess the capacity to remove more than 60% of contaminating pentachlorophenol (PCP) from soil and convert it into pentachloroanisole (Okeke et al., 1993), among other products. Furthermore, after 10 weeks growth on contaminated soil, 99% of PCP was biotransformed and pentachloroanisole had been eliminated by this time. Interestingly, phenol oxidase and Mn-peroxidase activities were much higher. The ability to absorb and degrade chlorophenols appears to be characteristic of many Basidiomycetes. Thus, in addition to L. edodes, mycelia of fungi such as Armillaria, Ganoderma, Pleurotus, Polyporus, Coprinus, and Volvariella were all able to remove PCP when grown in a batch cultivation system (Chiu et al., 1998). The capacities of the mushrooms to absorb and degrade the contaminant varied considerably: mycelia of Polyporus was best at absorbing PCP (31 g/kg), while Armillaria showed the highest capacity to break down the compound (13 g/ kg). The oyster mushroom Pleurotus ostreatus was able to remove up to 90% of the phenol content of a solid state bioreactor supplemented with olive milling wastes (OMW) (Seti et al., 1998). Zervakis et al., (1996) found that the tolerance level of two Pleurotus species (P eryngii and P pulmonarius) towards raw OMW ranged from 25 to 50%.

Pleurotus, Lentinus and Flammulina can remove the caffeine and degrade tannins from husk. The fermented husk by Pleurotus showed a decrease of about 61 and 80%, respectively for caffeine and tannins. It was confirmed that those mushrooms did not degrade caffeine, just accumulate it in the fruit body, but degrade tannins (Fan et al., 2000a,b,c). Other solid wastes, such as sugar-cane trash and bagasse as well as cork residues, may also present an environmental problem because of their toxic content and/or complex biological degradation, and these also are amenable to remediation using edible mushroom species, such as Pleurotus (Pani et al., 1998) and Lentinus and Ganoderma (Riu et al., 1997), respectively.

12.10 CONCLUSIONS Mushrooms serve as delicacies for human consumption and as nutriceuticals, as "food that also cures". Mushrooms, the fruiting bodies of basidiomycetous

fungi, contain substances of various kinds that are highly valued as medicines, flavoring and perfumes. Mushroom production can convert the huge lignocellulosic waste materials into a wide diversity of products (edible or medicinal food, feed and fertilizers), protecting and regenerating the environment. Nevertheless, the biological potential of mushroom is far from exploration. The species used for human, knowledge of induction of fruiting body and the mechanism of process of SSF are limited.

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Shah MM, Barr DP, Chung N & Aust SD, 1992, Use of white rot fungi in the degradation of environmental chemicals, Toxicology Letters, 64-65, 493-501. Soccol CR, 1995, da fermentaqio no estado s61ido na valoriza~io deresfduos agroindustriais, Fran9a-Flash Agricultura, 4, 3-4. Sonnenberg ASM, Baars JJP, Hendrickx PM & Kerrigan RW, 2005, Breeding mushroom: state of the art, Acta Edulis Fungi (Suppl.), 12, 163-173. Stamets P, 2000, Growing Gourmet and Medicinal Mushrooms, 3rd ed., Califomia, Ten Speed Press.

Stijve T, 1995, Arsenic in mushrooms. Coolia, 38, 181-190. Vetter J, 1994, Data on arsenic and cadmium contents of some common mushrooms, Toxicon, 32, 11-15. Weber A, Lehrberger G & Morteani G, 1997, Gold and arsenic in mushrooms, mosses and needles: Biochemical aspects of a middle ages geogenic dumping site in Oberviechtach, northern Oberpfaelzer Wald. Geol Bavar, 2, 29-50. Wood DA & Goodenough PW, 1977, Fruiting of A. bisporus: changes in extracellular enzyme activities during growth and fruiting. Archives of Microbiology, 114, 161-165. Yamanaka K, 1997, Production of cultivated edible mushroom. Food Reviews International, 13, 327-333. Yang GL, Ma L, Wang YW & Wang Y, 1993, Physiology and biochemistry of lignocellulose utilization by Pholiota narneko, Proc. First Int. Conf. Mushroom Biol. Mushroom Products, Vol. 17, Hong Kong, pp: 163-168. Yang XM, 1986, Cultivation of Edible Mushroom in China, Agriculture Printing House, Beijing, ER. China. Zervakis G, Yiatras P & Balis C, 1996, Edible mushrooms from olive oil mill wastes. International Biodeterioration and Biodegradation, 38, 237-243. Zheng Z & Shetty K, 2000, Solid-state production of polygalacturonase by Lentinus edodes using fruit processing wastes, Process Biochemistry, 35, 825-830.

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18.1. INTRODUCTION Gibberellins (GAs) are a group of diterpenoid acids that function as plant growth regulators influencing a range of developmental processes in higher plants. One of them, gibberellic acid (GA3), has received the greatest attention. It affects stem elongation, germination, elimination of dormancy, flowering, sex expression, enzyme induction and leaf and fruit senescence. GA3 is a highvalued plant growth regulator with various applications in agriculture (Arteca 1995). The industrial process currently used for the production of GA3 is based on submerged fermentation (SmF) techniques, using Gibberella fujikuroi, or Fusarium moniliforme. It is a typical secondary metabolite. Upon exhaustion of nitrogen sources, exponential ceases and secondary metabolism is triggered, with concomitant biosynthesis of GA3 (Borrow et al., 1955; Brtickner & Blechschmidt 1991). In spite of the use of the best process technology, the yield of GA3 is low. In fact, as early as 1979, it was stressed that the SmF process used for its production was approaching a saturation point beyond which cost reduction would be impossible. The presence of product in dilute form in SmF was recognized as a major obstacle in economic manufacture of the product, mainly due to the consequent higher costs of downstream processing and disposal of wastewater. Moreover, the cost of separation of the microbial cells from fermentation broth using centrifugation or microfiltration has been reported to involve between 48 and 76% of the total production cost of the microbial metabolite by submerged fermentation. (Jefferys 1970; Kumar & Lonsane 1989). Recognizing that it is essential to look beyond the conventional submerged technique and explore other alternatives to achieve a more economical processes, different studies have been carried out to decrease the production costs, using

several approaches such as screening of the fungi, optimization of the nutrients and culture conditions, development of alternative processes (immobilized cells, fed-batch culture) and minimization of the cost of extraction procedure (Bandelier et al., 1997). Another technique, solid-state fermentation (SSF), has been investigated to increase the yields of GA3 and also to decrease production costs (Kumar & Lonsane 1987a; Pastrana et al., 1995; Tomasini et al., 1997; Bandelier et al., 1997; Gelmi, et al., 2000). Indeed, the SSF technique has Shown a number of economic advantages over SmF process in the production of microbial biomass and metabolites and the valorization of agro-industrial by-products (Pandey 1992; Soccol 1996; Pandey et al., 2001; Soccol & Vandenberghe 2003). The present chapter will firstly briefly describe gibberellic acid physical, chemical and biological properties. Then, it will treat about this metabolite production by fermentation, considering its biosynthesis pathway, fermentation techniques, GA 3 formation physiology in fermentative process, factors influencing fermentation and newest advances carried out to decrease the production costs, with special focus on SSE Finally, a case study of GA3 production by G. fujikuroi, using mixed substrate of important Brazilian agro-industrial wastes as a practicable alternative for the production of GA3 by solid-state fermentation will be presented.

13.2. PROPERTIES AND USES OF GIBBERELLIC ACID The gibberellins (GAs) are all tetracyclic diterpenoid acids strictly related, representing an important group of plant growth hormones. Known gibberellins are identified by subscribed numbers Gan were "n" corresponds, approximately, the order of discovery. Gibberellic acid, which was the first gibberellin to be structurally characterized, is GA3 (Hill 1977; Arteca 1995). All gibberellins have an ent-gibberellane ring system and are divided in two main types based on the number of carbon atoms, the C20GAs which have a full complement of 20 carbon atoms and C19GAs in which the twentieth carbon atom has been lost by metabolism. Many structural modifications can be made to the ent-gibberellane ring system. This diversity accounts the large number of known GAs (Kumar & Lonsane 1989; Sponsel & Hedden 2004). Gibberellins are distributed widely through the plant Kingdom where they play an important role in plant growth and development. They have also been isolated from fungi and bacteria. Of the 131 presently known naturally occurring GAs, 105 have been found exclusively in higher plants (including angiosperms, gymnosperms and fems), 11 in the fungi only, and the rest from both the sources. Of them, gibberellic acid (GA3) has received the greatest attention. GA 3 regulates the growth tax in plants being extensively used to produce a series o f benefic effects useful in agriculture (Mander 2003; Leitch et al., 2003; Crow et al., 2006).

Gibberellic acid (C19H2206), chemically characterized as a tetracarbocyclic dihydroxy-g-lactonic acid containing two ethylene bonds and one free carboxylic acid group (Cross, 1954). It is a white crystalline powder, with a melting point of 233 -235 ~ soluble in alcohols, acetone, ethyl acetate, butyl acetate, while is soluble with difficulty in petroleum ether, benzene and chloroform. The product can't be decomposed in dry condition and it is rapidly decomposed in hot condition and in aqueous solutions. It's half live in diluted aqueous solutions is about 14 days at 20 ~ and 2 hours at 50 ~ (O'Neil, 2001). Plant tissues usually contain only about 0.001 to 1.0 mg of gibberellic acid equivalent per kilogram of fresh weight. Different gibberellins are usually found in the same plants, and their pattern can change depending on stage of development. Sensitivity to various effects caused by different gibberellins can differ depending on the plant genera, organs and development stage (Takahashi 1986; Brtickner & Blechschmidt 1991). GAs appear to be involved in every aspect of plant growth and development, but their most typical (and spectacular) property is the enhancement of stem growth. Active gibberellins show many physiological effects, each depending on the type of gibberellin present as well as the species of plant (Martin 1983; Mander 2003). Considering the numerous effects of gibberellins, it seems logical that they would be used in commercial applications. Their major uses are (Martin 1983; Taiz & Zeiger 1991; Mander 2003): 9

Management of fruit crops - most seedless table grapes are now grown with the application of GA3, inhibiting senescence of citrus fruit maintaining the rind in better condition; controlling skin disorder in "golden delicious" apples;

9

Production of ornamental plants - inducing to flower either earlier than usual, or in off-seasons. Sporadic flowering in some plants is often a problem with plant breeders, but may be ameliorated with GA applications;

9

Malting of barley: 2-3 days may be saved by the addition of 25-500 ~tg of GA3 for each kg of barley.

9

Extension of sugarcane: increase in grown and sugar yield.

13.3. GIBBERELLIC ACID PRODUCTION

13.3.1. The gibberellin biosynthesis pathway The biosynthesis of GAs in plants and in fungi has received a great deal of attention and has been the subject of several reviews in the last ten years (Mac Millan, 1997; Hedden 1999; Tudzynski 1999, 2005; Sponsel & Hedden 2004; Kawaide 2006). Gibberellins are diterpenes synthesized from acetyl CoA

via the mevalonic acid pathway. They all have either 19 or 20 carbon units grouped into either four or five ring systems (Sponsel 1995). GAs, like other diterpenoids, are produced from hydroxymethylglutaryl (HMG) coenzyme A (CoA) via mevalonic acid, isopentenyl diphosphate, geranyldiphosphate (GDP), farnesyl diphosphate (FDP) and geranylgeranyl diphosphate (GGDP), which is a precursor not only for GAs, but also for the carotenoid neurosporaxanthin and ubiquinones. Ent-kaurene, the first GA-specific intermediate, is produced in two cyclisation steps from GGDP via ent-copalyl diphosphate (CPP). Sequential oxidation of ent-kaurene at C-19 via ent-kaurenol and entkaurenal yields entkaurenoic acid, which is further oxidised to ent-7othydroxykaurenoic acid. A final oxidation at C-613, resulting in contraction of ring B, leads to formation of GA12-aldehyde. These steps of the pathway are identical in the fungus and in higher plants (Tudzynski, 2005). The subsequent steps in plants will not be described in this work. In G. fujikuroi, GA12aldehyde is first 3l]-hydroxylated to GAin-aldehyde, which is then oxidized at C-7 to form GA14. GA14 is then converted to the 19-carbon gibberellin GA4 by 20-oxidation. GA 4, the first biologically active GA, is desaturated to GA 7, which is then converted to GA3 by late 13-hydroxylation. GA1 is formed in a minor side reaction by 13-hydroxylation of GA4 (Tudzynski, 2005).

13.3.2. Fermentation techniques Fermentation is the industrial method practiced for the manufacture of GA3 preferentially with G fujikuroi or its imperfect stage F moniliforme (Borrow et al., 1955). Is possible produce it by chemical synthesis (Corey et al., 1978; Hook et al., 1980) or extraction from plants (Kende 1967) but these methods are not economically feasible. Liquid surface fermentation (LSF) was employed in earlier years for the production of GAs and its use was continued until 1955. It offers advantages such as no foam formation and no mechanical damage to mycelial cells, as compared to the submerged fermentation process. Although, due to disadvantages inherently present in LSF as production of a wide range of by-products, very low yield (40 to 60 mg GAs.Lsubstrate-1), prolonged incubation time ( 1 0 - 30 days) and prone to contamination this technique was abandoned for GA 3 production being substituted by submerged fermentation (SmF) (Kumar & Lonsane 1989; Brtickner & Blechschimidt, 1991). The presence of product in dilute form in SmF was recognized as a major obstacle in economic manufacture of the product, mainly due to the consequent higher costs of downstream processing and disposal of wastewater. Moreover, the cost of separation of the microbial cells from fermentation broth using centrifugation or microfiltration has been reported to involve between 48 and 76% of the total production cost of the microbial metabolite by submerged

fermentation (Vass & Jefferies 1979; Kumar & Lonsane 1987b; Bandelier et al., 1997). Recently different studies have been carded out to decrease GA3 production costs using several approaches as screening of fungi, optimization of the nutrients and culture conditions, use of agro-industrial residues as substrate, development of new processes (immobilizes cells, fed-batch culture) and minimization of the cost of the extraction procedure. Some of these listed in Table 1. Solid-state fermentation (SSF) has also been investigated to increase yields of GA3 and also minimize production and extraction costs (Kumar & Lonsane 1986, 1987a, 1987b, 1987c; 1988, 1990; Bandelier et al., 1997; Tomasini et al., 1997; Gelmi et al., 2000, 2002; Machado et al., 2001, 2002, 2004; Escamilla et al., 2000; Corona et al., 2005). Indeed, The SSF technique has shown a number of economic advantages over submerged fermentation processes in the production of microbial biomass and metabolites and in the valorization of agro-industrial by products (Pandey 1992; Pandey & Soccol 1998; Padey et al., 2000a,b; Soccol & Vandenberghe, 2003).

13.3.3. GA 3 formation physiology in fermentative process Fermentative production of gibberellins is a classic example of secondary metabolite production as the phases of growth can be clearly distinguished and related to nutritional and environmental states operating in the fermentor. Borrow et al., (1961; 1964a,b) have exhaustively studied this process and established producing and non-producing phases of the gibberellin fermentation process. TheJ'conventional lag phase in nitrogen-limited medium is undetectable as the /' strain requires little or no adaptation and growth in the fermentor starts quickly due to the use of vigorous mycelial cells as inoculum. Growth during the balanced phase is initially exponential and subsequently becoming linear. The uptake of glucose, nitrogen and other nutrients remains almost constant per unit increase in dry weight. This phase extends until exhaustion of one of the nutrients occurs subjecting the cells to a deceleration stage. No GA is produced in this phase. The following storage phase occurs with the presence of excess glucose and the exhaustion of nitrogen, causing an increase of dry weight due to accumulation of lipids (45%), carbohydrates (32%) and polyols. In this phase the production of gibberellins and other secondary metabolites begins and is continued in the presence of available glucose. The next maintenance phase, is operative between the maximum mycelial formation and the onset of terminal breakdown of mycelial components. Because it is the main gibberellinproducing phase, its continuation, even for several hundred hours, if glucose is present in excess, is of industrial importance. Except for the continued

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13.3.4. Nutritional factors influencing GA 3 production Glucose and sucrose have often been used as carbon source, but concentrations above 20% of glucose at the beginning of the fermentation should be avoided, as it causes catabolic repression (Borrow et al., 1964a). Many workers have used alternative carbon sources, such as maltose, mannitol, starch and plant meals or mixtures of fast and slowly utilized carbon sources, e.g. glycerol, glucose and galactose (Darken et al., 1959; Sanchez-Marrokin, 1963; Hemandez & Mendoza 1976; Maddox & Richert 1977; Kahlon & Malhotra 1986; Kumar & Lonsane 1989; Pastrana et al., 1995; Cihangir & AksSza 1997; Tomasini et al., 1997; Machado et al., 2001). The quality and quantity of nitrogen are very important for gibberellin fermentation because of the ammonium regulation of this process. All described media that guarantee high yields of GAs are low nitrogen media, as gibberellic acid production begins at, or soon after, nitrogen exhaustion (Borrow 1964a; Brtickner & Blechschmidt 1991; Tudzynski 1999). Favorable nitrogen sources are ammonium sulfate, ammonium chloride and slowly assimilable sources as glycine, ammonium tartarate, plant meals and corn step liquor (Kumar & Lonsane, 1989). The influence of C:N ratio is directly related with GA3 production metabolism. So, their values must provide initial active mycelial growth in a nitrogen-limited balanced medium, initiation of GA3 production after nitrogen exhaustion and extended metabolite production in the presence of sufficiently available carbon substrate. Therefore, the ratio normally used is high, between 6:1 and 45:1. In SmF is common the utilization of two stage processes, were the initial stage has C:N ratios between 10:1 and 25:1 and final stage varies from 25:1 and 200:1 (Borrow 1964a; Jefferies 1970; Kumar & Lonsane, 1989; Brtickner & Blechschmidt 1991; Tudzynski 1999). Besides carbon and nitrogen, magnesium, potassium, phosphate and sulfate are all needed in the biosynthesis of secondary metabolites. In spite of that, negligible information is available on these aspects in the microbial production of GAs. Normally the requirements are met efficiently using the salts combinations from Czapek-Dox and Raulin-Thom media. Trace element requirements are often met by impurities in commercial media (Borrow 1964a; Kumar & Lonsane, 1989; Brtickner & Blechschmidt 1991).

13.3.5. Environmental factors influencing GA 3 production pH variation is one of the most influent factors in the composition of the produced gibberellin mixture. For GA3 production, pH generally employed is between 3.5-5.8. The pH increase will lead a bigger production of GA4,7 and more acid media (pH < 3.5) will increase GA1 production (Kumar & Lonsane, 1989). In solid-state fermentation pH measurement is complex. Therefore are few works describing values used for GA3 production using this technique. Gelmi et al., 2000, using amberlite inert support established for studied system initial pH of 4.5, Tomasini et al., 1997 in a fermentation in cassava flour used initial pH 6.0 and Machado et al., (2001) with a mix substrate of coffee husk and cassava bagasse had better GA3 production with initial pH between 5.0-5.4. The effect of temperature on the GA3 production is dependent on the strain employed. Values between 25-34 ~ are reported (Hemandez & Mendoza 1976; Maddox & Richert 1977; Kahlon & Malhotra 1986; Jones & Pharis 1987; Nava-Saucedo et al., 1989; Kumar & Lonsane 1990; Pastrana et al., 1995; Cihangir & Aks6za 1997; Tomasini et al., 1997; Escamilla et al., 2000; Machado et al., 2002; Corona et al., 2005). Jeferys (1970), reported that the optimum temperature for growth of the strain is between 31-32~ while the production of GA3 was maximized at 29~ Since the biosynthesis of gibberellins involves many oxidative steps, a good aeration of fermentors is critical for an optimal production process. In fact, since the value of oxygen consumption for a growing mycelium in the exponential phase of growth remains constant, the demand of oxygen increases more or less exponentially (Tudzynski 1999). Jefferys 1970 suggests that the aeration must be as vigorous as possible, being reported, for SmF rotations between 150-1400 rpm. In in a feed-batch reactor with SSF Bandelier et al., 1997 used an air flow of 0.9 Lair.(h.kgdry matter)-1, Tomasini et al., 1997 of 0.42 Lair.(h.kgdry -1 matter) , and Gelmi et al., 2000 of 0.46 Lair.(h.kgdry matter) -1 in a glass column reactor. In this kind of reactor, Machado et al., 2004 determined that better GA 3 production should be achieved with a low aeration (0.24 Lair.h-l.gdry matter-1) in the first 72 h of fermentation and, after that, with a much more vigorous aeration (0.72 Lair.h-l.gdry matter-l).

13.4. GIBBERELLIC ACID PRODUCTION BY SOLID-STATE FERMENTATION ON MIXED SUBSTRATE OF COFFEE HUSK AND CASSAVA BAGASSE Although GA3 has been conventionally produced by submerged fermentation, it can also be produced by SSE Production through SSF route could be relative cheaper and may involve lower costs, if several approaches such as application of cheaper substrate viz. agro-industrial residues, optimization of the nutrients

and culture conditions, minimization of the cost of extraction procedure, etc. are applied. The residues of coffee industry represent about 50% of total mass and practically do not find any industrial or useful application. Instead, their disposal is a major environmental concern. These products are rich in organic matter, which could make them suitable for bioconversions processes. The principal difficulty for its utilization in fermentations is the presence of inhibitory metabolites, as phenolic compounds (tannins, chlorogenic acid and caffeic acid) and caffeine, which represents about 6-14% of coffee pulp dry weight. But if these difficulties are overcome useful products can be produced from these residues by cultivating microbes in submerged fermentation or solid-state fermentation (Elias 1978; Perraud-Gaime et al., 2001). Laboratory studies were carried out in 250 mL erlenmeyer flasks and in aerated columns using statistical experimental designs. The strains of G fujikuroi or F moniliforme used were maintained in potato dextrose agar (PDA) and were inoculated in Czapek Dox medium in rotatory shaker at 30~ for 4 days for developing the inoculum (Bandelier et al., 1997). The GA3 isolation was done with phosphate buffer, pH 8 and metabolite quantification by high performance liquid chromatography (Varian system composed of a pump, a diode array detector and a column C18. Mobile phase methanol:water- 40:60). Aqueous layer was separated and quantification was made by high performance liquid chromatography.

13.4.1. Selection of microorganism and treatment of substrate The first part of the work aimed at exploring the possibility of using coffee husk as substrate for the production of GA3 (Machado et al., 2001). Solidstate fermentation was compared with liquid fermentation (done in the aqueous extract of the substrate) with five strains of G fujikuroi and one of its imperfect state, F moniliforme. The conditions of fermentation were similar for both systems. The results showed that SSF resulted higher quantities of GA3 and that the strain G fujikuroi LPB-06 was best adapted in the chosen substrate. Hence, further works were conducted with the strain LPB-06 in solid-state fermentation. Because coffee husk has phenolic compounds that could interfere in GA3 production, pre-treatment of the substrate was studied. For this, different conditions of pre-treatment (alkaline hydrolysis), utilizing different concentrations of KOH and time of treatment were tested. With this study, a production of about 100 mg GA3.kg.dry matter -1 were reached in the best conditions" 45 minutes hydrolysis in aqueous solution of 5 g KOH/L.

13.4.2. Studies in Erlenmeyer flasks - optimization of fermentation conditions Studies were carried out on optimization of nutritional and physical factors, such as supplementation of minerals and nitrogen to the substrate, inital pH and moisture of substrate and incubations temperature, for improved yields of GA3 (Soccol et al., 2000; Machado et al., 2002). To change the original coffee husk C:N relation that was about 14:1, cassava bagasse was used. This residue contains a large amount of amide, resulting in a very high C:N relation (110:1). The cassava bagasse was added in six different concentrations, 15, 30, 45, 60, 75 and 90%. The best results were obtained in substrate enriched with 30% of cassava bagasse, resulting in a C:N relation of 43:1, and reaching a production of 230.6 mg GA3.kg.dry matter-1 an increase of almost 100% compared with a substrate 100% coffee husk. To test the influence of addition of certain salts, two statistical experimental designs were performed. In the first one, the parameters studied with 2 (7-4) experimental design were presence or absence of seven salts reported as useful in gibberellins production (KH2PO4; K2SO4; MgSO4; ZnSO4; CuSO4; FeSO 4 and (NH4)2SO4), in the concentrations suggested by Kumar & Lonsane (1989). The results demonstrated that there several salts, in spite of being indicated as useful in the production of GA3, had a negative effect. That is probably explained by their natural presence in coffee husk, and an addition becoming inhibitory for the growth and metabolism of the microorganism. The second optimization was done varying the concentration of the salts that had the best positive influence in the production of GA3:FeSO4 and (NH4)2SO4. The concentrations chosen varied between the highest and lowest values suggested by Kumar and Lonsane (1989). With this optimization, a production of 389 mg GA3.kg.dry matter-1 was achieved with the saline solution, consisting of 30 mg of FeSO4 and 10 mg of (NH4)2SO4.100 mL -1, as shown in the contour response (Figure 1). The physical parameters studied were incubation temperature, initial moisture and pH of substrate. To test the influence of these parameters, two experimental designs were performed. The first was a 2 (3-1) and under the best conditions of initials pH and moisture of substrate as 5.0 and 70%, respectively, and incubation temperature of 30~ 325 mg GA3.kg.dry matter-1 was produced. Observing the Pareto chart of effects (Figure 2), it is noticed that the variable of larger influence in the production of GA 3 is the pH, followed by the initial moisture of the substrate, both suggesting that larger values would take to an increasing of the answer variable.

Figure 1" Contour response to GA 3 production as a function of (NH4)2SO 4 and FeSO4 concentrations (levels given in mg GA3.kgdrymatter1).

Figure 2: Pareto chart of effects of first optimization of physical parameters in erlenmeyer flasks (response variable mg GA3.kgdrymatter-1)

According to Kumar & Lonsane (1989), the pH is one of the most important factors for gibberellins production, mainly for definition of produced gibberellin, suggesting concentrations among 3.5-5.8 for increase the GA3 production. The levels used in this experiment were 4.0, 4.5 and 5.0. It is well known that in SSF initial moisture of substrate is one of the most important parameters. According to Raimbault (1998), in most of SSF processes, values between 35 and 80% are used, depending on the microorganism and substrate. Among the used levels, temperature did not significantly influent in the GA3 production. Thus, in subsequent experiments, temperature was maintained at 29~ and higher values for initial pH and moisture were chosen; moisture and pH were 78% and 5.5, respectively. The results are shown in the contour response (Figure 3). A maximum of 492.5 mg GA3.kg.dry m a t t e r - 1 was produced using a mixed substrate comprising coffee husk and cassava bagasse (7:3, dry wt), supplemented with saline solutions containing (%) 0.03 FeSO 4 and 0.01 (NH4)2SO4, pH and moisture as 5.3 and 75%, respectively, and incubation temperature as 29~ the GA3 yield was almost 5 times more than the results without optimization.

13.4.3. Studies in packed-bed column bioreactor 13.4.3.1. Optimization of fermentation conditions Studies were carried out on optimization of gibberellic acid production in 14 glass columns placed in a temperature controlled water bath (29~ which were packed using pre-inoculated substrate with forced aeration as described by Raimbault & Alazard (1980). For this, two statistical experimental designs were performed, both varying the airflow rate and initial moisture of the substrate. The other parameters used were the ones determined in Erlenmeyer flasks: 7 days fermentation, mixed substrate comprising coffee husk and cassava bagasse (7:3, dry wt), supplemented with saline solutions containing (%) 0.03 FeSO4 and 0.01 (NH4)2SO4, pH 5.3. With aeration levels of 0.24, 0.48 and 0.72 Lair.h-l.gdrymatter1 and initial moisture of 68, 73 and 78 %, the best result obtained was of 373 mg GA3.kgdry m a t t e r - 1 with 78% of initial moisture and aeration of 0.2 Lair.h-l.gdry matter-1 9 Comparing the results obtained in Erlenmeyer flasks with the same initial moisture content, a value 25% lower was obtained. Observing Pareto chart of effects (Figure 4), it could be assumed that the aeration has a negative influence on fermentation. Nevertheless, this observation opposes with the theory, for as much the metabolite biosynthesis progress over growing oxidation levels. In fact, according to Tudsynski (1999) considering that the oxygen consumed for mycelium production might remain constant, the oxygen demand increases approximately in exponential way.

Figure 3: Contour response to GA3 production as a function of pH and initial moisture of substrate (levels given in mg GA3,kgdrymatter-1).

Figure 4: Pareto chart of effects of first optimization of moisture (%) and aeration (Lair.h-l,gdry matter 1) in packed-bed columns bioreactor (response variable mg GA3,kgdry matter 1)

Therefore, in the second optimization was tested the hypothesis that a vigorous aeration in the beginning of fermentation should be affecting the microorganisms growth and, as a result, the gibberellic acid production. Hence, a low aeration (0.24 Lair.h-1 .gsubstrate-1) was used in the first 72 h of fermentation and then the levels of 0.24, 0.72 and 1.20 Lair.h-l.gsubstrate-1 were tested. As shown in Pareto chart of effects for first optimization (Figure 4), the initial moisture had a positive influence on gibberellic acid production, hence, higher levels were experimented (78, 80 and 82%). With this, a better production of GA3 was achieved, reaching 625 mg GA3.kgary matter-1 with moisture and aeration of 78% and 0.72 Lair.h-l.gsubstrate 1 respectively (Figure 5). It was an improvement of 27% compared to Erlenmeyer flasks fermentation and of 68% compared with columns fermentation with constant aeration.

13.4.3.2. Kinetics of G fujikuroi growth and GA 3 production The present work was undertaken to study the kinetics of growth and production of GA 3 by G. fujikuroi, using coffee husk and cassava bagasse as substrate in aerated columns bioreactor (Machado et al., 2004). SSF was carried out in 14 glass columns placed in a temperature controlled water bath (29~ The air flow was set at 0.24 Lair.h-l.gsubstrate-1 for the first 72 h of SSF and was increased to 0.72 Lair.h-1 .gsubstrate -1 after that, After each 24 h, two columns were withdrawn as samples for analysis. A tendency of increasing moisture and water activity was observed during the course of fermentation. This behavior could be expected due to the use of saturated air for aeration during the process, resulting in significant mass transfer during the experiment. In Erlenmeyer flasks without aeration, with the same substrate the moisture decreased gradually (Soccol et al., 2000; Machado et al., 2002). As carbon compounds in the substrate are metabolized, they are converted into biomass and carbon dioxide. Production of carbon dioxide causes the weight loss of fermenting substrate, and the amount of weight lost can be correlated to the amount of growth that takes place (Raimbault, 1997). In this experiment, loss of weight reached 9% at 7th day of fermentation and followed the Monod model: a lag phase of 48 h, exponential phase 48-120 h and stabilization until 144 h. In Figure 6 the behavior of the system for biomass and GA 3 in the 7 days of fermentation is shown. Table 2 was made considering all data for the kinetic study. For all kinetic calculations, the 7th day of fermentation was not considered since GA3 production decreased after the 6th day. Although the substrate was of complex nature, the main carbon source for this fermentation was considered the non-reducing sugars, which came mainly from cassava bagasse starch. Therefore, in Table 2, these were the values considered in column "S". Biomass

Figure 5: Contour response to GA 3 production as a function of aeration and initial moisture of substrate in packed-bed columns bioreactor (levels given in mg GA3.kgdrymatter 1)

Table 2: Kinetic parameters in SSF for gibberellic acid production

Where: DM - dry matter (dry fermented substrate); X - biomass; S - substrate (nonreducing sugars); P - product (gibberellic acid); g - specific growth rate; r p - GA 3 production per hour

was calculated as twice the increasing protein assuming that 50% of fungal biomass was protein, and considering that the protein consumed in the substrate was proportional to the loss of weight of the substrate.

The phases of biomass evolution can be analyzed observing Figure 6. Evidently it followed a Monod model having a lag phase (days 0 and 1), where the specific growth rate was practically null, and represented a stage in which microbial biomass did not start to multiply; logarithmic or exponential growth phase (day 1 to 3), reaccelerated growth phase (after the 3rd day) when the biomass synthesis started to demit in the process. Stationary phase was not observed as that probably might reach after the 7th day. The maximum specific growth rate (~tmax) were determinate considering Monod Model between days 1 and 3 (logarithmic growth phase) and it was 0.041 h -1. This value was similar to those reported for other SSF-grown G. fujikuroi that varied between 0.12 and 0.57 (Gelmi et al., 2000), between 0.06 and 0.08 (Pastrana et al., 1995) and 0.018 (Bandelier et al., 1997). The main product of this fermentation was gibberellic acid. After 6 days concentration of 0.940 g GA3.kg dry matter-1 was achieved. Table 2 showed that the best production of the metabolite was between 4 and 6 days of fermentation, just after the logarithmic growth phase. This behavior is characteristic for secondary metabolites kinetics as gibberellic acid. In secondary metabolism, the desired product is usually not derived from the primary growth substrate but a product formed from the primary growth substrate acts as a substrate for the production of a secondary metabolite. The secondary metabolites are not essential for the organisms' own growth and reproduction. In fact, normally, they are produced in "stress" situations for the microorganism (Pandey et al., 2001). Because the metabolic rout of gibberellic acid production is known and it happens after several oxidation reactions, high yields of oxygen are required. Hence, the change of aeration rate in 4th day contributed for the raise in GA 3 production rate too. The kinetics parameters calculated were the maximum and the total rate of GA 3 production: rSmax= 0.254 g.kg-l.day -1 rTota1 = 0.154 g.kg-l.day -1. Kumar and Lonsane (1987) obtained a yield of 1.2 g GA3.kg dry matter-1 utilizing wheat bran in a pilot scale bioreactor after seven days of fermentation (rTota 1 = 0.171 g.kg-l.day -1) and Bandelier et al., (1997) in a similar system 3 g GA3.kg dry matter -1 after 10 days (rTota 1 = 0.3 g.kg-l.day-1). Gelmi et al., (2000) achieved 0.760 g GA3.kg dry matter -1 with vermiculite inert support after 5 days of fermentation (rTotal = 0.152 g.kg-l.day -1) and Tomasini et al., (1997) reported 0.24 g GA3.kg dry matter-1 after 3 days of fermentation with cassava flour as substrate (rTotal = 0,08 g.kg-l.day-1). Considering the same system, the concentration achieved was the best, having the advantage of the utilization of agro-industrial residues as substrates.

Ell

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Figure 6: Biomass and gibberellic acid production in SSF

It has been observed that after some time, the GA 3 decays in substrate (Tomasini et al., 1997; Bandelier et al., 1997; Machado et al., 2001). This phenomenon was probably due to GA3 decomposition in production system (Perrez et al., 1996; Gelmi et al., 2002).

13.4.3.3 Matematical modelling of G fujikuroi growth and GA 3 production The packed-bed columns bioreactor was connected to a gas chromatograph to make the exit gas analysis. With the respirometric data, a logarithmic correlation between accumulated CO2 and biomass production was determined and the mathematical model were compared with experimental data, leading to a better knowledge about the behavior of the system for future works in scaling up, and comparison with submerged fermentation (Machado 2004). On-line gas monitoring system measured the flow rate and gas composition of the outlet air stream from four columns. These columns were connected to a gas chromatograph linked to a personal computer, as described by Saucedo-Castafieda et al., (1994). The respirometric data were processed in Excel | in witch instantaneous curves of CO2 and 02 calculations were determined using the equations developed by Pandey et al., 2001. To determine the correlation between biomass and CO2,the logistic law (Ebune et al., 1995) was applied. This model (equations 1 and 2) describes the mycelial growth in logarithmic and stationary phases in solid-culture of substrate.

dX = IAX 1- m dt X =

Xm

1 + fie -*'m`

(1)

---) 3 -

xmxO -1

(2)

Where X is the biomass ( g b i o m a s s . k g d r y matter -1) at time t (h), Xm the maximum biomass concentration, ~l m the maximum specific growth rate (h-1) and Xo is the initial biomass concentration. From the experimental data of biomass using the Statistica program by StatSofl, -1 -1 h-1 X m = 2 4 . 3 8 gbiomass.kgdrymatter ; XO - 1.171 g b i o m a s s . k g d r y matter ; ~ m -- 0 . 0 3 7 were determined. The high regression coefficient (0.996) obtained and the good correspondence with the experimental values Xm = 24.18 gbiomass.kgdrymatter --1,, XO = 1.122 g gbiomass.kgdrymatter -1 ; ~lmax_. 0.0522 h-1 proved the adequacy of the model. In SSF processes, the amount of biomass produced at a particular period of time can be related to the 02 consume pattern, if the fraction employed in metabolites synthesis is known or irrelevant. Gibberellic acid synthesis has several oxidative stages. In this work, agro-industrial residues with different nature of carbon sources were used as substrate. Hence, its mathematical modeling relating to 02 consumed with biomass production was considered as of complex nature. Considering C02 was only produced by microbial respiration, this parameter seemed to be more directly related to biomass. Hence, using the model achieved a theoretical value of biomass was determined for every time that had a CO2 analysis. Finally, a regression of the curve accumulated CO2 vs. biomass was made, leading to a logarithmic correlation between these two variables (equation 3): X = 3.9872 In (CO: acum) + 23.824

(3)

The kinetic parameters of the microorganism growth were established with the values of experimental and estimated biomass. As shown in Figure 7, the two growth curves were very similar and followed a Monod model having the same lag phase (0 to 24 h) and exponential growth phase (24 to 48 h). The desacelerated growth phase started in both the cases after 48 h but the stationary phase appeared in experimental data after 120 h and was not found in estimated data. The maximum specific growth rates (mmax) were determined between 24 and 48 h (exponential growth phase) and were almost the same for experimental and estimated biomass (0.0522 h-1 and 0.0520 h-i, respectively). For the global

Figure 7: Growth curve for experimental and estimated biomass

specific growth rate ( / / ) , calculated between 0 and 144 h, the same value of 0.0206 h -1 was found.

13.5. C O N C L U S I O N S This work proved that with judicious selection of nutritional and physical factors, the yield of GA 3 can be much enhanced. In the presented case, the increase was almost 10 times. Also, it confirmed that a mixture of coffee husk and cassava bagasse could be a good substrate for the production of GA3 by SSE The biomass is a key variable in fermentation studies, since typically metabolic activities such as substrate consumption and product formation are strongly related to either or both of the growth rate and the actual biomass present. However, it is very often necessary to use indirect methods of determining biomass in SSF, such as the measurement of cell components, this being especially true for those processes involving fungi. Even with the difficulties that are encountered in SSF process, kinetic procedure can not be substituted by goodwill, subjectivism or even by the simple and overall process description. Mathematical models can be tools in the development of rational strategies for the design and optimization of operation of large-scale bioreactors.

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Cihangir N & Aksiiza N, 1997, Evaluation of some food industry wastes for production of gibberellic acid by fungal source, Environmental Technology, 18, 533-537. Cihangir N, 2002, Stimulation of the gibberellic acid synthesis by Aspergillus niger in submerged culture using a precursor, World Journal of Microbiology and Biotechnology, 3, 727-729. Corona A, Sanchez D, Agosin E, 2005, Effect of water activity on gibberellic acid production by Gibberella fujikuroi under solid-state fermentation conditions, Process Biochemistry, 40, 2655-2658. Cross BE, 1954, Gibberellic acid Part I, Journal of Chemical Society, 4670-4676. Crow JR, Thomson RJ & Mander LN, 2006, Synthesis and confirmation of structure for the gibberellin GA131 (18-hydroxy-GA4), Organic Biomolecular Chemistry, 4, 2532-2544. Darken MA, Jensen AL & Shu P, 1959, Applied Microbiology, 7, 301-303. Ebune A, AI-Asheh S & Duvnjak Z, 1995, Effects of phosphate, surfactants and glucose on phytase production and hydrolysis of phytic acid in canola meal by Aspergillus ficuum during solid state fermentation, Bioresources Technology, 54, 241-247. Elias LG, 1978, Composici6n quimica de la pulpa de caf6 y otros subproductos. In- Pulpa de Caf6 - composici6n tecnologfa y utilizaci6n, JE Brahan & R Bressani (eds), Instituto de Nutrici6n de Centro Am6rica Y Panama INCAP, Bogota, pp 19-27.

m

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Escamilla EM, Dendooven L, Magafia IP, Parra R & De La Torre M, 2000, Optimization of gibberellic acid production by immobilized Gibberella fujikuroi mycelium in fluidized bioreactors, Journal of Biotechnology, 76, 147-155. Gelmi C, Perez-Correa R, Gonzalez M & Agosin E, 2000, Solid substrate cultivation of Gibberella fujikuroi on an inert support, Process Biochemistry, 35, 1227-1233.

Gelmi C, Perez-Correa R & Agosin E, 2002, Modeling Gibberella fujikuroi growth and GA 3 production in solid-state fermentation, Process Biochemistry, 37, 1033-1040. Hedden P, 1999, Recent advances in gibberellin biosynthesis, Journal of Experimental Botany, 50, 553-563 Hern~indezE, Mendoza MD, 1976, Producci6n de ~icidogiber61ico por Gibberellafujikuroi en substratos que contienen pulpa de aceituna aceite de oliva o subproductos de la extracci6n de este t~ltimo, Revista de Agroquimica y Tecnologia de Alimientos, 16, 357-366. Hill TA, 1977, Hormonas Reguladoras del Crescimento Vegetal, Ediciones Omega S.A., Barcelona, 74 p. Holbrook AA, Edge WJW & Baily F, 1961, Spectrophotometric method for determination of gibberellic acid, Advances in Chemical Series, 28, 159-167. Hollmann D, Switalski J, Giepel S & Onken U, 1995, Extractive fermentation of gibberellic acid by Gibberella fujikuroi, Journal of Fermentation and Bioengineering, 79, 594-600. Hook JM Mander LN & Rudolf U, 1980, Journal of American Chemistry Society, 102, 6628-6629.

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Kumar PKR & Lonsane BK, 1987a, Potential of fed-batch culture in solid-state fermentation for production of gibberellic acid, Biotechnology Letters, 9, 179-182.

Kumar PKR & Lonsane BK, 1987b, Extraction of gibberellic acid from dry mouldy bran produced under solid-state fermentation, Process Biochemistry, 10, 138-143. Kurnar PKR & Lonsane BK, 1987c, Gibberellic acid by solid state fermentation: consistent and improvement yields, Biotechnology and Bioengineering, 30, 267-271.

Kumar PKR & Lonsane BK, 1988, Batch and fed-batch solid-state fermentations: kinetics of cell growth hydrolytic enzymes production and gibberellic acid production, Process Biochemistry, April, 43-47. Kumar PKR & Lonsane BK, 1989, Microbial Production of Gibberellins: state of the art. Advances in Applied Microbiology, 34, 29-139. Kumar PKR & Lonsane BK, 1990, Solid-state fermentation: physical and nutritional factors influencing gibberellic acid production, Applied Microbiology and Biotechnology, 34, 145-148. Lale G, Jogdand VV & Gadre RV, 2006, Morphological mutants of Gibberellafujikuroi for enhanced production of gibberellic acid, Journal of Applied Microbiology, 100, 65-72.

Leitch SK, Blake PS & Mander LN, 2003, Synthesis and structure determination of three new 12~-hydroxy C:0 gibberellins (GA127GA128and GAle9), ARKIVOC Journal, 7, 145-160. Lu ZX, Xie ZC & Kumakura M, 1995, Production of gibberellic acid in Gibberella fujikuroi adhered onto polymeric fibrous carriers, Process Biochemistry, 30, 661-665.

Machado CMM, Oliveira BH, Pandey A & Soccol CR, Coffee husk as substrate for the production of gibberellic acid by fermentation, 2001, In- Book on Coffee Biotechnology and Quality, T Sera, CR Soccol, A Pandey & S Roussos (eds), Kluwer Academic Publishers, Dorcrecht, pp 401-408. Machado CMM, Soccol CR & Pandey A, 2002, Gibberellic acid production by solid sate fermentation in coffee husk, Applied Biochemistry and Biotechnology, 102, 179-192.

Machado CMM, Oishi B, Pandey A & Soccol CR, 2004, Kinetics of Gibberellafujikuroi growth and gibberellic acid production by solid-state fermentation in a packed-bed column bioreactor, Biotechnology Progress, 20, 1449-1453. MacMillan J, 1997, Biosynthesis of the gibberellin plant hormones, Natural Product

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,:Ji~ilili Production of Antibiotics and . . . . . . . . . .i .. .. . . .~. ...... . . .:~. . . . . . Other Commercially Valuable !ii i i i !i!i!i i i!.i!. . . . . . . !i.i.!.i!.i i.!.i .i....... i.i!i i!i i !:ii :i Secondary i !!ii i i i Metabolites ..................

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.....................Javier

Barrios-Gonzdlez & Armando Mejia

ii!ii~iii~iil 14.1. INTRODUCTION Secondary metabolites are compounds produced mainly by actinomycetes and fungi, usually late in the growth cycle (idiophase). Although antibiotics are the best known secondary metabolites, there are others with an enormous range of other biological activities. Moreover, the last two decades have been a phase of rapid discovery of new activities and development of major compounds of use in different industrial fields, mainly: pharmaceutical and cosmetics, food, agriculture and farming. Some examples are: anti-inflammatory, hypotensive, antitumor, anticholestrolemic, but also insecticides, plant growth regulators and environmental friendly herbicides and pesticides. These compounds are usually produced by liquid submerged fermentation, but many of these metabolites could be advantageously produced by solid-state fermentation. Although solid-state fermentation (SSF) systems have been used in several oriental countries since antiquity, SSF has been transformed, in the last 25 years, for new purposes using new approaches of microbiology, biochemistry and biochemical engineering. This higher degree of control has allowed the use of SSF to produce sophisticated and valuable molecules like secondary metabolites (SMs). Ten or even five years ago reviews on this field pointed out that SSF was an emerging technology with great potential for the production of SMs at industrial scale. The authors commented that mycelial morphology associated with the microorganisms used for secondary metabolite production is well suited for growth on a solid support. Also that SSF presents advantages like" higher product yield, often in shorter times, higher product stability, lower energy requirements; while some disadvantages, like more complicated scale-ups as well as difficulties in monitoring and controlling process parameters, were also mentioned (Barrios-Gonz~lez & Mejfa 1996; Balakrishnan & Pandey 1996; Robinson et al., 2001).

Today, industrial SMs production by SSF is a reality. Some years ago an Indian company started industrial scale production of some secondary metabolites. It has since become a successful enterprise and the Food and Drug Administration (FDA) of the USA has approved the technology (SSF) developed by Biocon India for the production of fungal metabolites for human application (Suryanarayan 2003). In the near future the competition, between conventional SmF and SSF processes, promises to be tougher and more interesting. In the last 10 years the study of secondary metabolite (SM) production by SSF has been characterized not only by an increase in the number of publications, but also by the increase in the proportion of SMs with biological activities different from antibiotics. Another interesting feature of this stage is the surprisingly high productivity of SMs obtained in the processes designed in these studies. Ten years ago, a similar review described only one process with a production level above 7 mg/g (BarriosGonzfilez & Mejfa 1996), while higher yields are quite common in recent work (see Table 2). As was 10 years ago, SM production in solid culture was most often studied in SSF on natural substrates. This trend is represented by a considerable amount of pragmatic work directed to design high producing processes for particular SMs, most of them using agricultural products or wastes (SSF on natural substrates). Authors have looked for more efficient solid substrates for the production of particular SM. Generally, the same work is followed by the search of nutritional supplements and/or process conditions, often by sophisticated statistical methods. As a result, this new stage is characterized by a surprisingly high production yield of different SMs. SSF has also been studied using inert solid support materials, which greatly facilitates basic studies. However, production yields as high as the ones obtained in SSF on solid substrates are also reported in these systems. Basic studies have also been performed on simplified model SSF systems, like membrane cultures (on agar media in Petri dishes). However, Rahardjo et al., (2004) have warned for a careful use of this model system since it could be artificial and not describing the actual SSF. The authors showed that this kind of culture presents a different metabolism and kinetics, in relation with SSF. Many advantages of SSF are a consequence of the different physiology shown by fungi and other microorganisms on a solid substrate, in relation to the one presented in SmF. The molecular and physiological reasons underlying the different behavior of microorganisms in SSF are presently not well characterized, but a deeper understanding of this physiology is required to explore the possibilities for controlling or directing product formation in SSE This information would also be of key importance to design efficient methods for strain improvement for these processes.

14.2 SECONDARY METABOLISM 14.2.1 General aspects Secondary metabolites are compounds with varied and sophisticated chemical structures, produced by strains of certain microbial species, and by some plants. Although antibiotics are the best known SMs, there are other such metabolites with an enormous range of biological activities, hence acquiring actual or potential industrial importance. These compounds do not play a physiological role during exponential phase of growth. Moreover, they have been described as SMs in opposition to primary metabolites (like amino acids, nucleotides, lipids and carbohydrates), that are essential for growth. A characteristic of secondary metabolism is that the metabolites are usually not produced during the phase of rapid growth (trophophase), but are synthesized during a subsequent production stage (idiophase). From studies in liquid medium, it is now known that production of SMs starts when growth is limited by the exhaustion of one key nutrient: carbon, nitrogen or phosphate source. For example, penicillin biosynthesis by Penicillium chrysogenum starts when glucose is exhausted from the culture medium and the fungus starts consuming lactose, a less readily utilized sugar. Most SMs of economic importance are produced by actinomycetes, particularly of the genus Streptomyces, and by fungi.

14.2.2 Biosynthetic families Microbial SMs show an enormous diversity of chemical structures. However, their biosynthetic pathways link them to the more uniform network of primary metabolism. It has been shown that SMs are formed by pathways which branch off from primary metabolism at a relatively small number of points, which define broad biosynthetic categories or families (Barrios-Gonz~ilez et al., 2003). 1) Metabolites derived from shikimic acid (aromatic amino acids). Examples are ergot alkaloids and the antibiotics like candicidin. 2) Metabolites derived from amino acids.This family includes the 13 lactam antibiotics: penicillin, cephalosporins and cephamycins, as well as cyclic peptide antibiotics such as gramicidin or the immunosupressive agent cyclosporin. 3) Metabolites derived from Acetyl-CoA (and related compounds, including Krebs cycle intermediates). This family can be subdivided into polyketides and terpenes. Examples of the former group include the antibiotic

erythromycin and the hipocholesterolemic agent lovastatin. An example of the second group is the plant growth regulator gibberellic acid, as well as carotenoid pigments. 4) Metabolites derived from sugars. Examples of SM in this group are streptomycin and kanamycin.

14.2.3 Genetic regulation Since secondary biosynthetic routes are related to the primary metabolic pathways and use the same intermediates, regulatory mechanisms such as: induction, carbon catabolite regulation and/or feedback regulate their biosynthesis. These pathway-specific regulatory mechanisms operate in conjunction with an overall control which is linked to growth rate. In submerged fermentations (SmF), these regulatory mechanisms are by-passed (or taken advantage of) by environmental manipulations. Hence, an inducer such as methionine is added to cephalosporin fermentations, phosphate is restricted in chlortetracycline fermentation, and glucose is avoided in fermentations like penicillin or erythromycin. The fermentation processes of antibiotics regulated by carbon are now conduced with slowly utilized sources of carbon, generally lactose. When glucose is used, it is usually fed at a slow, continuous rate to avoid catabolite regulation. Also nitrogen sources like soybean meal are used to avoid nitrogen (ammonium) regulation. Although, the so called precursor effect is not a regulatory mechanism, in some cases, a precursor is used to increase one specific desirable metabolite, for example lysine is added as precursor and cofactor to stimulate cephamycin production by Streptomyces clavuligerus (Khetan et al., 1999; Barrios-Gonz~ilez et al., 2003). Studies on the molecular level have shown that genes of the biosynthetic pathways of SMs are clustered together in the genome of fungi and actinomycetes. In addition, studies have indicated that often a specific regulatory gene is embedded in these gene clusters. The co-regulation of these clusters can, in part, be explained by coordinated transcriptional control of biosynthetic genes by "narrow" or "broad" -domain transcription factors in fungi. The narrow pathway-specific regulators are usually found in the cluster and positively regulate gene expression. A typical example is AflR (aflatoxin regulator) (Woloshuk et al., 1994). Cluster regulators that are not found in the cluster itself include 2 regulators of cephalosporin production (Schmitt et al., 2004) and PENR1 for penicillins (Litzka et al., 1998).

Broad-domain factors are transcriptional factors that are important in integrating cellular responses to environmental parameters like C and N sources, temperature, etc. Responses to environmental signals are transmitted through (Cys2His2 zinc-finger) global transcription factors that mediate carbon (CreA), nitrogen and pH (PacC) signaling. Regulation by both narrow- and broad-domain transcription factors ensures that secondary metabolite pathways can respond to the demands of general cellular metabolism and the presence of specific pathway inducers (Keller et al., 2005).

14.2.4 New biological activities As mentioned above, the last two decades have been a phase of rapid discovery of new activities and development of major compounds of use in different industrial fields. This new era has been driven by modem strategies to find microbial SM. Earlier whole cell assay methods, like bioassays, are being replaced by new and sophisticated, target-directed, mode-of-action screens. In this way, culture broths of new isolates are tested in key enzymatic reactions or as antagonistic or agonistic of particular receptors. This new approach relies on the knowledge of the biochemical and molecular details of different diseases or physiological processes (Barrios-Gonz~ilez et al., 2003; Barrios-Gonzfilez et al., 2004). This growing wealth of bioactive compounds is usually produced by liquid submerged fermentation, but many of these metabolites could be advantageously produced by solid-state fermentation. Table 1 presents the biological activities and applications of some of the SMs that have been produced in high yields by SSE Other SMs that have been produced in this culture system include the coconut aroma compound, 6-pentyl-a-pyrone (Sarhy-Bagnon et al., 2000), and the cyclodepsipeptides dextruxins A and B; compounds that display insecticidal and antiviral activity (Liu & Tzeng 1999). Also, the novel tetramic acid antibiotic conoicetin, which shows a pronounced antibacterial and antifungal action, inhibiting even multidrugresistant strains of Staphylococcus aureus has been produced by SSE Interestingly, the producing fungus, C. ellipsoida, synthesizes this antibiotic only in SSF, although it grows well in SmF (Segreth et al., 2003).

14.3 MODERN TYPES OF SSF Two types of SSF systems can be distinguished depending on the nature of the solid phase used. 14.3.1 SSF on solid natural substrates This is the most commonly used (and most often described) system and involves

Table 1. Biological activities and applications of some fungal secondary metabolites that have been produced by SSF,

the cultivation of microorganisms on agricultural products, such as rice, wheat bran, etc.; this system is referred to as cultivation on solid substrates or on natural substrates. Cultivation on solid substrates uses materials that serve both as a support and as nutrients source. These materials are usually starchy, or lignocellulosic agricultural products such as grains and grains by-products, as well as agro corps and their residues such as cassava, potato, beans, sugar beet pulp, etc. This '~culture system has the advantage of using cheap unprocessed

or moderately processed agricultural raw materials. However, SSF on solid substrates has a disadvantage: the carbon source constitutes part of their physical structure. During the growth of microorganisms, the solid medium can be partially degraded, and as a result the physical characteristics of the medium change, particularly in long cultures like secondary metabolite fermentations. As a result, heat and mass transfer can be reduced (Ooijkaas et al., 2000; Barrios-Gonz~ilez & Mejfa 1996).

14.3.2 SSF on inert support This system involves the cultivation of microorganisms on an inert support impregnated with a liquid medium. In this SSF, the inert support serves not only as a reservoir of nutrients (in a liquid phase), but also as an anchor point for the microorganism. The abovementioned disadvantage can be overcome by the use of inert supports which have a relatively constant physical structure throughout the process, enabling improved control of heat and mass transfer. Another advantage of SSF on inert supports, relative to SSF on natural substrates, is its less complicated product recovery. Extra- or even intracellular products can be extracted quite easily from the inert support, hence the products can be obtained with less impurities. Perhaps the most important advantage of this system is that it facilitates basic studies, since medium composition can be precisely designed. In this way a defined medium can be used and the effect of different compounds, on production, tested. Besides, the liquid medium can be extracted from the inert support at any time of the culture and its components analyzed. From the applied side, this system allows the design of adequate production media, and mass balances for more-advanced process modeling and process control are more easily performed, since concentrations of all the nutrients in the production medium are known. On the other hand, SSF on inert support has been used with two variants: a) SSF on natural inert support, such as sugar cane bagasse; b) SSF on artificial inert support, such as polyurethane foam, polystyrene, amberlite, vermiculite, perlite, pozolano particles (volcanic material) and clay granules (Ooijkaas et al., 2000; Bafios et al., 2007a). The potential of SSF on impregnated inert supports as a commercial system has been analyzed. One conclusion is that the higher costs of defined media make solid substrate the preferred choice, especially for low-cost products. However, for high-added-value products, such as secondary metabolites, SSF on inert supports can be used because the medium costs are normally a fraction of the overall production costs. Ooijkaas et al., (2000) give, as an example, the cost calculation for spore production on defined media on inert support. The authors show that fermentation costs account for less than 20% of the

production costs. In addition, when downstream processing, formulation and registration are also considered, the medium costs will become even less important. Furthermore, it is known that downstream processing can markedly affect the overall production costs owing to recovery losses. Because downstIeam processing is simplified and improved with inert supports, it is anticipated that this will affect the overall production costs in a positive way, and consequently outweighing the higher medium costs (Ooijkaas et al., 2000). From the analysis of studies of SM production by SSF on solid substrates and on inert support, conclusions are beginning to emerge, often complementary, which allow a clearer vision of how to produce SMs on SSF, as well as its potential to the future.

14.4 DEVELOPMENT OF SSF PROCESSES FOR THE PRODUCTION OF SMS 14.4.1 Selection of solid substrates The possibility of altering the medium composition and investigating the influence of certain medium components is limited when using natural substrates. Modifying the type and concentration of the medium is indirectly carried out by changing the grain or agricultural residue, by mixing agricultural products with each other and/or by supplementation with nutrients solution. The solid substrate role is also related to physical structure and thus water retention capacity and porosity. Studies to develop a production system for a particular SM on SSF on solid substrate generally start by establishing the best (basic) solid substrate. This is done by comparing SM production yields on different agricultural products or residues, like: wheat bran, barley or wheat rawa. Analyzing the research work of the last years, that achieved high yields of fungal SMs (Table 2), it is possible to draw several conclusions about the choice of solid substrate. Wheat bran is the most frequently used solid substrate and also a very effective one. Five of the 10 more productive processes, for different fungal SMs, used WB. Moreover, it is has also been successful in processes with actinomycetes and (endospore forming) bacteria (Table 3), since 3 of the 7 more productive processes use wheat bran. This can be further illustrated by the case of rifamycin SV production process, where an impressive yield of 32 mg/g was achieved in 7 days (Krishna et al., 2003). Wheat rawa is a substrate that has been used lately as solid substrate for the production of some SM of fungi and actinomycetes with excellent results. The only example of the use of this substrate in the production of fungal metabolites is in the biosynthesis of cephalosporin C where a very high production

Table 2. Production yields, of fungal SMs, by SSF on different natural substrates and by SSF on inert supports.

Table 3. SM production, by actinomycetes and Bacillus sp, in SSF on different natural substrates.

(22.28 mg/g) was achieved in a relatively short period (5days) (Adinarayana et al., 2003). With actinomycetes a good example of the use of wheat rawa is the production of 17.15 mg of neomycin per g of dry medium by Adinarayana et al., (2003b), second highest in the actinomycets group. Kota & Sridhar (1999) used wheat rawa in combination (1:1) with cotton seed de-oiled cake, reaching a yield of 15 mg of cephamycin per g, also in 5 days. Few other solid substrates have given high yields. One case is the use of rice bran by Saykhedkar & Singhal (2004) to produce the antifungal griseofulvin

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(9.732 mg/g) in 9 days. The use of groundnut (peanut) shells as a solid substrate to produce 13.18 mg/g of tetracycline in 5 days is interesting (Asagbra et al., 2005). This substrate, because of its cellulosic nature and physical structure, approaches to an inert support, as does the solid substrate (corn-cob) used for the production of oxytetracyclin (7.5 mg/g in 8 days); both fermentations performed with actinomycetes. The use of rice as solid substrates has given good results only when the fungus Monascus sp. is used. Rice is a typical substrate for this fungus, since it is used since antiquity in a traditional fermentation to produce the Chinese food additive red rice. A relatively high production level of lovastatin (monacolin K) (12 mg/g) was obtained with the use of rice as a solid substrate by a strain of this fungus, although in a relatively long time (35 days) (Xu et al., 2004). Hence, rice has also been the solid substrate used in processes to produce biopigments (Carvalho et al.,, 2006) and in a process to produce lovastatin together with ~(-aminobutyric acid (a hypotensive drug) aiming to produce a multifunctional dietary supplement (Suet al., 2003). Logical substrates, for certain fungi, have worked well for the production of their SMs. In this way, ergot alkaloids production by Claviceps species was higher in SSF on rye than on impregnated bagasse (Trejo et al., 1992; Trejo et al., 1993; Barrios-Gonz~ilez & Mejia 1996). On the other hand, it is interesting to note that some particular SMs are not synthesized in some natural substrates. The study on nigerloxin production by SSF found that this metabolite was not produced when corncob, rice husks, bagasse or tapioca wastes were used as solid substrates. In other studies, where different solid substrates were compared, lovastatin production was higher in wheat bran, followed by barley and much less produced in gram bran or bagasse. Worst results were obtained using fruit waste like orange and pineapple epicarp (6 times lower production), which do not seem like a good option for SM production (Valera et al., 2005). In cephalosporin C, production was better with wheat rawa, and a bit lower with wheat bran. Comparatively lower yields were obtained with rice bran.

14.4.2 SSF on inert support systems The use of supports impregnated with liquid medium offers additional flexibility when designing the medium for optimal production of metabolites. SM production studies on inert support have used media recommended for SmF and optimized its overall concentration. The optimum is usually between 2 and 2.5 times the concentration used in SmF (Barrios-Gonz~lez et al., 1988, Dominguez et al., 2000, Sarhy-Bagnon et al., 2000, Bafios et al., 2006a). Once the concentration level is established, optimal concentration of individual nutrients can be determined

by, for example, central composite designs and response surface analysis methodology. In this way optimum values can be found with less number of experiments, as is done in SSF on solid substrate (see next section). Although SSF on inert support is very convenient for basic studies, previous review work already indicated that solid fermentation on impregnated bagasse (natural inert support) could be very productive, with the case of penicillin (13 mg/g) (Barrios-Gonz~ilez & Mej/a 1996; Barrios-Gonz~ilez et al., 1993). Very recent results show that artificial inert supports can have an excellent performance for the production of SMs. Bafios et al., (2006a) designed a lovastatin production process by SSF on artificial inert support that reached 19.95 mg/g in 7 days. These studies were performed with A.terreus TUB F-514, a wild strain isolated from a soil sample from Irak by Dr. Szakacs. The production of this strain, in SmF with exactly the same medium, was only 0.62 mg/ml. This strain was also tested in SSF on sugarcane bagasse impregnated with the same liquid medium, and final yield was 8.615 mg/g. It is yet not clear if the relatively better performance of the artificial inert support system is a general feature or if it depends on the particular fungus and metabolite. In a second stage of this work, A.terreus TUB F-514 was mutated and a mutant strain selected by rational screening methods (see Strain Improvement section). The new mutant was cultured in the artificial support system producing 27.89 mg of lovastatin per g (Bafios et al., 2006b). On the other hand, in our earlier studies on penicillin production by SSF, bagasse was impregnated with a concentrated liquid medium which included some insoluble components. Results indicated that penicillin production was strongly controlled by the proportions of bagasse, nutrients and water. Interestingly, high antibiotic production was obtained with different combinations, but low bagasse content (10 to 12%) of the solid medium was a common feature (see the end of section 4.4.3).

14.4.3 Optimization of nutritional factors

14.4.3.1 Selection of supplements In SSF on solid substrates, modification of the type and concentration of nutrients is done indirectly by selection of the solid substrate. However, a further and finer balance of the solid medium composition, for optimal metabolite production, is done by the addition of supplements. Besides complementing the C and N sources and minerals in the substrate, these supplements can contain inductors or precursors. C sources can be mono-, disaccharides or polysaccharides like starch. N sources are usually inorganic or organic with grains or cereal flours (mixed C and N sources). Typically, this stage is performed by testing a number of potential C, N and complex organic sources, as well

as minerals. Their impact on production can be evaluated, at a fixed concentration, by Plackett-Burman design, and compared with a control without supplements. In this way the "effective nutrients" are identified. In a second stage, the effective nutrients concentration is optimized by a response surface methodology (Murthy et al., 1999; Adinarayana et al., 2003a). Research work has shown that the optimization of supplements has played a key role in increasing production yields with different metabolites and solid substrates. During optimization of cephalosporin C production by SSF on wheat rawa, production of the antibiotic increased 216% after the optimization of additional C and N sources; while these operations accounted for a 94% increase in cyclosporin A production (Table 4). However, there are also examples of very high yields on wheat bran without supplements. Valera et al., (2005) indicated that for lovastatin production, supplementation with external C sources like sucrose and lactose and N sources such as ammonium sulphate and ammonium nitrate, inhibited the metabolite production. As can be seen in Tables 2, 3 and 4, although a great number of supplements have been tested, a relatively small group is giving good results. Often soluble starch has been successfully used as additional C source, as has yeast extract as N source. Also some cereal or grain flours like millet, ragi (finger millet) or jowar (sorghum) or seeds de-oiled cakes like peanut (groundnut) or sunflower de-oiled cakes have given good results as complex organic sources. Nevertheless, another conclusion is that each microorganism and metabolite has different requirements. These requirements go from no supplements for A. flavipeps in lovastatin fermentation, passing through the requirement of only trisodium citrate for nigerloxin production by A.niger to very complicated requirements for P. brevi-compactum in the production of mycophenolic acid. (these SSFs on wheat bran). Another aspect to have in mind when developing solid media for SM production is that studies in liquid culture have shown that C, N or P sources can have a negative effect on SM production, due to regulatory mechanisms such as carbon catabolite repression. Earlier work of our group, studying aflatoxin production on cassava SSF, showed that when the concentration of phosphate and ammonium were reduced in the solid medium, aflatoxin production increased proportionally (Barfios-Gonz~ilez et al., 1990). Since aflatoxin biosynthesis is regulated by nitrogen and phosphate, these results suggested that regulatory mechanisms were also active in SSE Making use of the advantage of SSF on inert support for basic studies, catabolite repression of penicillin biosynthesis was studied in SSF on sugarcane bagasse, and compared with parallel experiments in SmF. Regulatory thresholds were estimated in both systems by carefully correlating

Table 4. Impact of supplements optimization on secondary metabolites production by SSF

glucose uptake kinetics with the initiation of penicillin biosynthesis. Results (SmF = 28 - 20 g/l; SSF = 3 6 - 14 g/l) showed that penicillin biosynthesis is also regulated by carbon catabolite regulation in SSF and at similar thresholds as the ones observed in SmF (Garcfa et al., 1993). These studies indicate that mechanisms that regulate the biosynthesis of secondary metabolites also play a role in solid culture. Hence, glucose should be used carefully in fermentations regulated by C as inorganic N and phosphate sources in fermentations regulated by N or phosphate. Regulatory mechanisms in SSF could explain the success in the use of polysaccharides like starch and/or complex nitrogen organic supplements, that bypass these mechanisms by slowly dosing glucose or ammonium.

14.4.3.2 Use of inductors and precursors In liquid culture medium, the addition of a precursor (biosynthetic pathway intermediate) that is rate limiting, during antibiotic and other SM biosynthesis, brings about an increase in production and/or directs the synthesis preferentially towards one of the products of the related metabolites that would be formed otherwise. No specific studies on this subject have been performed in SSE however, the effect of triptophane on alkaloid production by SSF reported by Trejo et al., (1992), as well as the increase in tetracycline production by the addition of methionine indicates a precursor effect (Banfos-Gonz~ilelz &Mejfa 1996). In our own research on penicillin production, phenylacetic acid has been used as a precursor for penicillin G. HPLC analysis of extracts from SSF samples have shown that only penicillin G is produced and no evidence of the synthesis of other penicillins has been found (unpublished results). Recently,

Saykhedkar & Singhal (2004) reported that the addition of 0.1% of choline chloride, as a precursor, produced a 76% increase in the yield of griseofulvin in SSE This compound is a methyl donor and increases the rate of biosynthesis in liquid cultures. This information indicates that the precursor effect also manifests in SSF, so has to be considered with the supplements. On the other hand, it probable that barbital, added to rifamycin SSF (Krishna et al., 2003), was not necessary due to good aeration in this culture system. It was previously thought that barbital was a precursor in the synthesis of rifamycin V, since it increased its production (decreasing rifamycin W concentration in the culture broth) in industrial SmFs. In a previous work we showed that barbital effect was indirect by partially inhibiting respiratory chain, hence liberating more 02 towards biosynthesis, for the oxidation step leading to rifamycin B in a viscous industrial fermentation with low 02 transfer (Mejfa et al., 2003).

14.4.4 Optimization of environmental factors

14.4.4.1 Substrate pretreatment Natural substrates and even inert supports generally require some kind of pretreatment to make their chemical constituents more accessible and their physical structure more susceptible to mycelial penetration and adhesion. Moreover, it also improves water retention capacity (penetration and distribution). Early work in or laboratory was directed towards using sugar cane bagasse as an inert support for the production of penicillin by P.chrysogenum, but growth and production was extremely low. Microscopic observations, using colored water, indicated that the liquid was not penetrating the bagasse fibers. It was noticed that, after thermal pretreatment in the autoclave, water could penetrate the fibers. When this pretreated bagasse was used for SSF, abundant growth and antibiotic production was finally obtained (unpublished results). Solid substrates are generally moistened with distilled water (that can include salts solution) and then autoclaved from 20 minutes to 1 hour. A variation that can be used with wheat bran is acid pretreatment. Since the early studies on gibberellic acid, performed by Kumar & Lonsane (1987), the authors described an acid pretreatment: mixing the wheat bran with 60% mineral salts-acid solution of 0.2 N acidity (HC1) before autoclaving. Recent work of Krishna et al., (2003) showed that rifamycin SV production increased nearly 300% when the substrate was treated with 0.2 M HC1. Although similar growth was obtained when wheat bran was pretreated by moistening with distilled water before autoclave, antibiotic production was much lower. The authors comment that acid pretreatment probably influenced the breakdown of starch and protein, and improved the water distribution. Also Sekar et al., (1997) reported that cyclosporin A production in SSF was increased from 0.16 to 1.14 mg/g by acid pretreatment of the substrate (wheat bran).

14.4.4.2 Particle size After pretreatment, solid substrates are usually ground and sieved to a certain average particle size, For wheat bran particle sizes around 0.4 - 0.7 mm are usually employed in recent research. However, in early studies, Kumar & Lonsane (1987) reported an increase in gibberellic acid production with the use of 0.30.4 cm, in relation with smaller particles. With wheat rawa, Ellaiah et al., (2004) found coarse size (> 0.84 mm) to give the best results in neomycin production. 14.4.4.3 Moisture content and water activity Water is the main component of microorganisms, and in SSF has a key role in enzymes, nutrients and products diffusion through the solid matrix. However, the importance of this parameter appears to be more critical for SM formation, probably since initial conditions must generate an adequate environment for production phase (idiophase) after the rapid growth phase (Barfios-Gonz~ilez & Mejfa 1996). In SSF, the best initial moisture content (IMC) value depends on the nature of the substrate, mainly on its water retention capacity. Hence, different substrates will show different ranges of IMC for optimal metabolites production. Recent studies on SM production by SSF do not show very impressive production increases by optimizing IMC anymore. This is because the optimal ranges for the most usual substrates are already known, so the initial values are not far from the optimum. Thus, the study using wheat rawa obtained best production at 80% IMC, although different metabolites were studied using S.marinensis, S.clavuligerus and A.chrysogenum. Works with wheat bran show optimum values at around 70% (cyclosporin C and mycophenolic acid), while best nigerloxin and lovastatin production were obtained at 65% IMC. It is interesting to note that rifamycin SV SSF fermentation was carried out at 80% IMC with very high yields. Less common solid substrates showed optimum values at different IMC: groundnut shells 67% > corncob 65.5 > rice 53 to 65% >rice bran 50%. Water activity (aw) is related to the concentration of solutes in a liquid medium and represents the unbounded, and therefore available water. Through indirect calculations it has been proposed that in cassava SSF, Aw decreases during the culture (Oriol et al., 1988a; Oriol et al., 1988b). This has been recently confirmed in a wheat bran SSF system (Corona et al., 2005), showing that water availability for the microorganism decreases strongly during the solid culture of Gibberella fujikuroi. Conversely, higher gibberellic acid production yields were obtained at higher Aw values (1 - 0.96), although this last experiment was performed on a model system (agar culture on Petri dish). Interestingly, Sekar et al., (1998)

obtained a higher yield of cyclosporin A (1.92 mg/g), when the solid culture was incubated in a chamber with controlled relative humidity at the highest value (95%); while SSF incubated at lower values gave lower yields. Cultures incubated at 80% relative humidity only produced 0.98 mg/g and inspection of the solid culture indicated dryer zones where growth was poor. Oriol et al., (1988b), working with A. niger in SSF using sugarcane bagasse with absorbed liquid medium, showed that Aw controls growth rate in SSF, being this parameter higher at higher water activity values. The authors found that when IMC was varied between 40 and 75%, but keeping Aw of the medium constant, growth rate was not modified (m=0.4 h-l). However, BarriosGonz~ilez et al., (1988), using the same SSF system and keeping Aw constant, showed that IMC had a very important impact on penicillin production level in SSF. In the same work, experiments were performed in which nutrients concentration was increased (decreasing Aw), keeping IMC constant at 70%. It was found that the use of very concentrated media favors the antibiotic production in SSF. This was an important effect, since penicillin yields increased 5-fold in 2X concentrated medium (twice the recommended concentration for SmF). Conversely, the use of concentrated media negatively affected production in SmE In a later study, using the same system, it was observed that the same IMC could be reached by using different combinations of the solid phase (represented by the nutrients + support). And that this gave different penicillin production yields. It was observed that when using different combinations of support nutrients and water, higher penicillin yields were obtained in those conditions when support was kept low (10-12.5%). It was not clear why such different combinations could create a favorable environment for production during idiophase (Dom/nguez et al., 2001). Respirometric analysis (derivative form) showed that growth phase (trophophase) is observed like a steadily rising peak (CO2 production or 02 uptake). The transition to idiophase is seen when the rising peak falls, indicating that growth was limited by exhaustion of the key nutrient (in that case glucose). Idiophase is seen as a usually lower and wider second peak, it is during this period that secondary metabolites are synthesized. With the above mentioned information, it was possible to identify a high production respiratory profile. Results showed that a low and steady respiration profiles during idiophase (relatively horizontal and wide second peak) correlated with high penicillin production. While higher and variable respiration patterns in this phase were associated with low production conditions (Dominguez et al., 2000). These studies show that in SSF, like in liquid culture, idiophase starts when growth is limited by the exhaustion of a key nutrient and that .productive

idiophases are characterized by low and constant growth rates. The way to achieve this, in the case of penicillin SSF, was the use of conditions (low support proportion) that permit a slow but constant nutrients supply during idiophase, supporting slower but constant growth rates for longer periods. Very probably, similar respiration patterns are being obtained in the high yielding SSF processes for SMs described in previous sections. It is also possible that other SSF variables can also be manipulated to achieve this high production respiration pattern. On the other hand, a practical consideration on this subject is that evaporation is an effective mechanism for the removal of metabolic heat produced during the cultivation in large-scale SSF reactors (Guti6rrezRojas et al., 1996). However, evaporation can affect water activity (Aw) and might have and adverse effect on microbial metabolism (Weber et al., 1999; Larroche & Gros 1992; Oriol et al., 1988). Replenishment of the lost water is therefore required. In non-mixed systems this is impossible, so solid substrates should be able to release a large amount of water without affecting the Aw (Weber et al., 1999). The alternative is mixing of the medium, which enables the possibility of homogenous water addition besides promoting mass and heat transfer.

14.4.4.4 Aeration One strategy used to assure adequate oxygen availability to the solid culture, is diffusive aeration by using static shallow beds of solid medium. Working with wheat bran SSF, Sekar & Balaram (1999) found that when the thickness of the bed was increased from 1 to 2 cm, there was a significant reduction in yield of cyclosporin A; indicating a limitation in available oxygen and moisture (trays were incubated in controlled relative humidity chambers) as the depth increased. Rao et al., (2005) studied the area of exposed surface on production of nigerloxin, by varying the ratio of the quantity of wheat bran to the flask volume. The optimum yield of the product was obtained at a ratio of 1:20. The second aeration strategy is to employ forced aeration in more complex fermenters. Valera et al., (2005) studied this parameter in lovastatin production in a 2 1 reactor with a load of 200 g of solid culture. Lovastatin yield improved with airflow rate, reaching a maximum at 1 vvm. Carvalho et al., (2006) also found an optimum of 1 ml of air per gram of wet substrate per minute, in the production of Monascus pigments in rice SSF, using a system of aerated glass columns. Although in the latter study, higher aeration rates had a deleterious effect on production.

14.4.4.5 Mixing Mixing of the medium prevents heterogeneity of the solid medium composition and mycelial age. It breaks long mycelial nets, generating shorter mycelia in similar physiological state (Barrios-Gonz~ilez & Mejfa 1996). Mixing enables the possibility of homogenous water addition besides promoting mass and heat transfer. However, it has been reported that some fungi do not tolerate mixing in SSF (Mudgett 1986), possibly since this operation not only can break mycelia but can disturb mycelial attachment to the solid medium. Our group studied the effect of mixing on a penicillin production on impregnated bagasse (unpublished results and Barrios-Gonz~ilez & Mejfa 1996). Results showed a positive effect of manual mixing once a day on product formation (4, vs 5.7 mg/g) and, interestingly, an important increase in metabolic activity: 0.32 g of total CO2/g dry culture vs 0.44 g/g at 96 h (Fig.l). Since in these experiments mixing operation included unpacking each column fermenter, mixing and re packing, it was noticed that the solid culture showed moisture loss. When the experiment was performed restituting moisture loss, even higher penicillin production yields were reached (Fig. 2). Image analysis indicated that the microbial population was formed by shorter mycelia (approximately 200 vs 500 p), that presented higher branching frequency (branching every 67 _+ 25 vs 240 _ 70 Ia). In other words, a higher number of growing hyphal tips, giving rise to a higher metabolic activity and probably to a greater secretion surface, as well as greater physiological homogeneity of mycelia in the solid culture (Barrios-Gonz~ilez & Mejfa 1996). This way of creating a higher branching frequency is an alternative to the genetic approach (See Stain Improvement section). In a subsequent work, using a 2 Kg rotating drum-type solid fermenter, it was confirmed that mixing had a positive effect on penicillin production. However, highest production was obtained with a mixing time of 2 minutes per day (at 5 rpm), while production decreased slightly with 4 minutes per day, and no antibiotic was produced with 20 minutes of mixing per day (Miranda et al., 2003). Recently, Valera et al., (2005) also used intermittent stirring in a 2 1 reactor for the production of lovastatin. The case of griseofulvin is interesting since high antibiotic production (9.732 mg/g) was obtained by SSF on rice bran that was incubated under very severe agitation conditions, at 250 rpm in flasks (Saykhedar & Singhal 2004). This is quite uncommon and probably the only antecedent is the very early work carried out by Hesseltine and the group in NRRL with aflatoxin production on rice SSE The authors found a very high increase in production when a flask with rice fermentation was agitated in shaker (200 rpm) as if it contained liquid medium. A relatively very high concentration of aflatoxins was obtained in this way (1.5 mg/g) (Hesseltine 1972).

Fig. 1 Respiration (integrated form) of PenicilliumchrysogenumP2 during SSF; static (--), and mixing once a day (---).

Fig. 2 Penicillin production by SSF: mixed once every 24 h (e) and static (0).

14.4.4.6 pH It is considered that global pH of the liquid phase of a SSF can be considerably different than the local pH values on the solid surfaces where growth is taking place, due to the superficial charge effects and the ionic equilibrium modified by the effect of solute transport (Mudgett, 1986). A general procedure to for pH measurement in SSF is to determine global pH, after suspension of a sample in a 10 times grater volume of water. However, there are no comparative studies (SSF vs SmF) on the effect of pH on secondary metabolism. Very recent results in our laboratory indicated that this global value of pH in the solid culture relates very well with the pH sensed by the fungus in the SSE

Studying cephalosporin C biosynthesis in bagasse SSF, it was found that pH is a key parameter since it only allows biosynthesis in a relatively narrow range of pH values. If this control was also displayed in SmF this phenomenon could be used as a very precise biosensor to describe the microenvironment sensed by the fungus in SSE Comparative studies were performed, using the same strain and exactly the same medium composition. Results showed that behavior of pH was similar in both culture systems and, more important, that the antibiotic synthesis was also controlled by pH in SmF and by exactly the same range of pH (Cuadra et al., 2007).

14.4.4.7 Inoculum type and size In SSF inoculum must be distributed homogeneously and must be high enough to assure predominance of the strain. Two different strategies are commonly being used, that is, inoculating with spores or inoculating with mycelium that comes from a previous stage of liquid culture (seed culture). Among the studies in which spore inoculum was used, the work on alkaloid production on rye grains is interesting since the effect of inoculum size was determined (Trejo, 1992). Inoculum size was varied between 2 X 10 6 and 2 X 108 spores/g of dry medium. Results showed that, by increasing inoculum size, lag phase (or germination) was slightly reduced, while growth rate was increased. Optimum concentration for alkaloid production was 2 X 10 7 spores/g. Interestingly, lovastatin production by SSF on wheat bran was performed with a similar inoculum size i.e. 2.5 X 10 7 spores/g (Valera et al., 2005). Also, SSF with actinomycetes have been inoculated with spores. Asagbra et al., (2005) tested the effect of inoculum size on the-production of tetracycline on peanut shells SSE The authors found 108 spores/g to be optimal, while the use of 101~ and specially lower that 10 6 produced lower yields. Kota & Sridhar (1999) also found 108 spores/g to be an optimum inoculum level for the production of cephamycin C. On the other hand, SSFs for SMs production are often inoculated with mycelium generated in a previous stage of liquid seed culture. Using a strain of the fungus Achremonium chrysogenum, Adinarayana et al., (2003b) found that inoculating cephalosporin C SSF with 10% w/v of seed culture produced best results. In these experiments, increasing inoculum size from 5 to 10 % gave a production increase of 25%. In the case of cyclosporin A, Sekar et al., (1998) found that size and type of inoculum had significant impact on the immunosuppressant yield in SSF. The culture inoculated with mycelium (30%) was significantly higher than the culture inoculated with spores. However, it is important to note that spores were produced in SmF. Also that, as authors indicated, the fermentation inoculated with spores lagged approximately

2 days behind the one inoculated with mycelium, so yields might have not been so different if this culture had been sampled for 2 more days. Also, the authors did not test higher spore inoculum size. In any case, experiments performed in this work showed that increasing inoculum size (with seed culture) from 20 to 30% caused a 50% increase in cyclosporin A production. In a subsequent report, this group optimized the mycelium inoculum to 60% of seed liquid culture. Very high inoculum sizes with mycelium have also been used in fermentations with actinomycetes. Krishna et al., (2003) obtained very high rifamycin yields in SSF on wheat bran. The process was inoculated with 40% seed culture. Moreover, the yield obtained with this inoculum size was 42% higher than the one obtained in cultures inoculated with 10%. Ellaiah et al., (2004) also observed a strong influence of high (mycelial) inoculum size on the production of neomycin.

14.5 MICROBIAL PHYSIOLOGY IN SSF The special physiology displayed by fungi and other microorganisms in SSF is presently poorly characterized. In fact, many advantages of SSF are a consequence of the different physiology shown by microorganisms in solid medium, in relation to the one presented in SmE Several differences (and advantages) have been described for enzymes production: SSF provides higher productivities, less prone to problems with substrate inhibition and yields enzymes with a higher temperature or pH stability (Htilker et al., 2004). In relation with SMs production by SSF, a clear difference is higher production, often in shorter periods. Moreover, sometimes the SM is produced only by SSF, even though the organism also displays good growth in SmF (Segreth et al., 2003). The molecular and physiological reasons underlying the different behavior of microorganisms in solid medium are presently not well characterized. A few works have studied the physiological differences that arise during the growth of microbial cells in the two types of culture systems (Biesebeke et al., 2002). In Aspergillus oryzae, water activity seems to play an important role in this context. This is apparent from the accumulation of polyols: glycerol, erythritol and arabitol in SSF conditions. Furthermore, glycerol and erythritol dehydrogenases were induced by osmotic stress conditions (Ruijter et al., 2004). Molecular differences in gene expression, depending on the fermentation technique, have been described by Ishida et al., (2000). The authors found A.oryzae has two different genes that code for glucoamylase: glaA and glaB; and that the latter is expressed only under SSF conditions, while glaA is synthesized in SmF. gla B was induced by low Aw, high temperature and physical barriers to hyphal extension; while promoter motifs were identified. Although, in the experiments

performed by Biesebeke et al., (2005) glaB was induced by maltose rather than by low Aw. Very recently our group performed molecular studies, comparing lovastatin production by A.terreus, in SSF and SmF. Results showed that the expression of a gene related to the adaptation of the fungus to low Aw conditions (glaw) was approximately 5 times higher in SSE Also, that the expression of genes of lovastatin biosynthetic pathway was 3 times higher in SSF, and expressed for a longer period. Higher expression of the gene related to low Aw seemed to be a characteristic of the physiology of SSF and maybe related to higher expression of lovastatin genes. However, in this experiment a more concentrated medium (2.5 times) was used in SSF, in relation to the one used in SmE Hence, another experiment was performed where exactly the same concentration of medium was used in both systems (2.5X). Results indicated that glaw induction was due to medium concentration and not to the culture system. Yet, in these new conditions, lovastatin genes expression in SmF was 10 times lower than in SSF, indicating that high lovastatin (SM) production is characteristic of the special physiology of SSE However, this expression is not due to low Aw, but to other (unknown) factors of SSF (Barrios-Gonz~ilez et al., 2007). These results show that higher production in SSF is partially explained by higher transcription of the biosynthetic genes in SSF, but this might not be the only factor, since transcription was 3 times higher in SSF, while specific lovastatin production in SSF was 13 times higher than in SmF (see next section).

14.6 STRAINS FOR SSF One of the major positive aspects of SSF is that metabolites are, in many cases, produced at much higher yields than by SmF (Barrios-Gonz~ilez et al., 1998; Barrios-Gonz~lez et al., 1993; Barrios-Gonz~lez & Mejia 1996; Balakrishnan & Pandey 1996; Roibinson et al., 2001). Recent reports on SM production by SSF, where production in SmF has been determined too, also support this claim. The parameter used in Table 5 to make this comparison is "relative production", which is calculated simply by dividing production in SSF by production in SmF. This value gives a rough idea of how many times is production in SSF higher than production in SmF, and hence how efficiently is this particular strain expressing its production potential in solid culture (Barrios-Gonzfilez et al., 1993). This table shows that fungi, actinomycetes and even bacteria (Bacillus) can be very efficient for SM production in SSE Although different products and species are shown, relative productions are very high and range from 6.7 to 97. This value is lower in P.chrysogenum strains developed for SmF (last two examples). It is important to note that precise comparisons using the same medium composition can only be done in SSF on inert support. Earlier studies of our group indicated that biochemical and physiological responses of

Table 5. Production yield of some secondary metabolites by SSF and SmF.

many microorganisms differ greatly from those in SmF, leading to discrepancies in their production level displayed in both culture systems (Barrios-Gonz~ilez et al., 1993; Barrios-Gonz~ilez & Mejia 1996). In that work we studied penicillin production, of several strains of P.chrysogenum, in SmF and in SSE These were strains, of different production levels, that had been developed for SmF and included an industrial strain. Some data of those experiments was included at the end of Table 5 (see P.chrysogenum P2 & Industrial). The first conclusion of this study was that higher penicillin yields in SSF could be achieved with strains that were higher producers in liquid medium. In this way, 1.345 mg/g were obtained with Wisconsin 54-1255, a strain that produced only 0.56 mg/

ml in SmF, while 2.084 mg/g were reached in SSF with strain P2, which was a higher producer than the latter in SmF (0.83 mg/ml). Moreover, the industrial strain reached the highest production level of 13 mg/g in SSE However, this was in a certain sense, disappointing since it was not so far from the production displayed by this strain in SmF (9.804 mg/ml). When we calculated relative productions, from this data, we were able to draw a second conclusion: higher producing strains, developed for SmF, tend to express their production potential less efficiently in SSF (lower PS/PLs) than do lower producing strains, that are closer to the wild type and that show higher PS/PL values. This means that during the course of the genetic improvement programs the industrial hyperproducing strains, developed for SmF, have been loosing some (unknown) functions that are important for good adaptation and performance in solid medium (Barrios-Gonz~ilez et al., 1993). Some aspects of more recent research also support this conclusion. Some of the studies reported in the last years used wild isolates to produce SMs by SSF with surprisingly high production yields. One example is the 5.06 mg of nigerloxin/g reached by Rao et al., (2005) using a wild isolate of A.niger. Asagbra et al., (2005) compared tetracycline production by SSF of several collection ~trains with the antibiotic yield of a locally isolated strain (Streptomyces sp.). Since this wild isolate displayed higher tetracycline titers in this culture system the authors optimized conditions and obtained 13.18 mg of tetracycline in a 6 days fermentation. This production level by a wild isolate would be practically impossible in a SmF process. Furthermore, our group designed a lovastatin production process in SSF on artificial inert support that reached 19.95 mg/g in 7 days (Bafios et al., 2006a). This very high production level was achieved with A.terreus TUB F-514, a wild strain (isolated from a sample of soil from Irak by Dr. G. Szakacs). In this study the production of this strain in SmF was also determined. Lovastatin yield obtained in liquid medium was only 0.62 mg/ml, giving a relative production of 32; indicating that the strain is very well suited for solid culture. Of course this parameter is very broad since one value is mg of SM per g of dry solid medium that is divided by another one that is in mg of SM per ml of culture medium. In fact, from a physiological point of view, the only precise comparison can only be done by calculating specific production. Unfortunately, this parameter is seldom reported since information of biomass concentration in solid is needed. Due to the advantages of SSF on inert support, this work could report that each mg of mycelium from SSF produced 816 mg of lovastatin, while each mg of mycelium from SmF only produced 59 mg of lovastatin; a specific production more thanl3 times higher in SSE This indicates that these microbes present a different physiology in SSF, and one characteristic of this special physiology is the higher SM production in this culture system (see section 5).

14.7 STRAIN IMPROVEMENT FOR SSF The science and technology of genetically manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement. The microbial production strain can be regarded as the heart of a fermentation industry, so improvement of the production strain(s) offers the greatest opportunities for cost reduction without significant capital outlay (Parekh et al., 2000). Moreover, success in making and keeping a fermentation industry Competitive depends greatly on continuous improvement of the production strain(s). Improvement usually resides in increased yields of the desired metabolite. However, other strain characteristics can also be improved. Genetic improvement of the production strain has a long history of success in SmF industry, best exemplified by the improvement of penicillin production, in which modem reported titles are 50 g/l, an improvement of at least 4,000 fold over the original parent (Peberdy 1985). Strain improvement has been performed mainly by mutation and selection. However, other procedures like sexual and parasexual crosses and protoplast fusion have also been employed in the last 20 years. More recently, genetic engineering strategies are also starting to be applied to strain improvement (Barrios-Gonz~ilez et al., 2003).

14.7.1 Classical genetic methods In relation to SSF, although wild isolates perform surprisingly well in SSF, genetic improvement of these strains is also very important or even inevitable for the development of competitive SSF processes. In this sense, it is significant that, from the 10 more productive SSF processes for fungal SMs presented in Table 3, eight used an improved strain. In fact, in 3 of the cases the strain improvement was described in the publication. It is also meaningful that the 3 most productive SSF process for SMs of actinomycetes (Table 4) also used improved strains. In SSF, it is now clear that special strains, particularly suited for SSF, have to be generated for SM production (Barrios-Gonz~ilez et al., 1993) as well as for enzyme production (Shankaranand et al., 1992). What is not so clear is what methods and strategies should be used.

14.7.1.1 Starting from hyperproducing strains for SmF According to the conclusions of the above mentioned study with P.chrysogenum (Barrios-Gonz~ilez et al., 1993), one form of generating high yielding strains for SSF processes could be starting from an available hyperproducing strain for SmF and isolating mutants that can perform well in SSF (high PS/PL). Ideally, these mutants will combine high production capacity with efficient performance in SSE In this work, we started from P.chrysogenum P2, a strain for SmF, with a discrete SSF production level of 2.084 mg/g (PS/PL of 2.5). By screening spontaneous mutants it was possible to obtain mutant P2-4 which

produced 10.555 mg of penicillin/g in SSF, showing a relative production of 10.8 (times higher than in SmF) (Table 5). Mutants with even higher relative production (16.7) were also isolated in this study, but since they came from a low producer (Wisconsin), its final production in SSF was lower than that of P2-4. Apparently, the genes coding for the characteristics useful for adaptation to solid environment were not completely lost.

14.7.2 Genetic improvement of selected wild isolates A strategy that has produced excellent results is screening of the best wild isolates and then subject them to a mutation and selection program. In 1999, Murthy et al., evaluated four strains of Tolypocladium inflatum, deposited in the standard culture collection centers (ATCC), for production of cyclosporin A in SSF. They selected strain ATCC 34921, which produced 0.459 mg/g, and from it developed a mutant strain to produce high titers of this metabolite in SSE The authors carried out 3 sequential stages of mutation and selection, using UV light, epichlorohydin and protoplast generation and regeneration. The selected high yielding mutant strain DRCC 106 produced 1.031 mg of cyclosporin A/g, which represented a production increase of 125%. Under optimum fermentation conditions this strain produced 4.834 mg/g. Our group used this strategy in lovastatin production by SSF. A.terreus TUB F-514, was isolated and screened by Szak~ics et al., (1998). In our laboratory, a SSF process on artificial inert support was developed in which this strain produced 19.95 mg/g. In a second stage, the strain was mutated with UV light and a new mutant strain was isolated by a rational screening method (based on the theoretical environmental constraints found by microorganisms in SSF), that produced 27.89 mg/g also in 7 days. In fact, the mutant strain displayed a slightly higher relative production value of (34.7) than the parental (32) (Table 5).

14.7.3 Molecular genetic improvement It is important to advance in the understanding of the particular environment faced by fungi and actinomycetes when growing in a solid culture, and the characteristics or functions that allow microorganisms to adapt and perform better in solid culture. In this way researchers will be able to design efficient rational screening methods to obtain superior mutants for SSE Furthermore, if specific genes involved in its adaptation to the solid environment and/or that have an impact in its productivity are identified, molecular improvement could also be achieved. This would be done by cloning the genes and introducing several copies into the microorganism. Recently, Biesebeke et al., (2005) studied the relation between the number of hyphal tips and enzyme secretion during the growth of A.oryze in SSE The

authors reasoned that growth of the fungus on a solid substrate involves modification of the substrate by secreted enzymes and subsequent penetration of the hyphae in the substrate. Since enzymes secretion occurs around the apical and subapical region of the advancing hyphal tip, an increase in branching frequency (hyphal tips) should give a higher enzyme producing strain. To prove this, the authors cloned 2 genes related to branching frequency and made 2 constructions to disrupt the genes (pclA and pg/pi-tp). The disrupted strains displayed an increased hyphal branching and produced 50% more amylase, 100% more glucoamylase and 90% more protease than the parental A.oryzae strain in SSF on wheat kernels. Unfortunately enzyme production of these strain and the mutants was not evaluated in SmF, so it is not so clear if this is a molecular improvement method for SSF or if it is a general one. In another example of molecular improvement our group constructed a cosmid vector for fungi, and cloned the genes of the whole penicillin biosynthetic pathway. This construction was used to transform strains of P.crysogenum and penicillin production in SmF and SSF was evaluated. Preliminary results indicate that at least one transformant of strain P2 presented a 421% production increase to reach 12 mg/g in 92 h, while production increase in SmF was much lower.

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Saykhedkar SS & Singhal RS, 2004, Solid-state fermentation for production of griseofulvin on rice bran using Penicillium griseofulvum, Biotechnology Progress, 20 (4), 1280-1284. Segreth MP, Bonnefoy A, Bronstrup M, Knauf M, Schummer D, & Toil L, 2003, Conisetin a noavel from Coniochaeta ellipsoidea, Journal of Antibiotics, 56, 114-122. Rao KCS, Karanth NG, & Sattur AP, 2005, Production of nigerlosin, an enzyme inhibitor and a free radical scavenger, by Aspergillus niger using solid state fermentation, Process Biochemistry, 40, 2517-2522. Sekar C, Rajasekar VW, & Balaram K, 1997, Production of cyclosporin A by solid state fermentation, Bioprocess Engineering, 17, 257-259. Sekar C & Balaram K, 1998, Optimization studies on the production of cyclosporin A by solid state fermentation, Bioprocess Engineering, 18, 293-296. Sekar C & Balaraman K, 1999, Cyclosporin A production by Tolypocladium inflatum using solid state fermentation, Process Biochemistry, 18, 293-296. Shankarananda VS, Ramesh MY, & Lonsane BK, 1992, Idiosyncrasies of solid-state fermentation system in the biosynthesis of metabolites by some bacterial and fungal cultures, Process Biochemistry, 27, 33-36. Suryanarayan S, 2003, Current industrial practice in solid state fermentations for secondary metabolite production: the Biocon India experience, Biochemical Engineering Journal 13, 189-195. Szakfics G, Morovjfin G & Tengerdy RP, 1998, Production of lovastatin by a wild strain of Aspergillus terreus Biotechnology Letters, 20 (4), 411-415.

Taylor AP, 2004, Quantification of the contribution of surface outgrowth to biocatalysis in sol-gels: oxytetracycline production by Streptomyces rimosus, Biotechnology Letters, 26 (22), 1707-1711. Trejo MR, (1992) These de Doctorat Biologie Cellulaire-Microbiologie, Universit6 de Provence, Aix-Marseille I, France Trejo HMR, Lonsane BK, Raimbault M & Roussos S, 1993, Spectra of ergot alkaloids produced by Claviceps purpurea 1029c in solid-state fermentation system: influence of the composition of liquid medium used for impregnating sugar-cane pith bagasse, Process Biochemistry, 28 (1), 23-27.

Valera HR, Gomes J, Lakshmi S, Gururaja R, Suryanarayan S, & Kumar D, 2005, Lovastatin production by solid state fermentation using Aspergillus flavipes, Enzyme and Microbial Technology, 37, 521-526. Weber FJ, Tramper J & Rinzema A, 1999, A simplified material and energy balance approach for process development and scale-up of Coniothyrium minitans conidia production by solid-state cultivation in a packed-bed reactor, Biotechnology and Bioengineering, 65, 447-458.

Woloshuk CP, Foutz KR, Brewer JF, Bhatnagar D, Cleveland TE & Payne GA, 1994, Molecular characterization of aflR, a regulatory locus for aflatoxin biosynthesis, Applied Environmental Microbiology, 60 (7), 2408-2414. Xu G, Chen Y, Chen Y, Ling X, & Li X, 2004, Production of monacolin K in solid-state fermentation of Monascus sp. 9901 that does not produce citrinin, Proceedings of The First International Symposium on Insight into the world of indigenous fermented foods for Technol Development and Food Safety, Food and Fermentation Industries, 30 (10), 59-63. Yang SS & Swei WJ, 1996, Oxytetracycline production by Streptomyces rimosus in solid-state fermentation of corncob, World Journal of Microbiology and Biotechnology, 12 (1), 43-46. Yang SS & Wang JY, 1996, Morphogenesis, ATP content and oxytetracycline production by Streptomyces rimosus in solid substrate cultivation, Journal of Applied Bacteriology, 80, 545-550.

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i i i i i i i i lSumathy ilili Babitha, Ashok Pandey & i i i i i i i i iAdenise i !i Lorenci Woiciechowski 15.1 INTRODUCTION There is a marked trend towards the use of natural additives in the food and feed industry. In the case of natural pigments, there is a renewed interest in traditional technologies for the production of pigments such as carmine (from insects), annatto and curcuma (from plants) or Monascus (from a fungus). There has also been a lot of research on new alternatives, in the last few decades. Because of the intensive metabolite production that is possible in bioprocesses, there is a high interest in pigment production by fermentation. This chapter describes in brief the importance of natural pigments, presents some of the commercially successful, discusses the potential of other microorganisms for pigment production by SSF, and finally describes aspects of the production of Monascus by solid-state fermentation.

15.2 NATURAL PIGMENTS Natural pigments are generally regarded as a preferable alternative to synthetic additives, because of their vegetal or animal origin. The preference towards pigments of natural origin makes sense, because in terms of metabolic pathways, "natural" substances might be more easily transformed in our body, since we have many matabolic pathways in common with other organism. At the other side, several natural pigments present anti-oxidant properties, a useful property consistent with the trend towards the development of nutraceutical foods. This very antioxidant property (which means that the pigment itself is easily oxidized) makes some pigments unstable under extreme conditions of light, pH or temperature, when compared to its synthetic counterparts. However, the artificial pigments present two problems: first, there are several artificial pigments which were banned from the list of permitted color additives in the last few years, because of suspected toxicity or allergenicity; second, even synthetic additives proved harmless in adequate levels, it still appears as "artificial color" on the food p a c k a g e - which is currently, bad advertising.

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Natural organic pigments have been known for long. The most important pigments were plant extracts, such as curcuma and indigo, or animal extracts such as carmine. Until the 19th century, natural organic pigments were the most important either for food and cosmetic use or for the textiles industry (the paint industry, on the other side, had always counted on several inorganic pigments which are, however, toxic for humans). After 1850, with the synthesis of mauve by W. Perkins, there has been a flourishing of organic pigment industry and, as a complement, the weakening of traditional pigment agro industry such as indigo, in India. The great variety in synthetic organic pigments stimulated its use without any concern about its toxicity. In 1900, about 80 artificial pigments were used in foodstuffs. At that time, there was no regulation regarding the use or the purity of such substances; however, there was reasonable toxicity in some cases. Since 1971, the carcinogenic effect of several substances in the human body is being studied (Nazar6, 2001). Today, most synthetic pigments have its use forbidden for food and cosmetic use (FDA, 2002). Increasing doubts regarding the safety of several artificial colors has stimulated its substitution by natural color additives (Kim et al., 1995). Some of the artificial colors, such as azorubin or tartrazin, may cause allergies (Fabre et al., 1993); even the list of permitted artificial colors is constantly reduced due to new information about adverse effects of known pigments. There are 9 artificial color additives currently permitted in drugs, foods and cosmetics ("FD&C colors") in USA, 2 of which with restricted use. At the other side, there are 21 natural color additives (5 of which with restricted use), permitted for the same uses. These natural colors are from diverse origin, from fruit and vegetable juices to caramel (food-grade carbohydrates thermally processed) (FDA, 2002). The same situation prevails in other countries: the list of permitted color additives in the European Union is presented in Table 1 (Arlt, 2004). Analysis of Table 1 shows that while several natural pigments are permitted in as much as needed amounts in foods, none of the artificial pigments have derestricted use: they must be used at controlled levels. This indicates that the natural pigments market is rather promising, especially because the pigments now perhfitted do not span over the whole visible spectrum (Vargas et al., 2000). Finally, refrigeration, canning, dehydration, fuming, bottling and exposition to light, air apd extremes of humidity and temperature are factors which tend to alter the natural food color, making color additives essential for the recovery of product quality (Griffiths, 2005). At the moment there is an increasing demand for natural pigments in food industry, both from natural and vigetal sources. New sources of natural pigments such as betalains from beet and anthocyanins from grapes, and optimizing

Table 1 - Food colors permitted in the European Union

a - use "at quantum satis": as much as needed, but following good manufacturing practices

their extraction processes open new evenues for its application in food industry. Furthermore, it is estimated that up to 70% of the world plants have not yet been exploited towards their potential for production of natural substances, and that only 0.5% were totally investigated for. At the other side, the growing demand in the food industry supersedes the offer of traditionally produced pigments, and requires new alternatives- such as production of pigments by fermentation (RIA, 1999). The only pigments presented in Table 1, which are currently produced economically by fermentation are riboflavin and betacarotene.

15.3. MICROBIAL PIGMENTS- PRODUCTION AND MARKET Currently, the pigments produced by microorganisms and used commercially are riboflavin (vitamin B2, a yellow pigment permitted in most countries) by Eremothecium ashbyii and Ashbya gossypi; the pigments from Monascus (discussed later on) produced by M. purpureus and M. ruber; carotenoids (yellow pigments produced by several microorganisms, but currently produced commercially only from micro algae) such as [3-carotene (by Dunaliella salina and D. bardawil) and astaxanthin (by Haematococcus pluvialis), and ficobiliproteins such as phycocianin (a blue pigment used in food and cosmetics), produced by Spirulina sp. Indigoids, anthraquinones and naphtoquinones are pigments which hold potential use in the near future (Jacobson & Wasileski, 1994). In recent years, active search for microorganisms producing non-toxic metabolites has been performed by several researchers (Babitha et al., 2004; Pandey and Babitha, 2005; Downham & Collins, 2000; Mapari et al., 2005). Fungi are the most adequate microorganisms for pigment production in SSE The use of cyanobacteral cultures such as Spirulina in SSF is limited because light has a poor penetration on the substrates, and even though some of these microorganisms may be cultivated in heterotrophic cultures, light still is necessary for the stimulation of photo-pigments (chlorophyll and phycobilins) production. Bacteria, at the other side, may grow in solid substrates but usually show a better development in liquid media. There is an enormous amount of pigmented fungal metabolites. They frequently also present pharmacological activity, which reduces the possibilities for food use. Table 2 shows some fungal strains with potential for application in SSF production of pigments.

Table 2. Selected fungal strains with potential for pigment production in SSF

The market for natural pigments produced by bioprocesses is hard to estimate, either due to the lack of statistics of regional, low-technology products or to the fact that the production is pulverized over many small companies worldwide. In the specific case of Monascus pigments, the consumption of these pigments in Japan raised from 100 ton in 1981 to 600 ton in 1992, and was estimated as U$12 million (Lee et al., 1995; Hajjaj et al., 1997). Natural pigments frequently cost 5 to 10 times more than its synthetic equal (Spears, 1998). The best case is that of l-carotene produced by microalgae, which costs approximately U$1000/kg against U$ 500/kg produced by synthesis; despite the higher price, ~-carotene produced by fermentation may compete in markets where it is important that all the ingredients be "natural"; besides, the microbial pigment is a mixture of cis- and trans- isomers, with anti-cancer effects that synthetic I]-carotene, mostly cis-, does not show. The world market for pigments of natural sources (excluding nature-identical and caramels) was estimated in 1987, as U$ 35 million; in 2000, this market was around U$ 250 million (Downham & Collins, 2000). Based on this growth tendency (600% in thirteen years, against 200% for the whole color market in the same period), today the market for natural pigments (which excludes natureidentical and caramels) is probably on the order of U$ 350 to 600 million. The biggest markets for food pigments are Europe and United States. The utilization distribution is not proportional to the food consumption (or to the population), because pigments are used in processed foods: there is a potential demand for

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15.4. MONASCUS PRODUCTION BY SSF 15.4.1. Monascus products Easily encountered in diverse ecosystems, fungi from the genus Monascus are traditionally used in oriental countries, originally in China and Thailand, to prepare a fermented rice with strong red color, which finds several applications ranging from conferring color to products such as wine, cheese and meat, to medicinal uses and as a meat preservative (Wong & Koehler, 1981). While some Monascus metabolites are important due to their strong color, others show hypocholesteremic and antimicrobial properties. Because of their high potential as food color additives, this microorganism was studied with more intensity in the last decade (Martfnkova et al., 1995; Zhang et al., 2000, Babitha et al., 2006a, 2006b, 2007a, 2007b). Currently, several companies sell the dry, pulverized, fermented rice product as a nutritional supplement with ability to reduce cholesterol levels, and others sell the dried product or purified extracts as food colors. 15.4.2. Metabolites structure and biosynthesis Fungi of the genus Monascus produce a series of secondary metabolites, which include several azaphilone pigments with a similar polyketide structure. These pigments are produced as a mixture of red, orange and yellow compounds which are commonly used without further separation, although the main commercial interest lies on the red compounds. According to some authors, there are more than i0 pigments produced by the culture, but only some of them were elucidated structurally (Shin et al., 1998). The main Monascus pigments are illustrated in Figure 1. The orange pigments, monascorubrin and rubropunctatin, are synthesized in the cytosol from acetyl coenzyme A through a multi-enzymatic polyketide synthase complex. These pigments are not hydro-soluble and are unstable in extreme pH (Hajjaj et al., 2000), but present structures with high affinity to compounds containing primary amino groups (thus called aminophiles). Reactions with amino acids lead to formation of water-soluble red pigments, monascorubramine and rubropunctamine. The mechanism of yellow pigment formation is not yet clear; some authors consider that these are products of the alteration of orange pigments, as others believe it to be pigments with their own metabolic pathway (Lin & Demain, 1991; Jt]zlov~i et al., 1996). The polyketide pathway is fairly well known, but details of the biosynthesis of Monascus pigments are important in order to allow better manipulation of conditions and medium composition regarding

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Figure 1. Structures of Monascus sp. pigments fungal metabolism. There are reports of analogues to Monascus pigments produced by a non-Monascus fungus, Penicillium sp. AZ, using the similar metabolic pathways (Ogihara et al., 2000). Because of their affinity to amino groups, Monascus pigments are frequently associated with proteins (Wong & Koehler, 1981) or with the cell wall, forming a complex that may be of difficult extraction. Other authors consider that there may be a fixation of the pigments to lipids of the fungal biomass, so that the extraction would involve cell breakage and dissolution in an organic solvent (St. Martin, 1990). Also due to this affinity for amino groups, it is possible to convert orange lipid soluble pigments to red water soluble ones by reaction with amino acids and analog compounds in vitro (St. Martin et al., 1991). In that case, nitrogen from the amino group (from the amino acid or analog) takes the place of the oxygen of the ring on rubropunctatin or monascorubrin, yielding analogs of rubropunctamine or monascorubramine, but presenting a radical linked to the N in substitution to the H of the natural red pigments.

15.4.3. Monascus: Strains and Morphology The genus Monascus contains five main species pertaining to the family Monascaceae and to the class Ascomyceta (M. pilosus, M. sanguineous, M.

eremophilus, M. pallens and M. lunisporas) (Pitt & Hocking, 1997, Park et al., 2004), whose most important characteristic is the ability to produce secondary metabolites of polyketide structure (Jt~zlov~i et al., 1996), some of them with strong yellow, orange or red color. Most strains used for pigment production are ultimately species of M. pilosus, eventually referred to as M. purpureus or M. ruber. The colonies in potato-dextrose-agar (PDA) after 7 days are 20 to 30 mm in diameter, plane, eventually with small aerial development, sparse, with flocculent superficial texture, mycelium initially white (1 to 2 days), turning to orange and then to brick red as the culture develops, with formation of cleistotechia and aleurioconidia (Figure 2). There usually is the formation of soluble pigments that diffuse through the agar. The cleistotechia are spherical, from 30 to 60um in diameter, formed as a hyphal knot from a well defined stalk, with cell walls turning to brown with maturation; ascospores are ellipsoid, hyaline, 5-7x44,5um in size, with a smooth cell wall. There may be the formation of aleurioconidia in pedicels, laterally to hyphae, but most commonly terminal, sometimes growing isolated but more commonly in chains up to 10 cells, spherical to piryform, frequently rounding on maturation, with 10-14mm in diameter or 10-18• in size, with thick walls, smooth and brown. Most species produce also chlamidoconidia and arthrospores. The colonies of M. ruber grow more quickly than other species of Monascus (Pitt & Hocking, 1997).

Figure 2. Monascus cultures in PDA after 7 days incubation at 32~ Note the diffusion halo of water soluble pigments and the characteristic color of mycelium.

Source: Carvalho et al., 2004

15.4.5. Monascus cultivation conditions 15.4.5.1. Temperature The optimal temperature range for most Monascus species is 28-32~ with some strains requiting temperatures as low as 25~ and others as high as 37~ (Lin & Demain, 1991).

15.4.5.2. Presence of oxygen The strains of the genus Monascus are incapable of anaerobic growth using glucose as a substrate, but may grow in oxygen-limiting conditions. Under these conditions, there is a higher production of ethanol and CO2, but a lower production of pigments. In conditions of higher aeration, ethanol production decreases and pigment production increases. It was observed that increase in the partial pressure of CO2 increased the pigment production (Pastrana et al., 1995). In high glucose concentrations (above 20 g/L in liquid culture), a Crabtreelike effect occurrs, that is, the shift to a predominantly anaerobic metabolism, with production of ethanol, even in good aeration conditions (Chen & Johns, 1994; Rosenblitt et al., 2000, Carvalho 2004). With lower glucose concentrations or with other sugars, it is possible to divide production in two phases: initially glucose is converted to ethanol and biomass, and then ethanol is converted to biomass and pigment (Hamdi et al., 1996). In a defined liquid medium with oxygen-limiting conditions, red pigment production is growth-associated, whereas in oxygen excess pigment production may be inhibited by the effect of an unknown product (Hajjaj et al., 2000). The influence of oxygen concentration in solid substrate fermentation was also investigated; at 0.02atm CO2, an increase of the oxygen partial pressure up to 0.5 atm gave high pigment yields, whereas low CO2 pressures with 0.21atm 02 gave higher pigment yields (Han & Mudget, 1992).

15.4.5.3. pH Growth has been observed in a wide range of pH, from 2.5 to 8.0, with the ideal range being from 4.0 to 7.0 (Yongsmith et al., 1993). Within this range, the variation in growth is small, with a little better development at pH 4.0 (Lin & Demain, 1991; Chen & Johns, 1993). Although the growth was better at pH 4, the biomass yield was higher at pH 6.5 (Chen & Johns, 1993). The production of pigments is affected in a different way: at lower pH, there is predominance of yellow pigments, and at a higher pH, there is a predominance of red pigments. At pH 2.5, there was production mostly of a bright red pigment with a skeleton similar to the traditional pigments (Yongsmith et al., 1993); at pH 4 there was a stimulation of synthesis of ankaflavin (yellow pigment), and the total production of pigments increased with a increment in

pH until pH 5.5. Above this value, there was a decrease in pigment production (Lin & Demain, 1991), although the production of red pigments was higher than that of yellow ones. At pH 7, there was no more production of yellow pigments (Yongsmith et al., 1993). The change of pH during growth depends on nitrogen sources, in the first place, and also the carbon sources. Regardless of initial pH, the final pH tends to be the same (Jt]slov~i et al., 1996), usually in the range of 7 to 8 (Yongsmith et al., 1993).

15.4.5.4. Liquid vs. solid fermentation Although the traditional production of ang-kak for use as a coloring agent is conducted on a solid support (rice), most of the studies performed in laboratory have been done using liquid culture media, which present easily controllable conditions. Solid-state fermentation (SSF), however, gives a higher yield and productivity of pigment than liquid fermentation. A comparison was made between liquid and solid media of similar composition, the solid media obtained from the liquid by addition of a gelling agent, followed by the extrusion in rice-sized particles. The solid media thus prepared supported the production of up to three times more pigment than the corresponding liquid media, but the cultivations over rice were still superior (Johns & Stuart, 1991). 15.4.5.5. Humidity in SSF The traditional process of ang-kak production is the same used for other koji varieties (koji is the general name given to rice fermented by several fungal species, usually Aspergillus), hence the alternative name "red koji". The production process consists of maintaining non-agglutinating rice immersed in water up to 24h, and then steaming (autoclaving), addition of an inoculum (a portion of rice previously fermented) and addition of water at intervals to maintain humidity (Palo et al., 1960; Wong et al., 1981). The humidity needed in the rice fermentation varies according to the traditional description, but it is usually recommended that the humidity be enough to permit growth of mycelium through the grain, without disintegrating the grain. The ideal humidity for pigment production in solid substrates is around 56%, with pH 6 (Johns & Stuart, 1991).

15.4.5.6. Carbon s o u r c e s The most common way to produce Monascus in SSF is the cultivation of steamed rice. This is also the best natural substrate in terms of color production; however, several other carbon sources have been used as substrates for Monascus growth in liquid media, and these are presented here because they might be used as the medium of choice in SSF over suitable supports.

The most common carbon sources in formulated media are glucose, sucrose and starch. The highest growth is usually observed with glucose (St. Martin, 1990). The pigment production depends on several factors such as nature and concentration of the substrate, pH and nitrogen source. The volumetric production of pigments in submerged media is higher with starch and dextrin, while specific production is higher with maltose and almost as good with glucose (Lin & Demain, 1991), but that comparison between sugars must be done carefully, taking into account the concentrations. In glucose concentrations lower than 20g/L, growth and red pigment production are excellent; glucose concentrations higher than 20g/L lead to a Crabtree-like behavior, with ethanol production and reduced cell growth and pigment formation, even in the presence of oxygen. This indicates that there could be a repressive effect from glucose, and that the use of another sugar might avoid this effect. As a matter of fact, in high concentrations (50g/L), maltose is better than glucose, especially in the presence of peptones. These different concentrations for sugars show stronger effect on production of yellow rather than red pigments, although with maltose, red pigment formation is stimulated (Chen & Johns, 1994). A good alternative carbon source is ethanol (Jtlslov~i et al., 1994), which is naturally produced by the fungus in oxygen-limiting or excess glucose conditions (Pastrana et al., 1995; Hamdi et al., 1997). Since biomass formation is favored by the use of carbohydrates, the process could be performed in two steps (for example, maltose-ethanol) in order to enhance the efficiency of ethanol use for pigment production (Jt]slov~i, 1996). There are some contradictions on the effect of carbon sources: for instance, Lin (1991) did not encounter repressive effects for any carbon source, using concentrations of up to 10% carbohydrate. These contradictions may be possibly attributed to different organisms and aeration rates used.

15. 4.5. 7. Nitrogen sources Different N-sources used for Monascus growth range from inorganic nitrogen (ammonium and nitrates) to peptones. In the traditional ang-kak production, there is no need of addition of ~nitrogen sources, since rice has 5 to 8% proteins, (dry basis) (Franco, 1992). When using other substrates, the addition of a nitrogen source (especially organic nitrogen) stimulates pigment production. According to Lin (1991), the use of monosodium glutamate as a nitrogen source stimulates pigment production, which has been confirmed by other authors. The kind of pigment and its excretion by the cell are also related to the nitrogen source: organic nitrogen, as in amino acids, favors the formation of red pigments (Yongsmith et al., 1993; Jt]slov~i et al., 1996), and the use of peptones is better for pigment production and cell growth, as well as for pigment secretion at pH 6.5 (Chen & Johns, 1993), although the use of polypeptones favor the formation of yellow pigments (Jt]slov~i et al., 1996). It

is possible that the stimulating effect of amino acids on the production or liberation of pigments is caused by an enhancement in solubility, since the derivatives of Monascus pigments linked to amino acids, are more soluble than the original pigments. Studies demonstrate that the use of ammonia as a nitrogen source may favor the orange pigment production (Jtlslov~i et al., 1996). A comparison of the relative amounts and color quality of pigments produced using several amino acids show that yellow and orange pigments are unaffected by the nitrogen source, whereas red pigments differ in tone and solubility (Jung et al., 2003)

15.4.5.8. Other factors According to Lin (1991), high concentrations of phosphate and of magnesium sulphate inhibited pigment production and the growth was a crescent linear function of MgSO4 concentration, in the range from 0.5 to 16mM. The addition of corn oil stimulates (doubled) the pigment production, while the addition of 0.4% Tween 80 neither affect glucose uptake, nor retard the growth rate, but enhanced the pigment productivity (6 to 8 times, reaching 8535 units of absorbancy/g dry matter) (Chiu &Poon, 1993).

15.4.5. 9. Complex culture media Culture media for Monascus pigments production are very diverse, ranging from defined compositions to natural ones. Being a common contaminant in grains, and having been isolated from several substrates with a high solids concentration, Monascus grows in a wide variety of natural substrates. Some natural substrates already tested, besides rice and other cereals, are cassava starch (Yongsmith et al., 1993; Lee et al., 1995; Carvalho et al., 2001), prickly pear juice (Hamdi et al., 1996), and dairy milk (Kujumdzieva et al., 1997). In some cases, as in cassava starch, it is necessary to supplement these substrates with yeast extract and peptones, as vitamins and organic nitrogen supplements. The components of the complex culture media used were already discussed in the text, and include several sugars (most commonly glucose), oligoelements and organic nitrogen sources (amino acids, peptones) or inorganic nitrogen (ammonium, nitrates). A medium used in some works is composed of: glucose 40g/L; NH4NO3 3 g/L; KzHPO 4 6g/L; KHzPO 4 6g/L; MgSO4.7H20 0.5g/L; KC1 0.5g/L; FeSO4.7H20 10mg/L; ZnSOa.7H20 10mg/L; MnSOa.H20 3mg/L; final pH 6.3 (Wong 1981, Lin and Demain, 1991). 15.4.5.10. Culturing Monascus over rice For the production of Monascus pigments in SSF, best conditions are forced aeration (with water-saturated air) of 1NmL/min.g of wet substrate (56% humidity)

in an 8cm height bed, at 32~ for 7-8 days, after inoculation with 10g/kg of a spore suspension with at least 106CFU/mL. Under these conditions, a maximum specific growth velocity of 0.039 h-1 and a specific pigment production velocity of 27.5 AU/g biomass.h -1 were obtained. Aeration and humidity play a critical influence in pigment formation, and careful design of the fermenter must be done prior to scale-up; in tray fermentation, although forced aeration is not necessary, the bed height must be reduced to 2-3cm (Carvalho et al., 2006).

15.4.6. Production and extraction in SSF The production of Monascus in SSF is very straightforward, except for the fact that, depending on the fermentation conditions, the substrate may lose its rigidity, so that a suitable solid layer must be used (generally 2 to 15 cm). Figure 3 illustrates Monascus pigments production over rice, in trays.

Figure 3. Monascus production in trays

When only the coloring fraction is necessary, without substrate residues - for use in beverages, for an i n s t a n c e - the pigment must be extracted with an organic solvent, such as 60-70% ethyl alcohol (pure ethanol is less efficient; methanol is very efficient, but toxic). After the extraction, the solvent is evaporated and the pigment eventually processed. (Carvalho et al., 2006). Depending on extraction conditions, an adequate filler must be used to enhance processing.

15.4.7. Pigment analysis, toxicity and stability There are several studies on the toxicity of Monascus pigments, which are apparently safe in the quantities tested. The many years for which these pigments have been used suggested low or non-existent toxicity (Lin & Demain, 1991). Since the strains from the genus Monascus started to be studied systematically, it has been believed that the pigments produced also presented antibiotic properties; later, it was verified that this activity is due mainly to other substance, named monascidin A (Wong & Koehler, 1981). Further studies showed that this subs~lce was, in fact, citrinin, a mycotoxin with nephrotoxic action produced by several fungi (Blanc et al., 1995a), but that not all Monascus strains produced citrinin. Unfortunately, it seems that all good pigment producers also produce citrinin. Finally, studies over the toxicity of purified fractions of pigments showed that there was indeed an antibiotic activity for Monascus pigments, especially the orange ones and, in lower degree, the red ones (Martinkov~i et al., 1995). Although there is not yet a definitive conclusion about pigment toxicity and citrinin production in industrial processes, several actions may be taken to prevent or reduce this problem: the use of strains which produce less citrinin; the control of the nitrogen source (organic nitrogen sources favor red pigment production and decrease citrinin production); the control of cultivation conditions (aeration, pH, solid-substrate vs. liquid fermentation); the transformation of orange pigments into red, non toxic complexes, using amino acids (Blanc et al., 1995, 1995a; Jhzlov~i et al., 1996) and the extraction in low citrinin-solubility conditions, which may be achieved controlling pH, since citrinin is strongly acidic. There is much space for strain development, as may be noted by the examination of Table 3 (Carvalho, 2004)"

Table 3 - Pigment production and productivity, and citrinin production in rice for several Monascus strains.

avalues from Miyashira, 2003, bstrainunknown

Table 3 shows that there is a high diversity among different Monascus strains, with a large span in production, productivity and citrinin production. It is very likely that future production will be made with pigment overproducers which do not produce citrinin.

15.4.7.1. Analysis Pigment production by Monascus is usually evaluated by measuring absorbances for pigment solutions on the ranges near 400, 470 and 500nm for yellow, orange and red pigments, respectively (Johns & Stuart, 1991; Lin & Demain 1992). The ratio of absorbance at 500nm/absorbance at 400nm gives the ratio between red and yellow pigments (Wong & Koehler, 1981). In SSF, it is necessary to make a solvent extraction step, followed by absorbance reading. The amount of solvent used is, usually, 5mL solvent for each gram of fermented material, and the extraction time lh under agitation. Red pigment solubility is highest in an aqueous solution containing 60-70% ethanol (Carvalho, 2006). The fact that the yellow, orange and red pigments of Monascus are produced as a mixture probably affects the analysis by simple measure of absorbance. Nonetheless, the vast majority of authors estimate pigment production by this method, with pigment production ranging from hundreds of absorbance units/ ml culture media in submerged fermentations (e.g. 220 OD510/mL, in optimized conditions by Kim et al., [2002]), to thousands of absorbance units/g dry substrate, in SSF (e.g, 5430 ODs00/g dry matter, by Lin & Demain, [1992]). The best procedure for pigment analysis is liquid chromatography, wich allows to separate and quantify individual pigments; with this method, Hajjaj et al. (2000) considered 1 unit OD480correspondent to 15mg/L of red pigment with M = 498 g/mol. Naturally, this equivalence should not be applied to crude pigment extracts, wich may contain several substances with different absorbances. Biomass analysis may be adequately done via ergosterol analysis, rather than glucosamine (which may have interferences from pigments) (Carvalho et al., 2006)

15.4. 7'.2. Pigment use and stability Surprisingly, only few works deal on the stability of Monascus preparations, considering that several industries produce this pigment. Some documentation can be found at the producers such as Allok (www.allok.com). According to Lin & Demain (1992), these pigments are fairly stable to autoclaving and in a wide range of pH. According to Fabre et al., (1993), sausages or pates colored with red pigments of Monascus presented a residual color of 92 to 98% after three months at 4~ with good sensorial acceptance. However, the pigments

were unstable to light (only 20% residual color after 50 days) and heat (45% residual color after 2h at 100~ They are more stable at neutral or basic pH (Fabre et al., 1993, Lee & Chen, 2000). Table 4 shows the residual color of Monascus pigments incubated in aqueous solutions at several pH and temperatures.

Table 4. Residual color (in %) of aqueous pigment solutions, after 25h incubation at several pH and temperatures,

Source: Carvalho et al., 2005 As can be seen at Table 4, Monascus pigments should be used in food applications with neutral pH and moderate temperatures. However, the pigment stability is very good in lower water activity, such as in alcoholic beverages, pates and sauces.

References Arlt U, 1998, The legislation of food colors in Europe, Natcol: Basle,www.natcol.com, accessed in Nov, 2004. Babitha S, Sandhya C & Pandey A, 2004, Natural food colorants, Applied Botany Abstracts, 23 (4), 258-266. Babitha S, Sandhya C, Carvalho JC, Soccol CR & Pandey A, 2006a, Natural food colorants from Monascus purpureusln- Current Topics on Bioprocesses in Food Industry, C Larroche, A Pandey & CG Dussap (eds), Asiatech Publishers, Inc, New Delhi, pp 186-196. Babitha S, Soccol CR & Pandey A, 20006b, Jackfruit seed - A novel substrate for the production of Monascus pigments through solid-state fermentation, Food Technology and Biotechnology, 44, (4), 465-471. Babitha S, Soccol CR & Pandey A, 2000c, Effect of stress on growth, pigment production and morphology of Monascus sp. in solid cultures, Journal of Basic Microbiology. Babitha S, Soccol CR & Pandey A, 2000d, Solid-state fermentation for the production of Monascus pigments from jackfruit seed, Bioresource Technology. Blanc PJ, Loret O & Goma G, 1995, Production of citrinin by various species of Monascus, Biotechnology Letters, 17, 291-294.

Blanc PJ, Loret O & Goma G, 1995a, Characterization of monascidin A from Monascus as citrinin, International Journal of Food Microbiology, 27, 201-213. Carvalho JC, Soccol CR & Miyaoka MF, 2001, Produ~o de pigmentos de Monascus em meios ~ base de baga~o de mandioca. In: Anais do VII Encontro Regional Sul de Ci~nciae Tecnologia de Alimentos, Regional Paran~i-SBCTA-PR, ABM2-15. Carvalho JC, 2004, Desenvolvimento de bioprocesso para a produ~o de pigmentos a partir de Monascus por fermenta~o em substrato s61ido. Curitiba, 2004. Tese (Doutorado em Processos Biotecnol6gicos). Setor de Tecnologia- Universidade Federal do Paran~i. Carvalho JC, Pandey A, Oishi BO, Brand D, Rodriguez-Leon JA & Soccol CR, 2006, Relation between growth, respirometric analysis and biopigments production from Monascus by solid-state fermentation Biochemical Engineering Journal, 29, 262-269. Carvalho JC, Oishi BO, Pandey A & Soccol CR, 2005, Biopigments from Monascus: strains selection, citrinin production and color stability, Brazilian Archives of Biology and Technolgy, 48, 6. Chen MH & Johns MR, 1993, Effect of pH and nitrogen source on pigment production by Monascus purpureus, Applied Microbiology and Biotechnology, 40, 132-138. Chen MI-I & Johns MR, 1994, Effect of carbon source on ethanol and pigment production by Monascus purpureus, Enzyme and Microbial Technology, 16, 584-590. Chiu W &Poon YK, 1993, Submerged production of Monascus pigments, Mycologia, 85,214-218. Downham A & Collins P, 2000, Coloring our foods in the last and next millennium, International Journal of Food Science and Technology, 35, 5-22.

Fabre CE, Goma G & Blanc PJ, 1993, Production and food applications of the red pigments of Monascus rubber, Journal of Food Science, 58 5, 1099-1110. FDA-U.S. Food and Drug Administration Food Color Facts: 1998, http:// www.cfsan.fda.gov/~lrd/colorfac.html, accessed on 25/07/2002.

Franco G, 1992, Tabelas de Composi~ao Qufmica dos Alimentos 9a ed. Rio de Janeiro: Liv. Ed. Atheneu (1992). Griffiths JC, 2005, Food Technology, 59. l-lajjaj H, Klaebe A, Loret MO, Tzedakis T, Goma B & Blanc P, 1997, Applied and Environmental Microbiology, 63, 2671-2678. Hajjaj H, Blanc P, Groussac E, Uribelarrea JL, Goma G & Loubiere P, 2000, Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation, Enzyme and Microbial Technology, 27, 619-625. l-lamdi M, Blanc PJ & Goma G, 1996, Effect of aeration conditions on the production of red pigments by Monascus purpureus growth on pricey pear juice, Process Biochemistry, 31,543-547. Hamdi M, Blanc PJ & Goma G, 1996, A new process for red pigment production by submerged culture of Monascus purpureus, Bioprocess Engineering, 17, 75-79.

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Han O & Mudget RE, 1992, Effects of oxygen and carbon dioxide partial pressures on Monascus growth and pigment production in solid-state fermentations, Biotechnology Progress, 8, 5-10. Jacobson G & Wasileski J, 1994, In- Bioprocess production of flavor, fragrance, and color ingredients, 1st edn, John Wiley & Sons: New York.

Johns MR & Stuart DM, 1991, Production of pigments by Monascus purpureus in solid culture, Journal of Industrial Microbiology, 8, 23-38. Jung H, Kim C, Kim K Shin CS, 2003, Color characteristics of Monascus pigments derived by fermentation with various amino acids, Journal of Agricultural Food Chemistry, 51, 1302-1306. Juzlova P, Martinkova L, Lozinski J & Machek F, 1994, Ethanol as substrate for pigment production by the fungus Monascus purpureus, Enzyme and Microbial Technology, 11,996-1001. Juzlova P, Martinkova L & Kren V, 1996, Secondary metabolites of the fungus Monascus: a review, Journal of Industrial Microbiology, 16, 163-170. Kim HJ, Kim JH, Oh HJ & Shin CS, 2002, Morphology control of Monascus cells and scale-up of pigment fermentation, Process Biochemistry, 38, 649-655. Kujumdzieva AV, Hallet JN, Savov VA & Rasheva TV, 1997, Monascus purpureus strain producer of pigments and by-products, U.S. PAT. 5,627,068. Lee YK, Chen DC & Toshiomi Y, 1995, Production of Monascus pigments by a solidliquid state culture method, Journal of Fermentation and Bioengineering, 79, 516-518. Lee YK & Chen DC, 2000, Applications of Monascus pigments as food colorant, www.allok.com//literature, accessed in Oct 2000. Lin TF, 1991, Studies on the formation of Monascus red pigments, PhD Thesis MIT, Massachusetts. Lin TF & Demain AL, 1992, Formation of water-soluble Monascus red pigments by biological and semi-synthetic processes, Journal of Industrial Microbiology, 9, 173-179. Lin TF & Demain AL, 1991, Effect of nutrition of Monascus sp. on formation of red pigments, Applied Microbiology and Biotechnology, 36, 70-75. Mapari S, Nielsen KF, Larsen TO, Frisvad JC, Meyer AS & Thrane U, 2005, Exploring fungal biodiversity for the production of water-soluble pigments as potential natural food colorants, Current Opinion in Biotechnology, 16 (2), 231-238. Martinkova L, Juzlova P & Yeseley D, 1995, Biological activity of polyketide pigments produced by the fungus Monascus, Journal of Applied Bacteriology, 79, 609-616.

Miyashira GY, Rodrigues R & Kilkian BV, 2003, Sele~o de linhagens de Monascus sp.para cultivo semi-s61ido Florian6polis: proceedings of the XIV SINAFERM. Nazare RFR, 2001, Potencialidade de plantas amaz6nicas produtoras de corantes naturais, http://www.uesb.br/sbcn/amazonia.html, acessed on 08/06/01.

iiiiii!iii!iiii!ii!iliiiiiiiiiiiiiiiiiiiiiiiiii! i!iiiiii iiiiliii............. i....................... i l iliiiii iiii!iiiii!iii!i iliii.....i!iii iiiiiiiiiiiiii!iiii i....... Ogihara J, Kato J, Oishi K & Fujimoto Y, 2000, Biosynthesis of PP-V, a Monascorubramine Homologue, by Penicillium sp. AZ, Journal of Bioscience and Bioengineering, 6, 678-680.

Palo MA, Vidal-Abeva L & Maceda LM, 1960, A study on ang:kak and its production, Philippines Journal of Science, 89, 45-47. Pandey A & Babitha S, 2005, Microbial pigments, Advanced Biotech, 3 (8), 17-19. Park HG, Stamenova EK & Jong SC, 2004, Phylogenetic relationships of Monascus species inferred from the ITS and the partial b-tubulin gene, Botany Bulletin of Academy of Singapore, 45, 325-330.

Pastrana L, Blanc PJ, Santerre AL, Loret MO & Goma G, 1995, Production of red pigments by Monascus tuber in synthetic media with a strictly controlled nitrogen source, Process Biochemistry, 30, 333-341. Pitt JI & Hocking AD, 1997, Fungi and food spoilage, 2nd edn, Chapman & Hall: Londo RIA - no 591, May 1999.

Rosenblitt A, Agosin E, Delgado J & Correa RP, 2000, Solid substrate fermentation of Monascus purpureus: growth, carbon balance, and consistency analysis, Biotechnology Progress, 16, 152-162. Shin CS, Kim HJ, Kim MJ & Ju JY, 1998, Morphological change and enhanced pigment production of Monascus when cocultured with Saccharomyces cerevisiae or Aspergillus oryzae, Biotechnology and Bioengineering, 59, 576-581. Spears K, 1988, Trends in Biotechnology, Vol, 6, Developments in food colourings: the natural alternatives. St. Martin EJ, 1990, US PAT. 4, 927,760. St. Martin EJ, Kurek PR & Schumacher EF, 1991, US PAT. 5,013,565. Vargas FD, Jim6nez AR, & Lopez OP, 2000, Natural pigments" Carotenoids, Anthocyanins, and Betalains - Characteristics, Biosynthesis, Processing, and Stability, Critical Reviews in Food Science and Nutrition, 40, 173-289. Wong HC & Koehler P, 1981, Production and isolation of an antibiotic from Monascus purpureus and its relationship to pigment production, Journal of Food Science, 589592. Yongsmith B, Tabloka W, Yongmanitchai W & Bavavoda R, 1993, Culture conditions for yellow pigment formation by Monascus sp. KB 10 grown on cassava medium, World Journal of Microbiology and Biotechnology, 9, 85-90. Zhang ML, Peng CX & Zhou YF, 2000, US PAT. 6, 046, 022.

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Production of Aroma Compounds

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16.1 INTRODUCTION Odoriferous compounds named aromas are molecules with low molecular mass (M0.90), but some, especially yeasts and molds can tolerate drier conditions (as low as Aw--0.70). Molds are also strict aerobes, which means that they require air for their activity. They can tolerate more acidic conditions than most bacteria. Microorganisms in any given environment are in a constant competition and those for which the entire abiotic conditions are optimal become the dominant microbial population, whereas other groups are suppressed. Food preservation aims at changing the ideal conditions for the spoiling agents. Preservation alternatives include heat treatments, cold and frozen storage, drying, acidification, modified atmospheres, irradiation and application of preserving chemicals. Preservation of forage crops should fit the large bulk of the crops and should be cost effective. The most common methods of forage crop preservation include hay making and ensiling. The former is based on sun drying, while ensiling is based on anaerobic lactic acid solid-state fermentation. For both methods, the crops should be harvested at the optimal stage of maturation when their nutritional quality is best. A delay of even a week may lower their digestibility for ruminants because of advanced lignification of the cell wall. Hay making is suitable especially for thin crops which can dry quickly in sunny seasons. Losses of nutritional materials occur during hay making due continued respiration in the drying plants (Figure 1), and detachment of dry leaves which become brittle. In forage legumes (such as alfalfa, clover and vetch) the protein is present in the leaves and therefore, they are most susceptible to nutritional losses during racking and tedding of the drying crop. Rain is yet another factor which may interfere with drying or cause damage during storage. Silage can be made of almost any crop and is less weather dependent. It is more consistent than hay in nutrient content. However, silage making requires high capital investment in storage structures (silos) and machinery.

Figure 1. The effect of moisture content and ambient temperature during wilting on respiration losses (adapted from Honig, 1980). 19.3 RETROSPECTIVE OF SILAGE MAKING Ensiling has been practiced since ancient times. This is evidenced by an Egyptian mural that dates back to 1000-1500 BC that shows ensiling of whole-crop cereals. Silos were also found in ancient Carthage. Little is known about silage making in the Medieval Ages. Interest in ensiling of forage crops was renewed in Europe in the 19th century because it was similar to production of sauerkraut. In 1877 a French farmer, Auguste Goffart, published his results on ensiling of chopped whole-crop forage corn. This publication had a great impact on spreading the ensiling technology in Europe and North America. A setback to ensiling was afflicted with the publication of the book Sweet Silage (Fry, 1885) who advocated that silages reach 50~ before sealing in order to eliminate detrimental microorganisms. This practice which was used till the 1960s resulted in substantial losses in nutritional constituents. When more knowledge on silage biochemistry and microbiology was attained this mal practice was abandoned. In the 1920s Virtanen from Finland developed direct acidification of moist grass silage by using a mixture of hydrochloric and sulfuric acids. This process helped to reduce fermentation losses and proteolysis in such silages. For this achievement he was awarded the only Noble Prize in Agriculture so far. In the 20 th century tower and horizontal silos were developed as well as plastic sheeting for sealing. The recent trend is to use big plastic bales and sleeves for storing silage which permit flexibility in storage and use. Machinery that include harvesters, choppers and silage unloaders were developed. Numerous silage additives were

introduced, both chemical and biological (bacterial inoculants and enzymes) that improve the ensiling fermentation and preservation and in some cases also enhance animal performance. The current production of silage in Europe and North America is estimated at 200 million tons of dry matter, as compared with 300 million tons of hay. The estimated areas of crops harvested for silage total 43 million hectares (Wilkinson et al., 2003).

19.3.1. The ensiling process The biochemical and microbiological events that occur during ensiling can be divided into four distinct stages: Initial-aerobic, during filling of the while air is still present between the to that in the fresh crop (6.0-6.5). and proteolysis continue, as well as ,

.

.

silo and immediately after sealing, plant particles and the pH is similar During this stage plant respiration activity of aerobic microorganisms.

Fermentation, lasting several days to several weeks after oxygen is depleted, during which various lactic acid bacteria (LAB) develop and convert water-soluble carbohydrates (WSC) to organic acids, mainly lactic acid. As a result the pH decreases to 3.8-5.0. Stable, when relatively few changes occur in the sealed silage, given no air penetrates into the silage. Unloading for feeding, the silage is re-exposed to air and aerobic microorganism are re-activated and spoilage processes begin.

Each of these phases are characterized by biochemical and microbiological processes which are described in the following paragraphs.

19.3.2. Biochemical processes during ensiling The desired process during ensiling is the conversion of simple sugars (watersoluble carbohydrates, WSC) to organic acids, mainly lactic acid, through lactic acid fermentation. The acids suppress undesirable microbial activity and preserve the silage. The composition of the ensiled crop should promote LAB during ensiling. The crops composition depends on crop type, stage of maturity at harvest, fertilization and extent of wilting. The requirements for successful ensiling include adequate dry matter (DM) and WSC content that should be between 300 and 400 g kg -1 and at least 30-50 g kg -1 DM, respectively. The buffering capacity of the ensiled biomass should be low. The buffering capacity is a measure of a chemical/biochemical system to resist decrease in pH even when acids are present. High ash (mineral) and protein content contribute to

the buffeting capacity of the plants. For example, the buffeting capacity of legumes is higher than that of cereals (around 500 vs. 250 milli-equivalents NaOH kg-lDM, respectively, needed to increase the pH from 4.0 to 6.0). Therefore, it is more difficult to ensile legumes than cereals. In order to overcome the high buffeting capacity of legumes it is recommended to wilt legumes to about 500 g kg -1 DM prior to ensiling. This improves the WSC to buffeting capacity ratio. The resulting product is referred to as haylage. In the initial aerobic phase of ensiling the pH is still around 6.0 and sufficient air is present between the plant particles. These conditions enable proteolysis and respiration to take place by endogenous plant enzymes. When the plants are harvested only a small fraction of their surface area is exposed to light, and therefore, photosynthesis stops. Respiration in the plant continues which results in formation of ATP. Assuming hexoses are respired, the reaction can be summarized as follows" C6H1206 + 602 ~ 6CO2 + 6H20 + 0.690 Mcal mo1-1 glucose

Warm temperatures and high moisture content of the plants enhance respiration, which might be intensive during wilting. This process lowers the WSC pool necessary for the ensiling fermentation. Respiration and aerobic microbial activity at this stage results in heating (temperature might rise to >40~ and loss of nutrients in the form of CO2. Excessive heating leads to browning and Maillard type reactions which further decrease protein and WSC availability of the silage. Figure 1 describes the effects of moisture and ambient temperature during wilting on respiration losses. Proteolysis is yet another process that occurs during this phase. It results in breakdown of proteins to shorter peptides, free amino acids and eventually ammonia. The ammonia is formed by microbial activity. Proteolysis continues until the pH of the ensiled crop decreases below 5.0; it is affected by both temperature and moisture content of the herbage; high tannin content protects proteins against proteolysis. The extent of proteolysis might reach 20% of the total protein. Proteolysis reduces the amount of by-pass protein (protein that by-passes the rumen and reaches the intestine) which is important for the ruminant nutrition. Both respiration and proteolysis are undesirable processes in which losses occur and they should be minimized by rapid and efficient ensiling followed by a fast decrease in pH. During the fermentation stage of ensiling most reactions that occur are microbial and will be discussed in the following sections on silage microbiology. Due to

the pressure in the silo the plant cells are ruptured and their sap is released. The sap contains WSC which are necessary for the ensiling fermentation. During this stage a limited hydrolytic activity takes place which results in partial degradation of fructans and of cell-wall constituents, mainly pectin, and hemicellulose, which add oligosaccharides and increase the WSC pool. The extent and speed of decrease in pH depends on the crop composition, its DM and WSC content, buffeting capacity and microbial populations that develop. If the crop is very moist (>70% moisture) the decrease in pH is fast; and if very dry (0.97). A minimal concentration of nitrates (1-2 g N-NO 3 kg -1 DM) in the silage which is reduced to NO also inhibits clostridia.

Clostridium botulinum, a protein fermenting species, produces a very potent neuro-toxin, which is fatal to animals and humans. This bacterium may contaminate the ruminants ration through occasional carcasses of rodents or birds found sometimes in broiler litter or from grazing pastures fertilized with manure. In Israel, most cattle are vaccinated to control this problem. Yeasts are single-cell eukaryotes. During the anaerobic phases of ensiling they ferment sugars and produce ethanol and CO2. Under aerobic conditions lactateassimilating yeasts become active and they result in increase in pH and further aerobic spoilage. They are the most important spoilers of silage upon aerobic exposure, and in most silages they initiate the aerobic deterioration. Yeasts can be controlled in silage by volatile fatty acids (VFA, such as acetic, propionic and butyric acids) added directly or produced by selected bacteria (e.g., the heterofermentative L. buchneri) or by antimycotic chemicals. If the silage undergoes homolactic fermentation it is more susceptible to aerobic spoilage by fungi, because of the lack of inhibiting VFA. The most important yeast genera associated with silage include Candida, Hansenula, Pichia and

Saccharomyces.

Filamentous fungi (molds) are obligate aerobic microorganisms which produce micellium in the silage. They develop in the silage upon aerobic exposure, if the sealing is inadequate or during feedout. The most susceptible parts of silage to molding are the top and near the walls in bunker silos, or the outer

layers of baled silages. Molds result in change in color, odor and texture of the silage, and high losses. Certain molds may produce mycotoxins in the silage which are hazardous to the animals and humans. The mold genera associated with silage include Penicillium, Fusarium, Aspergillus and Geotrichum and Mucor. Acetic acid bacteria include acetobacter and gluconoacetobacter which sometimes initiate aerobic spoilage in corn silages. They are obligate aerobes which oxidize ethanol to acetic acid. The former can oxidize acetic and lactic acids to CO2 and water while the latter cannot.

19.3.4. Practical aspects of silage making Silage making involves a series of actions which start at the harvesting of the crop and ends at the feed-out step. Silage making is a sophisticated preservation method of forage crops which usually requires infrastructure (silos) and machinery. It is important that all actions should be performed in the optimal precise manner in order to insure the success of ensiling and preservation. Negligence at Critical Control Points might result in failure, spoilage and losses. Synchronization between the actions is a key factor for success. The steps involved in silage making include: harvesting, wilting, chopping, enrichment, transportation, compaction, sealing, feed-out.

19.3.4.1. Crops for silage Forage crops are ensiled in various parts of the world with various climate conditions. In every geographical region crops which are best adapted to the local conditions and give the highest profits to the farmers are grown. In general, forage crops should be harvested at the time point when their yields, ensiling properties and nutritional value are at peak. The major forage crops for silage worldwide include maize (Zea mays), alfalfa (Medicago sativa) and various grasses. In some parts of the world various cultivars of sorghum (Sorghum bicolor) are used for silage; In Israel wheat (Triticum aestivum) is popular. Corn is an optimal crop for silage with yields up to 20 tons of DM per hectare, with high nutritional value for ruminants (its digestibility is over 700 g kg -1 and it also serves as an energy source due to the starch in the grains, with net energy for lactation energy content of 1.5 Mcal kg-1). It is harvest for silage at about the half dent stage of maturity (which means that half of the starch in the grains hardened), at 300-400 g kg -1 DM and it can be ensiled very well without wilting. The final pH of well preserved corn silage is 3.7-4.0. Sorghum is suitable for semi-arid areas because of its drought resistance and is less fastidious than corn with regard to fertilization. Its yields are 10-15

tons of DM per hectare and the digestibility of BMR (brown mid-rib) cultivars approaches that of corn. Lodging of such lignin-reduced cultivars might be a problem. Alfalfa, a perennial legume, is very popular in area with abundant water. It is harvested for silage several times during the growing season at 10-20% bloom when nutritional value is maximal. If the alfalfa is harvested at a later stage its NDF (neutral detergent fiber) content increases and its nutritional value decreases. Its protein content is high, about 180 g kg -1 DM. However, the high protein and mineral and low WSC contents result in high buffeting capacity which make it difficult to ensile. In addition, its DM content at harvest is too low (only 150-250 g kg -1) and therefore, in order to avoid effluent losses and clostridial fermentation, alfalfa should be extensively wilted to about 500 g kg1 DM. Wilting also increases the relative WSC content in the crop. Such silage is referred to as 'haylage' in which the fermentation is slow and the final pH values are 4.5-5.0. Other forage legumes that can be ensiled include clover (Trifolium spp., red and white), field peas, vetch (Vicia sativa) and sulla (Hedysarum coronarium). Perennial grasses are common crops for ensiling and they include temperate and warm (tropical) species which are numerous. The former have a higher nutritional value. Examples for grasses in temperate climate zones are Italian ryegrass (Loluim multiflorum), perennial ryegrass (Lolium perene), cocksfoot (Dactylis glomerata), timothy (Phleum pretense) and meadow fescue (Festuca pratensis). Examples for tropical grasses include guineagrass (Panicum maximum), Bermuda grass (Cynodon spp.) and elephantgrass (napier grass, Pennisetum purpureum). As the grasses mature the rumen digestibility of the grass decreases because of lignification process. Grasses contain enough WSC needed for the ensiling fermentation, and their buffering capacity is low. Grass and legume are sometimes co-ensiled because they can be grown and managed together in the field, the mixtures have improved ensiling properties and higher nutritional value, as compared with silages made from the separate crops. In Israel it is not possible to grow mixed crops in the same field because of religious prohibition. Small grain cereals include wheat, barley, oat, rye and triticale. They are used as forage crops for ensiling and hay in areas with a short rain season. In Israel wheat for silage is very popular. It is grown on winter rain and harvested in spring at the milk ripening stage. Its yields at this stage are around are 10 tons of DM per hectare and its DM digestibility is 640-680 g kg -1. Table 2 summarizes the changes in whole crop wheat during maturation (Weinberg et al., 1991; Ashbell et al., 1997). Cell wall contents (NDF) peak at early milk

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Table 2. Changes in composition (g kg -1 DM) of whole crop wheat during maturation (adapted from Weinberg et al., 1991 and from Ashbell et al., 1997).

DM, dry matter; NDF, neutral detergent fiber; ADF, acid detergent fiver; ADL, acid detergent lignin; CP, crude protein; WSC, water-soluble carbohydrates.

stage of maturity and then it decreases due to the development of the starchy grains. The more mature the plants are, the more lignified they are, and therefore, their fiber digestibility decreases.

19.3.4.2. Harvesting The time of harvesting forage crops for silage affects the quality of the silage and the profit obtained by both crop growers and cattle farmers. The decision when to harvest is influenced by factors such as expected yields, nutritional value of the crops, ensiling characteristics and weather conditions. The later the harvest is the higher the yields. However, as mentioned earlier, as plants mature their digestibility decreases because of lignification processes. In addition, harvesting at a later maturity stage might result in a biomass which is too dry and its WSC content is too low. Plants which are too dry are resilient and therefore, difficult to compact in the silo and air can penetrate more easily. Fermentation of dry crops is slow, resulting in high ensiling losses and molding upon aerobic exposure. On the other hand, harvesting at an early stage may lead to moist plants that result in effluent and clostridial fermentation. Therefore, the time window for harvesting crops for silage is limited. For cereals the status of the starch in the grains is an indicator of maturity; the milk stage of maturity (when the grain is still semi-liquid) is recommended. For legumes and grasses the maturity is determined according to the extent of blooming (flowering). Such crops should be harvested between early and mid-bloom (10-50% flowering). The cutting height should be adjusted in order to avoid mixing with soil and debris which can damage the knives and decrease the nutritional value of the harvested biomass. The lower parts of the plants are inferior in quality and therefore, a higher cut of about 7 cm above ground is recommended.

19.3.4.3. Wilting Wilting refers to drying of the crops in the field before they are picked up. As mentioned above plants that are harvested with a high moisture content should be wilted in order to achieve the desirable DM content required for successful ensiling. Wilted plants should have an appropriate "thin" structure that enable rapid loss of moisture. Weather conditions should be favorable for rapid drying. Legumes and grasses are usually wilted before ensiling. In Israel, wheat for silage is usually wilted for a few hours and this practice is preferred over direct harvesting because of efficiency considerations of the harvesting combines.

19.3.4.4. Chopping Chopping refers to the action of cutting the plants to small pieces in order to enable their compaction (consolidation) in the silo with subsequent removal of air. Chopping increases the surface area of the biomass and some WSC-rich juice is released to the outer surface which enhances the lactic acid fermentation. The recommended particle length of the cuts is 10-30 mm; shorter pieces may result in excessive effluent and they may not supply long enough fibers which are necessary for the rumen functionality; pieces which are too long may inhibit adequate compaction in the silo, especially of dry plants which resist compaction, which enables air penetration into the silage and spoilage. Chopping is usually carried out at pick-up and the chopped particles are blown into the shipping wagon which follows the pick-up. As mentioned in the Microbiology section, chopping increases the LAB population in the ensiled biomass. It is possible to apply silage additives at this step which enables adequate mixing. The chopped forage is transported to the silo in trucks or wagons. Transportation costs depend on the distance of the field from the silo and truck size.

19.3.4.5. Filling and compaction Filling should be fast in order to exclude air quickly and avoid respiration and aerobic losses. It may take several days to fill a large silo. Bunker silos are filled in a series of slopped wedges which are compacted by wheel tractors which move slowly back and forth continuously during the filling operation. In tower silos compaction is accomplished by the weight of the forage. The DM density of a silage depends on silage DM content, silo dimensions and geometry and the intensity of compaction. The drier the biomass is, it is more resilient and the more difficult it is to compact it. The estimated DM density of a well-compacted wheat silage at 350 g kg -1 DM in a horizontal bunker silo is 230 kg m -3. Other density values found in the literature vary between 100 and 430 g kg DM m -3.

Figure 2. Compacting chopped crop in a bunker silo.

19.3.4.6. Sealing Sealing protects the top layer against air penetration. Sealing of bunker and stack silages is usually accomplished with polyethylene sheeting of various thickness (0.1-0.2 mm), and the thicker it is less air penetrates. The plastic sheeting is susceptible to physical tears, sometimes caused by birds and animals. It is possible to protect the sheeting with a layer of paper pulp or other materials that also keep it from flapping. Used tires are also spread over the sheeting to anchor it to the silage. 19.3.4. 7. Unloading In horizontal silos the face of the silage is exposed to air during unloading and aerobic spoilage is enhanced. It is possible to use scrapers or front-mounted bucket tractors for unloading. The latter tends to produce cracks in the face through which air can penetrate deeper into the silage, as compared with a smooth face obtained with a scraper. Block cutters leave the unloaded silage intact, and therefore, the blocks can be stored for several days. Experimental data reveals that air penetrates 1-2 m deep into the silage from the face. In order to minimize the time that the face is exposed to air it is recommended to renew it every three days in wide bunker silos, or to unload a depth of 20-30 cm everyday. The plastic cover should not be removed further than necessary during the daily unloading of the silage. In tower silos the unloading can be designed from the bottom or the top of the silo.

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19.3.4.8. Ensiling facilities Silage facilities include various types of silos in which the ensiled crops are fermented and stored until used for feeding. Two types of stationary silos can be used: tower silos made of concrete or metal, and horizontal bunker silos made of concrete floor and walls. The construction of such silos requires

large capital investments. Silo size should be designed according to feeding needs and the expected daily removal of silage from it. Other ensiling facilities include heavy duty plastic sleeves into which the chopped crop is pressed by a special machine. Stack silage consists of a long heap of chopped and consolidated crop which is prepared on the ground or on plastic sheeting and sealed with plastic sheeting. It is also possible to store silage in 0'5-0.75 ton bales. Bales are prepared by wrapping four to eight layers of polyethylene stretch around the units by a special machine. The crop (usually grass) is not chopped, and bale silage is sensitive to aerobic exposure. Round or square bales are available and they enable flexible use as these relatively small units that can be opened one at a time according to needs and they can be brought to pasture areas. In Israel, the recent trend is to construct large feeding centers which serve several farms and supply total mixed rations (TMR) which are formulated according to requirements of the animals and the feed which is available. Such feeding centers operate with relatively large bunker silos which contain several thousands tons of silage with a very fast turnover. Experimental data reveals that it is also possible to store silage in small plastic bags (10-20 kg ) which can be used by small holder cattle farmers in Africa and other areas that cannot afford the facilities required for common ensiling.

19.3.5. Additives for silage Silage making is a multi-step process which is affected by many factors which are related to the characteristics of the crops, to management practices and to weather conditions. As mentioned before the crops composition should be adequate and allow a rapid development of LAB under anaerobic conditions. Ensiling steps should be synchronized and avoid delays which are detrimental to the process. Cooperative weather conditions are necessary for undisturbed harvest and rapid wilting. These factors are not always optimal for the ensiling process and cannot be controlled. For these reasons, various additives for silage have been developed which should improve the silage fermentation and preservation and its nutritive value. Some farmers feel that it is beneficial to use additives "just to be on the safe side". It should be emphasized that additives may be helpful under suboptimal conditions, however, they cannot compensate for poor crop quality or inadequate silage management. In the beginning of the 20th century farmers in Europe used molasses to enhance the WSC content in low sugar crops. In 1929-~933 Virtanen from Finland developed the mineral acid additives that decrease the pH rapidly and arrest any microbial activity and thus preserve very moist silages of northern Europe (For that he won the only Nobel Prize in Agricultural Sciences so far). Since then, many more additives were developed, both chemical and biological.

The latter comprise bacterial inoculants and enzymes. It was estimated that in 2001 silage additives were used in over 50% of the silages produced in the USA (Hoard's Dairyman). Silage additives can be classified according to their effect on the silage fermentation. The additive categories include fermentation inhibitors, fermentation enhancers, inhibitors of aerobic deterioration, nutritional additives and moisture absorbents. Additives should be used according to the specific conditions and needs which exist in a given silage. Additives should be effective, easy to use, non-corrosive to machinery, non-pollutant, should not be detrimental to human or animal health and their use should be price effective. The following paragraphs shortly describe the various classes of additives.

19.3.5.1. Fermentation inhibitors It is aimed at partial or full inhibition of the microbial activity in the silage. These additives include sulfuric acid, formic acid and formaldehyde and they are used in moist silages resulting in a rapid decrease in pH. Their rate of application is 2-4 liter per ton (wet weight). Formic acid and formaldehyde bind proteins and so increase by-pass protein in ruminants. Formic acid is corrosive to machinery, increases effluent, it is volatile and inconvenient to use.

19.3.5.2. Fermentation enhancers It is aimed at speeding up the ensiling fermentation and so minimize losses. Such additives include molasses, bacterial inoculants and enzymes. Molasses directly add fermentable sugars and can be used in low-sugar crops. Bacterial inoculants comprise mainly homofermentative LAB, single or multi-strain. Species that are most often used include Lacobacillus plantarum, Pediococcus pentosaceus and Enterococcus faecium. They are applied at 105-106 microorganisms per g crop, and when established in the silage produce rapidly lactic acid which results in fast decrease in pH. In some studies it has been observed that feeding silages inoculated with specific LAB strains also improved animal performance (a probiotic effect?). This is manifested by a 5-10% increase in feed intake, weight gain, milk yield and feed efficiency. Enzymes include cell-wall degrading enzymes such as cellulases, hemicellulases and pectinases from fungal origin. Their optimal activity is at pH 4.5-5.0 which is compatible with silage conditions. Partial degradation of the cell-wall results in release of WSC necessary for the ensiling fermentation. In addition, silage digestibility is improved. However, high application rates are required for significant effects and these are usually not economical.

19.3.5.3. Inhibitors of aerobic deterioration These additives are aimed mainly at inhibiting yeasts and molds which are the major spoilage microorganisms upon aerobic exposure of silage. They include both chemicals and bacterial inoculants. Chemicals most often used are volatile fatty acids and sulfur based chemicals which produce in the silage sulfurdioxide. In Israel, some farmers use salt (NaC1) at 3 kg per m E on the top of silages to inhibit molds. Sometimes, a bacterial inoculant is used which includes Lactobacillus buchneri, a heterofermentative LAB that produces high concentrations of acetic acid in the silage to inhibit aerobic fungi. Another available bacterial additive includes Serratia rubidaea, which secretes an antifungal peptide. Silage additives which are available are numerous. Their labels claim various improvements of silage fermentation, enhanced aerobic stability and improved animal performance. At present there are no legal standards with regard to these claims. However, in various countries there is a voluntary process of testing silage additives in controlled experiments performed by independent research laboratories. An example is the UK Forage Additive Approval Scheme which sorts and approves silage additives according to their claimed actions, as follows: A~ Silo trials: improving fermentation, improving aerobic stability, reducing effluent,

reducing ensiling losses. B~ Animal trials" improving intake, improving digestibility, improving feed

efficiency and increasing milk production. Such schemes can help farmers choose silage additives according to their needs.

19.3.6. Nutritional aspects Silages supply energy, protein and fiber to ruminants. Fibers are necessary for proper function of the rumen, whereby the rumen microorganisms degrade fibers to volatile fatty acids which are absorbed through the rumen walls and serve as energy source for the animals. The nutritional quality of silage depends on the crops that are used to make it, the maturity of the crop at harvest, and on silage fermentation and management. Silage is different from fresh forage or hay by its low pH value and short particle size. The advantage of feeding silage is the ease it can be mixed into total mixed rations (TMR) due to the high moisture content and short particle size. On the other hand, short particle size may result in insufficient chewing and saliva secretion which helps to buffer the rumen. The digestibility of silages that are made from crops which are too mature is reduced due to high lignin content. The intake of silages which are too moist and poorly fermented is reduced, probably because of reduced palatability. High levels of acetic and butyric acids also reduce silage intake by the animals.

19.3.7. Silage and health Forage crops and their preserved forms are the first step in the food production chain for human beings. Their quality also affects animal health and wellbeing. Therefore, it is very important that silages should be safe for use. However, growing and preserving forage crops take place under field and farm conditions which are difficult to control and whereby many complex factors exist which might affect their safety. In the past, we have witnessed fatal incidences which were caused by feeding cattle various types of fodder, the most conspicuous case was the out break of bovine spongiform encelophathology ('the mad-cow disease') which was caused by feeding animal waste. In Israel, a fatal heart syndrome in beef cattle was caused by feeding broiler litter which contained high levels of maduromycin (Sygro| a poultry coccidiostat). Hazards to health associated with silage include detrimental microorganisms, toxic chemicals and excess of acidity. Excess of acidity might impair feed intake. The list of undesirable microorganisms in silage includes E. coli some strains of which are pathogenic to both animals and humans. Enteropathogenic strains of E. coli (such as O157:H7) may cause acute diarrhea in calves or mastiffs. Such strains may acquire acid resistance and survive in silage with pH values above 4.2. Listeria monocytogenes may develop in spoiled aerated parts of silages with pH values above 5.0, and in baled silages which are more susceptible to air ingress. The organism may cause various diseases in animals and humans, including meningitis, encephalitis and abortions. Sheep are the most affected animals by this microorganism. Clostridium botulinum may produce an extremely potent and fatal neurotoxin in silages with pH values above 4.5. The incidence of botulinum in silage is low and this microorganism may origin from dead small animals which find their way into the silage. Molds are yet another group of microorganisms which develop in silages that are exposed to air. Some molds may produce mycotoxins in the silage which cause various acute or chronic diseases. The genera that produce mycotoxins include Penicillum, Aspergillus and Fusarium. Nitrates, which some forage crops accumulate are toxic to animals. Under mild acid conditions prevailing in the silage during early phases of ensiling nitrates are reduced to nitric oxide (NO). Nitric oxide may then be oxidized to nitrogen dioxide (NO2) which forms nitrous and nitric acids. These substances cause respiratory irritation and affect humans ('silo-fillers disease') and animals alike. Other toxicants include phyto-estrogens that can cause fertility problems which may origin from foreign plans that contaminate the fields. Hazards are not always predictable. In order to ensure silage (and feed) safety the hygiene of the forage crops and silage should be maintained. Application of

Good Management Practices (GMP), which involves careful silage management can minimize the incidence of forage related health problems. HACCP (Hazard Analysis and Critical Control Points) focuses on prevention of problems at critical stages of production. Such control system should identify hazards that may occur during silage preparation and storage. Any new technology or change from the conventional practice should be assessed for potential risks. For example, new silage additives which should be carefully tested before approved for sale. Another example pertains to the safety of forage crops irrigation with recycled sewage water. Much research on chemical and microbiological aspects is conducted in Israel on this issue.

19.4 PRESERVATION OF AGRICULTURAL BY-PRODUCTS BY ENSILING Agricultural by-products are obtained from food processing, surplus fruit and vegetables, or animal excreta. Examples include hulls of various nuts, apple, grape and tomato pomace, beet, coffee pulp, citrus peels and pulp, bakery waste, broiler litter and more. These by-products can be utilized by ruminants for their energy, protein, mineral and fiber content. Agricultural by-products are usually cheap, and in many cases seasonal. During the season they might be produced in amounts too high to be used on the farm. Because of their high moisture, carbohydrate or nitrogen contents, they spoil rapidly. Therefore, they cannot be stored in their raw form for a long time. Accumulation of spoiling materials might cause serious environmental problems: effluent from moist heaps with high BOD values might pollute freshwater sources. Large piles of rotting material might emit noxious odors and attract insects and rodents. The benefit from utilization of by-products for animal fee~ing arises from both their nutritional value and from reducing the environmental load. Efficient utilization of by-products requires application of adequate preservation technologies which are also cost-effective. Ensiling is actually the only preservation method that can~ be applied (by-products are too bulky for preservation by sun-drying). Ensiling technology should be especially adjusted according to the composition and unique characteristics of each by-product. The research conducted on citrus peels and pulp is an example for the complexity of the problem. This by-product is very moist (>80% moisture), rich in sugars (20-30% in dry matter) and high in yeast population (104-106 cfu g-l). Storing the peels in sealed containers resulted in much effluent and high DM losses (up to 30%). The major fermentation product was ethanol (15% in DM) which reflected the yeast activity. Application of sorbic acid or blanching of the peels to 80~ reduced the yeast numbers and significantly minimized losses. However, such treatments were not economical relative to the value of the peels. For

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practical purposes in Israel fresh peels are fed immediately or mixed with dry broiler litter and the resulting mixtures are nutritionally balanced and more stable. The use of coffee pulp for ruminant feeding is another example. The pulp which remains after separation of the coffee beans accumulated in large amounts in coffee growing areas. Feeding coffee pulp to ruminants is limited due to high caffeine and tannin content which have a negative effect on the animals. Reducing the content of these constituents requires creative research solution. No practical solution has yet been reached.

19.5 SILAGE QUALITY Many factors affect silage quality, which include weather conditions at harvest and ensiling, plant species, stage of maturity, dry matter content of the crop and silage management (wilting, chopping, sealing, application of additives). The overall objective of ensiling is to maintain quality of the crops as close as possible to that of the fresh plants. Silage quality is assessed by preservation and nutritional parameters. The preservation quality of a silage can be assessed by sensory evaluation and by chemical and microbiological analysis. Sensorial appraisal is the primary and simplest indication of silage quality and it examines color, odor, texture, visible molds and sense of heat. Color usually turns into light green (khaki) due to loss of Mg-ion from the chlorophyll when the pH decreases; odor should be typical with no unpleasant notes; the silage particles should be free flowing and not sticky with no visible molds. Warm silages indicate aerobic spoiling activity. Laboratory analysis should include only relevant measurements according to silage type and characteristics. The basic chemical analysis includes pH, determination of lactic acid and volatile fermentation products (ethanol and volatile fatty acids) and ammonia nitrogen. The final pH value of the silage depends on the initial sugar content of the crop, its DM content and buffeting capacity. For cereal silages it should be below 4.0, for legumes 4.5-5.0. The ratio lactic : acetic acids should be at least 2:1, which indicates proper fermentation. Butyric acid is undesirable and it indicated clostridial secondary fermentation. Ammonia results from protein degradation in the silage and NH3-N should not exceed 10% of total N. Losses can be assessed by weight loss during ensiling or from the difference in the content of a specific constituent between the fresh and ensiled crop. Microbiological evaluation of the silage usually includes the enumeration of lactic acid bacteria, enterobacteria, yeast, molds and clostridia. These are determined in appropriate selective media which are incubated at specific temperatures.

The assessment of aerobic stability of the silage is based on two measurable parameters: the time it takes for the temperature to increase by l or 2~ above the ambient temperature, or CO2 production during aerobic exposure of the silage. The latter is measured in a 'bottle system' whereby the upper part contains the silage sample and the lower part contains 25% KOH which absorbs the CO2 (Figure 4). The nutritional quality of the silage is evaluated by various analyses: for example, ash content above the normal values may indicate soil contamination which is undesirable. Fiber level is an indicator of the maturity of the plants and correlates with silage digestibility. Fiber content is measured after refluxing in neutral or acid detergent solutions to obtain NDF (neutral-detergent fiber) and ADF (aciddetergent fiber), respectively. The former comprises cellulose, hemicellulose, lignin ash and some of the pectin whereas the latter does not include hemicellulose which is extracted in the acid solution. Thus, the difference between NDF and ADF gives an estimate of the hemicellulose. By further acid hydrolysis of the ADF fraction the cellulose is hydrolyzed and after ashing it is possible to obtain the lignin content (ADL, acid-detergent lignin). High ADF content correlates with poor digestibility. It is possible to apply traditional wet chemistry to the various analyses. The analytical novelty of the last decade is the development of NIRS (near infrared spectroscopy). It is based on spectra ('fingerprints') which are obtained from absorption of infra-red light (in the wavelength region from 750 to 2500 nm) by specific molecular groups in the analyzed commodity (such as carbonyl, System for aerobic stability determination

Figure 4. Bottle for aerobic stability test.

amine, carboxyl and hydroxyl). The optical data is then regressed against known parameters to produce the best correlations and the resulting equations are used to predict the concentration of various chemical constituents in unknown samples. The advantage of NIRS is the simplicity of sample preparation, the fastness of obtaining results and its high accuracy. Digestibility is a measure of the forage fraction which is absorbed and utilized by the animal. It is possible to measure the digestibility of any constituent of the forage, the most common digestibility measurements include dry matter, organic matter and NDF. Digestibility can be measured in vivo (involving animal feeding trials) or in vitro. The latter involves a two-stage incubation of dried samples in rumen liquor and with pepsin.

19.6. RESEARCH NEEDS New developments and changes are introduced into silage technology constantly which should be tested and approved for their efficacy. This is true for all aspects of ensiling. New crop cultivars are introduced for higher yields, resistance to drought, resistance to lodging or for seed rotation purposes. The new crops and cultivars should be studied for their ensiling characteristics, aerobic stability and nutritional value. New methods for storing silage are developed, such as bale silage which are more susceptible to aerobic spoilage than conventional silage. Data on such new technologies needs to be generated. The same applies to new silage additives. More basic aspects include the interactions between various microbial populations during ensiling. For example, how do lactic acid bacterial inoculants for silage exert beneficial (probiotic) effects on ruminants? For research purposes novel molecular techniques can be used which enable to follow up the fate of microorganisms in the silage or in the rumen. They include the use of genomic primers for specific strains or marking a target strain with a special trait. An example is a modified strain of E. coli which possesses the green fluorescence protein (GFP) and is resistant to kanamycin which was used to follow its fate in silage and hay. Ensiling experiments can be performed in different ways: in mini-silos which serve as models and enable to analyze replicate samples during ensiling (timecourse studies). Another way is to bury Dacron bags with control and treated silage samples in various sites in the silo on the day of ensiling. When the unloading front reaches the bags they can be retrieved and analyzed. Such experiments are performed in a 'true' silage environment. It is also possible to use Whole silos to test specific treatments (such as new additives) compared to control un-treated silos. Such experiments enable also to conduct animal

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Acknowledgements ,,,,

Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No 468-E, 2006 series.

Suggested readings Buxton DR, Muck RE & Harrison JH, 2003, Silage Science and Technology. Agronomy Monograph 42, American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc. Madison, WI, USA. McDonald P, Henderson AR & Heron SJE, 1991, The Biochemistry of Silage, 2nd edn, Chalcombe Publications, Marlow Bottom, Marlow, Bucks, UK. Van Soest P, 1994, Nutritional Ecology of the Ruminant, 2nd edn, Cornell University Press, Ithaca, NY, USA. Woolford M, 1984, The Silage Fermentation. Marcel Dekker, Inc., New York, NY, USA.

References Ashbell G, Weinberg ZG, Brukental I, Tabori K & Sharet N, 1997, Wheat silage: effect of cultivar and stage of maturity on yield and degradability in situ. Journal of Agricultural and Food Chemistry, 45 (3), 709-912. Fry G, 1885, Sweet ensilage, The Agricultural Press Co, London, UK. Goffart A, 1877, Manual of the culture and ensilage of maize and other fodder crops. Masson, Pads, France. Honig HH, 1980, Mechanical and respiration losses during pre-wilting of grass. In: Thomas, C. (ed.) Forage Conservation in the 80s, Proceedings of the British Grassland Society Occasional Symposium No I1, Brighton, UK, pp. 201-204. Weinberg ZG, Ashbell G, Hen Y & Harduf Z, 1991, Ensiling whole wheat for ruminant feeding at different stages of maturity. Animal Feed Science and Technology 32: 313320. Wilkinson JM, Bolsen KK & Lin C J, 2003, History of silage, In- Buxton DR, Muck RE & Harrison JH, (eds.), Silage Science and Technology, Agronomy Monograph 42, American Society of Agronomy, Inc., Crop Science Society of America, Inc., Soil Science Society of America, Inc. Madison, WI, USA. pp. 1-30. Zimmer E, 1980, Efficient silage systems. In: Thomas C (ed.), Forage Conservation in the 80s. Proceedings of the British Grassland Society Occasional Symposium No 11, (1979), Brighton, UK, pp. 186-197.

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20.1. INTRODUCTION "How will solid waste be managed?" is an important question demanding attention due to the rapid growth in solid waste generation worldwide. Urban conglomerations, with their ever-increasing population and consumerist lifestyle generate voluminous solid wastes. Apart from these sources, industries are other potential and relatively new source of solid wastes. Industrial growth is resulting in greater goods production, improved livelihood opportunities for urbanites but degraded environmental quality of cities. The key problems posed by industrial wastes include" a) Contamination of soils and surface and subsurface water by pollutants present in the sludge through leaching, b) The contamination of environment by heavy metals and chemicals, c) Colour imparted to the water bodies or soils and d) Odour problem inviting public attention. The ecologically and commercially sustainable management of industrial sludges is a major issue faced by all Indian industries due to stringent National disposal standards regardless of size or location of the industry. A number of solid waste disposal strategies have been adopted around the world. The major disposal technologies include, Open dumping, Hog feeding, Land filling, Land spreading, Composting, Lime stabilization, Thermal drying (including pelletisation) and Incineration. Each solution has benefits and limitations. No single solution has universal applicability. In such a scenario, there is an obvious need to reuse and recycle the solid wastes. In the past years, several new methods, mainly based on biological processes have been developed to convert solid wastes into energy and organic fertilizers. All these conversion processes create useful products that can be marketed. Waste management under these conditions gets a completely different image and perspective, compared to the actual state of the art. These methods include a) Vermicomposting, b) Composting, c) Biomethanation and, d) Ferti-irrigation.

The problem of solid waste disposal has further increased in cities because of shortage of dumping sites and strict environmental legislation, so scientists are seeking for management altematives, which are economically and ecologically sustainable. Vermicomposting can be an appropriate altemative for the safe, hygienic and cost effective disposal of organic fraction of solid waste (Kaviraj and Sharma, 2003). The transformation of solid wastes into vermicompost is of double interest: on the one hand, a waste is converted into value added product, and, on the other, it controls a pollutant that is a consequence of increasing population, urbanization and intensive agriculture (Kaushik and Garg, 2003). In 1881, Charles Darwin published his last book "The formation of vegetable mould through the action of worms with observation on their habits" shortly before his death. The book drew attention to the great importance of earthworms in the breakdown of dead plant material and the release of essential nutrients from it. However, only in last few decades, potential of earthworms for breaking down organic waste has been explored in depth and many large scale vermicomposting facilities have been developed all over the world with varying success. This chapter has been complied in order to enable the readers to derive information about potential of vermicomposting technology in solid waste management.

20.2. COMPOSTING, VERMICOMPOSTING AND VERMICULTURE Composting is bioconversion of organic matter by heterotrophic microorganisms (bacteria, fungi, actinomycetes and protozoa) into humus-like material called compost. The process occurs naturally provided the fight organisms, moisture, aerobic conditions, feed material and nutrients are available for microbial growth. By controlling these factors the composting process can occur at a much faster rate. Vermicomposting is the process by which worms are used to convert organic materials (usually wastes) into a humus-like material known as vermicompost. The goal is to process the material as quickly and efficiently as possible.

Yermiculture is the culture of earthworms. The goal is to continually increase the number of worms in order to obtain a sustainable harvest. The worms are either used to expand a vermicomposting operation or sold to customers who use them for the same or other purposes. If the goal is to produce vermicompost then we want to have maximum worm population density all of the time. If the goal is to produce worms then we keep the population density low enough so that reproductive rates are optimized.

20.3. PRINCIPLE OF VERMICOMPOSTING Certain species of earthworms can ingest organic waste rapidly and fragment them into much fine particles by passing them through gizzard. The earthworms maintain aerobic conditions in the vermicomposting process, ingest solids and convert a portion of it to earthworm biomass and respiration products and egest peat like material termed as worm castings (Loehr et al., 1985). Castings are much more fragmented, porous and microbially active than parent material (Edwards, 1988; Edwards and Bohlen, 1996) due to humification and increased decomposition. The earthworms derive their nourishment from the microorganisms involved in the waste decomposition; and organic waste to be decomposed. The earthworms and the microorganisms act symbiotically to accelerate and enhance the decomposition of the organic waste. The composition of the worm casting depends on the parent material. During this process, important plant nutrients such as nitrogen, potassium, phosphorus, calcium etc. present in the waste are converted through microbial action into forms that are much more soluble and available to plants than those in the parent substrate (Ndegwa and Thompson, 2001). Overall, the vermicomposting process is a result of the combined action of earthworms and microflora living in earthworm intestine and in the organic waste (Albanell et al., 1988).

20.4. EARTHWORMS: GENERAL INTRODUCTION The earthworm derives its name from the fact that it burrows and eats its way into the earth. Earthworms have been on the Earth for over 20 million years. There are 3627 species of earthworms distributed throughout the world. Aquatic worms are called as microdrilli and terrestrial earthworms are known as megadrilli. In India, there are about 509 species of earthworms, belonging to 67 genera. Besides these, more than 20 species from other countries have been introduced into India. These are known as 'peregrines'. Earthworm occur in diverse habitats, organic materials like manures, litter, compost etc are highly attractive for earthworms but they are also found in very hydrophilic environment close to both fresh and brackish water, some species can survive under snow (Sharma et al., 2005).

20.4.1. Classification of earthworm Kingdom: Animalia, Phylum: Annelida, Class: Oligochaeta, Order: Opisthopora, Family: Lumbricidae, Genus: A large number of genera have been described in literature, Species: A large number of species under each genus have been described in literature. Earthworms have also been classified on the basis of their ecological niche (Bouche, 1977) and feeding behaviour (Lee, 1985) [Fig. 1]

Fig 1. Classification of earthworms 20.4.2. Classification of earthworms based on ecological groups A brief discussion of different ecological groups of the earthworms is given below:

20.4.2.1 Epigeic species These species live above the mineral soil surface typically in the litter layers and plant debris and feed on them. These are phytophagous. Most of the species have insignificant role in humus formation and are not good for use in field conditions for soil reclamation. They have high reproductive rate and high cocoon production rate. However, their life span is relatively short. They show high metabolic activity and hence are particularly useful for vermicomposting. Examples are Eisenia foetida, Eisenia andrei, Eudrilus eugeniae, Perionyx excavatus and Drawida modesta.

20.4.2.2 Endogeic species These species inhabit mineral soil beneath the top soil surface generally forming horizontal tunnels to the soil surface. They feed on soil more or less enriched with organic matter. They are probably important in improvement of soil texture and structure (pedogenesis) and are not much beneficial in organic matter decomposition and recycling of plant nutrients. Their reproduction rate is moderate and they have shorter life span. Example is Octochaetona thurstoni.

20.4.2.3. Anecic species These are surface feeding earthworms that construct and live in permanent burrows in the mineral soil layers but come to the surface to feed on organic matter, mostly plant litter, and pull it into their burrows. They are important in burying surface litter. They are great help in incorporation of organic matter into the soil, and distribution and cycling of plant nutrients, and also in improvement of soil structure and texture (pedogenesis). These species have low cocoon production rate and limited reproductive capacity, but their life span is longer. Examples are Lampito mauritti, Lumbricus terrestris and Octochaetona serrata. A summary of characteristics used by Bouche to distinguish the earthworms on the basis of ecological niche is given in Table 1 (Gajalakshmi and Abbasi, 2004a).

20.4.3 Classification of earthworms based on feeding behaviour According to their feeding habits, earthworms are classified into detritivorous and geophagus (Lee, 1985). Detritivores feed at or near the soil surface mainly on plant litter or dead roots and other plant debris in the organic matter rich surface soil or on mammalian dung. These include epigeic and anecic forms. These are also called as humus formers. Geophagus feed deeper beneath the surface, ingesting large quantities of organically rich soil. These include endogeic forms. These are also called as humus feeders. For the purpose of vermicomposting of different organic wastes, generally epigeic species of earthworms are used widely in India (Ismail, 2005). It is generally known that the epigeic species Eudrilus eugeniae, Perionyx excavatus and Eisenia foetida have a potential as waste decomposers. In order to utilize these species successfully in vermicomposting and vermiculture all aspects of their biology and physical requirements must be known. The life-cycles of each of the three species are now well documented after intensive studies under controlled conditions. Venter and Reinecke (1988) presented studies on Eisenia foetida, Reinecke and Viljoen (1992) on Eudrilus eugeniae, and Hallatt et al., (1990) on Perionyx excavatus. From a comparison of the lifecycle it is evident that all three species are prolific breeders, maintaining a high reproduction rate under favourable conditions of temperature, moisture and food availability.

20.5. FOOD AND FEEDING HABITS OF EARTHWORMS Earthworms exhibit a high degree of niche diversity (Table 2). Surface dwellers largely feed upon leaf litter on soil surface. Burrow formers swallow soil and

Table 1. Summary of characteristics to classify the earthworms on the basis of ecological niche (Gajalakshmi and Abbasi, 2004a)

derive nutrition from it. The quantity and quality of food available in an ecosystem determines population size~ composition and diversity of earthworm community. In general, daily ingestion of feed varies from 100 to 300 mg/g of worm body weight. According to one estimate, an earthworm can consume 8 to 20 g dung/year. So at a population density of 1,20,000 adults/ha, dung consumption would be 17.20 tones/ha/year (Bhatnagar and Palta, 1996). In a temperate deciduous forest, annual leaf fall of approximately three tones/ha/year will be consumed just in threemonths (Satchell, 1983). These estimates thus amply indicate that earthworms are important in soil biota mixing and incorporating organic matter into soil. Some earthworms are able to selectively digest certain microorganisms (Dash et al., 1984).

20.6. BIOLOGY OF EARTHWORMS Earthworms are long, thread-like, elongated cylindrical, soft bodied worms with uniform ring like structures all along the length of their body. Earthworms vary greatly in size. In India some peregrine species like Microscotex phosphoreus (Duges) are only 20 mm long while some endemic geophagous worms such as Drawida grandus (Bourus) may reach up to one meter in length. The colour of earthworms generally ranges from a brownish- black tinge to purple with some exceptions. Generally the dorsal side of the worms is darker while ventral side is paler. Their bodies are segmented which are arranged linearly and outwardly

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highlighted by circular grooves which are called annuli. The number of segments varies from 80 -100 or more. The first segment into which the mouth opens is called as peristomium. On the dorsal surface of the peristomium is a lobe like structure called prostomium which overhangs the mouth. The last segment is called the anal segment and it has a perforation for the anus at the hind end. At the sides of the body on the ventral surface of each segment are four pairs of short, stubby brittles or setae. The setae provide action for movement and also enable the worms to cling to their burrows when predators try to pull them out. Earthworms possess both male and female gonads. At maturity, it develops swollen region behind the anterior which is called as clitellum. It deposits its eggs in a cocoon without the free larval stage. Cocoon production starts at the age of 6 weeks and continues till the end of 6 months. Under favourable conditions, one pair of earthworms can produce 100 cocoons in 6 weeks to 6 months (Ismail, 1997). Cocoon is a translucent, small, spherical protective capsule in which earthworms lay their eggs. The shape, size, colour and number of cocoons vary from species to species. The incubation period of a cocoon is roughly about 3-5 weeks, in temperate worms it ranges between 3-30 weeks and in tropical worms within 1-8 weeks. Quality of organic waste is one of the factors determining the onset and rate of reproduction (Garg et al., 2005). Epigeic earthworms swallow large quantities of decaying animal waste and plant litter. The quantity of food taken by a worm varies from 100 to 300 mg g-1 body weight day -1 (Edwards and Lofty, 1972). While the worm is feeding, the buccal chamber is everted and the food is drawn into the mouth by the sucking action of the muscular pharynx. The gizzard serves to break the food into fine particles which is then sent into the intestine where gastric juices act on the ingested food to digest proteins, fats and carbohydrates. The excreta is egested through the anus as castings (Ismail, 2005).

20.7. BASIC CHARACTERISTICS OF VERMICOMPOSTING EARTHWORM SPECIES The selection of suitable species for vermicomposting and consequential purpose/ utilization is necessary. Such worm species should be tolerant to diseases and its culturing techniques should be simple enough to adopt. Simple characteristic features, therefore, should be known. These are as detailed below: 1. Earthworm should be efficient converter of plant litter or animal waste to body proteins, so that its growth rates are high; 2. It should have high consumption, digestion and assimilation rate (vermicomposting qualities); 3. Earthworm should have wide adaptability (tolerance) to environmental factors (capability to live in varying temperature conditions). Eisenia

foetida has a wider tolerance for temperature than Eudrilus eugeniae and Perionyx excavatus. Eisenia foetida can be cultivated in areas with higher temperature (as high as 43~ as well as lower temperature ( 90%) is in organic form and only a small fraction is present in inorganic form (Baruah and Barthakaur, 1997). Under normal conditions only 0.5 - 2.5% of total nitrogen is converted into forms available to plants. Inorganic nitrogen mainly nitrate and ammonia are available nitrogen forms that are used by plants (Tan, 1996). Vermicomposting process causes significant increase in total nitrogen content of feed stocks after worm activity. According to Crawford (1983) nitrogen content in vermicompost is dependent on the initial nitrogen present in the feed stock and the degree of decomposition. Decrease in pH may be another important factor in nitrogen retention as this element is lost as volatile ammonia at higher pH values (Hartenstein and Hartenstein, 1981). Viel et al., (1987) have reported losses in organic carbon might be responsible for nitrogen addition. Addition of nitrogen in the form of mucus, nitrogenous excretory substances, growth stimulating hormones and enzymes from earthworms has also been reported (Tripathi and Bhardwaj, 2004). These nitrogen rich substances were not originally present in feed and might have contributed additional nitrogen content.

20.11.3. Organic carbon There is a decrease in the organic carbon content of the feed stock during vermicomposting due to loss of carbon as CO2 Suthar, (2006) has reported

that earthworms promote such microclimatic conditions in the vermireactors that increase the loss of organic carbon from substrates through microbial respiration. Garg et al., (2005) have reported a 58.4% reduction in organic carbon in cow dung and 55.4% reduction in horse dung after 90 days of vermicomposting. Kaviraj and Sharma (2003) have reported a 20-45% loss of organic carbon during vermicomposting of municipality or industrial wastes.

20.11.4. Ash content There is an increase in the ash content of the feed stock during vermicomposting. Increase in ash content can be attributed to the enhanced mineralization in the presence of earthworms (Gupta et al., 2006). They have reported an increase in the ash content of water hyacinth and cow dung mixed feed stock in range of 16.3 to 56.5%. 20.11.5. C" N ratio The ratio of carbon to nitrogen is important for the proper growth of any plant. The loss of carbon as carbon dioxide in the process of respiration and production of mucus and nitrogenous excrements enhance the level of nitrogen which lower the C: N ratio (Senapati et al., 1980). The C: N ratio is used as an index for maturity of organic wastes. According to Senesi (1989) decline in C: N ratio to donkey > buffalo > goat = cow = horse > camel. The biomass gain for E. foetida (live weight) per g dry weight of feed source was highest in sheep waste and smallest in camel waste. The mean number of cocoons produced per worm per day in sheep waste was 231% greater than cocoons produced per day in buffalo waste. The number of cocoons produced per earthworm per day in different dung materials was in the order: sheep > cow ~ horse = goat > camel > donkey > buffalo. They concluded that cow, horse, sheep and goat wastes supported the growth and reproduction of E. foetida, hence can be used as feed materials in large scale vermicomposting facilities. The buffalo, donkey and camel wastes can be used as feed in mixture with cow or sheep or goat wastes. The physico-chemical quality of the initial

feed mixtures and vermicompost produced from these wastes is give in Tables 5 and 6. The biochemical changes in fresh cow manure caused by the earthworm Eisenia andrei were measured over a period of 4 months, under controlled laboratory conditions by Atiyeh et al., (2000). Worms reduced the pH and decreased the moisture content in the manure. The C: N ratio of the manure with or without earthworms decreased progressively from 36 to 21. The ash and nitrogen contents increased greatly for a few weeks after the introduction of earthworms, reflecting a rapid breakdown of carbon compounds and mineralization by earthworms. CO2 evolution decreased rapidly indicating increased stability of the organic matter. The biology and population dynamics of Eudrilus eugeniae was studied in cattle waste solids by Dominguez et al., (2001) by growing in groups of 1, 2, 4, 8 or 16 hatchlings in 100 g of waste in incubators at 15 ~ 20 ~ 25 ~ and 30~ It was found that earthworm biomass production was temperature dependent, maximum production being attained at the two highest population density and highest temperature. The greatest organic waste to earthworm conversion ratio of 10:1 (10%) was recorded at the most dense earthworm population. The conclusion of this investigation was that Eudrilus eugeniae is a fast-growing and productive earthworm in animal waste, that is ideally suited as a source of animal feed protein as well as for rapid organic waste conversion. It is more productive in terms of rate of growth than other species and would seem to be a suitable candidate for vermicomposting systems. Effect of various substrates on the biomass production of Eudrilus eugeniae was studied by Mpoame and Nguekam (1994). Worm productivity was 119.9 g/kg of substrate with rabbit manure, 86.3 g/kg with sheep manure and 33.0 g/kg with cattle manure. Chicken manure was unsuitable because it allowed the worms to escape. Jambhekar (1992) reported the use of earthworms as a potential source to decompose organic wastes such as cow dung, poultry manure, horse dung, pig manure and sugarcane trash. The results revealed that C/N ratio of earthworm treated waste reduced to 15.9 as compared to 31.1 in an untreated waste, within one month. While at the end of 45 days, it was further reduced to 12.0 in earthworm treated waste.

20.12.2. Vermicomposting of industrial wastes The industrial wastes which are rich in organic matter and free from toxic substances or ions could be suitable substrates for vermicomposting. The different industrial wastes that are tested for vermicomposting have been encapsulated in Table 7. The use of earthworms in sludge management has been termed as

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vermistabilization. Butt (1993) showed that paper-mill sludge was a suitable feed for Lumbricus terrestris under laboratory conditions. By the addition of spent yeast from the brewing industry, the C: N ratio of this sludge could be adjusted according to the requirements. Elvira et al., (1996) studied the efficiency of Eisenia andrei (Bouche) in bioconverting paper-pulp mill sludge mixed with primary sewage sludge. The presence of earthworms accelerated mineralization of organic matter, favored breakdown of structural polysaccharides, and increased humification rate. Solid paper-mill sludge mixed with sewage sludge in a 3" 2 ratio resulted in the highest growth rate and the lowest mortality of E. andrei, whereas paper-mill sludge mixed with pig slurry exhibited a high mortality. High mortality was attributed to changes in the environmental characteristics (Elvira et al., 1996). Banu et al., (2001) reported the bio-management of paper mill sludge using an indigenous (Lampito mauritii) and two exotic (Eudrilus eugeniae and Eisenia foetida) earthworm species. Results indicated that 25% concentration of paper mill sludge with standard bedding material (Mangifera indica foliage (40%) + cow dung (40%) + Saw dust (20%)) was ideal. Eisenia foetida proved to be the best worm in paper mill sludge bio-management. Vermicomposting of solid textile mill sludge (STMS) mixed with cow dung, agricultural residues, biogas plant slurry and poultry droppings have been reported (Kaushik and Garg, 2003; Kaushik and Garg, 2004). Vermicomposting resulted in significant reduction in C: N ratio and increase in nitrogen content. Total K and Ca were lower in the final cast than the initial feed mixture (Table 8). Solid textile mill sludge can be potentially useful as raw substrate in vermicomposting if mixed with up to 30% with other organic wastes. Garg et al., (2006) have reported the growth and reproduction of E. foetida in a range of different feed mixtures containing anaerobically digested biogas plant slurry (BPS) and solid textile mill sludge mixture (STMS) (Fig 2). The maximum worm biomass was observed in 100% B PS and the lowest in the 60% B PS+ 40% STMS feed mixture. The maximum worm biomass attained in 100% CD was slightly lower than 100% BPS. Increasing percentage of STMS in the feed mixtures promoted a decrease in biomass gain of Eisenia foetida. The maximum worm biomass was attained in 10th or l lth week in all the feed mixtures except 60% BPS+ 40% S TMS feed mixture (8th week). The net weight gain by Eisenia foetida in 100% BPS was 2 - 4 folds higher than STMS containing feed mixtures.

20.12.3. Vermicomposting of municipal solid waste Management of garbage by vermicomposting in environment friendly manner has been reported by several workers. Narayana (2001) made studies on the feasibility of vermicomposting of vegetable waste of a town by earthworm Eudrilus eugeniae, and found that the final compost promoted seed germination

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using Perionyx excavatus. Maize residue was most suitable among all tested agricultural residues. Chaudhuri et al., (2003) conducted laboratory scale experiments to access the suitability of rubber leaf litter (Hevea brasiliensis) as vermiculture substrate for Perionyx excavatus, Eudrilus eugeniae and Eisenia foetida. The rubber waste was most suitable for Eudrilus eugeniae followed by Eisenia foetida and Perionyx excavatus. Vinceslas-Akpa and Loquet (1997) have reported the management of maple pruning waste using Eisenia spp. Mineralization of the organic fraction was more during the first month of vermicomposting. There was a concomitant decomposition of polysaccharides including cellulose. The degradation of aromatic compounds and lignin was started only after one month of the start of the experiment. Frederickson et al., (1997) explored the possibility of combining green waste composting and vermicomposting. They reported that combined systems were useful in two ways (1) enhanced stabilization of green waste by earthworms (2) initial thermophilic stages of windrow composting of green waste results in effective pathogen control while vermicomposting alone may not achieve. Gobi et al., (2001) studied the vermicomposting of coir pith by Eudrilus eugeniae and found that the NPK values increased significantly from its original value after vermicomposting. The lignin and cellulose content were lesser than initial amount. The peels of bitter cassava root, a major source of food carbohydrate in tropics, form toxic waste which is lethal to the soil invertebrates. Investigations by Mba (1996) highlighted the ability of Eudrilus eugeniae to partially detoxify the wastes and convert the toxic cassava peels into valuable vermicompost. Vermicomposting of Neem (Azadirachta indica) was studied by Gajalakshmi and Abbasi (2004b) using Eudrilus eugeniae. Contrary to the fears that n e e m ~ a powerful nematicide~might not be palatable to the annelids, the earthworms fed voraciously on the neem compost, converting upto 7% of the feed into vermicompost per day. Indeed the worms grew faster and reproduced more rapidly in the neem-fed vermireactors than in the reactors fed with mango leaf litter earlier studied by the authors (Gajalakshmi et al., 2005).

20.12.5. Vermicomposting of aquatic weed (water hyacinth) Water hyacinth (Eichhornia crassipes) continues to justify the sobriquet of the "world's worst weed" by defying all the attempts that have been made across the world of eradicating it or finding commercially viable methods of its largescale utilization. Vermicomposting potential of green and dry water hyacinth using Perionyx excavatus earthworm was tested by Sannigrahi et al., (2002). The results indicated faster composting in fresh conditions. Total chemical analysis showed that water hyacinth vermicompost contained about 1.4 to 1.6% nitrogen, 0.8 to 1.0% phosphorus, 1.6 to 2.0% potassium, 0.8 to 1.1%

fiJiiiJiiJii!fiJJfFiJfijJf!iiiiiiiiiiii!iiiiiiiii! iii!ii!iiiii!i calcium and 13.1 to 17.6% sulphur. Comparatively higher manure values were recorded in vermicompost prepared from dried materials than fresh material The viability of vermireactors fed with different proportions of water hyacinth (WH) and cow dung (CD) was assessed over six-months by Gajalakshmi et al., (2001). All reactors performed sustainably with a steadily rising vermicast output, worm zoomass, and number of offspring. It was observed that during the first 10 days of operation the vermicast output as well as growth in the worm zoomass was low. As the worms had been cultured to adulthood on CD before they were introduced to the predominantly WH-fed reactors, they had apparently taken some time to adapt to the new feed~ The potential of two epigeic species (Eudrilus eugeniae and Perionyx excavatus) and two anecic species (Lampito mauritii and Drawida willsi) of earthworms was assessed in terms of efficiency and substantiability of vermicomposting water hyacinth by Gajalakshmi et al., (2001). In different vermireactors, 75 g of 6:1 water hyacinth: cow dung as feed were introduced, vermicasts were produced with steadily increasing output in all the reactors. E. eugeniae was by far the most efficient producer of vermicasts, followed by the other epigeic P. excavatus. In terms of the efficiency of vermiconversion of water hyacinth with cow dung, the animal species followed the trend: E. eugeniae > P. excavatus > L. maritii > D. willsii. Similar trends were observed from increase in animal biomass and number of cocoons produced. Gajalakshmi et al., (2002) conducted a six months long trial on different water hyacinth fed vermireactor ~ (i) fresh whole plant, (ii) dried whole plants, (iii) chopped pieces of fresh plants, (iv) 'spent' weed taken from reactors after extracting volatile fatty acids (VFAs), (v) pre-composted fresh weed and (vi) pre-composted spent weed. The first four forms were studied with and without cow dung. The experiments revealed three trends (i) of the various forms of the weed assessed, the pre-composted forms were the most favoured as feed by Eudrilus eugeniae, while the fresh whole plant form was the least favoured, (ii) the different forms of spent weed were favoured over the corresponding forms of fresh weed, and (iii) blending of cow dung (~ 14% of the feed mass) with different forms of water hyacinth had a significant positive impact on vermicast output, growth in worm zoomass, and production of offspring relative to the corresponding unblended feed. The experiments confirmed that water hyacinth can be sustainably vermicomposted in any of the forms with E. eugeniae. An attempt to develop a system with which the aquatic weed water hyacinth can be economically processed to generate vermicompost in large quantity was made by Gajalakshmi et al., (2001). The weeds were first composted by

!iii!iiiiiiiiiii i!iiii ii3i high rate method and then subjected to vermicomposting in reactors operating at much larger density of earthworms. The composting step was accomplished in 20 days and the composted weeds were found to be vermicomposted three times as rapid as uncomposted water hyacinth. There was no earthworm mortality during the first four months in spite of high animal density in the reactors. The result also indicated that an increase in the surface to volume ratio of the reactor might further improve their efficiency. Gupta et al., (2006) developed different vermireactors containing water hyacinth and cow dung as the feed mixture. They concluded that if water hyacinth is mixed with up to 25% in cow dung on dry weight basis, the vermicompost quality is not affected (Table 9); but a higher percentage of water hyacinth in the feed mixture retarded the growth and fecundity of the worms used and also affected the nutritional quality of vermicompost.

20.12.6. Vermicomposting of sewage sludge The use of earthworms to manage sewage sludge is termed as vermistabilization (Hartenstein, 1978). In this process of sludge decomposition and stabilization about three times increase in rate of microbial growth as compared to noningested sludge has been reported (Neuhauser et al., 1988). Neuhauser et al., (1980) studied the growth of the earthworm Eisenia foetida in relation to population density and feed rationing. The maximum biomass attained by worms was significantly higher in activated sludge than other tested feeds (horse manure, cow manure). Benitez et al., (1999) studied the evolution of earthworms' biomass and changes in enzyme activities during sewage sludge vermicomposting using Eisenia foetida. With time, hydrolase (Glucosidase, urease, BAA hydrolysing protease and phosphatase) and dehydrogenase enzymes' activities decreased as available organic compounds (water-soluble carbon) decreased. Ndgewa et al., (2000) carried out pilot scale studies to investigate the effect of two important system design parameters, viz., the stocking density and the feeding rate for the vermicomposting of biosolids amended with paper mulch as bedding. A stocking density of 1.60 kg w o r m s / m 2 and a feeding rate of 1.25 kg feed/kgworm/day resulted in the highest bioconversion of the substrate into earthworm biomass. Physico-chemical changes affecting in activated sludge by the earthworm Eisenia foetida have been reported by Hartenstein and Hartenstein, (1981). The results showed that mineralization was accelerated 1-3 fold and 2% of the minerals were assimilated. The rate at which heavy metals were concentrated during sludge catabolism was also accelerated. Castings stabilized within 2 weeks. Nucleic acids, which can be used as an index of microbial biomass, were present at a greater concentration in the worm casts than in the sludge, while

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Fig 2. Growth curves of Eisenia foetida in different feed mixtures

in Phaseolus radiatus and Dolichos biflorus to a great extent over the control. The Institute of Natural Organic Agriculture (INORA) has developed techniques of vermicomposting of garbage waste by using high performance species of earthworms such as Eudrilus eugeniae, Perionyx excavatus, Eisenia foetida and Drawida willsi. Senapati et al., (1985) studied the vermicomposting of domestic garbage by Lampito mauritii for 75 days. Percentage decomposition of the garbage was 36% without earthworms and 54% in the presence of earthworms. Gandhi et al., (1997) compared the traditional composting with vermicomposting of domestic waste. The results showed that earthworms can thrive on kitchen waste containing vegetable and fruit peels as well as left over food. Huhta and Haimi (1988) explored the potential of vermicomposting of decomposable matter produced by a family of five persons; garden residues and contents of composting toilet using crushed bark as mixture material. They concluded that Eisenia foetida thrives well and reproduce in household garbage both in summer and winter conditions. Under appropriate conditions worm multiply and complete the process without further care. Das and Talukdar (2001) reported the combined use of earthworms and cellulolytic micro-organisms in decomposition of kitchen waste. A combined system consisting of composting and vermicomposting has been proposed by Graziano and Casalicchio (1987) to obtain valuable vermicompost from municipal garbage waste. Padma et al., (2002) reported that conversion of vegetable waste into vermicompost by Eudrilus eugeniae took approximately sixty days. Shinde et al., (1992) studied vermicomposting of vegetable waste by three different earthworm species, viz.,

Pheretima elongata, Eisenia foetida and Perionyx excavatus. The vermicompost so obtained from different species was having same fertilizer value. Gupta (1997) experimented with Octochaetona serrata species of earthworm for the vermicomposting of kitchen vegetable waste, cow dung, leaf litter cloth, paper and grasses. The earthworm accelerated the decomposition of different wastes in the range of 1 9 - 60%. Tripathi and Bhardwaj (2004) evaluated the vermicomposting potential of Eisenia foetida and Lampito mauritii for degrading kitchen waste amended with cow dung under tropical conditions. Both the species resulted in increased NPK and decreased C/N and C/P ratios after 150 days vermicomposting period. There was moderate mineralization and faster decomposition by Eisenia foetida in comparison to moderate mineralization and moderate decomposition by Lampito mauritii. The average number of cocoons and adults produced was greater by Eisenia foetida than by Lampito mauritii. Finally they concluded that Eisenia foetida may be a better adapted species for decomposition of kitchen waste mixed with cow dung under tropical conditions. Kaviraj and Sharma (2003) conducted a comparative study between exotic (Eisenia foetida) and local (Lampito mauritii) species of earthworms for the evaluation of their efficiency in vermicomposting the municipality solid waste. The results showed that Eisenia foetida was superior to Lampito mauritii in terms of organic carbon reduction, C/N ratio reduction, increase in electrical conductivity and potassium. But Lampito mauritii was able to modify the soil characteristics.

20.12.4. Vermicomposting of agricultural wastes and plant residues Agricultural wastes of diverse kinds can be used as a substrate for earthworm feeding and converting them into vermicompost. Wright (1972) reported that Lumbricus terrestris fed little on fresh apple leaves, but did so readily after the leaves had been leached in water. This was due to the fact that leaves become soft and easy to fragment. Lee (1985) reported enhanced decomposition of cellulose and hemicellulose in garden wastes using earthworm Eisenia andrei and Lumbricus rubellus. Vermicomposting of farm wastes, namely, mango leaves and local grass was carried out using two different species of earthworms, viz., Eisenia foetida and Eudrilus eugeniae, and various chemical parameters governing compost maturity have been reported (Talashilkar et al., 1999). Kale et al., (1982) studied the process of vermicomposting using two species of earthworms, Eudrilus eugeniae and Perionyx excavatus by introducing them into agricultural waste and sugarcane trash as feeding materials. They reported that decomposition of cellulose and lignin present in organic wastes was enhanced by earthworm. Manna et al., (1997) accessed the suitability of soybean straw, wheat straw; maize stover, chickpea straw and city refuse for vermicomposting

the phenolic content, which may potentially serve as an index of humification, was less concentrated.

20.13. EFFECT OF VERMICOMPOST ON PLANTS AND CROPS There is accumulating scientific evidence that vermicomposts can influence the growth and productivity of plants (Edwards, 1998). Abad et al., (2001) have prepared an inventory of materials suitable for use as growing media for ornamental potted plant production which included agro-industrial wastes such as fish or meat meal, bagasse, slaughterhouse waste, brewing waste etc. The beneficial effect of vermicompost on plants may be due to their physical and chemical properties such as particle size, porosity, water holding capacity, air capacity, electrical conductivity and pH which are even more important than the concentration of nutrients (Gouin, 1998). Atiyah et al., (2000) have reported the differences in the effect of vermicompost and composts on marigold and tomato plants. Plants were less responsive to the composts than vermicomposts. This difference in growth may be due to the fundamental differences between the composting and vermicomposting processes which use quite different microbial communities, with composting tending to result in the release of mineral nitrogen in the ammonium form, where as vermicomposting releases most of the nitrogen in the nitrate form (Edwards and Burrows, 1988), the form readily available for plant uptake. Kale et al., (1992) have reported an enhanced activity of the microbial populations (N-fixers, actinomycetes and spore formers) in a paddy field inoculated with vermicompost. There was higher level of total nitrogen in the experimental plots which may be due to the higher count of N-fixers. Garcia-Gomez et al., (2002) assayed the effect of two different vermicomposts prepared from brewing waste and olive mill waste on calendula and calceolaria plants and concluded that vermicompost so obtained was not phytotoxic and permitted the plants to grow disease free and with no weeds. Arbuscular mycorrhizal fungi (AMF) are an important component of the soil microflora, promoting nutrient uptake into plants in exchange for carbon compounds from the host (Sylvia et al., 1998). Few studies have tested the effect of vermicomposts on AMF. Kale et al., (1987) reported an increase in AMF colonization in Salvia and Aster roots growing in a garden soil mixture amended with vermicompost. Cavender et al., (2003) have also reported an increase in AMF colonization of roots of Sorghum bicolor after vermicompost application. In contrast, Sainz et al., (1998)found that amending mineral soils with vermicompost of urban wastes caused significant reduction in AMF colonization of clover and cucumber roots, although growth was increased. These antagonistic effects of vermicompost on AMF colonization could be due to physical and chemical properties than biological effects (Cavender et al., 2003).

Materechera (2002) reported the availability of nutrients to maize from earthworm casts collected from a grazing paddock in South Africa. The maize grown on earthworm casts had significantly higher plant height, stem diameter and higher uptake of the nutrients. He has suggested the addition of earthworm casts especially in small scale production systems where soils are mostly deficit in P, Mg, Zn and Mn. Atiyeh et al., (2000) reported that pig manure vermicompost could be a beneficial component of green house media when mixed up to 20% with the control. Mba (1996) reported that cassava peel vermicompost application in the fields enhanced aerial biomass production in chickpea but acidified the soil and depressed the Irrododrilus surface cast production. He has suggested that cassava peel vermicompost can be utilized in agricultural fields if cyanide toxin effects are eliminated from it. Jeyabal and Kuppuswamy (2001) established that integration of 50% N through vermicompost, 50% N through chemical fertilizers and biofertilizers, Azospirillum and phosphobacteria can be used in rice-legume cropping system with affecting crop yields and soil properties. Various green house and field studies have examined the effects of a variety of vermicomposts on a wide range of crops including cereals and legumes (Chan and Griffiths, 1988), vegetables (Edwards and Burrows, 1988; Wilson and Carlile, 1989; Subler et al., 1998; Atiyeh et al., 2000); ornamental and flowering plants (Edwards and Burrows, 1988; Atiyeh et al., 2000) and field crops (Mba, 1996). Most of these investigations have confirmed that vermicompost usually have significant beneficial effects on plant growth.

20.1,4. HARMFUL OR NEGATIVE ROLES OF EARTHWORMS Like all living creatures earthworms too are known to pose some problematic activities negative to our economic interests. It is reported that excessive accumulation of earthworms within potted plants disturbs root growth and nutrient supplies to plants. Some earthworm species have a role in spreading, development of parasites and pathogens like foot and mouth disease; besides acting as intermediate hosts of protozoans, cestodes and helminthes. Earthworms have also reported for spreading soil fungi and pathogens. Many experts from different parts of world have reported that usually surface dwelling earthworms have habits of pulling leaves and damage some plants like potted plants. In crops, tobacco crop and rice is damaged in China and Japan. It has also been reported that vegetables particularly leafy vegetables like lettuce etc in different parts of world are being damaged to some extent. Problem of earthworm abundance in Grass tufts, Golf courses, Lawns and in Air Port shoulders are faced all over world. In Air Fields earthworms attract all sorts of predatory complex that pose bird collision threats to aircrafts.

20.15. CONCLUSIONS The use of earthworms in management of soils is an ancient practice in India. Mannmaniam Sundaranar (a Tamil Scholar) in his literary masterpiece has detailed out the role of earthworms in soil management. On the academic front, there are a number of prominent university and institute-based researchers dealing with vermiculture in India and abroad. The vermicomposting research in India has mainly been on the utilization of animal wastes, city refuse and agricultural crop residues as feed. The other areas which require attention in future are use of industrial wastes and sewage sludge as feed for earthworms and effect of the produced vermicompost on the soil properties and crop production. Little attention has been paid by the workers/scientists in India on the utilization of local earthworm species in vermicomposting. Further research is required on the utilization of local earthworm species individually or in combination with other composting earthworms.

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in earthworm casts is more of microbial origin, Current Science, 79, 1158-1159. Wilson DP & Carlile WR, 1989, Plant growth in potting media containing worm worked duck waste. Acta Horticulturae, 238, 205-220. Wright MA, 1972, Factors governing ingestion by the earthworm Lumbricus terrestris (L.), with special reference to apple leaves, Annals of Applied Biology, 70, 175-188.

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Subject Index

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A actuators 152 aeration 34, 104, 120, 122, 129, 320, 480 aerial mycelia 383 aflatoxin 315 agitation 34 agro-industrial residues 412 aleurioconidia 344 amylases 14, 192, 382 apical 93 apple essence 370 aroma compounds 356, 416, 427 aseptic operation 20 astaxanthin 340 artificial color 337 azaphilone pigments 342 B bacterial spores 238 banana odor 363 beta carotene 340 binary interactions 92 biodegradation 5 biofuels 5 biogas 428 bioherbicides 5 biological delignification 266 detoxification 5,429 factors 22

sustainability 139 biomass determination 56 DNA 57 direct 56 ergosterol 57 glucosamine content 57 indirect 56 metabolic gas balance 58 protein 57 biopulping 5 bioremediation 5 biosurfactants 5 biotransformation 417 blastospores 242 blue cheese 380 breakfast cereals 381 budding phenomena 85 button mushrooms 256 butyric fermentation 452 C carotenoids 340 catabolite repression 5, 125 cellulases 187, 263,395 chamPignons 256 cheese making 3 chemical energy 117 water potential 77 chemotropism 92

514 Index citric acid 207,392, 416, 427 citrinin 350 cleistotechia 344 clostridial spores 452 coconut flavor 363 cold junction 146 communication network 158 composting 13,428, 469 conductive cooling 239 convective heat 105 curcuma 338 D delignification 266 differential pressure 148 diffusion coefficient 77 dynamic parameters 77, 81 E earthworms 470 anecic species 472 endogeic species 471 epigeic species 471 feeding behaviour 472 electronic spin resonance 103 energy balance 38 ensiling 13,443 losses 448 process 446 enthalpy 39 entomopathogenic fungi 232 exopectinase 100 extremo-thermophiles 29 F feed 379,427 fermenter 6 cement mixer 10 column 10, 126 deep trough 10 design 6 horizontal paddle 126 packed bed 129, 239, 289 protein production 10 rocking drum 127 rotating drum 10, 123 Zymotis 130 fersol 173

fiberization 139 fieldbus 159 firewire 159 flavored foods 370 flavors banana 363, 364 buttery flavors 364 coconut 363 floral 364 fruity 365 lemon 364 strawberry 364 flow sensor 148 food 379, 413 forage crops 443 preservation 444 fragrance 356, 417 fruit body 254 fruit pulps 435 fumaric acid 216 functional food 380 fungal aroma 101 production 101 fungal spores 237 fuzzy logic 171 G galactosidases 191 gamma decalactone 358 genetic algorithms 172 germination 96 gibberellic acid production 277 properties 278 gibberellins 277 biosynthesis pathway 279 fermentation 280 glucoamylase 22 gluconic acid 222, 394 glutaminase 192 growth phase 50 accelerated 51 desacelerated 51 exponential 51,52 logarithmic 51,52 negative 52

Index 515 stationary

51

H heat transfer 38 coefficient 39 hemicellulases 187, 395 hexoenzymes 138 hot junction 146 humidity sensor 150 humification 119 hydration 97 hydric stress 90 hydrophobic polymers 369 hydrostatic pressure 85 hyphal orientation 93 I idiophase 302 immersion bioreactor 132 indigo 338 industrial enzymes 183 processing 415,423 sensors 145 inert substrate 3, 19, 309, 422 inter-particle space 13 integrated sensor 147 inulinase 194 IK kinetics 48 measurements 56 velocity 50 koji 3, 6, 129, 183,371,380

mechanism 31 mathematical models 5,294 empirical equations 5 mechanistic equations 5 mesophiles 30 metabolic heat 38 microbial growth associated growth 68 non-associated 69 partially associated 68 microbial physiology 324 microbial respiration 120 miso 3 modeling 41 monascus pigments 337 mushroom 253,416,427,432 biological efficiency 258, 381 bioremediation 267 detoxification 267 mycorrhizal 255 parasitic 255 saprophytic 256 mycopesticides 5 mycotoxins 4 N negative growth rate 52 neural networks 171 nutrient diffusion 120

lactation energy 453 lactic acid 218, 391, 416,444 lactones 359 lag phase 51 ligninases 188 ligninolysis 266 lipase 100, 190, 386

O odoriferous compounds 356 oilcakes 430 orange aroma. 370 organic acids 205,417 optical density 56 orifice plate 149 osmoregulation 99 osmotic pressure 85, 90, 101,366 oxygen balance 62 uptake rate 63 oyster 256

M magnetic sensor mass transfer effects 21

P pH sensor 151 particle size 35 milling 36

k

150

516 Index pectinases 191,263, 384 peeling effect 138 penicillin 4 permselective membrane 370 pervaporation 370 phenolic antioxidants 381 photosynthesis 117 phycocianin 340 physical energy 21 physico-chemical factors 22 phytase 193, 395 pigments 334, 390 pilot plant 127 pineapple aroma 365 plafractor 135 polyketide 344 polygalacturonase 100 polymeric 27 prebiotic 380 primodia 258 probiotic 380 profibus 162 programming languages 168 proteolysis 447 protease 14, 189 protein enrichment 32 psychometric 42 psychrophiles 30 pulsed mixing 131

sensors 145 serial communication 158 shiitake 256 silage 445 additives 459 harvest 455 homolactic fermentation 450 microbiology 449 nutritional quality 465 preservation 450 quality 464 sensory evaluation 464 wilting 456 single-cell protein 4 sterility 20 solvent extraction 369 soy sauce 3 specific growth rate 40, 43, 48, 49, 70 maximum 42 maintenance coefficient 70 spores production 230 sporulation 96,233 static beds 129 steric interactions 92, 95 steroids transformations 4 stirred beds 131 strain improvement 328 substrate bed 21 surface area 13, 17, 18, 120

I1 radial growth 90 rate 93 ragi 380 redox enzymes 138 relative humidity 32 resistance thermometer detector respiration 21 respirometry 5 respiratory quotient 67 riboflavin 340 rotational diffusivity 103

T tannases 387 tempeh 3, 380 temperature sensor 147 thermal dispersion 149 thermistor 147 thermocouple 146 thermodynamics 31, 75 parameters 77 thermophiles 29 thermostable enzymes 385 transducer 148 transport phenomena 5, 22, 34 trophophase 304 turbine sensor 149 turgor pressure 85

$

secondary fermentation 452 metabolites 302 seebeck effect 146

147

V

Index 517 vapour pressure 31 vermicomposting 468 agricultural wastes 500 animal wastes 490 aquatic weed 495 benefits 476 earthworms 470 industrial wastes 491 municipal solid waste 493 principle 470 process 476 sewage sludge 497 types 483 vermiculture 4 6 9 vermistabilization 493 vinegar 4

viscosity 22 void fraction 35 X xylanases 394

W water activity 13, 17, 30, 32, 79, 90, 93, 151, 236,318 sensors 82 wilting 456 worldfip 163 Z zigbee 163 zymotis 130

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