The Chemistry and Processing of Wood and Plant Fibrous Material: Cellucon '94 Proceedings

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THE CHEMISTRY AND PROCESSING OF WOOD AND PLANT FIBROUS MATERIALS

THE CHEMISTRY AND PROCESSING OF WOOD AND PLANT FIBROUS MATERIALS Editors: JOHN F KENNEDY Director of Birmingham Carbohydrate and Protein Technology Group, Research Laboratory for the Chemistry of Bioactive Carbohydrates and Proteins, School of Chemistry, The University of Birmingham, Birmingham, England and Professor of Applied Chemistry, The North East Wales Institute of Higher Education, Wrexham, Clwyd, Wales GLYN 0 PHILLIPS Chairman of Research Transfer Ltd, (Newtech Innovation Centre), Professorial Fellow of The North East Wales Institute of Higher Education, Wrexham, Clwyd, Wales and Professor of Chemistry, The University of Salford, England PETER A WILLIAMS Head of the Multidisciplinary Research and Innovation Centre and the Centre of Expertise in Water Soluble Polymers, The North East Wales Institute of Higher Education, Wrexham, Clwyd, Wales

WOOI)HI~AI) PUJ3I~ISf-IING lAIl\/lI~I~ED

Oxford

Cambridge

New Delhi

Published by Woodhead Publishing Limited Abington Hall, Granta Park, Great Abington Cambridge CB21 6AH, UK www.woodheadpublishing.com Woodhead Publishing India Private Limited G-2, Vardaan House, 7/28 Ansari Road, Daryaganj New Delhi - 110002, India www.woodheadpublishing.com First published 1996 Reprinted 2001, 2004, 2005, 2010 © 1996, Woodhead Publishing Limited The authors have asserted their moral rights.

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the authors and the publisher cannot assume responsibility for the validity of all materials. Neither the authors nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage or liability directly or indirectly caused or alleged to be caused by this book. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming and recording, or by any information storage or retrieval system, without permission in writing from Woodhead Publishing Limited. The consent of Woodhead Publishing Limited does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from Woodhead Publishing Limited for such copying. Trademark notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 978-1-85573-305-3

Printed in the United Kingdom by CPI Antony Rowe

CELLUCON CONFERENCES AND THE CELLUCON TRUST Cellucon Conferences as an organisation was initiated in 1982, and Cellucon '84, which was the original conference, set out to establish the strength of British expertise in the field of cellulose and its derivatives. This laid the foundation for subsequent conferences in Wales (1986), Japan (1988), Wales (1989), Czechoslovakia (1990), USA (1991), Wales (1992), Sweden (1993) and Wales (1994). They have had truly international audiences drawn from the major industries involved in the production and use of cellulose pulp and derivatives of cellulose, plus representatives of academic institutions and government research centres. This diverse audience has allowed the crossfertilization of many ideas which has done much to give the cellulose field the higher profile that it rightly deserves, Cellucon Conferences are organised by The Cellucon Trust, an official UK charitable trust with worldwide objectives in education in wood and cellulosics. The Cellucon Trust is continuing to extend the knowledge of all aspects of cellulose worldwide. At least one book has been published from each Cellucon Conference as the proceedings thereof. This volume arises from the 1994 conference held in Bangor, Wales, UK, and the conferences planned to be held in Russia, France and Finland, etc, will generate further useful books in the area.

THE CELLUCON TRUST TRUSTEES AND DIRECTORS Prof G 0 Phillips (Chairman) Prof J F Kennedy (Deputy Chairman and Treasurer) Dr P A Williams (Secretary) Mr T Greenway Mr W B Painting Dr C A White

Research Transfer Ltd, UK The North East Wales Institute, UK and The University of Birmingham, UK The North East Wales Institute, UK Akzo Nobel Surface Chemistry Ltd, UK Hoechst (UK) Ltd, UK Fisons Scientific, UK v

CELLUCON CONFERENCES ORGANISING COMMITTEE Prof G 0 Phillips (Chairman) Prof J F Kennedy (Deputy Chairman and Treasurer) Dr P A Williams (Secretariat) Mr H Hughes (Secretariat) Mr P Bale Prof W B Banks Prof C Bucke Dr H L Chum Dr A Fowler Dr K Geddes Mr T Greenway Prof J Guthrie Dr H Hatakeyama Dr M B Huglin Dr P Levison Dr J Meadows Mr W B Painting Mr A Poyner Mr R Price Prof J Roberts Dr J F Webber Dr C A White

Research Transfer Ltd, UK The North East Wales Institute, UK and The University of Birmingham, UK The North East Wales Institute, UK The North East Wales Institute, UK Hercules Ltd, UK University of Wales, UK The University of Westminster, UK American Chemical Society (Cellulose, Paper and Textile Division), USA Courtaulds Ltd, UK Crown Berger Europe Ltd, UK Akzo Nobel Surface Chemistry Ltd, UK University of Leeds, UK Fukui Institute of Technology, Japan University of Salford, UK Whatman International Ltd, UK The North East Wales Institute, UK Hoechst (UK) Ltd, UK Consultant, UK Shotton Paper Co Ltd, UK Institute of Science and Technology, University of Manchester, UK The Forestry Authority, UK Fisons Scientific, UK

Cellucon Conferences are sponsored by: The Biochemical Society, UK - Chembiotech Ltd, UK - Hoechst (UK) Ltd, UK - The North East Wales Institute of Higher Education, UK - The University of Birmingham, UK - The University of Lund, Sweden - USAF European Office of Aerospace Research and Development - US Army Research, Development and Standardisation Group, UK - Welsh Development Agency - Whatman Specialty Products Division, UK.

Cellucon Conferences are supported by: The American Chemical Society (Cellulose, Paper and Textile Division) Aqualon (UK) Ltd - Akzo Nobel Surface Chemistry Ltd, UK - Courtaulds Chemicals and Plastics, UK - Ministry of Defence - Crown Berger Europe Ltd, UK - DOW Chemicals, UK - The Forestry Authority, UK - The Southern Regional Research Center, USA - Shotton Paper Company Ltd, UK - Syracuse Cellulose Conferences, USA. vi

THE CELLUCON CONFERENCES 1984 Cellucon '84 UK

CELLULOSE AND ITS DERIVATIVES Chemistry, Biochemistry and Applications

1986 Cellucon '86 UK

WOOD AND CELLULOSICS Industrial Technology, Biotechnology, Structure and Properties

1988 Cellucon '88 Japan

CELLULOSICS AND WOOD Fundamentals and Applications


CELLULOSE: SOURCES AND EXPLOITATION Industrial Utilisation, Biotechnology and Physico-Chemical Properties

1990 Cellucon '90 Czechoslovakia

CELLULOSE New Trends in the Complex Utilisation of Lignocellulosics (Phytomass)

1991 Cellucon '91 USA

CELLULOSE A Joint Meeting of: ACS Cellulose, Paper and Textile Division, The Cellucon Trust, and 11 th Syracuse Cellulose Conference


SELECTIVE PURIFICATION AND SEPARATION PROCESSES The Role of Cellulosic Materials

1993 Cellucon '93 Sweden CELLULOSE AND CELLULOSE DERIVATIVES Physico-Chemical Aspects and Industrial Applications 1994 Cellucon '94 UK

CHEMISTRY AND PROCESSING OF WOOD AND PLANT FIBROUS MATERIALS The Chemistry and Processing of Wood and Plant Fibrous Materials

The proceedings of each conference were formerly published by Ellis Horwood, Simon and Schuster International Group, Prentice Hall, Campus 400, Maylands Avenue, Hemel Hempstead, Herts, HP2 7EZ and are now published by Woodhead Publishing Limited, Abington Hall, Abington, Cambridge CBl 6AH. THE CELLUCON TRUST is a registered charity, UK Registration No: 328582 and a company limited by guarantee, UK Registration No: 2483804 with its registered offices at The Research Laboratory for the Chemistry of Reactive Carbohydrates and Proteins, The School of Chemistry, The University of Birmingham, Birmingham, B15 2IT, England. Vll

ACKNOWLEDGEMENTS This book arises from the International Conference - CELLUCON '94 which was held at the University of Wales, Bangor, UK. This meeting owed its success to the invaluable work of the Organising Committee.

MEMBERS OF THE LOCAL ORGANISING COMMITTEE - CELLUCON '94 Prof W B Banks (Chairman) University of Wales, Bangor, UK Dr HOmed (Secretariat) University of Wales, Bangor, UK Dr P A Williams (Secretariat) The North East Wales Institute, UK Mr H Hughes (Secretariat) The North East Wales Institute, UK Prof J F Kennedy The North East Wales Institute, UK and (Deputy Chairman and The University of Birmingham, UK Treasurer) Akzo Nobel Surface Chemistry Ltd, UK Mr T Greenway Whatman International Ltd, UK Dr P Levison The North East Wales Institute, UK Dr J Meadows Hoechst (UK) Ltd, UK Mr W B Painting Research Transfer Ltd, UK Prof G 0 Phillips Institute of Science and Technology, Prof J Roberts University of Manchester, UK

Vlll

CONTENTS Preface

XUl

PART 1: THE FIBRE AND NON FIBRE RESOURCES 1 Forestry - Sustainable production and processing J Evans 2 Changing patterns of global wood and fibre supplies . R J Cooper 3 Blackcurrant stems - An agri-waste with potential as a dilutent to existing tree-based fibre sources . D Stewart and R M Brennan 4 Phenolic acid dimers in barley straw cell walls D Stewart and I M Morrison 5 'Non wood plant fibres'. Availability in Kenya and need for maximum utilization . R M urali and J G M wangi 6 Current developments in plant derived gums and resins for the chemical industry in Kenya B Chikamai

PART 2: PULPING. 7 Advances in steam explosion pulping (SEP) . B V Kokta, Y Ben, J Doucet, A Ahmed and D A Sukhov 8 Kinetics and mechanism of wheat straw pulping . A A Baosman, G C Fettis, M J Ramsden and S J Smith 9 Ethanol pulping of pretreated non-wood fibre materials B Lonnberg, M EI-Sakhawy and T Hultholm 10 The chemical composition of tropical hardwoods and its influence on pulping processes . N A Darkwa 11 Pulping characteristics and mineral composition of 16 field crops cultivated in Finland . K A Pahkala, T J N Mela and L Laamanen 12 Screening, purification and characterization of novel xylanases used in pulp bleaching

1

3 13 25 31

37 49

61 63 81

99 III

119 127

B Cuevas, B Bodie, C Wang and M Koljonen 13

Delignification and bleaching of non-wood fibres with peroxycompounds D Stewart and I M Morrison ix

133

x Contents

14 15

Biobleacbing of pulp and paper mill black liquor in fluidized bed reactor tRng immobilized Phanerochaete chrysosporium BKMF 1767 S S Marwaha, R S Singh, P K Khanna and J F Kennedy ViscositylDP relationships for cellulose dissolved in cuprammonium and cupriethylene diamine solvents J H Morton

PART 3: PHYSICAL AND CHEMICAL PROCESSING OF FmRE AND FIBROUS PRODUCTS 16 Property enhancement of plant fibres for industrial use W B Banks 17 Physicochemical aspects of fibre processing . L Salmen and S Ljunggren 18 The effect of acetic anhydride treatments on the mechanical properties, hydrophobicity and dimensional stability of Russian Fifths and Scandinavian pine M J Ramsden, F S R Blake and N J Fey 19 Recovery of packaging laminate components to enhance waste management . E T Evans, M J Kay, N Kirkpatrick and D SWales 20 Celsol - Biotransformation of cellulose for fibre spinning M Vehilainen and P N ousiainen 21 Concurrent modification of wood with phthalic anhydride in composite manufacture R Salisbury, M Lawther and P Brown 22

23 24 25 26

Engineering composites from oriented natural fibres: A strategy. P E Humphrey Reactive cellulosefibres rather than reactive dyes . D M Lewis and Q G Fan The treatment of cotton cellulose with Trichoderma reese; engineered c e l l u l a s e s . . . A Cavaco-Paulo, L Almeida and D Bishop Characterisation of paperboard packages designed for liquid containment C J Harrold and J T Guthrie Biochemical investigation of cellulosic and lineous materials in museum collections M A Robson

PART 4: PHYSICAL AND CHEMICAL PROCESSING OF FIBRE AND NON FIBROUS PRODUcrS 27 Polymeric materials derived from the biomass A Gandini 28 High performance and highly functional polymeric materials from plant components H Hatakeyama

143 151

159 161 173

183

191 197 205 213 221 227 235

243

249 251

263

Contents

29 30

31 32 33 34

Preparation and physical properties of biodegradable polyurethanes derived from the lignin-polyester-polyol system S Hirose, K Kobashigawa and H Hatakeyama Viscoelastic properties of biodegradable polyurethanes derived from coffee grounds K Nakamura, Y Nishimura, T Hatakeyama and H Hatakeyama The fractional composition of polysaccharides in alkaline pretreated and steam pressure treated wheat straw R Sun, J M Lawther and W B Banks Effects of extraction conditions and alkali type on the yield and neutral sugar composition of wheat straw hemicellulose J M Lawther, R Sun and W B Banks Thermodestmction of cellulose and levoglucosone production G Dobele, G Rossinskaja, B Rone and V Yurkjane Star-shaped and crosslinked polyurethanes derived from lignins and oligoether isocyanates S Montanari, B Baradie, J-P Andreolety and A Gandini

PART 5: APPLICATIONS OF CELLULOSE, CELLULOSE DERIVATIVES, LIGNIN AND CELLULOSE-RELATED ENZYMES 35 The alkaline degradation of cellulose relating to the long term storage of radionuclides in cement J Shimizu, J F Kennedy, L L Lloyd and W Hasamudin 36 The use of cellulose and cellulose derivatives in immobilised systems for the removal of colour from textile effluents N Willmott, J T Guthrie, G Nelson and B Burdett 37 New polymer electrolytes based on modified polysaccharides C Schoenenberger, J F Le Nest and A Gandini 38 Thermal and FfIR studies of Tencel-g-co-Hema and Tencel-g-coHema carbanilates M A Kazaure, J T Guthrie and B B Dambatta 39 ESR as a method for monitoring lignins activity during the interaction with monomer and oligomer silicon containing compounds. T Dizhbite, G Telysheva and G Shulga 40 The regularities of lignosulphonate behavior on different interfaces and its alteration by purposeful modification G Telysheva, T Dizhbite, E Paegle and A Kizima 41 Some physicochemical properties of xylanolytic enzymes produced by Aspergillus fumigatus IMI 255091 . L A Hamilton and D A J Wase 42 Endoglucanase j3-D-glucosidase and xylanase induction in Dichomitus squalens (Karst.) Rid . E Resende, M Carolina and N T Rodeia Index

Xl

277 283 291 313 345 351

359 361 369

377 385

393 399 405 413

419

PREFACE This book illustrates what a remarkable resource is offered to us by wood and related plant materials. The aspects described are of an inter-disciplinary character, and will prove of great value to the wood chemist, biochemist and paper technologist. The subject is developed progressively. It starts with the production, management and changing patterns of global wood and fibre resources. Wood pulping is a traditional area of wood utilisation, but throughout the world there is a need for an injection of new technologies to utilise more fully all the lignocellulosic components and to provide improved environmental processes. Thus, steam explosion, ethanol pulping, the role of enzymic and other biological modifications offer exciting new vistas for both wood and non-wood fibrous materials, as do new synthetic pathways to innovative chemical derivatives. New high performance composite materials, chemically modified wood and related cellulosic products receive expert treatment in this volume, Both physical and chemical processing are dealt with, and new biochemical methods for treating wood are described. Increasingly, surplus cellulosic plant fibre wastes offer a raw material for high performance and functional polymers, which are also biodegradable. Vegetable biomass is now being recognised as a unique starting material. These proceedings also celebrate the tenth anniversary of The Cellucon Trust, specifically set up to promote research and communication in cellulose chemistry. The period has witnessed a renaissance in this subject, and The Cellucon Trust can be justifiably proud of its contribution. From the Cellucon and allied meetings with our Japanese, American, Slovakian and Scandinavian colleagues, ten volumes of diverse research findings have been published. New solvent systems have led to new cellulosic fibres, and new chemistry has provided cellulosics with new functional roles and product fields. It was appropriate that this anniversary meeting should have been held in Wales, the traditional home of Cellucon, and in particular the University of Wales, Bangor. Here forestry science and wood chemistry have been a long established speciality. A new established Chair in Wood Science has been inaugurated in Bangor and we are grateful to Professor W Barton Banks, the first holder of the Chair for organising a happy and innovative conference. Again I thank the Secretariat and members of The Cellucon Trust for their unfailing support. Glyn 0 Phillips Chairman, The Cellucon Trust XlII

Part 1: The fibre and non fibre resources

1 Forestry - sustainable production and • processing J Evans - British Forestry Commission, Alice Holt Lodge, Wrecclesham, Farnham, Surrey, aUIO 4LH, UK

Introduction The world's forest resources are under threat, both real and imagined. Owing to tropical deforestation, air pollution and the possible effects of climate change, the well being of trees and forests has risen very much up the agenda of public concern. The objective of this paper is to provide an overview which focuses on the changing nature of the resource, examines the rise in second growth and plantation forests, and addresses the question of sustainability of plantation forestry in particular. In these ways I hope to answer the question, how secure are the world's forests as a continuing fibre resource? Remarks concerning processing, which my title includes, will be confined to generalisations about log size and future wood quality reflecting the changing nature of the forest resource.

Forest Resources of the World Accurate data on forest areas by countries are notoriously difficult to obtain. Both questions of reporting accuracy and definition of what constitutes forest are amongst the largest sources of error. The Food and Agriculture Organisation of the United Nations (FAD) do publish statistics at approximately 10 year intervals and Table 1, which in part derives from FAD (1992,1993a & b), summarises the position for 1990.

3

4 The fibre and non fibre resources

Table 1:

Global and Regional Forest Statistics (1990)

Forest Mha

land

Deforestation Mha/y

New Planting M haly

Forest! capita (ha)

Africa

630

24

4.1

0.1

0.8

Asia & Pacific

350

35

3.9

2.1

0.2

L. America

820

44

7.4

0.4

2.0

15.4

2.6

Tropical

%

1,800

Americas

720

25

0.3

2.6

1.6

Europe & N. Africa

180

27

-

0.2

0.3

E. Asia

170

16

0.8?

2.0?

0.1

CIS & near East

890

35

-

2.2

2.6

Australasia

120

9

-

j.65 -

e

X

....J"O

...J~

wg

()~

••

0.60 I

0.06

I

1

I

I

• SEP 200 oC/2 minoC/4 • SEP 200 min

•

0.08 0.10 0.12 0.14 0.16

0.18

CELLULOSE I ordered Figure 16 Structural changes in CTMP, CMP and SEP of aspen.

0.17 0.16 0

w 0.15 a: w 0.14

0

a: 0.13 0

w 0.12

SEP

(J)

0

-J

~

0.11

...J

0.1

UJ

0.09

-J

o

0.08 0.07 100 110 120 130 140 150 160 170 180 190 200 210 TEMPERATURE (0 C) Figure 17 Effect of temperature on cellulose I.

Steam explosion pulping

0.78 0

RMP

0.76

w a: 0.74 w 0

CMP

a: 0.72 0 en 0

-W

0.7 0.68

CTMP SEP

en 0.66 0 -' :J 0.64 .....J .....J

w 0.62

o

0.6

100 110 120 130 140 150 160 170 180 190 200 210 TEMPERATURE (0 C)

Figure 18 Effect of temperature on cellulose II. In Figure 19, breaking length value of SEP is compared as a function of different cooking time as well as cooking severity. An increase in paper strength is observe with the rise of cooking time from 1 to 2 min at 195°C, when compared the pulp at the same ionic content as well as the same chemical pretreatment. At the cooking temperature 200°C, the cooking time of 1 min seems to be sufficient for properties increase because it leads to required CI increase and CII decrease (Figure 16). Effect of pulpin~ conditions on surface chemical composition as well as cQ'stallinity In comparative semi-industrial trials, Barbe et al. (3) showed that SEP exhibited much higher strength than CMP at comparable total ionic content and yield. In order to explain the fundamental factors which may contribute to the higher strength level of SEP, both SEP and CMP taken from the Barbe et al. (3) trials were examined by ESCA(ll). In Table 1, kraft aspen pulps were compared to water exploded aspen chips (SEP-H 20), SEP (8+1), and CMP (8+1). SEP (8+ 1) revealed a higher O/C content on the surface than CMP (8+ 1) did, which indicates better bonding surface characteristics. Lower percentages of C1and 01 (found mostly on lignin or non-cellulosics) for SEP, as opposed to CMP, also indicate a lower amount of lignin on the surface, even though the bulk percentages of lignin are the same for both SEP and CMP (10). Finally, the higher SIC found for SEP, compared to CMP, indicates a higher level of surface lignin sulfonation for steam explosion pulps. It seems that a higher percentage of carbohydrates and a higher percentage of sulfonated lignin on the fiber surface of SEP (compared to CMP) can partially explain superior adhesion when related to strength properties.

77

78 Pulping

8

EXPLOSION PULPING OF ASPEN: TOTAL IONIC CONTENT VERSUS BREAKING LENGTH AT CSF 100 - EFFECT OF OPERATING CONDITIONS

2000C I'

E

o =8%No ZS0 3 =40%O.o.C.

.~

~

190°C 2'

180°C 5'

J 8;::::

1700C II'

6

::I:

o

I~

tt!)

:z

lLLJ ,..J

y

4

o

:z

I

::lIl:::

x~

x-16°/oNo2S03 == 32°/00.D.C

12%No2S03=24%0.D.C.

8°1o N02S03 ==IG%OD.C.

oct

:~

~x

x 195°C

2

ltD

I'

o

195°C 2'

o

195°C 3' 200°C 75"

A

O-t---~--,r----,.----r----...----Y----r---

120

160

200

240

TOTAL IONIC CONTENT (mmol/kg)

Figure 19 Effect of operating conditions and total ionic content on the breaking length of pulp (SEP) at 100 CSF level.

Table 1 ESCA Spectroscopy of Ultra-High Yield Aspen Pulps Pulp

Yield (%)

Total Ionic Content (mmol/kg)

Sulfonic

Cl

01

(%)

(%)

0.59

20.0

7.7

OIC

(mmol/kg)

Kraft

SIC

BL (km)

Spec. ref. Energy (MJlkg)

SEP (8+1)

92

174

43

0.52

33.9

10.3

3.08

7.2

3.0

eMP (8+1)

89.9

190

53

0.41

43.1

14.3

2.22

4.7

9.7

SEP (H2O)

91

85

0.34

55.6

22.7

4.1

8+ 1: 8% Na2S03 + 1% NaOH; SEP: 195°C; 1 min; CMP: 150°C; 30 min

Steam explosion pulping

In Table 2, cellulose crystallinity I index as well as crystallite sizes were compared for aspen fibers, CTMP (5+5), CMP (8+1) and SEP (8+0) and SEP (8+1), all prepared in semi-industrials (3,6,7). It was shown (9) that CTMP or CMP aspen treatment increases C 1 cellulose crystallinity from 56% to 71.50/0 or 71.3%. In the case of SEP submitted to a temperature above the glass-transition of lignin, crystallinity C1 increases to 75.90/0 irrespective of the chemical treatment. It seems that crystallinity and crystallite sizes, being 21.4 A for aspen and 27 A, 27 A for CTMP or CMP and 31.5 or 31.5 for SEP (8+0) or SEP (8+0.5), are a function of temperature rather than the type of chemical treatment. Table 2 X-Ray Diffraction Parameters Aspen Fibers

CTMP 128°C; 10 min 5% Na2S03 5% NaOH

CMP 150°C; 30 min 8% Na2S03 1% NaOH

SEP 194°C; 1 min 8% Na2S03

SEP 194°C; 1 min 8% Na2S03 0.50/0 NaOH

Crystallinity Index (Cr.I) (%)

56.0

71.5

71.3

75.9

75.9

Crystallite Sizes (002) (A)

21.4

27

27

31.5

31.5

CONCLUSIONS The presence of NaOH, NaHC0 3 or MgC0 3, as opposed to Na2S03 alone or other chemical systems, leads to a higher sulfonate and carboxylate content, although the bulk lignin content of SEP pulps did not show much difference. The physical strength of paper and its brightness improves when the pulp has higher hydrophilic group content. The presence of NaOH, NaHC0 3 or MgC0 3, as opposed to Na2S03 alone or other chemical system trials, led to a lower relative specific refining energy consumption and better physical strength. In addition, the presence of NaHC0 3 or MgC0 3, compared to Na2S03 alone or with NaOH or other chemical systems, resulted in an increase in brightness of up to 4% as well as a 3% to 6% yield increase. Excellent paper strength and low specific refining energy similar to the one obtained with NaOH can be produced with either NaHC0 3 or Na2S03. The strength values of SEP compares well to those of low yield hardwood industrial kraft pulp (8). At the same time, NaHC0 3 can protect yields well over 90% and brightness levels 64%. Better bonding of SEP fibers may be at least partially explained by a higher O/C ratio on the fiber surface, by a higher level of SIC ratio as well as by a higher percentage of bonding CI crystallinity in SEP when compared to CMP.

79

80

Pulping

ACKNOWLEDGEMENT We wish to thank the NSERC, FCAR, Stake Technology Limited for their financial supports.

REFERENCES 1. Kokta, B.V., Process for Preparing Pulp for Papermaking, Can. pat. # 1,287,705 (Aug. 20, 1991) 2. Kokta, B.V., Ahmed, A., Zhan, H. and Barbe, M., "Explosion Pulping of Aspen" Paperija Puu-Paper and Timber, (9):1044-1055 (1989). 3. Barbe, M.C., Kokta, B.V, Lavallee, H.C. and Taylor, J.,"Aspen Pulping: A Comparison of Stake Explosion and Conventional Chemi-mechanical Pulping Process" Pulp and Paper Canada, 91(12), T395-T403, December 1990. 4. Katz, S., Beatson, R. and Scallan, A.M., "The Determination of Strong and Weak Acidic Acid Groups in Sulphite Pulps, Paprican PPR, 408 (1982). 5. Kokta, B.V. and Daneault, C., "Brightening Ultra-High-Yield Hardwood Pulps with Hydrogen Peroxide and Sodium Hydrosulfite" TAPPI, 69(9), 130133 (1986). • 6. Sukhov, D.A., Zhilkin, N.A., Valov, P.M. and Terentiev, O.A. "Cellulose structure in relation to paper properties" Tappi, 74(3) 201-204 (1991). 7. Kokta, B.V., Ahmed, A., Garceau, J.J. and Chen, R., "Progress of Steam Explosion Pulping: an overview, Lignocellulosics: Science, Technology, Development and Use, Kennedy, Phillips and Williams, Editors; Ellis Horwood Series in Polymer Science and Technology, pp. 171-212 (1991). 8. Kokta, B.V., "Steam Explosion Pulping of Aspen: Results from Semiindustrial Trials" Poplar Council of Canada Newsletter, 2. pp. 9-14, June (1991). 9. Carrasco, F., Kokta, B.V., Ahmed, A. and Garceau, J.J., "Ultra-High-Yield Pulping: Relation between Pulp Properties and Fiber Characteristics by Multiple Linear Regression." Preprint of 1991 Pulping Conference, pp.407417, Orlando, Nov. (1991). 10. Kokta, B.V., Ahmed, A., Garceau, J.J., Carrasco, F., Zhai, D. and Huang, G.Q., "Steam Explosion Pulping of spruce and Aspen: Optimization of the Process", Proceedings of 78th. Annual CPPA Meeting, vol. 1, A91-AI05, Montreal, Jan. (1992). 11. Hua, X, Kaliaguine, S., Kokta, B.V. and Adnot, A., "Surface Analysis of Explosion Pulp by ESCA", Wood Science and Technology, (in press 1994).

8 Kinetics and mechanism of wheat straw pulping A A Baosman, G C Fettis, M J Ramsden and S J Smith - Chemistry Department, University of York, Heslington, York YOI 5DD, England

2.1

ABSTRACT

This paper describes the first part of an intended in depth study of the kinetics of pulping Saudi Arabian wheat straw with caustic soda solution in a rotating steel reactor of 250 ml total capacity. The work is of current relevance in the UK because there is renewed interest in finding uses for waste straw which can no longer be burned in

fields after harvesting the grain crop. potential outlet.

Paper production from straw pulp is one

The extent of dissolution of lignin during pulping was measured in the temperature range 25°C to 170°C using the standard Klason method of analysis for lignin. In a few runs residual caustic soda was titrated with acid. Some NMR and IR studies were also done on straw before and after pulping and on lignin. In order to avoid all possibility of mechanical pulping it was necessary to cut the straw prior to reaction neatly into 2-3 em lengths and avoid grinding it for the kinetic studies. Titration of residual caustic gave variable results initially. This was shown to be due to strong absorption of some of the caustic by straw. Multiple washing by water was necessary in order to recover it quantitatively. The rate of delignification reaction was found to be first order with respect to lignin and 0.6 order with respect to caustic soda. Values were obtained for the rate constants 81

82

Pulping

at a number of temperatures and the activation energy was found to be 14 ± 3kJ mol". This low value is indicative of the rate controlling step being physical rather than chemical in nature, e.g. diffusion of caustic within the structure of the straw.

2.2

INTRODU:
1000

0.70 1.08 0.42 0.83 0.90

The kappa numbers indicating lignin content were lower for grass pulp than wood pulp. Grasses harvested during the growing period were easily cooked to kappa number 9-14 (Figure 1), which was lower than the kappa number for commercial birch sulphate pulp (17-20) [fable 2) and the kappa numbers for the other plants tested. Viscosity of the pulp made of grass, straw or hemp was similar to that of birch pulp.

122

Pulping

TALL FESCfJE

%

45 40 35 30 25 20 15 10 5 0

%

A

B

~pulp

30 25 20 15 10 5 0

D C -rejects

A

C B ga kappanumber

D

RED CLOVER 45 40 35 30 25

90 80 70 60 50

% 40 30

% 20

15 10 5 0

20 10 0

A

o pulp

B

A

D

C

• reJects

B

C

D

~ kappanumber

Figure 1. The effect of plant age on pulp yield, amount of rejects and kappa number in tall fescue and red clover. Samples taken A=at the beginning of flowering (June), B=at full flowering (July), C=at seed maturity (August), D=in following spring.

Table 3. Pulp yield, rejects, kappa number and viscosity for goat's rue and red clover after pulping in different amounts of NaOH. Species

NaOH- NaOH% residue

Pulp %

Rejects Kappa Viscosity LW % number mm

gil

Goat's rue

Red clover

16.0 20.0 24.0

2.6 6.5 11.7

13.7 18.3 22.5

24.2 15.7 11.6

45.5 38.2 34.7

790 970 920

1.01 0.92

16.0 20.0 24.0

0 4.7 9.7

23.9 22.8 24.8

13.4 9.7 7.7

63.4 48.5 46.2

850 890 930

0.70 0.87 0.89

Field crops

123

The amount of NaOH (16% of dry matter) used in trials was too low for dicotyledons. In the case of red clover and in goat's rue the pulp yield, amount of rejects and kappa number became more acceptable when the dose of cooking chemical was increased to 20 or 24% of dry matter (Table 3).

3.2 Mineral concentrations The mineral concentrations were higher in the non-wood species than in birch and the concentrations in grasses and cereals differed from those of dicotyledons (Table 4). The silica concentration of grasses ranged between 0.9 and 6.1 % and that of dicotyledons between 0.2 and 0.8%, being lowest in oilflax straw «0.1%). The ash content was lowest in straw of oilflax and hemp (3.8-3.9%). In some species plant age was important for mineral content (Figure 2). The effect of plant age on chemical and pulping properties of several non-wood plants has been discussed in detail in papers published earlier (9, 10).

Table 4. Mineral concentrations in dry matter of crop samples taken in 1990. Species, harvesting

Ash

Fe mg/kg

%

Reed canarygrass Tall fescue Meadow fescue Timothy Rye Oat Barley Wheat Reed Goat's rue Red clover Lucerne Oilflax Fibre hemp Nettle Turnip rape Rape straw Birch

Mn mg/kg

5.09 5.31 9.10 10.03

2.63 2.42 1.52 0.88 3.61 3.68 6.13

101.5 100.3 53.6 131.3 159.0 48.6

24.0 61.9 42.4 38.0 18.8 46.2 15.3

5.41

3.52

97.3

7.79 6.93 6.22 6.83 3.93 3.75 12.13

3.30 0.27 0.31 0.38

(1.)

~

~ 0

c.....

e

~

(5 c::

0 0: .;;;

c.....

0

c Q)

~.....

2.0E-6

C

~

>
o

[1] In the case of a cyclic anhydride, the carboxyl group formed remains attached to the wood, as shown, which explains the greater hygroscopicity of wood so modified. That the above representation is correct is shown by the use of the carboxyl group for grafting on further polymers (2).

Phthalic anhydride in boards The BioComposites centre, started working with PA in 1991. Phthalylated wood made into a board with phenolic resin was found to have greatly increase water resistance.

In an attempt to show that wood modification was a key component of this effect, we also made boards in which the PA was simply added in various ways to the board furnish. Unfortunately for our theory, these boards also showed improved properties. A full study was carried out using a resin (J2005A) which had proved to be satisfactory in earlier work. This was not the best resin, but one which was easy to work with and readily available. We chose to work with wood shred rather than fibre or flakes, for ease of handling.

Modification with phthalic anhydride

207

Outline of trial At this time, we were assuming an analogy between PA and acetic anhydride, so the amount of chemical used was quite high: 10%, 14% and 18%. Two series of boards were made, one with wood that was modified with PA beforehand, the other with PA being added with the resin. Sufficient boards of each type were made to provide enough material for British Standard tests of : thickness swell, linear expansion and water uptake from manufacture to conditioning at 65% relative humidity at 20°C from conditioning to 1 hour water soak at 20 0C from conditioning to 42 hour water soak at 20°C from conditioning to conditioning at 93% relative humidity at 20 0 e from conditioning to 2 hours boiling after V313 cyclical testing during repeated boiling and drying at 105°C weight loss due to V313 cyclical boiling internal bond strength at 65% relative humidity at 20 0 e at 93% relative humidity at 200C after 1 hour soak at 20°C followed by reconditioning after 24 hours soak at 20°C followed by reconditioning after 2 hours boiling followed by drying and reconditioning after V313 after cyclical boiling three-point bending tests at 65% relative humidity at 20 0 e after 24 hours soak at 20 0 e weight loss due to biological challenge by Coniophora puteana Gleophyllum trabeum Pleurotus ostreatus

Results All the properties were improved by PA, whether it was reacted with the wood before board manufacture or added with the resin. The figures for the physical testing are not presented here, because they have been superceded by later work. The results of the biological testing are shown in Tables 1 and 2, because no further trials have been made.

208 Physical and chemical processing of fibre and fibrous products

Table 1.

Table 2.

Weight loss due to fungal attack: wood modified by PA fungus\amount of PA

none

10%

14%

18%

Coniophora puteana

25.4

7.14

2.27

.44

Gleophyllum trabeum

4.8

0.15

-0.09

-0.35

Pleurotus ostreatus

13.34

-0.47

-0.56

-0.47

Weight loss % due to fungal attack: PA added with resin fungus\amount of PA

none

10%

14%

18%

Coniophora puteana

25.4

2.82

1.92

1.43

Gleophyllum trabeum

4.8

-0.57

-0.45

-0.06

Pleurotus ostreatus

13.34

-0.57

-1.03

-1.17

The physical testing results also showed that reaction during manufacture was nearly as good as prior modification, and also suggested that much lower levels of PA would also be effective. We therefore made boards using PA to replace some of the phenolic resin, and did a more restricted set of tests on them. The tests were not to British Standard, but were designed to give maximum information from the minimum of material. It was found that the best results were obtained by replacing about 20 - 25% of the resin (dry weight) by PA. The improvements obtained are summarised in the table.

Table 3.

Relative improvements achieved in chipboard properties by replacing about 20% of PF resin by PA Property IBS - conditioned IBS - boiled IBS- retention thickness swell - cold soak thickness swell - 2 hour boil bending strength - conditioned bending stiffitess - conditioned bending toughness - conditioned bending strength - boiled bending stiffness - boiled bending toughness - boiled

Improvement factor 2.1 14 9 1.2* 1.8* 1.3 1.3 1.6 1.5 1.5 1.9

* ratio of log reciprocal

swell


209

These are not minor improvements, and any proposed mechanism must be able to account for the scale of these effects. It was found that all the properties were highly correlated with initial IBS, and this property only is used in the graphs below.

Possible mechanisms PA could be acting as an accelerator for the resin, producing more complete cures before the wood starts to deteriorate. A series of trials showed (Figure 1) that PA does act as an accelerator, but not a very powerful one, and the properties of boards made with PA go on increasing with pressing for longer:

Effect of press time

onlBS 0.6 .....- - - - - - - - - - - - - - -...... 0.5

+----------=-"'=---.-.:::::::::~---t

0.4

-I--

---I

---,~L.._

Ci

[J

Q.

~0.3 +--~--~~--------~~ UJ ~

• 20%PA

0.2

+-_~-_-----::..-c::;;.---------I

0.1

-I-----""7"'--~~---------...;;~

noPA

• 20% PA, boiled no PA, boiled

0.0 .....- - . . . - . -......- -......- - - . . - - -......- ---'1 o 10 20 30 40 50 60 press time

at 200°C (mins)

Figure 1 PA could be acting to improve the resin in some other way. We investigated this by making boards with vermiculite replacing wood and also by testing resin-impregnated strips of glass fibre filter paper. PA was detrimental to all the properties except dry strength. However, when cellulose filter paper or newsprint was used to make the strips, PA improved the strength and stiffness of the strip, both when dry and when soaked.

Table 4

Effect of PA on properties of resin strips control = 100

support

dry strength

dry stifthess

wet strength

wet stiffness

glass fibre

136

99

96

79

cellulose

107

110

109

116

newsprint

III

108

212

201


The effect of PA on resin cannot explain the greatly increased water resistance of boards, so what about the effect upon the wood? A set of boards was pressed at 20OC for ten minutes with or without 2% PA, but with no resin. As made, these were of the same thickness and density. After conditioning, the boards bad picked up about the same amount of moisture, but the control boards had swelled more. On soaking in cold water, the control boards immediately started to swell at a rate of about 100/0 per minute, and continued to do so for 7 Y2 minutes, when measurements were stopped. The board with PA initially swelled at a much' slower rate, about 1% in the first minute, but after 4 minutes it too was swelling at 10% per minute. The overall delay amounted to about 1 minute. This difference may be chemical, or may be due to the PA decreasing to porosity of the board. In either case, such a minor difference cannot explain the greatly improved resistance of boards made with PA to two hours of boiling. A further set of boards made without resin was cut into blocks immediately after pressing and cooled in a dessicator. They were then quickly measured and mounted for internal bond measurement. The board containing PA was no stronger than the board without it. PA is clearly not acting as a binder. The effect of PA cannot be explained by its action on either wood or resin, it must involve an interaction between all three components. Boards are sensitive to water content. Perhaps the PA "locks up" the water in some way and stops it interfering. A series of boards have been made and tested, and the results are shown in the Figure 2. It is not possible to translate the curve for the boards with PA onto the control curve by a horizontal movement only ~ so PA is not acting only to reduce the effects of moisture.

Phthalic anhydride addition.

Effect of mattress moisture on 18S. '1.6 .....- - - - - - - - - - - - - - - - - -..

"1.4

+--------~------------4

1.2

-+---------~------------I

(i1.0

+----------~~---------I

0...

•

~O.8 +-----------~--------4

2%PA

~ 0.6 +-------~-----~=-------I

control

)(

(/)

0.4 -+-0.2

-..--;:l~---------I

~---------------===-~--I

------"'1.....-----.. .

0.0 ......- - - - -..... o 5

10

moisture content of mattress (%)

Figure 2

15


211

Chemical considerations The resin is in aqueous solution and has a high pH. The board is pressed at high temperature. In these conditions the PA cannot survive long before being hydrolysed to acid, and some will be converted to phthalate ion. On the other hand, phthalic acid is thermolabile, and could start to dehydrate to PA. A phthalate ion from di-sodium phthalate, on the other hand, would have to grab two protons from the alkaline surroundings, and hold onto them long enough to be dehydrated. This seems unlikely. Dehydration is a mechanism that is not available to terephthalic acid (the para isomer) either.

Spectroscopic evidence The reactions of PA, phthalic acid and sodium phthalate with wood components were also investigated using FTIR. There is good evidence of reaction of PA and phthalic acid with all components at 1800C or less. Sodium phthalate does not appear to react even with lignin. FTIR spectra show that when PA reacts with wood at 1600C only di-ester is formed, no acid is detectable. Phthalic acid reacts just as thoroughly at 1800C. This shows that benzenecarboxylic acids can react directly with wood, without forming an anhydride. Futher studies confirmed that terephthalic acid also reacts with wood at 1600C, but in this case acid groups remain. However, sodium phthalate shows no reaction even at 200 0C. That relevance of these reactions was confirmed since it was shown by spectroscopy that phthalate is also present exclusively as di-ester when PA is added to a board with the resin.

r

We conclude that equation 1], derived from studies using anhydrous solvents, does not represent the reaction of PA with wood when the two are simply heated together, or pressed together in a board. This can be confirmed by looking at the affinity of the product for water. Whereas a carboxyl group is more hygroscopic than the hydroxyl it replaces, a di-ester should be much less hygroscopic than two hydroxyls. The equilibrium moisture content at 65% r.h. of wood modified with 2% PA was found to be 7.5%, compared to 9.2% for untreated wood. Once the di-ester is formed, it is unlikely to react further in board pressing, but boards made with this material show the property enhancements. It therefore appears that we have a chemical reaction between PA and wood, and a physical interaction between the modified wood and the resin.

Comparison of different additives We would expect, from the reaction information, that phthalic acid should be comparable in its action to PA. Terephthalic acid may also have an effect, but di-sodium phthalate should have none. A series of boards, all using the same amount of resin, were made to test this. The results (Fig 3) are not in full agreement with the prediction. Terephthalic acid is harmful. Phthalic acid is comparable to PA. But di-sodium phthalate is also beneficial. Whatever the mechanism, it seems unlikely that wood modification is involved.


Comparison of additives.

Effect on dry and boiled 18S. 0.8 ~------------------------..

0.6

....-......-_4----------------------___

~

ca :i ~ 0.4 ....... .....---."""'....---+--4-------------------D.

tJ)

m 0.2

phthalic anhydride

phthalic acid

sodium phthalate

control

terephthalic acid

Figure 3

Summary PA greatly improves the properties of particleboard made with PF resin, both when it is used to pre-modify the wood and when it is added with the resin. There is little difference in the effects, so there is probably little difference in the mechanism. PA does not significantly improve the properties ofPF resin or of wood alone.

In solvent-free conditions, PA reacts with wood completely to give di-ester. The product is not hygroscopic. No further reaction with the resin is likely, so the board improvements must result from a physical interaction. Sodium phthalate also improves board properties, but does not react with wood. different mechanism must be involved, which probably also contributes when PAis used.

A

There is clearly much more work to be done before this system is fully explained.

We acknowledge the contribution of Dr H Earl at the start of this work, and the help of Dr D Gerrard ofBP with the spectroscopy.

References (1) R M Rowell, (1983) Chemical modification of wood. Forest Products Abstracts Vol.6 No.12,363-383 (2) H Matsuda, (1987) Preparation and untilisation of esterified wood bearing hydroxyl groups.

Wood Science and Technology 21:75-88

22 Engineering composites from oriented natural fibres: A strategy P E Humphrey - Oregon State University, Corvallis, Oregon, USA

1 INTRODUCTORY SYNOPSIS Most plant fibres which have rigid walls consist of highly evolved arrangements of lignocellulosic (LC) sub - elements. The impressive physical properties of these fibres are, however, rather poorly utilized in present-day composites. It will be argued here that such fibres could be used in some quite revolutionary ways, and looking to nature's solutions to her engineering problems may help us in this endeavour. The internal micro-structure and external shape of mammalian bones are, for example, tailored to the mechanical and biological demands placed upon them. It is this principle of judiciously manipulating overall shape and the spatial distribution of properties therein which will be contemplated here. New types of molded engineering components incorporating spatially oriented and modified LC-fibres could be the result. Products so derived may partially replace environmentally less attractive materials, including pressed steel, aluminium alloys and some polymers, in a diverse range of engineering applications. We will see that property control within the proposed engineered components may be achieved by manipulating a combination of the following: i) spatial distribution of fibre type; ii) spatial distribution of fibre orientation; iii) spatial distribution of localized conditions (thermodynamic, chemical, stress) to which pre-formed fibre networks are exposed during consolidation processes.

213


It is well known that the structures of conventional wood-based composites (mainly panel products) are greatly influenced by physical mechanisms that occur within them during their consolidation (hot pressing; item iii) above). These mechanisms include unsteady-state heat and moisture transfer, rheology (densification and stress relaxation) and adhesion. Some models which simulate material behavior during the pressing process will be summarized here, and their possible use (albeit in much modified form) to aid in developing ways to form the new molded components will be considered. Let us start by considering some general principles upon which the design of engineering components often depend, before going on to look at some possible ways of synthesizing new and useful objects from natural fibres.

2 Design rationale for engineering components: the way engineers usually do it Most components used in engineering applications are designed using shape as the principal variable. This usually follows some type of materials selection activity which is done in light of the anticipated demands in service versus the attributes of a range of candidate materials. Desired combinations of attributes are clearly diverse, usually including load bearing properties, density and cost, but also often including thermal properties, factors such as machinability, finishing, wear, and means of connection. In more forward looking design groups, some form of environmentally inclusive life-cycle analysis may be made when allocating cost (4,14). With the ever increasing demand for efficient designs, much attention of late has been given to optimizing shape, and this has led to the evolution of quite sophisticated design strategies using numerical methods of analysis. "Shape Optimal Design", or SOD, using FEM and, more recently, Hereditary algorithms (6) are, for example, now being used in diverse engineering fields as well as in the aerospace industry where they originated. Furthermore, greater care is being taken in identifying performance criteria for materials which are to be used in particular combinations of in-service demand. Pioneering in these approaches to materials selection is the work of Ashby and his co-workers (1). They have developed well founded rationales for identifying performance indices. Mass-to-strength and mass-tostiffness ratio manipulation under specific demand conditions are common concerns when using such approaches. Many combinations of factors (such as mechanical, thermal, aerodynamic, and vibrational ones) may, however, all come together to affect an optimal selection in this strategy .. Even in Ashby's approaches, almost all of the materials considered in the data base upon which the approaches depend are assumed to be homogeneous in nature, or at least orthotropic. This is in contrast to many materials and micro-structures which occur naturally in plants and animals and which perform highly specialized functions therein.

3 Nature's solutions to engineering design In the above, it is largely assumed that a homogeneous material structure, or a regularly repeating micro-structure, be employed and that the shape of the object is the only aspect that may be varied very much once a selection of material is made. One only needs to

Composites from natural fibres

215

consider the structure of even the most primitive plant genera to realize that nature's solution to the task of efficiency in design is much more sophisticated and leads to some elaborate multi-functional and tailored solutions (11). Microscopic examination of trees or animal bones or insect shells soon shows that the internal structure of such objects are elaborate and involve complex mixing of diverse cell types and orientations (12). Such structures lie within objects which themselves are not of simple geometric shape. In other words, through the power of natural selection, nature combines the concept of "shape optimal design" (SOD) with that of internal structure control, and thence spatial property distribution control. Together, these two concepts yield natural systems which are significantly more efficient and viable than almost any human construct. The concept of manipulating the spatial distribution of properties within an object will here be termed "Internal Property Distribution Control" or IPDC.

4 Can we combine SOD and IPDC in human-made composite components? Some attempts have, of course, already been made to control both shape and properties in human-made objects. This has mainly been in fibre re-enforced polymer products where mandrel windings or wovens of long strands of carbon, glass, boron etc are formed into relatively simple shapes. The winding or weaving or lay-up configurations within such objects are usually selected in the light of stresses anticipated in service. Spatial gradients of property change achievable in such approaches are, however, largely limited by the formation method and truly three-dimensional control over internal structure remains illusive. A couple of rather interesting examples of shape and property control are to be found in the wood utilization field. Both spatial property distribution (by lumber selection and subsequent placement) and shape are, as we well know, controlled in glu-Iaminated beam design. On a rather smaller dimensional scale, density profile (cross-sectional mass distribution) in hot pressed panel products is increasingly acknowledged as being an important factor in affecting the efficiency with which we tailor panel design to in-service demands (7). The transformed section of a panel may be likened to the shape of an "I" beam and thence, for example, effect its ability to span floor joists in a building. This particular paradigm will be taken further in the discussion to follow, in which an extension of the mechanisms used to affect density profiles in panel products will be considered for the development of new natural fibre based engineering components. As mentioned above, one may apply the principle using a diversity of raw materials -synthetic or elemental fibres, particles etc in conjunction with diverse matricizing agents and processing methods. Natural fibres do, however, have some special attributes which may offer us special opportunities. They lend themselves especially well to in-process modification; this is unlike most other fibrous materials which are relatively inert. Before looking at this aspect in a little more depth, let us first list some of the primary attributes of plant fibres (at least those with rigid walls) which may effect their behavior during processing and in subsequent service. For our purposes, it is convenient to boil down the fibres' attributes to the following: •

permeable to gases and liquids due to the presence of the lumen (and inter-fibre spaces in fibre networks);


•

wall accessible to, and reactive with, some molecules - particularly small polar ones and this makes the fibre amenable to modification in-situ;

•

wall therefore hygroscopic and this makes unmodified fibres dimensionally unstable (below wall saturation) and, more encouragingly, an active player in the convection of polar vapours and the bound diffusion of adsorbed molecules;

•

hygro-thermo-viscoelastic and this makes the fibre amenable to structural modification (deformation, densification) in-situ (particularly by manipulating thermodynamic and chemical conditions while applying stress);

•

anisotropic in terms of mechanical and translational properties (particularly axially versus transversely);

•

has significant specific (density corrected) axial load-bearing properties and toughness.

5 Structural changes that occur within wood-based composites during consolidation Our looking at the mechanisms operative within conventional wood-based panel products during pressing has provided some of the groundwork upon which to base processing innovations in the present strategy. For this reason, a very brief overview of the mechanisms and our modelling of them will be given here before going on to identify some opportunities for innovation The above listed fibre attributes should be remembered from henceforth. Heat and moisture move throughout conventional wood based composites during pressing. This is principally by means of thermal conduction and the convection of water vapour. The water vapour gradients are generated within the pore spaces of the hygroscopic fibre network since some form of phase balance is maintained localI y between water adsorbed in the fibre walls and that existing as vapour in the pore spaces; local energy content dictates the phase balance and temperature of the solid-gas combination. As a consequence of heat and moisture transfer and phase change, an ever changing spatial distribution of temperature, adsorbed (fibre wall) moisture and within--void partial pressure of water vapour occurs within the composite as pressing proceeds. Since the fibre network is hygro-thermo-viscoelastic, it densifies differently in different locations depending on the time-temperature-moisture history sustained and the stress applied from the platens of the press. As one would expect in flat pressed panels, the induced property gradients are most extreme in the compression direction (usually through the thickness) and the density profile is a consequence. It is this density profile which effects the flexural properties of the products, for there is usually a strong correlation between density (level of consolidation) and mechanical properties for assemblages of bonded natural fibres or particles. In the pressing process, rheological changes (which affect the density profile changes as pressing proceeds) directly influence the translation properties of the furnish material (principally permeability to water vapour and thermal conductivity). It is, however, temperature and moisture which influence the densification process. In other words, there is a high degree of linkage between the mechanisms that occur within the fibre network as pressing proceeds. For this reason, numerical methods of simulation have been used to

Composites from natural fibres

217

account for the interactions, and the resultant algorithms are now being used as tools to aid in optimizing the production of conventional composites and the development of new machines for their production (approaches such as continuous steam injection pressing). The interdependencies among mechanisms in wood-based composites during pressing are represented schematically as Figure 1. A range of example predictions of the models which simulate heat and moisture movement, rheological behavior and adhesion development are to be found in publications and ones in preparation (3,7). One typical set of model predictions for the thermodynamic (heat and moisture transfer) aspect of the system applied to the consolidation of a homogeneous mat of wood flakes is presented as Figure 2. Following this (Figure 3) is a predicted density profile development plot using the same model, but this time applied to the pressing of a homogeneous mat of thermo-mechanically produced wood fibres and fibre bundles. It is this plot that most clearly demonstrates the potential for using the modelling techniques to aid in controlling the structure (and thence properties) within natural fibre composites by manipulating pressing conditions.

HEAT AND MOISTURE TRANSFER Thermal conduction, phase change, and vapor convection affect local temperatures, MCs, and vapor pressures. Local balance between temperature, MC, and vapor pressure is maintained.

Figure 1. The mechanisms operative within dry-formed composites during pressing.


_--------,160

0.0 UPPER PLATEN

0.5

1.0 LOWER P\.ATEN

1--------,..f20 10

/:.(0.0

0.5

1.0

UPPER PLATEN

LOWER PLATEN

0.0 UPPER PLATEN

1.0 LOWER PLATEN

Figure 2. Predicted variations of cross-sectional (consolidation direction) distributions of temperature [A], vapour pressure [B], and adsorbed moisture (on a dry basis) [C], with simulated pressing time. These data relate to a mattress of Sitka spruce flakes, initially at 11 % moisture content, pressed to a mean density of 650 kg m? with 1600C platens (8).

Simulated Density Profile Development 900

Density (Kg/ m3)

Thickness

Elapsed Time (Sec.) o

Figure 3. Predicted cross-sectional (consolidation direction) density distributions with simulated pressing time. These data relate to a mattress of thermo-mechanically produced Douglas-fir fibres, initially at 11 % me, pressed to a mean density of 650kg m? with 160°C platens. An extended form of the simulation model used to generate the data of Fig. 2 was used here (5). Note: Total mattress thickness decreased from an initial value of 42-mm (150kg m") to a constant value of 16-mm (650-kg m") in the first 37 seconds of simulated pressing. This variation is not represented graphically here; equal mass was, however, conserved in each of the ten cross-sectional modelling regions.

Composites from natural fibres 219

In order to simulate such processes, a range of materials properties input data for networks of fibres, particles and the like has to be provided. A suite of miniaturized techniques have been developed to provide such data. These include, but are not limited to, techniques to explore the following: •

Gas permeability of natural fibre networks and particle mats as functions of flow direction and level of consolidation (density) (2);

•

Thermal conductivity as a function of fibre moisture content, direction and level of consolidation (density);

•

Rheological properties (viscoelastic with micro-fracture) as functions of fibre moisture content, temperature and level of consolidation (density) (13);

•

Bonding reactivity (for a given adhesive and adherend) as a function of temperature and moisure content (preliminary and simplistic at this stage!) (9,10).

6 A possible sequence of fibre processing stages to form the new materials The insights gained in studying the behaviour of conventional composites will aid in the formulation of the new ones. A strategy now under detailed development is including (but is not limited to) the stages listed below. •

Fibre separation and selection: This may be from naturally occurring populations (eg extracted from trees of a single specie), or by species-dependent ranges in properties. Natural and man-made fibres may ultimately be combined. Very rapid but accurate fibre sorting techniques are being considered.

•

Fibre modification prior to re-constitution: This may encompass a diversity of chemical and mechanical treatments to impart desirable properties to the fibres prior to their being further modified during the re-constitution phase to follow. Clearly, in present products, adhesive is the main additive at this stage (although fibres used in papers and the like are modified in a wider range of ways). In the proposed composites, alternative treatments may be warranted. For example, the application of very small quantities of a ferromagnetic element renders the fibres amenable to subsequent orientation in a magnetic field (see below.)

•

Creation of a pre-form which consists of fibres spatially oriented in two or three dimensions within a simple or complex shape: Preliminary experimentation suggests that magnetic means have potential (15), although a range of possibilities are being explored.

•

Consolidation of the pre-form to create a component with controlled shape and internal structure: It is proposed to consolidate the pre-form in a sealed pressing system to affect the thermodynamic and chemical environment in such a way as to trigger a


desirable range of reactions. In other words, concentrations of reactive vapours will be transtluxed through the porous matrix of fibres. This could stimulate reactions which affect localized densification, adhesion, dimensional stabilization and the like. Zoned injection and removal offers the potential for spatially controlled reactions and thence final structure.

CONCLUSIONS Biological parts are, in certain respects, the antithesis of most objects fabricated by engineers from commodity materials. The formidable power of natural selection tailors the former to very specific demands, while the latter often perform moderately well in a wider range of uses. With improvements in our design ability and the advent of sophisticated process control in manufacturing, it may become feasible to form highly tailored objects which resemble their natural counterparts. This opens up exciting design opportunities which may have aesthetic as well as functional ramifications.

REFERENCES (1) Ashby, M.F. 1992 Materials Selection in Mechanical Design. Pergamon Press, London. (2) Bolton, A.J. and P.E. Humphrey. 1994. The permeability of wood-based composites. Part I. A review of the literature and some unpublished work. Hotsforschung, 48(suppl.):95-100. (3) Bolton, A.J., P.E. Humphrey and P.K Kavvouras. 1988-1989. The hot pressing of dryformed wood-based composites. Parts 1 to VI. Ending with: Holl,{orschung,43(6):406-410. (4) Chapman, P.F. and Roberts, F. 1983. Model Resources and Energy. Butterworths, London. (5) Haselein, C. 199-. Modelling the formation of advanced natural fibre composites (provis. title). Doctoral Thesis, Oregon State University, Corvallis, Or., USA. (6) Hedberg.S. 1994. Emerging Generic Algorithms. AI Expert. 9 (9): 25-29. (7) Humphrey, P.E. 1992. Pressing issues in panel manufacture: internal behaviour during pressing. Proceedings of the 25th. Particleboard symposium, March 1991, Pullman, WA. (8) Humphrey, P.E., and A.J. Bolton. 1989. The hot pressing of dry-formed wood-based composites. Part Il, A simulation model for heat and moisture transfer, and some typical results. Holzforschung, 43(3): 199-206. (9) Humphrey, P.E. and D. Zavala. 1989. A technique to evaluate the bonding reactivity of thermosetting adhesives. J. Testing and Evaluation (ASTM), 17(6):323-328. (10) Humphrey, P.E. and Ren, S. 1989. Bonding kinetics of thermosetting adhesive systems used in wood-based composites: the combined effect of temperature and moisture. J. Adhesion Sci. and Technology, 3(5):397-413.

(11) Mattheck, C. 1990. Why they Grow, How they Grow:

the Mechanics of Trees.

ArboriculturalJournal. 14,1-17. (12) Neville, A.C. 1993. Biology of Fibrous Composites. Cambridge Univ. Press, Cambridge. (13) ReD, S. 1992. Thermo-hygro-rheological behavior of materials used in wood-based composites. Doctoral Thesis, Oregon State University, Corvallis Or., USA. (14) Van Griethuysen, A.J. 1987. New Applications of Materials. The Hague, Netherlands. (15) Zauscher, S. 1992. Orienting lignocellulosic fibres by means of a magnetic field. Masters Thesis, Oregon State University, Corvallis, Or., USA.

23 Reactive cellulosic fibres rather than reactive dyes D M Lewis and Q G Fan - Department of Colour Chemistry, Leeds University, Leeds LS2 9JT, UK

Abstract In order to overcome the disadvantages of reactive dyeings of cellulose substrates, the concept of reversing the conventional system by incorporating the nucleophilic group in the dye and the reactive group containing the electron deficient carbon atom in the fibres is explored with compound A: 2,4-dichloro-6-(2'-pyridinoethylamino)-s-triazine chloride (DCPEAT) and compound B: 2-chloro-4-(2'-pyridinoethylamino )-6-(4" vinylsulphonyl-anilino)-s-triazine chloride (CPVT). Cotton was pre-treated either by an exhaustion method or a pad-batch method. The dyeing was carried out using the bisaminoalkyl derivative ofC. I. Reactive Red 120. The results indicated that the modified cellulose dyeing process is successful with the quaternary reactive compounds, giving high uptake of dyes in the absence of salt and a high degree of dye-fibre covalent bonding.

1. Introduction Reactive dyes are extremely popular for dyeing cellulosic fibres because of the bright colours, wide shade ranges, easy application and more importantly their excellent wet fastness of the reactive dyeings. However, reactive dyes are vulnerable to hydrolysis under conditions where the dyes are applied to cellulose substrates thus lowering the fixation efficiency resulting in problems of coloured effluents with high salinity harmful to both the environment and the sewage system.

221


During the reactive dyeing process, two fundamental elements play an important role. They are: • an electron deficient reactive atomic centre, usually the carbon atom(s) adjacent to an electron withdrawing group in the dye structure; • an electron rich nucleophilic atomic centre, usually the hydroxyl group in cellulosic fibres or the amino group in the wool fibre. In order to overcome the disadvantages of reactive dyes, the concept [1] of reversing the above system by incorporating the nucleophilic group in the dye and the reactive group containing the electron deficient carbon atom in the fibre is further explored with two compounds having following structure:

,

CI

N--< W-CH2-CI12-NH~ 0N=(

CI- 'I

N

CI

compound A 2,4-dichloro-6-(2'-pyridinoethylaminoj-s-triazine chloride (DCPEAT)

compound B 2-chloro-4-(2' -pyridinoethylamino )-6-( 4" -vinylsulphonylanilino)-s-triazine chloride (CPVT) Cotton was activated to make it subsequently dyeable by amino or aminoalkyl dyes such as the bis-aminoalkyl derivative of C. I. Reactive Red 120 (MR120) of the following structure:

Dye: MR120 The practical application of the two reagents, DCPEAT and CPVT, involved two steps. Firstly, cotton was treated using one of the reagents under the same conditions as reactive dyeing. The activated cotton was then dyed with modified C. I. Reactive Red 120 (MR120).

Reactive cellulosic fibres

223

Once DCPEAT or CPVT is fixed onto the cotton, the substantivities of anionic dyes to the reactive cotton are greatly improved because of the charge attraction between the cationic pyridinium centre of the reagent and the anionic sulphonate group of the dye. In this way, high exhaustion of dye can be achieved. On the other hand, due to the resistance of amino or aminoalkyl dyes to hydrolysis, it can be expected that this modified cotton dyeing process could greatly decrease the waste of dye by raising the fixation efficiency of dyes. Overall, this system can render the following advantages over the conventional reactive dyeing procedure whilst maintaining the same high wet fastness profiles of the final dyeings: • • •

excellent pad-liquor stability, zero electrolyte use" less washing-off time.

2. Experimental 2.1 Pre-treatment of Cotton by a Long Liquor Process (Exhaustion) Cotton was treated with the reagents" either at pH 9 (DCPEAT) or pH 11.5 (CPVT), at a liquor ratio of20: 1 at 50°C for 90 min.

2.2 Pad-Batch Pre-treatment of Cotton Cotton was padded to 90% pickup with pad liquor containing either Na2C03 20 gil (DCPEAT) or Na3P04 10 gil (CPVT) and Sandozin NIE (Sz) 10 gil, then rolled, sealed and batched for 24 hours at 30°C. Following batching the samples were thoroughly rinsed in running cold water for 5 minutes.

2.3 Dyeing of Activated Cotton Dyeing was carried out using 20/0 owf MR 120 at pH 9 at a liquor ratio of 20: 1 at the boil for 1 hour. Unlike "standard" reactive dyeing" no salt was added.

2.4 Dyebath Exhaustion (OAt) Exhaustion values were obtained by measuring the absorbance of the dye liquor at the maximum absorbance wavelength (Amax = 520 nm for MR120) before and after dyeing.

2.5 Dye Fixation (%) This was obtained by measuring the K/S values of samples before and after soaping which was carried out by boiling in a solution containing Sandozin NIE (Sz) 5 gil and sodium carbonate 2 gil for 15 minutes.

224 Physical and chemical processing of fibre and fibrous products 3. Results and Discussion The results showing the effect of the pretreatment and subsequent reactive dyeing procedures on dye exhaustion and fixation are shown in Figures 1- 4.

100

~ o ........

80 60 40

20

o

untreated

4 %owf

6 %owf

8°A>owf

DCPEAT Concentration I

Figure 1

[J

EXh~usti~nOfDye. Fixation of Dye~

Effect of DCPEA T concentration (exhaust application) on dyeing

100r

~ o ........

1-----I

20 gIL

30 gIL

40 gIL

50 gIL

DCPEAT Concentration

~I=:J Exhaustion of Dye III Fixation of Dye Figure 2

Effect of DCPEAT concentration (pad-batch application) on dyeing

Reactive cellulosic fibres 225

;e 108~' o

~

60

4020 ~

O~--4 %owf

6 %owf

10%owf

8O/oowf

CPVT Concentration

Figure 3

Effect ofCPYT concentration (exhaust application) on dyeing

100 r '

\---

t

80l L

60t f

40t 20f i ~ L

OL-- 20 gIL

30 gIL

40 gIL

50 gIL

CPVT Concentration Exhaustion of Dye

Figure 4

~

Fixation of Dye-I _. .

-------~

Effect ofCPYT concentration (pad-batch application) on dyeing

It is obvious that the modified cotton dyeing process is successful especially where CPVT was applied by the exhaust method at concentration ~ 8% owf It is especially worth noting that the high degree of dye exhaustion and fixation (> 95%) was achieved in the absence of salt. The likely reaction scheme is shown in Scheme 1, in the case of CPVT application.


+

HO-eell

j

Scheme 1

4. Reference [1] I). M. Lewis and X. P. Lei, Book of Papers of American Association of Textile Chemists and Colorists, Annual Conference and Exhibition, Atlanta, (1992)259.

24 The treatment of cotton cellulose with Trichoderma reesei engineered cellulases A Cavaco-Paulo,* L Almeida* and D Bishop'[ - *Dep Eng Textil, Universidade do Minho, 4810 Guimaraes, Portugal; tDept Textiles & Fashion, De Montfort University, Leicester LEI 9BH, UK

I-INTRODUCTION Controlled enzymatic hydrolysis can provide ecologically acceptable routes to finishing cellulosic textiles (1). The most widely used application is the replacement of the stone washing process to produce the fashionable aged appearance of denims. Other cellulase treatments are used to improve the appearance of cotton fabrics by removing fuzz fibre and pills from the surface. Such processes also modify the fabric mechanical properties in ways which lead to the perception of improved handle, particularly of improved softness (2). Increasing use is being made of cellulases in domestic fabric washing products where they are claimed to aid detergency (3), as well as removing damaged fibrillar material from cotton fibre surfaces. This improves fabric appearance, colour brightness and softness (1). Cellulase enzymes have a specific catalytic action on 1,4-(3-D glycosidic bonds of the cellulose polymer, which apart from 6-8% of moisture, is essentially the sole constituent of scoured and bleached cotton. Cellulase, by definition, consists of a complex mixture of three major enzyme types: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and cellobiases (EC 3.2.1.21) (4). A general model for action of these enzyme components on cotton cellulose is that the endoglucanases (EGs) cause random hydrolytic chain scission at the most accessible points of long cellulose polymers, while the cellobiohydrolases (CBHs) split cellobiose from the "non reducing end" of cellulose molecules. The cellobiase hydrolyzes cellobiose to glucose. Improvement in purification and analysis techniques have lead to the isolation and study of the pure components and new models have been proposed. It is now clear that the mode of action of the pure cellulase components cannot be classified simply as "endo" or "exo" in type (5). A synergism between the different components has been observed, but the detailed mechanism of their action is not yet fully understood (5).

227


The cellulolytic complex of Trichoderma reesei is one of the most extensively investigated fungal enzyme systems (5). It is known to contain at least one cellobiase, CBH I, CBH II, EG I and EG Il. The genes of these hydrolases canbe manipulated such that some activities are deleted to produce new cellulase combinations (6). Furthermore it has been reported in recent literature (7, 8) that two more cellulase components known as EG III and EG V are always present in the crude mixtures from Trichoderma reesei. In the present work, cotton degradation was studied after short (as made in textile applications) and extended periods of cellulase hydrolysis. Comparisons were made between the effects of crude cellulase mixture from Trichoderma reesei (TC), and mixtures in which the activities of EO I and EO II (C-EGs) or the activities of CBH I and CBH II (C-CBHs) had been deleted. 2-EXPERIMENTAL Cellulases: All enzyme samples are from Primalco Ltd., Rajamaki, Finland. These were: CE 883042 (Total crude), CE 519/92 (Crude-EGs) and CE 523/92 (Crude-CBHs). Enzyme Activity: The activities per gram of crude were measured towards carboxymethylcellulose (CMC), phosphoric acid swollen Avicel (PASA) and cellobiose, at pH=5, as described previously (9). The activity towards scoured cotton fabric was also determined by measuring weight loss. Cotton Satnples: All samples used were 100% cotton fabrics after industrial scouring and bleaching. Treatments~

Shm1: The fabrics were treated with the enzyme (dilution factor, 1/500) using the ratio: l g of fibre to 20 ml of bath; buffer pH = 4.8 (acetate, 0.5 M), temperature 50 oC. The fabrics were treated during 30, 60, 120 and 240 minutes. The treatments were stopped by addition of a solution of sodium carbonate (10 %). The fabrics were washed after treatment with hot and cold water. The treatments were carried out in the stainless steel pots of a Linitest machine rotating at 65 rpm. Long: The fabrics were treated with the enzyme (dilution factor, 1/60 for the CrudeCBHs and 1/120 for the Total crude and Crude-EGs) using the ratio: l g of fibre to 50 ml of bath; buffer pH=4.8 (acetate, 0.5 M), temperature 50 0C. The fabrics were treated without agitation for 3, 6 and 13 days in a beaker inside an incubator. The fabrics were washed after treatment with hot and cold water. Weight Loss was determined by weighing the samples before and after treatment, after conditioning for 24 hours at 20°C and 65 % of relative humidity. Mean Chain Len~th: Mean chain lengths were determined by the weight difference taken from weight loss calculations and measuring in solution the reducing ends of the leaving sugars as cuprous neocuproine complex in alkaline media at 95 0C. Cotton Reducing Power: The reducing ends of the cotton fibres were also quantified via the cuprous neocuproine complex in alkaline media at 95 0C. Viscosity nleasurements: Fluidity and specific viscosity were measured as described (10), on cuproethylenediamine (CED) solutions of cotton cellulose. Breaking Load Loss (%) was measured relative to the untreated fabric in an Instron machine, model 4204. Bending Hysteresis of the Fabrics was measured on KES-FB2 from Kato Tech. Co, Ltd. Crystallinity Index by X-ray diffraction measurement was obtained by the method described by Chidambareswaran and others (11). The X-ray diagrams were obtained using a Philips Analytical PWI7IO diffractometer with a X-ray tube using Ni filtered Cu Ka,radiation and the limits were 10° and 3()O.

Trichoderma reesei 229

Scannin~ Electron Microscopy Photo~aphs (Leica Cambridge Stereoscan 360) were taken after 2 minutes of gold metalization (Bio-Rad SC 502). Enyironmental Scannin~ Electron Microscemy (Electron Scan, Mod 3) A video-tape was recorded during the drying of cotton fibres, which had never been dried after the cellulase treatment. The pressure of the chamber during drying was brought down from 6 Torr to 2,5 Torr.

3-RESULTS AND DISCUSSION The measured activities of C-CBHs towards CMC and of C-EGs towards PASA were greater than those of TC (tab. 1). These results illustrate the expected increments in classical endo and exo type activities for C-CBHs and for C-EGs respectively (12). While TC was found to have lower measured activity than C-EGs towards cellobiose, CMC and PASA, it caused consistently greater cotton weight loss than C-EGs in both long and short treatments (tab. 1 and fig. 1). This apparent contradiction shows that care should be exercised in predicting cellulase activity on cotton, from data obtained using other forms of cellulose or its derivatives. The result also points to the importance of synergy between the various components in the hydrolysis of cotton cellulose. Substrate \ Enz me Cellobiose a) CMC b) PASA b)

The deletion of CBH I and CBH II activity from the total crude mixture dramatically reduced the rate of cotton weight loss (fig. 1) thus confirming the importance of exo type activity in solubilization of the polymer. The deletion of EG I and EG II activity also caused some reduction in the rate of cotton weight-loss, and this is expected from the synergy between endo and exo type activity. Nevertheless, it is clear that C-EGs and TC can both bring about complete dissolution of cotton cellulose, albeit in somewhat longer reaction times, as shown by the SEM photos after 6 days of degradation (fig. 2a). The surprisingly high activity of C-EGs may possibly due to the previously reported synergy between the two CBH components (13) and, or, to some endo activity in our C-EGs. The latter may be accounted for either by remaining EGs such as EO III and V or by some endo characteristics of the CBHs themselves (5, 14). Weight Loss (%) - LONG TREATMENT 80

~------------.

--a--0~

Total Crude Crude-CBHs Crude-EGs

Weight Loss (%) - SHORT TREATMENT

3.....------------2

10 20 100 200 Time (days) Time (min) Figure 1 - Relation between weight-loss and treatment time.

300


In the short hydrolysis, the relative activity of C-CBHs is higher and the relative activity of C-EGs is lower, when compared with the longer hydrolysis. This seems to be due to the rotation of the reactor where the hydrolysis takes place. The agitation is known to increase the weight loss ( 15) and to have a synergistic action with an endo type cellulase (16). However the main structure of the fibres remains unchanged (fig. 2b).

Figure 2 a) Scanning electron microscopy photographs of long treated fibres

Figure 2 b) Scanning electron microscopy photographs of short treated fibres

SEM photos (fig. 2) showed that the short treatment caused the changes only at the fibre surface, while the long treatments also affected the internal structure of the fibre.

Trichoderma reesei

231

Mean Chain Length Leaving Sugars - SHORT TREATMENT

4...------------------.., --0--0-3

~

Total Crude Crode-CBHs Crode-EGs

2

o

100

200

300

Time (min) Figure 3 - Relation between mean chain length of the leaving sugars and treatment time.

In treatments with C-CBHs, the mean chain length of the leaving sugars (fig. 3) decreased from 3.4 (30 min) to 1.7 (240 min) showing endo character when compared with the other mixtures where the mean chain length was about 2 throughout. The results show that the soluble cellooligosaccharides resulting from TC and C-EGs hydrolysis are readily broken as they are produced to a mean length of 2, while those resulting from C-CBHs are slowly broken. The slow decrease of the mean chain length of the soluble cellooligosaccharides caused by C-CBHs is believed to be due to the lower cleavage frequencies of EGs compared with those of CBHs (14), on the soluble cellooligosaccharides. The low cellobiase activity of these cellulase mixtures is also reflected in the results. Specific Viscosity (0 sp , - LONG TREATMENT

Cotton Reducing Power (mg gli/g eel)

1 , 2 . . . - - - - - - - - - - - - -.....

0,7

~====~~=====n---""

1,0 0,8 0,6

0,4

- - 0 - Total Crude --0-- Crode-CBHs

0,6 Total Crude Crode-CBHs Crode-EGs

°

~

Crude-EGs

0,5 ............-........--..--...................-...........-...........--1 20

40

60

80

0

20

40

60

80

Weight Loss (%) Weight Loss (%) Figure 4 - Relation between cotton reducing power and between specific viscosity with weight-loss

In spite of causing low weight loss, the C-CBHs enzyme produced higher concentrations of terminal reducing groups in the cotton fibres and a large decrease in the viscosity of their CED solutions (fig. 4). TC and C-EGs also produced appreciable quantities of terminal reducing groups but the viscosity of CED solutions of the treated celluloses changed only slightly, even at a weight loss greater than 50 %. These results confirm the increased endo type activity of C-CBHs in causing random cellulose chain scission, (hence a large reduction in CED solution viscosity) and the sequential nature of exo type activity, (hence little change in mean polymer chain length or CED solution viscosity). These effects are further illustrated in figure 5, where an increasing slope of the plot of cotton fluidity versus cotton reducing power indicates increasing randomness of cellulolytic attack. Thus C-CBHs is shown to cause the most random hydrolysis and CEGs to cause the most localized attack, with TC having an intermediate effect. While the randomness of cellulolytic action of different cellulases is commonly measured in this


way by using CMC as a soluble substrate (17) these relationships have not previously been reported for cotton cellulose. Fluidity (lin sp 1,9

1,8

D

1,7

o

1,6

6

1,5 1,4

- LONG lREATMENT

~--~---------------.

e:===-.!r---6--__J

b ....

-+---.~......----r--r----r-~_,.-~....,.-_r_..,.___t

0,4

0,6

0,8

1,0

1,2

Cotton Reducing Power (mg gli/g eel) Figure 5 - Relation between cotton reducing power and specific viscosity

It is interesting to note that the relative randomness or localization of cellulase action on cotton and the extent of the produced damage (fig. 2) did not change the measured crystallinity of the fibre (tab. 2). A similar result has previously been observed for complete cellulases (18). Thus the present work tends to confmn the view that the action of cellulase is not confined initially to non-crystalline regions, irrespective of the cellulase components present. It has also been reported (19) that the hydrolysis of cellulose by cellulase concentrates of Trichoderma viride is first order with respect to substrate. This suggests uniform reactivity of (crystalline and amorphous) cellulose and therefore implies that no change in crystallinity should result from cellulase hydrolysis. 1 ABLE 2 - CrystallInIty Index (WeIght-Loss) Untreated 83% (0%)

TC C-CBHs C-EGs 240min 240min 240min 83% (23%) 83% (09%) 83% (1,8%)

TC

6 days 83% (63%)

C-CBHs 6 days 83% (3%)

C-EGs 6 days 81% (52%)

Cellulase hydrolysis reduced the tensile strength cotton fabrics as shown in figure 6. Recently a possible relation between loss in tensile strength and endo activity has been reported (16). The present results support this to the extent that C-CBHs caused much greater strength loss at low weight loss than the other cellulase mixtures (fig. 6). On the other hand C-EGs did not show reduced strength loss for a given weight loss in comparison with TC. This may however be due to the effects of the residual EGs (III and V) in the present C-EGs sample. Breaking Load Loss (%) - LONG lREATMENT Breaking Load Loss (%) - SHORT lREATMENI 100 40 --0-- Total Crude 80 30 ---0-- Crode-CBHs Total Crude Crode-CBHs Crode-EGs

60 40

~

Crode-EGs

20 10

20 0 .......-.................-.---.--...--........-....--.......

o

20

40

60

80

o .w-=~=!:~-....----.-J 0

1

2

Weight Loss (%) Weight Loss (%) Figure 6 - Relation between breaking load loss and weight -loss

3

Trichoderma reesei

233

We have observed before (16) that for short cellulase treatments the changes in bending hysteresis of cotton fabrics, related to internal friction of the fibrous assembly, are due to the formation of microfibrils on (or their removal from) the fibre sutface. The small changes in bending hysteresis, for the short treatment, are consistent with what can be seen in SEM photos (fig. 2b). The large decrease in bending hysteresis for higher weight loss (long treatments) observed here is mainly due to the reduction in the number of fibres in a yarn and reduction of its diameter, as shown by SEM photos (fig. 2a). In as far as low bending stiffness/hysteresis is correlated with perceived softness, TC and C-EGs would be preferred over C-CBHs for their cotton fabric softening effects. This is consistent with a previously reported observation (16) that endo activity with high mechanical action led to the formation of microfibrils on fibre surface and caused increased inter-fibre friction, and hence an increase in bending hysteresis.

TABLE 3 • Bending Hysteresis (gf.cm/cm) Untreated 0.050

TC 240min 0.043

C-CBHs 240m in 0.053

C-EGs 240min 0.045

TC 2 days 0.015

C-CBHs 2 davs 0.048

C-EGs 2 days 0.019

Examination of the wetting (swelling) and drying (collapsing) behavior of untreated and cellulase treated cotton fibres by ESEM revealed that the fibre twisting and retraction behavior in untreated fibres is absent after long cellulase treatments. It is therefore postulated that a cellulase treatment may be beneficial to achieving stable, fully-relaxed dimensions in knitted cotton textiles.

CONCLUSIONS Crude cellulases from Trichoderma reesei have been used to investigate the mechanisms of cellulase hydrolysis of cotton cellulose in the form of scoured and bleached fabric. Predictions of the general model for cellulase action were largely confirmed. An EG "rich" crude, from which CBH I and CBH II activity had been deleted showed typical endoglucanase activity, causing random chain scission which led to a rapid rise in fluidity and high strength loss but did not solubilize cotton. A crude, from which EG I and EO II activity had been deleted, caused less random hydrolysis (i. e. increased exo type activity) than the total crude mixture. Nevertheless, it solubilized cotton as effectively as the total crude mixture. This C-EGs did however retain some endo type activity which was possibly due to the endo character of both CBH I and II and also due to the undeleted EO ill and V. Since it is important to maintain fibre strength in most textile applications it does not appear to be appropriate to increase the endo activity of cellulases intended for cotton processing. There may however be advantages for low endoglucanase or endoglucanase free mixtures especially where substantial weight loss may be required to achieve softness or de-pilling without serious loss of strength. The short treatments, as used in textile applications, keep the changes at the fibre surface while the long ones affect the internal structure of the fibre. ESEM studies have revealed that the fibre retraction and twisting behavior of untreated fibres is absent after long treatments.

ACKNOWLEDGMENTS We thank Primalco Ltd for kindly supplying the special crude cellulase mixtures used in this work. We also thank Dr. C. Carr of UMIST and Mr. C. Gilpin of the Electron Microscopy Unit, Biological Sciences Department, Manchester University, for their


help with the ESEM work. The authors are grateful to the British Council/Portuguese Universities Rectors Council for the grants under the Treaty of Windsor scheme which enabled the development of this paper.

REFERENCES 1 .. Cavaco-Paulo, A. and Almeida, L., (1994) Nova Textil, 32, 34-38 2·· Almeida, L. and Cavaco-Paulo, A., (1993) Melliand Textilber., 74, 404-407 3 .. Murata, M., Hoshino, E., Yokosuka, M. and Suzuki, A., (1993) Jour. Am. Oil Chem. Soc., 70, 53-58 4 - Enzyme Nomenclature (1978), Academic Press, New York 5 .. Enari, T. and Niku-Paavola, M., (1987) CRC Critical Reviews in Biotechnology, CRC Press 5(3) 67-87 6 .. Nevalainen, H., Pentilla, M., Harkki, A., Teen. T. and Knowles J., (1991) In Molecular Industrial Mycology, chap. 6, 129-148, edited by S. Leong and R. Berka, Mercel Dekker 7 - Ward, M., Wu, S., Dauberman J., Weiss, G., Larenas, E., Bower, B., Rey, M., Clarkson, K and Bott R., (1993) In Trichorderma reesei Cellulases And Other Hydrolases, edited by P. Suominen, T. Reinikainen, Foundation for Biotechnical and Industrial Fermentation Research - Finland, 8, 153-158 8 - Saloheimo, A., Henrissat, B. and Pentilla, M., (1993) In Trichorderma reesei Cellulases And Other Hydro lases, edited by P. Suominen, T. Reinikainen, Foundation for Biotechnical and Industrial Fermentation Research - Finland, 8, 139-146 9 .. Evans, E., Wales, D., Bratt, R. and Sagar, B., (1992) J. Gen. Microbiol., 138, 1639-1646 10 - Standart Proposal UNE 57.039 11 - Chidambareswaran, P., Sreenivasan S. and Patil N., (1987) Textile Research Journal, April, 219-222 12 - Ghose, T., (1987) Pure and Applied Chem., 59, 257-268 13 - Nidetzky, B., Steiner W., Hayn, M. and Claeyssens, M., (1993) In Book of Abstracts ofTRICEL 93, A12, June 2-5, Majvik, Finland 14 - Biely, P., Vrsanka M. and Claeyssens, M., (1993) In Trichorderma reesei Cellulases And Other Hydro lases , edited by P. Suominen, T. Reinikainen, Foundation for Biotechnical and Industrial Fermentation Research - Finland, 8, 99-108 15 - Cavaco-Paulo, A. and Almeida, L., (1994) Biocatalysis, 10 353-360 16 - Cavaco-Paulo, Almeida, L., (1994), Paper presented at 204th ACS Meeting - Cell Div., S. Diego, CA. 17 - Ramos, L., Nazhad, M.and Saddler, J., (1993) Enz. Microb. Tee., 15,1-11 18 - Finch, P., (1985) In Cellulose Chemistry and its Applications, edited. by T. P. NeveU, S. H. Zeronian, pp. 312-343, John Wiley & Sons 19 - Sagar, B. F. (1985) In Cellulose and its Derivatives: Chemistry, Biochemistry and Applications, edited by J. F. Kennedy, G. O. Phillips, D. J. Wedlock & P. A. Williams, pp. 199-207, Ellis Horwood

25 Characterisation of paperboard packages designed for liquid containment C J Harrold and J T Guthrie - Polymer Group, Department of Colour Chemistry, University of Leeds, Leeds LS2 9JT, UK

Introduction Many types and forms of polymers are used extensively in today's packaging industry. The need for extended self life along with special packaging criteria, means that the technology required has to change to meet these needs. This is clearly evident in the packaging of liquid products. Liquid producers require packages that are cheap, strong, aesthetically acceptable, and environmentally sound. By using virgin and recycled paperboard in conjunction with advanced multi-layer polymer coatings, these criteria can be met by the packaging company. The main polymers that have been used to fulfil these requirements are: • Poly(propylene) • Poly(ethylene) • Ethylene-vinyl alcohol copolymer These polymers have been adapted, using new coating technology, to produce various types of coatings that give the barrier properties that enable many types of liquids to be contained. These coating formulations are now established for the packaging of low to medium 'aggressive' liquid products. However, the need to pack more 'chemically aggressive' liquids is growing and so new coating technologies will need to be developed by the packaging industries. This paper shows a range of results from various techniques that have been used to characterise three packaging systems. Characterisation of these products is a necessary step for future advancement. The results will show why and how more 'chemically aggressive liquids' escape from these types of cartons.

235


Experimental The cartons used in this study were manufactured by Field Group, Newcastle. The cartons were made of virgin paperboard manufactured by Enzo-Gutzeit OY, Finland. The paperboard was coated on both sides. The upper surface of the paperboard was coated with 20 grams of poly(ethylene). This surface was used for printed material on the outside of the carton. The lower surface was coated with a co-extrusion of poly(ethylene) and ethylene-vinyl alcohol copolymer. This surface was the barrier to liquid escape on the inside of the carton. The liquids used in this study were: • • • • • • • • •

Mr. Muscle window cleaner Werner & Mertz Frosch Spulmittel Kanzentrat cone. dishwash McBride liquid detergent code:64140 Werner & Mertz Frosch Essigreiniger bath/kitchen cleaner McBride Q-matic liquid detergent code 64239 Vernel fabric conditioner Reckitt & Colman Naison Verte fabric conditioner x 4 German softlan fabric conditioner Comfort fabric conditioner

The carton system and the liquids to be contained therein were characterised using various techniques available to the Colour Chemistry Department at the University of Leeds. The characterisation techniques used were:

•

Scanning Electron Microscopy (S.E.M.) - The Scanning Electron Microscopy used in the Department of Colour Chemistry is a JOEL 820 with x-ray mapping capabilities.

•

Differential Scanning Calorimetry (D.S.C.) - The instrument used was a DuPont 2000 unit. The Program used was:

•

Room temperature to 300°C at a rate of 1DoC per minute. A nitrogen gas purge of 3 D.2dm per minute was used.

•

Wetting Studies - Zisman and Kaelble Manipulations The 'critical surface tension' and the 'solid wetting tension' of the barrier coating surface was measured using the Zisman and Kaelble approaches. With the Kaelble method the contribution of polar and dispersive forces to the surface activity is measured. With the Zisman method the overall surface activity is measured. - Surface Tension Measurement Using a du Nouy tensiometer with a platinum ring the surface tension of the various liquids was measured.

•

Particle Size Analysis - The particle size of the test liquids was measured using a Coulter" multisizer. The experimental procedure used was: • • • •

1g of test liquid in 150g of electrolyte Electrolyte: lsoton 2, azide free, balanced solution Run time of 100 seconds Number difference and number cumulative methods for manipulation of data

Paperboard packages

•

237

Wicking Studies - In this analysis the paperboard was used as a chromatography plate. Samples of the coated paperboard were placed into the various test liquids and the rate of liquid uptake measured.

These techniques were employed to try and gain information on why certain liquids could not be contained in this type of carton system.

Results and Discussion The main technique used to examine the carton system was scanning electron microscopy. This high powered microscopy was used to give information on the micro structure of the coatings and seal areas within the carton. Figure 1 shows an example of a micrograph produced using this technique. Figure 1 shows how the barrier coating has fractured to reveal the cellulosic board underneath. In these areas it is evident that high stresses on the coating system cause failure. \

•\

\

Figure 1

Micrograph of a seal in a carton that has failed to contain Mr Muscle window cleaner

Differential scanning calorimetry was used to examine the thermal charactenstics of the barrier coating used on the inner surface of the cartons. The resulting thermograph can be seen in Figure 3. This thermograph shows temperature bands where the various layers within the barrier coating melt. These temperature are important in the carton sealing procedure. If too Iowa


temperature is used then polymer melt will not occur and seal areas will not be covered. If too high a temperature is used blistering of the polymer will occur. The temperatures shown in thermographs of the polymer coatings can be related to the temperatures measured in carton sealing. -0.2 -0.4 -0.6

'iii

~

I

-0.8

..

: :::a:

-1

ii:

akAui nment 103°C - Melt for poly(ethylene) Layers 124°C - Melt for ethylene-vinyl alcoholl oly(ethylene) co-extruded region 163°C - Melt for ethylene-vinyl alcohol polymer layer

-1.2 -1.4

+------+ I -----+-------+------+-----+--------f

o Figure 3

50

100

150

200

250

300

Temperature (OC) Thermograph of the co-extruded poly(ethylene)/ethylene-vinyl alcohol barrier coating

Another way of characterising the surface of the barrier coating is through wetting studies. These involve manipulation of measured surface properties to gain a critical value of surface tension for the polymer surface. The two manipulative techniques used were the Zisman plot and the Kaelble plot. The results obtained using these two methods can be seen in Table 1.

Table 1 Summary of Results showing the Critical surface tension, Ye, and the solid wetting tension,

rs, for the barrier coating.

Board

Zisman Critical Surface -1

Kaelble Solid Wetting -1

Tension (mNm ')

Tension (mNm ')

Unheated barrier coating

27.12

28.91

Flamed sealed barrier coating

28.85

32.35

The results in Table 1 clearly show that the barrier coating is 'activated' in the seal areas. That is, the wetting tension is higher so allowing a wider range of liquids to spontaneously wet the surface. This can be seen when we look at the surface tensions of the liquids to be contained, Table 2. Looking at Table 2, the liquids that cannot be contained all have surface tensions lower than Ye and 1s in the flame sealed areas of the barrier coating. Therefore, in these areas, the liquids will spread across the seals. Any defects in the sealed areas will then be open to failure. Another physio-chemical property to be measured was particle size. Table 3 shows the mear particle sizes of several test liquids used in this study.

Paperboard packages 239

Table 2 Surface tensions for a selection of the test liquids Liquid

Mr. Muscle window cleaner Werner & Mertz Frosch Spulmittel Kanzentrat conc. dishwash McBride liquid detergent code:64140 Werner & Mertz Frosch Essigreiniger bath/kitchen cleaner McBride Q-matic liquid detergent code 64239 Vernel fabric conditioner Reckitt & Colman Naison Verte fabric conditioner x 4 German softlan fabric conditioner Comfort fabric conditioner Fail Pass

Surface Tension (mNm- 1) 27.03 30.00 30.04 30.50 34.74 34.83 36.28 39.37 44.19

=Liquid can not be contained =Liquid can be contained

Table 3 Particle Size Results Test Liquids

Mean Particle Size

blm)

Mr.Muscle window cleaner

1.44

Werner & Mertz Frosch Spulmittel Kanzentrat conc. dishwash

1.05

McBride liquid detergent code:64140

1.70

Werner & Mertz Frosch Essigreiniger bath/kitchen cleaner

2.75

McBride Q-matic li_guid detergent code 64239

3.67

Vernel fabric conditioner

9.02

Reckitt & Colman Naison Verte fabric conditioner x 4

4.59

German softlan fabric conditioner

13.37

Comfort fabric conditioner

7.99

Fail Pass

=Liquid can not be contained =Liquid can be contained

From Table 3 we can see that the liquids with a mean particle size below 3 microns fait whereas liquids with a mean particle size above 3 microns pass. This suggests that a liquid containing a high proportion of very small particulates will be difficult to contain. The final characterisation technique used in this study was wicking analysis. This technique examines what happens after the liquid has passed through the defect in the barrier coating. It measures how quickly the liquid passes through the cellulosic board. An example of the results obtained with this technique can be seen in Figure 4. The wicking study showed that the liquids that were difficult to contain were also the liquids that wicked through the cellulosic paperboard quickly.


0.5

I!

~ c

........Uquid 1

0.4

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-Ir-Uquid3 ..... Uquid4 ..... Uquid5 -'-Uquid6 -+-Uquid7 -Uquid8

~ 0.3 ~

-r

O.2

t:

~

C

0

~-----+-----+-----+------+-----+---------; I

o

50

100

150

200

250

300

Time (Hours)

Figure 4 Results of a Wicking Study carried out on Enso SBS Board

Conclusions This study was carried out with two clear objectives. Firstly, to establish how carton failure occurs and with which types of liquids. Secondly, to investigate how the liquids that fail can be separated from liquids that pass by their physio-chemical nature. The results that have been obtained have shown that: • • • • • •

liquids escape the carton assembly via defects in the barrier coating the defects are caused by fracturing of the barrier coating in the seal areas and by poor polymer flow over the gusset point at the base of the carton the defects are a result of the mechanics of sealing of the carton rather than any inherent faults in the polymers used there is no trend between type of liquid packaged and carton failure the liquids that are most likely to fail have low surface tensions and/or small mean particle size distributions liquids that fail tend to wick through the paperboard quicker than liquids that pass

These observations show that to contain a wider range of liquids the mechanics of sealing the carton must be addressed. Until then it is possible to predict how 'aggressive' a liquid will be towards the carton assembly by measuring it's surface tension, mean particle size, and wicking rate. The results of these three tests can then be compared to results obtained for liquids that are known to pass or fail containment.

References [1] [2] [3] [4] [5] [6] [7] [8]

L.$tepek, Polymers as Materials for Packaging, Ellis Horwood Ltd, 1987. Barrier Polymers and Structures, Various. American Chemical Society, 1990. Reference Manual for the CaULTER® Multisizer, issue F. D.Campbell & J.R.White, Polymer Characterisation, Chapman & Hall, 1989. D.J.Shaw, Introduction to Colloid and Surface Chemistry. 3rd Edition, Butterworths, 1980. C.A.Finch, Poly(Vinyl Alcohol) - Developments. Wiley, 1992. l.H.Sperling, Introduction to Physical Polymer Science. 2nd Edition, Wiley, 1992. Physiochemical Aspects of Polymer Surfaces, Volume 1 & 2. Proceedings of the International Symposium on Physiochemical Aspects of Polymer Surfaces, August 2328, 1981, in New York City, New York. Plenum Press, 1983.

Paperboard packages

[9]

241

L.Mascia, Thermoplastics, Materials Engineering. 2nd Edition, Elsevier Applied Science, 1989.

26 Biochemical investigation of cellulosic and ligneous materials in museum collections M A Robson - Studio, 29 Park Avenue, Birmingham, BI8 5ND, UK

ABSTRACT

Cellulose is the main structural component of plant cell walls. It is recycled relatively quickly which emphasizes both its susceptibility to attack by organisms and the range of organisms able to do so and utilize the breakdown products. It occurs as the major constituent of wood (and thence by processing, to paper and chipboard), and similarly, as plant fibres used in textiles; for example flax and jute are phloem fibres, sisal and manila are leaf fibres and cotton is made up from seed fibres. Cellulose is often chemically modified in the production of derivative forms for particular uses. Such uses are textiles (rayon is regenerated cellulose), packaging films (such as cellophane), photographic film (cellulose nitrate and acetate), and as thickeners, fillers and extenders (eg. carboxymethyl cellulose) in adhesives foods and emulsion paints. Many of these forms of cellulose are susceptible to biological attack. This depends on the presence of suitable environmental conditions for colonization by organisms and also on the physical and chemical form of the cellulose which will vary depending on the type of product being made. Physical break up of cellulose fibres by grinding can increase susceptibility. When paper is manufactured the cellulose is delignified, yielding a more susceptible material. Lignin protects cellulose from microbial attack as shown by the greater resistance of sisal or jute fibres compared to cotton. (1) Enzymes released by organisms can continue to break down materials even when the cells which produced them are removed or no longer alive. 243


Numerous studies have been reported on the deterioration of works of art due to growth of fungi. Staining of wood does not significantly alter the strength of the wood but does reduce its value. Ethnographic artefacts in anthropological collections are collected because they are part of the description of an evolving race of men whose traditional skills may otherwise be lost. The organic nature of the raw materials used for their manufacture (eg. wood, fibre, leaves, bark, bamboo, rattan etc) are difficult to preserve and are particularly susceptible to deterioration by biological agents. Similarly, are collections of archival material, cellulosic textiles natural history specimens and photographic film. Museums are a special category of buildings, with respect to risk of damage caused by organisms. Good housekeeping and suitable environmental control, which should be the basis of all biodeterioration prevention are of paramount importance in the museum environment and cannot be over emphasised. An ecological approach is relevant to the biodeterioration of collections. Moisture content and nutrients are both important when considering remedial measures or predicting the susceptibility of an artefact to biodeterioration. Regular inspection of collections in storage and on display will safeguard against further biological infestations. INTRODUCTION Collection management of lignocellulosic materials in museums has in the past relied solely on the expertise of both the curator and conservator. Identification of the nature and extent of the conservation of such collections and the special problems and needs that exist, will today forge interdisciplinary alliances between the scientist, technologist, conservator and curator worldwide. Such collections consist of archaeological and historical wood, ethnographic material, natural history specimens, archival and library material and photographic documents and film. Traditional methods and techniques of preservation of cellulosic ethnographic material are time consuming. Infestation eradication is carried out where appropriate, and worm-eaten wood and moth-infested textiles are restored. Fungus-induced stains on archival and library material and natural history specimens are conserved. Periodic checks are carried out in the stores for insect attack, particularly silverfish on tapa or bark cloth, and the larvae of the female furniture beetle on objects made from wood, bamboo, cork, papier machie, cane and rushwork. Damp conditions can induce mildew and mould and will dissolve any binding adhesive in cloth and in any painted decoration.

Biochemical investigations in museum collections

245

DETERIORATION In time, all objects deteriorate because of the materials that are used in their manufacture gradually wear out or break down. Lignocellulosic collections are very susceptible to degradation and by handling. Such collections are altered almost imperceptibly by the environment in which they are kept. The main factors which affect materials are light, temperature, humidity and air pollution. The process of deterioration is inevitable and irreversible and is merely slowed down by controlling the environment. Light will fade dyes or pigments and embrittle fibres. A reduction in light level to 50 lux will help protect against such damage.

RELATIVE HUMIDITY Sensitive organic materials are also susceptible to relative humidity which relates to the amount of moisture in the air. Moisture content varies with temperature. Hot air retains more moisture than cold air. With a lowering of temperature the air dispels water as droplets which may form on cold surfaces. The term "relative humidity" is used as a measure of the moisture content of air. Relative humidity relates to the amount of moisture in a given quantity of air at a certain temperature, to the maximum amount of moisture the air can hold at that temperature. The organic materials in lignocellusosic collections contain a certain amount of water in their structure. This amount alters according to the amount of moisture in the atmosphere. If the air is very dry they will give up moisture, and if damp, they will absorb it. Cellulosic fibres and paper become brittle if the air is dry and wood will crack and warp. When the air is damp, most organic materials swell, although canvas and other textiles made from a twisted thread will shrink and become taut. If the amount of moisture in the air frequently fluctuates, the dimensions of the object will alter. This continuous change puts a strain on the structure of the material and it may start to break up. The strain is greater if there are several different materials combined together in one object as they change directions by different amounts. For example paint will flake off cellulosic fabrics such as canvas because it does not move as much as the canvas which will stretch and shrink with changes of relative humidity. Veneer will lift off wooden furniture because the veneer and the wood of the underlying structure swell and shrink different amounts in different directions. Moisture in the air also encourages mould to grow.


A steady relative humidity should be maintained between 50 and 60 per cent, preferably 55 per cent. Above 65 per cent, mould will grow; below 45 per cent organic materials become brittle and shrink.

TEMPERATURE The deterioration of a material is caused by chemical reactions taking place in it. If the room temperature is high these reactions will take place much faster. Mould and fungi flourish in warm temperatures and stagnant air. All materials expand and contract with changes of temperature but they do this to different degrees. Thus if different materials are used together in one object, the change in dimension can cause problems. The temperature of a room can affect an object more indirectly by altering the humidity of the air. The temperature should be as constant as possible throughout the day and night.

AIR POLLUTION Air pollution consists of fine dust and gases. Some gases such as sulphur dioxide, nitric oxide and nitrogen dioxide form acids with the moisture in the air which can harm materials such as textiles. Sunlight mixed with car fumes makes ozone which will destroy photographs, degrade textiles and cause some pigments to change colour. Dust not only soils the object but can be acidic. There is also internal pollution generated from materials or objects within the store or display case. For instance rubber flooring and some dyes in textiles give off sulphur compounds which will degrade cellulosic fibres; photocopiers produce ozone which is harmful to photographs; many composite woods such as block board and chipboard give off formaldehyde. (2) It is not easy to control these harmful effects of the environment as air conditioning may be cost-prohibitive. When installed, it will correct the relative humidity, pollution and the temperature but it is expensive to maintain.

DISCUSSION An ecological approach is relevant to the biodeteriation of such collections. Enzymes released by organisms can continue to break down materials even when the cells which produced them are removed or no longer alive.

Biochemical investigations in rnuseum collections

247

Degradation phenomena are being studied, new conservation methods developed and deacidification problems investigated. Chemical intervention with the use of biocidles, insecticides and furnigants for long-term eradication of fungus induced stains and beetle infested wood has highlighted potential dangers both to the operator and the artefact in the long-term. Freeze-drying techniques give promising results as an alternative form of treatment. Laser-stain removal and gamma eradication are being developed within health and safety guidelines. Information, imaging and communication technologies will over the next decade assist museums, libraries, galleries, archives and documentation centres in making their resources user-friendly and readily available to the student and scholar without having to handle the material. Suitable environmental controls, good housekeeping and regular inspections in spring or early summer when the beetle emerge will safeguard the curation of lignocellulosic material in museum collections.

CONCLUSIONS Moisture content and nutrients are both important when considering remedial measures or predicting the susceptibility of an artefact to biodeterioration. Biochemical investigation of lignocellulosic rnaterials in museum collections will provide the knowledge which enables the conservator to use minimal intervention. Such passive conservation is based not on remedial treatment of damage but on environmental inspection and control which will retard the natural life cycle of the destructive organisms and agents. The time spent on such in-house maintenance is well invested. The artefacts will therefore be displayed for a longer period in a more authentic condition.

ACKNOWLEDGEMENT The author wishes to thank the following for their discussion, encouragement and support: David Bailey, Leo Biek, Debbie Bolton, Pat Brown, Andrew Carson, Eileen Collins and Christopher Radbourne, Richard Green and Alan Mitchell, Anne Hudson, Hilde Smith and Pauline Timmins, and to Wendy Firmin, Editorial Critic, Bunny Warren and Cathie Mair, Secretariat, Kevin and Robert Brown, Directors of Advance Office Services, UK for practical assistance and professional advice.

REFERENCES 1.

,.,

Allsopp, D. (1986) Introduction to Biodelerioration, Edward Arnold, London. Plowden, A. and Halahan, F. (1987) Looking after Antiques, Pan Books Ltd, London.

Part 4: Physical and chemical processing of fibre and non fibrous products

27 Polymeric materials derived from the biomass A Gandini - Materiaux Polymeres, Ecole Francaise de Papeterie et des Industries Graphiques (INPG), BP 65, 38402 St. Martin d'Heres, France

INTRODUCTION The incessant production of organic molecules arising from the biological activities of the animal and vegetal realms, commonly called the biomass, forms the very essence of the perpetuation of our species (food) and has contributed to improve the quality of its life (energy, clothing, materials, health care, culture...). These natural compounds synthesized by living organisms cover a wide domain of molecular structures, topologies and sizes and are assembled in an extraordinary variety of supramolecular architectures. The traditional exploitation of these renewable resources are today complemented by recent efforts not only to ameliorate their yield, quality and variety but also the modes of their recovery, refining and transformation. In the specific context of materials, man has taken advantage of: (i) natural polymers e.g. in textile manufacturing, papermaking, the use of leathers and furs, the elaboration of elastomeric products, and of course all the applications of wood; (ii) oligomers and resins e.g. for paints, lacquers, skin tanning, inks, adhesives; and (iii) monomers and other small molecules e.g. as dyes and precursors to polymers and resinous products. Some of these technologies call upon little or no chemical modification of

251

252 Physical and chemical processing of fibre and non fibrous products

the original structures, as with wood, cellulosic fibers, natural rubber, tanning and indeed papermaking in which only lignins are chemically degraded. Others are more radical in that the natural products are submitted to rather drastic transformations in order to manufacture novel polymeric materials. This review deals only with the latter aspect, applied either to the bulk of the product e.g. in the polymerization of a terpene, or just to its surface, e.g. in the compatibilization of cellulosic fibers with a polypropylene matrix. Its scope is moreover limited to recent investigations which have shown that renewable resources can provide polymeric materials with special properties and promising applications. The extraordinary achievements of polymer science and technology in the last few decades are reflected in the multitude of materials available today. These are the result of a very intensive and well-financed research programme based mostly on petrochemistry. The strategic choice made in the aftermath of the second world war to privilege fossil rather than renewable sources for the elaboration of polymers has delayed the progress of the latter alternative for lack of adequate funding and therefore any product arising from the chemical exploitation of the biomass is bound to face an unfair comparison, both technical and economic, with petroleumbased counterparts. It seems thus premature to envisage the industrial production of materials for routine applications, i.e. polymers capable of competing with the standard "plastics", unless particularly simple and economic procedures are found to convert these ubiquitous raw materials produced by photosynthesis into polymers for everyday requirements. On the other hand, there is certainly much scope today for devoting sustained efforts to elaborate macromolecular structures possessing special properties, unique features and/ or potential added value, i.e, to prepare polymers by making good use of those chemical peculiarities of natural substances (or of their derivatives) which are not readily accessible from fossil-based counterparts. The examples discussed below attempt to show that these approaches are feasable and that polymer science can profit from the rational exploitation of monomers, oligomers and polymers derived from the biomass. A more extensive treatment of this topic has been published recently (1) and brought up to date in a series of forthcoming articles (2).

Polymeric materials

253

THE CHEMICAL MODIFICATION OF NATURAL POLYMERS Polysaccharides Cellulose owes its peculiar properties both to the regular and linear enchainment of anhydroglucose units and to the ease with which intermolecular hydrogen bondings can be organized along its polysaccharide chains. The disruption of the macromolecular regularity and/or of those strong interactions by reactions involving the hydroxy groups of the polymer is always accompanied by important changes in the mechanical, thermal and solubility behaviour as clearly illustrated for example by the major difference in the properties of cellulose and its esters and ethers. This type of chemical modification has been practiced for over a century and recent advances are modest in terms of the novelty associated with the ensuing materials, except for the possibility of preparing thermotropic and lyotropic liquid-crystalline polymers (3). Grafting processes induce of course much more important structural changes in polysaccharides. Much attention has been devoted to studying and later optimizing the mechanisms leading to the attachment of synthetic polymer strands onto the backbone of the natural polymer (4). The best results are obtained with free radical initiation, but two problems persist, viz. the relatively low grafting efficiencies and the formation of non-negligible amounts of homopolymer formed concomitantly with the branches. These drawbacks are sufficiently serious to have limited very drastically the number of industrial applications of grafted polysaccharides. Cellulose bearing polyacrylamide grafts shows interesting superabsorbent properties coupled with the additional advantage related to the potential biodegradability of the main chain. A recent study has shown that cellulosic derivatives can be used as basic components for the elaboration of polymer electrolytes (see Schoenenberger, Le Nest and Gandini, this book). In one specific instance cellulose ethers were grafted and crosslinked with polyether chains by condensation reactions involving mono- and di-functional macroisocyanates. The role of the cellulosic backbones in these structures is to improve their film-forming properties in order to allow the manufacture of solid state lithium batteries by continuous casting of the polymer electrolyte between the two electrodes. If on the one hand the bulk transformation of polysaccharides by chemical reaction has not provided many new materials in recent years,


the shift of attention towards the modification of the surface of cellulosic fibres in view of their utilization in composite materials has sparked numerous investigations and stimulating results. The number of conferences and ensuing papers devoted to this subject in the last few years (5-7) testify to its vitality. Several reasons justify the use of these natural fibers: (i) their excellent mechanical properties; (ii) their ready availability directly from numerous vegetal species or from papermaking (including recycled fibers) and other biomass refineries; (iii) their variety in terms of morphology, geometry and surface properties, again depending on the source and/or the separation processes used to isolate them; (iv) their renewable character and (v) their low cost in most instances. In specific applications, their low density with respect to glass fibers can also be a considerable advantage. These positive features are unfortunately accompanied by some problems related to the structure of cellulose, namely: (i) its modest temperature resistance which limits the application of these composites to below about 1S0°C and (ii) its hydrophilic character which can induce dimentional instabilities and a long-term drop in mechanical properties. Whereas the former disadvantage has no solution because it is intrinsic to the very chemistry of the polysaccharide chain, the latter can be overcome if the matrix is a very effective barrier to water diffusion. This explains the particular emphasis on research related to the use of polyolefins as the continuous phase in cellulosic composites. As with other more conventional composites, the problem of compatibilization between the cellulosic fibers and the synthetic matrix has been a major issue aimed at optimizing the mechanical properties of these novel materials. As an example, the quality of the interface cellulose/polyolefins can be improved by corona treatment (8) or specific chemical modification (9,10) of the fibers' surface or the matrix. The former process introduces polar groups following free radical activation, the second is based on reactions which can lead to chemical bonding between the two components as with certain surface treatments of glass fibers. Inverse gas chromatography has been particularly useful for the characterization of the cellulosic surfaces. Work in progress on this topic in our laboratory concentrates on two possible means of rendering the fiber/matrix interface more compatible. One approach consists in treating the fibers with alkenyl monomers bearing NCO groups in order to induce their condensation with the superficial OH functions and then suspending

Polymeric materials

255

the modified fibers in the monomer which is to form the matrix so that, during its polymerization, the double bonds fixed on their surface participate in the growth process giving a chemically-bound interface. The other procedure involves preparing copolymers between conventional monomers and comonomers bearing NCO groups and then adding the cellulosic fibers so that their superficial OH groups condense with the NCO functions of the copolymer matrix thus establishing a good interface. Up to 60% fibers can be added in these composites and the gains in mechanical properties in the systems where good adhesion has been insured between matrix and fibers are of about a factor of four in tensile modulus and a factor of two in tensile strength with respect to the values related to the pure polyolefin. Cellulose "whiskers" in the form of rodlike microfibrils obtained from animal sources are also a potential source of very good mechanical reinforcement. Chitin is the most abundant polysaccaharide from animal source found essentially in the shell of marine intervertebrates. Its main structural difference with respect to cellulose, starch and other vegetal polysaccharides is the presence of an N-acetyl side group on each unit. Its deacetylation leads to the formation of chitosan which is its primary amino derivative. The inclusion of these polymers derived from animal sources in the present context is justified by the interest of their use in a chemically modified form in materials (11). Apart from reactions with acids and isocyanates which involve both the OH and NH2 groups of these peculiar polysaccharides, the formation of Schiff bases with mono- and dialdehydes is specific to the presence of the latter functions. Chitin, chitosan and their numerous derivatives have a strong chelating character which makes them particulary interesting in applications such as ion collectors (recovery of traces of metals from solutions) and chromatographic substrates. They are also used as textile additives, dialysis membranes and in medical, surgical and pharmaceutical applications.

Wood The main reasons for the chemical modification of wood, apart from the specific treatments for its preservation against pests, are aimed at its plasticization and at the improvement of its dimensional stability and weathering, viz. the resistance to moisture, light and oxygen. The essence of these modifications (12) is the partial or total destruction of the


crystalline character of cellulose, e.g. the destruction of intermacromoleculer hydrogen bonds through the establishment of new moieties having a non-polar or a less pronounced polar character. Of course these processes concern the hydroxy groups and therefore also involve lignins and hemicelluloses. The reagents used for softening the wood structure include organic halides, anhydrides, oxiranes and isocyanates. The replacement of the OH groups gives rise to various new moieties (esters, ethers, urethanes), but more importantly to the inclusion in the macrostructure of aliphatic or aromatic groups of variable size. It follows that depending on the nature and specific structure of the reagent (e.g. aliphatic chain length attached to the reactive group) as well as the amount added with respect to the wood substrate, a whole variety of materials can be obtained ranging in mechanical and thermal properties from semicrystalline to amorphous, from rigid to elastomeric, from waterswelled to hydrophobic, from thermoset to thermoplastic. This last change implies that lignin has been partly depolymerized with the destruction of its three-dimensional topology. Two extreme examples show the range of applications of the materials obtained. The antishrink properties linked to the decrease in water affinity of wood can be greatly improved by partial acetylation which however does not destroy the main supramolecular features of the substrate (13). On the other hand, the esterification of wood with important proportions of long-chain aliphatic anhydrides gets rid of most hydrogen bonding, cellulose cristallynity and the crosslinked character of lignin. These drastic effects, coupled with the plasticizing role of the aliphatic chains produce a softened thermoplastic material which can be cast into films, moulded into objects and expanded to give foams (14).

THE POLYMERIZATION OF NATURAL MACROMONOMERS From the point of view of polymer science, one must distinguish between two major families of oligomeric compounds encountered in the biomass: (i) the structures which bear no reactive sites (e.g, saturated triglycerides) and are therefore inert to further chain growth to give polymeric materials, but which find useful applications as additives (e.g, in printing inks in replacement of mineral oils) or precursors to specific products (e.g. soaps); and (ii) structures which possess one of several moieties which can

Polymeric materials

257

be activated in chain or step polymerization reactions and which can therefore be used as macromonomers alone or in conjunction with more conventional monomers. The most relevant among the latter substances are drying oils, plant resins, hemicelluloses, tannins and lignin fragments (1,2). The applications of drying oils (e.g, linseed oil) and resins (e.g. rosin used as such or after specific chemical modifications) in paints, inks and other coating materials as well as their basic modes of polymerization are well established and represent in fact one of the best examples of the rational exploitation of renewable resources. Hemicelluloses have not found any major application as macromonomers, but must be considered important precursors to polymers via their transformation into furans as discussed in the next section. The "phenolic" macromonomers are examined briefly below. Tannins Tannins are oligomeric flavonoids present in the bark or wood of several trees. The traditional use as tanning agents remains the basic application of these natural products, but their intervention as macromonomers in the formulation of formaldehyde-based resins has been studied and successfully applied (15). The resultig resins can contain up to 65% tannins and display good adhesive properties, particularly adapted to plywood manufacturing. Lignins Lignins accompany cellulose and hemicelluloses in most manifestations of the biomass and are the second most important polymer produced by the biosynthesis. In the composite assembly of wood and plant macrostructures, the cellulosic fibers are the reiforcing element and lignins the crosslinked amorphous matrix. Traditional chemical pulping for papermaking as well as more recent biomass refinery technologies, e.g. organolv and steam explosion, provoke the degradation of lignins in order to solubilize the resulting fragments and isolate the cellulosic fibers. Depending on the vegetal species used and their mode of delignification, these fragments vary considerably in molecular mass and distribution, topology and detailed composition in terms of the relative proportion of characteristic "monomeric" units. It follows that, contrary to cellulose, the term "lignins" comprises a whole family of structures (16) which bear


however a common denominator: the presence of "phenylpropane" units, i.e. phenolic moieties with three aliphatic carbon atoms attached at their C-4 position. The other chemical feature common to all lignins is the presence of important proportions of aliphatic hydroxy groups. An attempt at representing an in situ lignin macromolecule is given in 'another paper in this book (see Montanari & Gandini): the oligomers obtained in the various splicing processes can have average DPs typically ranging from 10 to 100 and polydispersity indices going from about 2 all the way to more than 10, not to mention the varied chemical structure of the fragments in terms of functional groups. This lack of uniformity has been the major obstacle to the rational exploitation of lignins for polymeric materials. Thus, any degradation all the way down to "monomeric units" will provide a mixture of phenolic derivatives which would be difficult to use as well-defined chemicals unless economic separating procedures are devised. The use of lignin fragments as macromonomers seems a more reasonable approach, but even then only in formulations where the variety of structures and molecular masses do not affect the essential properties of the materials obtained. Three major strategies have been investigated in this context (1,2): (i) the chemical incorporation of lignins in formaldehyde-type resins in partial substitution of phenolic monomers; (ii) the grafting of lignins with alkenyl monomers; and (iii) the use of lignins in polycondensation reactions. Lignins, particularly those obtained by organosolv processes, participate in the growth of phenolic-type resins although of course steric hyndrance makes them less reactive than monomeric phenols. This incorporation can be improved by methylolation or phenolation (1,15,17). Grafting lignins by radical polymerization of alkenyl monomers is marred in some systems by the retarding role of the phenolic units, but interesting materials have been obtained with polyacrylamide strands (18). The third route to lignin-based polymers consists in exploiting the reactivity of phenolic and aliphatic OH groups borne by all lignins, albeit in different proportions. Polyesters, polyurethanes and polyethers have been obtained in this way as discussed in the paper by Montanari & Gandini in this book and the results obtained both on the reactivity of different lignins and on the properties of the materials indicate that this is the most promising way

Polymeric materials

259

of pursuing research on the valorization of this underprivileged natural product in the realm of materials science. POLYMERIZATION OF MONOMERS FROM THE BIOMASS Some simple structures produced by vegetal activities possess the chemical characteristics required for polyaddition or polycondensation. Other interesting monomers are obtained through the chemical exploitation of natural precursors.

Polyols Glycerol is a by-product of the industrial transformation of fats and oils. It is used in the manufacture of polyesters and polyurethanes. Some monosaccharides like sorbitol are also precursors to polyols for the elaboration of rigid polyurethanes. These crystalline sugars must first be transformed into a viscous liquid by oxipropylation in order to facilitate their rapid mixing with the polyisocyanate comonomers.

Terpenes Many essential oils contain unsaturated hydrocarbons which are dimeric structures of isoprene. These terpenes respond to cationic polymerization and give resinous oligomers which find applications in adhesives (tackifying agents), printing inks, paints and varnishes (1). Recent progress in cationic polymerization has opened the way to living systems, i.e. the possibility of obtaining polymers and copolymers with controlled structure and narrow DP distribution. The application of these criteria to the polymerization of terpenes should provide new materials with a wider range of properties.

Furanic monomers and polymers Saccharide structures in the form of monomeric, oligomeric and polymeric pentoses and hexoses can be converted into furanic derivatives by acid-catalyzed dehydration. In particular, virtually all agricultural wastes, e.g. corn cobs, rice hulls, .sugar-cane bagasse, contain sufficient amounts of C5 hemicelluloses to be interesting raw materials for the production of furfural. This heterocyclic aldehyde has been an industrial commodity for over fifty years, but its role as a first-generation synthon is


far from being exploited to its full potential. Most of it is converted into furfuryl alcohol and the rest provides a modest range of applications. Yet, research carried out in the last two decades (1,2,19,20) shows that many monomers can be prepared from it through simple and convenient routes and that the polymers and copolymers arising from them are an original family of materials. The mechanistic peculiarities of these polymerizations as well as the properties of the products obtained have been described in specific studies and summarized in appropriate reviews (1,19,20). Only some recent investigations will be briefly discussed here. 5-Methylfurfural, which is always obtained as a side product in the industrial synthesis of furfural, polymerizes by successive condensations between the methyl group and the aldehyde function in a strongly basic medium to give a conjugated macromolecular structure (21):

Nu

(n+l)

~~

~

O/y

n 0

-""'6

0.1

0

~

--

0.

0.

10 CG-PPG-MDI

O'----...I...--~----'-----'-------'O

o

0.2

0.4 0.6 0.8 CG/Polyol, gIg

1.0

Fig.6 The relationship between stress at break (<Jb) and Young's modulus (E) and CG/poly~1

ratio of CG-PPG-MDI system.

Viscoelastic properties of biodegradable .polyurethanes

289

variation of E values estimated by mechanical measurements carried out at 25 °C also accorded well with that of E' estimated by dynamic measurements. Both results indicate that the CG component functions as a hard segment and that PU samples in the glassy state are hardened with increasing CG content. Figure 7 shows the glass transition

140

temperature (Tg) estimated from DSC heating

120

curves of CG-PPG-MDI and CG-PEG-MDI

100

systems as a function of CG/polyol. The values of Tg increased with increasing CG/polyol ratio and agrees well with the

o

C)

I-

results of Figure 3, although the Tg values estimated from DSC curves were lower than tan () peak temperatures of a. dispersion. In general, it is known that Tg values measured by DSC corresponded to the starting temperature of tan ()peak measured by DMA.

•

80 60

",.

/

40

o / / CG-PEG-MDI o /

20

o -20

/'0

/

/

/

/

0

L . . - - _ - L - _ - - - ' - - _ - - - I_ _o o o L - _.......

o

0.2

0.4

0.6

0.8

1.0

CG/Polyol, gIg Fig.7 The relationship between Tg and CG/polyol ratio.

Conclusions From the above results it is concluded that: (1) PU films having a variety of viscoelastic properties can be derived from coffee grounds by reaction with MDI and polyols. (2) Coffee grounds act as a hard segment in PU's. (3) Glass transition temperature increased with increasing amount of CG. At the same time, the activation energy of a. dispersion increased, suggesting that the molecular chains including the hard segments are enhanced together with the soft segments. (4) Beside a. dispersion, two local mode relaxations, ~ and 'Y, were observed;

~

dispersion is attibuted to the local mode relaxation of the phenyl groups in PU and 'Y dispersion is attributed to the rotation of the methyl groups. (5) Dynamic modulus of CG-PPG-MDI system was higher than that of CG-PEG-MDI system because of the difference of functionality.

References 1. Hirose S., Yano S., Hatakeyama H. and Nakamura K., U. S. Pat., 4, 987, 213 (1991). 2. Hirose S., Hatakeyama H. and Nakamura K., Japanese Pat., 1,791,797 (1993). 3. Hirose S., Yano S. and Hatakeyama H., Japanese Pat., 1, 813, 561

(1994).

290

Physical and chemical processing of fibre and non fibrous products

4. Yoshida H., Morck R., Kringstad K. P. and Hatakeyama H., J. Appl. Polym. Sci., 40, 1819 (1990). 5. Reimann A., Morck R., Yoshida H., Hatakeyama H. and Kringstad K. P., J. Appl. Polym. Sci., 41,39 (1990). 6. Yoshida H., Morek R., Kringstad K. P. and Hatakeyama H., J. Appl. Polym. Sci., 34, 1187 (1987). 7. Hirose S., Yano S., Hatakeyama T. and Hatakeyama H., in "Lignin, Properties and Materials", ACS Symp. Ser., 397, W. G. Glasser and S. Sarkanen, Eds., p.382, American Chemical Society, Washington D.C. (1989). 8. Yano S.,

Hirose S. and

Hatakeyama

H., in

"Wood Processing

and

Utilizat.ion", J. F. Kennedy, G. O. Phillips and P. A.Williams Eds., p.269, Ellis Horwood, Chichester 9. Nakamura

K., Morek

(1989). R., Reimann A. and Hatakeyama

H., in "Wood

Processing and Utilization", J. F. Kennedy, G. O. Phillips and P.A. Williams Eds., P.175, Ellis Horwood, Chichester (1989). 10. Nakamura K., Morck R., Reimann A., Kringstad K. P. and Hatakeyama H., Polym. Adv. Technol., 2, 41 (1991). 11. Nakamura K., Hatakeyama T. and Hatakeyama H., Polym. Adv. Technol., 3, 151 (1992). 12. Nakamura

S., Todoki M., Nakamura

K. and Kanetsuna H.,

Thermo.

Chimica. Acta., 136, 163 (1988). 13. Hatakeyama H., Nakamura K. and Hatakeyama T., Pulp. Paper Mag. Canada,

6, TRIOS (1980). 14. Wada Y., "Kobunshi no Kotai Bussei", "Solid State Properties of polymers", p.69, p.260-271, p.278, p.293, p.298, p.300, Baifukan, Tokyo (1971). 15. Hirose S., Yoshida H., Hatakeyama T. and Hatakeyama H., "Viscoelasticity of Blomaterials", W.G. Glasser and H.Hatakeyama Eds., p.385, American Chern. Soc., Washington, DC (1992). 16. Kamimoto M., Hatakeyama T., Magoshi J., Mitsuhashi F. and Yokokawa H., "Shin Netsu Bunseki no Kiso to Oyo" , "Fundamentals and Applications of Thermal Analysis, New Edition", p.157, Riaraizu-sha, Tokyo (1989).

31 The fractional composition of polysaccharides in alkaline pre-treated and steam pressure treated wheat straw R Sun, J M Lawther and W B Banks* - The BioComposites Centre and *School of Agricultural & Forest Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, Wales

ABSTRACT

Wheat straw, pre-treated with 1.5% sodium hydroxide at 20°C for 6 h, was extracted with 0.250/0 ammonium oxalate for 4 h at 85°C, followed by steam pressure treatment at 120°C/2 bar for 7 h. The residue was then delignified using acidic sodium chlorite solution, followed by treatment with 24% potassium hydroxide with 2% boric acid for 2 h at 20 0 e for determination of hemicellulose and a-cellulose.

The yields were

determined gravimetrically and related to the control sample. Results showed that pretreatment with 1.50/0 sodium hydroxide significantly influenced the solubility of hemicellulose, whilst the steam pressure treatment had a pronounced effect on lignin solubility.

It was also found that xylose was the major sugar constituent in the

hemicellulose fraction and the hydrolysate obtained during NaOH pre-treatment, with

291


glucose and galactose as minor components. The content of arabinose was higher in the hydrolysate obtained from the pre-treatment than in the hemicellulose fraction, whereas xylose in hemicellulose was higher than that in the hydrolysate of the pretreatment.

The content of uronic acid in the hydrolysate obtained from the 1.5%

NaOH pre-treatment was higher than in the hemicellulose fraction. The range of molecular weights of both the hemicellulose and "pre-treatment" straw hydrolysate was also examined.

Key words: pre-treatment, wheat straw, steam pressure treatment, lignin, extraction, hemicellulose, pectic substances, sugar, uronic acids, molecular weight.

IN~rRODUCTION

Significant solubilisation of cell walls and hemicellulose has previously been ascribed to the synergistic effect of sodium hydroxide and steam pressure treatment of wheat straw (1). Rai and co-worker (2) have reported that the combined effect of alkali and steam

pressure treatment brought about dramatic changes in chemical composition as well as utilization of cellulose and hemicellulose of rice straw. The present investigation was aimed at determining the effect of sodium hydroxide and steam pressure treatments on the constituents of polysaccharides in wheat straw.

Fractional composition of polysaccharides

293

MATERIALS AND METHODS

Materials

Wheat straw was obtained from Silsoe Research Institute (Silsoe, Bedfordshire) and was ground using a Christie Laboratory mill to a 60-mesh size screen. The ground straw was then dried in a cabinet oven with air circulation for 16 h at 60°C and stored at 5°C until use. All chemicals were of analytical or reagent grade. All experiments were performed in duplicate and weights and yields are given on a dry, untreated, straw weight basis.

1.5% NaOH pre-treatment (figure 1)

The ground wheat straw was firstly pre-treated in air with 1.50/0 sodium hydroxide at 20°C for 6 h. After filtration on a nylon cloth, the residue was recovered, washed three times with water, twice with 960/0 ethanol and once with acetone, then dried in an oven for ) 6 h at 60°C and reweighed. The weight lost was defined as dry matter loss. The disappearances of hemicellulose and lignin were calculated from the control sample, which vias dipped only in distilled water for 6 h at 20°C.

The supernatant was

neutralized with dilute acetic acid and concentrated on a rotary evaporator under reduced pressure at 40°C against water to dry.

The resultant brown hydrolysate

obtained from the pre-treatment was kept in a fridge at O°C until analysis.


Extraction of pectic substances

Pectic polysaccharides were extracted from the residue of 1.5% NaOH pre-treated for 4 h at 85°(: using 0.25% ammonium oxalate (AO) according to the method described by Phatak et al (3).

The supernatants were extensively concentrated on a rotary

evaporator under diminished pressure at about 40°C to one-twentieth of their original volume, and then precipitated with 5 volumes of 96% ethanol for 24 h at 20°C. After filtration and drying in an air circulated 'oven for 16 h at 60°C, the resultant pectic substances or pectin fractions were stored in a fridge at DoC until analysis. The residue was rinsed once with water, twice with 96% ethanol and once with ether and then dried in an oven for 16 h at 60°C.

Steam pressure treatment

1.3 g of the residue from the 0.25% AO extraction was treated in a 100 ml capacity

autoclave at 120°C/2 bar for 7 h. The liquid/solid ratio was 25: 1. The reactor was placed in an oil bath which had previously been brought to a sufficiently high temperature so that the contents of the vessel could be heated to the reaction temperature within 5 min. When the desired temperature was reached timing was begun. The reactor was removed and placed into a water bath for rapid cooling at the end of the treatment period. After filtration, _the residue was washed three times with water, dried at 60°C for 16 h, reweighed and then stored at room temperature ready for extraction of polysaccharides.

The liquor from the water steam treatment was

concentrated to dryness on a rotary evaporator under reduced pressure at 40°C. The dried supernate was kept in a fridge at 5°C until analysis of polysaccharides solubilised during the steam pressure treatment process.


295

Delignification

Lignin content in wheat straw was determined according to the method described by Bagby et al (4), Collings et al (5) and Asensio et al (6).

The residue from above

procedures (1.0 g) was stirred with water (100 ml) and 100/0 acetic acid (8 ml), and delignified with sodium chlorite (NaCI02, 3.2 g) in flask fitted with a magnetic bar. The mixture was heated for I h at 75°C. More acid (4 ml) and sodium chlorite (1.6 g) were then added and the mixture heated for another hour. After 2 h the residue was filtered out on a nylon cloth and washed with water (three times), 96% ethanol (twice) and ether (once), then dried at 60°C for ]6 h and reweighed. The difference in weight was defined as sodium chlorite lignin.

Isolation of hemicellulose and cellulose

Hemicellulose was isolated from the remaining straw using an aqueous solution of 240;0 potassium hydroxide (0.9 g straw residue/I 00 ml extractant) and 2% boric acid, in air, for 2 h at 20°C. The residue was recovered, washed with water until alkali free and then with 5% acetic acid (once), water (once), ethanol (once) and acetone (once). It was then dried at 60°C for 16 h. The weight lost was taken as hemicellulose. The weight of the residue which remained after the alkaline extraction, corrected for ash content, was termed a-cellulose.

After neutalization with dilute acetic acid, the

supernatant was concentrated to a small volume under reduced pressure at a temperature not exceeding 40°C, and then precipitated with 5 volumes 96% ethanol for 24 h at 20°C. After filtration and drying in an air circulated oven for 16 h at 60°C, the resultant hemicellulose fraction was kept in a fridge at 0 °C until analysis.


Neutral sugar, uronic acid, methyl ester and acetyl content analyses

Preliminary identification of the neutral sugars in the hydrolysates of the steam pressure pre-treatment, pectic substances, hemicelJuloses and cellulose was obtained using thinlayer chromatography with ethylacetate-isopropanol-water (60:30: 10) as the solvent system. The visualization of sugars on the thin-layer chromatograms was achieved with 100/0 aqueous ammonium molybdate and heated to 100

0

e for

10 min according to the

method of Egon et al. (7).

The contents of the neutral sugars (arabinose, xylose, mannose, galactose and glucose) in the solubles of steam pressure treatment, pectic polysaccharides, hemicellulose, cellulose and the hydrolysate of pre-treatment were measured by gas chromatography after conversion to corresponding trimethylsilyl (TMS) ether derivatives. Methods are described in our other paper in this volume.

The uronic acids were assayed colorimetrically, as anhydrogalacturonic acid in pectin, or as glucuronic acid in hemicellulose and in the hydrolysates, using 3-phenylphenol color reagent according to the procedure outlined by Blumenkrantz and Asboe-Hanson (8) with a modification by Wedig and co-workers (9).

A Hewlett-Packard Diode

Array 8452A spectrophotometer was used to measure anhydrogalacturonic acid or glucuronic acid at a wavelength of 520 nm.

The value of rhamnose in pectin was determined by the quantitative colorimetric procedure of Gibbons (10) and Dische et al (11) after hydrolysis for 4 h in 2 N trifluoroacetic acid. Methyl ester content was determined according to the method described by Wood and Siddiqui (12). Acetic acid content was estimated using the

Fractional composition of polysaccharides 297

transesterification method outlined by Browing (13), with a small modification from

Whistler and Jeanes' procedure (14).

Measurement of physicochemical properties of pectin and hemicellulose

Viscosity was determined using a Brookfield Synchro-Lectric Viscometer (Model LV). A citrus pectin was used as a reference. Pectin samples (2%,w/v) were prepared in O.IM sodium phosphate buffer, pH 7.0, allowed to hydrate at 4°C for 16 h (15). Viscosity was then estimated (cps) at 25°C.

Optical rotation was measured on a polarimeter (Perkin Elmer, type 108) according to the methods described by McCready et al (16).

Pectin samples (1.00/0, w/v) were

prepared in double distilled water and centrifuged before measurement. A citrus pectin was also used as a reference.

The average molecular weights (M w) of pectin and hemicellulose were determined by gel permeation chromatography (17) on a PL aquagel-OH 50 column (300 x 7.7mm, Polymer Laboratories Ltd)., calibrated with PL pullulan polysaccharide standards (peak average molecular weights 667, 5800, 12200, 23700, 48000, 100000, 186000, and 386000, Polymer Laboratories Ltd). The pump was a Knauer HPLC pump 64., with a flow rate of 0.5 ml/min for measuring pectin and 0.1 ml/min for the hydrolysates and hemicellulose. The eluents were double distilled water for pectin and 0.02 N NaCI in 0.005 M sodium phosphate buffer, pH 7.5 for the hydrolysates and hemicellulose, respectively.

Detection was achieved by a Knauer differential refractometer.

column oven was maintained at 30°C.

The

Pectin samples were dissolved in double


distilled water at a concentration of 0.1%. The hydrolysates and hemicellulose were dissolved with 0.02N NaCI in 0.005 M sodium phosphate buffer, pH 7.5.

Gelling of AO extracted pectin samples was tested according to the procedure of Chang et al. (18). Pectin samples were prepared in distilled water at a concentration of

1.00/0 (w/v).

Wheat straw

I Dry at 60°C for 16 h. Dried wheat straw

IPre-treatment with 1.5% NaOH at 20°C for 6 h. Pre-treated wheat straw

I Addition of 0.25% ammonium oxalate at 85°C for 4 h. Depectinated wheat straw ITreatment with pressure steam at 120°C/2 bar for 7 h. Steam pressure treated straw Addition of acetic acid and sodium chlorite to pH 4.2-4.7 for delignification at 75°C for 2 h.

Lignin free sample 24% KOH and 2% boric acid at 20°C for 2 h alkaline extraction of hemicellulose.

Hemicellulose free sample The residue washed with water until alkaline free and 5% acetic acid(once ), water( once), ethanol(once), and acetone( once), then dried at 60°C for 16 h.

Cellulose Figure 1. Scheme for extraction and isolation of polysaccharides from sodium hydroxide pre-treated and steam pressure treated wheat straw


299

Nitrobenzene oxidation of lignin in wheat straw, pre-treated wheat straw, and extracted hemicellulose and cellulose

The method. for alkaline nitrobenzene oxidation of lignin remaining attached to/associated with hemicellulosic tractions and cellulose was based on the procedure published by Scalbert and Monties (19) with some modifications.

The method is

described in our other paper in this volume.

RESlJLTS AND DISCUSSION

The fractional composition of sodium hydroxide pre-treated and steam pressure treated wheat straw

Plant cell walls contain three types of structural polysaccharides, namely cellulose, hemicellulose and pectic substances. In wheat straw, cellulose and hemicellulose are the predominant components, comprising about 700/0 of dry mass. The third major cell wall component in straw is lignin, which composes of 14-170/0 (sodium chlorite lignin) of dry straw. The fractional composition of 1.50/0 sodium hydroxide pre-treated and steam pressure treated wheat straw is shown in table 1. The weight loss of dry matter in 1.5% sodium hydroxide pre-treatment (at 20°C for 6 h) process is

28.47%~

about

20% of lignin and 50% of hemicellulose are dissolved during treatment. More details of the neutral sugar composition of soluble hemicellulose in the pre-treatment are given elsewhere (20).


Table 1. The fractional composition of sodium hydroxide pre-treated and steam pressure treated wheat straw (0/0 dry weight)

The weight loss of 1.5% NaOH treatment 28.47(containing 3.56 lignin and 16.35 hemicellulose) Pectin

5.34

The weight loss of steam pressure treatment 10.81(containing 3.63 lignin and 5.07 hemicellulose) Lignin Hemicellulose

10.13 11.42

Cellulose

34.07

Ash

3.02

Total

103.26

The composition and physicochemical properties of pectin

The yield of anhydrogalacturonic acid, methoxyl, acetyl and ash contents of pectin extracted with 0.25% ammonium oxalate at 85°C for 4 h from the residue of NaOH pre-treated wheat straw are shown in table 2. A pectin value of 5.34% was obtained. The content of anhydrogalacturonic acid in extracted pectins was 24.52%.

The

methoxy content was low in the sample indicating that wheat straw pectin is a lowmethoxy pectin. The data also showed that ammonium oxalate extracted pectins have acetyl groups present in their structure. The acetyl content of pectin in the extract was about 4.64%. This indicated that pectic substances are only partially acetylated. Based


301

on study of sugar beet pectins, Rombouts and Thibault (21) concluded that most of the acetyl groups were associated with galacturonic acid residues rather than with neutral sugars.

In 1982, Chesson and Monro (22) observed that hot water soluble pectins

contained more acetyl groups as well as more neutral sugars than did oxalate soluble pectins. They also assumed that acetyl groups in pectins were primarily associated with galacturonic acid residues. The extracted pectin contained a high amount of ash, which was due to the pre-treatment with mineral hydroxide.

Table 2. The chemical composition(%) and functional properties of pectin extracted with 0.25% ammonium oxalate at 85°C for 4 h(1.5 g wheat straw / 100 ml extractant) from 1.5% NaOH pre-treated(at 20°C for 6 h) wheat straw.

Composition'! and functional propertiesa

0.250/0 ammonium oxalate extraction

Pectin yield

5.34

Anhydrogalacturonic acid

24.52

Methoxy content

4.76

AcetyI content

4.64

Avcrage molecular weight Optical rotation

laln25 °

17000 +70 3.05

Viscosity (cps) Ash (w/w)

15.68

aDate are expressed on a dry basis, and represent the mean of duplicate runs.

'Table 3 shows the neutral sugar composition of pectin extracted with 0.25% ammonium oxalate from NaOH pre-treated wheat straw.

As shown in table 3, the


extract was found to be rich in galactose, xylose and arabinose and low in rhamnose and glucose. Undoubtedly, xylose is a good indicator for hemicellulose and glucose for cellulose (23).

It appeared that an amount of hemicellulose was solubilized and

extracted during the 0.25% AD treatment process. Studies of sycamore suspension cell wall by Darvill et al. (24) also showed that the neutral and acidic pectic polysaccharides were covalently attached to the hemicellulose.

Table 3. The composition of neutral sugars (relative %) in pectin extracted with

0.25~/0

ammonium oxalate at 85°C for 4 h ( 1.5 g wheat straw / 100 ml extractant) from 1.5% NaOH pre-treated (at 20°C for 6 h) wheat straw.

Sugars

Content'vrclativc %)

Rhamnose

8.41

Arabinose

24.20

Xylose

18.17

Mannose

NDb

Galactose

37.36

Glucose

11.90

aDate are expressed on dry basis, and are the mean of duplicate analyses. bNot detectable.

The acidic polysaccharide has been identified as a pectin and contained galacturonic acid as the major sugar component with small amounts of galactose, arabinose, rhamnose, and xylose (25, 26). It is also important to point out that the galacturonic acid content of commercially available pectin extracted from citrus, apple peel, and


303

sugar beet pulp is remarkably different from the galacturonic acid content of wheat straw pectin isolated by the procedure described here, mainly in the much lower galacturonic acid content of wheat straw pectin.

The average molecular weight of pectin isolated by 0.25% AO peaked at 17, 000. Optical rotation of the pectin (1.0%, w/v) was about +70. The viscosity (2%, w/v) of pectin was determined at 3.05 cps. These values were much lower than those observed in citrus pectin.

The pH, molecular size, degree of methylation, and temperature

significantly affect the viscosity of wheat straw pectin.

However, this low viscosity

property of wheat straw pectin, which is similar to sugar beet pulp pectin, indicates a high potential for application in low-caloric, high fibre beverages (3).

Due to the presence of acetyl groups, low viscosity and low molecular weight, in extracted wheat straw pectin, no gel formation was observed for the extract at 1.00/0 level of addition to water (27). Citrus pectin at 1.00/0 formed a firm gel.

The weight loss and the content of neutral sugars of water soluble material during the steam pressure treatment

The dry weight loss during the steam pressure treatment was 10.81 %; this fraction was found to contain about 21 % of lignin and 15% of hemicellulose. This indicated that the NaOH pre-treatment significantly influenced the solubility of hemicellulose, whereas steam pressure treatment had a great effect on lignin dissolution. Rai and co-worker ( I) observed that sodium hydroxide pre-treatment of wheat straw followed by steam pressure treatment increased the solubilization of cell walls and hemicellulose. content of neutral sugars in the water soluble fraction obtained

The

during the steam


pressure treatment is shown in table 4.

Xylose was the major sugar constituent.

Galactose, arabinose and glucose were present in noticeable amounts.

From the

aforementioned data shown in table 4, it is also noteworthy that some portion of hemicellulose was removed during the steam pressure treatment.

Table 4. The content (0/0) of neutral sugars of water soluble material during the steam pressure treatment (120 0 C/2 bar for 7 h)

Neutral sugars''

Content(%)

Arabinose

2.7

Xylose

6.J

Mannose

o.~

Galactose

J.5

Glucose

l.~

aDetermined by hydrolysis with 2 N trifluoroacetic acid at 121 C for 2 h in sealed 0

pressure tube then by gas chromatography after conversion to trimethylsilyl(TMS)

ether derivatives(mean of duplicate analysis).

The neutral sugar composition of hemicellulose and cellulose

The hemicellulose fraction of wheat straw is thought to be composed mainly of {3 1-4 linked D-xylopyranose units with side chains of various lengths containing L-arabinose, D-glucuronic acid or its 4-0-methyl ether, D-galactose and possibly D-glucose (28). The relative sugar composition of hemicellulose, extracted from NaOH pre-treated and steam pressure treated wheat straw, is presented in table 5. Xylose was the major


305

constituent and comprised more than 800/0 of the total sugars in hemicellulose. Minor constituents were galactose, glucose and arabinose. Mannose is present only in trace amounts in extracted hemicellulose.

Compared with the relative sugar content of 1.50/0 NaOH pre-treated straw hydrolysate, the hemicellulose containing high content of xylose could be extracted only after treatment with chlorite and subsequent alkaline extraction.

This provides

evidence that hemicelluloses in wheat straw cell wall material were either bound to or shielded by lignin, preventing their extraction prior to delignification (29).

Table 5. The sugar composition(relative 0/0) of hemicellulose and cellulose extracted from sodium hydroxide pre-treated and steam pressure treated wheat straw.

Sugars

Hemicellulose

Cellulose

63

1.2

Xylose

83.3

2.8

Mannose

Traces

Arabinose

Galactose

3.6

Glucose

6.2

Traces 96.0

Table 5 also shows the composition of the neutral sugars in wheat straw cellulose after extraction with 240/0 KOH and 20/0 H3B03 for 2 h at 20°C.

It is evident that

O!-

cellulose is contaminated with hemicelluloses which have not been removed by the previous fractionation procedures. Treatment with 72% H2S04 (2 h, 20°C) and 3% H2S04 (6 h, 100°C) hydrolysed the cellulose and produced a neutral sugar

306


composition(relative %) of arabinose 1.2%, xylose 2.8%, glucose 96.00/0 and trace amounts of galactose.

The resistance to extraction by 240/0 KOH suggests that

hemicellulose is very strongly associated with the cellulose, analogous to the observation that xylans are often associated with lignified tissues.

The content of uronic acids and the range of molecular weights in hemicellulose and the hydrolysate of NaOH pre-treatment

The content of uronic acids in the hemicellulose fraction was very low; 1.00/0, whilst a relatively high content was detected in the hydrolysate of the sodium hydroxide pre-

treatment; 4.4%.

The ranges of the molecular weight of wheat straw hemicellulose extracted using 24% KOII and 2% H3B03 at 20°C for 2 hand 1.5% NaOH pre-treated straw hydrolysate are shown in figure 2. The 1.5% NaOH pre-treated straw hydrolysate gave a molecular weight about 18 000. In contrast, a sharp and symmetric peak of hemicellulose had

apparent low molecular weights, corresponding to 9 500. This result was in agreement with Whistler and co-workers' study (30) in early 1948.

They indicated that weak

alkaline solutions generally solublized hemicellulose B, the more acidic or branched fraction, to a greater extent than hemicellulose hemicellulose.

~

a more linear and less acidic

Therefore, hemicellulose B can be more or less selectively extracted

from wheat straw with very weak alkaline solutions, such as saturated lime water or low percentage sodium hydroxide solution (e. g. 1.5% NaOH pre-treatment). These studies have also shown that the hydrolysate of the pre-treatment with 1.5% NaOH contained more hemicellulose B, which had a relatively high content of uronic acid and high molecular weight, while the hemicellulose extracted with 240/0 KOH and 20/0


307

H3B03 at 20°C for 2 h from NaOH pre-treated and steam pressure treated wheat straw contained more hemicellulose A, which had a lower content of uronic acid, and was low molecular weight. In addition, the high molecular weight of the hydrolysates can probably be ascribed to the fact that some soluble lignin-polysaccharide complexes were extracted during the 1.5% NaOH pre-treatment process.

The content of phenolic acids and aldehydes of alkaline nitrobenzene oxidation lignin in wheat straw , pre-treated wheat straw , wheat straw hemicellulose and cellulose.

The FTIR spectra of wheat straw and pre-treated wheat straw (figure 3) appeared to be rather similar. However, on closer examination of the spectrum of wheat straw, it can be seen that there is a absorbance at 1720 crrr l.corresponding to ester linkage between cinnamic acids and lignin/polysaccharides in wheat straw cell walls, whereas those of pre-treated wheat straw nearly disappear peak at 1720 em-I. Figure 3 also show the FTIR spectra of hemicellulose (c) and cellulose (d) extracted from pre-treated (6 h, 20°C, 1.5% NaOH) and steam pressure treated (120°C/2 bar, 7 h) wheat straw. The very weak absorbances around 1510 cm- 1 (in hemicellulose) and 1410 cm- 1 (in cellulose) were due to aromatic groups in associated lignin. This figure indicated that the extracted hemicellulose and cellulose still contained some residual lignin.


Table 6. The content (%) of phenolic acids and aldehydes of alkaline nitrobenzene oxidation lignin in wheat straw, pre-treated wheat straw and wheat straw hemicellulose and cellulose.

Phenolic acids

Wheat

Pre-treated

straw

straw

Gallic acid

0.21

0.11

Protocatechuic acid

NDb

NO

And aldehydes''

Hemicellulose

Cellulose

0.0042

0.001~

NO

0.0056

p-Hydroxybenzoic acid

0.08

0.047

0.0038

0.0056

p-Hydroxybenzaldehyde

0.28

0.11

0.0060

0.0027

Vanillic acid

0.14

0.051

0.026

0.010

Vanillin

2.34

1.69

0.024

0.012

Syringic acid

0.20

ND

0.01.1

o.())

p-Coumaric acid

0.07

0.010

0.0050

0.020

Syringaldehyde

2.22

1.89

0.0012

0.012

Acetovanillone

0.16

0.083

0.00 10

ND

Ferulic acid

0.25

0.17

0.0012

0.0012

Cinnamic acid

0.05

0.023

0.0033

0.OO3H

0.0030

0.OO~5

0.092

0.11

Unknown Total

6.00

4.18

I

aDetermined by HPLC after alkaline nitrobenzene oxidation at 170°C for 2.5 h in steel autoclaves. bNot detectable.


-- a

--+-b

Q) (/)

c: o c, (/)

e (5

'0 Q) a;

o

......-

o----===:lI::=-=---------..-=-:~----------..,.:;:;::::o

6.4

7.6

7.0

8.5

8.2

8.8

9.4

9.1

V(Elution, ml)

Figure 2. The GPC range of the molecular weight of 1.5% NaOH hydrolysates (a) and hemicellulose (b) extracted from NaOH pre-treated and stearn pressure treated wheat

straw. Microns 2.6

2.8

3.0

3.5

-S..O

4..5

5.0

3500

6.0

7.0

1500

8

10

15 20

1000

500

Wavenumber

Figure 3. FTIR spectra of wheat straw (a), pre-treated (1.5% NaOH, 20°C, 6 h) wheat straw (b), hemicellulose (c) and cellulose (d) extracted from 1.5% NaOH pre-treated and steam pressure treated (120° C/2bar, 7 h) wheat straw.

309

310


The phenolic composition of wheat straw, pre-treated wheat straw, hemicellulose and cellulose extracted from 1.5% NaOH pre-treated and steam pressure treated wheat straw is summarised in table 6. The total phenolic contents in wheat straw, 1.5% NaOH pre-treated wheat straw, and extracted hemicellulose and cellulose were 6.00, 4.18, 0.092, and 0.11 %~ respectively.

The major components were found to be

vanillin and syringaldehyde in both wheat straw and 1.50/0 NaOH pre-treated wheat straw.

Hemicellulosic fraction showed higher contents of gallic acid,

p-

hydroxybenzaldehyde, vanillic acid and vanillin than that in cellulose. Protocatechuic acid was not detected in hemicellulose, whereas it appeared at a level of about 0.0056% in cellulose.

A=

20

10

0

4

12

16

21

26

Extraction time (h)

Figure 2. The effect of extracting time on the yield of hemicellulose and cellulose extracted using 10% KOH and 2% H3B03 at 20°C.

Figure 3 The effect of KOH concentration on the extraction yield of wheat straw hemicellulose and cellulose at 20°C for 2 h extraction with 2% H3B03.

Wheat straw hemicellulose

325

Figure 3 shows the different yields of hemicelluloses and celluloses, depending on the potassium hydroxide concentration, at 20°C for 2 h extraction with 2% boric acid, plotting the yield versus potassium hydroxide concentration for extraction at 5, 10, 15, 20, 24 and 30%(w/v). As expected, at lower concentrations of KOH, the extracting yield increased steeply with potassium hydroxide concentration until a constant value was reached due to the depletion of remaining hemicellulose in the sample. These values were about 27.79, 32.57, 33.57, 34.21, 34.23 and 34.93 at concentrations of 5, 10, 15, 20, 24 and 30% of KOH, respectively. In contrast to hemicellulose, the yield of cellulose was dramatically decreased at the low concentration of KOH and then dropped slowly to a constant value.

The results

obtained coincided with Wise and co-workers' study (20). They demonstrated that, in general, the yield of hemicellulose obtained depended upon the alkaline strength of the extractant with alkaline solutions above 10% giving only small increases in yield.

The yields of hemicellulose and cellulose extracted using 24% potassium hydroxide with 2 % boric acid for 2 h from wheat straw holocellulose was plotted in figure 4 against the extracting temperatures of 0, 10, 20 35 and 50°C.

The yields of

hemicellulose were 31.01, 33.43, 34.23, 35.85 and 36.54% at the respective temperatures. Thus, hemicellulose has a limited solubility in cold alkaline solutions, but, presumably, warm alkaline solutions may lead to hemicellulose degradation (17) even though the extractability still seems to increase above 50°C extraction for the conditions shown. The yield of cellulose decreased from 39.05 to 33.76% as the temperature increased from 0 to 50°C.


50

40

:E

30

C>

"ij) ~

e-

0

20

1= -

~ '"C

Q)

:;:

10

0

I

0

~----_

10

20

.._-_ .. __ ._.-

_

35

Temperature (C)

Figure 4. The effect of extracting temperature on the yield of wheat straw hemicellulose and cellulose with 24% KOH and 2% H3B03 at 20°C for 2 h.

Hemicelluloses can be extracted from holocellulose by alkali but a proportion of them, particularly the glucomannans, are exceedingly resistant to extraction.

The

addition of boric acid or borates to potassium hydroxide increases the dissolving power for glucomannans, mainly due to the formation of borate complexes with hydroxyl groups in cis-position (21, 22). The effect of boric acid concentration on the extraction yield of hemicellulose (20°C for 2 h) with 24% potassium hydroxide is shown in figure 5. Significant effectscan be seen. At the lower concentrations of boric acid, the yield of hemicellulose increased with acid concentration; this was particularly enhanced with the increase in rH3B03] from 1-2%.

Above 5%, the

yield of hemicellulose was observed to decrease, dropping from 35.0% to 33.74% at 12% boric acid. This is partly ascribed to the effective neutralisation of alkali as boric acid concentration increased to significant levels.

The"optimum extraction

concentration" of boric acid is therefore either 2 % or 5 %, depending upon which of


327

the following criteria are considered most important; consumption of boric acid or total hemicellulose yield.

50

1:L-----J

45

...-s:

40

.2> G)

~

e-

35

"C

~ "C

30

Qi

~ 25

20

5

0

12

Concentration (%)

Figure 5. The effect of H3B03 concentration on the extraction yield of wheat straw hemicellulose and cellulose at 20 0 C for 2 h extraction with 24 % KOH.

Figures 6 and 7 clearly show that the "hemicellulose dissolving power" of various alkalis, at the same concentrations, on the yield of hemicellulose varies widely. The yields of hemicelluloses extracted with 15% of Ca(OH)2, NH3H20, KOH, NaOH, and LiOH at 20°C for 2 h were 5.46, 17.84,33.59,34.80 and 35.08% respectively. In a comparison of the extracting power of 1M solutions of potassium, sodium and lithium hydroxide, an approximately equal effect of all three hydroxides was found. This is in agreement with studies of the extraction of xylans from softwood (21). Depending on the strength of alkalis, sodium hydroxide and lithium hydroxide are more effective than potassium hydroxide for the removal of hemicellulose. However, the preferred hydroxide is still potassium hydroxide, mainly because the potassium acetate formed during the neutralization of the alkali

328


Ca(OH)2

NH3.H20

KOH

NaOH

LiOH

Figure 6. The effect of different alkali on the extraction yield of wheat straw hemicellulose with 15 % of each alkali and 2 % H 3B03 at 20° C for 2 h.

35

30

25

P

s:

20

C)

"i) ~

e

15

"'C

~

10

"'C

Gi

>:

5

0 Ga{OH)2

NH3.H20

KOH

NaOH

LiOH

Figure 7. The effect of different alkali on the extraction yield of wheat straw hemicellulose with 1 M each of alkali and 0.2 M "3B03 solution at 20°C for 2 h.

extract is more soluble in the alcohol used for precipitation than sodium acetate (21). The yields of hemicellulose obtained using calcium hydroxide and liquid ammonia were markedly lower than for the alkali metal hydroxide solutions.

In general,


329

increase In the concentrations of the various alkalis resulted in increased hemicellulose yields.

The studies have shown that the yield of hemicellulose depends strongly on a number of factors. These include concentration and type of alkali, concentration of boric acid, temperature and time of extraction.

Effects of the time and temperature of extraction, concentrations of KOH and "3B03, and various alkalis on the sugar compositions of hemicellulose

Hemicellulose can be found in many structures and compositions.

The most

abundant in wheat straw are the arabinoxylans, which are heteropolymers of xylose units, linked by {3 (1~) glycosidic bond and arabinose residues linked to the main chain (23).

The relative sugar composition of hemicellulose fractions after

hydrolysis are presented in figures 8-11. The sugar analysis of hydrolysates showed that xylose was an extremely predominant component sugar in wheat straw hemicellulose and that it comprised more than 80% of the total sugars in hemicellulose. On the other hand, as was apparent from the figures, arabinose, galactose and glucose were present as minor sugar constituents of hemicellulose. Mannose was also detected in trace amounts. These results accord with those of Hatfield IS (24), in which he stated that in grasses most of the xylose and arabinose would be found in hemicellulose as pectic polysaccharides are present in relatively small amounts.

As can be seen, no big differences in neutral sugar contents were observed for the extractions at different periods with 10% KOH and 2% H3B03 from wheat straw in

330


figure 8. These suggest that extraction time does not greatly affect the nature of the solubilized polysaccharides.

The yield of xylose was increased from 80.00 to

86.12% with the increase of extraction time from 4 h to 26 h.

The yields of

arabinose, glucose and galactose decreased from 13.18 to 9.23%, 5.00 to 3.33%, and 1.78 to 1.23%, respectively during the same period. These data indicated that arabinose, glucose and galactose were readily dissolved with a short period of extraction, while more xylose needed long period of extraction.

100

80

--

lit---

=

=

---..... +

;

~

12

16

o 4

-------------~

..

21

Gal Glu

----

.

Ara

Xyl

----

--

----~~

26

Extraction time (h)

Figure 8. The effect of extracting time on the sugar composition (relative %) of hemicellulose extracted with 10% KOH and 2% H3B03 at 20°C from wheat straw.

The effect on the neutral sugar composition of the hemicellulose fraction of increasing potassium hydroxide concentration from 5 to 30% is shown in figure 9. Inspection of these figures shows that, in general, the relative amount of xylose present in hemicellulose decreases as the KOH concentration is increased from 530%. Yields of arabinose, glucose and galactose increase accordingly. However, it is interesting to note that a significant dip in the xylose yields curve occurs at a


331

100

-----.---

80

-

~

_ _ _ _ _ _ _ _ _...- -.---e----

-....

60

c: 0 E

...-

UJ

0

Q.

E

40

---

0 0

Ara Gal Glu Xyl

co

C)

::J

en

.

20

~

o 5

------

.

=1

....

15

10

. ....

-~

24

20

30

Concentration (%)

Figure 9. The effect of KOH concentration on the sugar composition (relative %) of hemicellulose extracted from wheat straw at 20° C with 2 % H3B03.

100

.---

•

•

80

~ ......., c:

Ara

60

Gal

0

E

fn

0 0-

GJu

40

E

Xyl

0 0

tiC) ::J

20

(J)

._--------

-----------

0 0

10

20

35

Temperature (C)

Figure 10. The effect of extracting temperature on the sugar composition (relative %) of hemicellulose extracted with 24% KOH and 2% H3B03 for 2 h from wheat straw.

[KOHl of around 15 %, with an obvious concomitant increase in relative yields of the other sugars. A hemicellulose fraction rich in arabinose, glucose and galactose


can hence be isolated using a specific extraction regime, nominally 15% KOH, 2% boric acid at 20°C.

In figure 10, the relative sugar compositions of hemicellulose extracted with 24 % potassium hydroxide and 2 % boric acid for 2 h are plotted against extracting temperature.

It is apparent that the amount of arabinose occurring in extracts

increases significantly as the temperature increases from O°C (5.42 %) to 50°C (11.92%).

100

80

.----

=

=

=

~.

-....

Glu

-+-

Gal

---

Xyl

Ara

0 1--------

o

o

...

...

y

... ~

...

-

-

coricentratidh

(%)

8

12

Figure 11. The effect of H3B03 concentration on the sugar composition (relative %) of hemicellulose extracted with 24% KOH at 20°C from wheat straw.

In figure 11, the relative contents of sugars in the hydrolysates of hemicelluloses extracted with 24 % potassium hydroxide and various concentrations of boric acid at 20°C for 2 h, are plotted as a function of the concentration of boric acid.

As

expected, as the boric acid concentration increased, the content of xylose dropped significantly (88.87 to 79.29%). The contents of arabinose and galactose increased

Wheat straw hemicellulose 333

from 6.03 to 8.28% and 1.43 to 1.97% respectively in the boric acid free and 1% boric acid extraction media. Glucose content was generally noted to increase with boric acid concentration. The relative neutral sugar compositions of hemicellulose extracted by the various alkalis with 2% boric acid at 20°C for 2 h (table 1) was very similar. In all cases xylose was the major component, and arabinose, glucose and galactose the minor components of the hemicelluloses.

The only significant difference between the

hemicellulose fractions was that the content of xylose was relatively low, and that of arabinose high in the calcium hydroxide extracts of hemicellulose compared with other alkali extraction.

The molar ratios of xylose:arabinose:galactose:glucose in

15% Ca(OH)2 and 2% H3B03 extracted hemicellulose were 51:10:1.1:2.4, whilst in 15% Li(OH) and 2% H3B03 extracts the corresponding values were 58:5.2:1.2:2.2, respectively.

These results, e.g. large amount of xylose with

relatively low levels of other neutral sugars, pointed to the presence of mainly xylan or arabinoxylan. In 1993 Fidalgo and co-workers (25) showed that the wheat straw hemicellulose contains an arabinoxylan and that a percentage of arabinose units were linked to lignin. In addition, they also revealed that in the alkali lignin fraction the percentage of arabinose units linked to lignin (as percent of total arabinose) was higher than the percentage of linked xylose. This is in agreement with the findings of Xiao-an et al. (26). There is a certain amount of ester bonding between phenolic components of lignin and xylose, arabinose, and uronic acids in the heteroxylans of hemicellulose (27); the amount of bonding appears to increase with plant maturity (28). Lignin inhibits digestion of hemicellulose by steric hindrance as well as by direct bonding to hemicellulose (6, 29).

Overall, xylose is the most resistant of

chemical constituents of hemicellulose to alkali extraction.


Table 1. The sugar composition(relative %) of hemicellulose extracted from wheat straw holocellulose using various alkalis at 20°C for 2 h.

Gal

Glu

77.3

1.9

4.3

14.7

76.8

2.6

6.0

15% NH3 + 2% H3B03

7.4

87.9

1.3

3.3

1M NH3 + 0.2M H3B03

7.4

87.8

1.4

3.4

15% KOH + 2% "3B03

10.4

82.6

2.2

5.0

1M KOH + 0.2M H3B03

10.9

83.6

1.3

3.8

15% NaOH + 2% H3B03

7.5

86.4

2.1

4.0

1M NaOH + 0.2M H3B03

7.0

86.6

2.2

4.3

15% LiOH + 2% "3B03

7.8

87.0

2.2

3.9

1M LiOH + 0.2M "3B03

8.2

87.0

1.4

3.4

Chemical concentration

Ara

Xyl

15% Ca(OH)2 + 2% H3B03

15.6

1M Ca(OH)2 + 0.2M H3B03

Man

Ta

T

a Abbreviation for trace.

The contents of uronic acids in hemicellulose

The content of uronic acids in hemicellulose fractions extracted using different concentrations of KOH at 20°C, for 2 h with 2% H3B03, are shown in table 2. Although a small component in hemicellulose, significant differences appeared at different concentrations of KOH extractions.

An increase of KOH concentration

from 5 to 24 %, led to a 4% decrease in uronic acid content.


Table 2. The content(%) of uronic acids in wheat straw hemicellulose fractions extracted using different concentration of KOH at 20°C for 2 h with 2% H3B03.

Uronic acids (%)

Concentration of KOH (%) 5

7.2

10

5.8

15

4.2

20

3.9

24

3.1

30

3.1

The average molecular weight of hemicellulose

The GPC determined molecular weights of wheat straw hemicellulose extracted using 5, 15, and 24% KOH, at 20

0

e for 2 h with 2%

H3B03, are shown in figure

12. The mean molecular weights of hemicelluloses extracted using 5%, 15% and 24% KOH with 2% H3B03 at 20 respectively (30).

0

e

for 2 h were 27000, 20000 and 12000,

This result is in approximate agreement with Aspinall and co-

worker's study (8). They illustrated that a molecular weight determination by the isothermal distillation method gave a value of 8000-11800

± 400 (degree of

polymerisation 47-76) for the methylated wheat straw xylan.

Wegener (21)

mentioned that extraction with dilute alkali solutions (e.g. 5% KOH) removed the more soluble xylans and galactoglucomannans while most of the glucomannan can be removed only with higher alkali concentrations of 16 or 24% potassium hydroxide or 17.5 % sodium hydroxide,

which is in accordance with our

experimental results. With the increase of alkali concentration from 5 to 24%, the


average molecular weights were decreased. Table 3 shows the average molecular weights (M w) of wheat straw hemicellulose fractions extracted using different concentrations of KOH at 20°C for 2 h with 2% H3B03. Corresponding M w for 5, 10, and 15% KOH extracted straw hemicellulose were 27000,

2‫סס‬oo

and

2‫סס‬oo

respectively. According to Whistler and co-workers' study (31) in early 1948, weak alkaline solutions generally solubilize hemicellulose B, the more acidic and/or branched portion, to a greater extent than hemicellulose A, which is more linear and less acidic nature.

Therefore, hemicellulose B can be more or less selectively

extracted from plant material with very weak alkaline solutions, such as saturated lime water or a low percentage of potassium hydroxide solution (e. g. 5 %). Forty years later, Wen and co-workers (17) studied the isolation and characterization of hemicellulose from sugar beet pulp and indicated that apparent molecular weights of hemicellulose A and B had two major carbohydrate peaks. molecular weight equal or greater than

15‫סס‬oo

daltons.

The first one had a The second major

carbohydrate peak had a molecular weight of 4‫סס‬oo daltons. Furthermore, they also mentioned that the elution profiles of hemicellulose B extracted with 5% NaOH were similar to that extracted with 10% NaOH, but the peak area of peak I was larger with 5% NaOH extraction than that for 10% NaOH and there was less tailing of the peak.

Hence,

they concluded that higher concentrations of NaOH caused

fragmentation of hemicellulose B. This result is in agreement with our experimental data, which shows that the average molecular weights of hemicellulose decreased from 27000 to 12000 with increase of extraction concentration of KOH from 5 to

30%, mainly due to the fragmentation at high concentration of KOH. In 1992, in a study of the structural and solution properties of corn cob heteroxylans, Ebringerova and co-workers (32) demonstrated that the molecular weight M w of heteroxylans determined by light scattering ranged up to approximately

35‫סס‬oo.

During the same


337

year, based on the analysis of wheat arabinoxylans from a large-scale isolation, Annison and co-workers (33) concluded that the pentosans had a high degree of polymerization with apparent molecular weights of

50‫סס‬oo

Da and 758000 Da.

Table 3. The average molecular weight of wheat straw hemicellulose fractions extracted using different concentration of KOH at 20°C for 2 h with 2% "3B03.

Concentration of KOH (%)

Molecular weight

5

27000

10

20000

15

20000

20

20000

24

12000

30

12000

The neutral sugar composition of cellulose

It is evident that a-cellulose is contaminated with hemicellulose and pectic substances which have not been extracted during the previous fractionation procedures. Treatment with 72% H2S04 (2 h, 20°C) and 3% H2S04 (6 h, 100°C) hydrolysed the "cellulose," producing neutral sugar composition (relative %) of arabinose 2.8, xylose 7.3, galactose 1.1 and glucose 88.8, with trace amounts of mannose. The resistance to extraction by 24% KOH suggests that hemicellulose and pectic substances are very strongly associated with the cellulose, which is similar to xylans and are often associated with lignified tissues.


According to the model proposed by Preston (34), hemicellulose and cellulose are closely connected and the cellulose mirofibrils are coated with hemicellulose polymers. Studies of sycamore suspension cell wall by Darvill et al. (35) suggested that the neutral and acidic pectic polysaccharides were covalently attached to the hemicellulose.

In 1986, based on a study of hindrance of hemicellulose and

cellulose hydrolysis by pectic substances using pectinase and cellulase, Shalom (36) confirmed that cellulose and hemicellulose in the' cell wall are sterically masked by the pectic substances. This surrounding effect may sterically hinder hemicellulose and cellulose hydrolysis.

The content of phenolic acids and aldehydes of alkaline nitrobenzene oxidation lignin in extracted wheat straw hemicellulose

The .FTIR spectra of hemicellulosic fractions extracted with 5% KOH and 2% H3B03 (a), 10% KOH and 2% H3B03 (b), and 24% KOH and 2% H3B03 (c) at 20°C for 2 h in figure 13 appeared to be rather similar.

However, on closer

examination of the spectrum of the hemicellulosic fraction (a) extracted with 5% KOH and 2% H3B03 at 20°C for 2 h, it can be seen that there are two weak peaks at 1626 and 1562cm-1, whereas that of fraction (c) extracted with 24% KOH and 2% H3B03 at 20°C for 2 h has a strongly single flattened peak at 1562cm- 1. The absorbance around 1626 em-lis a carbonyl stretching band due to para-substituted ketone or aryl aldehydes.

The small bands at 1330, 1220, 1155 and 84Ocm- 1

indicated syringl, guaiacyl ring breathing with CO stretching, aromatic CH in-plane deformation and aromatic C-H out of plane bending vibrations in wheat straw lignin. This figure indicated that the extracted hemicellulosic fractions still contained some residual lignin.


~a

-'b "'-c 12

Q) U)

c:

o a. U)

e

2

o

"0 Q) 1»1

c

0~~~~££~~~~~=*==±-_---~~~~~4 6.4

7.6

7.0

8.4

8.1

8.7

9.0

9.3

V (Elution vol, ml)

Figure 12. The range of the molecular weight of wheat straw hemicellulose extracted using 50/0 KOH (a), 15% KOH (b) and 24% KOH (c) at 20°C for 2 h with 2% H3B03'

2.6

2.8 I

3.0

3.5

,

4..0

Microns l.5 5.0

6.0

7.0

8

10

(8 ,

(c ]

auen Wsyenumber

Figure 13. FTIR spectra of wheat straw hemicellulose extracted with (a) 5% KOH and 2% H3B03, (b) 10% KOH and 2% H3B03 and (c) 24% KOH and 2% H3B03 at 20°C for 2 h.

15

20

340


The phenolic composition of the hemicellulosic fraction (c) extracted with 24% KGH and 2% H3B03 at 20°C for 2 h is summarised in table 4. The free and bound phenolic contents in the hemicellulosic fraction were 0.15 and 0.59%, respectively. The major components in bound phenolic acids and aldehydes were found to be phydroxybenzoic acid and vanillic acid, while the free phenolic content showed higher content of gallic acid. The characterisation of wheat straw lignin and lignin polysaccharide complexes is currently the subject of detailed further study in our laboratory.

Table 4. The composition of phenolic acids and aldehydes in wheat straw hemicellulose extracted with 24% KGH and 2% H3B03 at 20°C for 2 h.

Phenolic acids and aldehydes

Free a( %)

Gallic acid

0.15

Boundb ( % )

0.029

Protocatechuic acid

0.0082

p-Hydoxybenzoic acid

0.24

p-Hydroxybenzaldehyde

0.00094

0.019

Vanillic acid

0.16

Vanillin

0.085

Syringaldehyde

0.021

Ferulic acid

0.0032

Cinnamic acid

0.026

Total

0.15

0.59

aDetermined by HPLC without alkaline nitrobenzene oxidation. bDetermined by HPLC after alkaline nitrobenzene oxidation at 170°C for 2.5 h in steel autoclaves.


341

SUMMARY

Various procedures for extracting and isolating hemicellulose and cellulose from wheat straw holocellulose have been examined.

The optimal time for extracting

hemicellulose using 10% KOH and 2%H3B03 at 20°C was found to lie between 21 and 26 h. The suitable concentration of KOH for extracting hemicellulose at 20°C for 2 h was 24% and the temperature was around 20°C if 24% KOH was used in the extraction for 2 h with 2% H3B03. The most favourable concentration of H3B03 in the extractant of 24% KOH at 20°C for 2 h was 5%. potassium,

Aqueous solutions of

sodium and lithium hydroxide are appropriate for isolation of

hemicellulose from wheat straw holocellulose, but the preferred alkali was potassium hydroxide.

The molar ratios of xylose:arabinose:galactose:glucnse in 24% KOH

and 2% H3B03 for 2 h at 20°C extracted hemicellulose were 58:5.6:0.7:2.0 and in that the content of uronic acid was 3.1 %. The average molecular weight Mw of the hemicellulose was about 12000. The total content of phenolic acids and aldehydes in hemicellulose extracted with 24% KOH and 2% H3B03 at 20°C for 2 h was 0.64%.

ACKNOWLEDGEMENTS

The authors acknowledge the financial support for the research from LINK Collaborative Programme in Crops for Industrial Use.

We gratefully thank Dr.

James Bolton, Director of The BioComposites Centre, for the award of a research studentship to Runcang Sun. We are also grateful to Gwynn Lloyd Jones, Andy Mclauchlin, Sue Griffiths and Sara Hughes for their valuable suggestions and constructi ve discussion.


REFERENCES

1. Per Aman and Erik Nordkvist, Swedish J. Agric. Res. 13,5(1983). 2. M. G. Jackson, Anim. Feed Sci. Technol. 2, 105(1977). 3. A. Chesson, J. Sci. Food Agric. 32,745(1981). 4. K. C. B. Wilkie, Adv. Carbohydr. Chern. Biochem. 36,215(1979). 5. R. W. Jones, L. H. Krull, C. W. Blessin, andG. E. Inglett, Cereal Chemistry 56(5),441(1979). 6. Cindy L. Wedig, Edwin H. Jaster, and Kenneth J. Moore, J. Agric. Food Chern. 35,214(1987). 7. N. Rukma Reddy, James K. Palmer, and Merle D. Pierson, J. Agric. Food Chem.32, 840(1984). 8. G. O. Aspinall and R. S. Mahomed, Canad, J. Chern. 34, 1731(1954). 9. AOAC Official Methods of Analysis, 14th ed Association of official analytical Chemists, Washington, DC. (1984). 10. M. O. Bagby, G. H. Nelson, E. G. Helman, and T. F. Clark, Tappi 54(11)., 1876(1971). 11. G. F. Collings, M. T. Yokoyama, and W. G. Bergen, J. Dairy Sci. 61, 1156(1978). 12. Amparo Asensio and Eliseo Seoane, J. Natural Products 50(5),811(1987). 13. M. Mansoor Baig, Charles W. Burgin, and James J. Cerda, J. Agric. Food Chern. 30, 768(1982). 14. Mohammad A. Sabir, Frank W. Sosulki, and Neil W. Hamon, J. Agr. Food Chern. 23(1); 16(1975). 15. N. Blumenkrantz and G. Asboe-Hanson, Anal. Biochem. 54,484(1973). 16. Cindy L. Wedig, Edwin H. Jaster, and Kenneth J. Moore, J. Agric. Food


343

Chern. 35,214(1987).

17. L. F. Wen, K. C. Chang, G. Brown, and D.

o.

Gallaher, J. Food Sci. 53(3),

826(1988). 18. R. C. Sun, J. M. Lawther and W. B. Banks, Extraction, Isolation and Physicochemical Characterization of Weak Acidic Pectic Polysaccharides from Wheat Straw, to be published.

19. R. C. Sun, J. M. Lawther and W. B. Banks, Fractionation and Chacterization of Polysaccharides from Wheat Straw, to be published.

20. L. E. Wise, M. Murphy, and A. A. Dj Addieco, Paper Trade J. 122(2), 35( 1946). 21. Dietrich Fengel Gerd Wegener, "Wood Chemistry, Ultrastructure, Reactions," Academic Press, Walter de Gruyter Berlin, New York, 43(1989).

22. G. Gonzalez, J. Lopez-Santin, G Caminal, and C. Sold, Biotechnology and Bioengineering, Vol. XXVIII, 288( 1986).

23. B. L. Browning, "Methods of Wood Chemistry," Vol. II. Intersci. Publ., New York, London, 566(1967).

24. R. D. Hatfield, Agron J. 81,39(1989). 25. Maria L. Fidalgo, Maria C. Terron, Angel T. Martinez, Aldo E. Gonzalez, Francisco J. Gonzalez-Vila, and Guido C. Galletti, J. Agric. Food Chern. 41, 1621 ( 1993).

26. Xiao-an, L. Zhong-Zheng and T. Die-Sheng, Cellul. Chern. Technol. 23, 559 (1989). 27. F. E. II. Bartton, W. R. Windham, and

o.

S. Himmelsbach, J. Agric. Food

Chern. 30, 1119(1982).

28. R. W. Bailey, In Chemistry and Biochemistry of Herbage, (Ed.) G. W. Butler and R. W. Bailey, Academic Press, New York, 157(1993).


29. B. D. E. Gaillard, J. Agric. Sci. 59, 639(1962). 30. Yuliko Fulushi, Osamu Otsuru, and Masaakira Maeda, Carbohydrate Research 182, 313(1988). 31. R. L.Whistler, J. Bachrach, and D. R. Bowman, Arch. Biochem. 19 25(1948). 9

32. A. Ebringerova, Z. Hromadkova, J. Alfoldi, and G.Berth, Carbohydrate Polymers 19, 99(1992). 33. G. Annison, M. Choct, and N. W. ,Carbohydrate Polymers 19, 151 (1992). 34. R. P. Preston, "The Physical Biology of Plant Cell Walls," Chapman and Hall, London(1974). 35. A. Darvill, M. McNeil, P. Albersheim, and D. P. Delmer, "The Biochemistry of Plants," (Ed.) P. K. Stumpf and E. E. Conn, 1:91. Academic Press, New '~ork,

(1980).

36. Noach Ben-Shalom, J. Food Sci. 51(3), 720(1986).

33 Tbermodestruction of cellulose and levoglucosone production G Dobele, G Rossinskaja, B Rone and V Yurkjane - Institute of Wood Chemistry, Latvia, Riga

ABSTRACT The present study is dedicated to the process of the thermal transformation of cellulose under the action of phosphoric acid. The problems of the influence of concentration of phosphoric acid and the temperature of preliminary fixation during the impregnation of cellulose upon the development of the dehydration reactions and the formation of l,6-anhydrides levoglucosan and levoglucosenon have been considered.

INTRODUCTION The modification of cellulose with phosphoric acid ensures the change of the direction of the thermal destruction process, including the catalysis of the dehydration reactions and the change of the anhydroformation mechanism. Up to now, insufficient attention has been paid to the low temperature reactions of dehydration and depolymerization proceeding as a result of changing the conditions of the thermofixation of phosphoric acid in the cellulose matrix.

METHODS OF INVESTIGATION Sulphate index of

cellulose samples (6-cellulose content 98%, crystallinity 0.65) were the object of the

345


investigation. Phosphoric acid (1-10% from cellulose) was introduced by the impregnation method. The thermal fixation of the samples was realized in an inert atmosphere in the temperature range 100-200°C (heating rate 5°C/min). Pyrolysis was realized at subsequent temperature 350°C. The cellulose samples and the solid products of thermal treatment were analyzed by thermal analysis, IRand X-ray photoelectronic spectroscopies. The dynamics of the evolving of water was determined by stepwise pyrolytic gas chromatography (SPGC). 1,6-Anhydrosugars were determined by gas-liquid chromatography.

RESULTS AND DISCUSSION The introduction of phosphoric acid to the cellulose macromolecule results in a considerable change of the thermodestruction mechanism of a polymer: the amount of the volatile products of destruction is decreasing, the temperature of the main process of thermodestruction is decreasing, and the water yield is increasing (Table 1). Table 1. Characteristics of the cellulose thermodestruction in the presence of H3P04 (% in terms of oven dry matter)

%

10

Initial

Temperature

Mass

temperature of mass loss °C

of maximal rate of mass loss °C

loss of water up to up to 5l2Jl2JoC 500°C % %

260 200 170 170 160

325 280 270 260 245

91.4 74.3 68.6 68.2 64.8

Yield

14.2 24.0 25.8 28.6 31.9

If, during cellulose pyrolysis, levoglucosan is the major component of resin, and the levoglucosan yield does not exceed 0.6 %, then the pyrolysis of cellulose in the presence of phosphoric acid results in an increase of the levoglucosenon yield (Figs. 1 and 2). According to Shafizadeh [1], the maximum levoglucosan yield (8 ... 12%) has been obtained under conditions of isothermal heating of cellulose at 350°C in the presence of 1.5% phosphoric acid. The maximum formation of levoglucosenon (34%) has been registered

Thermodestruction of cellulose and levoglucosone

under pyrolytic gas chromatography conditions (430°C) upon the introduction of 1% phosphoric acid to the cellulose [2]. Yield, %

36

Fig.l Variations in water and levo-glucosenon yield during eel 1 u los e thermodestruction ( 350°C) . ( 1) levoglucosenon, (2) - water o

~

_ _---'_ _

-...J"---

o

H3P04, %

26

Yield, %

80

.. - - .. "

'-'"

•

3

+ .

16

10

- . -

&

- . . ..

.

~ e· • • . . . • • . • •

••••••••.•.•••••••••••••••••••••

Temperature, C

Fig.2 Variations in anhydrosugars yield during thermodestruction of cellulose. (1)- levoglucosan

(pyrolysis of initial cellulose) , (2)levoglucosenon (pyrolysis of cellulose with 5% H3P04)

In the present work, the influence of thermal fixation and concentration of the phosphoric acid introduced to the cellulose upon the formation of levoglucosenon and levoglucosan during the thermal destruction process has been studied. From

the data shown in

Figs. 1 and

2 and

Table 2 it

347


follows that both the main representatives of 1,S-anhydrides - levoglucosan and levoglucosenon are formed during the pyrolysis of the phosphoric acid containing cellulose samples. In this case, their total yield depends on the amount of the acid introduced to a small extent, varying within the range 10.3 ... 11.7%. The levoglucosenon/levoglucosan ratio in the total product, upon the introduction of less then 5% phosphoric acid to the cellulose, remains approximately equal, while at an increase of acid up to 10%~ the levoglucosenon yield increases. "hermofixation, during which the initial reactions of solid interaction in the system are being developed affects both tne total yield of 1,6-anhydrosugars and quantative distribution. The slow heating (up to 100°C) of the samples containing up to 5% acid hampers the formation of levoglucosenon during subsequent pyrolysis, and the ratio on nonhydrated and hydrated anhydrosugars increases up to 9.0 (2.4% acid). However, with the increase in the amount of the acid introduced, the hampering effect is inhibited, and at the acid content 10.7%, the specified ratio is 0.8 already. Table 2. Summary yield of 1,6-anhydrosugars (350°C) (% in terms of oven dry matter of cellulose) Amount of H3P04 introduced

Yield of 1,6 - anhydrosugars, % Temperature of thermofixation

JJ

°C

%

20 2.4 4.8 10.8

100

10.3 10.2 10.5 9.3 11.7 16.1

120

140

160

180

200

7.9 13.8 15.0 15.9 17.4

13.6 23.2 16.4

16.6

22.9 22.1

17.0 13.1

An increase in the thermal fixation temperature up to 140 and 160°C for cellulose samples containing 2.4% acid is accompanied by the drop of the levoglucosan/levoglucosenon ratio down to 2.0 and ~.3) respectively. After thermal fixation up to 200°C, the levoglucosan yield is 12.5%. The influence of thermal fixation with the activation of levoglucosenon formation for samples containing 4.8% phosphoric acid is expressed the most prominently. A sharp increase in the levoglucosenon / levoglucosan ratio up to 12.0 is observed in a thermal fixation temperature range of 140 ... 180°C, which

Thermodestruction of cellulose and levoglucosone

corresponds to a levoglucosenon yield of 21.4%. Further increase in the thermofixation temperature results in a decrease in anhydroformation during pyrolysis. For the maximum formation of levoglucosenon upon the introduction of 10.8% phosphoric acid (yield 19.1%) during the process of the fast pyrolysis of cellulose) a thermofixation of 160°C is sufficient. Hence, it has been established that during a thermofixation of 100-200°C, the interaction reaction in the system cellulose-phosphoric acid with the formation and decay of complexes, presumably of the main type, proceed more perfectly as compared to the impregnation process. In this case, the cellulose depolymerization process is activated, which promotes an increase in the yields of non-hydrated and hydrated anhydrosugars during subsequent pyrolysis: low temperature thermofixation (100°C) at a 2.4% content of phosphoric acid limits the development of the dehydration reactions. The levoglucosan and levoglucosenon yields during pyrolysis make up 9.1 and 1.1) respectively; low-temperature thermofixation (180°C) at a 4.8% content of phosphoric acid in the system provides the maximum development of the dehydration and depolymerization reactions. The levoglucosan and levoglucosenon yields during pyrolysis are 1.8 and 21.4%, respectively. The analysis of the experimental data of cellulose pyrolysis has shown that the impregnation with phosphoric acid inhibits the formation of the volatile products of destruction during the increase of the water fraction in their composition (Table 1) (gas-liquid chromatography results in the accumulation of the C=C and c=o bonds in the intermediate products of destruction (IR-spectroscopy). These results indicate that, during the formation of levoglucosenon, the intra- and intermolecular dehydration reactions are decisive. From the data of X-ray photoelectron spectroscopy of the products of the thermal destruction of cellulose (initial ones with the addition of phosphoric acid) (Table 3), the temperature limits of the existence of bonds in the structure: 20-180°C for hydrogen bonds; 18--350°C for mono-) di-) and triester bonds; 260-450°C for P-O-P bonds of polyphosphate groups; 450°C and more for phosphorus-hydrogen bonds have been established.

REFERENCES 1. Shafizadeh F., Furneaux R.H., Stevenson Carbohydrate Res., 1979, 71, 169. 2. Fung D.P.C. Wood Sci., 1976, 9, 55.

T.T.

349

* **

2740 220

28lJ..0 286.LJ.

286.11 287.1J.

1.440 81J.0 300 200

284.0 285.0

287.3

286.l&.

1050 4.50 112 90

680 1200 270

E""r· •••

eV 285 286.7 288.4.

284.0 285.0

imp/sec

C1.

P0 4

H~P04

H~PO.~

134..3

133.3

-

-

-

13lJ..l

-

-

-

13l!..1

-

-

13£&..5

eV

E.... ,-.....

3.8" H3

for sa.mples conta.1nina- 10. 8% for samples containina- 3.8%

450

350

260* 280**

20

ere

Temp. P'2l:'

20 50

-

-

80

-

-

-

20

-

20

imp/"sec

284.0 285.1 286.4287.4

284.0 285.2 286.5

284.0 285.0 286.2 287.2

eV 285.0 286.6 288.2

E.".r' • • •

C1

ll34 3L!O

2600 832

1120 700 180

800 360 160

1060

-

610 1120

1mp//sec

10. 8%

•••

-

-

133.6 135.5

-

134.1 135.6

-

-

134..2 135.4

-

eV 134.4.

E.... r

H:'3P0 4

Table 3. Characteristic of the RF-spectra of the cellulose sample contain1n~ phosphoric acid. p~ior to and after thermal treatment.

-

63 25

-

-

95 30

-

30

1mp/"sec

-

-

30

12/J.

P2"....

w

Vl

VI

~

0-

o

~ "'1

~

=

=t>

a

t:S

o

t:S 0t:S

~

~

sr

::::h

~

o

~.

~ ~

Q

~

[

~

s

~

t:S 0-

[

~

~

~

o

34 Star-shaped and crosslinked polyurethanes derived from lignins and oligoether isocyanates S Montanari, B Baradie, J-P Andreolety and A Gandini - Materiaux Polymeres, Ecole Francaise de Papeterie et des Industries Graphiques (INPG), BP65, 38402 8t Martin d'Heres, France

INTRODUCTION Lignins have stimulated considerable interest in recent years as potential additives and/or reagents for the elaboration of novel polymeric materials (1,2). The extraordinary abundance of this natural polymer, second only to cellulose, would obviously justify a more rational exploitation of its potential outside its use as a source of energy in paper mills. However, only relatively minor applications have been found for lignins, mostly in the form of lignosulfonates (2). Lignins obtained from various vegetal sources and biomass refinery processes, like the conventional pulping or the newer organosolv and steam-explosion technologies, can differ considerably in structure and molar masses (1,2), but have a single common qualitative feature in terms of their possible use as a macromonomer, namely the presence of both aliphatic and aromatic hydroxy moieties. All other functional groups, capable in principle of providing chain growth, are either too scarce or unreliable to be of general interest in that context. The overall content of OH groups and the relative abundance of phenolic and alcohol-type structures of course vary from lignin to lignin according to the type of species, but within reasonable limits. These variations in hydroxy population bear therefore little or no consequence on their potential exploitation as sites for condensation reactions which include essentially esterification, etherification and the formation of urethanes. 351


Our laboratory has been engaged in research efforts towards a more systematic valorisation of renewable resources for the elaboration of polymeric materials (see other paper by Gandini in this book) and has already investigated the synthesis and characterisation of polyesters derived from lignins (3,4) using acid dichlorides as co-reagents. The present communication describes a study of polyurethanes prepared with oligomeric mono- and di-isocyanates. Lignin-based crosslinked polyurethanes have already been prepared and characterized (1,2), but in order to ensure a good reactivity of the OH groups borne by the natural macromolecules, a chain-extension reaction with an oxirane, e.g. propylene oxide, was deemed necessary before carrying out the actual polycondensation with commercial diisocyanates. Our previous experience with polyesters had shown that this chain-extension reaction was not necessary because we found that the OH functions of unmodified lignins were in fact quite available for esterification under appropriate conditions (3,4). The novelty of the approach described here stems therefore from the application of the same principle, namely the use of lignins as macromonomers for the synthesis of polyurethanes, without any preliminary chemical modification. The other original aspect was the fact that the isocyanates chosen as complementary reagents were new mono- and di-functional macromonomers capable of introducing soft segments in the final polymeric structure. With the former, star-shaped topologies were obtained, whereas the latter produced networks. EXPERIMENTAL Most experiments were carried out with Alcell organosolv lignin (5) obtained by the ethanolic pulping of a mixture of hardwoods, kindly provided by Repap Co. This industrial product was thoroughly characterized after precipitation of its acetone solution in dilute HCI, to eliminate phenolic monomers and oligomers, thorough washing with water, and vacuum drying to constant weight. The elemental analysis gave C 67.0%, H 6.4% and 0 26.6%. The GPC and vapour-pressure osmometry, both in THF, yielded Mn=810, Mw=1930. From the 13CNMR spectrum of acetylated samples the following OH contents per C9 unit were determined: 0.22 primary aliphatic, 0.14 secondary aliphatic and 0.58 phenolic. Other analyses included FTIR spectroscopy which, apart from the typical features expected for all lignins, showed a weak absorption at 1705 crrr! denoting minor amounts of carbonyl moieties and DSC analyses which gave a Tg around 65°C. The lignin model compounds, namely ethanol (EtOH), guaiacol (G), 2,6-dimethoxyphenol (DMP) and 4-hydroxy-3-methoxy-benzyl alcohol

Star-shaped and crosslinked polyurethanes

353

(HMBA), were commercial samples (Aldrich), used as received. The model 2-methoxyethylisocyanate (MOEI), the oligopropylene oxide monoisocyanate (0 PO M, Mn=800) and the oligoethylene oxide diisocyanate (OEOD, Mn=700) were prepared from the corresponding commercial primary amines (Aldrich) by the reaction with bis(trichloromethyl)carbonate and characterized as described elsewhere (6). Apart from that of the Alcell lignin itself (L·OH), the structures of the reagents used in this work are shown below: CH 20H

08

OH

©r

OCH 3

CH 30

OCH3

OCH 3

(DMP)

(G)

OH

(HMBA)

(MOEI)

OCN~CH2TH-°teH3 CH 3

9

(OPOM)

OCN{CH2CH2-0tCH2-NCO 12 (OEOD)

Solvents were dried before use and all other reagents were purchased in their purest available form. Reactions were conducted under magnetic stirring in an inert atmosphere in THF at 20-45°C using dibutyltin dilaurate as catalyst. They were all homogeneous and could be followed by monitoring the evolution of the IR spectrum of the solution, and in particular the decrease in the intensity of the NCO peak at 2250 crrr l , The products were characterized by spectroscopic procedures, GPC, vapour-pressure osmometry and thermal analyses, with particular emphasis on 13C-NMR spectroscopy to examine their fine structural details.

RESULTS AND DISCUSSION Research on the reaction of NCO functions (electrophiles) with both aliphatic and aromatic OR groups (nucleophiles) is very well documented and has provided the basis for the controlled elaboration of polyurethanes. In particular, it is well established that aromatic


isocyanates are much more reactive than aliphatic counterparts and, between the two families of hydroxylic compounds, phenols are less reactive (less nucleophilic) than alcohols; moreover, among the latter compounds, primary functions react more readily than secondary ones which are in tum more reactive than tertiary ones mostly because of steric effects. Before studying the reaction of the oligoether isocyanates with lignin, it was decided to establish semiquantitative criteria concerning reactions of model compounds and work progressively towards the actual synthesis of the polymeric materials. The first combinations explored were those involving small molecules simulating both the various lignin OH moieties (EtOH, G, DMPand HMBA) and the NCO function attached to aliphatic oligoether chains (MOEI). Reactions occurred smoothly and went to completion albeit at different rates depending on the reactivity of the various hydroxy groups; with HMBA, the primary aliphatic OH condensed more readily than the phenolic counterparts. All these simple urethanes were fully characterized and no anomaly was detected with respect to the expected sructures. The next step consisted in increasing the size of the monoisocyanate, i.e. going from MOEI to OPOM while keeping the monomeric nature of the lignin models. All monofunctional hydroxy compounds reacted stoichiometrically with the macroisocyanate but at rates which depended on the nature and sterle situation of the OH groups, ethanol giving the fastest condensation and 2,6-dimethoxyphenol the slowest, as expected. With HMBA, the reaction of the aliphatic OH occurred normally and much more readily than that of the phenolic function. This was clearly confirmed by studying the interaction at a molar ratio of unity, i.e. with only half NCO groups available with respect to the total number of OH groups: the product obtained contained essentially the urethane moiety resulting from the condensation of the benzylic (aliphatic) hydroxy functions to the detriment of the phenolic counterparts. The characterization of the product arising from the stoichiometric reaction ([NCO]/[OH]=l) showed that only part of the phenolic OH groups had been condensed and that this interaction had become particularly slow after the condensation of the benzylic functions, so slow that the OPOM had been consumed by the notoriously sluggish self-condensation of NCO moieties. This behaviour can be rationalized in terms of a problem of steric hindrance intervening in addition to the intrinsic difference in reactivity between the two types of OH groups. In other words, on the one hand the primary aliphatic hydroxy functions reacted much more readily than the phenolic ones and thereafter the oligoether chains from the macroisocyanate wrapped around the phenolic ring thus establishing a

Star-shaped and crosslinked polyurethanes

355

steric obstacle to the reaction of the second OH group. To these considerations one must add the possible hydrogen bonding between the donor ether moieties from OPOM and the acidic phenolic OH functions. Having established some basic criteria concerning the reactivity of the typical lignin hydroxy groups towards monofunctional aliphatic isocyanates including linear oligoether structures, we moved to the preparation of star-shaped macromolecular polyurethanes based on OPOM and Alcell lignin. The fact of switching from monomeric models to the unmodified natural macromonomer posed again the basic question of whether steric problems might arise, i.e. of the actual availability of the OH groups towards the condensation reaction with the NCO functions borne by OPOM. The systems investigated differed by a single parameter, namely the initial [NCO]/[OH] ratio which varied from 0.15 to 1 in six steps. The infrared spectra taken as a function of time, showed without any ambiguity that the expected condensations were occurring as indicated by: (i) the progressive decrease, down to the actual disappearance, of the band at 2250 crrr l in the spectrum of the reaction medium indicating the consumption of the isocyanate functions; (ii) the appearance of a carbonyl band at 1740 crrr l (urethane functions) which increased as the reaction advanced; (iii) the progressive shifting of the band around 3430 cm- 1 to lower frequencies, i.e. about 3330 crrr-I because of the consumption of the OH groups and the formation of the corresponding NH functions; (iv) the substantial increase in the relative intensity of the large band around 1100 cm- 1 in the spectrum of the isolated product, caused by the C-O-C moieties from the oligoether chains being progressively appended to the lignin cores; and finally (v) the corresponding increase in the bands between 2800 and 2980 cm- 1 caused by the aliphatic C-H bonds from the oligopropylene oxide chains. A more detailed inspection of the structure of these products was achieved by analysing their quantitative 13C-NMR spectra against those of the starting reagents and of the model urethanes prepared in the first and second phase of this investigation. This closer look confirmed, on the one hand, the validity of the general conclusion reached from the IR study and provided, on the other hand, a way of assessing the relative reactivity of aliphatic and phenolic OH groups. The results obtained in the interaction of HMBA and OPOM were confirmed here in that the condensation occurred mostly on the former moieties as revealed by: (i) the systematic presence of a peak at 155.7 ppm, characteristic of the carbonyl carbon borne by an aliphatic urethane, whose intensity grew as the [NCO]j[OH] ratio was increased; (ii) the very modest, if any, contribution of the peak at 153.6 ppm, arising from the same carbon but in an aromatic urethane, which did not grow with increasing relative amounts of OPOM; (iii) the presence of a peak at 148 ppm, attributed to


the C3 (guaiacyl) and C3+C5 (syringyl) carbon atoms at ~-O-4 with respect to free phenolic functions, which showed little change as a function of the initial synthetic conditions; and (iv) the presence of a peak at 157.2 ppm, attributed to the carbon atoms of carbonyl functions in self-condensation products of OPOM, which grew as the [NCO]/[OH] ratio was increased. There seems to be little doubt that in the present context only the aliphatic OH groups display an adequate reactivity towards the oligoether monoisocyanate, for the reasons already invoked in the discussion of the results of the condensation between H MBA and the same macroisocyanate. This conclusion was corroborated by carrying out a reaction in stoichiometric conditions between the model MOEI and the Aleell lignin which gave a product characterized by the presence of both aliphatic and aromatic urethanes (resonances at 156.2 and 154.0 ppm, respectively) and the absence of detectable amounts of free phenolic moieties (no peak at 148 ppm). It remains to be seen whether the loss of reactivity of the phenolic OH groups is merely due to the sheer size effect of the isocyanate macromonomer or to concomitant structural features associated with its chain capable of blocking the reactivity by complexation or hydrogen bonding. Work is in progress to unravel this problem. The star-shaped polyurethanes obtained from the reactions of OPOM and Alcell Iignin were submitted to OPC and vapour pressure osmometry analyses which showed that their molar masses as well as the degree of polydispersity increased with an increasing initial proportion of macroisocyanate: thus Mn and M w went progressively from 810 and 1930, respectively for the unreacted lignin to about 1300 and 7000, respectively for the polyurethanes prepared under stoichiometric conditions. These results, analysed in terms of Ip going from 2.4 to more than 5, appear reasonable if one considers that the number of oligopropylene oxide chains which are attached to lignin is the higher the higher the number of available aliphatic OH groups per lignin molecule. The DSC thermograms of these products displayed glass transition temperatures which decreased as the number of oligoether branches per lignin core was increased as shown in Table 1. It is important to underline that lignins are soluble in polyethers like PEO and PPO so that the present star-shaped structures must be viewed as homogeneous systems down to the intramolecular level. Moreover, the two constituents of these materials have very low molar masses. It follows that one would indeed expect the variation of T g with composition to obey an additivity relationship, as is indeed the case, although the initial drop in Tg with 10% OPOM seems rather brutal. This feature could be attributed to both the introduction of the oligoether branches and the

Star-shaped and crosslinked polyurethanes 357

corresponding disappearance of the OH groups of the lignin, a possibility which is being checked by using model isocyanates. Another interesting change in the properties brought about by the progressive introduction of oligopropylene oxide chains as branches on the lignin core is the evolution of the solubility of the ensuing materials. Table 1 gives the extent of extract in diethyl ether as a function of branching, showing that one can readily solubilize lignins in such poor solvents by this type of chemical modification. Similar changes were found with respect to the hydrophilic character of the star-shaped polyurethanes and this feature bears important implications in terms of possible new uses of lignin-based materials, e.g. in water borne printing inks. [NCO]/[OH] Tg (OC) ~ Et20 sol [NCO]/[OH] Tg (OC) o (lignin) 0.4 5 -58 +64 0.1 0 55 0.8 -75 0.2 -30 -83 OPONH2

% Et20 sol. 75 100 100

Table 1. Properties of star-shaped lignin-OPOM materials. The final phase of this exploratory investigation called upon the reaction of Alcell lignin with the OEOD macrodiisocyanate. These polycondensations led to crosslinked polyurethanes containing however a certain proportion of soluble products arising from the self condensation of OEOD. Here, once again the reaction seems to occur mostly on the aliphatic OH groups of lignin and therefore it is not necesary to increase the proportion of NCO functions above that level to induce the bridging

among lignin macromolecules. Indeed, with [NCO]/[OH] as low as 0.2, we obtained about 50% of gelled material and with a ratio of 0.6, more than 80% of the product had crosslinked. The T g s of these networks after extraction with CH2Cl2 were considerably higher than those of the corresponding star-shaped thermoplastic counterparts: about 40°C for the product obtained with 20% NCO (23°C before extraction), 4°C for that corresponding to 60% NCO (-23°C before extraction) and finally -12°C for the network prepared with 80% NCO (-35°C before extraction). This trend is consistent with the stiffening role of lignin cores holding both ends of the oligoethylene oxide chains. The soluble portion, corresponding to branched structures and to 0 E 0 D selfcondensation products, played the role of plasticizing agent for the crosslinked materials. TGA thermograms indicated that all the lignin-based polyurethanes prepared in this investigation were stable up to about 250°C.


CONCLUSION As in our previous study on lignin-based polyesters (3,4), we again showed in this work that one can prepare star-shaped (A) and crosslinked (B) polyurethanes based on lignin and polyethers as shown below, without any preliminary modification of the natural macromonomer. However, contrary to polyesterifications (3,4) the present systems displayed a very pronounced difference in reactivity between the aliphatic and the phenolic OH groups of lignin. Work is in progress to gain a deeper insight into this issue and to extend the scope of this general topic to other materials in order to find novel applications for lignins.

(A)

REFERENCES 1. Glasser, W.G. and Kelley, S.S. (1987) in Encyclopedia of Polymer Science and Engineering, Wiley, N.Y., vo1.8, p. 795. 2. Gandini, A. (1992) in Comprehensive Polymer Science, Aggraval, S.L. and Russo, S. (Eds), Pergamon Press, Oxford, p. 527. 3. Guo, Z.X. and Gandini, A. (1991) Europ. Polym. J. 27,1177. 4. Guo, Z.X., Gandini, A. and PIa, F. (1992) Polym. Int. 27,17. 5. Pye, E.K. and Lora, J.H. (1991) Tappi J. 74(3), 113. 6. Callens, S., Le Nest, J.F., Gandini, A. and Armand M. (1991) Polym. Bull. 25, 443.

Part 5: Applications of cellulose, cellulose derivatives, lignin and cellulose-related enzymes

35 The alkaline degradation of cellulose relating to the longterm storage of radionuclides in cement J Shimizu,* J F Kennedy,* L L Lloyd** and W Hasamudin* *Birmingham Carbohydrate and Protein Technology Group, Research Laboratory for the Chemistry of Bioactive Carbohydrate and Proteins, School of Chemistry, The University of Birmingham, Birmingham B15 2TT, UK; **Chembiotech Ltd, University of Birmingham Research Park, Birmingham B15 2SQ, UK

ABSTRACT The pathways of alkaline hydrolysis of cellulose as a function of different exposures to alkali and the influence of the hydrolysates on the europium adsorption on calcite were investigated relating to the longterm storage of radionuclides in cement. Several non-volatile organic acids derived from alkali-treated cellulose were identified as the corresponding trimethylsilyl derivatives by using GC /GC-MS. Hydrolysates of cellulose in 0.1 M and 0.05 M NaOH solutions were different from those in saturated Ca(OH)2 solution. Alkaline hydrolysates of cellulose reduced the absorption of europium on calcite. INTRODUCTION Plans by various countries taking seriously the longterm (>100 years) storage of medium to low level radionuclide waste are being focused on encapsulation of the total waste in metal drums, and incarceration of the drums in cement below ground level [1][2]. Since much of the waste and cement is likely to have a cellulose carrier (pulp, paper, tissue and cotton), concern has been expressed about the possible leaking of the radionuclides by transport in the ground water to water supply sources, because degradation products [3][4][5] of cellulose in alkaline solution such as in the cement, by virtue of being small molecular-weight ionic compounds, could 361

362 Applications

form water soluble salts with radionuclides and possibly enhance [6] this transport dramatically. Accordingly a study of which products could be expected from the anaerobic alkaline degradation of cellulose is being undertaken. This paper describes possible alkaline degradation pathways together with liquid chromatography and GC-MS of the products as a function of different exposures to alkali. EXPERIMENTAL

Alkaline hydrolysis of cellulose Cellulose powder (25 g) (20 urn, Aldrich) was added to 0.05M NaOH, O.lM NaOH and saturated Ca(OH)2 solutions (250 ml). Each sample was then stored at room temperature for periods of 21 to 201 days under anaerobic condition. After degradation, the centrifuged hydrolysate supernatant solutions were neutralised with 1M HCI (c.a. 7-10 drops) and freeze-dried. Gas liquid chromatography of alkaline hydrolysates of cellulose Trimethylsilylation (TMS) was achieved by the following method [7]: 5 mg of freeze dried hydrolysate was added to 0.5 ml pyridine (Aldrich) containing 0.3% (w Iv) lithium perchlorate (LiCI04.3H20) (Aldrich) and the solution maintained at 400C for 2 hr. Trimethylsilylation was then carried out by the addition of 200 fll hexamethyldisylazane (Aldrich). and 100 JlI trimethylsilyl chloride (Aldrich). After a further 10 minutes at 400C the sample (2 J.11 injection) was subjected to gas phase chromatography on a Carlo Erba Instrument GC 8000 series with the following column specification: Alltech Econocap SE-30, 0.54 mm 1.0., 15 m length, film thickness 1.2 microns. Nitrogen, at a flow rate governed by a pressure of 10 kPa, was used as carrier gas, and a temperature program was used for the column (600C to 2200C with the rate of 10oC/min and then maintained at 220 0C for another 10 minutes). The elution profiles were obtained using a flame ionisation detection system. D-Glucitol was used as an external standard to evaluate elution positions for each peak. The same sample was then subjected to GC/MS by employing a Shimadzu Gas Chromatograph GC-14A for gas liquid chromatography and a Kratos Analytical MS 80 for mass spectrometry. Gel permeation chromatography of alkaline hydrolysates of cellulose Alkaline hydrolysates of cellulose were analysed using BioGel P2 gel permeation chromatography (Bio-Rad Laboratories). The water jacketed column (55 x 1.3 em) of the packing was equilibrated and maintained at 60 °C, and 0.1 M NaCI solution was used as the mobile phase (11 ml/hr). The

Radionuclides in cement

363

freeze-dried solid, dissolved in distilled water (100 mg/ml), was centrifuged at 10,000 g for 10 min at 10 °C to yield a water soluble fraction an aliquot (150 JlI) of which was injected into the BioGel P2 GPC column. Column eluents were continuously monitored using a spectrophotometer (Cecil CE-472) at 360 nm to monitor coloured fractions and using an automated L-cysteinesulphuric acid assay [8] at 420 nm with a heating bath and a colorimeter to monitor sugars. Europium adsorption test Sample solutions including alkaline hydrolystes of cellulose, isosaccharinate, methyl ~-D-glucopyranoside and a neutral cellulose oligosaccharides were added to 0.1 M NaOH solutions (20 ml) in centrifuge tubes, together with Ig/l CaC03 in 0.1 M NaOH. The solutions were spiked with 1 ml of a europium-152 in 0.1 M NaOH. The tubes were shaken overnight. Next day, they were centrifuged for 30 min at 50,OOOg. The supernatant was assayed for europium-152 in a gamma-counter (NaIdetector). From the difference in activity before and after adsorption, the distribution factor Kd could be calculated. The distribution factor is defined as: Ka = Ell -152 adsorbed (cpm I g) Eu -152 in solution (cpm / ml)

(mIl g)

The Kd values were compared with the Kd values for the systems where no degradation products were present. This Kd further denoted as Kd". The ratio Kd": Kd is the factor (reduction factor RF) by which the adsorption is reduced by adding a ligand. The relationship between the reduction factor and the ligand concentration (L) is given by:

where K* is the stability constant of the ML complex and [L] is the free (uncomplexed) ligand concentration. RESULT and DISCUSSION Gas liquid chromatogram of alkaline hydrolysates of cellulose Table 1 shows the identification of the degradation products from the alkaline degradation of cellulose after storage for 21 days. Relative retention times of the TMS derivatives were determined with the derivative of D-

364 Applications

glucitol as the reference. Identification of the mass spectra data were based on the published data [3][9][10] Glucometasaccharinic, isosaccharinic, 2-deoxytetronic and lactic acids were detected in the hydrolysates of degraded cellulose (day 21) in the three different conditions (0.1 M NaOH, 0.05 M NaOH and saturated Ca(OH)2). Since the degradations were conducted at ambient temperature, the number of products formed was not as many as the cellulose treated in higher concentration of alkali and extreme temperature [3]. Table 1 Products identified from the alkaline hydrolysate of cellulose under various alkaline conditions after day 21 Product Identified Alkaline Degradation Condition RTa Saturated 0.1 M 0.05M Ca(OH)2 NaOH NaOH 3-deoxy-arabino-hexonic 0.96 X X Xb (glucometasaccharinic) 3-deoxy-2-C-hydroxymethyl-D-threopentonic (~-D-isosaccharinic ) 3-deoxy-2-C-hydroxymethylD-erythro-pentonic (a- D-isosaccharinic) 3-deoxy-D-arabino-hexono-l,4-lactone 3-deoxy-D-ribo-hexono-l,4-1actone 3-deoxy-erythro-pentonic 2-deoxy-erythro-pentonic 3-deoxytetronic 2-deoxytetronic glycolic 2-deoxyglyceric 3-deoxyglyceric (lactic)

0.95

X

X

X

0.95

X

X

X

0.78 X 0.78 X 0.84 X 0.83 0.68 X 0.69 X X 0.45 X 0.41 X 0.43 X X a: GC retention time of TMS derivatives to D-glucitol derivative b: detected

X X

X

X

Gel permeation chromatography of alkaline hydrolysates of cellulose Figures 1, 2 and 3 show chromatograms of alkaline hydrolysates of cellulose exposed to 0.1 M NaOH, 0.05 M NaOH and saturated Ca(OH)2 solution respectively. According to the cysteine-sulphuric acid assay, Figures 1 and 2 show three major peaks. Based on studies by using standard materials (glucose, neutral and acidic oligosaccharides, sodium isosaccharinate, sodium gluconate, acetate, formate), first, second and third peaks corespond to high molecular weight peak (higher than 1800), intermediate molecular weight peak or acidic molecules including organic acids smaller than


Figure 1 P2 gel permeation chromatogram of alkaline hydrolysate of cellulose exposed to O.IM NaOH for 21 days

365

IMW acidic molecules

HMW

- - cysteine-sulphuric acid assay

monosaccharide

------ absorbance at 360 nm

I'""..

~ !~'

:

o

123 Elution time (hr)

.

r

4

Figure 2 P2 gel permeation chromatogram of alkaline hydrolysate of cellulose exposed to 0.05 M NaOH for 21 days - - cysteine-sulphuric acid assay ------ absorbance at 360 nm

/"'-_

o Figure 3 P2 gel permeation chromatogram of alkaline hydrolysate of cellulose exposed to saturated Ca(OH~for 21 days

..... _--_ ..'

1 2 3 Elution time (hr)

4

- - cysteine-sulphuric acid assay ------ absorbance at 360 nm

,

o

-

.

123 Elution time (hr)

4

366 Applications

monosaccharides (e.g. acetate, formate), neutral oligosaccharides and monosaccharides, respectively. In addition, possible neutral oligosaccharides also eluted between the second peak and the third one. According to the spectrophotometer monitor at 360 nm, coloured materials eluted in the high molecular weight and the intermediate molecular weight or acidic molecules position. The chromatogram (Figure 3) of the hydrolysate of cellulose in saturated Ca(OH)2 solution is different from those in both NaOH solutions: there is a relatively small monosaccharide peak, a trace amount of coloured materials and no high molecluar weight peak. This suggests the mechanism of alkaline hydrolysis of cellulose depends in part on the type of alkali. Europium adsorption test Figure 4 shows the results of the europium adsorption tests. Alkaline hydrolysates of cellulose exposed to 0.1 M NaOH solution for 201 days, stored in form of a non-neutralised and neutralised solution and freezedried solid, have a large effect on the adsorption of europium on calcite (CaC0 3). Figure 4. Europium Absorption Test

1200 1:ceUulose hydrolysate 2:cellulose hydrolysate, neutralised 3:ceUulose hydrolysate, freeze-dried 4:isosaccharinic acid 0.001 M 5:methyl· ~-D-glucopyranoside O.OOIM 6:ceUulose oligosaccharides (lOmglml)

1000

...

S

~

800

~u

600

=

i

~

.,= Q

400

~ Q

{12

..c < 200

0 1

2

3

4

5

6

However, isosaccharinic acid (a representative degradation product of alkaline hydrolysis of cellulose), methyl ~-D-glucopyranoside and alkaline hydrolysates of cellulose exposed to saturated Ca(OH)2 (data not shown) did not reduce the adsorption of europium on calcite (CaC03). However, cellooligosaccharides prepared by hydrolysing cellulose with trifloroacetic acid also reduced the adsorption of europium on calcite (CaC03). This result


367

may suggest that neutral hydrolysates such as cellooligosaccharides rather than acidic hydorolysates such as isosaccharinic acid are involved in the adsorption inhibition of europium on calcite. CONCLUSIONS

Several non-volatile organic acids derived from alkali-treated cellulose were identified using GC /GC-MS. Hydrolysates of cellulose in both NaOH solutions were different from those in saturated Ca(OH)2. Oligosabhccharides were possibly derived from cellulose in alkaline solutions. Alkaline hydrolysates of cellulose only in NaOH solutions have reduced the absorption of europium on CaC03 and it is noteworthy that neutral oligosaccharide could also reduce the absorption of europium on CaC03. The products of alkaline hydrolysate of cellulose (possibly neutral hydrolysates) specific to exposure to NaOH solutions must therefore be resposible for this efffect. The formation of various types of organic acids as identified in this study is expected to playa significant role in transporting the radionuclides from the storage compartment to the water course. This phenomenon might cause serious pollution problem by the radioactive material. ACKNOWLEDGEMENT

Authors thank Paul Scherrer Institut, Villigen PSI, Switzerland and Nationale Genossenschaft fur die Lagerung radioaktiver Abfalle, Wettingen, Switzerland for their europium absorption tests. REFERENCES

[1] [2]

[3] [4]

[5]

[6]

Nuclear Energy Agency, Excavation Response in Geological Repositories for Radioactive Waste, OEeD, Paris (1987) 11 Roy R., Radioactive Waste Disposal, The Waste Package (vol. 1), Pergamon Press Inc., New York (1982) 52 Johansson M. H. and Samuelson 0., Endwise degradation of hydrolysis in bicarbonate.], App. Poly. Sci (1978)., 22, 615 Alen R., Niemela K.and Sjostrom E., Gas-liquid chromatographic separation of hydroxy monocarboxlic acids dicarboxylic acids on a fused-silica capillary column, J. Chrornatogr. (1984), 301, 273 G. N. Richards and H. H. Sephton, The Alkaline Degradation of Polysaccharides Part 1 Soluble Products of the Action of Sodium Hydroxide on Cellulose J. Chern. Soc. ,(1957), 4492 M. Dozol, W. Krischer, P. Pottier and R. Simon (editors),"Leaching of Low and Medium Level Waste Packages Under Disposal 'Conditions" Graham & Trotman Ltd., London (1985) 15

368 Applications

Kennedy J. F., Selective detection ~nd quantitaive determination of the pentoses Chromatographia (1970)3, 316 [8] Kennedy J. F., Stevenson D. L., and White C. A., The behaviour and Starch-Related Oligosaccharides Series on Gel permeation Chromatography as a Function of Molecular Shapes, Starch/Starke (1988), 40 Nr.10,S. 396 [9] Petersson G., Mass spectrometry of aldonic and deoxyaldonic as trimethyl deivatives, Tetrahedron (1970) 26, 3413 [10] Niemela K. and Sjostrom E., Non-Oxidative and Oxidative Alkaline degradation of Pectic Acid, Carb. Res, (1985), 144, 93-99

[7J

36 The use of cellulose and cellulose derivatives in immobilised systems for the removal of colour from textile effluents N Willmott," J T Guthrie," G Nelson] and B Burdett] - *The Department of Colour Chemistry and Dyeing, The University of Leeds, Leeds, LS2 9JT and tThe British Textile Technology Group, 856 Wilmslow Road, Didsbury, Manchester M20 2RB, UK

ABSTRACT A strategy is outlined for the removal of reactive dye colour from textile wastewaters using bacteria, capable of dye degradation, attached to environmentally-acceptable cellulose copolymeric supports. A mixed population of bacteria have been isolated from dyehouse effluent and tested on ten different reactive azo dyes. All of the tests resulted in the removal of the original colour and the production of unidentified, colourless or differently coloured, species within the time-scale provided. Immobilisation of these bacterial colonies onto characterised cellulosic supports, as a route to enhance their dye degrading activity, is described.

INTRODUCTION Increasing public expectation of the quality of water in rivers and estuaries in the U.K. has led to complaints concerning the discharge of coloured effluent to our watercourses. Faced with new legislation, to be introduced on the 1st of January 1996 [1], and a lack of proven and economically feasible technologies for treating coloured effluents, some sectors of the dyeing and fmishing industry are under threat. Reactive dyes are very important commercially for the dyeing and printing of cellulosic fibres. They are characterised by brilliant shades, excellent wet-fastness and good stability to attack by light and chemicals. Thousands of tons of such organic dyes are wasted every year during manufacture and application [2].Losses of dye from reactive dyebaths can be as high as 50%, resulting in highly coloured, alkaline wastewater containing unreacted dye, hydrolysed dye and salt. At present, there is no commercially

369

370 Applications

available process for the complete removal of reactive dyes from a coloured, textile effluent. Most reactive dyestuffs are recalcitrant to the biodegradation processes currently used in sewage treatment works and their adsorption onto conventional biomass (bioelimination) is low. Approximately 90% of the reactive dyes in textile effluents passes through the receiving sewage treatment works into the adjoining watercourse [3]. For this investigation, a U.K. knitwear dyehouse supplied fresh samples of industrial textile effluent, containing mostly aqueous solutions of reactive dyes. The colour strength of the effluent that this company discharges and its use of large depths of shade often renders coagulation and flocculation treatment methods uneconomical. The aim of this work was to enhance the treatment capabilities of existing biological systems. Previous workers have found azo dye-assimilating bacteria in the sewer soils from dyehouses [4-9]. In this study, a mixed population of bacteria was isolated from textile effluent. This mixed population leads to the degradation of several reactive azo dyes under test conditions. All, or part, of the culture will be immobilised by entrapment, by encapsulation and by covalent linkage to a modified cellulosic support which should in principle stabilise, and perhaps enhance, the dye-degrading activity of the bacteria. It should also make the regeneration and recovery of the active culture from biological reactors much simpler. There are many different examples of immobilised biological species that exhibit biological activity. These include enzymes, antibodies, enzyme inhibitors, antigens and peptide hormones [10]. In recent years, whole cells have been immobilised, particularly for use as industrial catalysts. It is preferable that the immobilised cells are active but unable to proliferate. Novel cellulosic foams, cellulose wood pulp (both natural and chemically modified) and straw can be used as the biological supports for immobilisation. Ine extensions of the current study', regenerated cellulosic foams will be based on the Tencel system. These are prepared by the dispersion of a metal carbonate, or other metal salt, into a solution of cellulose in N-methylmorpholine N-oxide. On immersion in an acidic medium, the cellulosic component precipitates and carbon dioxide is released. The metal ions and the N-methylmorpholine N-oxide are extracted from the foams by washing. The nature of the process provides an even distribution of pores throughout the structure. This is important as the physical form of the biosupport must enable efficient transport of the dyestuffs and the dyestuff metabolites. Graft copolymerisation provides a general method for the modification of the chemical and physical properties of cellulose supports via the covalent linkage of side chains to the polymeric backbone [11]. The following points of interest with respect to the support are the subject of current study :

1. The 2. The 3. The 4. The 5. The 6. The 7. The

dyeability with reactive dyes. adsorption and absorption capabilities. hydrophilic/hydrophobic character. stabilisation of the microbial culture. physical and chemical form. thermal and chemical stability. biodegradability.

Removal of colour from textile effluents

371

8. The stability in continuous liquid media.

EXPERIMENTAL Materials

The reactive dyes used are shown in Table 1. Reactive Red 1 and Reactive Orange 1 were synthesised according to standard methods and purified by salting out from solution using sodium thiocyanate. The structure and purity of the two dyes was confirmed by FTIR spectroscopy and capillary electrophoresis. The bacterial culture used in this work was isolated from a textile effluent sample, using a streak plate technique and maintained on agar-soy broth slants at 3°C. The Agar no.2 and Tryptone Soy Broth were obtained from Lab M, Topley House, Wash Lane, Bury, England. All other chemicals (analar grade) were supplied by Aldrich Chemicals Co.(Gillingham,U.K.), BDH Ltd. (Poole, Dorset,U.K.), Vickers Laboratories Ltd. (Burleyin-Wharfedale, Nr.Leeds,U.K.) and Fluka Chemicals Ltd. (Gillingham, Dorset).

Colour Index Name Reactive Red 1 Reactive Orange 1 Reactive Red 120 Reactive Blue 217 Reactive Yellow 111 Reactive Yellow 125 Reactive Red 195 Reactive Yellow 145 Reactive Blue 221 Reactive Blue 222

Table 1 - Reactive dyes used in this study Dye Manufacturer Commercial Name laboratory synthesised laboratory synthesised ICI, Blakeley, U.K. Procion Red HE-3B LJ.Specialities,Wigan,U.K. Kayacelon React Dark Blue C-NR Drimarene Brilliant Yellow K-2GLK Sandoz, Leeds, U.K. Sandoz, Leeds, U.K. Drimarene Golden Yellow K-2R LJ .Specialities,Wigan,U.K. Sumifix Supra Red 3BF 150% LJ .Specialities,Wigan,U.K. Sumifix SupraYellow 3RF 150% LJ.Specialities,Wigan,U.K. Sumifix Supra Blue BRF 150 % L.J .Specialities,Wigan,U.K. Sumifix Supra Navy BF 150%

Analysis of industrial textile efnuent

The colour, turbidity, odour and pH of fresh effluent samples obtained from a knitwear dyehouse were monitored over a five week period. The conditions used in order to provide an effective analysis of the industrial textile effluent are shown in Table 2. Table 2 - Conditions used in the study of textile effluent samples Temperature / °C Volume / em" Air Supply A 20* none(sealed sample bottle) 1000 B 20 none(sealed glass jar) 180 C 20 limited(air gap in jar) 80 D 20 unlimited(open glass jar) 80 3-5# E none(sealed glass jar) 180 * sample on laboratory bench at room temperature (subject to night time variation) # sample stored in refridgerator

372 Applications

It is inevitable that the exact chemical composition of a textile effluent is undefined because of the number of different processes occurring within a dyehouse. Very few textile fmishing companies segregate liquid discharges. The varying nature of industrial textile effluent must therefore be a consideration in this work. The effluent samples used in this work contained mainly brown, black and navy blue reactive dyes in unknown concentrations. Each sample of effluent was assessed visually on a daily basis and the absorption spectrum of the filtered effluent was measured at various intervals using an SP8-400 PyeUnicham UV-visible spectrophotometer.

Bjodea=radatjon of reactive dyes A mixed population of bacteria was isolated from one of the effluent samples and stored on agar slants. The culture was tested on ten structurally different reactive azo dyes. Two of the dyes were synthesised and purified in the laboratory. The rest were unpurified commercial samples. The bacteria were grown anaerobically on agar plates containing 3 O.25g/dm3 of reactive dye and 30g/dm of Tryptone Soy Broth (a complex nutrient source). The plates were incubated at 30°C for 24 hours. Colour changes occurring in the plates during the test period may be the result of the protonation of the dye chromophore rather than the breakdown of the dye structure. This was tested by the addition of a few drops of citric acid and of hydrochloric acid separately to the top of the dyed agar. Purified Reactive Red 1 (Figure 1) was also tested under aerobic conditions on agar-soy broth plates. Both static and shaking broth cultures were also used with no air supply, with a limited air supply and with an unlimited air supply. Each of the samples was incubated at 30°C for 24 hours. The concentration of Reactive Red 1 in the plates was O.25g/dm3 and the concentration in the broth cultures was 1.4g/dm3 . Figure 1 - Reactive Red 1

Cl

OH I

~ Na OS 3

N--( N N-=(CI

NH--{

SO Na 3

RESULTS AND DISCUSSION Analysis of Industrial Textile Emuent A loss of colour was observed in all of the effluent samples during the timescale provided. The rate and extent of discoloration, greatest in test B, appeared to be dependent upon the conditions used. Limiting the supply of air to the samples resulted in a greater loss of


373

colour over a shorter time period, particularly when the effluent was stored at room temperature. The extent of discoloration decreased in the order: Test B, Test A, Test C, Test D, Test E for the range of testing conditions (Table 2) A-E. Removal of colour from the effluent was quantified by the decrease in the absorbance values of the sample across a range of wavelengths in the visible region of the spectrum. The rate of the disappearance of colour was greatest during the first seven days of the study. After this, the absorbance values (in the visible region) remained relatively constant. As the colour strength of the effluent samples fell, the rate of discoloration sharply decreased, suggesting that the production of the dye-degrading species was dependent upon the concentration of the biodegradable substrate present. Complete decolouration of the samples did not occur in the timescale provided. Discoloration of the samples was accompanied by the production of a strong sulphurous smell. There was also a reduction in the pH of the effluent (which seemed to be proportional to the extent of discoloration) and an increase in the turbidity of the solutions. The pH of the original effluent sample, which was very slightly turbid, was 8.7. Smaller volumes of the textile effluent exhibited a greater degree of discoloration than did the larger volumes. Discoloration may be due to the action of a biological species present in the textile effluent. A microorganism, or group of microorganisms, operating most effectively under anaerobic conditions, may degrade the dyes to produce differently coloured species. The growth of the microbial colony must be favoured by the warm laboratory temperatures, the exclusion of oxygen and the abundant supply of nutrients (including the reactive dyes) in the fresh effluent. An increase in the size of the microbial population through metabolism of these nutrients would diminish the concentration of nutrients and decrease the rate of growth. Hence, the rate of discoloration of the effluent would decrease with time.

Biodegradation of Reactiye Dyes The mixed population of bacteria removed the original colour from all of the agar plates incubated under anaerobic conditions. The colour loss was not due to protonation. Unidentified, colourless or differently coloured species, were produced during the timescale provided. Discoloration occurred in discrete zones underneath and around the bacterial growth. In some cases the zonal clearance of colour was quite dramatic, as shown in Plate 1. This may suggest that an extracellular biodegradation mechanism is not in operation. However, an extracellular process would be limited by the diffusion of the dyedegrading species through the bacterial cell wall and through the agar medium. Further investigation is required in this area. Under aerobic conditions, discoloration of Reactive Red 1 occurred in the zones around the bacterial growth as with the anaerobic plates. A pale yellow species was produced. The area of the clearance zone was smaller than when anaerobic conditions were used. There was a characteristic change in the hue of the broth cultures depending upon the air supply.This occurred with both the static and the shaken broth cultures. The original solutions were a deep red colour. When filtered, at the end of the incubation time,

374 Applications

the samples with no air supply were bright yellow, the samples with a limited air supply were moss green, and the samples left open to the air were orange-brown. The different colours may be explained by the reactive dye being broken down by a different degradation pathway, or by a varying extent, according to the amount of air available to the system.

Plate 1 - Plate containing 0.25g/dm 3 of Sumifix Supra Blue BRF 150%, 2% Agar no. 2 3 and 30g/dm of Tryptone Soy Broth. The plate was incubated under anaerobic conditions at 30°C for 24 hours.

CONCLUSIONS AND FURTHER WORK A mixed population of bacteria has been isolated and shown to cause the discoloration of ten different reactive azo dyes. The extent of discoloration is greatest when the bacteria are incubated under anaerobic conditions at 30°C for 24 hours. Separation and identification of the mixed bacterial population is the subject of further study. The dye-degrading activity of the immobilised system will be evaluated. Model reactive dyebath effluents of known chemical composition will be used to test the effectiveness of the immobilised treatment system. Real industrial textile effluents will then be used. Capillary electrophoresis will be used to identify the dye degradation by-products. Spectra of the effluent samples will be compared to those of authentic samples of the expected reactive dye metabolites.


375

REFERENCES [1] Skelly,K., An Industry Approach - Management of Colour in Effluent, DEMOS seminar, Brighouse, U.K., (June 1994). [2] Anliker, R., Colour Chemistry and the Environment, Rev.Prog. Col., 8, (1977), 61-62. [3] Pierce, J., Colour in Textile Effluent - the Origins of the Problem, l.S.D.C., 110, (April 1994),131-133. [4] Idaka, E. and Ogawa, T., l.S.D.C., (March 1978), 91-94. [5] Ogawa, T., Yatome, C. and Idaka, E., Biodegradation of p-Aminoazobenzene by Continuous Cultivation of Pseudomonas pseudomallei 13NA, l.S.D.C., 97, (October 1981),435-437. [6] Ogawa, T., Yatome, C., Idaka, E. and Kamiya, H., Biodegradation of Azo Acid Dyes by Continuous Cultivation of Pseudomonas Cepacia 13NA, l.S.D. C., 102, (1986), 12-14. [7] Yatome, C., Ogawa, T., Koga, D. and Idaka, Biodegradability of Azo and Triphenylmethane Dyes by Pseudomonas pseudomallei 13NA, l.S.D. C., 97, (1981), 166-169. [8] Yatome, C., Ogawa, T., Itoh, K., Sugiyama, A. and Idaka, E., Degradation of azo dyes by cell-free extract from Aeromonas hydrophila var. 24B, l.S.D. C., 103, (nov 1987), 395-398. [9] Yatome, C., Ogawa, T., Hishida, H. and Taguchi, T., Degradation of azo dyes by cellextract from Pseudomonas stutzeri, l.S.D. C., 106, (Sept 1990), 280-283. [10] Burck, S.D., Polymer, 16, (1975), 409. [11] Gil, M. H. M., Immobilisation of Proteins, Enzymes and Cells onto Graft Copolymeric Substrates, PhD Thesis(University ofLeeds), (1983). ACKNOWLEDGEMENTS This work is being funded by the Department of Trade and Industry, London, U.K., the Engineering and Physical Sciences Research Council, and the British Textile Technology Group, East Didsbury, Manchester, U.K. The project is part of the Postgraduate Training Partnership Scheme.

37 New polymer electrolytes based on modified polysaccharides C Schoenenberger, J F Le Nest and A Gandini - Materiaux Polymeres, Ecole Francaise de Papeterie et des Industries Graphiques (INPG), BP 65, 38402 St. Martin d'Heres, France

INTRODUCTION Polymer electrolytes have attracted much attention in recent years because of their potential applications in solid state batteries, sensors, electrochromic devices and supercapacitors. materials (1).

A recent review summarizes the main issues concerning these By definition an electrolyte must contain ionic species, usually in

considerable amounts, and allow their transport towards the corresponding electrodes. The replacement of traditional liquid electrolytes by polymeric counterparts requires structures which can dissolve the ions (good solvating properties) and displaying a high segmental mobility (low glass transition temperatures). Polyethers (preferably polyethylene oxide) have been found to respond adequately to these imperatives in conjunction with various salts, mostly lithium salts of strong acids. In order to optimize the performances of these systems, various generations of materials have been developed. Thus, following the tests on high molecular-weight linear polymers, it became necessary to minimize the crystallisation of the polyether chains and the creep of the electrolyte film by preparing crosslinked structures bearing short polyether chains between branching points. In fact the crystalline regions are non-conductive and therefore an obstacle to ionic transport and linear macromolecules flow when heated. Furthermore, these networks were later modified in order to limit the conductivity to the cationic species by fixing the anions to the polymer chains 377

378 Applications

through covalent bonds. In fact, for applications like batteries, the best results are achieved with Li/Li" as an energy storage couple without the intervention of electrode phenomena related to the anion which tend to decrease the current density. Numerous investigations related to the above synthetic aspects, but also to the full mechanical, electrochemical and thermal characterization aimed at understanding the mechanisms related to ionic transport were carried out in our laboratory and this provided sound fundamental criteria for the optimization of functional properties. In order to prepare viable devices with these electrolytes it is also essential to be able to cast them into films as thin as possible. This was our next challenge which was tackled by introducing into the basic polyether network structure an additional backbone consisting of polysaccharide chains, well known for their film-forming properties. The grafting and crosslinking reactions used to join the polysaccharide chains to the polyether strands were based on the formation of urethane links between the OH groups of the former and the NCO end-groups of especially-prepared oligothers with or without the intervention of an alternative crosslinking by the condensation among pendant Si(OEt)3 moieties in the presence of moisture. The present paper describes a study within this novel approach to polymer electrolytes (2).

EXPERIMENTAL The cellulosic derivative chosen for preparing neutral networks was a commercial hydroxyethylcellulose (HEC) which had a DP of 300 and 2.7 ethylene oxide units per anhydroglucose ring. The oligopropylene oxide monoisocyanate (OPOM, Mn=8oo) and the oligoethylene oxide diisocyanate (OEOD, Mn=7oo) were prepared following the synthetic procedure developed in our laboratory (3). 2-Methoxyethylisocyanate (MOEI), used as a model for the oligoether isocyanates, was prepared by the reaction of 2-methoxyethylamine with bis(trichloromethyl)carbonate according to the same procedure. The product was purified by distillation and successfully characterized by IR and NMR spectroscopy. 3-isocyanatopropyltriethoxysilane was used as purchased. The catalysts dibutyltin dilaurate and triethylamine were high-grade commercial products used without further treatment. The only appropriate reaction solvent for HEC was N,N-dimethylacetamide (DMAc) which was purified and dried before use. The HEC-based networks crosslinked by the urethane moieties were prepared at room temperature by mixing the cellulose derivative with OPOM and OEOD in

New polymer electrolytes

379

DMAc in the presence of catalytic amounts of dibutyltin dilaurate. The reaction mixture was poured into a sylilated glass mould in order to cast a membrane 0.5 mm thick. After the crosslinking had been completed, the membranes were submitted to a Soxhlet extraction with CC14 and thoroughly vacuum dried. The general structure of these networks is shown below. Their specific structure depended of course on the relative amounts of HEC and oligomeric mono- and di-isocyanate. Thus, both the crosslink density and the relative proportion of crosslinks and grafts could be varied.

oII

H~'8o;-\ I

O_C-N

0

0-

"" OH

OH

, \,

o

The networks based on alkoxysilane hydrolytic condensations were prepared by treating HEC first with a mixture of OPOM and 3-isocyanatopropyl triethoxysilane following the same procedure as above. To the resulting grafted polymer triethylamine was added and the solution poured into the mould. Crosslinking by a sol-gel mechanism in the presence of moisture gave the general structure shown below. Again, the specific topology of the networks could be varied as a function of the relative proportions of the reagents involved in this two-phases polycondensation.

380

Applications

The addition of LiCI04 or LiN(CF3S02)2 was carried out by soaking the membranes into a concentrated solution of the relevant salt in acetonitrile and letting the ionic species diffuse into the networks. The

ionomeric

networks

were

prepared

from

commercial

ethylhydroxyethylcellulose (EHEC) with a DP of 80, a DS with respect to ethylation of 2.7 (i.e. a small proportion of residual available OH groups) and an average number of ethylene oxide unit of ca. 1.5 per anhydroglucose ring.

This was first

treated with a stoichiometric amount of chlorosulfonylisocyanate in acetone at 5° under rigorously dried conditions and then with an excess of solid Li 2C03: .,.

EHECell-QH + OCN-SO:lO --...

Li 2C0 3

EHECell-trN-s~a --...

o

9

EHECell-QCN-SO:JLi+

g

This EHEC sulfonate with Li+ counterions was precipitated in water, thoroughly vacuum dried and used as a polyelectrolytic additive in place of lithium salts in the preparation of membranes based on HEC, as described above. All the materials obtained were characterized by FTIR spectroscopy, DSC, dynamic mechanical properties, ionic conductivity and cyclic voltammetry.

RESULTS AND DISCUSSION The kinetics of the condensation reaction between HEC and MOEI, OPOM and OEOD were studied by monitoring the decrease in the absorbance of the NCO peak around 2200 crrr las a function of time. All reactions went to completion and showed a second-order behaviour. Table 1 gives some typical values of the second-order rate constant for the three combinations.

The monomeric model isocyanate was


381

Table 1. Influence of the extent of substitution of the HEC hydroxy groups by OPOM and OEOD on the Tg and the second-order rate constant

OPOM

OEOD

lO5·k2

Tg

%[NCO)/[OH]

%[NCO)/[OH]

l.mol.s!

CC

10 25 50 75

0 0 0 0 0 10 20 40 60 40 10 30 10 30

49 44

-70 -74 -72 -74 -74 / -40 -50 -48 -66

100

0 0 0

0 20 25

25 50 50

22 21 31

-67 26 -69

7 10-3

6 10-3

-;'~

5 10- 3

bO

f-4

4 10-3

3 10-3

2 10-3 ~"'--'-.....Io...-I-"""""-~~~...&..o.....Io-~--'--"---"~-"""",""-"-"",,,,,--,-~~

0 2 3 4 5

6

[LiCIO 4 ] / mol.r t

Fig. 1 : Evolution of the inverse of Tg with salt concentration for a network prepared with OPOM and OEon ([NCO]/[OH]=O,8). The concentration relates to the polyether chains only.

382

Applications

considerably more reactive than the oligomeric counterparts probably because of steric hindrance with the latter. The reaction between HEC and a mixture of OPOM and OEOD gave a rate constant in the same range as with each individual macroisocyanate. Considering that one is dealing here with aliphatic isocyanates, i.e. with rather sluggish reagents, the results were satisfactory in terms of insuring the consumption of all the NCO groups by the OH function of HEC within a lapse of time of about 24 hours. Table 1 also reports a number of Tg values for various grafted and crosslinked HEC. Interestingly, the fact of appending oligopropylene oxide strands to HEC results in a constant Tg very close to that of the oligomer itself, independent of the grafting density, indicating that there is no interaction between the two polymeric structures which appear to be totally incompatible. The DSC tracings confirmed this situation in that the ~Cp associated with the glass transition was directly proportional to the extent of oligopropyleneoxide grafts. Unfortunately, the Tg of the starting HEC could not be determined because of the spread of this transition over an excessive temperature range, but values around 1300 have been reported for this polymer. Any physico-chemical affinity between the HEC chains and the oligopropylene grafts would have therefore resulted in a variable single Tg, decreasing with increasing grafting density. Crosslinked HEC/OEOD systems gave Tg values close to -50 0 when the proportion of reacted OH was high. This is again the value of oligoethylene oxide unperturbed by the.HEC 'chains except for the fact that they block the two ends of the oligomers. In other words, there is no detectable affinity between these two structures. Mixed topologies with grafts and interchain bridges gave Tgs between -50 and 0 -70 (Table 1), viz. very adequate values in terms of the use of these materials as polymer electrolytes (1). Networks prepared by the sol-gel technique gave Tgs in the same temperature domain. Addition of the lithium salts to these different networks provoked an increase in Tg (Fig. 1) with a linear relation between Tg-l and Li+ concentration with respect to the polyether chains only, as already encountered with simpler polyether-type structures (1). Thus, the polysaccharide chains do not intervene in the solvation of the cations, as observed previously in terms of the non-participation of polydimethylsiloxane chains in polyether-based networks prepared from them (4). Finally, networks containing lithium EHEC sulfonate gave a single Tg around -40°. The study of the dynamic mechanical properties of these elastomeric networks gave curves in which the elastic modulus dropped from 109 to about 106 Pa at the


glass transition, as shown in the example of Fig. 2.

383

The classical free-volume

treatment of the data through master curves provided very satisfactory values for the Cl(Tg) and C2(Tg) constants in the WLF equation, viz. 10 and 60 K respectively, in excellent agreement with similar values obtained previously with numerous other polyether-based networks (4). The ionic conductivity of these new polymeric electrolytes was measured as a function of temperature, LiCI04

concentration and network topology.

The best

values obtained in this investigation were about 3 10-4 Scm-I at 60° for a ratio [ether oxygen]/[Li+]=20, which is close to the optimum conductivities displayed by polyethylene oxide-based networks with the same salt. Fig. 3 shows Arrhenius-type plots for different salt concentrations. The typical non-linearity stems from the wellknown feature attributing the ionic mobility to segmental motions, i.e. a free volume behaviour, and not to activated jumps. In other words, one is dealing again here with the WLF law and indeed the corresponding treatment of the conductivity data according to that equation provided Cl(Tg) and C2(Tg) values of 14 and 50 K respectively, viz. practically the same values as those obtained from viscoelasticity. The mechanism of ionic transport in these systems is therefore of the same nature as that occurring in polyether-based networks without polysaccharide moieties (1,4), namely the cations and the mobile anions are subject to the intrinsic segmental motion of the polymer host. Moreover, the present results indicate that only the oligoethylene oxide bridges and the oligopropylene oxide grafts are responsible for these movements, since the polysaccharide chains show a negligible affinity for the ionic species. The cyclic voltammetry tests were conducted in the potential range of -0.5 to 3.5 V in a lithium cell. No electrochemical instability was detected at 90° for these systems. Apart from the various characterizations carried out on membranes about 0.5 mm thick, the film-forming aptitudes of these materials were tested and it was found that thicknesses ten times lower could be obtained with good mechanical properties. This is not a lower limit intrinsic to the structures studied, but rather the minimum obtained with very simple processing techniques. In conclusion, this novel family of polymer electrolytes based on cellulose derivatives displays all the positive features inherent to amorphous polyether networks with the added advantage of good film-forming properties. pursued and extended to other polysaccharides such as chitosan.

This study is being

384

Applications

Fig. 2 : Variation of the elastic modulus as a function of temperature at 5 Hz for a saltless network (HEC-OEOD, [NCO]/[OH]=O.4)

9

"~

0..

.5 UJ '-"

bO

8

..9

-20

-40

0

Tr

20

40

60

Fig. 3 : Arrhenius-type plot of the ionic conductivity of an HEC-OPOM-OEOD network ([NCOV[OH]=O.8) containing increasing amounts of LiClO4 .

.-

-3 -4

's ~

-5

•

O/Li=24

tj

•

O/Li=17

'6:0-6

•

O/Li=20

•

O/Li=lO

•

O/Li=5

CI)

.s .9

-7

-8 2.5

~~....1-.-011-""'--'--~~~"--"-_ _"-""'---"--

--"

3.5

REFERENCES 1.1£ Nest, J.F., Gandini, A. and Schoenenberger C. (1994) Trends Polym. Sci. 2,432. 2. Le Nest, J.F., Gandini, A., Xu, L. and Schoenenberger, C. (1993) Polym. Adv. Technol. 4, 92.

3. Callens, S., Le Nest, J.F., Gandini, A. and Armand, M. (1991) Polym. Bull. 25, 443. 4. Le Nest, J.F., Gandini, A. and Cheradame H. (1988) Br. Polym. J. 20, 253.

38 Thermal and FT IR studies of Tencel-gco-Bema and Tencel-g-co-Hema carbanilates M A Kazaure,* J T Guthrie and B B Dambatta* - *Department of Chemistry, Bayero University, PMB 3011; Kano, Nigeria; Department of Colour Chemistry and Dyeing, University of Leeds LS2 9JT, UK

ABSTRACT Tencel has been grafted with 2-hydroxyethyl methacrylate (HEMA) using the Ce(IV) technique for different. HEMA concentrations. The resultant copolymers were converted to their corresponding carbanilates. The structural compositions as well as the thermal behaviour were studied using FT-IR and DSC respectively. Results obtained showed that grafting occurred and there there is evidence of polymer-polymer compatibility.

INTRODUCTION The Ce(IV) initiation method was used to prepare copolymers of Tencel and HEMA using various concentration of the vinyl monomer with the view to producing products with low graft "add-on" having possible industrial utility. The introduction of Tencel by Courtauids pIc in the 90's into the fibre market and its subsequent proven capability of being an environmentally friendly regenerated cellulosic fabric, has necessitated research into the physical and chemical properties of the fabric. Graft copolymerisation reactions have been used to improve some of the physico-chemical properties of Tencel. As a result of the inherent insolubility of the copolymers formed, product characterisation requires that copolymers be converted to some derivative equivalent in order to obtain materials capable of solution characterisation. The method reported by Guthrie and Tune [1] was that of carbanilation. We now

385

386

Applications

report a study dealing with the structural composition as well as the thermal stability of selected copolymers and their corresponding carbanilates using FT-IR and thermal analytical techniques.

EXPERIMENTAL The Tencel was supplied by Courtaulds plc, Derby UK. HEMA, Cerium (IV) ammonium nitrate (CAN) and phenyl isocyanate (Aldrich Chemical Co.) were all used as received without any further purification. Pyridine was purified according to the method described by Ralph and Gilkerson [2]. The grafting reactions were conducted following the method reported by Hebeish at al [3]. Four different concentrations of the monomer were used based on the ratio of 1:1,1:2,1:3, and 1:4 (weight by weight) of Tencel:monomer concentration. Reactions were conducted over 3 hours at 60°C in the presence of O.IM CAN. A control was set up. After grafting, samples were thoroughly washed with methanol and Soxhlet extracted for 24 hours in methanol to remove homopolymer. Copolymers were dried under reduced pressure at 40°C and thus % graft add-on values were calculated. The resultant copolymers were subsequently carbanilated following the method outlined by Guthrie [4].

DSC DSC was conducted using a Du Pont Thermal Analyser 2000. Thus, the required amount of sample was placed into an aluminium pan with an empty pan on the reference platform. The apparatus was purged with nitrogen at a flow rate of 3.3 x 10-6 rrr' S-I. The heating range was between 25°C to 500°C at a rate of 10°C per minute. Restults were recorded as DSC thermograms.

FT-IR STUDIES A Perkin-Elmer 1740 FT-IR spectrophotometer was used in this study. Two different routes were followed for the fibrous materials: diffuse reflectance (DR) and attenuated total reflectance (ATR). In ATR, IR-radiation is made to enter a prism of a high refractive index, IR transmitting material, the radiation being totally internally reflected. At the same time Prism produces an evan escent wave which can interact with the sample in contact with the prism to produce a spectrum. In the DR mode, radiation from the spectrophotometer is focused onto the sample and reflected energy is collected and recorded as the spectrum. ATR produces a very short path length in the sample for (the IR light. This makes the technique ideal for highly absorbing materials such ~ fibres. The FT-IR spectra of the powdery carbanilates were acquired using the KBR technique.

Tencel-g-co-Hema

387

RESULTS Attenuated total reflectance spectroscopy and diffuse reflectance spectroscopy are versatile and powerful techniques, ideal for rapid quantitative and qualitative analyses as sample preparation is relatively simple to perform. The techniques are well suited for providing information about the surface properties or about the bulky nature of a material. We have conducted an investigation of the surface properties of Tencel, Tencel-g-co-HEMA and the corresponding carbanilates. Spectral data obtained from these techniques are summarised in Tables 1, 2, and 3 respectively. Assignments have been made although we suggest that overall interpretation should be carried out with caution.

Table 1

DR-IR absorption assignments (em") for Tencel, Tencel-g-co-HEMA (TgcH) (at different ratios of HEMA) and poly HEMA (PHEMA).

Groups stretching

Tencel

O-H (H bonded)

35673249

O-H (free OH)

3604

TgcH TgcH TgcH TgcH PHEM

(1:1) (1:2) (1:3) (1:4) A

3588- 3581- 3543- 3578- 3441 3258 3256 3225 3206 3604

-

1280

1279

1278

1279

1273

1180

1178

1178 1174

1157

C=O (ester)

1733

1736 1728

1733

1728

C-H (methylene)

2917 2949 2914 2928 2949

C-O-C (ester) C-O-C (ether)

1135

Tencel and Tencel-c-co-HEMA have common O-H stretching vibrations at regions above 3200 em" corresponding to hydrogen bonded substances and suggest that not all the -OH groups available for grafting have been grafted. This is of no great surprise as it has already been reported [4] that only a small proportion of OH groups on cellulosic materials are available for grafting. The presence of a small but sharp peak at 3630 em" for Tencel, however, indicates that it contains freer -OH functionality which is absent in the spectra of copolymers. The spectra of all the Tencel-g-co HEMA samples differ from that of Tencel by the methacrylate vibration

388

Applications

Table 2

ATR FT-IR absorption frequencies for Tencel, Tencel-g-co-HEMA (TgcH) at different ratio of HEMA and poly HEMA (PHEMA).

Groups stretching

Tencel

O-H (H bonded)

36873260

3687- 3686- 3687- 3687- 3441 3333 3333 3333 3333 1260 1259 1259 1259 1273

C-O-C (ester) C-O-C (ether)

TgcH TgcH TgcH TgcH PHEM

(1:1) (1:2) (1:3) (1:4) A

1157

1155 1155 1155 1155 1157

C=O (ester)

1714 1715 1714 1714 1728

C-H (methylene)

2920 2924 2923 2923 2949

depicted in the C-H vibration band (methylene), C-O-C vibration band (ester and ether) at 2922 em", 1252 em", 1150 em" and 1708 em" respectively for TgcH (1:1) which is invariably superimposable on all other copolymers. This is an indication that grafting has occurred and that not all OR groups have been utilised for grafting. The picture presented is in agreement with already calculated % graft add-on. The presence of C=O stretching vibration band at 1703 em" for all copolymers indicates and further confirms the mechanism and action of Ce(IV) on Tencel i.e, the formation of free radical to initiate copolymerisation, and aldehyde group formation on either position 2 or 4 of the anhydroglucose unit. The ATR method tends to show that Tencel and it copolymers all contain free OH groups, a result of a shift in the OH stretching vibration band to 3600 em" absorption region. Considering the partially crystalline nature of all cellulosic materials, this seems normal for Tencel. However, for the copolymers, it may be attributed to a free -OH vibration band corresponding to the ungrafted OR groups as well as the OH group on HEMA. The spectra of the carbanilate are almost indistinguishable and superimposable on each other as shown in Table 3. The main difference, however, occurs in the 1:3 and 1:4 ratio where the -CH-stretching band for the methylene unit is shifted to 3035 em:' probably as a result of higher HEMA content. The spectra of Tencel-g-co-HEMA and that of the corresponding carbanilate differ by the N-H bond stretching vibration bands between 3326 em" to 3387 em" and 3134 em" shown by all the carbanilates. Also, the OH stretching exhibited by Tencel and its copolymers

Tencel-g-co-Hema 389

Table 3

FT-IR absorption frequencies of Tencel-g-co-HEMA carbanilates (TgcHC), Poly HEMA carbanilate (PHEMAC) and Tencel tricarbanilate (TTC).

TgcHC TgcHC TgcHC TgcHC

Groups stretching

(1:1) (1:2)

TTC

(1:3) (1:4)

PHEMA C

3387 3327 3327

3326 3328

N -H stretch

3140 3140 3134 3134

3136 3135

C=O (ester)

1724

1728

1729

1730

C=C (Aromatic ring)

1536 1531

1554 1552

1537

1554

C-H (methylene)

2957 2957 3035 3035

2928 2928

C-O-C (ester or ether)

1221

1222

N-H (H bonded)

1729 1711

1220 1233

1233

is not found in the carbanilates (PHEMAC inclusive). carbanilation if not over carbanilation.

Table 4

1233

This could be a sign of

Thermal behaviour of Tencel(T), Tencel-g-co-HEMA(TgcH), Tencel tricarbanilate(TTC).

Sample%

Grafting

Temperature of endotherm (OC)

T

o

86, 346

TgcH

34.07

87, 353

TTC

o

230, 298, 354

and

390 Applications

Table 5

Thermal behaviour of Tencen-g-co-HEMA at different grafting levels

Sample

% grafting

Temperature of endotherm peak (OC)

TgcH(I:I)

34.07

87, 354

TgcH(I:2)

53.96

84,217, 357

TgcH(1:3)

66.32

84, 356

TgcH(l :4)

69.21

87,357

Table 6

Thermal behaviour of Tencel-g-co-HEMA carbanilates at different grafting levels

Sample

% grafting

Temperature of endotherm peak (OC)

TgcHC(I:I)

34.07

61,283, 349

TgcHC(I:2)

53.96

296, 352

TgcHC(I:3)

66.32

247, 358

TgcHC(I:4)

69.21

247, 359

PHEMAC

241,408

Results of the DSC analysis of Tencel (T), Tencel-g-co-HEMA (TgcH), Tencel-g-coHEMA carbanilates (TgcHC), and Tencel tricarbanilate are shown in Tables 4, 5 and 6. All peaks below 100°C correspond to the H20 peaks. This is as a result of the hygroscopic nature of cellulosic maerials (Tencel inclusive). Table 4 shows that Tencel melts at 346°C and this melt temperature increases to 354 °C with introduction of HEMA. The single peak could be an indication of polymer-polymer compatibility. Thermograrns of Teneel tricarbanilates portray the absence of water and show 3 peaks with the main peak at 298°C equivalent to the carbanilate melting behaviour. The small peak at 230°C could be due to carbanilation but no satisfactory explanation could be given for the other peak at 354°C other than the possible contamination by copolymeric species during measurements. From Table 5, we see that an increase in the Tencel:HEMA ratio does not have any significant influence on the melting behaviour of the copolymer. Rather, what is evident is the presence of

Tencel-g-co-Hema

391

the melting behaviour of the copolymer. Rather, what is evident is the presence of polymer-polymer compatibility portrayed by the single broad peak for all the four different ratios under investigation. The unusual peak at 217°C could probably be due to the formation of Tencel-CAN complex during copolymerisation. The thermograms for Tencel-g-co-HEMA carbanilates showed two basic endothermic peaks which correspond to the carbanilate and the copolymeric melting behaviour. Here also, the melting behaviour is generally independent of the amount of graft addon. Poly HEMA carbanilate showed two peaks with one similar to the carbanilate melting behaviour and the other at 408°C corresponding to the melting of PHEMA.

REFERENCES [1]

J.T. Guthrie, P.D. Tune; J. Polym. Sci. part A Polym. Chern. (1991), 13021312.

[2]

Ralph, E.D., W.A. Gilkerson, J. Chern. Soc. (1964) 86,4783.

[3]

Hebeish, A.M.1. Khalil, E. Alfy; J. Polym. Sci. Polym. 3137-3143.

[4]

J.T. Guthrie, PhD Thesis, University of Salford, (1971).

[5]

J.T. Guthrie, A. Hebeish; The chemistry and technology of cellulosic copolymers, Springer-Verlag, Heidelberg, (1981).

Chern. 19 (1981),

Acknowledgements: The authors wish to acknowledge the help given to M.A. Kazaure by the Bayero University Research Committee and Department of Colour Chemistry of the University of Leeds.

39 ESR as a method for monitoring lignins activity during the interaction with monomer and oligomer silicon containing compounds T Dizhbite, G Telysheva and G Shulga - Latvian State Institute of Wood Chemistry 27 Dzerbenes Str, Riga, Latvia, LV-I006

ABSTRACT

ESR was employed to study the interaction of lignins with silicon-organic compounds. The comparison of the ESR spectra of the co-reagents and products of interaction of lignosulphonates (LS) with the water-soluble oligomer sodium alumomethyl siloxanolate (SAHS) has demonstrated different characters of their interaction in acid and alkaline media. The observation of the behaviour of the lignin radicals as spin probes in a wide temperature range (77-448 K) made it possible to reveal a considerable decrease in the rigidity of the lignin matrix as a result of lignin silylation with monomeric silicon-organic compounds (hexamethylsilazane, phenylaminomethyl-(methyl)-diethoxysi lane).

393

394 Applications

I liTRODUCTI 011 The presence of conjugated systems in the structure of lignin in situ and the development of polyconjugated blocks upon the isolation of lignin during different wood processing ensure the stability of free radicals and make it possible to apply the high-sensitive method ESR for monitoring the lignin changes during the interaction with various co-reagents [1,2]. . The present work demonstrated the application of ESR for investigation of the interaction of lignins with siliconorganic compounds: sodium alumomethyl siloxanolate (SAHS), hexamethylsilazane (HHDS) and phenylaminomethyl(methyl)-diethoxysilane (AH-2).

EXPERIMBNTAL ISH

SPECTRA. The ESR spectra were recorded on a Radiometer RE-1306. The concentration of paramagnetic centers was measured using diphenylpicrylhydrazyl (N8t 5.63*10~1e spin) as a standard. A line in the ESR spectrum of Hn2 + in magnesium oxide served as a secondary standard. The absorption areas were determined by the double integration of the ESR spectra. The error of determining the concentrations of paramagnetic centers and the relative concentration error were 20% and 5%, respectively [3]. An average error of determining the line width was 0.02 mT.

=

THERMAL ARALYSIS. Thermal analysis was made using a derivatograph "HOH" under self-atmosphere conditions, the range of temperatures 20 ... 500°C, the rate of heating 10°C/min~ the standard - aluminium oxide. IR SPECTRA. IR spectra were determined using an IR-20 spectrometer within the range of frequencies 3700 - 450 om- 1 . The samples were pressed with KBr into pellets.

RESULTS AND DISCUSSION. The comparison of the ESR spectra both of co-reagents and products of interaction of lignosulphonates (LS) with SAHS has manifested the different characters of their interactions in acid and alkaline media. It has been shown that this difference is conditioned by the change of the co-reagents structure in media with different activity. In alkaline medium~ the SAHS aqueous solution is optically transparent and does not exhibit any paramagnetic properties. At the same time, in neutral and acid media, the UV-spectrum of SAHS has a specific set of absorption bands and its ESR-spectrum (pH 4.2) is a singlet line with a g-factor of 2.002, 5.5 mT wide. In

ESR as a method for monitoring lignins activity 395 this case. the concentration (Fig. la, Curve 1).

of

PMC

is

1020

spin/g

The ESR-spectra of the modified products in alkaline medium (Fig.lb, Curves 2 ... 4) are practically identical as regards their characteristics (g-factor, the width and form of the singlet line) and the ESR-spectrum of the initial lignosulphonate (Fig.lb, Curve 2). The differences manifest themselves only in rather a higher PMC content in modified samples which is probably caused by the stabilization of a part of the radicalsintermediates appearing during the LS self-oxidation process which are usually observed in alkaline medium [3]. This is obviously connected with the fact that the paramagnetic characteristics of the LS modified in alkaline media do not practically change with the increase in the modificator amount. On the contrary, six equidistant lines with a width equal to the g-factor 2.002 appear in the ESR-spectra of the LS products modified in acid medium (Fig.la, Curves 3 .. 5), in addition to the initial singlet line (Fig. la, Curve 2). The appearance of a sextet is caused by the formation of the new PMC whose uncoupled electron is delocalized on the aluminium atom (1=5/2) of the modificator [4] to a considerable extent. The newly formed PMC are stable for more than two months.

b

2

3

s

Fig.l ESR - spectra of SAHS (1) and LS initial (2) and modified in the acid (a) and alkaline (b) media at Ilass ratio LS/SAMS 1.0:0.1 (3); 1.0:0.5 (4); 1.0:1.0 (5)

396 Applications

It may be suggested that in acid medium, the donoracceptor interaction between aluminium in the SAHS molecule and the lignosulphonate donor groups results in a considerable rearrangement of electron density, both of the donor and acceptor, and is accompanied by the formation of stable paramagnetic centers. A visible increase in the concentration of the PHC yielding a six-linear spectrum of ESR at the transition of the LS/SAH5 mass ratio from 1.0:0.1 to 1.0:0.5 (29*10~1e and 20*10 A18 spin/g respectively) and the retaining of the PHC concentration at the same level (2a*10~1e spin/g) during further increase in the SAHS part shows that the appearance of new stable PHC is possible only up to specific LS/SAHS mass ratios, i.e. is limited by the presence of active centers in the LS molecule. The comparison of the IR-spectra of the initial LS, SAHS and the products of their interaction at different pH values has shown a considerable increase in adsorption in the ranges 590 ... 600 cm- 1 and 1030 ... 1050 cm- 1 (a new peak) characteristic for valent fluctuations of the Si-O bond in the 5i-0-C and 5i-0-5i groups for the products obtained in acid medium. The Si-O-C groups formed are probably rather stable in acid medium, are hydrolyzed in alkaline medium to a great extent [5], and therefore, they practically do not manifest themselves in the IR-spectra. The structural features detected by ESR and IR spectroscopic methods explain the difference of thermooxidative stability of L5 treated with 5AHS in media with different acidity. The thermal analysis data show that modification changes the character of LS thermodestruction significantly. In this case, the variations for acid and alkaline media are ambiguous. In both cases, a temperature shift of the beginning of LS thermal disintegration approximately by 40°C tOWards higher values is observed, i.e. the thermooxidation stability of modified LS is higher, as compared with the initial ones. However, the temperature corresponding the maximum mass loss rate increases from 250 to 299 0C only for the LS modification products in acid medium. No changes are observed for alkaline medium at a low consumption of the modifier, while at LS/5AHS ratios of 1.0:0.5 and 1.0:1.0 the maximum mass loss temperature even decreases.

ESR as a method for monitoring lignins activity

397

The mass loss at 50eoC indicates that the modification of LS in acid medium results in a considerable increase in thermooxidative stability already at a SAHS consumption of 10% from the LS mass. In this case, the absolute mass loss decreases by 16%, as compared with the initial LS. Further 5- and 10-fold increase in the SAHS consumption (from 10 up to 50 and 100% in terms of LS) results in an additional decrease in the absolute value of mass losses 1e and 19%. In fact. up to 500°C. the LS modified in alkaline medium lose the mass equal to the sum of the mass losses of the initial LS and SAHS registered for this temperature. Besides the presence of the stable paramagnetic centers of conjugated systems the ESR spectra of lignins is also conditioned by the presence of the free radicals stabilized by the lignin matrix [3]. In general the heating of the lignin samples in the range of the temperature approximating to the temperature of glass transition causes the decay of the free radicals stabilized by matrix rigidity. Therefore the change of the amount of PHC during the heating under the above mentioned conditions may give the information about state of lignin matrix after modification. This method was employed for characterization of lignin sylilated by HMDS. The heating of the modified lignin was accompanied by enhancing of PMC amount at observed decreasing of the matrix rigidity (data of thermomechanical investigation). This manifests that formation of the free radicals prevails their decay. Formed free radicals could participate in the process of further transformation of modified lignin [6]. The free radicals formed at low-temperature radiolysis of lignin can be used as a spin probe. In the case of investigation of modification of lignin with AH-2 this method allowed to establish the difference in the structure of the products obtained in various conditions [7].

Accelerations of the free radicals death of lignin modified at 200°C indicates on increasing in its structure the microregion content with enhanced molecular mobility (Fig.2). The conclusions made based on the ESR data were confirmed by other physico-chemical methods.

398 Applications

[R]/[Ro]

Fig.2 Temperature dependence of free radicals relative concentration in irradiated (at 19BaC) the lignin samples, previously modified with AH-2 at 130°C (1) and 200°C (2). O-------------I""----....a.------a._ _

...a.-...J

-100

-100

-50

0

50

100

temperature, C

SUMMARY

The application of the ESR method to the investigation of the interaction of lignin with different silicon-organic compounds has allowed to determine the specifity of the processes, taking place with the participation of free radicals and to investigate the microstructure of modified lignins using spin probe.

LITERATURE 1. Sarkanen K.V., Formation, Structure

Ludwig C.H., Lignins. Occurence, and Reactions, New-York, 1971, 916

p.

2. Westermark U., Samuelsson B. and Lundquist K. Proceedings "Seventh International Symposium on Wood and Pulping Chemistry", Vol.l, 1993, p.93. 3. Zarubin H.Ya., Wood Chemistry, 1984, N 5, p.3-19 4. Pshezecki S.Ya., Kotov A.I., Hilinchyk V.I., ESR of Free Radicals in the Radiation Chemistry, H., 1972., 485 p.

5. Telysheva G.M., Lebedeva G.N., Sergeeva V.N., Wood Chemistry., 1983., N 1., p.94-101. 6. Telysheva G.M., Pankova P., Cellulose Chemistry and Technology, 1989, N 6, p.701-721. 7. Telysheva G.M., Pankova P., Sergeeva V.N., Wood Chemistry, 1985; N 4, p.87-91.

40 The regularities of lignosulphonate behavior on different interfaces and its alteration by purposeful modification G Telysheva, T Dizhbite, E Paegle and A Kizima - Latvian State Institute of Wood Chemistry, LV-I006 27 Dzerbenes Str, Riga, Latvian Republic

ABSTRACT

The adsorption behavior of lignosulphonates (L5) and of modified L5 (MLS) with water soluble silicon-containing compounds were investigated within a wide pH range (i11) .

The general

regularities of MLS adsQrption on a solid surface were established to be analogous to those of L5, however MLS differed by enhanced values of maximum adsorption, owing to hydrophobic aspects of MLS molecule and insertion of new active centers.

The participation of macromolecular associates in adsorption has been shown experimentally. The multilayer nature of L5 and HLS adsorption is confirmed by the good agreement between the adsorption isotherms obtained experimentally with those calculated on the basis of the Aranovitch model of adsorption [1], which takes into account the lateral interaction of adsorbate molecules.

399

400 Applications The enhancement of dispersion activity of HLS compared with that of LS has been found owing to an increase in adsorption on kaolin and surface activity.

INTRODUCTION Lignosulphonates (LS) - polydisperse polyelectrolytes, the basic units of which consist of the C6 C3 structure, contain anionic groups of three types, namely: sulfonate, phenolic hydroxyl and carboxyl groups. Uncharged L5 molecules are coiled. The alteration of the degree of dissociation of the L5 ionogenic groups leads to changes in the macromolecule net electric charge and molecule size and conformation. The effective degree of LS dissociation is almost zero at pH 1, about 20% at pH 11 [2].ln the course of 5 and up to 80% at pH dissociation of ionogenic groups, the LS molecule extends due to the electrostatic repulsion between the neighbouring groups. Nowadays, LS are used as surfactants for many industrial applications. In order to optimize the performance of these polymers, numerous scientific investigations have been made to comprehend how different internal and external factors influence the L8 behaviour on different interfaces.

The present

work, is

background for

aimed

at the

purposeful alteration

development of of the

the

efficiency

of the LS activity on different interfaces modification with oligomeric water soluble containing compounds.

by the silicon

MATERIAL AND METHODS The initial sodium lignosulphonate contained 1.23 mequiv/g of sulphonate groups with pKa 1.6 and 1.08 mquiv/g of phenolic hydroxyl groups with pKa = 11. The solution pH was adjusted with sulfuric acid or sodium hydroxide, but the ionic strength was maintained constant. The modifier - sodium oligoheterosyloxanolate (OHS) - contained AI, with 5i/Al ratio of 3.

=

The surface tension (0) of the solutions was measured at 20°C by the Wilhelmy method, using a platinum plate and bidistilled water. The thickness of the monolayer (14 A) was taken from [3]. The formation and characteristics of the associates in solution were monitored by light-scattering on a multiparameter flow cytofluorimeter EPIC.

Lignosulphonate behavior on different interfaces 401

The value of LS adsorption on kaolin was calculated on the basis of the change of the surface tension of L5 solution. The calibration curve was obtained at pH 8.9 the equilibrium pH value of the kaolin - water system. The

rheological

determined

properties

of

suspensions

using a

viscosimeter Rheotest-2, The measuring system was a cone K-1 - plate.

type

were

RV2.

RESULTS AND DISCUSSION The surface tension (0) of initial L8 and modified L5 water solution clearly depends upon the water pha§e pH (Fig.l). Modification provides the decreasing of 0 over all the pH range, although the profiles of the concentration relations are similar, and the range of the maximal 0 depression occurs in acid media for both samples. 80 Sigma·JO.....3. N/m 70

60 50 40

~~

30 '--_ _

o

.. _

__ .. _ . ---A-_--'

- - - - t l . . - -_ _- - - - "

0.4

0.8

1.2

Concentration. %

----

1

-+-

2

3

--e-

4

Fig.l Concentration dependence of surface tension of aqueous solution of: (1) MLS, pH=5.0; (2) MLS, pH=8.9; (3) L5, pH=8.9; (4) L5, pH=4.5. The depression of 0 is determined, to a great extent, by the introduction of hydrophobic blocks to the L5 molecule. The shielding of the charge and blocking of the L3 ionogenic groups as a result of modification affects the molecules conformation on the interface. This can be observed when comparing the average values of the areas occupied by the molecules of the initial and modified L5.

402 Applications

From the plots of Gibbs isotherms it has been established that the values of the average areas occupied by the molecules of the initial L5 increase 3 times when the medium pH increases from 1 to 11. After modification, the pH of L5 changes from 4.5 to 8. It is supposed that at such pH, the molecule of the L5 under study occupies an area of approximately 160 1 2 on the interface, while the extrapolated area for the modified LS did not exceed 140 A2. The study of the L8 and HLS solutions prior to and after their adsorption on kaolin by light-scattering has shown that the associates formed participate in the formation of adsorption layers. For both L5 and HLS, the effective concentration of the associates in solution decreases considerably after the adsorption on kaolin, mainly at the expense off the decrease in the amount of the largest associates (Fig.2). 100 Relative iDlen8it

80

60 40 20

o

1

5

10

1

5

10

Partiole size. mkm (101 seale)

Particle size. mlcm (101 scale)

1

2

Fig.2. Particle size distribution histograms for the 0.3% L8 solution before (1) and after adsorption (2). The modification of L8 results in an increase in adsorption on kaolin. This is connected not only with hydrophobization, but also with the formation of positively charged centers in the LS molecule, since it is known that the edges of the kaolin crystallites have a negative charge. The re-arrangement of experimental isotherms in the co-ordinates of different adsorption models has shown that the Langmuir model is not suitable. Different grid models (BET, Anderson, Hjutig, Aranoyich) have been checked. It has been shown that the isotherms are linear within the most wide range in the co-ordinates of Aranovich's equation [1] which has the

Lignosulphonate behavior on different interfaces 403

variable parameter "z" taking into account the interaction among the adsorption molecules. The maximum constant of regression (0.9987) for the Aranovich model for the adsorbent/adsorbate systems under study may be achieved using the coordination number 11. The high coordination number confirms the previous suggestion concerning the formation of a film - a liquid layer of adsorbed molecules on the surface of solids [4]. All the models under study indicate a multilayer character of L5 and MLS adsorption on kaolin. The enthalpy and absolute Gibbs energy values in the case of MLS adsorption are considerably higher than in the case of L5. They are for MLS UHo 14.5 kJ/mol, UGo -2.2 kJ/mol and for L5 UHo 12.7 kJ/mol, UGo -0.3 kJ/mol. This indicates a high energetic advantage of ML5 surface layer formation. The maxim on the curves of the dependence of the mole heat capacity of the adsorption layer as a function of relative L5 concentration, calculated according the Aranovich model (Fig.3), perhaps, reflects the end of the formation of the adsorption layer and the beginning of filling a new layer, which is accompanied by the change of the system organization and rearrangement of the molecules in adsorption layers.

=

=

=

1 Heat ca.pacit.y, reI.units

=

200 Yield stress, Pa

0.6

150

.

100

...... ~ .....

0.8

0." i'

50

_

.

0.2

o o

Io.-_....L-.-_--a-_..-.&-_---&_---J

0.2

0.4

0.6

C/Cs -1

--+-2

0.8

Ot.--.----'----...I.-------"

o

0.05

0.1

Concentration, -1

0.15 ~

-+-2

Fig.3 (left) Variation of L5 heat capacity during the adsorption processes of L5 (1) and MLS (2) on the kaolin surface, the Aranovich model. Fig.4 (right) Dependence of the relative yield stress of kaolin suspension on L5 (1) and MLS (2) concentration. The comparison of the state of the maxima on these curves shows that the formation of a monolayer in the case of ML5 ends at concentrations lower than in the case of L5.

404 Applications

The sum of such factors as adsorption on kaolin and surface activity determines the enhancement of the dispersion activity of HLS as compared with the initial L5. This is obvious from the graphical dependence of the relation of the yield stress at a definite L5 concentration with the yield stress at a zero L8 concentration against L5 concentration in kaolin suspension (Fig.4). At a 0.1% concentration of HLS in solution, the kaolin suspension approaches complete deflocculation. SUMMARY

The general regularities of L5 and MLS adsorption behaviour are similar at the different interfaces. The participation of macromolecular associates in adsorption as well as multilayer nature of adsorption have been confirmed experimentally. The calculated thermodynamic parameters of adsorption have shown an energetic advantage of surface layer formation by MLS against L5. The enhanced surface activity and dispersion activity of MLS is explained by introduction of hydrophobic blocks and new positively charged centers as well as blocking of LS ionogenic groups.

REFERENCES 1. Aranovich G.L. J. of Phys. Chem., 1989, v.63, N9, p.2529 - 2533 (Russia). 2. Kontturi A.K., Kontturi K. J. Colloid Interface Sci., 1988, v.124, N1, p:328. 3. Fors K., Fremer K.E. Int. Symp. on Wood and Pulp. Chem. Stockholm, 1981, v.4, p.29-38. 4. Afanas'ev N.I., Telysheva G.M., Makarevich N.l., Khrol Y.S. Wood Chemistry, 1990, N2, p.85-92 (Latvia).

41 Some physiochemical properties of xylanolytic enzymes produced by Aspergillus fumigatus IMI 255091 L A Hamilton] and D A J Wase* - tChemical Technology Division, Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, Tennessee, 37891-6194, USA; *Environmental Biotechnology Division, School of Chemical Engineering, The University of Birmingham, Edgbaston, Birmingham, B12 2TT, UK

ABSTRACT The xylanolytic enzymes xylanase and f3-o-xylosidase from Aspergillus jumigatus IMI 255091 have temperature optima of 70°C and pH optima lying over a broad range between 4.0 and 7.0. Thermal stability of these enzymes and stability at various pH values suggest that several isoenzymes may exist such that among them there is a range of highly active enzymes which are highly tolerant of elevated temperatures and retain their activities over a broad range of pH values. The specificities of the enzymes for their respective assay substrates were determined: the KM values for xylanase and f3-oxylosidase were 11.7 mg mL- 1 and 0.1 mmol L-1 respectively. Vmax values for xylanase and f3-xylosidase were 0.4 and 0.3 mmol minI mL- I.

INTRODUCTION Vegetative plant materials are, in general, made up of three main lignocellulosic components: cellulose, hemicellulose and lignin. The relative fractions of these components can vary widely, depending on many variables, such as the source of the lignocellulosic, and the season and the time ofyear; similarly, the proportions of various constituents of each of these three materials can also vary. Nevertheless, in the majority of cases, o-xylan has been noted as an important constituent of the hemicellulose fraction. As a result, most investigations of the enzymatic degradation of this polymer have focussed on xylanase and f3-o-xylosidase enzymes. As o-xylan is branched, other enzymes such as a-L-arabinosidase and a-o-g1ucuronidase are also present to remove 405

406 Applications

substituent side-chains. However, in general terms, the degradation of o-xylan can be said to be dependent on the hydrolytic action ofxylanase and p-o-xylosidase [1, 2]. Aspergillusfumigatus IMI 255091 has previously been shown to produce xylanase and

p-o-xylosidase as well as a range of other xylanolytic enzymes [3, 4]. There has been considerable interest in such hemiceUulolytic enzymes, and their potential application in the sugar, alcohol, paper, pulp, feed-processing and food industries [5], and the activity of these enzymes produced by A. fumigatus is particularly high under normal assay conditions. However, the conditions under which activity is optimal are not clear. The purpose of this study was therefore to determine the physicochemical conditions at which greatest activity occurs. MATERIALS AND METHODS

Culture broths containing xylanase and p-o-xylosidase activities were obtained from shake-flask fermentations of A. fumigatus IMI 255091 using hay as lignocellulosic substrate. The liquid medium was as described previously by Hamilton and Wase [6]. Enzyme-containing broths were preparedby filtration ofthe culture broth from day 7 of the fermentation. The resulting broth was stored at 4°C with no detrimental effects to enzymic activity [7] and used for subsequent analyses.

XYLANASEACTIVITY This assay is based on hydrolysis of xylan to reducing sugars. The birchwood xylan used as substrate was prepared in the following manner. A 10 mg mL- 1 suspension of xylan in 50 mmol L-1 citrate buffer, pH 5.0, was autoclavedfor 10 minutes at 121°C, 15 psig, furnishing a fully dissolved solutionofxylan, ready for hydrolysis by xylanase. To 1.0 mL diluted enzyme, 1.0 mL xylan was added, mixed and incubated at 50°C. The reaction was stopped at various time intervals over a period of 15 minutes. The concentration of reducing sugars released was determined by the method described by Sumner and Graham [8]. Activity was expressed as mmols of xylose residues released per minute per mL ofenzyme(IU mL- 1 ).

P-D-XYWSIDASE ACTIVITY p-n-xylosidase activity was assayed by measuring the release of 4-nitrophenol from the substrate, 4-nitrophenyl p-o-xylopyranoside (pNPX). To 0.1 mL enzyme broth, 0.9 mL PNPX (1.0 mmol mL-1 in 50 mmol L-1 citrate buffer, pH 5.0) was added, mixed and incubated at 50°C. The reaction was stopped at various times by adding 1 mL sodium carbonate (2 mol L-1) . Activity was expressed as mmol of 4-nitrophenol released per mL of enzyme per minute (IU mL- 1 ).

EXPERIMENTAL pH and temperature optima The two substrates were hydrolysed at pH 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 using 0.05 mol mL-1 citrate buffer (pH 3.0-pH 6.0) and 0.05 mol mL-1 sodium phosphate buffer (pH 7.0-pH 8.0). Enzyme activitywas determined as previously described [9]. The optimum

Physiochemical properties of xylanolytic enzymes

407

hydrolysis temperature for each of the substrates was established by hydrolysing it at the following temperatures: 30°C, 40°C, 50°C, 60°C, 70°C and 80°C.

Temperature stability Glass vials containing the enzyme broths were incubated at 30°C, 50°C and 70°C. At various time intervals (0, 5, 10, 20, 30 minutes, 1, 2, 4, 8, 24 and 48 hours) aliquots of enzyme broth (0.6 mL ) were removed and xylanase and f3-o-xylosidase activities were determined as previously described [9].

Stability at various pH values Glass vials containing enzyme broths, adjusted to pH 3.0, 5.0 and 7.0 using the buffers described above, were incubated at 30°C. At various time intervals (0, 5, 10, 20, 30 minutes, 1, 2, 4, 8, 24 and 48 hours) aliquots of enzyme broth (0.6 mL) were removed and xylanase and f3-o-xylosidase activities were determined as previously described [9].

Determination of K M and VDIU The constants of Michaelis-Menten (KM and Vmax) were determined from plots of the Lineweaver-Burk type and confirmed using plots of the Eadie-Hofstee type. KM is the concentration of substrate when the rate of reaction is half its maximum rate; Vmax is the maximum reaction rate of substrate hydrolysis [6]. RESULTS AND DISCUSSION

Temperature and pH optima Typical bell shaped curves (not shown here) resulted for both xylanase and f3-nxylosidase enzymes (see Table 1 for temperature optima). Xylanase was active over a broad range of temperatures having at 30°C approximately 50% of the activity measured at 70°C, the optimum. In contrast, at 30°C, J3-o-xylosidase activity was unmeasurably low, but activity at 70°C was optimal for this enzyme, too. It is interesting to note that optimum activity at 70°C markedly exceeds that of 30°C, the normal temperature for cultivation of A. fumigatus. Indeed optima as high as this suggest that enzymes produced by this strain of A. fumigatus are extremely thermotolerant and as such may be suitable for use in the saccharification of lignocellulosics, although there are further factors such as pH and stability still to be considered. It has been shown previously that the cellulase complex of A. fumigatus has pH optima well to the acid side of neutrality [9]. This also seems to the case with f3-D-xylosidase, in which optimum activity occurs when the pH is 4.5 (see Table 1). However, the optimum pH for xylanase is not so clearly defined (see Table 1). Maximum activity was achieved when hydrolysis of birchwood xylan was carried out over a large range of pH values (from pH 4.0 to pH 7.0). Royer and Nakas [10] similarly showed that Trichoderma longibrachiatum xylanase activity was optimal between pH 4.8 and pH 5.8 and Hrmova et ale [11] also showed that xylanases from Aspergillus terreus and Aspergillus niger were highly active between pH 7.1 and pH 8.0. In contrast, the extracellular xylanase and f3-o-xylosidase of the hemicellulase complex of Aureobasidium pullulans had pH optima that were

408 Applications

different from the corresponding optima for A., fumigatus. To confuse matters further, Myburgh et al. [1] showed that xylanase activity from A. pullulans was optimal when the pH was 4.0 and that it was (3-o-xylosidase that possessed high activity over a broad range of pH (between 4.0 and 7.0). Overall, then, it is impossible to be specific in the case of hemicellulases, and tests must be used to establish optima for the particular microbial strain in question.

Table 1: Temperature and pH optima for xylanase and p-o-xylosidase enzymes produced by A. fumigatus IMI 255091.

Enzyme

Temperature Optimum

pH Optimum

Xylanase

70°C

4.0-7.0

J3-o-xylosidase

70°C

4.5

Thermal and pH Stability It has been mentioned that the xylanase and J3-o-xylosidase enzymes produced by A. fumigatus are highly active at elevated temperatures. At these temperatures, initial conversion rates may be at their highest but thermal denaturation of the enzyme may result in a process in which overall product yields are poor. Enzymes with enhanced stabilities are therefore highly advantageous. Considerable stability of the hemicellulase complex of A. fumigatus is demonstrated in Table 2. There was little loss of activity at 30°C. At 50°C, rapid loss of activity occurred initially, but 50% of the activity was retained, even after 48 hours (e.g. see Figure 1). In contrast, substantial deactivation of the enzymes occurred during incubation at 70°C. However, xylanase activity remained significant, with 20% of the original activity retained.

Table 2: Thermal stability ofxylanase and p-o-xylosidase at various hydrolysis temperatures over 48 hours. Temperature

% Activity Remaining After 48 Hours

Xylanase

p-o-Xylosidase

30°C

80

100

50°C

50

50

70°C

20

0

Figure 1 demonstrates how enzyme activity initially declined during the first few hours of incubation, and then levelled out to a constant value. This is also true for the cellulase enzyme complex produced by this strain of A. fumigatus, and was discussed previously by Wase et. al. [9]. Stewart and Heptinstall [12] showed the presence of isoenzymes for A. fumigatus IMI 246651 and so it would be reasonable to suggest that

Physiochemical properties of xylanolytic enzymes 409

the same is true for Ai fumigatus IMI 255091. That is, isoenzymes may exist such that amongst them there is a range of highly active to semi-active enzymes, some of which are highly tolerant of elevated temperatures. ~

~

~

C

• ...-4

120 100

>

80

60

• ...-4 ~

(I)

40

• ...-4

~ .......

~

20 0

0

0

10

20

30

40

50

Time (h) Figure 1: Thermal stability of xylanase (II, 30°C; . , 50°C; 0, 70°C). In each case, activity fell sharply during the initial few hours. However, after at most 5h exposure to the test temperature, the residual activity was constant for the remainder of the two-day period, suggesting initial dissimilation of unstable isoenzymes. Similar stability profiles resulted when either xylanase or 13-n-xylosidase was incubated at various pH values (see Figures 2 and 3). Thus, 13-n-xylosidase (Figure 2) retained 100% of its original activity at pH 5.0. Either side of this pH value there was initial deactivation; thus effects of incubation at pH 3.0 and pH 7.0 were essentially equal. Within a short time, activity approximately halved, then remained at this level. Although xylanase (Figure 3) was active over a broad range of pH values, the activity of the enzyme decreased almost immediately with time. Most activity was retained (ca. 60%) during incubation at pH 5.0 whereas most was lost at pH 3.0 (ca. 70%). ~

~

~

C

• ...-4

120 100

>

80

60

• ...-4 ~

(I)

40

• ...-4

~ .......

~

20 0

0

10

20

30

40

50

Time (h) Figure 2: p-n-xylosidase stability at various pH values (II pH 3; . , pH 5; 0, pH 7).

410 Applications

120 ~--------------. 100 80 60 40

20

O------------.. . . .--..A.---.-....--.... o 50 40 20 30 10 Time (h) Figure 3: Stability ofxylanase at various pH values (II, pH 3;., pH 5; C, pH 7). Myburgh et ale [1] studied the stability of xylanase and p-o-xylosidase from Aureobasidium pullulans NRRL Y 2311-1. between pH 4.0 and pH 8.0. They found that 100% ofthe xylanase activity was retained after 5 hours at pH 5.0. At pH values of 4.0 and 6.0 activity dropped to 700A. in the first 5 hours. At pH values higher than pH 6.0 the activity dropped in two stages. After 5 hours incubation the activity had dropped to 25% of the original activity then to zero when incubated up to 24 hours. The authors attributed this phenomenon to the presence of multiple forms of xylanase, each with a different stability from the others. The results obtained for A. jumigatus show a similar trend in that activity reaches a constant level also indicating the presence of multiple forms of xylanase. Like xylanase, A. jumigatus p-o-xylosidase retained activity even after 48 hours at pH 5.0. Similarly, the enzyme from Aureobasidium pullulans also retained maximum activity after 24 hours incubation at its optimum pH of 4.0 [1]. Although Myburgh et ale [1] did not study the effects of pH stability on p-o-xylosidase activity below pH 4.0, one suspects that the same trend would result. That is, with A. fumigatus J3-oxylosidase, optimum stability is at pH 5.0. At pH values greater than or lower than pH 5.0 the enzyme becomes less stable losing approximately the same amount of activity.

Substrate specificities of Iylanase and p-D-xylosidase Xylanase enzymes may be assayed using xylans from various sources. During this investigation it was decided to use birchwood xylan since it contains 90% xylose residues. The synthetic substrate, PNPX appears to be the substrate of choice in the literature. The Michaelis-Menten constants observed for xylanase and J3-n-xylosidase are summarised in Table 3.

Physiochemical properties of xylanolytic enzymes

411

The general variability of xylans and assay conditions for determining xylanase activity makes direct comparisons with xylanase enzymes from other sources difficult. However, the specificity of xylanase and J3-o-xylosidase appears to be measured in terms of which substrates are hydrolysed by separated and purified forms of xylanase and p-o-xylosidase. Problems also arise because of the broad specificity of one enzyme for several substrates. For example, Ozcan et ale [13] observed that the xylanase enzyme produced by the yeast Pichia stipitis was also able to hydrolyse PNPX. The rate at which PNPX was hydrolysed by xylanase was extremely slow compared with 13o-xylosidase. Table 3: The Michaelis-Menten constants for the hydrolytic action ofhemicellulases produced by A. jumigatus. Enzyme

KMmgmL- 1

Vmu nunol mL- 1 min- 1

Xylanase

11.69

0.42

(3-Xylosidase

0.10

0.34

GENERAL DISCUSSION Recently, xylanases and other hemicellulases have received increased attention due to their possible application in paper manufacturing. Xylanase pretreatment of paper pulps has been shown to aid the bleaching process, while preserving the cellulose fraction. Trials using these enzymes for prebleaching are currently being carried out [14, 15]. Enzymes that exhibit high temperature optima and stability and are active over neutral to alkaline pH values would clearly be highly desirable for such a process. For the xylanase and fl-o-xylosidase activities of A. fumigatus to be suitable for such a process in the paper pulp industry, they need to be potent and stable for long periods of time at 50°C and neutral pH. Our investigations have indeed shown that this is so. ACKNOWLEDGMENT Lesley A. Hamilton thanks S.E.R.C. (Science and Engineering Research Council) for a research studentship for this work.

REFERENCES [1] Myburgh J., Prior B.A. and Kilian S.G. Process Biochem., (1991), 26, 343-348. [2] Puis J. and Poutenan K. In "Enzyme systems for lignocellulose degradation". Ed. M.P. Coughlan. Elsevier Science Ltd., London, UK. (1989), Pp. 151-165. [3] Kim S.W. Ph.D. Thesis, (1989). University of Birmingham, Birmingham, UK. [4] Wase D.A.J. and Raymahasay S. In "Cellulose and its derivatives -chemistry, biochemistry and applications". Eds. J.F. Kennedy, G.O. Phillips, D.J. Wedlock and P.A. Williams. Ellis-Horwood, Chichester, UK. (1985), Ch. 49. [5] Gomes, J., Purkarthiofer, H. M., Kapplomiller, J.,Sinner, M. and Steiner, W. Appl. Microbiol. Biotechnol. (1993), 39, 700-707.

412 Applications

[6] Hamilton L.A. and Wase D.A.J. Process Biochem., (1991), 26, 287-292. [7] Holland T.M. Ph.D. Thesis, (1989). University ofBirmingham, Birmingham, UK. [8] Sumner J.B. and Graham V.A. J. Bioi. Chem., (1925), 65, 393-395. [9] Wase D.A.J., Hamilton L.A., Holland T.M., Kim S.W. and McManamey W.J. In "Cellulosics: materials for selective separations and other technologies". Eds. J.F. Kennedy, G.O. Phillips and P.A. Wtlliams. Ellis-Horwood, Chichester, UK. (1993), Ch. 30. [10] Royer J.C. and Nakas J.P. Enzyme Microbial Technol., (1989), 11, 405-410. [11] Hrmova M., Biely P. and Vrsanska M. Enzyme Microbial Technol., (1989), 11, 610-616. [12] Stewart J.C. and HeptinstaJl J. Methods in Enzymology, (1988), 160, 33-39. [13] Ozcan S., Kotter P. and Ciriacy M. Appl. Microbiol. Biotechnol., (1991), 36, 190195. [14] Grant R. Pulp Paper Int., (1991),33, 61-63 [15] Khasin A., Alchanati I. and Shoham Y. Appl. Environ. Microbiol., (1993), 59(6), 1725-1730.

42 Endoglucanase, !3-D-glucosidase and xylanase induction in Dichomitus squalens (Karst) Reid E Resende, M Carolino and N Teixeira Rodeia - Departamento de Biologia Vegetal, Bloeo C2, ~ piso, Faeuldade de Ciencias da Universidade de Lisboa, Campo Grande, 1700 Lisboa, Portugal

ABSTRACT The amount of endoglucanase, B-D-glucosidase and xylanase produced by the fungus D. squalens were found to be dependent on the source of carbon and on the presence of the Tween 80 in the growth medium. Growth on cotton cellulose enhanced the production of endoglucanase, B-D-glucosidase and xylanase in the culture filtrates relative to the other sources of carbon (Avicel cellulose, carboxymethylcellulose = CMC, paper mill sludge, sawdust of Pinus sp).The endoglucanase induced by CMC exhibits 760/0 of residual activity after 2 h at 80 oC, maintaining about 1000/0 activity after 1 h at 50 oC, pH 5.0; it has a half-life of 17 min at 70 oC, pH 5.0. This enzyme shows optimal pH activity at pH 5.0 and pH stability between 4.0 and 6.36 where it exhibits a residual activity of more than

76%.The B-D-glucosidase component was isolated by chromatography on DEAE Sephadex A-50.

INTRODUCTION Cellulases and xylanases are produced by a variety of fungi and bacteria. These enzymes hydrolyse glycosidic bonds in cellulose and xylan, two of the most abundant polysaccharides in nature. Both enzymes have potential applications in the bioconversion of lignocellulose to useful products.The purpose of the present study was to examine the effect of carboxymethylcellulose, sawdust of Pinus sp., Avicel cellulose, paper mill sludge, cotton cellulose, Tween 80 and hemicellulose - rich substrates on the production of cellulases and xylanases by Dichomitus squalens .

MATERIAL AND METHODS MATERIALS

Dichomitus squalens

(n~

571) belongs to the Mycology Center's culture collection of

413

414 Applications

the Faculty of Sciences of Lisbon. It was isolated from a stump of Pinus sp. The fungus has been maintained by sub-culture on potato dextrose agar. MElHODS

Cultivation methods 250 ml Erlenmeyers containing 100 ml of a basal liquid medium, Norkrans & Hammarstrorn (1963), enriched with biotin 5 ug 1-1 and thiamine 100)lg 1-1 and supplemented with different carbon source (Avicel cellulose - 1%; cotton fibre 2.76%; CMC - 1%; paper mill sludge - 1.5%; sawdust of pinus tree - 1.5%) were inoculated with mycelium discs and incubated at 28 0C. The effect of a surfactant on the production of cellulases or xylanases was evaluated adding 0.1 % of Tween 80 to each half series flasks containing cotton as carbon source.

Enzyme assays Endoglucanase and xylanase activities were assayed using the method of Wood & Bhat (1988). B-D-glucosidase activity was assayed using the method of Wood (1968). Protein is estimated using the method of Lowry et ale (1951) or by absorbance at 280 nm. Units of activity are expressed, in the case of endoglucanase, as the release of 1 umole of reducing sugar (glucose equivalent) per minute. In the case of B-Dglucosidase as the release of 1 umole of o-nitrophenol per minute and in the case of xylanase activity as the release of 1 umole of reducing sugar (xylose equivalent) per minute. Temperature and pH optima - the effect of pH, temperature and the half-life at 70 0C and pH 5.0 of the endoglucanase activity and stability were evaluated by the method of Parr (1983).

Enzyme purification All operations were performed at room temperature.The extracellular extract was concentrated in an ultrafiltration apparatus under nitrogen pressure (ultrafilters cut-off of PM-5 or PM-IO KDa membrane in an Amicon cell -50ml- under a constant pressure of 25 - 30 psi ). When the concentrated enzyme solution from the ultrafiltration step was chromatographed on DEAE - Sephadex A-50 column, endoglucanase and 'B-D-glucosidase activities were adsorbed on the column. These were eluted with a linear gradient composed of 0.1 M sodium acetate buffer, pH 4.0 and 1M NaCI in the same buffer.

RESULTS In Figure 1 where are represented B-D-glucosidase, endoglucanase and xylanase activities and the substrates utilized ( cotton fibre, Avicel cellulose, sludge of paper mill and sawdust of Pinus sp.) it is evident that cotton was the best inducer substrate for D .squalens produce all enzymes and in contrast the sawdust of pinus tree was the worst inducer substrate. In the same Figure we can observe the enzyme quantities produced with the time of culture. In Figure 2 we can observe that the surfactant Tween 80 promotes an higher liberation of all enzymes on the 5th week and in some cases with an increase higher than 100 %. In Figure 3 we represent the effect of temperature on the endoglucanase induced by CMC and it has a higher activity at 60 0 C although it was more stable at 50 0 C

Physiochemical properties of xylanolytic enzymes 415

E

.. 400

'0

E

C200

Cot. Avl. Slu. Sew.

Col.

=cotton fibre;

Avi.

Cot. Avl. Slu. Sew.

Cot. Avl. Slu. Sew.

= Aviccl cellulose; Slu.= sludge of paper min; Saw. =sawdust

Fig.I - Effect of cotton fibre, Avicel cellulose, sludge of paper mill and sawdust of pine tree on the (l-glucosidase, endoglucanase and xylanase enzyme activities.

i

..

1

n.,lucoeld •••

C

i

-. 'i

.. ..

X,'_nee•

I

i

I

1

TI",.

c•••".)

I

I

TIfft.

.

Wilh Tween RO (0)

t

(•••".,

1

TI", • • • • • • •,

Without Tween 80 (.)

Fig. 2.- Effect of the surfactant Tween RO on the production of endoglucanase 13glucosidase and xylanase enzymes from D.squlliells with cotton as carbon source.

(results not shown). We can conclude that this enzyme has a half-life of 17 min at 700C and pH 5.0. We also studied the effect of pH on the endoglucanase of the D.squalens induced by CMC and we can observe that the optimum pH level for activity was of S.O and for stability was 5.7 ( Fig. 4). When the enzyme was kept for I8h at pH values from 4.0 up to 6.36 the residual activity was higher than 76%. However pH below 4.0 or higher than 7.0 a rapid deactivation of the enzyme was promoted ( Fig. 4). In Figure 5, we can verify that the endoglucanase has a half-life of 17 minutes at 70 0 C and at pH 5.0. In Figure 6 we represent the separation of the enzymes using DEAE Sephadex A-50 and we can conclude that Sephadex permits a better separation

416

Applications

Therefore with Sephadex the B-D-glucosidase is separated from the endoglucanase being eluted the first enzyme between fraction 17 and 22 and the endoglucanase between fraction 26 and fraction 44, when CMC was the substrate inducer. Although when the cotton was the substrate they were separated one endoglucanase and one BD-glucosidase with inverse molecular weight from those present in CMC.

120

100

60 Enz. Act. (%) 40 ;----_r-----.-----r-...--__-

O-t------,r----r-~-__r_-~------.

20

40

60

80

3

4

5

_ 7

6

pH

Temp.(OC)

Fig. 3 - Effect of the temperature on the endoglucanase activity from D.squalens induced by CMC .

Fig. 4 - Effect of pH on the acuvity and the stability of one endoglucanase from D.squalens induced by CMC. 30

2,2

__ 1,8

.(,J

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