Industrial Biorefineries and White Biotechnology

INDUSTRIAL BIOREFINERIES AND WHITE BIOTECHNOLOGY

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INDUSTRIAL BIOREFINERIES AND WHITE BIOTECHNOLOGY Edited by

ASHOK PANDEY RAINER HÖFER MOHAMMAD TAHERZADEH K. MADHAVAN NAMPOOTHIRI CHRISTIAN LARROCHE

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK 225 Wyman Street, Waltham, MA 02451, USA Copyright © 2015 Elsevier B.V. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-444-63453-5 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress For Information on all Elsevier publications visit our website at http://store.elsevier.com/ Printed and bound in the USA

Front Cover Upper picture: Oleochemical Biorefinerie of Avril, Grand-Couronne/France, photo credit: Cédric Helsly, with kind permission Lower picture: Rapeseed cultivation in the Picardie region/France, photo credit: Philippe Montigny, with kind permission Both images have been taken from the website of Avril, Paris/France with kind permission.

CONTENTS

List of Contributors xiii Preface xvii

PART A: INDUSTRIAL BIOREFINERIES

1

1. Biorefinery Concepts in Comparison to Petrochemical Refineries Ed de Jong and Gerfried Jungmeier

3

1. Introduction 2. The Definition for Biorefinery 3. The Economic Value of Biomass Using Biorefining 4. Classification of Biorefineries 5. Conventional Biorefineries 6. Advanced Biorefineries 7. Whole Crop Biorefinery 8. Oleochemical Biorefinery 9. Lignocellulosic Feedstock Biorefinery 10. Syngas Platform Biorefinery (Thermochemical Biorefinery) 11. Next Generation Hydrocarbon Biorefinery 12. Green Biorefinery 13. Marine Biorefinery 14. Chain Development 15. Biorefinery Concepts in Comparison to Petrochemical Refineries 16. Biorefinery Complexity Index 17. Discussion and Conclusions References

3 5 7 9 11 12 12 13 13 14 14 15 16 16 17 24 27 30

2. Algal Biorefineries Yanna Liang, Tyler Kashdan, Christy Sterner, Lilli Dombrowski, Ingolf Petrick, Michael Kröger and Rainer Höfer

35

1. Introduction 2. Algal Research in the USA 3. Macroalgae 4. Microalgae 5. Downstream Processes 6. Products Produced from Algae at Commercial Scales 7. Conclusions References

36 38 46 48 55 69 83 84

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Contents

3A. Pulp Mills and Wood-Based Biorefineries Raimo Alén

91

1. General Aspects 2. Pulping Processes and Their By-Products 3. Pretreatments of Wood Chips Prior to Pulping 4. Thermochemical Conversion Methods 5. Conclusions References

91 96 109 113 119 120

3B. The Pine Biorefinery Platform Chemicals Value Chain Rainer Höfer 1. Introduction 2. Extractable Volatile Oils 3. The Tall Oil Value Chain 4. Conclusion References

4A. Sugar- and Starch-Based Biorefineries Rainer Höfer 1. Introduction 2. Sugar and Starch Crops 3. Sugarbeet Refining and Processing 4. Alcoholic Fermentation 5. The Ethanol-Based C2—Value Chain 6. Beyond C2 Platform Chemicals by Fermentation 7. Sucrochemistry 8. Starch Refining and Processing 9. Starch Uses 10. Conclusions Acknowledgment References

4B. Ethanol from Sugarcane in Brazil: Economic Perspectives Luiz Augusto Horta Nogueira and Rafael Silva Capaz 1. Introduction 2. Ethanol from Sugarcane in Brazil: Context and Evolution 3. Economic Aspects of Ethanol from Sugarcane in Brazil 4. Final Remarks References

127 127 130 136 151 152

157 158 159 179 183 190 192 201 205 211 227 228 228

237 237 238 240 244 245

Contents

5. Vegetable Oil Biorefineries Coraline Caullet and Jérôme Le Nôtre 1. Introduction 2. Vegetable Oil Feedstock 3. The Whole-Plant Biorefinery Concept—From Plants to Industrial Products 4. Industrial Vegetable Oil Biorefineries 5. Future Challenges of Industrialization 6. Conclusions and Perspectives References

6. Biogas Biorefineries Harald Lindorfer and Bettina Frauz 1. Introduction 2. Substrates for Biogas Production 3. Biogas Utilization 4. The Chemical Platform Methane 5. Fertilizer Production 6. Mass and Energy Balances 7. Other Biorefinery Concepts with Strong Focus on Biogas Production 8. Perspectives of Biogas Biorefineries References

247 247 249 252 264 266 268 268

271 271 275 280 284 284 288 291 292 293

7. Civilization Biorefineries: Efficient Utilization of Residue-Based Bioresources 295 Ina Körner 1. Introduction 2. Primary, Secondary, Tertiary, and Quaternary Bioresources 3. Civilization Biorefineries 4. Approaches Toward Civilization Biorefineries References

296 297 311 321 337

8. Biomass Pyrolysis for Hybrid Biorefineries Paul J. de Wild

341

1. Introduction 2. Pyrolysis-Based Fractionation of Biomass 3. Biomass Pyrolysis for Biorefineries 4. A Pyrolysis-Based Hybrid Biorefinery Concept 5. Conclusion References

341 342 348 360 365 365

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Contents

9. Single-Cell Biorefinery Qingsheng Qi and Quanfeng Liang 1. Introduction 2. Simultaneous Substrates Utilization in Single Cell 3. Coproduction in Single Cell 4. Single-Cell Biorefinery 5. Conclusion Acknowledgments References

PART B: WHITE BIOTECHNOLOGY

10. Biocatalysis Licia M. Pera, Mario D. Baigori, Ashok Pandey and Guillermo R. Castro 1. Introduction 2. Screening for Novel Biocatalyst 3. Development of Biocatalysts 4. Raw Materials 5. Reaction Media 6. Conclusions References

11. White Biotechnology for Organic Acids Guocheng Du, Long Liu and Jian Chen

369 369 371 375 381 384 384 384

389 391

391 392 394 403 404 404 405

409

1. Introduction 409 2. Conclusion 434 References 435

12. White Biotechnology for Amino Acids Murali Anusree and K. Madhavan Nampoothiri

445

1. Introduction 2. History and Evolutionary Route 3. Production Processes 4. Strain Improvement 5. Amino Acids in Detail 6. Alternative Sources for Amino Acid Production 7. Prospective and Outlook

445 446 447 451 454 466 466

Contents

Acknowledgment References

13. Industrial Enzymes Reeta R. Singhania, Anil K. Patel, Leya Thomas, Mandavi Goswami, Balendu S. Giri and Ashok Pandey 1. Introduction 2. Enzymes Classification 3. Microbial Enzyme Production 4. Industrial Application of Enzymes 5. Enzyme Immobilization 6. Global Enzyme Market Scenario 7. Conclusion References

14. White Biotechnology in Biosurfactants Kuttuvan Valappil Sajna, Rainer Höfer, Rajeev K. Sukumaran, Lalitha Devi Gottumukkala and Ashok Pandey 1. Introduction 2. Biosurfactants 3. White Biotechnology in Glycolipids Biosurfactants 4. White Biotechnology in Lipopeptide and Lipoprotein Biosurfactants 5. White Biotechnology in Polymeric Biosurfactants 6. Conclusion and Future Perspective Acknowledgment References

15. Exopolysaccharides from Prokaryotic Microorganisms—Promising Sources for White Biotechnology Processes Margarita Kambourova, E. Toksoy Oner and Annarita Poli 1. Introduction and Definition 2. Advantages and Disadvantages in Microbial Production of EPSs 3. Composition and Structure 4. EPS Properties and Structure–Function Relationships. Microbial Producers. Biofilms 5. Polysaccharide Roles in the Prokaryotic Cell 6. Synthetic Pathways 7. EPS Production 8. Commercially Important Properties and Industrial Applications of Market-Valued EPS

467 467

473

474 475 476 486 493 494 496 496

499

499 501 502 510 515 517 517 517

523 523 524 526 528 531 533 533 536

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Contents

9. New Microbial EPS. EPS from Extremophiles 546 10. Conclusion 547 References 547

16. White Biotechnology for Biopolymers Guo-Qiang Chen, Juanyu Zhang and Ying Wang 1. Introduction 2. Strains for Production of PHA 3. PHA Produced in Industrial Scale References

17. Microbial Poly-3-Hydroxybutyrate and Related Copolymers Raveendran Sindhu, Parameswaran Binod and Ashok Pandey 1. Introduction 2. PHB-Producing Microbes 3. Fermentation Strategies 4. Downstream Operations 5. Characterization Techniques 6. Strain Improvement, Mutation, and Metabolic Engineering 7. Substrate Manipulation for the Production of Various Classes of PHB 8. Applications 9. Conclusion and Perspectives References

555 555 559 560 572

575 576 578 582 586 588 592 595 599 601 601

18. White Biotechnology in Cosmetics 607 Kuttuvan Valappil Sajna, Lalitha Devi Gottumukkala, Rajeev K. Sukumaran and Ashok Pandey 1. Introduction 2. Functional Properties of Cosmetically Important Compounds 3. Classification of Biotechnologically Derived Cosmetic Ingredients 4. Conclusion References

19. Production and Extraction of Polysaccharides and Oligosaccharides and Their Use as New Food Additives Clarisse Nobre, Miguel Ângelo Cerqueira, Lígia Raquel Rodrigues, António Augusto Vicente and José António Teixeira 1. Introduction 2. Extraction, Production, and Purification of Polysaccharides and Oligosaccharides

608 610 614 644 644

653

653 656

Contents

3. Food Applications of Polysaccharides and Oligosaccharides 662 4. Health and Nutritional Benefits of Polysaccharides and Oligosaccharides 666 5. Regulation and Safety Aspects 668 6. Conclusions 670 Acknowledgments 670 References 670 Index 681

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LIST OF CONTRIBUTORS

Raimo Alén Laboratory of Applied Chemistry, University of Jyväskylä, Finland Murali Anusree Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India Mario D. Baigori Planta Piloto de Procesos Industriales Microbiológicos (PROIMI–CONICET), Tucumán, Argentina Parameswaran Binod Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India Rafael Silva Capaz Institute of Natural Resources, Federal University of Itajubá, Itajubá, Brazil Guillermo R. Castro Laboratory of Nanobiomaterials–Institute of Applied Biotechnology (CINDEFI), Department of Chemistry, School of Sciences, Universidad Nacional de La Plata–CONICET (CCT La Plata), Argentina Coraline Caullet SAS PIVERT, Parc Technologique des Rives de l’Oise, Venette, Compiègne cedex, France Miguel Ângelo Cerqueira Centre of Biological Engineering, University of Minho, Braga, Portugal Guo-Qiang Chen School of Life Sciences, Tsinghua University, Beijing, China Jian Chen Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu, China Ed de Jong Avantium Chemicals, Amsterdam, The Netherlands Paul J. de Wild Energy Research Centre of the Netherlands (ECN), Petten, The Netherlands Lilli Dombrowski Fakultät für Naturwissenschaften, Brandenburgische Technische Universität Cottbus-Senftenberg, Germany Guocheng Du Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu, China xiii

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List of Contributors

Bettina Frauz Schaumann BioEnergy GmbH, Pinneberg, Germany Balendu S. Giri Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India Mandavi Goswami Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India Lalitha Devi Gottumukkala Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India Rainer Höfer Editorial Ecosiris, Düsseldorf, Germany Gerfried Jungmeier Joanneum Research Forschungsgesellschaft mbH, Institute for Water, Energy and Sustainability, Graz, Austria Margarita Kambourova Laboratory of Extremophilic Bacteria, Institute of Microbiology, BAS, Sofia, Bulgaria Tyler Kashdan Department of Advanced Energy and Fuels Management, Southern Illinois University, Carbondale, IL, USA Ina Körner Hamburg University of Technology (TUHH), Hamburg, Germany; BioResourceInnovation (BRI), Hamburg, Germany Michael Kröger DBFZ Deutsches Biomasseforschungszentrum, Leipzig, Germany Jérôme Le Nôtre SAS PIVERT, Parc Technologique des Rives de l’Oise, Venette, Compiègne cedex, France Quanfeng Liang State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, P. R. China Yanna Liang Department of Civil and Environmental Engineering, Southern Illinois University, Carbondale, IL, USA Harald Lindorfer Schaumann BioEnergy GmbH, Pinneberg, Germany Long Liu Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu, China K. Madhavan Nampoothiri Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India


Clarisse Nobre Centre of Biological Engineering, University of Minho, Braga, Portugal Luiz Augusto Horta Nogueira Institute of Natural Resources, Federal University of Itajubá, Itajubá, Brazil E. Toksoy Oner Department of Bioengineering, Marmara University, Istanbul, Turkey Ashok Pandey Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India Anil K. Patel DBT-IOC Centre for Advanced Bio-Energy Research, Indian Oil Corporation, R & D Center, Faridabad, Haryana, India Licia M. Pera Planta Piloto de Procesos Industriales Microbiológicos (PROIMI–CONICET), Tucumán, Argentina Ingolf Petrick Fakultät für Naturwissenschaften, Brandenburgische Technische Universität Cottbus-Senftenberg, Germany Annarita Poli Institute of Biomolecular Chemistry (ICB), National Research Council (CNR), Pozzuoli, Italy Qingsheng Qi State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Jinan, P. R. China Lígia Raquel Rodrigues Centre of Biological Engineering, University of Minho, Braga, Portugal Kuttuvan Valappil Sajna Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India Raveendran Sindhu Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India Reeta R. Singhania DBT-IOC Centre for Advanced Bio-Energy Research, Indian Oil Corporation, R & D Center, Faridabad, Haryana, India Christy Sterner U.S. Department of Energy, Golden Field Office, Golden, CO, USA Rajeev K. Sukumaran Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India José António Teixeira Centre of Biological Engineering, University of Minho, Braga, Portugal

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Leya Thomas Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India António Augusto Vicente Centre of Biological Engineering, University of Minho, Braga, Portugal Ying Wang School of Life Sciences, Tsinghua University, Beijing, China Juanyu Zhang School of Life Sciences, Tsinghua University, Beijing, China

PREFACE

Although the beginnings are shrouded in the mists of human prehistory, viniculture1 as well as beer brewing2 and sourdough bread-making3 are early domestic technologies. In this sense, yeast microbes reckon among the earliest domesticated organisms and methods of White Biotechnology have accompanied mankind since the very beginnings of civilization. Renewable raw materials have been utilized by mankind through the millennia as food, to feed domesticated animals, to clothe themselves, or as firewood, construction material, and to make articles for daily use.The replacement of craft activity by power-driven machines such as steam engines that were fueled by the fossil raw material coal together with the associated changes in economic and social organization that began in Great Britain in the late eighteenth century represent the beginning of the Industrial Age, characterized inter alia by the improved logistics for people and goods by railways and steam ships.The triumph of fossil raw materials began when, in addition to coal, crude oil (also called petroleum) was discovered from the middle of the nineteenth century as a resource, first for lamp oil (in the USA widely sold as kerosene4) and since the early 1900s to produce appropriate hydrocarbon fractions that could fuel internal combustion engines, such as diesel engines (compression-ignition engines), Otto motors (spark-ignition engines), and combustion turbines (jet engines). However, untreated crude is virtually useless, just good to be burned thereby producing an awful smell and a great deal of smoke. Only in a refinery the complex mixture of hydrocarbon molecules in crude oil is separated and converted by fractionation, cracking, reforming, isomerization, hydrotreating operations into petroleum products, which can be used as fuels, lubricants, and as feedstock in petrochemical processes. Today, the fossil raw materials coal, crude oil, and natural gas remain the dominant world energy sources accounting for roughly 80% of world energy supply.5 However, the Club of Rome’s report published in the year 1972 together with the first oil crisis, which erupted in 1973, already created awareness that the fossil resources on which the industrial base depends are limited and will run out with no major change in the physical, economic, or social relationships of society.6 The message was further developed when in 1987, the Brundtland commission created the sustainable development concept.7 1 McGovern

PE. Ancient wine: the search for the origins of viniculture. Princeton: Princeton University Press; 2003. F, Zarnkow M. Das Bier: Eine Geschichte von Hopfen und Malz. München: C.H. Beck; 2014. 3 Dupaigne B. The history of bread. New York: Harry N. Abrams Publ; 1999. 4 Lucier P. Scientists & Swindlers. Baltimore: Johns Hopkins Univ. Press; 2008. 5 International Energy Agency. Key World Energy Statistics 2014. Paris: OECD/IEA; 2014. 6 Meadows DH, Meadows DL, Randers J, Behrens III WW. The Limits to Growth. New York: Universe Books; 1972. 7 Brundtland G, editor. Our Common Future, The World Commission on Environment and Development. Oxford: Oxford University Press; 1987. http://www.worldinbalance.net/agreements/1987-brundtland.html; http://www.undocuments.net/ocf-ov.htm. 2 Meuβdoerffer

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This c oncept was meant to provide a long-term balance between the environment, the economy, and the social well-being of humanity.8 As a result, in 1992, the UN Conference on Environment and Development (UNCED), more commonly known as the Rio Earth Summit, established a number of initiatives to promote the uptake of sustainable development worldwide. Contemporaneously, anthropogenic climate change emerged on the public agenda in the mid-to-late 1980s and year 1990, the first report of the Intergovernmental Panel on Climate Change (IPPC) ascertained that (besides the “natural greenhouse effect which already keeps the Earth warmer than it would otherwise be”) “emissions resulting from human activities are substantially increasing the atmospheric concentrations of the greenhouse gases such as carbon dioxide, methane, chlorofluorocarbons, and nitrous oxide. These increases will enhance the greenhouse effect, resulting on average in an additional warming of the Earth’s surface.”9 The steadily growing global energy demand5 on the one side and, on the other, the finite nature and instability of fossil fuel supply and, because of their exploitation, the everincreasing atmospheric concentration of the carbon dioxide greenhouse gas have initiated a turnaround away from fossil fuels toward the utilization of biomass as a renewable raw material and energy resource. Conceptually, the processing of biomass to produce fuels, power, heat, and value-added chemicals would be done analogous to today’s petroleum refineries in conversion facilities called biorefineries.10 Biomass comprises the entire terrestrial vegetation, defined as the “mass of live or dead organic matter”11 or, somewhat more specifically, as “the biodegradable fraction of products, waste and residues from biological origin from agriculture (including vegetal and animal substances), forestry and related industries including fisheries and aquaculture, as well as the biodegradable fraction of industrial and municipal waste.”12 The immense variety of natural resources requires a preselection of refinery feedstock and allows for a welladapted design of value chains. Insofar, biorefineries will distinguish from petrochemical refineries in order to conform to the complexity in composition and regional distribution of living matter at the same time linking with agriculture and arable farming as key elements for a secure supply and a genuine, large expansion of available biomass feedstock. Significant progress has been made during the last decade with 8 Höfer

R. History of the Sustainability Concept–Renaissance of Renewable Resources. In: Höfer R, editor. Sustainable solutions for modern economies, RSC Green Chem No. 4. Cambridge: RSC Publ; 2009. p. 1–11. 9 Houghton JT, Jenkins GJ, Ephraums JJ, editors. Climate change: the IPCC scientific assessment. Cambridge, New York, Port Chester, Melbourne, Sydney: Cambridge University Press; 1990. 10 Kamm B, Gruber PG, Kamm M, editors. Biorefineries–industrial processes and products. Weinheim:Wiley-VCH; 2006. 11 Global Terrestrial Observing System. Biomass. Rome: Food and Agriculture Organization of the United Nations (FAO); 2009. http://www.fao.org/gtos/doc/ecvs/t12/t12.pdf. 12 Directive 2009/28/EC of the European Parliament and of the Council of April 23, 2009 on the promotion of the use of energy from renewable sources and amending and subsequently repealing Directives 2001/77/EC and 2003/30/EC; http://eur-lex.europa.eu/legal-content/en/ALL/;ELX_SESSIONID=x71WJn1GR1wz616PdpLT7Td y5VpJdqBhXvWFhngNwFvfFcvWpYcg!-639629260?uri=CELEX:32009L0028.

Preface

regard to the industrial utilization of biomass and the manufacture of bio-based building blocks. Bio-based intermediates such as 1,3-propanediol, isobutanol, succinic acid, and 1,4-butanediol, which all were at laboratory level still in 2006 have meanwhile entered world-scale production.13 Part A of Industrial Biorefineries & White Biotechnology provides a comprehensive survey of biorefinery concepts and updated information about individual biomass refining unit operations, regional key aspects, and the road maps toward marketable products and energy in comparison to petrochemical refineries and process chains. Part B is dedicated to highlight White Biotechnology14 (also known as Industrial biotechnology or biotechnology applied to industrial processes) as a particularly promising gateway to a sustainable future. White biotechnology has positioned itself distinctly from Red biotechnology, which is aimed at medical processes and from Green biotechnology, which is biotechnology applied to agricultural processes such as genetically modified crops and plants.15 Part B of Industrial Biorefineries & White Biotechnology summarizes the achievements made by research and industry in microbial and enzymatic catalysis and throughout organic specialty chemicals, bioplastics, and in the utilization of biotechnology for food and personal care applications. The editors would like to thank all the authors, who by their origin and their academic or industrial spheres of activity showcase the global scope of modern chemistry, for their commitment and for bringing in their knowledge, their professional experience, and their expertise. Ashok Pandey Rainer Höfer Mohammad Taherzadeh K. Madhavan Nampoothiri Christian Larroche

13 Künkel A. Symbiosis

of chemistry and biology: biodegradable and renewable polymers. 3F-Talks: Functional Fibres and Films. RWTH Aachen: DWI-Leibnitz Institute; March 2015. 14 Haas T, Kircher M, Köhler T, Wich G, Schörken U, Hagen R. White Biotechnology. In: Höfer R, editor. Sustainable solutions for modern economies, RSC Green Chem No. 4, Cambridge: RSC Publ.; 2009. p. 436–478. 15 Soetaert W,Vandamme EJ, editors. Industrial biotechnology. Weinheim: Wiley-VCH; 2010.

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PART A

Industrial Biorefineries

1


CHAPTER 1

Biorefinery Concepts in Comparison to Petrochemical Refineries Ed de Jong1, Gerfried Jungmeier2

1Avantium Chemicals, Amsterdam, The Netherlands; 2Joanneum Research Forschungsgesellschaft mbH, Institute for Water, Energy and Sustainability, Graz, Austria

Contents 1. Introduction 3 2. The Definition for Biorefinery 5 3. The Economic Value of Biomass Using Biorefining 7 4. Classification of Biorefineries 9 5. Conventional Biorefineries 11 6. Advanced Biorefineries 12 7. Whole Crop Biorefinery 12 8. Oleochemical Biorefinery 13 9. Lignocellulosic Feedstock Biorefinery 13 10. Syngas Platform Biorefinery (Thermochemical Biorefinery) 14 11. Next Generation Hydrocarbon Biorefinery 14 12. Green Biorefinery 15 13. Marine Biorefinery 16 14. Chain Development 16 15. Biorefinery Concepts in Comparison to Petrochemical Refineries 17 16. Biorefinery Complexity Index 24 17. Discussion and Conclusions 27 References30

1. INTRODUCTION In recent years, substantial steps into the transition toward a biobased economy have been taken. Multiple drivers, some policy and geographically dependent, are steering an economy where material wastes are minimized, new bioproducts are replacing their fossil counterparts, greenhouse gas (GHG) emissions are reduced; while economic perspectives are developed supported by innovative policies. The recent extreme volatilities in prices (fossil oil, biomass raw materials) and very fluctuating demand ask for robust systems to be competitive in the long run. An economy based on innovative and costefficient use of biomass for the production of both biobased products and bioenergy should be driven by well-developed integrated biorefining systems. This will result in large additional volumes of biomass required, possibly causing increasing food and Industrial Biorefineries and White Biotechnology http://dx.doi.org/10.1016/B978-0-444-63453-5.00001-X

Copyright © 2015 Elsevier B.V. All rights reserved.

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Industrial Biorefineries and White Biotechnology

commodity prices, and undesired competition with production of food, feed, wooden products, paper, and so on. It may also have profound environmental implications including loss of (boreal and rain) forests, biodiversity, soil productivity, and (fresh) water availability. Accordingly, reforestation programs, sustainable management, conservation, and sustainable development of all types of forests in the long view cannot be limited to emerging economies such as Algeria (“barrage vert” in the Sahara) or Kenya (“Green Belt Movement” founded by Nobel Price Wangari Maathai) but need to be considered for deforested regions globally, including deforested regions in developed economies. Efficient and sustainable use of biomass resources, which is of paramount importance, can be enhanced by the use of biorefinery processes and their products, which will form the foundation of a future biobased economy. The ultimate goal should not just be to efficiently and sustainably make use of biomass for nonfood applications. It should also encompass increasing availability of biomass for nonfood applications by improved food chain efficiency in industrialized countries. The integration of agroenergy crops and biorefinery manufacturing technologies offers the potential for the development of sustainable biopower and biomaterials that will lead to a new manufacturing paradigm.1 International Energy Agency (IEA) Bioenergy is an organization setup in 1978 by the IEA with the aim of improving cooperation and information exchange between countries that have national programs in bioenergy research, development, and deployment.The IEA was established in November, 1974 within the framework of the Organisation for Economic Co-operation and Development (OECD) to implement an international energy program. It carries out a comprehensive program of energy cooperation among OECD member countries. Its aims include to promote: systems for coping with oil supply disruptions, rational energy policies, an oil market information system, improved energy supply and demand structures, and integrated environmental and energy policies. IEA Bioenergy’s vision is to achieve a substantial bioenergy contribution to future global energy demands by accelerating the production and use of environmentally sound, socially accepted, and cost-competitive bioenergy on a sustainable basis, thus providing increased security of supply while reducing GHG emissions from energy use (http://www.ieabioenergy.com/). For the period 2013–15 there are 10 Tasks operating under the IEA Bioenergy umbrella covering all major aspects of the bioenergy field. The relevance of biorefinery in a successful bioenergy research policy has been acknowledged by the establishment in 2007 of a specific IEA Bioenergy Task 42 on biorefineries, coproducing fuels, chemicals, power, and materials from biomass. The major objective of this Task is to assess the worldwide position and potential of the biorefinery concept, and to gather new insights that will indicate the possibilities for new competitive, sustainable, safe, and eco-efficient processing routes for the simultaneous manufacture of transportation biofuels, added-value chemicals, power and heat, and materials from biomass. This Task is covering an exciting field which can have a large

Biorefinery Concepts in Comparison to Petrochemical Refineries

impact both in environmental and technological innovation policies and practices. To open up the biorefinery-related potential, system and technology development is required. Research, development, and deployment (RD&D) programs can link industry, research institutes, universities, governmental bodies, and non-governmental organizations (NGOs), while market introduction strategies need to be developed. Major outputs of Task 42 (http://www.iea-bioenergy.task42-biorefineries.com/en/ ieabiorefinery.htm) include: • Biorefinery definition and biorefinery classification system2 • Country reports describing and mapping current processing potential of existing biorefineries in the participating countries, and assessment of biorefinery-related RD&D programs to help national governments defining their national biorefinery policy goals and related programs. • Bringing together key stakeholders (industry, policy, NGOs, research) normally operating in different market sectors (e.g., transportation fuels, chemicals, energy, etc.) in multidisciplinary partnerships to discuss common biorefinery-related topics, to foster necessary RD&D trajectories, and to accelerate the deployment of developed technologies. • Brochures, reports and publications on specific areas such as on Biobased Chemicals “Bio-based Chemicals – Value Added Products from Biorefineries” 3,6, “Green Building Blocks for Biobased Plastics and Biofuel-driven biorefineries 4 and ”A Selection of the most Promising Biorefinery Concepts to produce Large Volumes of Road Transportation Biofuels by 2025”.5 • Development of a “Biorefinery Fact Sheet” to document and report facts and figures of biorefineries in a common and compact format, consisting of a brief description, the classification scheme, mass and energy balance as well as a whole value chainbased sustainability assessment in comparison to conventional systems.7

2. THE DEFINITION FOR BIOREFINERY IEA Bioenergy Task 42 has developed the following definition for biorefinery as depicted in Figure 1.1: Biorefinery is the sustainable processing of biomass into a spectrum of marketable products and energy.

This means that biorefinery can be a facility, a process, a plant, or even a cluster of facilities. The IEA Bioenergy Task 42 has produced a brochure that gives an overview of the different kinds of biorefineries.7 The brochure illustrates at which scale (commercial, demonstration, or pilot) these biorefineries are currently operational. A main driver for the establishment of biorefineries is the sustainability aspect. All biorefineries should be assessed for the entire value chain on their environmental,

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Figure 1.1 Biorefinery and its role in the transformation of biomass.

economic, and social sustainability covering the whole life cycle (construction— operation—dismantling). This assessment should also take into account the possible consequences due to the competition for food and biomass resources, the impact on water use and quality, changes in land-use, soil carbon stock balance and fertility, net balance of GHGs, impact on biodiversity, potential toxicological risks, and energy efficiency. Impacts on international and regional dynamics, end users and consumer needs, and investment feasibility are also important aspects to take into consideration. As the sustainability assessment is not an absolute number, the sustainability assessment is made in comparison to conventional systems providing the same products and services. A biorefinery is the integral upstream, midstream, and downstream processing of biomass into a range of products. In the classification system IEA Bioenergy Task 42 (described in the next chapter) has differentiated between mechanical pretreatments (extraction, fractionation, separation), thermochemical conversions, chemical conversions, enzymatic conversions, and microbial (fermentation both aerobic, anaerobic) conversions. A biorefinery can use all kinds of biomass from forestry, agriculture, aquaculture, and residues from industry and households including wood, agricultural crops, organic residues (both plant and animal derived), forest residues, and aquatic biomass (algae and seaweeds). A biorefinery is not a completely new concept. Many of the traditional biomass converting technologies such as the sugar, starch, and pulp and paper industry can be (partly) considered as biorefineries. However, several economic and environmental drivers such as global warming, energy conservation, security of supply, and agricultural policies have also directed those industries to further improve their operations in a biorefinery manner. This should result in improved integration and optimization aspects of all the biorefinery subsystems.


A biorefinery should produce a spectrum of marketable products and energy. The products can be both intermediates and final products, and include food, feed, materials, and chemicals; whereas energy includes fuels, power, and/or heat. The main focus of biorefinery systems which will come into operation within the next years is on the production of transportation biofuels. The selection of the most interesting biofuels is based on the possibility that they can be mixed with gasoline, kerosene, diesel, and natural gas, reflecting the main advantage of using the already existing infrastructure in the transportation sector. The volume and prices of present and forecasted products should be market competitive. Biorefineries are expected to contribute to an increased competitiveness and wealth of the countries by responding to the need for supplying a wide range of biobased products and energy in an economically, socially, and environmentally sustainable manner. Biorefineries show promises both for industrialized and developing countries. New competences, new job opportunities, and new markets are also expected to elaborate. Furthermore, the development of biorefineries is expected to also contribute to the implementation of several European, North American, and global policies and initiatives. In principle two different motivations for biorefineries are distinguished in IEA Bioenergy Task 42: “product-driven” biorefineries, e.g., pulp and paper and “energy-driven” biorefineries, e.g., road transportation biofuels. The biorefinery definition demands that biorefineries should produce both nonenergetic and energetic outlets and applies to product-driven biorefinery processes that primarily generate biobased products (biomaterials, lubricants, chemicals, food, feed, etc.) and process residues that are almost always used to produce heat and power (for internal use or sale). In energy-driven biorefinery processes the biomass is primarily used for the production of secondary energy carriers (biofuels, power, and/or heat); process residues are used for heat and electricity, or are sold as feed in case of biodiesel and bioethanol in the current situation, or even better are upgraded to added-value biobased products to optimize economics and ecologics of the full biomass supply chain. Both primary products and energy-driven processes are considered as true biorefinery approaches provided that the final goal is the sustainable processing of biomass. Product volumes and prices should be competitive, so their market value should be maximized.

3. THE ECONOMIC VALUE OF BIOMASS USING BIOREFINING The economic value of biomass is determined by the revenue from the various products on the market and the production costs (e.g., capital and operation costs) of the various products. In most of the cases products with a relative high market value are associated with high production costs, and vice versa. In addition, also the size of the market is relevant for the economic feasibility of biorefining. In most of the cases, products with a high market value have a relative small market (e.g., speciality chemicals) and vice versa (e.g., liquid

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Table 1.1 Fossil-derived product substitution options (cost price per GJ end product) Fossil feedstock Biomass cost cost (€/GJ) (€/GJ end product)

Heat Power Transportation fuel Average bulk chemicals

3 (coal) 6 (coal) 8 (oil) 30 (oil)

4 22 10 75

transportation fuels). Economic values of fossil feedstocks to be possibly substituted by biomass show large differences (Table 1.1).The lowest values are attributed to heat production, whereas the highest values are associated with replacement of fossil-derived bulk chemicals. Because heat is mainly produced from the cheapest fossil fuel (coal), the material costs for the production of 1 GJ of heat will amount to €3 (assuming a 100% conversion efficiency). On the other hand the costs to convert biomass feedstock to heat via combustion are quite low compared to various biorefinery processes. Feedstock costs for power production approximate €6/GJ; fossil transportation fuels feedstock cost being around €8/ GJ. Production of 1 GJ of bulk chemicals requires an additional average of 3–4 GJ of conversion energy (usually harvested from fossil oil or natural gas) which may provide considerable cost increases especially when the price of natural gas is linked to that of fossil oil. Consequently, feedstock costs for bulk chemicals are estimated at €30/GJ.8,9 Comparing fossil with biomass cost prices reveals that high capital costs are found in power production and chemical synthesis processes. In theory, the former could be circumvented by directly converting biomass to power. Capital costs of producing chemical compounds could be seriously reduced by directly obtaining (most of) the required molecular structures from biomass. In such cases the economic value of biomass feedstocks grossly exceeds the value associated with their caloric value (which is only €3/ GJ). They could represent values of up to €75/GJ, provided that components could be obtained in a pure form. Assuming a biomass yield of 10–20 tonnes of dry weight per hectare per year and that the biomass will just be used for its caloric value, this would represent a value of €450–900/ha per year, values that are too low for farmers in Western Europe to make an acceptable standard of living. Things would be different if we could separate biomass into fractions that can be used to produce food, feed, biobased products (chemicals, materials), and/or bioenergy (fuels, power, and/or heat). As we saw above, separated biomass fractions can generate financial returns exceeding their caloric value alone. Assuming that 20% of biomass is suitable to produce chemical compounds, 40% to produce biofuels, and the remainder to produce power and heat, a biomass yield of 10–20 tonnes dry matter per hectare biomass yield potentially could generate €2000– 4000/ha, enough for farmers to make an acceptable standard of living.


4. CLASSIFICATION OF BIOREFINERIES In the past, biorefineries were classified based on a variety of different bases, such as: • Technological implementation status: conventional and advanced biorefineries; first, second, and third generation biorefineries. • Type of raw materials used: whole crop biorefineries (WCBRs), oleochemical biorefineries, lignocellulosic feedstock biorefineries, green biorefineries, and marine biorefineries. • Type of main intermediates produced: syngas platform biorefineries, sugar platform biorefineries. • Main type of conversion processes applied: thermochemical biorefineries, biochemical biorefineries, two platform concept biorefineries. Examples of those biorefineries can be found in Kamm and Kamm.10,11 However, an unambiguous classification system was lacking, but in 2008, IEA Bioenergy Task 42 developed a more appropriate biorefinery classification system.2,12,13 This system is based on a schematic representation of full biomass to end-products chains.The background for this biorefinery classification system is the current main driver in biorefinery development, i.e., efficient and cost-effective production of transportation biofuels, to increase the biofuel share in transportation sector, whereas for the coproduced biobased products additional economic and environmental benefits are gained. The classification approach consists on four main features which are able to identify, classify, and describe the different biorefinery systems, viz.: platforms, products (energy and biobased materials and chemicals), feedstocks, and conversion processes (Figure 1.2). The platforms (e.g., C5/C6 sugars, syngas, biogas) are intermediates which are able to connect different biorefinery systems and their processes. Platforms can also be already a final product. The number of involved platforms is an indication of the system complexity. The two biorefinery product groups are energy (e.g., bioethanol, biodiesel, synthetic biofuels) and products (e.g., chemicals, materials, food, and feed). The two main feedstock groups are “energy crops” from agriculture (e.g., starch crops, short rotation forestry) and “biomass residues” from agriculture, forestry, trade, and industry (e.g., straw, bark, wood chips from forest residues, used cooking oils, waste streams from biomass processing). In the classification system a differentiation was made between four main conversion processes, including: biochemical (e.g., fermentation, enzymatic conversion) (red squares), thermochemical (e.g., gasification, pyrolysis) (yellow squares), chemical (e.g., acid hydrolysis, synthesis, esterification) (blue squares), and mechanical processes (e.g., fractionation, pressing, size reduction) (white squares) (Figure 1.2). The biorefinery systems are classified by quoting the involved platforms, products, feedstocks, and—if necessary—the processes. Some examples of classifications are the following: • Oil biorefinery using oilseed crops for biodiesel, glycerin, and feed (Figure 1.7) • C6 sugar platform biorefinery for bioethanol and animal feed from starch crops

9

10 Industrial Biorefineries and White Biotechnology

Figure 1.2 Network on which the biorefinery system classification method is based.


• Syngas platform biorefinery for Fischer–Tropsch (FT) diesel and phenols from straw • C5 and C6 sugars, electricity and heat, lignin biorefinery using wood chips for bioethanol, electricity, heat, and phenols (Figure 1.8) A full overview of the platforms, products, feedstocks, and conversion processes is given in Figure 1.2.

5. CONVENTIONAL BIOREFINERIES Biorefining is not a new activity: production of vegetable oils, beer and wine requiring pretreatment, separation and conversion techniques developed thousands of years ago, and a Chinese official started paper production around AD 100. Industrial biorefining was initiated by the introduction of steam-driven paper machines in the nineteenth century. Most innovations are, however, related to developments in food production: crystalline sugar, potato starch (early and mid-nineteenth century), wheat and corn starch (early twentieth century) and, recently, soy oil, proteins, and vitamins. Industrial processing techniques, developed in Europe and North America, are applied worldwide and serve as examples of biorefining evolvement. Some are discussed here. Industrial potato starch production, sparked by the initiatives of the successful Dutch entrepreneur Scholten in 1839, was facilitated by the availability of clean water, good agricultural land and cheap transportation through canals (constructed for peat winning). He copied his first factory over 50 times in Dutch, German, and Polish agricultural areas, to be followed by many competitors including farmers cooperatives suffering from artificially reduced potato prices.14 Next to (modified) starch, they generated a range of products including thermoplastic starch-based biopolymers. Coproduct development was provoked by factory concentrations following Dutch legislation that demanded wastewater cleaning which thus far was fed into canals causing foam and odor production. The subsequent consolidation into larger plants facilitated the development of coproducts such as high-value protein for human consumption. To achieve this, an innovative process was developed to isolate high-quality native proteins from potato fruit juice. The protein fractions have all novel and unique properties for applications in food, cosmetics, and pharmaceuticals. Potato fibers initially used for animal feed, are now used as feedstocks for the production of higher value food products. Ethanol from fermentation of potato starch (called “Spiritus” or Kartoffelsprit in German) has not only been used to make vodka but was also blended up to 25% into transportation fuel until the 1950s.19 Modern European sugar production started when a British blockade of Napoleonistic France in 1810, provoked the search for feedstocks to replace sugar imports from the Caraibics. Already in 1801, Franz Achard had processed 250 tonnes of beet into crystalline sugar in Germany, introducing processing steps (extraction, filtration, evaporation, crystallization, centrifugation) that currently still are used.15 The process also yielded

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molasses and residual sugar that later served as feedstocks for industrial yeast production after 1840, and still later for ethanol production. Beet pulp continues to serve as a valuable component in cattle feed. Soybeans gained importance after World War II to substitute protein foods and generate edible oil.Today, soy is a leading crop in the USA, while Brazil, Argentina, and Paraguay are important exporting nations. Oil production starts with the cracking of the beans, adjusting their moisture content, rolling them into flakes, and extracting the oil with hexane. It is subsequently refined and blended, remaining husks being used as animal feed. Soybeans are used in many food products (margarines, butter, vegetarian burgers), as a source of vitamin E, in industrial products (oils, soap, cosmetics, inks, clothing), and—increasingly—as biodiesel feedstock.9

6. ADVANCED BIOREFINERIES Additional biorefineries may be introduced in a variety of market sectors in the short term (up to 2020) by the upgrading and extension of existing industrial infrastructures. New biorefinery concepts highlighted in this paragraph are, however, still mostly in the R&D, pilot or small-scale demonstration phase with commercialization being further away. It is expected that these new concepts will be implemented in the market in the medium term (2015–25) in different countries16 although current economic conditions (relatively low oil prices, credit crisis, and recessions in parts of the global economy) might cause severe delays in their market implementation of some of the biorefinery concepts. The most important concepts of the advanced biorefineries are discussed below.

7. WHOLE CROP BIOREFINERY In a WCBR, grain and straw fractions are processed into a portfolio of end products. It encompasses “dry” or “wet” milling and consequent fermentation and distilling of grains (wheat, rye, or maize). Wet milling starts with water-soaking the grain adding sulfur dioxide to soften the kernels and loosen the hulls, after which it is ground. It uses wellknown technologies and allows separation of starch, cellulose, oil, and proteins. Dry milling grinds whole grains (including germ and bran). After grinding, the flour is mixed with water to be treated with liquefying enzymes and, further, cooking the mash to breakdown the starch. This hydrolysis step can be eliminated by simultaneously adding saccharifying enzymes and fermenting yeast to the fermenter (simultaneous saccharification and fermentation). After fermentation, the mash (called beer) is sent through a multicolumn distillation system followed by concentration, purification, and dehydration of the alcohol. The residue mash (stillage) is separated into a solid (wet grains) and liquid (syrup) phase that can be combined and dried to produce “distiller’s dried grains


with solubles” (DDGS), to be used as cattle feed. Its nutritional characteristics and high vegetable fiber content make DDGS unsuited for other animal species and extension to the more lucrative poultry and pig feed markets continues to be a focus to create extra value for the DDGS fraction. The straw (including chaff, nodes, ears, and leaves) represents a lignocellulosic feedstock that may be further processed (see subsection “Lignocellulosic Feedstock Biorefinery”).

8. OLEOCHEMICAL BIOREFINERY An oleochemical biorefinery can be considered as a special example of a WCBR which combines biodiesel production with that of high added value vegetable oil-based products (Figure 1.7). It uses fatty acids, fatty esters, and glycerol from oil crops to produce the so-called platform (basic) chemicals, functional monomers, lubricants, and surfactants.17,18 Altering lipid profiles by breeding or improved crop management could provide new chemical functionalities thus increasing added value of industrial oilseed crops. In the long run, oleochemical biorefining might produce renewable feedstocks for fossil-based chemical refineries. The success of a biorefinery will ultimately depend on its integration with its existing fossil counterparts, and building blocks of oleochemical biorefineries are offering a neat interface. The NExBTL process of Neste Oil19,20 demonstrates how fossil and biorefineries might interact. Precursor feedstocks used to produce vegetable oil-based products also contain substantial amounts of lignocellulosic biomass, which can be used in a lignocellulosic feedstock biorefinery.

9. LIGNOCELLULOSIC FEEDSTOCK BIOREFINERY Lignocellulosic feedstock biorefinery encompasses refining lignocellulosic biomass (wood, straw, etc.) into intermediate outputs (cellulose, hemicellulose, lignin) to be processed into a spectrum of products and bioenergy.2,21 Lignocellulosic biomass is expected to become the future’s most important source of biomass and be widely available at moderate costs showing less competition with food and feed production. Below, different types of lignocellulosic feedstock biorefineries will be discussed. Lignocellulosic biomass is treated with among others acid or alkaline agents to release cellulose, hemicellulose, and lignin, the former being further converted with (enzymatic) hydrolysis into mainly glucose, mannose (C6), and xylose (C5).21 These C6 and sometimes C5 sugars are currently predominantly used as feedstock for fermentation to produce biofuels (ethanol, butanol, hydrogen) and/or added-value chemicals, lignin being applied for combined heat and power production to be used internally or sold. Future lignin applications include added-value chemicals such as phenolic components or composites21,22 while C6 and C5 sugars can also be used as feedstock for chemical catalytic conversions.3,21 The forest-based biorefinery encompasses full integration of biomass and

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other feedstocks (including energy) for simultaneous production of pulp (paper) fibers, chemicals, and energy.23,24 The pulp and paper industry25 can be considered as the first nonfood biorefinery, value-added coproducts including tall oil, rosin, vanillin, and lignosulfonates. Pulp and paper companies in industrialized countries are currently suffering from decreased demand in some sectors (e.g., newsprint), rising costs and increased competition from emerging countries, and production of value-added coproducts from underutilized streams and waste materials provide a viable survival strategy.The European Forest-based Technology Platform has defined research options for zero-waste woodbased biorefineries,26 and suggested that pulp mills produce bioproducts and biofuels from forest-based biomass and mill residues using advanced fractionation and conversion followed by sugar or syngas routes. Lignin, the most abundant by-product, has unique prerequisites to produce chemical platforms for renewable polymers, specialty chemicals, materials, and high-quality fuels.

10. SYNGAS PLATFORM BIOREFINERY (THERMOCHEMICAL BIOREFINERY) In this biorefinery type, lignocellulosic biomass is pretreated (size reduction, drying, and/ or torrefaction) to allow high-temperature and high-pressure entrained flow gasification into synthesis gas of mainly CO and H2.The syngas is cleaned in a high-temperature gas cleanup system, often applying steam reforming to modify its CO/H2 ratio following downstream synthesis requirements. The clean gas can be used to produce biofuels and/ or chemicals (FT diesel, dimethylether), a range of alcohols including bioethanol; and/ or a variety of base chemicals (ethylene, propylene, butadiene, etc.) using catalytic synthesis processes.27

11. NEXT GENERATION HYDROCARBON BIOREFINERY The essential role of chemistry, chemical catalysis, thermal processing, and engineering in the conversion of lignocellulosic biomass into green gasoline, green diesel, and green jet fuel was stressed in a National Science Foundation and the Department of Energy workshop held in 2007. While it took years of research and design to develop the modern petroleum industry,27 a similarly expansive and sustained effort is required to develop hydrocarbon biorefineries. Advances in nanoscience provide unprecedented options to control molecular chemistry and promises to accelerate development of biomass-to-fuels production technologies. Expertise of the chemistry, catalysis, and engineering communities—earlier instrumental in the development of fossil refining— is required for the rapid development of cost-effective hydrocarbon biorefineries. However, recent history with the companies Choren, Range Fuels, and Kior has taught that economically scaling up this technology is not straightforward.


Liquid phase catalytic processing is a promising biorefinery process that produces functionalized hydrocarbons from biomass-derived intermediates (e.g., intermediate hydroxymethylfurfural or HMF). Renewable furan derivatives can be used as substitute building blocks for fossil fuels, plastics, and fine chemicals,28–30 or to develop biofuels based on C5 and C6 carbohydrates (sugars, hemicellulose, cellulose). Currently, Avantium Chemicals in the Netherlands is developing chemical catalytic routes to generate furanics for renewable polymers, bulk and specialty chemicals, and biofuels.31–33

12. GREEN BIOREFINERY The use of grassland for cattle production in Europe is on the decline; however, it is felt that continued grass cultivation is essential to preserve valuable grassland landscapes. Green biorefineries, feeding grass or other green/fresh biomass to a cascade of processing stages, offer an innovative alternative. Essential is the mechanical grass (“green biomass”) fractionation into a liquid phase containing water-soluble compounds (lactic acid, amino acids) and a solid phase mainly consisting of fibers.34,35 Overall economic efficiency of the biorefinery is mainly determined by the economic return of the fibers. Green biorefineries can use a wide range of biomass including sugar beet or other leaves, clover, or lucerne to generate a highly diverse range of products. Mixed feedstocks (e.g., fresh and silage grass) sometimes constitute an intermediate between green and lignocellulosic biorefineries. Dutch researchers developed a biorefinery for grass and other leaf material (alfalfa, beet, etc.), costs for grass (€70–80/tonne) exceeding those of leaves (€50–70/tonne). Fibers (representing 30% of the products by weight) were valued at around €100/tonne, other components at an average of €800/tonne of dry grass, making the use of grass in potential very cost-effective.36 Fractionation of grass appeared, however, to be cumbersome, and therefore costly. So major improvements should be achieved in this area. The central part of the green biorefinery is a mechanical refiner37 where leaf material is broken so that fibers can be obtained in a rather pure form (containing less than 11% of the protein). The protein is recovered from the press-juice after heat coagulation and a separation step; the rest of the juice is concentrated by evaporation. Main products are proteins to be used as pig and poultry feed; fibers for building materials, insulation material, plant pots, biocomposites, packaging material and biofuel feedstock; and soluble components like amino acids (polymeric), sugars, organic acids, and minerals. Solubles are concentrated to be used as feed component or fermentation feedstock.9 European green biorefinery projects are running in Austria, Germany, Ireland, and the Netherlands, most emphasis being put on grass refining.9,34,35 The starting point is zero-waste and zero-emission extraction of valuable substances, all residues to be used in a biogas plant to realize energetically self-sufficient operation of the plant.

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13. MARINE BIOREFINERY The net global primary biomass production is equally divided between terrestrial and aquatic systems. So far, policies have focused mainly on terrestrial biomass, while marine sources like microalgae (diatoms: green, golden, and blue/green algae), and macroalgae (brown, red, and green seaweeds) and their derived products could provide a potential that is still not yet fully known. Diatoms are the dominant phytoplankton life form, probably representing the largest biomass potential on Earth, covering an estimated 100,000 species that often accumulate oils. Algae can, depending on species and growing conditions, accumulate significant amounts of oils, carbohydrates, starch, and vitamins. Green algae are a rich source of starch and oils, golden algae producing oils and carbohydrates. Marine crops have long been recognized for their GHG abatement potential, their ability to absorb CO2 possibly exceeding that of terrestrial species. More recently, they have been recognized as a potential source of biofuel feedstocks.9 However, cost of production/harvesting of biomass in all marine biorefineries is currently still too high to be a viable option for fuels and bulk chemicals applications.

14. CHAIN DEVELOPMENT Biorefineries can (under certain conditions) disregard economies of scale.38 Limitations in optimal plant size are caused by feedstock transportation needs: larger plants demanding larger distances to fulfill feedstock requirements year round. Long transportation distances are especially harmful for feedstocks with high concentrations of water (transport of which is expensive but not effective), minerals, or organic components (required to maintain local soil quality). In contrast to fossil feedstocks, that can generally be recovered following the exact timing of its demand (natural gas, often a by-product of oil production, being the exception to the rule), most biomass types (wood being the exception) are harvested only during a relatively short period of the year. Year round biomass availability requires expensive storage facilities, while crops with high water concentrations cannot be stored over long periods. Biorefinery systems should be designed in such a way that capital intensive operations can continue year round in central plants; collection, separation, and storage can be decentralized. By doing so, minimal investments and energy use are required to recycle minerals and soil components back to the fields. Specific fractions could then be transported to alternative biorefineries, further processing intermediate products derived from a range of crops. This enables robust multi-input single-output systems that can withstand fluctuations in harvested volume as well as price variations, varying the use of given crop components depending on market demand. Decentralized pretreatment units, further, allow efficient waste heat recovery generated by (fossil or) biomass sources, which often is not possible in central power generation facilities,


while also offering improved living conditions to rural areas and perspectives for developing economies.9 Decentralized preprocessing does, however, require additional capital and labor costs. This drawback can be overcome by improving the overall economics of the production chain by • Process automatization and telecontrol of the process, limiting labor inputs required for continuous process supervision. • Some steps are no longer required as mentioned above for recycling of the minerals. In the past this trade-off was never made because the waste products from the traditional biorefineries could be discarded at low cost, often without any treatment. Later governments ordered companies to cope with these environmental problems often at very high economic and energy costs. • If expensive equipment can be used year round, capital costs per unit product are considerably reduced as compared to seasonal operations such as potato starch, beet sugar, cane sugar, and cassava production. • Choice of unit operations that have low advantages of economies of scale. In many traditional biorefineries the very large volumes that are processed often result in the duplication of equipment because larger equipment cannot be built because of physical limitations. Sometimes one has the choice to use unit operations that show only small economy of scale benefits such as the usage of membrane processes instead of evaporation using heat for concentration purposes. Another strategy could be to convert the desired components in intermediates that can be recovered by crystallization/precipitation or even to leave the component in the process water and subsequently convert these components to biogas that can be used on site or fed to the grid.

15. BIOREFINERY CONCEPTS IN COMPARISON TO PETROCHEMICAL REFINERIES The production of biobased products could generate US$10–15 billion of revenue for the global chemical industry.3 The potential for chemical and polymer production from biomass has been comprehensively assessed in several reports and papers. In 2004, the US Department of Energy issued a report which listed 12 chemicals which it considered as potential building blocks for the future.39 This list was reviewed and updated in 2010.40 The economic production of transportation biofuels is often a challenge.The coproduction of chemicals, materials, food, and feed can generate the necessary added value. Recently a paper was published highlighting all biobased chemicals with immediate potential as biorefinery “value-added products”. The selected products are either demonstrating strong market growth or have significant industry investment in development and demonstration programs.3

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Often a comparison is made between traditional petrochemical refineries and biorefineries. Table 1.2 gives an overview of the major similarities and dissimilarities of petrochemical refineries and biorefineries while Figures 1.3 and 1.4 give an overview of the base petrochemicals and their major applications41 as well as the updated top 12 building blocks derived from biomass.39,40 Table 1.2 and Figures 1.3 and 1.4 clearly illustrate that starting materials, processes, and products are quite different. Table 1.2 Comparison of refineries and biorefineries regarding to feedstocks, building block composition, processes, and chemical intermediates produced at commercial scale Refinery Biorefinery

Feedstock

Feedstock relatively homogeneous

Low in oxygen content The weight of the product (mole/mole) generally increases with processing Some sulfur present Sometimes high in sulfur Building block composition (Bio)chemical processes

Chemical intermediates produced at commercial scale

Main building blocks: Ethylene, propylene, methane, benzene, toluene, xylene isomers. Almost exclusively chemical processes Introduction of heteroatoms (O, N, S) Relative homogeneous processes to arrive to building blocks: Steam cracking, catalytic reforming Wide range of conversion chemistries Many

Feedstock heterogeneous regarding bulk components e.g., carbohydrates, lignin, proteins, oils, extractives, and/or ash Most of the starting material present in polymeric form (cellulose, starch, proteins, lignin) High in oxygen content The weight of the product (mole/ mole) generally decreases with processing It is important to perceive the functionality in the starting material Low sulfur content Sometimes high in inorganics, especially silica Main building blocks: Glucose, xylose, fatty acids (e.g., oleic, stearic, sebacic) Combination of chemical and biotechnological processes Removal of oxygen Relative heterogeneous processes to arrive to building blocks Smaller range of conversion chemistries: Dehydration, hydrogenation, fermentation Few but increasing (e.g., ethanol, furfural, biodiesel, mono-ethanolglycol, lactic acid, succinic acid, …)

Biorefinery Concepts in Comparison to Petrochemical Refineries Naphtha and gas (petroleum)

BTX

p-Xylene 27.6 Mton/a

O

Toluene 18.4 Mton/a

Polybutadiene, rubbers

OH Benzene derivatives O

Foam polyuretanes O OH Terephtalic acid 22 Mton/a

NH Styrene 23 Mton/a

Caprolactam 3.2 Mton/a

O O

OH

O

HO O

Nylon 6

Acetone 5.1 Mton/a

Nylon 6.6

Phenol 8 Mton/a

Resins and chemicals

Bisphenol A 4 Mton/a

Plastics and polymers

Propylene derivatives OH

Polypropylene

HDPE, LDPE, LLDPE Ethylene derivatives

N Acrtylonitrilc 4.7 Mton/a Fibers, plastics, resins

HO CI

OH Ethylene glycol 17.8 Mton/a

Vinyl chloride 33 Mton/a

PET, antifreeze

Polyvinil chloride (PVC)

OH

OH Adipic acid 2 Mton/a

O

Propylene 71

1,4-butanediol 1 Mton/a O

Resins and chemicals

Solvent and chemicals

HO

OH

HO

Maleic anhydride 1.7 Mton/a Solvent and plastics

Polyesters fibers and films Polystyrene and resins

Ethylene 112 Mton/a

Butadiene 9.3 Mton/a

Benzene 36.5 Mton/a

O Ethylene oxide 2.85 Mton/a

Polyesters

OH Propylene glycol 1.2 Mton/a

Solvent O

OH

OH Isopropyl alcohol 1.8 Mton/a

Acrylic acid 1.6 Mton/a

Solvent and chemicals

Solvent

CI

O

Allyl chloride 0.33 Mton/a

Propylene oxide 4.8 Mton/a

Epoxy resins

Polyols and chemicals

Figure 1.3 Base petrochemicals, major applications, and global production in 2009.41

Figure 1.4 Proposed biobased platform molecules.39,40

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However, it might be still very attractive to integrate biomass processing in traditional refineries as a way to upgrade conventional refineries and this represents a modern systems version of a retrofit problem. Examples include the production of “green biodiesel,” the NExBTL process, and the catalytic cracking of pyrolytic lignin. Green biodiesel (Petrobras/H-BIO, UOP with ENI) is produced widely with the hydrogenation of plant oils (or animal fat) using hydrogen available at the refinery. Fortum Oil Oy uses the proprietary NExBTL process to produce an isoparaffinic fuel (not FAME) by hydrodeoxygenation (or catalytic hydrotreatment of vegetable oils or animal fats) still compatible with existing diesel engines (capacities range from 170 to 800 kT/year).41 Currently, Fortum Oil produces more than 2 million metric tonnes per year equivalent to 675 million gallons per annum distributed from its three worldwide facilities in Porvoo, Finland; Rotterdam, the Netherlands, and Singapore.42 The oil and syngas platforms in particular represent a number of opportunities of processing biomass or biomass-derived intermediates by utilizing existing petrochemical facilities, such as oil cracking, hydrotreating, gasification, and chemical synthesis. The resulting products include gasoline, diesel, olefins, alcohols, acids, waxes, and many other commodity chemicals derivable from syngas.41 The systematic development of such integrated scenarios could use a systems approach to differentiate between available feedstocks (biomass and fossil), processing routes (biomass, petrochemical refinery), and available chemicals. This comparator could produce scenarios for integration badly needed in reviewing the numerous options available in practice.41 Insofar biorefineries create a process chain that adds biomass as a resource alternative to coal, crude oil, or natural gas in order to create C2-, C3-, or C4-base chemical platforms.43 In principle, fossil and renewable resources can substitute each other and historically such replacements, more particularly the substitution of coal by crude oil and natural gas, have occurred. It is also important to keep in mind that crude oil and natural gas differ significantly in composition, depending on the origin (Tables 1.3 and 1.4).The increased usage of shale gas changes the ratio between C2, C3, and C4 building blocks produced and might create extra potential for biomass-derived C4 building blocks (e.g., succinic acid and butanediol).

Table 1.3 Typical approximate characteristics and properties and gasoline potential of various typical crude oils44 Crude source and Paraffins Aromatics Naphthenes Sulfur name % vol % vol % vol % wt

Nigerian light Saudi light Saudi heavy Venezuela heavy

37 63 60 35

9 19 15 12

54 18 25 53

0.2 2 2.1 2.3

Table 1.4 Typical approximate composition of natural and oil processing gases (percent by volume44) Type gas H2 CH4 C2H6 C3H4 C3H8 C3H6 C4H10

N2+CO2

C5+

n/a n/a

98 42

0.4 20

n/a n/a

0.15 17

n/a n/a

0.05 8

n/a n/a

1.4 10

n/a 3

5–6 12 n/a

10 5–7 74,2

3–5 5–7 15,6

3 16–18 5,5

16–20 0.5 n/a

6–11 7–8 2,1

42–46 0.2 n/a

5–6 4–5 n/a

n/a n/a 2,1

5–12 2–3


Natural gas Petroleumassociated gas Oil processing gases Catalytic cracking Pyrolysis Shale gas (typical)45

C4H8

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Biomass, compared to fossil feedstock, is first and foremost distinguished by the high content in oxygen (Table 1.5). The above biobased platform molecules (Figure 1.4) can be synthesized via chemical or biochemical manufacturing technologies. Biochemical technologies often use glucose as nutrient solution. By alcoholic fermentation, glucose is disintegrated yielding the C2-Platform molecule ethanol. Fuel-grade bioethanol is rather easily dehydrated to ethylene using an Al2O3/MgO or a zeolite catalyst in a bioethanol-to-ethylene process. Bioethylene that way becomes an alternative to ethylene sourced from steam cracking of petroleum fractions, natural gas, or shale gas as the point of origin for the C2 product tree (Figure 1.5). Table 1.5 CHO composition of crude oil, fats and oils, and lignocellulosic biomass Animal fats and Lignocellulose Crude oil vegetable oils (wood)

Carbon Hydrogen Oxygen

85–90% 10–14% 0–1,5%

76% 13% 11%

50% 6% 43%

Figure 1.5 Most important product trees derived from ethylene.46

Biorefinery Concepts in Comparison to Petrochemical Refineries dimerization (Figure 3)

isomerization

H 2O

H 2O

OH

OH

metathesis FischerTropsch catalyst

metabolic engineering method?

CH3OH

fermentation fermentation

biomass - cellulose - sugar cane - glucose - lignin - triglycerides

catalytic cracking

fermentation

CO/H2 (syngas) OH

fermentation OH

fermentation or biodiesel production HO

(mixtures of hydrocarbons)

gasification

H2O

OH

OH

OH

H2 / catalyst H2O

Figure 1.6 Possibilities for synthesis of propylene from biomass using fermentation, gasification, or cracking strategies.49

Starting from renewable raw materials the C3-Platform can be accessed both by chemical as well as biochemical routes (Figure 1.6). With biodiesel setup on a firm and permanent basis as transportation fuel glycerol as by-product has become a marketrelevant commodity and nucleus of a plethora of value-added chemicals.3,18,47 Transportation fuel manufacturing by hydrotreatment of vegetable oils and animal fats produces propane as coproduct.19,20,48 Other manufacturing concepts for biopropylene and biopropane include glycerol dehydration to acrolein,46 gasification of biomass to produce a syngas followed by synthesis of biomethanol, and methanol-to-olefins technology to produce propylene,50,51 fermentation of sugars to produce bioethanol, followed by dehydration to bioethylene, dimerization of ethylene to produce normal butenes, which are reacted with bioethylene via metathesis to produce propylene,52 direct conversion of glucose to propylene using the same artificial metabolic pathway that is used to produce bio-isobutene.53 Another important C3 building block, lactic acid, can be produced through both fermentation of carbohydrates and chemical conversion starting from glycerol.43,54 Lactic acid can be dehydrated yielding acrylic acid, can be reduced to 1,2-propanediol or can undergo polycondensation to polylactic acid.55 The C4-Platform is accessible by fermentation using corn or sugarcane bagasse as feedstock by acetone–butanol or acetone–butanol–ethanol (ABE) fermentation using Clostridium acetobutylicum or Clostridium beijerinckii under anaerobic conditions. This process has been industry standard since decades and produces the three solvents in a ratio ABE = 3:6:1.43,56 More recently, microbial fermentation technologies which genetically

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alter Escherichia coli to generate several higher chain alcohols from glucose, including 1-butanol, 2-methyl-1-butanol, and more particularly isobutanol have been developed. Acid catalyzed dehydration would convert isobutanol into a mixture of C4 olefins (1-butene, cis-2-butene, trans-2-butene, and isobutene) which then convert to a mixture of unreacted isobutene and 1,3-butadiene in a catalytic dehydrogenation reaction at elevated temperature and low pressure well known from petrochemical process technologies. Carbohydrate-based C4-building blocks do not only substitute crude oil-derived crack C4 intermediates but complement the platform and provide new opportunities for C4-based polymers. This is in particular the case for succinic acid (butanedioic acid). Although the bulk of the actual industrial production of succinic acid is made by hydrogenation of maleic anhydride and subsequent hydration, nearly 0,5 Mio mto biosuccinic acid using sugar-containing feedstock including glucose syrup from hydrolyzed starch, grain sorghum, or corn steep liquor from wet-milling have been established or are under construction.57,58 A major outlet for succinic acid is its conversion into 1,4 butanediol. Because of the wide variety of possibilities for a biorefinery configuration a quick preliminary assessment of the (bio)chemical processes at the laboratory stage is very useful.59 The proposed method enables a review of the processes within a broader sustainability context. It is inspired by green chemistry principles, technoeconomic analysis and some elements of environmental life-cycle assessment. This method evaluates a proposed (bio) chemical process against comparable existing processes using a multicriteria approach that integrates various economic and environmental indicators. An effort has been made to incorporate quantitative and qualitative information about the processes while making the method transparent and easy to implement based on information available at an early stage in process development. The idea is to provide a data-based assessment tool for chemists and engineers to develop sustainable chemistry.59,60

16. BIOREFINERY COMPLEXITY INDEX As indicated before, currently many different biorefinery concepts are being developed and implemented. Some of these biorefinery concepts are simple, using one feedstock (e.g., vegetable oil) and producing two or three products (e.g., biodiesel, animal feed, glycerine) with current available commercial technologies. However, other biorefinery concepts are sometimes very complex using many different feedstocks (e.g., algae, miscanthus, and wood chips from short rotation) to coproduce a broad spectrum of different products (e.g., bioethanol, phenol, omega-3 fatty acids, biodiesel) using technologies that still need to become commercial in the upcoming years. It is concluded that each of these different biorefinery concepts has a different degree of complexity, which makes it difficult for industry, decision-makers, and investors to decide, which of these concepts are the most promising options on the short, medium, and long term, and to judge on the technological and economic risks.


Therefore, IEA Bioenergy Task 42 has published a working document to present the current status of an approach to develop a “Biorefinery Complexity Index (BCI)” and to calculate the BCI for some selected biorefinery concepts.61 The approach was developed since 2010 and started with the analogy to the “Nelson’s complexity index” used for oil refineries. The Nelson’s (complexity) index was developed by Wilbur L. Nelson and published in the “Oil and Gas Journal” (1960–61) to quantify the costs of the refinery’s components. The Nelson’s index is an indicator for the investment intensity, the cost index of the refinery, the value addition potential of a refinery, the refinery’s ability to process feedstocks, such as high-sulfur crude, into value-added products. The higher the complexity of the refinery the more flexible it is. Based on the classification system of biorefineries as discussed above and the “Nelson’s complexity index” for oil refineries a BCI is under development. The following basic assumptions on the complexity of a biorefinery are used: 1. The number of different features of a biorefinery influences the complexity. The complexity increases by the number of features in a biorefinery. 2. The state of technology of a single feature influences the complexity.The complexity decreases the closer a technology is to a commercial application, meaning a high “Technology Readiness Level (TRL)” of a feature has lower technical and economic risks, and so a lower complexity. 3. For the products and feedstock the “Market Readiness Level” is applied in analogy to the TRL of the processes and platforms. Therefore only the TRL is used. 4. This leads to the basic assumption for the calculation procedure of the BCI that the complexity is directly linked to the number of features and the TRL of each single feature involved. 5. This means that the complexity of a commercial application, which means that all features are commercially available, is then only determined by the number of features; whereas in noncommercial application the TRL increase additionally the complexity of the biorefinery system. For each of the four features (platforms, feedstocks, products, and processes) of a biorefinery the TRL can be assessed using level description between 1 (“basic research”) to 9 (“system proven and ready for full commercial deployment”). Based on the TRL the feature complexity (FC) for each single feature of a biorefinery is calculated. With the number of features and the FC of each single feature the Feature Complexity Index (FCI) for each of the four features (platforms, feedstocks, products and processes) is calculated. The BCI is the sum of the four FCIs. To simplify the presentation the Biorefinery Complexity Profile (BCP) is introduced.The BCP is a compact format to present the complexity of a biorefinery by giving the BCI and the four FCIs of each feature. The BCP, which includes the BCI and the four FCIs has the following format: BCP: BCI(FCIplatforms/FCIFeedstocks/FCIProducts/FCIProcesses), with an example 8 (1/1/3/3) for a 1-platform (oil) biorefinery using oilseed crops for biodiesel, glycerin, and feed

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Figure 1.7 A 1-platform (oil) biorefinery using oilseed crops for biodiesel, glycerin, and feed with a Biorefinery Complexity Profile of 8 (1/1/3/3).

(Figure 1.7). In Figure 1.8 a second generation biorefinery such as 3-platform (C5 and C6 sugars, electricity and heat, lignin) biorefinery using wood chips for bioethanol, electricity, heat, and phenols has a BCP of 29 (8/1/4/16) is shown.61 The following conclusions on the BCI and BCP were drawn: 1. They give an indication for the relative comparison of different biorefinery concepts and their development potential. 2. They present a benchmark of the “complexity” of a biorefinery in terms of involved platforms, feedstocks, processes, and products, and their specific and overall “Technology Readiness Level.” 3. The higher the BCI the more beyond “state of the art” is the biorefinery. 4. The BCI of a biorefinery producing biodiesel from vegetable oil which is fully deployed, with 8 (1/1/3/3) is a benchmark to compare the complexity of other current and future biorefinery systems. 5. The BCI will change in the future if the TRL has changed, e.g., if a pilot plant, demonstration plant, and/or first-of-a-kind commercial plant will go into operation. 6. The BCP shows the most relevant features contributing to the complexity of a biorefinery. 7. The BCP of a biorefinery gives an indication on the technological and economic risks.


Figure 1.8 A 3-platform (C5 and C6 sugars, electricity and heat, lignin) biorefinery using wood chips for bioethanol, electricity, heat, and phenols with a Biorefinery Complexity Profile of 29 (8/1/4/16).

The first results and conclusions of a critical review by the country representatives in IEA Bioenergy Task 42 show that the “Biorefinery Complexity Index” adds additional relevant information on the assessment and comparison of different biorefinery systems. It was concluded that the results are potentially relevant for industry, decision-makers as well as investors as additional information is generated to assist them in their strategies to implement the most promising biorefinery systems by minimizing technical and economic risks.

17. DISCUSSION AND CONCLUSIONS Successful market implementation of integrated biorefineries requires reliable processing units combined with environmentally acceptable and economically profitable production chains. Development and implementation of the biorefinery concept should include crop cultivation and the selection of crops that maximize full chain performance. Table 1.6 gives an overview of the different biorefineries and their development stage. It should be mentioned that although the sugar and starch biorefineries are in full-scale operation, their development will get a new input due to the biobased economy demands for new products and certainly for reduction of costs. Further biorefinery improvement

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Table 1.6 Overview of the main characteristics of the different biorefineries Predominant Phase of Concept Type of feedstock technology development

Conventional biorefineries

Starch (corn, wheat, cassava) and sugar crops (sugarcane, sugar beet), wood

Whole crop biorefineries

Whole crop (including straw) cereals such as rye, wheat and maize Oil crops

Oleochemical biorefineries Lignocellulosic feedstock biorefineries Green biorefineries

Marine biorefineries

Lignocellulosicrich biomass: e.g., straw, chaff, reed, miscanthus, wood Wet biomass: green crops and leaves, such as grass, lucerne and clover, sugar beet leaf Aquatic biomass: microalgae and macroalgae (seaweed)

Products (selection)

Pretreatment, chemical and enzymatic hydrolysis, catalysis, fermentation, fractionation, separation Dry or wet milling, biochemical conversion

Commercial

Sugar, starch, oil, dietary fibers, pulp and paper

Pilot plant (and Demo)

Starch, ethanol, distiller’s dried grains with solubles

Pretreatment, chemical catalysis, fractionation, separation Pretreatment, chemical and enzymatic hydrolysis, catalysis, fermentation, separation Pretreatment, pressing, fractionation, separation, digestion

Pilot plant, Demo, commercial

Oil, glycerin, cattle feed

R&D/Pilot plant (EC), Demo (USA)

Cellulose, hemicelluloses, lignin

Pilot plant (and R&D)

Proteins, amino acids, lactic acid, fibers

Cell disruption, product extraction and separation

R&D, pilot plant and Demo

Oils, carbohydrates, vitamins

is expected to generate more feedstocks, technologies, and coproducts, inevitably offering all kinds of economic opportunities. Research and development will speed up agricultural and rural development, increase industrial development, and open existing and newly created markets. It can be foreseen, however, that biorefinery technologies will develop gradually over time, because the more fractions are obtained the more markets should be served. All these markets dictate that raw materials and intermediates are available at a rather constant supply and therefore prices. The build up of this raw material supply will take time. The current status of biorefineries is exemplified in a strengths, weaknesses, opportunities, and threats analysis of biorefineries are presented in Table 1.7.


Table 1.7 Strengths, weaknesses, opportunities, and threats (SWOT) analysis on biorefineries

Strengths • Adding value to the sustainable use of biomass. • Maximizing biomass conversion efficiency— minimizing raw material requirements. • Production of a spectrum of biobased products (food, feed, materials, chemicals) and bioenergy (fuels, power, and/or heat) feeding the full biobased economy. • Strong knowledge infrastructure available to tackle both nontechnical and technical issues potentially hindering the deployment trajectory. • Biorefinery is not new, in some market sectors (food, paper…) it is common practice.

Weaknesses • Broad undefined and unclassified area. • Involvement of stakeholders of different market sectors (agro, energy, chemical…) over full biomass value chain necessary. • Most promising biorefinery processes/concepts not clear. • Most promising biomass value chains, including current/future market volumes/prices, not clear. • Studying and concept development instead of real market implementation. • Variability of quality and energy density of biomass.

Opportunities • Makes a significant contribution to sustainable development. • Challenging national, European and global policy goals—international focus on sustainable use of biomass for the production of bioenergy. • International consensus on the fact that biomass availability is limited so that the raw materials should be used as efficiently as possible—i.e., development of multipurpose biorefineries in a framework of scarce raw materials and energy. • International development of a portfolio of biorefinery concepts, including composing technical processes. • Strengthening of the economic position of various market sectors (e.g., agriculture, forestry, chemical, and energy).

Threats • Economic change and drop in fossil fuel prices. • Fast implementation of other renewable energy technologies feeding the market requests. • No level playing field concerning biobased products and bioenergy (assessed to a higher standard). • Global, national, and regional availability and contractibility of raw materials (e.g., climate change, policies, logistics). • (High) investment capital for pilot and demo initiatives difficult to find, and existing industrial infrastructure is not depreciated yet. • Fluctuating (long-term) governmental policies. • Questioning of food/feed/fuels (land use competition) and sustainability of biomass production. • Goals of end users often focused upon single product.

Biorefineries can provide a significant contribution to sustainable development, generating added value to sustainable biomass use and producing a range of biobased products (food, feed, materials, chemicals, fuels, power, and/or heat) at the same time. This requires optimal biomass conversion efficiency, thus minimizing feedstock requirements while at the same time strengthening economic viability of (e.g., agriculture, forestry, chemical and energy) market sectors. As biomass availability is limited, it should be used

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efficiently, effectively producing materials and energy in multi-purpose biorefineries. The use of the “Biorefinery Complexity Index/BCI” might add additional relevant information on the assessment and comparison of different biorefinery systems. It is concluded that the BCI is potentially relevant for industry, decision makers as well as investors as additional information is generated to assist them in their strategies to implement the most promising biorefinery systems by minimizing technical and economic risks. The perceived conflict between energy and food production can be allayed by developing technologies based on lignocellulosic materials but it was discussed before that this currently results in a much higher BCI. Biorefining requires further innovation but offers opportunities to all economic sectors. Building a biobased economy can help to overcome present difficulties while laying the foundation of an environmentally benign industry. One of the key prerequisites of a successful biorefinery is to invite key stakeholders from separate backgrounds (agriculture/forestry, transportation fuels, chemicals, energy, etc.) to discuss common processing topics, foster necessary R&D trajectories and stimulate deployment of developed technologies in multi-disciplinary partnerships. Optimal economic and environmental performance can be further guaranteed by linking the most promising biobased products, that is, food, feed, (fiber-based) added-value materials and (functionalized and platform) chemicals with bioenergy production.

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32. van Putten R-J, van der Waal JC, de Jong E, Rasrendra CB, Heeres HJ, de Vries JG. Hydroxymethylfurfural, a versatile platform chemical made from renewable resources. Chem Rev 2013;113:1499–597. 33. de Jong E, Dam MA, Sipos L, Gruter G-JM. Furandicarboxylic acid (FDCA), a versatile building block for a very interesting class of polyesters. In: Smith PB, Gross R, editors. ACS symposium series “biobased monomers, polymers and materials”. 2012. p. 1–13. http://dx.doi.org/10.1021/bk-2012-1105.ch001. 34. Mandl MG. Status of green biorefining in Europe. Biofuels Bioprod Biorefining 2010;4:268–74. 35. Schaffenberger M, Ecker J, Koschuh W, Essl R, Mandl MG, Boechzelt HG, et al. Green biorefinery – production of amino acids from grass silage juice using an ion exchanger device at pilot scale. Chem Eng Trans 2012;29:505–10. 36. Sanders JPM. Renewable resources and biorefineries. September 19, 2005. Gent http://www.rrbconference. ugent.be/presentations/sanders%20Johan.pdf. [accessed 15.06.09]. 37. Hulst AC, Ketelaars J, Sanders J. Separating and recovering components from plants. US patent 6740342; 1999. 38. Sanders JPM, Van der Hoeven DA, Van Dijk C. Voorwaartse integratie in de akkerbouw. Utrecht (the Netherlands): InnovatieNetwerk; 2008. ISBN: 978-90-5059-352-6. 39. Werpy T, Petersen G. Top value added chemicals from biomass, volume 1 results of screening for potential candidates from sugars and synthesis gas. 2004. [Online 2004]. Available at: http://www1.eere.energy.gov/bio mass/pdfs/35523.pdf. 40. Bozell JJ, Petersen GR. Technology development for the production of biobased products from biorefinery carbohydrates – the US Department of Energy’s “Top 10” revisited. Green Chem 2010;12:539–54. 41. Kokossis AC, Yang A. On the use of systems technologies: a systematic approach for the synthesis and design of future biorefineries. Comput Chem Eng 2010;34(9):1397–405. 42. Neste Oil. NExBTL® Renewable diesel singapore plant tallow pathway description. http://www.arb. ca.gov/fuels/lcfs/2a2b/apps/neste-aus-rpt-031513.pdf [accessed 17.08.14]. 43. Pascault J-P, Höfer R, Fuertes P. Mono-, di, and oligosaccharides as precursors for polymer synthesis. In: Matyjaszewski K, Möller M, editors. Polymer science: a comprehensive reference. McGrath JE, Hickner MA, Höfer R, editors. Polymers for a sustainable environment and green energy, vol. 10. Amsterdam: Elsevier; Oxford: Waltham; p. 59–82. 44. Kraus RS. Exploration, drilling and production of oil and natural gas. In: Kraus RS, editor. 75. oil exploration and distribution. J.M. Stellman, editor-in-chief. Encyclopedia of occupational health and safety. Geneva: International Labor Organization; 2011. 45. Klaber KZ. Natural gas production in the Marcellus and Utica shales. In: Pittsburgh chemical day (Sept. 25) with reference to pace Global’s NiSource Gas Transmission and Storage presentation to WVONGA (May 6, 2010). 2012. 46. Bader HJ, Horn S, Fehrenbacher U, Grosshardt O, Kowollik K, Pohsner U, et al. Informationsserie Nachwachsende Rohstoffe 2009. 64 p. https://www.vci.de/vci/Downloads-vci/textheft_farbig.pdf. 47. Pagliaro M, Rossi M. The future of glycerol. 2nd ed. Cambridge: RSC Green Chemistry No. 8, RSC Publ.; 2010. 48. Huber GW, O’Connor P, Corma A. Processing biomass in conventional oil refineries: production of high quality diesel by hydrotreating vegetable oils in heavy vacuum oil mixtures. Appl Cat A Gen 2007;329:120–9. 49. Brandin J, Hulteberg C, Nilsson AL. Bio-propane from glycerol for biogas addition. Malmö: Rapport SGC 198, Svenskt Gastekniskt Center; 2008. 50. Keim W. C1 chemistry: potential and developments. Pure Appl Chem 1986;58:825–32. 51. Bare SR. Methanol to olefins (MTO): development of a commercial catalytic process. In: Modern methods in heterogeneous catalysis research, FHI lecture Nov. 30. 2007. http://www.fhi-berlin.mpg.de/acnew /department/pages/teaching/pages/teaching__wintersemester__2007_2008/bare_mto_301107.pdf. 52. Mathers RT. How well can renewable resources mimic commodity monomers and polymers? J Pol Sci A Pol Chem 2012;50:1–15. 53. Chayot R. Direct fermentation for isobutene, butadiene and propylene production: a highway to renewable plastics, synthetic rubber and fuels. In: AIChE proceedings metabolic engineering X. 2014. http:// www3.aiche.org/proceedings/Abstract.aspx?PaperID=354187. 54. Chen L, Ren S, Re XP. Lactic acid production from glycerol using CaO as solid base catalyst. Fuel Process Technol 2014;120:40–7.


55. Haas T, Kircher M, Köhler T,Wich G, Schörken U, Hagen R.White biotechnology. In: Höfer R, editor. Sustainable solutions for modern economies. Cambridge: RSC Green chemistry, No. 4, RSC Publ.; 2010. p. 436–74. 56. Qureshi N, Blaschek HP. Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101. J Ind Microbiol Biotechnol 2001;27:287–91. 57. de Guzman D. Chemical industry awaits for bio-succinic acid potential. ICIS Chemical Business; 2012. http:// www.icis.com/resources/news/2012/01/30/9527521/chemical-industry-awaits-for-bio-succinic-acidpotential/. 58. Cok B, Tsiropoulos I, Roes AL, Patel MK. Succinic acid production derived from carbohydrates: an energy and greenhouse gas assessment of a platform chemical toward a based economy. Biofuels Bioprod Bioref 2014;8:16–29. 59. Patel A, Meesters K, den Uil H, de Jong E, Blok K, Patel M. Sustainability assessment of novel chemical processes at early-stage: application to biobased processes. Energy Environ Sci 2012;5:8430–44. 60. Patel AD, Meesters K, den Uil H, de Jong E, Patel MK. Early stage comparative assessment of novel biobased processes. ChemSusChem 2013;6:1724–36. 61. Jungmeier G. The biorefinery complexity index. 2014. http://www.iea-bioenergy.task42-biorefineries.com/ upload_mm/6/2/f/ac61fa53-a1c0-4cbc-96f6-c9d19d668a14_BCI%20working%20document%2020 140709.pdf.

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CHAPTER 2

Algal Biorefineries Yanna Liang1, Tyler Kashdan2, Christy Sterner3, Lilli Dombrowski4, Ingolf Petrick4, Michael Kröger5, Rainer Höfer6 1Department

of Civil and Environmental Engineering, Southern Illinois University, Carbondale, IL, USA; 2Department of Advanced Energy and Fuels Management, Southern Illinois University, Carbondale, IL, USA; 3U.S. Department of Energy, Golden Field Office, Golden, CO, USA; 4Fakultät für Naturwissenschaften, Brandenburgische Technische Universität Cottbus-Senftenberg, Germany; 5DBFZ Deutsches Biomasseforschungszentrum, Leipzig, Germany; 6Editorial Ecosiris, Düsseldorf, Germany

Contents 1. Introduction 2. Algal Research in the USA 2.1 Aquatic Species Program 2.2 Recent Development

36 38 38 40

40 40

2.2.1 Legislation/Law 2.2.2 Planning

2.3 Conclusion 3. Macroalgae 3.1 Fundamentals Related to Macroalgae 3.2 Macroalgal Cultivation 4. Microalgae 4.1 Fundamentals Related to Microalgae 4.2 Microalgal Growth

46 46 46 47 48 48 49

49 50 51

4.2.1 Autotrophic Growth 4.2.2 Heterotrophic/Mixotrophic Growth 4.2.3 Algal Carbon Storage

4.3 Microalgal Culture Systems

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52 53

4.3.1 Open Ponds 4.3.2 Closed PBRs

5. Downstream Processes 5.1 Dewatering

55 55

55 56 57 58 58

5.1.1 Gravity Sedimentation 5.1.2 Flotation 5.1.3 Filtration 5.1.4 Centrifugation 5.1.5 Conclusions

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59 60 60 60

5.2 Drying 5.2.1 Drying by Solar Energy 5.2.2 Flash Dryers 5.2.3 Spray Dryers 5.2.4 Drum Dryers

Industrial Biorefineries and White Biotechnology http://dx.doi.org/10.1016/B978-0-444-63453-5.00002-1


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5.2.5 Conveyor Dryers 5.2.6 Freeze Dryers

61 61

5.3 Cell Disruption

61

62 63

5.3.1 Chemical Methods 5.3.2 Mechanical Methods

5.4 Extraction

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5.4.1 Solvent Extraction 5.4.2 Supercritical Fluid Extraction

5.5 Hydrothermal Processing 6. Products Produced from Algae at Commercial Scales 6.1 Traditional Uses of Macroalgae

67 69 69

70 71 72 72

6.1.1 Agar 6.1.2 Carrageenan 6.1.3 Funori 6.1.4 Alginates

6.2 Conventional Nonfuel Products from Microalgae

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6.2.1 Health Food 6.2.2 Polyunsaturated Fatty Acids 6.2.3 Squalene 6.2.4 Pigments

6.3 Nonconventional Products from Microalgae

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6.3.1 Biodiesel 6.3.2 Bioethanol 6.3.3 Terpene-Based Biofuels

7. Conclusions 83 References84

1. INTRODUCTION The use of algae dates back to thousands of years when people in China, Chad, and Mexico consumed cyanobacterium Nostoc, Arthrospira (Spirulina), and Aphanizomenon for food.1 Algal cultivation started with macroalgae “nori” (Porphyra) in 1640. Eighteen years later, agar production in Japan was reported.2 During the eighteenth until the early twentieth century, brown algae were processed to produce iodine and soda. Monoculture of microalgae Chlorella vulgaris was first achieved in 1890. In the early 1950s, as a result of searching for new alternative food and protein sources to address increase in world population, research on algae was broadly initiated. At the same time, mixed algal cultures were used to treat wastewater in a shallow, mechanically mixed, raceway-type open pond at University of California, Berkeley.3 Commercial large-scale culture of Chlorella was started in early 1960s in Japan by Nihon Chlorella. In the early 1970s, a facility in Lake Texcoco by Sosa Texcoco (Ecatepec, Estado de México) was established to culture and harvest Arthrospira.

Algal Biorefineries

By 1980, there were 46 large-scale factories in Asia producing more than 1000 kg of microalgae (mainly Chlorella).4 In the following years, large-scale production of microalgae was started in Australia, USA, Israel, and India. From 1978 to 1996, the US Department of Energy’s (DOE’s) Office of Fuels Development funded the Aquatic Species Program (ASP).5 The focus was to produce biodiesel from high lipid-content algae which grow in ponds and utilize waste CO2 from coal fired power plants. Though tremendous advances were made in the science of manipulating algal metabolism and the engineering of microalgae production systems, this program was terminated after two decades. The main reason was that it was not economically feasible to produce biodiesel from the desired route. Of course, compared with a gasoline price of $1.23/gallon in 1996, biodiesel produced from microalgae was not going to be viable. During recent years, starting from around 2005, however, algal research was resurrected. This is mainly due to several factors: the link between climate change and increased CO2 concentration in the atmosphere; exploding global demand for transportation fuels; concerns about “peak oil”; concerns about energy security owing to huge amount of oil import. In 2007, Chisti published a review paper: biodiesel from microalgae.6 In this paper, Chisti compared oil yield among soybean, corn, canola, jatropha, coconut, oil palm with microalgae. If corn oil was used to produce biodiesel to meet 50% of the all US’s transportation needs, then 846% of existing US cropping area was needed. Instead, if microalgal oil was the feedstock for making biodiesel, then only 2.5% of existing US cropping area was required. This calculation assumed that the oil content in microalgae was 30%.These numbers really fueled interests among a diverse population. From general public, scientists, engineers to government agencies, the enthusiasm to develop algal biofuel was extremely high. During the following years, a great number of start-up companies were founded to conduct algal research and development. However, as of now, commercial scale production of biofuel from microalgae has not been very successful though tremendous effort and money have been invested in this field. The businesses that are viable are those seeking to produce valuable products rather than fuels. To give a broad perspective on algal biorefinery, this chapter starts by describing the ASP program followed by recent funding provided by US DOE. It then introduces the basic physiology and chemistry of algae growth and cultivation for macroalgae and microalgae. For each group, following introduction of fundamental knowledge, methods for cultivation are provided. For microalgae which are the focus of this chapter, growth modes between autotrophic and heterotrophic, are presented and compared. Downstream processes for algae harvesting, concentrating and product extraction will also be provided. Finally, the description of products produced at commercial-levels from microalgae is divided into two big categories: nonfuels and fuels.

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2. ALGAL RESEARCH IN THE USA 2.1 Aquatic Species Program As part of a larger response to the energy crises of the early and mid-1970s, in 1978, the Carter Administration consolidated all federal energy activities under the newly established US DOE. The Administration also established what was then called the Solar Energy Research Institute (SERI) in Golden, Colorado, now called the National Renewable Energy Laboratory (NREL). SERI was a first-of-its-kind federal laboratory focused on the development of solar energy. As one of the various programs established to research all forms of solar energy, the Biofuels Program was initiated to investigate the use of plant life as a source of transportation fuels. In the same year, the Biofuels Program started the Aquatic Species Program (ASP) for developing renewable transportation fuels from algae.5 The ASP ran from 1978 to 1996. Early research (1978–1982) focused on hydrogen production from algae; however, in the early 1980s, the research focus switched to the production of biodiesel from algae. The ASP was led by NREL (SERI at that time) and included subcontracted research and development activities by private industry and universities around the country. The ASP’s primary focus was the production of biodiesel from high lipid content algae grown in open ponds utilizing waste CO2 from coal-fired power plants. With this in mind, the ASP systematically outlined the research into the following three main areas: 1. Applied Biology: collection, screening, characterization of high lipid producing strains as well as growth under extreme conditions (temperature, pH, and salinity); biochemistry and physiology of lipid product; and genetic engineering of algae; 2. Production Systems: validation of the open pond system for mass production (scales ranging from 100 g/L, >3 × seawater), produces β-carotene; (c) Haematococcus pluvialis, red color due to astaxanthin, a carotenoid used in aquaculture feeds and nutritional products; (d) Chlorella vulgaris, first microalgae produced commercially for human foods; (e) Amphora sp., as with most diatoms, requires large amounts of silicate, increasing production costs; (f) Nannochloropsis sp., grown in seawater, is now a popular species for biofuel/feed production; (g) Micractinium sp., grown in wastewater, can aggregate into large flocs (“bioflocculation”); (h) Botryococcus braunii, a unique hydrocarbon producing species (see oil droplets release); and (i) Anabaena cylindrica, a nitrogen-fixing cyanobacterium with potential for fertilizer production. Notes: (a–d) currently commercially grown species; (a) and (i) cyanobacteria (also known as blue-green algae); (b–d, g, h) green algae; (e) a diatom; and (f ) a Eustigmatophyte; (b, e, f) saltwater; and (c, d, g–i) freshwater or brackish water microalgae. (Adapted from Ref. 3.)

Microalgae have numerous commercial and industrial applications and use potentials, including production of food ingredients, bioplastics, chemical feedstock, pharmaceuticals, and algal fuel, and can also be used as a means of pollution control.

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6.2.1 Health Food 6.2.1.1 Chlorella

Chlorella is produced by more than 70 companies across the globe, from Taiwan, Japan, Germany, to USA (Table 2.8).The total annual mass of production is around 2000 dry tons with a market value of $44/kg (http://www.oilgae.com/non_fuel_products/chlorella. html).The largest producer has been Taiwan Chlorella Manufacturing Company (TCMC) (http://www.taiwanchlorella.com). This company was established in 1964 and has a goal to be “The Best Chlorella Manufacturer in the World.”TCMC grows Chlorella Sorokiniana, a freshwater green microalgae, in open ponds with an annual production of over 400 tons. Based on C. sorokiniana’s cellular composition which is rich in proteins, vitamins, minerals, Chlorella growth factor, and other beneficial substances, TCMC has developed a series of products ranging from Chlorella powder, Chlorella tablet to Chlorella extract fluid for use in the Asian cuisine. Algal flour with high percentage of accumulated algae oil/lipid as well as defatted algal flour which still contains proteins and carbohydrates have been developed as multifunctional food ingredients to be added to foods such as pasta or cookies, as a food supplement claimed to improve the nutritional quality of the daily diet. Chlorella can also be added to other foods including frozen desserts, cakes, sugar cookies, cheese, ice-cream, mayonnaise, pasta, and soft drinks.156 Additionally, and in order to avoid the unappealing green color, the unpleasant fishy or seaweed flavor commonly related to microalgae cultivated photoautotrophically, Chlorella protothecoides has been fermented in the dark on certain organic carbon sources rather than CO2.The resulting algal cells looked yellow and had less than 500 ppm of color generating impurities and/or lack an unpleasant odor. To further decrease color, classical mutagenesis was used. Screened mutants appeared to have

Table 2.8 Commercial products from microalage

Total production (ton/yr)

Major manufacturer

Algae

Purpose

Producer country

Chlorella

Human nutrition

Taiwan, Japan, Germany, USA

2000

Spirulina

China, India, USA, Japan, Myanmar

3000

Crypthecodinium Dunaliella

Human nutrition (protein/ linolenic acid) DHA oil Beta carotene

240 1200

Royal DSM BASF

Haematococcus

Astaxanthiin

USA Australia, Israel, USA, China USA, India, Israel

300

Cyanotech

Adapted from Ref. 4.

Taiwan Chlorella Manufacturing and Co. Dainippon Ink and Chemicals, Inc.

Algal Biorefineries

even low color. The algal cells were then lysed at different degrees and combined with different flavorant, odorant, or colorant to form a mixture. 6.2.1.2 Spirulina

Spirulina has been used for human nutrition for many years due to its high protein content and other nutrition value. In particular, this alga contains linolenic acid, an essential fatty acid that cannot be synthesized by humans. Annual production of Spirulina has been estimated to be around 3000 tons (Table 2.8).The largest producer is DIC Corporation (formerly Dainippon Ink and Chemicals), which is also the world’s first company to produce Spirulina at commercial scales (http://www.dlt-spl.co.jp/business/en/spirulina/).This company has two sites for Spirulina production. One is in Hainan (People’s Republic of China) and another one, Earthrise Farms, in California (USA). Annual production for these two plants is 450 and 350 tons/year, respectively. Other notable producers are: Cyanotech in Hawaii (USA) and Myanmar Spirulina Factory in Yangon, Myanmar. Spirulina requires high pH and bicarbonate concentration for growth. This selective requirement makes its commercial production easier since other microalgal strains cannot tolerate such harsh environmental conditions. 6.2.2 Polyunsaturated Fatty Acids Polyunsaturated fatty acids (PUFAs) refer to a group of fatty acids that have more than 18 carbon atoms and multiple unsaturated double bonds. If a carbon double bond is at the third position from the methyl or omega end, it is called an ω-3 fatty acid. Similarly, if the double bond is at the sixth position from the omega end, it is named as an ω-6 fatty acid. Both kinds of PUFAs cannot be synthesized by humans and animals.The conventional source is marine fish oil.Thus, fish oil has been a popular supplement for humans. But fish itself does not synthesize PUFAs either. Fish obtain these fatty acids either from their diet, marine microalgae, as is the case with prey fish like herring or sardines or, as is the case with fatty predatory fish, by eating prey fish that have accumulated PUFAs from phytoplankton. Accordingly, the extraction from algae to yield algae oil is a hotly debated alternative.157 To get PUFA-rich algae oil, the dried algae are submitted to solvent extraction using hexane, followed by winterization, acid refining, neutralization, and centrifugation. Alternatively, the extraction of oil from algae has been studied using supercritical carbon dioxide and ethanol as cosolvent.92 PUFAs have been recommended for adults as a dietary supplement since they are essential for proper functioning of our brains. Other research has shown that a diet rich in omega-3 fatty acids can even lower the risk of heart disease.158 In particular, for babies during the first 6 months of their life when developments of nervous system and visual abilities are fast, they are recommended to have baby milk formulas that contain PUFAs if they are not breastfed. To avoid any possible contamination of PUFAs extracted from fish, only those extracted from microalgae have been added to baby formulas. For this purpose, heterotrophic microalgae, Crypthecodinium cohnii and Schizochytrium limacinum SR21 have been grown in large-scale fermentors to produce docosahexaenoic acid (DHA) (C22:6). In USA, Martek has been a

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Figure 2.7 Chemical structure of squalene [6E,10E,14E,18E)-2,6,10,15,19,23-Hexamethyl2,6,10,14,18,22-tetracosahexaene]. (Picture taken from Pherobase.)

key producer of DHAs. Martek acquired OmegaTech in 2002. In 2003, Martek’s DHA productivity was 240 tons. But Martek itself was acquired by Royal DSM in 2010. Besides DHA, eicosapentaenoic acid (EPA, C20:5) has been developed from cultures of Phaeodactylum tricornutum by the University of Almeria in Spain.1 Globally, the total annual demand of EPA is estimated to be 300 tons.159 Regarding omega-6 PUFAs, linolenic acid (C18:3) from Spirulina and arachidonic acid (C20:4) (ARA) from Porphyridium have been investigated. Also Mortierella alpina has been identified to be an excellent production organism for ARA and is the only commercially used micro-organism for ARA manufacturing.160 6.2.3 Squalene The green microalga B. braunii grows as colonies of individual cells held together by a colony matrix that contains a large mixture of liquid hydrocarbons. B. braunii is further classified into four races; namely races A, B, L, and S, depending on the type of hydrocarbons synthesized. The B race produces triterpenoid hydrocarbons, squalene (Figure 2.7) and botryococcene, both of which are putative condensation products of farnesyl diphosphate as the major matrix components.161 Squalene is currently used in cosmetics, foods and the medical and pharmaceutical sectors. Conventionally, the major commercial source of squalene has been the liver oil of deep-sea sharks (Centrophorus spp.) and the steamer distillate of the physical refining of vegetable oils such as olive oil, palm oil, sunflower oil, soybean oil, or corn oil.162 6.2.4 Pigments

6.2.4.1 Carotenoids

Carotenoids (Figure 2.8) are yellow orange and red terpenoid pigments in microalgae. The two main compounds are β-carotene and astaxanthin. Carotenoids are mostly used as food colorants and supplements for human and animal feeds.81 Both β-carotene and astaxanthin can be synthesized through chemical reactions at a much lower price compared with those derived from microalgae. However, natural carotenoids have the

Algal Biorefineries

CH3

CH3 CH3

CH3

O HO

CH3

CH3

CH3

H3C CH3

CH3

CH3

β-carotene

CH3

CH3 CH3

H3C

CH3

astaxanthin

CH3

CH3

H3C

O

OH

CH3

Figure 2.8 Algal carotenoids.

Figure 2.9 BASF Betatene® unmixed open brine pond Dunaliella salina culture at Hutt Lagoon, Western Australia. (Reproduced with the kind permission of BASF.)

advantage of supplying the natural isomers in their natural ratio and the natural isomers of β-carotene are considered superior to the trans synthetic form.159 The main algal species for producing β-carotene has been Dunaliella (Figure 2.9). When grown under high salinity and high light intensity, Dunaliella cells can contain up to 14% of β-carotene. This feature makes its large-scale cultivation a reality. Companies in Australia, Israel, USA, and China have been engaged in growing this algal species over the years. The major producer is BASF.163 The alga that has been used to produce astaxanthin is Haematococcus, a freshwater one that normally grows in puddles, birdbaths and other shallow fresh water depressions.When cultured under the right conditions, such as the two-stage process as summarized by Spolaore,4 these cells can contain astaxanthin up to 3%. Even though the cost is much higher for producing the natural forms rather than the synthetic counterpart, natural astaxanthin has been used in diets for carp, chicken, and red sea bream due to enhanced natural pigment deposition. Commercial producers can be

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found in Hawaii, India, and Israel. In Israel, Algatechnologies, Ltd., produces AstaPure from cultures of Haematococcus pluvialis. In USA, Cyanotech grows the same species of microalgae and sells BioAstin as the product that has high content of astaxanthin. 6.2.4.2 Phycobiliproteins

In contrast to carotenoids which are lipophilic, phycobiliproteins are water soluble. As photosynthetic accessory pigments, these molecules are deeply colored, either red or blue, and proteinaceous. The major compounds in this group of pigment are phycocyanin and phycoerythrin.81 Due to their striking colors because of incorporated tetrapyrrole chromophores, these pigments have been used as natural colorants for foods, cosmetics, and pharmaceuticals. In addition, they have been used in clinical and/or research immunology owing to their powerful and highly sensitive fluorescent properties.164 Phycobiliproteins have been produced from Spirulina and red microalgae Porphyridium and Rohdella. In the case of Spirulina, DIC has a product called Lina blue which is used in chewing gum, ice slush, sweets, soft drink, dairy products, and wasabi. Recently, DIC developed a new product called Linablue HGE which has better heat and acid resistance to conventional ones (http://www.dlt-spl.co.jp/business/en/spirulina/linablue.html).

6.3 Nonconventional Products from Microalgae The capability of algae to grow practically anywhere: in saline water, in waste water as well as on desert land and thus their noncompetitive situation with arable land and growing food have created since decades a worldwide interest to use them as a biofuel. Additionally, the resurrection of algal research in the USA starting from around 2007 has led to formation of countless start-up companies aiming to produce biofuels from microalgae. All algae contain proteins, carbohydrates, lipids and nucleic acids in varying proportions. While the percentages vary with the type of algae, there are algae types that produce up to 50% and more algal oil by weight (Table 2.9). However, they do so only at low rates and productivity.Thus, development of algae biofuels has been predicted to rather become a long and difficult march, with high risks and uncertain outcomes.165 6.3.1 Biodiesel Like many other vegetable oils, algal oil is a triglyceride, also called triacylglycerol; that is fatty acid triesters of glycerine with different even-numbered, linear, saturated or unsaturated fatty acids.162 As such, they are highly viscous (about 35–60 cSt compared to 4cSt for diesel fuel at 40 °C).When used as a fuel for compression-ignition engines, the high viscosity leads to problems in pumping, poor atomization in the engine and incomplete combustion.Therefore, a reduction in viscosity is important to make high-viscous oil a suitable alternative fuel for diesel engines.167 The most appropriate way to adjust the viscosity of algal oil is by converting the triacylglycerides into lower molecular weight fatty acid methyl

Algal Biorefineries

Table 2.9 Chemical composition of algae on a dry matter basis Species of sample Proteins Carbohydrates

Lipids

Nucleic acid

Scenedesmus obliquus Scenedesmus quadricauda Scenedesmus dimorphus Chlamydomonas rheinhardii Chlorella vulgaris Chlorella pyrenoidosa Spirogyra sp. Dunaliella bioculata Dunaliella salina Euglena gracilis Prymnesium parvum Tetraselmis maculata Porphyridium cruentum Spirulina platensis Spirulina maxima Synechoccus sp. Anabaena cylindrica

12–14 1.9 16–40 21 14–22 2 11–21 8 6 14–20 22–38 3 9–14 4–9 6–7 11 4–7

3–6 – – – 4–5 – – – – – 1–2 – – 2–5 3–4.5 5 –

50–56 47 8–18 48 51–58 57 6–20 49 57 39–61 28–45 52 28–39 46–63 60–71 63 43–56

10–17 – 21–52 17 12–17 26 33–64 4 32 14–18 25–33 15 40–57 8–14 13–16 15 25–30

Taken from Ref. 166.

esters (FAME) standardized as biodiesel, when meeting the requirements of ASTM D 6751 and Euro Norm EN 14214, respectively. The fatty acid composition of the different microalgae species is significant and can have a crucial effect on the characteristics of the biodiesel produced. Algal oils are composed of saturated and unsaturated fatty acids with 12–22 carbon atoms with chain lengths and degree of unsaturation depending on microalgae species. More particularly, the relative intensity of PUFAs is species specific.168 Synthesis of FAME is performed by transesterification of algal oil and a short chain alcohol (usually methanol) in the presence of a catalyst (usually KOH or NaOH) (Figure 2.10). Although the theoretical alcohol:oil molar ratio is 3:1, the molar ratio of 6:1 is generally used to complete the reaction accurately. The relationship between the feedstock mass input and FAME mass output is about 1:1, which means that theoretically, 1 kg of oil results in about 1 kg of biodiesel. The entire mixture then settles. Glycerin is left at the bottom and FAME is left on top.The glycerin can be used to make soap or is refined and the FAME is washed and filtered. Algae oils with a high titer of free fatty acid may be esterified prior to transesterification by glycerine or methanol yielding crude oil or crude FAME to be mixed with algal oil raw material. The difficulties in efficient biodiesel production by autotrophic growth of microalgae lie in finding an algal strain with a high lipid content and fast growth rate. Alternatively, microalgae can be cultured in heterotrophic conditions39 instead of autotrophic growth in order to develop a cost-effective and sustainable algal oil production to boost lipid titers and produce tailored oils.

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dƌŝŐůǇĐĞƌŝĚĞнϯDĞK,їϯ&Dн'ůǇĐĞƌŝŶĞ 60 °C. However, when rosin is exposed to air over a prolonged period of time, the exposed surface darkens, i.e., the conjugated double bonds of abietic-type resin acids are easily oxidized by atmospheric oxygen forming hydroperoxide, epoxide, or hydroxyl impurities.TOR streams are therefore predominantly employed as chemical intermediaries and modified for their use in the production of adhesives and sealants, tackifiers, chewing gum bases, paper size, printing ink resins, rubber processing aids, disproportionated rosin soaps, rosin resin esters, and specialty applications.

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3.3.1 Rosin Soaps and Fortified Rosin Soaps The most important use of rosin is its ability to impart paper with resistance to penetration by water or aqueous liquids (“paper sizing”). The sizing of paper stops writing inks from blurring and feathering and helps paper bags retain their strength by resisting moisture absorption. To anchor rosin size on pulp fibers prior to paper formation rosin is transferred by saponification with NaOH in water-soluble sodium resinate and then precipitated by the addition of aluminum sulfate (alum) as a waterinsoluble aluminum resinate on the fiber surface (Figure 3B.22). A further improvement of the sizing is achieved by treating the native rosin with fumaric acid or maleic anhydride converting the abietic acid and related compounds to tricarboxylic species called “fortified rosin.” The chemistry of this process involves a tautomeric rearrangement of the main component of rosin, abietic acid, to give an isomer in which both double bonds are in the same ring (Levopimaric acid). This isomer then undergoes a Diels–Alder reaction and may subsequently be processed to yield an aqueous dispersion (so-called invert size).64 Maleinated rosins are oxidation-resistant, relatively high melting resins. Besides paper sizing they are used in the coating industry as binders in solvent-based and, after neutralization, in aqueous applications to increase hardness and improve wetting, adhesion and gloss of oil paints, alkyd resins, paper coatings, flexo and packaging inks, jet inks and adhesives.65 3.3.2 Disproportionated and (Per)hydrogenated Rosin Rosin soaps have played a crucial role in the development of the large scale synthetic rubber (SBR) production for the automotive tire industry. However, the conjugated

“Setting” of Rosin Soap H 3C

COOH

Soap form O O

CH3

O

CH3

CH3

Fortified Rosin

o

Precipitated form

o-

AI

3+

Anchoring group

o

OAI(OH)+

Fiber surface

M. Hubbe

Figure 3B.22 Internal paper sizing using rosin and fortified rosin. (Hubbe, M. Rosin Soap Size, MiniEncyclopedia of Papermaking Wet-End Chemistry, BioResources, NC State University, Dept. of Forest Biomaterials, Raleigh/USA, http://www4.ncsu.edu/∼hubbe/SOAP.htm; with kind permission.)

The Pine Biorefinery Platform Chemicals Value Chain

double bonds of abietic acid tend to capture free radicals and to inhibit the propagation of the polymerization reaction. Thus, when using rosin as an emulsifier, it is necessary to reduce the abietic acid fraction to less than 1% by conversion into resin acids having no conjugated diene structure. This can be achieved by the removal of two hydrogen atoms of abietic acid and rearrangement of the double bond system to form an aromatic nucleus and yield dehydroabietic acid in a reaction called dehydrogenation or disproportionation on the one hand or, on the other, by (per) hydrogenation of the conjugated double bonds yielding dihydro- and tetrahydroabietic acid (Figure 3B.23). Both reactions are generally carried out with a suitable catalyst such as Pd/C, Ni, iodine, or metal iodides at a temperature between 250 and 275 °C. The resin acids are then converted into the respective sodium or potassium soaps for their use as an emulsifier in making GR-S-type rubber.66 3.3.3 Dimerization Besides hydrogenation, dimerization of abietic acid using sulfuric acid as a catalyst is a way to make rosin less susceptible to oxidation.67 Dimerization of rosin (Figure 3B.24) increases its molecular weight and softening point, induces higher viscosity (molten and in solution), and improves stability. (a)

O II C–Oθ

CH3 CH3

CH3

H2 catalyst 250-275°C

CH3

HO2C

Abietic Acid

Dehydroabietic Acid

(b) H2

COOH Abietic Acid

catalyst

H2

COOH Dihydroabietic Acid

catalyst

COOH Tetrahydroabietic Acid

Figure 3B.23 Disproportionation and (per)hydrogenation of rosin. (a) Disproportionation. (b) (per) hydrogenation of rosin.

149

150


COOH palustric acid H COOH levopimaric acid

HOOC

+

Lewis acid COOH

COOH abietic acid

dimer abietic acid

COOH neoabietic acid

Figure 3B.24 Dimerization of rosin. (Adapted from Ref. 67.)

3.3.4 Rosin Esters The esterification of TOR with methanol leads to methyl rosinate. Appearance and stability are improved when hydrogenated rosin is used as raw material.The methyl ester of hydrogenated rosin is a light amber liquid resinous tackifier and plasticizer with a boiling point of 360–364 °C. It functions in fragrance compounds as a fixative, carrier, and compatibilizer and as a component in lipsticks and other personal care preparations. Ethylenglycol diabietate and diethylenglycol diabietate are sticky, yellow liquids used as a plasticizing component in hot melt adhesives and floor adhesives. Esterification of rosin, hydrogenated rosin, or dimerized rosin with glycerol (Figure 3B.25) or pentaerythritol takes place in the melt. Since the carboxyl group of rosin acids is sterically hindered temperatures in excess of 250 °C and continuous removal of the water are generally required in order to force the reaction toward completion eventually with the addition of an acid esterification catalyst or an alkaline metal oxide. A typical softening point for glycerol rosinate is 85 °C, and 105 °C for pentaerythritol rosinate. The difference in softening point affects their compatibility and hence adhesive performance. Glycerol esters of rosin are generally recognized as safe substances68 and approved by several FDA notifications as direct and indirect food additives.69 They are broadly used in manifold adhesive applications, such as pressure sensitive (PSA) tapes and labels, sealants in flooring and construction, lithographic ink resins, roadmarking resins, bookbinding, as well as other specialty applications.


C – OH HO – CH2

C – O – CH2

║

║

O

O

+

C –OH HO – CH

C – O – CH

║

O

C –OH HO – CH3

C – O – CH3

║

ROSIN ACID

3 H2O

║

O

O

+

║

GLYCEROL

TRI-ESTER

O

WATER

Figure 3B.25 Esterification reaction producing a typical rosin ester. (Eastman Tackifier Center, Rosin Esters, http://www.eastman.com/Markets/Tackifier_Center/Tackifier_Families/Rosin_Resins/Pages/Ro sin_Esters.aspx.)

4. CONCLUSION As already said in the Introduction, wood has accompanied mankind from the dawn of civilization as a source for light, fire, heat, and as construction material; not to forget the importance of living forests and rain forests for biodiversity and as lung of the planet. Naval stores can certainly claim a role as historical forerunners of modern biorefineries. After their disappearance, pulp and paper mills took over and must worry in turn now about their survival in a world of electronic communication and information storage. The growing concerns about finiteness of fossil resources, their contribution to global warming, and about the ecological impact of more and more demanding technologies for their extraction call again for a return to wood as a renewable resource for heat and power generation70 and for the thermochemical, chemical, or biochemical manufacturing of transportation fuel71 (see Chapter 3).

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CHAPTER 4A

Sugar- and Starch-Based Biorefineries Rainer Höfer Editorial Ecosiris, Düsseldorf, Germany

Contents 1. Introduction 2. Sugar and Starch Crops 2.1 Sugarcane 2.2 Sugarbeet 2.3 Sweet Sorghum and Grain Sorghum 2.4 Sugar Maple 2.5 Sugar Palms 2.6 Maize (Corn) 2.7 Wheat 2.8 Triticale 2.9 Barley 2.10 Rice 2.11 Cassava 2.12 Potatoes 2.13 Pulses 2.14 Sago Palm 2.15 Stevia 3. Sugarbeet Refining and Processing 3.1 Sugarbeet Supply 3.2 Extraction 3.3 Carbonation 3.4 Crystallization 3.5 Product 4. Alcoholic Fermentation 4.1 Alcoholic Fermentation for Beverages 4.2 Alcoholic Fermentation for Biofuels

158 159 159 160 162 163 164 166 168 171 171 172 173 175 176 177 179 179 181 181 181 182 183 183 184 186

186 187 188 188

4.2.1 Raw Materials 4.2.2 Fermentation 4.2.3 Distillation 4.2.4 Anhydrous Ethanol

4.3 Acetic Acid 5. The Ethanol-Based C2—Value Chain 5.1 Bioethanol to Ethylene 5.2 Ethylamine 5.3 Ethylesters 6. Beyond C2 Platform Chemicals by Fermentation Industrial Biorefineries and White Biotechnology http://dx.doi.org/10.1016/B978-0-444-63453-5.00005-7

189 190 190 191 192 192 Copyright © 2015 Elsevier B.V. All rights reserved.

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6.1 C3 Building Blocks

192

192 193 193

6.1.1 Lactic Acid 6.1.2 3-Hydroxypropionic Acid 6.1.3 Isopropanol

6.2 C4 Building Blocks

6.3 Higher Carboxylic Acids

6.3.1 Citric Acid

6.4 Amino Acids

194

6.2.1 n-Butanol194 6.2.2 Isobutanol 195 6.2.3 The Biosuccinic Acid Platform 196 6.2.4 The Pentosan Value Chain 197 6.2.5 2,5-Furandicarboxylic Acid (FDCA) and Polyethylene Furanoate (PEF) 198

6.4.1 Glutamic Acid

198 198

200 200

7. Sucrochemistry 7.1 Inverted Sugar Syrup 7.2 Gluconic Acid 7.3 Sucralose 7.4 Sucrose Esters 7.5 Sugar Polyols 8. Starch Refining and Processing 8.1 The Global Starch Market 8.2 Industrial Starch Production Processes

201 202 202 203 204 205 205 205 206

206 208 209 210 210 210

8.2.1 Corn Starch Processing 8.2.2 Wheat Starch Production 8.2.3 Rice Starch Production 8.2.4 Potato Starch Production 8.2.5 Tapioca Starch Production 8.2.6 Pea Starch Production

9. Starch Uses 9.1 Native Starch 9.2 Physical Treatment 9.3 Starch Graft Copolymers 9.4 Modified Starches

211 211 211 213 214

214 214 217

9.4.1 Starch Esters 9.4.2 Starch Ethers 9.4.3 Converted Starches

1 0. Conclusions 227 Acknowledgment228 References228

1. INTRODUCTION Plant carbohydrates are grouped as soluble sugars (such as sucrose from sugarcane), storage carbohydrates (such as starch from grains and tubers), and structural carbohydrates (which make up the plant cell wall such as cellulose, hemicellulose, lignin, and pectin). Sugar is an aliment commonly known as table sugar, cane sugar, or beet sugar and most commonly

Sugar- and Starch-Based Biorefineries

associated with sucrose. The current annual sugar production has reached 175 Mio mto. (2013/14). Thereby about a quarter is derived from sugarbeet, three quarters from sugar cane.1,2 The sucrose molecule is a disaccharide composed of the monosaccharides glucose and fructose. Sugars generally have their origin in the photosynthesis of plants.They are used as an energy source in living organisms and in biosynthesis. When monosaccharides are not immediately needed as an energy source they are often converted to storage forms, such as starch in many plants. Starch is a mixture of two polysaccharides: amylose and amylopectin. Starch can be hydrolyzed efficiently by acid treatment or by the use of enzymes yielding depolymerized starches with increased water solubility compared to the native starch. Sugarcane is indigenous to tropical Southeast Asia and has been cultivated there for millennia. The crop spread westward through India to the Persian Empire and Mesopotamia and later accompanied the Arab expansion around the Mediterranean. Nonetheless, sugar remained a luxury item for centuries. To sweeten food usually honey or fruits were used. In 1493, on his second voyage, Christopher Columbus carried sugarcane seedlings from the Canary Islands to the New World and in the following centuries the West Indies became the most important source for sugar supplies to Europe and North America. It took until 1747 before Andreas Sigismund Marggraf announced his discovery that sugar can be obtained also from the sugarbeet that is suitable for growing in temperate climate zones. Marggraf ’s student, Franz Carl Achard later devised an economical industrial method to extract the sugar in its pure crystalline form. He also developed selection–breeding methods that are still valid and was the first to establish a sugar refinery based on sugarbeet in Kunern in Lower Silesia (modern day Poland) in 1801.3 Starch came into the game as a source of sugar when Gottlieb Sigismund Constantin Kirchhoff in 1811 discovered that acid hydrolysis of potato starch yielded glucose, a discovery which would lead to today’s starch-derived sweetener industry. Shortly after, in the year 1819, H. Braconnot discovered that glucose also is formed by the treatment of wood with sulfuric acid. An industrial process of wood saccharification (Bergius Process) using wood treatment with hydrochloric acid was started in the interwar period between world war I and world war II under the leadership of the Th. Goldschmidt AG in Mannheim and has been conducting business until 1959.4

2. SUGAR AND STARCH CROPS 2.1 Sugarcane Sugarcane (Saccharum officinarum) is a large tropical grass that produces multiple stems or culms each of which consist of a series of nodes separated by internodes. Usually sugarcanes grow up to 6 m in height. The plant is mainly composed of a huge stalk, also known as a stem, which is stout, jointed, and fibrous and stores energy in form of sucrose in parenchyma cells and vascular tissues. The top of the stalk is relatively low in sucrose while the bottom of the stalk is high in sucrose. Sugarcane is primarily grown as a source of sugar providing around 70% of the world’s sugar demand. Modern sugarcane varieties

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Figure 4A.1 Sugarcane workers. (Painting made by Roland, Project Esperanza Art Shop, with kind permission; http://esperanzameanshope.org/home; Project Esperanza serves the Haitian immigrant population in Puerto Plata, Dominican Republic, through education, social aid, and community development)

that are cultivated for sugar production are complex interspecific hybrids (Saccharum spp.) that have arisen through intensive selective breeding. S. officinarum or the “noble canes” accumulate very high levels of sucrose in the stem but have poor disease resistance.The origins of S. officinarum are intimately associated with the activities of humans as S. officinarum is thought to be the product of complex introgression between Saccharum spontaneum, Eriathus arundinaceus, and Miscanthus sinensis, i.e., a purely cultivated or garden species with no members found in the wild (Figure 4A.1). Sugar cane stems are harvested when sucrose content in the stems is 12–20%. Sugar is initially extracted from the raw cane at sugarcane mills distributed throughout the growing region. The cane is shredded and the juice extracted by crushing. Clarified sugar juice is then concentrated by evaporating the water in the sap to produce “syrup”. The syrup then goes through multiple rounds of crystallization to extract the sucrose. It is boiled and the sucrose crystallizes from the remaining molasses fraction. The product of this step is known as massecuite. The massecuite is then centrifuged to separate the sucrose from the molasses.

2.2 Sugarbeet Rootcrops like beetroot, chard, and mangelwurzel share a common wild ancestor, the sea beet (Beta vulgaris maritima) native to the seacoasts of the Mediterranean. The sugarbeet as we know it today is derived from many years of selective breeding of these varieties after the discovery that beetroot was a source of sucrose and could be established as an economic source of sugar in Europe. In fact, sugarbeet is not a plant occurring in the wild. It is a product of breeding research initiated by F. C. Achard, and that way became a model for improving plant performance through genetics and breeding. The sugarbeet plant is also unique in history by its role as a catalyst in revolutionizing agriculture: Growing sugarbeets in Europe changed the previous small grain monoculture and


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Figure 4A.2 Sugarbeets —a powerful sugar producing crop. (Adapted from (a) and (b) Wach W. Sugar Biorefinery, Südzucker AG Mannheim/Ochsenfurt, IEA-Bioenergy Task 42 Biorefineries - German Workshop on Biorefineries, 15th September 2009, Worms; (c) Klartext, Zuckergewinnung: Alles wird verwertet, Forum Zucker, Bonn, [email protected]; with kind permission).

introduced the concept of crop rotation. This created the practice of green manure resulting in better soil fertility and reducing weed problems, while also supplying a source of food for livestock from beet tops, crowns, and pulp (Figure 4A.2). The sugarbeet plant consists of a conical, white, fleshy root and a rosette of leaves. Beets are planted in late March/early April and harvested in late September and October. The average weight of sugarbeet ranges between 0.5 and 3 kg. The leaves are numerous and broad and grow in a tuft from the crown of the beet. Sucrose is formed by photosynthesis in the leaves, and is then stored in the root. The root of the beet contains typically about 75% water, and inter alia between 12% and 21% sugar (sucrose), depending on the cultivar and growing conditions, and 0.7–0.9% inorganic components, and approx. 7% pulp.3 The pulp is insoluble in water and mainly composed of cellulose, hemicellulose, lignin, and pectinous substances. Its use, after drying, is for animal feed.

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2.3 Sweet Sorghum and Grain Sorghum Sorghum is a genus of grasses with about 30 species, which are raised for producing starch and proteins in the grains, sugar in the stalks and many of which are used as fodder plants, either cultivated or as part of pasture (Figure 4A.3). Being a native to the tropics and subtropics, sweet sorghum (Sorghum bicolor (L.) Moench) has been domesticated by man long ago and cultivated as source of sugar since time millennia. Owing to its origins the species is capable of growing in harsh environments under hot and dry climatic conditions. Sweet sorghum, also called “sorgo,” differs from grain sorghum (also called “milo”) in that it is grown for its stalk. Its grain yield (seed head) is low, and it is juicier and higher in sugar content (Figure 4A.4;Table 4A.1). The taller the plant and the thicker the stalk, the more juice the plant will produce. In fact, sweet sorghum stalks contain up to 75% juice, varying between 12% and 23% in sucrose and estimated by Chefs as pure culinary gold.5

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Figure 4A.3 Sorghum. (Credentials: (a) Illustration by John Burgoyne, http://www.johnburgoynestudi o.com/pages/faq (taken from Ref. 5); (b) ICRISAT’s Principal Sorghum Breeder BVS Reddy in a field of sweet sorghum; (c) Sorghum stalks range in diameter from 4.5 to 1.5 cm, with kind permission; The International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) is a nonprofit, nonpolitical organization that conducts agricultural research for development in Asia and sub-Saharan Africa with a wide array of partners throughout the world. (http://www.icrisat.org/index.htm))


Figure 4A.4 Sweet sorghum (in the background) growing behind grain sorghum (in the foreground); notice the differences in plant height, seed head size and stalk thickness (Sweet Sorghum Association, with kind permission).

Table 4A.1 Composition of sweet sorghum Parameter Quantity

Fermentable sugars Fiber Water Dissolved nonsugars Ash pH

13% 15% 68% 3.5% 0.5% 6.8%

Grain sorghum is shorter than sweet sorghum and has been bred for higher grain yields. It is suitable for harvesting with combines and used for both grain and forage production as a major feed for cattle. Sorghum is a perennial crop in areas that don’t have a winter freeze. Seeing it growing in the field, it is a grass similar to corn in vegetative appearance with wide blade-shaped leaves arcing out in a whirl around the thick stalk. Growth and development is similar to corn and other cereals. As such sorghum ranks fifth among the most important cereal crops of the world with 80% of the area devoted to sorghum being located within Africa and Asia.6,7

2.4 Sugar Maple Acer saccharum (sugar maple, also called rock maple), a native of North America, is a deciduous tree normally reaching heights of 25–35 m. In Canada the tree extends from the extreme southeast corner of Manitoba, through central Ontario, the southern third of Quebec and all of New Brunswick and Nova Scotia. Within the United States the species is found throughout New England, New York, Pennsylvania; from central New Jersey to the Appalachian Mountains; in North Carolina, Tennessee, Missouri; a small area of Kansas, Iowa, and the eastern two-thirds of Minnesota. Sugar maple is a landscape

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Figure 4A.5 Sugar maple.

standout of breathtaking beauty. Medium to dark-green leaves in springtime turn yellow, burnt orange or red in fall (Figure 4A.5). Maple syrup is usually made from the xylem sap of sugar maple, red maple, or black maple trees.These trees store starch in their trunks and roots before the winter; the starch is then converted to sugar that rises in the sap in the spring. Maple trees can be tapped by properly drilling holes in the trunk through the phloem, just inside the bark about 1 m from the ground. The tree must be about 40 years old before juice can be obtained. It then produces juice for 100 years. The collected sap with an average sucrose concentration of 2–4% is processed by boiling, concentrating and filtering to evaporate much of the water, leaving the concentrated syrup behind.8 Forty liters of maple sap are required to be boiled to produce only 1 L of pure syrup. The main sugar in pure maple syrup is sucrose. The darker grades of syrup additionally contain a small and variable amount of fructose and glucose.

2.5 Sugar Palms Between the eventually more than 3000 species of the palm family9 (Arecaceae) that occur naturally in the tropics and subtropics the main sugar yielding palms are the Asian Palmyra palm (Borassus flabellifer, also known as lontar palm, toddy palm or wine palm) and the sugar palm10 (Arenga pinnata, common names include arenga palm, black sugar palm) (Figure 4A.6). The sugar palm is a medium-sized palm, growing to 20 m tall, with a striking, round crown of tufted leaves. Tapping therefore is a physically taxing and risky business traditionally done by small, local sugar tappers that climb to the top of the palms to cut the flowering stalks and catch the oozing liquid sap (Figure 4A.7). Making palm sugar is an occupation that many villagers in Asia do after rice harvest to earn additional income. In some instances the tree itself is tapped rather than the flowering spikes, but this is an isolated production method. The sugar palm sap containing around


Figure 4A.6 Palmyra palm. (a) Main entrance of the Angkor Wat temple city girdled by Asian Palmyra Palms, the national flora emblem of Cambodia and highly respected in Tamil culture (Andrew Lih, en. wikipedia, CC-BY-SA-2.0); (b) Palmyra Palm in Angkor Wat. (Picture by Franz Xaver; http://commons.wiki media.org/wiki/File: Borassus_flabellifer.jpg; (a) and (b) licensed under the Creative Commons permission.)

Figure 4A.7 Sugar palm. (a) Arenga pinnata (Francisco Manuel Blanco, Flora de Filipinas: Segun el sistema sexual de Linneo, Imprenta de Sto. Thomas, Manila (1837)); (b) Tapping sugar palm, Batang Toru, North Sumatra; (c) Processing sugar palm sap for sugar. (See more at: http://blog.worldagroforestry.org/ index.php/2012/11/23/sugar-women-wine-money-men-and-orangutan/#sthash.xwwgIWFR.dpuf)

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10% sucrose11 is cleared primarily by heating and drunk fresh or fermented to yield palm wine. For the production of palm sugar, the juice is purified with lime, concentrated by boiling in open pans to thick syrup and then poured into containers, where it solidifies to a slightly porous, caramel-colored cake called jaggery. Cutting the flower buds (inflorescences) of the coconut palm in a similar way as sugar palm tapping yields coconut sugar (also commercialized as coco sugar or coconut palm sugar). It should be noticed that the oil palm (Elaeis guinensis) also contains around 10% sucrose12 and thus can be considered as a source of fermentable sugar. Date sugar (also miel de palma, palm honey, palm syrup) is obtained from the sweet fleshy fruit of the date palm (Phoenix canariensis) which contains up to 81% sugar consisting primarily of sucrose as the principal disaccharide.13

2.6 Maize (Corn) The history of modern-day maize (Zea mays subsp. mays) begins at the dawn of human agriculture in what is now Mexico. Actually, maize is, after wheat and rice, the most important cereal grain in the world (Figure 4A.8). Maize thrives best in a warm climate and is now grown with exceptional yield per unit area in most of the countries that have suitable climatic conditions. Its growth depends more on high summer temperatures than on a high mean temperature. It will ripen in a short hot summer and will withstand extreme heat. A large amount of water is needed

Figure 4A.8 World corn production (year 2012/2013).


during the growth of the maize. Its average maturing period is relatively short and this makes it possible to grow at fairly high latitudes within a global corn belt stretching between 40° South to 45° North in the USA, where approximately 1/3 of world production grow in Iowa, Illinois, Indiana, Michigan, Ohio, Nebraska, Kansas and part of Missouri. In Europe, maize growing reaches as far North as 46° on the West coast of France. From this point, the line runs across Europe reaching 50° in the East before falling, only to rise again in Russia approaching 52°. Great importance is attached to the use of sound and fresh grain to give high quality food and corn-based finished products (Figure 4A.9). Maize is a tall, determinate annual C4 plant14 varying in height from 1 to 4 m producing large, narrow, opposing leaves (about a tenth as wide as they are long), borne alternately along the length of a solid stem.15 An individual kernel of corn is a seed and largely comprised of three parts: the pericarp, the endosperm, and the germ (or embryo) (Figure 4A.10).16 The pericarp or hull of the corn kernel is a thin outer covering made up of two layers. Removal of this part of the corn kernel results in corn bran. The endosperm, which comprises up to 82% of the kernel’s dry weight, is the source of energy for the germinating seed. In all field corn, the endosperm is comprised of two types of starch: vitreous and floury. The proportion of these starches is controlled genetically. The floury endosperm is the softer starch, and as the kernel matures, this type of starch dries down to create the “dent” in the top of the kernel. The major chemical component of the maize

Figure 4A.9 Maize. (a) Zea mays—Köhler’s Medizinal-Pflanzen-283. In: Hermann Adolph Köhler, Köhler’s Medizinal-Pflanzen in naturgetreuen Abbildungen mit kurz erläuterndem Texte, Bd. 1–3, Gera: Verl. Franz Eugen Köhler (1883–1914); (b) Edge of a field of Corn or Maize north-eastern Lower Saxony, Germany. The plants are ± full-grown but still not quite ready for harvest. (Attribution: Christian Fischer; licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license)

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Industrial Biorefineries and White Biotechnology –Internal structures of a corn kernel. PERICARP

STARCH FLOURY ENDOSPERM (81.9%)

HULL AND FIBER (5.3%)

STARCH AND GLUTEN VITREOUS

GERM (11.9%)

TIP CAP (0.8%)

–Proximate chemical analysis of corn grain Compound

Average % Mass (dry basis)

Starch Hemicellulose Cellulose + Lignin Protein Fat Ash

71.7 6.2 3.3 9.5 4.3 1.4

Source: Watson, S.A. (1987). Structure and Composition. In Watson, S.A. and Ramstad, Paul E. (Ed.), Corn: Chemistry and Technology (pp. 53-82). Minneapolis, MN: American Association of Cereal Chemists

Figure 4A.10 Internal structure of a corn kernel and analysis of a corn grain. (Adapted from: http://bio web.sungrant.org/Technical/Biofuels/Technologies/Ethanol+Production/Ethanol+Wet+Grind+Processes/ Default.htm)

kernel is starch, which provides 72% of the kernel weight. Other carbohydrates are simple sugars present as glucose, sucrose and fructose in amounts that vary from 1% to 3% of the kernel. The starch in maize is made up of amylose and amylopectin (Figure 4A.11). In common maize amylose makes up 25–30% of the starch and amylopectin makes up 70–75%.

2.7 Wheat The agrarian revolution around 10,000 years ago has repeatedly been seen as the major transition in the human past, a changeover from the natural environment in control of humans, to humans in control of the natural environment. Wheat (Triticum spp.) is traditionally looked on as one of the founder crops domesticated in the Fertile Crescent enabling the emergence of city-based societies at the start of civilization because it was one of the first crops that could be easily cultivated on a large scale, and had the additional advantage of yielding a harvest that provides long-term storage of food.17 World history assumes a fundamental connection between wheat and western societies to such a degree


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Figure 4A.11 Chemical composition of amylose and amylopectin. (a) Structure of amylose; (b) Structure of amylopectin. ((a) and (b), Adapted from BeMiller JN, Whistler RL. Carbohydrates. Food Chemistry. 3rd ed. New York: Marcel Deker; 1996. pp. 157–223)

that the first assessment of any single epoch is based upon wheat culture vigor: healthy wheat culture means civilization is in ascendancy; unhealthy or extinct wheat culture means civilization is in decline. Today, wheat is grown on more land area than any other commercial crop and continues to be the most important food grain source for humans. Its production leads all crops, including rice, maize, and potatoes.18 Wheat grows best when temperatures are warm, about 25 °C, with maximum growth temperatures of 30–32 °C. The crop normally needs between 110 and 130 days between sowing and harvest, depending upon climate, seed type, and soil conditions (winter wheat lies dormant during a winter freeze).Wheat also needs a lot of sunshine, especially when the grains are filling. In this way, particularly in the stage of maturity, when the color of the crop takes on a golden color, wheat has dominated landscapes for centuries (Figure 4A.12).

Figure 4A.12 Wheat field. (popularscreensavers.com; http://www.v3wall.com/es/html/pic_down/192 0_1200/pic_down_45915_1920_1200.html)

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Technological advances in soil preparation and seed placement at planting time, use of crop rotation and fertilizers to improve plant growth, and advances in harvesting methods have all combined to promote wheat as a viable crop. Agricultural cultivation using horse collar leveraged plows (at about 3000 BC) was one of the first innovations that increased productivity (Figure 4A.13). Previously dominated by labor-intensive rural harvesting techniques, improved agricultural husbandry has more recently led to an extensive mechanization including the combine harvester performing three separate harvesting operations—reaping, threshing, and winnowing—into a single process (Figure 4A.13(b)(c)). The wheat grain is a tiny egg-shaped seed (Figure 4A.14). At the top is a tuft of hairs called the “beard” and at the other end, where the grain is attached to the stalk, is the germ—the embryo of the plant. The outside layers of the grain are loose and most of them are separated during threshing. The edible part of the grain consists of three layers: Bran forms the outer part of the grain. It consists of several layers. It is a good source of dietary fiber and B vitamins. The endosperm is the food reserve,

Figure 4A.13 Technological advances in wheat harvesting. (a) Traditional rural wheat harvesting, Maroc (1975) (Picture: Rainer Höfer); (b) Traditional rural wheat threshing: “Como paja que se lleva el viento” http://mirefranero.com/category/frases-cortas/page/35/; (c) John Deere harvesters, wheat harvesting in modern times (Posted in Uncategorized).


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Figure 4A.14 Wheat grain. (a) wheat grains; (b) Section of a grain of wheat; (c) wheat spike.

making up about 85% of the grain. It is composed mainly of starch. The germ, the most nutritious part of the grain, contains protein, fat, iron, and B vitamins. The average composition of wheat comprises 9–12% protein, 2–5% fat, 9% fiber, 13% water, and 58–70% starch.

2.8 Triticale Triticale is a cross-breed between a wheat as female and rye as a male partner.19 Taste and ingredients of triticale are between those of wheat and rye. Triticale has been bred to connect the unpretentiousness of the rye with the quality of wheat, therefore, the cultivation focuses on less favorable climate conditions.The starch content of tritical is similar to wheat and higher than rye.

2.9 Barley Barley (Hordeum vulgare L.) is a member of the grass family. As wheat it was one of the first plants to ever be domesticated as early as the ancient empires. Later in Medieval Europe it was commonly used as peasant food, ground into bread, and as fodder for livestock. Barley is a drought-resistant widely adaptable crop capable of living in various climates, where other grains can’t grow—from arctic latitudes and alpine altitudes to saline desert oases including tropical areas where it is grown as a winter crop. Barley can be identified because of the long awns which give the typical appearance of the barley field. With increasing maturity the ears tend to the ground, so that the ears of ripe barley form a round, hanging arch (Figure 4A.15). The mature barley grain comprises the embryo and the endosperm and is surrounded by a nonstarch cell layer (called aleurone) mainly consisting of lipids and protein. The endosperm provides a store of carbohydrates in the form of starch granules (about 80% by weight barley) embedded in a protein matrix and supports the initial growth of the germinating embryo. The grain is protected by an outer husk (peel) that covers the back

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Figure 4A.15 Barley. (a) Drawing of barley (original book source: Prof. Dr. Otto Wilhelm Thomé Flora von Deutschland, Österreich und der Schweiz 1885, Gera, Germany, www.biolib.de); (b) ripening barley on the field (http://tajagroproducts.com/countries/Luxembourg.html); (c) barley kernel structure. (home brewtalk.com)

of the grain and prevents water loss. The mean starch content is of 55%, i.e., barley is higher in protein but lower in starch compared with corn or sorghum. The grain is used for malt beverages and in cooking. Barley is of no importance as a raw material for industrial starch.

2.10 Rice Like wheat and barley, rice has been a source of food for people since thousands of years. Rice production originated in China, and was spread to South and Southeast Asia. Cultivated rice can be divided into two separate species: Oryza sativa (Asian rice) and Oryza glaberrima (African rice). In terms of nutritional value and value of production, rice is the most important crop in the world. For 3.3 billion people living in Asia, rice provides 35–80% of their total calorie intake. The rice grain consists of an outer protective covering, the hull, and the rice caryopsis or fruit (Figure 4A.16).The endosperm cells are thin-walled and packed with amyloplasts containing compound starch granules. Rice contains up to 75% starch depending on the variety. This means the plant is one of the main sources of starch worldwide. To isolate rice starch from dried broken rice with good recovery and low residual protein content, alkaline solutions (the so-called wet process20), or proteolytic enzymes are commonly used.21 Due to the fine particle size, rice starches are among the smallest of the vegetable powders, measuring

Sugar- and Starch-Based Biorefineries Component Hull Bran & Germ Starchy Endosperm

20 % 10 % 70 %

Hull Pericarp Seed Coat Nucellus Aleurone Layer

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Figure 4A.16 Rice kernel structure. (Adapted from Orthoefer, Food Technol 1996;50(12):62 taken from Ref. 91)

7–9 μm.This inherent fineness results in an extraordinary increase in surface area. As a result the rice based products exhibit an extraordinary soft-touch effect. Rice starch are thus ideal to be used in both decorative cosmetics as well as those for skin and hair care products. In Japan, it has been proposed to use rice (which is normally fermented into sake) but also rice washing drainage, rice bran, husks and straw as an ethanol biofuel source.22

2.11 Cassava Cassava (Manihot esculenta, also called manioc, mandioca, yuca, mogo, tapioca-root or kappa, depending on the region where it is grown) is a woody shrub of the Euphorbiaceae family. Native to South America it is extensively cultivated as an annual crop in tropical and subtropical regions for its edible starchy tuberous root (Figure 4A.17). The cassava root is long and tapered, with a firm homogeneous flesh (which is source of tapioca starch) encased in a detachable rind, about 1 mm thick, rough and brown on the outside. Two varieties of the cassava are of economic value: the bitter, or poisonous; and the sweet, or nonpoisonous (Table 4A.2). Farmers often prefer the bitter varieties because they deter pests, animals, and thieves. Because the volatile bitter constituents can be destroyed by heat in the process of preparation, both varieties yield a wholesome food. Cassava is harvested by hand by raising the lower part of the stem and pulling the roots out of the ground, then removing them from the base of the plant.The upper parts of the stems with the leaves are plucked off before harvest. The roots are then washed and the peel (skin and cortex) is removed to process only the central part of the root. The starch granules are locked in cells together with all the other constituents of the protoplasm (proteins, soluble carbohydrates, fats and so on), which can only be removed by

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a purification process in the watery phase. It is therefore necessary to rupture all cell walls in order to release the starch granules. This can be done by biochemical or mechanical action.The biochemical method allows the roots to ferment to a certain stage; then they are pounded to a pulp and the starch is washed from the pulp with water. This method does not give complete yields and the quality of the resulting starch is inferior. Mechanical action is carried out by slicing the roots and then rasping, grating or crushing them, which tears the flesh into a fine pulp. Cassava starch is a white to off-white powder with moisture below

Figure 4A.17 Cassava. (a) Cassava field; (b) Rural cassava processing; Hofmann, H. Wurzelbrot der Tropen, Natürlich: Das Magazin für ganzheitliches Leben, 1–2008, http://www.natuerlich-online.ch/; Maniokforschung ETH Zürich, http://www.cassava.ch/ (Copyright Photos: Christoph Heuberger, with kind permission); (c) and (d) Cassava Tubers (Amada44, Wikimedia Commons (03.Jan.2011); Permission is granted to copy, distribute and/or modify document under the terms of the GNU Free Documentation License, Version 1.2.) Table 4A.2 Typical composition of mature cassava roots Constituent %

Moisture Starch Sugars Protein Fats Fiber Ash

69.8 22.0 5.1 1.1 0.4 1.1 0.5


13%.The pH of the slurry in water is neutral. Cooked it forms a quite clear gel with a long and slightly stringy texture making it a preferred thickener in delicate foods and desserts. Cassava is used as an alternative starch source for the manufacture of bioethanol.23

2.12 Potatoes The potato (Solanum tuberosum L.) is an herbaceous perennial crop that grows about 60 cm high and belongs to the night shade family (Solanaceae). The word “potato” may refer either to the plant itself or the edible tuber. The tuber has a high nutritional value, and its easy adaptability to different climates has allowed it to spread from its origins in the highlands of the Peruvian Andes on the border between Bolivia and Peru carried by the Spanish to Europe, from there to all elevation zones in temperate regions of all the continents, and, lately, its production has been increasing most rapidly in the warm, humid, tropical Asian lowlands during the dry season.24 The potato plant bears white, pink, red, blue, or purple flowers with yellow stamens. After flowering, potato plants produce small green fruits that resemble green cherry tomato. The fruits contain the toxic alkaloid solanine and are therefore unsuitable for consumption. The part of the potato plant we eat is called the tuber, which is actually an enlarged, swollen underground stem, mainly composed of water (up to 80%). Each plant will produce multiple tubers. Potato tubers come in a variety of colors, but most common are red and white (Table 4A.3). Their shape can be round, oblong, flattened, or elongated. In former days, potato harvest meant hard drudgery. People had to work laboriously by hand to eat: digging in the soil, with a fork, gently lift out the potato nests, and collect the tubers. People had to work no matter whether the sun burned, whether it was raining, or autumn storms moved across the country. It was not until the late nineteenth century that mechanization helped agriculture. Initially horse carriages were used in potato fields. Today, modern harvesting machines perform work in a few hours, the same work that dozens of helpers do in a day (Figure 4A.18). Table 4A.3 Average constituents of potato (Solanum tuberosum L.) Constituents Percentage (wb)

Moisture Protein Fat Starch Nonstarch carbohydrates Reducing sugar Ash Carotene (average) Thiamine Riboflavin Ascorbic acid

50–81 1.0–2.4 1.8–6.4 8–29 0.5–7.5 0.5–2.5 0.9–1.4 4 mg/100 g 0.10 mg/100 g 0.06 mg/100 g 12 mg/100 g

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Figure 4A.18 Potatoes. (a) Drawing of a potato crop (Amédée Masclef, Atlas des plantes de France, Tome III, Paris: Klincksieck (1893) p. 234); (b,c) Potatoe harvesting and potatoe harvesting machine; Qingdao Hongzhu Agricultural Machinery Co., Qingdao.

The chemical composition of potatoes is very variable and is greatly influenced by variety, environment, and farming practices. Starch constitutes 65–80% of the dry weight of the tuber.25

2.13 Pulses Pulses (also called grain legumes) are annual plants belonging to the Leguminosae (alternatively Fabaceae) family which are grown primarily for their edible grains or seeds. These seeds are harvested mature yielding from 1 to 12 seeds of variable size, shape, and color within a pod. They are marketed dry, to be used as food or feed. Included in the pulses are: dry beans like pinto beans, kidney beans and navy beans, dry peas, yellow peas, lentils, and others (Figure 4A.19). Like many leguminous crops, pulses play a key role in crop rotation due to their ability to fix nitrogen. In fact, pulses are an important part of the human diet in many parts of the world because they are both a rich and inexpensive source of protein and also a


Figure 4A.19 (a) Pulses and (b) peas in the pod. ((a) Adapted from: Bekkering, E. Pulses in Canada, Catalog no. 96 325 X—No. 007; ISSN 0-662-35659-4, http://www.statcan.gc.ca/pub/96-325-x/2014001/ article/14041-eng.htm, Statistics Canada’s Open License Agreement; (b) Anhui Hausen Food Machinery co., LTD, Tianchang City in Anhui Province/China, with kind permission)

good source of vitamins, minerals, and carbohydrates. In addition, legumes have historically acted as a critical source of food insurance against times of famine, as they are considered virtually indestructible if properly dried. The major carbohydrate of pulse seeds is starch, which accounts for 22–45% of the dry matter, compared with cereals (up to 70%) and potatoes (around 20%).26 Pulse starch, in particular, peas starch is extracted from the Pisum sativum dry pea. It is characterized by a high amylose content (in the order of 35 -39%) and a low gel temperature, which explains its functional texturizing and film-forming properties. Pea starch is used for its gelling or thickening properties in many agri-food applications (meats, delicatessen, sauces, pastas, creams) and more specifically for its film-forming properties in certain coatings or other textured foods.

2.14 Sago Palm The sago palm (true sago palm, Metroxylon sagu) is native to the lowlands of western Papua New Guinea, the Moluccan archipelago, Peninsular Malaysia, and Sarawak but naturalized and grown today throughout tropical Southeast Asia.The sago palm is an extremely hardy plant which grows in swampy terrain, including acidic peat soils, submerged and saline soils, and moist climate with uniformly high sunshine, heat, and humidity. Young sago palms grow shrubby.The full-grown palm trees reach heights of up to 15 m and 60–70 cm trunk diameter.The vegetative phase in the sago palm lasts 7–15 years, during which excess photosynthate from the leaves is transported to the trunk and stored as starch in the pith, the inner portion of the trunk. At maturity the trunk is fully saturated with starch almost to the crown.The starch storage is at its peak just before the plant blooms and dies. A single palm yields up to 360 kg of starch. For the extraction of sago starch the palms are felled. The trunks are divided into sections, split lengthwise, and debarked. At domestic level the pith is rasped manually.The rasped mixture of fiber and pith is macerated by kneading.The starch granules in suspension are separated from the fibers by sieving. At industrial level the debarked trunk sections are fed into a mechanical rasper. This rasps the pith into finer

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pieces, which are fed into a hammer mill. The resulting aqueous starch slurry is made to pass through a series of centrifugal sieves to separate the coarse fibers. Further purification is achieved by separation in a series of cyclone-fluid-separators. Dewatering of starch is carried out using a rotary vacuum drum dryer followed by hot air drying to yield sago flower. Moist sago starch is used to make a popular native food, pearl sago. Besides the use as a foodstuff in the Southeast Asian cuisine, which is its main application, sago starch like other starches has a multitude of industrial uses as a hydrocolloid, stabilizer, and thickener, and as a feedstock for fermentation (Figure 4A.20).27

Figure 4A.20 Sago palm; sago starch extraction and processing. (a) Sago trunks mature and cut to log ready to be processed; (b) The first step of processing sago starch: Starch debarker machine doing peeling works (Pictures (a) and (b) Nee Seng Ngeng & Sons Sago Industries SDN. BHD., Sibu, Sarawak, Malaysia); (c) Sago palm, botanical illustration: Tom Lück, Wikimedia Commons auf Deutsch, licensed under the Creative Commons Attribution 3.0 unported license; (d) Sago starch extraction and processing chain (Biopolymer Research Group (BRG), University Teknologi Malaysia, Johor, Malaysia)


2.15 Stevia Stevia is a herbaceous perennial shrub that grows up to 1 m tall and has leaves 2–3 cm long. It belongs to the Aster family, which is indigenous to the northern regions of South America. Stevia is still found growing wild in the highlands of the Amambay and Iguaçu districts (a border area between Brazil and Paraguay). It is estimated that as many as 200 species of Stevia are native to South America; however, no other Stevia plants have exhibited the same intensity of sweetness as Stevia rebaudiana.28 The plant has been studied and scientifically described for the first time about 1889 by Moisés Santiago Bertoni. Stevia is a heat-loving plant and not frost resistant (Figure 4A.21). In tropical regions without frost the plant has a lifecycle of 4–6 years. Elsewhere stevia cultures must be created every year to be harvested between September and October, but at the latest before the first early frost. With extracts from the leaves having up to 300 times the sweetness of sugar but being virtually calorie-free, stevia has attracted attention with the rise in demand for low-carbohydrate, low-sugar sweeteners. Because stevia has a negligible effect on blood glucose it is attractive to people on low-carbohydrate diet. Stevia’s taste has a slower onset and longer duration than that of sugar and some of its extracts may have a bitter or licorice-like aftertaste. The most important single sweeteners in the S. rebaudiana leaves include (terms of weight fraction): • Stevioside (5–10%) • Rebaudioside A (2–4%) • Rebaudioside C (1–2%) • Dulcoside A (0.2–1%) Especially rebaudioside A has aroused the greed of the food industry: Rebaudioside A is closest to our idea of a sweet taste of all stevia ingredients and has nearly no bitter aftertaste. Chemically, the sweeteners stevioside and rebaudioside A are glycosides of the diterpene steviol. They are constructed by forming a glucose ester at steviol’s carboxyl group, and by forming an acetal at the opposite hydroxyl group with combinations of glucose and rhamnose; stevioside has two linked glucose molecules at the hydroxyl site, whereas rebaudioside A has three, with the middle glucose of the triplet connected to the central steviol structure.

3. SUGARBEET REFINING AND PROCESSING While all fruits and vegetables produce sugar, sugarbeet plants and sugar cane contain the most accessible stores of sucrose. Although sugar is not vital for human consumption, 70% of global sugar production is used for food, 18% for biofuels, and 12% for industrial and other applications.29

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Figure 4A.21 Stevia rebaudiana. (a) Stevia plant; (b) Stevia field (Picture (a) and (b): Foundation of Humanity and Development “F.H.D.” Guido Herrera, with kind permission); (c) Dry leaves of Stevia rebaudiana; (d) chemical structure: Stevioside; (e) chemical structure: Steviol; (f ) chemical structure: Rebaudioside A.


More generally spoken, sugarbeet processing is the production of sugar (sucrose) from sugarbeets. Byproducts of sugarbeet processing include pulp and molasses. Most of the molasses produced is processed further to remove the remaining sucrose. The pulp and most of the remaining molasses are mixed together, dried, and sold as livestock feed.

3.1 Sugarbeet Supply Despite the use of considerable mechanization in sowing, care, and harvesting, the cultivation of sugarbeet is still one of the most labor intensive areas in agriculture. The beets are harvested in the autumn and early winter by digging them out of the ground. They are either stored temporarily in clamps at the side of the field or transported directly to the factory by large trucks. In the sugar factory they are typically received by high-speed conveying and screening systems. The screening systems remove loose dirt from the beets and pinch the beet tops and leaves from the beet roots. The conveyors transport the beets to storage areas and then to the final cleaning and trash removal operations that precede the processing operations. Sugarbeet processing and later sugar processing at the boiling station are accompanied by foam due to surface active substances, such as proteins, sugarbeet pectin (SBP), ferulic acid, which are eluted during the transport and handling in water and stabilized by sugar.30 After cleaning, the beets are separated from the water, usually with a beet wheel, and are transported by drag chain and belt conveyor to the processing operations (Figure 4A.22).

3.2 Extraction The processing starts by slicing the beets into long, thin chips, called cossettes.The cossettes are conveyed to continuous diffusers, in which hot water is used to extract sucrose in a countercurrent flow between 50 and 80 °C until 99% of the sucrose are thus removed. The sugar-enriched water that flows from the outlet of the diffuser is called raw juice and contains between 10% and 15% sugar besides organic and inorganic impurities, so-called nonsugars. This raw juice proceeds to the juice purification operations. The processed cossettes or pulp leaving the diffuser are conveyed to the dried-pulp manufacture operations. Despite being a byproduct of sugarbeet processing, beet pulp itself is low in sugar and other nonstructural carbohydrates, but high in energy and fiber. Among other nutrients, it contains 10% protein, 0.8% calcium, and 0.5% phosphorus. Beet pulp is a valuable animal feedstuff or can be used as a feedstock for biogas manufacture (ref. Chpt. 6).31

3.3 Carbonation The juice must be cleaned up before it can be used for sugar production.This is done by a process known as carbonation. First, the juice passes through screens to remove any small cossette particles.Then the mixture is heated to 80–85 °C and proceeds to carbonation tanks. Here milk of lime [Ca(OH)2] is added to adsorb to the impurities and CO2

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Figure 4A.22 Flow chart—white sugar manufacture from beet. (Environmental Handbook Volume II: Agriculture, Mining/Energy, Trade/Industry, German Federal Ministry for Economic Cooperation and Development/Bundesministerium für wirtschaftliche Zusammenarbeit und Entwicklung GTZ/BMZ, 1995)

gas is bubbled through the mixture to precipitate the lime as insoluble calcium carbonate crystals. The small, insoluble crystals that are produced during carbonation settle in a clarifier and are filtered out together with the nonsugar impurities. After filtration, about 15% solids “thin juice” proceeds to a multistage evaporator which increases the sucrose concentration in the juice by removing water. After evaporation and treatment with decolorizing absorbents and filtration, the percentage of sucrose in the “thick juice” or evaporator syrup is 60–80% or higher with sucrose purity >90%.

3.4 Crystallization In the sugarhouse, sugar is produced from the thick juice obtained by the evaporation of thin juice. The thick juice is pumped to large boiling stations, where even more water is boiled off until conditions are right for sugar crystals to grow. The seed crystals are carefully grown through control of the vacuum, temperature, feed-liquor additions, and steam. When the crystals reach the desired size, the thick, brown juice, a mixture of liquor and crystals, known as massecuite, is spun in a centrifuge to separate the white sugar from the brown syrup.The syrup is returned to the boiling station and boiled again until there is no more sugar left to extract. The sugar crystals are then washed with pure hot water and are sent to the granulator, which is a combination rotary drum dryer and


cooler. After cooling, the sugar is screened and then either packaged or stored in large bins for future packaging. The liquid that was separated from the sugar crystals in the centrifugals is called syrup. This syrup serves as feed liquor for crystallization and is introduced back into the evaporator along with standard liquor and recycled wash water. The process is repeated once again, resulting in the production of molasses, which can be further desugarized using an ion exchange process called Quentin-Process or molasses desugarization. Molasses that is not desugarized can be used in the production of livestock feed or for other purposes.

3.5 Product The final sugar is white and ready for use, whether in the kitchen or by an industrial user such as a soft drink manufacturer. As for raw sugar production, because one cannot get all the sugar out of the juice, there is a sweet by-product made: beet molasses.This is usually turned into a cattle food or is sent to a fermentation plant such as a distillery where alcohol is made. Fulfilling differing consumer needs and expectations sugar is commercialized, for example, as cube sugar, sugar cones, rock candy, icing sugar, instant sugar, preserving or jelly sugar, brown sugar, burnt sugar or caramel or as a semifluid sugar syrup.

4. ALCOHOLIC FERMENTATION In alcoholic fermentation32 (also called ethanol fermentation) yeast species, such as Saccharomyces cerevisiae, convert carbohydrates, mainly sucrose or glucose, under anaerobic conditions into ethanol, and carbon dioxide (Figure 4A.23). First one glucose molecule breaks down into two pyruvate molecules in a process known as glycolysis.The energy from this exothermic reaction is used to bind inorganic phosphate (Pi) to ADP (adenosine diphosphate) forming ATP (adenosine triphosphate) and convert NAD+ (nicotinamide adenine dinucleotide) to NADH (reduced nicotinamide adenine dinucleotide): C6H12O6 + 2ADP + 2Pi + 2NAD+ → 2CH3C(O)C(O)O− + 2ATP + 2NADH + 2H2O + 2H+ The two pyruvates are then broken down into two acetaldehydes and give off two CO2 as metabolic waste products. The two acetaldehydes are then converted to two ethanol molecules by using the H− ions from NADH; converting NADH back into NAD+. Alcoholic fermentation has been used for brewing or winemaking since millennia. More recently, glucose fermentation has become a key technology for the manufacture of ethanol as a biofuel and, based on ethanol, a promising alternative to petroleum refining for the manufacture of low Mw chemical building blocks, such as ethylene.

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Figure 4A.23 Alcoholic fermentation. (Adapted from http://elinow-bioreview2.wikispaces.com/, under a Creative Commons Attribution Share-Alike 3.0 License)

4.1 Alcoholic Fermentation for Beverages Different principles of processing have been developed to obtain alcoholic beverages. The simplest way is to harvest and press ripe grapes and leave the fresh juice to ferment naturally, i.e., without addition of yeast solely by the effect of yeast fungi adhering to the outside of the grapes. The wide ranges of white and red wines that are known worldwide are all made according to this simple principle. When all sugars have been assimilated, the fermentation will stop. Usually, grape wines will contain 9–15 Vol% (alcohol by volume, ABV, or alc/vol, i.e., 72–120 g/l) ethanol.33 Within the European Union, the term “wine” in English and in translation is reserved exclusively for fermented juice of grapes. Within the United States wine may include the fermented juice of any fruit or agricultural product provided that it is between 7 and 24 Vol% ethanol and intended for nonindustrial use. Wines made from products besides grapes are usually named after the product from which they are produced or they feature traditional regional designations. Whereas wine is made by direct fermentation of fruit sugars the art of beer brewing relies on starchy cereals as feedstock. The most common starch source used in beer is malted grain. Grain is malted by soaking it in water, allowing it to begin germination, and then drying the partially germinated grain in a kiln. The germinated, and then dried, grains are called malt. Malting grain produces enzymes, particularly α-amylase (starch-liquefying enzyme), β-amylase (enzyme releasing maltose from starch and its dextrins), proteases, and phosphatases that convert starches in the grain into fermentable sugars. Nearly all beer includes barley malt as the majority of


the starch. The word “brewing” refers to the transformation of starch into fermentable sugars, in this case mainly maltose. After brewing, the resulting sugar solution is boiled with hops (providing desired bitter flavor) and inoculated with selected strains of brewer’s yeast, usually S. cerevisiae. Fermentation is sometimes carried out in two stages, primary and secondary. Once most of the alcohol has been produced during primary fermentation, the beer is transferred to a new vessel and allowed a period of secondary fermentation or maturation, after which the beer is bottled and pasteurized. Nearly all beer includes barley malt as the majority of the starch. Other malted and unmalted grains (including wheat, rice, oats, rye, and less frequently, corn and sorghum) may be used. The alcoholic strength of alcoholic beverages can be increased by relatively simple discontinuous or continuous distillation of the fermented mash (also known as burning) which leads to a distilled beverage (also called spirit, liquor, hard liquor, or a brand). Hard liquors are distinct as a drink due to distillation, and usually have an ABV over 35%. The plethora of alcoholic beverages is traditionally determined by regionally available raw materials and often characterized by proprietary brands and trademarked designations of origin. Some examples listed by raw materials: • Barley: Fermentation → Beer, ale; Fermentation + distillation → Scotch whiskey, Irish whiskey • Rye: Fermentation + distillation → American rye whiskey, Canadian rye whisky, Vodka (Poland), Korn, Doppelkorn (Germany), Akvavit34 (Denmark, Sweden) • Corn: Fermentation + distillation → Bourbon whiskey • Wheat: Fermentation → Bavarian Weissbier, Berliner Weisse, Dutch Witbier Fermentation + distillation → Weizenkorn (Germany) • Rice: Fermentation → Rice beer, Huangjiu (黃酒), Choujiu (China), Chungju (Korea), Sake (Japan) • Fruit: Fermentation → basically, almost any fruit that is suitable for human consumption can serve as raw material for wine; particularly well-known are elderberry, gooseberry, strawberry, feijoa (pineapple guava), plum, and passion fruit wines. The alcohol content of such “Obstweine” can be up to 18 Vol%. • Sugar Palms: Fermentation → Palm wine (also called toddy) Fermentation + distillation → Arrack • Grapes: Fermentation → Wine Fermentation + distillation → Brandy, Cognac, Armagnac (France), Weinbrand (Germany), coñac (Spain) • Apples: Fermentation → Äbbelwoi (Germany), cidre (France), sidra (Spain) Fermentation + distillation → Calvados • Plums: Fermentation → Plum wine Fermentation + distillation → Slivovitz

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• Potato: Fermentation + distillation → Kartoffelschnaps,35 Wodka (Germany), Akvavit22 (Norway) • Sugarcane: Fermentation + distillation → Rum (Caribbean), cachaça (Brazil) • Agava: Fermentation → Pulque (Mexico) Fermentation + distillation → Tequila (Mexico) • Morello cherry: Fermentation + distillation → Kirschwasser (Germany) • Anis: Maceration → Ouzo (Greece), Raki (Turkey), Pernot (France) • Wormwood: Maceration → Absinth • Honey: Fermentation → Mead

4.2 Alcoholic Fermentation for Biofuels Alcoholic fermentation is by far the most common technology for ethanol manufacture, accounting for more than 90% of all ethanol production; the residual amount being made by acid catalyzed hydration of ethylene in the gas phase at 300 °C and 60–70 atm in the presence of a catalyst consisting of solid silicon dioxide coated with phosphoric acid.36 CH2]CH2(g) + H2O(g) ↔ CH3CH2OH(g) Indeed, today, fermentation ethanol is mainly produced for gasoline; only a small share is consumed as alcoholic beverage, as a solvent and for other industrial applications. The biochemical principles that underlie the fermentation for ethanol fuel are the same as for alcoholic beverages. However, because ethanol is hygroscopic and easily picks up water from ambient air and the distribution system and because water is not miscible with gasoline, the water content of fuel ethanol must be limited, when blended with gasoline to reduce the risk of phase separation and to avoid engine stall due to “slugs” of water in the fuel lines interrupting fuel flow. For that reason, anhydrous ethanol (i.e., an ethyl alcohol that has a purity of at least 99%, exclusive of added denaturants) has traditionally been specified in most countries when ethanol is blended with gasoline to be used as motor fuel. 4.2.1 Raw Materials Ethanol can be produced from any source containing appreciable amounts of sugar or materials such as starch and cellulose that can be converted into sugar (Figure 4A.24). As already said, the disaccharide sucrose is readily fermented by S. cerevisiae after hydrolysis into glucose and fructose by the enzyme invertase which is naturally present in this yeast. When ethanol is produced from sugarcane or sugarbeet, the molasses, cane juice or beet syrups (thick juice) are directly used as raw material. Starch containing biomass, such as wheat, maize, triticale and barley, may be used as meal after grinding (dry milling) or undergo a wet milling operation to separate the starch from proteins, oil and fibers. For the processing of corn (maize) both technologies are used. Corn wet mills are generally larger in size than dry-grind facilities and achieve


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Figure 4A.24 Production of bioethanol. (Adapted from Dinjus E, Arnold U, Dahmen N, Höfer R, Wach W. Green fuels – sustainable solutions for transportation. In: Höfer, R. ed., Sustainable solutions for modern economies, RSC Green Chem. No 4, RSC Publ., Cambridge (2009) p. 125–163)

a more targeted separation of the grain in the individual components.37 In dry milling, the entire corn kernel is first processed into flour, which is referred to in the industry as “meal.” The same applies to other cereals. This meal is then slurried with water to form a mash (liquefaction). Enzymes are added to the mash to convert the starch in a process called saccharification to glucose syrup (ref. 9.4.3.2) as raw material for fermentation. A particularly abundant source of fermentable sugar is the cellulose and hemicellulose which exist in lignocellulosic biomass, a composite material of rigid cellulose fibers embedded in a cross-linked matrix of lignin and hemicellulose. Lignocellulosic biomass is often a waste material of the food processing and forest products industries that may be locally readily available. However, to make this sugar source available for fermentation, the lignocellulose composite structure must be degraded by pretreatment technologies38 such as the organosolv process or steam explosion, ammonia fiber explosion, acid hydrolysis, or ozone pretreatment and the cellulose and hemicellulose must be broken down into simple sugars by concentrated acid, dilute acid, or enzymatic hydrolysis. 4.2.2 Fermentation Industrial yeast (S. cerevisiae, also known as “brewers yeast” because it is also used for brewing beer and making bread) is used to convert sugars to ethanol and carbon dioxide

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(CO2). The fermentation process usually occurs in batches with each fermentation tank filled and fermentation completed before being drained and refilled with a new batch. Some facilities use continuous fermentation processes in which new fermentation material is continuously added and fermented product continuously removed. Continuous fermentation has greater reactor productivity, but there are fewer opportunities for contamination in batch reactors. 4.2.3 Distillation The fermented mash (now called beer) contains 8–12% ethanol by weight. For the ethanol to be usable as a fuel, the majority of the water must be removed. Most of the water is removed by distillation. The slurry form of material obtained from bottom of beer still is called “distillery slop.”39 It is dried to produce dried distillers grains with solubles used for cattle feed and fertilizer.The streams coming out at different sections of the column are aldehydes from top, fusel oil and ethanol mixture from middle and bottom stream with water. The middle stream is fed to a rectification column to produce a product called rectified spirit, a low-boiling water-ethanol azeotrop with maximum 96.5 Vol% ethanol and 3.5% water and a boiling point of 78.1 °C.This mixture is also called hydrous ethanol and can be used as a fuel alone, but unlike anhydrous ethanol, hydrous ethanol is not miscible in all ratios with gasoline, so the water fraction is typically removed in further treatment to burn in combination with gasoline in gasoline engines. 4.2.4 Anhydrous Ethanol Anhydrous ethanol, also known as absolute ethanol is a clear, colorless and homogeneous liquid consisting of at least 99.5% ethanol by volume at 15.6 °C. Several technologies have been developed to convert rectified spirit further to absolute alcohol or anhydrous ethanol.40 4.2.4.1 Azeotropic Distillation

Several compounds such as benzene, n-pentane, cyclohexane, hexane, heptane, isooctane, acetone, diethyl ether can be used as an entrainer to produce anhydrous ethanol.41 Of these, benzene and cyclohexane have been used most extensively. Presently, benzene is in disuse due to its carcinogenic nature, although it is still being employed in some countries. 4.2.4.2 Extractive Distillation with Liquid Solvent

Extractive distillation with liquid solvent is used commercially for the production of anhydrous ethanol from ethanol–water mixtures. This method uses a relatively nonvolatile liquid solvent, such as ethylene glycol, which is fed into a distillation column some trays above the ethanol feed tray. The presence of the solvent at relatively high concentration in the liquid on the trays alters the volatility of one of the feed components more than the other, so a separation of the feed components can be made in the column.


4.2.4.3 Extractive Distillation with Soluble Salt

The use of salts to break the ethanol–water azeotrope for industrial production of anhydrous ethanol from dilute solutions dates back to the patents registered in the period 1932–1934 which were the basis for the implementation of the HIAG process (HIAG-Verein, Holzverkohlungsindustrie AG, later Degussa, today Evonik). The HIAG process employed a 70–30 molten mixture of potassium and sodium acetate (fed into the hot reflux stream of the distillation column) as agent to break the azeotrope, and produced anhydrous ethanol. 4.2.4.4 Molecular Sieves

Molecular sieves or Molsieves have become the most popular means to dehydrate ethanol to absolute or nearly absolute levels and have more or less entirely replaced azeotropic distillation. Zeolite molecular sieves are usually employed. 4.2.4.5 Membrane Processes

Hyperfiltration (reverse osmosis), pervaporation, and gas/vapor permeation have been proposed for producing anhydrous ethanol. Several companies such as GFT Membrane Systems GmbH, Kalsep, and Lurgi have constructed membrane separation plants which utilize the pervaporation process or the vapor permeation process where flat membranes are used. 4.2.4.6 Future Technologies

High gravity fermentation, quick steeping, new strains of grain and improved enzymes, yeasts and process automation are at the horizon to further make dry milling of corn an attractive technology for bioethanol production.42

4.3 Acetic Acid It is known to mankind since prehistoric times that wine, beer, or other weak fermented liquors will become sour on exposure to the air. The underlying aerobic fermentation has been used ever since for the production of acetic acid, colloquially known as vinegar.Vinegar making, so as it is practiced by chefs around the world in the kitchen, as well as by the industry, involves a two-step process—the anaerobic yeast fermentation to yield ethanol (ref. 4.1, 4.2) followed by an aerobic bacterial fermentation process yielding acetic acid. Acetic acid fermentation is performed by the action of acetic acid bacteria (acetobacteraceae,AAB). AAB first oxidizes the ethanol to acetaldehyde, which is then converted to acetic acid: R–CH2OH → 2e− + 2H+ + R–CHO

R–CHO + H2O → R–COOH + 2e− + 2H+

Historically, small amounts of acetic acid were also produced as a result of destructive distillation (or carbonization) of hardwoods. Around 80% of acetic acid, however, is actually manufactured synthetically by methanol carbonylation using various catalysts, another 20% by acetaldehyde, liquid butane/naphtha oxidation, or methylacetate

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carbonylation yielding acetic anhydride. Dilute acetic acid (5–18%, vinegar) produced by fermentation represents only a small part of less than 5%.

5. THE ETHANOL-BASED C2—VALUE CHAIN Beyond the use of ethyl alcohol as a beverage, solvent or transportation fuel, the alcoholic fermentation of sucrose opens a pathway to an alternative C2 chemistry starting from ethanol as a reactive chemical intermediate and building block43 and applying technologies that are well-established unit operations in the chemical industry.

5.1 Bioethanol to Ethylene By inversion of the catalytic ethylene hydrolysis (Chapter 4.2) fuel-grade anhydrous bioethanol is rather easily dehydrated to ethylene at elevated temperature using, for example, a silicoaluminophosphate, HZSM-5 zeolite, or a heteropolyacid catalyst in a fixed bed or fluidized bed reactor (Figure 4A.25).44 The bioethylene produced can be integrated in existing downstream operations to produce C2-based intermediates and polymers,45 such as: • polymerization to polyethylene (PE); • alkylation of benzene yielding ethylbenzene followed by dehydrogenation yielding styrene C6H6 + C2H4 → C6H5CH2CH3 → C6H5CH]CH2 + H2 ȋƪǦȌ

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• chlorination or oxychlorination yielding dichloroethane as intermediate for the production of vinylchloride monomer (VCM) and PVC.46 CH2]CH2 + Cl2 → ClCH2–CH2Cl 2H2C]CH2 + 4HCl + O2 → 2ClCH2–CH2Cl + 2H2O Cl–CH2–CH2–Cl → H2C]CH–Cl + HCl • catalytic oxidation to yield acetaldehyde.47 [PdCl4]2− + C2H4 + H2O → CH3C(O)H + Pd + 2HCl + 2Cl− • oxidative acetoxylation with acetic acid using a Pd/Au catalyst to yield vinyl acetate (Wacker process) C2H4 + CH3C(O)OH + 1/2 O2 → CH3C(O)OCHCH2 + H2O inyl acetate monomer (VAM) is broadly used for the manufacture of polyvinyl acetate V (PVAc), polyvinyl alcohol (PVAl) and ethylene-vinyl acetate (EVA)-, vinyl acetateacrylic-, vinyl chloride-vinyl acetate (VC/VAc)-, and vinyl pyrrolidone-vinyl acetate (Vp/VAc)-copolymers. • oxidation to yield ethylene oxide (EO): 2C2H4 + O2 → 2C2H4O Industrially, this reaction proceeds at 200–300 °C and a pressure of 10 bar at a silver catalyst in tube bundle reactors. Alternatively, the direct conversion of ethanol using a Au/Li2O/Al2O3 catalyst to yield EO has been investigated.48 By ethoxylation of natural fatty alcohols from vegetable oils with biobased ethylene oxide fully biobased nonionic fatty alcohol polyglycol ether surfactants can be manufactured

ROH + n C2H4O → R(OC2H4)nOH

• Dimerization yielding 1-butene49 (IFP-SABIC alphabutol process) 2CH2]CH2 → CH3CH2CH]CH2 • Olefin metathesis (Olefin Conversion Technology, OCT) of ethylene and 2-butene (manufactured by isomerization of 1-butene) yielding propylene.50 CH2]CH2 + CH3CH]CHCH3 → 2CH3CH2CH]CH2

5.2 Ethylamine The direct amination of ethanol (from alcoholic fermentation) with ammonia yields ethylamine.51

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CH3CH2OH + NH3 → CH3CH2NH2 + H2O Ethylamine is also produced by reductive amination of acetaldehyde CH3C(O)H + NH3 + H2 → CH3CH2NH2 + H2O

5.3 Ethylesters Ethyl esters are formed by acid-catalyzed elimination of water from a carboxylic acid with ethanol

R–C(O)OH + CH3CH2OH → R–C(O)OCH2CH3 + H2O

In the main esterification process for acetic acid ethyl ester (ethyl acetate) a mixture of acetic acid and ethanol with a small amount of sulphuric acid is preheated and fed to an esterifying column where it is refluxed.The mixture removed goes to a second refluxing column where a ternary azeotrope containing 85% ethyl acetate is removed. Water is mixed with the distillate after which it separates into two layers. The top layer is fed to a refluxing column from which the residue containing 95% ethyl acetate is distilled to remove any impurities. Ethyl acetate and ethyl lactate are used as solvents. Some ethyl esters of carboxylic acids are used as flavor and aroma substances.

6. BEYOND C2 PLATFORM CHEMICALS BY FERMENTATION Fermentation processes can be a valuable alternative to the conventional chemical synthesis, particularly when the finished product contains specific and complex stereochemistry. Fermentation technology in the industrial synthesis of chemicals started to be used in the first decades of the twentieth century. Industrial production of citric acid by fermentation, achieved by Pfizer in 1923, was an early success in this field. However, it was only with the production of penicillin during the Second World War that the whole sector took off. Today, besides ethanol, the range of products that are produced by fermentation includes antibiotics, organic acids, amino acids, polysaccharides, vitamins, and enzymes.

6.1 C3 Building Blocks 6.1.1 Lactic Acid Although already discovered in 1780 by the Swedish chemist Carl Wilhelm Scheele, who isolated the lactic acid from sour milk, lactic acid has attracted more recently a great deal of attention due to its widespread applications, mainly in food, chemical, cosmetic, and pharmaceutical industries. Also, it has a great potential for the production of biodegradable and biocompatible polylactic acid52 (PLA) and, besides 3-hydroxypropionic acid, as an intermediate for sugar-based acrylic acid.53 Lactic acid production can be achieved either by chemical synthesis routes or by fermentative production (lactic acid fermentation). By the chemical synthesis route, a racemic mixture of dl-lactic acid is usually


produced. Commercial lactic acid is actually produced by fermentation. By using an appropriate bacterial or fungal strain both, L(+) or D(−) lactic acid can be produced specifically. Lactic acid bacteria are classified into homofermentative or heterofermentative. Homofermentative lactic acid bacteria produce virtually a pure lactic acid, whereas heterofermentative produce byproducts such as ethanol, formic, or acetic acid along with lactic acid. Besides Bacillus strains, Escherichia coli and Corynebacterium glutamicum, the main homofermentative LAB used for the production of lactic acid from different carbon sources are Lactococcus lactis, Lactobacillus delbrueckii, L. helveticus, or L. casei. Batch fermentation is the most commonly used fermentation process starting with the addition of lime or chalk, glucose, and water to the reactor. The process produces crude calcium lactate, as a result of pH neutralization, that must be precipitated and reacidified by a mineral acid such as sulfuric acid. These steps are considered as major economic hurdle for lactic acid production because it generates gypsum waste material.54 In order to get rid off this main issue, commercial lactic acid fermentation technologies have now been developed that use recombinant yeast instead of bacteria and therefore can be carried out under acidic conditions.55 6.1.2 3-Hydroxypropionic Acid 3-Hydroxypropionic acid (3-HP) is an organic acid that has a potential utility for specialty synthesis to malonic acid by oxidation, to esters by esterification reactions with alcohols, to 1,3-propanediol by reduction, and is actually the most promising pathway (alternatively to lactic acid) to be converted to acrylic acid. Actually, acrylic acid is produced first and foremost by the two-step, catalytic gas-phase oxidation of propylene (oxidizing primarily proylene to acrolein, and then, in a second stage, the reaction of acrolein to acrylic acid) where the propylene in turn is obtained by steam cracking of propane and butane (from natural gas and oil), steam cracking of naphtha (from crude oil) or catalytic cracking of gas oil (from oil) and propane (from shale gas). Although chemical synthesis routes have been described to produce 3-HP, biocatalytic routes are regarded as the more promising approach, particularly the use of genetically recombinant E. coli to ferment glucose syrup56 (or glycerol) to 3-HP which is then dehydrated in the presence of a strong acid catalyst, such as H3PO4, to form acrylic acid (Figure 4A.26). 6.1.3 Isopropanol Isopropanol is currently synthesized via three different methods: indirect hydration of propylene (also called the sulfuric acid process), direct hydration of propylene, and catalytic hydrogenation of acetone. Efforts have been made to produce isopropanol by utilizing the TA76 strain of metabolically engineered E. coli. After the alcohol accumulates in the culture, production drastically decreases. Isopropanol removal by gas stripping allows for the continuation of the conversion process.57 Further development of this process may result in an alternative route to propylene by the dehydration of bioisopropanol.

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Figure 4A.26 Acrylic acid synthesis routes. (Adapted from Ref. 53 a)

6.2 C4 Building Blocks 6.2.1 n-Butanol n-Butanol (1-butanol) is a four carbon straight chain alcohol with a boiling point of 118.8 °C (compared to iso-butanol (2-methyl-1-propanol) 108 °C; sec. butanol (2-butanol) 99 °C; tert. butanol (2-methyl-2-propanol) 83 °C). 1-Butanol is actually produced by hydroformylation of propene with syngas yielding a blend of n- and i-butanol depending on reaction conditions.The reaction first generates butanal (n-butyraldehyde) which is then reacted with H2 to form n-butanol and/or i-butanol. Biobutanol is produced through the anaerobic fermentation of corn by the ABE process (acetone butanol ethanol fermentation process) using the Weizman Organism Clostridium acetobutylicum or C. beijerinckii. The process produces ABE in a ratio of 3:6:1. The process also creates a recoverable amount of H2 and a number of other byproducts.58 Although the potential use as a biofuel has created considerable interest in biobutanol as compared to fossil fuel and bioethanol, the primary limitation associated with ABE fermentation are the toxic effects that butanol has on microorganisms. Accordingly, within the traditional ABE fermentation process, the concentration of butanol usually does not exceed 1.3%.


Therefore, the search for high butanol-tolerant microorganism strains as well as to employ in situ toxicity removal technologies (such as stripping, adsorption, and pervaporation) in order to overcome the toxicity problem is ongoing.59 Biobutanol similar to bioethanol can be dehydrated yielding 1-butene (instead of ethylene) and then by SHOP-, Guerbet- or Ziegler-Natta-technologies yield α-octene and higher α-oligomers to be converted into biobased SHOP-, Ziegler-, or Guerbet-alcohols.The Guerbet reaction is an organic reaction converting a primary aliphatic alcohol into its β-alkylated dimer alcohol with loss of one equivalent of water. This reaction requires a catalyst and elevated temperatures. 6.2.2 Isobutanol Similar to n-butanol, isobutanol is being investigated as a second generation biofuel because it does not readily absorb water from air (preventing the corrosion of engines and pipelines), the high energy density and the miscibility at any proportion with gasoline, meaning the fuel can “drop into” the existing petroleum infrastructure as a replacement fuel or major additive. Besides the fermentation pathway to n-butanol, bio-isobutanol can be produced by bioengineered organisms, such as cyanobacteria, E. coli, Bacillus subtilis, S. cerevisiae, or Ralstonia eutropha. Besides glucose, these organisms can use other carbon sources for its growth and energy supply: other monosaccharides such as fructose and ribose; disaccharides such as sucrose, mannose, and maltose; organic acids such as lactic acid, but also some amino acids such as l-glutamate.60 Isobutanol as a chemical intermediate gives access to aromatics like p-xylene from biomass by a reaction pathway consisting in sequentially dehydrating isobutanol in the presence of a dehydration catalyst to provide a C4 alkene such as isobutylene, dimerizing the C4 alkene to form one or more C8 alkenes such as 2,4,4-trimethylpentenes or 2,5-dimethylhexene, then dehydrocyclizing the C8 alkenes in the presence of a dehydrocyclization catalyst to selectively form renewable p-xylene in high overall yield. The p-xylene can then be oxidized to form terephthalic acid or terephthalate esters (Figure 4A.27).61

Figure 4A.27 The isobutanol value chain toward terephthalic acid.

Although the bioengineered organisms are being moved forward toward higher isobutanol productivity through genetic modifications, they have not yet achieved the ability to produce bio-isobutanol in quantities large enough for commercial use.62

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6.2.3 The Biosuccinic Acid Platform Succinic acid (butanedioic acid; historically known as spirit of amber) is a naturally occurring dicarboxylic acid, discovered by Georgius Agricola in 1546 by dry distillation of amber. Succinic acid is a natural component of the Krebs cycle (also called tricarboxylic acid (TCA) cycle and citric acid cycle). This metabolic process and thus the biosynthesis of succinic acid occur in most plants, animals, fungi, and many bacteria. Until recently, all succinic acid was produced from petroleum feedstocks by hydrogenation of maleic anhydride and subsequent hydration. However, ambitious projects are underway to produce this interesting platform chemical by White Biotechnology operations. Furthermore, fermentation of glucose to yield succinic acid has distinct advantages. While during bioethanol production, for example, 2 mol inert waste CO2 per mole glucose are formed, biosuccinic acid production is a CO2 fixing operation.63

Bioethanol fermentation: C6H12O6 → 2C2H5OH + 2CO2

Biosuccinic acid fermentation: C6H12O6 + 2CO2 → 2C4H6O4

Besides other carbohydrates, dextrose from maize and sucrose from sugarcane are evaluated as feedstocks for biosuccinic acid.Yeasts, filamentous fungi, including Penicillium simplicissimum, and a number of bacteria (Corynebacterium glutamicum, Enterococcus faecalis, Actinobacillus succinogenes, Mannheimia succiniciproducens, Anaerobiospirillum succiniciproducens, recombinant E. coli) have shown to accumulate succinic acid.The microorganisms are first propagated on fermentable sugar, nutrients, and ammonia. They are then mixed with a fermentable sugar feedstock in a fermenter, where a fermentation process produces the disodium or diammonium succinate. Several challenges must be overcome. Microorganisms that produce succinic acid are often not able to tolerate large concentrations of the acid or its salt. Another challenge is the narrow pH under which bacterial microorganisms can operate. Accordingly, different process variations have been developed: • low pH yeast fermentation with downstream processing (DSP) by direct crystallization (DC), • anaerobic fermentation to succinate salt at neutral pH and subsequent DSP by electrodialysis (ED), and • anaerobic fermentation producing diammonium succinate (AS) followed by acidification and formation of diammonium sulfate as coproduct in DSP. Life cycle assessments suggest that low pH yeast fermentation with direct crystallization is the most beneficial process to bio-based succinic acid from an environmental perspective (Figure 4A.28).64 On the one hand, the anaerobic fermentation producing diammonium succinate (AS) as intermediate permits the economical production of 2-pyrrolidone and N-methyl2-pyrrolidone without the prior isolation of succinic acid. 2-Pyrrolidone can be produced by the direct hydrogenation of AS in the presence of an active metal catalyst and a mixture

Sugar- and Starch-Based Biorefineries OH

HO

1.4-butanediol

OH HO HO

O

Esterification

O

MeOOC

COOMe

OH OH

Tetrahydrofuran

Fermentation

HOOC

O

-butyrolactone

Hydrogenation

COOH

O

Dehydration

Succinic Acid

HOOC

O Dehydrogenation Cyclization

O

COOH Fumaric Acid

O

COOH COOH Maleic Acid

Figure 4A.28 Succinic acid as a platform chemical. (Adapted from Ref. 43)

of 2-pyrrolidone and N-methyl-2-pyrrolidone is yielded by the catalytic hydrogenation of AS in the presence of methanol.65 On the other hand, the biosynthesis of succinic acid opens alternative pathways to a number of chemical commodities and specialty chemicals: Hydrogenation of succinic acid or its dialkylester leads to 1,4-butanediol—as alternative to established industrial manufacturing processes such as the Reppe process starting from acetylene and formaldehyde, Mitsubishi’s butadiene-acetic acid process, Kuraray’s propylene oxide/allyl alcohol technology, Toyo Soda’s dichlorobutene process, and the hydrogenation of dimethyl or diethyl maleate. The acid-catalyzed dehydration of 1,4-butanediol would yield tetrahydrofuran (THF). It is, however, noteworthy that THF is part of a well established value chain refining pentosan-containing agricultural wastes, such as corncobs, cottonseed hull bran, oat hulls, rice hulls, or bagasse.66 6.2.4 The Pentosan Value Chain Pentosans are hemicelluloses (polysaccharide components of plant cell walls of woody plant tissue other than cellulose; xylan is an example of a pentosan consisting of d-xylose units with 1β→4 linkages) or arabinoxylans (polysaccharides found in the bran of grasses and grains such as wheat, rye, and barley) (Figure 4A.29). In the conventional (Quaker Oats, now IFC) manufacturing process, the agricultural waste raw material is mixed with dilute sulfuric acid and the pentosans are hydrolyzed to pentoses (e.g., xylose), which are then cyclodehydrated to furfural. The furfural formed is recovered by steam distillation and fractionation and then submitted to decarbonylation and hydrogenation to yield THF.

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Renewable raw materials containing pentosans

+ H2SO4 + H2O → furan resins D-xylose

xylan - 3 H2O

furfuryl alcohol

furfural

2-methyl furan

- CO

furan

tetrahydrofuran

Figure 4A.29 The pentosan value chain.

6.2.5 2,5-Furandicarboxylic Acid (FDCA) and Polyethylene Furanoate (PEF) Since the first reported synthesis of FDCA by Rudolf Fittig in 1876 by reacting mucic (galactaric) acid with hydrobromic acid, furandicarboxylic acid has permanently been subject of research as a sugar-based chemical intermediate and polymer building block but has not made it to the industrial production. However, more recently a process has been discovered that allows for the economic conversion of carbohydrate biomass, such as corn starch, into fructose; the conversion of fructose into alkoxymethylfurfural (RMF) using a dehydration process in the presence of alcohols and an acid catalyst in a continuous flow fixed bed reactor at 175°-225°C (with intermediate formation of 5-hydroxymethylfurfural, 5-HMF); the catalytic oxidation of RMF in acetic acid to make 2,5-furandicarboxylic acid; and the polycondensation of FDCA with bio-based ethylene glycol into polyethylene furanoate (PEF) (Figure 4A.30). Thereby FDCA has become a potential compound to replace terephthalic acid and PEF the fully bio-based counterpart of polyethylene terephthalate (PET).

6.3 Higher Carboxylic Acids 6.3.1 Citric Acid Citric acid (2-hydroxypropane-1,2,3-tricarboxylic acid) is a weak organic tricarboxylic acid with three different values of pKa (3.1, 4.7, and 6.4). It can exist either in an anhydrous (water-free) form or as a monohydrate. At room temperature, citric acid is a white hygroscopic crystalline powder (Figure 4A.31). It is a primary metabolic product formed in the Krebs cycle used by all aerobic organisms to generate energy and as such found in small quantities in virtually all plants and animals.

Sugar- and Starch-Based Biorefineries Cellulose, Starch, Sucrose carbohydrate biomass

5-HMF glucose

alkoxymethyl furfural

fructose

2,5-FDCA ethanol

ethylene

ethylene oxide

ethylene glycol

Polyethylene furanoate (PEF)

Figure 4A.30 The Pathway to Polyethylene Furanoate (PEF) Adapted from: a.) Kröger, M.; Prüße, U.; Vorlop, K-D, A new approach for the production of 2,5-furandicarboxylic acid by in situ oxidation of 5-hydroxymethylfurfural starting from fructose, Topics in Catalysis (08-2000) 13(3):237-242; b.) Gruter, G.J.M.; Dautzenberg, F. Method for the synthesis of 5-alkoxymethyl furfural ethers and their use, US 8,133.289, 2007 (Furanix Technologies); c.) Sipos, L. A process for preparing a polymer having a 2,5-furandicarboxylate moiety within the polymer backbone and such (co)polymers, WO 2010077133, 2009 (Furanix Technologies); d.) Thiyagarajan,S.; Pukin, A.; van Haveren, J.; Lutz, M.; van Es, DS. Concurrent formation of furan-2,5- and furan-2,4-dicarboxylic acid: unexpected aspects of the Henkel reaction, RSC Adv., 2013, 3, 15678-15686; e.) Gotro, J. Polyethylene Furanoate (PEF): 100% Biobased Polymer to Compete with PET? (Apr. 8, 2013), http://polymerinnovationblog.com/polyethylene-furanoate-pef-100-biobased-polymer-to-compete-with-pet/; f.) Lanzafame, P.; Centia, G.; Perathoner, S. Catalysis for biomass and CO2 use through solar energy: opening new scenarios for a sustainable and low-carbon chemical production, Chem. Soc. Rev., 2014,43, 7562–7580.

Although industrial-scale citric acid production originally was based on the physical extraction from citrus fruit or lemon juice, resp., fermentation is the major route to commercial citric acid since Pfizer began industrial-level production using this technique. All substrates containing glucose, including sucrose, beet molasses, corn syrup, and dextrose from enzyme-treated starch, are well-suited for the manufacture of citric acid. Processes employed are surface fermentation in shallow pans or submerged fermentation in bubble column fermenters using Aspergillus niger mold and submerged fermentation by yeast (Candida guilliermondii, C. lipolytica), with A. niger still being the main industrial fermenting organism. After the mold is filtered out from the fermentation broth, citric acid is recovered by solvent extraction or more commonly by calcium citrate precipitation,

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Figure 4A.31 Citric acid.

followed by treatment with sulfuric acid to convert the calcium citrate to calcium sulfate and citric acid and then removing the calcium sulfate by filtration. The final processing involves crystallization, centrifuging, fluidized bed drying, and classification.The product is sold as an anhydrous or monohydrate acid.67 About 70% of total citric acid production is used as an acidulent, flavor enhancer and preservative in food and carbonated beverages, about 15% as detergent builder cobuilder or cleaner in liquid household detergents, detergent powders, hard-surface cleaners, and fabric softeners. Other applications include the pharmaceutical industry as antioxidant or in the form of iron citrate as a source of iron for the body; the use as chelating agent and pH adjuster in hair conditioners and shampoos or in metal finishing and cleaning; as a setting retarder in gypsum plaster; the use as starting material for a number of citrate esters, such as acetyl tributyl citrate, tributyl citrate, and tri-2-ethylhexyl citrate used as plasticizers for a variety of vinyl resins and films, or in some cellulose acetate and cellulose nitrate gums and resins.68

6.4 Amino Acids 6.4.1 Glutamic Acid Glutamic acid (α-aminoglutaric acid) is an example for a bulk amino acid which is exclusively produced by fermentation. Since Kyowa Hakko in 1956 started the first industrial production, the product has been continuously growing.69 Global production has reached 1.7 Mio mto. in the year 2007 and has risen by 2010 to over 2 Mio mto.The primary worldwide use of glutamic acid is as intermediate for the production of monosodium glutamate (MSG). MSG functions as umami flavor enhancer in food.Virtually all MSG is consumed in the production of foods. A negligible amount of MSG is used in animal feeds. Currently, China is the world’s largest MSG producing and consuming country. Chinese production and consumption accounted for approximately 73% and 67% of world production and consumption in 2009, respectively.70 MSG exists as stereoisomers but commercial MSG is made to contain over 99% of the naturally predominant l-glutamate form. Bacterial species such as C. glutamicum and Brevibacterium strains are used as bioconverters in the fermentation process (Figure 4A.32). Selection of raw materials is essential for economic amino acid production in general. According to a decentralized biorefinery concept amino acid producers are often located close to sugar or starch plants in order to decrease transport costs. Depending on geographical location of the manufacturing plant carbon sources like cane molasses, beet molasses, or starch hydrolysates from corn, potato, or cassava are used.While molasses are

Sugar- and Starch-Based Biorefineries Sugars in syrup are taken into the fermentative microorganism

Sugar cane Fermentation Glutamate is increased. Fermentative microorganisms are added. Sugars

Fermented broth Fermentation tank

Sugars

Excreted into the fermented broth Glutamate

Glutamate is accumulated.

Crystallization of mono sodium glutamate

The mono sodium glutamate crystal is dried.

Figure 4A.32 Production of mono sodium glutamate by fermentation. (Ajinomoto Co. Inc. with kind permission)

common in Europe, South America, and China, starch hydrolysate is the most important carbon source in North America.71 Starch hydrolysate from cassava (Manihot esculenta Crantz, popularly known as tapioca) is widespread in South-East Asia.72 Pure sugars are usually favorable compared with molasses because of unwanted side reactions and changing qualities of the complex media components. In addition, glutamic acid is isolated from the spent wash from molasses desugarization processes especially from Steffen waste; from the regeneration wash from deionization processes; or from the nonsugar fraction in the chromatographic separation of molasses into sugar and nonsugar fractions during crystal sugar manufacturing from sugarbeet in a sugar factory.3

7. SUCROCHEMISTRY The sucrose molecule, as it occurs in sugar crops such as sugarbeet and in table sugar is chemically a disaccharide consisting of α-d-glucose (dextrose) and β-d-fructose (levulose, fruit sugar). These two molecules are connected by an acetal oxygen bridge that forms an α,β-1,2-glycosidic bond, i.e., an α-d-glucopyranosyl-[α1→2]-β-dfructofuranose. The entirety of chemical reactions (but not the microbiological transformations) that convert the sucrose molecule was given the name “sucrochemistry.”1 However, enzymatic catalysis is applied for the industrial synthesis of sucrose isomers, such as isomaltulose (Palatinose™, Südzucker) and trehalulose. Although a sugar replacer itself, currently isomaltulose is mostly used as an intermediate for hydrogenation using a

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Raney nickel catalyst to yield the tooth friendly sweetener Isomalt® (Südzucker), chemically an equimolar composition of 6-o-α-d-glucopyranosido-d-sorbitol (1,6-GPS) and 1-o-α-d-glucopyranosido-d-mannitol-dihydrate (1,1-GPM-dihydrate).73

7.1 Inverted Sugar Syrup The sucrose molecule can be split into glucose and fructose by acid hydrolysis or enzymatically by treatment with invertase (from S. cerevisiae, also called β-fructofuranosidase) (Figure 4A.33).

Figure 4A.33 Sugar inversion. (Transferred from en.wikipedia by Ronhjones available under Creative Commons.)

Industry standard is the heterogeneous acid-catalyzed process by an acidic ion exchange resin in a fixed bed column.74 After hydrolysis, the 1:1 mixture of glucose and fructose is called invert sugar. Invert sugar is a colorless or slightly straw-colored viscous syrup. It is a natural humectant providing solubility and resistance to crystallization. This is why it is ideal as a sweetener for soft drinks. Its ability for controlling crystallization and creating a smoother mouth feel is the main reason for professional pastry chefs to use invert sugar in confectionary for preparations such as ganache and jellies and in the preparation of sorbets and ice cream. As invert sugar becomes brown when it is heated, enhancing its flavor it is also ideal for making caramel and other toffee sweeteners. The inverted sugar syrup can be further separated into glucose and a fructose fraction by a continuous chromatographic procedure.75

7.2 Gluconic Acid On the one hand, gluconic acid is obtained by mild continuous-flow oxidation of glucose using O2 promoted by supported Au, Pt, and Pd catalysts76; on the other, an enzymatic process for the oxidation of glucose into gluconic acid and hydrogen peroxide (H2O2) uses a combination of glucose oxidase and catalase enzymes which may be obtained from an A. niger strain (Figure 4A.34).77

Figure 4A.34 Production of gluconic acid via glucose oxidation.


Gluconic acid is used as an acidity regulator in food. It is also used in cleaning products where it dissolves mineral deposits especially in alkaline solution. The gluconate anion chelates Ca2+, Fe2+, Al3+, and other metals.

7.3 Sucralose Sucralose (also called 4,1′,6′-trichloro-4,1′,6′-trideoxygalactosucrose, E955) is technically a chlorinated carbohydrate, and it is made by selectively replacing three hydroxyl groups on the sugar molecule with three chlorine atoms (Figure 4A.35). This chlorination is achieved by different process variants on the basis of the original Tate & Lyle patent78 via selective protection of the primary alcohol groups followed by acetylation and then deprotection of the primary alcohol groups. Following an induced acetyl migration on one of the hydroxyl groups, the partially acetylated sugar is then chlorinated with a chlorinating agent such as phosphorus oxychloride, followed by removal of the acetyl groups to give sucralose.79 Sucralose is approximately 600 times sweeter than sugar and is not metabolized and excreted unchanged again in the body. It is calorie-free and can be used in tooth-friendly products. Sucralose distinguishes itself in particular in combination with other sweeteners, and is applicable through its high quality honey, its very good water solubility and its stability in a wide range of foods and beverages.

Figure 4A.35 Sucralose manufacturing process. (Adapted from Ref. 79)

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7.4 Sucrose Esters The limited solubility of sucrose, the temperature sensitivity and tendency to caramelization even below the melting point (185–186 °C) and the presence of three primary and five secondary hydroxyl groups, all of which can react, make the synthesis of sucrose esters80 a meticulous task. Completely or highly esterified sucrose octaacetate, a hygroscopic, white crystalline solid, is manufactured by reacting sucrose with glacial acetic acid or acetic acid anhydride. It has a bitter taste which has led to its use as bitterant and aversive agent. A noncrystallizing sucrose ester is sucrose acetate isobutyrate (SAIB) manufactured by reacting sucrose with acetic and butyric anhydride in the presence of an in situ formed mixture of sodium acetate and sodium isobutyrate as a catalyst (Figure 4A.36).81 SAIB is a stable liquid that is soluble in most alcohols, vegetable oils, animal fats, and vegetable-based waxes. It has a light color, as well as good thermal, hydrolytic, and color stability. It is used as an additive in nonalcoholic beverages, both carbonated and noncarbonated, and as a weighting agent that adds density to the beverage, in effect thickening the consistency. Outside the food industry SAIB is used as a plasticizer, pigment dispersant, flow improver and adhesion promoter in solvent based inks, lacquers, decorative coatings, lipsticks and nail polishes. Fatty acid esters of sucrose are manufactured by transesterification of fatty acid methyl ester (FAME) in dimethylformamide or dimethyl sulfoxide or solvent-free, alkaline or enzymatically catalyzed.82 The alcoholysis with FAME is catalyzed by in situ manufactured fatty acid soap, which also acts as an emulsifier for the fat-insoluble sucrose. Thereby, the sucrose reacts in the first stage with fatty acid methyl ester to a sucrose partialester; in the second stage, the further reaction to the sucrose monoesters, diesters, or to the fully saturated octaester occurs.83 Sucrose esters of fatty acids are known as emulsifiers in food technology, in cosmetics and pharmaceutical preparations. The octaester was developed as a noncalorific fat substitute. In alkyd coatings, sucrosepolyolesters of unsaturated fatty acids act as nonvolatile cross-linking agents and reactive diluents that are incorporated into the polymer film during autooxidation.84

Figure 4A.36 Sucrose acetate isobutyrate.


7.5 Sugar Polyols Thanks to their high functionality polyhydric alcohols such as unmodified sucrose, molasses, or corn syrup85 and sucrose-based polyether polyols play an important role for the production of rigid polyurethane foams.86 The high functionality of the sucrose polyols enables the making of rigid polyurethane foams with excellent dimensional stability to be used for thermal insulation, as well as for the production of building elements, particularly sandwich and composite elements.To produce sugar-based polyether polyols a concentrated aqueous sucrose solution or a suspension with other hydroxy- or amine-bearing liquid coinitiators and an oxyalkylation catalyst such as potassium hydroxide are used as feedstock. Alkylene oxide is added over a period of time until the reaction product is a liquid. At this stage the water is removed. The remaining alkylene oxide is then added until the desired polyether polyol is obtained.87

8. STARCH REFINING AND PROCESSING Virtually all green plants contain starch as storable form of energy, which makes starch next to cellulose and hemicelluloses the most abundant biopolymer on Earth. Plants store starch in different organs (fruit, seeds, rhizomes, and tubers) to prepare for the next growing season. The main raw material sources for starch production are maize, wheat, potatoes, rice, and cassava, from which tapioca starch is derived. Many other crops (ref. 2.6–2.14) are employed for special purposes or sporadically used in comparatively small quantities. The perishable nature of tubers and roots limits the markets of these raw materials to the region of production, whereas cereals like maize and wheat are globally traded commodities. Cereal corn mills can be regarded as a kind of prototype for whole crop biorefineries with so-called coproducts making a valuable contribution to the economy of the starch manufacturing plant. During processing, for example, all components of the maize grain, i.e., the germ, the hulls, gluten, and starch are valorized (ref. 8.2.1.1). In contrast to the cereal starch production the extraction of root and tuber starches does not deliver coproducts of comparable value. As the processes for the extraction are different for the mentioned crops the starch industry cannot easily switch from one source to the other in order to adapt to fluctuating market conditions both on the raw material and on the end product side.

8.1 The Global Starch Market The global starch production in 2010 was of more than 70 Mio mto of primary starch, of this, historically 32% are dry, native and modified starches and 68% are refined liquid starches.88,100 Global starch consumption is projected to reach 133.5 Mio mto by the year 2018, driven primarily by the diversity and sheer number of end-use applications in both food and nonfood industries,89 including native and modified starches, but also the large volume of starch that is converted into syrups for direct use as glucose and

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isoglucose (known in the USA as high fructose corn syrup, HFCS), and as substrates in the form of very high dextrose syrups (known as starch hydrolysates) for fermentation into organic chemicals, including ethanol. Raw material use is regionally quite unbalanced; the USA uses nearly exclusively corn; in South East Asia (Thailand and Indonesia) cassava cultivation has been advancing dynamically, China mainly uses corn and cassava, and Europe maize, potatoes, and wheat. On a global basis, starch production by raw material comprises approximately: maize starch 80%; wheat starch 8%; potatoes starch 5%; tapioca starch 5%; rice, sorghum, peas, and other 2%.88,90

8.2 Industrial Starch Production Processes Due to the seasonality and the very different biological structure of starch crops, starch production technologies depend on the type of raw material. Cereals can easily be stored in a grain elevator, which is why cereal starch can be produced throughout the year, whereas tuber and root-starch must be produced immediately after the harvest of the crop. 8.2.1 Corn Starch Processing In former days, corn used to be harvested by hand. Most farmers now use a grain combine that removes the cobs from the stalks and the kernels from the cobs. The combine catches the corn stalks, snapps off the ears, and removes the husk and the kernels from the cobs. The kernels are dried and stored either on the farm or in a grain elevator and from there are supplied to the starch manufacturer. For corn starch production maize may be processed by two basic methods known as “dry milling” and “wet milling.” Ethanol is the primary product of the dry milling process, and is also one of the products produced via wet milling technology (Figure 4A.37). 8.2.1.1 Wet Milling

The corn wet milling process is designed to efficiently separate the maize kernel into three principal parts (ref. Figure 4A.10): 1. the outer skin, called the bran or hull (8% of the corn grain); 2. the germ, containing the corn oil (12% of the grain); and 3. the endosperm, containing gluten and starch (80% of the grain). In the wet milling process (which means an excess of water to flour is used to make a slurry or batter) the shelled and cleaned corn is conveyed to steep tanks where it is soaked for 30–50 h at elevated temperature in an aqueous sulfurous acid solution resulting in the softening of the corn kernels. During the soaking, soluble nutrients are absorbed into the water. This water is later evaporated to concentrate these nutrients to become condensed corn fermented extractives. Continuing with the milling process the corn germ is now removed from the water soaked kernels by a series of mills and cyclone germ separators. The germ is dried and cold pressed before solvent extraction


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6DFFKDULILFDWLRQFRQYHUVLRQ 90% of the global soybean cultivation.2 The agricultural production stage11 includes the growth of the plant that starts with the selection of the species leading to the best yields of oil and lignocellulosic materials. Research on crop nutrition and disease control, optimizing rotation break, and possibilities of multiple cropping and intercropping as well as breeding of new cultivars is performed at this stage. Responsible farming practices12 (agriculture raisonnée in French) need to be incorporated in this connection. Second, the harvest, the logistic, and the storage of the plant have to be taken into account to optimize the efficiency of the biorefinery. In the wholeplant biorefinery concept, the harvest has to be optimized to recover all valuable fractions. Therefore, it is important to take into consideration the implementation of the agricultural mechanistic aspects and to innovate in the machines and tools used on the fields. This harvesting stage can also include the first stage of the biorefinery by separating seeds on one side and stems and leaves on the other. Compared to fossil resources, harvested biomass can be sensitive to environmental factors such as temperature, humidity, or microorganisms. Therefore, the storage of the biomass appears as a crucial stage to maintain the quality of the future products. The optimization of the storage and transportations facilities has to be studied to maintain the maximum output of the wholeplant biorefinery concept. Finally, the logistic stage takes an important part in the concept as well. For example, it is important to notice that, to minimize costs and increase profits, the choice of the location of a biorefinery unit and its size are often determined by the theoretical modeling of all the agricultural production steps.

3.2 The Biorefinery Stages Once the biomass has been delivered to the refinery, a succession of transformations can be applied to valorize two main types of compounds: the oil (composed of fatty acids and glycerol) (Figure 5.5),13 and the lignocellulose (Figure 5.6).14,15 After several biorefining stages, both fatty acids and glycerol can be considered as platform intermediates to give access to numerous products that can substitute chemicals or usage.

Vegetable oils or Animal fats

TRIGLYCERIDES Reaction of the fatty chain

Reaction of the carboxy group Basic chemicals H2 O

MeOH

Fatty acids Glycerol Esters: Plasticizers, lubricants, solvents, emollients etc.

Alcohols:

additives, bactericides, fungicides, emulsifiers etc. Acetalisation

Dicarboxylic acids, diols polymers, plasticizers, stabilizers

Epoxidation

Amines:

emulsifiers, lubricants, Flotation agents, surfactants, plasticizers gasoline or lubricating

Glycerol formal solvent

Dimerisation

Biodiesel

Ring opening Functionalised derivatives (S, N, OH etc.) Emulsifiers, lubricants, polymers, stabilizers

Oligo or polymerisation and esterification

Esterification or glycerolysis

Polyglycerols and esters surfactants, polymers

Mono, di glycerides Surfactants (food)

Hydrogenolysis 1,2 or 1,3 propanediol Solvent, antifreeze, additives, monomers (polyurethanes)

New oleochemicals agrochemiccals, fragrances, pharmaceuticals

Oxidative cleavage Dicarboxylic acids, diols polymers, plasticizers, stabilizers

Oxidative carbonylation or Transcarbonatation

Glyceryl carbonate Solvent, additive, monomer, chemical intermediate Internal dehydration Glycidol Stabilizers, demulsifiers, polymers (polyurethanes)

Figure 5.5 Overview of accessible products from triglycerides. (Adapted from Ref. 13.)

Polyaldehydes or polyacids polyamides, polyesters, plasticisers

Esterification Fatty esters New lubricants

Vegetable Oil Biorefineries

Oxidation Dihydroxiacetone (tanning agent,building block in organic synthesis) Glyceric acid (building block in organic synthesis)

Hydroformylation

Metathesis

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Figure 5.6 Overview of accessible products from lignocellulose by Ragauskas et al.14

3.2.1 Separation The first stage of the biorefinery is often the separation of the main components of the plant.16 Most of the time, this stage has been performed on the field by the harvesting unit. In the case of rapeseed, the seeds are separated from the rest of the plant (stem, leaves), whereas in the case of other plants such as sunflower, the seeds have to go through an additional stage to separate the husk from the seed kernel that is then used for oil recovery. The husk is then treated as a lignocellulosic fraction. 3.2.2 Fractionation The fractionation is a stage applied to the lignocellulosic biomass (stem, leaves, and eventually husks), where different chemical and/or biochemical treatments are applied. The first step is often a physical modification of the biomass to reduce the size of the biomass fragments and to improve the treatments by chemicals and/or enzymes.17 The main goal is to be able to separate the components of the biomass: cellulose, hemicellulose, and lignin. This hydrolytic stage can be performed using acids (or bases), or enzymes, or a combination of the two types of treatments. A complete fractionation stage will lead to the formation of lignin and sugars, usable for further modification stages. 3.2.3 Thermotreatments The lignocellulosic biomass can be converted directly to bioproducts without a fractionation stage. Heat is then applied to treat the biomass under different conditions: first, the biomass can be simply burned to produce energy18; pyrolysis under high temperatures and low level of oxygen, or gasification, will form syngas that can be used to produce heat and electricity, or in catalysis (or fermentation processes); application of heat and pressure, or liquefaction, can lead to the formation of biooil that can be further refined to be used, for instance, as fuels. Most of the time, those types of thermotreatments


produce energy or bioproducts of a relatively low value. Researchers are currently concentrating their efforts to produce high value bioproducts from thermochemical processes such as the recovery of phenols from the lignin fraction of biomass using supercritical carbon dioxide, for instance. Unfortunately, low yields are obtained so far, and high capital costs and safety issues could become drawbacks to industrialization. 3.2.4 Pressing/Trituration Extracting oil from seeds can be performed with a typical mechanical press. This technique however presents some limitations due to its low productivity. It is usually employed to extract valuable oils with high-quality grades. To allow industrialization, most of the time, seeds undergo a trituration treatment: after being washed, sometimes preheated, they are pressed using a worm-gear press with pressures reaching up to 500 bar, allowing a maximum oil recovery (up to 95%). The resulting press cake still contains 3–5% of oil that can be further extracted using an organic solvent (typically hexane). Countercurrent extraction is used to minimize the amount of solvent that reaches approximately 1.5 L to extract 1 L of oil. The solvent is then distilled off, at 70 °C in the case of hexane and reused for further extraction (hexane consumption reaches 1 L/ton of seeds). 3.2.5 Biotech Conversions

3.2.5.1 Valorization of Second Generation Carbohydrates Obtained from Fractionation

As for the chemical conversions, biochemical conversions constitute the last stage of the biorefinery concept for the production of bioproducts. Different intermediates obtained from the previous stages are used as feedstocks such as oil, sugars, or lignocellulose. The most developed conversion consists of the production of bioethanol by fermentation from carbohydrates. When sugars are obtained from lignocellulosic biomass, the bioethanol is considered to be of second generation. Of course, other bioproducts can be produced by fermentation and more advanced fermentation processes can even be envisaged such as those converting sugars to natural or unnatural fatty acids by metabolic engineering. 3.2.5.2 Valorization of Fatty Acids and Glycerol

In addition, biotech processes are also efficient in order to perform chemical modifications of vegetable fatty acids such as regioselective hydroxylation. Glycerol can also be used as feedstock in fermentation processes and lead to the production of chemicals of interest. More efforts could be developed to perform the depolymerization of lignin by biochemical ways. 3.2.6 Chemical Conversions

3.2.6.1 Valorization of Second-Generation Carbohydrates Obtained from Fractionation

Aromatics are a class of chemicals that are still difficult to obtain from biomass, and efforts are currently made to produce aromatics from lignin. Chemical depolymerization

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processes are still under research at the laboratory scale, and some efforts are focused on the use of supercritical carbon dioxide treatment to produce phenol, for instance. However, yields and selectivities remain low, and the capital costs linked to the high temperature and pressure needed are detrimental for industrial applications. Nevertheless, lignin can be treated as a substrate for binding or polymer applications.19 As an example, one can think of the postmodification of lignin that reacts as a polyol, with fatty acids to obtain macropolyol molecules. 3.2.6.2 Valorization of Fatty Acids and Glycerol

Fatty acids and glycerol could be considered as new platform intermediates for the synthesis of biobased products. Those products can have the same structure as bulk chemicals obtained today from petroleum or new intermediates with similar or new properties. In the oilseed biorefinery, chemical conversions are already applied on an industrial scale such as for the production of biofuels.20 Vegetable oils are indeed easily converted by transesterification to fatty acid esters that can be used in diesel engines. In this process, a large amount of glycerol is produced. Hydrodeoxygenation processes (also called hydrotreatment, decarboxylation, decarbonylation), often using catalysts, can be carried out on fatty acids to produce fuels such as biodiesel or biobased jet fuel. Finally, conversions, applied to oils, glycerol, or sugars, using chemical2,8 or biochemical21 reaction technologies can lead to the formation of bulk and fine chemicals, or materials. As an example at an industrial scale, we can mention the conversion of glycerol to epichlorhydrin, a precursor used for epoxy resins, performed for example by Solvay (Epicerol®-Process) and DOW (GTE-Technology). The global epichlorhydrin production reaches approximately 1 Mio mto, including the Epicerol® process of 100,000 tons in place in Thailand (another production site of similar capacity is being built in China) (Figure 5.7).22 Compared to the original process starting from petrolbased propylene, the new pathway is saving one conversion step and appears more sustainable with less dangerous reagents (HCl instead of chlorine gas) and less byproducts formation. The oleaginous and lignocellulosic biorefinery stages present today different levels of developments going from laboratory research, to pilot and demonstration level, and to commercialization (Figure 5.8). Some processes are already applied in commercial industrial production plants, but others are still in need of development to reach commercialization. The production of vegetable oils for food has been a commercial process since a long time ago. The same is true for the traditional use of fatty acid esters in cosmetics and detergents. However, the microbial production of fatty acids (natural and unnatural) is still in its infancy. New applications for vegetable triglyceride oils and crude tall oil (CTO) are also at a laboratory


Figure 5.7 Industrial glycerol conversion to epichlorhydrin.22

research level such as hydrotreatment23 (hydrodeoxygenation) processes leading to green diesel fuel and jet fuel production and their use for polymer syntheses. A 100,000 tons per year hydrotreatment biorefinery for the conversion of CTO into renewable dropin diesel biofuel built by UPM in Lappeenranta/SF is scheduled to go on stream this year.24 In other cases, such as the conversion of glycerol, a few transformations are reaching the commercial stage. Examples are the epichlorhydrin production for epoxy resins applications, the synthesis of polyglycerol esters based on natural or on synthetic glycerin, and dihydroxyacetone (DHA) for cosmetics uses. At the pilot level, glycerol is now transformed to acrolein, an important commodity chemical, or to diols that find applications in multiple areas (polymers, food, paints, cooling, etc.). New processes to valorize

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Figure 5.8 Relevant examples of products obtained from oilseed biomass and development levels. (Copyright © 2014, SAS PIVERT)

glycerol are still at the research level such as oxidative conversions to glycolic acid or glyceric acid. Cellulose from the vegetable oil biorefinery can be used for the production of second-generation ethanol (not depicted in the figure). Concerning lignin, gasification (syngas) and pyrolysis (biooil and biochar) processes are now reaching pilot and demonstration levels, and some of them are close to the commercial level. Other valorization methods of lignin are still at the primary level with, for instance, the aim of producing single aromatics. Hemicellulose, which after hydrolysis, results in a mixture of pentose sugars such as xylose and arabinose has been transformed into alkyl polypentosides via the reaction with fatty alcohols.25 It is important to mention that those conversions of the different fractions obtained from the vegetable oil biorefinery are not only performed by single chemical steps or via single biotechnological pathways but also a combination of them. Moreover, their simultaneous use in the same reaction vessel (hydride catalysis) is a challenging but promising area of research nowadays.

3.3 Main Markets of the Vegetable Oil Biorefinery The global vegetable oil production reaches between 150 and 200 Mio mto per year with around 80% used for food applications.1 The main crops used are, in decreasing order, soy, palm, rapeseed, and sunflower.


The oil resulting from the pressing stage is filtered and dried and can be used directly for human food consumption (80%), the remaining 20% being employed for bioproducts applications such as detergents, soaps, or biofuel. Nowadays, the market of vegetable oils toward new oils of better and healthier composition for human consumption is of real importance. Therefore, the biorefinery must also focus on the development of crops and fractionation processes that improve oil properties for food applications. In 2011, approximately 20 Mio mto of vegetable oil was transformed into biofuel, mainly biodiesel. The geographical production of oil-based biofuel is mainly located in Europe (8.6 Mio mto), the United States (1.5 Mio mto), and Brazil (0.5 Mio mto).26 From the biodiesel production, glycerol was produced at 1.9 Mio mto in 2010/2011 with an annual growth of 15%. In the biorefinery concept, the economic feasibility of biodiesel production should be based also on the revenues generated by the valorization of coproducts such as glycerol. Moreover, it is important to notice that, in Europe, the availability of other by-products (meals, cakes) leads to a decrease in the dependence of animal feed importation for the agroindustry. To allow the use of seed cakes as animal feed, the extracted press cake is treated with steam to remove the remaining 2% of hexane in the cake. As a consequence, the digestibility of the deoiled meals is decreased. The price of rapeseed cake varied between 200 and 400 dollars per tons in 2012–2015.1 Efforts are also ongoing to valorize press cakes by conversion of various products from the cake such as the proteic fraction that can be used to produce chemicals.27 Concerning the bioproducts used in the chemical industry, as already said before, epichlorhydrin from glycerol today has reached industrial-scale production resulting in a more or less complete disuse of from synthetic glycerin manufacturing. Other compounds such as acrolein or diols can be accessed from glycerol, and some examples are reaching industrial production, for example, 1,2-propanediol by Archer Daniels Midland (ADM).28 Glycerol has also been used for the production of DHA by biotechnological conversion with applications in cosmetics (Soliance and others). Other chemicals such as long chain alkanes from fatty acids for fuel application are still under development. The market of biomaterials from second-generation lignocellulose constitutes another market of the vegetable oil biorefinery. In France, this lignocellulose is mainly obtained from flax and hemp, but the whole-plant biorefinery concept can be applied to other crops such as rapeseed or sunflower. Applications of second-generation lignocellulose could be found in the paper industry after recovery of the cellulose fraction. Fibers can also be used directly in the formulation of composites resins to obtain materials with biobased origin and biodegradability properties.29 The market of these composites remains however a niche market.

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Lignin can enter the composition of plywood as a glue with some commercial applications but is relatively limited. One main drawback remains the price of second-generation lignin around 1 euro per kilogram and combined with policies and regulations such as in Europe, where carbon dioxide emissions have to be decreased by 60%, the main use of lignin is as fuel to produce energy on site. It needs also to be noted that lignin, when separated from lignocellulose, is an amorphous material that has lost the connective properties developed in the living nature.

4. INDUSTRIAL VEGETABLE OIL BIOREFINERIES The knowledge transfer from the research and development level to large-scale production constitutes the first criterion to the industrialization of the vegetable oil biorefinery. Thus, pilot facilities are necessary to be able to evaluate new and innovative technologies from vegetable oil biorefineries. Other important aspects have also to be considered to allow a viable industry such as the choice of the location of the biorefinery, the study of the social and economic characteristics, and the sustainability criteria.

4.1 Geographical Aspects The first level of choice for the location of a vegetable oil biorefinery could aim to minimize distances between the different actors and therefore to decrease the transportation costs and its associated greenhouse gas (GHG) emission. In that conjecture, a local or regional biorefinery appears more suitable than a biorefinery involving worldwide actors. The closeness of both raw material and product users could be favored. If those two criteria appear difficult to combine, the location of a biorefinery can be chosen in relation with transportation facilities. A whole plant biorefinery appears as a preferable concept, especially if the transformation units are located close to the crop production sites.Thus, the location of a biorefinery plant can be dictated by the nature of the oleaginous crop, and therefore, the place where its production, linked to the climate, is performed. Depending on the scale of production, the size of the biorefinery will be adapted. If possible the close-by proximity of bioproducts users will be ideal, but if it is not the case, it will be preferable to build a new biorefinery in an industrially rich area where heat integration can be performed. Moreover, this location will also be rich in transportation facilities. To be efficient, the territorial biorefinery should be adapted to multiple feedstocks and multiple bioproduct production in order to comply with crop management techniques. By opposition, another type of biorefinery, focused on transportation hubs such as the Port of Rotterdam or the Port of Ghent, is based on the same model as petrol.The type of biomass is often limited to a few crops and so is the number of large scale bioproducts. In this case, the long distance travels of the feedstocks can appear detrimental in their sustainability evaluation.


4.2 Socioeconomic Aspects The choice of the location of a vegetable oil biorefinery close to the production area of the corresponding crop will imply a positive effect on the rural development of the local community. New jobs will be created in places where exodus was observed during previous industrial revolutions based on coal or petrol. Both national and regional governing entities are usually favorable to the creation of biorefineries due to the aforementioned local improvements and also to global consequences such as a decrease of dependence toward other energy exporting countries. Another aspect of the development of biorefineries concerns the position that it should take in our future society. The first step should emerge from our education system, at an early stage, with as a consequence, a more responsible and sustainable behavior of our society. As importantly we should adapt the education programs in schools and universities toward the themes of bioeconomy and sustainable development.When successful on a large scale, biorefineries will create numerous jobs where highly trained and educated people will be needed. In this time of transition and while waiting for the emergence of new graduates and postgraduates, opportunities should be proposed to train experienced workers to the needs of the biorefinery industry. Concerning the economic aspects of the transition to biorefineries, industries have to adjust their technology to the characteristics of biomass feedstocks. Investments in developing new knowledge are needed, and when the research would be fruitful, it has to be implemented at pilot and demonstration levels before becoming commercial productions. Those different stages could imply high capital costs and that is why the use of already existing process units is preferred. Concerning the oleaginous biomass, oils are a feedstock treated on an industrial scale. However, their modifications to chemicals and materials involve the development of new technologies. This is true for the valorization of glycerol, but current efforts are focusing on conversions toward bulk chemicals that are usually produced from petroleum resources. Thus, their uses will be compatible with the current chemical industry. The lignocellulosic fraction of the oleaginous biomass can be valorized in different ways, and some transformations are already on a large scale (Section 3). The markets targeted by the products obtained from the vegetable oil biorefinery are wide with, for instance, human food, animal feed, fuels, bulk chemicals, cosmetics, paints and coatings, and materials for automotive or packaging applications. Two strategies can emerge for the market of biobased compounds: either the products obtained are the same as the existing ones produced by the petrol industry, or the biobased products are new. In the first case, the industrialized use of biobased intermediates is easy as soon as specifications, performance, and price are competitive with fossil-based feedstock. Producing new products from the vegetable oil biorefinery is more challenging but offers also new perspectives to capitalize on the

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unique chemical structures and properties of natural raw materials. The chemical industry is ready to accept and promote new products as long as their performance is demonstrated.

4.3 Environmental Aspects The vegetable oil biorefinery and the whole-plant concept has, of course, positive impacts on the environment compared to the petrol-based industry. However, sustainability studies have to be performed thoroughly and applied to each biobased product to be able to assess their consequences to our society.The important aspects to take into consideration are GHG emission, land use, water use, biodiversity, safety, and toxicity. Life cycle assessments give good overviews of all those aspects and could decide, together with economic studies, the viability of the industrialization of a biobased product. It is important to mention that a biobased product may not be biodegradable and its end of life has to be taken into consideration.

5. FUTURE CHALLENGES OF INDUSTRIALIZATION 5.1 Technical Innovations The different actors of the vegetable oil biorefinery are facing numerous challenges to become a viable industry. First, efforts have to be made for more efficient crop productions and biomass recovery to ensure an optimum feed to biorefineries. Oilseed crop yields could be improved and so could their resistance to diseases and predators. A better understanding of parameters such as weather forecast, hygrometry, land use, soil composition, etc. is needed, and new predictive tools have to be developed. Technical innovations also imply an improvement of all the tools needed to collect, separate, and pretreat biomass. For applications in the chemical industries, the main challenge of biomass compared to petrol consists of its high level of heteroatoms and especially oxygen atoms. Therefore, deoxygenation processes of vegetable oils need to be industrialized. In some cases, the presence of water can also be a challenge for known industrial processes and some biobased intermediates such as lignins are difficult to convert efficiently into valuable products. All those characteristics of biobased feedstocks and intermediates are the precise points where technical innovations have to be developed. Thus, academic and industrial chemists and biochemists have to adapt their processes to those new starting material to be able to convert them to bioproducts. Some of those would be similar to already known fossil-based products, but in the case of the production of completely new products, users have to adapt as well. Hence, safety regulations have to be addressed and new commercial campaigns have to be created. The vegetable oil biorefinery is also aiming to the development of more healthy products for human consumption and innovations in new crops are needed. In the case of fermentative oil productions


from lignocellulosic carbohydrates, new downstream processes are needed to recover oils and they could involve innovative technologies. Finally, technical innovations should also imply a better control and understanding of products’ end of life to achieve a maximum sustainability.

5.2 Economic Challenges To develop a viable biobased economy, industries are facing numerous challenges, and the first one is often linked to the difficulty to assess the market of a new product.Therefore, new tools for economic evaluation of biobased products have to be adapted. The production of feedstocks (volume, prices) has to be evaluated and guaranteed. At all stages of the biorefinery, high levels of investment are needed due to the novelty of technologies to be developed. Industrial actors need to ensure the feasibility of biomass conversions at different scales and before validating capital investments at commercial levels, pilot and demonstration processes have to be performed, either by the industries themselves but they will often promote the collaboration with research centers or by small- and medium-sized enterprises. Industries will also favor technologies that can be integrated to the facilities they already possess. Biomass conversion research has also to take into consideration this aspect in order to ease future valorization. Industries will face challenges to create new markets for the products obtained from the vegetable oil biorefinery.

5.3 Societal Adaptation Our future society will face the increase of the population and the associated needs for food and also for energy, transportation fuels, and chemicals. Even if novel alternatives to petrol such as shale gas are emerging, they are and always will not be sustainable. Biorefineries and especially the vegetable oil biorefinery appear as a solution, but only if all its actors are included in its development. Combined with other sustainable energy productions coming from wind power or solar energy, they will provide solutions to our climate issues and also to our economic difficulties linked with the creation of new jobs. Due to their location, biorefineries will provide a solution to rural exodus and therefore promote the local economy. Hence, our society has to adapt to the emergence of biorefineries by developing new education and training programs for the future biobased industry.

5.4 Sustainability Challenges The impact of the vegetable oil biorefinery on our environment has to take an important part of the future industrialization of biobased products. The oilseed crops production has a positive effect on GHG emissions, but its influence on land uses needs to be evaluated to reach a sustainable economy. Water could become a rare commodity in a large number of locations and the consequences of the emergence of vegetable oil biorefineries on water supply have to be studied. The biodiversity is an important factor to be

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taken into consideration and in the development of vegetable oil biorefineries, the choice of the crops is crucial and must take into consideration agroecological approaches in order to conciliate biodiversity preservation and vegetable oil production. Industrial actors, and their associates in the biomass conversion, are also facing sustainability challenges, and they should aim at the development of processes that are more energy efficient. For instance, the selectivity of processes has a direct influence on energy consumption and less by-product formation implies less separation steps. Finally, even in a biobased economy, the carbon cycle has to be closed and the end of life of biobased products has to be taken into account, especially when those sustainable products are not biodegradable.

6. CONCLUSIONS AND PERSPECTIVES We showed that oleaginous crops are particularly efficient to produce a large variety of oils and numerous by-products that can be valorized into multiple commodity chemicals. By applying the concept of the whole-plant biorefinery, triglycerides, fatty acids, glycerol, and lignocellulose are converted into chemicals that can find applications in several markets. The aim of the biorefineries is to contribute to the increase of sustainability of our chemical industry. The vegetable oil biorefinery will play an important role in that challenge.Targets such as the level of sustainable carbon of 14% by 2020 are within reach.To reach those targets, we believe that the whole-plant vegetable oil biorefinery should be applied at a territorial level involving multiple and local crops. Available technologies should be of different types and should lead to multiple products of interest for the chemical industry. Tranversality along the value chain should remain a key element of future biorefineries. Moreover, we believe that the development of the third generation oleaginous biorefinery will only be possible by gathering people of multiple skills toward the same goal to promote sustainability.

REFERENCES 1. FAO—Trade and Markets Division. Oilseeds market summary. http://www.fao.org/fileadmin/templa tes/est/COMM_MARKETS_MONITORING/Oilcrops/Documents/Food_outlook_oilseeds/Food _outlook_Nov_12.pdf; November 2012. [accessed 02.07.14]. 2. Hill K, Höfer R. Natural fats and oils. In: Höfer R, editor. Sustainable solutions for modern economies. Cambridge: RSC Publ.; 2009. p. 167–237. 3. a. Bogaart V. Glycerin market brief. Croda; 2009. http://www.npt.nl/cms/images/stories/Verslagen/ Presentatie_Bogaart_Croda_15042010.pdf. b. Quispe CAG, Coronado CJR, Carvalho Jr JA. Glycerol: production, consumption, prices, characterization and new trends in combustion. Renewable Sustainable Energy Rev Nov. 2013;27:475–93. 4. Kazmi A, Kamm B, Henke S, Theuvsen L, Höfer R. In: Kazmi A, editor. Advanced oil crop biorefineries. Cambridge: RSC Publ.; 2012. 5. http://www.institut-pivert.com.


6. a. McKevith B. Nutritional aspects of oilseeds. Nutr Bull 2005;30:13–26. b. O’Brien RD. In: O’Brien RD, editor. Fats and oils—formulating and processing for applications. 3rd ed. Boca Raton: CRC Press, Taylor & Francis Group; 2009. p. 1–744. 7. a. http://www.oilworld.biz. b. http://www.cetiom.fr. 8. Abraham TW, Höfer R. Lipid-based polymer building blocks and polymers. In: Matyjaszewski K, Möller M, editors. Polymer science: a comprehensive reference. Polymers for a sustainable environment and green energy, (McGrath JE, Hickner MA, Höfer R, vol. editors.) vol. 10. Amsterdam, Oxford,Waltham: Elsevier; 2012. p. 15–58. 9. British Nutrition Foundation (BNF). Unsaturated fatty acids. nutritional and physiological significance. In: The report of the British nutrition foundation’s task force. London: Chapman & Hall; 1992. 10. Morrison WH, Hamilton RJ, Kalu C. Sunflowerseed oil. In: Hamilton RJ, editor. Developments in oils and fats. US: Springer; 1995. p. 132–52. 11. Stamatelatou K, Turley D, Laybourne R, Flénet F, Quinsac A, Marriott R, et al. In: Kazmi A, editor. Advanced oil crop biorefineries. Cambridge: RSC Publ.; 2012. p. 48–101. 12. Thormahlen S. Sustainable solutions for nutrition: a consumer expectation. In: Höfer R, editor. Sustainable solutions for modern economies. Cambridge: RSC Publ.; 2009. p. 68–85. 13. Corma A, Iborra S,Velty A. Chemical routes for the transformation of biomass into chemicals. Chem Rev 2007;107:2411–502. 14. Ragauskas AJ, Beckham GT, Biddy MJ, Chandra R, Chen F, Davis MF, et al. Lignin valorization: improving lignin processing in the biorefinery. Science 2014;344. http://dx.doi.org/10.1126/science.1246843. 15. Zakzeski J, Bruijnincx PCA, Jongerius AL, Weckhuysen BM. The catalytic valorization of lignin for the production of renewable chemicals. Chem Rev 2010;110:3552–99. 16. Mulder W, Harmsen P, Sanders J, Carré P, Kamm B, Schönicke P, et al. Primary processing. In: Kazmi A, editor. Advanced oil crop biorefineries. Cambridge: RSC Publ.; 2012. p. 102–65. 17. Gu T, editor. Green biomass pretreatment for biofuels production, Springer briefs in green chemistry for sustainability. Dordrecht, Heidelberg, New York, London: Springer; 2013. 18. Kaltschmitt M, Thrän D. Biomass-based green energy generation. In: Höfer R, editor. Sustainable solutions for modern economies. Cambridge: RSC Publ.; 2009. p. 86–124. 19. Windeisen E, Wegener G. Lignin as building unit for polymers. In: Matyjaszewski K, Möller M, editors. Polymer science: a comprehensive reference. Polymers for a sustainable environment and Green energy, (McGrath JE, Hickner MA, Höfer R, vol. editors.) vol. 10. Amsterdam, Oxford, Waltham: Elsevier; 2012. p. 255–65. 20. Dinjus E, Arnold U, Dahmen N, Höfer R, Wach W. Green fuels—sustainable solutions for transportation. In: Höfer R, editor. Sustainable solutions for modern economies. Cambridge: RSC Publ.; 2009. p. 125–63. 21. Haas T, Kircher M, Köhler T,Wich G, Schörken U, Hagen R.White biotechnology. In: Höfer R, editor. Sustainable solutions for modern economies. Cambridge: RSC Publ.; 2009. p. 436–78. 22. a. Bell BM, Briggs JR, Campbell RM, Chambers SM, Gaarenstroom PD, Hippler JG, et al. Glycerin as a renewable feedstock for epichlorohydrin production. The GTE process. Clean 2008;36(8):657–61. b. Dobbelaere S. Biorefining opportunities in the biodiesel sector. Amsterdam 2010. http://www.biorefinteg.eu/fileadmin/bioref-integ/user/documents/6._Dobbelare_-_Biodiesel_sector_-_130910_.pdf. c. http://www.solvaychemicals.com/EN/Sustainability/Issues_Challenges/EPICEROL.aspx. d. Caulier T. Epicerol® and the environmental challenges of bio-based chemistry. In: 6th Workshop on fats and oils as renewable feedstock for the chemical industry, Karlsruhe. 2013. e. http://www.solvay.com/en/media/press_releases/20120228-epichlorohydrin.html. 23. a. Egeberg RG, Michaelsen NH, Skyum L. Novel hydrotreating technology for production of green diesel. Lyngby/ DK: Haldor Topsøe. http://www.topsoe.com/sites/default/files/novel_hydrotreating_technology_for_ production_of_green_diesel.ashx_.pdf [accessed 23.03.2015]. b. Kuronen M, Mikkonen S, Aakko P, Murtonen T. Hydrotreated vegetable oil as fuel for heavy duty diesel engines. Porvoo/SF: Neste Oil; 2007. c. Hartikka T, Kuronen M, Kiiski U. Technical performance of HVO (Hydrotreated vegetable oil) in diesel engines. SAE Technical Paper 2012-01-1585. 2012. http://dx.doi.org/10.4271/2012-01-1585. 24. Voegele E. Finland-based UPM plans crude tall oil renewable diesel plant. Biomass Mag February 10, 2012. http://biomassmagazine.com/articles/7617/finland-based-upm-plans-crude-tall-oil-renewablediesel-plant/?ref=brm.

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25. Rauter AP, Vogel P, Queneau Y, editors. Carbohydrates in sustainable development I. Berlin, Heidleberg: Springer; 2010. 26. http://www.ebb-eu.org. 27. Tuck CO, Perez E, Horvath I, Sheldon RA, Poliakoff M.Valorization of biomass: deriving more value from waste. Science 2012;337:695–9. 28. Vandeputte J. Le glycérol, “building blocks” majeur de la bioraffinerie oléagineuse. Oilseeds Fats Crops Lipids 2012;19:16–21. 29. Riedel U. Biocomposites: long-natural fiber-reinforced biopolymers. In: Matyjaszewski K, Möller M, editors. Polymer science: a comprehensive reference. Polymers for a sustainable environment and Green energy, (McGrath JE, Hickner MA, Höfer R, vol. editors.) vol. 10. Amsterdam, Oxford, Waltham: Elsevier; 2012. p. 295–315.

CHAPTER 6

Biogas Biorefineries Harald Lindorfer, Bettina Frauz Schaumann BioEnergy GmbH, Pinneberg, Germany

Contents 1. Introduction 271 2. Substrates for Biogas Production 275 3. Biogas Utilization 280 4. The Chemical Platform Methane 284 5. Fertilizer Production 284 6. Mass and Energy Balances 288 7. Other Biorefinery Concepts with Strong Focus on Biogas Production 291 8. Perspectives of Biogas Biorefineries 292 References293

1. INTRODUCTION Biogas is a product that is a part of the majority of biorefinery concepts. This is because of the favorable characteristics of anaerobic digestion, particularly the high energy yield from organic substances, the low cost technology of biogas production, the low demand for substrate composition and quality, and the wide application spectrum. Biorefineries in which biogas production is playing a substantial role often belong to one of the following groups: • Biogas biorefineries focusing on the production of biogas and fertilizers • using energy crops, agricultural residues, and residues from rural conservation (protected areas) • using waste substrates like food waste or food production waste • Starch crop biorefineries based on cereals, maize, potato, etc. • Sugar crop biorefineries based on sugar beet, sugar cane, etc. • Green biorefineries based on wet biomass like grass, clover, lucerne. • Civilization biorefineries processing all types of organic wastes from municipals (household wastes, wastewater), food industry restaurants, grocery stores, etc. In the biorefinery roadmap drawn up by the federal Government of Germany, biogas biorefineries are defined as follows1: “In a biogas biorefinery there is no separate component separation in primary refining; instead, a large proportion of the organic ingredients and components of the biomass are removed (with the notable exception of lignin), producing raw biogas.” Industrial Biorefineries and White Biotechnology http://dx.doi.org/10.1016/B978-0-444-63453-5.00008-2


271

272


Primary refining describes the treatments of the raw material before producing the platform chemicals, whereas secondary refining includes processes and treatments for the upgrading of the platform chemicals. Still, it is difficult to classify existing or projected facilities into one of these categories. This is because of the development of most biorefineries in a bottom-up approach. This means that existing biomass processing facilities (e.g., biogas or ethanol plants, sugar, starch, or oil producing plants) currently producing only one or a few products are switched into a biorefinery that aims to expand the number of usable biomass fractions and/or the produced products. As a consequence, the upcoming biorefineries will be often a conglomerate of different concepts. Biorefineries that are completely designed before the start of construction are characterized as topdown approaches in which the complete utilization of the plant biomass is the main focus. Top-down concepts are highly integrated systems for the production of a wide range of products for different markets producing zero waste. Figure 6.1 shows an overview of a typical biogas biorefinery. Generally, biogas biorefineries consist of three main units: • The substrate reception including pretreatment and feeding equipment • The digestion unit including one or more gas tight and heated anaerobic tanks connected by a biogas collection system • The digestate storage and possibly posttreatment units 3RZHU &+3

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The more complex the substrates are and the higher the content of inorganic impurities, the higher will be the demand of pretreatment steps. In food waste installations, hammer mills, screw presses, or centrifugal separators are used to separate packaging materials or other impurities from the organic parts, while in plants treating the Organic Fraction of Municipal Solid Wastes (OFMSW) sieving units are favored. Often, a hydrolysis or mix tank is installed to homogenize the incoming materials. If necessary, a hygienization unit needs to be installed. In agricultural plants, sometimes hammer mills or dissolvers are utilized before digestion to increase the digestibility of poor degradable substrates like litter or grass silage. In the digestion of solid substrates and high strength liquids, completely stirred tank reactors are preferred. Only in few agricultural and OFMSW applications percolation systems are gaining importance. With the use of completely stirred tanks, generally a postdigester is advantageous to increase the degradation rate and to reduce residual methane potential of the digestate. Especially in the digestion of fibrous substrates like cattle litter or grass silage long hydraulic retention times are favorable.2 So, most agricultural plants using that type of substrates consist of main and post digester. In the digestion of low strength liquid wastes or wastewaters, often more sophisticated reactor types like expanded granular sludge bed, upflowing anaerobic sludge bed, or fixed bed reactors are used to realize higher loading rates. In tanks with a membrane cover, the produced biogas is stored directly in the tanks. This type of biogas storage is generally used for desulfurization of the biogas by blowing in some oxygen. In steel digesters or tanks with a concrete roof, an external biogas storage and desulfurization are needed. After desiccation, the biogas is transported via a pipeline to the biogas using unit, for example, a combined heat and power plant (CHP) or to further upgrading (Biomethane). After digestion, the residual digestate is generally stored on site and used for fertilization of the surrounding agricultural land. The digestate of plants using only agricultural by-products, energy crops, or food production wastes shows a favorable chemical composition and is ready for direct land application. In installations using various organic wastes, a further treatment is often necessary. In this chapter, only biorefinery concepts in which biogas production is playing a substantial role are covered. During anaerobic digestion, organic material is decomposed into an oxygen-free atmosphere by bacteria that produce a gas, called biogas, and usually containing methane and carbon dioxide plus components of minor importance.5 Figure 6.2 shows a comparison of anaerobic and aerobic treatment of organic residues or industrial wastewater. The figure clearly underlines the advantages of the anaerobic treatment for nonlignocellulose substrates. Of their chemical energy content (mainly carbon and hydrogen), 70–90% is transformed into biogas. The amount of heat losses and bacterial biomass production are very low. Anaerobic digestion is the most favorable treatment for all organic substances when recycling of

273

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Figure 6.2 Comparison of anaerobic and aerobic treatment of liquid organic wastewater. Table 6.1 Composition of biogas

Methane Carbon dioxide Nitrogen Vapor Ammonia Hydrogen Hydrogen sulfide Oxygen Other compounds

Molecular formula

Biogas

CH4 CO2 N2 H2O NH3 H2 H2S O2

50–75% 25–50% 0 (−5%) 2–7% 80% PHA, namely PHBV can be achieved. While MCL PHA are mostly difficult to reach high cell density due to the heavy demand on

561

562

Ralstonia eutropha

No

PHB

Glucose

>200

>80%

Alcaligenes latus

No

PHB

Glucose or sucrose

>60

>75%

Escherichia coli

phbCAB + vgb

PHB

Glucose

>150

>80%

R. eutropha

No

PHBV

Glucose + Propionate

>160

>75%

R. eutropha Escherichia coli

No phbCAB

P3HB4HB

Glucose + 1,4-BD

>100

>75%

R. eutropha Aeromonas hydrophila

phaCAc No

PHBHHx PHBHHx

Fatty acids Lauric acid

>100 80% 50%

Pseudomonas putida Pseudomonas oleovorans Pseudomonas entomophila Bacillus spp. Halomonas spp.

No

MCL PHA

Fatty acids

∽45

>60%

Yes

Fatty acids

>20

>70%

Sucrose Glucose

>90 >100

>50% >80%

Halomonas spp.

Yes

MCL homoPHA PHB PHB or PHBV SCL–MCL PHA

Glucose + fat

>50

>60%

No Yes

Company

Tianjin North. Food, China Chemie Linz, btf, Austria Biomers, Germany Jiangsu LanTian, China ICI, UK Zhejiang Tianan, China Metabolix, USA Tianjin Green Biosci. China P&G, Kaneka, Japan P&G, Jiangmen Biotech Ctr, China Shandong Lukang, China ETH, Switzerland Baisheng, Shandong, China Biocycles, Brazil KDN, Qingdao, China Baisheng, Shandong, China

Note: CDW: Cell dry weight; vgb: Gene encoding Vitreoscilla hemoglobin; phbCAB: PHB synthesis genes encoding β-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase; Ac: Aeromonas caviae; 1,4-BD: 1,4-butanediol; phaCAc: PHA synthase gene phaC from Aeromonas caviae.


Table 16.2 Known bacterial strains used for pilot and large-scale production of various polyhydroxyalkanoates (PHA)2 DNA Strain manipulation PHA type C-source Final CDW (g/L) Final PHA (% CDW)

White Biotechnology for Biopolymers

oxygen by Pseudomonas spp. that are obligate aerobes (Table 16.2). For example, when Pseudomonas entomophila was used to make PHA using fatty acids as substrates, CDW went down to around 20 g/L due to the intensive foaming problem resulted from fatty acids (Table 16.2). Reduction on aeration to avoid foaming led to reduce CDW. Although fed-batch processes are efficient to achieve high cell density fermentation in most cases, it suffers from having to interrupt the fermentation process and cleaning up the entire fermentation system including resterilization. All these operations increase the complexity of the process, leading to high PHA production cost.

3.2 Continuous Process Continuous process offers the advantages of maintaining growth conditions constantly, allowing the cells to grow to relative high density and maintaining that density for a long period of time. Since conditions are constant, CDW, PHA content, and Mw as well as PHA monomer compositions can be maintained relatively stable and reproducible during the continuous processes. However, microbial contamination is a setback especially for continuous long fermentation processes that are more prone to attract infections. To avoid contamination, it is important to select microorganisms that are robust in growth and that growth conditions selectively favor the production strains. Recently, the author’s lab found that some Halomonas spp. were able to grow to a high CDW in the presence of high salt concentrations such as 35–80 g/L NaCl and high pH of 8–11.23 Since most nonhalophilic bacteria are not able to grow under the high NaCl conditions and high pH, these PHA-producing strain grow to overdominate others. Continuous fermentation processes were conducted using the Halomonas species TD01 for at least two weeks without contaminated by other microbes. At the end of the fermentation, CDW reached over 80 g/L containing around 75% PHA. The process has been optimized by the industries in the presence of seawater instead of NaCl aqueous solution to reach over 100 g/L CDW containing over 80% of PHA (unpublished results). Most importantly, the continuous fermentation process was run for at least two weeks without bacterial contamination under 60 g/L NaCl and pH 8–9 and under open (unsterile) conditions. Such a process can save not only fresh water but also energy cost as well as intensive labors for cleaning the fermentation systems. In the future, such process should be developed to use cellulose as substrate to avoid food versus fuels or food versus chemicals disputes (Figure 16.7).

3.3 Mixed Cultures Many studies on PHA production have been focused on industrial biotechnology-based production methods using pure culture technology and genetically modified microorganisms.8 Due to the high costs for sterilizing equipment and the substrate, as well as the batch-wise processing, PHA production is complicated and expensive.

563

564


Figure 16.7 Open, continuous, energy, and water-saving processes for production of bioplastics polyhydroxyalkanoates (PHA) and biofuels.

To lower the PHA production cost, some studies used a strategy called microbial community engineering for enrichment of PHA-producing biomass.24 They require nonsterile substrate and operational conditions. PHA-producing microorganisms selected from the natural environment, instead of a certain type of modal bacterium, are used for PHA production. Additionally, the PHA production process can be operated continuously.25 Several studies have investigated the possibility of implementing the microbial community engineering process for PHA production using synthetic wastewater. It has proven possible to enrich a stable microbial community for PHA production, while reaching a comparable productivity in terms of maximum PHA content (90% of CDW) and biomass specific production rate to pure culture process.26 The results demonstrate a possibility to produce valuable chemicals while treating wastewater. Agro-industrial waste streams instead of artificial substrates for PHA production were also used,27–29 including effluents of sugar factories, oil mills, wood mills, paper mills, or municipal wastes. However, the PHA storage capacity obtained from these studies was still significantly lower than the microbial enrichments selected on synthetic feedstock, reaching only PHA content around 55% of the dry weight.29–31 A study conducted by Albuquerque et al.25,27 obtained the currently best PHA storage capacity from real wastewater of 75% of the CDW. Nevertheless, the process should be further optimized to increase the PHA storage capacity of enrichments selected from agro-industrial wastewater.


Figure 16.8 Polyhydroxyalkanoates (PHA) can be produced from activated sludge generated from wastewater treatments. PHA-based biofuels including 3-hydroxybutyrate methyl ester (3HBME) and 3-hydroxyalkanoate methyl esters (3HAME) can also be formed from methyl-esterifying PHA.32

PHA production from wastewater opens a new way for not only low-cost material production but also for low cost production of PHA-based biofuels including 3-hydroxybutyrate methyl ester (3HBME) and 3-hydroxyalkanoate methyl esters (3HAME) (Figure 16.8).32 Palm oil could also be a raw material resource for PHA production. Plant oils are known to generate higher PHA yields due to higher carbon content per gram of oil compared to sugars.33 Among various oils, palm oil is being studied extensively for the production of various types of PHA. It has been confirmed that high yield production of PHA could be realized from palm oil and its by-products.The studies provide preliminary results on the efficiency of palm oil bioconversion into PHA and future implementation of these substrates for PHA production systems.33

3.4 Applications of PHA PHA applications as bioplastics, fine chemicals, implant biomaterials, medicines, and biofuels have been researched for many years. Bacterial PHA synthesis has been found

565

566


Figure 16.9 Polyhydroxyalkanoates and its related technologies are forming an industrial value chain ranging from fermentation, materials, fine chemicals, energy to medical fields.2

to be useful for improving robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield improvement on some fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ, PhaR, or PhaC have been found to be useful for achieving protein purification, specific drug targeting, and biosurfactants. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, energy to medical fields (Figure 16.9).2 Recently, it becomes possible to develop structurally controllable PHA for making PHA as a smart material.10,11,34

3.5 Environmentally Friendly Bioplastics for Packaging Purposes PHA were developed at the beginning as an environmentally friendly bioplastic for packaging purposes. ICI in the 1990s developed PHA with a trade name of Biopol, and was used as a shampoo bottles. Procter and Gambles (P&G) developed them with another name Nodax into a series of products ranging from coating sheets to nonwoven textiles to fibers. Metabolix trade named their PHA as Mirel. All these efforts have been targeted for bioplastic applications. In the late 1990s, many Chinese fermentation companies began to exploit their overcapacity for large-scale production of PHA (Table 16.3).2 At this time, Chinese companies are producing all types of PHA including poly-(R)-3-hydroxybutyrate (PHB),


Table 16.3 Known companies involved in polyhydroxyalkanoates (PHA) production and application developments2 Company Types of PHA Scale (t/a) Period Applications

ICI, UK Chemie Linz, Austria

PHBV PHB

300 20–100

1980s to 1990s 1980s

BTF, Austria

PHB

20–100

1990s

Biomers, Germany

PHB

Unknown

1990s to present

BASF, Germany

PHB, PHBV

Pilot scale

1980s to 2005

Metabolix, USA Tepha, USA

Several PHA Several PHA

1980s to present 1990s to present

ADM, USA (with Metabolix) P&G, USA

Several PHA

Unknown PHA medical implants 50,000

2005 to 2012

Packaging Packaging and drug delivery Packaging and drug delivery Packaging and drug delivery Blending with Ecoflex Packaging Medical bio-implants Raw materials

1980s to 2005

Packaging

Monsonto, USA

PHB, PHBV

1990s

Raw materials

Meredian, USA Kaneka, Japan (with P&G) Mitsubishi, Japan Biocycles, Brazil Bio-on, Italy Zhejiang Tian an, China Jiangmen Biotech Ctr, China Yikeman, Shandong, China Tianjin Northern food, China Shantou Lianyi, China


Contract manufacture Plant PHA production 10,000 Unknown

2007 to present 1990s to present

Raw materials Packaging

PHB PHB PHA (unclear) PHBV

10 100 10,000 2000

1990s 1990s to present 2008 to present 1990s to present

Packaging Raw materials Raw materials Raw materials

PHBHHx

Unknown

1990s

Raw materials

PHA (unclear) PHB

3000

2008 to present

Raw materials

Pilot scale

1990s

Raw materials

Several PHA

Pilot scale

1990s to 2005

Jiangsu Nan Tian, China Shenzhen O’Bioer, China Tianjin Green Bio-Science, China Shandong Lukang, China Qingdao VLand, China Shandong Baisheng, China

PHB

Pilot scale

1990s to present

Packaging and medicals Raw materials

Several PHA

Unknown

2004 to present

Unclear

P3HB4HB

10,000

2004 to present

Raw materials

Several PHA

Pilot scale

2005 to present

Raw materials


Pilot scale Pilot scale

2012 to present 2012 to present

Raw materials Raw materials

Several PHA

567

568


poly-4-hydroxybutyrate (P4HB), poly-3-hydroxyoctanoate (PHO) (up part left to right), random copolymers of (R)-3-hydroxybutyrate and (R)-3-hydroxyvalerate (PHBV), (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate (PHBHHx), and (R)3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB) (Figure 16.6). However, due to the high cost of production, PHA as an environmentally friendly plastic still have a very limited market. Much more efforts are still needed to lower the production cost to a level comparable to the petrochemical-based plastics.

3.6 Biofuels HAME derived from bacterial PHA was developed by the author’s lab,32 the PHA-based biofuel could provide a strong candidate for current biofuels or fuel additives market. The term biofuels is used for all types of biomass-derived fuels employed in the transportation sector. Most biofuels can be utilized either as a substitute for common fossil fuels such as gasoline and diesel or in blends with them. Other such as biomass-based natural gas requires changes in both vehicle construction and fuel distribution infrastructure.35 PHA universally synthesized by many bacteria grown in various carbon sources including wastewater that can be cleaved and reacted with methanol to form their corresponding methyl esters.32 3HBME or 3HAME improved the combustion heat of ethanol with 3HAME having more obvious effects. With the increased content of 3HBME or 3HAME in the ethanol, the combustion heats of blended fuels remained at the similar levels. This may enhance the possibility to use 3HAME as a biofuel because the addition of a low content on 3HAME can have similar effects.32 Since PHA can be obtained from wastewater treatment process, the PHA cost can be significantly reduced for biofuels purpose. Fuel ethanol has a combustion heat (CH) of over 26 kJ/g, higher than 21 kJ/g of 3HBME (Table 16.4), both of their CH are approximately half of that of gasoline.When blended with HBME, all CH showed a slight reduction, ranging from 2 to 6 kJ/g, depending on the amount of HBME or ethanol added to the gasoline (Table 16.4). For ethanol–gasoline blends, their CH decreased with increasing ethanol ratios in the blends, all the CH of the blends were lower than that of the pure gasoline, the highest CH of Table 16.4 Heats of combustion from blends of HBME–gasoline compared with that of ethanol–gasoline30 Sample Combustion heat (MJ/kg) Sample Combustion heat (MJ/kg)

HBME H5 H8.5 H10 H15 H20

21.11 ± 0.41 36.90 ± 0.52 37.34 ± 0.02 37.28 ± 0.09 36.54 ± 0.12 34.52 ± 0.25

Ethanol E5 E10 E15 E20 97#Gasoline

26.21 ± 0.10 38.66 ± 0.02 38.51 ± 0.23 38.43 ± 0.24 36.79 ± 0.23 40.07 ± 0.78

H denotes HBME–gasoline blends, E is ethanol–gasoline blends, and the next number stands for the volume ratio. For example, E5 means 5% (VV-1) HBME in gasoline.


the blend (E5) can be 96.5% of gasoline. However, for HBME–gasoline blends, the CH of the blends showed firstly a decrease, then increase to reach the maximum at nearly 8.5% before the CH decreased again at H15.36 These results clearly show that HBME, when mixed with gasoline in ratios of 8.5–10 (v/v)%, could have a positive synergistic effort on gasoline combustion as revealed by fuel related properties shown below, and this explains why reduction on CH for HBME– gasoline blends was less serious compared with that of ethanol–gasoline blends. PHA obtained from wastewater can also be cleaved and reacted with methanol to form their corresponding methyl esters. The resulting 3HBME and 3HAME improved the combustion heat of ethanol, with 3HAME having more obvious effects. In addition, 3HBME and 3HAME could also be used as fuel additives for other fuels such as propanol, butanol, gasoline, and diesel. Because the application of PHA does not require highly purified PHA, the production process appears to be much simpler.32 Therefore, the application of PHA as a biofuel allows the PHA industry to become a close cycle (Figure 16.10).

3.7 Medical Implants PHA have been studied for medical implants applications such as heart valves, vascular tissues, bone tissues, cartilage replacements, nerve conduits, as well as esophagus tissues. Many studies have been carried out in the author’s lab in the past 20+ years. Recently, mechanism on PHA-promoted tissue regeneration was revealed to relate cell responses to PHA biodegradation products and cell–material interactions mediated by microRNA.34,37 Very importantly, PHA implants were found not to cause carcinogenesis during long-term implantation.38 PHA have demonstrated great potentials in biomedical areas.39

Figure 16.10 Polyhydroxyalkanoates (PHA) production and application as bioplastics and biofuels will close the PHA application cycle, leading to reduced CO2 emission. Left picture: a1 and b1, combustion of fuel ethanol; b1 and b2, combustion of HBME.36

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3.8 Monomers as Chiral Intermediates As monomers of PHA, chiral 3-hydroxyalkanoic acids (3HAs) are important chemicals used as precursors or intermediates for the synthesis of various fine compounds including pharmaceuticals, antibiotics, food additive, fragrances, and vitamins.3,40,41 With over 150 different monomers, PHA are a rich source of chiral hydroxyalkanoic acids. PHA monomers can be divided into two groups according to their carbon-chain length: SCL 3HA contains 3–5 carbon atoms and MCL 3HA contains 6–14 carbon atoms. MCL 3HA have shown potential to be pharmaceuticals of high value.42 For example, 3-hydroxyhexanoic acid (3HHx) can be used as intermediate for synthesizing analogs of laulimalide, an anticancer chemical.43 3-hydroxyoctanoic acid (3HO) exhibits potential antimicrobial activities44; 3HO is also an intermediate for statins known as HMG-CoA reductase inhibitors.45 Myrmicacin, identified as 3-hydroxydecanoic acid (3HD), was found as a growth inhibitor against pollen, fungi, and bacteria.46,47 3-hydroxydodecanoic acid (3HDD) and 3-hydroxytetradecanoic acid (3HTD) are common constituent of lipid A in the cell surface of Gram-negative bacteria.48

3.9 Smart Materials The application of PHA as a low-cost biodegradable plastic has been hampered by its higher production cost and the difficulty to precisely control their structures and properties. Global efforts have been made to develop technology for lowering the PHA production cost. With the successful construction of β-oxidation weakened Pseudomonas spp. as PHA production platforms, we are now able to control the formation of homopolymers, random and block copolymers including monomer structures and ratios, this allows us to obtain PHA with consistent properties. At the same time, we can introduce various functional groups into the PHA site chains in a quantitative way, which provide more opportunities for site chain grafting. Functional PHA together with endless possibilities for grafting have provided us limitless ways of making new PHA, possibly with some high value-added functionalities, leading to smart materials.

3.10 Challenges for R&D To develop PHA into a commercially acceptable material, following efforts have to be made: first, feasible technology must be developed to reduce PHA production cost to a level competitive to petroleum plastics; secondly, value-added functionalities must be provided to PHA so that high production cost can be compensated by its high-end-applications. To reduce PHA production cost, following efforts should be made: 1. Achieving high cell density fermentation with high PHA content in CDW: This is important as a high cell density will allow effective cells and PHA recovery; 2. Grow cell in low cost substrates (or mixed substrates): Substrates contribute 50–60% of PHA production cost, low cost substrates such as sludge hydrolyzed products or


kitchen waste, or even hydrolyzed cellulose (if cheap), can be consumed by many PHA-producing organisms; 3. Grow cell in unsterile process in mixed substrates: Sterilization is an energy consuming process, maintaining sterilization throughout the fermentation process is costly and complicated; 4. Grow cell in continuous process in mixed substrates under unsterile conditions:This is a close to ideal situation, in which, if succeeded, will significantly save energy, reduce process complexity, and increase production efficiency; 5. Achieve ultrahigh PHA accumulation in the cells in continuous process in mixed substrates: If the cells contain over 95% PHA in their CDWs, the washing process will increase PHA content further to over 98% content, this ultrahigh PHA content provides a possibility to process the dry or even wet cells directly as plastic pellets in extruders; 6. Increase substrates to PHA conversion efficiency: Many substrates are consumed by the cells to maintain their metabolic activities, these substrates are often turned into CO2 and water. If the waste of substrates can be reduced, it helps to reduce production cost; 7. Achieve controllable cell flocculation for easy separation after continuous growth in mixed substrates: Downstream processing contributes 30–50% of PHA cost, controllable flocculation reduces cells and broth separation cost, thus leading to reduction on PHA production cost; 8. Achieve controllable PHA synthesis (homopolymers, random and block copolymers, functional polymers) in large scale: Microbial processes often produce inconsistent ratios of monomers in PHA copolymers. This significantly affects PHA applications. Technology must be developed to control the PHA micro and macrostructures so that all microbial PHA have consistent structures and properties; 9. Production and applications of chiral PHA monomers: Chiral PHA monomers are mostly difficult to make chemically. If a large-scale application on certain PHA monomers can be found, it will increase the PHA values; 10. Turning low cost PHA into functional PHA:This will save a lot of efforts to reduce PHA production cost. To achieve functional PHA, applications must be identified that make good use of unique PHA properties including biodegradability, biocompatibility, high Mw, and ease of making block copolymerization.

3.11 Future Prospective At this moment, PHA are far less competitive compared with its petrochemical peers. Beside its relative poor properties, high cost is the major factor preventing its successful large-scale marketing. Effects have been made by many labs around the globe to address the 10 challenges listed above. We foresee that ultrahigh PHA production strains with controllable flocculation properties are successfully grown to high cell density (>200 g/L) in a continuous

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fermentation process using low cost substrates including hydrolyzed cellulose, hydrolyzed sludge, or kitchen waste under unsterile conditions. PHA produced by such strains under these robust conditions have consistent structures and thus stable properties. The PHA produced in the above way have a cost similar to petroleum plastics, allowing PHA to compete with conventional plastics not only with its environmentally friendly features but also with its low cost and sustainability. By then PHA will have a significant share of the big pie belonging to conventional packaging materials. On the other hand, smart PHA materials with properties of shape memory, selective gas permeability, selective liquid permeability, temperature and/or pH responsibility, self-healing, controllable degradation and/or controllable porosity will be developed. These properties will add values to the microbial PHA, they should have a small but big margin market.

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17. Tsuge T, Watanabe S, Sato S, Hiraishi T, Abe H, Doi Y, Taguchi S.Variation in copolymer composition and molecular weight of polyhydroxyalkanoate generated by saturation mutagenesis of Aeromonascaviae PHA synthase. Macromol Biosci 2007;7:846–54. 18. Pascault J-P, Höfer R, Fuertes P. Mono-, di, and oligosaccharides as precursors for polymer synthesis. In: Matyjaszewski K, Möller M, editors. Polymer science: a comprehensive reference. McGrath JE, Hickner MA, Höfer R, editors. Polymers for a sustainable environment and green energy, vol. 10. Amsterdam, Oxford, Waltham: Elsevier; 2012. p. 59–82. 19. Wang Y,Yin Jin, Chen GQ. Microbial polyhydroxyalkanoates, Challenges and opportunities. Curr Opin Biotechnol 2014;30:59–36. 20. Global bioplastic packaging market by product type http://www.google.de/imgres?sa=X&biw=1280 &bih=538&tbm=isch&tbnid=jXijzrJU3w5C7M%3A&imgrefurl=http%3A%2F%2Fbiopol.free. fr%2Findex.php%2Fpolyhydroxyalkanoates-pha-and-bio-derived-pe-to-drive-bioplastic-packagingto-2020-a-marketstudy%2F&docid=nQ9qLd5yHiI2cM&imgurl=https%3A%2F%2 Fpira.revolutiondatacms.com%2Fuploads%2Fpublic%2Fimages%2FBusiness%252520Intelligence%2FPress%252520Re leases%2FBioplastics2020_CHART.jpg&w=487&h=402&ei=k3QAU8zREoa0tAaZloDIAQ&zoom =1&iact=rc&dur=564&page=1&start=0&ndsp=19&ved=0CGMQrQMwBA. 21. Wu Q, Sun SQ,Yu PHF, Chen AXZ, Chen GQ. Environmental dependence of microbial synthesis of polyhydroxyalkanoates. Acta Polym Sin 2000;6:751–6. 22. Reinecke L, Nicolas R, Schaffer S. Mikrobiologische Herstellung von C4-Körpern aus Saccharose und Kohlendioxid, DE 102010029973 (Evonik Degussa), 2010. 23. Tan D, Xue YS, Aibaidula G, Chen GQ. Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01. Bioresour Technol 2011;102:8130–6. 24. Johnson K, Jiang Y, Kleerebezem R, Muyzer G, van Loosdrecht MC. Enrichment of a mixed bacterial culture with a high polyhydroxyalkanoate storage capacity. Biomacromolecules 2009;10:670–6. 25. Albuquerque MGE, Bengtsson S, Martino V, Pollet E, Reis MAM. Eco-engineering of mixed microbial cultures to develop a cost-effective bioplastic (PHA) production process from a surplus feedstock - sugar molasses. J Biotechnol 2010;150:S70. 26. Jiang Y, Marang L, tamis J, van Loosdrecht MC, Dijkman H, Kleerebezem R. Waste to resource: converting paper mill wastewater to bioplastic. Water Res 2012;46:5517–30. 27. Albuquerque MGE, Concas S, Bengtsson S, Reis MAM. Mixed culture polyhydroxyalkanoates production from sugar molasses: the use of a 2-stage CSTR system for culture selection. Bioresour Technol 2010;101:7112–22. 28. Albuquerque MGE, Eiroa M,Torres C, Nunes BR, Reis MAM. Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses. J Biotechnol 2007;130:411–21. 29. Albuquerque MGE, Martino V, Pollet E, Averous L, Reis MAM. Mixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-rich streams: effect of substrate composition and feeding regime on PHA productivity, composition and properties. J Biotechnol 2011;151:66–76. 30. Bengtsson S, Hallquist J, Werker A, Welander T. Acidogenic fermentation of industrial wastewaters: effects of chemostat retention time and pH on volatile fatty acids production. Biochem Eng J 2008;40:492–9. 31. Bengtsson S, Pisco AR, Reis MAM, Lemos PC. Production of polyhydroxyalkanoates from fermented sugar cane molasses by a mixed culture enriched in glycogen accumulating organisms. J Biotechnol 2010;145:253–63. 32. Zhang XJ, Luo RC,Wang Z, Deng Y, Chen GQ. Applications of (R)-3-hydroxyalkanoate methyl esters derived from microbial polyhydroxyalkanoates as novel biofuel. Biomacromolecules 2009;10:707–11. 33. Sudesh K. Polyhydroxyalkanoates from Palm oil: biodegradable plastics (eBook/PDF). Springer; 2012. 34. Wang HH, Zhou XR, Liu Q, Chen GQ. Biosynthesis of Polyhydroxyalkanoate Homopolymers by Pseudomonas putida. Appl Microbiol Biotechnol 2011;89:1497–507. 35. Dinjus E, Arnold U, Dahmen N, Höfer R,Wach W. Green fuels – sustainable solutions for transportation. In: Höfer R, editor. Sustainable solutions for modern economies. Cambridge: RSC Publ; 2009. p. 125–63. 36. Wang SY, Liu MM, Xu Y, Zhang XJ, Chen GQ. Properties of a new gasoline oxygenate blend component: 3-hydroxybutyrate methyl ester produced from bacterial poly-3-hydroxybutyrate. Biomass Bioenerg 2010;34:1216–22.

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37. Wang Y, Jiang XL, Yang SS, Lin X, He Y, Yan C, et al. MicroRNAs in the regulation of mesenchymal stem cells interfacial behaviors on microgrooved surface pattern. Biomaterials 2011;32:9207–17. 38. Peng SW, Guo XY, Shang GG, Li J, Xu XY,You ML, et al. An assessment of the risks of carcinogenicity associated with polyhydroxyalkanoates through an analysis of DNA aneuploid and telomerase activity. Biomaterials 2011;32:2546–55. 39. Chen GQ, Wang Y. Medical applications of biopolyesters polyhydroxyalkanoates (PHA). Chin J Polym Sci 2013;31:719–36. 40. Ren Q, Grubelnik A, Hoerler M, Ruth K, Hartmann R, Felber H, et al. Bacterial poly(hydroxyalkanoates) as a source of chiral hydroxyalkanoic acids. Biomacromolecules 2005;6:2290–8. 41. Ren Q, Ruth K, Thony-Meyer L, Zinn M. Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives. Appl Microbiol Biotechnol 2010;87:41–52. 42. Takahashi K, Murakami T, Kamata A, Yumoto R, Higashi Y, Yata N. Pharmacokinetic analysis of the absorption enhancing action of decanoic acid and its derivatives in rats. Pharm Res 1994;11:388–92. 43. Faveau C, Mondon M, Gesson JP, Mahnke T, Gebhardt S, Koert U. Synthetic studies on a phenyllaulimalide analogue. Tetrahedron Lett 2006;47:8305–8. 44. Ruth K, Grubelnik A, Hartmann R, Egli T, Zinn M, Ren Q. Efficient production of (R)-3-hydroxycarboxylic acids by biotechnological conversion of polyhydroxyalkanoates and their purification. Biomacromolecules 2007;8:279–86. 45. Lee S, and Lee K. Method of preparing statins intermediates. Patent WO/2004/096789, 2004. 46. Iwanami Y. Myrmicacin, a new inhibitor for mitotic progression after metaphase. Protoplasma 1978;95:267–71. 47. Iizuka T, Iwadare T, Orito K. Antibacterial activity of myrmicacin and related compounds on pathogenic bacteria in silkworm larvae, Streptococcus faecalisAD-4. J Fac Agri., Hokkaido 1979;59:2–10. 48. Lüderitz O, Galanos C, Lehmann V, Mayer H, Rietschel ET, Weckesser J. Chemical structure and biological activities of lipid A’s from various bacterial families. Naturwissenschaften 1978;65:578–85.

CHAPTER 17

Microbial Poly-3-Hydroxybutyrate and Related Copolymers Raveendran Sindhu, Parameswaran Binod, Ashok Pandey Centre for Biofuels and Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India

Contents 1. Introduction 2. PHB-Producing Microbes 2.1 Screening of Microorganisms 2.2 PHB Production Using Wild-Type Bacteria 2.3 PHB Production Using Recombinant Bacteria 2.4 PHB Production Using Mixed or Cocultures 2.5 PHB Production by Cyanobacteria 3. Fermentation Strategies 3.1 Submerged Fermentation

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3.1.1 Batch 3.1.2 Fed-Batch 3.1.3 Continuous

3.2 Solid-State Fermentation 4. Downstream Operations 4.1 Chemical Methods 4.2 Biological Methods 5. Characterization Techniques 5.1 Fourier Transform Infrared Spectroscopy 5.2 Nuclear Magnetic Resonance 5.3 Thermogravimetric Analysis 5.4 Differential Scanning Calorimetry 5.5 Gel Permeation Chromatography 5.6 Atomic Force Microscopy 6. Strain Improvement, Mutation, and Metabolic Engineering 7. Substrate Manipulation for the Production of Various Classes of PHB 7.1 Importance of External Substrate Addition 7.2 Manipulation of Carbon Sources

7.2.1 Properties of PHB due to Carbon Manipulation

585 586 586 587 588 588 588 589 590 590 591 592 595 596 596 598

7.3 Effect of Nitrogen and Phosphorus 7.4 Inhibitor Addition 8. Applications 8.1 Medical

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8.1.1 Targeted Drug Delivery 8.1.2 Nanofibrous Matrices as Cell Supporting Materials

Industrial Biorefineries and White Biotechnology http://dx.doi.org/10.1016/B978-0-444-63453-5.00019-7


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8.1.3 Biomedical Application of PHB Sutures 8.1.4 Organic-Soluble Chitosan/PHB Ultrafine Fibers for Skin Regeneration 8.1.5 PHB Composite Materials for Bone Tissue Regeneration

600 600 600

8.2 Industrial

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8.2.1 Packaging Applications 8.2.2 Recombinant Protein Purification

9. Conclusion and Perspectives 601 References601

1. INTRODUCTION Pollution by plastics is one of the major concerns all over the world. The major factors that attract these synthetic polymers are that they are cheap, can easily produce from petrochemicals, and are very durable and flexible. These synthetic plastics are inevitable for day-to-day human life.The main disadvantages of these polymers are its nonbiodegradable characteristics and these are produced from nonrenewable natural resource. So there is a need for alternative sources of plastics that can be produced from renewable resources and it should be biodegradable. Several researchers are constantly involved in the search for alternative sources of plastics, and this has resulted the discovery of biodegradable polymers in several life forms such as plants, microbes, and animals. Among these, microbes are the preferred source because the culture condition and other bioprocess can be easily controlled and monitored. Several microorganisms such as bacteria can synthesize polymers like poly-3-hydroxybutyrate (PHB) that are biodegradable, and utilization of these biopolymers will help to reduce the dependence of petroleum derived plastics. These bacterial PHB can be produced from renewable resource that offers ecological advantages as compared to thermoplastics and elastomers produced from fossil carbon sources.1 PHB is a short chain length (SCL) polymer with several properties similar to polypropylene and it can be easily degraded aerobically or anaerobically. It is a crystalline material with high melting temperature and high degree of crystallinity. The physical and mechanical properties are similar to polypropylene, but PHB is stiff and brittle and the degree of brittleness depends on the degree of crystallinity, glass transition temperature, and microstructure. One of the main drawbacks of this polymer is that it is thermally unstable which will lead to decrease in viscosity and molar mass. The structure of PHB is given in Figure 17.1. The properties of PHB can be improved by blending with other polymers or by addition of lubricants or plasticizers or by suppression of crystallization and by lowering the glass transition temperature. The blending of PHB with other polymers allows a variety of copolymers to be produced with flexibility and tensile strength. Poly (hydroxybutyrate–valerate) (PHBV) is such a copolymer produced by certain bacteria when grown under specific carbon source. It is less stiff and tougher than PHB and can be used as packaging material (Figure 17.2).

Microbial Poly-3-Hydroxybutyrate and Related Copolymers

Figure 17.1 Structure of poly-3-hydroxybutyate.

&+ 2

+&

&+&+

2 &+&

2

&+

2 &+&

Q

Figure 17.2 Structure of PHBV.

Several microorganisms are known to accumulate PHB within the cells as an intracellular storage material for carbon and energy source. In microbes PHB synthesis takes place by a sequence of reactions catalyzed by three enzymes—3-ketothiolase, acetoacetylcoA reductase and PHB synthase.The first step is catalyzed by the enzyme 3-ketothiolase (E.C. 2.3.1.16) which condenses acetyl-CoA to acetoacetyl-CoA. This intermediate is reduced to D (-)-β-hydroxybutyryl-CoA by an Nicotinamide Adenine Dinucleotide Phosphate (NADPH)-dependent acetoacetyl-CoA reductase (E.C.1.1.1.36) and the enzyme PHB synthase catalyzes the head to tail polymerization of the monomer to PHB (Figure 17.3). PHB is naturally accumulating in a wide variety of microorganisms. Ralstonia eutropha is the well-known bacterium which accumulates PHB up to 80% of the cell dry weight utilizing various carbon sources. The PHB exists as water insoluble inclusions in bacteria and it shows mechanical properties similar to that of polypropylene.2 One of the main limitations for the bulk production of PHB is its high production costs as well as recovery costs. However with the development of genetic and metabolic engineering techniques allowed PHB biosynthesis in several recombinant bacteria and yeasts by improving the yields and thereby reducing the overall production costs.3 By implementing several metabolic engineering strategies like external substrate manipulation, inhibitor addition, recombinant gene expression, host cell genome manipulation, and protein engineering, it is possible to construct microbial plastic factories to produce biopolymers with desirable structure as well as properties.4 Utilization of inexpensive renewable carbon sources like plant oils, waste materials, and carbon dioxide is essential to reduce the production cost further. PHB finds applications in the development of biomedical devices and related products, as scaffolds in tissue engineering, development of vascular grafts and artificial heart valves and as a packaging material. One of the major limitations of using PHB is due to

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Acetyl-CoA β-ketothiolase Acetoacetyl-CoA Acetoacetyl CoA reductase (R)-3-Hydroxyacyl-CoA PHB synthase

PHB granule

Figure 17.3 Mechanism of poly-3-hydroxybutyrate synthesis within bacterial cell.

weak mechanical properties. Hence these polymers need to be blended with synthetic polymers for improved applications. Cost of production is one of the major limiting factors for large-scale production of PHB. Recent developments in gene manipulation, metabolic engineering, and utilization of cheaper sources as well as new fermentation strategies for production lead to the reduction of production cost. This chapter discusses about PHB and the related copolymers, its production, characterization, factors affecting production as well as applications in various sectors.

2. PHB-PRODUCING MICROBES Several species of bacteria, recombinant strains as well as cyanobacteria are known to produce PHB. Table 17.1 shows list of microbes producing PHB.

2.1 Screening of Microorganisms Screening and selection of microorganisms producing PHB is the first step in the bioprocess development of PHB. Several screening protocols have been developed to detect PHB in microbial cells. PHB confers some opacity to the bacterial cells thus allows a primary screening of PHB positive and negative colonies. This effect is only observed when the


Table 17.1 Microbes producing poly-3-hydroxybutyrate Microorganism PHB% References

Bacillus megaterium Bacillus mycoides Bacillus sphaericus NII 0838 Bacillus firmus NII 0830 Corynebacterium glutamicum Azotobacter vinelandii Chlorococcus Oscillatoria Anabaena Pseudoanabaena Synchocystis

45% 69.4% 31% 89% 36% – – – – – –

Chaijamrus and Udpuay8 Borah et al.10 Sindhu et al.11 Sindhu et al.12 Jo et al.19 Zanzig and Scholz17 Beyatli et al.37 Beyatli et al.37 Beyatli et al.37 Beyatli et al.37 Beyatli et al.37

difference is relatively high. The lipophilic dyes such as Sudan black, Nile blue, and Nile red have been used to distinguish between PHB accumulating and nonaccumulating strains. Nile red produces a strong orange fluorescence (emission maximum, 598 nm) with an excitation wavelength of 543 nm (maximum) upon binding to PHB granules.5 The oxazone form (Nile pink or Nile red) is probably formed from the basic oxazine dye Nile blue A by spontaneous oxidation in aqueous solution, thereby producing fluorescence.6 The lipophilic dyes must be dissolved in organic solvents such as ethanol (for Sudan black B) or acetone (for Nile red) and then are poured onto the agar plates. Since the cells are killed during the staining of the colonies, master plates have to be prepared. Spiekermann et al.7 developed a protocol for a viable colony staining method based on direct inclusion of the Nile red or Nile blue A in the agar medium. In such case the growth of the cells is not affected and the occurrence of PHB in the colonies can be directly monitored. This viable-colony staining method can be done with Nile red as well as Nile blue A. However, with Nile blue A, the intensity of the fluorescence was generally weaker and this can be enhanced by increasing the concentration of Nile blue A in the medium. The fluorescent microscopic image of PHB positive strain is shown in Figure 17.4.

2.2 PHB Production Using Wild-Type Bacteria Several wild-type bacterial strains were reported to produce PHB. Bacteria of the genera Bacillus, Ralstonia, Pseudomonas, Alcaligenes, Azotobacter, Halomonas, Corynebacteria, Lactobacillus, and Vibrio species were reported to produce PHB as a storage compound in response to nutrient imbalance caused by growth under conditions of excess carbon source and limitation of other nutrients like nitrogen or phosphorous. PHB is reported to be produced by several Bacillus species like Bacillus megaterium,8,9 Bacillus mycoides,10 Bacillus sphaericus NII 0838,11 Bacillus firmus NII 0830,12 Bacillus thuringiensis,13 Bacillus cereus,14 Azotobacter beijerinckii,15 Azotobacter chroococcum,16 Azotobacter vinelandii,17 Actinobacillus,18 Corynebacterium glutamicum19 etc.

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Figure 17.4 Primary screening profile of bacteria based on Nile blue staining.

2.3 PHB Production Using Recombinant Bacteria Recombinant microorganisms offer several characteristics for the production of PHB in a cost-effective manner. The costs can be reduced by including the use of cheap substrates or by enhancement of the product yield.20 Escherichia coli is a suitable host as a heterologous expression background for foreign genes that can be easily manipulated and improved by means of recombinant DNA methodologies. High-cell-density cultivation strategies for numerous E. coli strains are well established.21 Escherichia coli cells that accumulate large amounts of PHB become fragile, facilitating the isolation and purification of the biopolymer, and the bacterium does not express PHA (polyhydroxyalkanoate)degrading enzymes.22 Genes responsible for PHB biosynthesis (pha or phb genes) from a number of microorganisms, such as Cupriavidus necator, formerly called Alcaligenes eutrophus23; Pseudomonas aeruginosa24; Alcaligenes latus25; Thiocapsa pfennigii26; and Streptomyces aureofaciens27 have been introduced into E. coli. In most cases, the biosynthetic genes were expressed under the control of their native promoters, and the resulting recombinants were able to accumulate PHA from different carbon sources.22 de Almeida et al.28 conducted several studies where PHB production has been evaluated in E. coli and metabolic engineering has been carried out to improve productivity. Nikel et al.22 reported a mutation in arcA encoding, a protein that regulates aerobic respiration under microaerophilic conditions, resulted in accumulation of higher amounts of PHB in the cell. Low agitation has a positive effect on PHB synthesis in E. coli carrying phaCAB operon and phaP encoding phasin, a granule-associated protein. Several studies have been carried out by Kocharin et al.29 to transfer the PHB biosynthetic pathways to alternative hosts which lack enzymes involved in depolymerization. With the help of genetic and protein engineering techniques, it is possible to enhance polymer productivity. Recombinant E. coli are capable of producing ultrahigh molecular


weight PHB with superior properties after stretching of the material. Metabolically engineered Pseudomonas sp. 61-3 produced PHB copolymers which are similar to low density polyethylene (LDPE). Hydrogenase 3 and acetyl-CoA synthetase enzymes are very important in PHB synthesis. Wang et al.30 enhanced coproduction of hydrogen and PHB by recombinant E. coli by over expressing hydrogenase 3 and acetyl-CoA synthetase. Anaerobic metabolic pathways dedicated to coproduction of hydrogen and PHB were established due to the advantages of directing fluxes away from toxic compounds like acetate and formate to useful products. Overexpression of hydrogenase 3 resulted in an increase in PHB yield from 0.55 to 5.34 mg PHB/g glucose in MS medium with glucose and acetate as carbon source.

2.4 PHB Production Using Mixed or Cocultures One of the main disadvantages of using pure culture for the production of PHB includes the high cost of the pure substrates utilized and the cost of sterile precultivation of the bacteria utilized as well as the sterile operation of the final production process. Mixed or coculture systems are widely used in several fermentation processes. There are many fermentation systems, where microorganisms assimilate one substrate and convert it to an intermediate metabolite which is converted from other microorganisms to metabolitetarget product. Mixed or coculture systems will serve as an attractive addition to traditional pure culture-based technology for the production of PHB. To reduce their production cost, several efforts have been made for developing better bacterial strains and more efficient fermentation as well as recovery processes.The use of mixed cultures and cheap substrates can reduce the production cost of PHB. Accumulation of PHB by mixed cultures occurs under transient conditions caused mainly by intermittent feeding and variation in the electron donor/acceptor presence. The maximum capacity for PHB storage and the PHB production rate are dependent on the substrate and the operating conditions used.31 Ganduri et al.32 reported better PHB productivity through coculturing Lactobacillus delbrueckii and R. eutropha. PHB concentrations of 12 g/L and 40 g/L were reported for laboratory experiments and fed-batch fermentation, respectively.The study revealed that equally high yield is possible in simple batch fermentations by controlling the mixing intensity. Shalin et al.33 reported utilization of mixed microbial cultures for PHB production. Bacillus firmus NII 0830 was used for the production of PHB since it accumulates a large amount of PHB and a second organism L. delbrueckii NII 0925 was used to provide lactic acid. Enhanced economy, simple process control, nonrequirement of mono-septic processing are some of the advantages of using mixed culture system. Tohayama et al.34 reported the effect of controlling lactate concentration and periodic change in dissolved oxygen concentration (DOC) affects PHB production using a mixed culture of L. delbrueckii and R. eutropha. Here, the glucose was converted to lactate by L. delbrueckii and the lactate was converted to PHB by R. eutropha.

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The results indicate that periodic fermentation resulted in superior PHB yield with high PHB productivity when DOCs were controlled to be constant at less than 1 ppm or 3 ppm. Short- and long-term temperature effects on aerobic PHB producing mixed cultures were evaluated by Johnson et al.35 Temperature coefficient related to PHB metabolism was higher than short-term temperature changes. The specific growth rate on acetate decreased with increase in temperature. At higher temperature (30 °C), the culture produced 84% PHB. At lower temperature (15 °C), the production was decreased and the feast phase was longer and growth occurred predominantly on acetate rather than on stored PHB.

2.5 PHB Production by Cyanobacteria Several phototrophic bacteria produce PHB as an intracellular reserve for reducing power.These microorganisms accumulate more glycogen than PHB. Its synthesis is stimulated when reducing equivalents are excess or quickly interrupted when balanced growth is permitted; here PHB acts as a buffer system for regulating the intracellular redox balance.36 Several cyanobacteria were reported to produce PHB. This includes Chlorococcus, Oscillatoria, Anabaena, Pseudoanaebaena, and Synechocystis.37 Sharma and Mallick38 explored the potential of cyanobacteria for the production of PHB. Due to their minimal nutrient requirement and ability to grow in waste waters in presence of sunlight and CO2 serves as an alternative source for PHB production since the biomass is converted to PHB by solar energy.The study revealed that by media engineering, PHB production was improved fourfold. Phosphorous limitations as well as exogenous supply of carbon sources like acetate, glucose, maltose, fructose, and ethanol were found to have a positive effect on PHB accumulation.

3. FERMENTATION STRATEGIES 3.1 Submerged Fermentation 3.1.1 Batch Several reports were available on utilizing batch cultures for production of PHB. A nitrogen limitation condition is not ideal for batch fermentation employing microorganisms which cannot grow under nitrogen limiting conditions. Jiang et al.39 investigated the use of lactate and a lactate and acetate mixture for enrichment of PHB production by mixed cultures in sequencing batch reactors. The mixed cultures enriched on lactate can accumulate over 90% PHB within 6 h, which is the best result reported for a bacterial culture in terms of final PHB content and the biomass specific PHB production rate. The second mixed culture enriched on lactate and acetate produced 84% PHB after 8 h. The study revealed that the use of different substrates has no significant effect on the functionality of PHB production process.


PHB production using R. eutropha ATCC 17697 and A. latus ATCC 29712 was evaluated using a bioreactor as batch by Azhar et al.40 Maximum sugar consumption and sugar utilization efficiency were attained after 100 h; ammonia was completely assimilated after 80 h. The cell dry weight after 100 and 80 h were 10.18 and 8.73 g/L, respectively. PHB synthesis from a mutant strain A. vinelandii using glucose in a batch reactor was optimized by Dhanasekar et al.41 The initial medium pH significantly affects the productivity. Incubation temperature does not have a significant role in PHB production. Substrate concentration significantly affects the productivity as well as the PHB yield. It was observed that maximum productivity was observed with short fermentation period and lag phase were also minimized. 3.1.2 Fed-Batch Fed-batch cultivation is one of the best methods to produce high cell density with high PHB content. The important strategy for fed-batch fermentation is to feed the growth limiting substrates at the same rate as the rate of substrate is utilized by the organism.This helps in preventing the formation of by-products that are produced when the substrate is excess and leads to production of product of interest. PHB production from glycerol by Zobella denitrificans MW1 using high cell density fed-batch fermentation was reported by Ibrahim and Steinbuchel.42 The strain showed an increased PHB content at a low concentration of ammonium chloride. In this method, a much higher concentration of PHB (54.3 g/L) and highest cell density (81.2 g/L) were obtained in the presence of 20 g/L NaCl with optimized feeding of glycerol and ammonia to support both cell growth and polymer accumulation over a period of 50 h. PHB production by Saccharophagus degradans using raw starch as carbon source in a fed-batch culture was evaluated by González-Garcia et al.43 The strain accumulated 21.35% and 17.46% of PHB using glucose and starch as sole carbon source.The physical properties of these polymers were similar. Patnaik44 reported fed-batch optimization of PHB synthesis through mechanistic, cybernetic, and neural approaches. Enhancement of PHB productivity was investigated by applying two artificial neural networks to a bioreactor with finite dispersion and noise in feed streams. One network filtered the noise and other controlled the filtered feed rates of carbon and nitrogen sources. The study revealed that neural optimization doubled the maximum PHB concentration in fed-batch fermentation with R. eutropha by optimizing the time dependent feed rates. High cell density fed-batch fermentation of A. eutrophus was carried out for the production of PHB in a 60 L fermentor by Ryu et al.45 The PHB production was carried out by maintaining constant pH using NH4OH solution and PHB accumulation was induced by phosphate limitation. The fed-batch fermentation resulted in final cell concentration of 281 g/L and PHB concentration of 232 g/L and a productivity of 3.14 g/L/h.

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Yeo et al.46 reported enhancement of PHB production by a two stage supplementation of carbon sources and continuous feeding of NH4Cl. The study revealed that gluconate was used to maximize specific growth rate during initial stage of growth while glucose was used to maximize PHB synthesis. The sequential feeding of gluconate and glucose resulted in 50% enhancement in PHB production when compared to glucose alone.The glucose-grown culture showed a higher level of NADPH during the NH4Clexausted PHB accumulation stage than was observed in the gluconate-grown culture, which reflects that the reason of higher PHB production observed when glucose was used as a carbon source. NH4Cl feeding following the depletion of initial NH4Cl resulted in elevated levels of both ATP and NADPH, which increased the PHB biosynthesis rate, and also in a decrease in the level of NADH, which reflected the alleviation of the inhibitory effects on the cells caused by nitrogen depletion. A recombinant E. coli K24K strain was constructed and evaluated for PHB production from whey and corn steep liquor as main carbon and nitrogen source by Nikel et al.22 PHB was efficiently produced by the recombinant bacteria grown aerobically in fed-batch cultures in a laboratory-scale bioreactor in semisynthetic medium supplemented with agro-industrial by-products. The cells accumulated 72.9% of PHB with a productivity of 2.13 g/L/h. Quillaguiaman et al.47 achieved high PHB content and volumetric productivity by fed-batch culture of Halomonas boliviensis. Shake flask cultivation was carried out in minimal medium and growth was supported with supplementation of aspartic acid, glycine, or glutamine. Addition of glutamine resulted in high cell weight and when it was replaced with monosodium glutamate there was no change in cell density. 3.1.3 Continuous Continuous fermentation is a fermentation strategy having the possibility of achieving high productivity with strains having high maximum specific growth rate, which can be operated as a single stage or multistage process. An early nutrient limitation would result in low biomass with high PHB content while late induction of nutrient limitation would result in high biomass but with less PHB content. Hughes and Richardson48 patented a fermentation process for the production of PHB from Alcaligenes sp. continuously in a fermentor. A medium containing nutrient salts, carbon and energy source, and a water-soluble compound that is assimilated by microbes was supplied continuously removing an equal amount of medium containing bacterial cells from the medium thereby maintaining the amount of aqueous medium in the vessel at constant level. PHB is produced in R. eutropha under unbalanced growth conditions, hence a twostage continuous culture system to be adopted. In the first stage maximum cell biomass were produced and in the second stage PHB was produced. The PHB content and productivity were 47.6% and 1.43 g/L/h, respectively.


Henderson and Jones49 reported growth of A. eutrophus in a continuous system under various conditions of dilution rate, nutrient limitation, and carbon substrate. The study revealed that maximum PHB content, productivity, and carbon substrate utilization rate when grown on glucose medium were maximal at low dilution rate under ammonia limitation. The PHB content decreased in a linear manner as a function of dilution rate. The strain showed highest growth rate when grown on media containing lactate. The study revealed that A. eutrophus cannot regulate the rate at which it takes up excess carbon substrate which is solely required for growth especially during growth on lactate at low dilution rate thereby producing PHB as a means of avoiding harmful effects of high concentration of intracellular metabolites. A two-stage continuous culture system where a phase-wise optimization was carried out which maximizes the residual biomass growth rate in the first stage followed by a PHB production rate in the second stage was reported by Lee et al.50 Maximum PHB productivity of 2.86 g/L/h was obtained in this process. Khanna and Srivastava51 reported members of the genus Wautersia uptake excessive carbon from the medium and accumulate PHB. Continuous cultivation of Wautersia eutropha was carried out in a 7 L reactor. Reactor was operated in a batch mode for initial 15 h followed by a fed-batch mode for sufficient biomass and PHB production, followed by a continuous mode so that PHB production was continuously maintained and released from the reactor.

3.2 Solid-State Fermentation Few reports are available on PHB production by solid-state fermentation (SSF). Ramadas et al.52 have developed a novel SSF bioprocess in which polyurethane foam (PUF) was used as a physical inert support for the production of PHB by B. sphaericus NII 0838. Media engineering for optimal PHB production was carried out using response surface methodology (RSM) adopting a Box–Behnken design. The factors optimized by RSM were inoculum size, pH, and (NH4)2SO4 concentration. Under optimized conditions—6.5% inoculums size, 1.7% (w/v) (NH4)2SO4, and pH 9.0, PHB production and biomass were 0.169 and 0.4 g/g PUF, respectively. This is the first report on PHB production by SSF using PUF as an inert support. The results demonstrate that SSF can be used as an alternative strategy for the production of PHB. The major inherent problem associated with PHB production in SSF systems is the biomass retrieval of bacterial cells. This limitation can, however, be overcome by using PUF as an inert support. Oleivera et al.53 reported PHB production by R. eutropha using agro-industrial residues. The PHB productivity and content were 4.9 mg/g medium in 60 h and 39%, respectively. Production was carried out using soy cake alone or supplemented with sugarcane molasses. The results obtained showed that the biopolymer obtained by SSF has similar properties as commercial PHB, except for the higher molar mass and the

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lower degree of crystallinity. Thus, the present data indicate that SSF is an interesting alternative for the production of PHB, allowing the production of biopolymers with adequate properties from low-cost, renewable resources. SSF process provides a biopolymer that is identical to a commercial PHB produced by submerged fermentation, as well as to other PHB data reported in literature. The only differences noted for the polymers produced by SSF were a higher molar mass and a lower degree of crystallinity, which both represent advantages for the SSF process, since these properties enable a broader range of applications for the PHB produced by this method.

4. DOWNSTREAM OPERATIONS The extraction as well as purification of the PHB is one of the key steps in the bioprocess. An ideal purification method is that which leads to high purity and recovery level at a low production cost.

4.1 Chemical Methods The common chemical methods for extraction of PHB are solvent extraction, chemical digestion using sodium hypochlorite, sodium hypochlorite and chloroform, surfactants, surfactant hypochlorite digestion, surfactant chelate digestion, chelate hydrogen peroxide treatment, selective dissolution of nonpolymer cell mass by proton etc. Solvent extraction method is one of the oldest techniques adopted for extraction of PHB. The mechanism of action of solvent is that it modifies the cell membrane permeability and then solubilize PHB. This process was first adopted by Lemoigne and Baptist for the extraction of PHB from B. megaterium and Rhodospirillum rubrum.54 Several solvent mixtures were tried for the separation of PHB from the solvent by solvent evaporation or by precipitation in a nonsolvent. Vanlautem and Gilain55 used liquid halogenated solvents like chloroethanes and chloropropanes for the extraction of PHB. This method was found to be superior when compared to extraction using other solvents like tetrahydrofuran methyl cyanide, tetrahydrofuran ethyl cyanide, and acetic anhydride. Noda56 developed a method with a mixture of PHB solvent and nonsolvent, the insoluble biomass is separated leaving behind a suspension of precipitated PHB in the nonsolvent. Another method for extraction of PHB is by using sodium hypochlorite digestion method.57 With this method, 86% purity was observed with R. eutropha and 93% purity with E. coli. One of the major drawbacks of this method is that it causes degradation of PHB resulting in 50% reduction in molecular weight. Hahn et al.58 developed a novel strategy for PHB extraction by combining the advantage of both differential digestions by hypochlorite and solvent extraction. Adopting this method for extraction of PHB from R. eutropha, three separate phases were obtained, an upper phase of hypochlorite solution, a middle phase of non-PHB cell materials and


undisrupted cells, and the chloroform phase contains the PHB. The polymer is recovered by precipitation in a nonsolvent and filtration and by adopting this method, the degradation of polymer is significantly reduced. Dong and Sun59 adopted a combination of surfactant and hypochlorite process for extraction of PHB. By this method, polymer can be extracted to a purity of 98%. In this method, a freezing pretreatment was also used for cell lysis. The main advantages of this method are its low operating cost and limited degradation of polymer. Chen et al.60 reported that by adding a chelate to the surfactant increase the release of polymer. Addition of chelate destabilizes the outer membrane forming complexes with divalent cations. This in turn cause changes in inner membrane and improves cell lysis and gives a high purity polymer. The advantages of this method are high purity of the product and low environmental pollution. One of the main drawbacks of this method is generation of large volume of waste water during the product recovery step. Liddell and Locke61 developed a combination of chelate treatment with hydrogen peroxide for the extraction of PHB produced by R. eutropha. For the recovery of polymer, first a heat pretreatment was carried out followed by treatment with chelating agent like diethylene triamine pentamethylene phosphonic acid and hydrogen peroxide. By adopting this method polymer can be recovered with a purity of 99.5%. A novel strategy for selective dissolution of nonpolymer cell mass by protons in aqueous solutions and crystallization of the polymer was developed by Yu and Chen.62 Polymer extracted by this method showed high purity and high yield. Studies revealed that this method is much cheaper than conventional methods used for recovering polymer and reduces the overall cost of polymer recovery by 90%. Fiorese et al.63 developed a novel method for recovery of PHB from C. necator biomass by solvent extraction with 1, 2-propylene carbonate. Process parameters like temperature, contact time, precipitation period, and pH affects the extraction efficiency and polymer properties. The highest yield of 90% and purity of 84% were obtained at temperature of 130 °C, contact time of 30 min, and precipitation period of 48 h. Under these conditions, high molecular weight PHB was obtained and physical properties like glass transition temperature, melting temperature, melting enthalpy, and crystallinity were not affected. The polymer yield did not improve with the heat/pH treatment; this treatment increased the PHB molecular weight and purity.

4.2 Biological Methods Holmes and Lim64 developed the enzymatic extraction process for the extraction of PHB. Proteolytic enzymes were used for this process.The process involves a heat treatment followed by enzymatic hydrolysis, surfactant treatment, and decolorization with hydrogen peroxide. The advantage of this pretreatment is that the recovery rate was higher and the major drawback of this technology is the high cost of the enzyme.

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Harrison et al.65 extracted polymer from R. eutropha upon treatment with lytic enzymes of Cytophaga sp. without any mechanical processing and 90% purity was achieved by this method. Lu et al., 200666 developed a combined method involving enzyme and sodium hypochlorite for extraction of polymer from Burkholderia sp. PTU9. A recovery of 89% was achieved by this process. De Koning and Witholt67 reported a combined process involving sequential treatment with heat, alcalase, and SDS assisted by Ethylene Diamine Tetraacetic Acid (EDTA) treatment for the recovery of the polymer from Pseudomonas. A recovery rate of 95% was achieved by this process. Kapritchkoff et al.68 observed that for enzymatic recovery and purification of PHB from R. eutropha, proteases were the most suitable enzymes for non-PHB biomass solubilization and purification. Due to high efficiency and low-cost, pancreatin makes an ideal candidate for large-scale applications.

5. CHARACTERIZATION TECHNIQUES Characterization techniques were used to determine molecular mass, molecular structure, morphology, thermal as well as mechanical properties. Various techniques commonly used for characterization of PHB include FTIR, NMR, DSC, TGA, GPC, and AFM.

5.1 Fourier Transform Infrared Spectroscopy Fourier transform infrared spectroscopy (FTIR) is a technique which is used to obtain infrared spectrum of absorption, emission, and photoconductivity of solid, liquid, and gas. It is used to detect different functional groups in PHB. FTIR spectrum is recorded between 4000 and 400 cm−1. For FTIR analysis, the polymer was dissolved in chloroform and layered on a NaCl crystal and after evaporation of chloroform, the polymer film was subjected to FTIR. The spectrum of PHB shows peaks at 1724 cm−1 and 1279 cm−1, which corresponds to specific rotations around carbon atoms. The peak at 1724 cm−1 corresponds to C–O stretch of the ester group present in the molecular chain of highly ordered structure and the adsorption band at 1279 cm−1 corresponds to ester bonding.69 Figure 17.5 shows FTIR spectrum of PHB.

5.2 Nuclear Magnetic Resonance Nuclear magnetic resonance (NMR) is a physical process in which nuclei in a magnetic field absorb and reemit electromagnetic radiation. Analysis of NMR spectra allows the determination of polymer composition, and the distribution of monomer units can be deduced from the diad and triad sequences by NMR spectral analysis. For characterization of polymer, the extracted polymer will be dissolved in CDCl3 followed by NMR analysis. The NMR spectrum for PHB shows three characteristic signals. A doublet at 1.53 ppm represented the methyl group (CH3) coupled to one proton while a doublet of


quadruplet at 2.75 ppm resulted from methylene group (CH2) adjacent to an asymmetric carbon atom bearing a single proton.The third signal was a multiplet at 5.52 ppm, which was attributed to a methyne group (CH). Figure 17.6 shows NMR spectrum of PHB.

5.3 Thermogravimetric Analysis Thermal degradation of the polymer was studied using thermogravimetric analysis (TGA). Here, the change in physical and chemical properties of the polymer was

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Figure 17.5 FTIR spectrum of poly-3-hydroxybutyrate.

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Figure 17.6 NMR spectrum of poly-3-hydroxybutyrate.

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30.05C 498.04C -2.483mg -98.532%

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Figure 17.7 TGA curve of poly-3-hydroxybutyrate.

measured as a function of increasing temperature. PHB is very brittle and has low melting temperature. Several studies revealed that blending of PHB with other polymers is advantageous in terms of cost reduction with improved properties when compared to PHB alone.70 Figure 17.7 shows the thermal degradation profile of PHB.

5.4 Differential Scanning Calorimetry Differential scanning calorimetry (DSC) is a thermoanalytical technique used to study the thermal properties of the polymer using a differential scanning calorimeter. In this process, the difference in the amount of heat required to increase the temperature of a sample and reference is measured as a function of temperature. Sample and reference will be maintained at same temperature throughout the experiment. DSC curves were plotted based on heat flux versus temperature or time. Thermal transitions of polymer can be determined by this technique. DSC is widely used for the decomposition behavior determination of the polymer. Figure 17.8 shows the DSC curves of PHB.

5.5 Gel Permeation Chromatography Gel permeation chromatography (GPC) is a type of size exclusion chromatography technique used for determination of molecular weight of polymers, since molecular mass is an important factor determining the physical properties of polymers. Tetrahydrofuran is used


Figure 17.8 DSC curve of poly-3-hydroxybutyrate.

to dissolve the polymers prior to determination of molecular mass using GPC. For analysis, two columns HT3 and HT5 and a refractive index detector with chloroform as the elution solvent were used. Several factors affect the molecular weight of PHB. Effect of substrate and culture conditions affecting the molecular weight of PHB was reported by Chen and Page.71 The protocol adopted for extraction of the polymer can also lead to loss of molecular mass of polymer.72

5.6 Atomic Force Microscopy Atomic force microscopy (AFM) is a novel method for imaging the surface architecture of cells and cellular components. It provides a real-time three-dimensional images under natural conditions and gives high resolution images of surface topography. A study conducted by Sudesh et al.73 revealed that AFM can be used to observe directly and characterize proteins associated with native polymer granules. The study revealed three-dimensional images of proteins associated with native polymer granules as well as PHB single crystals. One of the main advantages of using AFM as imaging tool is that minimum sample pretreatment is required and it will not damage the sample. Immunogold labeling will help to understand the molecular mechanism of PHB granule formation, mobilization, and regulatory machinery involved. Figure 17.9 shows AFM image of PHB matrix.

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Figure 17.9 Atomic force microscopic image of poly-3-hydroxybutyrate matrix.

6. STRAIN IMPROVEMENT, MUTATION, AND METABOLIC ENGINEERING Recombinant E. coli serves as a good candidate for PHB production.The main drawback is the cost associated with Luria-Bertani medium, ampicillin, and requirement of pure oxygen. Hence, large-scale production is not practically feasible. Utilization of cheaper carbon source makes the process economically viable. Liu et al.74 reported PHB production from molasses by recombinant E. coli. The fermentation with molasses was economically viable when compared to that of glucose. The PHB content and productivity were 80% w/w and 1 g/L/h, respectively. The study revealed that molasses concentration had a significant effect on PHB synthesis and cell growth. A fed-batch strategy was adopted to overcome substrate inhibition as well as to improve cell growth and PHB production. PHB synthesis was evaluated using a recombinant E. coli arcA mutant using glycerol as sole carbon source by Nikel et al.75 Casein amino acids showed a significant effect on PHB production. The study revealed that microaerophilic fed-batch cultivation leads to a 2.57-fold increase in volumetric productivity when compared to batch cultivation. Lee76 reported PHB production from several recombinant E. coli strains harboring A. eutrophus PHA biosynthesis genes utilizing xylose from cotton seed hydrolyzate or soy bean hydrolyzate as sole carbon source.The study revealed that there is a twofold increase in PHB production when the medium is supplemented with a small amount of cotton seed hydrolyzate or soy bean hydrolyzate.The PHB concentration and PHB content were


4.4 g/L and 73.9%, respectively. Among the various recombinant E. coli strains, tested E. coli TG1 (pSYL107) showed highest productivity using cheap hemicelluloses hydrolyzate. A new fermentation strategy using cell recycle membrane system was developed by Ahn et al.77 for the efficient production of PHB from whey by recombinant E. coli strain CGSC 4401 harboring the A. latus PHA biosynthesis genes. The working volume of fermentation was constantly maintained by cell recycle and by fed-batch cultivation employing an external membrane module. The PHB concentration and PHB content were 168 g/L and 87%, respectively. Slater et al.78 reported cloning and expression of A. eutrophus H16 PHB pathway in E. coli. Library was constructed in cosmid pVK 102. The study revealed the relation between PHB transcriptional control and transcriptional control in other regulations, and PHB biosynthetic pathway is controlled by nitrogen and oxygen limitation. Cosmid clone was subcloned and the PHB biosynthetic pathway gene with a fragment size of 5.2 kb was cloned into multicopy vectors, which can direct the PHB synthesis in E. coli to 80% dry weight. To endow the superior PHB biosynthetic machinery to E. coli, Choi et al.25 cloned the PHA biosynthetic genes from A. latus. Three PHA biosynthetic genes that form an operon consisting of PHA synthase, β-ketothiolase, and reductase genes were constitutively expressed from the natural promoter in E. coli. Recombinant E. coli strains accumulated more amount of PHB when compared to R. eutropha. The PHB productivity was 4.36 g/L/h. This improvement should allow recombinant E. coli to be used for the production of PHB with a high level of economic competitiveness. This study provided a strategy of enhancing PHB productivity by developing recombinant E. coli strains harboring the more efficient PHA biosynthesis machinery of A. latus. The effect of different amino acids supplements on the synthesis of PHB by recombinant E. coli was evaluated by Mahishi and Rawal.79 The study revealed that when the basal medium is supplemented with amino acids, except glycine and valine, all other amino acid supplements enhanced PHB accumulation in recombinant E. coli harboring PHB synthesizing genes from S. aureofaciens. Cysteine, isoleucine, or methionine supplementation increased PHB accumulation by 60, 45, and 61%, respectively. Amino acid biosynthetic enzyme activities in several pathways are repressed by end product supplementation. End product inhibition in the cysteine biosynthetic pathway controls the carbon flow due to sensitivity of serine transacetylase to cysteine. Hence, supplementation of cysteine favors a change in carbon flux that eliminates the requirement of acetyl-CoA for serine transacetylation which in turn provides more carbon source and acetyl-CoA for PHB synthesis. Degradation of methionine and isoleucine yields succinyl CoA, an intermediate of tricarboxylic acid cycle and allows more acetyl-CoA to enter the PHB biosynthetic pathway. Effect of anaerobic promoters on the microaerobic production of PHB in recombinant E. coli was reported by Wei et al.80 Nine anaerobic promoters were cloned and constructed upstream of PHB synthesis genes phbCAB from R. eutropha for the microaerobic production of PHB in recombinant E. coli. Among the various promoters,

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alcohol dehydrogenase was found to be most effective. Recombinant E. coli strain showed a PHB accumulation of 48% after 48 h of static culture while with native promoter produced only 30% PHB. The molecular weights of PHB produced under microaerophilic conditions were found to be higher. The study conducted by de Almeida et al28 revealed that aeration affects PHB synthesis from glucose and glycerol in recombinant E. coli carrying phaBAC and phaP of Azotobacter sp. FA8 grown on glycerol at low agitation accumulated more PHB and ethanol than at high agitation. When glucose is used low agitation led to a decrease in PHB formation. Small variations in available oxygen can lead to significant changes in metabolism of E. coli cultures, which reflect the metabolic adjustments that take place to optimize cell growth in facultative aerobe, vary when using glycerol or glucose affecting the synthesis of products. Heterologous expression of the bacterial PHB biosynthesis pathway in Saccharomyces cerevisiae involves the utilization of acetyl-CoA, an intermediate of the central carbon metabolism, as precursor and NADPH, a redox cofactor used during anabolic metabolism, as a required cofactor for the catalyzing enzymes in the PHB biosynthesis pathway. Provision of acetyl-CoA and NADPH by alteration of the endogenous pathways and/ or implementation of a heterologous gene/pathway was investigated with the aim to improve PHB production in S. cerevisiae. Engineering of the central carbon and redox metabolism substantially improve PHB production. By adopting metabolic engineering, microorganisms can be engineered to produce new products with higher yield and productivities. Kocharin et al.29 expressed the bacterial PHB pathway in S. cerevisiae and evaluated the effect of engineering the formation of acetyl-CoA, which is an intermediate of the central carbon metabolism and precursor of PHB biosynthetic pathway for heterologous PHB production by yeast. Kocharin et al.29 engineered the acetyl-CoA metabolism by cotransformation of a plasmid containing genes for native S. cerevisiae alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), acetyl-CoA acetyltransferase (ERG10), and a Salmonella enterica acetyl-CoA synthetase variant (acsL641P), resulting in acetoacetyl-CoA overproduction, together with a plasmid containing the PHB pathway genes coding for acetyl-CoA acetyltransferase (phaA), NADPH-linked acetoacetyl-CoA reductase (phaB), and PHB polymerase (phaC) from R. eutropha H16. Enhancement of acetyl-CoA production by coexpression of genes on the acetyl-CoA boost plasmid improved the productivity of PHB during growth on glucose and further enhanced the productivity of PHB approximately 16.5 times bioreactor cultivations and reduce the flux from acetyl-CoA to lipids. Li et al.81 over expressed Nicotinamide Adenine Dinucleotide (NAD) kinase to enhance the accumulation of PHB in recombinant E. coli harboring PHB synthesis pathway by an accelerated supply of NADPH, one of the important factors affecting PHB production. The study revealed that NAD kinase in E. coli harboring the PHB synthesis operon could increase the accumulation of PHB to 16–35% weight compared


to controls. The availability of NADPH is an important factor for improving PHB productivity. The NAD kinase overexpression enhanced PHB production in recombinant E. coli harboring the PHB synthesis operon due to effective supply of NADPH. Mutation can be carried out by chemical methods, UV radiation, or by genetic elements such as transposons. UV mutagenesis seems to be one of the promising methods for strain improvement. The study conducted by Ugwu et al.82 revealed that UV mutagenesis can be successfully used in generating mutants for extracellular production of PHB. This is the first report on the use of mutants for extracellular production of (R)3-HB. Production of (R)-3-HB using acetoacetate seems to be an interesting biosynthetic route in C. necator. Mutational effects of PHB polymerase on PHB accumulation was reported by Taguchi et al.83 The assay system consists of a PCR-mediated random mutagenesis and two assay procedures based on plate assay and High Performance Liquid Chromatography (HPLC) assay based on the conversion of PHB to crotonic acid.The level of PHB accumulation, an activity estimation of the R. eutropha polymerase would be efficiently achieved by monitoring the level of PHB accumulation using this in vivo assay system.

7. SUBSTRATE MANIPULATION FOR THE PRODUCTION OF VARIOUS CLASSES OF PHB PHB production by wild as well as recombinant strains consists of a cell growth phase and a PHB production phase. A nutrient-rich medium will be used to obtain high cell mass in the early cell growth phase followed by a PHB production phase, where the cell growth is limited by depletion of some nutrients like nitrogen, phosphorous, magnesium, or oxygen which favor the metabolic shift for biosynthesis of PHB. These biopolymers have been drawing much attention as promising substitutes for chemically synthesized polymers due to their similar mechanical properties to petroleum-derived plastics and complete biodegradability. There are several reports about PHAs consisted of both SCL and MCL (medium chain length) monomer units (SCL-MCL-PHA). The physical properties of PHAs are highly dependent upon their monomer units, and therefore, biodegradable polymers having a wide range of properties can be made by incorporating different monomer units. The monomeric composition of PHB can be engineered using various metabolic engineering and substrate manipulation approaches. The composition of the monomer in a copolymer depends on the hosts PHB synthase as well as on the hydroxyacyl-CoA thioester precursors supplied to the enzyme, which in turn depend on the metabolic pathways operating in the cell and on the external carbon source.The primary objective of metabolic engineering strategy is to include various controlling factors that determine polymer material properties like monomeric composition, chain length, and copolymer microstructure for optimizing yield. Pathway engineering for PHB production

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offers the opportunity to synthesize novel polymers with desirable properties in lowcost, high-productivity fermentations.

7.1 Importance of External Substrate Addition The simplest metabolic engineering approach for the production of PHB copolymers is to manipulate the carbon sources in the culture medium.This strategy has been exploited to modify polymer composition by varying the feed ratio of different substrate precursors. Currently, there have been novel applications of this strategy to generate unusual sulfur-containing polymers. Ewering et al.84 reported a recombinant R. eutropha producing poly (3-hydroxy-S-propyl-o-thioalkanoate) copolymers containing thioether linkages in their side chains when fed with alkylthioalkanoates (thio fatty acids). The intermittent addition of valerate to wild R. eutropha leads to formation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (P (3HB-co-3HV)).

7.2 Manipulation of Carbon Sources Carbon sources play a major role in PHB copolymer biosynthesis. The addition of various carbon sources in different concentration will enable to engineer the polymer with different physical properties. Various microorganisms like R. eutropha, A. latus, and Pseudomonas sp. were reported to produce PHA by the condensation or modification of acetyl-CoAs generated from sugars. Ralstonia eutropha as well as other bacteria are capable of accumulating PHB from sugars except for R. rubrum, where two acetyl-CoA moieties are condensed to form acetoacetyl-CoA by β-ketothiolase (phaA). Acetoacetyl-CoA is reduced to (R)-3hydroxybutyryl-CoA by an NADPH-dependent reductase (phaB). PHA synthase (phaC) finally links (R)-3-hydroxybutyryl-CoA to the growing chain of PHB. In R. rubrum, NADH-linked acetoacetyl-CoA reductase reduces acetoacetyl-CoA to (S)-hydroxybutyrylCoA. Two crotonases convert (S)-hydroxybutyryl-CoA to (R)-hydroxybutyryl-CoA, and finally polymerized by PHA synthase to PHA. Many Pseudomonas belonging to the rRNA homology group I except Pseudomonas oleovorans, use 3-hydroxyacyl-ACPs generated from fatty acid biosynthesis pathway as precursors for PHA production. The enzyme 3-hydroxydecanoyl-ACP CoA transacylase coded by phaG gene was cloned from Pseudomonas putida will serve as intermediate for fatty acid biosynthesis pathway for PHA production from sugars. The phaG gene present in P. oleovorans cannot synthesize MCL-PHAs since the expression of this gene is suppressed at transcriptional level resulting in no MCL-PHAs synthesis from glucose or gluconate. A schematic representation of bacterial PHB synthesis from glycerol is shown in Figure 17.10. Pseudomonas oleovorans and most pseudomonads belonging to the rRNA homology group I can accumulate MCL-PHAs using 3-hydroxyacyl-CoA intermediates of β-oxidation pathway when grown on various alkanes, alkanols, or fatty acids. Aeromonas sp. utilize β-oxidation pathway to supply PHA precursors, especially 3HB and

Microbial Poly-3-Hydroxybutyrate and Related Copolymers Bacterial cell

glycerol NAD+ NADH2 Dihydroxyacetone ATP ADP Dihydroxyacetone phosphate ATP ADP

NAD+ NADH2

Phosphoenolpyruvate ADP ATP Pyruvate CO2 Acetyl-CoA

PHB granule (R)-3-Hydroxyacyl-CoA

Acetoacetyl CoA reductase Acetoacetyl-CoA β-ketothiolase

PHB synthase

Figure 17.10 Schematic representation of bacterial poly-3-hydroxybutyrate synthesis from glycerol.

3HHx monomers from various fatty acids. The composition of the PHA formed by pseudomonads is highly dependent on the structure of the carbon substrate especially when the PHA precursors are supplied from β-oxidation pathway. Generally, the chain length of monomers in PHAs is the same as that of carbon sources or shortened by 2, 4, or 6 carbon atoms. The three putative enzymes involved in providing hydroxyacylCoA from β-oxidation pathway include hydratase, epimerase and 3-ketoacyl-CoA reductases. Tsuge et al.85 reported cloning of the (R)-specific enoyl-CoA hydratase genes from Aeromonas caviae and P. aeruginosa, revealed that hydratase will supply 3-hydroxyacylCoAs from β-oxidation pathway. Two expression plasmids for phaJ1 (Pa) and phaJ2 (Pa) were constructed and introduced into E. coli DH5α strain. The recombinants harboring phaJ1 (Pa) or phaJ2 (Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities for SCL enoyl-CoA or MCL enoyl-CoA. The coexpression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14–29 wt% of cell dry weight from dodecanoate as a sole carbon source. This study revealed that phaJ1 (Pa) and phaJ2 (Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle. The recombinant E. coli harboring R. eutropha PHA biosynthesis genes could accumulate PHB from glucose; E. coli has been metabolically engineered to produce various PHAs. The general metabolic pathway for the production of PHB from fatty acids is shown in Figure 17.11.

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Industrial Biorefineries and White Biotechnology RCH2CH2COOH acyl-CoA Synthetase

HS—CoA H2O

RCH2CH2CO—SCoA (Acyl-CoA) acyl-CoA dehydrogenase

FAD FADH2

RCH:CHCO—SCoA (trans-2,3-dehyroacyl-CoA) H 2O

enoyl-CoA hydratase R H

OH

CO—SCoA (L-3-hydroxyacyl-CoA)

3-hydroxyacyl-CoA epimerase R H

CO—SCoA (D-3-hydroxyacyl-CoA)

OH

PHA Synthase R

H

O

O

n

Figure 17.11 Poly-3-hydroxybutyrate synthetic pathway from fatty acids.

7.2.1 Properties of PHB due to Carbon Manipulation The material property of PHB is similar to polypropylene but its high melting temperature of 170 °C makes the processing of PHB difficult. P (3HB-co-3HV) copolymer is less stiff and tougher and shows higher elongation to break and reduced melting point ranging from 160 °C to 100 °C based on polymer composition (0–25 mol% 3HV). MCL-PHAs can be used as a biodegradable rubber and coating material due to a much lower crystallinity and higher elasticity. PHAs consisting of both SCL- and MCL-monomer units have been produced by several bacteria. Among those, poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (P (3HB-co-3HHx)) and poly (3-hydroxybutyrate-co-3-hydroxyalkanoate) (P (3HB-co3HA)) have the superior mechanical properties similar to LDPE depending on the monomer composition. The superior properties of SCL-MCL-PHA copolymers are attracting various industrial applications.

7.3 Effect of Nitrogen and Phosphorus Wang and Lee86 reported a nutrient limitation strategy to increase PHB content for the development of economically attractive process. Among the various nutrient limitation


condition including N, P, Mg, and S, nitrogen limitation was found to be the best strategy since it allowed the greatest enhancement of PHB production. Nitrogen-limited conditions lead to an increase in residual cell concentration. During fed-batch culture of A. latus, two feeding strategies during nitrogen limitation were applied.87 Since the DOC response was more sensitive than the pH response on the carbon depletion, the DO-stat was used during actively growing stage. After nitrogen limitation, feeding strategy was changed from the DO-stat method to the optimally determined feeding profile due to the no apparent DO increase upon carbon depletion under nitrogen-limited condition.The feeding profile was determined experimentally by calculating the sucrose consumption rate during the nitrogen- limited period. Nitrogen limitation was applied at a cell concentration of 76 g/L, which has been suggested to be optimal for both high cell and PHA concentration from fedbatch cultures of R. eutropha. Sucrose concentration was maintained within 5–20 g/L. After 8 h of incubation under nitrogen limitation, cell concentration, PHB concentration, and PHB content reached were 111.7 g/L, 98.7 g/L, and 88 wt%, respectively, resulting in the productivity of 4.94 g/L/h.The highest productivity of 5.13 g/L/h was obtained at 16 h.86

7.4 Inhibitor Addition PHA synthesis can be altered by the addition of inhibitors. These compounds have particularly useful for incorporating MCL monomers derived from β-oxidation into PHAs. Acrylate was used to inhibit β-oxidation in wild-type R. eutropha during growth on octanoate so that the bacteria accumulated a copolymer of 3-hydroxypropionate, 3HB, 3-hydroxyhexanoate, and 3-hydroxyoctanoate (P (3HP-co-3HB-co-3HHX-co-3HO)) containing both SCL and MCL monomers.

8. APPLICATIONS 8.1 Medical 8.1.1 Targeted Drug Delivery Arsenic trioxide loaded biocompatible PHB–PVA1 nanoparticles (60,000 Da), and dextran derivatives such as dextran sulfate and cationic dextran are the most commonly used in cosmetic industry. Dextran derivatives form a thin film by adsorbing to skin or hair and moisturize. Dextran sulfate is manufactured by sulfonation of dextran while cationic dextran is salt of dextran with anionic or amphoteric surfactants. Dextran can be used to formulate the novel cosmetic delivery agent in modern cosmetic formulations. Dextran-70 has been reported to be used in formulation of bioadhesive patches for wrinkle reduction and facial augmentation.114 Dextran sulfate was used in preparation of biocompatible polymer layer for self-standing cosmetic sheet, which is a novel delivery method for active ingredients to skin. Benefit of cosmetic sheet is overall skin improvement, as it can absorb sebum, control perspiration and odor of skin.115 Dextran sulfate has blood flow promoting activity as it easily penetrates the epidermis and improves nitric oxide synthesis in the keratinocytes, which makes it useful in skin and hair cosmetics.116 In hair cosmetics, apart from improving blood flow, dextran also stabilizes the alcohol in oil type emulsion and provides ease of application.117 Cationic dextran such as dextran hydroxypropyl trimethyl ammonium chloride is used in rinse-off hair conditioner which

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protects, nourishes, and improves the appearance of hair.118 Carboxymethyl dextran is a well popular dextran, which is prepared by substituting one or more hydroxyl group of dextran with carboxymethyl group by treating with carboxymethylating agent in alkaline solution. Native or carboxymethyl dextran can be used to coat body powder in which dextran leaves a protective coating on skin and provides a smooth and matte finish.119 3.5.3 Gellan Gum Gellan gum is an exopolysaccharide of glucose, glucuronic acid, and rhamnose with a molecular weight of 5 × 105 Da produced by Sphingomonas elodea. A unique property of gellan gum is its thermostability and viscosity of the solution remains stable over a wide range of temperature. Gellan gum is widely used in shampoos, body washes, sprayable sunscreens, etc. Gellan gum acts as emulsion stabilizer by preventing the separation of oil and water. In cosmetic formulations, gellan gum acts as suspending agent by stabilizing the emulsion and keeping it uniform against temperature fluctuations. Gellan gum is a gelling agent, but not a thickening agent as it does not change the rheology of solution as efficient as the other gums. Because of this property, gellan forms a thin gel that makes it useful in shampoos and body washes. Apart from that, gellan gum gives a nice sensation on application to skin. Structural modification of gellan gum changes the functional properties and its performance. A completely deacylated gellan gum forms a rigid brittle gel, while gellan gum of low acyl and high glyceryl forms an elastic gel.120A hair styling gel had been formulated with partially deacylated gellan gum which gives a good hold and fix to hair throughout the day.121 3.5.4 Welan Gum Welan gum is an exopolysaccharide produced by Alcaligenes species. It has similar structure of gellan with additional side chain of l-rhamnose or mannose. Like gellan, it is stable over a wide range of temperature (up to 150 °C) and pH (2–12). It exhibits increased viscosity at low shear rate. By altering the metabolic flux of pathway of welan production by pH control process, welan production reached a maximum of 25 g/L.122 In cosmetic formulations, welan serves as rheology modifier and emulsion stabilizer. Like gellan gum, it forms a film and promotes the slow absorption of active ingredients. Though it is safe to use, it is still not reported to be used in any personal care products.123 3.5.5 Rhamsan Rhamsan gum is a polysaccharide produced by Alcaligenes and Sphingomonas species. It has the same backbone structure of gellan with disaccharide side chain of β-d-glucose. The advantage of using rhamsan over the other gums is its low concentration that is

White Biotechnology in Cosmetics

sufficient to achieve the desired viscosity. Viscosity remains stable over a wide range of temperature and pH. Rhamsan gum exhibits high suspension capability than plant-based gums and can be used to deliver active ingredients to skin or mucosa in cosmaceuticals.124 Rhamsan can be used as thickener in creams, lotions, and other cosmetic products. It was reported to be biocompatible with other additives such as antitanning or skin cleansing compounds.125,126 It was also used to produce nonallergic cosmetic adhesive or latex for preparation of false eyelashes.127 Structural modification of rhamsan gum with a nonpolymer siloxane graft made it an excellent conditioner for damaged hair.128 Rhamsan gum was used as a gelling agent in cosmetic gel preparations used for coloring skin and hair.129 3.5.6 β-Glucan β-Glucans are heterogeneous group of polysaccharides produced by a variety of sources such as bacteria, fungi, and plants. Depending on the source and extraction process, they vary in their structural and functional properties. β-Glucan from yeast contains β-1,3 and β-1,6 glycosidic linkage and has high biological activity than the plant-derived glucans. They are produced commercially from baker’s yeast Saccharomyces cerevisiae. Among higher fungi, L. edodes and Pleurotus ostreatus are important sources of β-glucan. Nonspecific immune stimulation is the most important biological activity exhibited by β-glucan. Apart from the biological activity, water holding, oil binding, and rheological properties of β-glucan make it an effective ingredient in cosmetic composition. Petravić Tominac et al. elaborately discussed cosmetic applications of β-glucan. It can be used as thickener and emollient in cosmetic preparation. It can also be used as one of the photoprotective agent in sunscreen lotion, because of its free-radical scavenging activity and ability to induce epidermal macrophage action that results in skin protection. β-Glucan is an anti-irritant in lactic acid-based skin creams. It was reported for its hair growth promoting action by activating hair follicle and promoting hair regeneration. β-Glucan acts synergistically with antiaging compounds, which helps in rejuvenating skin and reduces the visible signs of aging.130 Carboxy methyl (CM) glucan is the modified yeast glucan with improved water solubility. Structural modification improved the functional properties of glucan, making it highly useful in cosmetic formulation. Topical application of CM glucan offers substantial protection from UV rays by inhibiting the depletion of antioxidant molecule on skin. CM glucan also accelerates skin recovery by promoting the growth of keratinocytes.131 β-1,3 d-glucan produced mainly by soil-dwelling bacteria includes linear β-(1,3) glucan called curdlan, side-chain-branched β-(1-3,1-2) glucans, and cyclic β-(1-3,1-6) glucans. Curdlan is mainly produced by Agrobacterium species while the rest of the β-1,3 d-glucan were produced by Bradyrhizobium species.132 Curdlan exhibits unique gelation behavior and viscoelastic properties on the basis of concentration and temperature of

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setting. Aqueous dispersion of curdlan forms a thermo irreversible elastic gel by heating above 80 °C.133 A hair dressing cosmetic preparation was formulated with curdlan as hair gelling polymer. Curdlan when applied to hair becomes gel on blowing with hot air of temperature 60–80 °C, which helps to set the hair leaving a smooth finish.134 Curdlan can also be used along with other hydrocolloids in thickening compositions for cosmetic purpose and gives rise to good skin feeling and nonstickiness.135 3.5.7 Schizophyllan Schizophyllan is a nonionic polysaccharide produced by submerged fermentation of basidiomycete Schizophyllum commune. Structurally, it is glucan having β-(1-3)-linked backbone with β-(1-6)-linked glucose side chains. Schizophyllan exhibits unique rheological characteristics depending on the concentration of polymer and shear rate. Schizophyllan exhibits Newtonian behavior up to certain critical shear rate and then follows a shear thinning behavior i.e., critical shear rate decreases with increasing concentration of polymer.136 Schizophyllan can be used as stabilizer and rheology modifier in cosmetic preparations as it stabilizes the oil-in-water emulsion along with surfactants. Schizophyllan is available in market as mushroom β-glucan (MC-Glucan) which is used as antiinflammatory agent in sunscreen lotion. Schizophyllan can alleviate skin irritation and also enhances skin immunity.137 3.5.8 Scleroglucan Fungi, Sclerotium species produce nonionic exopolysaccharide called scleroglucan which has a structure similar to schizophyllan. High thermal stability, remarkable rheology, and compatibility are interesting characteristics of scleroglucan. It exhibits shear thinning behavior with good yield value and is an effective suspending and stabilizing agent. Scleroglucan is quite compatible with surfactants, thickening agents, and all other ingredients in cosmetics. It has good water retention property along with light skin feel. All this properties make it an effective ingredient in personal care products.138 3.5.9 Levan Levan is a heterogeneous class of fructan polymer obtained from plants and microbes. Microbes belonging to genera Bacillus, Streptococcus, and Corynebacterium were reported to produce levan.139 Because of the unique backbone, levan exhibits different functional properties that distinguish it from other biopolymer. As it has low intrinsic viscosity and does not swell in water, levan is not an ideal thickening or gelling agent; instead, it can be used to give a smooth finish to the product with a good skin feel.140 Kim et al. discussed the cosmaceutical activities of levan produced by Zymomonas mobilis. Levan exhibits excellent moisturizing activity same as that of hyaluronic acid. Levan has anti-inflammatory activity that reduces skin irritation and along with its cell proliferative activity, it can be used in formulation of cosmaceuticals for photo-protection of sensitive skin.139,141


3.5.10 Pullulan Pullulan is a water-soluble glucan gum produced by black yeast Aureobasidium pullulans. Hayashibara, a major commercial supplier of pullulan, produces it through aerobic fermentation of starch syrup. Pullulan can increase viscosity of the solution without forming gel. Pullulan can be used as thickener or binding agent in cosmetic compositions. Pullulan is a good adhesive agent with film forming property and could be used to provide an instant skin tightening effect in antiaging creams. In cleansing products, it gives the smooth texture, retains the foam, and helps in easy rinse off with water. Hence, it can also be used to manufacture face masks or facial packs that can be washed away with water easily.142,143 3.5.11 Chitin and Chitosan Chitin is a structural polysaccharide found in the species such as yeast, fungi, insects, and marine invertebrates. Structurally, Chitin is a homopolymer of N-acetyl glucosamine with β-(1,4) linkages, while chitosan is deacetylated chitin. Chitin is produced commercially from shellfish waste through chemical treatment. Since chitin extraction using chemical process is energy-consuming and generates environmentally toxic waste, alternative treatments using microbial enzymes are recommended. Chitosan is produced from chitin by enzyme chitin deacetylase from fungi such as Aspergillus nidulans. Mucor rouxii was found to have chitosan in its cell wall at significant level which makes it potential source for chitosan extraction.144 Chitin and chitosan are widely used in skin and hair care products.They are excellent hydrating agents which moisturize and protect the skin from drying. Both chitin and chitosan have film-forming ability, which makes them a good carrier in cosmetics and fix ingredients on skin for long-lasting effects.145 Modified chitin such as chitin nanofibril can increase fibroblast turnover with improved collagen synthesis that can be implicated in scar-reducing cosmetic preparations.146 Chitin–glucan complex, extracted from the A. niger mycelium was well tolerated by skin and was used in skin care cream for improved skin hydration and to diminish signs of skin aging.147 3.5.12 Hyaluronic Acid Hyaluronic acid (HA) is the high-cost biopolymer widely used in cosmetics. HA is a polymer of N-acetyl glucosamine with alternate β-(1,3) and β-(1,4) glycosidic linkages. In aqueous dispersion, even low concentration of HA behaves in non-Newtonian manner due to its random coil interaction between molecules. HA forms a stable gel at the concentration of above 1.5%. As HA is abundant in animals, animal source like rooster comb can be used to extract it. Disadvantage in commercial production of animalderived HA is stringent downstream processing to remove proteoglycans attached to HA and other potential contaminants such as prions. In this scenario, microbial production of hyaluronic acid is gaining attention. It is secreted as extracellular capsule material by

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Streptococci species. Streptococcus equi is a commercially used microbe to produce hyaluronic acid using complex medium.148 Vazquez et al., proposed a seafood by-products-based culture medium for the production of hyaluronic acid by Streptococcus zooepidemicus. The medium contains mussel processing waste as carbon source and tuna peptone as protein substrate and thus can be a cheap alternative to synthetic peptone-based medium.149 Now, genetically modified strain with high productivity of hyaluronic acid is in place to meet the challenges with conventional fermentation. Because of the excellent water holding capacity and viscoelastic properties, hyaluronic acid can be considered as highly effective humectants to hydrate the skin. Since the natural function of hyaluronic acid is to protect and repair the skin, the topical application of HA is found to have scar repair, skin rejuvenation, and hair regeneration functions. Hyaluronic acid is well reported in antiaging cosmetic composition as it easily penetrates the skin, softens the skin, and restores the skin elasticity. Currently, hyaluronic acid-containing dermal fillers are in market which gives instant face lift and youthful look to the aged skin.150,151 Topical application of hyaluronic acid can regulate the molecular mechanisms such as hyperproliferation, growth factor production, and extracellular matrix deposition in the processes of wound healing. Hyaluronic acid can be used to treat acne and keloid scarring and helps to give an even skin contour.152,153

3.6 Polypeptides and Proteins Advances in skin care and aesthetic science promoted the inclusion of many polypeptides and proteins in cosmetic formulation for their superior skin conditioning properties. Some widely used polypeptides and proteins are described below. 3.6.1 λ-Polyglutamic Acid λ-Polyglutamic acid (PGA) is an anionic biopolymer produced by Bacilli species and its commercial production is a well-established fermentation process. Unique water absorption ability, biocompatibility, and biodegradability made PGA useful for hydrogel preparation that has been used for controlled release of drugs and artificial scaffold materials.154 Because of the excellent water holding capacity, PGA can be used in moisturizing formulations to increase the wettability of skin, decrease transepidermal water loss and helps in protecting the skin from drying. Apart from moisturizing, PGA hydrogel improves skin elasticity and gives a light and smooth skin feel with long lasting effectiveness.155,156 Dermal fillers containing PGA were reported to be used to smoothen away wrinkles and other aesthetic defects. PGA-based dermal fillers can be an inexpensive solution to hyaluronic acid or collagen-based dermal fillers. As PGA is quite inert and biocompatible, PGA-based cosmetic formulation was found to be less allergic than hyaluronic acid- or collagen-based cosmetic composition. Another important cosmaceutical property of PGA is its skin whitening effect via antimelanogenesis by inhibiting tyrosinase enzyme.157,158


3.6.2 Collagen Collagen is one of the structural proteins in the extracellular matrix and exists as elongated proteins in skin. Collagen has triple helix conformation with inter- and intramolecular hydrogen bonding and disulphide linkages. The abundant amino acids present in collagen are glycine, proline, and hydroxylproline, out of which hydroxylproline protects the collagen from proteolytic attack.159 The role of collagen in skin is to provide tensile strength and elasticity. As the age advances, skin shows wrinkles and sagginess due to the reduced synthesis of collagen, which is correlated to fibroblast aging and loss of mechanical properties of collagen.160 Many exogenous factors such as smoking, stress, and administration of steroids can also affect the collagen level as the hormone cortisol can degrade collagen. Commercial grade collagen is mainly derived from animal source such as bovine and chicken through different preparatory processes. It can also be derived from human, only through donors. Collagen extracted from bovine lime split waste through pepsin digestion exhibits the same triple helix conformation and mechanical properties of native collagen.161 Since mainly animal source is being used, production should meet stringent manufacturing process and should pass good quality assurance. Extracted collagen product should be pure, free of all viral or prion contaminants, and exhibit the same mechanical properties of native collagen.162 An attractive alternative for expensive production of collagen from animal source is fermentation technology.Yeasts such as S. cerevisiae, Pichia pastoris, Hansenula polymorpha and bacteria such as E. coli and Bacillus brevis were reported as recombinant hosts for the production of human collagen, gelatin, and hydroxylated collagen. Out of these, recombinant P. pastoris is the highest producer, as it can accumulate recombinant collagen close to 1.5 g/L.163 Collagen is used as dermal filler to reduce wrinkles, correct acne scars, and for lip augmentation. The advantage of using recombinant collagen over bovine collagen is its no allergic response and long-lasting effect contributed by its high molecular weight.164 Collagen is a good humectant because of its water holding capacity and hence can be used in formulation of moisturizing creams. Since it is a high-molecular weight compound, it cannot penetrate through the skin, but can increase the skin hydration by staying on the skin surface.165 An in vivo study showed that topical application of collagen like peptide had excellent antiaging properties by reducing the intensity of wrinkles.166 Craven et al. reported that wrinkle formation in photodamaged skin is correlated with the depletion of collagen VII, which leads to weakened dermal and epidermal junction integrity.167 Topical applications of compounds such as retinoic acid can restore the collagen formation and thus reduce wrinkles.168 Though many cosmetic products containing collagen are in market, the skin rejuvenation effect by topical applications of collagen is still questioned due to the limited penetration of high-molecular weight compounds through stratum corneum.

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3.6.3 Keratin and Keratin Hydrolysate Keratin is the group of structural protein present in skin, hair, nail, feather, and wool. Keratin is a very robust and inert biopolymer. Structurally, it is a superhelix comprised of α helix and β pleated polypeptides linked by hydrogen bonds and disulphide bridges. Animal waste from poultry industry, meat processing and fur processing industry are usual commercial sources of keratin. Keratin is used in large quantities in feed industry, cosmetic industry, fertilizer industry, cement industry, and ceramic industry.169 Keratin is widely used in hair care products. Keraplast Technologies, a leading keratin supplier makes use of keratin derived from pure New Zealand wool. On applying to damaged hair, keratin binds strongly, helps to rebuild the keratin, and thus repairs the damaged hair. It also protects and conditions the hair giving a healthy luster and shine to hair. Keratin hair treatments are available to treat frizzy hair and give smooth silky hair.170 Aesthetic procedures such as coloring, straightening, and permanent waving are the causes of hair damage. Harsh chemical treatments during these aesthetic procedures result in damage to cuticle layer, loss of tensile strength and hence hair becomes susceptible to breakage, which together results in unmanageable, dry, and dull-looking hair. Application of keratin restores the mechanical properties and moisture content of the hair, which were diminished due to harsh treatment of hair.171 Keratin hydrolysates are produced by microbial degradation of keratin-rich material such as poultry feathers.Though many bacteria, actinomycetes, and fungi are reported to produce keratinase enzyme, B. subtilis and Bacillus licheniformis are only commercial feasible organisms due to their high enzyme titer and efficient utilization of poultry waste such as feathers. Versazyme is a commercially available keratinolytic enzyme from BioResource International, Inc, and is used in manufacture of animal feed.172 Microbial or enzymatic-derived keratin hydrolysate is more suitable as a cosmaceutical agent than chemical-derived keratin hydrolysate as it contains low-molecular weight proteins and peptides. Keratin hydrolysate produced by B. subtilis is used in shampoos and rinse-off conditioners. In this composition, keratin hydrolysate penetrates deep into hair cuticle, results in sealing of cuticles, and improves the hydration of hair, which overall leads to improved health and appearance of hair.173 High-molecular weight keratin poypeptides are proposed as hair setting or fixating agents.174 The mechanism of action is by forming molecular bridges between cystein residues of keratinaceous film and hair follicle due to drying and stabilization of the film where hair can be set in the desired way.175 The use of keratin hydrolysate in eye lash makeup composition not only improves the appearance of eye lashes but also helps to lengthen and strengthen the eye lashes.176 3.6.4 Botulinum Toxin Introduction of botulinum toxin is a major breakthrough in the area of antiaging cosmaceuticals and lead to increased consumer interest in the microbial products as miraculous cosmetic ingredients. Botulinum toxin is a dipeptide of 150 kD size produced by


Clostridium botulinum, which can cause fatal paralytic poisoning called botulism. There are seven types of toxins designated as type A, B, C, D, E, F, and G produced by different strains of C. botulinum. The potential use of botulinum toxin as a wrinkle-correcting agent was discovered by Jean Carruthers and Alastair Carruthers. They reported that C. botulinum A exotoxin can be used to treat glabellar frown lines with lasting effect from 3 to 11 months.177 Then after, a tremendous growth in usage of botulinum toxin happened in antiaging therapy and other aesthetic procedures. Injection of botulinum toxin to the skin inhibits the acetylcholine release at the neuromuscular junction and suppresses the passage of signals from nerve to muscle. This overall leads to the paralysis of facial muscle and eliminates the wrinkles. The paralyzed muscle will not be able to contract which results in the relaxation and smoothening of wrinkles. Botulinum can eliminate all form of wrinkles such as crow’s feet, brow furrow, smile lines, and frown lines.The treatment is less aggressive and more effective than aesthetic procedures such as dermabrasion and laser resurfacing. The procedure does not require anesthesia and is less time taking. Disadvantages are its reversible effect and possibility of side effects.178,179 Since botulinum is a neurotoxin, large-scale production should meet biosafety level 3 containment and strict handling to avoid the possible human intoxication. Fermentation is carried out in complex medium containing casein hydrolysate, yeast extract, and glucose for 4 days. Then, botulinum toxin is precipitated with 3 N sulfuric acid followed by purification with DEAE-Sephadex column. Botulinum toxin was crystallized then and purified by additional steps using various affinity and size exclusion chromatography. Purity and quality were checked by animal testing. Production and purification steps vary with Clostridium strain used and botulinum type to be produced.180 There are different commercially available botulinum toxins in different names (Table 18.5). Out of that, Botox is the most popular one which is usually referred generically instead of botulinum.Type A is commonly used as a wrinkle reducing agent as its effect lasts longer than that of type B. Still type B is preferred as it gives instant result. Type B botulinum was also recommended in the case, where chronic regular usage of type A results in immune response in the users.178,181 Table 18.5 List of commercially available botulinum brands with the type of botulinum toxins and manufacturers Product Botulinum type Manufacturer

Botox® Dysport® Myobloc® Xeomin® Neurobloc®

Type A Type A Type B Type A Type B

Allergan Inc., USA Ipsen Ltd, UK Elan Pharmaceuticals Inc., USA Merz Pharmaceutical, USA Solstice Neurosciences Inc., USA

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3.6.5 Human Growth Factor Human growth factors are considered as the sensational molecules in cosmetic industry due to their tremendous skin care activities and are being routinely used in all high-end cosmetics. Human growth factors are group of proteins that mediate various cellular activities in human body. There are different types of growth factors and their function varies with the cell type and cellular process. Growth factors have important role in cell differentiation, tissue morphogenesis, angiogenesis, and wound healing, thus playing an important role in maintaining homeostasis. Topical application of individual growth factor is proved to promote wound healing on skin. Brown et al. reported that epidermal growth factor (EGF) increased the epidermal regeneration and enhanced the rate of healing in partial thickness skin wounds.182 According to Fitzpatrick, a photodamaged skin behaves like a chronic wound. The treatment of photodamaged skin includes removal of damaged skin and regeneration of new epidermal and dermal layer. Human trial with the multiple growth factor derived from fibroblasts showed its effectiveness in treating photodamaged skin by increasing the collagen formation and epidermal thickening.183 Growth factor can also be used for skin rejuvenation or reversing the cutaneous aging. As the endogenous titer of growth factor is decreased due to reduced skin cell turnover during skin aging, exogenous supplementation of growth factor can promote skin repair and revitalize the skin. A combination of growth factors along with antioxidants, matrix building agents, and skin conditioning agents can be effective in skin rejuvenation treatment as this combination can mimic physiological condition of the skin. A cosmetic composition based on this concept was launched in market which is called TNS (Tissue Nutrient Solution) Recovery Complex System with NouriCel-MD™. This product claimed to promote the disappearance of wrinkles and pigmentation and at the same time improving skin firmness and texture.184 Human growth factors used in cosmaceuticals are mainly produced by biotechnological means.The luxury skin care brand called Revive uses recombinant EGF in their range of skin creams called renewal creams manufactured via renewal epidermal science technology. Revive renewal creams claim to restore the youthful appearance and could be considered as a substitute for the aesthetic procedures that are currently being adopted for the skin rejuvenation.185 Skin Actives, a US-based company markets heterologously expressed EGF in E. coli to use as skin conditioning agent.186 Another source of growth factor is cultured human fetal fibroblasts. 3D in vivo optical skin imaging showed the visible reduction in wrinkles and skin roughness by topical application of skin cream containing human growth factors and cytokines derived from cultured fetal fibroblasts.187 Various growth factors and their functions are explained in Table 18.6.184,188–190 3.6.6 Enzymes The overall objective of applying cosmetics is to stimulate certain biochemical pathways that help in improving aesthetic appearance of skin. Supplementation of enzyme to the


Table 18.6 Important growth factors used in cosmetics and their functions Growth factors Functions

References

Epidermal growth factor (EGF)

Wound healing, regulates collagenase secretion Regulate wound epithelization Stimulate wound healing and modulate ECM remodeling Stimulate wound healing, angiogenesis

182

Fibroblast proliferation Mitogenesis, regulate inflammation and collagen synthesis Endothelial cell and fibroblast mitogenesis Mitogenesis and wound healing

184 184

Keratinocyte growth factor (KGF) Transforming growth factor beta (TGF-beta) Vascular endothelial growth factor (VEGF) Fibroblast growth factors (FGF) Platelet-derived growth factors (PDGFs) Insulin-like growth factors (IGFs) Hepatocyte growth factor (HGF)

188 189 184,190

184 184

cosmetic preparation that can promote a specific biochemical pathway is beneficial and leads to improved performance than the original cosmetic preparation. There are three kinds of enzymes mainly used in cosmetics—protease, lipase, and superoxide dismutase. 3.6.6.1 Protease

Protease degrades the protein into peptides and amino acids. In skin care products, proteases are mainly targeted to promote exfoliation, which is shedding off superficial horny keratinized layer and to increase the absorption of water and other ingredients in the cosmetics. Protease can also be used in dermal peels preparation. In hair, excessive protein deposit can be removed by applying the composition containing protease to make the hair smooth. Bromelain, papain, chymotrypsin are the examples of herbal proteases used. Seki et al. reported that subtilisin, a serine protease produced by B. licheniformis is an effective skin exfoliator. Subtilisin-based dermal peel is safe, less harsh, and more efficient than α hydroxy-based chemical peels.191 Stratum corneum thiol proteases mediate desquamation (cell dissociation) and degradation of desmosomes on skin. Because of its human skin origin and effectiveness, stratum corneum thiol proteases can be used as ideal skin care agents to alleviate dry skin and acne. Commercial prospective of stratum corneum thiol proteases is possible via recombinant DNA technology by producing them in heterologous host.192 3.6.6.2 Lipases

Lipases are involved in breakdown of lipids. Excess oil buildup on face is a major skin condition that causes acne. Oily skin will not be aesthetically appealing as the makeup applied will not stay for a long time. Application of lipase onto skin will remove the excess oil buildup and results in skin balancing. Ansorge-Schumacher et al. discussed the cosmetic applications of lipases. Lipases are used as cleansing agent to remove dirt and

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shed off dead skin cells. Lipases can also be used in anticellulite treatment and body slimming product where it aids the breakdown of fat deposits. Functional perfumes using the lipases were proposed for the controlled release of active compounds and slow release of scent.193 Commercial source of lipids are fungi like A. niger, Mucor miehei, and various Rhizopus species. 3.6.6.3 Superoxide Dismutase

Superoxide dismutases (SODs) prevent free radical damage caused by photoirradiation and harmful pollutants. Due to antioxidant activity of SODs, topical application of SODs protects the skin from aging and photodamage. SOD improves the cellular respiration, maintains the integral keratinous structure, promotes skin elasticity, and gives a smooth feel to skin.194 Currently cosmetic products use SODs from plant source. A eukaryotic expression cassette containing SOD gene under SV40 promoter was introduced to human HeLa cell and mouse L cell for production of SOD, which can be commercially viable technology when compared to laborious extraction and purification of SODs from plant source.195 A synergistic action of yeast SOD and fennel peroxidase can give a full spectrum free-radical scavenging activity, which results in reduction of skin redness and erythema associated with skin exposed to solar radiation.196 Apart from the above mentioned enzyme, there are many enzymes used in cosmetics. Hyaluronidase, a hyaluronic acid-degrading enzyme is produced by bacteria belonging to the genera Staphylococus, Streptococci, and Clostridia. Hyaluronidase is used as dermal corrector to erase off excess dermal filler during dermatological procedures. Human placental alkaline phosphatase, which promotes the cell survival and proliferation of damaged skin tissue, can be a potential candidate to revitalize aged skin.197 Biotechnological production of human placental alkaline phosphatase using recombinant DNA technology can be considered as a safe and ethical choice for use of placental-derived enzyme.

3.7 Essential Fatty Acids, Sterols, and Lipid Derivatives 3.7.1 Polyunsaturated Fatty Acids Fatty acids are the integral part of skin and help to maintain a healthy and youthful skin. Topical application of fatty acids, especially polyunsaturated fatty acids (PUFA) has been proved to have potential impact on skin and hair. Primary role of fatty acids in cosmetic composition is its moisturizing function. Fatty acids function as emollients, which on application to skin form a hydrophobic layer and prevent the water loss from skin, eventually reduce the skin dryness and make the skin smooth and soft. Apart from that, PUFA are powerful antioxidants and can be used to alleviate oxidative damage associated with sun exposure. They were reported to inhibit collagen breakdown and eliminate aging skin symptoms like wrinkles.198 Fatty acids mediate various cell signaling pathways and regulate various biochemical processes on skin. ω-3 fatty acids are involved in synthesis of prostaglandin, which


regulates inflammatory responses. Hence, ω-3 fatty acids can be used as anti-irritating agent in harsh aesthetic procedures such as chemical peel. It can also be used as soothing agent in night creams to reduce photo-induced inflammations and other damages e.g., γ-linolenic acid.198 ω-3 fatty acids and ω-6 fatty acids are reported to be produced by fungi and marine microbes. Marine microalgae are commercially used for the production of PUFA, as they can accumulate very high level of PUFA. Metabolic engineering by inactivating peroxisome gene resulted in high eicosapentaenoic acid yield by the organism Yarrowia lipolytica.199 Docosahexaenoic acid (DHA) is commercially produced by Crypthecodinium Cohnii, a marine red algae by heterotrophic cultivation, where 30% of the total lipid accumulated is DHA.Various cosmetic products such as face mask, bathing solution are available with the extract derived from C. Cohnii.200 Xiamen Huison Biotech Co., Ltd, a commercial producer of DHA is using the algae Schizochytrium, which can accumulate nearly 25% DHA/g biomass.201 3.7.2 Squalene Squalene is a widely used ingredient in cosmetics due to its multiple skin and hair actions. Squalene is an isoprenoid present in cell membrane of animals. Shark liver used to be a major source of this hydrocarbon. Since deep sea shark faces extinction due to large-scale hunting for the extraction of squalene, now squalene production from plants or microbes have been encouraged. Marine microalgae Schizochytrium mangrovei and Aurantiochytrium mangrovei and yeast such as S. cerevisiae, Torulaspora delbrueckii, Pseudozyma sp. JCC 207 were reported to produce squalene. A recombinant E. coli strain with high yield of squalene (11.8 mg/L) was engineered by heterologous expression of hopanoid genes from Streptomyces peucetius along with modulating methylerythritol 4-phosphate pathway.202–207 Important properties of squalene are its lubricating activity and its oxidative stability. Squalene is an excellent emollient; it acts by penetrating deep into the skin and hydrates the skin. Since it has free-radical scavenging activity, it can stabilize the cosmetic composition and thus increase the shelf life of products.208 The biological function of squalene is to protect the skin from oxidative damage. Topical application of squalene suppresses singlet O2 production which is generated on exposure to solar rays and thus reduces the skin irritation. So, squalene can be used in sun protection lotion. Squalene is used to formulate lipid emulsions and nanostructured lipid carriers, which are the vehicles to deliver active components to skin in cosmetic dermatology.209 Because of the antimicrobial property, a composition containing squalene can be used to treat acne, skin inflammation, and minor skin wounds. A derivative of squalene called squalane, where all the double bonds are saturated by hydrogenation, has better scope of applications than the squalene due to its improved oxidative stability.

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3.7.3 Ceramides Ceramides are heterogeneous class of lipids present in stratum corneum of the skin epidermis. Structurally, they are sphingolipids where fatty acid is connected to sphingosine via amide linkage. Along with other lipids present in stratum corneum, ceramides form a highly ordered lamellar structure which is involved in skin barrier functions. Ceramides are involved in regulating transepidermal water loss through skin. Decrease in ceramide content or change in ceramides pattern can lead to various skin disorders. Skin appearance can be improved by topical supplementation of ceramides or any agent that can promote the lipid synthetic capability of epidermis.210 Ceramide-containing skin care and hair care products are the latest trends in cosmetic field. Though plants and animals are good source of ceramides, they are not a viable source as the extraction process is laborious and not so economical. So the ceramide used in cosmetic field are synthetic, hence many new ceramide derivatives are present in market. Some companies use fermentation technology to produce ceramides. Yeast such as Wickerhamomyces ciferrii (formerly known as Pichia ciferrii), S. cerevisiae KCCM 50,515, Yarrowia lioplytica were reported to produce ceramides. Evonik Industries AG personal care, markets comoferm, a ceramide solution. It employs a patented fermentation technology to produce phytosphingosine that can be converted to a range of human skin identical ceramides. Genetically modified yeast strain with improved productivity of sphingoid base was also reported.211–213 Ceramide is a high value cosmaceutical ingredient and is active at very low concentrations.Topical application of ceramides moisturizes the skin and hair and protects them from drying. The hair which has been sensitized by aesthetic procedure such as straightening shows some undesirable appearances such as lack of luster and will turn unmanageable due to loss of ceramides and proteins from hair strands. Shampoos, conditioners, and hair serums containing ceramides are used to treat this damage, where ceramide will go and bind to each hair strand and acts as a barrier that limits the leakage of protein, strengthen the cuticular cohesion, and prevent further damage.214 Determination of skin hydration by Corneometer showed that ceramide gives long lasting hydration. Apart from hydrating, ceramide also has protective function as it causes less transepidermal water loss and decreases the irritation associated with exposure to synthetic surfactants.214 3.7.4 Biosurfactants Biosurfactants are microbial surface active compounds and have tremendous applications in cosmetics due to their high surface activity and multifarious bioactivities. Surfactants are widely used in cosmetics for stabilizing the oil–water emulsion and uniformly dispersing various components.The key feature of surfactants used in cosmetic formulation is their skin friendly nature, which means that the surfactant is safe and compatible with the skin. The advantages of biosurfactants over chemical surfactants are their


biodegradability and eco-friendly nature. The major class of biosurfactants used in cosmetics is glycolipids which include sophorolipids, rhamnolipids, and mannosylerythritol lipids. Sophorolipids are widely used in cosmetic preparation. Sopholiance S, antibacterial agent and sebum regulator is a cosmetic ingredient marketed by a French company called Soliance. It is produced in large scale by fermentation of Candida bombicola in the medium containing glucose and methyl rapeseedate. In vitro studies have shown that soliance can inhibit the growth of Propionibacterium acnes and other acne-causing bacteria. In vivo studies have shown that soliance can regulate sebum secretion and reduce the formation of papule and pustule on skin. It can be used in the preparation of face and body cleanser, deodorant, and as oil control agent to give a good makeup hold and limit skin shine.215 Borzeix et al. reported the antioxidant and antielastase activity of sophorolipids which showed their potential in formulation of antiaging cosmaceuticals and sun protection creams.216 Rhamnolipids can act as effective emulsifier in cosmetic composition and are being used in various skin and hair care preparations. Apart from emulsification, other properties attributed by rhamnolipids are foaming and lubrication. Rhamnolipid-containing formulations can be used for problematic skin, where rhamnolipids control excess oil secretion. Rhamnolipids keep the product uniform by controlling the rheology of the formulation and hydrate the skin.217 Cosmetic composition containing rhamnolipids as active ingredients was proved to reduce signs of aging such as wrinkles. They can be formulated in any form such as liquids, suspension, cream, powder, lotion, or ointment.218 Another potential biosurfactant used in cosmetic industry is mannosylerythritol lipids. Morita et al. reported the excellent moisturizing activity of MEL-A which is comparable to ceramide. Mechanism of action could be the efficient penetration of biosurfactant through the epidermis and improvement in moisture retention by forming liquid crystals. MEL-A can be a potential alternative to ceramides with the advantage of economical production through fermentation technology.219 MEL-A can be an efficient hair care agent since it can repair the damaged hair and improve the mechanical properties of the hair. MEL-A was proposed to be absorbed onto hair, form a lamellar structure, and nourish hair, compensating the loss of hair surface lipid, methyleicosanoic acid during the harsh chemical treatment.220 Kanebo Cosmetics had successfully developed MEL-B-based cosmetic products such as skin lotion and cream. Both water-in-oil emulsion and oil-in-water emulsion can be prepared by MEL-B.221 Bacillus subtilis produces lipopeptide biosurfactant called surfactin which has excellent surface activity. Unlike other biosurfactants, surfactin is highly hydrophilic and forms transparent gel which can be exploited in skin care formulation.221 Emulsan, a high-molecular weight biosurfactant produced by Acinetobacter calcoaceticus, exhibits high emulsifying activity. Emulsan can be used in soaps and shampoos

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preparation as it can act as cleansing or degreasing agent, while improving the cosmetic appearance of hair and skin.222

4. CONCLUSION Personal care industry is mainly driven by innovation in manufacturing and formulation of cosmetics and the potential benefits attributed. Environmental and health impacts of the cosmetic formulations are other key factors that have drawn consumer interest recently. White biotechnology is the economical and eco-friendly solution for the production of bioactive compounds that can replace the traditional synthetic compounds in cosmetic industry and provide a highly efficient skin or hair care functions. Different cosmaceutical functions of the bioactive molecules and production aspects are discussed in this book chapter. However, more research is still needed to explore the potential biomolecules with interesting dermatological function from different microbial genera. A knowledge gap is very clear in the cosmetic science as the possible mechanisms behind different cosmaceutical functions of commonly used biomolecules are still missing.

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CHAPTER 19

Production and Extraction of Polysaccharides and Oligosaccharides and Their Use as New Food Additives Clarisse Nobre, Miguel Ângelo Cerqueira, Lígia Raquel Rodrigues, António Augusto Vicente, José António Teixeira Centre of Biological Engineering, University of Minho, Braga, Portugal

Contents 1. Introduction 653 2. Extraction, Production, and Purification of Polysaccharides and Oligosaccharides 656 3. Food Applications of Polysaccharides and Oligosaccharides 662 4. Health and Nutritional Benefits of Polysaccharides and Oligosaccharides 666 5. Regulation and Safety Aspects 668 6. Conclusions 670 Acknowledgments670 References670

1. INTRODUCTION Carbohydrates can be classified into sugars, oligosaccharides, and polysaccharides. They are identified according to their chemical structure and the nature of the constituent monosaccharides.1 They can be linear or branched with different degrees of ramification. These chemical differences lead to the changes in their solubility, viscosity, gelling behavior, as well as their digestibility. The diverse sources (i.e., plant, microbial, animal, and algal) from which a polysaccharide can be obtained greatly increase these differences, making their identification, characterization, and standardization a challenging task.2a In food industry, polysaccharides can be divided in two main groups: starch derived products and nonstarch polysaccharides. Starch and their derivatives without any doubt lead the polysaccharides type used in the food industry. Due to their high availability, nutritional and technological properties, starches are in the genesis of a great number of food products known worldwide.2,3 Starches can present many differences according to their origin, preparation method, source of the crops and possible genetic modification, molecular weight and composition, influencing their properties when applied as a food ingredient, or Industrial Biorefineries and White Biotechnology http://dx.doi.org/10.1016/B978-0-444-63453-5.00021-5


653

654


additive. Obtained from the corn, wheat, potato, and rice (ref. Chpt. 4A), commercial sources of starch are composed mostly by amylose and amylopectin in a ratio of approximately 1:3.4 Modified starches appear to overcome some of the limitations of starches in their natural form, enabling the change of some of its properties, such as viscosity and digestibility.5,6 Nonstarch polysaccharides are represented by the polysaccharides obtained from the plant (e.g., guar gum2b, locust bean gum, pectin), microbial (e.g., xanthan2c and gellan gum), animal (e.g., chitosan2d and gelatin2e), and seaweeds (ref. Chpt. 2) (e.g., alginate2f and carrageenan) sources. They present several applications in the food industry, namely as gelling agent, thickener, emulsifier, antimicrobial, film forming capacity, and barrier to oil uptake.2a,7–9 Besides the commercially available polysaccharides, many others with potential application in the food industry may be obtained from nontraditional sources.Table 19.1 summarizes some examples of the polysaccharides obtained from nontraditional sources potentially useful for the food industry, however still without legal approval for commercial use. Table 19.1 Polysaccharides obtained from non-traditional sources with potential use in food industry Solvents and methodology of Polysaccharide Functionality and Species extraction structure properties References

Caesalpinia pulcherrima, Adenanthera pavonina, and Gleditsia triacanthos Sophora japonica Gracilaria birdiae Gleditsia sinensis Tricholoma mongolicum Imai

Aegle marmelos

Cold water and ethanol

Galactomannan

Thickener, film forming properties

8,10

Hot water, ethanol, and sulfuric acid Hot water and ethanol Hot water, ethanol, and sulfuric acid Ultrasonicmicrowave synergistic extraction with water Hot water, acetic acid, and acetone

Galactomannan

Thickener

11

Agar

Antioxidant and gelling properties Gelling properties

12

–

Antioxidant properties

14

–

Emulsifier

15

Galactomannan

13

Production and Extraction of Polysaccharides and Oligosaccharides

Polysaccharides, after chemical, physical, or biological degradation, give origin to oligosaccharides, which can also be synthesized by chemical glycosylation and de novo synthesis, or by enzymatic catalysis.16–18 A broad range of oligosaccharides has been studied in the last years due to their potential prebiotic activity. However, taking into account the evidence from in vivo studies, only fructans (inulin and fructooligosaccharides (FOS)) and galactooligosaccharides (GOS) fulfill all the criteria required to be considered prebiotics.19 Other emerging prebiotics that can stimulate the growth of bifidobacteria include isomaltooligosaccharides (IMO), maltooligosaccharides (MOS), xylooligosaccharides (XOS), soybean oligosaccharides (SOS), glucooligosaccharides, lactosucrose (LS), lactulose, raffinose, and stachyose.20–23 In vitro and feeding trial data suggest a prebiotic effect of these carbohydrates, although till date, there are no published clinical data from human studies.24 Other oligosaccharides are being studied regarding their potential prebiotic activity, including germinated barley foodstuffs, gentiooligosaccharides, mannanoligosaccharides, chitooligosaccharides, arabinoxylooligosaccharides, oligosaccharides from melibiose, pectic oligosaccharides, oligodextrans, palatinose, polydextrose, gluconic acid, glutamine, and hemicelluloses-rich subtracts, lactose, resistant starch and its derivatives, as well as sugar alcohols.20,21,25 Prebiotics are obtained either by extraction from the plants, such as inulin from chicory roots; by enzymatic hydrolysis of plant polysaccharides, such as XOS; or by transgalactosylation reactions catalyzed by an enzyme, such as GOS and FOS. Fructans are naturally present in more than 36,000 plants and vegetables.26 Most of the species belong to the Compositae or Liliales families.27 Chicory, Jerusalem artichoke, garlic, and onion appear as the ones with high contents in fructans.28 Other examples of the plants, fruits, and vegetables containing FOS include asparagus, wheat, bananas, rye, leeks, dahlia, carambola, june plum, and blue agave.20,27,29 However, due to the low FOS contents in these foods, and since they are season limited, the industrial production of FOS is performed by enzymatic processes. Fructans are a category of carbohydrates that include inulin and oligofructose. The inulin degree of polymerization (DP) varies between 2 and 60 units with an average (DPav) of 12 units, while oligofructose comprises short-chain molecules, including FOS from hydrolyzed inulin that can be a mixture of GFn and FFn molecules (2 95% oligosaccharides); Orafti Synergy 1 (powder, mix of oligofructose, and inulin); Orafti ST (powder, inulin > 90%); Orafti GR (Granulated powder, >90% inulin); Orafti HP (powder, >99.5% inulin); Orafti HP-Gel (Instant powder, >99.5% inulin) Fibrulose L60 (syrup, >60% of oligofructose); Fibrulose L85 (syrup, >85% of oligofructose); Fibrulose F97 (powder, >95% of oligofructose); Fibruline S20 (powder, >90% of inulin); Fibruline DS2 (powder, >98% of inulin); Fibruline Instant (powder, >90% of inulin); Fibruline XL (powder, >99% of inulin) Frutafit HD (powder, ≥90% oligofructose/inulin, highly dispersible native inulin); Frutafit IQ (powder, ≥90% oligofructose/inulin, instant quality native inulin); Frutafit CLR (powder, ≥85% oligofructose/inulin, inulin with high solubility); Frutafit TEX! (powder, ≥99.5% oligofructose/inulin); Frutalose OFP (powder, 92% oligofructose/inulin); Frutalose L85 (syrup, 85% oligofructose/inulin); Frutalose L90/L92 (syrup, 92% oligofructose/inulin

China France

BENEOOrafti SA

Belgium

Cosucra SA

Belgium

Sensus C.V.

Netherlands


Inulin and oligofructose From chicory roots

Meioligo FOS P (powder, 95% oligosaccharides); Meioligo CR (powder, >97% of 1-kestose); Meioligo G (syrup, 75% (w/v) of solids. Oligosaccharides, ≈55% of solids) (also distributed as Oligomax G-Syrup. By Zytex Biotech Pvt. Ltd., India) NutraFlora scFOS (powder, 95% oligosaccharides) Oligo-sugar (syrup, 70% (w/v) of solids. Oligosaccharides, ≈60% of solids); Oligo-sugar (powder, 23% oligosaccharides for animal feed) Powder P (Powder, ≥ 95% FOS)

664

Table 19.6 Fructans, GOS, and lactulose currently produced and available in the market84,139 Type of fructan Product name

From Blue Agave hearts

Olifructine-SP (powder, >85% fructans)

GOS

Oligomate 55 (syrup, 75% (w/v) of solids. GOS > 55% of solids); Oligomate 55P (powder, >99% GOS); TOS-100 (powder, >99% GOS) Cup-Oligo H70 (syrup, 75% (w/v) of solids. GOS, ≈70% of solids); Cup-Oligo P (powder, 70% GOS) Garakutoorigo [origomeito] 55N (syrup)

Lactulose

FOS, fructooligosaccharides; GOS, galactooligosaccharides.

Mexico

Nissin Sugar Manufacturing San-ei Sucrochemical FrieslandCampina Domo Clasado Solvay Pharmaceuticals GmbH

Japan

Morinaga Milk Industry Co.

Japan

Japan Netherlands UK Germany Japan


Vininal GOS (GOS, ≈60% of solids); TOS-Syrup (syrup, 75% (w/v) of solids) Bimuno® Duphalac (syrup, 72% (w/v) of solids); Bifiteral (syrup, 66.7% (w/v) of solids); Chronulac, Cephulac Lactulose (powder >95% lactulose) MLS-50 (syrup, 70% (w/v) of solids); MLP-40 (powder, 41% lactulose); MLC-A (powder, anhydride, 98% lactulose)

Nutriagaves de Mexico S.A. de C.V. (Namex) Yakult Honsha

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Individual FOS and GOS molecules with purities varying between 80% and 99% are only available for the analytical purposes. The main companies supplying the oligosaccharides are Sigma–Aldrich, Megazyme, and Wako Chemicals GmbH.

4. HEALTH AND NUTRITIONAL BENEFITS OF POLYSACCHARIDES AND OLIGOSACCHARIDES Besides the traditional use of polysaccharides, described above, in recent years, the scientific community and industries have been exploring a great number of health benefits associated to their consumption. Some of the health benefits reported for the polysaccharides and oligosaccharides are related with their use as dietary fiber and as prebiotic sources, leading to a reduction of cholesterol and glucose blood levels of consumers. The definition of dietary fiber constitutes one of the challenges of the scientific and regulatory communities. Initially presented as a substance obtained from the cell wall material, considering the origin of the materials, dietary fiber now includes all indigestible carbohydrates, highlighting their physiological behavior.139 The European Food Safety Authority (EFSA) Panel on Dietetic Products, Nutrition, and Allergies suggests two categories of carbohydrates, namely the glycemic carbohydrates referring to the carbohydrates digested and absorbed in the human small intestine (e.g., monosaccharides, disaccharides, MOS, and starch), and dietary fiber that represents nondigestible carbohydrates (e.g., cellulose, hemicelluloses, pectins, gums, mucilages, β-glucans, FOS, GOS, and resistant starch).140 A prebiotic is defined as “a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health.”30 In order to be considered as prebiotics, carbohydrates must fulfill three criteria, namely (1) resistance to gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption; (2) fermentation by intestinal microflora; and (3) selective stimulation of the growth and/or activity of those intestinal bacteria that contribute to health and well-being.19 Currently, it is known that carbohydrates can directly influence the physiological and metabolic processes in humans, and that this influence can reduce some of the problems associated with the appearance of the disease in a direct or indirect way. Several studies have been conducted in order to prove the link between the carbohydrates consumption and diseases in humans, presenting strong evidence that they are related, although the mechanisms involved in such activities are not completely understood.141 Some studies have evaluated the effects of polysaccharides in some physiological responses. Lindström et al.142 studied the effect of four polysaccharides, namely scleroglucan (fungal exopolysaccharide), xanthan gum, gellan, and native dextran on plasma lipids and caecal formation of short-chain fatty acids (SCFA) in mice. Results showed


that all the polysaccharides evaluated led to an increase of caecal tissue weight and an increase of SCFA production, thus predicting their influence in the maintenance of a healthy gastrointestinal tract. In 1995, Ellis et al.143 showed that guar gum could be used to reduce glucose absorption, insulin, and gastric inhibitory polypeptide secretion rates in the pigs. Some tests made in humans showed that hydrolyzed guar gum could be used to decrease the serum cholesterol, and the free fatty acid and glucose concentrations, when consumed in 15 g/day quantities, indicating that the hydrolyzed guar gum could improve lipid metabolism in humans.144 Most of the health benefits associated with the daily intake of prebiotics are linked to the end products formed during their fermentation by specific bacteria such as Bifidobacteria and Lactobacillus. The major end products of metabolism are strong acids as SCFA, mainly acetate and propionate that lower the pH of the colon producing an antibacterial effect.145 The selective fermentation of FOS and GOS, with consequent inhibition of pathogenic growth, has been extensively confirmed in several human studies.146 Clinical investigations concerning the health claims related with prebiotics intake include blood sugar and lipids regulation147–154; increase of mineral absorption, particularly calcium and biomarkers of bone health, which consequently reduces the risk of osteoporosis155–158; decrease of colon cancer risk159–163; prevention of gastrointestinal health by the decrease of the risk of intestinal infectious diseases,164–166 constipation,167,168 diarrhea,169,170 and inflammatory bowel disease171–173; stimulation of the immune system by vitamin B production, blood ammonia reduction, and promotion of normal gut microbiota renewal after antibiotic therapy133,174–177; and regulation of metabolic disorders related to obesity by promoting satiety and reducing food intake.178–180 The health benefits of prebiotic oligosaccharides have been extensively reviewed.57,137,181–185 The evidence for the above benefits varies significantly, and although many in vitro and in vivo studies have already provided scientific evidence of these benefits, more clinical trials are needed to prove it unequivocally for humans.21,24,186 Health and nutritional claims are used by the industry to add value to their products, informing the consumers the advantages of consuming them, thus influencing the consumer’s choice for a specific food product. Thus, the indication of health or nutritional benefits in food labels implies that the product respects a defined number of conditions.187 The USA started to regulate health claims in the 1990s, requiring that all packaged foods use the adequate nutrition labeling and the health claims defined by the Secretary of Health and Human Services.188 In 2006, the existing regulation on the nutrition and health claims was adopted by the European Council and Parliament that further harmonized rules for the use of nutrition claims such as “low fat,” “high fiber” or health claims such as “reducing blood cholesterol.”187 Recently, the European Commission published a list of the permitted health claims and corresponding nutrient, substance, food, or food category.189 In the entire list, there

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are some 12 polysaccharides and oligosaccharides presenting one or more health claims as is the case for chitosan, guar gum, glucomannan, β-glucan, resistant starch, and arabinoxylan produced from wheat endosperm.

5. REGULATION AND SAFETY ASPECTS Regulation is a very interesting and complex topic in food industry. The use of ingredients and food additives from natural sources, processing technologies used by the industry, amounts used, quality and safety of the products, and the way that consumers see the labeling are some of the aspects that the authorities have to consider upon approval of their use for human consumption. One of the hard tasks for the polysaccharides and oligosaccharides regulation is their definition as ingredients and/or food additives. In Japan, FOS, GOS, soybean, palatinose, lactulose, LS, XOS, and IMO are regulated as “Foods for specified health use” since early 1990s.133 Some of the carbohydrates already used in the food can be considered an ingredient; however, the same polysaccharide or/ and oligosaccharide if applied in a food product with a specific functionality can be considered a food additive. Being so, it is important to define and understand the main differences between a food ingredient and a food additive, which are several times mentioned as the same. Food and Agriculture Organization (FAO) and the Codex Alimentarius define food additive as “any substance not normally consumed as a food by itself and not normally used as a typical ingredient of the food, whether or not it has nutritive value, the intentional addition of which to food for a technological (including organoleptic) purpose in the manufacture, processing, preparation, treatment, packing, packaging, transport or holding of such food results, or may be reasonably expected to result, (directly or indirectly) in it or its by-products becoming a component of or otherwise affecting the characteristics of such foods.The term does not include ‘contaminants’ or substances added to food for maintaining or improving nutritional qualities,” while ingredient has been defined as “any substance, including a food additive, used in the manufacture or preparation of a food and present in the final product although possibly in a modified form.”190 Food regulations differ from country to country, and although several countries like USA, Canada, Australia, and European countries are involved in the development of food regulation, FAO of the United Nations and the World Health Organization (WHO) through the Codex Alimentarius Commission present standardized guidelines for the food industry and a list of accepted food additives in which the polysaccharides and oligosaccharides are included.191 In the USA, FDA has the primary legal responsibility for determining the use and safety of a food additive, and has also published an extensive list of food additives that can be used by the food industry.192 They mention that any substance that is reasonably expected to become a component of food is a food additive, and it should be subject to a premarket approval, unless the substance is generally recognized as safe among


experts.188 The evaluation of the safety of a substance by FDA considers (1) the composition and properties of the substance, (2) the amount that would typically be consumed, (3) immediate and long-term health effects, and (4) various safety factors. This evaluation will enable the determination of a safety level intake for the food additive used.193 In the EU, a substance can be used in food if it has been found that it is harmless to the consumers and that a technological need for its use is in place. Currently, the Regulation (EC) No 1333/2008 of the European Parliament indicates, through an extensive list, the food additives that can be used by the food industry.44,140 In this diploma, food additives are defined as “substances that are not normally consumed as food itself but are added to food intentionally for a technological purpose (…) such as the preservation of food.” In this definition, there are some examples that should not be considered as food additives, such as salt replacers, vitamins, and minerals. In the EU, the applications for authorization of a new food additive or to the modification of an already authorized food additive are based on the procedures described in the EFSA guide.194 As an example, if a polysaccharide is ought to be used as a food additive, the applications for authorization should follow the next steps: 1. Provide information on the chemistry and specifications of the new polysaccharide, potential hazards during the production and use, and methodologies for its identification in food, e.g., if the new polysaccharide is obtained from a botanical source, indications on the part of the plant used (e.g., seed), growth and harvesting conditions, as well as molecular weight, solubility, and viscosity are some of the information that should be given. The purity and the limits of any impurity present in the polysaccharide and their methods of analysis should also be included in the document (e.g., heavy metals). The presence of impurities can be related with the extraction methodology (e.g., solvents, temperature) and can, therefore, vary from source to source and it should be described. Moreover, the methodologies to identify the polysaccharide in the foods where it is to be used should be indicated and it should be applicable to all the food categories to which the substance may be added; 2. Provide existing authorizations and previous evaluations; e.g., if the additive proposed is a polysaccharide that has already been evaluated, the information from the previous submission should be given such as the name of the organization which carried out the evaluation and when the evaluation was undertaken; 3. Present the proposed uses and the kind of exposure to which consumers can be subjected with this new additive; e.g., if dealing with a polysaccharide extracted from a plant, the document should report the final use and the levels at which the substance will be added to the final food products. In this case, also the residues that could be generated in the extraction methodology used should be indicated. Further, it is important to provide information about the total exposure from all the possible sources (i.e., food products, food contact material, pharmaceutical, or cosmetic products); 4. Toxicological studies should be performed in vitro, and in vivo using laboratory animals evaluating the safety of the new additives, where toxicokinetics, genotoxicity,

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and toxicity should be assessed. In this case, the new additives are evaluated from the less to the more complex toxicological test and should continue only if the obtained data in the initial tests (i.e., absorption models and bioavailability models) indicate that the compounds are absorbed.

6. CONCLUSIONS The consumers demand for higher quality, safer, and healthier food products is one of the main challenges imposed to the food industry. Several strategies are being developed worldwide that include the application of innovative technologies and the use of novel products, achieving new and improved functionalities on food products. Polysaccharides and oligosaccharides, widely used in the food industry for several applications, have recently gained an increasing interest as possible high-value functional ingredients. The introduction of new polysaccharides and oligosaccharides in the food sector is limited by the technological and regulatory factors such as: • the yields of extraction and production, degree of purification, and production rates obtained; • the investment that is required to obtain their approval as food additives; • the difficulty of proving their health and nutritional benefits due to the timeconsuming and expensive clinical trials, including the in vivo studies in humans, needed to confirm the proposed claims. Finally, it must be pointed out the importance that consumers have in the application of these compounds in food products, i.e., clear information on the benefits of using polysaccharides and oligosaccharides must be provided to the consumers so that they perceive these products as natural, healthy, and safe.

ACKNOWLEDGMENTS The authors, Clarisse Nobre and Miguel A. Cerqueira are recipient of fellowship supported by the Fundação para a Ciência e Tecnologia (SFRH/BPD/87498/2012 and SFRH/BPD/72753/2010, respectively), POPH-QREN and FSE (FCT, Portugal). The authors thank the FCT Strategic Project PEst-OE/EQB/ LA0023/2013 and the Project “BioInd—Biotechnology and Bioengineering for improved Industrial and Agro-Food processes,” REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2—O Novo Norte), QREN, FEDER.

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INDEX Note: Page numbers with “f ” and “t” denote figures and tables.

A Abengoa Bioenergy New Technologies (ABNT), 349 Abietic acid, 148–149 AbitibiBowater kraft mill, 102 Absidia, 421 Absinth, 186 Absolute ethanol. See Anhydrous ethanol Acer saccharum. See Sugar maple Acetaldehyde, 191, 379, 432–433 Acetic acid, 105, 189–190, 409–412 extraction plant, 363–364, 364f Acetobacteraceae (AAB), 189 Acetone–butanol–ethanol (ABE) fermentation, 23–24, 194–195, 369–370, 380 Acetyl-CoA synthetase, 581 Achard, Franz Carl, 11–12 Acid hydrolysis, 362–363 for production of fermentable sugars, 353f Acid sulfite pulping, 97–98, 106–107 Acid thinning of starch, 217 Acidianus ambivalens, 428 Acidianus brierleyi, 428 Acidic pretreatments of wood chips, 112–113, 112t Acidithiobacillus ferrooxidans, 541 Acinetobacter calcoaceticus, 515, 529, 643–644 Acrolein, 261–262 Acrylic acid, 142, 193 synthesis routes, 194f Actinobacillus, 579 Actinobacillus succinogenes, 416 Acylglycerols, 248 ADP, 392 Advanced biorefineries, 12 Aerial views evaluation, civilization biorefinery data from, 327 Aeribacillus pallidus, 547 Aerobic digestion, for biogas, 273, 274f Aeromonas, 596–597 Aeromonas caviae, 597 Agar, 70–71, 424–425 chemical composition of, 70f

Agarobiose, 70 Agaropectin, 70 Agarose, 70 Aging, intrinsic and extrinsic, 611, 612f Agricultural residues, biogas production from, 276–278, 280t Agriculture industry, application of exopolysaccharides in, 540–541 Agrobacterium, 631–632 Agrobacterium tumefaciens, 625–626 Ajinomoto, 201f Akvavit, 185, 186 Alanine, 448 Alcaligenes, 579, 584, 621, 630–631 Alcaligenes eutrophus, 580, 583, 585, 592–593 Alcaligenes faecalis, 428 Alcaligenes latus, 580, 583, 593, 596, 599 Alcoholic fermentation, 183–190, 184f acetic acid, 189–190 for beverages, 184–186 for biofuels, 186–189 raw materials, 186–187 Alder-ene reaction. See Ene reaction Algae, 16, 403 chemical composition on dry matter basis, 79t pyrolysis of, 345–348 Algae Raceway Production System, 39 Algal biorefineries, 35–90 algal research in United States, 38–46 Aquatic Species Program, 38–40 legislation/law, 40 planning, 40 conventional nonfuel products from microalgae, 72–78, 73f, 74t health food, 74–75 pigments, 76–78 polyunsaturated fatty acids, 75–76 squalene, 76 macroalgae, 46–48 cultivation, 47–48 fundamentals related to, 46–47 microalgae, 48–55

681

682

Index

Algal biorefineries (Continued ) cell disruption, 61–64, 62t culture systems, 52–55, 52t dewatering, 55–58, 56t, 59t downstream processes, 55–69 drying, 58–61, 59t extraction, 64–67 fundamentals related to, 48–49 growth, 49–52 hydrothermal processing, 67–69, 68f, 68t nonconventional products from microalgae, 78–83 biodiesel, 78–80 bioethanol, 80–82 terpene-based biofuels, 82–83 traditional uses of macroalgae, 69–72 agar, 70–71 alginates, 72 carrageenan, 71–72 funori, 72 Algatechnologies, Ltd., 77–78 Algenol, 81–82 Alginates, 72, 538, 543 structure of, 72f Alginic acid, 72 Aliphatic carboxylic acids, 98–99, 103–105, 104f Alkaline phosphatase, 492 Alkaline pretreatments of wood chips, 110–111, 111t Alkaline proteases, 491 Alkaline sulfite-AQ-methanol (ASAM), 107–108 Alkyd resins, tall oil, 139–140 Alkyl polyglucosides (APGs), 225–227, 226f α amino adipate (AAA), 447, 458 α-aminoglutaric acid. See Glutamic acid α-amylase, 476–477, 486–489, 493 α-chymotrypsin (CHT), 400 α-HAs. See Hydroxy acids α-ketoglutarate, 454 α-ketoglutaric acid (a-KG), 417–420 chemical structure of, 418f production in yeast, 419f yield by different strains, 420t α-linoleic acid, 250–252 α,ω-dimer diamine (DDA), 144 α,ω-dimer diol, 144 α-pinene, 131–135 camphene synthesis from, 133f conversion to pine oil, 131f α-tocopherol. See Vitamin E

Aluminum, 137–138 Amber acid. See Succinic acid (SA) American Recovery and Reinvestment Act of 2009, 40 Amidoamines of tall oil fatty acids, 138–139 Amino acid efflux, engineering of, 453 Amino acids, 200–201, 492 in cosmetic formulation, 620–621, 620t glutamic acid, 200–201 and PHB, 593 Amino acids, white biotechnology for, 445–472 alternative sources for production, 466 aromatic amino acids, 464–466 aspartate family, 461f glutamic acid, 454–457 history and evolutionary route, 446–447 lysine, 457–461 methionine, 461–463 production processes, 447–451 microbial fermentation, 448–451 prospective and outlook, 466–467 strain improvement, 451–454 amino acid efflux, 453 anaplerotic pathways or precursor supply chain, 453 central carbon metabolism, 453 for higher energy efficiency, 453 metabolic engineering, 452–454 modifying enzymes in terminal pathways, 453 NADPH metabolism, 453 random mutagenesis, 452 for substrate broadening, 454 threonine, 463–464 Ammonium nitrate, 422 Ammonium sulfate, 422 Ammonium tallate, 137–138 Amylases, 489–491 Amylopectin, 158–159, 167–168, 211 chemical composition of, 169f Amylose, 158–159, 167–168 chemical composition of, 169f Amylum. See Starch Anabaena, 582 Anaerobic digestion for biogas, 273–275, 274f, 277–278, 332–333, 332f role in civilization biorefineries, 315–319 Anaerobic promoters, and PHB, 593–594 Anaerobiospirillum succiniciproducens, 416

Index

Anaplerotic pathways, and amino acid production, 453 Ancient Rome, waste/wastewater management in, 297–298 Anhydrous ethanol, 188–189 azeotropic distillation, 188 extractive distillation future technologies, 189 with liquid solvent, 188 membrane processes, 189 molecular sieves, 189 with soluble salt, 189 Animal feed industry, application of enzymes in, 490–491 Ankistrodesmus falcatus, 64 Antiaging cosmetics, 611–613, 613t Antioxidants, 610, 610f, 611t Aphanizomenon, 36 Aphanizomenon flos aquae, 621 Aquathermolysis, 362–363 Aquatic Species Program (ASP), 37–40, 46 Arabinogalactan, 488 Arabinose, 371–372, 465 Arabinoxylan, 403, 488 Archaeoglobus fulgidus, 530 Arenga palm. See Sugar palms Arenga pinnata. See Sugar palms Arginine, 447 Aromatic amino acids, 464–466 biosynthesis pathway, 465f Aromatics, 259–260 Arrack, 185 Arthrobacter, 541 Arthrobacter paraffineus, 418, 421 Arthrospira, 36–37 Arthrospira (Spirulina) maxima, 67 Ascophyllum nodosum, 617 Ascorbic acid. See Vitamin C Ashbya gossypii, 623–624 Asian Palmyra palm, 164–166, 165f Aspartate, 463 Aspartate semialdehyde, 461–462 Aspartic acid, 448 Aspartokinase, 462 Aspergillus, 476–478, 481, 615–616, 619–620 Aspergillus aculeatus, 421 Aspergillus awamori, 421 Aspergillus carbonarius, 421 Aspergillus flavus, 421 Aspergillus foetidus, 421

Aspergillus fonsecaeus, 421 Aspergillus japonicus, 658 Aspergillus luchensis, 421 Aspergillus nidulans, 421, 633 Aspergillus niger, 199–200, 202, 223, 392, 402–403, 421–425, 427–428, 615, 618, 633, 639–640, 658 Aspergillus oryzae, 466 Aspergillus phoenicis, 421 Aspergillus saitoi, 421 Aspergillus unguis, 393 Aspergillus wentii, 421 Asphalt emulsions, 137–138 AstaPure, 77–78 Astaxanthin, 66, 76–78 in cosmetic formulation, 617 Atlas Powder Co., 225 ATP, 392, 423–424 Aurantiochytrium mangrovei, 641 Aureobasidium pullulans, 633, 658 Austria, food wastes in, 323 Autohydrolysis, 112 Autotrophic microalgae, cultivation of, 49–50 Auxenochlorella protothecoides, 64 Auxotrophic mutations, for amino acid production, 452 Avantium Chemicals, 15 Azeotropic distillation, 188 Azotobacter, 579 Azotobacter beijerinckii, 579 Azotobacter chroococcum, 579 Azotobacter vinelandii, 528, 535–536, 579, 583

B Bacillus, 192–193, 418, 446, 477–478, 541, 546, 579, 632, 634 Bacillus brevis, 635 Bacillus cereus, 579 Bacillus circulans, 220 Bacillus firmus, 579, 581, 599–600 Bacillus licheniformis, 421, 476–477, 514–515, 544–546, 636, 639 Bacillus macerans, 220 Bacillus megaterium, 579, 586 Bacillus mycoides, 579 Bacillus sphaericus, 579, 585 Bacillus subtilis, 195, 382–383, 401, 503–504, 510, 514, 533, 621, 623–624, 636, 643–644 Bacillus thermodenitrificans, 547 Bacillus thuringiensis, 579

683

684

Index

Baking industry, application of enzymes in, 488 Ball mill, for microalgae, 63, 65 Bark, chemical composition of, 94t Barley, 171–172, 172f, 490–491 Base petrochemicals, 19f BASF, 495–496 Beer, 188 Beet, 11–12 Benzene, 188 BERBION project, 325–326 Beta vulgaris maritima. See Sea beet β-amylase, 486–488 β-carotene, 67, 76–77, 622 β-galactosidase, 488–489 β-glucan, 490–491 in cosmetics, 631–632 β-glucanase, 489–491 β-HA. See Hydroxy acids Betaine, 621 β-pinene, 131–135 flavor and fragrance chemicals from, 132f Betatene®, 77, 77f Betula pendula. See Silver birch B-factor iterative test, 401 Beverages, alcoholic fermentation for, 184–186 Bifidobacterium, 535–536, 667 BioAstin, 77–78 Biobased Chemicals Report, 5 Biobased platform molecules, 19f, 22 Biocatalysis, 389–408, 494 development of biocatalysts, 394–403 cross-linked enzymes, 397–398 enzyme immobilization, 394–400, 394f hybrid enzymes, 400–401 multienzyme reactions, 401–402 nanobiocatalysis, 398–400 pretreatments, 402–403 by submerged fermentation, 402 raw materials, 403–404 reaction media, 404 screening for novel biocatalyst, 392–394 chromogenic and fluorogenic assays, 392–393 fermentation assays, 393 metagenomic assays, 393–394 Biochemical conversion, in pyrolysis-based hybrid biorefineries, 362–363 Biocoil, 54 Biodiesel, 36–38, 78–80, 80f, 277–278 Bioenergy, 237–238

Bioethanol, 80–82, 81f, 187f, 190–191, 291, 349–350, 403, 491–492 Bioethylene, 22 Biofilms, role of exopolysaccharides in, 528–531 Biofuel carbon debt, 46 Biofuel-driven Biorefineries, 5 Biofuels, 7 alcoholic fermentation for, 186–189 anhydrous ethanol, 188–189 distillation, 188 fermentation, 187–188 raw materials, 186–187 application of enzymes for, 491–492 application of PHAs for, 568–569, 568t, 569f Biofungicides, 504 Biogas characteristics of, 275t composition of, 274t upgrading technologies, 282, 283t Biogas biorefineries, 271–294 chemical platform methane, 284 fertilizer production, 284–288 digestate composition of typical substrate mixes, 285t digestate treatment strategies, 286f digestate utilization and upgrading strategies, 287t mass and energy balances, 288–291, 289f–290f other biorefinery concepts with focus on, 291 perspectives of, 292–293 schema of, 272f substrates for biogas production, 275–279, 276t utilization of biogas, 280–282, 281t Bio-hythane, 275 Bioliq® process, 357–359, 359f bioliqSyncrude®, 358–359 Bioliquid refinery, 354, 356f Biolys, 450–451 Biomass economic value of, 7–8, 8t processing of, 6 types of, 6 Biomass to liquid (BTL) process, 117–118 Biomedical applications of exopolysaccharides, 543–545, 543t–544t Biomethane, 280–282, 284 Biooil, 342–344, 354, 356f, 365 compound classes in, 343t

Index

Bioplastic packaging application of PHAs for, 566–568 market, 559f Biopol®, 557 Biopolymers, white biotechnology for, 555–574 classification, 556 PHA produced and research companies, 558t PHA produced in industrial scale, 560–572, 561f applications of, 565–566, 566f, 567t biofuels, 568–569, 568t, 569f challenges for R&D, 570–571 continuous process, 563, 564f environmentally friendly bioplastics for packaging purposes, 566–568 fed-batch process, 561–563 future prospective, 571–572 medical implants, 569 mixed cultures, 563–565, 565f monomers as chiral intermediates, 570 smart materials, 570 strains for production of PHA, 559–560, 560f, 562t Biopropane, 23 Biopropylene, 23 Biorefineries, 6f advanced, 12 algal biorefineries, 35–90 biogas biorefineries, 271–294 chain development, 16–17 characteristics of, 28t civilization biorefineries, 295–340 classification of, 9–11, 10f conventional, 11–12 definition of, 5–7 green biorefinery, 15 lignocellulosic feedstock biorefinery, 13–14 marine biorefinery, 16 next generation hydrocarbon biorefinery, 14–15 oleochemical biorefinery, 13 pine biorefinery, 127–156 single-cell biorefinery, 369–388 sugar- and starch-based biorefineries, 157–236 SWOT analysis on, 29t syngas platform biorefinery, 14 vegetable oil biorefineries, 247–270 vs. petrochemical refineries, 17–24, 18t, 19f, 20t–21t whole crop biorefineries, 12–13 wood-based biorefineries, 91–126

Biorefinery Complexity Index (BCI), 24–27, 29–30 Biorefinery Complexity Profile (BCP), 25–26, 26f–27f Biorefinery Fact Sheet, 5 Bioremediation, 503–504, 516, 530, 541 Bioresources, 297–310 definition, categorization, and basic utilization chain, 302 need for classification of, 306–308 existing coding systems, 307–308 general bioresource coding system, 306–307, 307f paper chain, 308–310, 309f primary bioresources, 302–304, 303f quaternary bioresources, 303f, 305 secondary bioresources, 303f, 304 tertiary bioresources, 303f, 304–305 waste and wastewater management, 297–300, 297f collection, 301 definition and categorization, 300–301 Biosensors, 400, 492 Biosuccinic acid, 196–197, 197f Biosurfactants, white biotechnology in, 499–522 in cosmetics, 642–644 glycolipids, 502–509 mannosylerythritol lipids, 508–509 rhamnolipids, 503–505 sophorolipids, 505–508 lipopeptides and lipoproteins, 510–515 lichenysin, 514–515 polymyxin, 512–514 surfactin, 510–511 manufacturers and users, 502t polymeric biosurfactants, 515–517 emulsan, 515–516 liposan, 516–517 Biotech conversions, in vegetable oil biorefineries, 259 Biotin, 454 Bisulfite pulping, 107 Bitumen emulsions, 137–138 Black liquor, kraft, 95, 98 -based by-products, 100–105 composition of, 98t fractionation process, 103f Black liquor gasification (BLG), 116–117 Black sugar palm. See Sugar palms

685

686

Index

Blakeslea trispora, 622 Bligh and Dryer method, 65 Block copolymers, 556, 557f Blood stain removal, 509f Blue dextran, 545–546 Borassus flabellifer. See Asian Palmyra palm Borneol, 132–133 Borregaard LignoTech, 108 Botryococcenes, 82–83 structural formula of, 83f Botryococcus braunii, 66–69, 76, 82–83 Botrytis, 421 Botulinum toxin, in cosmetics, 636–637 commercially available brands, 637t Bradyrhizobium, 631–632 Branched fatty acids, 140–141, 140f Brandy, 185 Brazil, ethanol production from sugarcane in, 237–246 context and evaluation, 238–240 cost breakdown, 245f economic aspects, 240–244 flow sheet, 241f productivity of sugarcane ethanol mills, 239f sugarcane agroindustry production, 239f sugarcane mills, 238f Brevibacterium, 200, 462 Brevibacterium heali, 462 Brewers yeast. See Industrial yeast Brewing, 184–185 British gums, 222 Brown algae, 36 Brown macroalgae, 46–47, 347–348 Burkholderia, 588 Burning, in alcoholic fermentation, 185 Butanedioic acid. See Succinic acid (SA) Butanol, 113, 138, 381 1-Butene, 191 Byssochlamys, 428

C C3 building blocks, 192–193 3-hydroxypropionic acid, 193 isopropanol, 193 lactic acid, 192–193 C4 building blocks, 194–198 biosuccinic acid, 196–197 isobutanol, 195 n-butanol, 194–195

C2 value chain ethanol-based, 190–192, 190f Calvados, 185 Camphene, 132–133, 133f Camphor, 132–133 Candida, 418, 506–507, 509 Candida antarctica, 404 Candida apicola, 505 Candida bombicola, 505–506, 643 Candida catenula, 421 Candida citroformans, 421 Candida famata, 623–624 Candida glabrata, 418 Candida guilliermondii, 421 Candida intermedia, 421 Candida lipolytica, 516 Candida oleophila, 421 Candida parapsilosis, 421 Candida rugosa, 402–403 Candida shehatae, 374 Candida tropicalis, 421 Candida utilis, 615 Canthaxanthin, 66 Carbocationic polymerization, polyterpene resin production by, 133–135, 135f Carbohydrates, 653 Carbon catabolite repression (CCR), 369, 372–374, 373f Carbon dioxide (CO2), 66–67, 280–282 Carbon storage, of microalgae, 51–52 Carbonation, in sugarbeet processing, 181–182 Carboxy methyl (CM) glucan, 631 Carboxyl group, reactions of tall oil fatty acid at, 138–139 Carboxylesterase A (CesA), 401 Carboxymethyl dextran, 629–630 Carboxymethyl starch, 215 Carotenoids, 76–78, 77f in cosmetic formulation, 614–617 Carrageenan, 71–72 Carrier-type immobilization, 394–395 Cascade bioprocess, 449–450 Cascade use efficiency (CUE), 325 Casein amino acids, 592 Cassava, 173–175, 174f composition of roots, 174t Catalase, 489 Catalysis. See Biocatalysis Catalytic liquefaction, 117–118

Index

Catalytic-site deactivation, 485–486 Cationic dextran, 629–630 Cationic starch, 215, 216f Cattle farms, 276–277 C21-diacid, 142 Cell disruption of microalgae, 61–64, 62t chemical methods, 62–63 mechanical methods, 63–64 ball mill, 63 high-pressure homogenizers, 63 PEF technologies, 64 SFF technologies, 64 ultrasound, 63–64 Cell structures of macroalgae, 46–47 Cell walls of microalgae, 51 Cellobiose, 371 Cellulases, 475–477, 481, 489–490 Cellulose, 13–14, 262, 488, 543 pyrolysis of, 342, 344–346, 350–351, 362–363, 365 thermal stability regimes for, 344f Central carbon metabolism, and amino acid production, 453 Centrifugation, for microalgae, 58 Centrophorus spp., 76 Ceramides, in cosmetics, 642 Chaetoceros gracilis, 64 Chain development, in biorefineries, 16–17 Charcoal, 342–344, 350 Chemical conversions, in vegetable oil biorefineries, 259–262 Chemical flocculation, for microalgae, 57–58 Chemical oxidation of starch, 222–223 Chemical platform methane, 284 Chemical pulping, 92, 95, 97, 105–109 organosolv pulping, 107–108 possibilities for sulfite spent liquor-based by-products, 108–109 sulfite pulping, 105–107 Chemical synthesis, for amino acid production, 447 Chemistry sector, application of enzymes in, 492–493 Chiral intermediates monomers as, 570 Chitin, in cosmetics, 633 Chitinases, 493 Chitosan, in cosmetics, 633 Chlamydomonas, 81 Chlamydomonas reinhardtii, 82–83 Chlorella, 36–37, 65, 74–75

Chlorella protothecoides, 65, 74–75 Chlorella pyrenoidosa, 622–623 Chlorella sorokiniana, 74–75 Chlorella vulgaris, 36, 55–56, 63, 66, 69 Chlorellai, 81 Chlorinated carbohydrate. See Sucralose (3-Chloro 2-hydroxypropyl)-N,N, N-trimethylammonium chloride (CHPTAC), 214–215 Chlorococcus, 582 Chloroflexus aurantiacus, 428–429 Chlorophyceae, 46–47 Cholesterol, 492 Chondrus crispus, 71, 71f Choren, 14 Chorismate, 465 2-Choro-4-nitrophenyl ferulate, 392 Chromatographic separation, 450–451 Chromobacterium viscosum, 402–403 Chromogenic assays, 392–393 Chromohalobacter salexigens, 535–536 Chymosin, 488–489 Cinnamomum camphora, 80 Circinella, 431 Circular Flow Economy and Waste Management Law (Germany), 299 Circular open ponds, for microalgae, 52–53 Citrate synthase, 425 Citric acid, 198–200, 200f, 380–381, 409–412, 420–428, 618 commercial production, 619f production, 426t Citric acid cycle. See Krebs cycle Civilization biorefineries, 311–321 basic needs for prevalent implementation of steps toward, 334–337, 336f bioresource inventories in Bergedorf district, Hamburg, 326–328 inventory methods, 326–328 inventory results, 328 bioresource inventory and collection, 313–315, 314t case selections, 325–326 coupling of wastewater, waste, and energy management in Hamburg, 329–334 biogas generation, 332–333 digestate utilization, 333–334 inventory, 329–332 definition, 311–312

687

688

Index

Civilization biorefineries (Continued ) processing chains, 311–312 regional network, 311 unit locations, 312, 313f demand on information, 321–324 food losses till consumer level, 322, 323f food wastes at consumer level, 322–324 inventories and data provision, 321–322 distribution, 321 evaluation schemes, 324–325 exchanging industrial outputs for mutual benefits in Kalundborg, 334 principal process chain, 312f principal setup and elements, 311f products, 320–321, 320f unit process and technology inventory, 315–319 technologies for tertiary and quaternary bioresources, 315–316 treatment processes used in Hamburg, Germany, 316–319 Closed photobioreactors, for microalgae, 52t, 53–55 Clostridia., 640 Clostridium acetobutylicum, 23–24, 194–195, 369–370, 380, 623–624 Clostridium beijerinckii, 23–24, 194–195 Clostridium botulinum, 636–637 Clostridium botulinum A exotoxin, 637 Clostridium thermocellum, 466 CMP, 392 Cobalt, 137–138 Codex Alimentarius, 668 Coding system, bioresource, 306–308, 307f Coenobia, 56 Coenzyme Q10/ubiquinone, in cosmetics, 625–626 Cognac, 185 Coke smelting, 127–128 Cold-water-swelling (CWS) starches, 212 Colistin. See Polymyxin E Collagen, in cosmetics, 635 Collection of waste/wastewater, 301 Colletotrichum orbiculare, 504 Colonial microalgae, 48 Combi-cross-linked enzymes aggregates, 398 Combined heat and power plant (CHP), 273, 276–278, 281–282, 320 Compagnie Industrielle de la Matière Végétale (CIMV), 349–350 Companies, civilization biorefinery data from, 326

Compositae, 655 Composting, 316 Compressed natural gas (CNG), 281–282 Computer-aided solvent screening, 404 Conditionally essential fatty acids, 250–252 Confederation of European Paper Industries (CEPI), 310 code for recovered paper, 307, 307f Conjugated linoleic acid (CLA), 139–140 structure of, 140f Contact lens cleaning, 492 Contamination, in open pond systems, 53 Conventional biorefineries, 11–12 Converted starches, 217–227 acid thinning, 217 chemical or enzymatic oxidation, 222–223 cyclodextrins, 220, 221f dextrinization, 222 enzymatic conversion, 217–219, 218t Fischer glycosidation, 223–224 hydrogenation, 223–225 thermochemical conversion, 222 Conveyor dryers, for microalgae, 61 Cooligomerization of tall oil fatty acid, 140–141, 140f Coproduct development, 11 Coproduction in single cells, 375–381 downstream processing, 379–380 to maintain in vivo metabolism, 375–379 strategies, 376t–377t suitable and value-added product portfolio, 380–381 Corn. See Maize Corn bagasse, 23–24 Corynebacterium, 421, 446, 452–453, 462–463, 466, 579, 621, 632 Corynebacterium diphtheriae, 447 Corynebacterium glutamicum, 192–193, 200, 372, 379, 418–420, 446–447, 450–451, 455f, 456–461, 463–464, 466, 579, 621 Corynebacterium lilium, 462 Cosmaceuticals, 608 Cosmetic ingredients, biotechnologically derived amino acids, 620–621 applications of exopolysaccharides in, 541–542 astaxanthin, 617 β-glucan, 631–632 betaine, 621 biosurfactants, 642–644

Index

botulinum toxin, 636–637 ceramides, 642 chitin and chitosan, 633 coenzyme Q10/ubiquinone, 625–626 collagen, 635 dextran, 629–630 ellagic acids, 615 enzymes, 638–640 ferulic acid, 616 gallic acid, 615–616 gellan gum, 630 human growth factor, 638 hyaluronic acid, 633–634 hydroxy acids, 617–619 keratin and keratin hydrolysate, 636 kojic acid, 619–620 levan, 638 mycosporine and MAAs, 621 phlorotannins, 617 λ-polyglutamic acid, 634 polyunsaturated fatty acids, 640–641 pullulan, 633 resveratrol, 616 rhamsan gum, 630–631 schizophyllan, 632 scleroglucan, 632 squalene, 641 vitamin A and its derivatives, 622 vitamin B2, 623–624 vitamin B5, 624 vitamin B3/nicotinic acid, 624–625 vitamin C, 622–623 vitamin E, 623 welan gum, 630 xanthan gum, 626–629 Cosmetically important compounds antiaging activity, 611–613, 613t antioxidant activity, 610 hair care activity, 614 photo-protection activity, 613–614 skin lightening activity, 611 uses and potential side effects, 609t Crocosphaera watsonii, 535–536 Croda, 225 Crops. See specific entries Cross metathesis, 141 Cross-linked enzyme crystals (CLECs), 397–398 Cross-linked enzymes aggregates (CLEAs), 397–398 Cross-linked enzymes (CLEs), 395, 397–398, 397f

Crude glycerol, 278, 380–381 Crude oil, 20 characteristics and properties and gasoline potential of, 20t CHO composition of, 22t recovery, 504, 542 Crude sulfate/sulfite turpentine (CST), 130, 136 Crude tall oil (CTO), 136–137, 146–147, 260–261 soap, 100–101, 118–119 Crude turpentine, 130 Crypthecodinium cohnii, 64, 75–76, 641 Crystallization, of sugarbeet processing, 182–183 Cultivation, of macroalgae, 47–48 Culture systems, for microalgae, 52–55 Cunninghamella, 431 Cupriavidus necator, 580 Curdlan, 537–538, 543, 545–546, 631–632 Cyanobacteria, 51, 195 Cyanotech, 75, 77–78 Cyclodextrins (CD), 220, 221f Cyclohexane, 188 Cytochrome P450 monooxygenase, 506–507 Cytophaga, 588

D Dahl, Carl F., 128 Dairy industry, application of enzymes in, 488–489 manure, 382 Date palm, 164–166 Date sugar, 164–166 Decanters, for microalgae, 58 Deep eutectic solvents, 404 Dehydroabietic acid, 148–149 Dehydrogenation, 148–149 Denaturation, in enzyme production, 485–486 Dentures, 493 3-Deoxy-pentonic acid, 105 Derivatization, 395–396 Desmodesmus, 69 Destructive distillation of pine, 127–128, 129f Detergent industry, application of enzymes in, 489–490 Dewatering of microalgae, 55–58 centrifugation, 58 filtration, 57–58 flotation, 56–57 gravity sedimentation, 55–56, 56t methods, assessment of, 59t

689

690

Index

Dexpanthenol, 624 Dextran, 534, 537–538, 544–546 in cosmetics, 629–630 Dextran-70, 629–630 Dextran sulfate, 629–630 Dextrinization of starch, 222 Diamines, 145–146 Diaminopimelate (DAP), 447, 458 Diatoms, 16, 49 DIC Corporation, 75, 78 Dichloroethane, 191 3,4-Dideoxy-pentonic acid, 105 Diels–Alder reaction, 148 with tall oil fatty acid, 141–142, 142f Dietary fiber, 666 Diethyl oxalate, 418 Diethyl succinate, 418 Diethylenetriamine (DETA), 138, 145–146 Diethylenglycol diabietate, 150 Dihydroabietic acid, 148–149 Dimer acid, 143, 145–146 -based polyamides, 145 Dimer diisocyanate (DDI), 144–145 Dimer fatty acids, 143–146, 143f–146f Dimerization of rosin, 149, 150f of tall oil fatty acid, 143, 143f Dimethylallyl diphosphate, 82–83 7,8-Dimethyl-10-(D-19-ribityl) isoalloxazine. See Vitamin B2 Dimethylsulfoniopropionate (DMSP), 428 Direct liquefaction, 117–118 Disproportionated rosin, 148–149, 149f Distillation, in alcoholic fermentation, 188 Distilled dimer acid, 143 Distiller’s dried grains with solubles (DDGS), 12–13 Distillery slop, 188 DNA -modifying enzymes, 493 polymerases, 493 technology, application of enzymes in, 493 Docosahexaenoic acid (DHA), 250–252, 641 Domestic wastewater, 300–301 Dough process. See Martin process DOW, 260 Drum dryers, for microalgae, 60–61 Dry milling, 12–13, 186–187 maize, 206

Drying of microalgae, 58–61 conveyor dryers, 61 drum dryers, 60–61 flash dryers, 60 freeze dryers, 61 methods, comparison of, 59t by solar energy, 59–60 spray dryers, 60 Drying oils, 250–252 Dunaliella, 77, 81 Dunaliella salina, 50, 53, 67, 622 Dunaliella tertiolecta, 67–69 Dutch Biomass Technology Group (BTG), 354, 356f, 357

E Ecklonia cava, 617 Ecological applications of exopolysaccharides, 540–541 Economic value of biomass, 7–8, 8t Eicosapentaenoic acid (EPA), 75–76 Elaeis guinensis. See Oil palm Electrodialysis, 104–105 Electroneutral starch ethers, 214–215 Ellagic acids, in cosmetic formulation, 615 Ellagotannins, 615 EMPYRO project, 357 Emulsan, 515–516, 515f, 643–644 Ene reaction with tall oil fatty acid, 141–142, 142f Energy balance, in biogas biorefineries, 288–291, 289f–290f Energy crops, for biogas, 277, 280t Energy Independence and Security Act of 2007 (EISA), 40 Energy management in Jenfelder Au, Hamburg, 329–334 Energy Research Center of the Netherlands (ECN), 354, 355f Energy-driven biorefineries, 7 Enhanced oil recovery (EOR) technology, 504, 516 Enterobacter, 541 Enterobacter cloacae, 529, 541 Environmental aspects of industrial vegetable oil biorefineries, 266 Environmental Protection Agency (EPA) waste code, 307f, 308 Enzymes conversion of starch, 217–219, 218t

Index

in cosmetics, 638–640 entrapment, 396 immobilization, 394–400, 394f cross-linked enzymes, 397–398 nanobiocatalysis, 398–400 techniques, 395t industrial, 473–498 modification, for amino acid production, 453 oxidation of starch, 222–223 pretreatments, for biocatalysis, 402–403 synthesis, for amino acid production, 447 Epicerol® process, 260 Epichlorhydrin, 260–263, 261f Epidermal growth factor (EGF), 638 Epoxy curing agents, 145, 145f 2,3-Epoxypropyl trimethylammonium chloride, 214–215 Eremothecium ashbyi, 623–624 Erythritol, 380–381 Escherichia coli, 23–24, 192–193, 195, 372–374, 378–380, 382–383, 403, 416, 428–429, 446, 448, 462–464, 466, 482–483, 513, 580, 584, 592, 621, 625–626, 635, 641 glucose-specific phosphotransferase system in, 373f Essential fatty acids (EFAs), 250–252 in cosmetics, 640–644 Esterification of tall oil rosin, 150, 151f Esters sorbitan, 224–225, 224f starch, 214, 214f tall oil fatty acid, 138–139, 151f tall oil rosin, 150 Ethanol, 11, 39–40, 82, 113, 186, 187f, 277–278, 371, 379–380, 403, 492 -based C2 value chain, 190–192, 190f bioethanol to ethylene, 190–191 ethyl esters, 192 ethylamine, 191–192 from sugarcane in Brazil, 237–246 Ethanol fermentation. See Alcoholic fermentation Ethene, 140 Ethenolysis of tall oil fatty acid, 141, 141f Ethers, starch, 214–215 Ethyl esters, 192 Ethylamine, 191–192 Ethylbenzene, 190 Ethylene, 190–191 product trees derived from, 22f

Ethylene glycol, 188 Ethylene lactic acid. See 3-Hydroxypropionic acid (3-HP) Ethylene oxide (EO), 191 Ethylene-vinyl acetate (EVA), 191 Ethylenglycol diabietate, 150 2-Ethylhexanol, 138 Euglena gracilis, 623 Eukaryotic microalgae, 48–49 Eupenicillium, 421 European Food Safety Authority (EFSA) guide, 666, 669–670 European Forest-based Technology Platform, 13–14 European Landfill Directive, 316 European Union (EU) regulation, for food additives, 669 European waste catalogue (EWC), 300 European Waste Directive, 300 Eurostat, 300, 322–323 Evonik, 189, 451, 460–461, 463–464, 507–508, 642 Exopolysaccharides (EPSs), 523–554 advantages and disadvantages in microbial production of, 524–526 composition and structure, 526–527 from extremophiles, 546–547 market-valued, properties and industrial applications of, 536–546 agricultural and ecological applications, 540–541 cosmetic and personal care applications, 541–542 food applications, 538–540 miscellaneous applications, 545–546 oil industry application, 542–543 pharmaceuticals and biomedical applications, 543–545 production, 533–536 genetics and regulation of microbial polysaccharide expression, 535–536 microbial production conditions, 533–535 properties and structure–function relationships, 528–531 pseudoplastic behavior, 627f roles in prokaryotic cell, 531–532, 531f synthetic pathways, 533 Extracellular DNA (eDNA), 529–530 Extractable volatile oils, from pine tree, 130–136

691

692

Index

Extraction from microalgae, 64–67 solvent extraction, 64–65 supercritical fluid extraction, 65–67 Extractive distillation with liquid solvent, 188 with soluble salt, 189 Extremophiles, exopolysaccharides from, 546–547

F Fast pyrolysis, 114–115, 342–347, 350–354, 358, 362–363 Fatty acid methyl esters (FAMEs), 78–79, 115–116, 204 Fatty acids, valorization of, 259–262 Feature Complexity Index (FCI), 25–26 Feedstock pyrolysis-based hybrid biorefineries, 360–362 vegetable oil, 249–252 Fermentable sugars, acid hydrolysis and pyrolysis for production of, 353f Fermentation, 192–201, 413–417, 422–423, 425, 431–434, 445–446 amino acids, 200–201 glutamic acid, 200–201 assays, 393 C3 building blocks, 192–193 3-hydroxypropionic acid, 193 isopropanol, 193 lactic acid, 192–193 C4 building blocks, 194–198 biosuccinic acid, 196–197 isobutanol, 195 n-butanol, 194–195 enzymes, 478–479, 479f exopolysaccharides, 533–534 glutamic acid, 454, 456 higher carboxylic acids, 198–200 citric acid, 198–200 sulfite spent liquors, 108–109. See also Alcoholic fermentation; Solid-state fermentation (SSF); Submerged fermentation (SmF) Fertilizer production, 284–288 digestate composition of typical substrate mixes, 285t digestate treatment strategies, 286f digestate utilization and upgrading strategies, 287t Ferulic acid, in cosmetic formulation, 616 Feruloyl esterases, 392 5-O-Feruolyl-a-L-arabino-furanoside, 392

Filamentous microalgae, 48 Filtration, for microalgae, 57–58 First-generation biofuels, 115–116 Fischer glycosidation, 223–224 Fischer–Tropsch process, 114 Fish oil, 75 Flash dryers, for microalgae, 60 Flash pyrolysis, 347–348 Flashing light effect, 50 Flat plate reactors, for microalgae, 54–55 Flex-fuel cars, 240 Flocculants, for microalgae, 56, 56t Flocculation, 541 Flotation, for microalgae, 56–57 Fluidized bed technology, 362–363 Fluorogenic assays, 392–393 Food additive, 668–669 Food and Agriculture Organization (FAO), 668 Food and Drug Administration (FDA), 668–669 Food industry application of enzymes in, 486–489 application of exopolysaccharides in, 538–540, 539t losses, 322, 323f production residues, biogas production from, 275–276, 279t wastes, 322–324, 324t Forest residue, chemical composition of, 94t Forest-based biorefinery, 13–14 Formic acid, 105 Fortified rosin soaps, 148, 148f Fortum Oil Oy, 20 Fossil feedstocks, 22 CHO composition of, 22t economic value of, 8t Fractionation in pyrolysis-based hybrid biorefineries, 360–362 in vegetable oil biorefineries, 258 of biomass, pyrolysis-based, 342–348 algae, 345–348 macroalgae, 347–348 microalgae, 347 value-added products from lignocellulosic biomass pyrolysis, 344–345, 345t–346t Freeze dryers, for microalgae, 61 Froth flotation process, 132–133, 134f Fructans, 655. See also Fructooligosaccharides (FOS); Inulin

Index

available in market, 664t–665t -type homopolysaccharides, 533 Fructansucrases, 533 Fructooligosaccharides (FOS), 655–658, 667 available in market, 664t–665t food application, 663 yields of, 661t Fructose syrups, 488 Fruit juice industry, application of enzymes in, 489 FTases, 658 Fucose, 529, 544 Fucus vesiculosus, 617 Fumaria officinalis, 430 Fumaric acid, 430–432 Funding Opportunity Announcements (FOAs), 41t–45t Funori, 72 2,5-Furandicarboxylic acid (FDCA), 198 Furanix Technologies, 199f Furfural, 350–351 extraction plant, 363–364, 364f Fusarium moniliforme, 393

G Galactooligosaccharides (GOS), 655–658, 667 available in market, 664t–665t food application, 663 yields of, 659t–660t Gallic acid, in cosmetic formulation, 615–616 γ-carrageenan, chemical structure of, 71f γ-linolenic acid (GLA), 67, 250–252 Gasification, 114 black liquor, 116–117 use in kraft pulp mill, 116f Gasoline in Brazil, 240, 242 prices of, 242, 243f GDP, 392 GEA Westfalia Three-Phase Technology, 209 Gelatin, 424–425 Gelatinization, 211–212, 219 Gellan, 528, 534, 536–538, 540, 543, 545–546, 656, 666–667 in cosmetics, 630 Gelrite, 541 Genetics of microbial polysaccharide expression, 535–536 Genome shuffling, 412 Geobacillus tepidamans, 532, 547 Geobacillus thermantarcticus, 546

Geobacillus thermodenitrificans, 544–545 Geographical aspects of industrial vegetable oil biorefineries, 264 Geotrichum, 428 Germany food wastes in, 323 waste history of, 299. See also Hamburg (Germany) Global analysis and modification of substrates coutilization system, 374–375 Global market of enzymes, 494–496, 495f of starch, 205–206 Glucanases, 491 Glucans, 488 -type homopolysaccharides, 533 Glucansucrases, 533 Glucoamylase, 476–477, 486–488, 493 Glucoisosaccharinic acid, 105 Gluconacetobacter diazotrophicus, 533 Gluconate, 584 Gluconic acid (GA), 202–203, 202f, 223, 423 Gluconobacter, 622–623 Gluconobacter oxydans, 402 Glucooligosaccharides, 655 Glucose, 13–14, 369, 371–375, 403, 431, 462–463, 492, 584 syrups, 217 composition, 219t Glucose isomerase, 477 Glucose oxidase (GOD), 400, 488, 492–493 Glutamate, 454–456, 455f Glutamic acid, 200–201, 454–457 Glutathione, 381–382 Glycemic carbohydrates, 666 Glycerin, 79 Glycerol, 23, 260–263, 261f, 429 esters of rosin, 150 valorization of, 259–262 Glycerol carbonate, 403–404 Glycerol rosinate, 150 Glycine, 447 Glycolic acid, 105 Glycolipids, 502–509 Glycolysis, 183 Glycoside enzymes, 658 Glycosyl transferases, 392, 492 Glyoxylic acid, 418 Graft polymers, 556, 557f

693

694

Index

Grafting from method, 399–400 Grafting onto method, 399 Grain legumes. See Pulses Grain sorghum, 162–163 Grass silage, 291 Gravity sedimentation, for microalgae, 55–56, 56t Green algae, 16 Green biodiesel, 20 Green biorefineries, 15, 291 Green Building Blocks for Biobased Plastics, 5 Green detergents, 508–509 Green macroalgae, 46–47 Green microalgae, 51 Greenhouse gas (GHG) emissions, in biogas biorefineries, 288–291, 289f–290f Growth of microalgae, 49–52 autotrophic, 49–50 carbon storage, 51–52 heterotrophic/mixotrophic, 50–51 GTE-Technology, 260 Guar gum, 656 Guerbet reaction, 194–195 Gum turpentine, 130

H Haematococcus, 77–78 Haematococcus pluvialis, 77–78, 617 Hair cosmetics, 614 Haloferax denitrificans, 527 Haloferax mediterranei, 527 Halomonas, 532, 542–543, 563, 579 Halomonas alkaliantarctica, 546 Halomonas anticariensis, 547 Halomonas boliviensis, 584 Halomonas eurihalina, 529, 547 Halomonas levan, 547 Halomonas maura, 547 Halomonas smyrnensis, 526–527, 535–536, 547 Halomonas ventosae, 547 Hamburg (Germany) bioresource inventories in Bergedorf district, 326–328 inventory methods, 326–328, 328f inventory results, 328 bioresource treatment processes used in, 316–319, 317t utilization efficiency, 318f coupling of wastewater, waste, and energy management in Jenfelder Au, 329–334 biogas generation, 332–333, 332f

digestate utilization, 332f, 333–334 inventory, 329–332, 331t HAMBURG WATER Cycle®, 330f Hansenula anomala, 421 Hansenula polymorpha, 635 Happy Elephants™, 505–506 Hard liquor, 185 Hardwood kraft pulping, 99–100 Health food, from microalgae, 74–75 Heme-containing catalase enzyme, 492 Hemicellulose, 262 pyrolysis of, 342, 344–346, 350–353, 362–363 thermal stability regimes for, 344f Hemicelluloses, 13–14, 107, 109, 113, 488 Henkel, 213 Heterofermentative lactic acid bacteria, 192–193 Heterologous substrate catabolic pathway, 371–372 Heteronuclear Multiple-Bond Correlation (HMBC), 527 Heterotrophic microalgae, cultivation of, 50–51 HIAG process, 189 High fructose corn syrup (HFCS), 205–206 High fructose starch-based syrups (HFSS), 219 High oleic sunflower oil (HOSO), 256 High Rate Pond (HRP) design, 39 Higher carboxylic acids, 198–200 citric acid, 198–200, 200f High-pressure homogenizers, for microalgae, 63 Homofermentative lactic acid bacteria, 192–193 Homopolymers, 556, 557f Hordeum vulgare L. See Barley Hot-water extraction, 110 Household cleaners, 505–506 Human growth factor, in cosmetics, 638, 639t Hyaluronan, 543 Hyaluronic acid, in cosmetics, 633–634 Hyaluronic acid (HA), 538, 541–542 Hyaluronidase, 640 Hybrid biorefineries, pyrolysis-based, 341–368 Hybrid enzymes, 400–401 Hydracrylic acid. See 3-Hydroxypropionic acid (3-HP) Hydrated ethanol, in Brazil, 240, 243 prices of, 242, 243f production cost, 244f Hydrocarbon biorefinery, 14–15 Hydrochloric acid, 392–393 Hydrodeoxygenation, 20, 260–261 Hydrogen, 280–281, 284, 379–380 Hydrogen cyanide, 432–433

Index

Hydrogen peroxide, 492 Hydrogenase 3, 581 Hydrogenated rosin, 148–149, 149f Hydrogenated starch hydrolysate (HSH), 223–224 Hydrogenation of starch, 223–225 Hydrolysates, hardwood/softwood, composition of, 112t Hydrolysis starch, 218t in sulfite pulping, 106 Hydrothermal carbonization, 67, 284–285 Hydrothermal liquefaction (HTL), 46 Hydrothermal processing in pyrolysis-based hybrid biorefineries, 362 of microalgae, 67–69, 68f, 68t Hydrous ethanol, 188 Hydroxy acids, 105 in cosmetic formulation, 617–619 3-Hydroxyaldehydrate (3-HPA), 375–378 3-Hydroxyalkanoate methyl esters (3HAME), 565, 565f, 568–569 Hydroxyalkanoates, 555–574 3-Hydroxyalkanoic acids (3HAs), 570 2-Hydroxybutanoic acid, 105 3-Hydroxybutyrate methyl ester (3HBME), 565, 565f, 568–569, 568t 3-Hydroxybutyronitrile, 392 3-Hydroxydecanoic acid (3HD), 570 3-Hydroxydodecanoic acid (3HDD), 570 Hydroxyethyl starch (HES), 214–215, 215f 3-Hydroxyhexanoic acid (3HHx), 570 5-Hydroxymethylfurfural (HMF), 15 2-Hydroxymethyl-5-hydroxy-γ-pyrone. See Kojic acid 3-Hydroxyoctanoic acid (3HO), 570 2-Hydroxypropane-1,2,3-tricarboxylic acid. See Citric acid 2-Hydroxypropanoic acid. See Lactic acid 3-Hydroxypropionaldehyde (3-HPA), 430 3-Hydroxypropionic acid (3-HP), 193, 375–378, 428–430 Hydroxypropyl starch (HPS), 214–215 3-Hydroxytetradecanoic acid (3HTD), 570 HZSM-5 zeolite, 190

I Immobilized enzymes, 493–494 In silico modeling, 401 In vivo metabolism, coproduction in single cell to maintain, 375–379

Inbicon Biomass Refinery, 349–350 Incineration, 315, 318–319 Indirect liquefaction, 117–118 Industrial biocatalysis, 476 Industrial enzymes, 473–498 application, 486–493 animal feed industry, 490–491 baking industry, 488 biofuel from biomass, 491–492 chemistry and pharma sectors, 492–493 dairy industry, 488–489 detergent industry, 489–490 DNA technology, 493 food industry, 486–489 fruit juice industry, 489 leather industry, 491 personal care products, 492–493 pulp and paper industry, 490 specialty enzymes, 492 starch industry, 486–488 textile industry, 489 classification, 475–476, 475t existing commercial process vs. enzymatic processes, 475–476 downstream processing, 484–485, 485f enzyme immobilization, 493–494 global enzyme market scenario, 494–496, 495f microbial enzyme production, 476–486, 477t product formulation, 485–486 production technology, 478–482 solid-state fermentation, 479f, 481–482 submerged fermentation, 479–480, 479f sale by sector, 495f strain improvement, 482–484 mutation, 482 protein engineering, 483–484 recombinant DNA technology, 482–483 Industrial vegetable oil biorefineries, 264–266 environmental aspects, 266 geographical aspects, 264 socioeconomic aspects, 265–266 Industrial wastewater, 300 Industrial yeast, 187–188 Insulin, 482–483 Integrated forest biorefinery (IFBR), 92 Integrated multitrophic aquaculture (IMTA), 47 International Energy Agency (IEA) Bioenergy, 4–5 Bioenergy Task, 4–7, 9, 25, 42 Inulin, 655 available in market, 664t–665t

695

696

Index

Inverted sugar syrup, 202, 202f Irish moss, 71, 71f Isoborneol, 132–133 Isobutanol, 195 Isomalt®, 201–202 Isomaltooligosaccharides (IMO), 655, 663 Isomaltulose, 201–202 Isoparaffinic fuel, 20 Isopentenyl pyrophosphate, 82–83 Isoprenoids, 82–83 Isopropanol, 193 Isosorbide, 225 Isostearic acid, 144, 144f Isostearyl alcohol, 144 Iturin A, 382–383

J Juncus maritimus, 403

K Kalundborg (Denmark), exchanging industrial outputs for mutual benefits in, 334, 335t Kalundborg SYMBIOSIS, 326 Kampungs, 298 Kaneka Surfactin™, 511 Kappa. See Cassava Karlsruhe Institute of Technology (KIT), 357–359 bioliq® plant, 358f Kelcogels, 541–542 Keratin, in cosmetics, 636 Keratin hydrolysate, in cosmetics, 636 Kinases, 493 Kior, 14 Kirchhoff, Gottlieb Sigismund Constantin, 159, 219 Kirschwasser, 186 Kitchen waste collection chain, 313, 315f Klebsiella oxytoca, 220 Klebsiella pneumoniae, 429–430 Kluyveromyces, 478 Kojic acid, in cosmetic formulation, 619–620 Kraft black liquor, 116–118 gasification, 116–117 liquefaction, 117–118 Kraft pulping, 92, 93f, 95, 97–105 behavior of primary feedstock constituents, 98–100 black liquor-based by-products, 100–105 aliphatic carboxylic acids, 103–105, 104f

extractives—turpentine and tall oil, 100–101 lignin, 102–103 composition of black liquor, 98t pretreatment stages in, 110f Krebs cycle, 196, 198, 378, 417, 454 KREIS project, 326, 329 Kyowa Hakko, 200

L Laccase, 489 Lactase, 488–489 Lactate, 492 and poly-3-hydroxybutyrate production, 582 Lactate and acetate mixture, and PHB production, 582 Lactic acid, 23, 105, 192–193, 409–412, 432–434, 454 producing microbes, 434t Lactic acid bacteria (LAB), 192–193, 533 Lactobacillus, 533, 579, 616, 667 Lactobacillus brevis, 374 Lactobacillus buchneri, 374 Lactobacillus bulgaricus, 528 Lactobacillus casei, 192–193 Lactobacillus delbrueckii, 192–193, 528, 581 Lactobacillus helveticus, 192–193 Lactobacillus plantarum, 416 Lactobacillus reuteri, 533 Lactococcus lactis, 192–193 Lactonic sophorolipids, 506 Lactose, 492 Lactosucrose (LS), 655 Lactulose, 655 available in market, 664t–665t λ-polyglutamic acid, in cosmetics, 634 Laminaria japonica, 403 Laminaria saccharina, 347–348 Land-based pond system, for macroalgae, 47–48 Landfill gas, 273–274 Landfilling, 316 Large bags, for microalgae, 54 L-aspartic acid, 432 Latvian State Institute of Wood Chemistry (LSIWC), 350–351 integrated biorefinery system, 352f Laurics, 250–252 Leather industry, application of enzymes in, 475–476, 491 Legionella, 530

Index

Legislation, regarding algal research in United States, 40 Legumes, 176–177 Lentinus edodes, 615, 631 Leucine, 447 Leuconostoc, 533 Leuconostoc mesenteroides, 533, 629 Levan, 526–527, 534, 537, 541–543, 546 in cosmetics, 638 Levoglucosan, 350–351, 365 Levopimaric acid, 148 Levulinic acid, 113 Lichenysin, 514–515, 514f Ligases, 493 Light saturation, role in autotrophic growth of microalgae, 49–50 Lignin, 13–14, 98, 102–103, 259–260, 262, 264 pyrolysis of, 342, 344–346, 350–351, 362–365 thermal stability regimes for, 344f Lignin Biorefinery Approach (LIBRA), 360, 360f LignoBoost process, 102 Lignocellulosic biomass, 371, 491–492 accessible products from, 257f CHO composition of, 22t feedstock biorefinery, 13–14 pyrolysis European biorefineries, 349–350 hybrid refinery, 361f value-added products from, 344–345, 345t–346t Lignosulfonates, 105–106, 108 Liliales, 655 Limonene, 82–83, 131–132 Lina blue, 78 Linablue HGE, 78 Linoleic acid, 75–76, 139–140, 142–143, 250–252 structure of, 140f Lipases, 490–491 -containing enzymes, 492 in cosmetics, 639–640 Lipid derivatives, 640–644 Lipid trigger, 38–39 Lipolytic enzymes, 491 Lipopeptides, 382–383, 510–515 Lipoproteins, 510–515 Liposan, 516–517 Liquefaction, 219 kraft black liquor, 117–118 of microalgae, 67–69

use in kraft pulp mill, 116f Liquid phase catalytic processing, 15 Liquid Sorbitol, 223–224 Literature, civilization biorefinery data from, 327 L-malic acid, 432 Locust bean gum, 656 Long chain fatty acids starch esters of, 214f Lontar palm. See Asian Palmyra palm Lysine, 447–448, 457–461 biosynthetic pathways, 458f

M Macroalgae, 46–48 cultivation, 47–48 fundamentals related to, 46–47 pyrolysis of, 347–348 traditional uses of, 69–72 agar, 70–71 alginates, 72 carrageenan, 71–72 funori, 72 Magnesium, 137–138 Maize, 166–168, 167f in alcohol fermentation, 186–187 kernel, 168f production, 166f starch processing, 206–208, 207f dry milling, 208 wet milling, 206–207 Maleic anhydride, 430 Malted grain, 184–185 Maltodextrins, 217 Maltooligosaccharides (MOS), 655 Maltose, 462–463 syrup, 219 Mandioca. See Cassava Manganese, 137–138 Manihot esculenta. See Cassava Manioc. See Cassava Mannheimia succiniciproducens, 416 Mannose (C6), 13–14 Mannosylerythritol lipids, 508–509, 508f–509f, 508t, 643 Maple syrup, 164 Marggraf, Sigismund, 159 Mariculture, 47 Marine biorefinery, 16 Marine crops, 16

697

698

Index

Martek, 75–76 Martin process, 208–209 Mass balance, in biogas biorefineries, 288–291, 289f Massecuite, 160, 182–183 Mechanical grass fractionation, 15 Medical implants, 569 Meerwein–Ponndorf–Verley (MPV) reduction, 132 MEL-A, 643 Melanin, 611, 612f MEL-B, 643 Mesoporous materials, 399 immobilization of enzymes in, 399t Mesopotamia, 296 Metabolic engineering for amino acid production, 452–454 amino acid efflux, 453 anaplerotic pathways or precursor supply chain, 453 central carbon metabolism, 453 for higher energy efficiency, 453 modifying enzymes in terminal pathways, 453 NADPH metabolism, 453 of propionibacteria, 415 for substrate broadening, 454 Metabolic regulation for simultaneous substrates utilization, 372–374, 373f Metagenomic assays, 393–394 Metallosphaera sedula, 428 Methane, 280–281 as platform chemical, 284 Methanol, 492 Methionine, 447, 461–463 Methyl rosinate, 150 Methylerythritol phosphate (MEP) pathway, 82–83 Metroxylon sagu. See Sago palm Mevalonate (MVA) pathway, 82–83 Microalgae, 36–37, 48–55 biodiesel production from, 37 cell disruption, 61–64, 62t chemical methods, 62–63 mechanical methods, 63–64 conventional nonfuel products, 72–78, 73f, 74t health food, 74–75 pigments, 76–78 polyunsaturated fatty acids, 75–76 squalene, 76 culture systems, 52–55 closed PBRs, 52t, 53–55 open ponds, 52–53, 52t

dewatering, 55–58 centrifugation, 58 filtration, 57–58 flotation, 56–57 gravity sedimentation, 55–56, 56t methods, assessment of, 59t downstream processes, 55–69 drying, 58–61 conveyor dryers, 61 drum dryers, 60–61 flash dryers, 60 freeze dryers, 61 methods, 59t by solar energy, 59–60 spray dryers, 60 extraction, 64–67 solvent extraction, 64–65 supercritical fluid extraction, 65–67 fundamentals related to, 48–49 growth, 49–52 algal carbon storage, 51–52 autotrophic, 49–50 heterotrophic/mixotrophic, 50–51 hydrothermal processing, 67–69, 68f, 68t nonconventional products from, 73f, 78–83 biodiesel, 78–80 bioethanol, 80–82 terpene-based biofuels, 82–83 pyrolysis of, 347 recently funded projects in United States related to, 41t–45t Microbial fermentation for amino acid production, 445–446, 448–451 for organic acids, 418, 433–434 Microflotation, for microalgae, 57 mineralit® GmbH, 60–61 Mixotrophic microalgae, cultivation of, 50–51 Modified starches, 214–227 Mogo. See Cassava Molasses, 592 desugarization, 183 Molecular sieves, 189 Monomers as chiral intermediates, 570 Monosodium glutamate (MSG), 200, 201f, 446, 451, 456–457 Monoterpenes, 100 Mortierella alpina, 75–76 Mucor, 431 Mucor miehei, 639–640

Index

Mucor piriformis, 421 Mucor rouxii, 633 Multichain use efficiency (MUE), 325 Multienzyme reactions, 401–402 Municipal solid waste (MSW), 300–301 Mutagenesis, for amino acid production, 452 Mutation of strain for enzyme production, 482 Myanmar Spirulina Factory, 75 Mycosporine, in cosmetic formulation, 621 Mycosporine-like amino acids (MAAs), in cosmetic formulation, 621 Myrmicacin, 570

N NAD kinase, 594–595 NADPH metabolism, and amino acid production, 453 Nannochloropsis oculata, 67 Nanobiocatalysis, 398–400 Nanofabrication, 398 National Algal Biofuels Technology Roadmap, 40 National Renewable Energy Laboratory (NREL), 38, 46 Native dextran, 666–667 Native starch, 211 Natural gas methane, 284 Natural gases, composition of, 21t n-Butanol, 194–195 Nelson’s complexity index, 25 Neolithic transition, 296, 296f Neste Oil, 13 Neutral proteinase, 489 NExBTL process, 13, 20 Next generation hydrocarbon biorefinery, 14–15 Next-generation sequencing (NGS) technologies, 535–536 Nicotinamide, 624–625 Nicotinic acid. See Vitamin B3 Nile blue, 578–579 Nile red, 578–579 Nitrogen, role in citric acid production, 422 N-methyl-2-pyrrolidone, 196–197 Nodax, 566 No-distill ethanol fermentation, 379 Nonconventional products from microalgae, 78–83 Nondigestible carbohydrates, 666 Nondrying oils, 250–252 Nonfuel products from microalgae, 72–78, 73f, 74t Nonreactive polyamides, 145–146, 146f

Nonstarch polysaccharides, 653–654 Nonsugars, 181 Nonylphenol ethoxylates (NPE), 499–500 Norway spruce (Picea abies), 106t Nostoc, 36, 65 Novel biocatalyst, screening for, 392–394 chromogenic and fluorogenic assays, 392–393 fermentation assays, 393 metagenomic assays, 393–394 n-paraffin, 418 Nuclear magnetic resonance (NMR) techniques, 527 Nuclear Overhauser Effect Spectroscopy (NOESY), 527 Nucleases, 493 Nutracosmaceuticals. See Vitamins

O Octyl epoxy tallate (OET), 138 Offshore cultivation of macroalgae, 47–48 Oil crops, 249 composition, 249–252 oil composition, 250–252, 254t whole-plant composition, 249–250 production areas, 250t Oil drilling, 136–137, 136f Oil industry application, 542–543 Oil of turpentine. See Turpentine Oil palm, 164–166 Oil processing gases, composition of, 21t Oilseeds, 249 biomass, products obtained from, 262f world production, 251f–253f Olefin metathesis, 191 Oleic acid, 139–140, 143 Oleics, 250–252 Oleochemicals, 500–501 biorefinery, 13 Oligosaccharides, 653–680 extraction, production, and purification methodologies, 654t, 656–662 concerns, 656 downstream techniques, 662t traditional methods, 656 food applications, 657t, 662–666 health and nutritional benefits, 666–668 regulation and safety aspects, 668–670 Omega-3 fatty acids, 75–76, 640–641 Omega-6 fatty acids, 75–76, 641

699

700

Index

Omics, 415, 428 Open ponds, for microalgae, 52–53, 52t Organic acids, 380–381 used in cosmetics, 617–620 white biotechnology for, 409–444 α-ketoglutaric acid, 417–420 citric acid, 420–428 fumaric acid, 430–432 3-hydroxypropionic acid, 428–430 lactic acid, 432–434 productions and applications, 410t–411t propionic acid, 412–415, 414t succinic acid, 415–417 Organic carbon, 51 Organic Fraction of Municipal Solid Wastes (OFMSW), 273, 275, 278–279 Organic wastes, biogas production from, 278–279, 279t Organic wastewater, anaerobic and aerobic treatment of, 274f Organisation for Economic Co-operation and Development (OECD), 4–5 Organosolv fractionation, 362–363 Organosolv pulping, 107–108 OriginOil, 57 Oryza glaberrima (African rice), 172 Oryza sativa (Asian rice), 172 Oscillatoria, 582 Oxalic acid, 423 Oxygen, role in citric acid production, 423–424

P Paenibacillus, 512, 514 Paenibacillus amylolyticus, 512 Paenibacillus polymyxa, 512–513 Palatinose™, 201–202 Palm oil, 565 Palm oil mill effluent (POME), 278 Pantothenic acid. See Vitamin B5 Papain, 489 Paper chain, interrelations between bioresources in, 308–310, 309f Paper industry. See Pulp and paper industry; Pulp mills Paper sizing, 148, 148f Paracoccus denitrificans, 625–626 Peas, 177f starch, 176–177, 210–211 Pectinase, 489

Pediastrum, 48 Peeling, in kraft pulping, 98–99 Penicillin, 454 Penicillium, 481, 615–616 Penicillium glaucum, 421 Penicillium janthinellum, 421 Penicillium purpurogenum, 658 Penicillium restrictum, 421 Pentaerythritol rosinate, 150 Pentaerythritol tetratallate, 138 Pentosans, 197, 198f Perfluorinated surfactants, 499–500 Perfluorooctane sulfonate (PFOS), 499–500 Peripheral blood mononuclear cells (PBMC), 544–545 Peroxidases, 492 Personal care products applications of exopolysaccharides for, 541–542 application of enzymes for, 492–493 Personal interviews, civilization biorefinery data from, 327 Petrobras, 20, 242 Petrochemical refineries, 227 base petrochemicals, 19f biobased platform molecules, 19f product trees derived from ethylene, 22f vs. biorefineries, 17–24, 18t Petroleum industry, use of rhamnolipids in, 504 Phaeodactylum tricornutum, 62, 75–76 Phaeophyceae, 46–47 Pharmaceuticals application of enzymes in, 492–493 applications of exopolysaccharides in, 543–545, 543t–544t P3HB4HB, 556–557, 566–568 PHBHHx, 566–568 PHBV, 556–557, 566–568 Phenylalanine, 448, 465 Phlorotannins, in cosmetic formulation, 617 Phoenix canariensis. See Date palm Phosphate, role in citric acid production, 422–423 Phosphoenol pyruvate carboxykinase, 459–460 Phosphofructokinase, 425 Phosphotransferase system (PTS), 369 Photoautotrophic microalgae, cultivation of, 49–50 Photobioreactors (PBRs), for microalgae, 50, 52t, 53–55, 82 Photoinhibition, 49–50 Photo-protective cosmetics, 613–614

Index

Phycobiliproteins, 78 Phycocyanin, 78 Phycoerythrin, 78 Physical treatment of starches, 211–212 Phytase, 491 Phytic acid, 491 Phytophthora, 504 Phytophthora capsici, 504 Phytoplanktons. See Microalgae Picea abies. See Norway spruce Pichia, 418 Pichia naganishii, 618 Pichia pastoris, 635 Pigments, 76–78 Pine balsam. See Pine resin Pine biorefinery platform chemicals value chain, 127–156 crude tall oil, 136–137 extractable volatile oils, 130–136 tall oil fatty acid, 137–146 cooligomerization, 140–141 Diels–Alder and ene reactions, 141–142 dimer fatty acids, 143–146 esters and other derivatives involving carboxyl group, 138–139 ethenolysis, 141 soaps, 137–138 tall oil alkyd resins, 139–140 tall oil resin, 146–150 dimerization, 149 disproportionated and (per)hydrogenated rosin, 148–149 rosin esters, 150 rosin soaps and fortified rosin soaps, 148 tall oil value chain, 136–150 Pine oil, 100, 131–132 Pine oleoresin. See Pine resin Pine resin, 130 Pisum sativum, 176–177 Pitch (softwoods), 490 Plackett–Burman experimental design, 402–403 PLAHB, 556–557 Pleurotus ostreatus, 631 p-nitrophenyl, 392–393 Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (P (3HB-co-3HV)), 596 Poly (hydroxy-butyrate–valerate) (PHBV), 576 structure of, 577f Polyesterpolyols, 146

Polyethylene (PE), 190 Polyethylene furanoate (PEF), 198, 199f Poly-γ-glutamic acid (c-PGA), 382–383 Polyhydroxy acids, 617 Polyhydroxy bionic acids, 617 Polyhydroxyalkanoates (PHAs), 373–374, 380, 555–574 classification, 556 produced in industrial scale, 560–572, 561f applications of, 565–566, 566f, 567t biofuels, 568–569, 568t, 569f challenges for R&D, 570–571 continuous process, 563, 564f environmentally friendly bioplastic packaging, 566–568 fed-batch process, 561–563 future prospective, 571–572 medical implants, 569 mixed cultures, 563–565, 565f monomers as chiral intermediates, 570 smart materials, 570 strains for production of, 559–560, 560f, 562t produced and research companies, 558t Poly-3-hydroxybutyrate (PHB), 373–374, 378–379, 575–606 advantages, 576 application, 577–578 biomedical application, 600 bone tissue regeneration, 600 nanofibrous matrices as cell supporting materials, 600 organic-soluble chitosan/PHB fibers for skin regeneration, 600 in packaging, 600 recombinant protein purification, 601 targeted drug delivery, 599–600 characterization of atomic force microscopy (AFM), 591, 592f differential scanning calorimetry (DSC), 590, 591f Fourier transform infrared spectroscopy (FTIR), 588, 589f gel permeation chromatography (GPC), 590–591 nuclear magnetic resonance (NMR), 588–589, 589f thermogravimetric analysis (TGA), 589–590, 590f downstream operation

701

702

Index

Poly-3-hydroxybutyrate (PHB) (Continued ) biological methods, 587–588 chemical methods, 586–587 fermentation, solid-state, 585–586 fermentation, submerged batch, 582–583 continuous fermentation, 584–585 fed-batch, 583–584 mechanical properties, 577 mechanism of synthesis, 578f metabolic engineering, 594–596 mutation, 592, 595 production, 578–582, 579t, 595–599 and carbon sources, 596–598 by cyanobacteria, 582 effect of nitrogen and phosphorus, 598–599 external substrate addition, 596 from fatty acids, 598f inhibitors addition, 599 limitations of, 577, 581, 592 screening of microorganisms, 578–579, 580f using mixed/cocultures, 581–582 using recombinant bacteria, 580–581 using wild-type bacteria, 579 properties, due to carbon manipulation, 598 strain improvement, 592–595 structure of, 577f synthesis from glycerol, 597f Poly(3-hydroxybutyrate) (P3HB), 556–557 Poly(4-hydroxybutyrate) (P4HB), 556–557, 566–568 Poly-3-hydroxydecanoate (PHD), 556–557 Poly-3-hydroxydodecanoate (PHDD), 556–557 Poly-3-hydroxyheptanoate (PHHp), 556–557 Poly-3-hydroxyhexanoate (PHHx), 556–557 Poly-3-hydroxynonanoate (PHN), 556–557 Poly-3-hydroxyoctanoate (PHO), 556–557, 566–568 Poly(3-hydroxyvalerate) (P3HV), 556–557 Polylactic acid, 432–433 Poly(lactide) (PLA), 556–557 Polymerase chain reaction, 483 Polymeric biosurfactants, 515–517 Polymyxin, 512–514, 512f Polymyxin E, 512 Polypeptides, in cosmetics, 634–640 Polyphenols, in cosmetic formulation, 614–617 Poly-(R)-3-hydroxybutyrate (PHB), 566 Polysaccharides, 422, 653–680 in cosmetics, 626–634 exopolysaccharides, 523–554

extraction, production, and purification methodologies, 654t, 656–662 concerns, 656 traditional methods, 656 food applications, 657t, 662–666 health and nutritional benefits, 666–668 obtained from non-traditional sources, 654t regulation and safety aspects, 668–670 Polyterpene resins, 133–136, 135f Polyunsaturated fatty acids (PUFAs), 75–76 in cosmetics, 640–641 Polyvinyl acetate (PVAc), 191 Polyvinyl alcohol (PVAl), 191 Porphyra umbilicalis, 621 Porphyridium, 75–76, 78 Porphyridium cruentum, 62 Potassium tallate, 137–138 Potato, 175–176, 176f fibers, 11 starch production, 11, 208–209 constituents, 175t Prebiotics, 655, 666–667 Precursor supply chain, and amino acid production, 453 Pregelatinized starches, 211–212 Pressing, in vegetable oil biorefineries, 259 Primary bioresources, 302–304, 303f Primary feedstock constituents, in kraft pulping, 98–100 Primary refining, in biogas biorefineries, 272 Primary use efficiency (PUE), 325 Processed primary bioresources, 303–304 Processing chains, of civilization biorefineries, 311–312, 312f Product-driven biorefineries, 7 Products, of biorefinery, 7 PROESA® process, 349–350, 491–492 Prokaryotic microalgae, 48–49 Prokaryotic microorganisms, exopolysaccharides from, 523–554 1,2-Propanediol, 263 1,3-Propanediol (PDO), 375–378, 430 Propene, 140 Propionaldehyde dehydrogenase (PduP), 430 Propionibacteria, 413–415 Propionibacterium, 618 Propionibacterium acidipropionici, 413–415 Propionibacterium acnes, 643 Propionibacterium freudenreichii, 413–415 Propionibacterium jensenii, 413, 415

Index

Propionibacterium thoenii, 413 Propionic acid (PA), 380–381, 409–415 representative production methods, 414t synthesis pathways for, 413f Propionobacter acne, 506 PropTera™, 504 Propylene, 191 synthesis of, 23f Proteases, 490–491 in cosmetics, 639 Protein engineering, 400–401 for enzyme production, 483–484 Proteinases, 488, 491 -containing enzymes, 492 Proteins, in cosmetics, 634–640 Proteolysis, 485–486 Proteolytic enzymes, 491 for PHB extraction, 587 Prototheca, 80 Prototheca moriformis, 80 Prototheca portoricensis, 80 Prototheca stagnora, 80 Prototheca wickerhamii, 80 Prototheca zopfii, 80 Pseudoanaebaena, 582 Pseudomonas, 541, 544–546, 570, 579–581, 588, 596, 618–619 Pseudomonas aeruginosa, 382–383, 503–504, 528–530, 535–536, 580, 597 Pseudomonas dacunhae, 448 Pseudomonas entomophila, 561–563 Pseudomonas fluorescens, 418 Pseudomonas oleovorans, 527, 596–597 Pseudomonas putida, 596 Pseudomonas syringae, 380 Pseudozyma, 508–509, 641 Pseudozyma antarctica, 509 Public bodies, civilization biorefinery data from, 326–327 Public convenience places, 297–298 Pullulan, 533–534, 538, 541–545 in cosmetics, 633 Pullulanase, 486–488 Pulp and paper industry, 13–14, 128, 349 application of enzymes in, 490 Pulp mills, 91–126 chemical pulping methods, 105–109 organosolv pulping, 107–108 possibilities for sulfite spent liquor-based by-products, 108–109

sulfite pulping, 105–107 commercial pulping methods, 96t kraft pulping, 98–105 aliphatic carboxylic acids, 103–105 behavior of primary feedstock constituents, 98–100 extractives—turpentine and tall oil, 100–101 lignin, 102–103 possibilities for black liquor-based by-products, 100–105 pretreatment of wood chips prior to pulping, 109–113 acidic pretreatments, 112–113 alkaline pretreatments, 110–111 pulping processes and by-products, 96–109 thermochemical conversion methods, 113–119 crude tall oil soap, 118–119 gasification, 116–117 kraft black liquor, 116–118 liquefaction, 117–118 Pulping processes, 130f Pulsed electric field (PEF) technologies, for microalgae, 64 Pulses, 176–177, 177f Pyrodextrins, 222 Pyrolysis, 114–115, 284–285 use in kraft pulp mill, 116f Pyrolysis oil. See Biooil Pyrolysis-based hybrid biorefineries, 341–368 compound classes in biooil, 343t European lignocellulosic biorefineries, 349–350 feedstock and feedstock fractionation, 360–362 fractionation of biomass, 342–348 algae, 345–348 macroalgae, 347–348 microalgae, 347 value-added products from lignocellulosic biomass pyrolysis, 344–345, 345t–346t modes of pyrolysis, 343t as peripheral unit for biorefinery side streams, 360 as primary/central unit in biorefinery, 350–359 current developments, 354–359 historical background, 350–354 product recovery, 363–365 production of fermentable sugars, 353f thermochemical and biochemical conversions, 362–363 2-Pyrrolidone, 196–197 Pyruvate carboxylase, 459–460

703

704

Index

Pyruvate kinase, 425 Pythium, 504

Q Quaternary bioresources, 303f, 305 technologies for, 315–316 Quentin-Process, 183 Questionnaires, civilization biorefinery data from, 327 Quorum sensing (QS), 530

R Raceway ponds, for microalgae, 52–53 Radioactive decontamination, 511, 511f Raffinose, 655 Ralstonia, 579 Ralstonia eutropha, 195, 561–563, 577, 581, 583–586, 588, 593, 596 Random copolymers, 556, 557f Random mutagenesis, for amino acid production, 452 Range Fuels, 14 Rapeseed, 252, 256, 258, 263, 278 Raw juice, 181 Raw materials, for biocatalysis, 403–404 Reaction media, for biocatalysis, 404 Reactive oxygen species (ROS), 610 Reactive polyamides, dimer acid-based, 145 Recombinant DNA (rDNA), for enzyme production, 482–483 Rectified spirit, 188 Red macroalgae, 46–47 Redox balancing strategy, 371 Reflection, in autotrophic growth of microalgae, 50 Reforestation, 3–4 Regional network, of civilization biorefineries, 311 Reichstein process, 622–623 Renewable Fuels Standard (RFS), 40 Resazurin, 392 Resorufin, 392 Restriction enzymes, 493 Resveratrol, 616 Retama retam, 403 Revive, 638 Rhamnolipids (RLs), 503–505, 505f, 643 Rhamsan gum, in cosmetics, 630–631 Rhizobium meliloti, 621 Rhizopus, 431–434, 615–616, 639–640 Rhizopus arrhizus, 430–431

Rhizopus delemar, 431–432 Rhizopus oryzae, 431–432 Rhodobacter sphaeroides, 621, 625–626 Rhodococcus erythropolis, 392 Rhodospirillum rubrum, 586 Rhodotorula, 618 Riboflavin. See Vitamin B2 Rice, 172–173 kernel, 173f starch production, 209 Rock maple. See Sugar maple Rododphyceae, 46–47 Rohdella, 78 Rosin esters, 150 Rosin soaps, 148, 148f Royal DSM, 75–76 Rum, 186

S Saccharification, 186–187, 219 Saccharomyces cerevisiae, 183–185, 195, 202, 371, 403, 416, 428, 594, 631, 635, 641–642 Saccharomycopsis lipolytica, 421 Saccharophagus degradans, 583 Saccharum officinarum. See Sugarcane S-adenosylmethionine (SAM), 381–382 Sago palm, 177–178, 178f Sake, 185 Salicylic acid. See Hydroxy acids Salipiger mucosus, 529 Salting out crystallization, 450–451 Sapindus saponaria, 500–501 Sapogenin, 500–501 Saponaria officinalis, 500–501 Saponins, 500–501 Scenedesmus, 48, 81 Scenedesmus dimorphus, 65 Scenedesmus obliquus, 56, 63–64 Schizochytrium limacinum, 75–76 Schizochytrium mangrovei, 641 Schizophyllan, in cosmetics, 632 Schizophyllum commune, 632 Scleroglucan, 542, 666–667 in cosmetics, 632 Sea beet, 160–161 Seaweeds. See Macroalgae Second generation carbohydrates, valorization of, 259–260 Secondary bioresources, 303f, 304

Index

Secondary refining, in biogas biorefineries, 272 Second-generation lignocellulose, 263 Serratia, 446, 618–619 Serratia marcescens, 418, 464 Sewage-grown algae, 57–58 Shading, in autotrophic growth of microalgae, 50 Shale gas, 20 Shear thinning, 626 Shell higher olefin (SHOP) process, 141 Shikimic acid, 402, 465 Short-chain fatty acids (SCFA), 666–667 (S)-3-hydroxybutyric acid, 392 Silver birch acid sulfite spent liquors, 106t alkaline pretreatment of, 111t Simultaneous substrates utilization in single cell, 371–375 global analysis and modification of substrates coutilization system, 374–375 heterologous substrate catabolic pathway, 371–372 metabolic regulation for, 372–374, 373f SIMUP algorithm, 374–375 Single enzyme nanoparticles (SEN), 400, 400f Single-cell biorefinery, 369–388 concept, 370f, 382t coproduction in single cell, 375–381 downstream processing, 379–380 to maintain in vivo metabolism, 375–379 strategies, 376t–377t suitable and value-added product portfolio, 380–381 simultaneous substrates utilization in single cell, 371–375 global analysis and modification of substrates coutilization system, 374–375 heterologous substrate catabolic pathway, 371–372 metabolic regulation for, 372–374 Single-droplet technique, 116 Site-directed mutagenesis, 483–484 Skin lightening cosmetics, 611 Slaughterhouse wastes, biogas production from, 275–276 Slow pyrolysis, 114–115, 127–128, 345–347, 354 Smart materials, 570 Soapberry trees, 500–501 Soaps, tall oil fatty acid, 137–138 Soapwort, 500–501

Socioeconomic aspects of industrial vegetable oil biorefineries, 265–266 Soda pulping, 97 Sodium glutamate, 418 Sodium hypochlorite digestion method, for PHB extraction, 586 Sodium sulfate, 104–105 Sodium tallate, 137–138 Softwood kraft pulping, 99–100, 110 Solanum tuberosum L. See Potatoes Solar energy, drying of microalgae by, 59–60 Solar Energy Research Institute (SERI), 38 Solazyme, 80 Solid-state cultivation, 393 Solid-state fermentation (SSF), 382–383, 425, 456, 658 for enzymes, 479, 479f, 481–482 Solvay, 260 Solvent extraction method from microalgae, 64–65 for PHB extraction, 586 Sopholiance S, 643 Sophorolipids, 505–508, 506f–507f, 643 Sorbitan esters, 224–225, 224f Sorbitol, 223–224, 224f Sorghum, 162–163, 162f Sorghum bicolor (L.). See Sweet sorghum Soxhlet extraction from microalgae, 65 Soybean oligosaccharides (SOS), 655, 663 Soybeans, 12 Span®, 225 Specialty enzymes, 492 Spherezyme technique, 397–398 Sphingans, 528 Sphingomonas, 528, 630–631 Sphingomonas elodea, 528, 630 Sphingomonas paucimobilis, 529 Spirit (liquor), 185 Spirit of amber. See Succinic acid Spirit of turpentine. See Turpentine Spirulina, 63, 69, 75, 78, 81 Spirulina platensis, 64, 69 Spray dryers, for microalgae, 60 Squalene, 76 chemical structure of, 76f in cosmetics, 641 Stachyose, 655 Staphylococcus, 530, 640 Staphylococcus aureus, 621

705

706

Index

Staphylococcus epidermidis, 530 Starch, 159 crops, 159–179 refining and processing, 205–211 corn, 206–208, 207f global market, 205–206 industrial production processes, 206–211 pea, 210–211 potato, 210 rice, 209 tapioca, 210 wheat, 208–209 uses, 211–227 converted starches, 217–227 esters, 214 ethers, 214–215 modified starches, 214–227 native starch, 211 physical treatment, 211–212 starch graft copolymers, 213 Starch acrylonitrile graft copolymers, 213 Starch crop biorefineries, 291 Starch derived polysaccharides, 653–654 Starch graft copolymers, 213, 213f Starch industry, application of enzymes in, 486–488 Starch-based biodegradable plastics, 212 Starch-based biorefineries, 157–236 Starch-based superabsorbent polymers (SAPs), 213 Steam distillation, 363–365 of pine, 127–128, 129f, 131–132 Sterols, 640–644 Stevia, 179 Stevia rebaudiana, 179, 180f Stonewashing, 489 Strains for amino acid production, 451–454 amino acid efflux, 453 anaplerotic pathways or precursor supply chain, 453 central carbon metabolism, 453 for higher energy efficiency, 453 modifying enzymes in terminal pathways, 453 NADPH metabolism, 453 for substrate broadening, 454 for enzyme production, 482–484 mutation, 482 protein engineering, 483–484 recombinant DNA technology, 482–483

for polyhydroxyalkanoates production, 559–560, 560f random mutagenesis, 452 Strengths, weaknesses, opportunities, and threats (SWOT) analysis on biorefineries, 29t Streptococcus, 530, 533, 632–634, 640 Streptococcus epizooticus, 538 Streptococcus equi, 633–634 Streptococcus mutans, 533 Streptococcus zooepidemicus, 379, 633–634 Streptomyces, 477–478 Streptomyces aureofaciens, 580, 593 Streptomyces peucetius, 641 Styrene, 190 Submerged fermentation (SmF), 658 biocatalyst development from, 402 for citric acid production, 422 for enzymes, 479–480, 479f Suboptimal conditions, in autotrophic growth of microalgae, 50 Substrate broadening, and amino acid production, 454 Substrates for biogas production, 275–279, 276t Subtilisin, 476–477 Succinate, 382–383 Succinic acid (SA), 23–24, 196–197, 197f, 373–374, 409–412, 415–417, 454 productions, 417t Sucralose, 203, 203f Sucrochemistry, 201–205 gluconic acid, 202–203, 202f inverted sugar syrup, 202, 202f sucralose, 203, 203f sucrose esters, 204 sugar polyols, 205 Sucrose, 431, 492 Sucrose acetate isobutyrate (SAIB), 204, 204f Sucrose esters, 204 Sudan black, 578–579 Südzucker, 201–202 Sugar crops, 159–179 Sugar maple, 163–164, 164f Sugar palms, 164–166, 165f Sugar polyols, 205 Sugar production, 11–12 Sugar-based biorefineries, 157–236, 291 Sugarbeet, 159–161, 161f refining and processing, 179–183, 182f carbonation, 181–182

Index

crystallization, 182–183 extraction, 181 product, 183 supply, 181 Sugarcane, 159–160, 160f bagasse, 23–24 ethanol from, 237–246 value chain, 242f Sulfite pulping, 105–107 Sulfite spent liquor-based by-products, 108–109 Sulfolobus, 532 Sulfolobus acidocaldarius, 374 Sulfolobus metallicus, 428 Sulfolobus solfataricus, 530 Sulfonation, in sulfite pulping, 106 Sulfuric acid, 104–105, 193 Sun flower, 256 Supercritical carbon dioxide (scCO2), for extraction from microalgae, 66–67 Supercritical fluid extraction from microalgae, 65–67 Superoxide dismutases (SODs), in cosmetics, 640 Supersonic fluid feed (SFF) technologies, for microalgae, 64 Surfactants biosurfactants, 499–522 in cosmetics, 642–643 Surfactin, 510–511, 510f–512f Sustainability of biorefineries, 5–6 Sweet sorghum, 162–163, 163f composition of, 163t Sweet Sorghum Association, 163f Synechococcus, 81–82 Synechocystis, 582 Syngas, 284 in gasification, 114 Syngas platform biorefinery, 14 Syrup, 160 sugarbeet, 182–183

T Taiwan Chlorella Manufacturing Company (TCMC), 74–75 Talaromyces, 421 Tall diesel, 138 Tall oil, 100–101 alkyd resins, 139–140, 140f Tall oil fatty acid (TOFA), 137–146 composition of, 101t cooligomerization, 140–141

Diels–Alder and ene reactions, 141–142 dimer fatty acids, 143–146 esters and other derivatives involving carboxyl group, 138–139 ethenolysis, 141 polyamides, 138, 139f soaps, 137–138 tall oil alkyd resins, 139–140 Tall oil fatty amines, 138 Tall oil rosin (TOR), 100–101, 146–150 composition of, 101t dimerization, 149 disproportionated and (per)hydrogenated rosin, 148–149 resin acids in, 147f rosin esters, 150 rosin soaps and fortified rosin soaps, 148 Tandem reactions, 402 Tanneries, application of enzymes in, 475–476 Tapioca, 210 Tapioca-root. See Cassava Teflon, 54 Tequila, 186 Terpenes -based biofuels, 82–83 in cosmetic formulation, 614–617 Terpineol, 132–133 Tertiary bioresources, 303f, 304–305 technologies for, 315–316 Tetraethylenepentamine (TEPA), 138 Tetrahydroabietic acid, 148–149 Tetrahydrofuran (THF), 197, 196–197 Textile industry, application of enzymes in, 475–476, 489 Thermoanaerobic bacteria, 375 Thermochemical biorefinery, 14, 348, 351f, 355f Thermochemical conversion in pulp mills, 113–119, 116f conversion routes for harvesting residues, 115f crude tall oil soap, 118–119 kraft black liquor, 116–118 in pyrolysis-based hybrid biorefineries, 362–363 of starch, 220 Thermococcus, 532 Thermococcus litoralis, 530 Thermogravimetric analysis, 116

707

708

Index

Thermoplastic polyamides, 146, 146f Thermotoga, 532 Thermotreatments, in vegetable oil biorefineries, 258–259 Thermus aquaticus, 530 Thiocapsa pfennigii, 580 Three-phase technology, 209 Threonine, 446, 463–464 Tilghman, Benjamin Chew, 128 Timber procurement, 93, 94f Timber rafting, 94f Tissue engineering, 544 Tissue plasminogen activator (tPA), 379 TNS Recovery Complex System, 638 Tocotrienols, in cosmetics, 623 Toddy palm. See Asian Palmyra palm Toilet systems, 297–298 Tolypothirx, 65 Torrefaction, 115, 342–344 Torulaspora delbrueckii, 641 Transesterification, 118 Transglutaminase, 488 Transparent Technologies Private Ltd., 60 Transportation biofuels, 7, 23 Trehalose-6-phosphate synthase A (T6PSA), 425 Triacylglycerol, 78–79 Tricanter method, 209 Tricarboxylic acid (TCA) cycle. See Krebs cycle Trichoderma, 428, 476–478, 481, 484 Trichoderma reesei, 393, 482 Trichoderma viride, 421 Triethylenetetramine (TETA), 138, 145 Triglycerides, accessible products from, 257f Trimer acid, 143, 146 Trimer triol, 144 Trimethylolpropane trioleate, 138 Triterpene, 500–501 Triticale, 171 Triticum spp. See Wheat Trituration, in vegetable oil biorefineries, 259 True sago palm. See Sago palm Tryptophan, 446–447 Tubular reactors, for microalgae, 54 Turpentine, 100–101, 130 composition of, 101t ingredients of, 131f value chain, 134f Turpentine oil. See Turpentine Tyrosine, 465

U Ubiquinone. See Coenzyme Q10 UDP, 392 Ultrasound, for microalgae, 63–65 Umbellularia californica, 80 Undistilled dimer, 143 Unicellular microalgae, 48 Unit locations, of civilization biorefineries, 312, 313f United States, algal research in, 38–46 Aquatic Species Program, 38–40 legislation/law, 40 planning, 40 recently funded projects related to microalgae, 41t–45t University of Waterloo, 354 Unmixed open ponds, for microalgae, 52–53 UPM BioVerno, 119 Urban wastewater, 300 Urban Wastewater Treatment Directive, 300 US Department of Energy (DOE), 17 Biomass Program, 40, 46 Office of Fuels Development, 37 Ustulina vulgaris, 421

V Valorization of fatty acids and glycerol, 259–262 of second generation carbohydrates, 259–260 Value-added products of biorefineries, 13–14, 17 Vanillin, 108 Vegetable oil biorefineries, 247–270 feedstock, 249–252 crops composition, 249–252 main crops and worldwide production, 249, 251f–253f oil composition, 250–252 whole-plant composition, 249–250 future challenges of industrialization, 266–268 economic challenges, 267 societal adaptation, 267 sustainability challenges, 267–268 technical innovations, 266–267 industrial vegetable oil biorefineries, 264–266 environmental aspects, 266 geographical aspects, 264 socioeconomic aspects, 265–266 whole-plant biorefinery concept, 252–264, 255f biotech conversions, 259

Index

chemical conversions, 259–262 from field to biorefinery unit, 256 fractionation, 258 main markets of, 262–264 pressing/trituration, 259 separation, 258 stages, 256–262 thermotreatments, 258–259 Versazyme, 636 Vertical column reactors, for microalgae, 54–55 Vibrio, 530, 579 Vibrio costicola, 621 Vinegar. See Acetic acid Vinyl acetate, 191 Vinyl acetate monomer (VAM), 191 Vinyl acetate-acrylic, 191 Vinyl chloride-vinyl acetate (VC/VAc), 191 Vinyl pyrrolidone-vinyl acetate (Vp/VAc), 191 Vinylchloride monomer (VCM), 191 Virgibacillus spp., 541 Virgin primary bioresources, 302–303 Vitamin A and its derivatives, in cosmetics, 622 Vitamin B2, 381 in cosmetics, 623–624, 625f Vitamin B5, in cosmetics, 624 Vitamin B3/nicotinic acid, in cosmetics, 624–625 Vitamin C, in cosmetics, 622–623 Vitamin E, in cosmetics, 623 von Carlowitz, Hannβ Carl, 127–128

W Wacker process, 191 Waste, definition of, 300 Waste analysis, civilization biorefinery data from, 327–328 Waste Disposal Act (Germany), 299–300 Waste Framework Directive (Germany), 300 Waste management in Jenfelder Au, Hamburg, 329–334 Waste sorting, civilization biorefinery data from, 327–328 Waste/wastewater management, 297–300, 297f collection, 301 definition and categorization, 300–301 in Jenfelder Au, Hamburg, 329–334 PHA production from, 565, 565f, 569 wastewater treatment, 316, 319 Water-slurry process, 215 Wautersia, 585

Wautersia eutropha, 585 Weissella, 533 Welan gum, in cosmetics, 630 Wet milling, 12–13, 65 maize, 206 rice, 209 Wheat, 168–171, 169f, 403 gluten, 456 grain, 171f harvesting, 170f starch production, 208–209 Martin process, 208–209 three-phase technology, 209 Whiskey, 185 White biotechnology for amino acids, 445–472 for biopolymers, 555–574 in biosurfactants, 499–522 in cosmetics, 607–652 exopolysaccharides from prokaryotic microorganisms, 523–554 for organic acids, 409–444 polysaccharides and oligosaccharides, 653–680 poly-3-hydroxybutyrate, 575–606 White dextrins, 222 White liquor, 95 White spirit, 131–132 White sugar, 182f Whole crop biorefineries (WCBRs), 12–13 Whole genome sequencing (WGS), 535–536 Whole-plant biorefinery, for oil crops, 252–264, 255f biotech conversions, 259 valorization of fatty acids and glycerol, 259 valorization of second generation carbohydrates, 259 chemical conversions, 259–262 valorization of fatty acids and glycerol, 260–262 valorization of second generation carbohydrates, 259–260 from field to biorefinery unit, 256 fractionation, 258 main markets of, 262–264 pressing/trituration, 259 products obtained from oilseed biomass, 262f separation, 258 stages, 256–262 thermotreatments, 258–259

709

710

Index

Wickerhamomyces ciferrii, 642 Wine, 164–166, 183–185, 189, 474, 489 Wine palm. See Asian Palmyra palm Wodka, 186 Wood, chemical composition of, 94t Wood chips, pretreatment prior to pulping, 109–113, 110f acidic pretreatments, 112–113, 112t alkaline pretreatments, 110–111, 111t Wood turpentine. See Turpentine Wood-based biorefineries, 91–126 World Water Works Inc., 57 Worldwide productionof oilseeds, 249, 251f–252f

X Xanthan, 528, 533–534, 536–542, 545, 656, 666–667 in cosmetics, 626–629, 628f Xanthomonas campestris, 380, 535–536, 626–627 Xanthophyllomyces dendrorhous, 617 X-ray crystallography, 483

Xylanase, 484, 488–491 Xylanosomes, 399–400 Xyloisosaccharinic acid, 105 Xylooligosaccharides (XOS), 655, 663 Xylose, 13–14, 371–375, 465

Y Yarrowia lipolytica, 401, 418–421, 516–517, 641–642 Yeasts, 419f, 421 Yellow dextrins, 222 Yuca. See Cassava

Z Zea mays. See Maize Zeaxanthin, 66 Zero-waste wood-based biorefineries, 13–14 Zobella denitrificans MW1, 583 Zonix™, 504 Zooglea, 541 Zymomonas mobilis, 533, 632