Technologies for Biochemical Conversion of Biomass [1st Edition] 9780128025949, 9780128024171

Table of contents :
Content:
Front matter,Copyright,PrefaceEntitled to full textChapter 1 - Introduction, Pages 1-10
Chapter 2 - Pandect of Practice Unit and Process Engineering of Biomass Biochemical Conversion, Pages 11-19
Chapter 3 - Pretreatment Strategies for Biochemical Conversion of Biomass, Pages 21-64
Chapter 4 - Enzymatic Hydrolysis of Pretreated Biomass, Pages 65-99
Chapter 5 - Microbial Cell Refining for Biomass Conversion, Pages 101-135
Chapter 6 - Sugar Strategies for Biomass Biochemical Conversion, Pages 137-164
Chapter 7 - Microbial Fermentation Strategies for Biomass Conversion, Pages 165-196
Chapter 8 - Posttreatment Strategies for Biomass Conversion, Pages 197-217
Chapter 9 - Coproducts Generated from Biomass Conversion Processes, Pages 219-264
Index, Pages 265-277

Citation preview

Technologies for Biochemical Conversion of Biomass

Hongzhang Chen

Professor at the Institute of Process Engineering Chinese Academy of Sciences, Beijing, China

Lan Wang

Professor at the Institute of Process Engineering Chinese Academy of Sciences, Beijing, China

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom © 2017 Metallurgical Industry Press. Published by Elsevier Inc. 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. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-802417-1 For information on all Academic Press publications visit our website at https://www.elsevier.com/

Publisher: Jonathan Simpson Acquisition Editor: Simon Tian Editorial Project Manager: Naomi Robertson Production Project Manager: Julie-Ann Stansfield Designer: Greg Harris Typeset by Thomson Digital

Preface Today, mankind is moving from a civilization based on industry to one which recognizes the importance of the ecosystem. To realize sustainable development, it is a prerequisite to find a clean substitute for the oil resources. Biomass, the solar energy stored in chemical form in plant, has attracted both industrial and academic interest as a promising clean and renewable resource. Biomass conversion technology owns significant advantages over other conversion technologies for being environment friendly, effectively, and operation condition moderately. Therefore, it attracts more attentions in comparison to the physical and chemical conversion technologies. Over the past 20 years, the author has been devoted to the study of biomass conversion and product development. And, luckily, breakthroughs were made in the setup of a steam explosion–based pretreatment platform, solid-phased–enzyme–fermentation–separation coupling platform, and large-scale-solid-phased pure cultivation platform. All those platforms have been applied in industrialization productions and have received extensive acceptance from colleagues and enterprises. In the process of research and application, we proposed the notion of biomass biochemical conversion technology platform, which not only guided engineers to realize the biochemical conversion of biomass, but was also devoted to the development of related disciplines. With the development of research on biomass, related works have been published continuously. However, most of them focus on specified techniques for oriented biomass conversion, but only a few have provided a systematic and comprehensive review on the techniques employed. The author has summarized the existing research routes and results for the construction of biomass conversion platforms. It is expected to promote the conversion and use of biomass resources. The first two chapters review the unit operations of biomass conversion. Chapters 3–8 discuss conversion platforms: pretreatment platform, enzyme platform, cell refinery platform, sugar platform, fermentation platform, and posttreatment platform. Chapter 9 discusses polygeneration of the bioconversion products. The research on biomass biochemical conversion was supported by the National Key Basic Research Development Program of China (973 project, Nos 2004CB719700 and 2011CB707400) and the National High Technology Research and Development Program (863 Program, 2012AA021302). In xi

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a­ddition, the works of my coworkers and students were essential preconditions for completing this book. Dr. Menglei Xia, Dr. Zhimin Zhao, Dr. Zhihua Liu, Master Lanzhi Qin, Master Meixue Shao, Dr. Guanhua Wang, Dr. Wenjie Sui, Dr. Yuzhen Zhang, Dr. Guanhua Li, Master Yingyi Duan, Dr. Litong Ma, Dr. Zhiguo Zhang, Dr. Qin He, and Dr. Ning Wang participated in writing this book. Dr. Lan Wang participated in the revision and review of the book. Many references of our predecessors and colleagues have been cited in the book. I wish to express my sincere thanks to all of them. Some errors may exist in this book. I sincerely hope to receive criticism and guidance from readers in this regard. Hongzhang Chen State Key Laboratory of Biochemical Engineering Institute of Process Engineering Chinese Academy of Sciences St. Zhongguancun, Beijing, Peoples’s Republic of China June, 2015

Chapter 1

Introduction Chapter Outline 1.1 The Concept of Biomass 1.2 Biomass Conversion Methods 1.2.1 Biomass Physical Conversion Technologies 1.2.2 Biomass Chemical Conversion Technologies 1.2.3 Biomass Biochemical Conversion Technologies

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1.3 Role and Status of Biochemical Conversion Technologies of Biomass 1.4 Overview of Biochemical Conversion Platform of Biomass 1.5 Prospects of Biochemical Conversion of Biomass Industry References

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1.1  THE CONCEPT OF BIOMASS Biomass is a solar energy resource that has been utilized by humans for a long time. Solar energy is saved in the form of chemical energy through plant photosynthesis, and thus things like oxygen for breathing, plants and animals for food, wood for building and making fire, and clothes for cover and warmth appeared. However, it has been only 50 years since biomass was truly defined by humans. Biomass in English was first used in 1934 (it was defined as living weight in Merriam-Webster). According to the foreign retrospective database, biomass was defined as what it is nowadays in the Journal of Plant and Soil of America (Tergas & Popenoe, 1971). In 1976, four years after the 1972 oil crisis, an article (Tergas & Popenoe, 1971) introducing biochemical engineering proposed that wasted biomass could be reused as a kind of raw material. In 1979, an article (Crutzen, Heidt, & Krasnec, 1979) in Nature pointed out that the combustion of biomass produced polluted gases, while in 1980, the college of Process Engineering of Agriculture at the University of Netherlands indicated that biomass could be regarded as the source of energy materials (Bruin, 1980). In 1981, Oak Ridge National Laboratory of America started the security assessment of biomass energy technologies (Watson & Etnier, 1981). Since then, reports Technologies for Biochemical Conversion of Biomass © 2017 Metallurgical Industry Press. Published by Elsevier Inc.

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and research about biomass energy have come to the stage (Mes-Hartree & Saddler, 1983; Miller & Fellows, 1981; Schwarzenbach & Hegetschweiler, 1982; Stout, 1982; Zadražil & Brunnert, 1982; HongZhang & ZuoHu, 2000; Chen & Qiu, 2010). If we set the year 1980 as the origin of biomass utilization research, it has been over 30 years since then, which is also the time that a child takes to pay back to his or her family and society from birth. Within this time, biochemical conversion technology of biomass has developed rapidly, similar to the payback to family and society by the grown-up child. Research into biochemical conversion technology of biomass also began its journey to industrial application. The definition of biomass according to the US Department of Energy includes any animal and plant organisms. In particular, biomass in the USA includes agriculture and forestry waste, municipal solid waste, industry waste, and terrestrial and aquatic crops. The definition of biomass according to China’s Renewable Energy Association covers all kinds of organisms formed through plant photosynthesis, including all animals, plants, and microorganisms. Plants are autotrophs (producers), while animals are heterotrophic organisms (consumers). Humans selected those animals and plants for their own benefit during their survival process, and used animals the most. Regarding plants, humans used fruits that are rich in starch, protein, fat, and vitamins. Humans did not look for ways to use the other parts of plants, as there was no urgent need to do so. The biomass mentioned in this book refers to the lignocellulosic waste of plants besides those used for food and medicine.

1.2  BIOMASS CONVERSION METHODS Lignocelluloses are mainly composed of cell walls of dead cells. The main components of cell walls are cellulose, hemicellulose, and lignin, while pectin is the main component of the intercellular layer. Hemicellulose and lignin are connected to each other by chemical bonds, and the three components are connected by hydrogen bonds, resulting in the tight cell walls based on the skeleton formed by multistage fiber structure. In order to utilize lignocellulosic materials, the first step is to destroy the existing cell walls. There are various utilization technologies of biomass, but they can come down to three overall categories: physical conversion technologies, chemical conversion technologies, and biochemical conversion technologies.

1.2.1  Biomass Physical Conversion Technologies Biomass physical conversion technologies refer to the modification and processing of biomass to produce high-value products, thus realizing the valueadded utilization of lignocellulosic materials. The main products of physical conversion technologies include sheets, construction materials, and lignocellulosic composites.

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The technology of biomass artificial sheets includes preparation of raw materials, mixing, molding, and posttreatment (Duan, He, & Shang, 2009). Nontimber lignocellulosic materials that are fit for biomass artificial sheets include bagasse, wheat straw, straw, corncob, cotton stalk, and shive. Biomass artificial sheets, especially the nontimber lignocellulosic biomass artificial sheets, have positive significance for reducing the consumption of forest resources and protecting the environment. Biomass construction materials include biomass wall materials (Cui, Cui, & Bao, 2006), of which light straw magnesia cement springboard (Zhang, Liu, & Xun[J], 2011) and straw board (Zhu, Wang, & Liu, 2010) are the main categories; there are also others, such as corn straw insulation materials (Ding, Ren, & Zheng, 2014). The processing of these wall materials is similar to that of lignocellulosic boards, and thus the wall materials have properties of lightness, sound insulation, earthquake resistance, and anticorrosiveness. Artificial boards and wall materials are rudimentary forms of lignocellulosic materials usage. At present, humans utilize lignocelluloses for processing directly, while the manufacture of artificial board, using cellulose extracted from lignocellulosic materials, has fewer application examples. Woody biomasses are mostly used to produce biomass composites (Li, 2008). There are three forms of woody materials: laminated composite, hybrid composite, and penetration composite. Laminated composite is formed by glue lamination and pressurized glue of certain shaped sheets; it has a layered structure and a certain size and shape. Hybrid composite is obtained from the mixing of wood or woody materials as the base with other materials, such as inorganic materials and minerals, or the mixing within lignocellulosic materials; this is then pressurized into boards. Penetration composite is made by filtering a substance (inorganic materials, organic materials, and metal elements) into wood or woody materials, and then using deposition or chemical reaction to improve wood properties or give wood a certain function. Therefore, we can see that the physical conversion of plants mainly uses their tight physical structure and then converts them into materials. The products are utilized in daily life and production. However, physical conversion can hardly convert biomass into renewable products that could displace petroleum products, thus such technology cannot satisfy the needs of clean energy and chemicals nowadays.

1.2.2  Biomass Chemical Conversion Technologies Biomass chemical conversion technologies used to be applied in the pulp and paper industry. As the problems of energy and environment arise, people pay much attention to the research and application of biomass. There are many biomass chemical conversion technologies, including combustion, carbonation, gasification, thermal decomposition, and hydrothermal liquefaction technology.

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The traditional paper industry mainly adopted an acid-based chemical pretreatment approach to obtain cellulose in biomass for pulp preparation. However, due to the irreplaceability of paper production, we will not discuss the item here. Combustion of chemical conversion technologies is carried out to utilize the intense heat released during oxidation directly or convert it to power. Hemicellulose can be decomposed severely below 300°C. Cellulose can complete the decomposition process among 300–350°C. When the temperature exceeds 500°C, lignin begins to decompose. Such technology has a long history and is relatively cost-efficient. However, it generates a lot of greenhouse gases, such as SO2. Carbonation of chemical conversion technologies, a sort of ancient technology of biomass conversion, is to heat biomass to obtain gas, liquid, and solid products under air-free or air-limited conditions. Mechanism charcoal (Gao, Ma, & Shen, 2010) is also called artificial carbon or molding charcoal; this is molded under high temperature and high pressure, and then pyrolysis carbonized to get solid carbon products. By-products like tar and crude vinegar are obtained by condensation, recycling, and processing the gas mixture that is generated from pyrolysis. Tar, containing a high degree phenolic substance and many organic substances, is a raw material from which to extract aromatic compounds. Tar can also reconcile with the residue to produce No. 200 highgrade gasoline, or mix with coal as a coal-fired boiler fuel. Crude vinegar is not only a kind of chemical raw material, can also be used to help produce antimildew agents, insect-resistant agents, and antibacterial agents; in addition, it could increase efficiency when used with pesticides and reduce pesticide residues. Gasification of chemical conversion technologies (Shi & Hua, 2007) is carried out to convert the combustible parts of biomass into flammable gas (mainly hydrogen, carbon monoxide, and methane) at a high temperature using oxygen or oxygenates in the air as gasification agents, due to the properties of high volatile components, high carbon activity, and low sulfur and ash of biomass. It was proposed primarily by Ghaly to produce fuel gas of low density. According to its application, gasification can be divided into pressure gasification and gasification, whose principles are the same, while the latter has higher requirements for equipment, operations, and maintenance. Thermal decomposition technology is decomposes biomass materials into at least two components. Rapid thermal decomposition refers to the process of increasing the heating rate during thermal decomposition of raw materials, leading to instantaneous thermal decomposition at several hundred degrees Celsius. Thermal decomposition fluid, wood vinegar, quick carbide, and anhydrosugar are produced by thermal decomposition of biomass. Thermal decomposition fluid and quick carbide can be used in fuels. Wood vinegar can be used in smoked liquid, exterminator, and pesticide substitutes. Anhydrosugar can be used in polymer materials to produce biodegradable plastics. Hydrothermal liquefaction technology decomposes biomass in water at a high temperature and pressure. Similar to thermal decomposition technology, gas, liquid, and solid products can be obtained through hydrothermal liquefaction.

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The light component in the liquid phase (wood vinegar) dissolves in water, while heavy components mix with solid phase; thus three types of mixture were obtained gaseous, aqueous, and oleic (a mixture of oil and carbon). It is concluded that biomass chemical conversion technologies require relatively fierce conditions. Except for power generation, the products obtained have lower purity and cannot replace oil products as fine chemicals; nor can they be used as universal raw materials for industry to meet the demand of energy and environment.

1.2.3  Biomass Biochemical Conversion Technologies Biomass biochemical conversion technologies refer to the conversion of biomass into corresponding products through certain physical, chemical, and biological pretreatments. Pretreatments in the biochemical conversion technologies of biomass aim to help reach ideal conversion effects, not to produce final products, which is the essential difference between the aforementioned physical and chemical conversion of biomass. In addition, biochemical conversion technologies of biomass are more moderate than the other two. Biomass can be turned into different products, such as hydrogen, biogas, ethanol, acetone, butanol, organic acids (pyruvate, lactate, oxalic acid, levulinic acid, citric acid), 2,3-butanediol, 1,4-butanediol, isobutanol, xylitol, mannitol, and xanthan gum by selecting different microorganisms in the process of biochemical conversion (Chen, 2010). On the one hand, such products can synthetize replacements of petroleum-based products. On the other hand, the products can replace products derived from grains, such as ethanol. Compared with other conversion technologies, biomass biochemical conversion technologies are moderate, pure, clean, and efficient. Moreover, biomass can be turned into various intermediates by screening different enzymes or microorganisms through biochemical conversion technologies, thus providing many platform substances for the conversion of renewable materials, fuels, and chemicals. As a result, people pay much attention to biochemical conversion technologies of biomass.

1.3  ROLE AND STATUS OF BIOCHEMICAL CONVERSION TECHNOLOGIES OF BIOMASS We can see that through such a moderate way as biochemical conversion technologies of biomass, replacements of petroleum-based products can be obtained, which is potentially a new model to develop ecological agriculture so as to realize a circular economy. It will play an important role in solving energy, environment, and rural issues. Hence, biochemical conversion technologies of biomass are important to long-term development and society stability. The role and status of biochemical conversion technologies of biomass are reflected in the following aspects.

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1. It is the base of human living. Since oil was discovered and first exploited and used by humans, oil-based products have played an important role in many aspects of human life and production, especially the role of energy during production. However, according to BP statistics in 2010, oil can only be exploited for 45.7 years, natural gas for 62.8 years, and coal for 119 years. The world energy prospects of BP for 2030 indicate that the consumption of liquid fuel will increase from 39.433 Gt in 2010 to 46.711 Gt in 2030, while the yield of biofuel will increase from 575 Mt in 2010 to 2351 Mt in 2030. Meanwhile, compared with grains, lignocelluloses are cheaper and richer on earth than the raw materials used to produce biofuel. Therefore, it is important in terms of the survival of humans to turn biomass of lignocelluloses, especially agriculture and forestry waste, into raw materials that can substitute for petroleum. 2. It is an important way to make humans live in harmony with nature. In the long-term history of humans’ development, although they created an industrial civilization at the cost of cheap oil, coal, and natural gas as their capital, humans have caused a series of problems like the greenhouse effect and human health problems due to environmental pollution because of their ignorance of the carrying capacity of the earth. If we want to turn such a development pattern around, especially in terms of reducing our industrial system’s dependence on oil, an important way is to develop a new industrial chain that could replace petroleum-based products. Biochemical conversion technologies of biomass have been applied in the industrial system because we can get biobased energy, materials, and chemicals that can replace petroleum-based products through these technologies. Biochemical conversion technologies of biomass are clean and their materials are renewable biomass. The development of biobased products brings the intermediate products of ecological cycle to serve humans using the process of ecological transformation in nature; thus it is an important way to make humans live in harmony with nature. 3. It is an essential method to change the role of agriculture and increase farmers’ income. Agriculture has played the role of food supplier in society. Agriculture cannot pay for education, health care, and other fundamental needs (e.g., marriage expenses and pensions) in farmers’ lives any more, although the production of grains suffered little effect of natural disasters or the price of grains are protected by the government. Consequently, producing biobased products, which adds new roles of farm products as energy and materials through biomass by biochemical conversion technologies of biomass, can increase farmers’ income in two aspects. The first aspect is that the agriculture and forestry waste, which used to abandoned, can be sold as products to increase farmers’ income; the other aspect is the rise of new industries of biochemical conversion technologies of biomass, especially the ever-rising private enterprises, which increase employment opportunities for farmers, thus increasing farmers’ nonfarm income.

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4. It will open a new growth pole of economics. With rising energy prices and people’s attention to environment, oil-reliant economy growth and the industrial chain will be replaced by clean development patterns like a renewable biomass economy. Products derived from biochemical conversion technologies of biomass, a kind of regenerative resource that can replace oil-based products, have a large potential market. Biochemical conversion technologies of biomass are technology-intensive and capital-intensive industries, so they will promote optimization and upgrading of industrial structures, resulting in a new growth pole of economics.

1.4  OVERVIEW OF BIOCHEMICAL CONVERSION PLATFORM OF BIOMASS Plants form a natural defense mechanism, tight structure, and complicated components, in order to resist the invasion of microorganisms and pests and diseases during the process of evolution. To utilize biomass through biochemical conversion technologies, we can turn plants into sugars, which can be employed by many microorganisms, and then turn the sugar into different small molecular products by sugar conversion. Because of the complexity of polysaccharides in biomass, the components of sugar are diverse. So, the products are mixtures of different sugars, which need posttreatments for fermentation products so as to develop final products that can meet industrial needs. This implies that systematic industry chains that contain many unit operations are needed for the realization of plants’ biochemical conversion. Biochemical conversion platform of biomass is an integrated multidisciplinary technology system. However, integrated technology will undoubtedly increase the cost, which puts forward a challenge for the economic efficiency of the biochemical conversion process. Biomass in plants contains nonsugar components like lignin, except for polysaccharides. So, if we just utilize the sugar while treating the lignin as waste, this neither meets the requirements of cleaner production, nor utilizes resources efficiently. Therefore, it is necessary to turn lignin into corresponding products through physical, chemical, and biochemical methods. Moreover, due to the complexity of polysaccharides, turning different sugars into a single product can not only increase the cost of conversion, but also lead to market risk brought by sole products, and this cannot meet the need for multiproducts. Hence, biochemical conversion of polysaccharide in plants should also be multidirectional. Building a technical platform integrating different operations is important for industrialization of biomass biochemical conversion technologies. H. Z. Chen’s team has set up a technical platform of operations needed from materials to products based on the researches for more than 20 years. They raise the idea of grading refining polygeneration after study of heterogeneous composition of biomass (Fujian, Hongzhang, & Zuohu, 2001; Sun & Chen, 2008; Chen & Liu, 2007; Chen, Xu, & Tian, 2002) (Fig. 1.1).

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FIGURE 1.1  System of integration of biochemical conversion technologies of biomass (Chen & Qiu, 2010; Chen and Qiu, 2007; Jin and Chen, 2006, 2007).

1.5  PROSPECTS OF BIOCHEMICAL CONVERSION OF BIOMASS INDUSTRY Biochemical conversion of biomass industry is an effective way to solve problems of energy, because plants are the only corporeal category of renewable energy form. It is also an effective path to solve issues of agriculture, for the traditional grain-based agriculture will take up new roles as energy agriculture and material agriculture. Moreover, conversion of agriculture and forestry waste will increase farmers’ income, and improve the rural environment as well. It is a path to guarantee that humans can live in harmony with nature, as biomass can replace oil and thus reduce greenhouse gas release. Many countries have developed renewable energy positively under the construction of the established development goal of renewable energy. The European Union Summit passed a “20–20–20” strategy in the form of “Renewable Directive” in December, 2008. The strategy stipulated that greenhouse gas release should have been reduced by 20% by the year 2020 compared with 1990; the ratio of renewable energy consumption should have been raised to 20%, of which biology liquid fuel consumption of transportation energy should have been reached 10%. China also promised that consumption of renewable energy should have taken up 15% of total energy consumption by the year 2020. Biochemical conversion technologies of biomass will release their potential abilities in the development of industrialized ecological agriculture and circular economy, on the basis of their advantages of being moderate, clean, and efficient (Charlton, Elias, & Fish, 2009).

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REFERENCES Bruin, S. (1980). Biomass as a source of energy. Biotechnology Letters, 2, 231–238. Charlton, A., Elias, R., Fish, S., et al. (2009). The biorefining opportunities in Wales: understanding the scope for building a sustainable, biorenewable economy using plant biomass. Chemical Engineering Research and Design, 87, 1147–1161. Chen, H. Z., & Liu, L. Y. (2007). Unpolluted fractionation of wheat straw by steam explosion and ethanol extraction. Bioresource Technology, 98, 666–676. Chen, H. Z. (2010). Process engineering of bio-based products. Beijing: Chemical Industrial Press (in Chinese). Chen, H., & Qiu, W. (2007). The crucial problems and recent advance on producing fuel alcohol by fermentation of straw. Progress in Chemistry, 19(z2), 1116–1121. Chen, H. Z., & Qiu, W. H. (2010). Key technologies for bioethanol production from lignocellulose. Biotechnology Advances, 28, 556–562. Chen, H. Z., Xu, F. J., Tian, Z. H., et al. (2002). A novel industrial-level reactor with two dynamic changes of air for solid-state fermentation. Journal Of Bioscience and Bioengineering, 93, 211–214. Crutzen, P. J., Heidt, L. E., Krasnec, J. P., et al. (1979). Biomass burning as a source of atmospheric gases CO, H2, N2O, NO, CH3Cl and COS. Nature, 282, 253–256. Cui, Y. Z., Cui, Q., & Bao, W. (2006). Plant straw cement plate and group limo production technology (rudin). Journal of Wall Materials Innovation and Building Energy Conservation, 27–31 (in Chinese). Ding, Z. L., Ren, D. L., & Zheng, F. S. (2014). Research of cornstalk preparing building decoration composite panels. Journal of Agricultural Mechanization Research, 17, 47–49. Duan, H. Y., He, X. C., & Shang, D. J. (2009). Development status and prospective of our country’s industry of straw based panel. Journal of Agricultural Mechanization Research, 15, 18–22 (in Chinese). Fujian, X., Hongzhang, C., & Zuohu, L. (2001). Solid-state production of lignin peroxidase (LiP) and manganese peroxidase (MnP) by Phanerochaete chrysosporium using steam-exploded straw as substrate. Bioresource Technology, 80, 149–151. Gao, H., Ma, Y. H., & Shen, Z. G. (2010). Utilization status and prospects of straw material carbonization of Anhui Province. Journal of Shijiazhuang Railway Institute, 8612–8613 (in Chinese). HongZhang, C., & ZuoHu, L. (2000). Studies on ethanol extraction of steam exploded wheat straw. Chemistry and Industry of Forest Products, 20, 33–39. Jin, S., & Chen, H. (2006). Superfine grinding of steam-exploded rice straw and its enzymatic hydrolysis. Biochemical Engineering Journal, 30(3), 225–230. Jin, S., & Chen, H. (2007). Fractionation of fibrous fraction from steam-exploded rice straw. Process Biochemistry, 42(2), 188–192. Li, J. (2008). Biomass composite materials. Beijing: Science Press (in Chinese). Mes-Hartree, M., & Saddler, J. (1983). The nature of inhibitory materials present in pretreated lignocellulosic substrates which inhibit the enzymatic hydrolysis of cellulose. Biotechnology Letters, 5, 531–536. Miller, I. J., & Fellows, S. K. (1981). Liquefaction of biomass as a source of fuels or chemicals. Nature, 289, 389–399. Schwarzenbach, F. H., & Hegetschweiler, T. (1982). Wood as biomass for energy: results of a problem analysis. New Trends in Research and Utilization of Solar Energy through Biological Systems. Springer, 28–33.

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Shi, Z. P., & Hua, Z. Z. (2007). Manual of biomass and bioenergy. Beijng: Chemical Industrial Press (in Chinese). Stout, B. A. (1982). Agricultural biomass for fuel. New trends in research and utilization of solar energy through biological systems. Springer, pp. 73–79. Sun, F. B., & Chen, H. Z. (2008). Enhanced enzymatic hydrolysis of wheat straw by aqueous glycerol pretreatment. Bioresource Technology, 99, 6156–6161. Tergas, L. E., & Popenoe, H. L. (1971). Young secondary vegetation and soil interactions in Izabal, Guatemala. Plant and Soil, 34, 675–690. Watson, A., & Etnier, E. (1981). Health and safety implications of alternative energy technologies I. Geothermal and biomass. Environmental Management, 5(4), 313–327. Zadražil, F., & Brunnert, H. (1982). Solid state fermentation of lignocellulose containing plant residues with Sporotrichum pulverulentum Nov. and Dichomitus squalens (Karst.) Reid. European Journal of Applied Microbiology and Biotechnology, 16, 45–51. Zhang, C. S., Liu, X. J., & Xun[J], H. S. (2011). Research of construction rubbish-strawMagnesium cement wall thermal insulation material. Journal of Agricultural Mechanization Research, 1, 78–80 (in Chinese). Zhu, X. D., Wang, F. H., & Liu, Y. (2010). Research of manufacturing technology of anti-burning straw boards. Journal of Agricultural Mechanization Research, 13, 130–134 (in Chinese).

Chapter 2

Pandect of Practice Unit and Process Engineering of Biomass Biochemical Conversion Chapter Outline 2.1 Characteristics of Biomass Materials 2.1.1 Complexity of Biomass Materials 2.1.2 Technology Demands by the Biomaterials Complexity 2.2 Unit Operations of Biochemical Conversion Technologies of Biomass 2.2.1 Pretreatment Unit Operation of Biochemical Conversion of Biomass

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2.2.2 Saccharification Unit Operation of Biochemical Conversion of Biomass 15 2.2.3 Fermentation Unit Operation of Biochemical Conversion of Biomass 16 2.2.4 Posttreatment Unit Operation of Biochemical Conversion of Biomass 16 2.3 Process Engineering of Biochemical Conversion of Biomass and its Integration 17 References 18

2.1  CHARACTERISTICS OF BIOMASS MATERIALS 2.1.1  Complexity of Biomass Materials The complexity of biomass materials is the key to restricting its utilization, which has been presented fully in the book Process Engineering of Bio-based Products (in Chinese) written by Chen (2010). Taking plants as an example, biomass materials contain four parts: carbohydrate (sugar, starch, cellulose, and hemicellulose), lignin (polyphenylphenols), esters, and protein. However, the content of structural materials differs among the categories of plants. Even in the same plant, such contents can be different according to different growth periods, place of production, and different parts of the plant. Technologies for Biochemical Conversion of Biomass © 2017 Metallurgical Industry Press. Published by Elsevier Inc.

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Besides structural materials, there are lots of plant-based and commercially viable natural products, such as herbal active ingredients—alkaloids, glycosides, flavonoids, terpenes, organic acids, and polysaccharide compounds. There are also many important industrial products, such as lacquer, which is natural resin paint. Lacquer is processed by a kind of white viscous emulsion obtained from the phloem of cut sumac bark. Oleoresins obtained from pinaceae mason pines or other plant boughs can yield gum resin after removing volatiles by distillation. Gum resin mainly contains monoterpene, sesquiterpene, and diterpene components. Gum resin and its modified products, after deep processing, are widely used in paint, adhesives, ink, paper, rubber, food additives, and bioproducts. Natural rubber is also an important industrial raw material. We should not just consider whether products themselves can satisfy a certain need; we should also select corresponding plant materials and their conversion routes during products processing because of the complexity of plant materials. Only after that can products, raw materials, and the productive process be linked organically. We should break the traditional concept of sole products, and take full advantage of all parts of biomass under the instruction of full utilization of biomass, fractionation, and sequential use theory (Chen & Li, 2002). Under such conditions, biomass can be turned into various kinds of products. In order to realize the above goals, we should primarily aim for a breakthrough on the critical technologies of utilization of plant resources. A combination of biotreatment and eco-utilization technologies will further improve the conversion efficiency of mass and energy, enhance the product economy, and reduce costs. New technologies and processes will raise the ratio of biomass in renewable resources. Meanwhile, perfect production and service systems contribute to the environmental protection and sustainable development of national economy. Modern biotechnology, information technology, and engineering technology could be integrated together to improve existing technology and products. The screening of microorganisms and construction of efficient engineering bacteria in fermentation engineering, and development of efficient machinery and equipment, are necessary to improve process efficiency and product quality. Levels of economic development will vary in areas depending on the resources available. Modern technologies and traditional agriculture can be integrated. Based on the principles of unity, harmony, recycling, and regeneration, plant utilization and technical support systems can be built using a systems engineering approach, which may enable a positive cycle of environment and rural economy. Benefits to economy, zoology and society could then be achieved.

2.1.2  Technology Demands by the Biomaterials Complexity Plants mainly contain cellulose, hemicellulose, and lignin, which interweave with each other to form a complicated structure. Such a structure leads to the restriction of one component’s degradation by other components. For example,

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the steric hindrance of lignin to cellulose and hemicelluloses means that cellulase cannot fully degrade cellulose materials. The main components of straw are macromolecular compounds which have stable chemical properties, which are insoluble in water or other common organic solvents. At a normal temperature, straw cannot be hydrolyzed by dilute acid and dilute alkali. When straw is hydrolyzed directly by cellulase, the sugar yield is very low, less than 20% of the theoretical yield. Additionally, most of the products are pentose and hexose, generated by easily hydrolyzed hemicellulases. Under the conditions of excessive cellulase, the enzyme production rate is less than 10% of untreated materials. The three main components—cellulose, hemicellulose, and lignin of untreated straw—are tightly linked with each other. Their chemical structure and properties are totally different. Therefore, it is hard to utilize them effectively. For that reason, straw should be properly pretreated to change or destroy partial structure. Besides its various components, plants also have complicated and uneven multilevel structures. Taking straw as an example, it can be divided into leaves, sheath, burl, internode, panicle, stubble, and root at the organ level. At the tissue level, it can be divided into vascular tissue, parenchyma, epidermal tissue, and bands of fibrous tissue. In the cell level, it can be divided into fibroblasts, parenchyma cells, epidermal cells, duct cells, and sclereid. (1) The structure of straw is inhomogenous, and the chemical components and fiber morphology are different among various parts. In particular, the characteristic of fiber of straw is even more obvious than some broadleaf wood fiber. This indicates that such parts of straw have the potential to be used at high value. Different parts of organs in straw were not separated during harvest, so the whole straw has a variety of organs and tissues. (2) There are differences of chemical components of straws. Straw contains lots of hemicelluloses and ashes (more than 1%). (3) There are differences of characteristic of fiber morphology of straws. The contents of tiny fibers and parenchyma cells are as high as 40–50%, while the content of fibrocytes is just at the range of 40–70%. The structural features and components of various organs, tissues, and cells are different of straw biomass, so there are various conversion approaches. For example, the skins of cornstalk can be divided into two parts—the outer layer is the cortex, mainly containing epidermic cells; and the inner layer is fibrous layer, mainly containing cellulose, which is the primary source of paper fiber from gramineous plant materials. In addition, the ash content is low in the skins of cornstalk. Therefore, removing the outer layer of the corn stalk skins could improve the yield of cellulose. The core of corn stalk is mainly a vascular bundle surrounded by lots of parenchyma cells, so the contents of cellulose, hemicellulose, and lignin are relatively high. At the same time, the existence of so many parenchyma cells gives the core a loose structure that has a strong water absorption capacity, so it is suitable to be the fermentation carrier of some macrofungi. The leaves of corn stalk mainly contain epidermis and mesophyll, and a large number of silicon cells are distributed on the epidermis. Therefore, its ash content is the highest in the whole corn stalk. There are not so many bundle cells

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surrounded by mesophyll cells in mesophyll, so the content of cellulose in leaves is relatively low. The fact that leaves can curl and open are not only related to the motor cells, but also to the low degree of lignifications. Hence, leaves are more palatable as cattle food compared to other parts of corn stalk. The burl inflates in a circle around the stem, which has a high degree of lignification. So we can say that the high content of lignin is related to the structure of burl. As a consequence, the structure feature and properties of straw makes it hard to be used in a high-value way directly. Cellulose, hemicellulose, and lignin within unpretreated straw are tightly crossed together. Therefore, only by proper pretreatment, fractionation, separation of components, destroying, or changing partial structure of straw can we use it in a high-value way (Chen & Qiu, 2007).

2.2  UNIT OPERATIONS OF BIOCHEMICAL CONVERSION TECHNOLOGIES OF BIOMASS The key to the bioconversion process of biomass is to make the substrates contact with biocatalysts under certain conditions and turn substrates into products directionally. There are three premises to achieve this goal effectively. Firstly, it is essential to improve the mass transfer rate of biocatalysts so as to make the biocatalysts contact thoroughly with substrates. Secondly, a high heat transfer rate of the system is needed to make a proper condition for bioconversion. Finally, the unicity of substrates and controllability of the reaction process are indispensable to realize orient conversion. Therefore, it is essential to improve the rate of bioreaction in view of improving the rate of three-transmit process, because the process of biochemical conversion of biomass is virtually the integrated process of mass transfer, heat transfer, momentum transfer, and biocatalysis process. In addition, unit operations should be designed in view of the three-transmit process in order to improve biological conversion efficiency on the basis of the intrinsic characteristics of raw materials.

2.2.1  Pretreatment Unit Operation of Biochemical Conversion of Biomass Biomass forms its tight structure and protective barrier against outside invasion in its evolutionary process. So it is necessary to destroy its natural structure for subsequent chemical and biotreatment. The purpose of pretreatment unit operations of biochemical conversion of biomass is improving mass transfer coefficient of fluid within materials and the accessibility of substrates. The improvement of the mass transfer coefficient will provide benefits for not only the biochemical conversion process but also subsequent separation of components. On the one hand, improvement of materials’ mass transfer coefficient will increase the rate of chemical reagents, biocatalysts, or microorganisms transiting inside substrates. On the other hand, improvement of materials’ mass transfer coefficient will increase diffusion rate

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of products and reduce product inhibition, so as to promote the reaction in the positive direction. Improving the accessibility of substrates is done mainly to realize the fractionation of purpose components within biomass selectively, or to fully expose objective functional groups in order to make the chemical reagents, biocatalysts, or microorganisms contact with substrates effectively. During the process of pretreatment unit operation of biochemical conversion of biomass, some substances that are bad for the successor biochemical conversion—inhibitors—may be generated (Palmqvist & Hahn-Hagerdal, 2000). Hence, the generation mechanism of inhibitors in the pretreatment process of biomass should be revealed to establish pretreatment technologies that could reduce or avoid the generation of inhibitors, which will better serve the following biochemical conversion process. The construction of pretreatment platform of biomass should be based on the component structure of biomass and instructed by the conversion goals of different kinds of biomass.

2.2.2  Saccharification Unit Operation of Biochemical Conversion of Biomass The main point of biochemical conversion of biomass to produce products is to turn polysaccharides into monomers, which is also called saccharification traditionally. Monomers could be turned into other products or treated as products directly. The saccharification unit operation of biochemical conversion of biomass aims to fully turn the polysaccharides inside the biomass into available monomers. The polysaccharides inside biomass are cellulose and hemicellulose. Glucose, the monomer of cellulose, is the main carbon source of microbes. Meanwhile, it was indicated that the degradation products of hemicellulose could also be used as a carbon source by microbes (Zhang, Wang, & Zhang, 2004). The unit operations of biochemical conversion of biomass can be realized by either an enzyme platform or refining factory of cells. Since there are many components such as cellulose, hemicellulose, lignin, and cutin in biomass substrates, the process of saccharification by enzyme platform needs the combined action of a multienzyme system. So the construction of enzyme platform contains the process of preparation of cellulase and hemicellulase, as well as the screening process of enzyme preparations. The refining factory of cells can either be single saccharification process, or a cooperative process of saccharification and fermentation. The construction of cells refining factory contains the following processes—analyzing the genome of microbes which could degrade biomass, constructing artificial cells through genetic engineering, analyzing metabolic flux of microbes, and controlling the metabolic flux to better serve the process of biochemical conversion of biomass. The sugar platform of biochemical conversion of biomass is constructed based on the metabolic flux of microbes. A monomer is not only the product

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of microbial metabolism, but also an important source material of many small molecular substances. Accordingly, understanding metabolic flux of microbes is key to constructing the sugar platform of biochemical conversion of biomass. Sugar platform of biochemical conversion of biomass mainly serves the successor fermentation application. Therefore, the construction of sugar platform contains degradation of macromolecular polysaccharide into monomers through enzymes or refining factory of cells, as well as elimination of inhibitors generated from pretreatment through physical or chemical methods to increase the productivity of fermentation products.

2.2.3  Fermentation Unit Operation of Biochemical Conversion of Biomass The fermentation unit operation of biochemical conversion of biomass is the last process to turn biomass into final products through biochemical conversion. The goal of this unit operation is to turn monomers derived from cellulose and hemicellulose degradation into other universal small molecules for chemicals. The fermentation unit operation of biochemical conversion of biomass consists of the fractional hydrolysis fermentation process, simultaneous saccharification and fermentation process, simultaneous saccharification and cofermentation process, and consolidated bioprocessing process. Those four conversion processes are divided into solid-state fermentation and submerged fermentation. No matter which kind of fermentation is adopted, the key is to control conditions during fermentation process to satisfy the demand for microbial growth and to avoid the generation of inhibitors. As for solid-state fermentation, the key is to control conditions during the fermentation process according to the law of microbial growth and metabolism, and also on the basis of intrinsic features of biomass solid materials.

2.2.4  Posttreatment Unit Operation of Biochemical Conversion of Biomass Biobased products need to be separated and purified from the fermentation system after they are prepared through biochemical conversion of biomass, so a posttreatment platform is also necessary for the biochemical conversion process of biomass. The posttreatment unit operation of biochemical conversion of biomass consists of separation and purification, whose main aim is to improve product yield with the guarantee of product purity. Fermentation products accumulate in the fermentation system, which will inhibit the growth and metabolism of microbes. Hence, the separation of products during the reaction process can also reduce product inhibition.

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2.3  PROCESS ENGINEERING OF BIOCHEMICAL CONVERSION OF BIOMASS AND ITS INTEGRATION Biomass material is complex while it is extremely abundant in the earth. As a consequence, it is necessary to integrate various technologies on the basis of components separation to utilize biomass materials fully. From a macro point of view, the so-called integration of various technologies is virtually an application of chemical fundamental theory in bioconversion. Integration refers to an aggregation or combination of relatively independent parts to form a unity, which resulted in a scale effect or cluster effect. Li (Li & Xiang, 1999) pointed out that integration is to combine two or more factors (units or subsystems) to form an organic system, which is not the simple superposition of the factors, but their organic synthesis. That is to say, integration is a combination or construction based on certain rules, which aims to improve the whole function of an organic system. The combination of various technologies involved in the biomass industry is a good example of such organic integration. The modularization of biomass application technologies refers to an integration of technologies based on processing intent and their status in conversion. Application of biomass involves pretreatment of raw materials, preparation and compatibility of enzymes, biomass degradation, bioconversion, and products’ separation and purification. Each process contains many methods and technologies, which are selected based on engineering needs. Therefore, the whole industry of bioconversion comes down to several platforms—pretreatment platform, enzyme platform, sugar platform, cells refining factory platform, and posttreatment platform. These platforms themselves are the integration of various technologies. Moreover, in the construction of the biomass application industry, especially in the process of forming an industrial chain, these platform technologies integrate with each other. In view of biomass applications, modularization and process integration implement the basic concept of process engineering. Platform compounds, the basic organic compounds with large tonnage, have characteristics of abundant sources, cheap prices, and plentiful applications. Platform compounds can be treated as either products or raw materials. A large amount of high-value products with huge market potential can be synthesized from these platform compounds, such as methane, ethanol, ethylene, nancic acid, and so on (Cen, 2005). Many platform compounds were prepared during the era of coal chemical industry before the 20th century, such as benzene, acetylene, methane, and so on. After the middle of 20th century, the platform compounds from the petrochemical industry (ethylene, propylene, butadiene, benzene, methylbenzene, and xylene) gradually displaced products derived from the coal chemical industry. Since the beginning of the 21st century, oil prices have risen rapidly with the gradual exhaustion of fossil fuels. Therefore, the plentiful and renewable biomass materials will undoubtedly play an important role in the future, and also create opportunities for the development of reproducible new platform compounds, such as biomass. Compared with oil and other

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FIGURE 2.1  Scheme of platform compounds preparation from biomass.

fossil fuels, reproducible biomass materials, such as straw, are cheaper. More importantly, the production of new platform compounds from biomass via biochemical conversion technology has been more environmentally friendly. For this reason, it is a promising route. As shown in Fig. 2.1 (Chen & Wang, 2008), biomass (starch, hemicellulose, and cellulose) are degraded into middle platform compounds, such as biological synthesis gas and sugar (glucose and xylose), via thermochemical, chemical, or biological methods. Then these middle platform compounds are used to synthesize platform compounds, such as ethanol, glycerinum, and nancic acid, via biological and chemical methods.

REFERENCES Cen, P. L. (2005). Research and development of new platform compounds of the high oil price times. Conference Proceedings of Chinese bioresource technology and sugar engineering. Chen, H. Z. (2010). Process engineering of bio-based products. Beijing: Chemical Industrial Press (in Chinese). Chen, H. Z., & Li, Z.H. (2002). Cellulose material microorganisms and biomass fuel availability. Chemical Technology Market, 24(5), 25–29.

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Chen, H. Z., & Qiu, W. H. (2007). Key problems and research progress of ethanol fermentation by straw. Progress in Chemistry, 19, 1116–1121. Chen, H. Z., & Wang, L. (2008). Research progress of the key process in the production of bio-based products and the integration of eco-industrial chain-proposal of bio-products process engineering. Journal of Process Engineering, 8, 676–681. Li, H. F., & Xiang, Z. C. (1999). The theory of management integration. China Soft Science, 3, 87–89. Palmqvist, E., & Hahn-Hagerdal, B. (2000). Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification. Bioresource Technology, 74, 17–24. Zhang, Y. -h., Wang, J., Zhang, W., et al. (2004). Research progress of hemicellulose fermentation to produce fuel alcohol. Liquor-making Science & Technology, 5, 72–74.

Chapter 3

Pretreatment Strategies for Biochemical Conversion of Biomass Chapter Outline 3.1 Biomass Recalcitrance for Biochemical Conversion 3.1.1 Application of Biomass Recalcitrance in Life and Production 3.1.2 Proposal of Biomass Recalcitrance for Biochemical Conversion 3.1.3 Definition of Biomass Recalcitrance for Biochemical Conversion 3.1.4 Analysis of Biomass Recalcitrance for Biochemical Conversion 3.1.5 Research on Biomass Recalcitrance for Biochemical Conversion 3.2 Biomass Pretreatment Platform of Biochemical Conversion— Overview 3.2.1 Natural Biochemical Conversion Process of Biomass 3.2.2 Development of Biomass Artificial Degradation 3.3 Pretreatment Mechanism and Application of Biomass Biochemical Conversion

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3.3.1 Chemical Pretreatment Mechanism of Biomass Biochemical Conversion 40 3.3.2 Physical Pretreatment Mechanism of Biomass Biochemical Conversion 47 3.3.3 Biological Pretreatment Mechanism of Biomass Biochemical Conversion 50 3.4 Pretreatment Fractionation Technology for Biomass Biochemical Conversion 51 3.4.1 Steam Explosion Pretreatment for Biomass Biochemical Conversion 51 3.4.2 Tissue Fractionation Techniques for Biomass Biochemical Conversion 52 3.4.3 Cell Fractionation Techniques for Biomass Biochemical Conversion 52 3.4.4 Components Fractionation Technologies Before Biomass Biochemical Conversion 53

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Technologies for Biochemical Conversion of Biomass 3.5 Features of Biomass Biochemical Fractionation Conversion Pretreatment Technology 56 3.6 Evaluation of Biomass Biochemical Fractionation Conversion Pretreatment Technology 56 3.6.1 Evaluation Standards of Biomass Biochemical

Fractionation Conversion Pretreatment Technology 56 3.6.2 Comparison of Biomass Biochemical Fractionation Conversion Pretreatment Technology 57 References 59

3.1  BIOMASS RECALCITRANCE FOR BIOCHEMICAL CONVERSION In the process of evolution, lignocellulosic biomass formed a tight structure and lignified tissue as a framework to fight back against the invasion of microorganisms and to absorb sunlight adequately. The tight structures of biomass tissue make wood, bamboo, and rice straw available for human applications in the past, but difficult for biochemical conversion nowadays. Therefore, pretreatment is necessary to improve the conversion yield and total efficiency.

3.1.1  Application of Biomass Recalcitrance in Life and Production For a long time, humans made good use of the tight structure of plants formed during evolution in life and production. Humans had started to use the wood to build houses, since they moved from caves to nests. Today, wood is still an integral material for the building industry. In the 1980s, many roofs, doors, and windows of farm buildings are made of wood (Fig. 3.1) and people could live in these buildings with no painting for decades. In these buildings, the roof was always covered by reeds and cement. In the 1960s and 1970s, the enclosure walls of farmhouse were mostly made of adobe, mixed with some straw (Fig. 3.1). Wood is mainly used in the construction of buildings, since it contains much more lignin compared to herbs, so it can resist microbial degradation and last for decades. In production, in addition to the edible part of plant crops selected by human beings for farming, the other parts are also increasingly used, such as windmills, trolley, bamboo rake, bamboo, straw fences, sorghum fences, etc. (Fig. 3.2). The straw biomass produced from the common production, even if it is exposed to air, sunlight, and water for a long time, can still be used without degradation in the long term. In everyday life (Fig. 3.3), these plant biomasses, such as beds, desks, broom, and carved wood, are difficult for microorganisms to degrade in a natural environment.

FIGURE 3.1  Plant biomass in architecture.

FIGURE 3.2  Plant biomass in production.

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FIGURE 3.3  Plant biomass in daily life.

3.1.2  Proposal of Biomass Recalcitrance for Biochemical Conversion As energy, environment and human development problems have surfaced, the clean conversion technologies of renewable resources have become the focus of applied and academic fields. Due to the abundance and low cost of agricultural and forestry waste, plant biomass has great application potential. It mainly contains lignin, cellulose, and hemicellulose, and can be converted into bio-based materials, bio-based chemicals, and bio-based energy; it is thus an alternative to petroleum products in common industrial materials. However, in the bioconversion of plant biomass to bio-based products, it is difficult to degrade plant biomass by microorganisms or enzymes. To achieve the desired conversion requires a high amount of inoculum or enzymes so that the cost of conversion is economically feasible. To characterize the resistance of plant biomass to bioconversion, the biomass recalcitrance was proposed. The monograph on biomass recalcitrance (Michael E. Himmel, “Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy”) was published in 2008. The word “recalcitrance” originated in 1843; its original meaning was “kicking back,” which refers to the resistance of plants (Schultz, Craig, & Cox-Foster, 1994; van Wordragen & Dons, 1992).

3.1.3  Definition of Biomass Recalcitrance for Biochemical Conversion At the micro level, biomass recalcitrance was defined by Michael E. Himmel as follows: multiple resistance of plant material to microbial and enzymatic degradation (Himmel, 2009). He also summarized biomass recalcitrance from eight different aspects:

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1. the plant epidermis system, especially the keratin; 2. the arrangement and density of vascular bundles; 4. the relative content of sclerenchyma cells; 4. the degree of lignification; 5. the warty layers covered the secondary wall; 6. the complex composition and heterogeneous structure of the cell wall, such as microfibers and the matrix polymer; 7. the reaction resistance of enzymes on insoluble materials; 8. the fermentation inhibitors contained in cell wall or generated in conversion process. The cognation on biomass recalcitrance is currently only an assumption, which has not been objectively verified and analyzed in depth. Essentially, it can be attributed to two aspects: physical barriers and chemical barriers. The physical barrier is composed of epidermis systems, vascular bundles, sclerenchyma, lignification, a tumor layer, and cell wall composition, and is essentially due to the presence of keratin, wax, and lignin, to hinder the accessibility of enzymes with the substrate. The chemical barriers refer to the invalid adsorption of enzymes on insoluble substrates and the influence of inhibitors on enzymatic activity. Enzyme activity is weakened or stopped due to the hydrogen bonds and chemical bonds with cellulase. Biomass recalcitrance, on the one hand, it needs to verify the correctness of assumption; on the other hand, it needs to analyze the degree of resistance of plant biomass to degradation from a quantitative point of view. On these bases, the effects of various pretreatment technologies on each recalcitrance level should be evaluated to determine the economic, efficient, clean, and operable preprocessing techniques or integrated technologies.

3.1.4  Analysis of Biomass Recalcitrance for Biochemical Conversion Biomass recalcitrance is gradually formed to adapt to environmental changes in the long-term evolution process. In the prephanerozoic era, which lasted 30 million years, life on Earth was present in the aquatic environment, that is to say, the entire biosphere was included in the hydrosphere. Terrestrial life first appeared about 400 million years ago in the Middle Ordovician to Late Ordovician period, and achieved prosperity in the latest tenth of Earth’s history. The establishment of terrestrial ecosystems and the emergence of vascular plants are impossible to separate from evolution (Chen & Yang, 1994). Vascular plants are one of the most exotic biological groups on earth. In the current biosphere, they account for 97% of the total biomass and include about 30 million kinds of organism. Vascular plants, bryophytes, algae, and cyanobacteria, etc. together as primary producers support the huge terrestrial ecosystems.

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Vascular plants refer to photosynthetic and autotrophic organisms on land with a woody vascular system. Vascular plants and bryophytes without vascular bundles have complex individual development processes, so they are called embryophyta collectively. Lignification is the skeletonization of a plant. The skeletonization of animals and plants began in the late Proterozoic to early Cambrian period, about 500 million years ago. This skeletonization is signed by the emergency of calcareous algae in plants, which means the generation of an exoskeleton. The second skeletonization occurred in the middle or late Ordovician period and is signed by the origin of a lignified vascular system. The evolution from thalloid plants to vascular plants is the adaptive changing process from an aquatic environment to a terrestrial environment. This process involves innovations of plants’ intrinsic structures and physiological functions. The innovations gave plants new features including: 1. the ability to adjust and control water balance between the internal and external of a plant, which helps it to adapt to the drought environment inland; 2. strong mechanical support, which makes a plant upright on the land without an aqueous medium; 3. an effective transportation system of water and nutrients, which makes a plant absorb moisture and nutrients from soil effectively; and 4. the ability to resist ultraviolet radiation, meaning a plant can be exposed to sunlight. Vascular plants can adapt to and even take advantage of the special environmental conditions of land. The emergency of the cuticle is the important structural feature of vascular plants, and it can reduce water loss of plants. The cuticle layer on superficial cells is a polymer composed of alcohols and acids. It can effectively prevent water evaporation from the surface. However, the cuticle layer can also prevent CO2 from diffusing into the plant tissue. The generation of stoma is the adaptive evolution associated with cuticle layer. The subsidiary cells of stoma regulate the opening and closing of stoma by changing their degree of expansion, so they can effectively control water evaporation and CO2 diffusion. The competition for light and the broadened dispersal of germ cells effectively promote the evolution of tall plants. With this increase in height, water and nutrient transport become difficult and tall plants require more mechanical support. These factors contribute to the evolution of the vascular system. Initially, it is the emergence of lignifications, tracheids, and sieve cells which are conducive to nutrient transportation, and then vascular systems with transportation and supporting functions appeared. Cuticle, stomata, the vascular system, lignification, and plant growth are all related to the evolution of vascular plants. These series of evolutionary changes contribute to the vascular plants adapted to a terrestrial environment.

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Meanwhile, the cuticle, vascular system, and lignification become strong barriers to microbial degradation of vascular plants. According to the classification method proposed by Sachs (1875) (Wright, 1883), a plant’s mature tissues are divided into a dermal tissue system, vascular tissue system, and ground tissue system. The dermal tissue system can be divided into the epidermis and periderm; the vascular system can be divided into xylem and phloem; and the tissue-based system can be divided into parenchyma tissue including secretory, collenchymas (Liu, 2010), and sclerenchyma.

3.1.4.1  Dermal Tissue System Dermal tissue systems include the periderm and epidermis; periderm mainly exist in woody plants, and in this section, specifically refers to the epidermis. The epidermis is composed of a layer of epidermis cells, which cover the primary structure of the plant but does not exist on the external surface of root cap and apical meristem. Epidermal cells are generally living parenchyma cells (Liu, 2010) of various shapes, but are usually rectangular or irregular flat cells. The epidermis consists of these rectangular or irregular flat cells, additionally with half-moon guard cells and hairy epidermal cells. The outer wall of epidermal cells is often thickened and cornified, so the outer wall is covered with a cuticle layer. Liu (2010) also called this the cuticular membrane. The cuticle is mainly composed of cutin and wax. In 1847, the cuticle structure model was proposed by von Mohl (Li & Wu, 1993). There are many researches on structural models of cuticle layer. In one model, the cuticle layer is divided into three layers (Fig. 3.4): the first layer (outermost layer) is the epicuticular wax, the second layer is the cutin immersed in wax, and the third layer is a complex layer made up of cutin, wax, and polysaccharides (Li & Wu, 1993).

FIGURE 3.4  Plant cell wall cuticle. 1, wax on the surface of cuticle; 2, cutin immersed in wax; 3, mixed layer of cutin, wax, and polysaccharides; 4, middle layer; 5, primary and secondary cell wall; 6, epidermal cell.

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FIGURE 3.5  Plant cell wall. 1, Cuticle; 2, epidermal cell; 3, guard cell; 4, stoma lacune; 5, air chamber; 6, chloroplast.

The epidermal cells are arranged in a continuous layer tightly and slits form in many epidermal cells (Fig. 3.5). On both sides of the slit there exist halfmoon cells, which are called guard cells (Liu, 2010). The guard cells and the gap between guard cells together are called stoma (Fig. 3.6). The stoma area is sixth-millionth of the cuticle area (Li & Wu, 1993); the upper limit of stoma diameter is 0.9 nm, which can be passed by nonelectrolytes, small molecules, and hydrated ions. When the cuticle is treated with chloroform and removed with the fat-soluble substance, its water permeability increases by 2–3 orders of magnitude, indicating that 100–1,000 times the amount of stoma was exposed due to the removal of fat-soluble substances. The hair-like appendages called epidermal hair often exist outside the epidermal cells of various organs (Fig. 3.7). These may be alive or dead (Liu, 2010). Some cell walls of epidermal hair are completely hornified, which blocks the evaporation of moisture through the epidermal hair. The cuticle layer at the surface of the epidermal system is only connected with one layer of epidermis cells. In the pretreatment process of lignocellulosic feedstocks, even the energy-extensive comminution is difficult to reach the

FIGURE 3.6  Epidermal cell and stoma.

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FIGURE 3.7  Epidermal hair.

cellular level. During chemical and biological pretreatments, the existence of the cuticle layer reduces the availability of multi-components. Thus, in the utilization of lignocellulosic resources, particularly through the enzymatic hydrolysis and fermentation for bio-based fuels, the cuticle layer of the epidermis is the first barrier. There are two ways to disrupt these barriers: removal and degradation.

3.1.4.2  Vascular Tissue System Vascular bundles of gramineous plants usually have two distribution patterns: one is to arrange in two laps in which the outer circle is bigger and the inner circle is smaller, such as wheat, barley, and rice; another style is to scatter in the stem and not arrange in a circular shape, which is true for corn, sorghum, and sugar cane. The first kind of vascular bundles are shown in Fig. 3.8: the outermost layer of the stem is epidermis; the sclerenchyma tissue stay within the epidermis. The thick layers of parenchyma tissue within the parenchyma layer has a large cavity (a hollow stalk with fewer materials to maximize the amount of material mechanical performance). In the thick tissue distribution of the inner circle and a circle of smaller green tissue of vascular bundles (i.e., containing chloroplasts parenchyma bundle). Green tissue bundles and epidermis are arranged directly adjacent to each other, with a smaller interval of vascular bundles, distributed around a large vascular bundles in the parenchyma. Some aquatic species (rice) in the parenchyma layer are also distributed around the gas chamber (ventilation organization); the gas chamber is located between the two laps of vascular bundles. ­Scattered distribution of vascular species in the stem structure is shown in Fig. 3.9: an amount of stem epidermis layer of thick-walled parenchyma (i.e., hardening parenchyma); and the hardening of thin-walled parenchyma tissue, distributed within the parenchyma of the scattered vascular bundles. The epidermis (2) is

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FIGURE 3.8  Crosscutting map of wheat stem. 1, cuticle; 2, epidermis; 3, green strands; 4, fibers; 5, bundle sheath; 6, primary phloem; 7, primary xylem; 8, parenchymal tissues; 9, big cavity.

located in the small outer periphery of the stem are arranged closely, in the center of the distribution of the larger stems which are arranged loose. Hardening refers to thickening and lignification of the cell wall. Vascular system (Liu, 2010): In the plants or plant organs, phloem is combined with xylem to form a continuous tissue system throughout the entire plant or plant organs. Vascular bundles (Liu, 2010) (Fig. 3.10): In plant stems, leaves, flowers, and fruits, the xylem and phloem bundles are combined to form the fascicular structure. Xylem (Liu, 2010): Vessels and tracheids orient their alignment to form the xylem.

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FIGURE 3.9  Crosscutting map of maize stem. 1, Epidermis; 2, stoma; 3, parenchymal tissues; 4, primary phloem; 5, primary xylem; 6, bundle sheath of parenchymal tissues; 7, hardened parenchymal tissues.

FIGURE 3.10  Rip cutting of vascular tissue and surrounding sheath (×100).

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FIGURE 3.11  Crosscutting of vascular tissue and surrounding sheath (×100). 1, Sieve tube; 2, conduit; 3, air cavity; 4, bundle sheath.

Protoxylem (Liu, 2010): The earliest developed xylem originated from procambia. It has quite thin and elongated vessels and tracheids, which is located in the lower half of the Y shape. Metaxylem (Liu, 2010): The xylem developed from procambia in the late time. It contains coarse vessels and tracheids that are not elongated and is located in the upper half of the Y shape. Primary xylem (Liu, 2010): Protoxylem and metaxylem are mature tissues generated by shoot apical meristem, and such primary tissues are collectively called primary xylem, which is arranged as a Y shape on the horizontal section. Vessel (Fig. 3.11) (Liu, 2010): This is a hollow tube ringed by many dead cells. During the formation of a vessel, sidewalls are thickened and lignified, and the cell wall between cells are digested by enzymes, finally resulting in the hollow tube. There are five kind of thickened vessel secondary walls, thus five kinds of vessels: annular vessels, spiral vessels, scalariform vessels, reticulated vessels, and pitted vessels. Tracheid (Fig. 3.12): Each long prismatic tracheid is a water conduction unit, which is overlapped and linked by tracheid ends. Water is transferred through the pits on walls. Its content is relatively low in angiosperms. Vessel and tracheid: Transport of water and inorganic salts. Phloem (Liu, 2010): This is formed by the sieve tube, companioncells, parenchyma cells and fibers in plants or plant organs, and it is composed of screens. Protophloem (Liu, 2010): The phloem originally developed from cambium, and is squeezed outside the primary phloem and next to the bundle sheath. Metaphloem (Liu, 2010): The phloem developed from cambium in the late period is inside the primary phloem and next to the metaxylem.

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FIGURE 3.12  Tracheid. (A) Early wood tracheid; (B) late wood tracheid; and (C) spiral thickening.

Primary phloem (Liu, 2010): Protophloem and metaphloem are primary tissues and usually called primary phloem. Sieve tube (Liu, 2010): This is formed by live cells with long prismatic shape or long cylindrical shape connected with each other. There is no lignified cell wall in sieve tube molecule. Companion cell (Liu, 2010): This refers to the small parenchyma cells next to the sieve molecule. This cell is in long prismatic shape and much smaller than the sieve cell. Sieve cell (Liu, 2010): Transport of organic matters (photosynthetic products, hormones, etc.). Cavity (Himmel, 2009): This is contained in the vessels of protoxylem and is the early location of protoxylem occupied. It can be concluded from the above concepts that the secondary cell wall of vessel cells is thickened and lignified, thus xylem (referring to vessels in gramineous plants) turns out to be the barrier to biodegradation of lignocellulosic feedstocks.

3.1.4.3  Basic Organization Systems In herbaceous plant, basic organization systems are mainly composed of parenchyma and sclerenchyma. Parenchyma (Liu, 2010) (Fig. 3.13): This is mainly related to nutrition, but secretory cells, transfer cells, and companion cells have nothing to do with nutrition. Parenchyma cells mainly contain thin cell walls but some have lignified thick cell walls, for example, the parenchymal cell distributed in xylem. Sclerenchyma (Liu, 2010): This is normally composed of dead cells, with thick and lignified cell walls. Cells in sclerenchyma have various shapes and according to their morphology features, can be divided into sclereid and fiber. Sclereid cell has a short body and the length is several times less than its width; a fiber cell has a long body with a high length–width ratio.

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FIGURE 3.13  Parenchymal cells of corn stalk pith (×100).

Sclerotic cells are in various shapes; one that has a more or less equal-diameter polyhedrons is called a stone cell. Sclerotic cells have lignified and thick cell walls. On the cell walls there are lots of circular, simple pits. Sclerotic cells are usually transferred from parenchymalcells by sclerification; they can also be produced by meristematic cells. Sclerification is the thickening process of secondary walls in parenchymalcells. Fiber (Liu, 2010): In sclerenchyma (belonging to mechanical tissue), cells with high length–width ratio are fibers, which are contained in xylem and phloem. Wood fibers (Liu, 2010): This refers to the fibers in the xylem. Pits on cell wall of wood fiber are bordered pits or single pits. Pits are in a lens-shape or slit-shape. Extraxylary fiber (Liu, 2010): This refers to fibers outside the xylem, such as fibers located in the phloem, around vascular bundles, on both sides or one side of the cortex and vein, in monocotyledons. The cell walls of extraxylary fibers are usually thick and some lignified, and they have single pits whose surface view is often lens-shaped or slit-shaped. Fiber: This is spindle-shaped and sharp at both ends. Its cell wall is thick and often lignified. Fibers rarely exist alone and often are gathered into bundles. Bundle sheath (Fig. 3.11) (Liu, 2010): This refers to sclerenchyma tissues covered around vascular bundles of corn stems. Fibers belong to sclerenchyma with thick and lignified cell walls. Hence their utilization firstly needs to reduce the content of lignin and thus can improve the accessibility of cellulose. As most parenchyma cell walls only contain cellulose, they are therefore suitable for enzymatic conversion to bio-based fuels.

3.1.5  Research on Biomass Recalcitrance for Biochemical Conversion To deconstruct the natural biomass recalcitrance, physical, chemical, and biological pretreatments are developed in the utilization process of biomass. Series compositional and structural changes occur during processing, producing new

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FIGURE 3.14  Formation path of secondary barrier against degradation (Palmqvist & Hahn-Hägerdal, 2000).

substances, which have been verified to have inhibiting effects on the bioconversion process. To distinguish these inhibitory effects during pretreatment process with the natural barriers, the new concept of secondary biomass recalcitrance is proposed. The secondary biomass recalcitrance refers to the resistance characteristics against microorganism and enzymes, which are generated in the pretreatment process, including the fermentation inhibitors produced in enzymatic hydrolysis. The formation path of second recalcitrance is as follows (Fig. 3.14): Secondary recalcitrance was found during the comparative evaluation of a variety of biomass pretreatment processes. The reported inhibitors caused by the secondary recalcitrance include (Palmqvist & Hahn-Hägerdal, 2000; Fang, Huang, & Xia, 2005; Wang, Wang, & Zhang, 2009; Wang & Chen, 2011): formic acid, acetic acid, levulinic acid, furfural, hydroxymethyl furfural, vanillin, and other lignin degradation products (including phenolic acid). In addition, studies have shown that during the biomass pretreatment process, macromolecules formed by lignin and carbohydrates also have inhibitory effects. Therefore, the pretreatment of plant biomass should not only increase the sugar yield, but also reduce the inhibitory effects of secondary recalcitrance according to the utilization of sugar.

3.2  BIOMASS PRETREATMENT PLATFORM OF BIOCHEMICAL CONVERSION—OVERVIEW 3.2.1  Natural Biochemical Conversion Process of Biomass 3.2.1.1  Natural Biochemical Conversion of Biomass Resources Naturally, human cultivate plants selectively; some parts are used, the other parts are abandoned, and these abandoned parts have a negative impact on

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normal farming. They are mostly erased by burning (for example, large quantities of corn stalk), while the natural biochemical conversion process is mainly degraded dead twigs and withered leaves in artificial or original forests. There are three main sources of dead twigs and withered leaves. The first comes from the natural pruning process in forests. Natural pruning refers to the phenomenon of the branches gradually starting to litter after young trees’ canopy due to insufficient light. The second is due to the damage of forest pests. The third cause is bad weather, which breaks branches and tears down leaves. Natural biochemical conversion of biomass resources is necessary for the development of soil and the forest biosphere, and the amount of biomass required needs to be analyzed from a different perspective, especially that of soil nutrition. Studies have shown that the litter layer can hinder the natural regeneration of the forest (Wang, Li, & Yu, 2008). Thus in the conditions of ensuring the soil quality, transferring the natural degradation into artificially accelerated does not only complete natural ecology, but also meets the requirements of human development. The natural transformation process completes a step in the carbon cycle, and also provides a reference for artificial conversion of plant biomass. Screening microorganisms from natural habitats has become an important source of bacterial strains. The death process, and biochemical conversion processes of biomass in nature, has also become a source of wisdom in the artificial use of plant biomass ways and methods.

3.2.1.2  Natural Death Process of Biomass For plant roots in the soil, the death of cells is essential during the growth and development of many eukaryotic organisms. Since the organism itself controls the start and execution of cell death, this type of cell death is called programmed cell death (PCD). Two examples of PCD are aging and hypersensitivity. Tubular molecules are examples of plant developmental PCD. Among mature plant cells, catheters, fibers, hardened cells, and cork cells are dead cells (Chen, 2008); epidermal cells, parenchyma cells, sieve cells, and companion cells are living cells. Therefore, after the plant biomass harvest at different storage period, due to living cells containing cytoplasmics and organelles, and wherein enzymes are active, those factors result in a change in water content during storage, as well as from the catalytic enzyme, resulting in changes in the composition and structure, thus affecting its chemical conversion properties. Straw during storage, due to the different storage stages and storage methods, results in obvious changes in moisture, causing changes in the natural growth of microbial communities (Singh, Honig, & Wermke, 1996). Study on the changes of composition and structure in the degradation process with different storage methods and the effect on straw barrier will provide a reference for artificial straw and will be helpful for efficient storage in the industrial application of lignocellulosic feedstock.

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3.2.1.3  Natural Biochemical Conversion Process of Biomass and its Implications for Artificial Biochemical Conversion Process Natural decomposition of biomass is a process which combines physical effects, chemical effects, biological effects, etc., and can be briefly summarized in two stages : pretreatment and, solid-state fermentation. Natural preconditioning involves the physical, chemical, and biological processes, for a role in the process is often not simply classified as a treatment effect. In long-term evolution in nature, organisms formed a natural barrier structure to resist degradation of other organism; therefore, degradation of biomass needs pretreatment to change the physical structure or chemical composition, to make it easier for the biomass to be decomposed by microorganisms. Natural pretreatment effects include dissolution process of (water leaching) soluble compounds, and the mechanical grinding process of insoluble compounds and the damaging effects of microbial growth and metabolism. Natural preconditioning is an important factor to obtain carbon and energy from biomass of microorganisms, altering the availability of the biomass by preconditioning, particles contained in the biomass, such as surface area, porosity, etc.; other insoluble nutrient transformation occurs in soluble substances, such as depolymerization and hydrolysis reactions. Take soil animals as an example: they mechanically break biomasses, and tear some of the larger volume of the complete pyrolysis of biomass into smaller pieces, which increases the efficient size of the use of biomasses. Meanwhile, soil animals produce microbial proteins and growth factors can then be used to promote the growth of microorganisms. Natural solid-state fermentation is essentially a mixed fermentation process. Mutual promotions and inhibitions between fungi themselves, and between fungi and germs, even between actinomyces, work together to complete the catabolism of biomass. Microorganisms with different ecological habits throughout the decomposition process to saprophytic parasitic interactions succession order. Microflora in a nutrition organic matrix, are interdependent, and mutual inhibition constituting a saprophytic food chain, community composition, and showed a significant number of dynamic changes. The biochemical conversion processes of plant biomass under natural conditions have the following conditions for the use of biomass: 1. The storage of plant biomass should avoid the leaching of water and try to keep the plant intact to prevent microbial invasion. 2. Different microbial enzymes are needed for different stages of degradation, therefore, for the different purposes of biomass bioconversion, a suitable enzyme or microorganism should be chosen. 3. Degradation and residues of plant biomass in different habitats are different, therefore, the resistant barrier of plant biomass is a relative definition. 4. The pretreatment process of plant biomassis a concentration of the natural degradation process, therefore, it can be learned from the physical chemistry of biological processes under different habitats.

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3.2.2  Development of Biomass Artificial Degradation The lignocellulose in early times was used to complement the shortage of food and fuel due to congestion of populations in many developing countries (Klyosov, 1986). Lignocellulose was mainly applied by three methods. The first focused on a single product with a single pretreatment technology before 1990 (Kawamori, Morikawa, & Ado, 1986; Taniguchi, Kometani, & Tanaka, 1982a; Zadra il, 1977; Wayman & Parekh, 1988), and then an integrated pretreatment technology appeared for a single product (Carrasco & Roy, 1992; Maekawa, 1996; Holtzapple, Jun, & Ashok, 1991). In the 21st century, humans began to care about the problems of environment, energy, and rural development. Therefore, integrated pretreatment for multiproducts was developed in order to find an alternative to petroleum. However, back in 1987 (Klyosov, 1986), COALBAR Company in Brazil had used lignocellulose for both ethanol and charcoal. Thus the division above is not so strict, but research focuses are different in different stages.

3.2.2.1  Single Pretreatment Technology for a Single Product Before 1990, lignocellulose was only converted to ethanol with a single pretreatment technology; this technology included physical, chemical, thermal, and biological methods. Physical methods mainly include mechanical grinding and gamma-ray pretreatment (Bono, Gas, & Boudet, 1985). Chemical methods refer to dilute acid or alkali pretreatment (Kawamori et al., 1986), ozone pretreatment (Bono et al., 1985), and zinc chloride pretreatment (Cao, Xu, & Chen, 1995). Dilute acid pretreatment includes dilute sulfuric acid pretreatment and dilute hydrochloric acid pretreatment, hypochlorite pretreatment (David & Atarhouch, 1987), peracetic acid pretreatment (Taniguchi, Tanaka, & Matsuno, 1982b), and sulfur dioxide pretreatment (Wayman & Parekh, 1988). There are also single pretreatments used for a single product nowadays, such as fumaric acid, maleic (Kootstra, Beeftink, & Scott, 2009), and ionic liquid (Liu & Chen, 2006). All the aforementioned researches aimed to improve the yield of enzyme hydrolysis. Phosphoric acid was also applied to improve lignocellulose edibility (Deschamps, Ramos, & Fontana, 1996). Thermal methods included steam explosion pretreatment (Grous, Converse, & Grethlein, 1986), hot water pretreatment (van Walsum, Allen, & Spencer, 1996), and microwave pretreatment (Ooshima, Aso, & Harano, 1984). Organic solvent pretreatment (Lipinsky & Kresovich, 1982) and ammonia freeze explosion pretreatment (Holtzapple et al., 1991) were also applied, which could be categorized as a thermochemical method. For hot water pretreatment, two-stage hydrolysis at high temperature was applied to avoid the formation of furfural (Torget & Teh-An, 1994). The biological method was mainly used to hydrolyze lignin with microorganisms (Hatakka, 1983), reducing inefficacious absorption and enhancing the

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enzyme hydrolysis rate. Microorganism pretreatment was also researched to produce a single cell protein (Taniguchi et al., 1982a) and feed (Zadra il, 1977). Industrial application for single pretreatment for single product was backed to 1913 (Klyosov, 1986). In Georgetown, South Carolina, pin mill waste was pretreated with 2% sulfuric acid at 175°C in rotary steam heated digesters, producing 5,000 gallons of ethanol per day. Subsequently, a second factory was built in Fullerton, Louisiana. However, both factories did not become profitable till the middle 1920s. Thus it can be seen that lignocellulose conversion into single product with a single pretreatment method could hardly be used in industrialization nowadays because of high feedstock prices and strong environmental consciousness.

3.2.2.2  Integrated Pretreatment for a Single Product Early in 1986 (Klyosov, 1986), steam explosion pretreatment was believed to be one of the most economical and effective technologies. Pretreatment methods integrated with steam explosion include methanol, peroxide hydrogen, sodium hydroxide (Maekawa, 1996), and ammonia pretreatment (Carrasco, 1992). Moreover, in order to enhance biogas production in anaerobic digestion, corn stalk was pretreated with sodium hydroxide integrated with green oxygen or 1,4-dihydroxy anthraquinone (Ping, Xiujin, & Hairong, 2010), and rice straw was pretreated with grinding and calcium hydroxide (Cui, Zhu, & Wang, 2011). For fiber preparation, ultrasonic integrated chemical pretreatment was applied for poplar, obtaining fibers of 5–10 nm. Chemical pretreatment includes two processes: delignification with sodium hypochlorite and removal of gel, as well as hemicelluloses with sodium hydroxide (Chen, Yu, & Liu, 2011). Though the price of petroleum is rising and environmental consciousness is becoming stronger and stronger, the technology to produce ethanol or biogas still lacks competiveness due to economic problems. Thus, how to reduce cost is a key step of conversion. 3.2.2.3  Inevitability of Integrated Pretreatment for Multiproducts After the Second World War, only one biofuel company in the Soviet Union succeeded as a commercial operation. The essential reason was that lignocellulose was considered as multifunctional biomass and converted into ethanol (cellulose), yeast, furfural (hemicellulose), and fuel (lignin). Until 1986, this company owned 40 full-scale plants with a maximum capacity of 1,000 tons of wood material per plant per day. The production from this technology was 1.5 million tons of fodder yeasts and 195 million liters of ethanol. It proved that integrated pretreatment for multiproducts was the only way for the industrialization of lignocellulose refining. As early as 1986 (Klyosov, 1986), integrated pretreatment for multiproducts was proposed as an effective way to use lignocellulose, which was considered as wastes with large amounts. In 1993 (Wyman & Goodman, 1993), an economic

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model revealed that multiproducts from lignocellulose could decrease the cost of product. However, an effective pretreatment method had not been reported before. It was believed that bioproducts would become alternatives of those made from petroleum. However, lignocellulose refining technology could hardly be industrialized due to the lack of competitiveness, though related technologies have been studied for nearly 100 years and the price of petroleum is still rising now. Related researches mainly focus on biofuels, but in the long term, lignocellulose would be the only raw material for chemicals while petroleum is running out. Therefore, the importance of biochemicals should not be ignored. Even biochemicals could not take the place of petroleum; the multiproduct concept can make the industrialization of lignocelluloses refining more beneficial and cost efficient.

3.3  PRETREATMENT MECHANISM AND APPLICATION OF BIOMASS BIOCHEMICAL CONVERSION 3.3.1  Chemical Pretreatment Mechanism of Biomass Biochemical Conversion Acid-base chemistry of plant biomass processing has long been applied in the pulp and paper industry. Similar to the pulping industry, lignin fractions should be removed before the biomass biochemical conversion process, during which hemicellulose is also removed inevitably. Therefore, the mechanism of an acidbase pretreatment in the pulping industry is used as a reference to elaborate biomass biochemical pretreatment.

3.3.1.1  Mass Transfer Process of Chemicals During chemical acid treatment of plant biomass, one of the main factors affecting the treatment effect is the transfer rate of acid solution. For different plant biomass raw materials, caused by their different organizational structures, there is a difference in the internal pore structure, and this affects the acid-base fluid delivery paths. Table 3.1 lists the different delivery paths of pharmaceuticals in herbs, leaf wood, and needle wood materials (Zhan, 2011; He & Li, 2001). 3.3.1.2  Degradation Mechanism and Removal Order of Lignin Under Acid–Alkali Treatment 1. Delignification during alkaline treatment In the alkaline cooking process, a characteristic of delignification is that lignin macromolecules must be broken into small molecules, so that they can be dissolved in the solution. Therefore, the alkali delignification process is the cleavage reaction of various bonds between the units of lignin macromolecules. Meanwhile, the broken lignin molecules cannot be condensed into macromolecules any more.

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TABLE 3.1 Solvent Transfer Process in Different Kinds of Biomass Herbaceous plant

Hard wood

Soft wood

Structure

Vessel, sieve tube, parenchymal cell

Vessel, tracheid (fiber), ray cell

Tracheid (fiber), ray cell

Solution transfer process

Vessel→parenchymal cell (or sieve tube); compared with wood, the herbaceous plant is loose structure, low penetration resistance and fast diffusion.

Sapwood: vessel→pit→wood fiber; or fracture in other cells; heartwood: penetration is slow as thylose in vessel; high temperature can be used to enhance penetration.

End of tracheid→cell lumen→pit on cell wall→tracheid→ cross-field pit→ray cell→tracheid.

Solvent is usually penetrated from cell lumen to S3, S1, and cell corner. Pits in tracheid of early wood are more that those of late wood. Major impact factors



Composition and pH of solvent, temperature, pressure, material kind, and chip size.

The main linkages between the structural units of lignin macromolecules are ether bonds, as well as carbon–carbon bonds and ester bonds, which exist in grass materials. During alkaline pretreatment, different chemical bonds would show various reaction properties. a. Alkaline fracture of the linkages between phenolic α-aryl ether bonds and α-alkyl ether bonds Such a linkage is easy to break. During pretreatment, OH- and phenolic hydroxyl group (acidic) react with each other and the phenoxide generated is rearranged to facilitate the breakage of oxygen and α-C in the ether bond and phenylpropane. Whether the lignin molecules become smaller after the breakage of α-alkyl ether bonds and α-aryl ether bonds depends on the specific structures. For instance, after the breakage of α-aryl ether bonds and α-alkyl ether bonds in phenyl coumaric structure and pinoresinol structure, the lignin molecules do not getting smaller. The nonphenolic α-aryl ether linkage is very stable. b. Alkaline fracture of the phenolic β-aryl ether bonds The main reaction during alkaline treatment of phenolic β-aryl ether linkages is the elimination reaction of β-proton and β-formaldehyde. Thus, most chemical bonds cannot fracture unless the epoxide is formed through the nucleophilic reaction between OH- and α-C atoms (alkaline fracture). In the pulping industry, sulfate digestion is adopted, during

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which the HS− or S2− ions showed stronger electronegativity, as well as the nucleophilic attack ability, thus the phenolic β-aryl ether bonds are easily fractured through the rapid formation of cyclic sulfide. c. Alkaline fracture of nonphenolic β-aryl ether bonds The β-aryl ether bond is very stable, and only fractures under the following conditions: (1) For the nonphenolic β-aryl ether bonds connected on the α-hydroxy, the α-hydroxy is easily ionized in alkali liquor, thus generated oxygen ions would attack β-C atoms to form epocides, which facilitates the fracture of β-aryl ether bonds. (2) When α-hydroxy exists together with the nonphenolic β-aryl ether bonds, α-hydroxy could facilitate the formation of cyclic sulfides and thus lead to the fracture of β-aryl ether bonds. d. Fracture of the aryl–alkyl/alkyl–alkyl carbon–carbon bonds The aryl–aryl carbon–carbon bond is quite stable. These carbon–carbon bonds may be fractured under some specific conditions, leading to the changes of lignin molecule. Such reactions would rarely occur during alkaline pretreatment. e. Fracture of aryl–alkyl ether bonds Removal of methyl methoxy makes no difference to the decrease of lignin molecule. But it is the main reaction of methanol or methyl mercaptan formation during the high-temperature alkali treatment, and the methylmercaptan generated will cause air pollution. f. The condensation reaction during high-temperature alkali treatment The condensation of Cα-Aγ becomes the dominant reaction affecting the dissolution of lignin. The reaction starts from the methylene quinone structure. When NaOH is sufficient, delignification occurs; when alkali is insufficient, the condensation reaction occurs. Condensation reactions occur between the fractured lignin fractions to form larger lignin molecules, which are even harder to dissolve. Other condensation reactions, such as the Cβ–Cγ condensation and the condensation between phenolic structural units/breakage products and formaldehyde, often occur in the black liquor, which has little influence on the dissolution of lignin. 2. Delignification during acid treatment During acidic sulfite pulping, the main reaction that happens to lignin comes from hydrion and hydrated sulfur dioxide. The major part of sulfonation is Cα, Cγ occasionally, which increases the solubility of lignin. Overall, the β-aryl ether bond and methyl–aryl ether bond, phenolic type or nonphenolic type, are very stable during acidic sulfite digesting. However, the fracture reaction starts from the Cα atom, and with the reaction of hydrated sulfur dioxide forms sulfo groups. The fragmentation effect of lignin occur during acidic sulfite pulping is very noteworthy. Although the α-aryl ether bonds account for only 6–8% of the needle bush lignin fractions, their fracture would result in considerable fragmentation effects.

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During acidic sulfite digesting, sulfonation reaction and condensation reaction compete with each other due to the same reaction location of Cα. Thus, it is necessary to accelerate the sulfonation reaction to avoid condensation. During alkaline and acidic sulfite digesting, the main product of lignin chromophoric groups is diarylbenzenes. Catechol is obtained due to the removal of the methoxy group, and it is then oxidized into diquinone or forms dark compounds with metal ions. Finally, the stilbene structure is formed. 3. The sequence of lignin removal during acid–alkali treatment Generally, the sequence of lignin removal is S3-S2-S1-P-ML. Pretreatment liquor first enters the cell lumen through pits, thus S3 becomes the primary part for lignin removal. The degradation of hemicellulose and lignin in the secondary cell wall helps to form a porous structure, and then the lignin in the intercellular layer begins to be removed. Finally, for wood materials, lignin fractions in the secondary cell wall are left. For grass materials, the residual lignin fractions exist in the intercellular layer and the secondary cell wall. On one hand, taking wheat straw as an example, the lignin content in the intercellular layer is 2.45-fold of that in the secondary cell wall. On the other hand, due to the structural difference of monomers and the different delivery paths of pharmaceuticals, the degradation rate of lignin in different parts differs during the acid–alkali treatment process (Zhan, 2011).

3.3.1.3  Degradation Mechanisms of Carbohydrates Under Acids or Alkaline Treatments 1. Carbohydrates degradation in the alkaline process Under alkaline conditions, the cellulose and hemicellulose degradation reactions occur. a. The reactions of cellulose Peeling reaction: Reducing groups in the glucose end falls one by one. Under alkaline conditions, the reducing end is not stable and falls from the cellulose molecular by β-alkoxy elimination reaction. Terminal reaction: The alkali unstable reducing end groups become stable by converting to alkali-stable α-partial variant cellulose or β-partial variable cellulose, therefore stopping the reaction. Alkaline hydrolysis of cellulose: Under the conditions of high temperature and strong alkali, cellulose macromolecules will break into two or more molecules, and the reducing end increases from one to two or more, which promotes the peeling reaction. b. Hemicellulose reactions Acetyl group-off reaction: Under high temperature and strong alkali conditions, deacetylation reaction is the fastest and most complete reaction. Overall reaction of hemicellulose: Under high temperature and strong alkali conditions, hemicellulose activity is much higher than that of the active fiber, which is easy to be dissolved and decomposed. Hemicellulose structure has changed after the treatment. Because xylan in

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hemicellulose is more stable than mannose and polydextrosein in an alkaline solution, its degradation rate is much lower. Response and retention of xylanase: 4-O-methylglucoseuronic acid chain of xylanase molecular are partly or totally removed, so that the polymerization degree of polyethylene decreases. However, if the xylan has a branched chain, the branched molecules can impede the transfer of xylan to the outside of the fiber cell wall. Hexenuronic acid (HexA): In the alkaline treatment process, xylan chain group 4-O-methyl-glucuronic acid in the hemicellulose converts to 4-deoxy-hexene-4-uronic acid under high temperature and strong alkali conditions. With the dose of alkali used, HexA content decreased. c. The carbohydrates degradation course in the alkali treatment process The peeling reaction of carbohydrates starts at 100°C. Within the range of 100–150°C, peeling reaction is the major reaction. Within the range of 150–160°C, hydrolysis reaction is the main reaction. During the alkali treatment process, the degradation rate of different components are different: alduronic acid and mannose dissolve out within 100°C; within the range of 100–150°C, besides alduronic acid and mannose, galactose and arabinose also began to dissolve. Xylose will not dissolve unless the temperature is higher than 160°C. 2. Carbohydrate degradation in acid treatment process Under the acid treatment, mainly acid hydrolysis reactions occurred. Polymerization of cellulose and hemicellulose is greatly reduced. a. Reactions of cellulose and hemicellulose Acid hydrolysis: Acid hydrolysis mainly refers to the cleavage of (1–4)-βglycoside bond or other glycoside bonds. Carbohydrate firstly degrades into oligosaccharides, and then degrades further into monosaccharides. The higher the temperature and acid concentrations, the fiercer degrade reaction will be. The acidic oxidative decomposition reaction: The aldehyde ends of hemicellulose and cellulose are easily oxidized into saccharic acids under the treatment of sulfite. The monosaccharides produced can further degrade and hexose can degrade into organic acids, while pentose degrades into furfural. b. The reaction process of cellulose and hemicellulose Reaction process of hemicellulose: Acid hydrolysis of hemicellulose is highly related to its type and structure, polymerization degree, as well as number and length of branched chains. Acetyl groups in hemicellulose and furanarabinose mainly leave the hemicellulose molecular from the xylose chain under acidic conditions. However, poly-4-O-methyl glucose aldehyde xylose remains. At the same condition, the dissolving rate of hemicellulose is much higher than that of lignin. Sugars almost do not hydrolyze under 100°C; sugars hydrolyze fastly within 100∼200°C. During the degradation process, hemicellulose does not hydrolyze into

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monosaccharides directly. Instead, it firstly transfers into the solvent and starts to hydrolyze only when the H+ concentration is high enough. Reaction process of cellulose: Under high temperature and acidic conditions, little cellulose gets dissolved; however, glycosidic bonds tend to break, and the polymerization degree of cellulose decreases.

3.3.1.4  Hot Water Treatment Liquid hot water pretreatment, also known as pressure pyrolysis (Mosier, Wyman, & Dale, 2005) 40–60% of lignocellulosic biomass are dissolved under treatment of 200–230°F high-pressure water for 15 min. Almost all hemicellulose and 35–60% lignin are removed, and 4–22% cellulose is degraded (Mok & Antal, 1992). Hot water breaks the hemiacetal bonds within the biomass and generates acids. Therefore, water is also acidic (Weil, Sarikaya, & Rau, 1997) at high temperatures. In that case, polysaccharides, especially hemicellulose, can be hydrolyzed to monosaccharides, and parts of the monosaccharides further hydrolyzed into aldehyde, which suppresses the fermentation of microorganisms (Palmqvist & Hahn-Hägerdal, 2000). We can use alkali (such as KOH) to adjust pH of water to 5–7, and control the chemical reactions in the pretreatment. Lignocellulosic particles can be separated in a hot water pretreatment process (Weil et al., 1997), and cellulose separated has a high hydrolysis ability (Weil, Brewer, & Hendrickson, 1998). There are three types of reactors for hot water pretreatment: cocurrent, counter-current, and flow-through. In the cocurrent reactor, it flows in the same direction; in the counter-current reactor, the material and water flow in opposite direction; in the flow-through, hot water firstly passes a static bed equipped with lignocellulose, solving the lignocellulose, then runs out of the reactor. 3.3.1.5  Organic Solvents Treatment Cellulose solvents can be categorized into organic solvents (such as Cadoxen, CMCS) and inorganic solvents (such as high concentration of sulfuric acid, hydrochloric acid, phosphoric acid). The solvent treatment is mainly based on the principle of the dissolution in the similar material structure, that is, components of the lignocellulose are dissolved into a solvent to separate them from each other. Cellulose solvent treatment causes changes in crystal structure, thus greatly improving the rate of hydrolysis and the degree of hydrolysis (Fan, Gharpuray, & Lee, 1987). The application of organic solvents for separation of lignocellulose components mainly refers to those in the pulp and paper industry. In the late 19th century, there were reports on the usage of ethanol to separate lignin for papermaking. This method has advantages of low prices and easy recovery (Pan, Arato, & Gilkes, 2005; Stockburger, 1993). Nowadays, the organic solvents widely used include ethanol, methanol and glycol alcohol, methyl acetate, ethyl acetate, etc. (Mei-yun, 2004).

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Nowadays, some countries have carried out deep research on the solvent treatment method; these countries include United States, Canada, Germany, Sweden, Finland, and Japan. In 1985, Lora and Aziz (1985) proposed the application of organic solvents in batch cooking for slurry making, which greatly improve the development of Alcohol Cellulose Technology (Aziz & Sarkanen, 1989). This process is suitable for maple, poplar, and birch, and other hardwood pulp. Repap Company in Canada has employed this method for many years (Cronlund & Powers, 1992). The study of solvant pulping method in China is very recent but has developed fast. In 2001, Meiyun Zhang applied the ethanol treatment method in Chinese alpine rush and set up the optimal operation condition. The optimum conditions were: ethanol concentration, 55%; cooking temperature, 180°C; solid–liquid ratio, l: 10; holding time, 120 min; fine pulp yield, 53.18%; kappa value, 38.13; residual lignin rate, 4.64%. His research provides a theoretical basis for the industrial application of ethanol pulping technology in China. Xuegang Luo et al. (Cronlund & Powers, 1992) used ethyl acetate and acetic acid to degrade lignin. When cooked under 150–170°C for 2 h, pulp fibers obtained not only contained less lignin, but were easy to bleach. The kind of component separation technique has good economic and social benefits and fully considers the needs of environmental protection and reutilization of natural renewable resources. The method has the following advantages (Jimenez, Perez, & Lopez, 2002): (1) a variety of wood fiber applicability; (2) consumes less electricity, water and chemicals; (3) low economic cost, less investment; (4) less environmental pollution and almost zero emissions; (5) the solvent is easy for recycling; (6) good treatment effect; (7) byproducts are easy to extract and easy for utilization; (8) pulp obtained is easy to bleach; (9) pulp obtained contains low content of lignin; and (10) good beating performance. However, the organic solvent method also has disadvantages, which hinders its industrialization process: (1) as the organic solvent mainly are small molecules with low boiling point, the organic solvent are always volatile, flammable, or toxic, so the requirement for production equipment is very high; (2) due to the low boiling point of the solvent, to reach the target temperature (160–220°C) often requires high pressure, which brings high-voltage operational risk; and (3) the traditional method to wash the fibers after pretreatment cannot be used, as lignin is easy to redeposit and absorb on the fiber in traditional washing. These deficiencies brought big challenges to the production process, equipment, and operation. Recently, there has been a new type of cellulose solvent—ionic liquids (ionic liquids), which demonstrated that chloro-1-butyl-methylimidazole and 1-allyl-3-methyl imidazole can dissolve cellulose without pretreatment (Swatloski, Spear, & Holbrey, 2002; Ren, Wu, & Zhang, 2003), However, the hydrolysis capability of the regenerated cellulose has not been reported. Chen Hongzhang studied homemade ionic liquids, and applied it to the pretreatment of lignocellulosic feedstock. He also studied the impact of the steam explosion—ionic

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liquid on the pretreatment of lignocellulosic feedstock, which will be given in Section 3.4.4.1.

3.3.1.6  Ozone Treatment Ozone can be used to break down the structure of lignin and hemicellulose within lignocellulosic feedstock (Sun, Chen, & Wang, 2003). In this method, a large extent of lignin gets degraded, hemicellulose suffered a slight attack, and cellulose was hardly affected. The advantage of this method is: lignin can be effectively removed; no inhibitory substances are generated for further reaction; and the reaction can be carried out at room temperature and pressure. However, due to the large amount of ozone needed, the cost of the whole process is high. 3.3.1.7  Wet Oxidation Treatment The wet oxidation method was proposed in the 1980s. Under the conditions of high temperature and pressure, water and oxygen participate in reactions together. With the presence of water and oxygen, lignin is degraded by peroxidase. The treatment can enhance the sensitivity of materials to enzymatic hydrolysis. Hungary Eniko et al. (Varga, Schmidt, & Réczey, 2003) used the wet oxidation method for the treatment of 60 g/L corn stover (the treatment conditions are: 195°C, 15 min, 1.2 × 103 kPa, 2 g/L Na2CO3). Results showed that 60% of hemicellulose and 30% of wood fiber were dissolved, and 90% of cellulose was separated with solid form. The enzymatic conversion rate of cellulose was about 85%.

3.3.2  Physical Pretreatment Mechanism of Biomass Biochemical Conversion 3.3.2.1  Mechanical Comminution Plant tissue includes a variety types of cells, which shows significant differences in chemical composition and physical properties (Gordon, Lomax, & Dalgarno, 1985). A cell’s toughness is determined by cellulose, hemicellulose, and lignin in its cell walls (Jouany, 1991). Choong (1996) found that toughness of Castanopsis fissa leaf can be predicted by volume fraction of cell walls and neutral detergent fiber (NDF). Drapala, Raymond, & Crampton (1947) and Pigden (Crampton & Maynard, 1938) considered that plant particle size and shape after mechanically pulverization can reflect the distribution and concentration of lignin. The crushing equipment commonly used are ball milling, compression milling, double roll crushers, grinder with momentum flow pattern, and wet and frozen colloid mill crushing. The disadvantages of mechanical grinding are the high-energy consumption and high grinding costs. 3.3.2.2  Ultrafine Grinding Ultrafine grinding technology is a new technology developed since the 1970s with modern high-tech. In this method, material with the granularity of

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0.5–5.0 mm can be crushed into ultrafine powder within 10–25 µm. After ultrafine grinding, the original chemical properties of material remain unchanged, but the particle size and crystal structure are improved. The ground powder has special physical and chemical properties, which common particles do not usually have: uniform size, large specific surface area, increased porosity, good dispersion, adsorption, solubility, and chemical reactivity. An ultrafine mill is currently the most widely used ultrafine grinding equipment. Its advantages include high speed, no accompanying heat generation, fewer chemical reactions, and the maximizing of the retention of components’ bioactivity and physical and chemical properties. Therefore, it is more suitable for substances with low melting points (Gai & Xu, 1997; Jin & Chen, 2006; Sheng, Liu, & Tu, 2003; Pan, Wang, & Liu, 2004). In recent years, ultrafine grinding technology has obtained much attention in the fields of food, medicine, household chemicals, paper, medicine, etc. (Hui_xing, 2001; Zhang & Liu, 2002). The disadvantages of ultrafine grinding technology are that the mechanical grinding energy consumption and cost is high. The energy consumption of grinding depends on the size and nature of the material (Gai & Xu, 1997; Jin & Chen, 2006).

3.3.2.3  High-energy Radiation Treatment γ-Radiation (ionizing radiation) is commonly used to destroy straw cell walls and other agricultural waste composition (Al-Masri & Zarkawi, 1994). It is also very efficient in decreasing the polymerization degree of fibers or the removing lignin (Sandev & Karaivanov, 1979). Ionizing radiation is of benefit to the depolymerization (Focher, Marzetti, & Cattaneo, 1981) of cellulose. It can also loosen the structure of cellulose; it affects the structure of cellulose, thereby increasing the activity and accessibility of the cellulose. Thus, in the production of viscose fibers, radiation treatment can dissolve the pulp, and enhance the ability of cellulose to convert into viscose. For example, Fischer, Rennert, and Wilke (1990) used a 1 MeV electron accelerator to generate high-energy electrons for the radiation treatment of sulfite pulp. The results showed that the pulps treated with high-energy electron showed better uniformity and the reaction capacity with carbon disulfide. γ rays generated by 60Co has similar treatment effect with high-energy accelerated electrons (Focher et al., 1981; Stepanik, Rajagopal, & Ewing, 1998). Treatment with high doses of radiation can reduce the content of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid-insoluble lignin (ADL), and reduce sugar in the cell walls of straw, thereby increasing the digestibility of straw (Baer, Leonhardt, & Flachowsky, 1980; Leonhardt, Baer, & Hennig, 1983; Gralak, Krasicka, & Kulasek, 1989; Al-Masri & Guenter, 1993). Low doses of radiation can be used for sterilizing agricultural byproducts; Kume, Ito, and Ishigaki (1990) reported that a dose of 15 kGy was enough to kill all aerobic strains. A dose of 5–6 kGy can reduce fungi on the shell of the compressed fibers bed under the detection level. Malek, Chowdhury, and Matsuhashi (1994) reported that γ-rays from 30 kGy are needed to kill the aerobic bacteria in the

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straw. Kim, Yook, and Byun (2000) also found that 5–10 kGy γ radiation can effectively reduce microbial contamination in herbs.

3.3.2.4  Microwave Treatment Microwaves are electromagnetic waves with a frequency of 300 MHz to 300 GHz (wavelength 1 m–1 mm). Microwave treatment can change the hydrogen bonds between the cellulose molecules. Cellulose powders after treatment are not swollen and have high activity and accessibility. The effect of microwave treatment was significantly better than conventional heat treatment; Zhu et al. (Zhu, Wu, & Yu, 2005a,b; Shengdong, Ziniu, & Yuanxin, 2005; Zhu, Wu, & Yu, 2006) used three treatment methods, microwave/alkali, microwave/acid/base, and microwave/ acid/base/H2O2, to improve the hydrolysis rate of straw, and extracted xylose from the liquid. It was found that the substrate treated with microwave/acid/alkali/H2O2 has the highest weight loss and fiber content, and the highest enzymatic hydrolysis rate. Xylose recovery experiments showed that xylose cannot be recovered from the microwave/alkali-treated liquid, and xylose crystals can be obtained from the microwave/acid/base and microwave/acid/alkali/H2O2-treated liquid. However, due to the high cost of the process, it is difficult to use in industry. 3.3.2.5  Supercritical Processing 1. Supercritical CO2 processing Supercritical carbon dioxide (SC-CO2) has obvious advantages: economic, clean, environment-friendly, easy to recycle, etc. In recent years, SC-CO2 has often been used as an extraction solvent. Ritter and Campbell (1991) treated pine with SC-CO2, and found no change in pine morphology. They concluded that SC-CO2 was not an effective pretreatment method for lignocellulosic feedstock. However, there were also reports that SC-CO2 treatment could increase the permeability of Douglas fir (Demessie, Hassan, & Levien, 1995). There were also reports that the enzymatic hydrolysis rate of cellulosic and lignocellulosic feedstock were significantly increased when treated by SCCO2 (Zheng, Lin, & Wen, 1995). Kim and Hong (2001) pretreated aspen and ponderosa by SC-CO2. The treatment condition is described as follows: humidity range, 0–73% (w/w); pressure, 21.37–27.58 MPa; temperature, 112–165°C; holding time, 10–60 minutes; the reducing sugars obtained are significantly higher than control groups. 2. Supercritical water Cellulose degraded in supercritical water (P > 22.09 MPa, T > 374°C). The main products are erythrose, dihydroxyacetone, fructose, glucose, glyceraldehyde, methylglyoxal, and oligosaccharides. The reaction pathway of cellulose hydrolysis reaction in supercritical water has been elucidated (Sasaki, Kabyemela, & Malaluan, 1998; Sasaki, Fang, & Fukushima, 2000). Cellulose is first decomposed into oligosaccharides and glucose, and glucose is further degraded into fructose by isomerization. Glucose and fructose can

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both be decomposed into erythrose and glycolaldehyde or dihydroxyacetone and glyceraldehyde. Glyceraldehyde can convert to dihydroxyacetone, and these two compounds can be dehydrated to methylglyoxal. Methylglyoxal, glycolaldehyde, and erythrosecan further degrade into smaller molecules, mainly 1–3 carbon acids, aldehydes, and alcohols. When below the supercritical temperature, cellulose hydrolysis reaction takes 10 seconds to achieve complete conversion of cellulose, and the major product is glucose. In contrast, when above the supercritical temperature, the cellulose hydrolysis reaction only takes 0.05 seconds to be completed, and the main products are glucose, fructose, and oligosaccharides. Hydrolysis kinetics show that the degradation rate of cellulose is very high when above the supercritical temperature, but when below the supercritical temperature, the glucose degradation rate is higher than the degradation rate of cellulose. The cellulose degradation reaction occurs at the surface of cellulose (Feng, Van Der Kooi, & de Swaan Arons, 2004). 3. Other supercritical treatment Kiran and Balkan (1994) employed acetic acid–water, acetic acid-super critical CO2, and acetic acid–water to extract lignin under high pressure. They found that acetic acid–water system has the highest delignification rate (73 mol% (90 vol%) acetic acid delignification rate of 95%), followed by acetic acid–supercritical CO2 and acetic acid–water–supercritical CO2 systems. Machado et al. applied 1,4-dioxane-CO2, at 160–180°C, under 170 MPa for delignification and found that the extracting composition affects the selectivity, and higher CO2 will enhance the selectivity of hemicellulose extraction. The pure 1,4-dioxane owns the highest lignin extraction rate. Temperature has little effect, but cellulose start to degrade at 180°C (Machado, Sardinha, & de Azevedo, 1994). Reyes, Bandyopadhyay, and McCoy (1989) used supercritical alcohol and isopropyl alcohol to remove the lignin in the wood. They found that delignification rate increased with the reaction temperature and pressure. Delignification above the supercritical temperature is much higher than that under the supercritical temperature.

3.3.3  Biological Pretreatment Mechanism of Biomass Biochemical Conversion Biological treatment is carried out to remove lignin by microorganisms, releasing the wrap effect on cellulose. The corresponding study currently still stays in the experimental stage. Although many microorganisms can produce lignin-degrading enzymes, the enzyme activity is low, so it is difficult to be applied. A wood-rotting fungus has high-lignin decomposition ability. Microorganisms commonly used to degrade lignin are white-rot fungus, brown-rot and soft-rot fungi bacteria; white-rot fungus is the most effective. Currently, a number of white-rot fungi, such as Phanerochaete chrysosporium, Ceriporia

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lacerata, Cyathus stercolerus, Ceriporiopsis subvermispora, Pycnoporus cinnarbarinus, and Pleurotus ostreaus have high delignification ability (Kumar & Wyman, 2009; Shi, Chinn, & Sharma-Shivappa, 2008; Shi, Sharma-Shivappa, & Chinn, 2009). The white-rot fungus can not only decompose lignin, but also decompose cellulose and hemicellulose. Therefore, white-rot fungi on one hand decompose lignin; on the other hand, they lead to the loss of cellulose and hemicellulose. Thus, the separation of bacteria which only decompose lignin without biosynthesis ability of cellulase and hemicellulase is very important. Other enzymes capable of degrading lignin are: polyphenol oxidase, laccase, and catalase (Chen, 2005). Brown-rot fungi can change the nature of lignin but cannot decompose lignin; soft-rot fungi shows a low ability to decompose lignin. Biological treatment is usually carried out in relatively mild conditions, has little side effects, and produces little inhibitors. However, due to the low decomposition activity, the processing is very long (generally several weeks). Thus, there is a long time for the practical application. From the perspective of cost and equipment, microbial pretreatment shows unique advantages. We can treat lignin by specific enzymes to increase the lignin-decomposing rate. However, the study remains in the experimental stage.

3.4  PRETREATMENT FRACTIONATION TECHNOLOGY FOR BIOMASS BIOCHEMICAL CONVERSION 3.4.1  Steam Explosion Pretreatment for Biomass Biochemical Conversion Based on the composition features of straw, Hongzhang Chen invented the low pollution steam explosion technology (with pressure down from 3.0–1.5 Mpa) and revealed the autologous hydrolysis mechanism of steam-exploded straw. The steam explosion process does not need any chemicals, only to control the water content in the straw. Water acts as an acid catalyst at a high temperature in the steam explosion process (Mosier et al., 2005). Eventually, more than 80% of the hemicellulose can be separated and cellulose hydrolysis rates can rise above 90% (Chen & Liu, 2007). Laser-treated sugarcane bagasse at 216°C for 4 minutes had a conversion rate of cellulose of 67% (Laser, Schulman, & Allen, 2002). Hongzhang Chen has enlarged the explosive devices into 50 m3 with industrial scale, which is the largest in the world. Furthermore, the formed steam explosion technology platform has been widely used in paper mulberry bark ungluing (Chen, Peng, & Zhang, 2009), hemp degumming (Chen et al., 2003), peanut oil preparation (Chen, Wang, & Chen, 2011), herbal extracts (Yuan & Chen, 2005), and flavonoids preparation (Fu, Chen, & Wang, 2008). On the basis of research and application, the characteristics of steam explosion pretreatment can be summarized as follows: 1. Steam explosion can partly hydrolyze hemicellulose, reducing the complexity of cell wall components.

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2. During steam explosion, steam within and among the cells spurts out at certain moment to destroy the connection of cell wall and tissue. As a result, the tight structure of tissue and cell wall is loosened. With the hydrolysis of hemicellulose, the porous structure of lignocellulose is formed via steam explosion. Therefore, the specific surface area increases, which could enhance the mass transfer rate and improve the enzyme accessibility. 3. Product purity is enhanced after separation of hemicellulose and water-soluble components. 4. Steam explosion pretreatment is clear, effective, and efficient, with no chemicals added. 5. Steam explosion is easy to operate and spread. Therefore, lignocellulose pretreated by steam explosion is suitable for further physical, chemical, and biological pretreatment, which is the reason for steam explosion to become a research focus. Meantime, steam explosion integrated pretreatment explains the connotation of integrated pretreatment technology in this paper.

3.4.2  Tissue Fractionation Techniques for Biomass Biochemical Conversion Due to the difference in compositions of plant organs, it is helpful to achieve high-value utilization of biomass. In the process of steam explosion, the linkage among straw epidermal tissue, mechanical tissue (vascular tissue), and basic organization (parenchyma) are broken, which is of benefit to further separation. Therefore, it is feasible to introduce the mature fibroblast grading equipmentPaul screening instrument into pretreatment process of lignocellulose. After steam explosion and Paul Sieving, fibrous tissue cells were mainly distributed within 28 meshes, which are mainly epidermal tissue and vascular tissue (mechanical tissue). Parenchyma (ground tissue) cells were mainly distributed within 200 meshes. The combination of steam explosion and sieving pretreatment achieved the separation of plant tissue to some extent.

3.4.3  Cell Fractionation Techniques for Biomass Biochemical Conversion 3.4.3.1  Steam Explosion—Superfine Separation Technology Straw has been separated on the organization level via the steam explosion pretreatment. However, different cells of different tissues are still closely linked. In addition, a problem in the hydrolysis process of lignocellulosic is that enzymes can not sufficiently make contact with the substrate, thus increasing the specific surface area of the substrate will help to improve digestion rate. Currently, the ultrafine grinding technology has attracted much attention from various fields. When employed in the pretreatment of straw, it can efficiently

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break the linkage among cells and increase the specific surface area of the substrate. Meanwhile, it does not consume more energy than the traditional gridding method. Therefore, we invented the techniques combining steam explosion and ultrafine grinding. Comparing the combination of steam-explosion with common grinding equipment, parenchyma cells and epidermal cells were increased by 9.4% and 4.4%, and fiber cells were reduced by 13.4% in the steam-exploded straw superfine powder. Fiber cells increased by 2.3%, parenchyma cells were reduced by 15.7%, and duct cells were reduced by 50% in the steam-exploded straw residue. This illustrates that the combination of steam explosion and ultrafine grinding can achieve cell separation efficiently (Jin & Chen, 2006).

3.4.3.2  Steam Explosion—Wet Ultrafine Separation Technology Steam explosion—part wet superfine fiber fractionation is another useful method for lignocellulose pretreatment. The fiber cell content of the treated substrate is more than 60%. Compared with the steam explosion-superfine separation technology, this method has advantages on cell separation. Compared with the original straw and steam exploded but unseparated straw (Jin & Chen, 2007), cellulose content in fibrous tissue steam is significant after the treatment of higher explosion—wet ultrafine fractionation. The fibrocyte content is 63.1%, which is 37.8% higher than that of untreated. Cellulose content in the fiber texture is 65.6%, which is 74.9% higher than that of untreated. Therefore, steam explosion—wet ultrafine separation technology is efficient in the separation of cellulose cells.

3.4.4  Components Fractionation Technologies Before Biomass Biochemical Conversion 3.4.4.1  Combination of Steam Explosion and Ionic Liquid Pretreatment Compared with the organic solvent and electrolyte treatment method, there were some advantages of ionic liquid, which can be summarized as follows: 1. It needs nearly no steam pressure, thus avoiding the pollution of volatile organic solvent. 2. It has better chemical and physical stability, and a broad range in which temperature is stable (from lower than 25–300°C). 3. By adjusting the acidity, ionic liquid could dissolve almost all the common materials, including inorganic mineral and organic polymers. It was regarded as one of the three green solvents together with supercritical carbon dioxide and aqueous two-phase. Previous experiments (Liu & Chen, 2006) showed that the contents of cellulose, hemicellulose, and lignin of wheat straw decreased after steam explosion combined

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with ionic liquid pretreatment. When adding NaOH, the solubility of steam exploded straw in [BMIM]Cl (where [emim]Cl is the 1-butyl-3-methylimidazolium chloride) were significantly increased. In contrast, 1% sulfuric acid could decrease the content of hemicellulose and cellulose, but increase the content of lignin.

3.4.4.2  Steam Explosion—Alkali Hydrogen Peroxide Integrated Pretreatment Research revealed that lignin could be separated by alkali oxygenation. Chen et al. (2008) and Cara et al. (2006) reported that lignin in lignocellulose could be removed by hydrogen peroxide oxidation. Therefore, steam explosion combined hydrogen peroxide pretreatment method was studied. After steam explosion combined alkali hydrogen peroxide pretreatment, the content of hemicellulose and lignin in corn straw decreased from 33.5% to 24% and 22% to 8%, respectively, and cellulose content reduced from 63% to 25.4%. 3.4.4.3  Steam Explosion—Glycerin Combination Pretreatment Because the organic solvent separation method has advantages of cleanness, recyclability, etc., it is necessary to explore the possibility of solvent separation method in straw separation treatment. Solvents with low-boiling point are easy to evaporate and explode, but a solvent with large molecules is usually very expensive. Sun and Chen (2008) compared the small solvent with highbowing points, which included formic acid, propionic acid, ethylene glycol, butylene glycol, and glycerin. Results showed that steam explosion-glycerin combination pretreatment was very efficient. After treatment, 92% of cellulose remained. At the same time, more than 90% of hemicellulose and 7% of lignin were removed (Chen & Liu, 2007; Chen & Li, 2000). 3.4.4.4  Steam Explosion—Ethanol Integrated Pretreatment Ethanol treatment causes hemicellulose to be lost. However, we can firstly degrade hemicellulose in straw by steam explosion. Then, we remove the hemicellose by watering and remove lignin by ethanol. Finally, we use the remaining cellulose for ethanol fermentation. Results showed that after four-time water extraction, the recovery rate of hemicellulose was 80%, and most of that was xylose. Treated by low-temperature distillation, the recovery rate of ethanol was 88.4% and concentration was 42.2%, which can be recycled (Hongzhang & Liying, 2007; Chen & Li, 2000). 3.4.4.5  Steam Explosion—Electricity Catalyzing Integrated Pretreatment Electricity catalyzing is being used in wastewater pretreatment due to its advantages of nonchemical reagent, without second pollution, and being easy to operate. Pulp could be bleached by redox effect of electricity catalyzing because the redox effect catalyzed lignin to destruct its structure; consequently, lignin was removed and pulp was bleached. Therefore, steam explosion combined

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with electricity catalyzing pretreatment was developed. With this pretreatment, hemicellulose in lignocellulose was hydrolyzed in the steam explosion process and lignin was later degraded in the electricity catalyzing process. As a result, hemicellulose, lignin, and cellulose were separated and the treated materials could be converted into high-value products. Compared with the raw corn straw, the components content of core straw pretreated by steam explosion combined electricity catalyzing at the voltage 1.5, 2.5, and 5V decreased as follows: lignin content decreased by 6.9, 12.7, and 20.0%, respectively, cellulose content decreased by 1.0, 2.8, and 4.3%, respectively, hemicellulose decreased by 10.1, 14.6, and 16.9%, respectively. However, the soluble content increased by 50.0, 84.2, and 111.8%, respectively at voltage 1.5, 2.5, and 5V. This revealed that electricity catalyzing pretreatment degraded lignin, hemicellulose, and cellulose, leading to an increase in soluble component content. Glucose content in corn straw hydrolysate increased by 25.4% at the voltage 1.5V after steam explosion combined electricity catalyzing pretreatment.

3.4.4.6  Steam Explosion Combined Laccase Pretreatment Lignin-carbohydrate complex (LCC) inhibits enzyme hydrolysis rate. So it is important to separate LCC before hydrolysis. Enzyme pretreatment was mild and specific. Therefore, an enzyme combination was researched to remove LCC. The enzyme combination included laccase and feruloyl esterase or xylanase. Steam explosion material was pressed by screwing to separate the solid and liquid. Then, enzyme combination was put into the solid part. When laccase and feruloyl esterase were used, hydrolysis rates of cellulose, hemicellulose, and lignin of corn straw increased by 5.65, 10.97, and 33.93%, respectively, compared with that of steam explosion corn straw. Hydrolysis products of lignin and hemicellulose could be converted to other products with high value. This pretreatment was high-rate, efficient, mild, and specific. Moreover, water wastage was decreased by screwing the press instead of washing. Importantly, the increased hydrolysis rate could provide a high sugar content for subsequent fermentation. 3.4.4.7  Steam Explosion—Mechanical Carding Combination Pretreatment Chen and Fu (2011) established a steam explosion—dry carding classification method, which could separate the vascular tissue and parenchymatous tissue, increase the uniformity of lignocellulosic materials, and thus provide a basis for refining high-yield grade. Our research showed that issues including the core of straw, leaves, and skin can be separated with 30% moisture content and under the steam explosion condition: pressure at 1.5 MPa and lasted for 5 min. Compared to flow sorting, steam explosion—dry and steam explosion increases carding separation level from 1.08 to 1.25. After carding and grading, hydrolysis rates of bark and leaf parenchyma were increased by 1.65 and 1.41 times. Scanning electron microscopy showed that the steam explosion-mechanical carding enables cells’ vascular tissue and

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parenchyma to separate from each other. After separation, cells’ weight ratio between vascular tissue and parenchyma issue was about 3:2. The graded tissue was further treated with ethanol-catalytication, and reacted with 50% ethanol for 2 h at 180°C. The pulp yield was increased to 65%, higher than that of untreated (55%) and the lignin content decreased by less than 7%. Steam explosion-carding dry straw is an effective way to achieve organizational classification.

3.5  FEATURES OF BIOMASS BIOCHEMICAL FRACTIONATION CONVERSION PRETREATMENT TECHNOLOGY Compared with the single pretreatment technology, features of integrated lignocellulose pretreatment technologies can be summarized as follows: 1. Realized the target orientation of biomass full utilization. Achieved multidirectional conversion of various components of raw material, which can increase the value of by-products, and reduce waste in pretreatment process; 2. Made full use of structure heterogeneity of plant biomass composition at different levels, especially at the cellular level. Some tissue cells are more easy to separate after steam explosion-mechanical carding treatment, such as vascular tissue cells and parenchyma cells. By selective separation, these issues are used for spinning and papermaking. Furthermore, the parenchymatous cells can be used for bioenergy fermentation. 3. Wide adaptability. As more parameters can be adjusted in the integrated pretreatment method, the process has more flexibility for different species, and different production needs. 4. Process integration. Based on the structural characteristics of the lignocellulosic materials, biomass biochemical fractionation conversion pretreatment technology integrates the technologies from the paper-making industry, textile industry, sheet metal industry, and especially the petroleum refining industry for lignocellulose degradation. For example, the concepts from the petroleum refining industry guided the researchers to selectively use lignocellulosic feedstock for high-value products. 5. Optimize the integration. Every technology has its advantages and disadvantages. In technology integration, the shortcomings of single technologies are reduced and their advantages complement each other.

3.6  EVALUATION OF BIOMASS BIOCHEMICAL FRACTIONATION CONVERSION PRETREATMENT TECHNOLOGY 3.6.1  Evaluation Standards of Biomass Biochemical Fractionation Conversion Pretreatment Technology In research and industrial production, it is necessary to set up the evaluation standards for biomass biochemical fractionation conversion pretreatment

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technology. The technologies must follow the requirement of clear production, have a correct theory base and have high feasibility for industry practice. They can be summarized in detail as follows (Chen, 2004): 1. Theoretical correctness. We must ensure that preprocessing technique is based on the structure and composition of plant biomass, the multigeneration-oriented, established according to the physical chemistry of cellulose, hemicellulose, lignin, and other components. The raw materials can be fully utilized according to their nature features. 2. Technical feasibility. Firstly, technical solutions should be built on the industrialization device, including the preparation of equipment, maintenance, and parameter control; secondly, the feasibility of technical solutions: the operating unit should be established on certain theoretical guidance, including control methods of the technology and process parameters used in the process route; finally, the stability of the process: the process is not sensitive to certain parameters and is able to meet product requirements within a wide range. 3. Economic rationality. This mainly refers to the equipment usage; technique investment must have economic rationality, including material flow and energy flow in the production. 4. Operational feasibility. First, the operation must be safe, which means that equipment and process operations must be safe to operating personnel, whether in the short or long term; second, operation controllability, which means operation is less influenced by the human and has a certain degree of stability; finally, the technique is simple and easy to learn. 5. Environmentally friendly. In the pretreatment process, the waste produced must meet the requirement of environment protection. 6. The model must be easy to replicate. As the most important feature of plant biomass is wide distribution, the best preprocessing techniques can be processed in a village or town, reducing the volume of transport or storage, and then the substrate converted to the corresponding product on a large scale. Another effect of this mode is to contribute to the development of private enterprise, therefore advancing the urbanization process, and increasing employment opportunities and farmers’ incomes.

3.6.2  Comparison of Biomass Biochemical Fractionation Conversion Pretreatment Technology The different pretreatment methods were compared from six aspects as shown in Table 3.2. It can be seen that steam explosion-ethanol, steam explosion-alkali hydrogen peroxide, and steam explosion-mechanical carding are effective methods for substrate separation. The steam explosion-alkali extraction-machinery carding integrated pretreatment technology has being used on an industrial scale in Laihe Chemical Co., LTD in Songyuan, Jilin province in north China. The production line handles 300,000 tons of corn stover and produces 50,000 tons of acetone-butanol-ethanol, and 30,000 tons of high-purity lignin (which can be

58

Fractionation technology

Theory correctness

Technical feasibility

Economic rationality

Operational feasibility

Process environmental protection

Generalization performance

Total

Super grinding separation technology

3

2

1

3

1

3

13

Wet ultrafine separation

3

2

1

3

1

3

13

Ionic liquid

3

2

1

3

1

3

13

Alkaline hydrogen peroxide

3

3

3

3

2

3

17

Glycerin

3

1

2

3

3

3

15

Ethanol

3

3

3

3

3

3

18

Electrocatalysis

3

3

2

1

2

2

13

Laccase

3

1

1

1

3

2

11

Mechanical carding

3

3

3

3

2

3

17

3, Good; 2, general; 1, weak.

Technologies for Biochemical Conversion of Biomass

TABLE 3.2 Comparison of Fractionation Technologies

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converted into 20,000 tons of phenolic resin glue), and 120,000 tons cellulose (which can be converted into 50,000 tons of biopolyether polyols) per year. It has significant economic advantages compared with the existing butanol projects.

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Chapter 4

Enzymatic Hydrolysis of Pretreated Biomass Chapter Outline 4.1 Reviews on the Enzymes Participating in the Biomass Degradation Process 4.1.1 Cellulase 4.1.2 Hemicellulase 4.1.3 Lignin-Degradating Enzymes 4.1.4 Cellulase Cofactors 4.2 The Setup of Biomass Invertase Platform 4.2.1 The Enzyme Production 4.2.2 Chemical Modification Enzymes 4.2.3 Enzyme Engineering 4.3 Enzymes Conversion Platform for Biomass 4.3.1 Enzyme Synergy 4.3.2 Multicomponent Enzyme System 4.3.3 Fiber Bodies

65 66 67 72 74 75 75 81 83 87 87 87 88

4.3.4 Optimization of the CBH–EG–BG System 4.3.5 Design of the Multienzyme Complex 4.3.6 Enzyme Reactor 4.4 Economic Analysis of Cellulase Production 4.4.1 Relations Between Particularity of Cellulose and Production Costs 4.4.2 Relationship Between Process Indicators and Production Costs 4.4.3 The Major Policy to Reduce Cellulose (or Conversion) Enzyme Economic Costs References

88 88 89 91

91

92

94 96

4.1  REVIEWS ON THE ENZYMES PARTICIPATING IN THE BIOMASS DEGRADATION PROCESS The essential enzymes for the biomass degradation process mainly include cellulase, hemicellulase, the lignin degradation enzyme system, and cutin enzyme and cellulose enzyme cofactors. Establishment of the biomass biochemical transformation platform mainly refers to the preparation and application of various enzymes, especially application of the synergy effect between different enzymes. The biomass degradation process requires enzymes cofactor, cutin enzyme, and cellulose enzyme solution. The biomass biochemical invertase platform mainly Technologies for Biochemical Conversion of Biomass © 2017 Metallurgical Industry Press. Published by Elsevier Inc.

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refers to the preparation and application of various enzymes, especially the synergy effect among different enzymes.

4.1.1 Cellulase Enzymes with cellulose degradation activity are mainly produced by aerobic fungi and anaerobic bacteria. In the tropics, termites and some insects can also degrade cellulose, and most of them coexist with the cellulolytic microbe. Cattle, sheep, deer, and other ruminants are also important cellulose decomposers, in whose rumens cellulose gets degraded by coexisting bacteria (Jing, 2010). Because of the strong interactions, such as hydrogen bonding and hydrophobic accumulation between adjacent molecular chains in the natural cellulose crystal area, natural cellulose is difficult to degrade. Therefore, the specific activity of single cellulose enzyme is much lower than others. When acting on insoluble substrates, the reaction rate is no more linearly related to time and enzyme, the reason for which remains unknown. Early researches held views that the cellulose could only destroy cellulose crystalline structure and C1enzymes with hydrolysis activity and Cx enzymes for β-1,4 glycosidic bond decomposition did not exist. After the 1970s, due to the development of biochemistry and molecular biology, the fungal cellulose enzyme system represented by Trichoderma reesei was elucidated gradually (ZhenJiang, 2007). Fungal cellulases generally contain the following three components: (1) 1,4β-d glucan hydrolase. These enzymes can randomly degrade the β-1,4 glycosidic bond within the cellulose molecules; (2) 1,4-β-d-glucan cellobiohydrolase enzymes (1,4-β-d-Glucan cellobiohydrolases, CBHs, EC 3.2.1.91,) referred to the cellulose exonuclease (exoglucanases), which can cut the glycoside keys at the reducing or nonreducing end of the molecule and generate cellobiose; (3) β-glucosidase (β-d-glucosideglucohydrolases, β-glucosidases, EC 3.2.1.21, referred to as BG), these enzymes degrade the cellobiose into individual glucose molecules (Gao, 2003). There are three hypotheses on the mechanism of each cellulase component: the C1–Cx hypothesis, sequential action hypothesis, and synergy model (Gao, 2003; Lian & Wang, 2007). Reese proposed the C1–Cx hypothesis to explain the action mode of cellulase enzymes in 1950 (Reese, Siu, & Levinson, 1950). The hypothesis holds that firstly, the C1 enzyme acts the crystalline of the cellulose and makes it adaptable for the C2 enzyme. The C1enzyme can randomly hydrolyze crystalline cellulose, glucose, soluble cellulose derivatives, and β-1,4-oligomer; β-glucosidase converts cellobiose, and cellotriose sugar into glucose. In the hydrolysis of natural cellulose (high crystallinity cellulose), the C1 and Cx enzymes coordinate to act in different stages: the first stage is that C1 enzyme acts on the crystalline cellulose, but there is only conversion, not hydrolysis. Then Cx enzyme hydrolysizes converted amorphous cellulose partly into soluble monosaccharides.

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FIGURE 4.1  The possible methods of cellulose hydrolysis by cellulose enzymes (Riedel et al., 1997).

FIGURE 4.2  The synergistic degradation model.

C1

Cx

Crystalline cellulose → amorphous cellulose → glucosaccharase cellobiose  → glucose However, Reese et al. proposed that C1 and Cx hydrolyze cellulose in different phases, that is, it is only when C1, Cx, and β-glucosidase coexist that natural cellulose can be hydrolyzed. Yet, it has not been verified by the experiments, since if we firstly let C1 enzyme act on the substrate and then separated C1 and added Cx, the crystalline cellulose could not be hydrolyzed. Sequential action hypothesis on the mechanism of action of cellulase holds that exoglucanase (CBHI and CBHII) firstly hydrolyzes insoluble cellulose fiber into soluble dextrin and cellobiose and then the endoglucanase (EGI and EGII) acts on the fiber dextrin to generate cellobiose. Finally, the BG decomposes the cellobiose into two glucoses, which is shown in Fig. 4.1. Currently, the synergy model is generally accepted for the explanation of degradation mechanism of cellulose, which is shown in Fig. 4.2 (Woodward, 1991).

4.1.2 Hemicellulase Hemicellulase usually refers to the enzyme mixtures acting on noncellulose and nonpectin biomass. They can be divided into two categories: depolymerase and debranching enzymes. Depolymerase acts on sugar chain backbones and also

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can be divided into two categories: enzyme I have the endo activity acting in the middle of polymers; enzymes II have the exoactivity acting from the end of polymers. In fact, many enzymes own two kinds of activities at the same time. In addition, a series of oligosaccharide-producing enzyme systems can be achieved by combination of endo- and exoenzymes, such as β-glucosaccharase (3.2.1.21), β-xylan glucosidase (3.2.1.37), and β-mannosidase (3.2.1.25). Due to its low polymerization degree, it is difficult to distinguish between the endoand exoaction mode. The debranching enzyme is a kind of auxiliary enzyme, which can be divided into enzymes acting on ester bonds and glycoside bonds. The former ones include α-l-arabinofuranosidase (3.2.1.550) and α-glucuronidase (3.2.1.39); the latter include acetyl xylan esterase (3.2.1.72) and ferulic acid enzyme (3.2.1.73), which mainly act on xylan. It is reported that hemicellulase enzymes also contain the enzymes acting on acetylated polysaccharide glucomannan, including glucomannan and galactomannan. Only part of hemicellulose enzymes exhibit cross activities toward different hemicellulose, most of which possess strict specificity, and only acting on particular sequence of oligosaccharides. For example, β-xylosidase is suitable for the hydrolysis of xylobiose, and also active for xylotriose and xylo-oligosaccharide, gentiobiose, and cellobiose; feruloyl esterase acts on the ferulic acid ester of the arabinose in normal circumstances, in addition, it also acts on coumaric acid ester in some cases. Owing to the high heterogeneity structure of hemicellulose, different sugar molecules conformation numbers, nonsugar components, and the complicated connection bonds, this article does not conduct an in-depth discussion of specific enzymes. Instead, we list the basic activity, degradation effects on the biomass stocks, and the potential use of the hemicellulose enzymes (Merle, 2010).

4.1.2.1 Depolymerase 4.1.2.1.1 Xylanase Xylan synthesis from different plants depends on poly β-(1–4)-xylopyranosyl backbone, and depolymerase acts on β-(1–4) or β-(1–3) bonds between xylans. Under the effect of these two enzymes, xylanase anomeric carbon configuration of the reducing end is retained. Most enzymes belong to two different categories of structural xylanase glycosyl hydrolase family: GH10 owns high molecular weight/low isoelectric point, and GH11 has low molecular weight/high isoelectric point. The former has a wider range of catalytic functions, and higher efficiency on the hydrolysis of xylan with a high degree of substitition. It is reported that xylanase has a cellulose-binding domain. Most xylanase is characterized by identifying the types of xylan hydrolysis and the final product series. The major products of most endoxylanase are xylobiose and xylotriose. Most endoxylenes can hydrolyze unsubstituted xylose and the hydrolysis of xylan side chains mainly relies on special xylanase.

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Exoxylanase (3.2.1.37, 3.2.1.72, 3.2.1.56) usually has a hydrolysis specificity. β-Xylanase (EC 3.2.1.37) is more efficient on the high xylan. In addition, EC 3.2.1.72 is used for β-(1–3)-connected xyloside in most cases and exhibits limited degraded activity. Comparing with the endo xylanase, the enzyme protein of exoxylanase is usually larger (the molecular weight is larger than 100 kDa) and composed by two or more subunits. Due to the low content, little exoxylanase has been identified (Yang, Yao, & Fan, 2005). 4.1.2.1.2 Mannanase Endomannanase randomly hydrolyzes the mannan and β-d-1,4 mannoside bonds, such as glucomannan, galactomannan, and glucomannan. Compared with xylanase, only a few of mannanase have been identified. Mannanase from T. reesei (a kind of bacteria) has a similar mutidomain structure of cellulose hydrolysis enzymes. The most common hydrolysis products of galactomannoza and glucomannan are mannobiose, manninotriose, and an oligosaccharides mixture. Hydrolysis products depend on the substitution degree and distribution of bonds. Endonucleases release oligomer mannose and the further degradation requires β-mannosidase (1,4-β-d-mannanase, EC 3.2.1.25) and β-glucosidase (EC 3.2.1.21). β-Mannosidase and β-glucosidase catalyze the hydrolysis of oligomeric mannose and remove mannose or glucose from the nonreducing ends continuously. β-Xylosidase from T. reesei and β-mannosidase from Aspergillus niger can hydrolyze xylan and mannan, respectively, and release them by continuous exocutting effects (Zhao, Xue, & Ma, 2009). 4.1.2.1.3  β-Glucanase Endoglucanase, which is often considered to be a cellulase family member, has a higher affinity for cellulose and also acts on xylan and mixed β-(1–3,1–4)glucan. Just like endoxylanase, β-glucosidase can break the internal β-(1–4) or β-(1–3) bonds within glucose chains, and produce a reducing and a nonreducing end. EC 3.2.1.4 is one of the most important β-glucosidase; the β-d-(1,3) endoglucanase (EC 3.2.1.39) can act on the β-(1–4) glycosidic bond and its activity is limited on interconnected β-glucan. Endo-1,3 (4)-β-glucanase (EC 3.2.1.6) also has an activity of endoglycosyl hydrolase. For cellulases and endoglucanases, a lot of researches have been carried out on fungus T. reesei. Such fungi produce a variety of cellulase, which act on cellulose synergistically. In T. reesei, Cel7Bis a major endoglucanase, accounting for 6–10% of total T. reesei cellulose. They have broad acitivity toward solid and soluble substrate, such as CMC, xylan, and glucomannan. Furthermore, endoglucanase Cel5A is also active toward solid and soluble substrate (CMC, mannan), but cannot act on xylan. These enzymes account for 10% of the total T. reesei cellulose. Little endoglucanase (Cel2A, Cel45A) has been reported with a variety of specific activity and can hydrolyze solid or soluble substrates (Li, 2004).

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4.1.2.1.4 Xyloglucanase Xyloglucanase is the main hemicellulose polymer during the growth of primary cell wall in plants and is hard to distinguish from cellulose and xylan. Xyloglucan is closely linked with cellulose microfibril though hydrogen bonds, providing loading network for cytoderm and protecting cell wall from disintegration under osmotic pressure. Nowadays, a lot of researches focus on finding key enzymes with controlling and modification effects during the cell walls amplification process. Though the branching degree of xylan is not complicated, xylose on xylan and other substituents makes enzyme digestion of xylan more difficult than cellulose and β-glucan. Xylose can form side chains with d-pyran galactose and l-fucose, but rarely with l-arabfuranose. It is reported that the enzymes, which can digest plant cell walls such as endoglucanase, xylan-endoglycosyltransferase, and exoglycosidase (taking α-fucosidase, β-galactosidase, for example), can digest xyloglucan. Parts of cellulose can also digest xyloglucan skeleton. Endoglucanases including xyloglucanase and xyloglucan specificity form a new kind of polysaccharide degrading enzymes, which can attack the main carbon chain, even replacing the anhydroglucose unit. Some glucanase only acts on specific xylose replacement type, and other enzymes have a broader substrate scope. A xyloglucan from A. niger can work on a variety of β-glucan, and shows the highest activity toward Tamarindus xyloglucan. Now a new kind of enzyme derived from plant is found to modify xyloglucan through endocuting, hydrolysis, and glycosyltransferase effects (Merle, 2010).

4.1.2.2  Debranching Enzyme The glycoside lateral groups connected by xylan and glucomannan are mainly wiped off by α-glucuronidase, α-arabinfuranosidease, and α-d-galactosidase. The acetyl groups and hydroxycinnamic acid substituent group connected to xylan are removed by acetyl xylan esterases and ferulic acid/p-coumaric acid esterases. Usually substrate exhibits a feedback inhibition effect on the enzymes. There are significant differences among side debranching enzymes: some of them only hydrolyzed short chain oligosaccharides, which are mainly produced by backbone depolymerization endonuclease; others were able to remove branches on the whole polymer. But the latter is more suitable for the majority of oligomeric substrates. In the presence of auxiliary enzymes, synergy effects between the hemicellulases components can promote endoxylanase activity. 4.1.2.2.1  α-Glucuronidase α-Glucuronidase (3.2.1.139) catalyze hydrolysis of xylan into glucuronic acid or 4-O-methyl-glucuronic acid. It is reported that the enzyme substrate can be long chain xylan, and also can be the substrate with only one xylose at the nonreducing end. A membrane-bound bacterial enzyme only act on soluble oligosaccharide derived from xylan. For wheat xylan substrate, the synergistic action of α-glucuronidase and endoxylanase produces highly dissociative 4-O-methylglucuronic acid (Pei, Yemin, & Azure, 2003b).

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4.1.2.2.2  α-Arabinosidase α-Arabinosidase can cleave arabinose side chains from the xylan backbone, which synergizes with ferulic acid and acetyl xylanase. 4.1.2.2.3  α-d-Galactosidase (Pei, Yemin, & Azure, 2003a) α-d-Galactosidase mainly acts on the α-galactose side chain on the 0–6 positions of mannose skeleton units and can hydrolyze mannosanin wood, especially for galactomannan and galactoglucomannan. Although this enzyme is important for coniferous wood pulp, the corresponding researches are very limited (Li, 2004; Yang & Lee, 2006). 4.1.2.2.4  Acetyl Xylan Esterase The acetyl group is present in many of the hemicellulose and has the highest number in glucomannan and galactomannan. Cereals and broadleaf wood have higher levels than coniferous wood in acetylation of xylan. Acetylation reactions in coniferous wood mostly occurs in glucomannan. The primary role of acetylation is to maintain the solubility and hydrability of hemicellulose. The removal of acetyl group from xylanand galactomannan will cause a sharp decline in solubility for polymers. The release of acetyl group from the main chain will affect microbial growth (e.g., reduced pH). The release of acetyl group inhibits the growth of microorganisms, which is a considerable problem in the process of biomass conversion (Chen Yan, 2010). 4.1.2.2.5  Ferulic Acid Esterase Ferulic acid mainly presents in cereal, broadleaf wood, and pectin. In the xylan, they connected by an ester bond to the 2C of the side chains of arabinose backbone. The main function is to provide a three-dimensional stable structure for polymerization. Ferulic acid esterase (FAEs) can act on ferulic acid and coumaric acid. Some FAEs can react with polymer or xylo oligosaccharides. FAEs also act on xylan and pectin (Ceng Wei, 2009).

4.1.2.3  Hemicellulase for Different Biomass Feedstocks Xylan composition varies with the type of substrate, and shows the biggest difference among coniferous wood, broadleaf wood, and herbs. Debranching enzymes include arabinosidase, FAE, coumaric acid esterase, and xylosidase. Furthermore, the removal of synergy effect between the side chain and the polymer backbone can enhance degradation rate of endoxylanase. Therefore, xylan treated by a mixture of depolymerase and debranching enzymes is much easier to degrade than those treated by acetyl-xylanase (Poutanen & Sundberg, 1988; Saha, 2000). Xylan in coniferous wood is mainly composed of β-(1–4)-d-xylopyranose and the side chain is rich in 4-O-methyl-glucuronic acid. The ratio reported between uronic acid and xylose depends on the species and extraction methods, which ranges from (2:10) to (7:10). α-l-arabinofuranosyl units are linked to the

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backbone by 1,3-glycosidicbond. Unlike the xylan in hardwood and herbs, xylanin coniferous wood does not contain acetyl xylan groups. Therefore, the corresponding debranching enzyme does not contain acetyl esterase, which is mainly composed of α-glucosaccharase and α-l-arabinofuranosidase (Puls, 1997). Xylan in broad-leaved wood is mainly composed of β-d-xylopyranose units, which also may contain methyl-4-O-α-d-glucuronic acid and acetyl side chains. 4-O-methyl glucuronic acid is linked to xylose backbone through O-(1–2)-glycosidic bond, an ester bond mainly in the C-2/C-3 hydroxy group. For broadleaf trees and herbs, the debranching enzymes mainly include acetyl esterase, arabinofuranosidase, and FAE. For softwood, it requires a higher α-glucosidase activity. In addition, the specificity of depolymerase for side chain and structure is often related to the residues in the side chain. Endoxylanase randomly cuts 1,4-βd-xyloside within the backbone and exhibits high specificity toward the type of sugar, the chemical bond, and the substituent. Xylan hydrolysis is the dynamic equilibrium of three processes (removal of the side chain groups from the polymer backbone, reducing the length of the chain, and hydrolyzing the oligosaccharides into free monosaccharides. To achieve the best enzymatic efficiency, the enzymes need to work synergistically. Glucomannan and galactose–glucomannan (GCM) are the major hemicellulose in coniferous wood. The structure of GCM changes with the species and cell wall positions, and is usually linked to the backbone made of β-(1–4)-dmannopyranose and β-(1–4)-d-glucopyranose. Endomannanase and β-glucan are the main backbone depolymerase. β-Mannosidase and β-glucosidase only work in the hydrolysis of oligosaccharides (Puls, 1997). The minority of hemicellulose in biomass include arabinogalactan, xyloglucan, and β-glucan. Arabinogalactan mainly exists in coniferous wood composed by linear β-(1–3)-d-galactopyranose. Although β-(1–4)-arabinogalactan is also found, it is mainly in the highly substituted C-6 position. As a debranching enzyme, β-glucuronidase can assist the polymerization of β-galactan. Xyloglucanase found to degrade the β-(1-4)-d-glucopyranose skeleton were mainly from the GH74 family. Among the enzymes, α-xylosidase has the widest range of applications (Tenkanen, 1998). In addition, different preprocessing methods caused a different impact on the chemical structure of the hemicellulose, thus leading to different requirement for different types of enzymes: for example, the hydrolysis of polysaccharide in birchneeds xylanase, β-enzymes, and acetyl xylan esterase. The only usage of xylanase can only get 10% of the product. So, the selection of degrading enzymes must be based on the structural features of substrate.

4.1.3  Lignin-Degradating Enzymes Lignin-degradating enzyme systems are very complex. In recent years, many scholars have carried out studies on the catalytic mechanism of lignin decomposition. These enzyme systems include extracellular peroxidase, 1ignin

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peroxidase (LiP), manganese peroxidase, (MnP), and extracellular phenol oxidase-laccase (laccase, LaC). In addition, aryl-alcohol oxidase (AAO), gyoxaloxidase (GLOX), glucose oxidase (gucose-l-oxidase), phenol oxidase, and so on took part in or have some impact on degradation process of lignin. Furthermore, the bacteria can produce two new enzymes: FAE and coumaric acid esterase, which act on lignocellulosic material and produce ferulic acid and p-coumaric acid. These two enzymes work synergistically with xylanase for the decomposition of hemicellulose lignin polymer without the production of mineralized lignin (Howard, Abotsi, & Jansen van Rensburg, 2003).

4.1.3.1  Lignin Peroxidase and Manganese Peroxidase Both LiP and MnP are extracellular iron-containing heme proteins with glycosyl, are known as heme peroxidase. LiP is a glycoprotein, consisting of 10 long protein chains and a short protein chain, which is similar to other peroxidases, running a typical catalytic cycle; MnP is a kind of glycoprotein, which were also composed of ten long protein chains and a short protein chain. The activity center of LiP consists of a heme group together with two Ca2+ for stabilizing of the structure. The activity center of MnP is basically the same as LiP, but has two more Mn2+. The main difference between the two is that carbon ends of LiP are between two propionates in heme, but carbon ends of MnP are separated from the heme. In addition, MnP owns five disulfide bonds but only for LiP. In the catalytic process of lignin degradation, LiP and MnP capture an electron from the phenolor a nonphenolic compound, changing the latter into a cation group and leading the bond cleavage of the lignin. LiP mainly oxidates phenol into phenoxy oxidation residues; MnP also mainly oxidates phenol into phenoxy oxidation residues, but the concrete roles of the enzymes have not been identified. 4.1.3.2  Laccase (LaC) Laccase, a copper-containing polyphenol oxidase, can be divided into two categories: rhus laccase and fungal laccases. Those enzymes are mainly from lacquer and fungi. Due to high sugar content in fungal laccases, it was not until 1998 that humans obtained the first laccase crystal from Coprinus cinereus. Laccase can directly use O2 as a substrate without the presence of H2O2 and other second substrates. In this process, laccase itself loses the electrons or protons from hydroxy and transforms into free radicals. Laccase can also transform lignin into phenoxy radicals, which can further take part in copolymerization or homopolymerization reactions. These properties have made laccase gain a growing concern in the comprehensive utilization of lignin. In addition to LiP, MnP, and LaC, there are also glucose oxidases, glyoxal oxidases, mellow oxidases, and catalase involved in the lignin degradation process. But so far, the specific role of each enzyme is not entirely clear in lignin degradation (Chi & Yin, 2007; Puls, 1997).

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4.1.4  Cellulase Cofactors For a long time, the study of cellulase has mainly focused on glycoside hydrolase. Despite a lot of exploration, there has still been no solution to the problem of the degradation of the natural crystalline cellulose due to ignorance of the role of supermolecular structure of cellulase in the enzymolysis process. Researches from the perspective of matter and energy found that the dense structure formed by hydrogen bonds is the bottleneck in enzymatic of cellulose. The first stage of cellulose hydrolysis is the separation of oligomerization from the solid cellulose surface, which is the rate-limiting step of the hydrolysis. In the cellulose hydrolysis process, in addition to the synergy between the three cellulases, there are participation of other proteins. These proteins, which can promote the hydrolysis process, are called the cofactor of cellulase (Lu, Chen, & Ma, 2006). Reese put forward the existence of a hydrogen enzyme in 1950, which could destroy the crystal structure of cellulase and enhance the accessibility of cellulose. Cosgove discovered the expansion protein in plants, which is considered to be most likely to play the role of “hydrogen enzyme.” In the process of plant growth, expansion protein can induce the wall to loosen and expand in a reversible manner. This process causes the sliding of polymers by breaking the hydrogen bonds within the polymer network. Expanding protein itself does not control glycoside hydrolase activity, but can weaken the strength of the filter paper, showing synergistic effects with cellulase in the hydrolysis of filter paper. Saloheimo found Swollenin protein owning the similar gene sequences with plant expansion proteins. In the hydrolysis of filter paper and cotton experiments, Swollenin proteins can destroy the structure of the cellulosic substrate, without reducing sugar (Saloheimo, Paloheimo, & Hakola, 2002; Levasseur, Saloheimo, & Navarro, 2006). Shandong University in China also conducted fruitful researches on Swollenin protein, including gene clone, protein structure analysis, and heterologous expression (Yao, 2007). In addition, researchers also found a number of cofactors of nonhydrolysis cellulase. Gao Peiji et al. (Liu, Fan, & Sushi, 2008) separated a protein, which could weaken the absorption strength of cotton cellulose in hydrogen zone, lead to the expansion of cotton cellulose and chitin, and produce no reducing sugars. All these results matched the characteristics of Swollenin protein. Qiu applied laccase in the promotion of enzymatic digestibility for lignocellulose substrate. With the synergistic effect between laccase and cellulase, the reducing sugar content was increased by 37.9% and the ethanol production was increased by 13.8%. Analysis found that lignin got partly degraded after laccase treatment. This kind of ring-opening reaction transforms the substrate surface into a net structure, so that the accessibility of cellulase to the substrate has been enhanced significantly (Qiu & Chen, 2008).

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4.2  THE SETUP OF BIOMASS INVERTASE PLATFORM 4.2.1  The Enzyme Production Since bacterial α-amylases could be produced from scale fermentation, fermentation has became the main method for enzyme production. Depending on the cultivation modes, fermentation can be divided into submerged fermentation, solid-state fermentation, and immobilized cell culture. At present, the prevailing way is the submerged fermentation. Compared with traditional enzymes extracted craft from plant, the fermentation method has advantages in short production cycle, high enzyme yield, and less impact on the environment. However, it involves high requirements on the equipment and production craft (Chen, 2008). This section mainly focuses on fermentation technology for microbial enzymes.

4.2.1.1  Preparation of Cellulase Production 1. Producing strains Bacteria, fungi, and actinomycetes can produce cellulase, among which fungi Trichoderma has been widely studied and applied due to the high production ability of cellulase and high enzyme activity. The most researched fungi include T. reesei, Trichoderma koningii, and Trichoderma viride. In addition to Trichoderma, Actinomycetes are also the ideal strain due to their simple structure, which is easy for genetic analysis. A lot of researchers are committing to Actinomycetes enzyme production research. Mutagenesis is the main source of excellent fermentation bacteria for cellulase. Taking Trichoderma TH (Trichoderma pseudokon-ingii) as the starting strain, mutant UVIII obtained by UV could tolerate high concentrations of glucose, and owned 100-fold higher sensitivity to the inducer than the parent strain. At the meantime, the glucose absorption capacity declined significantly, making the bacteria partially remove the repression of glucose inhibition. With the development of modern molecular biology, genetic engineering bacteria has also become a source of cellulose fermentation bacteria (Wei & Li, 2008; Zhang, Liu, & Gao, 2009). 2. The fermentation medium Cellulase is a kind of inducible enzyme, whose biosynthesis was regulated by inducer. The fermentation medium usually choose the splintered, pretreated, and plant fiber contained raw, waste paper, vinasse as the main inducers and major carbon source together with nitrogen source and suitable inorganic salts. Research shows that adding 15% wheat bran, 2% urea, 1% ammonium sulfate, and 0.15% of KH2PO4 in brewers’ grains can improve enzyme activity and protein content significantly in the product. For liquid fermentation, these materials are made into liquid medium supplemented with excess water. In most researches, dry materials accounted for 3% of the amount of liquid medium. Xiaobin Yu et al. optimized liquid fermentation

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medium of Reesei cellulase WX2112 using response surface method and determined the best combination: 3.18% of soybean meal, 2.95% of wheat bran, 0.25% of KH2PO4, and 3.79% of avicel. The filter paper activity could reach a maximum of 10.53 IU/mL. 3. Brief introduction to the fermentation process a. Solid state fermentation Solid-state fermentation method could use straw powder and waste paper as raw materials. The materials are always mixed with koji, loaded under the curtain, or spread into a thin layer (thickness 1 cm), and ferments at a certain temperature in the culture room and humidity (RH 90∼100%). Its main feature is that there is no free water in the fermentation system, and microbes grow in the solid substrate with sufficient humidity and the fermentation environment is close to the natural state of microbial growth habit. It has many advantages such as more kinds of enzymes being produced, conducive to the degradation of natural cellulose, low investment, low energy consumption, high output, ease of operation, high recovery rate, no foam, and less environmental pollution. However, solid-state fermentation is susceptible to bacterial contamination; it is thus difficult to separate and purify the cellulase production, and tough to remove the pigment (Chen & Xujian, 2008). The temperature is the primary factor for solid-state fermentation. Optimization of media and culture conditions are an important method to reduce enzyme costs, increase enzyme activity, and realize the industrial production. Generally, it is considered best to adjust the starting pH of the medium to acidic range, which will help the growth of fungi and inhibit the bacteria breeding in solid-state fermentation. Hongzhang Chen and coworkers (Xu, Chen, & Li, 2002a; Xu, Chen, & Shao, 2002b) proposed a novel fermentation model named gas double-dynamic solidstate fermentation: under the optimum pressure pulse range, pulse frequency, and the circulation gas rate, fermentation temperature can get better controlled and the maximum temperature gradient is 0.12°C/cm; water activity get well maintained within steam-exploded straw as fermentation substrate; the fermentation period of dynamic (60 h) is a third shorter than the static fermentation period (84 h) and the enzyme activity increased by onefold from 10.182 to 20.36 IU/g. Under the pulsation pressure, microbial grows uniformly in the solid material layer, while there is no cell growth in the middle layer of the material under static solid-state fermentation condition. Gas double-dynamic solid-state fermentation lays the foundation for large-scale production of cellulase. b. Liquid fermentation method Liquid submerged fermentation is also known as full fermentation. In this method, straw and other raw materials are crushed and sent to a closed fermenter with stirring paddle and ventilation systems, cultivated with strain, and then sufficiently stirred with sterile air or self-priming

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the airflow. Gas and liquid can get as far as possible to contact to help the fermentation. Its main features are easy-to-control culture conditions, the avoidance of infection, and high production efficiency. As one of modern biotechnologies, submerged fermentation has become an important research and technology both domestically and internationally. Submerged fermentation generally uses a closed fermenter with a stirring paddle and ventilation system. The whole process from the medium sterile, medium cooling to fermentation are carried out in the same fermenter. Most fermentation time is about 70 h, and the temperature is generally less than 60°C. Inoculation quantity in liquid fermentation [generally 2∼10% (v/v)] is significantly lower than solid state fermentation. Zhang Dongyan studied the suitable fermentation conditions of Viride A S1313711, Trichoderma A S1312774, Trichoderma A S1313032, and Trichoderma ACC131167 for enzyme production. The results showed that the optimum temperature is 28°C, and appropriate initial pH for enzyme production is 4.5–5.5. The optimal parameters of Gibberella fujikuroi for cellulase production are as following: inoculation size is 5%, the incubation time is 120 h, the culture temperature is 28∼37°C, and initial pH value is 5–6 (Zeng & Wang, 2009).

4.2.1.2  Hemicellulase Production Studies on xylanase is primarily from fungi and bacteria. In most cases, the best activity is achieved at or near the temperature (about 40–60°C), neutral conditions (mainly bacteria xylanase) or slightly acidic conditions. However, some xylanase also been reported to have activity under extreme pH and temperature. In fact, it has been reported that xylanase remains active at a temperature of 5–105°C within pH 2.0–11.0. Some studies show that xylanase remained active when the concentration of NaCl up to 30%. These enzymes are ways that microorganisms adapt to extreme environments. Among these enzymes, thermophilic, basophils, and eosinophils xylanase been extensively studied. Conversely, the study on cold-adapted xylanase is much rarer (Shi, 2011). Xylanase derived from fungi has higher enzyme activity, such as T. reesei rut, C-30,851 IU/mL; Fusarium oxysporum, 245 IU/mL; Thermoascus aurantiacus, 208 IU/mL; A. niger, 283 IU/g; and Melanocarpus albomyces, 9300 U/g. The enzyme activities are not strictly comparable because the detecting step and the reaction conditions are not the same, such as temperature, incubation time, and substrate used. The stability of xylanase from fungal reported in alkaline and high temperature environment is not good except for M. albomyces. This enzyme exhibits good stability and high activity at pH 10.0, 70°C in solid-state fermentation, whose half-life is about 2 h. The strains producing alkali resistance, high temperature, and high xylanase activity include: Bacillus sp. Sam3, xylanase activity of 131 IU/mL, pH 7.0–9.0, 60–70°C; Bacillus sp. NCIM59, xylanase activity 500 IU/mL, pH 7.0–10.0, 60–70°C; Bacillus cirulans Ab16, xylanase activity 50 IU/mL, pH 5.0–9.0,

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55–80°C, and C. absonum CFR-702, xylanase activity 420 IU/mL, pH 6. 0–9.0, 78–85°C (Quan & Zhao, 2010).

4.2.1.3  Preparation of Lignin-Degrading Enzyme 1. Producing strains MnP is widely present in the white-rot fungus, such as the famous biopulping insect species, wax bacteria. It is easy to detect the presence of MnP when white-rot fungus lives on the lignocellulosic substrates. Almost all the Basidiomycetes, which cause wood rot and habitat in the soil degradation layer, can produce this enzyme. These Basidiomycetes mainly belong to wood decay fungi, including Corticiaceae, Stereaceae, Hericiaceae, Ganodermataceae, Hymenochaetaceae, Polyporaceae, Strophariaceae, Tricholomataceae, and so on. However, bacteria, yeasts, filamentous fungi, and mycorrhizal fungi do not produce MnP. Laccase (EC 1.10.3.2) is present in a wide range of fungi strains including Basidiomycetes, Ascomycetes, and Deuteromycetes, and some insects, bacteria, and plants also produce laccase. Laccase owns monomer, dimer, and tetramer forms with monomer as the general form. Laccase from the white-rot fungus owns the following characteristics: molecular weight is between 60∼80 kDa, glycosylated, containing 15–20% carbohydrate, owning an acidic isoelectric point (the pH value is between 3.5 and 7.0). Laccase can be produced by most of the white-rot fungus stains, but except Phanerochaete chrysosporium, which is well-known, for the lack of laccase production ability. The lignin peroxidase was first discovered in P. chrysosporium. At present, only a few white-rot fungi can produce LiP. The molecular weight of LiP is about 40 kDa, glycosylated, having an acidic isoelectric point. In addition, they contain a heme iron protoporphyrin IX (Chi & Yin, 2007). 2. Fermenting the substrate External factors affect the production of lignin-degrading enzymes, which are carbon, nitrogen, oxygen, and trace elements. Among them, the fungus P. chrysosporium is the more researched is generally believed to produce LiP and MnP but no laccase. However, literatures reported that P. chrysosporium can produce a small amount of lignin peroxidase and laccase under severely controlled concentration of nitrogen. Lignin-degrading enzymes are produced in an oxygen environment. When using glucose as a carbon source, the lignin-degrading enzyme synthesis was inhibited in P. chrysosporium. While using cellulose as a carbon source and cellulose is completely consumed and no oxygen is required, strains began to generate LiP. The right amount of manganese can improve LiP activity of P. chrysosporium. Mn is an element required in MnP synthesis; the amount of Cu2+ can promote the synthesis of strain laccase. Inducers and surfactant have an important role in the synthesis of lignin degrading enzymes. The inducer studied most was resveratrol. Different strains have the corresponding optimum surface agents. Polyethylene can stimulate

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the synthesis of lignin peroxidase, and 3,4-dichlorophenol can maintain the activity of LiP and MnP at a high level over a period of time. In the fermentation process of P. chrysosporium, adding HgCl2 can improve stability and activity of peroxidase, especially LiP. When using ferulic acid (FA, 4-hydroxy-3-methoxycinnamicacid) as substrate, LiP will be out of activity. p-Coumaric acid and guaiacol can improve laccase production of white-rot fungus Trametes sp. I-62 significantly. 2,5-benzyl alcohol is the best laccase inducer, followed by 3,5-dimethoxybenzyl, and 3,4-benzyl alcohol was the worst laccase inducer (Hui, 2006). Chen Hongzhang used steam-exploded wheat straw as a substrate for lignin enzyme production through solid-state fermentation. Cellulosic feedstocks, especially grasses, have been partially degraded after steam explosion treatment and the degradation products can replace expensive resveratrol as lignin gene expression inducer. Substrate produced from lignin peroxidase is rich in lignin. Cellulose-rich substrate is conducive to the synthesis of laccase (Lu, Zhang, & Hong, 2005). 3. The fermentation process introduction The fermentation process of microbial enzyme production is closely related with environmental factors. Fermentation methods have a great influence on the laccase production of white-rot fungi. According to the training methods, it can be divided into two categories: liquid fermentation and solid fermentation. 4.2.1.3.1  Liquid Fermentation Liquid fermentation refers to the process of mycelial growth and enzyme production in liquid medium containing certain nutrients. Since this method can facilitate industrial production and application, therefore, the reports of enzymes production in white-rot fungi through liquid are relatively common. Most studies have shown that adding an appropriate amount of inducing agent or Cu2+ in liquid medium can promote laccase production from white-rot fungus significantly. In addition, some metal elements, such as copper, manganese, iron, zinc, and so on, are an integral part or enzyme reactive group, which cannot be ignored. Many studies (Birhanli & Yesilada, 2006; Lorenzo, Moldes, & Sanromán, 2006; Rosales, Couto, & Sanromán, 2007; Gassara, Brar, & Tyagi, 2010) have confirmed that adding a certain amount of Cu2+ in the medium can promote laccase production of white-rot fungus significantly. 4.2.1.3.2  Solid-state Fermentation Solid-state fermentation is a method that uses solid medium to culture mycelial and harvest enzymes on the substrate surface in the absence of or containing a small amount of liquid. With the continuous development and in-depth study, fungal enzyme production by solid fermentation has attracted more and more scholars’ attention. Research and practice show that solid-state fermentation

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of white-rot fungi can get higher laccase production compared to the liquid fermentation. Solid fermentation supplies more oxygen circulation, which is conducive to the growth of microorganisms. Furthermore, the process is static, so the energy consumption of mechanical power can be omitted, and the subsequent process is relatively simple. These methods are close to the wild state, in particularly suitable for enzyme production. Scale expansion of white rot fungi solid-state fermentation (SSF) has also obtained more and more attention from researchers. Mass transfer system laid great impact on laccase yield. When using barley bran as noninert substrates, disc reactor is more suitable for white-rot fungus Trametes versicolor for laccase production (Couto, Sanromán, & Hofer, 2004; Couto, Moldes, & Liébanas, 2003). The maximum laccase activity reached 3.5 IU/mL, six times those in the other two fermenters. When using orange peel as a substrate in solid-state fermentation, the disc reactor is also applicable to white-rot fungus Trametes hirsuta for laccase production with the highest activity as 12 IU/mL. Because the disc reactor is better than the other two solid-state fermenters, scholars believe that the mass transfer would produce a certain shear force in the submerged and expanded bed reactors, affecting cell growth and enzymes biosynthesis. Therefore, design and mass transfer system is a key factor affecting the efficiency of enzyme production. At present, reports about the application of solid fermentation for laccase production from the white-rot strain are relatively rare. This is mainly because the solid-state fermentation tank is facing some insurmountable problems. In addition to equipment design and mass transfer system, fermentation process parameters include pH, temperature, aeration, and oxygen delivery, and humidity, which are difficult to control. Therefore, the current study focused on the improvement of existing solid-state fermentation systems or design of novel fermentation equipment, such as new RITA (Recipient Immersion Temporaire Automatique) batch immersion systems (Boehmer, Suhardi, & Bley, 2006). RITA immerses mycelium growing on the solid substrate into liquid medium. The advantages of this reactor are that it not only avoids the mechanical shearing effect on bacteria, but also is able to overcome the metabolites and organic dyes for cell growth and enzyme inhibition. Another way for efficient production of laccase from white-rot fungi is to seek a suitable carrier for the heterologous gene expression. The presence of glycosylin laccase can promote the hydrolysis of laccase protein, compared to other industrial production oxidoreductases; expression of white-rot fungi laccase in the heterologous host is very difficult. Therefore, the present study on laccase production is mainly concentrated on the optimization of fermentation conditions. In recent years, from the study of laccase production from white-rot fungi, either liquid or solid fermentation, a new feature has emerged that the natural substances is being used, especially the use of lignocellulosic organic byproducts generated in industrial and agricultural production, as the culture

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substrate. These organics include wheat bran, bagasse, rice straw, wheat straw, wood chips, straw skin, lees, soybean meal, corn cob, deinking sludge, vines, and olive mill wastewater. Most of them are usually rich in carbohydrates, able to provide nutrients for bacteria growth and metabolism. Furthermore, these organics also contain a certain amount of lignin, cellulose, and hemicellulose, regarded as an inducer of laccase production in white-rot fungi. It is reported that wheat bran also contains a lacquer enzyme-inducing substances-ferulic acid (approximately 0.4–1.0%) with a strong inducing effect on laccase production. Although so far there are no reports on other natural substrates containing special inducing substances, the feasibility of these natural organic matters as fermentation substrate for white-rot fungus has been widely recognized.

4.2.1.4  Cutinase Production With nitrophenyl butyrate as the screening substrate, a Thermophilic bacterium strain producing cutinase was screened. Under the suitable culture conditions, cutinase activity reached 19.8 U/mL. By pNPB hydrolase activity detection, pure natural cutinase was separated from fermentation supernatant of Thermophilic sp. (T. fusca) by sequential treatment of ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography separation. Thermophilic sp. cutinase has good thermal stability and pH stability. The optimum temperature is 60°C and the optimum pH for the reaction is 8.0, which is in line with the required clean production characteristics of the textile industry (Li, Liu, & Chen, 2009).

4.2.2  Chemical Modification Enzymes Chemical modification aims at changing the properties of natural enzymes for excellent characteristics and wider applications. Generally, the conformational change may occur following the chemical modification of the native enzyme and improve the enzyme activity and stability. We can say that the enzyme chemically modification theory provides an experimental basis and proof for the relationships between the structure and function of biological macromolecules. Chemical modification is very effective to improve the enzymatic properties and enlarge its application range. Experimental studies have shown that the appropriate chemical modifier and modification conditions can not only retain the enzymatic activity, but also can improve enzyme stability to heat, acids, alkalis, and organic solvents and change enzyme substrate and the optimum pH. There are a lot of enzyme chemical modification methods, but the basic principle is the use of chemical characteristics that the modifier possessed, directly or indirectly, reacting with amino acid residues in enzymes molecules, thus transforming into the enzyme molecule. When the enzyme is chemically modified, the following must be noted: (1) after the enzyme treatment by pH, oxidation, or reduction, the total amino acid analysis should be carried out, in particular, those are difficult to analyze

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(such as tryptophan, methionine); (2) because the modifying agent selected for the model compounds or enzymes do not necessarily apply to others, therefore one should find out whether other enzymes have been modified; (3) controlling the modification reaction conditions, such as buffer components; (4) identify whether there is a significant difference between optimum pH of the natural enzyme and optimum pH of enzyme after modification. Until now a variety of types of protein modification agents has been developed, including: (1) small molecules, such as acetyl imidazole, haloacetic acid, N-ethylmaleimide, carbodiimides, coke, diethyl tetranitro methane; modifying agent; and (2) macromolecules such as polyethylene glycol (PEG), polyamino acids, acetic acid/propionic acid copolymer, carboxymethyl cellulose, polyvinyl pyrrolidone, dextran, and cyclodextrin. Whether small molecule or macromolecular modifying agent, they can chemically react with a specific amino acid residue to form a covalent bond.

4.2.2.1  Chemically Modified Cellulases Maleic anhydride is often used as a chemical modifier for preparing alkali cellulose using acidic cellulase as substrate. Maleic anhydrides electively modified lysine group of cellulase, which extended cellulose tolerance to pH from 8.5 to 10.0. Using 0.5 mol/L maleic anhydride under reaction conditions pH 8.0, 2∼4°C with cellulase 25 min, the half-life of the modified enzyme under pH 8.0, 9.0, 10.0, and 11.0 was 13.08, 121.58, 126.0, and 187.30 h, respectively. Therefore, enzyme stability is greatly improved. It is reported that the modified maleic anhydride and N–bromosuccinimide cellulases are also good modifier, and the half-life of the modified enzyme at 30, 50, and 85°C are up to 120 min. The performance of the resulting cellulase was also superior to the original detergent enzymes. The cellulase modified by PEG owns significant enhancement on the thermal stability. Therefore, the capacity to resist heat also increased. At the same time, the affinity between the modified enzyme and the substrate also become higher than the natural enzyme (Li, 2007b). 4.2.2.2  Chemical Modification of Laccase With deep understanding on the structure and function of laccase, it was found that lysine (lys) residues is not the active center of laccase, which provides a theoretical basis for the chemical modification of laccase. At present, PEG, dextran, phthalic anhydride (PA), and citraconic anhydride (CA) have been applied to laccase chemical modification, and the effects of modification were also studied. Hua et al. modified laccase with PA in order to improve its stability against high temperature and acidic pH. The modification ratio of amino lysine groups of laccase is determined as 63.8% by TNBS method. It is shown that the modified laccase maintained the secondary and tertiary structure of its native counterpart but has a higher affinity to ABTS and a significantly improved thermal

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stability, as indicated by the extension of the half-life of enzyme activity at 55°C from 192.5 to 532.4 min. The suitable pH range is also expanded from pH 5.9∼7.8 to pH 4.5∼8.4. The degradation ratio of anthracene is increased twofold, making the modified laccase promise for the removal of PAHs. Native laccase was applied widely in industry. It was becoming very important and urgent because native laccase was amenable to activity loss under its application conditions. A commercial native laccase named DeniLite IIS with wide application has been researched. This laccase was modified with different anhydrides, such as phthalic anhydride (PA), succinic anhydride (SA), and maleicanhydride (MA). SA was confirmed as a best modification reagent for laccase according to enzymatic; activity and stability enhancement of modified laccase. The influences of three factors of phosphate buffer pH and SA concentration and modification time on enzymatic activity and stability were studied by L9 (34) orthogonal experiment. The results showed that no interaction existed between these three factors. The optimal conditions for modification were that phosphate buffer pH was 7.5, SA concentration was 2 mmol/L and modification time was 1.0 h. SA modification of laccase under the optimal conditions caused enzymatic activity increased by 50% and thermal stability at 50°C for 30 min enhanced by 15%. This research offered a suitable method for property reform of native laccase (Ya-hong, Gao, & Zheng, 2011).

4.2.3  Enzyme Engineering With the establishment and development of recombinant DNA technology, people remove dependence on natural enzymes in large part. Developments of genetic engineering enable people to obtain a variety of natural enzyme genes by cloning, and highly express them in heterologous microbial receptors, in the end massively producing them by fermentation technology. This method can greatly reduce the cost of the enzyme product, and also makes the production of rare enzymes easier.

4.2.3.1  Gene Cloning Most of the earlier enzyme-cloning method was achieved by constructing is a library, such as DNA libraries and cDNA libraries. A DNA library takes genomes of the starting strain as research object, by random digestion of genome into varying sizes of fragments, random digestion fragments of varying sizes, and inserted them into the cloning vector. After positive clones were screened, the objective gene can be determined. A cDNA library is a combination of cloned cDNA (complementary DNA) fragments inserted into a collection of host cells, which together constitute some portion of the transcriptome of the organism. cDNA is produced from fully transcribed mRNA found in the nucleus and therefore contains only the expressed genes of an organism. Similarly, tissue-specific cDNA libraries can be produced. In eukaryotic cells the mature mRNA is already spliced, hence the cDNA produced lacks introns and can be

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readily expressed in a bacterial cell. While information in cDNA libraries is a powerful and useful tool since gene products are easily identified, the libraries lack information about enhancers, introns, and other regulatory elements found in a genomic DNA library. With the fast development of network technology and gene synthesis technology, the current cloning method is more simple and straightforward. Cloning methods commonly used as following: (1) the synthetic method. According to the reported nucleotide sequence, researchers synthesized these genes in labs. (2) Specific primer amplification method. This refers to the method that designs the specific primer based on the nucleotide sequence and amplifies the similar gene from same species. 1. Cloning and expression cellulase gene So far, more than 7,000 cellulose enzyme gene sequences and the corresponding amino acid sequences has been reported and the 3D structures of more than 500 celluloses have been published. These data are published in Gen Bank, EMBL, DDBJ, and so on. At the same time, almost all of the cloned cellulase genes have been expressed in E. coli (Li & Zhang, 2011). Nearly more than 100 cellulase and xylanase genes can be cloned and expressed in E. coli, mainly endoglucanase and β-glucanase. Researches on cellulase synthesis regulation, cellulose degradation mechanisms, and new enzyme molecule construction can be achieved by cloning methods. There are no operation challenges on DNA extraction, digestion, plasmid recombination, and transformation. 2. Cloning and expression xylanase genes At the time of writing, more than 300 kinds of different xylanase genes sources have been reported, of which more than 100 kinds have been cloned and expressed in suitable hosts. Endoxylanase genes derived from Penicillium owns 10 sequences, among which five belong to the Family 10, four belong to Family 11, and the other belongs to Family 7. Four inner endoxylanase genes have been reported from Penicillium funiculosum, three of which owns CBM domains. XynA gene encodes xylanase/cellobiohydrolase which is similar to family 7. XynB genes belong to Family 11 and xynD belong to Family 10. These enzymes include a catalytic domain, and a serine-aspartic acid-rich connecting region and a prediction CBM domain. All of the xylanase-encoding genes described above have been expressed in Pichia yeast, E. coli, fungi, Aspergillus, Saccharomyces cerevisiae, and other expression systems. The optimum pH of xylanase derived from Penicillium is between 2.0 and 7.0, and the optimum temperature is between 40 and 60°C (Liu & Gao, 1998). 3. Cloning and expression of mannanase gene Since Henrissat (1991) divided the mannanase into glycoside hydrolase Family 5 and Family 26, many researchers have studied the sequences of

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mannanase gene. Currently, there are hundreds of pieces of β-mannanase gene information in gene banks, most of which have been cloned in bacteria, fungi, and plants. Some mannanases can maintain its activity at extreme environments, such as the optimum pH of MAN5A P13 is 1.5 and optimum pH of MANN from Aspergillus sulphureus is 2.4. The production of Man 1 from Yarrowia lipolytica reached 5.9 g/L (1575 U/mL). β-Mannanase gene from fungal is the most cloned and expressed. This gene can be isolated from Agaricus bisporus, Armillariella tabescens EJLY2098, Aspergillus fumigatus, P. chrysosporium, Aspergillus aculeatus, and so on. They have been expressed in Pichia yeast, S. cerevisiae, A. niger, and other heterologous expression systems (Cai, 2011).

4.2.3.2  Site-directed Mutagenesis of the Enzyme Molecule When researching the natural enzyme and the mutant, it is usual first to obtain the information on molecular characteristics, space structure, and relationship be­tween structure and functions. This information can be obtained through biochemistry, spectroscopy, and crystallography technologies. Then this information is used for enzyme molecular modification, which is called rational designment of enzyme molecular. Rational designment of enzyme molecular mainly includes chemical modification and site-specific mutagenesis. In contrast, the modification without enzyme information was called nonrational designment of enzyme molecular, which includes determinate evolution and hybrid evolutionary. The foundation of rational designment is the scientific knowledge of protein construction for engineering prediction. When using this strategy, it is essential to obtain the information of protein structure, especially the catalytic site and the relationship between the structures and functions. In the typical case, the data efficiency of enzyme or homological protein determined the protein to modify. First, we should understand the role of the amino acid residues in the enzyme, then carry out the modification with site-specific mutagenesis, secondary structure elements, even the exchange of structural threshold. Finally, we can analyze the characteristics of mutant enzyme. In recent years, the rational designment strategy has been widely used in the research of relationship between enzyme function and the specific amino acid residue. Until now protein, engineering has been carried out on the cellulase from different glucoside hydrolase (GH) families, including GH5, GH6, GH7, GH8, GH9, and GH45. 1. Site-directed mutagenesis of endocellulase Application of site-directed mutagenesis techniques on Thermobifida fusca and Bacillus N4 transformation can be changed the amino acid sequences within the cellulose enzyme, thus increasing the adapt range of pH, reducing product inhibition, and increasing enzyme activity. In addition, the use of molecular biology techniques to build hybrid enzyme also made considerable progress. Studies on the extreme heat archaeal

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enzyme EGPh implied that the enzyme lacks a cellulose-binding domain, and E324 is the active center of the enzyme. 2. Site-directed mutagenesis of exoglucan cellobiohydrolase The goal of rational design on endoglucanase is mainly to increase enzyme activity and stability (Zhang, Zhang, & Gao, 2006). Von Ossowski found that the outer ring from G245 to Y252 of Cel7 A of T. reesei have an impact on the enzyme activity. Wohlfahrt applied site-directed mutagenesis techniques onto T. reesei, replacing amide-carboxylic acids with carboxylic acid and making the mutant melting temperature changes. The modification improved the enzyme stability under alkaline conditions significantly. Though the catalytic rate on cellotetraose has not been alerted, the half-life of the two mutant enzymes under basic conditions increased by 2–4 times. Therefore, carboxylic acid can effectively change the pH-dependent properties in the protein engineering. 3. Site-directed mutagenesis of β-d-glucosidase Compared to exoglucanase enzymes, the rational design on the endoglucanase was relatively less. Fukuda applied site-directed mutagenesis on G294 of on the Bgl I from Aspergillus oryzae, and obtained three mutants, G294, FG294, and WG294Y, whose enzyme activities were increased by 1.5, 1.5, and 1.6 times higher than the wild, respectively (Li et al., 2009).

4.2.3.3  Enzyme-directed Evolution The relationship between protein structure and function is very complex, and the current understanding on the relationship is still very limited. When the knowledge of the molecular structure is very poor, the directed evolution become a powerful tool. Directed evolution can simulate the natural evolutionary mechanism without prior knowledge of the spatial structure of the protein. Its core is a random mutation plus selection, namely by PCR or other methods for gene random mutation or recombination, and make it expressed in the host cell, and then a high-throughput screen the mutant enzymes. For many cellulase enzymes, the three-dimensional structure has still not been resolved. Therefore, directed evolution has become a powerful tool for cellulase modification. Scholars have carried out a number of studies, and obtained a large number of mutant enzymes with improved activity, increased stability, or other properties (Ping, Liu, & Xue, 2009). Thermoanaer-obacter tengcongensis MB4 endoglucanase Cel5A pretreated by error PCR was screened by the Congo red plate and obtained two enzymes with 135 and 193% folds activities of the wild enzyme. Endoglucanase (EngD) from mesophilic Clostridium cellulovorans recombined with CelE from Clostridium thermocellum and obtained a mutant enzyme with twofold increase on the enzyme activity at 50°C. In the study of directed evolution of cellulase, researches on xonuclease are relatively less. This is mainly because the current exonuclease activity assay

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has not been unified, convenient, and fast, which restricts the throughput of mutations screening (Zhang et al., 2006; Li, 2007a).

4.2.3.4  Fusion Enzyme Fusion enzyme refers to the fusion protein composed by two or more enzymes (Chen, 2008). A fusion enzyme belongs to hybrid enzymes. It combines the advantages of different enzymes and has obvious heterosis. Amino acids of fusion enzyme have differences in static electricity, acidity, acidity and ion; therefore, they have special characteristics that normal proteins do not have. There are two ways to produce fusion enzymes: one is nonrational designment, mainly by building the library, and then screening out the fusion. Another method is rational designment, fusing the enzyme based on the key information of enzyme structure and functions. The fusion protein is usually conducted among homologs, because less similarity of the protein sequences increases the probability of fusion failure. The characteristics of the enzyme will change during the fusion treatment, including kinetic parameters, thermal stability, and optimum basic characteristics such as pH changes more easily. Fusion enzyme construction strategy can be divided into three categories: secondary structure integration, function domain integration, and whole protein integration. It is a corollary to develop the fusion protein engineering technology. This technology is based on protein project and computer science, which makes the synthetic enzyme strategy possible.

4.3  ENZYMES CONVERSION PLATFORM FOR BIOMASS 4.3.1  Enzyme Synergy Evolutionary selection pressure makes the microorganisms living on biomass use enzymes to degrade the biomass. Two collaborative system have been discovered: one is multicomponent cellulase secreted by fungus/bacteria and the other is cellulose secreted by anaerobic strains (Gruber, Kamm, & Kamm, 2007; Yinbo, 2011).

4.3.2  Multicomponent Enzyme System There are two synergy forms between fungal cellulase (Mosier, Hall, & Ladisch, 1999): one is the synergy between exocellulase and endocellulase; and the other is the synergy between exocellulase and exocellulase (Goyal, Ghosh, & Eveleigh, 1991; Wood & McCrae, 1979). Chanzy, Henrissat, and Vuong, (1984) proved CBHI enzyme can randomly bond to molecule chains of cellulose by studies of colloidal gold. Herissat et al. found that the ability of EGI for hydrolysis of microcrystalline cellulose is very low, and CBH I has no activity to CMC, but on microcrystalline cellulose. EG I and CBH I can synergistically degrade filter paper, microcrystalline cellulose,

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microcrystalline cellulose, and homogenized bacterial cellulose, but cannot degrade CMC synergistically. CBH I and EG I or EC II degrade insoluble cellulose synergistically, but the optimal ratio between the enzymes was different. Nisizawa found that CMC with a high degree of substitution has higher exonuclease activity than CMC with a low degree of substitution, suggesting that the low activity of CBH Ion CMC was related with substituent groups. T. reesei is a representative fungal cellulase. As an efficient cellulose producer, it is capable of secreting a large amount of cellulase, including CBH I, or Cel7A (60%), CBH II, or Cel6A (15%), EG I or Cel7B, EG II, or Cel5A (20%), and other minor components (such as EG III or Cel3A, XynI or Xyn11A, and swollenin). The difference between CBH I and CBH II is whether can satisfy the degradation requirement of cellulose substrate.

4.3.3  Fiber Bodies C. thermocellum is a typical hydrolyzing enzyme system of bacteria. This cellulase system consists of an approximately 200 KDa molecular, which has special structures including protein scaffold, anchoring threshold, cellulose binding threshold, and many cohesin domains. Molecular mass of fibrous bodies ranges from 50 to 5,000 Da and many fiber bodies also form polyfiber bodies through anchoring interactions. The catalytic domain of fiber bodies is similar to multicomponent fiber cellulase secreted by fungus. However, comparing with the multicomponent fiber cellulase, the hydrolysis efficiency of fiber bodies was much higher (Chen, 2011; Yinbo, 2011). In summary, an efficient industrial cellulase enzyme system should include a variety of functional enzymes, and these enzymes must be able to perform synergistically.

4.3.4  Optimization of the CBH–EG–BG System Since cellobiose is not only an inhibitor for CBH, but also is the precursor of fermentable sugars. Therefore, a complete cellulase system requires the use of BG to hydrolyze cellobiose. How to achieve mutual balance among CBH–EG– BG is the key issue to improve hydrolysis efficiency. Researches showed that T. reesei could secrete at least two kinds of BG enzymes under the abduction of cellulose. At the same time, comparing with the closed system, the filtration equipment, which gets rid of small molecules, can improve the efficiency of cellulose hydrolysis. Cellobiose is an inhibitor of CBH, the accumulation of which can decrease the hydrolysis rate (Gruber et al., 2007).

4.3.5  Design of the Multienzyme Complex Studies have shown that the enzyme synergy can help to build a more efficient industrial enzyme system. However, there is a difference on the characteristics

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TABLE 4.1 Compound Enzymes and Their Enzymatic Hydrolysis Effects for Different Substrates Hydrolysis yield (mg.g DM)

Abf II (%)

Abf III (%)

β-xyl

Xyl III (%)

Arabinose

Xylose

Watersoluble Arab xylan in wheat

20

20

40

20

343.7

548.7

Waterinsoluble Arab xylan in wheat

25

25

25

25

162.5

286.5

Vinasse

10

40

50

51.47

91.58

Substrate

between enzymes. So there are still challenges for man-made enzyme synergy systems. We just make a brief introduction on the design of the multienzyme complex. This can be divided into four steps: (1) the selection of the enzymes; (2) the consistency evaluation among different enzymes; (3) determining of the mixing ratio among enzymes; and (4) the optimization of technological parameters. Taking the hydrolysis of arabinose and xylose in wheat as an example, the minimal enzyme cocktail (MEC) includes four key enzymes. A slight difference on the substrate structures leads to different requirement on the compound proportion and amount of the enzymes used. The different compound enzymes and their hydrolysis results for different substrates are given in Table 4.1. Note: Abf II and Xyl III from H. insolens, Abf III from M. giganteus, βxyl from T. reesei. The total amount of the water-soluble enzyme protein and water-insoluble substrate used is a 0.55 g/kg (DM), and the total amount of the enzyme used for the vinasse is 0.45∼0.55 g/kg (DM).

4.3.6  Enzyme Reactor An enzyme reactor is the equipment used for the reactions between enzymes and substrates. Enzyme reactors can be divided into different types according to different methods. The reactor characteristics and their corresponding applications are listed in Table 4.2 (Lehe, 2006; Chen, 2008). In practical applications, enzyme reactors are chosen based on the enzyme, substrate and product characteristics, operating conditions, and operation requirements. Free enzyme-catalyzed reactions are usually carried

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TABLE 4.2 Introduction of Enzyme Reactors Type

Operation mode

The main characteristics

Homogeneous enzyme reactor

Agitator tank

Batch, batch

Using blender mixing

Ultrafiltration membrane reactor

Batch, batch and continuous

Membrane allows only low molecular compounds and enzyme pass through, suitable for large molecules substrates

Immobilized enzyme reactor

The drum

Partial, half batch and continuous

Immobilized enzyme suspended in the solution

Fixed bed/ packed bed

Continuous

Fluidized bed

Batch and continuous

Membrane reactor

Continuous

Bubbling tower

Partial, half batch and continuous

Suitable for the reactions with gas

out in the stirred reactor; for the low yield but expensive enzymes, enzyme recovery is very important. So, it is very suitable to use a membrane reactor; for the enzyme reactions gas involved, we can choose the bubble reactors. For the high temperature resistance enzymes, it is better to choose the jet reactor. If the molecular weight of the product is small, it is better to choose the membrane reactor, because micromolecules can easily pass through the membrane and get separated with the macromolecular substrate. However, the traditional membrane reactor has an obvious disadvantage: the concentration of the reducing sugar obtained is very low, so it is not suitable for the sequential treatment. Chen designed an ultrafiltration membrane bioreactor (Fig. 4.3) consisting of six enzymitic vessels which were used to increase the reducing sugar concentration. The effects of enzyme loading, the number of enzymatic vessel, and the dilution rate on enzymatic hydrolysis of steam-exploded rice straw were studied. The results show that the best condition is: 20 FPU/g, four enzymatic vessels, and 0.75 h−1 (D). Compared with the traditional batch reaction, the conversion of the substrate increased significantly from 18∼21% to 39.5%. Using the ultrafiltration membrane bioreactor consisting of one enzymatic vessel, the production of reducing sugar increased from 0.25 to 0.4 g/g, and the final reducing sugar concentration in the product steam increased from 4.56 to 27.23 g/L.

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FIGURE 4.3  1, Thermostatic water bath; 2∼5, enzymatic membrane tank; 6, peristaltic pump 7, membrane module; 8, sugar tank; 9, pressure control valve.

4.4  ECONOMIC ANALYSIS OF CELLULASE PRODUCTION 4.4.1  Relations Between Particularity of Cellulose and Production Costs As macromolecular proteins with catalytic activity, the function of cellulase and the stability of the integrity depends on its native conformation, which is very sensitive to temperature, the operating strength, and pH environment. Therefore, the properties put up high requirement on the cellulose production, which increases the production cost. 1. The low production from the producing strains The current conversion rate of substrate of industrial strains is, on average, between 6% and 10%, which means that more than 90% of the raw material cannot be converted. Meanwhile, the enzymatic activity of native cellulose is low. The enzymatic activity of industrial grade is generally 2,000∼5,000 U/g. However, laboratory grade of cellulose activity is commonly about 10,000 U/g or more, indicating that there is plenty of scope for industrial fermentation to be improved. 2. Fermentation conditions Different from the general chemical production, the cellulase fermentation requires high control of operation conditions, such as dissolved oxygen, temperature, pH, and other sophisticated requirements. Any unsuitable operation conditions will result in activity loss of the product. At the same time, the average growth phase of cellulase producing strain is 72 h and the average fermentation phase lasts about 4 d. The longer fermentation period

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leads to a continued consumption on sterile air, water, electricity, and manpower, which constitute an important part of production costs. In a production cycle, the fermentation process consumes most of the steam, water, electricity, and other energies. 3. Enzyme recovery The enzyme recovery efficiency is also a key factor to determine the overall production cost. Currently, the enzyme recovery in industry production was 70% and the deactivation rate is about 10–30%. In other words, due to the limitations of the recovery techniques nowadays, 30–40% of cellulase cannot be converted into a commodity. Therefore, improving the separation efficiency methods is key to improve production efficiency and reduce production costs.

4.4.2  Relationship Between Process Indicators and Production Costs In this section, we will take a cellulose factory with annual output of 480 tons as an example, to analyze the relationship between process indicators and production costs. The aim is to determine the limiting factors leading to high production cost of cellulase.

4.4.2.1  Relationship Between Production Costs and the Fermentation Period Fermentation period is directly related to the cost of production; it is not only related to the annual production capacity of the factory, but also is the most important factor determining the consumption of water, electricity, labor and other expenses. The current average period of cellulase fermentation is four days (including seed cultivation, fermentation processes, products recovery, etc.). If using short-fermentation period strain or new control technology to compress the fermentation period, the cost of inputs can be greatly reduced. 4.4.2.2  The Relationship Between Fermentation Yield and Cost Fermentation yield is also a key factor in the decision of cellulose production cost. The current conversation rate of substrate is about 6.5%. If this rate was increase 1–7.5%, raw material input costs can be greatly reduced. Table 4.3 shows the comparison of the raw material costing. If the conversion rate increased by 1%, as we can see, a cost of raw material of at least 2 million Yuan can be saved. At the same time, due to the reduction in the consumption of raw materials, we can save at least about 5,000 tons of water, 1.78 × 106 kw/h factory power, 7.4 × 104 ton steam, which add up to about 2.95 million Yuan in total and equal 19.6 and 10.2% of profit and total cost, respectively.

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TABLE 4.3 List of Fermentation Yield and Cost Relationships The unit price Consumption/t Consumption/t

A total of 480 tons

The raw material

500

5.17

4.4807

165,432

Wheat bran

1,000

1.293

1.1206

82,752

Yeast extract

1,500

0.012

0.0104

1,152

Lignocellulose

2,800

0.24

0.208

43,008

Peptone

2,500

0.072

0.0624

11,520

MgSO4 · 7H2O

5,000

0.0072

0.0062

2,400

KH2PO4

8,000

0.096

0.0832

49,440

CaCl2

1,250

0.0072

0.00624

960

Tween-80

15,000

0.176

0.152533

169,440

Ammonium sulfate

1,200

1.573

1.363267

120,960

Bubble enemy

700

6.667

5.778067

299,040

Project

Name

The raw material

4.4.2.3  The Relationship Between Enzyme Activity and Production Costs Compared with the conversion rate of the substrate, enzyme activity impacts great the production cost. If the enzyme activity were increased from the current average level (4600∼4700 U/g) by 1000 U/g, the total cost could be reduced by 3,000,000 Yuan (calculation method as aforementioned). In addition, the market price of cellulose shows a linear relationship with enzyme activity. In other words, if the current level of production (4600 U/g) increased to 5600 U/g, the market price will be higher than the original price 5600/4600 = 1.22 times, so profit growth will increase more than 1.22 times. 4.4.2.4  The Relationship Between the Filtration Method and the Production Cost Product separation and purification is one of the major parts of the cost. For existing separation methods, the costs mainly arise from two aspects: raw materials and energy consumption. First, in the enzyme recovery process, it requires ammonium sulfate to precipitate the enzyme cellulase. The total enzyme loss rate in this process is about 20% of the total enzyme. To product 480 t cellulose enzyme consumes at least 591 t ammonium sulfate, which is about 7.8% of the total cost. Feed cellulases must use ethanol as the precipitating agent. To product 480 t cellulose enzyme consumes at least 1870 t ethanol, which is about 21.4% of the total cost.

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Therefore, if we found a new separation method using a cheaper alternative to ammonium sulfate or ethanol, input costs could be greatly decreased. Moreover, because the cellulase is very sensitive to the environment, if the separation conditions (temperature, pH, etc.) are not suitable, it will result in the loss of enzymatic activity. Under existing conditions, the enzyme recovery rate in the precipitation section is only 90%, the recovery rate of the filtering portion is 95% (the highest level), and the loss of the enzyme activity was 7–10%. Therefore, after the whole separation process, the loss rate of the enzyme is as high as 20–23%. If we can reduce the loss of the enzyme activity or increase recovery efficiency, the inputs will be greatly saved.

4.4.3  The Major Policy to Reduce Cellulose (or Conversion) Enzyme Economic Costs Through the above analysis, it can be seen that the product cost can be saved from at least four aspects: (1) increase the rate of substrate conversion; (2) enhance the enzyme activity; (3) improve the product recovery; and (4) compress the fermentation cycle. Table 4.4 summarizes the achievements in enzyme development in recent years.

TABLE 4.4 The Main Strategy to Reduce the Cellulase Cost and the Corresponding Effects Source of enzyme gene

Expression host

Enzyme type

Enzyme activity

Thermonospora YX

E. coli

Endoglucanase

5.8 µmol/min/ mg × 103

B. subtilisDR

E. coli

Endoglucanae

0.82 U/mL

Xylella fastidiosa

E. coli

Endoglucanase

2.39 µKat

Azoarcussp. strain BH72

E. coli

Exoglucanase

30 U/mg of protein

Ruminococcus flavefaciens

E. coli

Endoglucanase

19.4 µg/min/mg

Bacillus sp. strain KSM-64

B. subtilis

Endoglucanase

21,700 U/L

Thermomonospora fusca

S. lividans

Endoglucanase

10 U/mL

A. tubingensis

K. lactis

Endoglucanase

—

Cryptococcus sp. S-2

P. pastoris

Endoglucanase

4.36 U/mg of protein

T. reesei

P. pastoris

Exoglucanase-7

—

T. reesei

P. pastoris

Exoglucanase-1

—

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TABLE 4.4 The Main Strategy to Reduce the Cellulase Cost and the Corresponding Effects (cont.) Source of enzyme gene

Expression host

Enzyme type

Enzyme activity

T. reesei QM9414

S. cerevisiae

Endoglucanase

—

T. reesei QM9414

S. pombe

Endoglucanase

—

T. reesei

S. pombe

Endoglucanase-2

Exoglucanase-3.81 U/ mg of protein

T. reesei

Y. lipolytica

Endoglucanase-1

Endoglucanase-0.1 U/ mg of protein

A. fumigatus Z5

P. pastoris

β-Glucosidase

101.775.2 U/mg of protein

Candida wickerhamii

S. cerevisiae

β-Gluocosidase

—

P. chrysosporium

P. pastoris

β-Glucosidase

52 U/mg of protein

Commercial mixture

Supplier

Reported enzyme activities (U/mL unless otherwise specified

Celluclast 1.5 L FG

Novozyme

65 FPU, 12 β-glucosidase, 660 xylanase (bwx) 60.7 FPU, 6.5 β-glucosidase

Novozyme 188

Novozyme

8.5 FPU, 665 β-glucosidase, 123 xylanase (osx), 29.3 α-arabinofuranosidase, 16.6 β-xylosidase, 116 α-galactosidase, 0.6 feruroyl esterase (U/mg) 1 endoglucanase, 0.35 exoglucanase, 14.75βglucosidase, 10 β-xylanase, 0.22 β-xylosidase, 0.09 α-arabinofuranosidase 0.1 FPU, 661 β-glucosidase

Spezyme CP

Genencor

58.2 FPU/ml, 128 β-glucosidase, 2622 xylanase(osx), 22.6 α-arabinofuranosidase, 7.3 β-xylosidase, 0.39 α-galactosidase (U/mg) 1.4 FPU, 21.8 CMCase, 0.09 exoglucanase, 1.82 β-glucosidase, 15 β-xylanase, 0.56 βxylosidase, 0.38 α-arabinofuranosidase, 55.2 FPU, 15.4 β-glucosidase

Multifect (xylanase)

Genencor

0.77 FPU, 35.9 β-glucosidase, 25,203 xylanase (osx), 9.44 α-arabinofuranosidase, 22.6 β-xylosidase, 2.39 α-galactosidase, 1.3p-coumaroyl esterase (U/mg) 6.3 endoglucanase, 0.46 exoglucanase, 3.3 β-glucosidase, 209 β-xylanase, 4.9 β-xylosidase, 3.21 α-arabinofuranosidase (Continued)

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TABLE 4.4 The Main Strategy to Reduce the Cellulase Cost and the Corresponding Effects (cont.) Commercial mixture

Supplier

Reported enzyme activities (U/mL unless otherwise specified

Accelerase 1000

Genencor

93 FPU, 7.3 CMCase, 1632 β-glucosidase, 849-xylanase 67.3 FPU, 84.2β-glucosidase

Primafast Luna CL

Genencor

Endoglucanases, no exoglucanase

GC220

Genencor

92.8 FPU, 99.7 β-glucosidase, 2782 xylanase 9(osx), 3.06 α-arabinofuranosidase, 7.3 β-xylosidase, 3.9 α-galactosidase

Shearzyme

Novozyme

27 FPU, 5.0 β-glucosidase, 2293 xylanase (bwx)

NS50013

Novozyme

63 FPU, 8 β-glucosidase, 1117 xylanase (bwx)

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Gassara, F., Brar, S. K., Tyagi, R., et al. (2010). Screening of agro-industrial wastes to produce ligninolytic enzymes by Phanerochaete chrysosporium. Biochemical Engineering Journal, 49, 388–394. Goyal, A., Ghosh, B., & Eveleigh, D. (1991). Characteristics of fungal cellulases. Bioresource Technology, 36, 37–50. Gruber, P. R., Kamm, B., & Kamm, M. (2007). Biological refining, industrial processes and products. Beijing: Chemical Industry Press. Henrissat, B. (1991). A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochemical Journal, 293(Pt 3)(Part 2), 781–788. Howard, R. L., Abotsi, E., Jansen van Rensburg, E. L., et al. (2003). Lignocellulose biotechnology: issues of bioconversion and enzyme production. African Journal of Biotechnology, 2, 602–619. Hui, Z. (2006). The research progress of lignin degradation enzyme system. Tianjin Agricultural Science, 12(3), 8–12. Jing, X. (2010). The research progress of cellulose enzyme. Chemical Industry and Engineering, 31(5), 46–49. Lehe, M. (2006). Modern enzyme engineering. Beijing: Chemical Industry Press. Levasseur, A., Saloheimo, M., Navarro, D., et al. (2006). Production of a chimeric enzyme tool associating the Trichoderma reesei swollen in with the Aspergillus niger feruloyl esterase A for release of ferulic acid. Applied Microbiology and Biotechnology, 73, 872–880. Li, Y. -H. (2004). The research progress of beta glycosidase enzymes. Journal of Anhui Agricultural University, 29(4), 421–425. Li, B. (2007a). Study on chemical modification of cellulase and its application on extraction of diosgenin. Tianjin: Tianjin University. Li, B. (2007b). Study on chemical modification of cellulose and its application on extraction of diosgenin. Tianjin University, 3, 5–10. Li, W., & Zhang, G. (2011). The cellulose enzyme gene engineering research progress. Biotechnology, 8, 51–54. Li, J., Liu, L., Chen, H., et al. (2009). The research progress of cutinase. The Journal of Biological Engineering, 25(12), 1829–1837. Lian, Y., & Wang, L. (2007). The research progress of cellulose enzyme degradation mechanism of cellulose. Weiming Xu Journal of Ningbo University, 20(1), 78–82. Liu, W., & Gao, P. (1998). Molecular biology of hemicellulase. Science and Technology of Cellulose, 1, 9–15. Liu, F., Fan, J., & Sushi, Z. Z. (2008). The research process of bioconversion of cellulose. Anhui Agricultural Science, 36(17), 7059–7060. Lorenzo, M., Moldes, D., & Sanromán, M. Á. (2006). Effect of heavy metals on the production of several laccase isoenzymes by Trametes versicolor and on their ability to decolourise dyes. Chemosphere, 63, 912–917. Lu, G., Zhang, Z., & Hong, W. (2005). The research progress of lignin enzymes solid-state fermentation. Feed Industry, 29–33. Lu, D., Chen, H., & Ma, R. (2006). Effect of straw apoplast protein on cellulase activity. Journal of Biological Engineering, 22(2), 257–262. Merle, M. E. (2010). Biomass degradation resistance barrier. Beijing: Chemical Press. Mosier, N. S., Hall, P., Ladisch, C. M., et al. (1999). Reaction kinetics, molecular action, and mechanisms of cellulolytic proteins. Recent Progress in Bioconversion of Lignocellulosics, 23–40. Pei, J. -J., Yemin, X., & Azure, S. (2003a). The research progress of Arab glycosidase. Microbiology, 15, 190–193. Pei, J. -j., Yemin, X., & Azure, S. (2003b). The research progress of α-glucuronic acid enzymes. Microbiology, 30, 91–94.

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Chapter 5

Microbial Cell Refining for Biomass Conversion Chapter Outline 5.1 Biorefinery 5.2 Cell Refining 5.2.1 Metagenomics of Cell Refining 5.2.2 Metabolic Engineering of Cell Refining 5.2.3 Systems Biology Technology of Cell Refining 5.2.4 Cell Processing and Synthetic Biology 5.3 Totipotent Cell Function: Artificial Cell 5.3.1 Multienzyme Reaction System 5.3.2 Carrier Immobilization

101 103 104

108

111 114 117 118 120

5.4 Intercellular Synergy 5.4.1 Characteristics of Mixed Fermentation 5.4.2 Precautions of Mixed Fermentation 5.5 Construction of a Cell Refining Factory 5.5.1 Modification of Microbial Cell Properties 5.5.2 Design of Cell Refining Factory 5.5.3 Construction of Cell Refining 5.5.4 Optimization of Cell Refining Factory References

123 123 125 126

127 130 130 131 133

5.1 BIOREFINERY In 1982, the concept of biorefinery was first proposed in Science, in which agricultural waste, plant-based starch, and lignocellulosic materials were used as raw materials to produce chemicals, fuels, and bio-based materials. Biorefinery was defined by US National Renewable Energy Laboratory as something using biomass as feedstock, combining biomass conversion processes and equipment comprehensively to produce fuel, electric energy, and chemical products. Due to the current crisis of energy, resources, and worsening environmental issues, the concept of a biorefinery has become a strategic research around the world, leading to the launch of national biorefinery plans in many countries (Kamm, Gruber, & Kamm, 2007). Technologies for Biochemical Conversion of Biomass © 2017 Metallurgical Industry Press. Published by Elsevier Inc.

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Microbial cell factories are the core of biorefinery technologies and the platform and material basis of cell refining. Cell refining is the process during which renewable resources were used as raw materials for energy, drug, and chemical materials production for human by microbial cell transformations. The first electron microscope made people understand that the eukaryotic cell is a complex open system consisted by cell film and passages, whose internal structure is similar to a factory. In the biological world, the specific surface area, transforming ability, reproduction rate, and other indicators of microorganisms, exceeded above all creatures, and thus microorganisms have a strong self-regulation and environmental adaptability. Based on these characteristics, “microbial cell factories” successfully realize the cell refining which serves human needs, such as Actinomycetes. sp. (antibiotics production), Penicillium. sp. (penicillin production), Corynebacterium glutamicum (lysine and glutamic acid production), and Aspergillus niger (citric acid production). In order to exploit microbes’ potential more efficiently, scholars screen finer strains to produce better products by utilized known metabolic regulation mechanism to transform various microorganisms based on well-known metabolic mechanism. The process is shown in Fig. 5.1 (http://news.sciencenet.cn/sbhtmlnews/2010/5/232185.html). Future biochemical conversion of biomass is a technology combination of bioconversion and chemical cleavage, including improved pretreatment and fractionation of lignocelluloses, optimized design of reactors for conversion of renewable feedstock, and improved biological catalysts and catalytic processes. Cell refining is a typical cross-domain of interdisciplinary research and

FIGURE 5.1  Schematic diagram of the cell refining process.

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its development needs joint efforts of experts from biology, chemistry, and engineering disciplines and other areas of technology, which integrated the refining process effectively. Up to 200 billion tons C are fixed annually on the earth through photosynthesis of plants, with the energy of 3 × 1018 kJ, about 10 times the energy consumed worldwide per year. Among them, straw is the largest biomass resources efficiently collected. More than 700 million tons straw were collected in China per year, which is expected to be conversed into raw material of bio-based chemical products to solve resources and energy crisis currently in China. However, although scientists around the world have studied this aspect for many years, the high-value utilization of straw resources has still been not achieved. The main obstacle is the technical and economic problems of straw conversion, which is a world scientific problem and engineering challenge. Sugar and starch were the main raw materials in traditional fermentation industry. The current rise of straw and other biomass refining is aimed to substitute the glucose starch with the glucose derived from cellulose. To replace the traditional structure of the petrochemical industry by straw and other biomass industry, the issue to use biomass as the new commonly used feedstock of biological and chemical industry should be broken through. Chen, Li, & Liu (2011) had analyzed the differences of chemical compositions, physical properties, and cellulose hydrolysis of straw from the level of molecule, cell, and tissue, revealing that the nonuniformity of straw is the primary cause of the difficulty of value-added-use of straw resource (Chen & Qiu, 2007, 2010). They have developed traditional straw utilization patterns based on the whole plant into a value-added conversion system with comprehensive biomass utilization by component fractionation and stratified multistage conversion (Jin & Chen, 2006, 2007; Chen, 2010). Based on their systematic reach they indicated the research direction for the high-value utilization of straw resources by providing a sophisticated scientific theory and engineering application of straw component fractionation—stratified multistage conversion for the establishment of a new biomass feedstock refining industry (Chen, 2006, 2008a,b), creating a new eco-industrial model of straw and other biomass refining for biobased products.

5.2  CELL REFINING The production processes of the cell refining platform include input output of organic raw materials, activation of genetic information, formation of a metabolic network, formation of metabolic material, and output of product. The microbial cell factory is the basic platform to achieve the goal of biomass biochemical conversion. However, the enzyme species of the wild-type microorganism in nature do not perform effectively and are not favored by industrial production. In order to transform microbes into microbial cell factories for biorefining, the latest technologies and methods, including genomics, metabolic engineering, systems biology, and synthetic biology, should be the tool for analysis of the essence of microbial gene, protein, and metabolic processes network,

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developing a better understanding of the microbial metabolism’s ability from the molecular, cellular, and ecosystem level. Based on this, scholars could redistribute the material and energy flows of microbial cell metabolism through the reorganization and optimization of manual control and fully exploit the microbial capabilities of decomposition, transformation, and chemical synthesis, and effectively prepare bioenergy and alternative petrochemical raw materials. Thus, the abundant biomass resources could truly become a substitute of petroleum industrial raw materials, and efficiently be prepared to bioenergy (biogas, biohydrogen, bioethanol, biodiesel, etc.) and platform chemicals (ethylene, lactic acid, fumaric acid, butyl acid, furfural, 1,3-propanediol). Metabolism is the basic feature of life phenomena. As a three-dimensional cell factory, the major metabolic pathways of microbes are related directly to the utilization of raw materials and product synthesis. However, the intracellular metabolic pathways are not independent and in complex metabolic network by various regulation. Regulation is a complex process at the transcriptional level and translation level. The existing regulatory system of microorganisms must be adapted, changed, or modified to improve the efficiency of cell factories. Genetic and environmental factors determine the microbial conversion capabilities. From a genetic perspective, the result of long-term evolution makes metabolic function a benefit for the microbial body. The metabolic processes are in the most economical state and do not excessively accumulate unfavorable metabolites. However, cell refining requires microbial cells to change the original metabolic pathways and accumulate a large number of expected metabolites (secondary metabolites). Thus, in order to achieve the rational regulation of microbial metabolic function, an in-depth understanding of the molecular mechanism of metabolic pathways, the microbial genomes and their interactions with the corresponding enzymes is required.

5.2.1  Metagenomics of Cell Refining Metagenome, also known as metagenomics or environmental microbial genomes, is the sum of the entire tiny DNA in biological environment (currently mainly bacteria and fungi). Metagenomics aims to study the microbial population genomics in environmental samples without the microbial cultivation stage. During the analysis process, the total DNA in the environment was directly extracted. Genetic screening and sequencing functional analysis were employed to study microbial diversity, population structure, evolutionary relationships, microbial functional activity relationships, and mutual cooperation relations between microbes and the environment. The appearance of metagenomic technology makes people aware of studying uncultured microorganisms, which account for more than 99% of all microorganisms, significantly enhancing the detectable space of microbial genes. Overall, the current application of metagenomic techniques can be divided into two aspects: developing the required function proteins by screening functional genes; and exploring the interactions

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between microorganisms and the surrounding environments through the analysis of metagenomic library to understand the microbial world more objectively and comprehensively.

5.2.1.1  Basic Strategy of Metagenomics The basic strategy of metagenomics is to: 1. extract the total DNA or mRNA from the environmental samples as completely as possible; 2. use appropriate vectors to clone the genomic DNA into the mode microorganisms to establish a macro genomic DNA library or a cDNA library; and 3. give new traits to host cells by exogenous gene or screen based on some known DNA sequence to find a new target gene clusters or biologically active substances. Based on the general process of screening new genetic resources and expressed products from Rumen microbes, the construction policies of metagenomic library are summarized in Fig. 5.2 (Wang, An, & Liu, 2010). Metagenomics analysis will enable us to deeply understand the diversity of rumen microorganisms, population structure in the system, evolutionary relationships, functional activity, and mutual cooperation relations, and to build control means of multiscale anaerobic digestion process based on reactor operation, changes of active bacteria, and functional changes in gene expression, providing a guiding role for the anaerobic microbial screening and biomass anaerobic conversion process control.

5.2.1.2  Construction of a Metagenomic Library Construction of a metagenomic library follows the basic principles and techniques of molecular cloning. Some special purpose steps and strategies should be taken according to the specific characteristics of the environment samples and the goals for database building. It is one of the key processes for the construction of metagenomic library to obtain the high purity, high molecular weight, and high concentrations of total DNA from environmental samples, which needs to extract the total DNA from the samples entirely so as to maintain its large fragments of DNA and to obtain the complete target gene or gene cluster. Currently the main methods used for DNA separation and extraction from environmental samples, such as soil are the situ lysis and ectopic lysis methods. Environmental samples in the situ lysis method (also known as the direct lysis method) were suspended in lysis buffer directly for extraction and purification, which is suitable for plasmid or phage as cloning vectors. This method has the advantages of easy operation, low cost, and high DNA extraction rate, while a smaller fragment of DNA is extracted (1∼50 kb) due to the strong mechanical shear. Ectopic adhesion lysis isolates microbial cells in samples of soil and sediment first, followed by a more moderate approach to extract DNA, known as the

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FIGURE 5.2  Construction and screening process of animal gastrointestinal metagenomic library.

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microbial cell extraction method. For the vector, the construction of a metagenomic library currently uses a plasmid, cosmid vector, and bacterial artificial chromosome. A bacterial artificial chromosome vector can clone large fragments of DNA, increasing the chance for complete guidance molecules, such as genes or gene cluster encoding antibiotic synthesis pathway, which shows a considerable advantage in the cloning and expression of macrogenome. To improve the expression level of the gene so as to facilitate the activity detection of recombinant clone, the expression vector was employed by some researchers to construct a metagenomic library directly. In addition, the shuttle vector can expand the host range in favor of expression of exogenous genes. Screening factors of the transformation efficiency, the stability of the recombinant vector and the expression of macro gene and target traits should be taken into consideration in selecting a host strain (e.g., antibacterial activity). The results showed that the active substances produced by different types of microbial species are significantly different. Therefore, the target host strain should be chosen according to the purpose, for example, 70% of the antibiotics from actinomycetes, Streptomyces sp., should be selected as a host bacteria to look for antibacterial, antitumor active substances while E. coli is used for the new enzyme screening.

5.2.1.3  Screening of a Metagenomic Library Currently metagenomic library screening methods are divided into sequencebased screening, function-based screening, compound structure screening, and substrate-induced gene expression screening. Screening based on the sequence is the process of designing a probe or PCR primers according to the sequence of associated functional genes to obtain a target gene from the gene library. Compound structure screening is the process of screening target genes by comparing the chromatogram of fermentation broth or extracting solutions from host cell with/without transferred exogenous gene. However, the substances screened by this method may not have the activity. Substrateinduced gene expression is the screen process using substance-induced catabolic gene clones, which can be used for the screening of enzyme activity. Metagenomics technology fills the blank of uncultured microbial research and has become a focus of international microorganism study, forming a new path to find new genes, develop new biologically active substances, and study the diversity of microbial communities (Zehr, Bench, & Carter, 2008; Mori, Mizuta, & Suenaga, 2008). Metagenomics technique does not rely on training while extracting genomic genetic material directly from the natural environment microorganisms to analysis the microbial population, which boosts better awareness of the knowledge of microbial population ecology and evolution, maximizes the excavation of microbial resources. The technology makes the study of environmental microbes focus not only on “determining what they are,” but also on “determining what they can do” without the training process (Wexler, Bond, & Richardson, 2005; Xu, Duan, & Zhou, 2006).

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Of course, the study of metagenomics faces many challenges. A metagenomic library consists of uncultured microbial genetic information, rather than the bacteria itself. The genetic background of these microorganisms is unknown and unable to study regulation of gene expression. It is possible that an exogenous gene is not expressed or undetectably expressed in the host cell. Thus, the metagenomic library screening may miss many natural products of environmental microbes, which will affect the results of the metagenomic study. However, with the improvement of metagenomic technology, progress will continue to enhance its application value.

5.2.2  Metabolic Engineering of Cell Refining The advent of genetic engineering has greatly promoted the development of microbial fermentation industry, which makes genetic modification of specific enzyme reactions in the microbial metabolic pathway possible. However, the microbial fermentation involves the synergistic action of multiple enzymatic reactions in the metabolic network of microorganisms. Genetic transformation of a single (or multiple) enzymatic reaction is not sufficient to improve fermentation performance. Extremely rich biodiversity of microorganisms determines their metabolites with diversity. With the acquirement of sufficient sequence information, metabolic pathways of these organisms will be reconstructed, providing effective strategies for us to develop these creatures. In 1991, Jay Bailey and Gregory Stephanopoulos first proposed a metabolic engineering concept: “using recombinant DNA technology to manipulate enzymes, transport, and regulatory functions, thereby to improve the cell activity.” With the development of technology, the definition of metabolic engineering has been corrected continually by later scholars. The essence of metabolic engineering is the quantitative analysis and control of the metabolic flux for the metabolic reconstruction to maximize the yield of the desired metabolites. Unlike traditional mutation breeding techniques, it is a purposeful and rational reconstruction, involving physiology, molecular biology, biochemistry, and biological pathway engineering and other disciplines. The main contents of metabolic engineering theory involves: 1. biosynthesis and metabolism network theory; 2. metabolic flux analysis, nodal analysis; 3. carbon resources, respiratory system, redox redesign; and 4. action mechanism of center metabolism and related metabolic analysis. It is worth noting that the two differences of metabolic engineering compared with genetic engineering are as follows. First, metabolic engineering is the systematic study of cell metabolism networks, putting more emphasis on the “integration” role of multiple enzymatic reactions. Second, after the completion of genetically engineered metabolic pathways, metabolic engineering should analyze the physiological changes in

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cell metabolism fluxes in detail to decide the next target for genetic transformation and constantly improve the physiological properties of cells (fermentation capacity) through multiple cycles. The completion of more and more biological gene sequencing and the progress of functional genomics research introduce the metabolic engineering into a new era. However, how to regulate a cell metabolism network effectively to improve cell performance is always the core of metabolic engineering and the microorganisms’ cell refining. Principles and methods of metabolic engineering based on the systematic analysis of metabolic networks is to transform the cell metabolism system using recombinant DNA technology, which includes three key steps: first, genetic modifications: construction of recombinant bacteria for improved performance; second, metabolic analysis: analysis of recombinant bacteria metabolic pathways, especially comparing its performance with the original strain performance; third, the design strategy: the design of genetic engineering goal for the next step. These three steps constitute one cycle, which is shown in Fig. 5.3 (Li, 2009). Metabolic engineering research usually consists of two parts; one is the analysis for cell systems. A cell metabolic network is a very complex system where the metabolism of cells can affect gene expression. In contrast,

FIGURE 5.3  Principles and methods of metabolic engineering.

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the expression of the gene controls the associated enzymes in the metabolic pathway and influences the choice of metabolic flux. Therefore, the analysis of metabolic engineering includes not only the intracellular metabolic flux, but also the intracellular gene expression by using transcriptomics and proteomics tools, which could reveal the correlation of gene expression and metabolic pathways. Another part is to genetically transform of engineered strains, change the metabolic flow, open up new metabolic pathways, and build superior strains by the results of the analysis (Zhang, 2009).

5.2.2.1  Metabolic Network Theory The theory of metabolic networks takes the biochemical reactions of cells as a whole network. The network of the cell metabolism consists of reaction system catalyzed by thousands of enzyme, membrane transport systems, and signal transmission systems with fine adjustment and coordination. The metabolite of shunt metabolic network is called a node and a small number of nodes that play a decisive role for the end products are called main nodes. 5.2.2.2  Metabolic Flux and Intermediate Products Analysis Metabolic flux analysis is an important means of metabolic analysis. It is assumed that the material and the energy inside the cell are in a steady state. The applications of extracellular substances concentration determination, radiolabeling, and isotopic tracing make the metabolic flux analysis easier. Through the flow analysis of cell under different situations, such as changes of the culture environment, removal of suppression, increase or decrease of enzyme activity, the node type and the optimal way could be determined. Also through the ways, the results of gene modification and the maximum theoretical yield rate could be estimated. For simple reaction system, satisfactory results could be obtained through accurate analysis and balanced calculation of all metabolic network. However, for more complex metabolic systems, pathways analysis becomes difficult. With the rapid development of computer technology, the methods to analyze the metabolic become diverse and promote a higher degree of automation. Thus, the flow to the final product from the metabolic intermediates will ultimately determine the yield of the final products. Metabolic flow changes of intermediary metabolites will be inhibited by cells and cause severe interference of intracellular function. Since some intermediate metabolites play an important role in cell signaling and the regulation system, the role of intermediate metabolites in the cell must not be overlooked during the metabolic transformation. 5.2.2.3  Metabolic Control Analysis Metabolic flux analysis reveals a static distribution of metabolism, and metabolic control analysis aims to the instability of internal and external environment for the cells, revealing the dynamic changes of cellular metabolism. The

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coefficient of elasticity and flow control are the two main indicators of metabolic control analysis. The elasticity coefficient reveals the effect of metabolic changes on the reaction rates. These two factors are interrelated, and may be measured directly or indirectly (Zhao, Wang, & Chen, 2004). The flow control coefficient is the metabolic flow changes of a branch flow caused by changes of the enzyme amount and is used to measure the control degree of a step enzymatic reaction on the entire reaction system. On the basis of microbiology and in-depth research of the genome, metabolic engineering techniques study the metabolic pathways of biochemical conversion of specific compounds and develop microbial primary metabolites, secondary metabolites, and important biobased chemicals. In addition, conducting functional studies of biocatalysis, optimizing the strains for essential enzyme protein production, modifying gene of enzyme, and developing new industrial enzymes are all the content of metabolic engineering. Galazka et al. transferred transport protein CDT1 and CDT2 of cellobiose and oligosaccharides from Neurospora crassa into yeast and realized the utilization of cellobiose by yeast. The yeast could take in cellobiose to produce ethanol directly by this modification process, which improved the efficiency of the whole sugar fermentation of cellulose and provided a new idea for large-scale production of biofuels (Galazka, Tian, & Beeson, 2010). Complex interactions between enzymes, regulators, and metabolites in microbial cells lead to extremely difficult work in optimizing metabolic pathways, which is facing serious challenges in metabolic engineering. The appearance of new methods of genomics, transcriptomics, and metabolomics greatly promotes the optimization of metabolic pathways. In particular, the development of systems biology provides a global scale for a profound understanding of the physiological and metabolic characteristics of microorganisms, creating an unprecedented opportunity for the development of metabolic engineering.

5.2.3  Systems Biology Technology of Cell Refining Achieving high yields of the target metabolite, a high yield and high intensity of production organic unity based on a better awareness of the regulation mechanism of the microorganism metabolism is the core content for cell refining biochemical biomass conversion technologies and has very important significance. The cell is a tightly regulated, multichannel, and multilevel network system to a large extent against external disturbance, which undoubtedly reduces the effects of genetically modification. In order to make further progress in metabolic engineering, deepening the understanding of the physiological activity patterns of cells is required. With the wide application of high-throughput experimental techniques, the gradual improvement of bioinformatics methods, and enhanced capacity of excavating valuable information from large data, the understanding of physiological activity law in microbial cells is deepened and

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the systems biology emerged. This systems biology enables metabolic pathway engineering from the local level to the overall level, and metabolic engineering has thus entered a new stage of development, which is called the system biotechnology era. Systematic biology is the study of all the components of biological systems (gene, mRNA, protein, etc.), and the subjects of mutual relations between the components under these specific conditions. In other words, the previous experimental biology concerns only with the case of genes and proteins, and systematic biology will have studied all relationships among genes and all proteins (Zhao, Bai, & Li, 2010). Whole research is the biggest feature to use the systems biology approach to study inherent physiological changes in microbes, microbial interactions, and relationships with the external environment interactions between microbes. This method can explore the whole domain of microbial biosynthesis regulatory genes and provide a more comprehensive theoretical basis for species improvement, remodeling and microbial genome expression, and systems regulation. Traditional methods of strain transformation are random and time-consuming. As in-depth understanding of metabolic pathways and regulatory mechanisms, metabolic engineering achieves the metabolic network optimization by overexpression or knocking down the key metabolic pathway genes. However, it is difficult to achieve the desired results by multiple genes to determine a trait, particularly when there are not suitable target genes to operate for less knowledge about the control mechanism. Development of reverse metabolic engineering is used to analyze metabolic pathway of mutants with improved performance and find the key targets to improve the physiological performance of strain, in order to achieve the targeted reconstruction of a metabolic pathway. This technology also achieves some success, but is still insufficient. In recent years, cell genome modification and a genome transcription project have provided a new way for bacteria transformation. These directed evolution technologies used in microbial metabolic engineering enable us to regulate gene expression levels more finely, and change the transcriptional level of multiple intracellular genes simultaneously. However, it still involves the nondirectiveness of mutations and the screening of vast mutants. Moreover, due to the interaction and the regulatory mechanisms of metabolic networks inside the cell, some genes which do not relate to the metabolic pathways, the expression level, or expression products may also have a significant impact on the optimization of metabolic networks, while overexpression a single gene or a few genes may cause an imbalance of a metabolic network as a whole, thus affecting the success of metabolic engineering. To find the key genes affecting metabolic optimized pathways and to achieve the balance of the regulation of metabolic networks are important research goals in metabolic engineering operations. The development of systematic biology provides people with a comprehensive understanding of the cell from the genome-scale metabolic network view, including the structural genes, which are

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compositions of the metabolic pathway, the complex regulatory mechanisms of cell metabolism, as well as the influence of genetic and environmental perturbations on the overall metabolism of the cells. Systematic biology technology thereby establishes an omics-scale metabolic model, which could evaluate and predict the possible effects of genetic engineering operations and guide the operation methods of metabolic engineering through the metabolic network analysis. Finally, physiological function of cells could be improved, including the yield and efficiency of bioethanol, biobutanol, organic acids, amino acids, and other fermentation products, and obtaining the species processing flow that meets the need of industry. Typical cellular metabolic engineering strategies in cell refining include the following three steps: 1. Construction of the initial engineered strain. This stage is similar to the traditional metabolic engineering strategy previously mentioned: transforming local metabolic pathways by analyzing the structure of the local metabolic network (such as reducing by-products by knocking out the competition pathway), and optimizing the physiology performance of a cell (such as lifting product toxicity and feedback inhibition effect). The schematic routes of the strain transformation process guided by systematic biology are shown in Fig. 5.4. 2. Systems analysis at genome level and computer simulation of metabolic analysis. As mentioned previously, high-throughput omics analysis techniques can effectively identify the new target genes and pathways that improve the production capacity of cell fermentation. At the same time, by using the metabolic network model at the genomic level, some other new target genes could also be simulated and analyzed. It should be emphasized that the target genes identified by these two systems analysis methods are not related to the local metabolism pathway; the traditional metabolic analysis makes it hard to identify them.

FIGURE 5.4  Strain modification process by systems biology.

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3. Optimization of industrial fermentation processes. The first and second rounds of microbial fermentation are carried out under laboratory conditions, which have many differences in the fermentation performance compared to large-scale industrial fermentation. Scale-up is often associated with the generation of high concentration by-products, and therefore the next round of metabolic engineering to optimize the fermentation ability of bacteria is still necessary. In recent years, there has been great progress to utilize information acquired from systematic biology research to guide metabolic engineering operations and directional design of a new metabolic pathway for the biochemical conversion of biomass in refining cells. Succinic acid in the krebs cycle is a platform compound formed by the reduction of oxaloacetate. Hong et al. determined all of the 2,314,078 gene sequences of Mannheimia succiniciproducens survival in rumen with hypoxia and a carbon dioxide-rich environment. They not only defined the genetic map of the bacteria, but also elucidated the main metabolic pathways of bacteria adapted to survive in the rumen. The bacteria can produce large amounts of succinic acid accompanied by the generation of some other organic acids. In order to improve the production of succinic acid, based on the results of the complete genome sequence, a metabolic model containing 373 reactions and 352 metabolites was constructed. The metabolic flux analysis showed that the carbon dioxide and phosphoenolpyruvate carboxylation into oxaloacetate were equally important to cell growth. Based on the results, from the view of the genome, improvement strategies of M. succiniciproducens were proposed (Hong, Kim, & Lee, 2004). Saccharomyces cerevisiae is the most commonly used industry host for fuel ethanol production and also one of the microbial model organisms. Jens Nilson and his research group (Bro, Regenberg, & Forster, 2006) built a biological model system of S. cerevisiae in genome scale. They designed a metabolism engineering operating strategy to reduce the yield of glycerol and improve the ethanol yield. Further metabolic engineering study showed that overexpression of glyceraldehyde-3-phosphate dehydrogenase gene in S. cerevisiae decreased the glycerol yield by 40% and increased the ethanol yield by 3%. In the mixed sugar fermentation system of engineered strain with overexpression of the gene, the production rate of ethanol increased by 25% with substrate of glucose, and xylose.

5.2.4  Cell Processing and Synthetic Biology Synthetic biology is evolved from people’s knowledge and understanding of life over a vast length of time, which is based on deepening understanding of the structure and function information about the genes and proteins and other basic constituent elements of life. Synthetic biology research emerged in response to innovative thinking and technology with the material of natural biological elements and support platforms of molecular biology and genetic engineering and other modern biotechnology. In 2000, the first successful artificial manufacture,

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a circuit similar gene regulatory networks, marked the birth of synthetic biology (Gardner, Cantor, & Collins, 2000; Elowitz & Leibler, 2000). But the real access of synthetic biology to the public was from the research reports of US biologist Craig Venter (Gibson, Glass, & Lartigue, 2010). A mycoplasma genome of 1080 kb was synthesized in the laboratory and then implanted into the goat mycoplasma cell without genetic material, creating the first “artificial single-cell organisms,” which declared that the birth of the first gene does not rely on natural gene template and the synthetic bacteria that had the ability to self-replicate. Genetic engineering technology is the core of synthetic biology. Based on the techniques of molecular biology, genomics, bioinformatics, and systems biology, synthetic biology designs, transforms, and reconstructs biological molecules, biological components, bioreactor systems, metabolic pathways, and processes, as well as cells with vitality, individuals, and other biological systems, and provides a biological way of manufacturing low-cost biological drugs, chemicals, functional materials, or energy alternatives (Wang, Peng, & Hu, 2011). Synthetic biology is different to traditional biology, which studies the internal structure of organism by anatomy. Synthetic biology is the study from basic organism elements to the artificial life system. Synthetic biology and genetic engineering are different in the connotation. Genetic engineering is the continuation, transformation, and transfer of the genes from one species to another while the aim of synthetic biology is to assemble the various components to create artificial biological systems and make the components able to run in vivo as the circuit and complete a variety of biological functions (Liu, Du, & Zhao, 2011). The goal of synthetic biology research is very clear: the synthesis of new life forms, or transformation of existing life forms to achieve new functionality. To achieve these goals, scientists explored from different levels and aspects. Currently, synthetic biology research is focused on three aspects (Zhang, Chang, & Wang, 2010): (1) standardization of biological components and the design and construction of biological modules; (2) minimal genome research; and (3) design, synthesis, and assembly of the genome. 1. Standardization of biological modules and design and construction of biological components The standardization of biological modules is to design and construct biological components (including promoters, terminators, protein encoding DNA sequences, etc.) according to certain standards or specifications, and to describe in detail about the biological components. When “parts” of synthetic biology—the parameters of biological components—are determined and standardized, the design and building of biological modules will become easy and reliable. The interaction relationship between inhibition and promoter of cell exhibits similar characteristics with switches and oscillators in the circuit system. Based on such characteristics, the design and assembly

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of the standardization biological element in different levels can generate similar systems of circuit. Such biological systems, the controllability and predictability of output can be improved to some extent. Biological module construction includes design and construction of new biological components, devices, or systems, and also includes redesign and transformation of existing biological systems in nature. Design or synthesis of some natural and nonnatural substances is also considered as one of the contents of synthetic biology, such as artificial synthesis of ribosomes. 2. Minimal genome research A suitable carrier cell is required for the expression of new biological modules. Ideal carrier cells should have a streamlined genomic structure to reduce the complexity and improve the controllability and maneuverability of the designed system. The minimal genome is the minimum number of genes necessary for maintaining the growth and reproduction of the cells under the optimum conditions. Therefore, the core of minimal genome research is to determine the essential genes. According to the necessity of genetic information, we can purposefully streamline the existing genome and remove nonessential genomic fragments. In addition, we can also redesign, synthesize, and assemble essential genes. These two methods are now recognized as two strategies to achieve the smallest genome construction. Of course, this “minimum” is a relative concept. In addition, there is no universal minimal genome that can be used for various biological applications. With the development of DNA sequencing technology, more and more microbial genomes have been sequenced, which provides an important foundation for genomes minimization. 3. Design, synthesis, and assembly of genome Synthetic biology research, whether construction of biological modules or minimal genome research, is the operation process of the genome sequencing. To achieve “engineered life,” the genome manipulation techniques, particularly genome sequencing technology and DNA synthesis technology, are essential. Genome sequencing technology allows us to “read” life’s “bible” and helps us understand the complex living systems. The establishment and improvement of the technical system is an important prerequisite for the development of synthetic biology. In summary, the different synthetic biology researches interact with each other: the design and construction of biological components and standardization of biological modules allow us to realize purposeful design and transformation of life forms on the basis of an in-depth understanding of the complex life system; minimal functional genomics research provides the ideal expression vector for new designed biological modules; synthetic genome technology provides a solid technical support for previous two implementations. The mutual reinforcement of the three researches ultimately achieves new life forms of specific function and practical value.

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Currently, industrial applications of synthetic biology have showed good prospects for development in the pharmaceutical, bioenergy, chemicals, and agriculture. The application of synthetic biology is an extension of the “cell factory” concept. Synthetic biology can address issues from energy, materials, environmental protection, and other social problems by the approach of artificial cell construction. According to the synthetic biology principles, Zhang, Evans, and Mielenz (2007b) constructed a new catalytic system with 13 known enzymes, which use starch and water to produce hydrogen under mild conditions. The hydrogen is then used for electricity production by fuel cells, which realized the application of biohydrogen in the automotive fields. Keasling and coworkers transformed E. Coli by the methods of synthetic biology. The transformed E. Coli could produce complex biofuels (such as fatty esters, fatty alcohols, and waxes) by monosaccharide fermentation. Moreover, the transformed organism also had the ability to secrete hemicellulase to turn the hemicellulose into biofuels (Steen, Kang, & Bokinsky, 2010). Liao et al. reconstructed the isobutanol production pathway in E. coli and synthesize branched-chain higher alcohol biofuels using glucose (Atsumi, Hanai, & Liao, 2008). In fact, the expression and regulation of genes and the complex metabolic networks inside the cells are as fine as spider webs and a small change will affect the whole body. The complexity of the expression and function of cellular gene and metabolism networks are much higher than the circuit board. Thus, even in today’s highly developed life sciences, the transplantation of streamlined minimal genome into mycoplasma without genetic materials is not successful. There is still a long way to go for synthetic biology.

5.3  TOTIPOTENT CELL FUNCTION: ARTIFICIAL CELL The biochemical conversion of biomass refining cell platform totipotent cell is to convert the substrate into a specific product with functional totipotent. The essence of the process is enzymatic catalysis in biological systems. Microbial cells have been rapidly applied to industry production for its diversity and simplicity. However, there are still some problems, for example: the permeability of the substrate membrane, which affects the final conversion rate; the side reactions leading to the degradation of substrate or products by passing reaction and the accumulation of byproducts. These problems, to some extent, limit the application of microbial whole cell transformation in industry. From a molecular perspective, genes and proteins are essential to perform the original cell functions, and proteins are the carrier of cell function. Therefore, to build any type of cell, the proteins must be concentrated on. The enzymes are the executor of cell refining activities. Biomolecules, such as enzymes, are parceled or fixed in a semipermeable membrane that forms the artificial cells.

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Enzymatic transformation and microbial transformation are important biological pathways in which energy, chemicals, and materials were produced with renewable biomass resources. Compared with the microbial processes, enzyme catalytic processes have advantages of clear transformation pathways, less byproducts, simple operation, high yield, and easy process integration and optimization. By extracting enzymes involved in the desired product from microbial for the catalytic reaction, the production of byproducts can be limited, and the conversion efficiency of the objective product may be improved. Enzymatic processes have been widely used in industry, but there are still obstacles limiting broader enzyme use (Ma, Wang, & Su, 2009): (1) it is usually difficult to maintain the long-term stability of the enzymes in vivo because of the unsuitable extracellular environment, and it is easy to lose activity especially in the oil phase or oil–water interface; (2) the reaction rate is slower than that of chemical reaction; (3) since the enzyme requires extraction and purification and the repeat utilization is low, the cost for enzyme is high and even more so when the coenzymes are required in the system; and (4) the industrial applications of enzymatic process has been mostly confined to a single enzyme-catalyzed system, and multienzyme systems are still difficult, which limits the development of more high-value-added products. In order to realize large-scale industrial applications of enzymes, the goal of efficient utilization of enzyme must be achieved. This requires not only the highest activity of a single enzyme play in each system (water phase, oil phase, the oil–water interface, etc.) but also the reuse of the enzyme and the coordination of multienzyme reactions and the coenzyme regeneration.

5.3.1  Multienzyme Reaction System Most of the existing artificial biotransformations have been completed by a single enzyme and single cell, leading to the deficient efficiency. In nature, the biological energy conversion utilization is completed by multienzymes or cell systems, which is common in nature. Organism metabolism and biological symbiosis in nature are maintained by a variety of multienzyme systems. It is an efficient and accurate system, maintaining a high level of equilibrium between a series of enzymatic reactions. A microorganism multienzyme system in the catalytic process exhibits high selectivity, high efficiency, and a high degree of coordination, which provides a reference for establishing a multienzyme reaction system and optimizes the transformation of biocatalytic processes. The multienzyme reaction system has been used in a multistep completion of enzyme-catalyzed biochemical reactions in order to improve the overall yield of the reaction, shorten the reaction time, and reduce raw material and energy consumption. At present, there are many reports about the use of multienzyme systems in biological synthesis of products, such as amino acids, organic acids, optically active alcohol ketones, nucleic acids, steroids, covering the fields of medicine, food, environmental protection. However, in domestic situations, it is only

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applied in starch, protein hydrolysis, and ethanol fermentation. Therefore, the multienzyme coupling reaction system has good development potential. In biotransformation, according to the different sources of the enzyme, the multienzyme system can be divided into two types: the self-coupling reaction system and the interspecies coupling reaction system (Xie, Zhou, & Gao, 2004).

5.3.1.1  A Self-coupling Reaction System The self-coupling reaction system refers to achieving the coupling of an enzyme reaction with the cooperation of multiple enzymes in microorganism. This system is to obtain a single strain which has high multiple enzyme activities by screening manually, or to obtain the high activity strain by recombinant expression of different enzyme genes derived from different source in the same strain, so as to achieve the coupling of the intracellular response and the efficient conversion of the target product. Only one kind of microorganism was used in the self-coupling reaction system with the demand of a variety of enzymes with high activity in the microorganism. Moreover, the overall increase of the activity of multiple enzymes is necessary for improvement of the efficiency of the entire system. Although the coexpression of different enzymes can be achieved in the same strain by gene recombination technology, it is quite hard to transform due to the conservative nature of the genetic mechanisms of microbial cells. Thus the application of the self-coupling multienzyme reaction system has been limited. 5.3.1.2  Interspecies Coupling Reaction System The interspecies coupling reaction system refers to the fact that the enzymes required for the reaction exist in various microorganisms, which constructs coupling reactions among heterogeneous microorganisms. The interspecific coupling reaction systems can be divided into two kinds: cofactor regeneration coupling system and substrate coupling reaction system. The cofactor regeneration coupling reaction is only completed with the participation of cofactor ATP and NAD(P)H and performed through the coupling reaction of cofactor regeneration. In the substrate coupling multienzyme reaction system, it could be started from the compounds with extensive sources and low cost, and a synthetic intermediate is obtained by enzyme catalysis. Then the intermediates are catalyzed by another enzyme to obtain the desired product. The system can reduce the separation and extraction steps of intermediates, shorten the reaction process, and then greatly reduce the separation costs and environmental pollution. The interspecies coupling reaction system compared with the self-coupling multienzyme reaction system has the following advantages. First, nicotinamide is the key coenzyme of electron transfer process in biological metabolism in the construction of the cofactor regeneration system and the metabolism and reproduction is precisely regulated by the microorganism itself. In a single cell, the nicotinamide acts on each reactions with a fixed ratio and it is hard to ensure

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recycling by using single cell self-coupling multienzyme systems. As for the interspecies coupling multienzyme reaction system, there is no correlation between growth and metabolism of different microorganisms. It is possible to make a successful coupling reaction among the multienzymes by regulation. The successful application of nicotinamide cofactor regeneration system realizes the large-scale preparation of chiral alcohol. Second, the enzyme required for the reactions are from different microorganisms, the choice of microorganisms required for building multienzyme coupling system has great diversity and flexibility. Meanwhile, the interspecies coupling reaction system can increase the activity of specific enzyme of different microorganisms by gene recombination technology and lower technical difficulty. The current research about multienzyme coupling reaction systems focuses on interspecies coupling reaction system for its better development potential. Learning from a highly coordinated multienzyme reaction system of microorganisms in nature, the multienzyme system will tend to a highly coordination of various enzymatic reactions by using genetic engineering technology and directed enzyme evolution technology, which will also promote the industrial process of the multienzyme system’s application in amino acids, organic acids, and pharmaceutical intermediates and other fine chemicals.

5.3.2  Carrier Immobilization Cell immobilization is developed on the basis of immobilized enzyme technology. The free cells are fixed on the defined spatial region (a suitable insoluble carrier) by physical or chemical methods, remaining activity of enzymes and can be used repeatedly. In 1959, Hattori and Furusaka used resin to adsorb E. Coli to realize the immobilization of cells for the first time, which showed broad development prospects in the fields of energy, environment, food, medicine, and chemistry. Cell immobilization is gradually applied in biochemical biomass conversion. Shen et al. coimmobilized Aspergillus niger spores with rich cellobiase and Lactobacillus delbrueckii in calcium alginate gel beads, coupling fixed-cell systems and enzymatic hydrolysis system of cellulosic feedstock. With this new bioreactor for lactic acid fermentation, the results of repeated batch reactions assay showed that synergistic coimmobilized cells have sustained, stable, and efficient lactic acid production capacityand can be used repeatedly (Shen & Xia, 2008).

5.3.2.1  Conventional Immobilization Methods Currently microbial immobilization methods are mainly adsorption, embedding, cross-linking, and covalent binding methods (Wang, Huang, & Luo, 2007). The adsorption method uses charge between the electrostatic microbial cells and carriers to immobilize microorganism cells. The method is simple; reaction conditions are mild; the process of fixing microbial cells has little influence on its activity. However, the combination is not firm, cell is easy to shed, and the number of fixed cells is limited by the type of carrier and its surface area.

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The embedding method is to embed the microbial cells in a water-insoluble polymer gel pore network space formed by polymerization, precipitation, or changing the solvent, temperature, or pH. A gel polymer network can prevent the leakage of the cell, allowing diffusion of the substrate and the product. This immobilization method has the advantages of easy operation, is simple to operate, fine multienzyme systems maintaining performance, and less impact on the activity of microbial cells, which makes it one of the most widely studied methods for immobilized microorganism. In the cross-linking immobilization method, the microorganism cells react with two or more polyfunctional reagent to form a covalent bond. The operation conditions are intense and have a great influence on the activity of microbial cells. In the covalent bonding methods, the functional groups (such as amino, carboxyl, mercapto, hydroxy, imidazolyl, and phenolic groups, etc.) on the surface of microorganisms form chemical bonds with carrier chemical groups. The linkages between microbial cells and the carrier are very strong; however, the reaction conditions are intense and hard to control.

5.3.2.2  Choice of Carrier There are multiple characters that make ideal microbial immobilization carriers. First, long life, high mechanical strength, high capacity, low price. Second, not easy to be biodegraded; simple immobilization process, easy to shape at room temperature, nontoxic to microorganisms during immobilization process and after immobilization. Third, good biochemical and thermodynamic stability; good matrix permeability; good precipitation separation; no interference in the function of biological molecules. The key of cell immobilization technology is the performance of the immobilization carrier material. Immobilization carrier materials currently used are mainly organic polymer carriers, inorganic carriers, and composite carriers (Qu & Yue, 2007). 5.3.2.2.1  Organic Polymer Carrier Organic polymer carriers include natural polymer carriers and synthetic organic polymer carriers. 1. Natural polymer carriers are generally nontoxic to microorganisms, have good mass transfer performance, but low strength and can easily be broken down by microorganisms in anaerobic conditions. Common natural polymer carriers are agar, gelatin, carrageenan, sponges, chitin, alginate, and chitosan. The agar-embeding cell method is simple and nontoxic to cells, with a larger clearance, allowing the diffusion of high molecular substances, but the mechanical strength and chemical stability is not good. Alginate is a widely used immobilization carrier. It has good chemical stability, nontoxic, high efficiency, and is suitable for fixing living cells or sensitive cells, but it cannot resist phosphate and Na+, K+, Mg2+, and other cations with high concentrations, and is broken easily and dissolved. The

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gel strength is not enough and it is hard to repeat the use of immobilized cells. 2. Synthetic organic polymer gel carriers show good resistance to antimicrobial decomposition, high mechanical strength, and good chemical stability. However, the mass transfer performance is poor and the cell activity will reduce in the embedding process. Common synthetic organic polymer gel carriers are polyacrylamides, light-curing resins, polyvinyl alcohol, and polyacrylic acid gel. Polyacrylamide (ACAM) gel has good mechanical, chemical, and thermal stability. Due to the toxicity of acrylamide monomer to cells and heat release of cross-linking process, cells often inactivated during immobilization process. Thus, there are few studies about the use of ACAM as embedding agents. However, the cells can overcome this disadvantage by first being embedded with agar and then ACAM, which is called secondary fixation. Polyvinyl alcohol (PVA) has high gel strength, good chemical stability, and strong antimicrobial decomposition performance. Compared with the ACAM gel, its biological toxicity is very small and causes little harm to the activity of cells. It is the most widely studied immobilization carrier. 5.3.2.2.2  Inorganic Carrier and Composite Carrier The inorganic carriers, such as porous ceramic beads, crushed red brick, sand, porous glass, kaolin, diatomaceous earth, activated carbon, and alumina, usually have a porous structure, and fix microorganisms or cells with effect of adsorption and charge. Those carriers have advantages of high mechanical strength, nontoxicity to cells, and high resistance to biodegradation. Composite carriers are the combination of organic carrier materials and inorganic carrier materials, and have the advantage of performance complementarity. 5.3.2.2.3  Microcapsule Immobilization Among most immobilization systems, microcapsule immobilization has taken much attention (Ma, Lin, & Yao, 2010). A suitable liquid environment is wrapped by a layer of microcapsule membrane. Various cells grow coordinately in the internal to achieve a variety of biochemical reactions. The outer membrane has the functions of isolation, protection, and mass transfer. Thus, the microcapsule system is like a virtual “cell factory,” absorbing nutrients from the outside, synthesizing specific products by the internal complex metabolic reactions, and finally discharging the products outside the membrane. The cellulose sulfate sodium/ploy-dimethyl-dially-ammonium-chloride microcapsule system has good biocompatibility, simple preparation, good physical and chemical properties stability, and good mechanical strength. By using this system to study immobilization and culture of varied cells with the products including ethanol, lactic acid, glutamic acid, 1,3-propanediol, and thrombolytic enzymes, it was found that the system was especially suitable for anaerobic culture.

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5.4  INTERCELLULAR SYNERGY Microbes are the core of industrial biotechnology. Fermentation requires microorganism strains with good performance. Early research on microbial fermentation transformation was screening of natural high-yield strains, followed by obtaining a mutant strain with increased fermentation capacity by chemical mutagenesis and high-throughput screening technologies. The great limitations of these traditional strain breeding technologies was that the microbial fermentation products of mutant species are very limited, which are ethanol, acetone, butanol, glycerol, organic acids, amino acids and antibiotics, and other metabolites. Therefore, researchers began to pay attention to the mixed culture of transformation process. In the platform of a refining cell, two or more microbial cells play a role of synchronization for competitive advantage, and the effect is much better than that of individual microorganism cells. The specific phenomenon is called synergistic effect between the microbial cells, which is the mixed culture of microorganisms. In long-term experiments and production practice, people gradually find that many important biochemical processes must be completed by two or more cocultured microbes. Microbial fermentation is widely used in the production practice, which can replace the production of single fermentation in many cases. Some new products are possibly produced during the mixed culture process because the mixed microbial fermentation is a biological hybrid system and the microorganisms in the system have a coordinating role on the growth and metabolism. Single strain fermentation has low utilization efficiency of raw materials, equipment, and energy while multistrain mixed fermentation can overcome these shortcomings. However, study on mixed fermentation and optimization of culture conditions is limited.

5.4.1  Characteristics of Mixed Fermentation 1. To obtain certain products those are not available for single fermentation Pure fermentation accounts for a large proportion of the modern fermentation industry, but in long-term production practice, it is found that some products can only be the product of multistrains mixed fermentation. The two-step fermentation process has been widely used in the production of vitamin C and the second step, from l-sorbose to keto-l-gulonic acid, is a mixed fermentation process of two bacteria. One bacterium is Gluconobacter oxydans and the other bacterium is Bacillus megaterium. The traits of the two bacteria are different, and their role is also not the same. Either bacteria cultured alone is not enough to produce cologne acids, or only two bacteria mixed fermentation can successfully complete the conversion process of cologne acids. 2. To promote the product yield Mixed fermentation uses symbiotic effects or nutritional interactions between two or more bacteria, and overcomes the cumulative adverse effects caused by accumulation of intermediate. After combinations of different strains,

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the activity of enzymes is greatly increased. This is because the mixed fermentation takes the advantage of effects from different strains, improves the enzyme production capacity, and increases the yield of products. Cellulase production by mixed fermentation proves to be an effective way to utilize natural cellulose resources. Fungi are used as cellulase production strains in industry and the produced cellulase is generally secreted into the medium. It could be easy to obtain cell-free enzyme products by filtration and centrifugation. Taking cellulose production by fungal mixed fermentation as an example, the mechanism of mixed fermentation is summarized (Zheng, 2011) as follows: a. Weakening feedback inhibition of enzymes by mixed fermentation During the enzymatic hydrolysis of cellulose for the production of glucose, glucose and cellobiose have strong feedback inhibition on cellulase, which affects the rate and extent of hydrolysis of cellulose. If the bacteria that can break down the cellulose and the bacteria which can utilize glucose and cellobiose are mixed together during one fermentation process, the feedback inhibition from glucose and cellobiose is greatly diminished. So, for the decomposition of cellulose, mixed fermentation is faster and more thorough than single pure culture fermentation. Si-Mei et al. studied the effects of Candida sp. on the activity of cellulase and amylase during solid-state fermentation of Aspergillus niger and Aaspergillus fumigatus. The results showed that the inoculation of small amounts of Candida sp. can greatly improve the activity of cellulase and amylase because yeast use cellobiose and other small molecule sugars formed by hydrolysis, reducing the inhibition of cellulase and amylase synthesis by cellobiose, and thereby increasing the activity of fermentation products (Si-Mei, Xue, & Cai, 2002). b. Enhancing total enzyme activity by enzymes complementary In the study of fungal cellulase fermentation process, it is found that although Trichoderma sp. is recognized as the best cellulase-producing bacteria, there are still disadvantages in two aspects, one of which is the suspected toxicity and the other is the low activity of β-glucosidase. Many Aspergillus sp., such as Aspergillus niger, can produce β-glucosidase with high activity and are recognized as strong and safe strains for cellulase production. The studies of mixed culture of Trichoderma sp. And Aspergillus sp. indicate not only that β-glucosidase activity increased but also that the endo-cellulase and cellobiohydrolase activity are improved. c. Mutualism Cellulase-producing strains mostly include Trichoderma koningii, Trichoderma viride, Aspergillus fumigatus, and Aspergillus niger; lignindecomposing bacteria include Sporotrichum sp. and white-rot bacteria; protein enhancing bacteria are usually yeast, such as Candida utilis, Candida tropicalis, and S. cerevisiae. The mutualism of strains puts

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the enzymes in a coordinating ratio and greatly improves their activity. Pu et al. cocultured nitrogen-fixing bacteria (Azotobacter sp.) and cellulose decomposing bacteria (Trichoderma sp.) and observed that the growth of the two strains and nitrogen fixation were higher than these of a single culture. The reason is that nitrogen-fixing bacteria can not only fix atmospheric nitrogen but also produce different vitamins and auxin, which can stimulate crop growth and development and strengthen other rhizosphere microbial life activity (Pu, Zhong, & Zheng, 2000). About half of the organic matter in soil is cellulose; cellulose-decomposing bacteria can break down these substances into simpler substances which can be used as a carbon source by nitrogen-fixing bacteria. d. Increasing yield Tu et al. studied the effects of two mixed Aspergillus strains on the activity of three cellulase components. The results showed that the two fungi were inoculated by a certain percentage, the activity of three cellulase components would greatly improve than single fermentation. The activity of FPase, microcrystalline cellulase, and carboxymethyl cellulase increased by 2.2∼51.1%, 20.7∼332.6%, and 29.4∼29.6%, respectively (Tu, Xue, & Si-Mei, 2004).

5.4.2  Precautions of Mixed Fermentation Compared with single microorganism fermentation, mixed fermentation can take advantage of microbial diversity to achieve synergy among bacteria and improve the conversion rate and yield as much as possible. However, the following details must be carefully considered (Song, Zhou, & Chen, 2008; Zhang & Hou, 2010).

5.4.2.1  Strain Combination The combination of mixed fermentation bacteria usually includes cellulosedecomposing bacteria, lignin-decomposing bacteria, and protein-enhancing bacteria. Cellulase-producing strains mostly include Trichoderma koningii, Trichoderma viride, Aspergillus fumigatus, and Aspergillus niger; lignindecomposing bacteria include Sporotrichum sp. and white-rot bacteria; proteinenhancing bacteria are usually yeast, such as Candida utilis, Candida tropicalis, and S. cerevisiae. It is necessary to pay attention to the compatibility of different strains and try to take advantages of microbial strains with similar habits during strain combination. 5.4.2.2  Different Fermentation Conditions Fermentation temperature, pH, time, moisture, and other factors and their interactions have a significant influence on fermentation. Temperature is the primary factor for solid-state fermentation. The starting medium pH and moisture content must first be adjusted according to the characteristics of the strains used and

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raw materials; then the temperature suitable for the growth and reproduction of microorganisms should be set; finally the best culture time for higher yield of aim products should be determined.

5.4.2.3  Pretreatment of Fermentation Feedstock Since the feedstock for general mixed fermentation is lignocellulosic biomass resources, the pretreatment of feedstock is necessary to separate cellulose, hemicellulose, and lignin for efficient enzymatic reactions. The pretreatments usually used include cooking, acid/alkali treatment, organic solvent-treatment, steam explosion, and the wet oxidation methods. In addition, the nutritional content of the lignocellulosic feedstock does not fully meet the needs of microbial growth and reproduction, so we have to add the appropriate carbon source (such as bran) and nitrogen (such as urea) in order to increase enzyme activity and protein content of the product. From the point of the industrialization, the conditions for mixed fermentation are simpler and easier to operate compared with pure strain fermentation. However, the mixed fermentation system is more complex than pure strain fermentation and the existing studies should be more thorough and extensive.

5.5  CONSTRUCTION OF A CELL REFINING FACTORY Cell refining makes use of complex microbial metabolic networks and regulatory networks, decomposes biomass, and synthesizes a range of energy products to replace fossil and chemical products. A microbial cell factory is the core technique of cell refining. The so-called “factory” is the place that can produce or manufacture a product. Therefore, the “factory” in a general sense should have specific production lines, and the corresponding power and other auxiliary systems, and is normally operated in certain management programs. The elements of a “factory” are designed based on what people want. The production line and auxiliary system are designed based on needs and regulated by the production schedule. “Cell factories” also have corresponding components (Zhang, Li, & Ma, 2007a). The so-called cell plants aim to discover the nature of genes, proteins, and metabolic networks, and reallocate material flow and energy flow systems of microbial cell metabolism by artificial restructuring and optimization. Microbes are the most widely distributed species on earth, and their metabolic diversity is determined by species diversity and genetic diversity. Microbes play a critical role in the earth’s irreplaceable material cycle and have formed varied mechanisms for nutrition taking in long-term evolution. Microorganisms degrade the biomass by secreted enzymes and convert biomass to bioenergy (biohydrogen, bioethanol, biodiesel, etc.), important chemical raw materials (such as ethylene, 1,3-propylene glycol, etc.), polymers, and other products. Of course, the compositions of biomass materials are abundant, including a variety of substances with hydroxyl, carbonyl, benzene, and other oxygen-containing

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groups. Compared with petroleum feedstock containing —(CH2)n— linear polymeric structure, biomass can provide more opportunity for development of new products, which are more conducive to the produce of various chemicals by chemical transformation (Chen & Wang, 2008), shown in Fig. 5.5: The production capacity of a biological cell factory is the most important factor to determine its ability to compete with petrochemical manufacturing. An efficient cell factory must have the following six important characteristics: 1. The cell should have a clear physiological genetic background and good genetic stability, as well as being easy to genetically transformation. Therefore, the construction of cell plants is mostly based on model microorganisms (such as E. coli) whose genetic background is relatively clear. 2. Fast growth, strong anabolic ability, high production yield, high production rate, and yield. Thus, it is possible to reduce the costs of biological manufacture. 3. Simple fermentation process, reducing the equipment cost, and operating costs. 4. Utilization of simple inorganic salt medium and inexpensive carbon sources to thereby reduce production costs. 5. Using cellulose as raw material. 6. Superior physiological performance, tolerance to high temperature, high osmotic pressure, high product concentration, and low pH.

5.5.1  Modification of Microbial Cell Properties The biorefining is parallel to petroleum refining, converting biomass to fuel, materials, or compounds platforms other types of chemical with the help of the natural ability of the microorganism and recombinant microorganism cells and a series of biochemical pathways (similar to unit operation, such as petroleum refinery, cracking, hydrocracking, reforming). It needs to modify the characteristics of microbial cell to meet industrial production needs.

5.5.1.1  Optimization of Strain Tolerance and Reduction of Metabolic By-products Synthesis The microbial fermentation production process inevitably suffers some inhibition of adverse environmental factors on product synthesis. The resistance of cells to an adverse environment is a very complex phenotype. By transcriptome comparison of strains grown in different environments, genes that are closely related to the phenotype could be found, allowing researchers to better enhance the strain tolerance to adverse environmental by metabolic engineering. Hirasawa et al. (Hirasawa, Yoshikawa, & Nakakura, 2007) compared the transcriptome differences in two strains of S. cerevisiae which had different resistances to ethanol. By using cluster analysis, they found that the expression level of tryptophan biosynthesis genes has a close relationship with ethanol tolerance. Overexpression of tryptophan biosynthesis genes could make

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FIGURE 5.5  Products from cell refining factory.

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strains with low tolerance to ethanol have 5% (V/V) ethanol tolerance ability and exogenous tryptophan addition also enhanced expression of tryptophan permease and increased ethanol tolerance. Hirasawa et al. (Hirasawa, Nakakura, & Yoshikawa, 2006) also analyzed transcription of two S. cerevisiae strains with different osmotic tolerance under high salt conditions and found that the sodium ion pump and copper metallothionein gene were closely related to cell osmotic resistance. Increasing the expression level of these genes can significantly improve osmotic resistance of strains. Glycerin is a by-product when S. cerevisiae was employed for ethanol production by anaerobic fermentation. Under anaerobic conditions, the formation of glycerin converted NADH into NAD+. The glycerol-3-phosphate dehydrogenase encoded by the gene GDP2 and GDP1 converted dihydroxyacetone phosphate into glycerol-3-phosphate. The breaking of GDP2 gene reduced the production of glycerol but slowed down the cell growth rate. In addition, strains with GDP1 and GDP2 gene double deletion cannot grow under anaerobic conditions. Thus, Nissen et al. constructed a new way to produce NAD+ in Azotobactervinelandii with GDP1 and GDP2 double gene deletion, thereby reducing the formation of glycerol by 40% (Nissen, Kielland-Brandt, & Nielsen, 2000).

5.5.1.2  Expanding Range of Substrate Utilization Expanding the range of substrates utilization has significant importance for the production of biobased chemicals by biomass, phenotype of fast utilization (especially under anaerobic conditions) of xylose, galactose, and other substrates in some microorganisms are related to the complex changes in gene expression. Bro, Knudsen, and Regenberg (2005) analyzed S. cerevisiae strains with different galactose uptake rate by transcriptome and identified PGM2 genes encoding glucose phosphate mutase, which were taken as new targets. By enhancing the expression of the gene, the galactose uptake rate of engineered bacteria was increased by 70%. The study also showed that transcriptome analysis of strains with different substrate consumption rates would greatly improve the efficiency of obtaining useful information. Bengtsson, Jeppsson, and Sonderegger (2008) analyzed the transcription differences between the normal group and four S. cerevisiae strains with different xylose-utilizing ability, and found that the expression of 13 genes in the four strains had changed. In the normal strains with corresponding overexpression or deletion of these different genes, it was found that there were five genes that could effectively improve the xylose utilization capacity of the strain. 5.5.1.3  Production of Heterologous Metabolites Eliasson, Christensson, and Wahlbom (2000) separated XYL1 and XYL2 genes from P. stipitis. Encoding of the two genes was dependent on NAD(P) H-dependent xylose reductase and NADH-dependent xylitol dehydrogenase. After being inserted into the S. cerevisiae, the endogenous XKS1 gene (encoding the xylulose kinase) was overexpressed, which allowed the S. cerevisiae strain TMB3001, growing under anaerobic fermentation and producing ethanol.

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Meanwhile, the overexpression resulted in a reduction of xylose utilization and the ATP/ADP ratio (Toivari, Aristidou, & Ruohonen, 2001).

5.5.2  Design of Cell Refining Factory In order to obtain efficient microbial cell factories, the three steps of design, build, and optimization are needed. After clarifying the product goals, the first question is how to design a highly efficient cell factory to produce the desired product. Most microbial fermentation products are a mixture of compounds, so it needs to be reformed on the basis of systematic research on microbial metabolic networks. The most effective way of systematic research on microbial metabolic networks is to construct metabolic network models. Determination of microbial genome sequences and the development of a gene function annotation tool allow us to construct a genomic-level metabolic network model, which greatly enhances our ability to analyze the metabolic network structure. So far, scientists have completed approximately 20 microbial metabolic network models. These models enable us to recognize the complexity of microbial metabolic networks in the systemic level, to predict the physiological properties of the cells and the genetic changes or the metabolic response of cells after environmental disturbances, and to simulate the genetically engineered target genes, and provide a good foundation for the cell plant design.

5.5.3  Construction of Cell Refining Establishment of the metabolic network model and computer simulation analysis provides the basis for the design of the cells’ factory. The specific implementations of cells construction will be completed by genetic modification. Common means of genetic manipulations include gene knockout, gene effective expression, gene integration, gene expression regulation (transcriptional level and posttranscriptional level); gene knockout and integration are two powerful tools widely used in building microbial cell factories. Knockout can be used in inactivation competition pathway, so that more metabolic flux flows into the target products. Integration of the gene can introduce a fragment of an exogenous gene into the cells’ factory; the foreign gene can assign the cells a new capacity, synthesis of new product, and enhance the resistance of cells to enable the production of products which could not be previously synthesized. Regulation of gene expression is another common genetic transformation strategy. By constructing a library of promoters with different expression levels, and controlling the posttranscriptional process, such as termination of transcription, mRNA degradation, and translation initiation, the intensity of gene expression can be regulated to control the distribution of metabolic flux and then more effectively to synthesize the target products. Li et al. achieved the conversion of nonmethylated plasmid in Clostridium acetobutylicum by inactivation of restriction modification systems. They further

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analyzed the existing deficiencies of the homologous recombination based on nonreplicating plasmid, homologous recombination based on replicating plasmid, antisense RNA technology, and type II intron gene inactivation technologies, and pointed out that improving the conversion efficiency is the key breakthrough of the genetically engineered operating system of Clostridium acetobutylicum. Finally, they provided suggestions about how to develop an efficient genetic manipulation system of C. acetobutylicum (Jia, Zhu, & Zhang, 2011). The microbial DNA transformation method is to import foreign genes and the current microbial transformations are mainly chemical and electrical transformation methods. These methods have shortcomings, such as the long time required for preparation of competent cells, cell activity loss during treatment, and incubation after treatment. Tan et al. (Tan, Wang, & Zhao, 2010) developed a sepiolitebased microbial DNA transformation method. Sepiolite is a mineral nanomaterial, which is cheap, abundant, and harmless. This transformation method does not require the preparation of competent and incubation after treatment, to obtain a higher conversion rate than the conversion of calcium transformation process. The genome mixed group is a new method for cell factories transformation. Genomes from different strain sources can be fully recombined by means of protoplast fusion cycle, which increases the opportunity to integrate the mutation into recombinants. Yang, Fan, and Xiao (2010) investigated the effect of genome mixed group changes during multiple rounds fusion process of Bacillus subtilis. By comparing the mixed result of Streptomyces coelicolor and Lactobacillus sp. genome groups, and combination with computer simulation of cycle fusion process, they demonstrated that the high frequency recombination technique of microbial cells is the basis on which to improve more sufficient genome mixed effects of Bacillus subtilis. Surface display is a valuable genetic manipulation technique, which allows the expression of exogenous peptides to show on the cell surface in the form of fusion protein. Spore surface display technology as a microbial surface display technology has attracted much attention from researchers because of its unique advantages, such as heterologous proteins expressed without transmembrane process and the resistance of spore. Xu, Wang, and Ma (2010) described the physiological structure and the formation of spores, construction principles of spore surface display system, and types of spore surface display system currently constructed. Bacillus subtilis surface display technology will get more attention in the field of biocatalysis and cell factories due to its safety feature. In addition, Bacillus subtilis also provides an important reference for the industrial production of high-value-added compounds.

5.5.4  Optimization of Cell Refining Factory By designing and constructing the plant factories, the primary cells’ factory can be obtained. However, its production capacity and physical properties have many shortcomings. A cells’ factory is an artificially designed microbial

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metabolism system. The introduction of multicell genes or an entire metabolic pathway may lead to some problems, such as metabolic imbalance, accumulation of intermediate metabolic products, and cannot achieve the requirements of industrial production. Thus, it is necessary to optimize the cell plants. Jiang, Li, and Ma (2010) described the coordination and optimization of multigene expression strategies from levels of transcription and translation. Components optimization by adding artificial scaffold protein is a new optimization technique of cell plants that has been recently developed. The technique can precisely control and optimize stoichiometric ratio of multiple enzymes in the metabolic pathway, balance the flow of each part in the pathway, shorten the space between each enzyme, and form the substrate channel, thereby greatly enhancing the efficiency of biosynthesis in the cell plant. Microbial synthesis of high-value-added fine chemicals is a relatively mature technology. These processes usually have fine produce condition and are in a small-scale production. Therefore, biosynthesis has certain advantages. The biorefinery process for bulk chemicals production is featured by large amounts of output and simple medium composition and operation process. These requirements reduce the nutritional needs of the industrial strains and improve their tolerance to substrates and products, enhancing industrial adaptability of cell factories. In recent years, the researches of extreme environmental microbes have laid a foundation for the industrial adaptability transformation of cell factories. However, to fully meet industry needs and realize industrial applications is still an important direction for future research on plant cells. In order to realize the applications of cell plants in the field of biorefinery, further studying and improving industrial adaptability of cell plants are always necessary, which could really develop cell plant manufacture into a high-yield, efficient, and economical modern industrial biotechnology industry. Among them, the further industrial development of biochemical transformation is inseparable from the core production unit-bioreactors. The reactions in the bioreactor occur at multiple scales, such as the genetic characteristics at a molecular level, the regulation of cell metabolism at a cell level, and mixing and transfer at a reactor level. Therefore, how to use the multiparameter detection technology of bioreactor and online computer control and data processing technology to associate phenotypic data with genetic structure relate to metabolic regulation, is not only important content of process optimizing and amplification of reactors but also the original intellectual property technologies. In summary, the development of efficient microbial cell plants can greatly enhance the production capacity and physiological performance of current industrial microorganisms, and reduce production costs and expand the products varieties of biomanufacturing. With the rapid development of new technologies, microbial cell plant techniques will also be more perfect and greatly promote the industrial upgrading of traditional petrochemical manufacturing industries, making tremendous contributions to the sustainable development of human society.

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Jiang, T., Li, L., Ma, C., et al. (2010). Strategies for regulating multiple genes in microbial cell factories. Chinese Journal of Biotechnology, 26, 1419–1425. Jin, S. Y., & Chen, H. Z. (2006). Superfine grinding of steam-exploded rice straw and its enzymatic hydrolysis. Biochemical Engineering Journal, 30, 225–230. Jin, S. Y., & Chen, H. Z. (2007). Fractionation of fibrous fraction from steam-exploded rice straw. Process Biochemistry, 42, 188–192. Kamm, B., Gruber, P. R., & Kamm, M. (2007). Biorefineries–industrial processes and products. Beijing: Chemical Industry Press. Li, Y. (2009). Metabolic engineering: an evolving technology for strain improvement. Chinese Journal of Biotechnology, 25, 1281–1284. Liu, D., Du, J., Zhao, G., et al. (2011). Application of synthetic biology in medicine and energy. CIESC Journal, 62, 2391–2397. Ma, G., Wang, P., & Su, Z. (2009). Nanoscience and enzyme. China Basic Science, 5, 49–54. Ma, Q., Lin, D., & Yao, S. (2010). Immobilization of mixed bacteria by microcapsulation for hydrogen production—a trial of pseudo “Cell Factory”. Chinese Journal of Biotechnology, 10, 1444–1450. Mori, T., Mizuta, S., Suenaga, H., et al. (2008). Metagenomic screening for bleomycin resistance genes. Applied and Environmental Microbiology, 74(21), 6803–6805. Nissen, T. L., Kielland-Brandt, M. C., Nielsen, J., et al. (2000). Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metabolic Engineering, 2, 69–77. Pu, Y., Zhong, Y., & Zheng, Z. (2000). The effect s of mixed culturing on the growth and nitrogen fixation of nitrogen-fixating bacteria and cellulolytic organism. Amino Acids & Biotic Resources, 22, 15–18. Qu, W., & Yue, X. (2007). Cell immobilization technique and its research progress. Shaanxi Journal of Agricultural Sciences, 6, 121–123. Shen, X., & Xia, L. (2008). Synergetic saccharification and lactic acid fermentation by coimmobilized cells using cellulosic materials. Journal of Chemical Industry and Engineering, 1, 167–172. Si-Mei, R., Xue, Q., & Cai, Y. (2002). The effect of mixed fermentation on cellulase and amylase activities. Journal of Northwest Sci-Tech University of Agriculture and Forestry, 30, 69–73. Song, P., Zhou, M., & Chen, W. (2008). A study on the fermentation process of microbial fertilizer with complex microbial community. Shanghai Environmental Science, 4, 156–161. Steen, E. J., Kang, Y., Bokinsky, G., et al. (2010). Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature, 463, 559–562. Tan, H., Wang, L., & Zhao, Z. (2010). Mechanism of DNA transformation based on mineral nanofibers and method improvement. Chinese Journal of Biotechnology, 26, 1379–1384. Toivari, M. H., Aristidou, A., Ruohonen, L., et al. (2001). Conversion of xylose to ethanol by recombinant Saccharomyces cerevisiae: importance of xylulokinase (XKS1) and oxygen availability. Metabolic Engineering, 3, 236–249. Tu, X., Xue, Q., Si-Mei, R., et al. (2004). Effects of mixed poly-fermentation on cellulase activity. Industrial Microbiology, 34, 30–34. Wang, C., Huang, B., & Luo, H. (2007). Immobilized microbe technology and its application. Yunnan Chemical Technology, 34, 79–82. Wang, J., An, P., & Liu, J. (2010). The new progress of metagenomics in the research of rumen microbial metabolization. Chinese Journal of Animal Nutrition, 3, 527–535. Wang, Z., Peng, J., Hu, Y., et al. (2011). Advance of synthetic biology in industrial application. Scientia Sinica Chimica, 41, 709–716.

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Chapter 6

Sugar Strategies for Biomass Biochemical Conversion Chapter Outline 6.1 Totipotency of Glucose 138 6.1.1 Catabolism of Glucose 138 6.1.2 Primary Metabolites and Secondary Metabolites 141 6.2 The Preparation of Xylose 143 6.3 The Preparation of Glucose 145 6.3.1 Pretreatment of Lignocellulosic Biomass 145 6.3.2 Hydrolysis of Cellulose 147 6.4 Preparation of Xylose 149 6.4.1 Industry Overview of Xylose Preparation 149 6.4.2 Industrial Process of Xylose Preparation 150 6.5 Pathway Mechanisms of Inhibitor and Solutions 152 6.5.1 The Inhibitor and its Mechanism 152 6.5.2 Detoxification Process 154

6.6 Economic Analysis of Sugar Platform Compounds in the Biochemical Conversion of Biomass 6.6.1 Sources and History of Sugar Platform Materials 6.6.2 Characteristics Analysis of Sugar Platform Compounds 6.6.3 Fermentability Analysis of Sugar Platform Compounds 6.6.4 Economic Analysis of Sugar Platform Compounds 6.6.5 Conclusions References

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The “sugar platform” is biomass with over 75% sugar making up its carbohydrate content, usable as a carbon source in industrial chemicals production (Chen, 2011). Sugars used in the fermentation industry platform are mainly from agricultural feed. Starch and starchy materials are always the raw material in the fermentation industry; and the glucose degraded from the starch is the most universal carbon source for microorganisms. Almost all common industrial strains of microorganism can utilize glucose as energy and as a carbon source to synthesize the cell skeleton and the extracellular products. In nature, the content of glucose in cellulose is much greater than in starch. Not only can nutrient levels be reduced but also agricultural and forestry waste can be fully utilized, when the glucose contained in cellulose can be used as raw Technologies for Biochemical Conversion of Biomass © 2017 Metallurgical Industry Press. Published by Elsevier Inc.

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FIGURE 6.1  Sugar platform of biomass and transformation

material for industrial production. Theoretically, as long as glucose can be produced from cellulose by pretreatment and hydrolysis (acid hydrolysis, enzymatic hydrolysis), a new sugar platform can be established for the fermentation industry, which has already brought profound changes in the fermentation process. Besides this, the lig­nocellulosic biomass contains high levels of pentose, which is also a potential “carbon pool” for the fermentation industry. Although not all microorganisms have the ability to take advantage of the pentose, utilization of pentose should be still paid much attention because of the widespread nature of the pentose phosphate pathway (PPP). As a part of the fu­ture conversion of biomass sugars, platform-building pentose-utilizing bacteria and the cofermentation of glucose and pentose represent the most important future direction for fermentation industry research (Fig. 6.1; NB xylose is a type of pentose).

6.1  TOTIPOTENCY OF GLUCOSE Glucose is the optimal carbon source for microbial fermentation. Most industrial microbes can use glucose to grow and reproduce. The microorganism can obtain reproducing power and acetyl coenzyme A and other substances to maintain metabolism and synthetic products by the decomposition of glucose. Therefore, glucose as a degraded form of lignocellulosic cellulose can be used as a platform to produce biofuel, such as ethanol (Chen, 2008, 2011).

6.1.1  Catabolism of Glucose The metabolism pathway of glucose in the microorganism can be divided into the Embden–Meverhef–Parnus pathway (EMP), the hexose monophosphate

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pathway (HMP), the Entner–Doudorof pathway (ED), and the phosphoketolase pathway (PK) (Wang, 2002).

6.1.1.1  Embden–Meverhef–Parnus pathway The EMP pathway is also known as the glycolytic pathway. In the EMP pathway, the glucose is transformed into 1,6-diphosphate, enzyme aldehyde. 1,6-diphosphate is decomposed into two 3C compounds by the fructose diphosphate, and then converted into two molecules of pyruvate. The EMP pathway process consists of the following 10 consecutive reaction steps. 1. glucose + ATP→glucose-6-phosphate + ADP 2. glucose-6-phosphate→6-phosphoric acid fructose 3. 6-phosphoric acid fructose + ATP→1,6-diphosphate fructose + ADP 4. 1,6-diphosphate fructose→dihydroxyacetone phosphate + glyceraldehyde 3-phosphate 5. dihydroxyacetonephosphate→glyceraldehyde 3-phosphate 6. glyceraldehyde 3-phosphate + NAD + H3PO4→1, 3-2 glyceric acid phosphate + NADH 7. 1,3-bisphosphoglyceric acid + ADP→glycerate 3-phosphate + ATP 8. glycerate 3-phosphate→glycerate 2-phosphate 9. glycerate 2-phosphate→phosphoenolpyruvic acid + H2O 10. phosphoenolpyruvic acid + ADP→pyruvic acid + ATP C6H12O6 + 2NAD + 2(ADP + Pi)→2CH3COCOOH + 2ATP + 2NADH2 In the EMP pathway, 6-phosphate glucose is transformed into pyruvic acid. It is the main metabolic pathway in many microorganisms. Microorganisms get energy (ATP), reducing power, and metabolic intermediates via the EMP pathway. Under anaerobic conditions the anaerobic microorganisms transform pyruvic acid into lactic acid, acetaldehyde, and alcohol by glycolysis. Under aerobic conditions, the aerobic microorganisms transform pyruvic acids into CO2 via Kreb’s cycle, or generation of citric acid, iso-citric acid, malic acid.

6.1.1.2  Tricarboxylic Acid Cycle CH3COSCoA + 2O2 + 12(ADP + Pi)→2CO2 + H2O + 12ATP + CoA Glucose can be transformed into CO2 and H2O by the EMP and TCA pathway 1. C6H12O6 + 2NAD + 2(ADP + Pi)→2CH3COCOOH +2ATP + 2NADH2 2NADH2 + O2 + 6(ADP + Pi)→2NAD + 2H2O + 6ATP 2. 2CH3COCOOH + 2NAD+ + 2CoA→2CH3COSCoA + 2CO2 + 2NADH2 2NADH2 + O2 + 6(ADP + Pi)→2NAD+ + 2H2O + 6ATP

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3. 2H3COSCoA + 4O2 + 24(ADP + Pi)→4CO2 + 2H2O + 24ATP + 2CoA C6H12O6 + 6O2 + 38(ADP + Pi)→6CO2 + 6H2O + 38ATP

6.1.1.3  HMP Pathway The HMP pathway is also known as the pentose phosphate cycle. In this approach, the glucose is transformed into glucose-6-phosphate acid by phosphorylation, and then the glucose is transformed into CO2 and pentose phosphate. The further metabolism of pentose phosphate is complex. Two phosphate hexose molecules and one phosphotriose are generated from the three pentose phosphate molecules catalyzed by transketolase, and triosephosphate is transformed into pyruvate via the EMP pathway. Complete HMP pathway: glucose-6-phosphate + 7H2O + 12NADP→6CO2 + 12NADPH2 + H3PO4 Incomplete HMP pathway: glucose-6-phosphate + 7H2O + 12NADP→CH3COCOOH + 3CO2  + 6NADPH2 + ATP The generated glyceraldehyde 3-phosphate can be transformed into pyruvic acid via the EMP pathway. A large number of C3, C4, C5, C6, and C7 precursors are produced via the HMP pathway for cell metabolism, such as synthesis of nucleic acid, coenzyme, histidine, aromatic amino acid, amino acid, and so on. The HMP and EMP pathways exist in nearly all the microbial cells. At present, the HMP pathway is the sole metabolism only in Acetobacter suboxydans.

6.1.1.4  ED Pathway The ED Pathway is also known as the 2-keto-3-deoxy-6-phosphate gluconate pathway. Glucose is transformed into glucose-6-phosphate, glucose-6-phosphate acid, 2-keto-3-deoxy-6-phosphate, gluconic acid, and then formed into pyruvate, glyceraldehyde 3-phosphate and 3-glyceraldehyde phosphate by the EMP pathway. C6H12O6 + ADP + Pi + NADP + NAD→2CH3COCOOH  + ATP + NADPH2 + NADH2 The ED pathway is an anaerobic metabolism pathway of sugar, and widely distributed in the Gram-negative bacteria, such as Pseudomonas saccharophila, Zymomonas mobilos, Pseudomonas aeruginasa, and so on. The ED pathway and HMP pathway are in microorganisms simultaneously, yet exist individually.

6.1.1.5  PK Pathway The PK pathway is always known as the ketone phosphate enzyme pathway. Where there are no EMP, HMP, and ED pathways, there is often PK. The ketone

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phosphate enzyme can be divided into the pentose ketone phosphate enzyme and the hexose ketone phosphate enzyme. Some heterofermentative lactic bacteria, such as Leuconostoc, Lactobacillus, Leuconostoc mesenteulides, Lactobacillus brevie, Lactobacillus manitopoeum, have the enzymatic activity of the hexose ketone phosphate enzyme yet without transketolase. Only one ATP is generated from the transformation, which is half of the EMP pathway. Approximately equal amounts of acetic acid, ethanol, and CO2 are generated during the process. C6H12O6 + ADP + Pi→CH3CHOHCOOH + CH3CH2OH + CO2 + ATP

6.1.2  Primary Metabolites and Secondary Metabolites 6.1.2.1  Primary Metabolites Glucose can be transformed into essential nutrition and energy by anabolic metabolism when it is absorbed into the microorganism to maintain life activities. The decomposition products and the polymeric products formed during the process, such as polysaccharides, proteins, nucleic acids, and esters, are called primary metabolites. The common primary metabolites are amino acids, nucleosides, and the enzyme or coenzyme. Primary metabolites are always useful biochemical products, for example: 1. Acetic acid (Wang & Jin, 2000) Acetobacter bacteria are a kind of microorganism that has the fermenting ability to produce acetic acid. Some of them are aerobic microorganisms, such as Acetobacter aceti, Acetobacter oxydans, and so on. Some of them are anaerobic microorganisms, such as Clostriolium themoacidophilus and Acetobacter xylinum. Ethanol is oxidized to form acetic acid by acetobacter bacteria under aerobic conditions CH3CH2OH + O2→CH3COOH + H2O

Acetic acid is obtained by chemical synthesis in industrial production, however, aerobic acetic acid fermentation is also inevitable. Vinegar production mainly depends on aerobic acetic acid fermentation. Ethanol is oxidized to form acetic acid by acetobacter bacteria under aerobic conditions. 2. Citric acid (Wang & Jin, 2000) Citric acid is widely used in citrate, flavors, beverages, and other manufacturing industry, and plays an important role in the food industry. Citric acid is the intermediate in the aerobic citric acid cycle. There are a lot of strains of bacteria that have the ability to produce citric acid. The most common citric-acid producing strains are Aspergillus niger and Aspergillus oryzae. 3. Ethanol Ethanol fermentation is the basis of the alcohol industry, and has a close relationship with alcohol drinks. Ethanol production from lignocellulosic

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biomass is also the “hot spot” of bioenergy, which is an important part of the solution for the energy crisis. The microorganisms most widely used for ethanol production are yeast and other bacteria, such as Zymononas mobilis and Pseudomonas saccharophila. Under aerobic conditions glucose is transformed into pyruvic acid via the EMP pathway in yeast, and then the pyruvic acid is transformed into ethanol and CO2 by the key bypyruvate decarboxylase. C6H12O6 + 2ADP + 2Pi→2CH3CH2OH + 2CO2 + 2ATP

4. Lactic acid (Wang & Jin, 2000; Zhang, 2007) Lactic acid is one of the most common products in the modern fermentation industry. The lactic acid fermentation process can be divided into homologous lactic acid fermentation and heterogenous lactic acid fermentation. Lactic acid is the only product of homologous lactic acid fermentation, but the products of heterogenous lactic acid fermentation are ethanol, acetic acid, and CO2, beside lactic acid. 1. Homologous lactic acid fermentation The microorganisms used in homologous lactic acid fermentation are called homologous lactic acid bacteria, such as Diplococcus, Streptococcus, Lactobacillus. Lactobacillus delhruckii and Lactobacillus bulgaricus are the common industrial fermentation strains. Hexose is the main carbon source in homologous lactic acid fermentation; the hexose is transformed into pyruvic acid in the EMP pathway, and then into lactic acid by lactic dehydrogenase. C6H12O6 + 2ADP + 2Pi→2CH3CHOHCOOH + 2ATP 2. Heterologous lactic acid fermentation Heterologous lactic acid fermentation is part of the PK pathway. Leuconostos mesentewides, Leuconostoc dextranicum, Lactabacillus brevi, and Lactobacillus lycopersici are the commonly used strains. Glucose is transformed into lactic acid, ethanol, and water in the pathway, and meanwhile one ATP is generated. C6H12O6 + ADP + Pi→CH3CHOHCOOH + CH3CH2OH + CO2 + ATP In Lactobacillus bifidus and Bifidobacterium bifidus, the glucose is transformed into two molecules of lactic acid, three molecules of acetic acid, and five molecules of ATP. 2C6H12O6 + 5ADP + 5Pi→2CH3CHOHCOOH + 3CH3COOH + 5ATP Lactic acid fermentation is widely used in the production of traditional foods, such as pickles, sauerkraut, yogurt, cheese, for which the main organic acid is lactic acid. Apart from its use in the food industry, lactic acid

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is also used to synthesize biodegradable plastics, which can replace fossil resources.

6.1.2.2  Secondary Metabolites Secondary metabolism is the process whereby microorganisms use primary metabolites as a precursor to the synthesis of secondary metabolites in certain growth periods of microorganisms. The secondary metabolites are not necessary and have no apparent physiological function (Berdy, 2005). Secondary metabolites in small quantities have no significant effect on microbial life processes. Results showed that blocking secondary metabolic pathways has no effect on cell growth and reproduction, therefore they are not necessary for the organism’s growth and reproduction. Secondary metabolites can be divided into several kinds: 1. Antibiotics, the secondary metabolites or derivative compounds produced by some microorganisms, which can inhibit or kill microorganisms. Antibiotics have been widely used in clinical contexts. 2. Toxins, substances produced by some microorganisms during the metabolic process. Toxins are harmful or fatal to humans or to other microorganisms. 3. Hormones, certain substances produced by microorganisms that can stimulate the growth of other microorganisms or of sexual organs. 4. Pigments, produced by many microorganisms in their growth process.

6.2  THE PREPARATION OF XYLOSE Xylose is a kind of pentose; its molecular formula is C5H10O5 and its structural formula is shown in Fig. 6.2. Hemicellulose is a polysaccharide of d-xylose in nature. The industrial production of xylose has been gradually maturing. Xylose is often used as a food sweetener and is the source of xylitol. The utilization of xylose and hemicellulose has been paid much attention with the development of high-value utilization of biomass. As a widespread natural sugar unit, the utilization of xylose as a carbon resource in the fermentation industry has become

FIGURE 6.2  Structure of xylose

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a “hot spot.” The technology of the fermentation of xylose to produce xylitol is mature. Acetone–butanol fermentation from xylose is also feasible. The use of xylose to produce ethanol is a global research hot spot. Consequently, the crisis of energy and food can be solved, if xylose is used as a carbon source in fermentation. Commonly, industrial microorganisms can metabolize glucose for their growth, but not all industrial microorganisms are able to utilize xylose. For example, in bioethanol production, the most widely used ethanol-producing bacterium is yeast (Saccharomyces cerevisiae). Yeast does not have the ability to rapidly metabolize xylose. To solve this problem, many researchers try to transform the existing microbial metabolism process in order to obtain optimal strains by genetic engineering (Hamacher, Becker, & Gardonyi, 2002). The metabolic pathway of xylose in vivo can be briefly stated as follows: first, the xylose is transported into the cell by a special membrane, then xylose is transformed into xylulose by the action of an enzyme, and then the xylulose is metabolized by the PPP. The bottlenecks in the utilization of xylose in many microorganisms are the transport of xylose and the transformation of xylose to xylulose. Conversion of xylose to xylulose in vivo can be divided into two types. First, xylose is reduced to xylitol by xylose reductase, xylitol is transformed into xylulose by dehydrogenase, and then the generated xylulose enters the PPP (Dumon, Song, & Bozonnet, 2011). This pathway can be easily achieved in vivo in yeast. Several researchers have expressed the S. cerevisiae xylose reductase gene and the xylitol dehydrogenase gene in Pichia yeast. However, even though it can get through this pathway of metabolism of xylose to ethanol, the effect is far from satisfactory. Another system of metabolic pathways of xylose is via the expressed isomerase gene in microorganisms (Dumon et al., 2011). Xylose isomerase genes were present in some bacteria and lower fungi, and xylose can be directly converted to xylulose by xylose isomerase. Although this pathway is also paid much attention, it has only recently been successful, because some isomerase gene from the bacterium are difficult to express in yeast. In 2003, Kuyper expressed the xylose isomerase gene of anaerobic fungi Piromyces sp. in a yeast (Kuyper, Harhangi, & Stave, 2003). These developments mean that the prospect for fermentation using xylose as a carbon source is very good, but strains need further study to achieve industrial fermentation (Dumon et al., 2011). Stephens, Christen, and Fuchs (2006) discovered the third metabolic pathway of xylose in Caulobacter crescentus (Fig. 6.3), the NAD-dependent xylose dehydrogenase (XDH) pathway. The xylose is transformed into d-xylonolactone by xylose dehydrogenase, the d-xylonolactone is transformed into d-xylonate by xylonolactonase, the d-xylonate is transformed into 2-keto-3-deoxidization valeric acid by a dehydration reaction, the 2-keto-3-deoxidization valeric acid is transformed into α-ketoglutarate, and the generated α-ketoglutarate enters the TCA pathway.

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FIGURE 6.3  Xylose metabolic pathways (Zhang et al., 2010)

6.3  THE PREPARATION OF GLUCOSE The main obstacle to the preparation of monosaccharide from lignocellulosic biomass is the structural complexity and recalcitrance of lignocellulosic biomass. So a breakthrough is required, using two complementary ideas to destroy structure and improve accessibility and enzyme-specific activity by pretreatment.

6.3.1  Pretreatment of Lignocellulosic Biomass The main components of lignocellulosic biomass are cellulose, hemicellulose, and lignin. They interact to form a complex network structure. The complex structure hinders the enzymatic hydrolysis of cellulose. Cellulose, a linear homopolymer of anhydroglucose units, is composed of crystalline and amorphous domains. In crystalline domains, there is a strong hydrogen bonding among adjacent chains of cellulose and weak hydrophobic interactions between cellulose sheets. Compared to the amorphous domains, the crystalline domains

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are more recalcitrant to enzyme attack, playing a major role in determining the rate of enzymatic hydrolysis. Thus, the pretreatment mainly removes hemicellulose and lignin and disrupts the crystalline structure of cellulose, which is a critical costly step (Chen & Qiu, 2010; Galbe & Zacchi, 2007). The purpose of the pretreatment is to remove lignin and hemicellulose, reduce cellulose crystallinity, and increase the porosity of the materials (Zhang, Ma, & Hong, 2010). An effective pretreatment must meet the following requirements: improve enzyme binding; avoid degradation and loss of sugar; enhance hydrolysis and fermentation of material; avoid high cost. Currently, pretreatment methods can be roughly divided into four different categories: physical, chemical, physical-chemical, and biological. Physical pretreatment includes grinding, crushing, and radiation (Mosier, Wyman, & Dale, 2005). Grinding and crushing are the methods that crush the lignocellulosic biomass, disrupt the bonding connection between the lignin, hemicellulose, and cellulose, and lower the polymerization degree, which is all beneficial to the enzymatic hydrolysis of cellulose. However, the energy consumption of physical pretreatment is high, so it is not popular in cellulose production. Radiation pretreatment includes microwave pretreatment, γ-ray pretreatment, and electron radiation pretreatment. Although these pretreatments could enhance the enzymatic hydrolysis of cellulose, the process is complex and expensive. In short physical pretreatment is simple and causes no pollution, but the energy consumption and cost is high. Chemical pretreatments include acid pretreatment, alkali pretreatment, ozone pretreatment, ammonia pretreatment, and organic solvent pretreatment (Mosier et al., 2005). These methods could swell the cellulose, hemicellulose, and lignin, destroy the crystalline cellulose and separate the three components from one another. Acid pretreatment can be divided into concentrated acid pretreatment and dilute acid pretreatment, and the dilute acid pretreatment is more popular than concentrated acid pretreatment. For example, cellulosic materials were pretreated by 1% dilute acid at 106–110°C for a few hours. After pretreatment, hemicellulose could be hydrolyzed into simple sugars, the average polymerization degree of cellulose was decreased, and yet the rate of enzymatic hydrolysis was significantly increased (Mosier et al., 2005). Alkali treatment could weaken the interaction of ester bonding and hydrogen bonding between hemicellulose and lignin. NaOH treatment has a strong effect of delignification (Hu, Wang, & Wen, 2008). For ammonia treatment, the lignocellulosic biomass is immersed in the ammonia solution with a cellulose mass fraction of 10% for 24∼48 h to remove most of the lignin (Kim, Kim, & Sunwoo, 2003). Ozone pretreatment could degrade the lignin and hemicellulose. The advantages of the ozone pretreatment are no pollution and a mild reaction condition. However, this pretreatment is expensive (Sun & Cheng, 2002). The organic solvents commonly used in organic solvent pretreatment are methanol, ethanol, acetone, ethylene glycol and triethylene alcohol, organic acids, and so on. Chen proposed and explored the atmospheric aqueous glycerol autocatalytic pretreatment (AAGAOP) to improve

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the enzymatic hydrolysis of lignocellulosic biomass. The results showed that the AAGAOP technique, as a novel strategy, enhanced the enzymatic hydrolysis of lignocellulosic biomass by removing the chemically composed barrier and altering the physically structural barrier (Sun & Chen, 2008). Physical-chemical pretreatment is the combination of physical and chemical methods. The typical physical and chemical treatment is steam explosion (SE). SE is one of the most extensively studied pretreatments, which combines mechanical tearing and chemical degradation effects. It combines physical tearing and chemical high-temperature cooking. The saturated steam can penetrate into the pores of stalks, and all pores tear in a rapid decompression process; chemical hightemperature cooking plays a role in the degradation of hemicellulose and lignin, as well as in the softening and exposing of the cellulose (Chen & Liu, 2007). Biological pretreatment with white rot fungi has shown remarkable advantages compared with physical/chemical pretreatments; these include simple technique, low energy requirement, no generated inhibitors, and low investment. White rotten fungi, such as Pharerochacte Chrysosprium (Shi, Chinn, & SharmaShivappa, 2008) and Coriolus versicolor are the potential industrial strains (Zhang, Xu, & Wang, 2007a), as they can produce enzymes to degrade the lignin.

6.3.2  Hydrolysis of Cellulose The cellulose hydrolysis method includes two categories, one being acid hydrolysis, and the other enzymatic hydrolysis. Acid hydrolysis technology appeared early and is mature. Enzymatic hydrolysis is mild, environmentally friendly, and will set the direction of future development, but the cost is high at present. Acid hydrolysis is the process that breaks the glycosidic bonding between the cellulose molecules by HCl with H2SO4 as a catalyst to form monosaccharides. Under the action of hydrogen ions and suitable temperature, the glycosidic linkages are hydrolyzed and the polymerization degree of polysaccharide is lowered, to the point where it even becomes monosaccharide. The reaction equation can be expressed as follows: (C6H10O5)n + nH2O→nC6H12O6 According to the kinds and concentration of acid, acid hydrolysis can be divided into inorganic acid hydrolysis and organic acid hydrolysis. The early hydrolysis method is by inorganic acid, and the cost is cheap. The most commonly used acids are sulfuric acid and hydrochloric acid. Here are some examples of the typical sulfuric acid hydrolysis process. Cen, Wu, and Zhang (1993) hydrolyzed nettle stalks by using concentrated sulfuric acid; for this the best hydrolysis conditions are 70% sulfuric acid, temperature is around 40∼50°C, solid–liquid ratio is 5%, particle size is 20∼40 mesh, and reaction time is 10∼20 min. Under optimal conditions almost all of the hemicellulose and cellulose in nettle stalks is hydrolyzed (Cen et al., 1993). Masada resource companies and Arkenol companies have invested in the concentrated

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acid hydrolysis process of cellulose to produce ethanol (Mielenz, 2001). Generally, sulfuric acid hydrolysis employs technology that was researched early on (Zhang, Yang, & Lv, 2007b). Dilute acid hydrolysis is a process whereby the acid treatment is used to pretreat lignocellulose materials. Dilute acid hydrolysis is mostly focused on the sulfuric acid catalyzed process. Other acids that may be used are hydrochloric acid, phosphoric acid, and organic acid, etc. Early dilute acid hydrolysis conditions were 0.5%∼0.5% acid concentration and temperature around 170∼200°C. In the 1980s, with the development of reactors, materials, and temperature control, the reaction temperature was increased to 200°C. In recent years, the reaction temperature has gone up to 230°C (Li, Ren, & Qi, 2009). The dilute acid method is one of the most effective lignocellulose pretreatment methods. This method can not only destroy the crystal structure of cellulose in raw material, but can also facilitate the fraction of hemicellulose from the lignocellulosic biomass (Zhang et al., 2007b). Another method is enzyme hydrolysis of cellulose to produce sugar. Compared to acid hydrolysis, enzymatic hydrolysis under mild conditions is environmentally friendly. Currently, cellulase is the world’s third largest industrial enzyme, and has been widely used in the fields of plant agricultural products processing, food brewing, the textile industry, paper recycling, enzyme detergents, and animal feed additives. The enzymes used in the industry come mainly from fungi, especially from Trichoderma reesei fermented cellulase (Henrissat, Driguez, & Viet, 1985). The mechanism of transforming cellulose into glucose by fungal cellulase has been studied by many scholars. Reese, Siu, and Levinson (1950) proposed that first the C1 enzyme acted on the crystalline cellulose to generate amorphous cellulose, secondly the formed amorphous cellulose was then further Cxenzymatic hydrolyzed into soluble glucose; in other words, the C1 reaction is a prerequisite step for Cx enzymatic hydrolysis.

Chen and Li (2000) proposed that C1 be considered as the combination of the CMC enzyme, the CBH enzyme, and the CB enzyme, and that it plays an important role on the crystalline regions. Currently, the generally accepted enzymatic mechanism is the synergistic process model (Fig. 6.4). First, the endocellulase acts on the cellulose to form a new amorphous cellulose-terminal (nonreducing end or reducing end), and then cellulose exonuclease is cut from the reducing or nonreducing end of the outer side to produce cellobiose (or glucose) (Woodward, 1991). In the enzymatic hydrolysis process of lignocellulosic biomass, the proportion of cellulase cost is relatively high, even up to half the total cost, so the key breakthrough point in industrialization of cellulosic ethanol and other bio-based products is the use of cellulase. Many efforts have been made to improve the hydrolysis efficiency of cellulose (Table 6.1).

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FIGURE 6.4  Synergetic degradation model (Woodward, 1991)

TABLE 6.1 The Synergetic Treatment (Liu, Sun, & Pei, 2011) Synergetic treatment

Mechanism and characteristics

Ultrasonication

Improving the cellulase molecule’s kinetic energy to increase the frequency of enzyme molecule collision, enhance reaction speed, and enhance the rate of cellulase update of the substrate surface.

Addition of reaction agent

Adding some substances, such as proteins, surfactants, polymers, etc., to enzymatic hydrolysis to increase the enzymatic hydrolysis of the sugar yield. However, the mechanism is not clear, the results presumably having shown that these substances reduced invalid adsorption and increased the availability of enzymes and enzyme stability.

Compound enzyme

An enzyme complex preparation composed of different types of enzyme can reduce cellobiose, the product of sugar and hemicellulose hydrolyzate inhibition of the enzymatic hydrolysis process; may degrade hemicellulose, lignin, and other components at the same time, so cellulase enzymes can also be increased.

6.4  PREPARATION OF XYLOSE The degradation of hemicelluloses is much easier than that of cellulose, and xylose can be obtained more easily by acid and other pretreatments. Xylose has also become an important industrial product, so the xylose industrial production process is introduced in this section to discuss the preparation breakthrough.

6.4.1  Industry Overview of Xylose Preparation In recent years, fermentation using xylose as raw material has been paid much attention, and the use of lignocellulose biomass to prepare xylose is already a mature technology. In the 1990s, xylose was widely used as an important chemical material. The main functions of xylose can be stated as follows (Tan & Huang, 2006): (1) cannot be absorbed by the human body; (2) promotes growth of bifidobacteria and is therefore beneficial to human health; (3) cannot be used by oral microorganisms; and (4) has some physiological functions of dietary fiber, such as lipid-lowering, lowering cholesterol and preventing colon cancer (Chen, 2001).

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TABLE 6.2 The Chemical Composition of Raw Materials (Tan & Huang, 2006) Raw materials

Cellulose (%)

Hemicellulose (%)

Lignin (%)

Corncob

32∼36

35∼40

25

Bagasse

40∼45

24∼25

24

Cotton hull

35∼44

25∼28

28

Rice hull

35∼44

16∼22

24∼32

Wheat straw

36∼40

17∼20

14∼15

Tea hull

38∼43

25∼28

24∼32

Meanwhile, xylose is also a raw material for producing xylitol by hydrogenation. Xylose is widely used in other areas, such as food, medicine, chemicals, leather, and dyes. Countries producing xylose include Russia, the USA, Japan, Finland, and Italy (Tan & Huang, 2006). In China, the raw materials used to produce xylose are corn cobs, bagasse, rice husks. These raw materials have high hemicellulose content, large output, and cost little (Table 6.2).

6.4.2  Industrial Process of Xylose Preparation The main xylose preparation method currently used in the industry is acid hydrolysis. The inorganic acid is added to the raw material that is rich in hemicellulose, the mixture is treated by high temperature, and as a result, the hemicellulose in the raw material can be hydrolyzed into xylose. The preparation process of xylose by deacidification can be formulated as follows (Tan & Huang, 2006). Raw materials are pretreated, then undergo acid hydrolysis, discoloration, concentration, crystallization, and finally form separated xylose crystals. The pretreatment process of the raw material is removal of gum, pectin, ash, and other ingredients; and then the raw material is hydrolyzed into monosaccharides by an acid catalyst, such as inorganic acid. The neutralization step is to remove the major inorganic acid hydrolyzate; the bleaching process is to remove pigments and impurities from the xylose mother liquor by activated carbon; the crystallization process is to remove the small amount of acid ingredient by evaporation, and control the wood sugar concentration and crystallization time so as to get xylose crystals. The deacidification process is the traditional extraction process of xylose, it is simple, involves low acid consumption, is easy to perform, and needs less investment. However, CaSO4 produced in the deacidification process is difficult to remove by chemical methods; as with lime and hydrolyzate, the local pH value may cause some variability in the xylose, affecting product quality.

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The ion exchange process is similar to the deacidification method, except that the acid is removed by ion exchange, instead of being neutralized by calcium hydroxide. Xylose is stable under acidic conditions, but unstable under alkaline conditions. Using ion exchange to neutralize this reaction not only solves the problems of the deacidification process, such as equipment fouling, but also improves the quality of hydrolyzate. Here is an example where a company used corncob as a raw material to produce xylose (Qin, 1996): 1. Pretreatment of corncob Filtering process: Screening by water and winnowing to remove impurities in raw materials. Raw material crushing: The moisture content of corn cob is lower than 12.18%; a pulverizer is used to keep particle size ≤5 mm. Water pretreatment: The pulverized materials were added to the stainless steel kettle and heated at 120°C for 120 min with stirring to remove gum, pectin, ash, and other impurities. 2. Hydrolysis: The pretreated corncob is put into the pot for hydrolysis. The hydrolysis operation process can be one of two types, dilute acid hydrolysis, and dilute acid with high pressure acid hydrolysis. In high-pressure hydrolysis, sulfuric acid concentration is 1.5%–2.0%, temperature is around 100∼105°C. In low-pressure hydrolysis the acid concentration is 0.5%–0.7%, temperature is around 120∼125°C. 3. Neutralization: The main purpose is to remove sulfuric acid. Lime or calcium carbonate is often used as a neutralizer. According to experience, when the hydrolyzate pH is 1.0∼1.5, then adjusted to pH 2.8∼3.0 by adding a neutralizing agent, residual sulfuric acid is only 0.05%–0.1%. In this case, the inorganic acid solution has been substantially removed. When the pH reaches 4.0, all the inorganic acid is removed completely. 4. Decolorization: Decolorization is the removal of the pigment from the hydrolysis. The commonly used bleaching agents are activated clay, activated carbon, coke, and other types of lignin. Bleaching agents (coke lignin or activated charcoal) can be recycled and reused after treatment. 5. Concentration: One purpose of the concentrated syrup is to increase the sugar concentration (around 35%∼40%), while the acid in the hydrolysis is evaporated; in addition, the calcium sulfate precipitation is beneficial in removing impurities by ion exchange. 6. Impurity: The sugar solution concentrated by evaporation after the preceding step also contains impurities, such as ash, acid points, nitrogencontaining solids, colloids, and pigments, which should be removed by ion exchange purification. The purity is up to 95%. Using cation resin No. 732 and No. 717 strong base anion resin porous joint treatment, the volume ratio can be selected as male:female = 1:1.5. 7. Crystallization: Xylose solution is concentrated under reduced pressure into the finished tank, the vacuum of the system is higher than 99 KPa, the liquid

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temperature should be controlled below 75°C. The solution was again concentrated by evaporation to 1/4 volume. When the material is put into the mold when it is hot, after the xylose solution is cooled to room temperature, white pure crystals of xylose will be precipitated. The crystal suspension centrifuge expels divisible liquor, that was xylose crystals. The recycled mother liquor is purified again, after a due process of dilution and bleaching treatment. 8. Drying: The xylose is dried, in a drying room with temperature lower than 100°C, until the moisture content is lower than 0.5%.

6.5  PATHWAY MECHANISMS OF INHIBITOR AND SOLUTIONS The degradation of cellulose and hemicellulose in lignocellulosic materials to build a sugars platform for further biotransformation as a carbon resource is a very promising pattern. Theoretically, the cellulose and hemicellulose can be hydrolyzed into monosaccharides. However, in research and in production trials, results showed that compared with the starch substrate, the utilization of lignocellulosic feedstock can solve many practical problems. The inhibitor generated in the pretreatment process is essential step in establishing a platform conversion route.

6.5.1  The Inhibitor and its Mechanism As previously mentioned, the degradation of cellulose and hemicellulose to monosaccharide can be divided into two categories: acid hydrolysis and enzymatic hydrolysis. In acid hydrolysis the raw material is treated by mineral acids (such as sulfuric acid, hydrochloric acid) at high temperature and pressure for some time, and the cellulose and hemicellulose are degraded into monosaccharides. In this process, acid breaks the glycosidic bond and plays a catalytic role in generating furans. Enzymatic hydrolysis is more moderate than acid hydrolysis, and generates fewer hydrolysis by-products. However, owing to the complicated structure of lignocellulosic biomass and its complex composition, pretreatment is needed to destroy the natural structure, improve the digestion, and facilitate the multicomponent utilization of lignocellulosic biomass. The pretreatment conditions are often more intense, and many products are generated during the pretreatment process. For example, in the hot water or steam (including SE) process, an acetyl group may be removed from hemicellulose to produce acetic acid at high temperature; this process also brings catalytic degradation and furans into effect. In short, it is known that by-products from the degradation and conversion of cellulose and hemicellulose include the previously described organic acids and furans. From the literature and experience, these two by-products significantly inhibit microbial growth (Palmqvist & Hahn-Hägerdal, 2000). The inhibitors in the hydrolysis of spruce wood as shown in Fig. 6.5. Moreover, the typical organic acid by-product obtained by hydrolysis pretreatment is generally weak acid, and such materials often inhibit microorganism growth so obviously that many weak acids are used as preservatives in the food

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FIGURE 6.5  The inhibitors in the hydrolysis of spruce wood (Palmqvist & HahnHägerdal, 2000a)

industry. Compared with strong acid, the dissociation degree of weak acid is low, and undissociated weak acid is fat-soluble, so that it can enter the interior of the cell through the cell membrane and the cytoplasm pH changes, which may be an important reason for weak acid’s inhibiting the growth of microorganisms. After pretreatment and hydrolysis, lignin will be transformed into different complex products, many of them phenols (Palmqvist & Hahn-Hägerdal, 2000). For fermentation, they include some of the important polyphenols that inhibit ingredients. Among them, the toxicity of low molecular weight phenolic compounds is high (Buchert, Puls, & Poutanen, 1989; Clark & Mackie, 1984). Generally, polyphenols can undermine the integrity of biological membranes, make cell membranes electively lose the carrier film, and damage the role of membrane proteins (Heipieper, Weber, & Sikkema, 1994). Unfortunately, so far, there is still a lack of detailed study and analysis of phenolic compounds in hydrolyzate liquor, and its action mechanism is also little understood. This is mainly due to the complexity of the raw material, and that the process is often very severe, so that the hydrolyzate has very complex components, reproducibility is poor and the pretreatment and hydrolysis processes are also complicated. Many studies using model compounds, such as vanillic acid, vanillin 4-hydroxy benzoic acid, as inhibitors of fermentation, affect the classification of substances in evaluating their conclusions but these are used only as references. With a great change in the concentration of hydrolyzate, there may be interactions of different phenols and polyphenols with the other ingredients. According to the literature, the inhibition of phenolic substances for S. cerevisiae is obvious. Ando et al. (Ando, Arai, & Kiyoto, 1986) reported that 1 g/L 4-hydroxybenzaldehydecan makes the ethanol production of S. cerevisiae be reduced by 30%. We have studied the effect of phenolic compounds, using vanillin as a model compound, on gum fermentation, and results showed that when vanillin concentrations were greater than 1g/L, microbial growth was completely inhibited (Fig. 6.6).

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FIGURE 6.6  The effect of vanillin on gum fermentation

The inhibition effects of different components reported earlier on the microbial fermentation are not always uniform, owing to variations in microorganisms and fermentation conditions. According to some literature and our findings, polyphenols in the hydrolyzate may be the most important inhibitor substances (Palmqvist & Hahn-Hägerdal, 2000). In short, the roles and mechanisms of the inhibitor component formed in the lignocellulose pretreatment and hydrolysis process need to be further studied.

6.5.2  Detoxification Process In order to enable the growth of microorganisms and increase in production, and reduce the inhibitor content, several measurements must be introduced. For example, some inhibitors greatly inhibited the growth of microorganisms, therefore, increasing the inoculum, to achieve the maximum amount of microbial biomass of the fermentation in a short time, is one possible method for reducing toxic hazards (Palmqvist, Hahn-Hagerdal, & Galbe, 1996). However, in most cases, simply increasing the inoculum may not be enough, we may need to introduce some detoxification methods to reduce the lignocellulosic hydrolyzate inhibitor content. The inhibitory hydrolyzate component can be removed by several methods, some of which may be relatively simple industrial applications. Common detoxification methods can be divided into three categories, physical detoxification, chemical detoxification, and biological detoxification (Palmqvist & Hahn-Hägerdal, 2000):

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1. Physical detoxification. Physical detoxification methods include physical rotary evaporation, an extraction method for removing inhibiting ingredients. The rotary evaporator method mainly removes some volatile components, the nonvolatile components can be removed by extraction. The detoxification effect of the rotary evaporator method is limited, because the boiling points of most inhibitors are not low. Extracting by organic solvent, such as diethyl ether or ethyl acetate, under suitable conditions is an effective detoxification method, it can extract a portion of the weak acids, furfural, and phenols (Palmqvist & Hahn-Hägerdal, 2000). 2. Chemical detoxification. Relatively speaking, the chemical method is more mature and is also suitable for industrial applications. For acid hydrolysis, alkaline treatment (overliming) is an effective and simple means of chemical treatment. This method uses calcium hydroxide as the neutralizing agent. The pH of the hydrolyzate is adjusted to 9–10, and then adjusted to 5.5 by sulfuric acid. Results reported in the literature showed that switching to sodium hydroxide can also have a detoxification effect, and better results are obtained by using sodium hydroxide. The alkaline treatment detoxification mechanism has two difficult aspects. One is that in producing calcium sulfate, precipitation may have a flocculation effect, the other one is that the inhibitors are not stable in high pH conditions (Palmqvist & Hahn-Hägerdal, 2000). In addition to overliming, sulfite-reducing agent treatment may also play a role in detoxification, and this method can reduce the concentration of furan derivatives (Palmqvist & Hahn-Hägerdal, 2000). The two sulfite overbased system may be better. Olsson, Hahn Hagerdal, and Zacchi (1995) adjusted the pH of hydrolysis to 3.1 with Ca(OH)2 and then adjusted it to 10.5 by adding 1 g/L sulfite, for 30 min at 90°C, which detoxification effect is better than the effect of alkaline treatment. 3. Biological detoxification. Biological detoxification is a process in which hydrolyzate treated by microbials, such as white rot fungi and soft rot fungi, have the ability to degrade lignin (Palmqvist & Hahn-Hägerdal, 2000). White rot fungi can secrete related enzymes, such as laccase, to degrade lignin. Hydrolysis by laccase can also be considered a direct treatment system. Jonsson, Palmqvist, and Nilvebrant (1998) reported that laccase treatment did not reduce the aromatic ring, but increased the content of high molecular weight substances. It can be concluded that small molecules of phenol and phenolic were oxidation polymerized, so that the toxicity of the hydrolyzate was reduced. Some scholars use soft rot fungi to treat hydrolyzate. The mechanism of this method is different from the mechanism of white rot fungi, which should be studied further. Compared to chemical detoxification and physical detoxification, biological detoxification is easier to manipulate. Peroxidase or laccase, for example, can degrade the inhibitor without any other operations, in mild condition. Owing to the relatively high cost of the enzyme, the immobilized enzyme may represent a better strategy.

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6.6  ECONOMIC ANALYSIS OF SUGAR PLATFORM COMPOUNDS IN THE BIOCHEMICAL CONVERSION OF BIOMASS 6.6.1  Sources and History of Sugar Platform Materials From the perspective of fermentable carbon cost, the development of sugar platform materials has passed through three stages: sucrose materials, starchy materials, and lignocellulosic biomass. Of which, the industrial fermentation technologies of sucrose materials and starchy materials are quite mature, while the utilization of lignocellulosic biomass is still in the initial stage (Bai, Anderson, & Moo-Young, 2008). Commonly used raw materials of sugar in the fermentation industry include sugarcane, sugar beet, molasses, and sugar grass. Sugarcane is rich in sugar and water, being an important source of raw materials for sugar in the fermentation industry. Compared with sugarcane, sugar grass juice provides sugar containing quite a bit of reducing sugar. Molasses is a viscous, brown semifluid, which is the by-product of the sugar refining industry. Molasses can be divided into sugarcane molasses, sugar beet molasses, grape molasses, and maize molasses, and all contain lots of fermentable sugar, which is regarded as an important source of sugar platform compounds. With the development of technologies, besides the sugars that can be directly used, humans begin to obtain sugars that can be used by microorganisms directly or indirectly from noncarbohydrates; one of these sugar sources is starch. Starchy materials commonly used in the fermentation industry include grains, potatoes, wild plants, and by-products of agrotechny. Rice, maize, and wheat provide starchy materials and high annual yields. Generally speaking, starch mainly exists in the seeds and tubers of plants. For example, the starch content of rice is about 62%–86%, wheat 57%–75%, and potatoes more than 90%. At present, such grains are the main raw materials used in the fermentation industry. However, one of the disadvantages of using grains as the main resource for the production of sugar is the issue of the reasonable distribution of resources in view of the fact that grains are also food for humans. Even if nongrain starchy materials are used, problems around occupying arable land still exist. Owing to the aggravation of the energy crisis and environmental pollution, more and more researchers are paying attention to the high-value utilization of renewable resources, especially lignocellulosic biomass (Hahn-Hägerdal, Galbe, & Gorwa-Grauslund, 2006). Lignocellulosic biomass is regarded as the traditional waste of agroforestry, and thus utilization of lignocellulose can not only reduce the waste of resources, it can also bring out extraneous income, realizing the “multi win” of resource, environment, and economy.

6.6.2  Characteristics Analysis of Sugar Platform Compounds Sugar raw materials, such as sugarcane and sugar beet, are widely distributed in China and contain lots of carbohydrates, which can be easily used in

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fermentation. Besides abundant carbohydrates, there is quite a lot of protein and cane wax in sugarcane, lots of pectin in sugar beet and 5%–12% colloidal substance and 10%–12% ash in molasses, which makes it essential to adopt proper pretreatment methods to improve fermentation performance when using such sugar materials in industry. Starchy materials are widely cultivated in China and the starch within them can be fermented directly to obtain sugars, which have been the most commonly used raw materials for the fermentation industry. The technological process of the production of glucose from starch can be determined as: starch→size mixing→ liquidation→saccharification→refinery of saccharified liquid→glucose. As an agriculture-based country, the grain yield of China has been increasing for several years and the total grain yield has reached 601.93 million tons with yearon-year growth of 2.1%. Meanwhile, China’s grain consumption is large owing to its considerable population, and the demand for grains in other fields has been increased with the increase in categories of fermentation products and the enlargement of industrial scale. Therefore, the total amount of starchy materials for industrial application is limited. It is necessary, therefore, to seek new sources of sugar platform compounds so as to avoid the situation of competition with grain as a human food. Generally speaking, “lignocellulosic biomass” refers to the plant resource containing wood and crop stalks, and a kind of chemical energy preserved in plants through photosynthesis. China has abundant lignocellulose resources and the annual yields of cornstalks, wheat stalks, and rice waste could reach 220, 110, and 18 million tons, respectively (Chen, Qin, & Li, 2014). However, the utilization of lignocellulose is limited by its compact structure, which leads to a situation in which stalks are usually thrown away or only burned for heating and lighting. At present, one of the important fields for the application of lignocellulose is paper making (Singh, Sulaiman, & Hashim, 2010). In addition, the paper quality demand strains the utilization of lignocellulose, leading to inefficient use of lignocellulosic biomass. Therefore, on the basis of reasonable allocation of resources, a series of physical, chemical, and biological methods should be employed to break down the rigid structure of cellulose, making it an important source of sugar platform compounds. Compared with starchy materials, lignocellulose, as a kind of green, renewable, and environmentally friendly raw material, has the properties of wide distribution and low cost, which makes it a potential source of raw materials for the construction of a sugar platform (Li & Chen, 2014). As shown in Section 6.2, lignocellulose is an important source of xylose, besides glucose, that can be used by most microorganisms. Xylose, a kind of pentose, mainly exists in hemicellulose among the lignocellulosic materials, where cellulose and hemicellulose account for almost 50% of the lignocellulosic biomass. Thus, it is feasible to produce xylose using lignocellulose in both theoretical and economic terms. The production of substantial quantities of pentose is an important difference in lignocellulose, compared with the other two kinds of sugar platform compounds.

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6.6.3  Fermentability Analysis of Sugar Platform Compounds Fermentability refers to the effects of some unfavorable components in the substrate on the fermentation process. For example, an impurity or inhibitor in one substrate may influence the growth of microorganisms, some of which might even terminate the fermentation process. Obviously, we can say that the fermentability of such substrate is poor. Molasses has a complicated composition, containing favorable components, but some that are hard to use or to be converted by microorganisms, and some of the unfavorable components will inhibit the growth and metabolism of microorganisms. For example, hydrogen production from molasses usually used mixed fermentation technology instead of single fermentation (Ueno, Otsuka, & Morimoto, 1996). Starch is a polymer composed of glucose, which can be hydrolyzed into monosaccharides, showing a high fermentablity. At present, starchy materials used in the fermentation industry mainly include cassava and maize, and Fig. 6.7 clearly shows the technological process of syrup production using maize as raw material.

FIGURE 6.7  Flow chart of syrup production using maize starch

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Lignocellulose has a promising prospect in industry because the available sugar components in lignocellulose account for 50%. However, owing to the natural resistance to degradation (including structural complexity and recalcitrance) of lignocellulose, direct utilization of lignocellulose is impossible without pretreatment (Himmel, Ding, & Johnson, 2007). The network formed by cellulose, hemicellulose, and lignin prevents the enzymes from making contact with the substrate, thus it is essential to break the tight structure with certain pretreatment methods in order to make enzymes form better contact with the substrate. It should be pointed out that during such processes, some inhibitors will be generated in the pretreatment process, which will be unfavorable for subsequent utilization. Besides, compared with those raw materials that are composed of almost pure sugar units, such as starch and molasses, there are still considerable amounts of substances that cannot be used by microorganisms in lignocellulosic materials after pretreatment, leading to low product concentration and complicated postprocessing technologies.

6.6.4  Economic Analysis of Sugar Platform Compounds The economic analysis of raw materials involves the total cost analysis of raw materials collection, unit operations and spendable preparations in the transformation from raw materials to end products. For the conversion process of sugar platform compounds, economic analysis refers to the raw materials cost, pretreatment cost, and enzyme cost. Currently, fermentable sugars in industry mainly include sucrose and starch, but the supply of these raw materials cannot satisfy industrial requirements. Typically, molasses can be accumulated in a large-scale sugar processing factory and used as cheap industrial raw materials for fermentation. Hence, more and more researchers are paying attention to the industrial application of lignocellulose. From the perspective of technological process, the advantages of lignocellulose as a sugar platform compound are not obvious because a series of pretreatment operations is needed to break the compact structure of lignocellulose, whose consumption accounts for quite a large proportion of the whole cost. However, lignocellulose contains abundant hexose and pentose and thus can produce many kinds of end products. Fig. 6.8 lists the categories of industrial products obtained from biomass. In the arena of industrial production, the cost of raw materials is an important step in the whole technological path. As a cheap and widely distributed material that conforms to the sustainable development concept, lignocellulose has huge potential to be a new sugar platform compound. Fuel ethanol production has been rapidly developed in the past few decades. In view of the problems in the technological path, America and some countries in Europe began to study the economic feasibility of cellulosic ethanol in the 1990s and modified the evaluation system promptly in response to new problems arising every year (Kling, Neto, & Ferrara, 1987). Here, we will illustrate the existing problems of ethanol production in several fermentation systems and simply compare the cost of raw materials.

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FIGURE 6.8  Chemicals obtained from the biomass conversion process

6.6.4.1  Raw Materials Cost Sugar grass, 30,000–40,000 Yuan/t, contains about 56% sugar components. Starchy materials, such as maize, 2000–3000 Yuan/t, contain 70% starch with 95%–100% saccharification yield. The sugar units in lignocellulose are cellulose and hemicellulose, which account for 50% of the total mass of lignocellulose. Most lignocellulose comes from agroforestry wastes and costs less, with an average price of 300 Yuan/t. Calculated according to the highest fermentation concentration in the production of 1 t ethanol, the raw materials costs of sugar grass and maize are 67 and 3.5 times that of lignocellulose, respectively.

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6.6.4.2  Fixed Assets Cost Fermentation technologies using sugar materials and starchy materials are quite mature, thus it is easy to establish a production line and purchase the necessary equipment. Research into cellulosic ethanol production is still in the initial stage, and the lack of the most appropriate facilities leads to a relatively high fixed assets cost. 6.6.4.3  Pretreatment Cost Sugar grass should be pressed to obtain mixed juice, which is used to produce ethanol through flocculation and multieffect distillation. For starchy materials, such as maize, certain methods should be adopted to extract starch from raw materials and convert it to saccharides, because most microorganisms cannot utilize starch directly. Present data show that 1.5 t maize, 200 kW h electricity, and 0.3 t coal are needed to produce 1 t ethanol (Chen & Fu, 2012). Maize starch should be liquefied, saccharified, and fermented to produce ethanol. In this process, the steam consumption of gelatinization in the saccharified phase accounts for 20%–30% of the total steam consumption. While using lignocellulose to produce ethanol, acid hydrolysis and SE pretreatments should be adopted to break its compact structure and the theoretical steam consumption of 1 t lignocellulosic biomass is 0.3–0.5 t (Kling et al., 1987). 6.6.4.4  Labor Cost The technology for industrial production of ethanol using sugar grass and starchy materials is mature, with the complete production line and a high degree of automation well established, saving considerable labor costs. The technology for using lignocellulose to produce ethanol is not mature, so that appropriate devices and technologies are lacking, leading to high labor costs. To conclude, the main costs for the fermentation of ethanol using maize starch are for the raw materials, while those for lignocellulose fermentation are for assorted devices and labor. Table 6.3 makes a summary of the economic costs of the two kinds of industrial modes. TABLE 6.3 Comparison of Production Costs of Fuel Ethanol (McAloon, Taylor, & Yee, 2000) Parameters

Starch process

Lignocelluloses process

Feedstock

78.16%

35.29%

Labor and supplies

14.94%

16.18%

Variable operating costs

11.49%

41.91%

Depreciation capital

27.59%

14.70%

Coproducts

−32.18%

−8.09%

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6.6.5 Conclusions Lignocellulose contains quite a lot of xylose besides considerable amounts of glucose and is regarded as a potential source of sugar platform compound. To obtain cheap and abundant lignocellulosic sugars, it is essential to establish a pretreatment system with the properties of low energy consumption, simple mode of operation, and large handling capacity to break the network structure, improve the accessibility of enzymes, and increase conversion efficiency. The present technologies of cellulosic ethanol production are not mature and some problems remain in the areas of industrial development and acquisition of profits. But it is undeniable that the large-scale production of ethanol will resolve the environmental problems caused by overuse of fossil fuels and fully utilize resources, conforming to the sustainable development concept. If energy efficiency is defined as the specific value of energy input and energy output, Gnansounou and Dauriat (2010) reported that the energy efficiency of power generation using solid fuels was 30%. In fact, the energy efficiency of ethanol production from willow, corn stover, and spruce reaches 25%, 25%, and 31%, respectively. Thus, lignocellulosic biomass will show more and more advantages in the energy field and become a new energy source in the future.

REFERENCES Ando, S., Arai, I., Kiyoto, K., et al. (1986). Identification of aromatic monomers in steam-exploded poplar and their influences on ethanol fermentation by Saccharomyces cerevisiae. Journal of Fermentation Technology, 64, 567–570. Bai, F., Anderson, W., & Moo-Young, M. (2008). Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnology Advances, 26, 89–105. Berdy, J. (2005). Bioactive microbial metabolites. The Journal of Antibiotics, 58(1), 1–26. Buchert, J., Puls, J., & Poutanen, K. (1989). The use of steamed hemicellulose as substrate in microbial conversions. Applied Biochemistry and Biotechnology, 20, 309–318. Cen, P., Wu, J., & Zhang, J. (1993). Study on the hydrolytic kinetics of lignocellulose material by concentrated sulfuric acid. Chemical Reaction Engineering and Technology, 9, 34–41. Chen, H. (2008). Biomass science and engineering. Beijing: Chemical Industry Press. Chen, H. (2011). Biotechnology of lignocellulose. Beijing: Chemical Industry Press. Chen, H., & Fu, X. (2012). Theory and application of the feedstock refinery of fermentation industry. Beijing: Science Press. Chen, H., & Li, Z. (2000). Factors of enzymic hydrolysis for cellulose and adsorption of cellulase. Chemical Reaction Engineering and Technology, 16, 30–35. Chen, H., Qin, L., & Li, D. (2014). Aerobic and anaerobic sequential culture fermentation (AASF) to produce bio-hydrogen from steam-exploded cornstalk. International Journal of Hydrogen Energy, 39, 8992–8999. Chen, H., & Liu, L. (2007). Technology of steam explosion: principles and applications. Beijing: Chemical Industry Press. Chen, H. Z., & Qiu, W. H. (2010). Key technologies for bioethanol production from lignocellulose. Biotechnology Advances, 28, 556–562. Chen, J. (2001). Prospects of xylose production and application. Anhui Chemical Engineering, 27, 13–14.

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Clark, T., & Mackie, K. (1984). Fermentation inhibitors in wood hydrolysates derived from the softwood Pinus radiata. Journal of Chemical Technology and Biotechnology, 34, 101–110. Dumon, C., Song, L., Bozonnet, S., et al. (2011). Progress and future prospects for pentose-specific biocatalysts in biorefining. Process Biochemistry, 47(3), 346–357. Galbe, M., & Zacchi, G. (2007). Pretreatment of lignocellulosic materials for efficient bioethanol production. Biofuels, 108, 41–65. Gnansounou, E., & Dauriat, A. (2010). Techno-economic analysis of lignocellulosic ethanol: a review. Bioresource Technology, 101, 4980–4991. Hahn-Hägerdal, B., Galbe, M., Gorwa-Grauslund, M. -F., et al. (2006). Bio-ethanol–the fuel of tomorrow from the residues of today. Trends in Biotechnology, 24, 549–556. Hamacher, T., Becker, J., Gardonyi, M., et al. (2002). Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization. Microbiology, 148, 2783. Heipieper, H., Weber, F., Sikkema, J., et al. (1994). Mechanisms of resistance of whole cells to toxic organic solvents. Trends in Biotechnology, 12, 409–415. Henrissat, B., Driguez, H., Viet, C., et al. (1985). Synergism of cellulases from Trichoderma reesei in the degradation of cellulose. Nature Biotechnology, 3, 722–726. Himmel, M. E., Ding, S. -Y., Johnson, D. K., et al. (2007). Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science, 315, 804–807. Hu, Z., Wang, Y., & Wen, Z. (2008). Alkali (NaOH) pretreatment of switchgrass by radio frequencybased dielectric heating. Biotechnology for Fuels and Chemicals, 148(1–3), 589–599. Jonsson, L., Palmqvist, E., Nilvebrant, N., et al. (1998). Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor. Applied Microbiology and Biotechnology, 49, 691–697. Kim, T. H., Kim, J. S., Sunwoo, C., et al. (2003). Pretreatment of corn stover by aqueous ammonia. Bioresource Technology, 90, 39–47. Kling, S., Neto, C. C., Ferrara, M., et al. (1987). Enhancement of enzymatic hydrolysis of sugar cane bagasse by steam explosion pretreatment. Biotechnology and Bioengineering, 29, 1035–1039. Kuyper, M., Harhangi, H. R., Stave, A. K., et al. (2003). High level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae. FEMS Yeast Research, 4, 69–78. Li, G., & Chen, H. (2014). Synergistic mechanism of steam explosion combined with fungal treatment by Phellinus baumii for the pretreatment of corn stalk. Biomass and Bioenergy, 67, 1–7. Li, Y., Ren, X., Qi, W., et al. (2009). A review of dilute acid hydrolysis of lignocellulosic biomass. Journal of Jilin Institute of Chemical Technology, 26, 29–34. Liu, Y., Sun, J., Pei, H., et al. (2011). Research progress on improving the efficiency of enzymatic hydrolysis of lignocellulose. China Brewing, 5, 16–20. McAloon, A., Taylor, F., Yee, W., et al. (2000). Determining the cost of producing ethanol from corn starch and lignocellulosic feedstocks. National Renewable Energy Laboratory Report. Mielenz, J. (2001). Ethanol production from biomass: technology and commercialization status. Current Opinion in Microbiology, 4, 324–329. Mosier, N., Wyman, C., Dale, B., et al. (2005). Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresource Technology 96 (6) 673–686. Olsson, L., Hahn Hagerdal, B., & Zacchi, G. (1995). Kinetics of ethanol production by recombinant Escherichia coli KO11. Biotechnology and Bioengineering, 45, 356–365. Palmqvist, E., & Hahn-Hägerdal, B. (2000a). Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresource Technology, 74, 25–33. Palmqvist, E., & Hahn-Hägerdal, B. (2000b). Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification. Bioresource Technology, 74, 17–24.

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Palmqvist, E., Hahn-Hagerdal, B., Galbe, M., et al. (1996). Design and operation of a bench-scale process development unit for the production of ethanol from lignocellulosics. Bioresource Technology, 58, 171–179. Qin, Y. (1996). Process and economy of xylose production. Fine Chemicals, 3, 24–26. Reese, E., Siu, R., & Levinson, H. (1950). The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis. Journal of Bacteriology, 59(4), 485–497. Shi, J., Chinn, M. S., & Sharma-Shivappa, R. R. (2008). Microbial pretreatment of cotton stalks by solid state cultivation of Phanerochaete chrysosporium. Bioresource Technology, 99, 6556–6564. Singh, P., Sulaiman, O., Hashim, R., et al. (2010). Biopulping of lignocellulosic material using different fungal species: a review. Reviews in Environmental Science and Bio/Technology, 9, 141–151. Stephens, C., Christen, B., Fuchs, T., et al. (2006). Genetic analysis of a novel pathway for d-xylose metabolism in Caulobacter crescentus. Journal of Bacteriology, 189(5), 2181–2185. Sun, F. -B., & Chen, H. -Z. (2008). Enhanced enzymatic hydrolysis of wheat straw by aqueous glycerol pretreatment. Bioresource Technology, 99, 6156–6161. Sun, Y., & Cheng, J. (2002). Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology, 83(1), 1–11. Tan, S., & Huang, C. (2006). Research progres s on extract technics of xylose. Food Science and Technology, 12, 103–105. Ueno, Y., Otsuka, S., & Morimoto, M. (1996). Hydrogen production from industrial wastewater by anaerobic microflora in chemostat culture. Journal of Fermentation and Bioengineering, 82, 194–197. Wang, B., & Jin, Q. (2000). Handbook of organic acid fermentation production and application. Beijing: China Light Industry Press. Wang, J. (2002). Biochemistry. Beijing: Higher Education Press. Woodward, J. (1991). Synergism in cellulase systems. Bioresource Technology, 36(1), 67–75. Zhang, G. (2007). Lactic acid bacteria: fundamental, technology and application. Beijing: Chemical Industry Press. Zhang, X., Xu, C., & Wang, H. (2007a). Pretreatment of bamboo residues with Coriolus versicolor for enzymatic hydrolysis. Journal of Bioscience and Bioengineering, 104, 149–151. Zhang, Y., Ma, R., Hong, H., et al. (2010). Metabolic engineering for microbial production of ethanol from xylose: a review. Chinese Journal of Biotechnology, 26, 1436–1443. Zhang, Y., Yang, J., Lv, X., et al. (2007b). Research processes in acid hydrolysis of lignocellulosic biomass. World SCI-TECH R&D (p. 29).

Chapter 7

Microbial Fermentation Strategies for Biomass Conversion Chapter Outline 7.1 Simultaneous Saccharification and Fermentation 167 7.1.1 Characterizations of Simultaneous Saccharification and Fermentation 167 7.1.2 Inhibition Factors for Simultaneous Saccharification and Fermentation 170 7.2 Advantages of and Breakthrough in the Simultaneous Saccharification and Cofermentation Process 171 7.2.1 Simultaneous Saccharification and Cofermentation 171 7.2.2 Combination Bioconversion of Lignocellulose 172 7.3 Consolidated Biomass Processing 175 7.4 Coculture Fermentation 178 7.5 Advantages of Solid-State Fermentation 179 7.5.1 Traditional SolidState Fermentation Technology 180 7.5.2 New Clean Technology for Solid-State Fermentation 183

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7.5.3 Demonstration of Cellulosic Ethanol Fermentation 7.5.4 Application and Development Prospects of Solid-State Fermentation 7.6 Economic Analysis of the High-Solids Biomass Conversion Process 7.6.1 Particularity of Enzymatic Hydrolysis and Fermentation of Biomass 7.6.2 Mixing Enhancement of Enzymatic Hydrolysis and Fermentation of Biomass 7.6.3 Stirring Power of Enzymatic Hydrolysis and Fermentation of Biomass 7.6.4 Stirring Power of Enzymatic Hydrolysis and Fermentation at Different Solid Loadings References

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FIGURE 7.1  Different bioprocesses of biomass conversion. Simultaneous saccharification and fermentation (SSF); simultaneous saccharification and cofermentation (SScF); separate hydrolysis and fermentation (SHF); consolidated bioprocessing (CBP).

After the pretreatment of lignocellulose, the next steps include four typical biological processes: cellulase and hemicellulase production, enzymatic hydrolysis of cellulose, hexose fermentation, and pentose fermentation. If the four processes are combined, the process is very cumbersome and the production cycle becomes longer. Meanwhile, the multistep fermentation process increases equipment investment costs. The lignocellulosic ethanol production process is divided into the following subprocesses, if the aforementioned four processes are in the same reactor (Fig. 7.1). Several processes are compared, and ethanol fermentation from lignocellulose taken as the example for study of biochemical conversion of a biomass fermentation platform. Finally, the development prospects of solid-state fermentation processes are introduced. Traditional research on ethanol production from lignocellulose was generally achieved by “two-step fermentation.” Enzymatic hydrolysis was first performed and then fermentation followed. Enzymatic hydrolysis following the fermentation process has been quite successful, but from an economic and technical point of view, “one-step fermentation” with the coupling of enzymatic hydrolysis and fermentation has greater potential. The step of enzymatic hydrolysis and fermentation takes place in the same reactor. Fermentation is classified as solid or liquid fermentation, according to the matrix state. This chapter first describes “one-step fermentation”: simultaneous saccharification and fermentation (SSF), the combination of biological conversion processes, consolidated bioprocessing (CBP), and coculture fermentation technology. Then, the advantages and prospects of solid-state fermentation technology are analyzed based on the author’s research.

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7.1  SIMULTANEOUS SACCHARIFICATION AND FERMENTATION 7.1.1  Characterizations of Simultaneous Saccharification and Fermentation In order to overcome the product’s feedback inhibition, Gauss, Suzuki, and Takagi (1976) proposed that saccharification and ethanol fermentation of cellulose be conducted in the same fermenter. The SSF process is shown in Fig. 7.2. Cellulose hydrolysis and fermentation of hexoses were conducted in the same reactor, and glucose generated during cellulose hydrolysis was rapidly utilized by microorganisms. Feedback inhibition of glucose was removed, which improved the efficiency of enzymatic hydrolysis and reduced the amount of cellulase used for hydrolysis. The desired reaction apparatus is reduced, and the likelihood of contamination is also reduced. Glucose and lignin are not separated during SSF, which eliminates the loss of sugar, reduces the number of reactors, and hence decreases investment costs (by about 20%). In addition, SSF can be applied for synergy fermentation of hexose and pentose. SSF has obvious advantages in the detoxification process. Through studies on the influence factors of SSF, results showed that enzymatic hydrolysis remains a major limiting factor for SSF. The reason for this phenomenon is that the optimum temperature of hydrolysis and fermentation is inconsistent (Chen, Li, & Chen, 1999). The optimum temperature for enzymatic hydrolysis is generally about 50°C, while the optimum fermentation temperature of Saccharomyces cerevisiae is generally about 30°C. Selected thermaltolerant yeast is helpful for SSF technology. The key issue of SSF technology

FIGURE 7.2  The systems of simultaneous saccharification and fermentation (SSF) of biomass (Hahn-Hägerdal, Galbe, & Gorwa-Grauslund, 2006).

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is the choice of the most suitable yeast. Yeast such as S. cerevisiae, Candida brassicae, is commonly used during SSF study. The best cellulase for SSF technology is currently secreted from the mutagenic Trichoderma reesei strain. Since the 1970s, Natick Army laboratories and Rutgers University have used the wild type Trichoderma viride (T. viride QM6a) as a starting strain, and they also obtained the high-yielding cellulase strains of QM9414, Rut-C30, and Rut-NG-14. The species name was then changed to T. reesei, in honor of E. T. Reese. The cellulase activity of these strains has been 10–15 times higher than that of the wild strain, which is now recognized as the most dynamic cellulase. In addition to yeast and T. reesei cellulase, used for the SSF process, a number of other species and enzymes, such as the combination of cellulase enzyme produced by Thermophilic Micromonospora (Thermomonospora sp.) and thermophilic cellulose Clostridium (Clostridium thermocellum), were also studied. The most noteworthy fermentation bacteria are swimming Micromonospora (Z. moqilis). The rate of ethanol production by Zymomonas moqilis is approximately three times higher than by the yeast, and ethanol yield was also slightly higher than with yeast, with the theoretical yield up to 96%∼97%. The glucose metabolism pathway was Endurance’s pathway, one molecule of glucose would produce two molecules of ethanol, two molecules of carbon dioxide and one molecule of ATP. The optimum pH for ethanol production was 4.5–6.0 for Z. moqilis, which is in accordance with the optimum pH of cellulase production by T. reesei. Its optimum growth temperature and fermentation temperatures are 30°C. The characteristic distinguishing it from yeast is that in response to the appropriate increase in temperature or the appropriate limited nutrient, the cell will stop growing, but fermentation will still proceed as usual. For example, at 37°C, ethanol yield is the same as at 30°C, but cell growth has stopped at this temperature. This feature of Z. moqilis can further improve the conversion rate of substrate (i.e., ethanol yield). For a relatively long period of cellulose fermentation, this is important in economics. We should pay attention to two issues during the SSF process. The first problem arises if the optimum enzymatic hydrolysis temperature and the optimum yeast fermentation temperature are uniform, (they are generally 50°C and 30°C, respectively). The best temperature of cellulase produced by T. reesei was 45∼50°C, while the general fermentation temperature of yeast is less than 30°C, and yeast will stop growth and fermentation above 37°C. If fermentation is conducted below 30°C, enzyme activity will greatly reduce. Since enzymatic hydrolysis is the rate-limiting step during SSF, so reducing the SSF fermentation temperature is not a good solution. In order to do both, the SSF temperature generally used is 37∼38°C, but this still cannot produce optimal enzymatic hydrolysis and fermentation conditions. Therefore, the selection of thermal- and ethanol-tolerant yeast during SSF is an important research topic. To solve the above problem, people are studying the process and other aspects of breeding. For instance, thermal-tolerant yeast and bacteria replace traditional yeast, which increases the reaction temperature. In this way the optimum fermentation temperature approaches the temperature of enzymatic hydrolysis,

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which increases the hydrolyzing rate of cellulose. Szczodrak in 1988 selected 58 kinds from 12 different thermal-tolerant yeast genera, and the growth and fermentation ability of these yeasts at temperatures above 40°C were tested. It was found that Fabospora fragilis CCY51-1-l could convert 140 g/L glucose into 56 g/L ethanol at 43°C, in which process the ethanol conversion rate reached 74% of its theoretical value. However, when the temperature rose to 46°C, the fermentation capacity significantly decreased, and ethanol conversion was only 46% (Lv, 2009; Szczodrak & Targoński, 1988). At present, normal yeast fermented at 40∼46°C has been initially successful in breeding, as evinced in Kluyveromyces marxianus, Kluyveromyces fragilis, Fabospora fragilis, and so on. The fermentation temperature of these three strains was almost exactly the optimum temperature of the enzyme. In addition to selecting thermal-tolerant yeast, the Japanese proposed using the normal yeast fermentation method for SSF, and so this is called the “oriental fermentation method.” This method also has been relatively successful, but the fermentation time is too long. SSF was achieved by using improved devices and process. Xiao Xin from the Institute of Process Engineering proposed dispersion, coupling, and parallel system for bioconversion of cellulosic ethanol production (Xiao & Li, 2000). The installation is shown in Fig. 7.3. In the three phases of this system, saccharification, fermentation, and separation of alcohol were carried out. In the saccharification part, hydrolysis can be carried out at a higher temperature, and hydrolyzate can be separated into enzymes and enzymatic hydrolyzate through the nuclear pore membrane. The cellulase component is returned to saccharification and continues to hydrolysis, sugar continues into the fermentation segment. This will not only solve the inconsistencies of reaction temperature and fermentation temperature, but also remove the glucose inhibition effect on the enzyme. Similarly, in the fermentation part, the yeast cells and fermentation broth are separated by a membrane, the yeast

FIGURE 7.3  Ethanol production system with dispersion, coupling, and parallel sections.

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cells return and continue fermentation, and the fermentation broth can be separated for obtaining ethanol by distillation, which removes the ethanol inhibition effect on yeast. Using this system for hydrolysis of cellulose, the cellulose conversion rate reached 81%, while the final conversion rate of cellulose hydrolysis by the general procedure was 66%. The efficiency of the former is 3.9 times that of the latter. Ethanol concentration, fermentation rate and cellulose conversion yield during cellulose ethanol production by this system were 8.14%, 0.66 g/(L·h) and 80.1%, respectively, which is 1.8, 1.3, and 1.7 times the equivalent in SSF. Another incompatibility problem of microbe and enzymes should be noted during SSF fermentations. Cell lysates or cell secretions (such as proteases) could damage the cellulase, and certain components of the crude enzyme preparation may affect cell growth, as well as the use of sugar and ethanol yield. The factors of SSF should include as follows (He, 1990): 1. Enzyme concentration. The higher enzyme concentration results in higher ethanol production, especially in the low enzyme concentration range. 2. The initial concentration of yeast cells. When cell concentration is around 2.5∼10 × 107 cells/mL in inoculation, ethanol production is not affected. 3. Temperature and pH. Optimum temperature and pH are different for cellulase and yeast. 4. Pretreatment. Pretreatment of cellulosic material before hydrolysis is usually necessary. Without pretreatment, fermentation efficiency and conversion rate are often low, but the pretreatment process is often the major cause of rising costs. 5. Substrate concentration. The high substrate concentration generally results in an increase in ethanol concentration and production. But the degradation rate of the substrate decreases slightly, generally speaking, which is beneficial for the fermentation. However, it is difficult for the water to penetrate the material when the substrate concentration is high, which has made cultivation difficult. One solution is to put the substrate and enzyme into the reactor during the fermentation process, which can effectively improve the ethanol concentration. In the general SSF process, the pretreated liquid enriched with pentose was fermented alone. With the new development of simultaneous microbial fermentation of glucose and xylose, the industrial development of simultaneous saccharification and cofermentation (SSCF) (combination of bioconversion) was proposed. The sugar solution from pretreatment and the treated cellulose are put in the same reactor, further simplifying the process.

7.1.2  Inhibition Factors for Simultaneous Saccharification and Fermentation Some inhibitory factors of SSF include xylose inhibition and the uncoordinated temperatures of saccharification and fermentation. During SSF, xylose generated from hemicellulose will remain in the reaction solution. When the xylose concentration reaches 5%, the inhibition of the cellulase by xylose is up to 10%. Strains

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such as Candida yeast and Pachysolen tannophilus can convert the xylose into ethanol and this will eliminate the xylose inhibition effect. Many research projects focus on finding a strain which is able to ferment glucose and xylose at the same time. Ethanol production was increased by 30–38% and 10–30% compared with pure bacterial fermentation of glucose and xylose, respectively (Chen, 2006). The complex structure of lignocelluloses leads to the very complicated process of utilization of material by microbe. Chen, Xu, and Li (2007) studied SSF and alcohol-fed batch SSF for ethanol production from different cellulosic feedstocks. They found that lignin inhibited SSF of cellulose, and that hemicelluloses have a dual role in the SSF of cellulose. On the one hand, the presence of hemicelluloses reduces the crystallinity of cellulose, which is favorable for SSF. On the other hand, the accumulation of xylose and xylose-oligosaccharides, produced from hemicellulose hydrolysates, and which cannot be used by yeast, will inhibit the enzymatic hydrolysis of hemicelluloses and cellulose and further reduce the ethanol production rate. However, promoting the role of hemicellulose is the major effort during SSF, and the inhibition effect is obvious during the late stages of simultaneous saccharification and batch-fed simultaneous saccharification fermentation. Therefore, the removal of lignin and the retention of hemicellulose from feedstock will help improve the ethanol production rate in SSF. The hydrolysis rate of cellulose and the ethanol yield can be improved by using fermentation strains and cellulase in the same reactor. Factors include enzyme concentration, temperature, pH, substrate concentration, and pretreatment can affect the bioconversion process. In normal enzymatic hydrolysis, glucose and cellobiose produced from the hydrolysis of cellulose will inhibit cellulase. In the SSF process, hydrolysis products were continuously used by ethanol fermentation strains, resulting in reduced sugar concentration, which removed the inhibition effect. This may accelerate the hydrolysis rate, shorten the fermentation time, increase cellulose hydrolysis, and ethanol yield (converted per gram degraded cellulose to ethanol grams), and obtain a higher ethanol concentration. In addition, during the traditional saccharification fermentation process, cellobiose produced from enzymatic hydrolysis makes up a large proportion of the fermentation liquid, which cannot be generally utilized by yeast. Therefore, the ethanol conversion yield is relatively low. During the SSF process, since glucose is used continuously, the cellobiase of the cellulase system maintains a high enzyme activity, cellobiose is immediately hydrolyzed into glucose and then further used, which improves the ethanol yield.

7.2  ADVANTAGES OF AND BREAKTHROUGH IN THE SIMULTANEOUS SACCHARIFICATION AND COFERMENTATION PROCESS 7.2.1  Simultaneous Saccharification and Cofermentation The definition of microbial transformation is the conversion process of a substance (substrate) into another substance (product) by microorganism. The process is one or several chemical reactions catalyzed by one or several specific

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extracellular or intracellular enzymes produced though the microorganism. In short, this process is the integrated technology of using microorganisms, or microbial enzyme synthesis. A particular microorganism can convert a specific substrate into a specific product, which used the enzyme as catalysts. Therefore, the difference between enzymatic conversion and microbial conversion is that the former is the chemical reaction catalyzed by a single enzyme. In order to achieve this, we need to provide the conditions such that the microorganism is capable of biosynthesis of these enzymes for the latter. Therefore, from this perspective, this seems to be true biotransformation. In addition, although the enzymes for biotransformation are from the microorganisms, they can also be derived from animals and plants. For a specific biotransformation process, whether using the microbial conversion technology or the enzymatic conversion technology, many factors should be considered in its implementation, such as cost, environmental impact, technical equipment, and quality requirements. In the study of the microorganism (or enzyme) transformation process, many issues, such as substrate selection, microorganisms’ conversion capability, conversion route, or conversion reaction selection need to be considered. The most important topic is the search for suitable microorganisms and how to improve the transformation ability of this organism. The discovery of a new enzyme, or a new response, in looking at the design of a new microbial conversion process provides a clue. To find the biocatalyst that can appropriately be used as microbial enzymes, an effective approach is the screening of new strains of microorganisms or enzymes. Combined biotransformation means the transformation of a precursor compound by one or more kinds of microorganisms or enzymes with particular conversion functions to obtain a diverse chemical structure, which is an effective means of finding new derivatives from known compounds. In some ways, combined biotransformation is more effective than chemical synthesis. Simultaneous saccharification and cofermentation is a combined biotransformation process in which the hexose and pentose are converted together after lignocellulose hydrolysis.

7.2.2  Combination Bioconversion of Lignocellulose Lignocellulose is a very attractive material for industrial ethanol production. Lignocelluloses mainly include 35–50% of cellulose, 20–35% of hemicellulose and 10–15% of lignin. The hydrolysis products of lignocelluloses are rich in sugars, including glucose, xylose, arabinose, mannose, galactose. Hexoses are easily converted into ethanol by microorganism fermentation, while xylose accounting for 10–40% of the total sugar content cannot be fully utilized. Therefore, if an engineering bacterium could be found that could convert the mixed sugars into ethanol, ethanol production would increase by 25% in theory (Li, Tiao, & Pan, 2003).

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Simultaneous saccharification and cofermentation means that enzymatic hydrolysis of raw material and fermentation of hexose and pentose were simultaneous, carried out by mixed strains or a xylose metabolic engineering strain. After the pretreatment of lignocelluloses, xylose produced from hemicelluloses was not separated with glucose, which was subsequently fermented for ethanol production. Compared with the previous two processes, SSCF not only reduces the feedback inhibition of the hydrolysis products but also integrates pentose fermentation into hexose fermentation. As a result, SSCF can improve the ethanol yield and reduce the capital cost. The most important breakthrough point of SSF is the discovery of efficient strains which can convert hexose and pentose simultaneously into ethanol. The main methods may include strain selection and genetic engineering.

7.2.2.1  Strain Screening Koskinen obtained two thermophilic anaerobic bacteria, named K17 and K15. The fermentation of lignocellulose by two thermophilic anaerobic bacteria was conducted. Glucose and xylose were simultaneously used for ethanol and H2 production. The ethanol inhibition effect for strain K17 is still not significant at 4% ethanol volume fraction (Li, Zhang, & Luo, 2009; Koskinen, Beck, & Örlygsson, 2008). Ryabova et al. screened deficient vitamin B2 mutants from the Pichia yeast genus. The strain can utilize xylose and cellobiose for ethanol production at a high temperature of 45°C, which increases overall ethanol yield from lignocellulose (Li et al., 2009; Ryabova, Chmil, & Sibirny, 2003). Kim et al. utilized pretreated barley hull using ammonia, adding 3% of dextran, 4% of xylanase and 15 FPU/g of enzyme to produce ethanol through SSF by recombinant Escherichia coli KO11. Final ethanol concentration was up to 24.1 g/L, reaching 89.4% of the maximum theoretical yield (Kim, Taylor, & Hicks, 2008; Wang, Wang, & Chen, 2010). Zhang et al. established a dynamic model to predict fuel ethanol production by SSF based on S. cerevisiae RW B222 and commercial cellulase. This model describes the glucose and xylose absorption competition in the enzymatic hydrolysis reaction of cellulose and xylan (Zhang, Shao, & Lynd, 2009b). Zhang, Deng, and Lu (2009a) obtained the Klebsiella pneumoniae XJ-Li strain, which can convert glucose and xylose into 2,3-butanediol. Medium composition and fermentation conditions were optimized and simple and feasible metabolic regulation methods were explored based on five or six carbon sugar cometabolism characteristics. The results showed that taking 60 g/L glucose and 40 g/L xylose as carbon sources, 5.75 g/L NH4H2PO4 as nitrogen, 2,3-butanediol concentrations reached up to 19.24 g/L at pH 5.5 and 38°C. pH regulation and exogenous regulation by adding vitamin C were decided upon. 2,3-butanediol production was increased by 16.4% by adjusting the pH to 5.5; 2,3-butanediol production can increase by 44.3% with the addition of 60 mg/L vitamin C to regulate the redox state of the culture medium. Final 2,3-butanediol concentration reached up to 33.47 g/L in batch fermentation for 48 h.

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7.2.2.2  Gene Engineering Currently, the ability of microorganisms (mainly S. cerevisiae) to utilize pentose is poor, which has hindered the application of SSCF. This should be the main research project for SSCF (Olofsson, Wiman, & Lidén, 2010). In general, hexose is mainly glucose, while pentose is mainly xylose, in lignocellulosic biomass. In most fungal and bacterial cells, xylose can be converted into 5-pxylulose by a series of biochemical reactions. It then can produce glycolytic intermediates through the pentose phosphate pathway (PPP) and eventually produce ethanol. Xylose reductase, xylitol dehydrogenase (fungus), and xylose isomerase (bacteria) are the most crucial to this process. Genetic engineering research is focusing on importing the code gene of the first two enzymes or third enzyme into the strains, and hence strains can convert xylose into ethanol. Currently, many research studies on genetic recombination microbes have reported on the use of xylose for ethanol production. S. cerevisiae, in use as the most common ethanol fermentation strain, had a higher ethanol yield and production rate under optimal conditions (0.45 g ethanol/g biomass, and 1.3 g ethanol/g cell·h). It can tolerate the ethanol and inhibitor well. S. cerevisiae that could tolerate 100 g/L ethanol has been reported. However, the wild S. cerevisiae cannot use xylose to produce ethanol. It can only convert xylose into a small amount of xylitol. For cofermentation of glucose and xylose, Karhumaa, Sanchez, and Hahn-Hägerdal (2007) imported the encoding gene of xylose and xylitol dehydro genase into the genome of S. cerevisiae, and obtained the recombinant strains giving an ethanol production rate of 0.13 g/(L·h). Yet xylose reductase relies on the substrate NADPH, making an incomplete cycle. This leads to accumulation of xylitol in the fermentation process. Petschacher and Nidetzky (2008) used fixed point mutation technology to make xylose reductase, taking NADH as cosubstrate. Compared with the scenario with no mutation of recombinant strains, ethanol production with recombinant yeast containing mutations of xylose reductase increased by 42% with an initial xylose concentration of 20 g/L. Kuyper, Winkler, and Dijken (2004) introduced the heterologous expression of xylose isomerase in S. cerevisiae, and gene recombinant strains which showed ethanol production of 0.42 g ethanol/g xylose were ultimately obtained. Owing to glucose metabolism using the 2-keto-3-DNA-6-phosphate gluconic acid cracking process (ED), Zymomonas mobilis produces less ATP, and hence leads to less cell growth. Compared with S. cerevisiae, Z. mobilis can use more glucose to produce ethanol. Furthermore, ethanol tolerance for Z. mobilis can reach 120 g/L (Dien, Cotta, & Jeffries, 2003). The main drawback of Z. mobilis is that it is sensitive to the inhibitor, and also cannot use the pentose. Therefore, Z. mobilis is also regarded as the strain for gene engineering construction. Earlier studies mainly focused on transferring the xylose isomerase and xylitol kinase genes into Z. mobilis. However, owing to the low activity levels of transketolase and transacylase, the recombinant strain cannot grow in a culture medium with xylose as the sole carbon source. Zhang, Eddy, and Deanda (1995) imported the coding gene of xylose and the two operons of the PPP approach into Z. mobilis,

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and obtained an effective modified strain, which can ferment glucose and xylose. The National Renewable Energy Laboratory (NREL) in the USA has conducted many research studies in SSF (SSCF), and the gene modified Z. mobilis was used for cofermentation of glucose and xylose. Compared with the ethanol yield using glucose fermentation and xylose sugar fermentation strains, the ethanol yield of cofermentation increased by 30∼38% and 10∼30%, respectively. The transport protein of xylose is the same as that of glucose, but the affinity of protein with glucose is about 200 times that with xylose, so that xylose use is limited. This is another problem of recombinant strains (Kötter & Ciriacy, 1993). Therefore, the addition of a cellulose enzyme can improve xylose conversion efficiency. Olofsson et al. (2010) controlled the release of cellulose by adjusting the dosage of cellulose and the absorption of glucose by bacteria, such that the xylose absorption rate increased from 40% to 80%. In addition, competitive inhibition can be further reduced by prefermentation of glucose. Bertilsson, Olofsson, and Lidén (2009) improved the utilization efficiency of xylose by prefermentation of glucose before enzymatic hydrolysis saccharification. Öhgren, Bengtsson, and Gorwa-Grauslund (2006) found that keeping the fermented liquid glucose fermentation at low concentrations is beneficial to xylose fermentation using the feed strategy of fermentation. Chandrakant and Bisaria (1998) reported other strategies for improving xylose utilization, such as adding xylose isomerase, enzyme and cell immobilization, and inoculation of xylose fermentation microorganisms with glucose fermentation combination. Fu, Peiris, and Markham (2009) used Z. mobilis and Pichia stipitis to coferment glucose and xylose, and ethanol production reached up to 0.49∼0.50 g/g. The optimized process can be achieved through the establishment of a mathematical model. Zhang et al. (2009b) introduced the mathematical model of SSCF, and pointed out that the hydrolysis constant of cellulose in hydrolysis saccharification and ethanol production, and microbial ethanol tolerance in fermentation are important. However, these strains are mainly screened out using soluble sugar as the substrate. In this condition, the sugar concentration is higher and the strain can grow fast, which is not consistent with the SSCF process with a low sugar concentration and a high substrate concentration. A good microorganism that can ferment soluble sugar is not necessarily a good SSCF strain. A good SSCF strain not only requires cofermentation of pentose and hexose in SSCF, but also tolerates ethanol and other inhibitors. Compared with SSF, SSCF further simplifies equipment requirements and shortens the fermentation period. Thus, the research into this process is attracting more and more interest, and there are good prospects for its application.

7.3  CONSOLIDATED BIOMASS PROCESSING Consolidated biomass processing (CBP), formerly known as direct microbial conversion, can combine the process of cellulase and hemicellulase production, cellulose hydrolysis, and ethanol fermentation by a microorganism. Some

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microorganisms in nature (such as clostridium and rumen bacteria) have the capacity of direct conversion of biomass to ethanol. These bacteria are anaerobic strains, which can hydrolyze cellulose and degrade sugars to produce ethanol. Direct microbial conversion combining three processes (the production of cellulase, cellulase saccharification, and the fermentation of pentose and hexose) can reduce the reactor cost. Compared with the traditional ethanol production process, the capital cost of CBP can be reduced by 25%. At present, CBP has attracted international interest, and many scientists have launched a variety of studies. Yet resistance of strains to ethanol concentration in CBP is low, and there are a variety of by-products. Moreover, ethanol concentration and yield are low as well. The core technology of liginocellulose conversion by CBP is to breed microorganisms or strains which can directly convert the lignocellulose to ethanol and other chemical products. At present, the microorganisms in CBP include thermal fiber end spore fungus, thermal hydrogen sulfide Clostridium and a thermal anaerobic bacillus (Chen, 2007). Some strains of Clostridium and rumen bacteria (Rumen bacterium) also have such characteristics. This direct conversion process by the cellulose hydrolysis and fermentation in CBP can further simplify the fermentation process. It also reduces bacterial pollution, especially in fermentation by thermophilic bacteria. Christakopoulos, Macris, and Kekos (1989) found that Fusarium oxysporum F3, cellulase producing strains, can directly convert glucose, xylose, cellobiose, and cellulose into ethanol. The pH is 5.5 and 6 in aerobic and anaerobic phases, respectively. Ethanol concentrations were 9.6 and 14.5 g/L, corresponding to 89.2% and 89.2% of the theoretical yield, respectively (Wang et al., 2010; Christakopoulos et al., 1989). C. thermocellum is another typical strain, strictly an anaerobic thermophile. Its optimal growth temperature is about 60°C. It has a high cellulose degradation rate and growth rate, and it can directly produce ethanol from cellulose. Owing to the higher fermentation temperature, the fermentation process is not easily polluted and ethanol is easily separated by means of a vacuum. Thus, these should be beneficial in overcoming product inhibition, especially to continuous fermentation. At present, the main problem of strains for direct fermentation of cellulose to produce ethanol is poor ethanol tolerance. So, it is difficult to get high ethanol concentration in the biomass conversion process. The characteristics of wild type C. thermocellum can be summarized as follows: in 1% cellulose medium, the ethanol and acetic acid production proportion was 1:1; ethanol concentration reached 1∼2 g/L after fermentation for 24 h; the reducing sugar accumulation was 3∼4 g/L and the cellulose degradation rate was 70∼90% after fermentation for 72∼96 h. Theoretically, Clostridium thermocellum can convert one glucose molecule into two ethanol molecules. It also degrades xylose, but it cannot utilize other hemicellulose degradation products, such as mannose, arabinose, galactose (Bender, Vatcharapijarn, & Jeffries, 1985). Although the fermentation performance of wild type C. thermocellum strains is poor, the fermentation process can be improved through mutagenesis,

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domestication and breeding methods, as well as the improvement of technological and culture conditions. For example, the ethanol tolerance of S-7 strains is improved through domestication mutagenesis at Massachusetts Institute of Technology. Ethanol concentration increased from 0.8% to 6.5%. The ratio of ethanol to acetate in products increased from 1:1 to 5:1. Ethanol concentration was more than 5 g/L and the reducing sugar accumulation was 24 g/L after fermentation for 40 h under the condition of substrate concentration of 40 g/L and pH adjustment. A change in process conditions can significantly alter fermentation results. Experimental results at Cornell University showed that the high sugar concentration of continuous fermentation can dramatically increase the ethanol concentration (Zertuche & Zall, 1985). In 2005, Balusu et al. used Clostridium cellulolyticum SS19 for anaerobic decomposition of cellulose to produce ethanol. Ethanol production is 0.41 g/g substrate while the yield was 81% when the concentrations of medium filter paper, corn starch, cysteine hydrochloride, and ferrous sulfate were 4.5, 8.0, 0.25, 0.01 g/L, respectively (Wang et al., 2010; Balusu, Paduru, & Kuravi, 2005). Yuan, Ren, and Ren (2008) obtained Kluyveromyces marxianus YX01, which performed well in ethanol fermentation and inulinase production. The new technology of one-step ethanol fermentation was evaluated by combining enzyme production, saccharification, and ethanol fermentation taking inulin as substrate. The fermentation was also carried out taking Jerusalem artichokes as material to directly produce ethanol. The optimal fermentation temperature was investigated in a shake flask, and ventilation and the effect of substrate concentration was also investigated in the 2.5 L fermentation tank. Experimental results showed that the optimal fermentation temperature was 35°C. In ventilation for 50 and 100 mL/min, the strain grew faster, and the fermentation time was shortened, while the sugar alcohol conversion rate increased significantly under stuffy conditions. When the concentration of inulin was 235 g/L, ethanol concentration was 92.2 g/L and ethanol yield was 0.436 g ethanol/g sugar, which was 85.5% of the theoretical value. On this basis, the Jerusalem artichokes planted in coastal tidal flats by sea water irrigation were used as the substrate, with direct fermentation of Jerusalem artichoke powder at a concentration of 280 g/L in a batch type feeding method obtained 84.0 g/L ethanol concentration, and ethanol yield was 0.405 g ethanol/g sugar, which was 80.0% of the theoretical value. CBP metabolic engineering can also be obtained using the following two methods: 1. Use cellulolytic or genetically engineered strains to produce ethanol. In 2010, Steen, Kang, and Bokinsky (2010) published an article in Nature, on how they modified E. coli by genetic engineering and metabolic engineering making it produce fatty acid esters, fatty alcohols and wax, etc. from biomass sugars. They also introduced the hemicellulase gene into the engineered strain, making it convert hemicellulose directly into final purpose synthetic products.

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2. Screening strains. In 2002, a new type of Clostridium phytofermentans was selected from the soil by the University of Massachusetts (Blanchard, Leschine, & Petit, 2009), and named as Q bacteria. This strain can use a variety of biomass raw materials. At the same time, the ethanol conversion rate was high, it had fewer by-products, and its ethanol tolerance was high. The group further identified that the degradation mechanism of cellulose fiber by Q bacteria was different from that of filamentous fungi, and this is expected to bring a breakthrough in the research of CBP. Overall, CBP is helpful in reducing the cost of the bioconversion process, which is attracting more and more attention in the research field (Xu, Shen, & Bao, 2010).

7.4  COCULTURE FERMENTATION Pure culture fermentation of cellulose hydrolysis strains further simplifies the process, but it has some shortcomings, as follows: 1. The slow growth rate of the strain and the long fermentation period. Especially under strictly anaerobic conditions, the delay phase of the strain in pure culture is very long. 2. The ethanol yield is low. The accumulation of reducing sugar is high at the end of fermentation. Some strains cannot use any other reducing sugar except for glucose. 3. The ethanol concentration is low, and the cost of product separation is high. For these reasons, pure culture fermentation by an anaerobic strain is not efficient in producing ethanol. One solution is to use mixed culture fermentation. This method involves using a strain to hydrolyze cellulose, and having its hydrolysis product used by another fermentation strain to produce ethanol. Culture fermentation using a cellulose hydrolysis strain combined with a glycolysis strain can further improve the hydrolysis fermentation rate, cellulose conversion, and ethanol yield and concentration. The key requirements of fermentation are to control oxygen content in the culture medium, achieve a high substrate concentration, reduce by-products, and facilitate strain breeding. Currently, the best-known strain used in cellulose hydrolysis fermentation is C. thermocellum. For example, the cocultivation of this strain and another thermophiles, Clostridium thermosaccharolyticum can improve the conversion performance of biomass. The former can convert the cellulose into glucose and cellobiose, and can also convert xylan into xylose. Cellobiose and glucose can be used by C. thermocellum. Although C. thermosaccharolyticum cannot hydrolyze cellulose, it can use the hydrolysate of cellulose, hemicelluloses, and cellobiose. Because natural cellulose is associated with a large number of hemicelluloses (its main ingredient is xylose), both strains can fully use natural cellulose material. The mutation strains of C. thermosaccharolyticum HG-4 obtained by Massachusetts Institute of Technology can generate a 4:1 ratio of ethanol and acetic acid. Because of the immediate use of reducing sugar

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without any reducing sugar accumulation in the fermented liquid, coculture fermentation accelerates the rate of cellulose degradation and increases ethanol concentration and yield. In the coculture fermentation system, the factors to be considered were as follows: (1) pH control in the process; (2) strictly anaerobic conditions; (3) high substrate concentration and small number of by-products. Coculture fermentation greatly simplifies the two-step fermentation process. It also makes up for some deficiencies of pure culture fermentation. Owing to the fast growth and metabolism of a strain, especially with thermophiles, the fermentation process was further accelerated. It therefore has the advantage of overcoming the long fermentation period of cellulose. Thermophiles also have the advantages of preventing bacterial contamination and reducing product cost. Therefore, this is an increasingly popular research area in cellulosic ethanol fermentation. The common characteristic of the aforementioned processes is that the traditional two main steps in the process of cellulose hydrolysis and fermentation are combined into one step. At the same time, enzyme recycling is also omitted. So the process is greatly simplified. Simplifying the process can not only reduce the production cost, but also decrease the risk of contamination (He, 1990).

7.5  ADVANTAGES OF SOLID-STATE FERMENTATION Fermentation technology includes submerged fermentation (SmF) and solid-state fermentation. The history of solid-state fermentation dates from several thousand years ago. Solid-state fermentation technology was used to prepare fermented foods thousands of years ago by the Chinese. Research on solid-state fermentation in organic acids, alcohol, biologically active substances, flavor compounds, and other classes of compounds has been developing rapidly in recent years, but these studies are in the laboratory research stage. Since 1945 and the success of penicillin liquid fermentation, liquid fermentation technology has developed fast. Pure solid-state fermentation (single strain) had been difficult to achieve in the past, while it was easy to implement in liquid fermentation. Solid-state fermentation can only be done with small machinery, even a few cubic meters is more than the size of the devices used for solid-state fermentation. It is difficult to amplify this to hundreds of cubic meters. However, with time, liquid fermentation also encountered obstacles. The large wastewater requirement is hindering its further development. Liquid fermentation requires a lot of water, and the concentration of products in the fermentation tank is often less than 10%. China has become the world’s largest fermentation industrial country. China is a water-scarce country, and more than two thirds of Chinese cities are facing a water crisis. In recent years, two thirds of rural areas are also experiencing some drought. In order to solve the oil crisis, the Chinese government is paying more attention to the fermentation industry, including ethanol, and other fuel and chemical fermentation. The fermentation industry will be further expanded by the fermentation of renewable biomass

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to biofuels. Therefore, the water shortage for fermentation becomes a problem, which can be solved by bioengineering works. Solid-state fermentation is an effective method for solving the current crisis. Solid-state fermentation can utilize industrial and agricultural waste, and municipal solid waste to produce value-added products without wastewater discharge. It is expected to make an important contribution to sustainable development. Compared to liquid fermentation, the disadvantages of solid-state fermentation are inefficient mass transfer and heat transfer. It is difficult to achieve large-scale industrial production of solid-state fermentation. The main reasons for this include easy contamination, low substrate utilization rate, lack of a mass transfer and a heat transfer model, and difficult reactor design. In addition to China, solid-state fermentation was also studied in Japan and some European countries. Overall, more work was done in strain breeding and mixed strains fermentation. The development of reactor and single strain fermentation still need further research.

7.5.1  Traditional Solid-State Fermentation Technology 7.5.1.1  Advantages and Disadvantages of Traditional Solid-State Fermentation Technology Although solid-state fermentation has unique advantages compared with liquid fermentation, there are also many deficiencies in solid-state fermentation. By limiting technological development in the past for a long period of time, solid-state fermentation technology has stayed in a relatively primitive state. Even in modern industrial production, people still use traditional solid-state fermentation technology. Traditional solid-state fermentation is synonymous with backwardness in industrial fermentation processes. Even in textbooks or biochemical fermentation engineering works, solid-state fermentation is rarely mentioned. Key conditions of modern fermentation technology are pure culture and large-scale fermentation. In the past decade, research into and application of solid-state fermentation technology has been developing rapidly. With the impact of science and technology and sustainable development, domestic and international attention on the gradual development of solid-state fermentation research has made great progress. Solid-state fermentation has been divided into traditional and modern solidstate fermentation. These are the advantages of solid-state fermentation: 1. The medium is cheap. Cereals, wheat bran, and other agricultural products can be used. 2. Special products can be produced. For example, red pigments, whose production is enhanced by solid-state fermentation. 3. The purification, recovery, and disposal of downstream processes in solidstate fermentation are usually simpler than in liquid fermentation.

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4. Solid-state fermentation can produce food with a special flavor and improve nutritional value. For example, tempeh can be used as a substitute for meat, and its amino acid and fatty acids can be easily digested. 5. There is no wastewater discharge in solid-state fermentation. There are some deficiencies in solid-state fermentation. Solid-state fermentation is a near-natural state fermentation, and there are many differences between it and submerged fermentation (SmF), in which the most significant feature is the low water activity and nonuniformity. Cell growth, and secretion of absorbed nutrients and metabolites are not uniform throughout, so that the detection and control of the fermentation parameters are more difficult. A number of biosensors used in liquid fermentation cannot be applied to solidstate fermentation. It is difficult to achieve large-scale industrial production of solid-state fermentation. The main reasons include easy contamination, low substrate utilization rate, the lack of solid-state fermentation reactor design, and so on.

7.5.1.2  Difficulties of Lignocellulosic Biomass Utilization Solid-state fermentation generally uses agricultural waste, such as wood cellulose and other biomass resources, as fermentation substrate. Lignocellulose is very cheap, being the main by-product of agriculture. However, only a few microorganisms have the capacity for cellulose hydrolysis. Because of the arrangement of the supramolecular structure of lignocellulose, it is very difficult to use. This difficulty is not only because of the tight cohesion of cellulose and lignin, but also because the cellulose molecular crystal structure of cellulose fibers in their base location. Popular lignocellulosic substrates include wheat straw, corn stover, rice straw, wheat bran, sugarcane bagasse, and wood. Studies aim to increase the protein content of the matrix as a ruminant animal feed, or to produce cellulase or other enzymes. 7.5.1.2.1  Enhancing Pretreatment of the Cellulosic Hydrolyzate Of the aforementioned substrates, only bagasse does not need to be pretreated, because it has been well broken in the process of pressing. Others require a variety of pretreatments. Wheat bran is pretreated at 121°C for 15–30 min. Corn, rice, and wheat straw will need to be pretreated under certain conditions. Steam treatment (150∼200°C, 10∼30 min) works very well in promoting solid-state fermentation for microbial cellulose degradation. Combinations of NaOH treatment with high-pressure cooking and steam treatment have similar effects. This pretreatment can dissolve hemicellulose and lignin, while the cellulose crystalline regions are damaged. This can greatly increase the cellulase enzyme’s contact with the cellulose. Several common pretreatment methods were compared and the results clearly indicate that these methods promote the degradation of cellulose, but the difference between these methods is relatively small.

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In these studies, most of the lignocellulosic substrate is ground to particles about 1 mm in diameter. This increases the specific surface area of the substrate and can produce broken cells. The broken cell walls and the exposed inner surface of the biomass are more vulnerable to microbial degradation. The actual exposed surface area of the cellulose fiber to the total amount of actual existence of cellulose has more sense. Moreover, the exposed surface area must be more sensitive to microbial attack. The pore size present in the lignocellulosic matrix is an important factor influencing the accessibility of cellulose to cellulase enzymes. Increasing the pore size of the matrix is also an important object of the pretreatment. Not all of the surface areas have the same sensitivity for the cellulase. Generally, the inner wall is more vulnerable to attack by enzymes than the outer layer. This explains why an attack on microbial cells always starts from the inside of the cell wall. If the pore diameter of the substrate is sufficiently large, reducing the particle diameter of the matrix is no longer efficient, because such a substrate already has a sufficiently large accessible surface area. At this time, the rate of degradation is influenced mainly by the mass transfer rate in the microporous intermediate. A disadvantage of the substrate after polishing is that it may lead to compaction within a small space and a poor gas diffusion substrate. 7.5.1.2.2  Strengthening the Lignin Degradation Process In recent years, the use of lignin in solid-state fermentation has begun to receive attention with respect to the use of lignocellulosic biomass. Its purpose is to produce feed for ruminants. The aim of the process is that the lignin is easily removed while the other matrix components, such as cellulose and hemicelluloses, are kept with rumen microbes. However, there are usually losses of cellulose and hemicellulose in this process. Recent studies have focused on white-rot fungus, which is a kind of Basidiomycetes. Key factors affecting lignin degradation are temperature, pH, water content, and the concentration of carbon dioxide and oxygen. Generally, carbon dioxide suppresses the degradation of lignin, while oxygen promotes its degradation. Oxygen and nitrogen may be the important factors influencing the utilization of the lignin and nonlignin matrices (mainly cellulose and hemicellulose). Some studies suggest that 50% of the oxygen concentration may stimulate the preferential removal of lignin. However, the effect of 100% oxygen concentration on the lignin removal is only a slight improvement over that of air. Generally, the addition of nitrogen can promote the growth of microorganisms and improve the nonlignin matrix conversion while inhibiting the use of lignin. In general, the degradation of lignin represents a week’s delay in the solidstate fermentation process. The incubation time will be prolonged to about two weeks, or even eight weeks or longer. In the solid-state fermentation of a matrix comprising cellulose, some easily degradable substrate is lost. A lignin degradation test showed that the digestibility of straw was significantly improved.

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Lignocellulose is often ground into particles of 1–2 mm diameter. This facilitates the destruction of the matrix structure and the increase of surface area. Acid and alkali pretreatment is generally used to promote the use of cellulose. If its purpose is to promote the degradation of lignin, acid and alkali treatment is generally not used. In fact, even cooking can inhibit lignin degradation. Cooking can increase the effective nutrient content while suppressing the use of lignin. Adding a small amount of sugar may be helpful in lignin degradation, because lignin metabolism is a cometabolism rather than simply as a carbon and energy source. It was found that the addition of different monosaccharides, such as glucose and xylose, stimulates the hydrolysis of lignin and cellulose degradation. Xylose can shorten the lag phase and reduce the use of hemicellulose. Under certain circumstances, appropriate treatments and simple pretreatments are necessary, especially considering the solid waste generated in a tropical environment.

7.5.2  New Clean Technology for Solid-State Fermentation 7.5.2.1  Gas Double-Dynamic Solid-State Fermentation Owing to the poor heat and mass transfer performance of traditional solid-state fermentation, a new gas dynamic fermentation process designed based on the results of the basic solid-state fermentation research was proposed. In this process, mechanical agitation was not included, and the gaseous phase of the solid-state fermentation process was only controlled. On the one hand, the pressure pulsation was rising and falling; on the other hand, the gas phase in the reactor was flowing with the progress of fermentation, which improved heat transfer and oxygen transfer in the solid-state fermentation process, promoted cell growth and metabolism and achieved pure culture. Solid-state fermentation technology has the advantage of saving water and energy, but it is difficult to overcome the large heat and mass transfer resistance problem, which easily leads to the death of localized cells. Meanwhile large-scale pure fermentation was difficult to achieve. The main features of gas double-dynamic solid-state fermentation are as follows: 1. Gas double-dynamic solid-state fermentation is different from traditional solid-state fermentation. It uses the method of pressure pulsation cycle change to enhance the solid-state fermentation. Through the study of the pressure pulse period, the gas distribution plate, and recycling rates, gas double-dynamic solid-state fermentation effectively strengthens heat transfer and oxygen transfer in the solid-state fermentation reactor, eliminating the temperature gradient of the solid bed and avoiding localized cell death; 2. on the basis of pressure pulsation, air exchange between multiple fermentation reactors was realized, forming a gas phase double dynamic (pressure pulsation and air circulation), which further improved the temperature and humidity distribution in solid-state fermentation. So cell growth and metabolism have been optimized;

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3. the supporting device and sterile inoculation operation method was invented to suit gas double-dynamic solid-state fermentation. This solved the bacterial contamination problem in large-scale operation of the solid-state fermentation process, and so achieved a pure fermentation; 4. the 50 m3 and 100 m3 production scale of the gas double-dynamic solid-state fermentation reactor was developed, and used in the production of cellulase, bassiana, Metarhizium, Bacillus thuringiensis, and other preparations. Compared with traditional solid-state fermentation, product yield increased more than two times. Compared with liquid fermentation, energy consumption decreased by 87.5% at the same yield, while avoiding a lot of organic wastewater emissions; 5. semicontinuous extraction solid-state fermentation, adsorption carrier solidstate fermentation, tobacco aging fermentation, periodic stimulation solidstate fermentation, and gas-coupled solid-state fermentation system were invented. The gas double-dynamic solid-state fermentation technology was extended to develop the fermentation technology for saving water suited to China’s water-shortage prone regions. In recent years, the Institute of Process Engineering, Chinese Academy of Sciences has cooperated with a number of enterprises to achieve the scale-up of the gas double-dynamic solid-state fermentation reactor. The industrial production processes of cellulase, anisopliae, Metarhizium, B. thuringiensis, and other preparations were established using the gas double-dynamic solid-state fermentation reactor. According to statistics, sales revenue (as at December 2006) amounted to 384,495,700 Yuan, of which profits and taxes reached 133,094,900 Yuan. Fermentation time was shortened by about one third by gas double-dynamic solid-state fermentation technology. Variable temperature operation can increase bacterial activity, which also plays a role in the optimization of complex bacteria combinations. Therefore, this offers a new method of fermentation technology improvement for traditional liquor, food flavoring, red koji, pectinase, feed additives, tobacco aging, and biogas production. Gas double-dynamic solid-state fermentation technology was different from the traditional solid-state fermentation technology and modern SmF technology. Correspondingly, the large-scale pure solid-state fermentation technique was called modern solid-state fermentation technology. The essential differences between gas double-dynamic solid-state fermentation technology and traditional solid-state fermentation technology are that for gas double-dynamic solid-state fermentation technology, a pure culture in the strict sense was achieved, the scale of industrial production was realized, periodic stimulation increased the fermentation titer by 3–5 times, temperature, humidity and pH was controlled during the fermentation; and no waste was discharged; although the investment of equipment was much higher than for traditional solid-state fermentation, it was much lower than for SmF. The successful development of gas double-dynamic solid-state fermentation technology marks the maturation of modern solid-state fermentation technology.

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With further improvement and refinement of the new gas double-dynamic solidstate fermentation technology system, it will break the dominance of SmF technology. According to existing practice test and production results, this modern solid-state fermentation technology can apply to large-scale pure cultivation of bacteria, fungi, or actinomycetes. Modern solid-state fermentation technology has broad application and unlimited prospects, which can be broadly grouped into the following three aspects. 1. Break the monopoly of SmF in modern fermentation industry technology There are many existing liquid fermentation production processes, which can be replaced by this modern solid-state fermentation technology. Typical examples include Bt fermentation, cellulase fermentation, pectinase fermentation, nitrogen-fixing bacteria fermentation, GA fermentation, and riboflavin fermentation. Almost the whole enzyme preparation can be produced by solid-state fermentation. There are also other agricultural antibiotics, organic acids, etc., which can be produced by solid-state ­fermentation. 2. Open up new biotech industry A typical example is seen when we consider bassiana, and Metarhizium fermentation bacteria, which do not produce spores in liquid; while spores would generate in solid-state fermentation. Therefore, modern solid-state fermentation technology is suitable for these strains. More importantly, the bioconversion of lignocelluloses with the full use of biomass is a major issue for human sustainable development. Modern solid-state fermentation technology will eventually overcome these technical and economic issues. It can create many new industries: (1) the cleaning industry; (2) the efficient organic fertilizer industry; (3) the bacterial protein feed industry; (4) the feed additives industry. 3. Improve solid-state fermentation technology Fermentation time was shortened by about one third by gas double-dynamic solid-state fermentation technology, and variable temperature operation can often increase bacterial activity. Therefore, it offers a new kind of fermentation technology to improve the traditional liquor, food flavor, red koji, pectinase, feed additives, tobacco aging, and biogas production.

7.5.2.2  New Technology of Straw Solid-State Hydrolysis and Fermentation Coupling Ethanol Separation In the process of “enzymatic hydrolysis for cellulose ethanol,” there is a significant product inhibition effect of cellulase. solid-state fermentation can solve the problem. However, the solid-state fermentation of cellulose has the disadvantages of low glucose and ethanol concentration, high cellulase dosage, large water use and difficult comprehensive utilization. Solid cellulose hydrolysis coupled with liquid fermentation technology can substantially improve the efficiency of cellulose hydrolysis and ethanol

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fermentation and reduce the cost of cellulosic ethanol. China registered patents for this technology in 2003. Airlift ethanol fermentation coupling separation technology is proposed based on the combination of gas-liquid dual circulation tower fermenter, vacuum reflow, CO2 stripping, circulation and mixing, and activated carbon adsorption. It has the following advantages: reduction of excess free cellulase dosage in liquid; reduction of wastewater in solid-state hydrolysis and fermentation; saccharification and fermentation is conducted in a different area in the same reactors, facilitating the optimum temperature for saccharification and fermentation; the achievement of a combination of the saccharification–fermentation–ethanol separation processes, reducing the cost of ethanol production from straw.

7.5.3  Demonstration of Cellulosic Ethanol Fermentation Lignocellulose is the most abundant renewable resource on earth, and can be derived from industrial and agricultural waste, forestry waste and urban waste (Liu, Qin, & Pang, 2013; Qin, Liu, & Jin, 2013). Fuel ethanol from lignocellulosic biomass has attracted attention from all over the world. In the United States, Verenium Corporation has the first demonstration factory of cellulosic ethanol, with an annual output of 5,299,000 L ethanol; it was put into operation in 2008. In addition, a demonstration project of the industrialization of cellulosic ethanol, supported by the US Department of Agriculture and Energy, was established by Abengoa using maize straw as raw material, Broin Companies using the whole corn (including straw) as the raw material, and Iogen Corporation using wheat straw as raw material (Du & Feng, 2009). On April 12, 2011, Poul Ruben Andersen, then bioenergy marketing director of Novozymes, said that a cellulosic ethanol factory with 40,000 tons annual production capacity was expected to be completed in 2012 by its partner M&G Group of Italy. The factory takes wheat straw, energy crops, and other biomass as raw material for ethanol production. This was the first commercial scale production facility not only in Europe, but in the world, marking the commercialization stage of cellulosic ethanol from after laboratory and formal pilot stage. Fuel ethanol in China has had a late start, but its development is rapid. At present, China is the world’s third largest fuel ethanol producer after Brazil and America. China has made a number of key technical breakthroughs in the technology research and industrialization of cellulosic ethanol. In 2008, an ethanol project with an annual output of 5,000 t straw was completed and started in trial operation by Henan Tianguan Group, including a device with an annual output of 10,000 t cellulase and 5,000 t ethanol, respectively. East China University of Science and Technology began to study fuel ethanol technology of agricultural and forestry wastes from the “eight to five” period, and a pilot plant with an annual output of 600 t acid hydrolysis for cellulosic ethanol production has been built, identified by the Ministry of Science and Technology. The project uses sawdust and rice husk as raw materials, and the cost is around 6000 Yuan/t.

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FIGURE 7.4  5 m3 steam explosion reactors.

Chen Hongzhang has been committed to research into cellulose conversion for the past ten years. Very fruitful research projects on solid-state fermentation technology industrialization, straw composition separation, and total biomass utilization were conducted by Chen. A demonstration engineering production line with an annual output of 3000 t of straw fuel ethanol and its comprehensive utilization system were established by Chen with the Corporation of Shandong Zesheng Bioengineering Technology Co., Ltd in 2006. The nonpolluting steam explosion technology, solid-state fermentation of cellulose, high concentration fermentation of straw cellulose, and ethanol separation process were coupled in this project, and 110 m3 ethanol fermentation project (expected annual production of ethanol 3000 t) was established. The production line consists of a 5 m3 steam explosion tank (Fig. 7.4), a 100 m3 cellulase solid-state fermentation tank (Fig. 7.5), a 110 m3 solid-state fermentation device (Fig. 7.6), and an ethanol fermentation coupled adsorption tower (Fig. 7.7). The operating results of the whole process showed that the ethanol yield passed 15%, ethanol concentration after activated carbon adsorption and desorption was above 69.8%, the straw cellulose conversion rate was more than 70%, and the ethanol production cost was about 4200–5000 Yuan/t. This project provided industrial-scale amplification parameters for a million tons fuel-grade ethanol production by straw enzymolysis and fermentation. Ethanol production from straw lignocellulose is a recognized technical problem, but is also one of the most promising technologies. At present, international research is in the planning stage for a kiloton cellulosic ethanol demonstration project. Chen Hongzhang et al. have established a demonstration project for the “new technology of straw enzymolysis and fermentation fuel ethanol and its industrialization.” A nonpolluting straw steam explosion system was created with independent intellectual property rights; a new technology of gas double-dynamic solid-state fermentation was also established. The operation of the demonstration project confirmed that the fuel ethanol production line is feasible, laying the foundation for large-scale, industrialized, low-cost production of fuel ethanol.

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FIGURE 7.5  100 m3 Celluloses solid-state fermentation reactors.

7.5.4  Application and Development Prospects of Solid-State Fermentation Research into solid-state fermentation of organic acids, ethanol, bioactive substances, flavoring substances, and other compounds has developed rapidly in recent years, but it is still at the stage of laboratory research (Chen, Liu, & Dai, 2014). At present, many industrial and agricultural residues and urban garbage have become a public nuisance, with adverse effects on the environment. As the understanding of the solid-state fermentation mechanism deepens, these materials could be transformed for useful or harmless products by modern

FIGURE 7.6  110 m3 Solid-state simultaneous saccharification and fermentation reactors.

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FIGURE 7.7  Ethanol fermentation adsorption tower.

solid-state fermentation technology. It can be helpful in solving environmental problems and addressing the resource crisis. As with cellulose ethanol fermentation, alcohol production, using nonfood crops and crop straw lignocellulosic materials as a fermentation substrate, by solid-state fermentation can not only solve the food crisis and reduce production costs, but also reduce waste water discharge. At the same time, the fermentation residue can be used as feed. To meet economic and environmental considerations, solid-state fermentation uses lignocellulosic biomass as the main substrate and this has practical significance. The insufficient mass transfer and heat transfer, easy contamination, low utilization rate of substrate, noncomplete detection means and other shortcomings hindered the development of solid-state fermentation. In view of the aforementioned problems, the development trend of solid-state fermentation has these aspects: 1. Accumulated products of solid-state fermentation form feedback inhibition for fermentation strains, so the tolerance of solid-state fermentation strains should be high. The strains, screening and breeding need to be strengthened; 2. Mixed fermentation by limited strains. Natural fermentation is not easy to control, and its strains are complex, product quality and yield are unstable, and fermentation efficiency is low. Pure fermentation is easy to contaminate. Combination fermentation by different strains can not only enrich the types of fermentation strains, but also could screen functional strains of flora through interactions between the strains.

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3. For the development of a solid-state fermentation reactor, enhancement of heat and mass transfer should be further studied. Scale-up and engineering of the solid-state fermentation reactor should also be paid attention. At the same time, it is important to transform the traditional extensive type reactor to the precise control type. 4. Separation technology. The study of separation technology, especially the research on online separation technology, should be systematically conducted. It has substantial significance, especially in the aspect of relieving product feedback inhibition.

7.6  ECONOMIC ANALYSIS OF THE HIGH-SOLIDS BIOMASS CONVERSION PROCESS Lignocellulose as raw material to produce fuel ethanol has been increasingly popular because it is widely available, environmentally friendly, and reduces the dependence on fossil fuels (Chen & Liu, 2014; Modenbach & Nokes, 2013). The production process of ethanol from lignocellulose mainly includes three unit operations: pretreatment, hydrolysis, and fermentation. As described in this chapter, the technology for producing fuel ethanol from lignocellulose consists of different processes, mainly solid-state fermentation, SSCF, separate hydrolysis and fermentation (SHF), and CBP. The main reason for the difficulty of fuel ethanol industrial production from lignocelluloses is the high economic cost of the conversion process. Generally the cost of production is divided into four parts: equipment and running costs, the cost of raw materials (including biomass raw materials and a variety of chemicals, etc.), personnel, operating cost, and transportation costs of different materials. Equipment, especially running cost (mainly operating energy consumption), is the main factor influencing the fuel ethanol production scale.

7.6.1  Particularity of Enzymatic Hydrolysis and Fermentation of Biomass Biomass straw conversion has its own particularity in the biological conversion process of biobased products, so the corresponding mode of mixing and energy consumption has its own requirements (Modenbach & Nokes, 2013; Chen & Liu, 2014; Modenbach & Nokes, 2012). During traditional SmF, the fermentation substrates exist in liquid form or as very small particles. In the biomass conversion process, biomass is in the form of solids. On one hand, in a relatively low ratio of solid to liquid, it presents a state with larger solid particles suspended in the liquid; on the other, with a higher ratio of solid to liquid, the reaction system entirely presents a solid state. So the method for strengthening the biomass conversion process is not the same as that of the traditional SmF. The comprehensive analysis of strengthened methods and energy consumption in enzymatic hydrolysis and the fermentation process of biomass straw will help to achieve an efficient production process.

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7.6.2  Mixing Enhancement of Enzymatic Hydrolysis and Fermentation of Biomass The power of the agitator is closely related to the flow state of the fluid in the stirred tank. Therefore, all factors that affect fluid flow state also influence the stirring power. The factors can be roughly divided as follows: the geometric parameters of the stirrer, operating parameters of the stirrer, stirring tank geometric parameters, and physical parameters of the medium. However, the main classification includes the geometric parameters of oars and slot, and the operating parameters affecting the physical properties of liquid. The reason for mixing power consumption is to overcome the resistance of the fluid. The output power of the mixer P0 is associated with the following factors: the diameter of the reaction tank D (m), the diameter of the mixer d (m), the height of the liquid column HL(m), the stirring speed n (r/min), the liquid viscosity µ (Pa·s), the liquid density ρ (kg/m3), the gravitational acceleration g (m/s2) and the form of agitator and the structure of the reactor. Because the diameter of the reactor and the height of the liquid have a certain proportional relationship with the diameter of the mixer, it meets the following equation: P0 = f (n, d , ρ , µ , g) x

 ρ nd 2   n 2 d  P0 = Kρ n3 d 5   µ   g  Because N p =

y

P0 , we can obtain the equation: ρ n3 d 5 N p = K Re x Fr y

Where Np is a numeral for stirring power; Re is the Reynolds number; Fr is the root number; x and y are undetermined coefficients.

7.6.3  Stirring Power of Enzymatic Hydrolysis and Fermentation of Biomass During the enzymatic hydrolysis process, we assume that the process was carried out in a vertical circular groove without a baffle slot and the impeller is the futaba impeller, depending on the equation of Nagata Shinji: p



b

 103 + 1.2 × Re 0.66   H   0.35+ D  P A N p = 30 5 = + B 3 (sinθ )1.2    10 + 3.2 × Re 0.66   D  ρn d Re 2   b d  Where A, coefficient, A = 14 +   670  − 0.6 + 185  ;  D  D  

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B, coefficient, B

2   d   d 1.3− 4  − 0.5 −1.14    D D   = 10 ;

2   b d   b  P, index, p = 1.1 + 4   − 2.5  − 0.5 − 7    ;      D  D D  B, the width of the impeller, m; H, the depth of the liquid layer, m; D, agitation tank diameter, m; d, mixer propeller diameter, m; θ, folding angle of leaf blade, θ = 90° for oars.

1. If b/D ≤ 0.3, P is small, and can be neglected; 2. During the enzymatic hydrolysis process, the solid–liquid mixture is high viscosity fluid, and the flow state of the mixing fluid is the laminar flow. Re value is small, so the first item of the equation of Nagata Shinji is dominant in the formula, and the second item is too small, and can be neglected. So the formula can be presented as follows: Np =

P0 A ≈ 3 5 ρn d Re

P0 ≈

A ρ n 3 d 5 ≈ Aµ n 2 d 3 Re

During the enzymatic hydrolysis process, the diameter of the reaction tank D (m), the diameter of the mixer d (m), the height of the liquid column HL (m), and the form of the agitator and the structure of the reactor are each a certain value. Stirring speed n (r/min), the mixing properties of the medium, liquid viscosity µ (Pa·s), and liquid density ρ (kg/m3) are the main factors. The fluid medium has different properties according to the different raw materials and the ratio of solid to liquid. During the enzymatic hydrolysis process, stirring speed is a certain value, therefore the stirring power is a function of the viscosity of the fluid. During the enzymatic hydrolysis process, the solid–liquid mixture can be regarded as solid particles suspended in the liquid, it is high viscosity fluid and consists of heterogeneous systems. Generally, under the condition of high solid–liquid ratio, the mixing liquid flow state is laminar flow. Average viscosity µm can be used to present the original liquid viscosity, and we set the ε as the ratio of solid particles to liquid volume. If ε ≤ 1, µm = µ(1 + 2.5ε); If ε ≥ 1, µm = µ(1 + 4.5ε).

7.6.4  Stirring Power of Enzymatic Hydrolysis and Fermentation at Different Solid Loadings During the enzymatic hydrolysis process, the solid–liquid mixture is high viscosity fluid and consists of heterogeneous systems. With the increase in solid

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TABLE 7.1 Stirring Power of Enzymatic Hydrolysis and Fermentation of Biomass at Different Solid Loadings The mass ratio of solid to liquid (%)

The volume ratio of solid to liquid (ε)

Average viscosity (µm)

Increased power (%)

Glucose concentration (g/L)

Ethanol concentration (g/L)

5

0.26

1.65

—

22

10

10

0.56

2.38

44

44

20

15

0.88

3.21

94

66

30

20

1.25

6.63

300

88

40

25

1.67

8.50

415

110

50

30

2.14

10.64

545

132

60

Increased power levels at different solid loadings were compared with that at 5% solid.

loadings, fluid properties obviously change. The bulk density of agricultural straw is about 0.04–0.20 t/m3. During the enzymatic hydrolysis process, the average viscosity changes with the solid–liquid ratio, and the required power can be calculated as in Table 7.1. Increased power levels at different solid loadings were compared with the glucan conversion and the ethanol yields, based on 80% and 90% of theoretical values, respectively. During the enzymatic hydrolysis process, viscosity increased with the increase of solid loading, and hence the power increased. Solid loading is between 5% and 15% during traditional enzymatic hydrolysis. Table 7.1 shows that the viscosity obviously increases when the solid loading is over 20%, and the power also increased by 300%. Compared with that at 5% solid loading, the power increased by 415% and 545% at 25% and 30% solid loading, respectively. The increase of solid loading obviously affected the viscosity of the hydrolysis system. Additionally, the power was also used to overcome the resistance force, which is surface tension, by interface area during the mixing. With the increase of solid loading, the resistance force increased. It follows that the required power level should be increased. Because ethanol concentration of 40 g/L is an effective value for reduction of the separation cost, a high solid loadings is necessary. The increase of solid loading can increase the glucose concentration during enzymatic hydrolysis and hence the ethanol concentration during fermentation, which can significantly reduce the capital cost of ethanol separation. However, there is a contradiction between the power of mixing and ethanol concentration. Taking into account the power of mixing, traditional mixing is not suitable for the high solid conversion process

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of biomass, and the reduction of energy consumption is an urgent demand of lignocellulosic ethanol. To sum up, owing to the shortage of social energy, the high solids conversion of lignocelluloses into ethanol has important practical significance and has attracted much attention in the academic world. There has been plenty of serious research on the conversion of lignocelluloses and stirring in high solid mixtures, but most of the research work is still at laboratory level and the industrialization of ethanol production has not been reported until now. Future research should focus on the design and construction of a strengthened system. From the macroeconomic viewpoint, high solids, the conversion of lignocellulose and process energy consumption should be systematically studied. At the same time, the analysis of different strengthening mechanisms in the design of new reactors and stirring methods should be carried out, to reduce the process capital cost.

REFERENCES Balusu, R., Paduru, R. R., Kuravi, S., et al. (2005). Optimization of critical medium components using response surface methodology for ethanol production from cellulosic biomass by Clostridium thermocellum SS19. Process Biochemistry, 40, 3025–3030. Bender, J., Vatcharapijarn, Y., & Jeffries, T. (1985). Characteristics and adaptability of some new isolates of Clostridium thermocellum. Applied and Environmental Microbiology, 49, 475–477. Bertilsson, M., Olofsson, K., & Lidén, G. (2009). Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce. Biotechnol Biofuels, 2. Blanchard, J., Leschine, S., Petit, E., et al. (2009). Methods and compositions for improving the production of fuels in microorganisms. Google Patents. Chandrakant, P., & Bisaria, V. (1998). Simultaneous bioconversion of cellulose and hemicellulose to ethanol. Critical Reviews in Biotechnology, 18, 295–331. Chen, H. (2006). Ecological high value conversion of straw resources: theory and application. Beijing: Chemical Industry Press. Chen, H. -Z., & Liu, Z. -H. (2014). Multilevel composition fractionation process for high-value utilization of wheat straw cellulose. Biotechnology for Biofuels, 7, 137. Chen, H., Li, Z., & Chen, J. (1999). Solid state simultaneous saccharification and fermentation of steam exploded biomass. Journal of Food Science and Biotechnology, 5, 78–81. Chen, H., Xu, J., & Li, Z. (2007). Temperature cycling to improve the ethanol production with solid state simultaneous saccharification and fermentation. Applied Biochemistry and Microbiology, 43, 57–60. Chen, H. -Z., Liu, Z. -H., & Dai, S. -H. (2014). A novel solid state fermentation coupled with gas stripping enhancing the sweet sorghum stalk conversion performance for bioethanol. Biotechnol Biofuels, 7, 53. Chen, M. (2007). Key technology research of ethanol production from corn stover. Hangzhou: Zhejiang University. Christakopoulos, P., Macris, B., & Kekos, D. (1989). Direct fermentation of cellulose to ethanol by Fusarium oxysporum. Enzyme and Microbial Technology, 11, 236–239. Dien, B., Cotta, M., & Jeffries, T. (2003). Bacteria engineered for fuel ethanol production: current status. Applied Microbiology and Biotechnology, 63, 258–266. Du, F., & Feng, W. (2009). Progress in alcohol production from straw: a demonstration project. Modern Chemical Industry, 29(1), 16–19.

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Fu, N., Peiris, P., Markham, J., et al. (2009). A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures. Enzyme and Microbial Technology, 45, 210–217. Gauss, W. F., Suzuki, S., & Takagi, M. (1976). Manufacture of alcohol from cellulosic materials using plural ferments. Google Patents. Hahn-Hägerdal, B., Galbe, M., Gorwa-Grauslund, M. -F., et al. (2006). Bio-ethanol—the fuel of tomorrow from the residues of today. Trends in Biotechnology, 24, 549–556. He, Y. (1990). Current status of one step lignocellulosic ethanol production. Jounral of Northwest Institute of Light Industry, 8, 95–101. Karhumaa, K., Sanchez, R. G., Hahn-Hägerdal, B., et al. (2007). Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae. Microbial Cell Factories, 6, 5. Kim, T. H., Taylor, F., & Hicks, K. B. (2008). Bioethanol production from barley hull using SAA (soaking in aqueous ammonia) pretreatment. Bioresource Technology, 99, 5694–5702. Koskinen, P. E., Beck, S. R., Örlygsson, J., et al. (2008). Ethanol and hydrogen production by two thermophilic, anaerobic bacteria isolated from Icelandic geothermal areas. Biotechnology and Bioengineering, 101, 679–690. Kötter, P., & Ciriacy, M. (1993). Xylose fermentation by Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 38, 776–783. Kuyper, M., Winkler, A. A., Dijken, J. P., et al. (2004). Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle. FEMS Yeast Research, 4, 655–664. Li, X., Tiao, S., & Pan, Y. (2003). Strains study progress of co-fermentation of hexose and pentose for ethanol production. Journal of Microbiology, 30, 101–105. Li, X., Zhang, Y., & Luo, X. (2009). Research progress of lignocellulosic ethanol. Modern Chemistry, 20–26. Liu, Z. -H., Qin, L., Pang, F., et al. (2013). Effects of biomass particle size on steam explosion pretreatment performance for improving the enzyme digestibility of corn stover. Industrial Crops and Products, 44, 176–184. Lv, X. (2009). Key problems of ethanol production from lignocellulosic biomass. Tianjin: Tianjin University. Modenbach, A. A., & Nokes, S. E. (2012). The use of high-solids loadings in biomass pretreatment—a review. Biotechnology and Bioengineering, 109, 1430–1442. Modenbach, A. A., & Nokes, S. E. (2013). Enzymatic hydrolysis of biomass at high-solids loadings—a review. Biomass and Bioenergy, 56, 526–544. Öhgren, K., Bengtsson, O., Gorwa-Grauslund, M. F., et al. (2006). Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400. Journal of Biotechnology, 126, 488–498. Olofsson, K., Wiman, M., & Lidén, G. (2010). Controlled feeding of cellulases improves conversion of xylose in simultaneous saccharification and co-fermentation for bioethanol production. Journal of Biotechnology, 145, 168–175. Petschacher, B., & Nidetzky, B. (2008). Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae. Microbial Cell Factories, 7, 9. Qin, L., Liu, Z. -H., Jin, M., et al. (2013). High temperature aqueous ammonia pretreatment and post-washing enhance the high solids enzymatic hydrolysis of corn stover. Bioresource Technology, 146, 504–511.

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Ryabova, O. B., Chmil, O. M., & Sibirny, A. A. (2003). Xylose and cellobiose fermentation to ethanol by the thermotolerant methylotrophic yeast Hansenula polymorpha. FEMS Yeast Research, 4, 157–164. Steen, E. J., Kang, Y., Bokinsky, G., et al. (2010). Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature, 463, 559–562. Szczodrak, J., & Targoński, Z. (1988). Selection of thermotolerant yeast strains for simultaneous saccharification and fermentation of cellulose. Biotechnology and Bioengineering, 31, 300–303. Wang, C., Wang, Y., Chen, J., et al. (2010). Ethanol production process from lignocellulose biomass. Biotechnology Information, 2, 012. Xiao, X., & Li, Z. (2000). Bioconversion of cellulose to ethanol intensified by separation, coupling and parallel operation. Engineering Chemistry & Metallurgy, 21(3), 283–287. Xu, L., Shen, Y., & Bao, X. (2010). Consolidated bioprocessing stratergy for lignocellulosic ethanol by Saccharomyces cerevisiae. Chinese Journal of Biotechnology, 26, 870–879. Yuan, W., Ren, J., Ren, X., et al. (2008). One step fermetantion of Jerusalem artichoke to ethanol. Chinese Journal of Biotechnology, 24, 1931–1936. Zertuche, L., & Zall, R. R. (1985). Optimizing alcohol production from whey using computer technology. Biotechnology and Bioengineering, 27, 547–554. Zhang, G., Deng, H., Lu, J., et al. (2009a). Bio-synthesis 2,3-butyl glycol from double substrate of glucose and xylose. Journal of Process Engineering, 9, 1174–1177. Zhang, J., Shao, X., & Lynd, L. R. (2009b). Simultaneous saccharification and co-fermentation of paper sludge to ethanol by Saccharomyces cerevisiae RWB222. Part II: Investigation of discrepancies between predicted and observed performance at high solids concentration. Biotechnology and Bioengineering, 104, 932–938. Zhang, M., Eddy, C., Deanda, K., et al. (1995). Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis. Science, 267, 240–243.

Chapter 8

Posttreatment Strategies for Biomass Conversion Chapter Outline 8.1 Principles of Posttreatment for Biomass Biochemical Transformation 199 8.2 Operation Units of Posttreatment for Biomass Biochemical Transformation 199 8.2.1 Pretreatment of Fermentation Broth and Solid–Liquid Separation 199 8.2.2 Biological Product Isolation and Purification Technologies 200 8.2.3 Advantages and Limitations of Single Posttreatment 202 8.3 Integration of Posttreatments for Biomass Biochemical Transformation 202 8.3.1 Integrated Bioreaction– Separation Process 203

8.3.2 Integrated Separation– Separation Processes 8.4 Example of Posttreatment for Biochemical Product, 2,3-Butanediol 8.5 Technical and Economic Analysis of Posttreatment for Biomass Biochemical Transformation 8.5.1 The Particularity of Biomass Biochemical Transformation Products 8.5.2 Technical and Economic Analysis of Posttreatment for Biomass Biochemical Transformation References

208

211

213

213

214 216

The biochemical production process consists of strain breeding, cell culture (fermentation), pretreatment, concentration, product complement, and purification. Commonly, the process before cell culture is called the “upstream process” while others are called “downstream processes” or “biological isolation and purification processes” (Chen & Wang, 2008). The “upstream process” and “downstream processes” must be compatible and coordinated so that the whole process can be optimized for biotechnology industrialization (Mei, Yao, & Lin, 1999). In general, downstream processes consist of four stages: (1) pretreatment of the fermentation broth and solid–liquid separation, (2) initial purification (extraction), (3) further purification (refining), and (4) final purification.

Technologies for Biochemical Conversion of Biomass © 2017 Metallurgical Industry Press. Published by Elsevier Inc.

197

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Among these, isolation and purification account for a large proportion of the total cost. For example, in the production of items made by traditional fermentation processes, such as antibiotics, acetic acid, and citric acid, isolation and purification account for 60% of the total production investment (Yu, Tang, & Wu, 2003). Currently, methods used for preliminary purification mainly include adsorption, ion exchange, precipitation, solvent extraction, aqueous twophase extraction, supercritical fluid extraction, reverse micellar extraction, and the membrane filtration method. Methods used in refining include chromatography, crystallization, and so on. Differing characteristics, such as size, electrostatic charge, hydrophobicity, solubility, and arrangement of specific chemical functional groups, lay the foundation for isolation and purification (Garcia, Bonen, & Ramirez-Vick, 2004). The achievement of effective and simple isolation and purification is a challenge in biochemical engineering. The general process is shown in Fig. 8.1. To choose optimal technologies and combine them sensibly, it is necessary to pay careful attention to the product properties.

FIGURE 8.1  Biological isolation and purification processes (Jin, Feng, & Su, 1998).

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199

8.1  PRINCIPLES OF POSTTREATMENT FOR BIOMASS BIOCHEMICAL TRANSFORMATION 1. Sample volume should be reduced as early as possible. Because of separation, cost is closely related to sample volume, and so reducing sample volume as early as possible saves money. In practice, reducing sample volume means removing water by evaporation. In the case of an easily decomposed target product, or one with a boiling point higher than that of water, evaporation should be avoided, while precipitation, extraction, adsorption, or affinity can be used. 2. Ensure the method employed gives high-resolution results. 3. Kiss principle (keep it simple, stupid): minimize separation steps, and simplify the separation process. 4. Extract the target product as early as possible. This can reduce separation steps and thus simplify the separation process. As impurities could lead to enzyme degradation or product denaturation, extracting the target product early could also improve the quality of the product. Crystallization and precipitation methods employ this principle, which makes them very economical in obtaining the crude product. Although preliminary crystallization or precipitation cannot achieve the requisite product purity, dissolving or recrystallization can be further used to purify the product. 5. The product inhibition effect in the bioreactor should be minimized. Sometimes high concentration or impurities in the product may seriously inhibit the cells’ production efficiency. Hence, reasonable integration of biological reaction processes with biological separation processes is ­beneficial in obtaining higher production efficiency and thus product concentration.

8.2  OPERATION UNITS OF POSTTREATMENT FOR BIOMASS BIOCHEMICAL TRANSFORMATION 8.2.1  Pretreatment of Fermentation Broth and Solid–Liquid Separation The first essential step of biological separation and purification is enriching or removing the cells to transfer the target product into the liquid phase and to remove other suspended particles and soluble impurities to improve the properties of the filtrate, for example by reducing viscosity. The aim of pretreatment of the fermentation broth is to change its properties for solid–liquid separation. For example, using acidification, heating could reduce the viscosity of the fermentation broth and adding a flocculating agent could make the cells or dissolved macromolecules condense into larger particles. For extracellular products, pH adjustment is often used to transfer the target product into the liquid phase. For intracellular products, on the other hand, it is better to collect the cells first, and then break them so that biochemical substances are released into the liquid phase. Then the cell debris should be separated to obtain the liquid containing biological material for use in subsequent operations.

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8.2.1.1  Pretreatment of Fermentation Broth Coagulation and flocculation technology can effectively change the dispersion state of cells, bacteria, and proteins, making them gather and thus increasing the volume for filtration; so these techniques are commonly used in the pretreatment of a fermentation broth containing small cells or with large viscosity. At present, the flocculant most commonly used is a synthetic polymer, such as polyacrylamide or polyethylene imine. An inorganic polymer flocculant is also good, for example, polymeric aluminum or iron salts. In addition, a natural organic polymer flocculants, for example, chitosan or dextran polysaccharides, as well as gelatin, glue, and sodium alginate, are also used. Bioflocculants are a class of substances with flocculation functions produced by microorganisms, of which the major components are glycoproteins, glycosaminoglycans, cellulose, nucleic acids, and other polymer materials. Compared with the chemically synthesized flocculants, the biggest advantages of bioflocculants and natural flocculants are being safe, nontoxic, and environmentally sound. Hybrid proteins can also be removed by isoelectric precipitation, heat denaturation, and adsorption methods. 8.2.1.2  Solid–Liquid Separation of Fermentation Broth The purpose of solid–liquid separation has two main aspects: first, to collect cells and strains and to obtain intracellular products by removing the liquid; secondly, to collect liquid containing the target product by separating and then flocculating the suspended solids, such as cells, strains, cell debris, and proteins. The common methods of solid–liquid separation are filtration and centrifugation. Conventional filtration equipment for biochemical substance separation consists of the frame filter and vacuum filter drum. Centrifugation can be divided according to its speed into three modes, normal speed (low speed), speed, and overspeed. Compared with conventional filtration, centrifugation has the advantages of high efficiency, good hygiene during operation, and others which make it suitable for performing the separation process on a large scale, although device investment cost is high and power consumption is large. 8.2.1.3  Cells Breaking Technology In order to extract intracellular biochemical substances, cells or strains should be collected first and then broken. Cell breaking methods can be grouped into two major categories, mechanical methods and nonmechanical methods. In mechanical methods, pressure, grinding, or ultrasonic waves are used to produce shearing force on the cell walls to break them. Enzymatic, osmotic shock, freezing and thawing, drying, and chemical methods are commonly used as nonmechanical methods, of which the enzymatic and chemical methods are the most widely used.

8.2.2  Biological Product Isolation and Purification Technologies 8.2.2.1 Precipitation Precipitation refers to the process whereby solute from the liquid phase becomes solid phase. By changing the physical and chemical parameters of the

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solution, one can change the solubility of the various components in the solution to separate out the target ingredient. Precipitation is the oldest method for biological substances’ separation and purification, and it is still widely used in industry and laboratories. As the action of the concentrated form is often greater than the effect of the purified form, precipitation is generally used as a preliminary separation method for removing the precipitated biological materials in the fermentation broth from which the bacterial cells or cell debris have already been removed. Then other methods can be used to further enhance the purity. Precipitation includes salting, isoelectric point precipitation, organic solvent precipitation, nonionic polymeric precipitation, polyelectrolyte precipitation, etc. As it has advantages, such as low cost, high yield, effective concentration efficiency up to 10–50 times the concentration ration, and simple operation, the precipitation method is widely used in downstream processing.

8.2.2.2  Membrane Filtration Membrane filtration is the method using pressure as driving force, responding to the membrane selective property to separate the different components in a liquid; this method includes microfiltration (MF), ultrafiltration (UF), nanofiltration (NF), and reverse osmosis (RO). The core of membrane filtration is the membrane itself, which must be permeable. That is, some substances can pass through the membrane, while others are hindered. Membrane filtration is mainly used for filtering the fermentation broth and cell collection and purification. 8.2.2.3  Solvent Extraction Solvent extraction is the process in which a compound transfers from one solvent to another owing to the difference in solubility or distribution coefficient between these two immiscible (or slightly soluble) solvents. Compared with other separation methods, it gives a better separation effect than chemical precipitation, and a higher degree of selectivity and faster mass transfer than the ion exchange method. Compared with distillation, solvent extraction has advantages such as low energy consumption, large production capacity, fast action, easy continuous operation and ease of automation. In recent years, a series of new separation techniques has been developed combining solvent extraction technology with other technologies to adapt to the development of DNA restriction and genetic engineering techniques, such as reverse micelles extraction, supercritical fluid extraction, liquid membrane extraction, etc. 8.2.2.4  Ion Exchange Ion exchange mainly uses synthetic material, namely ion exchangers, as a sorbent, to adsorb the valuable ions. In biological industry, the classical ion exchanger is ion exchange resin, which is widely used in the extraction of antibiotics, amino acids, organic acids, and other small molecules. The ion exchange method has advantages, such as low cost, simple equipment, easy operation, and no or little use

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of organic solvents. Meanwhile, it has its drawbacks, such as a long production cycle, sometimes poor product quality, and large pH changes in the production process. On top of this, it is not always possible to find suitable resin.

8.2.2.5 Adsorption Adsorption is widely used in biochemical engineering. Selective adsorption was applied early in the isolation and purification of enzymes, proteins, nucleotides, antibiotics, and amino acids. The early adsorbents are mainly kaolin, alumina, acid clay, gel ion exchange resins, activated carbon, molecular sieves, and cellulose. However, these adsorbents have the disadvantages of low absorption capacity and easy inactivation. It has been noted that the adsorbent must be recyclable, and used for hundreds or even thousands of times to be economical. Therefore, adsorbents require good physical and chemical stability and the regeneration process must be simple and fast. In recent years, some synthetic organic macroporous adsorbents, called macroreticular polymeric adsorbents have met these requirements, especially on an industrial scale. The adsorption method generally has the following advantages: (1) no or little use of organic solvents, (2) easy operation and safe equipment, and (3) small pH changes in the production process, making it suitable for substances with poor stability. However, adsorption also has disadvantages, such as poor adsorption selectivity, low yield, unstable performance, especially of inorganic adsorbents, discontinuous operation, high labor intensity; and some adsorbents, such as toner, are not environmentally sound. Therefore, at one time adsorption was almost replaced by other methods. With the development of gel type adsorbents and macroreticular polymeric adsorbents, adsorption came back into favor and was reapplied in biochemical engineering.

8.2.3  Advantages and Limitations of Single Posttreatment Single posttreatment has the main advantage of being easy to control. But for the whole process, the following drawbacks have been exposed. (1) many procedures, large amount of cumulative loss and low product yield. If the yield of every procedure was 90% of what can be expected, the final yield after six procedures would be only 53% of the total; (2) long operation time and frequent change of product buffer system easily lead to the inactivation of the target product; and this would reduce the yield; (3) large investment in equipment and separation medium increases the cost of the final products (Jin et al., 1998).

8.3  INTEGRATION OF POSTTREATMENTS FOR BIOMASS BIOCHEMICAL TRANSFORMATION Process integration is an important research direction. Based on the achievements of process integration technology in chemical engineering, its application in biochemical engineering is regarded as a promising means of promoting the industrialization of biochemical engineering. Process integration of biochemical

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separation means the effective combination of several units to achieve a more efficient separation technique. Rational process integration could simplify the process flow, reduce material and energy consumption and improve production efficiency (Chen, 2004). Process integration of biochemical separation includes an integrated bioreaction–separation process and an integrated separation–separation process.

8.3.1  Integrated Bioreaction–Separation Process There is generally inhibition of a product or by-product to bioreaction, which reduces the biocatalyst’s activity, inhibits cell growth and thus reduces the efficiency of the process. If the product or by-product was removed or consumed to eliminate the inhibition effect, long-term maintenance of biocatalyst activity and high-density cell growth could be realized and a high target product yield could be achieved. The integrated bioreaction–separation process, which is called “in situ product removal” (ISPR), extractive fermentation, or, in A. Freeman’s term, bioconversion, means the selective removal of the product or by-product from the cells or biocatalysts (Wang, He, & Ouyang, 1999). In the bioreaction process, the aim is to obtain as much product as possible, with a high rate of bioreaction. Timely removal of the product could mitigate the inhibition effect and realize the goal, and this is the initial motivation for developing an integrated bioreaction–separation process.

8.3.1.1  Brief Introduction to the Integrated Bioreaction–Separation Process Integrated bioreaction–separation processes have been classified by different researchers with different perspectives. A. Freeman et al. divided them into internal product removal and external product removal, from the view point of reactor structure. Chang-Ho Park et al., on the other hand, divided them into the integrated bioreaction membrane-based separation process and the integrated bioreaction nonmembrane-based separation process, affirming the position of membrane separation technology in the integrated process. Generally, integrated bioreaction–separation processes have the following three characteristics: 1. The structures of bioreactors are specific for integrated processes; 2. The methods of timely removal of products are various, but the program must be designed rationally with regard to product properties and the biological reaction system; and 3. As a new technology, the integrated process is applicable to various biological reactions. The integrated bioreaction–separation process has the following advantages: it mitigates the inhibition effect upon the product or by-product; it increases product yield; it makes the process continuous and stable by separating the nonfermentable substrates and aging cells; it simplifies the posttreatment process;

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and it reduces the investment and operation cost. However, the main drawback of the integrated bioreaction–separation process is that control of single unit operation cannot be achieved precisely. Since the separation process begins when the product concentration reaches a certain value, the operation processes become complex in the integrated process. In addition, a sterile condition is difficult to maintain in the integrated process and the metabolites secreted by the cells may be removed along with the product (Garcia et al., 2004). Because the separation is integrated in the bioreaction process, the environment of bioreaction changes, which may lead to change of the reaction kinetics and the mechanism of metabolic regulation. Typically, for example, the cells grow fast owing to the timely removal of the inhibited product or by-product, resulting in a cell density 10–20 times higher than that occurring in conventional fermentation. Additionally, the accumulation of some by-products that haven’t been removed may also change the reaction kinetics and mechanism of metabolic regulation. Therefore, influence on the bioreaction system should also be taken into consideration in the integrated process. The key to the integrated bioreaction–separation process is to choose a suitable separation technology to remove the product or by-product in situ. The following factors should be taken into consideration when choosing a separation technology. 1. The separation technology should be biocompatible. That is, separation should have no negative effect on the bioreaction, for instance, not leading to the inactivation, denaturation or death of the biocatalysts or cells, or changing the mechanism of metabolic regulation. 2. The choice of separation technology should be based on a full consideration of the physical, chemical, and biological properties of the product or byproduct. 3. Attention should be paid to the fluid properties. Hydrodynamic properties influence mass transfer in the separation process, thus affecting separation speed and efficiency. For example, non-Newtonian fluids with high viscosity cannot be separated by membrane technology. 4. Engineering and economic factors also should be considered. Technology with high engineering feasibility, low operating cost, stable performance, and long service life is regarded as ideal. At present, developed separation technology methods include vacuum fermentation, gas stripping, pervaporation, membrane distillation, membrane permeation, extraction, sedimentation, and crystallization.

8.3.1.2  Integrated Bioreaction–Membrane Separation Technology The shortcomings of traditional separation methods, such as their leading to biocatalyst inactivation, could be overcome by using membrane technology. Bioreaction and membrane separation could be coupled either in a device or in different devices.

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8.3.1.2.1  Bioreaction and Membrane Separation Coupled in Different Devices This method situates bioreaction and membrane separation in different devices. Bioreaction is conducted in a bioreactor (fermenter, enzyme reactor, etc.) and the mixture is then transferred to the membrane area for separation. The components for retention are recycled to be utilized and reactants (medium, enzyme, cells, etc.) are added into the bioreactor for the subsequent reaction. The permeated liquid is treated for further separation. The integrated system is shown in Fig. 8.2. This method is flexible. The bioreaction and membrane separation do not influence each other and could both be conducted in optimal conditions. Additionally, since the adoption of membrane separation, the efficiency of bioreaction has increased significantly. 1. Membrane separation Enzymatic hydrolysis of protein using this coupling system has two obvious advantages: the enzyme can be used repeatedly; product molecules can be controlled by choosing a semipermeable membrane. Ordinary intermittent hydrolysis has several disadvantages: the reaction needs to be terminated by heating or pH adjusted to achieve enzyme inactivation. Therefore, enzyme consumption is large. The end product is inhibitory to the enzyme, gradually decreasing the reaction rate. Products are uneven in quality, and reproducibility is poor. 2. Pervaporation (Wang et al., 2005) As a new type of membrane separation and clean production technology, pervaporation membrane technology coupled with fermentation to produce fuel ethanol can improve production efficiency, reduce energy consumption, and reduce pollution. It does not call for a third component. The equipment is simple in structure, with high separation efficiency and low energy consumption. Compared with the traditional batch fermentation, ultrafiltration–

FIGURE 8.2  Integrated system of bioreaction and membrane separation coupled in ­different devices (Wang, Li, & Zhang, 2005). 1 Substrate tank; 2 bioreactor; 3 pump; 4 membrane-separation device; 5 permeated liquid tank containing target product.

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cell cycle fermentation–fermentation processes, fermentation–pervaporation membrane technology has the following advantages: 1. The pervaporation membrane is dense, and its spread mechanism is dissolution–diffusion. Membrane plugging and membrane pollution are not likely to occur as long as the film retains excellent performance and good convective mass transfer is ensured near the membrane surface. Therefore, the membrane can work stably in the long term. 2. The inhibition effect on yeast cells can be reduced and even eliminated by separating ethanol from the fermented liquid in situ. Therefore, proper yeast cell concentration and high biological activity in the reactor are maintained to achieve high-density fermentation and a high sugar conversion rate of raw materials. 3. The fractionation can be cooled and condensed directly to obtain high concentration ethanol, which reduces the energy consumption of ethanol production. The energy consumption of pervaporation membrane technology occupies 1/10∼1/3 of the energy consumption in traditional distillation. There are no “three wastes” and thus environmental pollution is avoided. At the same time, the wastewater treatment process in traditional distillation technology can be eliminated. 4. Continuous fermentation has been shown to greatly reduce reactor volume. Automatic control becomes easy, to ensure stable operation. 5. The material with high concentration of sugar can be adopted as feedstock, so as to reduce the water consumption of the fermentation process and further reduce the energy consumption. The industrial fermentation process for producing anhydrous ethanol consists mainly of three steps. First, the raw material, after pretreatment and saccharification, is fermented to low concentration ethanol by microorganisms. Secondly, the low concentration ethanol is distilled to 95% ethanol (mass fraction). Thirdly, anhydrous ethanol with mass fraction above 99.5% is produced. Generally, the alcohol permselective pervaporation membrane is coupled with fermentation in the first step. The water permselective pervaporation membrane is used in the third step. By coupling the pervaporation membrane and ethanol fermentation, ethanol is selectively removed while yeast cells and glucose are intercepted in the fermenter to improve the volume production and concentration of ethanol (Zhang, Yu, & Yuan, 1997).

8.3.1.2.2  Bioreaction and Membrane Separation Coupled in the Same Devices In contrast, the biological catalyst (cells or enzymes) can be fixed on a semipermeable membrane in an appropriate manner. The substrate or medium flows on one side of the membrane, and react on the membrane when in contact with the biological catalyst, while the products flow through the membrane to the other side. This device is called a membrane bioreactor, in which biological reaction

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FIGURE 8.3  Coupled reaction and separation in the membrane system (Wang et al., 2005). 1 Culture medium and the substrate reservoir; 2 pump; 3 membrane bioreactor, 4 product storage tank.

and product separation occur simultaneously on a semipermeable membrane. The system is shown in Fig. 8.3. Tan et al. successfully achieved the compound lipase membrane for fat hydrolysis. The oil phase and the water phase were divided by the membrane to avoid emulsification by direct contact between the two phases. The reaction occurred on the membrane surface and the products were taken away two phases in time. Reaction and separation occurred at the same time, solving the difficult separation problem caused by emulsification in the enzymatic hydrolysis of fat (Tan, Zhang, & Wang, 2000).

8.3.1.3  Integrated Bioreaction–Gas Stripping Technique Product feedback inhibition is an inherent problem in the fermentation processes of liquid biofuel (ethanol, butanol) production. Ethanol’s toxic effect on strains limits the final concentration of ethanol, resulting in high levels of energy consumption, and a large amount of wastewater in the subsequent purification processes. In order to increase the sugar concentration in the fermented liquid and decrease product feedback inhibition, much research focused on the online separation of ethanol and butanol, including liquid–liquid extraction (Ishizaki, Michiwaki, & Crabbe, 1999), pervaporation (Liu, Liu, & Feng, 2005), membrane distillation (Banat & Al-Shannag, 2000), and gas stripping (Qureshi & Blaschek, 2001; Ezeji, Qureshi, & Blaschek, 2003; Ezeji, Qureshi, & Blaschek, 2004). Gas stripping is a relatively simple separation technology. It has the advantages of smaller investment, without causing damage to microorganisms or taking away nutrients from the medium while removing the product (ethanol, butanol) (Qureshi & Blaschek, 2001). Currently, nitrogen, carbon dioxide, and hydrogen are usually used as inert gases for ethanol or butanol gas stripping. On the basis of the existing gas stripping technology, a method of CO2 gas stripping coupling with the activated carbon adsorption method was put forward by our team for online separation of ethanol, which included at least two parallel adsorption towers and a CO2 circulation pump. This technique is a combination

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of a gas lift loop flow tower fermenter, vacuum reflow, CO2 gas stripping, circulation and mixing, and activated carbon adsorption. When the ethanol concentration is higher than 5%, the CO2 circulation pump starts to work. CO2 and the entrained ethanol flow into one of the adsorption towers and ethanol is adsorbed. When the first adsorption tower is saturated, the flow is switched to another adsorption tower. The ethanol is recovered by heating the ethanol-saturated adsorption towers. In ethanol fermentation processes using lignocellulosic biomass, the coupled solid-phase enzymolysis, liquid fermentation of ethanol, and adsorption separation help to reduce the cellulase dosage by 50%∼60% (w/w) and thus reduce the cost. Wastewater after fermentation reduces by 30%∼40%. Saccharification and fermentation are conducted in different areas although in the same reactor. Therefore, the temperatures for saccharification and fermentation could be coordinated to reach their own optimal conditions. Finally, ethanol concentration increases by 60% (v/v), and the ethanol concentration after activated carbon desorption reaches 69.8% higher (Chen & Qiu, 2010; Chen, 2008).

8.3.1.4  Integrated Bioreaction–Precipitation Technology Production of malic acid sodium from fumaric acid sodium under the action of the fumaric acid enzyme is a typical reversible reaction. The conversion rate is 70%∼80%, and malic acid content in the reaction system is 10% by the traditional method. By using the optimized high concentration of the ammonium fumarate system, the conversion rate increases to 88%∼90%, and the malic acid content increases to 20%, which is twice that of the traditional method. However, the cost is still higher than that of chemical synthesis of dl-malic acid. Based on the solubility difference, l-malic acid calcium salt precipitate from the solution, leads to the reaction’s constantly varying in the direction of product generation. The conversion rate of this method was as high as 99.9%, which helps to reduce the separation cost significantly (Hu, Ouyang, & Shen, 2001).

8.3.2  Integrated Separation–Separation Processes In terms of development trends, biochemical separation technology research aims to shorten downstream processes and improve the efficiency of single operation. Since the 1980s, in order to simplify biochemical separation processes and improve the efficiency of purification and recovery, researchers have put forward the concept of process integration, whereby several conventional unit operation tasks are completed in one operation. By reducing the number of purification steps, yield was increased, equipment investment and area were reduced, and the cost of separation was also reduced. Therefore, process integration is an attractive option for separation. Integrated separation processes coupled with different separation processes could integrate the advantages of various methods, and this is suitable for special system separation. Integrated separation–separation processes can be divided into biological separation unit integration and separation technology integration. Biological separation unit integration combines the original processes

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efficiently to reduce the number of separation steps and thus increase production efficiency. Separation technology integration combines two or more separation technologies using different separation principles by means of newly developed technology, to greatly improve separation efficiency. Based on this idea, a group of integrated separation processes, such as extractive crystallization, adsorptive distillation, electrophoresis, affinity extraction, affinity precipitation, affinity membrane separation, and expanded bed adsorption, were developed.

8.3.2.1  Aqueous Two-Phase Affinity Partitioning Technology Affinity is a kind of biological separation method, of which the advantages include fewer separation steps and high specificity, while the main disadvantage is that the samples need a series of pretreatments, such as filtration. As for aqueous twophase partitioning technology, large quantity of samples of solid–liquid mixture can be treated directly, but its specificity is poorer. By combining these two methods, a new aqueous two-phase affinity partitioning technology with high efficiency and specificity can be developed. The aqueous two-phase affinity system can be divided into three types according to the ligands, which are the group affinity ligands, dye affinity ligands, and biological affinity ligands. Aqueous two-phase affinity partitioning technology has been developing fast in recent years. There are more than 10 kinds of affinity ligands, which connect with PEG and dozens of separated substances. For example, Kroner used an aqueous two-phase affinity system consisting of PEG and Dextran to separate glucose-6-phosphorylation dehydrogenase, the partition coefficient of which increased from 0.18∼0.73 to 193. Ulrich used phosphate ester PEG–phosphate to extract β-interferon, the partition coefficient of which increased from about 1 to 630 (Mei et al., 1999). Aqueous two-phase affinity precipitation technology was developed based on the characters of methylacrylic acid and methyl methacrylate copolymer, which precipitate under alkaline conditions and dissolve again under acidic conditions. This process will be described later. First, the copolymer is added to the fermented liquid. The pH is adjusted to alkaline and the copolymer will precipitate, while the objective product is adsorbed. Then the precipitated ­copolymer is separated and put into the aqueous two-phase system. The pH is adjusted to acidic and the copolymer precipitation begins to dissolve. The objective product transfers to the upper phase of the aqueous two-phase system. This technology can realize highly efficient and selective separation. For example, immunoglobulin is used as ligand in the purification of protein A. First, the ligand is fixed as copolymer, which is distributed in the upper phase of the PEG–PES aqueous two-phase system. Owing to the specificity reaction, protein A accumulates in the upper phase. The pH is adjusted to make the copolymer precipitate. Then the precipitation is washed with detergent to obtain protein A. 8.3.2.2  Affinity Membrane Separation Technology Affinity membrane separation technology is a new method combining affinity chromatography and membrane separation. Ligands are coupled on the

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membrane. The separation procedures include adsorption, washing, elution, and regeneration. When the samples contact the membrane, the target protein is adsorbed on the membrane while the impurities go through the membrane. Then the target protein is washed by dissociative eluent and the ligands are regenerated. This technology shows great potential because it can integrate the steps of clarification, concentration, and purification into a whole and can couple with a biological reactor to set up a new reaction–separation process. Not only can affinity membrane separation technology apply to samples with a low concentration of biological products, but it can also achieve concentration during the purification process. In addition, it has the advantages of simple operation and equipment. It has already been applied in the separation of a monoclonal antibody, a polyclonal antibody, the trypsin inhibitor, etc., as well as the purification of antigens, antibodies, recombinant proteins, serum albumin, trypsin, chymotrypsin, interferon, etc. As a new separation technology, affinity membrane separation technology is on the rise and will constitute a powerful method for separation and purification of biological macromolecular substances (Mei et al., 1999).

8.3.2.3  Expanded Bed Adsorption Technology Expanded bed is a fluidized bed with adsorbent in a stable state. In contrast to the process of packed bed chromatography, inflation of the adsorbent layer occurs with the flow of liquid raw materials in the expanded bed. The extent of inflation depends on adsorbent density and fluid velocity. When the sedimentation velocity of the adsorbent equals the rising speed of fluid, the expanded bed reaches equilibrium. Owing to the expansion of the adsorbents, the gaps between them are enlarged. Thus, solid particles in the raw liquid, such as cells and cellular debris, can pass through the expanded bed while the target substance is adsorbed on the adsorbent. Therefore, the target protein is separated from the fermented liquid containing bacteria, cell debris, etc. directly. The operation of the expanded bed consists of balance, adsorption, washing, elution, and renaturation cleaning. It integrates the clarification, concentration, and initial purification of raw liquid into a unit operation, which reduces the number of steps in the operation and improves separation efficiency and the product yield. This technique has aroused widespread interest (Mei et al., 1999). Barnfield Frej applied the expanded bed with cation exchanger to extract recombinant human interleukin after renaturation from the breaking of E. coli liquid; the one-step yield reached 97% and the purification multiple was 4.35. Johansson used the expanded bed to extract recombinant verdigris cell exotoxin A from the E. coli bacterial periplasm. It took 2.5 h to deal with 4.5 kg cells. The specific activity of exotoxin A was 0.06 mg/mg protein and the recovery rate was 79%. For the conventional packed bed chromatography, dealing with the same quantity of cells took 8∼10 h, which was three times what was required for the expansion bed. Although the specific activity was a little higher (0.1 mg/ mg protein), the recovery rate (73%) was lower than that of the expansion bed. Genetech used the expanded bed to extract monoclonal antibodies from CHO

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cell cultures. The handling capacity was 7324 L. All cells were removed, the antibody concentrated five times and the recovery rate was as high as 99%. Chang and Chase utilized anionic ligands DEAE and dye ligands Red H-E7B to separate glucose-6-phosphate dehydrogenase, respectively. Owing to the affinity effect of dye with glucose-6-phosphate dehydrogenase, the purification multiple of dye was 8.6 times higher than that of DEAE. Noda et al. applied the expanded bed with bed column diameter of 1000 mm and 150 L STREAMLINE SP to extract recombinant human serum albumin from P. pastoris cultures, of which the handling capacity was 2000 L. The overall yield was 87.1%, which was consistent with the pilot result. Using heat treatment and expanded bed adsorption could replace the traditional five steps operation, not only reducing the operating time, but also increasing the yield by 30%. Maurizi et al. used STREAMLINE SP expansion bed chromatography and anion exchange chromatography (Mono Q) to replace centrifuge, filter, and cation exchange chromatography (S Sepharose) and anion exchange chromatography (Mono Q) to extract recombinant human interleukin 1 receptor blocking antibodies (IL-lra) from Bacillus subtilis fermented liquid. The product purity reached 90%∼92% and the recovery rate was 85%. These persuasive examples demonstrated the wide application of expansion bed technology in the biological field.

8.4  EXAMPLE OF POSTTREATMENT FOR BIOCHEMICAL PRODUCT, 2,3-BUTANEDIOL 2,3-butanediol is a kind of hydrophilic polyol, which can be produced by lignocellulose fermentation. The extraction of 2,3-butanediol from the fermented liquid represents a bottleneck in its industrialization. The composition of 2,3-butanediol fermented liquid is complicated; it contains bacteria, biological macromolecules, such as proteins, nucleic acids, and polysaccharides, and small molecules, such as ethanol, acetic acid, lactic acid, monosaccharide, and organic and inorganic salt. Besides, the concentration of 2,3-butanediol is low (usually 8%∼10%) and its boiling point high. Therefore, conventional distillation, steam distillation, or reverse flow formulation need high energy consumption; and heat transfer efficiency is decreased and the product yield low owing to the existence of biological macromolecules in the fermented liquid. The traditional separation of 2,3-butanediol includes solid–liquid separation, primary separation, and final separation. The solid substances in the fermented liquid are usually removed by flocculation, filtration, or centrifugation. Then primary separation (extraction, salting out, pervaporation, etc.) is conducted to obtain a solution with most of the impurities removed. Finally, the purified product is obtained by distillation. 2,3-butanediol fermentation mainly utilizes a variety of waste or nongrain raw material as the substrate. Therefore, besides the product, bacteria and protein, both insoluble and jelly, coming from the raw materials also exist in the fermented liquid. These impurities make filter blockage likely and thus

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increase the difficulty of solid–liquid separation. Xiu Zhilong et al. (Zhang, Sun, & Xiu, 2008) applied the chitosan flocculation method to realize solid– liquid separation for the 2,3-butanediol fermentation system. The flocculation rate reached more than 98% under optimum operating conditions. The retention rate of 2,3-butanediol was about 99% and the protein removal rate was 71%. Meanwhile, the supernatant after flocculation was clear and transparent. The bacteria after flocculation could be used again and transformation ability equaled that before flocculation. Primary separation is conducted after solid–liquid separation by solvent extraction, salting out, aqueous two-phase extraction, etc. to get to the 2,3-butanediol crude products with most impurities removed. Xiu, Li, and Zhang (2007) developed a series of new aqueous two-phase systems using hydrophilic organic solvent/inorganic salt to separate 2,3-butanediol from the fermented liquid. Not only were the distribution coefficient and the recovery rate of 2,3-butanediol much higher than those of the traditional aqueous two-phase system developed by Ghosh and Swaminathan (2003, 2004), but it could also be directly applied to the fermented liquid, which combined solid–liquid separation and primary separation into one step, simplifying the operation. An ethanol/ potassium aqueous two-phase system was used to extract 2,3-butanediol from the fermented liquid, and the recovery rate reached more than 90%. Not only could most of the proteins and substrates be removed from the alcohol phase, but also organic acid by-products could be removed. Pyruvic acid, citric acid, malic acid, fumaric acid, succinic acid, and some of the lactic acid and acetic acid were removed (Liu, Jiang, & Wang, 2009). Therefore, this new aqueous two-phase system is very beneficial for subsequent distillation, and so is a highly efficient separation technology with potential for industrialization. Huang He et al. successfully developed a separation method using hydrophobic silica zeolite to adsorb 2,3-butanediol from the fermented liquid. After pretreatment the fermented liquid was treated with the hydrophobic silica zeolite to adsorb 2,3-butanediol. Then anhydrous ethanol was used for stripping. After the ethanol removal, the target product was obtained. This method has the advantages of simple process, high separation efficiency, and low energy consumption, and thus has a good prospect of industrial application. Purified 2,3-butanediol can be obtained through distillation and countercurrent extraction. Industrial-scale counter-current extraction devices were successfully established in the USA and Canada as early as 1945 and 1948, respectively. However, high levels of energy consumption prevent its wide application in industry. Reverse osmosis coupled with distillation technology could effectively reduce energy consumption. Qureshi, Meagher, and Hutkins (1994) used Teflon membrane with a microporous structure for vacuum membrane distillation. Water vapor passed through the membrane while 2,3-butanediol was prevented. The concentration of 2,3-butanediol reached as high as 430 g/L by this method. Shao and Kumar (2009a) used polydimethysiloxane to separate 2,3-butanediol from the fermented liquid analog

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(water:butanol:2,3-butanediol = 7.4:12.0:80.6%, w:w:w) using butanol as solvent. The batch operation, allowed easy selection, and 2,3-butanediol with purity of 98% could be obtained. During continuous operation, the recovery rate was lower than 52% when purity reached 98%. Huang, Ji, and Li (2008) developed a method using hydrophobic silica zeolite to adsorb 2,3-butanediol from the fermented liquid. Then anhydrous ethanol was used for elution and the target product was obtained after removal of ethanol. Shao and Kumar (2009b) mixed zeolite particles into silicon rubber membrane evenly to improve the membrane composition. When zeolite accounted for 80% (w:w) of the blend membrane, the recovery of 2,3-butanediol increased from 47.4% to 62.8%. It can be seen from the example of purification of 2,3-butanediol that each separation technology has its advantages and disadvantages. It is difficult to achieve the required purification by using a single kind of separation and purification technology. Therefore, it is necessary to develop the traditional technology or combine it with some new technologies to improve yield, reduce energy consumption, and reduce the whole separation cost (Dai, Sun, & Sun, 2010).

8.5  TECHNICAL AND ECONOMIC ANALYSIS OF POSTTREATMENT FOR BIOMASS BIOCHEMICAL TRANSFORMATION 8.5.1  The Particularity of Biomass Biochemical Transformation Products As the biomass raw materials are mostly agricultural and forest by-products, which are natural substances, their components are complex. Biochemical transformation is regarded as a polygeneration process. In a unit operation, a component is always utilized, while others are seen as inert ingredients. Therefore, the concentration of product is low owing to the low concentration of substance. In addition, most biomass materials contain pigments, which come from lignin and substances with aromatic ring degradation (Li & Wu, 2006). Lin and Kringstad (1970) suggested that quinone was the main chromophore in lignin. Quinone was generated in the formation and degradation of lignin, and was responsible for the lignin’s color. Flavonoids and anthocyanins are also contained in some plants. These pigments can be extracted in the transformation process, which influences the biomass transformation. For example, some pigments would inhibit cellulose hydrolysis and fermentation performance. Therefore, it is necessary to remove these pigments from the materials using separation methods, such as macroporous resin adsorption, electrodialysis, ion exchange, precipitation. The particularity of biomass biochemical transformation compared with conventional chemical synthesis is shown in Table 8.1.

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TABLE 8.1 The Particularity of Biomass Biochemical Transformation Compared With Conventional Chemical Synthesis Biomass biochemical transformation

Conventional chemical synthesis

Materials

Agricultural and forest by-products

Chemical reagents

Transformation process

Low concentration of substance, inert ingredients, and pigments

Simple material components, high conversion rate

Products

Low concentration of products, many impurities

High concentration of products, fewer by-products

Separation

High requirement for separation process design

Conventional separation methods

8.5.2  Technical and Economic Analysis of Posttreatment for Biomass Biochemical Transformation Low concentration of products and many impurities lead to low equipment efficiency and high cost. To overcome this problem, Yang, Ding, and Chen (2005) developed an enzymolysis–fermentation–separation coupled technique for cellulose enzymolysis and ethanol fermentation. The coupled system includes an enzymolysis zone and a fermentation zone. The enzymolysis zone consists of the enzymolysis tank and a hollow fiber ultrafiltration membrane module, while the fermentation zone consists of a fermentation tank and a pervaporation device. The temperature is controlled at 50°C and 35°C respectively to make the enzymolysis and fermentation proceed at the optimal temperature. Straw is enzymolyzed in the enzymolysis tank while enzymatic hydrolysate circulates in the enzymolysis zone. Cellulase and sugars are separated by the ultrafiltration membrane. Cellulase is entrained by the enzymatic hydrolysate back into the enzymolysis zone for continuous enzymolysis while the sugars enter the fermentation tank for fermentation by immobilized yeast. The feedback inhibition effect on enzymolysis of sugars is therefore reduced. The pervaporation device is started at a certain time for online ethanol separation to eliminate the inhibitory effect of ethanol on cellulase and the strains. Results indicated that ethanol concentration increased by 8.6% after 72 h of fermentation in the enzymolysis and fermentation circulating devices without online ethanol separation, compared with that in isothermal simultaneous saccharification and fermentation. Again, ethanol concentration increased 2.4 times in enzymolysis and fermentation circulating devices with online ethanol separation, compared with that in isothermal simultaneous saccharification and fermentation. This means that online ethanol separation produces 2.1 times higher ethanol concentration.

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Therefore, the low concentration of ethanol is effectively mitigated and production cost is reduced. A detailed analysis is conducted as follows: 1. Fixed assets For 1 t/batch ethanol production, the capacity of the evaporation device needs to reach 1 t with separation after fermentation. While for separation coupling with fermentation, the evaporation device is started for 2 h at intervals of 16 h. The capacity of the evaporation device is calculated as follows: 16 + 2 ∗ 1 t = 0.25 t 72

Therefore, the production process is divided by the timely separation of ethanol, and this reduces the equipment investment. 2. Electricity consumption Electricity consumption is almost equivalent under the two strategies. 3. Labor cost As the strategy of separation after fermentation prolonged the production cycle, the labor cost increased accordingly. 4. Cost of raw materials Ethanol concentration increased by 2.1 times in enzymolysis and fermentation circulating devices with online ethanol separation, compared with that in devices with separation after fermentation. Therefore, the reduced cost of raw materials is calculated as follows.  1 − 1  ∗ 100% = 67.7%   3.1  To conclude, the technical and economic comparison of online separation and separation after fermentation is illustrated in Table 8.2. TABLE 8.2 The Technical and Economic Comparison of Online Separation and Separation After Fermentation Online separation

Separation after fermentation

Equipment investment

Production is divided, equipment investment is reduced

Large quantity of treating samples requires large items of equipment

Operation cost

Integrated equipment, convenient operation and management, low operation cost

Cycle is prolonged, operation cost is increased

Materials cost

Increased product concentration reduces the materials cost

Materials cost is relatively high

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REFERENCES Banat, F., & Al-Shannag, M. (2000). Recovery of dilute acetone-butanol-ethanol (ABE) solvents from aqueous solutions via membrane distillation. Bioprocess and Biosystems Engineering, 23, 643–649. Chen, H. (2004). Bioprocess engineering and equipments. Beijing: Chemical Industruy Press. Chen, H. (2008). Biomass science and engineering. Beijing: Chemical Industry Press. Chen, H., & Qiu, W. (2010). Key technologies for bioethanol production from lignocellulose. Biotechnology Advances, 28, 556–562. Chen, H., & Wang, L. (2008). Conversion technology and uitilization of biomass energy (VIII)— Principle and application for biomass bioconversion technology. Biomass Chemical Engineering, 42, 67–72. Dai, J., Sun, Y., Sun, L., et al. (2010). Research progress of bio-based chemical 2,3-butanediol. The Chinese Journal of Process Engineering, 10, 200–208. Ezeji, T., Qureshi, N., & Blaschek, H. (2003). Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping. World Journal of Microbiology and Biotechnology, 19, 595–603. Ezeji, T., Qureshi, N., & Blaschek, H. (2004). Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping. Applied Microbiology and Biotechnology, 63, 653–658. Garcia, Bonen, Ramirez-Vick, et al. (2004). Bioseparation process science. Beijing: Tsinghua University Press. Ghosh, S., & Swaminathan, T. (2003). Optimization of process variables for the extractive fermentation of 2,3-butanediol by Klebsiella oxytoca in aqueous two-phase system using response surface methodology. Chemical and Biochemical Engineering Quarterly, 17, 319–326. Ghosh, S., & Swaminathan, T. (2004). Optimization of the phase system composition of aqueous two-phase system for extraction of 2,3-butanediol by theoretical formulation and using response surface methodology. Chemical and Biochemical Engineering Quarterly, 18, 263–272. Hu, Y., Ouyang, P., Shen, S., et al. (2001). Study on the optimal conditions in simultaneous reaction and separation for l-malic acid production. Chinese Journal of Biotechnology, 17, 503–505. Huang, H., Ji. X., & Li, S. (2008). A method to utilize hydrophobic silicon zeolite for adsorption separation of 2,3-butanediol from the fermentation broth. China, CN200810024865.6. Ishizaki, A., Michiwaki, S., Crabbe, E., et al. (1999). Extractive acetone-butanol-ethanol fermentation using methylated crude palm oil as extractant in batch culture of Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564). Journal of Bioscience and Bioengineering, 87, 352–356. Jin, Y., Feng, X., & Su, Z. (1998). Expanded bed adsorption and its application in biochemical engineering. Chemical Industry and Engineering Progress, 17, 45–50. Li, X., & Wu, S. (2006). Research development of the reaction characteristic between lignin quinonoid chromophoric group and hydrogen peroxide. Journal of Cellulose Science and Technology, 14(4), 55–59. Lin, S., & Kringstad, K. (1970). Photosensitive groups in lignin and lignin model compounds. Tappi, 53, 658–663. Liu, F., Liu, L., & Feng, X. (2005). Separation of acetone-butanol-ethanol (ABE) from dilute aqueous solutions by pervaporation. Separation and Purification Technology, 42, 273–282. Liu, G., Jiang, B., Wang, Y., et al. (2009). Aqueous two-phase extraction of 2,3-butanediol by ethanol/potassium carbonate system from Dioscorea zingibe rensis fermentative broths. Journal of the Chemical Industry and Engineering Society of China, 60, 2798–2804. Mei, L., Yao, S., Lin, D., et al. (1999). New trends in biological separation process research—high effective integration. Chemical Engineering, 27, 38–41.

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Qureshi, N., & Blaschek, H. (2001). Recovery of butanol from fermentation broth by gas stripping. Renewable Energy, 22, 557–564. Qureshi, N., Meagher, M., & Hutkins, R. (1994). Recovery of 2,3-butanediol by vacuum membrane distillation. Separation Science and Technology, 29, 1733–1748. Shao, P., & Kumar, A. (2009a). Recovery of 2,3-butanediol from water by a solvent extraction and pervaporation separation scheme. Journal of Membrane Science, 329, 160–168. Shao, P., & Kumar, A. (2009b). Separation of 1-butanol/2,3-butanediol using ZSM-5 zeolite-filled polydimethylsiloxane membranes. Journal of Membrane Science, 339, 143–150. Tan, T., Zhang, H., & Wang, F. (2000). Preparation of PVA/chitosan lipase membrane and its application in synthesis of monoglyceride. Journal of Chemical Industry and Engineering, 18(s1), 325–331. Wang, S., Li, Y., & Zhang, X. (2005). Combinations of bioreactor and membrane separation. Jiangxi Science, 23, 185–190. Wang, X., He, R., & Ouyang, P. (1999). Research review on integrated bioreaction-separation process. Jiangsu Chemical Industry, 27, 7–11. Xiu, Z., Li, Z., & Zhang, J. (2007). A aqueous two-phase extraction method of 2,3-butanediol from fermentative broths. China, CN200710010203.9. Yang, S., Ding, W., & Chen, H. (2005). Enzymatic hydrolysis of steam-exploded rice straw in membrane bioreactor. Environmental Science, 26(5), 161–163. Yu, J., Tang, X., Wu, X., et al. (2003). The new biological processes and technologies. Beijing: Chemical Industry Press. Zhang, J., Sun, L., & Xiu, Z. (2008). Removal of Klebsiella pneumoniae cells from 2,3-butanediol fermentation broth by flocculation and reuse of cells in flocs. The Chinese Journal of Process Engineering, 8, 779–783. Zhang, W., Yu, X., Yuan, Q., et al. (1997). Ethanol fermentation -pervaporation process with complete cell recycle. Membrane Science and Technology, 17, 42–47.

Chapter 9

Coproducts Generated from Biomass Conversion Processes Chapter Outline 9.1 Characters of Raw Materials and the Necessity of the Polygeneration Mode 220 9.1.1 The Characters of Raw Biomass Materials 220 9.1.2 Problems in the Process of Biomass Exploitation 221 9.1.3 The Necessity of Polygeneration in Biomass Biochemical Transformation 221 9.2 The Breakthrough of Key Polygeneration Bonding Techniques 222 9.2.1 Nonpolluting Steam Explosion and its Platform of Component Separation Technologies 222 9.2.2 Platform of Water and Energy Saving Solid Pure Fermentation 222 9.2.3 Platform of Straw Solid-State Enzymolysis Coupled with Liquid-State Fermentation 223 9.2.4 Platform of Polysaccharide Fermentation Coupled with Membrane Recycling Enzymolysis 223

Technologies for Biochemical Conversion of Biomass © 2017 Metallurgical Industry Press. Published by Elsevier Inc.

9.3 Cleaner Production and Polygeneration 224 9.3.1 The Concept and Connotation of Cleaner Production 224 9.3.2 Cleaner Production and Polygeneration of Biomass Resource 225 9.4 Eco-Industry and Polygeneration 227 9.4.1 Biochemical Engineering and Industrial Ecology 227 9.4.2 Industrial Ecology Theory 228 9.4.3 Industrial Ecology Research Methods 233 9.4.4 Eco-Industrial and Process Integration 238 9.4.5 Technology Paradigm of the Ecology Industry and Polygeneration 242 9.5 Circular Economy and Polygeneration 247 9.5.1 Concepts and Technical Characteristics of the Circular Economy 248 9.5.2 Polygeneration of a Circular Economy and Biomass Resources 251

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9.6 Low Carbon Economy and Biological Products 252 9.6.1 Low Carbon Economy 252 9.6.2 The Low Carbon Economy and Biological Refining 253 9.6.3 The Low Carbon Economy and Bio-based Products Polygeneration 254 9.7 The Economic Analysis of Polygeneration of Biomass Biochemical Transformation 257

9.7.1 Economic Importance of Biomass Polygeneration 257 9.7.2 New Biomass Platform Chemicals 259 9.7.3 Economic Comparison of Bio-based Polygeneration Products to Replace Traditional Platform Chemicals 260 References 263

9.1  CHARACTERS OF RAW MATERIALS AND THE NECESSITY OF THE POLYGENERATION MODE 9.1.1  The Characters of Raw Biomass Materials The main reason why biomass is not utilized efficiently is its own character. Straw and wood are both lignocelluloses, consisting of cellulose, hemicelluloses and lignin. However, there are differences between straw and wood in structure and chemical composition, as well as their characters when transforming. The traditional process of biological transformation involves treating the straw as material with a single character, mainly utilizing the cellulose in it, which makes it hard to adapt this process to high-value utilization in industry. In fact, the biological structure of straw is not uniform. The chemical composition and form of cellulose are different in stem, stalk, leaves, spike, and sheath. In corn straw, the rind and leaves are compact in structure, while its core is loose. With regard to cellular composition, the parenchyma cell content is least in the rind, and 60% and 70% respectively in leaves and core. As to chemical composition, the cellulose content is highest in the rind, while the hemicellulose content is highest in the leaves. Lignin is abundant in the rind and the cob. The nonuniformity of structure results in differences in different parts when transforming. The transform characters and products change according to the different structures and compositions in the straw. Meanwhile, there are differences in enzymolysis, physics, and chemistry performance among different parts, tissues, and cells. For example, the nonuniformity of structure and properties in corn results in nonuniformity in the enzymatic hydrolyzation rate. After enzymatic hydrolyzation continuing for 24 h, the rate may reach 88.23% in the core of corn straw with the highest value, while the rate is about 28.33% under the same condition in the leaves. The fiber shows different characters in different portions. There are fibers with high quality in parts of the leaves and rind, which have similar character to the wood fiber. In order to solve the problems of the use of a single biological transformation method during the process of straw transformation, it is vital to recognize the nonuniformity of straw. The combination of biological transformation

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technology and straw component separation technology means the technology of wood utilization can be avoided when transforming the straw raw materials into liquid fuel. Realizing the total utilization of straw is useful and can lower the cost of transformation (Chen & Qiu, 2007).

9.1.2  Problems in the Process of Biomass Exploitation It is impossible nowadays to overlook the economic aspects of the technology connected with biomass refinery. There are three main reasons: (1) work on the development of optimal technology focuses only on single cellulose composition and the application of a single technology, while the integration of the pretreatment system and research into coupling technology are lacking. As a result, there are significant problems of environmental pollution, waste of resources and high cost. For example, during production of cellulosic ethanol, the emphasis is on utilizing the cellulose component, while the other fractions, hemicelluloses, lignin, and protein, are not utilized fully. The utilization of cellulose alone to produce ethanol can not only increase the cost but also cause waste of resources and pollution of the environment. The economic efficiency of the cellulose ethanol industry is a difficulty urgently needing to be solved. (2) The extraction methods are expensive and horribly polluting. The effective constituents of plants need to be extracted from the complex homogeneous phase or the heterogeneous phase, then purified and refined through separation and removal of impurities. Some traditional separation technologies such as filtration, sedimentation, centrifugal separation, distillation, extraction, chroma crystallization, absorption, molecular distillation, ultrafiltration, elec and reverse osmosis contribute greatly as separation methods to the task of extraction of plants, effective constituents. (3) The ratio of material utilization and production yield is low. This low ratio results in low production yield. Some extraction methods use refined material in order to obtain products with a high production yield, but these methods also cause the rate of multipurpose materials utilization to decline and waste to increase. This in turn increases the cost of production.

9.1.3  The Necessity of Polygeneration in Biomass Biochemical Transformation The utilization of biomass resources presents problems at several levels in both science and technology, which cannot be solved by research into a single area. The characteristics and utilization value of plant resources should be considered as a whole. It is in this light that research projects should be undertaken, and likewise the development and utilization of various components and manufacturing techniques should be regarded as one system, that is, the polygeneration system of biomass. Improving the level of resource utilization in the context of total utilization of biomass can result in economically feasible, intelligent, and efficient resource utilization.

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The essence of biomass polygeneration technology is taking biomass as raw material, through integrating various biomass transforming technologies, to gain numerous high-additional value fuels (such as bioethanol, butanol, biodiesel, hydrogen, methane, and so on), chemicals (furfural, levulinic acid, xylitol, xanthan gum, oxalic acid, lactic acid, Bio-PE, and so on), and materials (cellulose acetate, carboxymethyl cellulose, biological plate, and so on). The aim of polygeneration technology is the maximum utilization of resource and total product efficiency with minimal pollutant discharge.

9.2  THE BREAKTHROUGH OF KEY POLYGENERATION BONDING TECHNIQUES 9.2.1  Nonpolluting Steam Explosion and its Platform of Component Separation Technologies During steam explosion, there is no need to add any chemicals. If only the water content of straw is controlled, over 80% of hemicelluloses can be separated and the enzymatic hydrolysis rate of straw cellulose can reach more than 90% (Chen & Liu, 2007b; Chen & Li, 2002). With improvements arising out of laboratory investigation, the technology’s scale of engineering grew to 50 cm3. At present, a series of innovative methods, such as clean pulping, clean marijuana degumming, humic acid, and active xylooligosaccharide production from straw using steam explosion technology, have been developed successfully. In addition, fibrous tissue separation through steam explosion and mechanical carding emerges as a reality. The technological platform of straw fibrous tissue separation through steam explosion and wet ultrafine grinding technology is established (Chen & Liu, 2007a).

9.2.2  Platform of Water and Energy Saving Solid Pure Fermentation Fermentation technology consists of liquid-state and solid-state fermentation. Compared with solid-state fermentation, liquid-state fermentation can realize pure fermentation and industrial amplification easily, but it also produces wastewater. Solid-state fermentation has the advantages of saving water and energy, but it is difficult to overcome the problems of hard resistance in heat and mass transfer, which can easily result in partial microbe death and a low fermentation production rate. Meanwhile, it is hard to realize pure fermentation on a large scale and easy to infect other microbes. Gas double-dynamic solid-state fermentation is a new technology invented to overcome these problems. During this process, no mechanical stirring is added artificially, and the gas state is the only one to be controlled during solid-state fermentation. On the one hand, the air pressure is in an up-and-down pulsation pattern; on the other, the gas phase of the bioreactor is in flow, which improves heat and oxygen transfer during fermentation and motivates microbe growth

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and metabolites, achieving pure cultivation. The 100 m3 gas double-dynamic solid-state fermentation bioreactor is now the largest solid-state fermentation system in the world. With this bioreactor, taking steam explosion corn straw as the main raw material to produce cellulase can realize cellulase activity as 120 FPA/g dry medium through five batches of experiments, and the highest value can reach 210 FPA/g dry medium. The new bioreactor achieved cellulase production on a large scale and at low cost (Chen & Qiu, 2010). The new gas double-dynamic solid-state fermentation technology can shorten fermentation time by one third and temperature oscillatory operation can improve microbe activity, which is beneficial to the optimization of multiple microorganism combination (Chen, 2013).

9.2.3  Platform of Straw Solid-State Enzymolysis Coupled with Liquid-State Fermentation The coupling technology of cellulose solid-state enzymolysis and liquid-state fermentation can improve cellulose enzymolysis efficiency and ethanol fermentation efficiency, solving the problems of excessive water use and discordant temperature in enzymolysis and fermentation during cellulose liquid diastatic fermentation synchronously, and lowering the cost of cellulose enzymolysis to produce ethanol. The new airlift high-duty ethanol fermentation and separation coupling technology is an assembly of an airlift bicirculating tower-type fermenter, vacuum reflow, CO2 air lift, circulation, and mixture, with activated carbon adsorption, which realizes the triple-coupling of enzymolysis saccharification, liquid fermentation ethanol, and adsorption and separation. This equipment lowers the usage amount of cellulase to 15I U/g straw and reduces the quantity of wastewater in the residuum. Meanwhile, saccharification and fermentation occuring in different regions of the same bioreactor can be convenient for coordinating the optimum temperatures of saccharification (50°C) and fermentation (37°C); it also overcomes the deficiency of solid state for fast ethanol fermentation, with an ethanol yield of 15%, straw cellulose transfer rate of 80%, and active carbon adsorbing and desorbing ethanol concentration of 50%, which lower the production cost of straw-fermented fuel ethanol (Chen & Qiu, 2010).

9.2.4  Platform of Polysaccharide Fermentation Coupled with Membrane Recycling Enzymolysis The usage cost of cellulase accounts for 50–60% of the total cost of biomass transformation, which is the main obstacle inhibiting the industrialization of lignocellulose (such as straw) enzymolysis. Using a membrane bioreactor to hydrolyze steam explosion straw and recycle and reuse cellulase is a relatively perfect pathway. Product inhibition can be removed, hydrolysis yield can be improved and cellulase can be reused, if using an ultrafiltration membrane with

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proper molecular weight to hold back cellulase and unhydrolyzed cellulose, while the hydrolysis product can penetrate the membrane. The concentration of reducing sugar is low using a traditional membrane bioreactor for cellulose enzymolysis, which is bad for the follow-up process. The final reducing sugar concentration can be improved if several enzymatic vessels are in series to enhance the concentration of substrate in the membrane bioreactor. Chen et al. (Yang, Ding, & Chen, 2006) studied how to use the membrane bioreactor to improve the reducing sugar concentration with steam explosion straw as raw material. The results showed that the makeup of enzymolysis units is four enzymolysis pots and the dilution rate is 0.075 h−1. When the enzymolysis time is 24 h, the total conversion rate of steam explosion straw can reach 39.5%, which is one time more than traditional batch enzymolysis. The yield of reducing sugar improved by 60% compared with membrane bioreactor with one enzymolysis pot. The final reducing sugar average concentration increased from 4.56 to 27.23 g/L.

9.3  CLEANER PRODUCTION AND POLYGENERATION 9.3.1  The Concept and Connotation of Cleaner Production The cleaner production mentioned in the Cleaner Production Promotion Law refers to the measures of improving design, using clean energy and raw materials, utilizing advanced technology and equipment, improving management and integrated utilization, reducing pollution from the source, and improving the level of resource utilization, in order to eliminate harm caused to human health and environment. The United Nations Environment Programme (UNEP) defines cleaner production as follows: cleaner production is a new idea, which the continuous application of an integrated, preventive strategy applied to processes, products and services in pursuit of economic, social, health, safety and environmental benefits. It improves production efficiency and reduces the risks of humans and the environment (Li & Ma, 2009). According to the definition of cleaner production, its core connotation is to implement source reduction or to control the overall process of production and service. Cutting down the production of pollutants from the source, in fact, is to make more materials into products, which is an active and preventative strategy, resulting in saving time; controlling the overall process of production and service entails choosing the raw materials, technology and equipment, supervising the process, improving the quality of staff, managing scientifically and efficiently, and controlling the overall process of reusing waste. In all, cleaner production has three aspects: 1. Clean energy. This refers to a conventional source of energy with clean utilization, the utilization of renewable energy sources, and the various types of energy-saving technology.

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2. Clean producing process. This refers to less or no use of poisonous and harmful raw materials; no instances of poisonous and harmful intermediate products; reduction or elimination of dangerous factors during the production process, such as high temperature, high pressure, low temperature, low pressure, inflammable materials in hazardous contexts, explosives, strong noise and vibration; less or no waste; very efficient equipment; recycling of raw materials (inside and outside the factory); simple and reliable operation and optimization; thorough quantitative management. 3. Clean products. This refers to the saving of raw materials and energy, less use of expensive and rare materials and more use of secondary resources as raw materials; no components harmful to human health and the environment during or after using; products are easy to recycle and reuse; proper packaging; truly functional products which are energy saving, water saving, with low noise when in use and a long service life; and products should be easy to dispose of and biodegradable (Lei & Shen, 2007).

9.3.2  Cleaner Production and Polygeneration of Biomass Resource During polygeneration of biomass resource, as regards raw material, the biomass resource refers to reproducible or recycled organic substances, such as crop, forests, other plants, and their remains. Its most important feature is reproducibility, and it can constantly meet production needs. In order to analyze biological resource availability, the biological resource can be divided into agricultural biomass, forest biomass, and industrial waste. Agricultural biomass includes agricultural wastes, livestock waste, energy crops, and so on. The source of agricultural wastes is wide, including by-products after reaping, such as crop straw; livestock waste, including animal dung, which can produce methane; energy crops includes renascent herbs (such as switchgrass and reed), fuelwood, oil crops (such as rapeseed and sunflower seeds), saccharine, and starchy crops. Forestry biomass includes wastes from the process of combustion and fell, trim, and cleaning of wood. The industrial waste comes from the wood-processing industry and the food-processing industry, including sawdust, shell, kernel, and bagasse. The papermaking black liquid comes from the stewing process during pulping, heat can be gained from boiler combustion, and useful chemicals can be recycled. Cleaner production involves not only knowledge of natural science and social science in specific applications, but is also constantly updated with people’s ideas, which is a new initiative and production mode; and concepts must be updated as following: first, the concept of end controlling pollution is transferred to the whole process of production control. With the acceleration of industrialization, the drawbacks of end controlling become increasingly apparent. The large treatment facility investment and high operating costs raise production costs and reduce incomes. Secondly, end control is difficult

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to complete, often resulting in the transfer of pollutants, for example in flue gas desulfurization and dust removal, forming a large number of wastes, and the large-scale wastewater treatment produces a huge quantity of sludge. End control does not use resources efficiently, and cannot stop the waste of natural resources. Cleaner production can overcome these drawbacks, and strive to eliminate waste generation from the start. Thirdly, the concept of the traditional mode of production is updated to the new concept of cleaner production. In order to obtain products, the traditional mode of production on the one hand, takes the resources available from the environment, and on the other hand, pours useless waste into the environment. Because of this, the world suffered serious environmental pollution and ecological destruction. Cleaner production should minimize material consumption and minimize the waste. Fourthly, the update of the concept of extensive to intensive types. The production process should be optimized to save energy and reduce pollution, resulting in cleaner production. Fifthly, the update of the concept of production with environmental pollution to that of one of sustainable development. Promotion of cleaner production can reduce resource consumption and waste generation in a large areas, and can lead to the restoration of damaged environments, which is beneficial to sustainable development (Ouyang, 2003). In view of the abundant straw resources, Hongzhang Chen from the Institute of Process Engineering, Chinese Academy of Sciences, proposed and validated new ideas of “full utilization of biomass straw,” “straw eco-industry,” “layered, multilevel utilization,” and “fast and efficient component separation.” These ideas update technical routes relying on a single technology or a single component, achieving layered and multilevel use of straw components, according to multidisciplinary, multitechnology, and multiproduct combinations of ecological engineering principles. Through a multidisciplinary and multitech integration, a new technology of economic straw utilization is created. Features of this technical system, which should be efficient, comprehensive, and appropriate are described as follows: (1) as regards the multicomponent and structural complexity of straw, the three components of cellulose, hemicellulose, and lignin must be separated fast, economically, and effectively. This is the key to achieving full utilization of cellulose material biomass, and it is not only the premise of large-scale industrialization, but also the new requirements of subsequent biotransformation; (2) Effective utilization of lignin and hemicellulose is important to reduce costs and achieve comprehensively multigenerational and cellulosic cleaner production; (3) It is not appropriate to concentrate on a single product, but to establish a full multiindustrial use model for straw biomass according to ecological engineering principles, emphasizing the multilayer, multilevel, and recycling pathway, and moderate scale; (4) Straw should be treated as an abundant renewable resource, and not just as an object of environmental governance. An ecoindustrial park could be established with straw as raw material to achieve cleaner production.

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9.4  ECO-INDUSTRY AND POLYGENERATION 9.4.1  Biochemical Engineering and Industrial Ecology With social development, environmental and ecological issues have increasingly become the focus of attention, and the ecology perspective has gradually penetrated into industrial fields. In September, 1989, the United States science magazine Scientific American published an article written by Robert Frosch and Nicolas Gallopoulos about “sustainable industrial development strategy,” which came up with the point of view that industry can use a new mode of production for the first time. The authors propose that an industrial ecosystem can run in a circular mode, like a biological ecosystem, that matter and energy are continuously cycled in a food chain running like this: plants → herbivores → carnivores → microbes → plants. The concept of industrial ecology was proposed. Based on this idea, the basic principles of industrial ecology, the 4R technical principles (Reduce, Reuse, Recycle, Recover) were put forward. “Reduce” to reducing the production and consumption of materials and energy in the process flow at the input; “Reuse” is achieved by the means of by-product exchange and multilevel and comprehensive utilization of materials; “Recycle” relies mainly on recycling conversion technologies, processes, and energy integration technology; “Recover” requires the utilization of renewable resources as raw materials for industrial processing process. Based on the principles of 4R technology, a series of research methods can be formed in the field of industrial ecology, such as industrial metabolism—research methods for raw materials, multiscale cleaner production; research methods for the reaction process, life cycle assessment (LCA); research methods for the product, systems integration of energy and matter; research methods for the whole process, eco-industrial park construction; and research methods for regional industrial systems. Research methods are also the goal that biochemical engineering researchers are pursuing. Biochemical engineering originated from the biological and industrial sector. The proposed industrial ecology provides a theoretical basis for ecological and industrial development, and the combination of ecological strategies and industrialization promotes the development of biochemical engineering in a new stage. In this sense, the traditional subject areas of biochemical engineering cannot meet the needs of development, and new theoretical guidance is required. On the basis of the combination of modern biotechnology and industrial ecology basic theory, we propose a new concept of ecological biochemical engineering (Chen, 2008). In nature, biology uses matter and energy for production and consumption, and emissions from one type of organism are the nutrients for other organisms, forming a continuous material flow, natural digestion and purification, which is the ecological balance. According to this principle the ecological industry is being established and developed. In industrial production, people simulate ecological principles and design a production process in which waste is raw material for the next production process, so that the raw materials and energy

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involved in the production process are used hierarchically. The aim is to form a continuous material flow and energy flow, seek the full use of substances, promote a virtuous cycle and produce more goods for society. This industrial production system is called eco-industry. Eco-industrial production must be clean, green industry. It places more emphasis on raw materials utilization, forming a raw material logistics chain. Eco-industrial production has better economic efficiency, reflects scientific and technological progress, and therefore is more competitive in the market for sustainable industrial development. Use of resources during traditional industrial production focuses on product. The final product only takes up 20–30% for total raw investment, and irrational use of resources results in environmental pollution. Take ajinomoto production for example, only one third of the raw material is converted into the ajinomoto, and two thirds of the materials generate wastewater with high acid concentration containing 12% solids with COD of 70,000–80,000L−1. Eco-industry involves the rational use of raw materials, producing socially desirable products. The same raw materials not only ensure the original production, but are also used to produce more varieties of goods. For example, in the production of ajinomoto, product quality and quantity of monosodium glutamate can still be guaranteed. Two thirds of the unused feedstock was converted into single-cell protein, fermented protein feed, and ammonium sulfate (fertilizer, chemicals) in eco-industry.

9.4.2  Industrial Ecology Theory In the traditional industrial system, every manufacturing process is independent of other processes, including consumption of raw materials, products, and waste output. This industrial system is overly simplistic, and an industrial ecosystem can run in a circular way, just like a biological ecosystem. There are three divisions of organisms by function in biological ecosystems. Some organisms survive on sunlight, water, and minerals; some species depend not only on air and minerals, but also on the consumption of other species to sustain life, while eliminating wastes which become the food of other species. Some species in the third class turn wastes into minerals, and some consume each other in a complex network of processes to achieve metabolism. Similarly, in the industrial ecosystem, each industrial process must be interdependent and interrelated with other industrial processes, which is an idealized model of development. Although existing resources and ideas cannot achieve perfect results, the idea has allowed people to envision a newer development direction—the industrial ecosystem (Frosch & Gallopoulos, 1989; Lu & Ye, 2000). The focus of industrial ecology research is on a reasonable approach to make industrial systems achieve good ecological carrying capacity and how to design energy flow and material flow between the environment and industrial systems by the principles of sustainable development, changing the existing and open

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industrial systems to closed systems, and promoting ordering and rationalizing uses of substances and energy (Liu & Zhou, 2001). Different researchers have summarized industrial ecology research, and many other overviews in the field can be divided into the following six aspects (Chen & Jiang, 2002): 1. Material and energy flow studies: also known as industrial metabolism, research on industrial systems, regional and global material flows quantification, the impact on natural ecosystems, and technical methods to reduce these impacts; 2. dematerialization and decarbonization: looking for approaches to reduce the amount of raw materials and energy required absolutely or relatively in industrial economic activity, such as reducing the allocation of resources, extending the product life cycle, and using nonfossil fuel for production; 3. technological change and the environment: research and development theory and technology for accelerating the evolution of the industrial system; 4. life cycle planning, design, and assessment: evaluation of the environmental load of products from raw materials acquisition to production, use until the final treatment; identification and quantification of the consumption of energy and matter and the release of pollution; evaluation of the impact of this release and consumption on the environment, and finally devising of methods to reduce these impacts; 5. eco-redesign: seeking a new concept of product design with requirements that in the product design stage, the ecological and economic balance and possible impact of products on the environment should be considered, in order to produce products with minimal impact on the environment during their entire life cycle, to establish sustainable production and consumption systems; 6. eco-industrial parks: rational plans of resource share among enterprises in terms of raw materials and the actualization of energy exchange; pollutants from an enterprise become resources of another corporate, with this system achieving minimized use of substances and zero emissions. Major research areas in industrial ecology are components of industrial systems and the relationship between the environment and industrial systems; and the main points of this research include the following three aspects: (1) discussion of an analysis perspective on the various components of industrial systems and coordinated development of their integration with the biosphere and the effort to build a pattern of sustainable development; (2) discussion of the metabolism and flow of the basic elements of the current industrial system and mobile networks of material flow and energy flow, and optimize existing industrial systems; (3) explore the development power of industrial systems—the significant power of technology in industrial systems. Long-term development and evolution of key technologies and types is a decisive (but not the only) factor of the industrial system, which is conducive to further mixing of the existing industrial structure, and more conducive to the trend in formation of new combinations (Hao, 2000).

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For different research perspectives, different industrial ecology theories arose, among which several are influential theories.

9.4.2.1  Three Evolutionary Theories of Industrial System Ecosystem The ecosystem evolves from the open ecological systems of original life forms into semiopen ecological systems, to achieve perfect closed ecological systems. The single-component material flow in open ecological processes (shown in Fig. 9.1) does not rely on other forms of life, which model exists only in the case of great wealth of resources, where the existence of life basically does not harm available resources. With the growth in the numbers and types of life form, it became difficult with existing resources to maintain simple metabolic consumption. Different populations began to mix in different ways to form semiopen ecological systems (Fig. 9.1). Semiopen ecological systems are evolved under internal pressure generated by the interactions of life forms, and are richer than the open systems in efficiency, but waste generated causes tremendous pressure on the environment. Resources have not been used with the greatest effectiveness, and nature has been evolving for a long time, so that the biological ecosystem has evolved into a state of almost total material recycling. In this system, resources and wastes are relative: the waste of one species may be the resource of another species, and this is called a closed ecological system (Fig. 9.1). Inside this biological ecosystem, material is recycled closed; only solar energy is an eternal energy input (Lu & Ye, 2000). The industrial system, as an important part of the ecosystem, is bound to go through or is going through a similar process: the great development of the industrial revolution, with its environmental destruction and waste of resources, has produced industrial systems which are constantly adjusting internal and external relations, evolving from open to semiopen or even closed. The current industrial system is only linear superposition of substances without mutual relationship; and these are called first class eco-industrial systems, being similar

FIGURE 9.1  Schematics of three kinds of ecosystems. (A) Open ecological system, (B) semiopen ecological system, (C) closed ecological system.

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FIGURE 9.2  The model of ideal industrial systems.

to open ecosystems. Compared with first-class eco-industrial systems, resources become limited in second-class eco-industrial systems. Although the rate of resource utilization improved greatly, the flow of substance and energy is still one-way. Second-class ecosystems cannot be maintained in the long run, and waste increased inevitably with the reduction of resources. In order to transform into a sustainable development system, the eco-industrial system evolved into running a full cycle, and resources and waste cannot be distinguished in this form, which is called the third-class eco-industrial system. The ideal industrial systems (Fig. 9.2) should be close to the third-class eco-industrial system (Hao, 2000).

9.4.2.2  Theory of Ecological Restructure The theory of ecological restructuring has four aspects: (1) reuse of waste as a resource; (2) closed circulatory system and minimization of the use of consumable material; (3) dematerialization of industrial products and economic activity (service-oriented); (4) decarbonization of energy. Ecological restructuring of industrial systems has an effect on levels of macro, meso, and micro. At the macro level, it improves the material and energy efficiency of the whole economy; at the meso level, that is, the level of enterprises and production units, it reexamines the product and the manufacturing process, in particular, to reduce waste; at the micro level, by optimizing the reaction process at the molecular level, it improves the efficiency of the reaction and design of the most simple chemical synthesis (Hao, 2000). The effects of the four aspects of ecological restructuring on the three levels above are not the same (Li, Hu, & Shen, 2003). 9.4.2.2.1  Waste Recycling The systematic use of waste as a resource is the cause of the development of industrial ecology. From an ecological point of view, there is no waste in the true

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sense, but a lack of population consuming this section of resources. The garbage dump is not useless, but the accumulated resources are temporarily unusable. Only in a sound industrial system can the use of resources be maximized, in order to make waste into a valuable resource. 9.4.2.2.2  Closed Substance Cycle Systems Recycling is extremely important, and has the advantage of forming a stable circulatory system, and even reducing the flow of substances. Industrial recycling should have certain basic features—natural circulation of energy self-supply, which is the problem for resource recovery now. For the purposes of the recovery system, resource input is often greater than resource output, and sources of capital and technology for the recycling industry are limited. To this end, the government should strengthen investment and technology development, so that recycling becomes the means of preserving material and conserving energy. 9.4.2.2.3  Prevent Contamination of Consumption Metabolic analysis clearly shows that many industrial products are expendable in use: packaging materials, lubricants, solvents, flocculants, soap, whitening and detergents, paints, pigments, pesticides, and so on. Most toxic metals, such as arsenic, cadmium, chromium, copper and aluminum, mercury, silver, and zinc are contained in different products, which are also consumed in normal aging and using. For the phenomenon of consumption emissions, the main strategy is prevention. (1) Improved materials, that is, incorporation of materials which can prevent consumable emissions in the use of everyday consumer goods. (2) Recycling: for example, the United States chemical giant DOW Chemical Company recently introduced a new concept in chlorinated solvents, “molecular hire.” DOW users no longer buy a molecule itself, but its functions. They return the solvent to DOW after use and it is regenerated by DOW. (3) Alternative or ban; “alternative” means that harmless compounds replace toxic substances, and “ban” means that when the risk of toxic consumed substances is too great and other methods cannot solve the problem, the use of such toxic materials should be banned. 9.4.2.2.4  Dematerialization of Products and Services Currently, the world population is growing rapidly, and if we want to both enjoy a high standard of living under such conditions and reduce the impact on the environment to a minimum, the only method is to obtain more services and products on the basis of the same amount of material or less. This is the idea of dematerialization, whose aim is to improve resource productivity. In order to produce the same amount of product, we now use fewer materials and less energy, this decrease being due mainly to advances in technology. For example, the average weight of the car chassis has been greatly reduced, mainly because of the use of a variety of polymeric materials to replace steel, and this

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replacement of substance is called “substance conversion.” There is another very important factor of dematerialization, which can be referred to as the “information alternative.” In agriculture, for example, as regards prevention, the usage of various pesticides is always increased to ensure their effect. Whereas an information system that combines real-time observation of pests and early warning mechanisms can allow farmers to use the amount of pesticide needed at the right time. In short, with the continuous development of new materials and improved recycling technologies, a large number of ecologically helpful tendencies of products and production methods will be further strengthened. 9.4.2.2.5  Energy Decarbonization Since the Industrial Revolution began, carbon emitted in the consumption of minerals in the form of hydrocarbon has been the predominant element, and hydrocarbons (coal, oil, and natural gas) have accounted for more than 70% of global mining resources. However, mineral resources are the source of many problems: the greenhouse effect, smog, red tide, and acid rain. In recent decades, consumption of carbon coming from mined ore is growing, but mainly in developing countries. In terms of the world’s energy consumption, hydrocarbons will be widely dominant in the long term, and therefore, an energy decarbonization strategy is a bad strategy for reduction. In the long term, solar, hydro, nuclear, and hydrogen fuel are ideal energy carriers.

9.4.2.3  Theory of Industrial Biotechnology Community In natural ecosystems, different biomes have always formed close relationships based on certain characteristics, forming ecosystems with unique structures and functions. This idea can be extended to the industrial system in order to optimize the combination of industrial activities, to achieve the optimization of material and energy flows, and reasonable utilization. There are more significant examples of industrial symbiosis, an industrial ecology parks, and an industrial ecology community.

9.4.3  Industrial Ecology Research Methods Industrial ecology research examines the processes of industrial ecology in the view of ecological theoretical perspectives, and the relationship between industrial activities and the ecological environment, in order to adjust and improve the current principles and methods of industrial ecological chain structure, and establish a new closed material loop, so that industrial ecosystems and the biosphere can be compatible and survive. Industrial ecology includes analysis of system structure changes through supply chain network analysis (similar to a food web) and material balance accounting methods, for functional simulation and industrial stream analysis looking at the (input stream and the output stream) to study metabolic mechanisms and control methods of industrial ecosystems. Systems analysis is the core method of industrial ecology, and life

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FIGURE 9.3  Technical framework of life cycle assessment (LCA).

cycle assessment and industrial metabolic analysis developed on the basis of this constitute an effective method currently widely used in industrial ecology (Cao, 2004) (Fig. 9.3).

9.4.3.1  Life Cycle Assessment 9.4.3.1.1  Life Cycle Assessment Overview Life cycle assessment (LCA) is an evaluation process of the product, process, or activity from the collection and processing of raw materials to production, transportation, sale, use, recycling, conservation, recycling, and final disposal, that is, the entire life cycle of the system, in terms of its environmental impact (Consoli, Allen, & Boustead, 1993). The definition of IS014040 for LCA is: methods of summary and evaluation of the environmental and potential impact of a product, process (or service) system, and outputs all throughout their life cycle. LCA is a major theoretical basis and analytical method of industrial ecology. Although the LCA is mainly used in the evaluation of products and product systems, it has also been widely used in the field of industrial metabolism analysis and construction of eco-industrial parks. 9.4.3.1.2  Implementation Steps of LCA Implementation steps are shown in Fig. 9.4. 9.4.3.1.3  Determination of Purpose and Scope The first step of the LCA is to determine the study’s purpose and define the study’s scope. The purpose, scope, and intent of the study involve the application of geographical breadth of research, broad time span, and quality of data needed as well as other factors which will affect the direction and depth of research. The purpose should include a clear explanation of the reason for LCA studies and the future application of the results. In order to ensure the breadth and depth of

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FIGURE 9.4  Implementation steps of LCA.

study to meet the stated objectives, the scope of the study should be defined in detail, including the system boundary, the time boundary, methods, data types, and assumptions. The life evaluation process usually requires a lot of data, and the data within a period are not valid for the entire LCA, so that consideration is required as to whether the data collected at different times are still representative. In addition, the timeliness of the data about LCA needs to be considered. 9.4.3.1.4  Inventory Analysis Inventory analysis is an expression of the basic LCA data, and is the basis of LCA. Inventory analysis is quantitative data analysis of resource, energy consumption, or emissions to the environment throughout their life-cycle stage, of product, process, or activity resources. The core of inventory analysis is to establish input and output of a production system in units of expressed product features. Inventory analysis is an iterative process, generally including preparation for data collection, data collection, calculation procedures, and the distribution method of inventory analysis, inventory analysis results, and other processes. Inventory analysis can deal with detailed inventory for the inputs and outputs of the studied production system of each process unit, providing detailed data to support the diagnostic process, logistics, energy flow, and waste streams. Meanwhile, inventory analysis is also the basic phase affecting evaluation. 9.4.3.1.5  Impact Assessment In order to apply life cycle assessment in a variety of decision-making processes, the potential impact of the exchange environment must be taken into account in assessing the relative importance of various environmental exchanges as well as the contribution of each stage of production, or each product component, and this can be called the life cycle impact assessment (LCIA). As part of the entire LCA, the LCIA can be used to: identify opportunities to improve production systems and to help determine prioritization; characterize or establish a frame of reference for the product system or unit process, through the establishment of a series of parameters on product type systems, and it can provide environmental data or information to support decision-makers.

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9.4.3.2  Materials and Energy Flows Analysis As one of the important industrial ecology research areas, materials and energy flow analysis came into being with the in-depth study of sustainable development; and during the research on the interaction of the economic system with the natural environment, especially with reference to industrial systems in the 1980s, and it has been important to implement management tools in environmental activity. Material and energy flow analysis is on the raw materials and energy flows of the industrial system, including analysis from the extraction of raw materials to production, consumption, and final disposal. Whether industrial metabolism or raw material and energy flow analysis, studies are on theory and methods of raw material and energy flow, impact on the economy and natural ecosystems, and theories, methods, and techniques for reducing these impacts within globally and locally in industrial systems, as well as the production process. The main points of raw material and energy flow analysis are: the human economy system is only a subsystem of the natural ecosystem (Fig. 9.5), whose material and energy flows are similar to raw materials and energy flows of natural ecosystems, and which is a flow process about transferring the principle and energy into products and wastes. This process will inevitably have an impact on the natural environment, and the impact strength depends on the strength of raw materials and energy use. Raw material and energy flow analysis is carried out to understand the relationship between material and the economy and the environment, during extraction, use, recycling, and disposal, that is, the entire life cycle, and to find opportunities

FIGURE 9.5  Economic subsystem of global ecosystem.

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to reduce waste at every stage during these processes, reduce resource consumption, and minimize environmental impact. The basic frameworks are: 1. The industrial system is a subsystem of the global ecosystem, and the material and energy flows should be observed and analyzed in terms of an approach to an ecosystem. The use of waste and materials is designed with the goal of harmonizing human systems with the ecological background to minimize the impact on the environment. 2. Emphatically consider the impact of economic activities on biogeochemical cycles of N, C, S, P, and other nutrients, and the impact on the global flow of metal elements. 3. Holistic and systematic approaches are taken to characterize and quantify the flow of raw materials and energy. Raw materials, and the energy inputs and outputs of industrial systems should be subject to tracking and evaluation during the entire life cycle from raw material extraction, manufacturing for consumer use, recovery, recycling process, to disposal, in order to understand the full cycle. 4. Flow analysis of raw material and energy can be carried out on different spatial scales: the effects of human activities on global material cycles can be studied on a national or regional scale. The flow of raw materials and energy within or between departments can be studied on the departmental scale. 5. Raw material and energy flow analysis can encourage people to creatively identify the efficient use of raw materials and waste elimination and other issues. For example, the balance of ecosystem energy inputs and outputs, the adjustment of industrial policy from the perspective of evolution, the establishment of eco-industrial parks, and so on. 6. Research on strategies and methods to reduce the use intensity of raw materials; and current research topics in this area are: reduction of toxic and hazardous waste, reduction and replacement of pollution sources, recycling, remanufacturing, green design, and extension of producer responsibility. Environmental issues are directly related to the material and energy flows of the economic system, and the raw material and energy flow analysis is a systematic approach to understanding raw material extraction, use and single termination disposal (waste or recycling). It seeks to identify the quantitative relationship between material and energy flows of the economic system and environmental problems, and thus provide the basis for solving environmental problems. Currently, different studies use different models, and the main methods are the following: 1. Mass Balance The mass balance method is a research method conducted by describing the flow of some particular factor at a given time point, including the loss to the environment, estimating the input and output of the raw material flow system at each stage, and proposing all views of path analysis connected with materials. In the 1990s, the US Bureau of Minerals completed a study using this method, including As, Cd, Pb, Hg, W, Zn, and other metals and

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minerals. These studies trace the flow path of each mineral raw material, to explore opportunities to reduce waste, in order to make more efficient use of resources; meanwhile, they evaluate and quantify production, use, recycling, and lost data; also, to assess the use, processing, recycling, and disposal of minerals, including lost to air, land, and water. 2. Input–Output Analysis (IOA) The thought of IOA was first proposed by the French economist, Quesnay, in the 1700s, and it has developed since then to become the standard economic tool. IOA is usually related to required raw materials and predicted consumption of some product based on national economic statistics, which can trace the consumption of basic material and energy.

9.4.4  Eco-Industrial and Process Integration High-tech biotechnology is an emerging industry. Although many products of biotechnology are still in the research and testing phase, and the development of commercialization is fraught with difficulties and setbacks, its industrial development trend is irreversible. It will become a new growth point for the promotion of socioeconomic development. In the biotechnology industry, a variety of disciplines need to be blended, generally combined with engineering disciplines. Many successful examples of bioindustrial products show that the industrialization of biological products is closely related to the traditional chemical industry. For historical reasons, people pay more attention to biotechnology laboratory work from the past. Nowadays, the development of biotechnology is rapid, but what seems to be missing now is the technology to transfer the lab results into commercial products serving mankind. The industrialization technology of bioengineering products represents a bottleneck for the rapid development of biotechnology. Because of lagging industrial technology, a lot of good biological products always stay in the lab and cannot become a commodity, or although a product becomes a commodity, it cannot really give back to people because of the high cost of production. Given the success of process integration technology in the chemical industry, the development of an integrated technology of biological processes will become an important route to the solution of the problem of bio-products industry. Process integration refers to advanced production technology, which combined two or more process steps or production techniques organically together simultaneously in a manufacturing process or device. The aim is to simplify the sequence of integration processes, improve production efficiency, and reduce investment and production costs. Whether you look at chemical engineering or biological engineering, they are inseparable from the process of integration technology. A biological system is an integrated system of various reactions; even in the simplest of Escherichia coli thousands of reactions also occurred for cell synthesis and metabolite generation; a biological system is also an integrated

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system with reaction and mass separate, where various nutrients pass into the cell through the cell wall, and some metabolites are needed to release into the extracellular area to prevent intracellular accumulation. In traditional industrial fermentation, the product needs to go through the five basic steps of enzyme production, enzyme separation, enzyme hydrolysis of biological macromolecules, cell culture, and the isolated target product; while in genetically engineered protein production, including the basic steps: enzyme production, enzyme separation, enzyme hydrolysis of biological macromolecules, genetic engineering cell culture, induced expression of the product, cell disruption, product separation, renaturation, and purification; apart from slowness of the process and the low generation efficiency, the following problems may also exist: product or substrate inhibition during enzyme production, inactivation of product or enzyme during the separation process; product or substrate inhibition during cell growth or enzymatic hydrolysis, target product loss in cell disruption, and the condition matching and mutual constraints associated with various isolation and purification methods. Integrated bioprocess technology is the integration of several different methods of the reaction or separation steps discussed earlier, in a reactor or in a process, which will not only be able to simplify the process and improve production efficiency, but can also solve the product inhibition, deactivation, and match operating conditions and constraints as well as other issues (Chen, 2008). Biological process integration technology mainly includes the following aspects.

9.4.4.1  Biological Reaction and its Integration 1. Integration of hydrolysis and fermentation: enzymatic hydrolysis and microbial culture can be carried out simultaneously in a reactor; because small molecules produced by the enzymatic hydrolysis can be utilized by the microorganisms in a timely manner, a reactor or step of enzyme separation purification can be reduced, avoiding the inhibition of enzymatic hydrolyzation. 2. The building of new species having an integrated tendency: with the applications of metabolic engineering and recombinant DNA technology, a hydrolase gene is associated into a target microorganism, constituting bacteria producing both hydrolase and target fermentation production. 9.4.4.2  Integration of Biological Reaction and the Separation Process 1. Coupling of reaction and separation: the inhibition product or by-product obtained from biological reaction is separated from the system, eliminating the product inhibition on the catalyst, and increasing the biological reaction rate. 2. Bioreactor with separation factor: with the tools of molecular biology and metabolic engineering, on the basis of understanding the mechanism of fermentation processes and microbial metabolic pathways, the substance with similar properties to those of the target product is reduced by adjusting the conditions for the biological reaction, reducing the burden on the subsequent separation process.

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3. Enrichment of the desired product during the reaction: in a system where the product inhibition on microbial growth is smaller, using the differences in the nature of the target product and other impurities, the concentration of desired product improves by means of physical chemistry and biology in the reactor, or the concentration of the target product increases, or the amount of liquid reduces. By the culture method as microcapsules for cell immobilization, the target of macromolecular product concentration in the capsule can be achieved.

9.4.4.3  Integrated Biological Separation Process 1. Integrated biological process unit: through new and efficient separation technology, relevant units of processes achieve effective combination, effective reducing steps, and increased production efficiency. 2. Integrated set of separation: through the use of existing and newly developed biochemical separation techniques, or integration of two or more separation techniques based on different separation principles into a more effective separation technique, separation efficiency increased greatly. 9.4.4.4  Integration of Bioreaction and Modeling and Control of the Process Integration technology of bioreaction and modeling and control of the process is a better way to solve some special cases in biological systems. As the cell culture system is very complex, there will be a lot of unfavorable factors for the production target product. However, there are certain rules, which are difficult to resolve through general regulation and control. When the reaction pathways of biological systems and reaction laws are understood, the negative factors can be minimized with a precise control method. Such a set can form the integration of bioreaction and modeling and control. Work in this area is conducted in large companies around the world, which have achieved very good results. However, owing to the high level of technological confidentiality, the relevant papers and reports are few. Owing to the relative weakness of corporate research, in addition to the production of baker’s yeast and a few sets of production equipment for the introduction of antibiotics, the application of technology in this area is still lacking. In short, biological process integration technology is a direction of technology development in the bioproducts industry, and the strengthened research in this area will greatly promote the industrialization process in biotechnology. China has included the development of biotechnology as an important task of the century, and the industrialization of biotechnology is the ultimate goal in achieving the rapid development of biotechnology. High speed and efficient transformation of laboratory results into products as well as strengthening the technical content of the biotechnology industry are imperative. In the 20th century, the process industry has made tremendous progress and increased its development around the world; cell technology has reached a high level, and production scale and efficiency have improved continuously.

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Nonetheless, the contradiction between development and pollution and the search for sustainable development remain unresolved problems. As a new field of process engineering, process integration from a broader perspective integrates some new technologies and processes together, possibly having the solutions to the issues of high quality, energy saving, environmental protection, and sustainable development of the process industry from the source, which are the hotspots in process engineering research. Many organizations around the world have studied these matters. Among them are 16 multinational companies coordinated by the Centre for Process Integration at the British UMIST, as well as the Integration Committee at the International Energy Agency (IEA) which are the main process integration research centers in the world at present. Studies of process integration began in the late 1970s, initially mainly used for system energy saving, and developed into a systematic approach for the analysis and design of heat exchanger networks. Pinch technology has been widely used in the field of process industry, and much industrial practice shows that pinch technology plays an important role in improving system energy efficiency, and reducing investment and operating costs. On the basis of the integrated heat exchanger network and ideological pinch technology, whose field of application has gradually extended to improve utilization of raw materials, reduce emissions and operating procedures, and so on. Currently, the range of process integration is mainly within the macro-scale, shown in Fig. 9.6. The most simplified level is integrated within the single production process; at the next, energy and material integration among different processes under consideration; the highest level involves considering the development and coordination of the process industry, society, and environment, constituting eco-industry. Of these, the integrations on the levels of process and enterprise are more mature. In contrast, the process integration of industrial

FIGURE 9.6  Process integration at different levels.

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ecology research is in its infancy. With the development of process integration technology, its application scale expands to keep smaller molecules and a longer chemical supply chain. The process integration approach is not limited to pinch analysis, mathematical programming, and artificial intelligence techniques, but also includes the crossover and combination of these two methods with thermodynamic methods that have been introduced in the process of integration. At present, the integration process refers to the design optimization from the system perspective, with comprehensive integration around the material flow, energy flow, and information flow of the chemical system, providing direct support with methods and tools for process development. In this case, new concepts and techniques of process integration have been proposed, such as mass exchange network, process integration taking into consideration environmental impact, multigeneration systems, industrial ecology, and chemical supply chain integration (Chen, 2008).

9.4.5  Technology Paradigm of the Ecology Industry and Polygeneration An artificial ecosystem is composed of a variety of structural units. Through different combinations, these structural units form structural chains with different lengths and functions, and in various combinations these chains form a structural net. At the moment, the reason why many system developments are unsustainable is that the system structure is irrational. Such irrationality, on the one hand, can mean that a structural unit is improperly selected; on the other, it gives rise to the uncoordinated relationship between structural chains by unit and the structure mesh, which not only causes ecological balance disorders of the system, but also results in the blocking of system energy flow and interruption of the material cycle, and so affects the normal functioning of the system. The key to solving this problem is to design the system architecture for different situations affecting different systems. In this chapter, corn ethanol ecological engineering in Zhaodong City, Heilongjiang Province was taken as an example, with a study of the effect of system architecture design from three aspects, structural elements, individual chains, and the net’s effects (Chen, 2008).

9.4.5.1  Production Profile of Corn Ethanol Zhaodong City, Heilongjiang Province, is the main producing center for corn, and corn perennial plantings account for 68% of the city’s total area of arable land, with a total production of over 100 million tons, accounting for over 80% of the total grain output. In order to achieve scaling and industrialization of corn resource utilization, and an increase of productivity, in 1994, Zhaodong established corn-processing enterprises with the Jinyu Company as a leader in the processing and production of alcohol, with a processing capacity of 450 kt. Beside the output of alcohol, many by-products are also obtained such as CO2, distillers’ grains, corn germ and wastewater, among others. These products have

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a certain practical value, but direct emissions will bring great harm to the environment. Therefore, resource utilization, improvement of economic efficiency, and reduction of pressure on the environment have become the prime target of local companies and governments. The goal cannot be solved by traditional ideas and methods alone, and new breakthroughs must be sought. Ecological engineering and industrial ecology theory provide a good theoretical basis and methodology guidance for solving this problem.

9.4.5.2  Corn Ethanol Ecological Engineering Design Research on artificial ecosystem structure showed that the system function is completed by multilevel structure relationships of the structural elements, structural chains, and structural nets. Therefore, ecological engineering structural design must also be carried out from those three levels. The structural element is the basic unit or element of the system, and is the conceptual base of system components; the structural chain is the basic structural system, completing certain functions through a series of contacts; the structural net is the network structure relationship made up of different structural chains connected in certain ways. 9.4.5.2.1  Design of Structural Unit The design of a structural unit is the independent design process for the units of the production system after the conforming of the main target. It includes design for the type of product, size, and the utilization methods of related products. Accordance with the existing scale of operation, the processing of 45 kt corn in Zhaodong can produce 130 kt ethanol, 113 kt CO2, 70 kt distillers dried grains, 70 kt corn germ, and 1.80 Mt distillery waste. CO2, distillers dried grains, corn germ, and distillery waste belong to the structural unit consisting of corn processing systems, which need to be designed. 1. Corn germ utilization. The alcohol production process can produce 70 kt/a of corn germ. Based on the corn oil yield, 70 kt corn germ is designed to produce edible corn oil, and the designed production level was 13.4 kt. 2. CO2 utilization. CO2 and methane can directly synthesize acetic acid, and acetic acid is an important chemical intermediate and chemical reaction solvent, of which the maximum use is in the production of vinyl acetate monomer (VAM), and VAM can be used to produce protective coatings, adhesives, and plastics. The second largest, also the fastest growing use of acetic acid is in preparing purified terephthalic acid (PTA), and other uses include the production of acetic anhydride, propyl acetate/butyl acetate, and ethyl acetate. Thus, acetic acid industry is an effective way for the utilization of CO2. 3. Vinasse and waste utilization. Storage of vinasse and waste is difficult, owing to their high yields. Therefore, they are combined with cornmeal and used to produce pellets which are easily stored and transported, and the designed quantity is 120 kt. These products are supplied to farmers and farms

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run on a scale where the capacity is for the slaughter of 200,000. Farms were later built with an area of 35,000 m2, and their annual pig population reached 14,000 in 1997, with 2500 sows. Because fly ash is difficult to handle in the company power plant; pig manure has large manure moisture and odor concentration; it is also inconvenient to transport and use, and farmers are not very enthusiastic and so a new type of organic and inorganic fertilizer was designed using a combination of manure, inorganic fertilizer, and fly ash, in order to achieve value-added products and full and rational use of substances. This project has been put into production. 4. Design of distillery waste utilization. (1) Production of single-cell protein. According to experimental analysis, wastewater in vinasse contains 2500mg/L COD, 112mg/L nitrogen-containing, 30mg/L phosphoruscontaining, and 2.5–3.0% total solids with insoluble solids suspended of approximately 2.0–2.5% and soluble solids of 0.5%. Because it contains a lot of organic material, it can be used as a single-cell protein medium. Fungus mycelium contains 50–56% crude proteins, and is a high-quality protein like yeast. It can be used as a feed additive, and can be harvested only by filtration products. Single-cell protein culture tests carried out on the distillery waste showed that: single-cell protein yield from alcohol waste is generally up to 1.5–2%, and by this method the removal of COD at 30,000–40,000 mg/L is up to about 83% of the wastewater, after which treated wastewater COD and BOD are significantly reduced. (2) Waste storage aeration. After the above treatment, the COD concentration of wastewater is still about 6000 mg/L. According to the status of Zhaodong, with rich land and a relatively large proportion of saline, in order to achieve comprehensive wastewater management and get the appropriate benefits, depending on the actual terrain, eight water tanks were designed with a total volume of 78.66 × 104 m2. The water storage capacity was nearly 80 × 104 m3; wastewater storage accounted for 44.44% of the company’s total emissions, accepting and processing the wastewater equivalent of all wastewater produced by the entire company in nearly 6 months. The purpose of storage is to reduce the amount and concentration of organic substances in the hot season by biochemical processes. The wastewater was in storage in the cryogenic season, and was treated biologically when the temperature was suitable to achieve the goals of reuse and reduction of the environmental burden. Jilin fruit plants obtained more satisfactory results using the same method. (3) Pisciculture. The land north of Jinyu Company is all-saline with flat fine soil, and good antileakage performance, and so is easily transformed into a fish pond. Combined with the company’s development plan, four fish ponds with depth of 2–3 m were designed in 1996 to be built with an area of about 13,000 hm2 and a total capacity of 260,000 m2. To explore the use of wastewater for fish and the possibility of regarding fish as system structural units, the company conducted a test in mid-August to late September of 1997. Depending on technical processing requirements, six treatments were designed, each of which has a

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repetition. The depth of the test pools is 120 cm, and their radius is 47.5 cm. Unless otherwise stated, the concentrations of wastewater treatment added were 6000 BOD/L. The added amount accounted for 1% of the total amount of water once a day, and the chief detective indicators were COD, biomass, and dissolved oxygen (DO), when COD and biomass measurements were measured once a day, and DO measured once every five days; six treatments were included: control (No. 1, added the same amount of water as in the other groups), pure water distillers grains (No. 2), introduced species (No. 3, the proportion of added bacteria being 2% of the pool volume), open aerator (No. 4), added fertilizer (No. 5, 0.3 mg/L urea-based), and fermented alcohol wastewater (COD