Biotechnology in the Chemical Industry: Towards a Green and Sustainable Future 0128184027, 9780128184028

Table of contents :
Cover
BIOTECHNOLOGY
IN THE CHEMICAL
INDUSTRY:
Towards a Green and
Sustainable Future
Copyright
Dedication
List of figures
List of tables
Preface
Acknowledgments
1
General background and introduction
The impact of industrial biotechnology
Products of industrial biotechnology
Bulk chemicals
Biofuels and bioenergy
Fine and speciality chemicals
New materials
References
Relevant websites
Further reading
2
Industrial sustainability and biotechnology
References
Relevant websites
3
Renewable energy versus fossil resources
Renewable raw materials for the industry
References
Relevant websites
4
Chemical industry
Classification of chemical industry sectors
Basic chemicals
Speciality chemicals
Consumer chemicals
Life science products
References
Relevant websites
5
Bioprocesses in industrial biotechnology
Fermentation processes
Types of fermentation processes
Submerged fermentation
Surface fermentation
Solid-state fermentation (SSF)
Factors affecting enzyme production in SSF systems
Enzymatic processes
References
Relevant websites
Further reading
6
Application of biotechnology in chemical industry
Bulk chemicals
Citric acid
Lactic acid
Propane-1,3-diol (1,3-PDO)
Amino acids
Lysine
l -Glutamic acid
Food additives and food supplements
Starch modification, production of sweetener, and glucose syrups
Baking
Dairy products
Brewing
Distilling potable alcohol
Feed additives
Phytase enzymes
Nonstarch polysaccharides (NSP) degrading enzymes
Proteases
Mode(s) of action of enzymes
Agrochemicals
Fertilizers
Crop protection
Biocolorants
Flavors and aroma compounds
Solvents
Speciality products
Fermentation
Enzymes
Personal care products
Superoxide dismutase
Peroxidase
Tyrosinase
Proteases
Lipases
Hyaluronidase
Soaps and detergents
Bioplastics and other biopolymers
Biobased polymers
Biodegradable polymers
Biofuels
Bioethanol
Other biofuels made by assistance from enzymes
Processing of oil and fats
Bioremediation
Bioprocessing of pulp and paper
Biodebarking
Biobleaching
Deinking
Production of dissolving pulp
Fiber modification
Removal of shives
Retting of flax fibers
Biological solutions to processing problems
Slime control
Stickies control
Enzymatic modification of starch for surface sizing
Bioremediation of pulp and paper mill effluents
Organic synthesis
Transgenic plants
References
Relevant websites
Further reading
7
The economical and ecological advantages of industrial biotechnology
References
Relevant websites
8
Efforts made by different countries toward industrial biotechnology
Reference
Relevant websites
9
Major challenges facing industrial biotechnology
References
Relevant websites
Further reading
10
Future perspectives
References
Relevant websites
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
Back Cover

Citation preview

BIOTECHNOLOGY IN THE CHEMICAL INDUSTRY

BIOTECHNOLOGY IN THE CHEMICAL INDUSTRY Towards a Green and Sustainable Future Pratima Bajpai

Consultant-Pulp and Paper, Kanpur, India

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States © 2020 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. 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: 978-0-12-818402-8 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Susan Dennis Acquisition Editor: Kostas KI Marinakis Editorial Project Manager: Redding Morse Production Project Manager: Vijayaraj Purushothaman Cover Designer: Greg Harris Typeset by SPi Global, India

Dedication “Dedicated to my beloved parents and family,” for their love, endless support, encouragement and sacrifices.

List of figures FIG. 1.1  The industrial biotechnology value chain. FIG. 2.1  The triple P bottom line (DSM, 2004). FIG. 5.1  (A) Stirred tank fermenter. (B) Air-lift fermenter with an internal loop cycle of

FIG. 6.1  FIG. 6.2  FIG. 6.3  FIG. 6.4  FIG. 6.5  FIG. 6.6  FIG. 6.7 



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fluid flow and with an external recirculation pump. (C) Fluidized bed bioreactor https://www.kisspng.com/free/­fluidized-bed-reactor.html. (D) Fixed bed bioreactor. (E) Bubble column reactor. Parts A, B and E: Reproduced with permission from Najafpour (2007). Part D: Reproduced with permission from essentialchemicalindustry.org. 40-42 Production of L-lysine HCl via a conventional fermentation process (www.chemengonline.com/l-lysine-hcl-production-glucose/). 72 Growth in L-lysine production over the past several decades (www.chemengonline.com/l-lysine-hcl-production-glucose/). 72 Structure of food-grade pigments. Reproduced with permission from Venil et. al., 2013. 100 Stages of bioethanol fuel production. Reproduced with permission from Achinas and Euverink, 2016. 146 Different types of forest biomass. Reproduced with permission from Achinas and Euverink, 2016. 147 Schematic of a biochemical cellulosic ethanol production process. Reproduced with permission from Achinas and Euverink, 2016. 147 An overview of combinatory methodologies for bioremediation. Reproduced with permission from Sharma et al., 2018. 156

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List of tables TABLE 1.1 TABLE 1.2 TABLE 1.3 TABLE 1.4 TABLE 1.5 TABLE 1.6 TABLE 1.7 TABLE 3.1 TABLE 3.2 TABLE 3.3 TABLE 3.4 TABLE 4.1 TABLE 4.2 TABLE 5.1 TABLE 5.2 TABLE 5.3 TABLE 5.4 TABLE 5.5 TABLE 5.6 TABLE 5.7 TABLE 5.8 TABLE 5.9 TABLE 5.10 TABLE 5.11 TABLE 5.12 TABLE 5.13 TABLE 5.14 TABLE 6.1 TABLE 6.2 TABLE 6.3 TABLE 6.4 TABLE 6.5 TABLE 6.6 TABLE 6.7 TABLE 6.8 TABLE 6.9 TABLE 6.10 TABLE 6.11 TABLE 6.12 TABLE 6.13 TABLE 6.14 TABLE 6.15 TABLE 6.16 TABLE 6.17 TABLE 6.18



Industrialization of biology—benefits. 2 Color code differentiating the main areas of biotechnology. 2 Some historical developments in industrial biotechnology. 3 Environmental and economic benefits of biotechnology. 5 Defining technologies of modern biotechnology. 5 A vision for 2025. 8 Advantages of biotechnology in chemical production. 11 Renewable energy. 24 Fossil fuels. 24 Average world market price of some fossil and renewable resources. 27 Industrial sectors supplying the most important renewable raw materials. 29 Products from the chemical industry in 2014 by category (%). 33 Subsegments of the chemical industry. 33 Advantages of bioprocesses. 38 Different types of sparger. 43 Advantages of submerged fermentation over surface fermentation. 44 Disadvantages of submerged fermentation over surface fermentation. 44 Advantages of solid-state fermentation over submerged fermentation. 46 Disadvantages of SSF. 47 Traditional SSF processes. 48 Products produced by SSF technology. 48 Types of solid-state fermenters. 49 Major factors affecting microbial synthesis of enzymes in a SSF system. 50 Advantages of using enzymes. 51 Specificity of enzymes. 52 A selection of enzyme types used in industrial processes. 53 Enzyme classes and types of reactions. 54 Important bioprocessing systems for the production of commodity chemicals. 59 Product groups currently produced commercially using fermentation. 59 Products in developmental stage. 60 Uses of citric acid. 62 Microorganisms with the ability to produce citric acid. 63 Speciality foods produced by lactic acid fermentation. 65 Uses of lactic acid. 66 Uses of L-lysine. 71 Companies producing glutamic acid. 75 Enzymes used in food industry. 77 Environmental benefits of using glucoamylase enzymes for the conversion of starch to glucose. 80 Type of feed enzymes. 84 Advantages of supplementing feed with enzymes. 84 Prerequisite of enzymes used in animal nutrition. 84 Mode of action of different feed enzymes. 86 Biofertilizers and soil conditioners used in agriculture. 87 Biopesticides. 88 Types of biopesticides. 89

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xii TABLE 6.19 TABLE 6.20 TABLE 6.21 TABLE 6.22 TABLE 6.23 TABLE 6.24 TABLE 6.25 TABLE 6.26 TABLE 6.27 TABLE 6.28 TABLE 6.29 TABLE 6.30 TABLE 6.31 TABLE 6.32 TABLE 6.33 TABLE 6.34 TABLE 6.35 TABLE 6.36 TABLE 6.37 TABLE 6.38 TABLE 6.39 TABLE 6.40 TABLE 6.41 TABLE 6.42 TABLE 6.43 TABLE 6.44 TABLE 6.45 TABLE 6.46 TABLE 6.47 TABLE 6.48 TABLE 6.49 TABLE 6.50 TABLE 6.51 TABLE 6.52 TABLE 6.53 TABLE 6.54 TABLE 6.55 TABLE 6.56 TABLE 6.57 TABLE 6.58 TABLE 6.59 TABLE 6.60 TABLE 6.61 TABLE 6.62 TABLE 6.63 TABLE 6.64 TABLE 6.65 TABLE 7.1 TABLE 7.2 TABLE 7.3 TABLE 7.4 TABLE 8.1 TABLE 8.2 TABLE 9.1

List of tables

Biopesticides of botanical origin. Biopesticides from microorganisms. Advantages of using biopesticides. Few commercial biocontrol products for use against soilborne crop diseases. Substances with pest control properties. Possible reasons for the use of colorants in food substances. Naturally derived colors from plants sources. Naturally derived colors from microorganisms. Microorganisms producing pigments. Application of biocolors. Application of bacterial pigments. Production of food colors from plants using biotechnological methods. Important features of some biocolors. Benefits of biocolorants. Limitations of biocolorants. Flavors from plant cell culture. Food aroma compounds produced by microorganisms. Enzymes used for the production of aroma compounds. Solid-state fermentation applications in food aroma production. Use of solvents in different sectors. Organic chemicals utilized as solvents. Methods to produce solvents from biomass. Biobased solvents. Industrial biodegradable solvents. Advantages of using biobased solvents. Few established fermentation products. Vitamins produced by biotechnological methods. Microorganisms producing vitamin B12. Few selected biopharmaceuticals. A few examples of enzymes in industry. Use of enzymes in cosmetics. Four classes of enzymes are generally used in detergents. Advantages of using enzymes in detergents. Enzymes used in detergents (laundry and dishwash). Types of bioplastics. Market drivers. Bioplastic opportunities. First-generation and second-generation feedstocks for bioethanol. Enzymes involved in biofuel production. Enzymes used in the processing of fats and oils. Advantages of bioremediation. The disadvantages of bioremediation. Microbial enzymes involved in bioremediation. Biotechnological processes for removing VOCs from gases. Enzymes used in the bioremediation of different harmful chemical compounds. Advantages of transgenic plants. Major commercial players in plant biotechnology. The impact of industrial biotechnology on the environment. The impact of industrial biotechnology on the economy. The impact of industrial biotechnology on society. Companies using industrial biotechnology for improving manufacturing processes. Recent investments and developments in biotechnology sector in India. Major initiatives taken in India. Reasons for slow commercialization of biotechnology.

89 90 90 91 96 97 97 98 98 102 102 103 103 104 104 106 107 108 109 112 112 113 113 114 114 119 120 121 122 123 129 133 134 135 139 139 139 145 146 150 153 156 157 158 158 170 170 196 196 197 199 207 207 210



TABLE 9.2 TABLE 9.3 TABLE 9.4

List of tables

xiii

Challenges facing industrial biotechnology. 211 Comparison of industrial biotechnology and chemical technology. 212 Problems to be solved for making industrial biotechnology competitive to chemical technology. 212 TABLE 10.1 Industrial biotechnological applications with a high probability of reaching the market by 2030. 219

Preface sustainable processing relative to conventional operations. Economic drivers are the main factor for increasing acceptance of bioprocessing and bioproducts, but sustainability considerations are playing an increasing role. This book focuses on achievements and future prospects for biotechnology in sustainable production of goods and services, especially those that are derived at present mostly from the traditional chemical industry. It looks into the future impact of industrial biotechnology and lays out the major research areas, which must be addressed to move from a flourishing set of scientific disciplines to a major contributor to a successful future knowledge-based economy. It focuses in particular on the research needed to underpin three broad topics: biomass, bioprocesses, and bioproducts, including bioenergy.

Industrial biotechnology is the modern use and application of biotechnology for the sustainable processing and production of chemicals, materials, and fuels. It uses enzymes and microorganisms to make in a wide range of industrial sectors including chemicals, pharmaceuticals, food and feed, detergents, paper and pulp, textiles, energy, materials, and polymers. Mankind has already benefited from biotechnology for a long time, but with the advance of new technologies and a much deeper understanding of cell metabolism and materials science, many new opportunities have been identified, and others are continuing to emerge. The application of biotechnology across various industry sectors has invariably led to both economic and environmental benefits including less expensive processing, enhanced product quality, entirely new products, and environmentally



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Acknowledgments I am grateful for the help of many people, companies, and publishers for providing information and granting permission to use their material. Some excerpts were taken from Sunar K, Kumar U, Deshmukh S (2016). Recent Applications of Enzymes in Personal Care Products. In: Dhillon, G. Singh, Kaur, S. (Eds.), Agro-Industrial Wastes as Feedstock for Enzyme Production: Apply and Exploit the Emerging and Valuable Use Options of Waste Biomass. Academic Press, 279–298. ISBN: 9780128023921, with kind permission. Some excerpts were taken from Kirk O, Borchert TV, Fuglsang CC (2012). Industrial enzyme applications. Curr Opin Biotechnol;13:345–51, with kind permission. Gavrilescu M, Chisti Y (2005). Biotechnology—a sustainable alternative for chemical industry. Biotechnol Adv;23:471– 99, with kind permission. Ahmad P, Ashraf M, Younis M, Hu X, Kumar A, Akram NA, Al-Qurainy F (2012). Role of transgenic plants in agriculture and biopharming. Biotechnol Adv;30:524–540, with kind permission. Woodley JM, Breuer M, Mink D (2013). A future perspective on the role of industrial biotechnology for chemicals production. Chem Eng Res Des;91(10):2029–36, with kind permission. Bajpai Pratima (2018). Biermann’s Handbook of Pulp and Paper: Volume 1: Raw Material and Pulp Making, with kind permission.



Bajpai Pratima (2018). Biermann’s Handbook of Pulp and Paper Volume 2: Paper and Board Making, with kind permission. Bajpai Pratima (2015). Pulp and Paper Industry 1st edition—Chemicals, with kind permission. Bajpai Pratima (2009). Xylanases in “Encyclopedia of Microbiology, Third Edition”, (M. Schaechter and J. Lederberg, eds) Vol. 4. Academic Press, San Diego, pp. 600–612, with kind permission. Bajpai Pratima (2018). Biotechnology for Pulp and Paper Processing, Springer Nature, with kind permission. Novozymes (2011). Enzymes at work http://www.novozymes.com/en/about-us/ brochures/Documents, with kind permission. Bajpai Pratima (2013). Pulp and paper bioprocessing. Encycl Ind Biotechnol, pp. 1–17, with kind permission. Erickson B, Nelson JE, Winters P (2012). Perspective on opportunities in Industrial biotechnology in renewable chemicals. Biotechnol J;7(2):176–185. doi:https://doi.org/10.1002/ biot.201100069, with kind permission. Soetaert W, Vandamme E (2006). The impact of industrial biotechnology. Biotechnol J;1:756–69. doi:https://doi.org/10.1002/ biot.200600066, with kind permission. Bajpai P (2018). Industrial Enzymes—An Update, first ed. Bookboon. ISBN: 978-87403-2129-6, p. 118, with kind permission. Chen GQ (2012). New challenges and opportunities for industrial biotechnology. Microb. Cell Fact;11:111. doi:https://doi. org/10.1186/1475-2859-11-111, with kind permission.

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C H A P T E R

1 General background and introduction O U T L I N E 1.1 The impact of industrial biotechnology 1.2 Products of industrial biotechnology 1.2.1 Bulk chemicals 1.2.2 Biofuels and bioenergy

1.2.3 Fine and speciality chemicals 1.2.4 New materials

8 9 9 10

10 10

References

12

Relevant websites

13

Further reading

14

For many decades, chemical manufacturers have depended on complex and often environmentally damaging processes for synthesizing a variety of diverse products from petroleum and natural gas, to plastics and process chemicals, to vitamins and nylon. The chemical industry is facing increasing pressure for making chemical production more ecologically sound because of its dependence on fossil resources, its environmentally harmful production processes, and generation of toxic waste and by-products. But due to innovations in the recent years, biotechnology has entered into the industrial sector. This has created a new field generally referred to as “white biotechnology” or “green chemistry” (Kafarski, 2012). The industrialization of biology offers several advantages at both the national and international scale (Table 1.1) (National Research Council, 2015). The international biotechnology market has grown in the last decade (Barcelos et al., 2018). This is fueled by the following factors (Barcelos et al., 2018): - Economic recovery - Increased research funding - Government endeavors The United States is the first country in the development and application of biotechnology, but developing countries like Brazil and India are also making significant efforts in promoting biotechnology, especially agricultural and industrial biotechnology (Ramos et al., 2016).

Biotechnology in the Chemical Industry https://doi.org/10.1016/B978-0-12-818402-8.00001-X

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© 2020 Elsevier Inc. All rights reserved.

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1.  General background and introduction

TABLE 1.1  Industrialization of biology—benefits. Driving the innovation economy and sustainable economic growth Enabling sustainable and next-generation manufacturing Contributing to the solutions to some of the societal grand challenges of our time, like helping to provide clean, affordable, and sustainable energy Creation of new skills and jobs to benefit generations of today and tomorrow Based on National Research Council, 2015. Industrialization of Biology: A Roadmap to Accelerate the Advanced Manufacturing of Chemicals. The National Academies Press, Washington, DC, https://doi. org/10.17226/19001.

The main areas of biotechnology are differentiated according to the color code developed by Kafarski (2012) (Table 1.2). Industrial biotechnology (also referred to as white biotechnology) uses biological systems for producing chemicals, materials, and energy. This technology uses biocatalysis (the use of enzymes to catalyze chemical reactions) and fermentation technology (use of microorganisms), in combination with breakthroughs in enzyme and metabolic engineering and molecular genetics (Tang and Zhao, 2009; Carole et al., 2004; Philp et al., 2013; Barcelos et al., 2018; DSM, 2004; European Union, 2003; EuropaBio, 2003; OECD Report, 2001; William et al., 1999). Industrial biotechnology usually results in cleaner processes with minimum generation of waste and use of energy. Some of the historical developments in industrial biotechnology are presented in Table 1.3. Red biotechnology focuses on medical sector, whereas green biotechnology focuses on genetically modified crops. The three major drivers for industrial biotechnology are listed later (Bull et al., 1998): - Economic (market forces) - Governmental policy - Science and technology Fig. 1.1 shows the industrial biotechnology value chain. Raw materials, including crops and organic by-products, from agricultural sources and households are converted into sugars. From these sugars, desired product can be produced TABLE 1.2  Color code differentiating the main areas of biotechnology. White (industrial) Green (agricultural) Blue (marine and freshwater) Red (pharmaceutical) Brown (desert biotechnology) Purple (patents and inventions) Yellow (insect biotechnology, an emerging field of applied entomology that covers the use of insects in drug discovery) Based on Kafarski, P., 2012. Rainbow code of biotechnology. Chemik 66, 814–816.



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TABLE 1.3  Some historical developments in industrial biotechnology. Pre-1940s Solvents Baker's yeast Organic acids Amino acids Pre-1980s Antibiotics Prebiotics Biopolymers Single cell proteins Enzymes Biosurfactants Enzymes Post-1980s Biopharmaceuticals Recombinant proteins Monoclonal antibodies Plant bioactive compounds Recombinant vaccines Biofuels Based on Singh, R.S., 2014. Industrial biotechnology: an overview. In: Singh, R.S., Pandey, A., Larroche, C. (Eds.), Advances in Industrial Biotechnology. IK International Publishing House Pvt. Ltd, India, pp. 1–35.

Biochemicals

Agricultural (by) products

Physico-chemical treatment and/or enzymatic treatment

Sugars

Fine chemicals

Microorganisms or enzymes

Biomaterials

Biofuels

Bulk chemicals

FIG. 1.1  The industrial biotechnology value chain. Reproduced with permission from DSM, 2004. Industrial (White) Biotechnology—An Effective Route to Increase EU Innovation and Sustainable Growth. Position Document on Industrial Biotechnology in Europe and the Netherlands, http://www.sustentabilidad.uai.edu.ar/pdf/tec/industrial_white_biotech.pdf, 20 pp.

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by tailor-made microorganisms or enzymes. Many countries have a good hold in this value chain, due to the presence of several important key players in the chemical and food industry and agribusiness. Biotechnological processing uses enzymes and microorganisms for making products in a several industries including chemicals, pharmaceuticals, food and feed, detergents, paper and pulp, textiles, energy, materials, and polymers (Hatti-Kaul et al., 2007; Chen, 2012; Chen and Kazlauskas, 2011; Industrial Biotechnology Industry Report, 2011). Humankind has already benefited from biotechnology for a long time, but due to the advancement of new techniques and a thorough understanding of cell metabolism and materials science, several new opportunities have been identified, and others are continuously emerging. The term “white biotechnology,” proposed by European Union, is also gaining momentum now. This covers the field of industrial biotechnology, with “white” also referring to the favorable environmental aspects associated with the use of industrial biotechnology (Soetaert and Vandamme, 2006; Dale, 2003; Bachmann, 2003; Sørup et al., 1998; Brundtland, 1987; William et al., 1999; Eggersdorfer et al., 1992; Campbell, 1998; Okkerse and Van Bekkum, 1999; Vandamme et al., 2006; Wilke, 1995; Demain, 2000; Lens et al., 2005; Griffiths, 2001; Carlson, 2003; Ahmann and Dorgan, 2007; Bang et al., 2009; Philp, 2011; Singh, 2011; BCC Research, 2011a, b; Kircher, 2010; Carole et al., 2004; Philp et al., 2013; Hatti-Kaul et al., 2007; Chen, 2012; Chen and Kazlauskas, 2011; Industrial Biotechnology Industry Report, 2011; Erickson et al., 2012; Tang and Zhao, 2009). “White biotechnology actually has a lot to offer to our society. Now, it is our challenge to develop and exploit that on time” (DSM, 2004). Researchers are exploring methods for producing chemicals from renewable resources without depending on the conventional chemical methods. As an alternative to this, they plan to explore specially designed microorganisms and new molecules for producing products from biomass substrates. The cost can be reduced by reducing generation of waste and emission of greenhouse gases, energy consumption, and effectively aligning economic incentives with broader social and environmental objectives (www.eesi.org/articles/view/ can-white-biotechnology-help-create-a-green-chemicals-industry). Biotechnology is an all-round technology providing powerful routes for cleaning industrial products and processes and is playing a powerful role. Biotechnology is at an early stage in the chemical industry. But it has the potential as a major driver to the future of industry. Industrial biotechnology has been identified as a major emerging technology area. It is the application of scientific and engineering principles to the processing of materials by biological agents and can provide the process tools for biobased production of chemicals. Biotechnology has led to cleaner processes with reduced generation of wastes and reduced energy consumption in some areas. Biotechnology is found to be capable of producing huge wealth and affecting every important sector of the economy. “Industrial penetration of biotechnology is increasing as a result of advances in recombinant DNA technologies. It has already significantly affected healthcare; production and processing of food; agriculture and forestry; environmental protection; and production of chemicals and materials. New products from industrial biotechnology show more functionality; the more sophisticated chemistries enabled by biological catalysts lend themselves to “smarter” products for the consumer at little high cost” (Ghisalba et  al., 2009). Table  1.4 presents the benefits of biotechnology, and Table 1.5 presents the defining technologies of modern biotechnology.



1.  General background and introduction

5

TABLE 1.4  Environmental and economic benefits of biotechnology. Substantially reduced dependence on nonrenewable fuels and other resources Sustainable production of existing and novel products Safely destroys accumulated pollutants for remediation of the environment Better economics of production Reduced potential for pollution of industrial processes and products Based on Gavrilescu, M., Chisti, Y., 2005. Biotechnology—a sustainable alternative for chemical industry. Biotechnol. Adv. 23, 471–499.

TABLE 1.5  Defining technologies of modern biotechnology. Genetic engineering Metabolic engineering Cells of animals and plants Culture of recombinant microorganisms Hybridoma technology Bioelectronics Bioseparations and bioreactor technologies Nanobiotechnology Protein engineering Transgenic animals and plants Tissue and organ engineering Immunological assays Genomics and proteomics Based on Gavrilescu, M., Chisti, Y., 2005. Biotechnology—a sustainable alternative for chemical industry. Biotechnol. Adv. 23, 471–499.

Industrial biotechnology is one of the most promising approaches to pollution prevention, resource conservation, and cost reduction (Industrial Biotechnology Industry Report, 2011). It is often referred to as the third wave in biotechnology. “If developed to its full potential, industrial biotechnology may have a larger impact on the world as compared to health care and agricultural biotechnology. It offers businesses a way to reduce costs and create new markets while saving the environment. Also, since many of its products do not need the lengthy review times that drug products must undergo, it's a faster, easier pathway to the market. Today, new industrial processes can be taken from lab study to commercial application in two to five years, in comparison to up to a decade for drugs” (www.bio.org). “The application of biotechnology to industrial processes is not only transforming how we produce products but is also providing new products which could not even be imagined a few years ago. Because industrial biotechnology is so new, its advantages are not well known or understood by industry, policymakers or consumers” (www.bio.org).

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Industrial biotechnology has integrated product improvements with prevention of pollution from the very beginning. Industrial biotechnology has solved the phosphate water pollution problems in the 1970s, which was caused by the use of phosphates in laundry detergent. Biotechnology companies developed enzymes that were able to remove stains from clothes better than phosphates. This allowed the replacement of a polluting material with a nonpolluting biobased product; the performance of the end product also improved. This new method substantially reduced phosphate related algal blooms in surface waters around the globe and allowed consumers to get their clothes cleaner with reduced temperature of wash water and energy savings. “Primitive industrial biotechnology actually dates back to at least 6000 B.C. when Neolithic cultures fermented grapes to produce wine, and Babylonians used microbial yeasts for making beer. Over time, mankind's knowledge of fermentation increased, allowing the production of cheese, yogurt, vinegar, and other food products. In the 1800s, Louis Pasteur proved that fermentation was the result of microbial activity. Later in 1928, Sir Alexander Fleming extracted penicillin from mold. In the 1940s, large-scale fermentation methods were developed for producing industrial quantities of this wonder drug. After World War II, the biotechnology revolution started, giving rise to modern industrial biotechnology” (www.bio.org). For a long time, industrial biotechnology has been producing enzymes for the manufacturing sector and also for daily use. For example, meat-tenderizing enzyme and some contact lens cleaning fluids contain enzymes that remove sticky protein deposits. Industrial biotechnology involves the enzymes, produced by microorganisms. These are specialized proteins and have evolved in nature to be high performing biocatalysts that facilitate and accelerate complex biochemical reactions. These incredible enzyme catalysts make industrial biotechnology a powerful new technology. Industrial biotechnology actually works with nature for maximizing and optimizing existing biochemical pathways that can be utilized in manufacturing. The industrial biotechnology revolution rides on several developments in three areas of study of detailed information obtained from the cell genomics, proteomics, and bioinformatics. As a result, researchers can use novel methods to different types of microorganisms—bacteria, yeasts, fungi, marine diatoms, and protozoa. Industrial biotech companies use certain specially designed methods for finding and improving enzymes in nature. Information from genomic studies on microorganisms is helping scientists to capitalize on the wealth of genetic diversity in microbial populations. Scientists first search for microorganisms producing enzymes in the natural environment and then use DNA probes for searching at the molecular level for genes producing enzymes with specific biocatalytic abilities. After isolation, the enzymes can be identified and characterized for their ability for functioning in specific industrial processes. Biotechnology methods can be used to improve these enzymes if needed. Several biocatalytic tools are becoming available rapidly for industrial applications due to the significant advances in biotechnological methods. In several cases, the biocatalytic or whole-cell processes are so new that most chemical engineers and product development specialists in the private sector are not yet aware that they are available for deployment. “This is a good example of a technology gap where there is a lag between availability and widespread use of a new technology. This gap must be overcome to accelerate progress in developing more economic and sustainable manufacturing processes through the integration of biotechnology” (www.bio.org).



1.  General background and introduction

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According to Carlson (2012), in the United States, biobased product markets are significant, represented more than 2.2% of gross domestic product or more than $353 billion in economic activity in 2012. “To date White biotechnology has had its greatest economic impact in human health and in agriculture, bio-based chemicals are neither entirely new, nor are they a historic artifact. Current global bio-based chemical and polymer production is already estimated to be about 50 million tons each year, and bioprocessing techniques (such as fermentation, baking, and tanning) have been used throughout much of human industrial history. Agilent Technologies estimates that United States business-to-business revenues from industrial biotechnology alone reached at least $125 billion in 2012” (Solomon, 2013; www.nap.edu). “Bio-based chemical applications accounted for about $66 billion of that activity with biofuels adding another $30 billion. Lux Research estimates that industrial chemicals made through synthetic biology represent a $1.5 billion market in 2013 and that this likely will expand at a 15%–25% annual growth rate for the foreseeable future” (Cha, 2013). Based on a 2009 Organization for Economic Cooperation and Development (OECD) analysis, a USDA report shows that biobased chemicals will comprise higher than 10% of the chemical market in 2014 (Golden and Handfield, 2014). Several companies, including DSM, DuPont, Novozymes, and BASF are using biotechnology for replacing old production processes. DSM is conducting commercial production of succinic acid. The name of the product is Biosuccinium. It is sold globally since 2012, and this technology is also offered under license to value chain partners and coproducers (https:// reverdia.com/about/company-overview/). Biosuccinium is an important biobased chemical building block that offers the chemical industry a good alternative to various oil-based diacids. This product is based on a patented technology with a best environmental footprint and has a diverse range of applications in packaging, footwear, and also cosmetics. In 2012, Reverdia started the world’s first dedicated, large-scale plant for succinic acid production from renewable raw materials in Cassano, Spinola, Italy. This plant has a capacity of 10,000 tonnes/year and used yeast. Since that time, the company has surpassed its planned technical milestones. It has grown up from a biotechnology company to a codevelopment powerhouse. “For supporting the emerging biomaterials industry, the United States government formed the BioPreferred procurement program in the Farm Security and Rural Investment Act of 2002. Expanded under the Food, Conservation, and Energy Act of 2008, the bill gives federal procurement preference to bioproducts, including those obtained from white biotechnology. In addition, the legislation creates a voluntary labeling program, set to be unveiled in 2009, to identify items that meet USDA-determined thresholds for the inclusion of biobased materials. To date, BioPreferred has identified 2541 products across 33 item types that meet the required threshold. Applications of white biotechnology have already started achieving reductions in carbon emissions and fossil fuel use. Production of plastics with biobased innovations already stands at 1 billion pounds (out of an 80 billion pound market). A 2004 industry report projected that if all plastic production were met using biotechnology, United States oil consumption would reduce by 90–145 million barrels per year. DSM has estimated that moving to biobased chemical production across the industry could account for up to 20% of the global Kyoto greenhouse gas emissions reduction target. This significant carbon reduction potential, coupled with its ability to reduce costs for chemical producers and reduce dependence on fossil fuels, marks white biotechnology as a promising option in efforts to harmonize economic goals with climate goals. An important question remains whether this

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TABLE 1.6  A vision for 2025. Using biotechnology, an increasing number of chemicals and materials are expected to be produced by the year 2025. Biotechnological processes will be used to produce chemicals and materials that are difficult to produce conventionally or to make existing products in a more efficient way Industrial biotechnology will enable a range of industries for manufacturing products economically and in an environmentally sustainable way Renewable resources can be used eco-efficiently by using biotechnology Rural biorefineries would replace port-based oil refineries wherever economically feasible Energy obtained from biomass based on biotechnology is expected to account for an increasing share of European energy consumption by 2025 Green biotechnology will make a significant contribution to the efficient production of biomass raw materials The industry in Europe will be innovative and competitive with sustained cooperation and support between the research community, industry, agriculture and society Based on https://www.europabio.org/sites/default/files/industrial_or_white_biotechnology_-_research_for_europe.pdf.

new chemical industry will help advance additional, major environmental and public health and safety goals, as well” (www.eesi.org). For the future competitiveness of European industry, the development and use of industrial biotechnology is important. It provides a sound technological base for the sustainable society of the future. Table 1.6 shows vision a vision for 2025. OECD study (The Bioeconomy to 2030: designing a policy agenda, http://www.oecd.org/ futures/bioeconomy/2030) reports, “the industrial applications of biotechnology in 2030 will be responsible for 39% of the economic value generated by Biotechnology, which shows the healthy investment in research and development expected in this area” (www.bio.org).

1.1  The impact of industrial biotechnology The environment benefits as biotechnological processes are efficient users of renewable resources generating little end-of-pipe waste, which may be used as input into a further biological process. Also, shifting from chemical to biological processes can lead to substantial reductions in the release of carbon dioxide, reduction in energy, and water consumption. Economic benefits can be also achieved as biotechnology allows the use of more efficient processes that consume less energy. Already, fermentation and enzymatic processes are generally used in the manufacturing of fine chemicals, vitamins, pharmaceutical intermediates, and flavors. They are also making their entry into larger-volume segments such as polymers, bulk chemicals and biofuels, and several other industrial segments. Some reports by BCC Inc. (2005) and Freedonia Group Inc. (2005) predict annual growth rates of about 5% for fermentation products in comparison with 2%–3% for overall chemical production in the coming years, but other reports such as the one by McKinsey and Company (2003) predict much higher growth rates and estimate biotechnology to be used in the production of up to 10% of all chemicals sold in the next 10 years. Although there is a difference in the numbers, all studies show that industrial biotechnology will play a very important role in the chemical and other manufacturing industries in the future. Both these environmental and economic



1.2  Products of industrial biotechnology

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advantages will contribute toward a more sustainable society, with better opportunities for creation of job and retention, and less dependence on fossil fuels (https://www.europabio. org/sites/default/files/industrial_or_white_biotechnology_-_research_for_europe.pdf).

1.2  Products of industrial biotechnology Industrial biotechnology can be used for producing a wide variety of useful products. These fall under the categories of bulk chemicals, fine chemicals, pharmaceuticals, food additives and supplements, colorants, vitamins, pesticides, bioplastics, solvents, bulk chemicals, and biofuels. These products range from very cheap bulk chemicals (for instance, ethanol) to extremely expensive fine chemicals (for instance, vitamin B12) (Soetaert and Vandamme, 2006). Industrial biotechnology is already being used in the production of fine chemicals and pharmaceuticals, but bulk chemicals, biofuels, and bioplastics are also being produced to a great extent by the use of industrial biotechnology (Wilke, 1995; Demain, 2000). In few cases, a polymer building block is produced from fossil resources using enzymes. In other cases, a completely biodegradable bioplastic can be produced from renewable raw materials; for example, the biodegradable bioplastic PLA can be produced from corn. Industrial biotechnology can be used in a single step in a chemical synthesis route or replace a complete cascade of chemical synthesis steps with a single fermentation or biocatalysis step. Some examples are presented later: Biotechnology can replace the existing chemical processes and also allows the production of new products. In the fine chemicals sector, several products can be produced using biotechnology. In this sector, highly specific and mild reaction conditions of enzymes and cellular processes provide quality and efficiency benefits. But still, there are certain challenges to be faced for allowing increased use of biological processes in this sector and also several others. These—which must be addressed via the Strategic Research Agenda—include the sensitivity of many biocatalysts to high concentrations of substrate and product concentrations and their liking for an aqueous environment. Actually, in the aqueous requirement, most of the chemical products have very limited solubility in water (https://www.europabio.org/ sites/default/files/industrial_or_white_biotechnology_-_research_for_europe.pdf). A brief description of some industrial sectors is presented later. This shows the potential offered by biotechnological processes:

1.2.1  Bulk chemicals Several important chemicals are produced in very high volumes by using fermentation processes. These include citric acid, l-glutamic acid, and vitamin C. Petrochemical resources are becoming more expensive; therefore, this range is expected to increase significantly. New bulk polymers such as biodegradable plastics and monomers (e.g., 1,3-propanediol for novel polyester production) are also becoming available, and there is very good scope for further development based on tailored enzymes and microorganisms. Looking ahead, new polymers having better improved functionality can be manufactured using biotechnology. This would allow the production not possible using the traditional processes. These could include improved liquid crystals and materials having very high temperature resistance and mechanical properties.

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1.2.2  Biofuels and bioenergy Europe is dependent on energy imports to a greater extent, and local sources of energy will play an important role in reducing this reliance. The European Union is using biofuels to a small extent, but by the year, 2020 has planned to increase biofuel substitution of both petrol and diesel to 20%. A biotechnological process to use lignocellulosic waste materials such as straw and corn cobs as a substrate for bioethanol would be a major step forward, as would a biological process for producing biodiesel. Biomass can be used for producing methane and can be also used as a source of hydrogen in future.

1.2.3  Fine and speciality chemicals Speciality chemicals often require complex and inefficient chemical processes for their production. Therefore, it is not surprising that efficient and simpler bioprocesses are very important for this sector. Currently, industrial biotechnology is used to a large extent in the field of fine chemicals and pharmaceutical (15%). Further development is in progress. The most important fine chemicals are antibiotics and their intermediates. The global antibiotics market size is expected to reach USD 62.06 billion by 2025 according to a new report by Grand View Research, Inc. (https://www.grandviewresearch.com/press-release/global-antibiotic-market). Antibiotics are almost entirely produced by fermentation processes with the help of certain selected microorganisms. “The structural complexity of most antibiotics is so great that chemical synthesis has never been a serious alternative. Only in the case of few semisynthetic antibiotics are the building blocks obtained by fermentation and are chemically modified later to obtain new antibiotic derivatives with better efficacy. These days, chemical modifications are replaced by biotech methods to an increasing extent, with outstanding environmental and economic benefits” (Soetaert and Vandamme, 2006). Captopril is another example in the pharmaceutical sector. It is a angiotensin-converting enzyme (ACE) inhibitor used for treating high blood pressure. Captopril is built from d-βhydroxy-isobutyric acid and l-proline. These building blocks are produced by fermentation, with the yeast Candida rugosa and the bacterium Corynebacterium sp., respectively. Both building blocks are then linked using the conventional chemistry, which results in Captopril. In the fine chemical sector, Lonza has developed a biotech route, which starts with conversion of 3-cyanopyridine to nicotinamide (niacin or vitamin B3), nicotinic acid, and 6- hydroxynicotinic acid. Currently, these intermediate products for several chemical syntheses are produced using industrial biotechnology. Conversions are done by using enzymatic hydrolysis with nitrile hydratase from Rhodococcus bacteria or by bioconversion with living bacterial cells. The yields are almost quantitative, and the reactions are very specific.

1.2.4  New materials “In the longer term, nature will serve as the inspiration for novel materials and manufacturing processes, for instance more efficient solar cell using controlled transport phenomena. Bio-based performance and nano-composite materials will derive their properties from their specific nano- (or micro-) scale structure, or will be produced using the principles of natural self-organisation” (www.europabio.org).



1.2  Products of industrial biotechnology

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Introduction of using biotech process steps into chemical syntheses often results in substantial environmental benefits presented later: - - - -

Substantially reduced waste generation Reduced energy requirement Decreased use of solvents Elimination of dangerous intermediate products

But these environmental benefits are not the main reason for the technology switch. The process improvements and the cost reduction are almost always the driving force for such decision. The environmental benefits are a desirable side effect. By themselves, they are not enough to inspire decision makers for introducing a new technology (with associated failure risk). “The way industrial biotechnology combines both economic and ecological progress is quite typical: the increased efficiency and reduced production cost of such biotechnological processes almost always results in a greatly reduced ecological impact and generally leads to an improved competitiveness. In a 2001 OECD report, 21 such case studies were presented (Griffiths, 2001). Each case study convincingly shows the economic and ecological advantages offered by industrial biotechnology. It should be noted that in most cases, the processes described have been implemented in industrial practice and are competitive, and in no way limited to theoretical studies or research projects” (Soetaert and Vandamme, 2006). Table 1.7 shows the advantages of biotechnology in chemical production.

TABLE 1.7  Advantages of biotechnology in chemical production. Cleaner production since fewer wastes will be generated. It can eliminate environmental concerns over the disposal of chemical processing wastes Increased product yield Reaction steps will be reduced, usually compressed into one synthesis and one isolation step in a biotech process. The outcome is a 75% saving in capital equipment costs and a 50% cut in operation cost Cost-effective routes and new chemical entities possible Low-cost raw materials. Use of cellulose and biomass will reduce the cost. We will see the first biorefineries in a few years Innovative, can provide new strategy for value creation No by-products generated that are having undesirable color or odor High regio-/stereoselectivity of biocatalytic reactions Using plant biomass, as feedstock means feedstock grows, and it consumes CO2—one of the greenhouse gases Use of plant biomass if successfully done will provide primary feedstock and energy. Today, at least 5 billion kilograms of commodity chemicals are produced annually in the United States using plant biomass as the primary feedstock Unlike many chemical reactions that require very high temperatures and pressures, reactions using biological molecules work best at ambient temperatures under 100°F, atmospheric pressure, and water-based solutions. Therefore, manufacturing processes that use biological molecules can lower the amount of energy needed to drive reactions Based on biotechsupportbase.com/2014/02/15/impact-of-biotechnology-on-chemical-induatry.

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References Ahmann, D., Dorgan, J., 2007. Bioengineering for Pollution Prevention through Development of Biobased Energy and Materials: State of the Science Report. U.S. Environmental Protection Agency, Office of Research and Development, National Center for Environmental Research. EPA/600/R-01/028. Bachmann, R., 2003. Industrial Biotechnology—New Value-Creation Opportunities. . McKinsey and Co., Presentation at the Bio-Conference, New York. Bang, J., Follér, A., Buttazzoni, M., 2009. Industrial Biotechnology: More Than Green Fuel in a Dirty Economy? WWF Denmark. Barcelos, M.C.S., Lupki, F.B., Campolina, G.A., Nelson, D.L., Molina, G., 2018. The colors of biotechnology: general overview and developments of white, green and blue areas. FEMS Microbiol. Lett. 365 (21), 1. https://doi. org/10.1093/femsle/fny239. BCC Research, 2011a. Microbial Products: Technologies, Applications and Global Markets. BCC Research. BIO086A. BCC Research, 2011b. Enzymes in Industrial Applications: Global Markets. BCC Research. BIO030F. BCC Report, 2005. World Market for Fermentation Ingredients. Study GA-103R by Business Communications Company Inc, Norwalk. Brundtland, C.G., 1987. Our common future. The World Commission on Environmental Development. Oxford University Press, Oxford. Bull, A.T., Marrs, B.L., Kurane, R., 1998. Biotechnology for Clean Industrial Products and Processes. Towards Industrial Sustainability. Paris, OECD Publications. Campbell, C., 1998. The future of oil. Energy Explor. Exploit. 16, 125–152. Carlson, R., 2003. The pace and proliferation of biological technologies. Biosec. Bioterror. 1 (3), 203–214. Carlson, R., 2012. Synthesis. The U.S. Bioeconomy in 2012 Reached $350 Billion in Revenues, or About 2.5% of GDP. http://www.synthesis.cc/2014/01/the-us-bioeconomy-in-2012. Carole, T.M., Pellegrino, J., Paster, M.D., 2004. Opportunities in the industrial biobased products industry. Appl. Biochem. Biotechnol. 113–116, 871–885. Cha, A.E., 2013. Companies Rush to Build ‘Biofactories’ for Medicines, Flavorings and Fuels. The Washington Post. http://www.washingtonpost.com/national/health-science/companies-rush-to-build-biofactories-for-medicines-flavorings-andfuels/2013/10/24/f439dc3a-3032-11e3-8906-3daa2bcde110_story.html. Chen, G.Q., 2012. New challenges and opportunities for industrial biotechnology. Microb. Cell Factories 11, 111. https://doi.org/10.1186/1475-2859-11-111. Chen, G.Q., Kazlauskas, R., 2011. Chemical biotechnology in progress. Curr. Opin. Biotechnol. 22, 1–2. https://doi. org/10.1016/j.copbio.2010.12.002. Dale, B.E., 2003. “Greening” the chemical industry: research and development priorities for biobased industrial products. J. Chem. Technol. Biotechnol. 78, 1093–1103. Demain, A.L., 2000. Small bugs, big business: the economic power of the microbe. Biotechnol. Adv. 18, 499–514. DSM, 2004. Industrial (White) Biotechnology—An Effective Route to Increase EU Innovation and Sustainable Growth. Position Document on Industrial Biotechnology in Europe and the Netherlands. 20 pp. http://www. sustentabilidad.uai.edu.ar/pdf/tec/industrial_white_biotech.pdf. Eggersdorfer, M., Meyer, J., Eckes, P., 1992. Use of renewable resources for non-food materials. FEMS Microbiol. Rev. 103, 355–364. Erickson, B., Nelson, J.E., Winters, P., 2012. Perspective on opportunities in industrial biotechnology in renewable chemicals. Biotechnol. J. 7, 176–185. EuropaBio, 2003. White Biotechnology: Gateway to a More Sustainable Future. http://www.europabio.org/upload/ documents/wb_100403/Innenseiten_final_screen.pdf. European Union, 2003. Action Plan to Boost Research Efforts in Europe, April 2003, IP/03/584. http://europa.eu.int/comm/research/era/3pct/pdf/press-rel-en.pdf. Freedonia Group Inc, 2005. Fermentation Chemicals. Industry Study 1921 by The Freedonia Group Inc, Cleveland. Golden, J.S., Handfield, R.B., 2014. Why Biobased? Opportunities in the Emerging Bioeconomy. U.S. Department of Agriculture, Washington, DC. Griffiths, M., 2001. The Application of Biotechnology to Industrial Sustainability, OECD-Report. http://www1.oecd. org/publications/e-book/9301061e.pdf.



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Ghisalba, O., Meyer, H.P., Wohlgemuth, R., 2009. Industrial biotransformation. In: Flickinger, M.C. (Ed.), Encyclopedia of Industrial Biotechnology. Wiley. Hatti-Kaul, R., Törnvall, U., Gustafsson, L., Börjesson, P., 2007. Industrial biotechnology for the production of biobased chemicals—a cradle to grave perspective. Trends Biotechnol. 26 (3), 119–124. Industrial Biotechnology Industry Report, 2011. BBSRC support for bioenergy and industrial biotechnology. Recommendations to encourage UK science and technology for the energy and chemicals industries. Ind. Biotechnol. 7, 41–52. Kafarski, P., 2012. Rainbow code of biotechnology. Chemik 66, 814–816. Kircher, M., 2010. Discussion Paper—Session Trends in Technology and Applications, OECD Workshop on “Outlook on Industrial Biotechnology.” OECD Working Party on Biotechnology, Vienna. Lens, P., Westerman, P., Haberbauer, M., Moreno, A. (Eds.), 2005. Biofuels for Fuel Cells: Renewable Energy From Biomass Fermentation. IWA Publishing, Pittsburg. McKinsey and Company, 2003. In: Bachmann R., McKinsey & Company, Industrial Biotech—New Value-Creation Opportunities. Presentation at the Bio-Conference, New York. National Research Council, 2015. Industrialization of Biology: A Roadmap to Accelerate the Advanced Manufacturing of Chemicals. The National Academies Press, Washington, DC. https://doi.org/10.17226/19001. OECD Report, 2001. The Application of Biotechnology to Industrial Sustainability. http://www1.oecd.org/publications/e-book/9301061e.pdf. Okkerse, H., Van Bekkum, H., 1999. From fossil to green. Green Chem., 107–114. Philp, J., 2011. OECD Outlook on Industrial Biotechnology. OECD Directorate for Science, Technology and Industry. DSTI/STP/BIO(2011)3. Philp, J.C., Ritchie, R.J., Allan, J.E.M., 2013. Biobased chemicals. The convergence of green chemistry with industrial biotechnology. Trends Biotechnol. 31 (4), 219–222. Ramos, M.V., Melo, D.F., Silva, A.L.C., 2016. Biotecnologia: A Ciência, O Bacharelado, a Demanda Socioeconomica. UFC. Singh, R., 2011. Facts, growth, and opportunities in industrial biotechnology. Org. Process Res. Dev. 15, 175–179. Soetaert, W., Vandamme, E., 2006. The impact of industrial biotechnology. Biotechnol. J. 1, 756–769. https://doi. org/10.1002/biot.200600066. Solomon, D., 2013. Industrial Views on Synthetic Biology. Presented at Tooling the U.S. Bioeconomy: Synthetic Biology Conference. ACS Science & the Congress Project, Washington, DC. Sørup, P., Tils, C., Wolf, O., 1998. Biocatalysis: State of the Art in Europe, IPTS-Report. ftp://ftp.jrc.es/pub/EURdoc/ eur18680en.pdf. Tang, W.L., Zhao, H., 2009. Industrial biotechnology: tools and applications. Biotechnol. J. 4 (12), 1725–1739. https:// doi.org/10.1002/biot.200900127. Vandamme, E., Cerdobbel, A., Soetaert, W., 2006. Biocatalysis on the rise. Biocatalysis on the rise. Part I. Applications. Chem. Today 24, 57–61. Wilke, D., 1995. What should and what can biotechnology contribute to chemical bulk production? FEMS Microbiol. Rev. 16, 89–100. William, H., Scouten, W.H., Petersen, G., 1999. New Biocatalysts: Essential Tools for a Sustainable 21st Century Chemical Industry. CCR-Report. http://www.ccrhq.org/vision/index/roadmaps/New%20Biocatalysts.pdf.

Relevant websites www.eesi.org/articles/view/can-white-biotechnology-help-create-a-green-chemicals-industry. www.europabio.org. www.bio.org. www.nap.edu. https://reverdia.com/about/company-overview. www.europabio.org/sites/default/files/industrial_or_white_biotechnology_-_research_for_europe.pdf. www.nap.edu. www.grandviewresearch.com/press-release/global-antibiotic-market. www.europabio.org.

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www.sustentabilidad.uai.edu.ar/pdf/tec/industrial_white_biotech.pdf. biotechsupportbase.com.

Further reading Gavrilescu, M., Chisti, Y., 2005. Biotechnology—a sustainable alternative for chemical industry. Biotechnol. Adv. 23, 471–499. Singh, R.S., 2014. Industrial biotechnology: an overview. In: Singh, R.S., Pandey, A., Larroche, C. (Eds.), Advances in Industrial Biotechnology. IK International Publishing House Pvt. Ltd, India, pp. 1–35.

C H A P T E R

2 Industrial sustainability and biotechnology “The concept of sustainable development was launched by the World Commission on Environment and Development in the report—Our common future in 1987 and reinforced by the UN Earth Summit in Rio de Janeiro in 1992 Development” (Brundtland, 1987). Sustainability is a balancing act. It meets the requirements of the present without compromising the welfare of next generations. It is actually a process of change in which the use of resources, the direction of investments, the orientation of technological development, and institutional change are all in harmony and increase both the present and future potential for meeting the requirements of human beings and aspirations (www.gust.edu.kw). In the 21st century, sustainability refers to the requirement to develop the sustainable models important for both the human being and planet Earth to survive. “Sustainable development is no small undertaking for international companies looking to meet energy, food and environmental requirements of today without compromising the Earth's resources or its future. Many have been touting the value of biotechnology in ensuring a sustainable future for years. Sustainable development requires a framework for integrating environmental policies and development strategies in a global context. Increasingly, sustainability considerations will shape future technological, socio-economic, political, and cultural change to define the boundaries of what is acceptable” (Hall and Roome, 1996). “Sustainable development has been increasing in importance. Biotech companies across the board recognize the importance of making products, whether it’s flat-screen televisions or drought-resistant seeds or new drug therapies, in a manner more sustainable than they’ve done it in the past.” “Sustainable development provides a framework for integrating environmental policies and development strategies. It is expected to make an increasing contribution to the future shaping of global technological, socio-economic, political and cultural change and will define the boundaries of what is possible and desirable. While no single blueprint for sustainable development is likely to be agreed, sustainability should be regarded as a global imperative” (Hall and Roome, 1996; www.oecd.org). Sustainable industrial development is aimed at continuous innovation, improvement, and the use of “clean” technologies to bring a radical change in pollution levels and utilization of resources. New methods are required for managing increasing industrialization

Biotechnology in the Chemical Industry https://doi.org/10.1016/B978-0-12-818402-8.00002-1

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and ­urbanization globally in a sustainable manner. As all stages of a life cycle of product or process may have an effect on the environment, design principles based on a comprehensive strategy to new processes, which take into consideration all aspects, from selection and quantities of raw materials for improving the recovery during waste management, can be used for reducing the environmental impact. An ecofriendly process would, in principle, have (a) reduced consumption of energy and nonrenewable resources (particularly fossil fuel feedstocks) relative to the products or services delivered (b) reduced generation or elimination of waste (including materials and energy recycling and use). Therefore, the basic aim of any strategy designed for achieving such a process would be to maximize both the above strategies from raw materials from production, to consumption, finally to disposal of products. Biotechnology can make a substantial contribution for obtaining these objectives. Compared with traditional process, sustainable production processes and systems should be more profitable as they would require less wasteful use of materials and energy. This would result in reduced generation of pollutants including the greenhouse gases and would also allow more efficient use of renewable resources, for reducing the dependence on nonrenewable resources (Zosel, 1994; Van Berkel, 2000; Gavrilescu, 2004; Gavrilescu and Nicu, 2004; Gavrilescu and Chisti, 2005). Sustainability demands products that work well and, in comparison with their conventional counterparts, are long lasting, easily recyclable, less toxic, and biodegradable at the end of their useful life. Such type of products would be obtained as much as possible from renewable raw materials and contribute less to emission of greenhouse gases. “Between 1960s and 1990s, industrial production attempted to reduce its adverse impact by treating effluent and removing pollutants from an already damaged environment. Designing industrial processes and technologies which prevented pollution in the first place did not become a priority (Council Directive, 1996; Allen and Sinclair Rosselot, 1997; World Bank, 1999; EPA, 2003). Newer industries like microelectronics, telecommunications and biotechnology are already less resource intensive as compared to the traditional heavy industry (Kristensen, 1986; OECD, 1989; Rigaux, 1997), but this alone does not assure sustainability. Industry is truly sustainable only when it is economically viable, environmentally compatible, and socially responsible (OECD, 1998; UNEP, 1999; Wong, 2001). Models of sustainability have been discussed in several documents published by the Organization for Economic Cooperation and Development” (www.oecd.org) (OECD, 1989, 1994, 1995, 1998). A whole range of technologies have been already developed. These include the following: • • • • •

Renewable energies New materials Environmentally friendly chemicals Transport and processing systems Proper monitoring and control methods

“Biotechnology” is expected to play a significant role in most of these fields, but will they, in every situation, be efficient enough to justify the investment? A thorough evaluation of current approaches and results is required for determining this (Soetaert and Vandamme, 2006; Zechendorf, 1999). “The sustainable development is based on the conviction that it should be possible for increasing the basic standard of growing world population, without unnecessarily exhausting



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our finite natural resources and further degrading the environment in which we live (www. comsats.org). Emerging biotechnologies, based on new scientific discoveries, offer new options for striking a balance between developmental requirements and environmental conservation. A wider diffusion of the technology can be seen as the key to directing its positive impacts onto the world's society as a whole” (www.comsats.org). Biotechnology is developing in several sectors at a fast pace that improve the efficacy of the way in which products and services are provided. But the transfer and development of biotechnology, in an environmentally friendly manner, needs a range of conditions, including capital inputs that, in the case of several developing countries, are not available readily. Biotechnology plays a very important role in the sustainable development of different sectors. “Sustainability is fast becoming the corner stone of economy of many countries, both developed and developing. Such a development, in turn, depends upon the use of new technologies, innovations, entrepreneurship and utilization of inexhaustible supply of renewable resources. Biotechnology is one such technology, which has rapidly developed over the past few decades and has great potential for solving many problems pertaining to Agriculture, Industry, Environment and Health, which have direct relevance to sustainable development. These features and potentials of biotechnology have generated great interest among the developing countries, many of which have embarked on various programs in biotechnology at various levels. In the majority of developing countries, agriculture is the mainstay of the economy. Any improvement in agricultural productivity directly helps in improvement of the economy. The role of new biotechnology in agriculture has been described as a precursor to another Green Revolution that would help eliminate the world’s hunger. The conventional methods of genetic improvement resulted in significant increase in grain production over the past few decades. In addition, industrial processes based on biotechnology, are often economical as they consume less energy and use raw material more effectively. The largest contribution of biotechnology is in the pharmaceutical industry, where various drugs are being produced by genetically engineered microorganisms (GEMs). The best known examples are human insulin, interferon and growth hormones.” Of the several different methods that biotechnology embraces, none apply across all industrial sectors. Recognizing its strategic value, several countries are now making and implementing integrated plans for using biotechnology for industrial regeneration, creation of job, and social progress (Roberts et al., 1999; Liese et al., 2000; Rigaux, 1997). Biotechnology is all-round and has been evaluated as a major technology for a sustainable chemical industry (Lievonen, 1999; OECD, 2001). Industries that earlier never considered biological sciences as affecting their business are finding ways of using biotechnology to get the benefits. Biotechnology presents entirely new opportunities for sustainable production of existing and new products and services. Environmental concerns are helping the use of biotechnology in industry. This technology removes pollutants from the environment and also prevents pollution in the first place. Processes based on biocatalyst have an important role to play in this context. Biocatalysts operate at lower temperatures, generating waste that is less toxic. The emissions are fewer and less by-products are produced in comparison with conventional chemical processes. New biocatalysts with better selectivity and improved performance for use in various manufacturing and waste degrading processes are now available (Abramovicz, 1990; Poppe and Novak, 1992; Roberts et al., 1999). As the biocatalysts are selective, these reduce the requirement for purifying the product from by-products, thereby reducing energy requirement and environmental impact. Not similar to nonbiological catalysts, biocatalysts are self-replicating.

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Biological processes appear to be appealing because they use the renewable resources— sunlight, water, and carbon dioxide—for producing a range of molecules using processes requiring low energy consumption. These processes have been fine-tuned by evolution to provide effective, high-fidelity synthesis of products having low toxicity. Biotechnology provides renewable bioenergy and can yield new methods for monitoring the environment. This technology is being extensively used particularly in the production of biopharmaceuticals. In addition to provide novel routes to well-established products, biotechnology is being used for producing novel products. Biotechnology is being interfaced with other emerging disciplines; new areas are created such as nanobiotechnology and bioelectronics. Biotechnology has a great influence on agriculture, healthcare, medical diagnostics, environmental protection, criminal investigation, and food processing (Xiang and Chen, 2000; D’Orazio, 2003; Gavrilescu and Chisti, 2005). This is just a mere shadow of the future expected impact of biotechnology in industrial production and sustainability. The use of biotechnology in processing and production of chemicals, for increased sustainability, is presented below: Biotechnology offers one important route to development of clean products and processes; it provides powerful and versatile tools, which is able to compete with chemical and physical methods of reducing material and energy consumption and the production of wastes and emissions. The application of biotechnology in industry has still to occur, but several examples of its ability to produce cleaner and competitive products and processes are now available especially through the development and use of biocatalysts. The introduction of clean process does not necessarily require a complete change in manufacturing strategy or the refitting of plant. Upgradation of existing manufacturing processes by using biotechnology stages shows the opportunities for such an intermediate technology. Nonetheless, for biotechnology to achieve its full potential as a basis for clean industrial products and processes beyond its existing applications, innovative research and development will be needed. “The chemical industry produces a broad range of compounds that can roughly be divided into the following groups: fine chemicals, pharmaceutical products, bulk chemicals, plastics and fuels. The chemical industry is a very important production sector, but at the same time a big user of fossil resources and a substantial source of waste. Researchers, chemists and chemical engineers face major challenges for developing sustainable chemical processes that respect the environment, improve our quality of life and at the same time are competitive in the marketplace” (Soetaert and Vandamme, 2006; Miller and Nagarajan, 2000; Rogers et al., 2005; Tonelli et al., 2013; www.massey.ac.nz). This includes the development of new production processes. These reduce or eliminate the use of risky or hazardous substances, reduce energy consumption and generation of waste, and start as much as possible from renewable raw materials. The final goal is the development of a clean technology, starting from renewable raw materials and energy, with reduced generation of waste, and high productivity and competitiveness. Sustainable chemistry is based on a range of different technologies, which range from the following: • More effective traditional processes • Use of efficient catalysts



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2.  Industrial sustainability and biotechnology

• Use of innovative separation methods, for instance, membrane processes and recycling technology • Use of industrial biotechnology Biotechnology is affecting the chemical sector to a large extent. This shows that biotechnology is naturally suitable for sustainable chemistry (William et al., 1999; Europabio, 2003; O’Connell et al., 2009; Malik et al., 1995; Zechendorf, 1999; Gerngross, 1999). The use of renewable resources appears to be difficult in traditional chemical processes; industrial biotechnology can handle these renewable resources with great ease. Reduced generation of waste and energy consumption, the use of harmless renewable resources, and the high efficiency guarantee the sustainability of this technology. Industrial biotechnological processes increasingly penetrate the chemical industry, with very positive results regarding sustainability and also industrial competitiveness. It should be stressed that “industrial biotechnology is not the only technology in this quest for sustainability. The most sustainable chemistry consists of an interplay between different technologies. Best results can be obtained by using a suitable combination of conventional chemical technology and industrial biotechnology. New processes increasingly consist of combi-syntheses, consisting of several chemical and biotechnological steps. Also, innovative separation technologies such as membrane technology and the use of super-critical solvents are being increasingly used. These are helping to increase the eco-efficiency of this green chemistry” (www.sustentabilidad.uai.edu.ar). Recently, several leading companies operating in white biotechnology joined forces with independent third parties for conducting an assessment of the potential impact of white biotechnology. Detailed case studies were combined with a market analysis by McKinsey and Company for estimating an impact on the three elements of sustainable development: people, planet, and profit (“Triple P”) (Fig. 2.1).

Profit Economically viable

Drivers Cost reduction Novel products Hurdles Regulations Futher technology development Feedstock prices Investments

Drivers Knowledge-based quality jobs Responsiblity

Drivers Less energy Less waste

SUSTAINABILITY

Hurdles Unawareness Acceptance

Hurdles Futher technology development Waste management

People

Planet

Socially responsible

Environmentally sound

FIG. 2.1  The triple P bottom line (DSM, 2004). Reproduced with permission.

20

2.  Industrial sustainability and biotechnology

The analysis shows that the social, environmental, and economic benefits of white biotechnology go together. If all stakeholders work together in a self-reinforcing cycle, white biotechnology could create new jobs, while reducing the impact on the environment and even creating economic value (DSM, 2004; lrd.yahooapis.com).

References Abramovicz, D.A., 1990. Biocatalysis. Kluwer, Dordrecht. Allen, D., Sinclair Rosselot, K., 1997. Pollution Prevention for Chemical Processes. Wiley, New York. Brundtland, G., 1987. Our Common Future. Oxford University Press, Oxford. Council Directive, 1996. 96/61/EC concerning integrated pollution prevention and control. Off. J. EC L257, 26. D’Orazio, P., 2003. Biosensors in clinical chemistry. Clin. Chim. Acta 334, 41–69. DSM, 2004. Industrial (White) Biotechnology—An Effective Route to Increase EU Innovation and Sustainable Growth. Position Document on Industrial Biotechnology in Europe and the Netherlands. 20 pp. http://www. sustentabilidad.uai.edu.ar/pdf/tec/industrial_white_biotech.pdf. EPA, 2003. An organizational guide to pollution prevention. U.S. Environmental Protection Agency Office of Research and Development, National Risk Management Research Laboratory. Center for Environmental Research Information, Cincinnati, OH. Europabio, 2003. White Biotechnology: Gateway to a More Sustainable Future Author The European association for Bioindustries. 26 pp. Gavrilescu, M., 2004. Cleaner production as a tool for sustainable development. Environ. Eng. Manag. J. 3, 45–70. Gavrilescu, M., Nicu, M., 2004. Source Reduction and Waste Minimization. Ecozone Press, Iasi, Romania. Gavrilescu, M., Chisti, Y., 2005. Biotechnology—a sustainable alternative for chemical industry. Biotechnol. Adv. 23, 471–499. Gerngross, T., 1999. Can biotechnology move us toward a sustainable society? Nat. Biotechnol. 17, 541–544. Hall, S., Roome, N., 1996. Strategic choices and sustainable strategies. In: Groenewegen, P. (Ed.), The Greening of Industry: Resource Guide and Bibliography. Island Press, Washington, DC, p. 9. Kristensen, R., 1986. Biotechnology and the Future Economic Development. Institute for Future Studies, Copenhagen. Liese, A., Seelbach, K., Wandrey, C., 2000. Industrial Biotransformations. A Collection of Processes. Wiley-VCH, Weinheim. Lievonen, J., 1999. Technological Opportunities in Biotechnology. VTT, Group for Technological Studies, Espoo, Finland. Malik, K.A., Nasim, A., Khalid, A.M., 1995. Biotechnology for Sustainable Development. Faisalabad, Pakistan, National Institute for Biotechnology and Genetic Engineering (NIBGE). Miller, J.A., Nagarajan, V., 2000. The impact of biotechnology on the chemical industry in the 21st century. Trends Biotechnol. 18, 190–191. O’Connell, D., Braid, A., Raison, J., Cowie, A., 2009. Progress towards sustainability frameworks for biofuels, bioenergy and other bio-based products. In: Paper Presented at the OECD Workshop on Best Practices in Assessing the Environmental and Economic Sustainability of Bio-Based Products, Montreal, July. OECD, 1989. Biotechnology: Economic and Wider Impacts. OECD, Paris. OECD, 1994. Biotechnology for a Clean Environment: Prevention, Detection, Remediation. OECD, Paris. OECD, 1995. Technologies for Cleaner Production and Products. OECD, Paris. OECD, 1998. Biotechnology for Clean Industrial Products and Processes. Towards Industrial Sustainability. OECD, Paris. OECD Report, 2001. The Application of Biotechnology to Industrial Sustainability. http://www1.oecd.org/publications/e-book/9301061e.pdf. Poppe, L., Novak, L., 1992. Selective Biocatalysis: A Synthetic Approach. Wiley-VCH, Weinheim. Rigaux, F., 1997. Industrial Biotechnology in the Atlantic Provinces. From Emergence to Development? The Canadian Institute for Research on Regional Development, Toronto. Roberts, S.M., Casy, G., Nielsen, M.-S., Phyhian, S., Todd, C., Wiggins, K., 1999. Biocatalysts for Fine Chemical Synthesis. John Wiley and Sons Ltd., Hoboken, NJ, ISBN: 0-471-97901-5. Rogers, P.L., Jeon, Y.J., Svenson, C.J., 2005. Application of biotechnology to industrial sustainability. Process Saf. Environ. Protect. 83 (B6), 499–503. https://doi.org/10.1205/psep.05005.



Relevant websites

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Soetaert, W., Vandamme, E., 2006. The impact of industrial biotechnology. Biotechnol. J. 1, 756–769. https://doi. org/10.1002/biot.200600066. Tonelli, F., Evans, S., Taticchi, P., 2013. Industrial sustainability: challenges, perspectives, actions. Int. J. Bus. Innov. Res. 7 (2), 143–163. UNEP, 1999. International Cleaner Production Information Clearinghouse, CD Version 1. United Nations Environment Programme, Division of Technology, Industry and Economics, Paris. www.emcentre.com/ unepweb/. Van Berkel, R., 2000. Cleaner Production for Process Industries. Plenary Lecture. Chemeca, Perth, WA. William, H., Scouten, W.H., Petersen, G., 1999. New Biocatalysts: Essential Tools for a Sustainable 21st Century Chemical Industry. CCR-Report, 1999. http://www.ccrhq.org/vision/index/roadmaps/New%20Biocatalysts.pdf. Wong, M., 2001. Industrial Sustainability (IS) and Product Service System (PSS). A Strategy Decision Support Tool for Consumer Goods Firm. PhD Report, University of Cambridge, UK. World Bank, 1999. Pollution Prevention and Abatement Handbook. The World Bank Group, Washington, DC. Xiang, C.C., Chen, Y., 2000. cDNA microarray technology and its applications. Biotechnol. Adv. 18, 35–46. Zechendorf, B., 1999. Sustainable development: How can biotechnology contribute? Trends Biotechnol. 17 (6), 219–225. Zosel, T., 1994. Pollution prevention in the chemical industry. In: Edgerly, D. (Ed.), Opportunities for Innovation: Pollution Prevention. National Institute of Standards and Technology, Gaithersburg, USA, pp. 13–25.

Relevant websites www.oecd.org. www.comsats.org. www.massey.ac.nz. lrd.yahooapis.com. www.sustentabilidad.uai.edu.ar.

C H A P T E R

3 Renewable energy versus fossil resources O U T L I N E 3.1 Renewable raw materials for the industry

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A renewable resource can be used repeatedly and replaced naturally. Renewable energy is evolving expeditiously, whereas fossil fuels are going the way of the dinosaur. The distinction between the renewable energy and fossil fuels does not look so complicated. Renewable energy is derived from natural sources that can be replaced during an average human lifetime (Jering and Gunther, 2010; Tyson Stevens, 2018; Dahlson-Rutherford, 2013; Panwar et al., 2011; Demirbas, 2006; Bilgen et al., 2004). Table 3.1 shows different types of renewable energy. Examples are shown in Table 3.2. Natural gas is a fossil fuel. When it is burned, carbon dioxide is released. In the case of biomass, things usually get a little more complicated. When wood is burnt, more carbon dioxide is released in comparison with burning coal. Burning of wood is a common method of energy generation from biomass. Wood is classified as a renewable resource because trees can be replenished (www.amigoenergy.com). Fossil fuels are nonrenewable resources, because they have taken several thousands of years to get formed. These resources cannot be replenished once these are used. In addition, carbon dioxide—a greenhouse gas—is released when fossil fuels are burnt. This has an adverse impact on the environment and also impacts human health and contributes to climate change. These issues are provoking the world to explore alternate sources of energy that are renewable and are not much harmful. Furthermore, the slow exhaustion of traditional fossil fuel resources has motivated companies to develop more challenging reserves. These untraditional resources usually have higher cost of production. There is a higher risk of environmental impact too.

Biotechnology in the Chemical Industry https://doi.org/10.1016/B978-0-12-818402-8.00003-3

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© 2020 Elsevier Inc. All rights reserved.

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3.  Renewable energy versus fossil resources

TABLE 3.1  Renewable energy. Solar Wind power Hydroelectric energy Biomass is the term for energy from plants Hydrogen and fuel cells Geothermal power Other forms of energy

TABLE 3.2  Fossil fuels. Coal: formed from ferns plants and trees that hardened due to pressure and heat Oil: formed from smaller organisms like zooplankton and algae. Intense amounts of pressure caused this complex organic matter to decompose into oil Natural gas: undergoes the same process as oil; however, the process is longer and subject to higher amounts of heat and pressure causing further decomposition

“Even though natural gas increased its market share significantly over the past decade, renewable energy grew faster than any fossil fuel. In 2016, renewable energy generation in the United States grew to a record 22 gigawatts of capacity—burying fossil fuel growth” (www. amigoenergy.com). “The energy produced by renewables is just as affordable as energy produced by fossil fuels, if not cheaper in some cases. Some solar panel projects can even produce power at roughly half the cost of fossil fuels like coal. That is a lot of potential savings. Renewable energy is expected to get cheaper over time” (www.amigoenergy.com). One of the most noteworthy attribute of renewable energy is that it is available in abundance (Gavrilescu and Chisti, 2005). It is unlimited. Renewable energy resources are clean sources of energy having a significantly reduced negative environmental impact as compared with traditional fossil energy technologies. Most of the investment in the area of renewable energy has been made on materials and personnel for building and maintaining the facilities, rather than importing expensive energy. Technological developments have been made in the area of mass communication. People are becoming conservant with the disadvantages of burning fossil fuels. Renewable energy is a high-priority area. It is clean and sustainable. This has forced the human beings to take advantage. Researchers are continuously researching this area. They are exploring new techniques to exploit these energy sources in an effective manner. Global warming is becoming a serious problem. It results from burning of coal, oil, and natural gas. It is detrimental for the planet and the living creatures. Furthermore, fossil fuels cause several unfortunate accidents in the past. To bring to an end to this devastation, we should use renewable resources. This is due to the fact that they are cleaner and do not produce harmful gases. Furthermore, fossil fuels are limited. They will be exhausted one day. So, before this crucial stage comes up, energy experts should maintain



3.  Renewable energy versus fossil resources

25

a positive view and must make high endeavors for replacing fossils fuels with renewable energy resources as the major sources of producing electricity. Renewable energy is available in abundance. It will be potentially inexpensive, once this technology and its present infrastructure are improved. “The major sources of renewable energy include solar, wind, biomass, geothermal, hydropower, and tidal energy. Nonrenewable energy, such as coal, natural gas and oil, require expensive explorations and potentially dangerous mining and drilling, and they will become more expensive as supplies get exhausted and energy demand increases. Renewable energy produces only small levels of carbon dioxide emissions and therefore helps to fight climate change caused by burning of fossil fuel. Renewable energy sector is relatively new in most countries and this can attract several companies to invest. This will generate new jobs for the unemployed people. This will be beneficial particularly for the developing countries. This will boost their economies. Renewable energy can make the electricity prices stable. This is due to the reason that their cost is dependent upon the initial capital investment and is free of the fluctuating costs of coal, oil and natural gas” (Shahzad, 2015). The price of oil is dependent on many factors that also include political stability in several regions of the world. In the past, political disagreement has caused acute energy crises. Renewable energy can be produced locally, and so, it will not be affected by distant political disturbances. Several safety concerns are associated with fossil fuels, such as explosions on oil platforms and collapsing coal mines. These concerns do not exist with renewable energy. Coal, natural gas, and oil reserves are limited. An unknown and insufficient amount of each resource is buried deep underground or under the ocean (Shahzad, 2015). “As more of these reserves are harvested, finding new sources shall become more complicated and more expensive, and using them becomes tougher and sometimes risky as well. Trivial reserves, such as oil sands, require the burning of huge amounts of natural gas to process them into exploitable oil. Drilling under the ocean floor can lead to calamitous accidents, such as the famous British Petroleum Oil Spill in 2010. On the contrary, renewable energy is as effortless to find as wind or sunlight. Renewable energy is much cleaner than fossil fuels. Coal mining and petroleum exploration produce solid toxic wastes, such as mercury, lead and other heavy metals. The burning of coal to produce electricity uses large quantities of water which often discharges arsenic and lead compounds into surface waters and releases carbon dioxide, sulphur dioxide, nitrogen oxides and mercury into the air. Gasoline and other products of petroleum cause similar pollution” (Shahzad, 2015; www.iteejournal.org). These pollutants cause several problems listed later (Shahzad, 2015): • Respiratory diseases and death • Produce acid rain, which damages buildings and ruins fragile ecosystems • Depletion of the ozone layer through global warming “Renewable energy is cleaner than fossil fuels. Since the start of the Industrial Revolution, the earth’s temperature has increased at an increasing rate, raising oceanic water levels in its wake. Not only do fossil fuels heat the earth, they produce unhealthy by-products like air pollution, which adversely affects the lung health. Renewable energy, on the other hand, typically releases less carbon dioxide than fossil fuels. In fact, renewables like solar and wind power—apart from construction and maintenance—don’t release any carbon dioxide at all. When comparing renewable energy to fossil fuels, renewable energy generation is cleaner,

26

3.  Renewable energy versus fossil resources

easier to sustain over time, expanding more rapidly, and sometimes even inexpensive than fossil fuels” (www.amigoenergy.com). The use of renewable resources for industrial applications is not new (Eggersdorfer et al., 1992). People have used these materials from the first civilizations onward. To meet the basic requirements, people have used raw materials from plant and animals; natural fibers for clothing, wood for heating, animal fat for lighting, natural dyes for textiles and art works, etc. The initial industrial activities were also mostly based on the use of renewable resources, and this trend continued until the industrial revolution. In the 19th century, there was a radical change, brought about by the development of carbochemistry (the chemistry of carbon, particularly the chemical transformation of coal into industrially useful materials) and in the 20th century by the emergence of petrochemistry. The use of renewable raw materials reduced substantially, due to the very low prices for petrochemical resources. During this period, the developing chemical industry was almost systematically based on petrochemical feedstocks. These days, a large part of the chemical industry is based on these resources, and our energy needs are also largely met by fossil fuels such as coal, petroleum, and natural gas. More than 90% of all organic chemical substances produced in Europe (including fuel) are based on fossil resources. Nonetheless, several important industries are derived from renewable resources. Most of the fibers used in the textile industry are natural fibers (cotton, wool, flax, etc.), the oleochemical industry supplies our daily requirements for soap and detergents based on vegetable oils, the building industry is even now using a lot of wood and other natural fibers as construction material, etc. Furthermore, petrochemistry does not offer a good alternative for the use of renewable resources in several areas. For instance, most of the antibiotics are produced by fermentation, from sugars. About half of our drugs are still isolated from living organisms. The oil crisis between 1973 and 1979, when Organization of the Petroleum Exporting Countries (OPEC) increased the oil prices from 2 to 30 USD per barrel gave rise to a renewed interest in renewable resources. “Due to this crisis, serious concern grew about our increasing dependence on fossil resources and the fact that these are not available infinitely. This concern was largely channeled politically into the energy question and resulted in several studies concerning the development of alternative energy sources. The results of these studies showed that renewable raw materials were not yet competitive, and the interest for renewable raw materials rapidly disappeared when the oil price dropped again and the economy turned back to business as usual. In the nineties, the discussions around sustainable development and the greenhouse effect and also the emergence of the green political parties provided new impulses. The problems related to the food surpluses in the European Union were also an important driving force. Because of the high costs arising from these food surpluses, the European Union strongly intervened into the European Common Agricultural Policy (CAP). For this purpose, the European Union developed the set-aside” land concept in 1992. According to this principle, subsidies were given to farmers for not planting anything on parts of their land, in order to limit overproduction. Then, within the European Common Agricultural Policy, possibilities were created to use this land for nonfood applications. Thus, farmers could earn additional revenue from this land” (Soetaert and Vandamme, 2006; www.europa-bio.be). “With the increasing awareness and concern about industrial waste and its effects on the environment, the need arose for better biodegradable intermediates and final end products. These biodegradable products can naturally degrade into components that are absorbed back



3.  Renewable energy versus fossil resources

27

into the natural cycle, in contrast to persistent products that do not (or only after an unacceptably long period) disappear from the environment or from the food chain. Biodegradability was the focal point of many products and these were frequently based on renewable resources, in view of their intrinsic biodegradability. Such applications are, for example, chemical substances that will almost certainly end up in the environment, like lubricating oils for tree saws and agricultural machinery, detergents, etc. Green detergents like alkylpolyglucosides have already achieved a significant market share and are made entirely from renewable resources (fatty acid alcohols and glucose)” (Soetaert and Vandamme, 2006; www.europa-bio.be). The crude oil reserves in the world will not last forever (Campbell, 1998). Regarding fossil reserves, we are now faced with the contradictory situation that, while crude oil (petroleum) is being exhausted at a rapid pace, the proven oil reserves have remained the same for 30 years as a result of new oil finds. These proven oil reserves are located in increasingly difficult to reach places. So, the cost for extracting the crude oil increases, reflected in the high oil prices. By contrast, agricultural raw materials such as wheat and corn are becoming inexpensive as a result of the increasing agricultural yields. This trend is expected to continue for some time, also as a result of the realizations of the “green” biotechnology. This trend may be disturbed by the transitory effects of market imbalances and politics, but for several applications, the economic balance is tipping toward the use of renewable resources, also in bulk chemical sector. Renewable resources are less expensive than fossil resources (Table 3.3). “Agricultural byproducts such as straw are even 10 times less expensive than petroleum. It is also quite remarkable that the current world market prices for petroleum and sugar are about the same, inspite of the fact that sugar is a very pure (99.8%) and refined product and petroleum is a nonrefined crude raw material, consisting of a very complex mixture of hydrocarbons and other compounds. On an energy base, as renewable resources have about half the energy content of fossil resources, renewable and fossil resources are roughly equal in price. It is increasingly becoming clear that we are faced with a long-term trend in the price of petroleum instead of a transitional effect. For the simple reason of the price of raw materials, it is clear that the use of renewable raw materials has significant growth perspectives” (Soetaert and Vandamme, 2006).

TABLE 3.3  Average world market price of some fossil and renewable resources. Price (Euro/ton) Fossil resources Petroleum Coal Ethylene

250 40 500

Renewable resources Corn/wheat Straw Sugar

100 20 250

Based on Soetaert, W., Vandamme, E., 2006. The impact of industrial biotechnology. Biotechnol. J. 1, 756–769. 10.1002/ biot.200600066.

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Fossil fuel generation costs USD 0.05–0.17 per kilowatt-hour in G20 countries, including the United States, the United Kingdom, Russia, Japan, India, and Germany. But by 2020, the cost of renewables is expected to be USD 0.03–0.10 per kilowatt-hour, with the price of onshore wind power and solar photovoltaic projects expected to be as low as USD 0.03 per kilowatt-hour by 2019 (Kyree Leary, 2018). In the future, we expect to see that renewable energy becomes a real competitor in the fossil fuel industry. A new report recently published by the International Renewable Energy Agency (IRENA) has predicted that the cost of renewable energy will experience significant price reduction by 2020, putting it on par with or inexpensive than fossil fuels (www.businessinsider.com/renewable-energy-will-be-cheaper-than-fossil-fuels-by-2020).

3.1  Renewable raw materials for the industry Consumers are increasingly interested in products based on renewable raw materials because these are healthier and more natural, having a positive environmental impact. One of the main challenges facing the industry is the transition to sustainable operations. Industries are taking initiatives for reducing resource intensities or footprints and by adopting safer materials and processes. Many brand owners and retailers are therefore seeking to position themselves accordingly by defining strategies and goals for using renewable raw materials. In Europe, for instance, the use of renewable resources is also being driven by the European Commission’s measures to cut carbon dioxide emissions and to support the bioeconomy; similar programs exist in other regions. Renewable raw materials are preferred for two reasons: - These respond to the market pull resulting from consumer and retailer demand. - Renewable resources make it possible for developing products having novel functionalities and molecules that would otherwise not be accessible or less well accessible through fossil-based routes. Renewable raw materials can contribute to sustainable development by reducing carbon dioxide emissions and replacing fossil raw materials, but they are not per se sustainable. Several issues such as competition with food, land use, and biodiversity are playing a significant role in the debate about renewable resources. “Renewable resources are mainly based on the use of biomass and have a biological origin. Its fundamental basis is the plant growth and production, which takes place by the photosynthesis process, and perhaps via the intermediate step of animal production resulting in a large variety of available biomass. The total annual biomass production on our planet is estimated at 170 billion tons and consists of ~75% carbohydrates (sugars), 20% lignins, and 5% of other substances such as oils and fats, proteins, terpenes, alkaloids, etc. Of this biomass production, 6 billion tons (3.5%) are currently being utilised for human needs, distributed as: − 3.7 billion tons (62%) for human food use, possibly via animal breeding as an intermediate step. − 2 billion tons of wood (33%) for energy use, paper and construction requirements. − 300 million tons (5%) to meet the human requirements for technical (nonfood) raw materials (clothing, detergents, chemicals).



3.1  Renewable raw materials for the industry

29

The rest of biomass production is used in the natural ecosystems (feed for wild animals), is lost when biomass is obtained for humans (particularly by burning) or is lost as a result of the natural mineralization processes” (Soetaert and Vandamme, 2006; Campbell, 1998; www. europa-bio.be). The renewable raw materials are provided by agriculture and forestry. The animal breeding sector and fisheries also contribute mainly animal fat, but are not much important in view of the low conversion efficiencies of plant to animal (about 10%–25%). Several methods can be used to industrially convert this available biomass into renewable raw materials or energy carriers. This is often connected to the food sector, due to the reason that food ingredients and renewable raw materials for industrial application can be made in the same factory from the same agricultural raw materials. For instance, sugar or glucose are produced for human food use and are also the most important raw materials for industrial fermentation processes. The industrial sectors that supply the most important renewable raw materials are listed in Table 3.4 (Soetaert and Vandamme, 2006). “Although fractionation technology and enzyme technology are different in nature, the interaction between them is particularly decisive for success. For instance, the fractionation technology is strongly affected by using the hydrolytic enzymes. The obtained pure basic products (sugar, starch, cellulose, oils) are then converted into a very broad range of products, using physical, chemical and biotechnological processes. For example, starch and cellulose are chemically modified to derivatives that find several uses in our daily lives. Sugars like sucrose and glucose are chemically coupled to oleo-chemicals for obtaining detergents and emulsifiers. With respect to industrial biotechnological processes, the fermentation technology needs to be partiularly mentioned. This important technology makes use of microorganisms (bacteria, yeasts, and fungi) for converting basic raw materials such as sugars and oils into an almost unlimited range of products. By simple use of another production organism, the raw material can be converted to totally different products, ranging from products with a TABLE 3.4  Industrial sectors supplying the most important renewable raw materials. Sugar and starch sector: Produces carbohydrates such as sugar, glucose, starch, and molasses from plant raw materials such as sugar beet, sugar cane, wheat, corn, potatoes, sweet cassava, and rice Oil and fat processing sector: Produces several oleochemical intermediates such as triglycerides, fatty acids, fatty alcohols, and glycerol from plant raw materials like rape seeds, soybeans, palm oil, coconuts, and animal fats Wood processing sector, particularly the cellulose and paper industry: Produces mainly cellulose, paper, and lignins from wood. These industries process plant raw materials to break them down into separate components such as sugar, starch, cellulose, glucose, proteins, oils, and lignins. They make use of two technological pillars: Fractionation technology: This technology is mainly based on physical and chemical separation methods to separate agricultural raw materials into their separate components Enzymatic technology: This aspect of industrial biotechnology intervenes during the transformation of agricultural raw materials. In practice, mainly hydrolytic enzymes are used, for example, amylases, which hydrolyse starch to glucose Based on Soetaert, W., Vandamme, E., 2006. The impact of industrial biotechnology. Biotechnol. J. 1, 756–769. 10.1002/biot.200600066.

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chemical structure that is very close to the raw material (example gluconic acid from glucose) to products that have essentially nothing in common with the starting material (for example, antibiotics, enzymes, etc.). These different process steps, implying the use of different types of technologies often takes place within the same factory or industry complex. These are usually referred to as biorefineries, similar to the petrochemical crude oil refineries” (Soetaert and Vandamme, 2006).

References Bilgen, S., Kaygusuz, K., Sari, A., 2004. Renewable energy for a clean and sustainable future. Energy Sources A: Recov. Util. Environ. Eff. 26 (12), 1119–1129. Campbell, C., 1998. The future of oil. Energy Explor. Exploit. 16, 125–152. Dahlson-Rutherford, C., 2013. Renewable Raw Materials in the Industrial Chemical Industry. vol. 10. ESSAI. Article 15, http://dc.cod.edu/essai/vol10/iss1/15. Demirbas, A., 2006. Global renewable energy resources. Energy Sources A: Recov. Util. Environ. Eff. 28 (8), 779–792. Eggersdorfer, M., Meyer, J., Eckes, P., 1992. Use of renewable resources for non-food materials. FEMS Microbiol. Rev. 103, 355–364. Jering, A., Gunther, J., 2010. Use of Renewable Raw Materials: Presented to the ETC/SCP 2010 Report. European Environment Agency. Gavrilescu, M., Chisti, Y., 2005. Biotechnology—a sustainable alternative for chemical industry. Biotechnol. Adv. 23, 471–499. Kyree Leary, 2018. Renewable Energy Will Be Cheaper Than Fossil Fuels by 2020. www.businessinsider.com/ renewable-energy-will-be-cheaper-than-fossil-fuels-by-2020. Panwar, N., Kaushik, S., Kothari, S., 2011. Role of renewable energy sources in environmental protection: a review. Renew. Sust. Energ. Rev. 15 (3), 1513–1524. Shahzad, U., 2015. The need for renewable energy sources. Int. J. Inform. Technol. Electric. Eng. 4, 16–19. www.iteejournal.org/archive/vol4no4/v4n4_4.pdf. Soetaert, W., Vandamme, E., 2006. The impact of industrial biotechnology. Biotechnol. J. 1, 756–769. https://doi. org/10.1002/biot.200600066. Tyson Stevens, 2018. Renewable Energy vs Fossil Fuels: 5 Essential Facts. www.amigoenergy.com/blog/ renewable-energy-vs-fossil-fuels/.

Relevant websites www.amigoenergy.com. www.iteejournal.org. www.europa-bio.be. www.businessinsider.com/renewable-energy-will-be-cheaper-than-fossil-fuels-by-2020.

C H A P T E R

4 Chemical industry O U T L I N E 4.1 Classification of chemical industry sectors 4.1.1 Basic chemicals 4.1.2 Speciality chemicals 4.1.3 Consumer chemicals

4.1.4 Life science products 33 33 34 35

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Chemical industry is diverse. Chemicals produced by the chemical industry are used for making almost every man-made product and are playing a very important role in the lives of people around us (Chenier, 2002). Such products offer innumerable advantages, which make the life comfortable for people. The chemical industry includes basic chemicals, speciality chemicals, consumer care products, and life science products. This industry is also a major economic force providing jobs to millions of people around the world and generates billions of dollars in shareholder value and tax revenues for governments (www.oecd.org). The chemical industry is facing intense pressure for changing current working practices in favor of greener alternatives (Ulrich et al., 2000; Matlack, 2001; Carpenter et al., 2002; Poliakoff et al., 2002; Sherman, 2004; Asano et al., 2004; Micheal McCoy, 2006; Shreve and Brink, 1977; Gavrilescu and Chisti, 2005). There have been amazing changes in the chemical industry over the last decade or so, not only in Europe and the United States but also in China, India, and the remaining Asia and Brazil. The chemical industry is very large and competitive. It would grow at a fast rate in the future. The industry products account for a large share of the overall global chemical industry. This industry consists of a very diverse and complex range of products (https://organic-chemistry.chemistryconferences.org/.../global-chemical-industry-analy). “The global chemicals market is in excellent shape. This is being shown basically in the large increase in global revenue from EUR 1622 billion to EUR 3534 billion during 2005–15 (equivalent to an average annual growth rate of more than eight percent). This shows the

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4.  Chemical industry

global chemical industry is not negatively affected by the financial crisis of 2008. Chemical companies all over the world are in fact recording growing revenue and profit levels—and also mostly double-digit operating margins—since 2010. This development has been reinforced by the low-interest rate policies of the most influential central banks worldwide. Excellent credit ratings have allowed companies to get new finance at reduced interest rates and get new loans easily. This approach has been used by a number of chemical companies to sharpen their portfolios through mergers and acquisitions transactions and equity investment” (www.gtai.de). “Global market for speciality chemicals is projected to touch US$1.2 trillion by 2022 from an estimated US$970 billion in 2017. Adhesives and Sealants, Agrochemicals, Coatings and Engineering Thermoplastics constitute the largest segments in the global market for Speciality Chemicals, together cornering an estimated share of nearly 50% in 2016” (https://globenewswire.com/.../2017/.../Speciality-Chemicals-A-Global-Market-Overvie). “With increasing competition worldwide, innovation remains very important in finding new ways for the industry for satisfying its increasingly sophisticated, demanding and ­environmentally-conscious consumers” (www.essentialchemicalindustry.org). The chemical industries produce an extensive array of commodity industrial chemicals used for making other products to speciality chemicals designed for special use. These products can range from large bulk chemicals used to produce plastics to small bottles of cleaning solutions used by households. Several chemical companies have technical knowledge in research and process engineering, huge capital and management capacity, and skilled and competent labors. The companies involved in producing this diverse range of products also vary significantly (www.oecd.org; www.chemicalspolicy.org). The chemical industry is also a major employer. The industry is becoming more productive, and production processes are becoming highly automated; therefore, the world employment levels in the industry have dropped in the recent years. “Given the complexity of the processes and the constant need for innovation, the chemicals industry is research intensive. Most companies allot 4%–6% of their annual sales for R&D although the percentage of revenue spent on research varies from one branch to another” (OECD, 2001; www.chemicalspolicy.org). “Companies specializing in large-volume basic chemicals that have been widely used for many years do not spend much, whereas competition in the newer sectors can be met only by intensive research efforts. Research expenses are highest for the life sciences companies and lowest for producers of commodity chemicals” (www.chemicalspolicy.org). The companies that are involved in producing this vast array of products also vary considerably. “Some chemical companies are ranked amongst the largest industrial companies in the world - the top ten chemical companies had revenues in the range of USD10-30 billion.” These firms provide employment to several thousands of people, and they have several manufacturing sites that are located throughout the world. Other chemical companies manufacture only a few products at one site and are relatively small in size (Fortune, 2000; SOCMA, 2000). “The chemical industry is a catalyst for economic growth worldwide. It is a mainstay in global manufacturing superpowers such as United States, the European Union, India, China, and other developing economies. Chemical manufacturing is one of the most consistent and steadfast industries that are spurring on the progress of developing nations. The entire world depends on the multitude of products produced by the chemical industry, so the demand is constant. Humanity depends on medicine, food, water



33

4.1  Classification of chemical industry sectors

TABLE 4.1  Products from the chemical industry in 2014 by category (%). United States

Europe

Basic chemicals

61

60

Speciality chemicals

24

28

Consumer chemicals

15

12

Facts and Figures, CEFIC; 2016 Guide to the Business of Chemistry, American Chemistry Council, 2015 www.essentialchemicalindustry.org/ the-chemical-industry/the-chemical-industry.html.

TABLE 4.2  Subsegments of the chemical industry. • Inorganic chemicals industry • Organic chemical industry • Chemical wholesaling industry Chemical product manufacturing industry • Fertilizer manufacturing industry • Pesticide manufacturing industry • Soap and cleaning compound manufacturing industry • Dye and Pigment Manufacturing Industry • Generic pharmaceutical manufacturing industry Based on www.technofunc.com/index.php/domain-knowledge/chemicals-industry/item/sectors-of-chemical-industry.

for ­surviving and technology are fueled by innovation in chemistry. The combined impact of the chemical manufacturing, the incredible economic boost, and the products that are being manufactured is truly making the world a better place” (blog.mixerdirect.com/ understanding-the-chemical-industry). The chemical industry is facing many challenges in this century, which should be overcome. Through this, the industry would be able to help society for maintaining and improving the living standard and do so in a sustainable way. Tables 4.1 and 4.2 show the products from the chemical industry in 2014 by category (%) and subsegments of the chemical industry.

4.1  Classification of chemical industry sectors 4.1.1  Basic chemicals Basic chemicals are also termed commodity chemicals and represent a mature market. The basic chemical industry has large plants and is using the continuous processes. These plants require high energy input, and the profit is low. The industry is highly cyclical due to fluctuations in capacity utilization and the prices of the substrates. “The products of the industry are mostly used in processing applications in pulp and paper, textile, oil refining,

34

4.  Chemical industry

­ etals recovery and as raw materials for producing other basic chemicals, speciality chemm icals, and consumer products, including manufactured goods (textiles, automobiles, etc.)” (Swift, 1999; www.technofunc.com/index.php/domain-knowledge/chemicals-industry/ item/sectors-of-chemical-industry; www.precaution.org/lib/07/innovest.pdf; www.americanchemistry.com/2017-Elements-of-the-Business-of-Chemistry.pdf). The production of basic chemicals is highly capital intensive. In addition to this, production of basic chemicals is usually in large scale, and the requirement of energy is high. Basic chemicals include petrochemicals, polymers, and basic inorganics. Several million tons of basic chemicals are produced each year. Whereas most of the merchandise itself is relatively cheap and abundant, the basic chemical sector as a whole is extremely important. Petrochemicals are products traditionally obtained from oil, but there has been an increase in goods produced from coal, natural gas, and also biomass. This can be exemplified by the fact that few countries are producing ethanol using a combination of oil and natural gas, whereas other countries have started to produce it from coal. Another example is polyethene, which can be obtained not only from oil and gas but also from biomass. Several basic chemicals are not sold directly to the consumer. They are often sold in large quantities to clients in other sectors of the chemical industry, where they are used as ingredients in manufactured goods sold to the public. For instance, ammonia is natural gas. Some companies purchase it for producing nitric acid. Then ammonia is reacted with nitric acid for producing ammonium nitrate, which is used in fertilizer. Ammonia is also used to produce hydrogen cyanide, and hydrogen cyanide is a major component in methyl 2-methylpropenoate, which is a starting material for make acrylic polymers. Plastic resins that are another basic chemical are facing an upward trend due to their use as a replacement for older materials in the automobile industry, the construction industry, and the packaging industry (blog.mixerdirect.com).

4.1.2  Speciality chemicals Speciality chemicals are designed on the basis of their performance and function (blog.mixerdirect.com). Speciality chemicals are also called “performance chemicals” or “specialties.” These are differentiated—and often technologically advanced—products. They are produced in lower volumes in comparison with basic chemicals and are used for a certain specific purpose (for instance, as a functional ingredient or as processing aids in the production of a wide array of products). Specialties allow customers in reducing overall system costs, improve product performance, and optimize manufacturing processing in increasing yield through custom solutions. They are usually sold for what they do, rather than for what they contain (www.americanchemistry.com). “Long-term growth prospects for speciality chemicals are generally more dynamic as compared to basic chemicals. Several specialty markets exist, including - manufacturing industries Automobiles, cosmetics and other consumer products, electronics, food, foundries, lubricants, paper, plastic products, rubber products, etc. - nonmanufacturing industries Oil recovery, construction, and electric utilities. Furthermore, the speciality chemicals focus along markets, several of which are maturing and becoming increasingly international” (www.americanchemistry.com).



4.1  Classification of chemical industry sectors

35

“Speciality chemicals include a diverse range of products, and are used in everything from agricultural applications to dyes for textiles. Biofuels, biotechnology, pharmaceuticals, colorants (dyes and pigments), composites, crop protection products, edibles fats/oils, nanomaterials, paints, coatings, adhesives, and surfactants are some of the most common speciality chemicals. Emerging sciences such as biotechnology and alternative energy are two examples of sectors that rely on speciality chemicals because of the unique requirements of their fields. Whereas there are very many different types of speciality chemicals used in a wide variety of different applications, they all have live up to their name by being specifically designed to serve a particular purpose. Speciality chemicals include some of the most innovative ­products in the chemical industry as a whole. Clients can have a specialty chemical manufacturer engineer a one-of-a-kind product that provides a solution to their problem. Speciality chemicals are sold on the basis of their performance or function, rather than their composition. They can be single-chemical entities or formulations whose composition sharply affects the performance and processing of the customer's product” (blog.mixerdirect.com).

4.1.3  Consumer chemicals Consumer chemicals are found in the shelves of stores and can be found in millions of homes in the world. Consumer care products are one of the oldest segments of the chemistry business dating back thousands of years (ancient Babylonians were the first producers of soap). These are the formulated products, using what is often simple chemistry, but show a high degree of differentiation along branding lines. Expenditure on research and development is increasing, and several of these products are becoming high tech in nature. Consumer care products are usually formulated in batch-type operations, although some products, for example, detergents, are being produced in large dedicated plants. Formulating involves mixing, dispersion, and equipment for filling rather than reactors for chemical conversions. “Personal hygiene products constitute the bulk of consumer chemical manufacturing. Consumer chemicals include soaps like hand soap, bar soap, body wash, shampoo, and other hair care products. Dental hygiene products-toothpaste, mouthwash, and others are also a category of consumer chemical products. The cosmetics and fragrances market is one of the most profitable and successful sectors, as they are often considered a necessity by several people in the world. Vitamins and daily health supplements belong here, too. One of the other mainstays of consumer chemical manufacturing is household cleaning products like cleaning sprays, carpet cleaner, wood polish, dish soap, dishwasher detergents, laundry detergents, and several more items fall under this category” (blog.mixerdirect.com).

4.1.4  Life science products Life science includes companies in the areas of biotechnology, pharmaceuticals, biomedical technologies, and life system technologies. Life sciences (about 30% of the dollar output of the chemistry business) include differentiated chemical and biological substances, pharmaceuticals, diagnostics, animal health products, vitamins, pesticides, products for protection of crops, and modern biotechnology. Generally, batch processes are used in formulating operations where quality control and a clean environment are crucial. Technological advantages are extremely important, and the highest research and development expenditure is involved in this sector.

36

4.  Chemical industry

Life science chemicals induce specific outcomes in humans, animals, plants, and other life forms. Life science products are generally produced using very high specifications and are closely observed by governmental agencies such as the Food and Drug Administration. Crop protection chemicals are about 10% of this category and include herbicides, insecticides, and fungicides. Subsegments of the chemicals industry are presented in Table 4.2. They help the learner to understand various subclassifications and also major products and manufacturing operations in the chemical domain.

References Asano, K., Ono, A., Hashimoto, S., Inoue, T., Kanno, J., 2004. Screening of endocrine disrupting chemicals using a surface plasmon resonance sensor. Anal. Sci. 20, 611–616. Carpenter, D.O., Arcaro, K., Spink, D.C., 2002. Understanding the human health effects of chemical mixtures. Environ. Health Perspect. 110 (Suppl), 25–42. CEFIC, 2016. Guide to the Business of Chemistry. 2015. American Chemistry Council. Chenier, P.J., 2002. Introduction to the chemical industry: an overview. In: Survey of Industrial Chemistry. Topics in Applied Chemistry. Springer, Boston, MA. Fortune (Fortune Magazine) (2000), Fortune 5000 Web Site, http://www.fortune.com/fortune/fortune500/. Gavrilescu, M., Chisti, Y., 2005. Biotechnology—a sustainable alternative for chemical industry. Biotechnol. Adv. 23, 471–499. Matlack, A.S., 2001. Introduction to Green Chemistry. Dekker, New York. Micheal McCoy, 2006. Facts & figures of the chemical industry. Chem. Eng. News 84 (28), 35–72. OECD, 2001. Environmental Outlook for the Chemicals Industry. OECD Environment Directorate, Environment, Health and Safety Division, Paris. Poliakoff, M., Fitzpatrick, J.M., Farren, T.R., Paul, T., Anastas, P.T., 2002. Green chemistry: science and politics of change. Science 297, 807–810. Shreve, R.N., Brink Jr., J.A., 1977. The Chemical Process Industries, fourth ed McGraw Hill, New York. Sherman, D., 2004. Industrial biotechnology and the chemical Industry’s sustainability challenge. In: Paper Presented at the World Congress on Industrial Biotechnology and Bioprocessing, Orlando, FL. SOCMA (Synthetic Organic Chemical Manufacturers Association, United States), 2000. Web Site. http://socma. rd.net/services.html. Swift, T.K., 1999. Where is the chemical industry going. J. Natl. Assoc. Bus. Econ. Ulrich, E.M., Caperell-Grant, A., Jung, S.-H., Hites, R.A., Bigsby, R.M., 2000. Environmentally relevant xenoestrogen tissue concentrations correlated to biological responses in mice. Environ. Health Perspect. 108, 973–977.

Relevant websites https://organic-chemistry.chemistryconferences.org/.../global-chemical-industry-analy. www.oecd.org. www.gtai.de. https://globenewswire.com/.../2017/.../Specialty-Chemicals-A-Global-Market-Overvie. www.essentialchemicalindustry.org. www.chemicalspolicy.org. blog.mixerdirect.com/understanding-the-chemical-industry. www.technofunc.com/index.php/domain-knowledge/chemicals-industry/item/sectors-of-chemical-industry. www.precaution.org/lib/07/innovest.pdf. www.americanchemistry.com. blog.mixerdirect.com. www.essentialchemicalindustry.org/the-chemical-industry/the-chemical-industry.html. www.technofunc.com/index.php/domain-knowledge/chemicals-industry/item/sectors-of-chemical-industry.

C H A P T E R

5 Bioprocesses in industrial biotechnology O U T L I N E 5.1 Fermentation processes 5.1.1 Types of fermentation processes

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5.2 Enzymatic processes

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References

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Relevant websites

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Further reading

56

Bioprocess uses living cells or their components (e.g., enzymes and chloroplast) for obtaining desired products. This deals with designing and developing equipment and processes to manufacture products in the area of agriculture, chemicals, food, feed, pharmaceuticals, nutraceuticals, and polymers and paper from biological materials and treatment of wastewater (www.omicsonline.org; Singh, 2014). Bioprocess engineering combines biology, mathematics, and industrial design and ­involves designing of bioreactors and study of mode of operations of bioreactor. It also involves the study of different biotechnological processes used in industries for manufacturing biological products and optimizing the yield and the quality of the final product. Bioprocess engineering includes the work of industrial, mechanical, and electrical engineers in applying principles of their areas to processes based on using alive cells or subcomponent of such cells. This area is the subdiscipline within biotechnology that translates the discoveries of life science into practical products, processes, or systems that is able to serve the societal needs. Bioprocess engineering is used in the production of biopharmaceuticals and also in the production of alcohol, amino acids, organic acids, antibiotics, and several other specialty products. Bioprocessing includes the following: • Design and operation of bioreactors, sterilizers, and equipments for recovery of products • Development of systems for process automation and control • Layout of well-organized and safe biotech industries

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5.  Bioprocesses in industrial biotechnology

TABLE 5.1  Advantages of bioprocesses. Usually require lower temperature, pressure, and pH Can use renewable resources as raw material Greater quantities can be produced with less energy consumption Based on Singh, R.S., 2014. Industrial biotechnology: an overview. In: Singh, R.S., Pandey, A., Larroche, C. (Eds.), Advances in Industrial Biotechnology. IK International Publishing House Pvt. Ltd, India, pp. 1–35.

• Study on the optimization of yield of final product on an industrial scale • Maintenance of the product quality The objective of biotechnology is to apply molecular biology and cell manipulation for the processing of biological materials on a large scale. This objective can be achieved by pairing biotechnology with bioprocess development, which is very important part of several food, chemical, and pharmaceutical industries. Bioprocess uses microorganisms, animal cell and plant cell, and cell components, for instance, enzymes, for the production of new products and waste management. The use of microorganisms for transferring biological material for the production of fermented foods has its origins from ancient times. Since that time, bioprocesses are being developed for huge range of commercial products, ranging from relatively inexpensive materials like industrial alcohol and organic solvents to expensive specialty chemicals like antibiotics, therapeutic proteins, and vaccines. With the refining of technology and instrumentation, bioprocesses can be used in other areas where chemical processes are now being used. Bioprocesses offer many benefits over traditional chemical methods of production because they use living cells or enzymes (Table 5.1).

5.1  Fermentation processes The term “fermentation” is the deliberate use of microorganisms for producing useful products. Bacteria, yeast, and fungi are used for producing biomass, enzymes, primary and secondary metabolites, recombinant products, and products of biotransformation commercially. Modern industrial fermentation processes used in the food and beverage industry can be described according to different perspectives (Paulová et al., 2013). These processes generally use bioreactors, which can be classified with respect to the following: • • • • • •

Mode of feeding of the bioreactor, which can be batch, fed-batch, and continuous Immobilization of the biocatalyst (free cells or enzymes) Type of fermentation—submerged or solid substrate Use of pure or mixed culture Type of mixing in the bioreactor—mechanical, pneumatic, and hydraulic agitation Type of process—aerobic, microaerobic, and anaerobic

The decision as to which bioprocess should be implemented in any particular process involves considering the benefits and drawbacks of a particular setup. This includes examining the properties and availability of the basic raw materials, operating costs and investment, sustainability, availability of a competent manpower, and also the desired productivity and return on investment. In the commercial applications, each bioprocess system needs to



5.1  Fermentation processes

39

­ perate efficiently; the important criterion for the selection of a bioprocess remains the minio mum for capital costs per unit of product obtained. At the same time, with proper design and operation, the issues concerning by-product and wastewater management cannot be avoided in the commercial processes.

5.1.1  Types of fermentation processes 5.1.1.1  Submerged fermentation This method involves manufacturing biomolecules in which enzymes or microorganisms are submerged in a liquid, oil, or a nutrient broth. The process is used for several purposes, usually to manufacture industrial products. Submerged fermentation involves growing the microorganism in a liquid medium in which several nutrients are either dissolved or suspended as particulate solids in the medium (Frost and Moss, 1987). Submerged fermentations are used for the production of enzymes from microorganisms. In the industry, mostly submerged fermentation is used, as the submerged fermenter is amenable to engineering control and design and the space is saved. In this method, the enzymes and other reactive compounds are submerged in a liquid. The process is used for several purposes, generally in manufacturing of industrial products. Submerged cultivation ensures a controlled environment for the efficient production of high-quality end products and for obtaining maximum productivity and yield. The bioreactors are operated in batch, fed-batch, or continuous mode to grow different types of microorganisms producing a diverse range of products (Najafpour, 2007; Erickson, 2011; Garcia-Ochoa et al., 2011; Paulová et al., 2013; Stanbury and Whitaker, 1995; Zimmerman et al., 2009). Different methods of submerged fermentation of microorganisms in bioreactors are presented in the succeeding text, and the characteristic features, advantages, and disadvantages of each cultivation method are discussed. The relevant applications for batch, fed-batch, and continuous fermentation of microorganisms in liquid media used in the production of different types of products are dealt. “Fermentation takes place in large vessels having volumes of up to 1,000 cubic metres. The fermentation media uses renewable raw materials like maize, sugars and soya and is decontaminated. Most industrial enzymes are extracellular; these are secreted by microorganisms into the fermentation medium for breaking down the carbon and nitrogen sources. Batch-fed and continuous fermentation processes are generally used. In the fed-batch process, sterilized nutrients are introduced in the fermenter during the growth of the biomass. In the continuous process, sterilized liquid nutrients are introduced into the fermenter at the same flow rate as the fermentation broth leaving the system. Different parameters such as temperature, pH, oxygen consumption and carbon dioxide formation are measured and controlled for optimizing the fermentation process” (Paulová et al., 2013; ecoursesonline.icar.gov.in; www.biocon.com). Next, the enzymes are recovered from the fermentation broth, and the insoluble products, which are the microbial cells, are removed. This is generally performed by centrifugation. The industrial enzymes are mostly extracellular. These are secreted by cells in the fermentation broth. They are left in the broth after the removal of the biomass. The enzymes left in the broth are then concentrated by using evaporation, membrane filtration, or crystallization techniques depending on their final application. If pure enzymes are needed, they are generally separated using gel or ion exchange chromatography. Submerged type of fermenters is of different types (Ali et al., 2018) (Fig. 5.1A–E). These fermenters may be grouped in different ways:

40

5.  Bioprocesses in industrial biotechnology

Motor Air outlet Foam breaker Flat blade disc impeller Baffle Gas sparger

Air inlet Drain valve

(A)

Effluent gas

Outlet gas

Nozzle Inlet air

Recirculation loop

Pumb

Inlet air

(B) FIG. 5.1  (A) Stirred tank fermenter. (B) Air-lift fermenter with an internal loop cycle of fluid flow and with an external recirculation pump.

Gas

Solid

Gas bubble

Solid Solid particle

Gas

Distributor

(C)

FIG.  5.1, cont’d  (C) Fluidized bed bioreactor https://www.kisspng.com/free/fluidized-bed-reactor.html. (D) Fixed bed bioreactor. (Continued)

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5.  Bioprocesses in industrial biotechnology

Exhaust gas

Air sparger Air

(E) FIG. 5.1, cont’d  (E) Bubble column reactor. Parts A, B and E: Reproduced with permission from Najafpour (2007). Part D: Reproduced with permission from essentialchemicalindustry.org.

• Shape or configuration • Aerated or anaerobic • Batch or continuous Aerated stirred tank batch fermenter is mostly used. Aerated stirred tank batch fermenter This type of fermenter is an upstanding closed cylindrical tank. It is fitted with the following: - - - - - - -

One or more baffles attached to the side of the wall A water jacket or coil for heating and or cooling A device for forced aeration (known as sparger) A mechanical agitator usually a pair or more impellers Means of introducing organisms and nutrients Means of taking samples Outlets for exhaust gases

Modern fermenters are highly automated. These can continuously monitor, control, and record pH, dissolved oxygen, oxidation reduction potential, effluent oxygen and carbon



5.1  Fermentation processes

43

­ ioxide, and chemical components. Further diagrams of stirred tank fermenters are shown in d the succeeding text: Aeration system (Sparger) “Sparger is used to introduce air into fermenter. Aeration provides enough oxygen to the organism in the fermenter. Fine bubble aerators should be used. Large bubbles have less surface area in comparison to smaller bubbles which will facilitate oxygen transfer to a large extent. Agitation is not needed when aeration provides sufficient agitation which is the case of Air-lift fermenter. But this is possible for medium having low viscosity and low total solids. For aeration to provide agitation, the vessel height and diameter ratio (aspect ratio) must be 5:1. Air supply to sparger should be supplied through filter” (Najafpour, 2007; Guieysse et al., 2011; ecoursesonline.icar.gov.in). There are different types of sparger as presented in Table 5.2. 5.1.1.2  Surface fermentation “In this method, the microorganisms are grown on the surface of a liquid or solid substrate. These methods are very complicated and are not used much in the industry. Aspergillus niger forms a mycelium layer on the liquid surface of the aluminum or stainless steel trays. These trays are stacked in fermentation rooms which are provided with filtered air. This supplies oxygen and control the temperature of fermentation. Surface fermentation can be easily controlled and implemented. It does not need aeration or agitation of the fermentation broth, therefore it requires no instrumentation for aeration and agitation. The separation of citric acid from the mycelium is easy because the microorganism does not get dispersed into the

TABLE 5.2  Different types of sparger. Porous sparger Made of sintered glass, ceramics, or metal. It is used only in lab-scale nonagitated vessel. The size of the bubble formed is 10–100 times larger than pore size. There is a pressure drop across the sparger, and the holes tend to be blocked by growth, which is the limitation of porous sparger Used only in lab-scale nonagitated vessel Orifice sparger It is a perforated pipe kept below the impeller in the form of crosses or rings. The size should be about ¾ of impeller diameter. Air holes drilled on the under surfaces of the tubes and the holes should be at least 6-mm diameter. This type of sparger is used mostly with agitation. It is also used without agitation in some cases like yeast manufacture, effluent treatment, and production of SCP Used in small stirred fermenter Nozzle sparger It is single open/partially closed pipe positioned centrally below the impeller. When air is passed through this pipe, there is lower pressure loss and does not get blocked. Mostly used in large scale Combined sparger agitator This is air supply via hallow agitator shaft. The air is emitted through holes in the disc or blades of agitator Based on https://bioprocessing.weebly.com/fermentation.html.

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5.  Bioprocesses in industrial biotechnology

TABLE 5.3  Advantages of submerged fermentation over surface fermentation. Operation is simple Investment costs are lower Better process control Fermentation time is less Reduced floor space requirements Lower labor costs Easier maintenance of aseptic conditions Based on https://bioprocessing.weebly.com/fermentation.html.

TABLE 5.4  Disadvantages of submerged fermentation over surface fermentation. Higher expenses for equipment High consumption of electrical energy Process is very sensitive to short interruptions in aeration and sensitive to infections, which results in yield loss and breakdown of respective batches Based on https://bioprocessing.weebly.com/fermentation.html.

medium. Only the temperature and humidity of the fermentation chamber needs to be controlled. It can be used easily in small plants and also in the third world countries. During surface fermentation, the broth gets concentrated because of high evaporation rate during fermentation. Therefore expenses and losses during recovery and purification are less. However, surface fermentation has the following drawbacks” (ecoursesonline.icar.gov.in): - Capital investment costs are high. - In the developing countries, personnel expenses are high with very high wages. - Fermentation time is long resulting in low productivity. Submerged fermentation is preferred over surface fermentation for the several reasons shown in Table 5.3. However, it shows some disadvantages compared with surface fermentation (Table 5.4). 5.1.1.3  Solid-state fermentation (SSF) The origin of SSF can be traced back to bread making process in ancient Egypt. This process is used in the production of many industrial and pharmaceutical products, food, and fuel. It can be used as an alternative to submerged fermentation. In Japan, it has existed for many years and is known as koji fermentation (Holker and Lenz, 2005; Chisti, 1999; Paulová et al., 2013; Barrios-Gonzáles, 2012; Raimbault, 1998). This process uses microorganisms in a controlled environment for producing enzymes, fuel, and other important products. This



5.1  Fermentation processes

45

type of fermentation occurs in the absence of free water and is a much more simple process as compared with submerged fermentation. It requires very little energy, produces very high volumetric productivity, and is similar to the natural environment of many fungi. The volumetric productivity can be higher by eight times in comparison with that of submerged fermentation; the downstream process is much simpler than submerged fermentation (Chundakkadu Krishna, 2005). This process involves a solid matrix such as rice bran, which is placed on a medium to alongside microorganisms creating a substrate. This is stored at temperature, between 5°C and 95°C for 1–5 days. Agitation using constant or intermittent rotation is done. This type of fermentation plays an important role to grow filamentous fungi. The air is allowed to come in contact with the mycelium by smearing the mycelium. This is quite important as the fungi can be decomposed in their natural environment as they are on the ground. This allows the growth of filamentous fungi in conditions that represent their natural ­conditions. The growth of mold is enhanced by using substrates, which have a reduced water level. Wheat bran is the most common substrates used. It is important to monitor the air flow rate as this has an effect on water and oxygen levels and also any changes in temperature. Moisture levels are important for the growth of filamentous fungi, and the moisture content should be maintained at a specific level (Soccol et al., 2017). Sterilization of the environment is not needed in the case of SSF; this is because the fermentation substrate initiates sterilization, and the microorganisms inhibit micro flora from growing (www.ukessays.com; Chisti, 1999; Barrios-Gonzáles, 2012; Paulová et al., 2013; Chundakkadu Krishna, 2005; Soccol et al., 2017; Bhargav et al., 2008; Thomas et al.,2013; Singhania et al., 2009; Sato and Sudo, 1999; Pandey, 2001; Holker and Lenz, 2005). SSF also include several widely known microbial processes such as composting, soil growth, silage production, wood rotting, and mushroom cultivation (Pandey et al., 1999; Arora et al., 2018; Subramaniyam and Vimala, 2012; Hölker et al., 2004). “Several western foods such as mold-ripened cheese, bread, sausage and many foods of Asian origin including miso, tempeh and soy sauce are produced using this process. Beverages derived from SSF processes include ontjom in Indonesia, shao-hsing wine and kaoliang (sorghum) liquor in China and sake in Japan. SSF is used for the production of bioproducts from microorganisms under conditions of low moisture. The medium used for SSF is usually a solid substrate (e.g., rice bran, wheat bran, or grain), which requires no processing. In order to optimize water activity requirements, which are of major importance for growth, it is necessary to take into account the water sorption properties of the solid substrate during the fermentation. In view of the low water content, fewer problems due to contamination are observed. The power requirements are lower than submerged fermentation. Inadequate mixing, limitations of nutrient diffusion, metabolic heat accumulation, and ineffective process control renders SSF generally applicable for low value products with less monitoring and control. There exists a potential for conducting SSF on inert substrate supports impregnated with defined media for the production of high value products. It involves the growth of microorganisms on moist solid particles, in situations in which the spaces between the particles contain a continuous gas phase and a minimum of visible water. Although droplets of water may be present between the particles, and there may be thin films of water at the particle surface, the inter-particle water phase is discontinuous and most of the inter- particle space is filled by the gas phase. The majority of the water in the system is absorbed within the moist solid-particles” (Pandey et al., 1999; Arora et al., 2018).

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The term “solid-substrate fermentation” denotes any fermentation process that involves solids, including suspensions of solid particles in a continuous liquid phase and even trickling filters (Pandey et al., 1999; Arora et al., 2018; Subramaniyam and Vimala, 2012; Hölker et al., 2004). An example of SSF is koji fermentation. Aspergillus oryzae is known in English as koji. It is a filamentous fungus. In Japanese and Chinese preparations, A oryzae is used in the fermentation of soybeans. Another use of A oryzae is to saccharify rice and also potatoes in alcohol production. It is also used for producing rice vinegars and making Japanese drinks such as sake and huangjiu. High starch-containing products such as rice and manioc are used for producing sake and other alcoholic drinks as opposed to using malted barley or grapes. These koji molds are able to ferment the starch ingredients into simple sugars. Saccharomyces yeasts are not able to break down these starches. A oryzae was domesticated up to 2000 years ago. Several properties of A oryzae make it an important component of alcohol production and rice saccharification. For example, it can produce amylase enzymes and has a small amount of tyrosinase. Another advantage is its growth rate. The mycelia grow rapidly onto rice kernels for rice saccharification. A oryzae produces a nice smell and has a range of flavors, and the production of harmful color substances is low. The koji fungus is popular in Japan and is used for the production of sake and also soy sauce and other important Japanese foods. Advantages of SSF over submerged fermentation are shown in Table 5.5. In contrast, the environment in SSF can be quite stressful to the microorganism (Table 5.6). The major problem is to control the temperature during the fermentation process. Heat is produced during the metabolic activities of microorganisms, as the substrate used has low

TABLE 5.5  Advantages of solid-state fermentation over submerged fermentation. Downstream processing is easier Volumetric productivity is high Reduced energy requirements Might be easier to meet aeration requirements Resembles the natural habitat of some microorganisms The fungal hyphae are immersed in a liquid medium and therefore do not face the risk of desiccation Temperature control is generally not difficult; the organism is exposed to a constant temperature throughout its growth cycle The availability of oxygen to the biomass can be controlled well The availability of the nutrients to the microorganism can be controlled within relatively narrow limits through the feeding of nutrients pH control is easy Possible to use bioreactors that provide a low-shear environment, such as bubble columns or air-lift bioreactors Based on Pandey, A., Selvakumar, P., Soccol, C.R., Singh, P., 1999. Solid state fermentation for the production of industrial enzymes. Curr. Sci. 77(1), 149-162; Pandey, A., 1992. Recent process developments in solid-state fermentation. Process Biochem. 27(2), 109–117; and Soccol, R.C., Ferreira da Costa, E.S., Letti, L.A.J., Karp, S.G., Woiciechowski, A.L., Porto de Souza Vandenberghe, L., 2017. Recent Developments and Innovations in Solid State Fermentation, http://www.journals.elsevier.com/biotechnology-research-and-innovation/.



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TABLE 5.6  Disadvantages of SSF. Temperatures can rise to values that are above the optimum for growth because of the insufficient removal of waste metabolic heat Fungal hyphae are exposed to an air phase, which can desiccate them Oxygen is freely available at the surface of the particle, but there may be severe restrictions in the supply of oxygen to a significant proportion of the biomass that is within a biofilm at the surface or penetrating into the particle The availability of nutrients to the microorganism may be poor, even when the average nutrient concentration within the substrate particle is high Concentration gradients of nutrients are large within the particles; movement of the particles of the solid substrate can cause impact and shear damage Hyphae can suffer damage in fungal processes pH control is difficult under some circumstances Based on Pandey, A., Selvakumar, P., Soccol, C.R., Singh, P., 1999. Solid state fermentation for the production of industrial enzymes. Curr. Sci. 77(1), 149-162; Pandey, A., 1992. Recent process developments in solid-state fermentation. Process Biochem. 27(2), 109–117; and Soccol, R.C., Ferreira da Costa, E.S., Letti, L.A.J., Karp, S.G., Woiciechowski, A.L., Porto de Souza Vandenberghe, L., 2017. Recent Developments and Innovations in Solid State Fermentation, http://www.journals.elsevier.com/biotechnology-research-and-innovation/.

thermal conductivity, heat removal will be slow. When the heat produced goes beyond certain level, product denaturation results and affects the growth of microorganism, finally ending up in reduction in yield and the product quality. But there are certain examples in which, despite being more problematic, SSF may be advantageous (Pandey et al., 1999; Pandey, 2001): • When the product requires to be in a solid form (e.g., in the case of fermented foods). • When a certain product is produced under the conditions of SSF or, if produced in both SLF and SSF, is produced in much higher amount in SSF. For instance, certain enzymes get induced in SSF, and some fungi only sporulate when grown in SSF, in which the hyphae are exposed directly to an air phase. Monascus pigment and many fungal spores are produced in much higher yields in SSF. • If genetically unmodified organisms are used in a process for the production of such a product, then SSF may be the only option. • When the fermentation process is carried out by unskilled workers. Some SSF processes can be relatively resistant to being overtaken by contaminants. • When the product is produced in both SSF and SLF, the product produced in SSF has desirable properties which the product produced in SLF lacks. For instance, spore-based fungal biopesticides produced in SSF processes are generally more resistant to adverse conditions as compared with those produced in SLF and so more effective when spread in the field. • When it is crucial to use a solid waste for avoiding the environmental impacts that would be caused by its direct disposal. This is likely to become an important consideration as the ever increasing population puts an increasing pressure on the environment. Few examples of traditional SSF processes are presented in Table 5.7. Products produced by SSF technology are listed in Table 5.8.

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TABLE 5.7  Traditional SSF processes. Tempe involves growing the fungus Rhizopus oligosporus on cooked soybeans The koji step of soy sauce manufacture involves growing the fungus Aspergillus oryzae on cooked soybeans “Ang-kak” or “red rice,” which involves growing the fungus Monascus purpureus on cooked rice Based on Pandey, A., Selvakumar, P., Soccol, C.R., Singh, P., 1999. Solid state fermentation for the production of industrial enzymes. Curr. Sci. 77(1), 149-162; Pandey, A., 1992. Recent process developments in solid-state fermentation. Process Biochem. 27(2), 109–117; and Soccol, R.C., Ferreira da Costa, E.S., Letti, L.A.J., Karp, S.G., Woiciechowski, A.L., Porto de Souza Vandenberghe, L., 2017. Recent Developments and Innovations in Solid State Fermentation, http://www.journals.elsevier.com/biotechnology-research-and-innovation/.

TABLE 5.8  Products produced by SSF technology. Enzymes—amylases, proteases, lipases, pectinases, tannases, cellulases, rennet, etc. Pigments Aromas and flavor compounds Organics such as ethanol, oxalic acid, citric acid, and lactic acid Gibberellic acid Protein-enriched agricultural residues for use as animal feeds Animal feeds with reduced levels of toxins or with improved digestibility Antibiotics, such as penicillin and oxytetracycline Biological control agents, including bioinsecticides and bioherbicides Spore inocula—spore inoculum of Penicillium roqueforti for blue cheese production Based on Pandey, A., Selvakumar, P., Soccol, C.R., Singh, P., 1999. Solid state fermentation for the production of industrial enzymes. Curr. Sci. 77(1), 149-162; Pandey, A., 1992. Recent process developments in solid-state fermentation. Process Biochem. 27(2), 109–117; and Soccol, R.C., Ferreira da Costa, E.S., Letti, L.A.J., Karp, S.G., Woiciechowski, A.L., Porto de Souza Vandenberghe, L., 2017. Recent Developments and Innovations in Solid State Fermentation, http://www.journals.elsevier.com/biotechnology-research-and-innovation/.

Bacteria, yeast, and fungi are able to grow on solid substrates and are used in SSF processes. Bacterial SSF fermentations are not used much for large-scale production of enzyme, but are very important in the fermented food industry. Filamentous fungi are best adapted for SSF and used all over the world. Filamentous fungi are important group of microorganisms for enzyme production in SSF. The hyphal mode of growth gives an advantage to filamentous fungi over unicellular microbes in the colonization of solid substrates and the consumption of available nutrients. The filamentous fungi can penetrate solid substrates. Hydrolytic enzymes are excreted at the hyphal tip, without large dilution. This makes the action of hydrolytic enzymes very efficient and allows penetration into most solid substrates. This is quite important for the growth of the fungi. Fungi cannot transport macromolecular substrates across the cell wall, so these molecules must be hydrolyzed externally into soluble units, which can be transported into the cell. Solid-state fermenters are of the six types (Table 5.9): “The simplest type of SSF reactor is the tray bioreactor. In this bioreactor a thin layer of substrate is spread over a large horizontal area. There is no forced aeration, although the base of the tray may be perforated and air forced around the tray. If any mixing is needed, it is



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TABLE 5.9  Types of solid-state fermenters. Tray bioreactor Packed bed bioreactor Rotary drum bioreactor Swing solid-state bioreactor Stirred vessel bioreactor Air-solid fluidized bed bioreactor Based on Pandey, A., Selvakumar, P., Soccol, C.R., Singh, P., 1999. Solid state fermentation for the production of industrial enzymes. Curr. Sci. 77(1), 149-162; Pandey, A., 1992. Recent process developments in solid-state fermentation. Process Biochem. 27(2), 109–117; and Soccol, R.C., Ferreira da Costa, E.S., Letti, L.A.J., Karp, S.G., Woiciechowski, A.L., Porto de Souza Vandenberghe, L., 2017. Recent Developments and Innovations in Solid State Fermentation, http://www.journals.elsevier.com/biotechnology-research-and-innovation/.

by simple automatic devices or manual. Internal temperature may vary with ambient temperature; or the tray may be placed in a room where temperature is controlled. Tray bioreactors have been used successfully at laboratory, pilot, semicommercial and commercial scale” (Soccol et al., 2017). As with liquid cultures, SSF requires sterile technique. To inoculate a solid carrier, a small volume of liquid culture is added to the solid material. This is followed by extensive mixing. The carrier is then incubated in controlled environmental conditions of temperature and humidity until it is ready for harvesting. This process appears simple, but there is actually an abundance of variables to select when developing a successful SSF process. The rate of inoculation, moisture level in the carrier, growth conditions, and fermentation time are all major parameters influencing final titer and product formation. The selection of solid substrate is also a huge factor that can make or break SSF. The nutrient profile of the carrier is important for growth. Physical parameters such as porosity and water absorption should be considered. This list of variables only scratches the surface of what goes into SSF and does not even include factors that affect how easily cells can be harvested. Because of the complexity involved in designing and optimizing SSF, it is often not used at the laboratory scale. However, it can be an efficient way of producing products on an industrial scale (bitesizebio.com). 5.1.1.3.1  Factors affecting enzyme production in SSF systems

The factors affecting microbial synthesis of enzymes in a SSF system are presented in Table 5.10. “Recent trends on SSF is focusing on application of SSF for the development of bioprocesses for instance bioremediation and biodegradation of hazardous compounds, biological treatment of agroindustrial residues, biotransformation of crops and crop residues for nutritional enrichment, biopulping, production of value-added products like biologically active secondary metabolites, including antibiotics, alkaloids, plant growth factors, enzymes, organic acids, biopesticides, including mycopesticides and bioherbicides, biosurfactants, ­ ­biofuel, aroma compounds, etc.” (Soccol et al., 2017). “SSF systems, during the last two decades were termed as a ‘low-technology’ system appear to be a promising ones for the production of value-added ‘low volume–high cost’

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TABLE 5.10  Major factors affecting microbial synthesis of enzymes in a SSF system. Selection of a suitable substrate and microorganism Pretreatment of the substrate Relative humidity Particle size of the substrate Water content and aw of the substrate Type and size of the inoculum Control of temperature of fermenting matter Period of cultivation Maintenance of uniformity in SSF system Oxygen consumption rate and carbon dioxide evolution rate Based on Pandey, A., Selvakumar, P., Soccol, C.R., Singh, P., 1999. Solid state fermentation for the production of industrial enzymes. Curr. Sci. 77(1), 149-162; Pandey, A., 1992. Recent process developments in solid-state fermentation. Process Biochem. 27(2), 109–117; and Soccol, R.C., Ferreira da Costa, E.S., Letti, L.A.J., Karp, S.G., Woiciechowski, A.L., Porto de Souza Vandenberghe, L., 2017. Recent Developments and Innovations in Solid State Fermentation, http://www.journals.elsevier.com/biotechnology-research-and-innovation/.

products such as biopharmaceuticals. SSF processes offer several advantages in bioremediation and biological detoxification of hazardous and toxic compounds” (Soccol et al., 2017; Pandey, 2001). Both submerged and SSF are successful techniques for producing several valuable products. There are several reasons as to why SSF is the way forward. One of these is the current economic situation that immediately means that cost efficiency is a major element in the future. SSF offers higher energy efficiency and a lower water consumption as compared with submerged fermentation. Another reason that SSF is important is because of the higher awareness of environment protection. As SSF has a reduced energy consumption, it has less ill effects on the environment. It also has a lower production of effluent, which so reduces the risk of environmental pollution (www.ukessays.com).

5.2  Enzymatic processes Enzymatic processes show potential for applications in industrial biocatalysis, biosensing, and biomedical engineering (Bajpai, 2018; Binod et al., 2013; van Beilen and Li, 2002; Godfrey and West, 1996; Panke and Wubbolts, 2002; OECD, 1998; Adrio and Demain, 2014; Schafer et  al., 2002; Schmid et  al., 2001; Leisola et  al., 2002; Gurung et  al., 2013; Novozymes, 2011; Choi et al., 2015; Singh, 2016). These reactions are the basis of the metabolism of all living organisms and provide significant opportunities for industry for carrying out efficient and economical biocatalytic conversions (Kirk et al., 2002). “Enzyme catalyzed processes are gradually replacing chemical processes in many areas of industry. Several chemical transformation processes used in several industries have inherent disadvantages from an environmental and commercial angle. The reactions which are nonspecific may result in poor product yields. High temperatures and or high pressures required



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to drive reactions result in high energy costs and may need large volumes of cooling water downstream. Harsh and hazardous processes involving high pressures, high temperatures, acidity, or alkalinity need high capital investment, and specially designed equipment and control systems. Undesirable by-products may prove costly or difficult to dispose of. High chemicals and energy consumption and also harmful by-products have an adverse impact on the environment. In several cases, most of these drawbacks can be almost eliminated by the use of enzymes. Enzyme reactions are usually conducted under mild conditions, they are highly specific, and the reaction rates are high. Industrial enzymes are obtained from biological systems; they contribute to sustainable development as they are isolated from microorganisms which are fermented using primarily renewable resources” (Bajpai, 2018). “The developments in the area of biotechnology, particularly genetics and protein engineering, has opened a new era of enzyme applications in several industrial processes and is experiencing major research and development initiatives. This has, resulted not only in the development of several new products but also improvement in the process and performance of several existing processes” (Bajpai, 2018). In the presence of an enzyme, a chemical reaction occurs at a much higher rate, but the enzyme is not consumed by the reaction. Their ability to perform very specific chemical transformations has made them increasingly popular in industries where less specific chemical processes produce undesirable by-products. Purity and predictability are of special importance in manufacturing food where by-products may be harmful or affect flavor, and due to their specificity, pharmaceutical companies favor biotransformations in the development of novel therapeutic agents. In addition, enzymes are chiral catalysts, which mean that they can be used to produce optically active, homochiral compounds of a kind that are usually difficult in making use of traditional organic chemistry. In the recent years, a greater awareness of conservation issues are forcing polluting industries to consider alternative, cleaner methods, so there is now substantial growth of biotechnology outside of the pharmaceutical and food industries. Enzymes have many advantages for use in industrial processes (Table 5.11). One of the most important properties of enzymes that makes them so important as diagnostic and research tools is the specificity. A few enzymes show absolute specificity; that is, they will catalyze only a particular type of reaction. Other enzymes will be specific for a particular type of chemical bond or functional group. Generally, there are four distinct types of specificity (Table 5.12).

TABLE 5.11  Advantages of using enzymes. 1. They are of natural origin and are nontoxic 2. They have great specificity of action and hence can bring about reactions not otherwise easily carried out 3. They work best under mild conditions of moderate temperature and near neutral pH, thus not requiring drastic conditions of high temperature, high pressure, and high acidity/alkaline, which necessitate special expensive equipment 4. They act rapidly at relatively low concentrations, and the rate of reaction can be readily controlled by adjusting temperature, pH, and amount of enzyme employed 5. They are easily inactivated when reaction completed as far as desired Based on Novozymes, 2011. Enzymes At Work, http://www.novozymes.com/en/about-us/brochures/Documents.

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TABLE 5.12  Specificity of enzymes. Absolute specificity—enzyme will catalyze only one reaction Group specificity—enzyme will act only on molecules that have specific functional groups, such as amino, phosphate, and methyl groups Linkage specificity—enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure Stereochemical specificity—enzyme will act on a particular steric or optical isomer Based on Bajpai, P., 2018. Industrial Enzymes—An Update, first ed. Bookboon. ISBN: 978-87-403-2129-6, p. 118.

Because of these advantages, several industries are very much interested in adapting enzymatic methods to the requirements of their processes. Enzymes are useful in various areas of applications like textile industry, pulp and paper industry, starch industry, detergent industry, leather industry, food and feed industry, fine chemicals, pharmaceuticals, chiral substances, and biofuel production (Kirk et  al., 2002). The use of enzymes in animal nutrition is very important. This area is growing, particularly for pig and poultry nutrition. Feed enzymes offer the advantage of degrading specific feed components, which are found harmful or of no value to the livestock. In cosmetic products, it is used for skin peeling, and future applications may be skin protection. Notable medications of enzymes are digestive aids, for wound cleaning, lysis of vein thromboses, acute therapy of myocardial infarction, and as support in the therapy of certain types of leukemia. Enzymes can be also used in chemical analysis and as a research tool in the life sciences. Table 5.13 presents a small selection of enzyme types currently used in industrial processes listed according to class. The classes are defined in Table 5.14. • Laccase enzyme is used in a chlorine-free denim bleaching process, which also enables a new fashion look. • Glucosyltransferase enzyme is used in the food industry for the production of functional sweeteners. • Hydrolases are the most widely used class of industrial enzymes. Several applications are described in later sections of the book. • An alpha-acetolactate decarboxylase is used for reducing the maturation period after the fermentation process in beer brewing. • Glucose isomerase enzyme is used to convert glucose into fructose, which increases the sweetness of the syrup. Matching an enzyme with a process is the greatest challenge to a research and development program. Often, an industrial plant can be modified to accommodate the limitations of an enzyme, but this is quite costly, and the best approach is to find an enzyme more suited to the existing process. Increasingly, new organisms are being found living in unusual environments, and these are proving an excellent source of novel enzymes. Living organisms are now generally divided into three groups: the eukaryotes, the bacteria, and the archaebacteria. The eukaryotes, which include all animals and plants, are limited in their ability to withstand hostile conditions such as extreme ranges of temperature or pH. Some worms that can live above 60°C have been found living around deep ocean volcanic vents, but these are exceptional. Bacteria and archaebacteria are not so constrained and can thrive in quite unbelievable



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TABLE 5.13  A selection of enzyme types used in industrial processes. EC 1: Oxidoreductases Catalases Glucose oxidases Laccases EC 2: Transferases Glucosyltransferases EC 3: Hydrolases Amylases Cellulases Lipases Mannanases Pectinases Phytases Proteases Pullulanases Xylanase EC 4: Lyases Pectate lyases Alpha-acetolactate decarboxylases EC 5: Isomerases Glucose isomerases Epimerases Mutases Lyases Topoisomerases EC 6: Ligases Argininosuccinate Glutathione synthase Based on Webb (1992).

conditions, from freezing to boiling water and from an acidic pH 2 to alkaline pH 12. Archaea Pyrococcus furiosa grows optimally at around 113°C and finds it too cold if temperatures fall to 100°C. These are the organisms of the future in biotechnology. Many industrial processes are designed to run at high temperatures where chemical reactions are faster and viscosity is reduced. By using enzymes with optimal activities at these temperatures, changes to existing industrial plants can be reduced. Moreover, problems with contamination are reduced, and less cooling is required where the reactions are exothermic. Enzymes contribute to clean industrial products and processes. They show several advantages over chemicals. Enzymes can be produced from renewable resources and are in turn degraded by microbes in nature. Various industries have replaced old processes using chemicals that cause detrimental effects on the environment and equipment with new processes that use biodegradable enzymes under less corrosive conditions. Currently, industrial enzymes are manufactured by three major suppliers, Novozymes A/S (headquartered in Denmark), Genencor International Inc. (headquartered in the United States), and DSM N.V (headquartered in the Netherlands).

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TABLE 5.14  Enzyme classes and types of reactions. Enzyme commission number

Class of enzyme

Reaction profile

EC 1

Oxidoreductases

These enzymes catalyze redox reactions, that is, reactions involving the transfer of electrons from one molecule to another. In biological systems, we often see the removal of hydrogen atoms from a substrate. Typical enzymes catalyzing such reactions are called dehydrogenases For example, alcohol dehydrogenase catalyzes reactions of the type R-CH2OH + A → R-CHO + AH2, where A is a hydrogen acceptor molecule. Other examples of oxidoreductases are oxidases and laccases, both catalyzing the oxidation of various substrates by dioxygen, and peroxidases, catalyzing oxidations by hydrogen peroxide. Catalases are a special type, catalyzing the disproportionation reaction 2H2O2 → O2 + 2H2O, whereby hydrogen peroxide is both oxidized and reduced at the same time

EC 2

Transferases

Enzymes in this class catalyze the transfer of groups of atoms from one molecule to another or from one position in a molecule to other positions in the same molecule. Common types are acyltransferases and glycosyltransferases. Cyclodextrin glycosyltransferase (CGTase) is one such enzyme type, which moves glucose residues within polysaccharide chains in a reaction that forms cyclic glucose oligomers (cyclodextrins)

EC 3

Hydrolases

Hydrolases catalyze hydrolysis, the cleavage of substrates by water. The reactions include the cleavage of peptide bonds in proteins by proteases, glycosidic bonds in carbohydrates by a variety of carbohydrases and ester bonds in lipids by lipases. In general, larger molecules are broken down to smaller fragments by hydrolases

EC 4

Lyases

Lyases catalyze the addition of groups to double bonds or the formation of double bonds though the removal of groups. Thus, bonds are cleaved by a mechanism different from hydrolysis. Pectate lyases, for example, split the glycosidic linkages in pectin in an elimination reaction leaving a glucuronic acid residue with a double bond

EC 5

Isomerases

Isomerases catalyze rearrangements of atoms within the same molecule; for example, glucose isomerase will convert glucose to fructose

EC 6

Ligases

Ligases join molecules together with covalent bonds in biosynthetic reactions. Such reactions require the input of energy by the concurrent hydrolysis of a diphosphate bond in ATP, a fact that makes this kind of enzyme difficult to apply commercially

Based on Webb (1992) and Novozymes, 2011. Enzymes At Work, http://www.novozymes.com/en/about-us/brochures/Documents.

Presently, almost 4000 enzymes are known, and of these, approximately 200 microbial original types are used commercially. However, only about 20 enzymes are produced on truly industrial scale (Li et al., 2012). With the improved understanding of the enzyme production biochemistry, fermentation processes, and recovery methods, an increasing number of industrial enzymes can be foreseeable. The world enzyme demand is satisfied by about 12 major producers and 400 minor suppliers. Nearly 75% of the total enzymes are produced by three top enzyme companies, that is, Denmark-based Novozymes, US-based DuPont (through the May 2011 acquisition of Denmark-based Danisco), and Switzerland-based Roche. The market is highly competitive, has small profit margins, and is technologically intensive.

References 55

The total market for industrial enzymes reached $3.3 billion in 2010 (BBC Research, 2011; Global Industrial Enzymes Market Research News, 2011; Sarrouh et al., 2012). Of these, industrial enzymes are typically used as bulk enzymes in the detergent, textile, and pulp and paper industries and in the biofuels industry, among others. Usage for leather and bioethanol is responsible for the highest sales figures. Technical enzymes had revenues of nearly $1.2 billion in 2011. The highest sales are expected to be in the biofuel (bioethanol) market (Freedonia Group, 2011).

References Adrio, J.L., Demain, A.L., 2014. Microbial enzymes: tools for biotechnological processes. Biomolecules 4, 117–139. https://doi.org/10.3390/biom4010117. Ali, S., Rafique, A., Ahmed, M., Sakandar, S., 2018. Different types of industrial fermentors and their associated operations for the mass production of metabolites. Eur. J. Pharm. Med. Res. 5 (5), 109–119. Arora, S., Rani, R., Ghosh, S., 2018. Bioreactors in solid state fermentation technology: design, applications and engineering aspects. J. Biotechnol. 269, 16–34. Bajpai, P., 2018. Industrial Enzymes—An Update, first ed. Bookboon. ISBN: 978-87-403-2129-6118. Barrios-Gonzáles, J., 2012. Solid-state fermentation: physiology of solid medium, its molecular basis and applications. Process Biochem. 47, 175–185. BBC Research Report BIO030 F, 2011. Enzymes in Industrial Applications: Global Markets. BBC Research, Wellesley, MA, USA. Bhargav, S., Panda, B.P., Ali, M., Javed, S., 2008. Solid-state fermentation: an overview. Chem. Biochem. Eng. Q. 22, 49–70. Binod, P., Palkhiwala, P., Gaikaiwari, R., Namppthiri, K.M., Duggal, A., Dey, K., Pandey, A., 2013. Industrial enzymes—present status and future perspectives for India. J. Sci. Ind. Res. 72, 271–286. Chisti, Y., 1999. Solid substrate fermentations, enzyme production, food enrichment. In: Flickinger, M.C., Drew, S.W. (Eds.), Encyclopedia of Bioprocess Technology—Fermentation, Biocatalysis and Bioseparation. Wiley & Sons, pp. 2446–2460. Choi, J.M., Han, S.S., Kim, H.S., 2015. Industrial applications of enzyme biocatalysis: current status and future aspect. Biotechnol. Adv. 33, 1443–1454. Chundakkadu Krishna, 2005. Solid-state fermentation systems—an overview. Crit. Rev. Biotechnol. 25 (1–2), 1–30. Erickson, L.E., 2011. Bioreactors for commodity products. In: Moo-Young, M., Butler, M., Webb, C., et  al. (Eds.), Comprehensive Biotechnology, second ed. Elsevier BV, Amsterdam, pp. 179–197. Freedonia Group, 2011. World Enzymes. Freedonia Group, Cleveland, OH, USA. Frost, G.M., Moss, D.A., 1987. In: Kennedy, J.F. (Ed.), Biotechnology. vol. 79. Verlag Chemie, Weinheim, pp. 65–211. Garcia-Ochoa, F., Santos, V.E., Gomez, E., 2011. Stirred tank bioreactors. In: Moo-Young, M., Butler, M., Webb, C., et al. (Eds.), Comprehensive Biotechnology, second ed. Elsevier BV, Amsterdam, pp. 179–197. Global Industrial Enzymes Market Research News, 2011. Report—Global Industrial Enzymes Market: An Analysis. Godfrey, T., West, S., 1996. Industrial Enzymology. Macmillan Press Ltd, London. Guieysse, B., Quijano, G., Munoz, R., 2011. Airlift bioreactors. In: Moo-Young, M., Butler, M., Webb, C., et al. (Eds.), Comprehensive Biotechnology, second ed. Elsevier B.V, Amsterdam, pp. 199–212. Gurung, N., Ray, S., Bose, S., Rai, V., 2013. A broader view: microbial enzymes and their relevance in industries, medicine, and beyond. Biomed. Res. Int. 329121. https://doi.org/10.1155/2013/329121. Holker, U., Lenz, J., 2005. Solid-state fermentation—are there any biotechnological advantages? Curr. Opin. Microbiol. 8, 301–306. Hölker, U., Hofer, M., Lenz, J., 2004. Biotechnological advantages of laboratory-scale solid-state fermentation with fungi. Appl. Microbiol. Biotechnol. 64, 175–186. https://doi.org/10.1007/s00253-003-1504-3. Kirk, O., Borchert, T.V., Fuglsang, C.C., 2002. Industrial enzyme applications. Curr. Opin. Biotechnol. 13, 345. Leisola, M., Jokela, J., Pastinen, O., Turunen, O., Schoemaker, H., 2002. Industrial use of enzymes. In: Hänninen, O.O.P., Atalay, M. (Eds.), Encyclopedia of Life Support Systems (EOLSS). EOLSS, Oxford, UK, pp. 1–25. Li, S., Yang, X., Yang, S., Zhu, M., Wang, X., 2012. Technology prospecting on enzymes: application, marketing and engineering. Comput. Struct. Biotechnol. J. 2, e201209017. Najafpour, G., 2007. Biochemical Engineering and Biotechnology, first ed. Elsevier. Novozymes, 2011. Enzymes At Work. http://www.novozymes.com/en/about-us/brochures/Documents. OECD, 1998. Biotechnology for Clean Industrial Products and Processes. OECD, Paris, France.

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Pandey, A., 2001. Solid-State Fermentation in Biotechnology: Fundamentals and Applications. Asiatech Publishers. Pandey, A., Selvakumar, P., Soccol, C.R., Singh, P., 1999. Solid state fermentation for the production of industrial enzymes. Curr. Sci. 77 (1), 149–162. Panke, S., Wubbolts, M.G., 2002. Enzyme technology and bioprocess engineering. Curr. Opin. Biotechnol. 2002 (13), 111–116. Paulová, L., Patáková, P., Brányik, T., 2013. Advanced fermentation processes. In: Teixeira, J., Vincente, A.A. (Eds.), Engineering Aspects of Food Biotechnology Chapter: Advanced Fermentation Processes. Taylor & Francis Group Editors, pp. 89–110. https://doi.org/10.1201/b15426-6. Raimbault, M., 1998. General and microbiological aspects of solid substrate fermentation. Electron. J. Biotechnol. 1, 174–188. Sarrouh, B., Santos, T.M., Miyoshi, A., Dias, R., Azevedo, V., 2012. Up-to-date insight on industrial enzymes applications and global market. J. Bioprocess Biotech. S4, 002. https://doi.org/10.4172/2155-9821.S4-002. Sato, K., Sudo, S., 1999. Small-scale solid-state fermentations. Manual Ind. Microbiol. Biotechnol. 2, 61–63. Schafer, T., Kirk, O., Borchert, T.V., Fuglsang, C.C., Pedersen, S., Salmon, S., Olsen, H.S., Deinhammer, R., Lund, H., 2002. Enzymes for technical applications. In: Fahnestock, S.R., Steinbüchel, S.R. (Eds.), Biopolymers. Wiley-VCH, Weinheim, Germany, pp. 377–437. Schmid, A., Dordick, J.S., Hauer, B., Kiener, A., Wubbolts, M., Witholt, B., 2001. Industrial biocatalysis today and tomorrow. Nature 409, 258–268. Singh, R.S., 2014. Industrial biotechnology: an overview. In: Singh, R.S., Pandey, A., Larroche, C. (Eds.), Advances in Industrial Biotechnology. IK International Publishing House Pvt. Ltd, India, pp. 1–35. Singh, R., Kumar, M., Mittal, A., Kumar, P., 2016. Microbial enzymes: industrial progress in 21st century. 3. Biotech 6, 1–15. Singhania, R.R., Patel, A.K., Soccol, C.R., Pandey, A., 2009. Recent advances in solid-state fermentation. Biochem. Eng. J. 44, 13–18. Soccol, R.C., Ferreira da Costa, E.S., LAJ, L., Karp, S.G., Woiciechowski, A.L., Porto de Souza Vandenberghe, L., 2017. Recent Developments and Innovations in Solid State Fermentation. http://www.journals.elsevier.com/ biotechnology-research-and-innovation/. Stanbury, P.F., Whitaker, A., 1995. Principles of Fermentation Technology, second ed. Press, Pergamon. Subramaniyam, R., Vimala, R., 2012. Solid state and submerged fermentation for the production of bioactive substances: a comparative study. Int. J. Sci. Nat. 3, 480–486. Thomas, L., Larroche, C., Pandey, A., 2013. Current developments in solid-state fermentation. Biochem. Eng. J. 81, 146–161. van Beilen, J.B., Li, Z., 2002. Enzyme technology: an overview. Curr. Opin. Biotechnol. 13, 338–344. Webb, E.C., 1992. Enzyme nomenclature. Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the Nomenclature and Classification of Enzymes, sixth ed. Academic Press, San Diego, CA, 863 pp. Zimmerman, W.B., Hewakandamby, B.N., Tesar, V., Bandulasena, H.C.H., Omotowa, O.A., 2009. On the design and simulation of an airlift loop bioreactor with microbubble generation by fluidic oscillation. Food Bioprod. Process. 87 (3), 215–227. https://doi.org/10.1016/j.fbp.2009.03.006.

Relevant websites www.ukessays.com. bitesizebio.com. ecoursesonline.icar.gov.in. www.biocon.com. www.kisspng.com/free/fluidized-bed-reactor.html.

Further reading Pandey, A., 1992. Recent process developments in solid-state fermentation. Process Biochem. 27 (2), 109–117.

C H A P T E R

6 Application of biotechnology in chemical industry O U T L I N E 6.1 Bulk chemicals 6.1.1 Citric acid 6.1.2 Lactic acid 6.1.3 Propane-1,3-diol (1,3-PDO) 6.1.4 Amino acids 6.1.5 Lysine 6.1.6 l-Glutamic acid 6.2 Food additives and food supplements 6.2.1 Starch modification, production of sweetener, and glucose syrups 6.2.2 Baking 6.2.3 Dairy products 6.2.4 Brewing 6.2.5 Distilling potable alcohol

58 61 64 67 69 70 73 75 79 80 81 81 82

6.3 Feed additives 6.3.1 Phytase enzymes 6.3.2 Nonstarch polysaccharides (NSP) degrading enzymes 6.3.3 Proteases 6.3.4 Mode(s) of action of enzymes

82 83

6.4 Agrochemicals 6.4.1 Fertilizers 6.4.2 Crop protection

86 86 88

Biotechnology in the Chemical Industry https://doi.org/10.1016/B978-0-12-818402-8.00006-9

84 85 85

6.5 Biocolorants

96

6.6 Flavors and aroma compounds

105

6.7 Solvents

111

6.8 Speciality products 6.8.1 Fermentation 6.8.2 Enzymes

117 118 123

6.9 Personal care products 6.9.1 Superoxide dismutase 6.9.2 Peroxidase 6.9.3 Tyrosinase 6.9.4 Proteases 6.9.5 Lipases 6.9.6 Hyaluronidase

128 130 130 131 131 132 132

6.10 Soaps and detergents

133

6.11 Bioplastics and other biopolymers 138 6.11.1 Biobased polymers 138 6.11.2 Biodegradable polymers 138 6.12 Biofuels 6.12.1 Bioethanol 6.12.2 Other biofuels made by assistance from enzymes

143 143

6.13 Processing of oil and fats

150

57

149

© 2020 Elsevier Inc. All rights reserved.

58

6.  Application of biotechnology in chemical industry

6.14 Bioremediation

152

6.15 Bioprocessing of pulp and paper 6.15.1 Biodebarking 6.15.2 Biobleaching 6.15.3 Deinking 6.15.4 Production of dissolving pulp 6.15.5 Fiber modification 6.15.6 Removal of shives 6.15.7 Retting of flax fibers

159 160 160 161 162 162 163 163

6.15.8 Biological solutions to processing problems 163 6.16 Organic synthesis

166

6.17 Transgenic plants

168

References

171

Relevant websites

191

Further reading

192

6.1  Bulk chemicals Petrochemicals are derived from petroleum, a nonrenewable resource with finite amounts in the Earth, such as oil and natural gas. Petrochemicals will become costlier as oil becomes scarce. Greenhouse gases, which lead to global warming, are released when the petrochemicals are used. Use of biotechnology for producing chemicals would reduce our dependence on natural gas and oil. This would also result in reduced environmental impact of the chemical industry. The chemical industry has been using traditional biotechnological processes for several years. These processes include the production of enzymes, ethanol, organic acids, antibiotics, amino acids, and vitamins (Moo-Young, 1984; Poppe and Novak, 1992; Rehm et  al., 1993; Chisti, 1999; Flickinger and Drew, 1999; Herfried, 2000; Demain, 2000; Spier, 2000; Schmid, 2003). Traditional biotechnology is also extensively used in the production of fermented foods and treatment of waste (Nout, 1992; Moo-Young and Chisti, 1994; Jördening and Winter, 2004). Developments in the area of genetic engineering and other biotechnologies have considerably increased the application of biotechnology and deal with several constraints of biocatalysts of the pre-GMO era (Ranganathan, 1976; Liese et al., 2000; Schügerl and Bellqardt, 2000). Some chemicals, for example, citric acid, for many years are being manufactured on a commercial scale using biotechnological processes, as the chemical processes are complex and costly. Other noteworthy examples are described here, but there are several other processes still in the developmental stage, with chemicals being produced in small reactors on a small scale. Extensive research is being done for making these reactions more efficient and cost-effective. Fermentation processes are already being used for producing several important chemicals at very high volumes. Citric acid, lactic acid, 1,3-propanediol (1,3-PDO), and amino acids are the important chemicals produced by fermentation. As petrochemical feedstocks become more expensive, this range would increase. Table 6.1 shows potentially important bioprocessing systems for the production of commodity chemicals Table 6.2 shows product groups currently produced commercially using fermentation, and Table 6.3 shows products in developmental stage. (www.essentialchemicalindustry.org/.../biotechnology-in-the-chemical-industry.html) New bulk polymers such as biodegradable plastics and monomers (e.g., 1,3-PDO for novel polyester production) have also come onstream, and there is enormous scope for further ­development based on tailored enzymes and microorganisms. Looking to the future,



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TABLE 6.1  Important bioprocessing systems for the production of commodity chemicals. Clostridium thermocellum

Glucose, lactic acid

Ethanol, acetic acid

Clostridium thermosaccharolyticum

Lactic acid

Glucose, xylose, ethanol, acetic acid

Clostridium thermohydrosulfuricum

Glucose, xylose

Ethanol, acetic acid, lactic acid

Zymomonas mobilis

Glucose

Ethanol

Schizosaccharomyces pombe

Xylulose

Ethanol

Saccharomyces cerevisiae

Glucose

Ethanol

Saccharomyces cerevisiae

Glucose

Ethanol

Kluyveromyces lactis

Xylulose

Ethanol

Pachysolen tannophilus

Glucose, xylose

Ethanol

Thermobacteroidessaccharolyticum

Glucose, xylose

Ethanol

Thermoanaerobacter ethanolicus

Glucose, xylose

Ethanol, acetic acid, lactic acid

Aeromonas hydrophila

Xylose

Ethanol, 2,3-butanediol

Aspergillus

Glucose

Citric acid

Aerobacter aerogenes

Glucose

2,3-butanediol

Bacillus polymyxa

Glucose

2,3-butanediol

Dunaliella sp.

Carbon dioxide

Glycerol

Based on the Office of Technology Assessment (OTA), 1984. Commercial Biotechnology: An International Analysis. OTA-BA-218. U.S. Government Printing Office, Washington, DC.

TABLE 6.2  Product groups currently produced commercially using fermentation. Alcohols • Ethanol • Butanol • BDO • Acetone Amino acids • MSG • Lysine • Threonine • Tryptophan Organic acids • Citric • Lactic • Succinic Biogas • Methane

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6.  Application of biotechnology in chemical industry

TABLE 6.2  Product groups currently produced commercially using fermentation—cont’d Polymers • Xanthan • PHA Vitamins • Vitamin C • Vitamin B2 • Vitamin B12 Antibiotics • Beta-lactam • Tetracycline • Clavulanic acid Industrial enzymes • Amylase • Cellulase • Lipase • Protease Based on www2.deloitte.com/content/dam/Deloitte/nl/Documents/ manufacturing/deloitte-nl-manufacturing-opportunities-for-thefermentation-based-chemical-industry-2014.pdf

TABLE 6.3  Products in developmental stage. Alkanes • Nonane • Tetradecane Olefins • Butadiene • Isoprene • Propene • Farnesene Amines • Histamine • Tyramine Esters • Malonyl-ACP Dyes • Various dyes (e.g., indigo) Microbial oils • Biodiesel Based on Deloitte, 2014. Opportunities for the Fermentation-Based Chemical Industry. An Analysis of the Market Potential and Competitiveness of North-West Europe. Opportunities for the fermentation-based chemical industry: an analysis of the market potential and competitiveness of North-West Europe www2.deloitte.com/content/dam/ Deloitte/nl/Documents/manufacturing/deloitte-nl-manufacturing-opportunities-for-thefermentation-based-chemical-industry-2014.pdf



6.1  Bulk chemicals

61

­ iotechnology will allow the production of new polymers having better functionality that is b not possible by using the traditional processes. These could include improved liquid crystals and materials with excellent mechanical properties or temperature resistance. Chemical companies such as Monsanto and DuPont are using processes based on biotechnology and are generating substantial proportions of their income through biotechnology (Scheper, 1999; Bommarius, 2004). These companies were earlier using traditional methods exclusively using petrochemical-based products. Important bulk chemicals such as ethyl alcohol and cellulose esters are being already produced from renewable agricultural raw materials in the United States. New processes and renewable raw materials for other bulk chemicals that are presently obtained from petrochemical feedstocks are in the final stages of development. The examples are succinic acid and ethylene glycol. During 1990s, biotechnology used for cleaner production contributed about 60% of total sales for fine chemicals and 5%–11% for pharmaceuticals (OECD, 1989). Some well-established biotechnology products being manufactured in commercial scale are Bioethanol, Monosodium glutamate (MSG), Citric acid, Lysine, Lactic acid, Food processing enzymes, Vitamin C, Gluconic acid, Antibiotics, Feed enzymes, Xanthan, Threonine Hydroxyphenylalanine, 6-Aminopoenicillanic acid, Nicotinamide, d-p-hydroxyphenylglycine, Vitamin F, 7-Aminocephalosporinic acid, Aspartame, Methionine, Dextran 200, Vitamin B12, and Provitamin D2. These products have been around for a long time, but several are now being manufactured using engineered biocatalysts. Biocatalysis and metabolic engineering are the major areas of biotechnology which are driving transformation of the traditional chemical industry are biocatalysis and metabolic engineering (Poppe and Novak, 1992; Kim et al., 2000). Molecular biology and genetic engineering have been used for producing many modified enzymes with better properties in comparison to their natural counterparts. Metabolic engineering, or manipulation of metabolic pathways at molecular level is providing microorganisms and transgenic crops and animals with new and better abilities for producing chemicals. (Bruggink, 1996; Eriksson, 1997; Poppe and Novak, 1992; Kim et al., 2000 ur-www1.massey.ac.nz)

The following section discusses few important biotechnology products produced by fermentation.

6.1.1  Citric acid Citric acid is one of the most important natural organic acids.

Citric acid (2-hydroxy-propane-1,2,3-tricarboxylic acid)

Citric acid is largely being used in several industries. There is a worldwide demand for citric acid because of its applications in different industries (Sawant et al., 2018). “The name ‘citric acid’ is derived from the Latin word ‘Citrus’. It is obtained by metabolic pathways which are performed in living cells via tri-carboxylic acid cycle. Citric acid is non-toxic and have a sour taste. It is considered as a Generally Recognized As Safe (GRAS) compound. It is found in citrus fruits such as oranges, berries, limes, lemons, tangerines and grapes. Citric

62

6.  Application of biotechnology in chemical industry

TABLE 6.4  Uses of citric acid. Beverages

51%

Detergents and soap

16%

Food

18%

Pharmaceuticals and cosmetics

8%

Industrial uses

7%

acid is commonly used in food industry as a preservative, souring agent, antioxidant, emulsifier, etc. Because of these reasons, citric acid is used as a common ingredient in several food products and is in high demand all over the world for daily use” (www.jmbfs.org; Swain et al., 2012; Soccol et al., 2006; Radwan et al., 2010; Vasanthabharathi et al., 2013; Kapoor et al., 1983; Papagianni, 2007; Hamdy, 2013; Kareem and Rehman, 2011; Murad et al., 2003; Kumar et al., 2003). Table 6.4 shows uses of citric acid in different industries (Table 6.4). More than 70% of citric acid is used in food industry only, and the remaining 30% is used in the chemical, pharmaceutical, medical, and other industries (Soccol et al., 2003). Citric acid is consumed in food industry to a large extent because of its many beneficial properties (ElHussein et al., 2009; Yalcin et al., 2010). Citric acid is occupying a major position in the global market because of its application in several industries (Ali et al., 2011, 2016). Citric acid enhances the flavor and sourness. The acid extends the shelf life of the food products because it inhibits microbial growth. These factors are expected to increase the product demand in the next few years. The global citric acid market is expected to reach USD 3.83 billion by 2025, according to a study by Grand View Research, Inc. (www.grandviewresearch.com/press-release/global-citric-acid-market). High demand in food and beverage application is expected to drive the market in the next few years. High demand for citric acid for food preservation is expected to be a major driver for the industry growth. Furthermore, the growing need in pharmaceutical industry for digestive medicines is expected to positively drive the market in the next few years. The annual production of citric acid was 700 thousand tonnes in 1993, 1.4 million tonnes in 2004, 1.6 million tonnes in 2008, and 1.8 million tonnes in 2010 (Yalcin et al., 2010; Addo et al., 2016). The market in the United States was worth USD 448.4 million in 2016 (www.grandviewresearch.com/industry-analysis/citric-acid-market). In the food processing industry, citric acid is used for increasing the shelf life of the convenience foods. Well-established food processing industry in the United States is expected to have a positive effect on the industry trends. In North America, the regulations for the use of citric acid in nutraceuticals, nutricosmetics, and dietary supplements, particularly for infants, are strict. This is expected to create a challenge to regional manufacturers. Stagnation in terms of innovation in products for infant formulations is seen (www.grandviewresearch.com). Companies are increasing their production capacities for catering the increasing product demand. Jungbunzlauer, a major producer of biodegradable ingredients, is c­ onstructing a new citric acid plant in Austria (www.grandviewresearch.com/industry-analysis/citric-acid-market). There are several microorganisms with the ability to produce citric acid (Table 6.5). Aspergillus niger is an important producer of citric acid (Selvankumar et al., 2014; Singh et al., 2016; Alnassar et al., 2016; Ali et al., 2011). Commercial production of citric acid has been conducted using the fermentation process. The production is increased by optimizing the



6.1  Bulk chemicals

63

TABLE 6.5  Microorganisms with the ability to produce citric acid. Bacillus subtilis Bacillus licheniformis Corynebacterium spp. Aspergillus niger Aspergillus flavus Mucor piriformis Trichoderma viride Penicillium janthinellum Candida tropicalis Candida lipolytica Candida intermedia Based on Kapoor, K.K., Chaudhary, K., Tauro, P. 1983. Citric acid. In: Reed, G. (Ed.) Prescott and Dunn’s Industrial Microbiology. MacMillan Publishers Ltd., UK, pp. 709–747; Papagianni, M., 2007. Advances in citric acid fermentation by Aspergillus niger: biochemical aspects, membrane transport and modeling. Biotechnol. Adv. 25, 244–263.

environmental parameters. Genetic manipulation of microorganisms is also performed (Ali et al., 2016). Microorganisms are able to use inexpensive raw waste materials and produce value-added products (Singh et al., 2016). Microbial fermentation may be carried out by using the following methods (Gupta et al., 2015): (1) Submerged fermentation (2) Surface fermentation (3) Solid substrate fermentation Solid surface fermentation is a good alternative to conventional submerged fermentation as discussed in Chapter 5. In this process, microorganisms use inexpensive waste materials as substrates. The waste materials produced in food industries are utilized as substrates by microorganisms for the production of citric acid. In food, beverages, milk, and sugar processing industries, several biodegradable organic wastes are produced. These include the following (Hang and Woodams, 1984; Hang, 1998; Murad et al., 2003; Kumar et al., 2003; Hamdy, 2013; Kareem and Rehman, 2011): - Sugarcane bagasse - Grape pomace - Apple pomace - Pineapple pomace - Vegetables - Tapioca - Coconut husk - Banana peels - Citrus peels - Whey - Decaying fruits

64

6.  Application of biotechnology in chemical industry

Such nutrient-rich and inexpensive wastes can be used as substrates by microorganisms for the production of citric acid. The major production method involves using the fungus Aspergillus niger, which is grown in solutions of sucrose or glucose. The citric acid produced is precipitated with calcium hydroxide to form calcium citrate. This salt is filtered off, and the acid is regenerated with sulfuric acid.

6.1.2  Lactic acid

2-Hydroxypropanoic acid (lactic acid)

Lactic acid is an important platform chemical for producing polylactic acid (PLA) and other value-added products (Haffar et al., 2014). It is naturally produced by different types of microorganisms—bacteria, yeast, and filamentous fungi (Steinkraus, 1992). Generally, bacteria ferment C5 and C6 sugars to lactic acid by either homofermentative or heterofermentative mode. Xylose isomerase, phosphoketolase, transaldolase, and l- and d-lactate dehydrogenases are the major enzymes affecting lactic acid production. Genetic manipulation of microbial strains is usually required for producing lactic acid from unconventional carbon feedstocks. Lactic acid is produced by the fermentation of glucose. Several lactic acid bacteria (LAB) such as Lactobacillus fermentum, Lactobacillus buchneri, and Lactobacillus fructovorans produce a mixture of d- and l-lactic acid. Some lactic acid bacteria such as Lactobacillus bulgaricus, Lactobacillus coryniformis subsp. torquensy, and Leuconostoc mesenteroides subsp. mesenteroides produce highly pure d-lactic acid, and LAB such as Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus mali produce mainly l-lactic acid. The commercial production utilizes homolactic acid bacteria such as Lactobacillus delbrueckii, Lactobacillus bulgaricus, and Lactobacillus leichmannii (Badal, 2003). Several types of carbohydrate sources such as molasses, corn syrup, whey, glucose, and sucrose are used for the production of lactic acid. Lactic acid fermentation is a product-inhibited fermentation. The product recovery costs in downstream processing are quite high. Table 6.6 shows speciality foods produced by lactic acid fermentation. Batch fermentation process is generally used for the production of lactic acid (Vijayakumar et al., 2008). “Fermentation conditions are different for each industrial method but are generally in the range of 45–60°C having a pH of 5.0–6.5 for Lactobacillus delbrueckii and 43°C with a pH of 6.0–7.0 for Lactobacillus bulgaricus. The acid produced is neutralized by calcium hydroxide or calcium carbonate (Zhou et al., 2003). Fermentation takes 24–48 hours under optimal conditions. The lactic acid yield after the fermentation is 90–95wt% based on the initial ­substrate concentration. Fermentation rate depends on pH, temperature, initial substrate concentration and concentration of nitrogenous nutrients. There has been a considerable interest in solving the issues such as PLA weakens at high temperature with an objective for increasing the use of this renewable plastic. Hydrolysis reaction of methyl lactate is used for improving the performance of batch reactive distillation for producing lactic acid. LAB have complex nutrient requirements. Industrial wastes of food having high moisture content have been considered for commercial production of lactic acid. Product recovery is an important step in lactic acid production which is associated with separation and purification of lactic acid



6.1  Bulk chemicals

65

TABLE 6.6  Speciality foods produced by lactic acid fermentation. Western world Yogurt, sourdough breads, sauerkraut, cucumber pickles, and olives Middle East Pickled vegetables Korea Kimchi (fermented mixture of Chinese cabbage, radishes, red pepper, garlic, and ginger) Russia Kefir Egypt Laban rayab and laban zeer (fermented milks), kishk (fermented cereal and milk mixture) Nigeria Gari (fermented cassava) South Africa Magou (fermented maize porridge) Thailand Nham (fermented fresh pork) Philippines Balao-balao (fermented rice and shrimp mixture) Based on Steinkraus, K.H., 1992. Lactic Acid Fermentations. In: Applications of Biotechnology to Traditional Fermented Foods. National Research Council. National Academy Press, Washington, DC, pp. 43–51.

from fermentation broth. A traditional process for lactic acid production by lactose fermentation involves the purification steps which are important for obtaining the pure lactic acid. Alternative methods to this industrialized process are being studied. Several studies on lactic acid purification have been conducted by using several different methods for separation such as ion exchange, reactive extraction, membrane technology, distillation and electrodialysis” (Zhou et al., 2003;González et al., 2008; Chakkrit, 2010; Randhawa et al., 2012;Edreder et al., 2010; Omay and Guvenilir, 2012). LAB are the important microorganisms used in food fermentations (Jones et al., 2016). They contribute to the taste and texture of fermented products and inhibit the decaying of food by producing substances that inhibit the growth (Juturu and Wu, 2016). LAB are involved in the making of cheese, cultured butter, yogurt, sour cream, sausage, cucumber pickles, olives, and sauerkraut, but some species can spoil the taste of beer, wine, and processed meats. The production of lactic acid is around 350 000 ton per year and worldwide growth is expected to be 12– 15% per year. Hydrogenation of lactic acid into 1,3-PDO (a commodity chemical) is an industrially important reaction. 1,3-PDO finds several applications, mainly as a solvent for the production of polyester resins, drugs, cosmetics, food, de-icing fluid and antifreeze. Presently 1,3-PDO is synthesized by the hydration of propene oxide using Chromium based catalysts. This production route involves hydro peroxidation chemistry. This has several environmental problems because of the toxicity of Chromium catalysts. Lactic acid hydrogenation provides a viable green alternative for the synthesis of 1,3-PDO. (Jones et al., 2016; Wasewar et al., 2004; Corma et al., 2007; orca.cf.ac.uk)

66

6.  Application of biotechnology in chemical industry

TABLE 6.7  Uses of lactic acid. Polymers

39%

Food and beverages

35%

Personal care

13%

Solvents and industrial uses

13%

Table 6.7 shows uses of lactic acid. The lactic acid market is growing steadily and rapidly. The market capacity was 800,000 tons in 2013. The United States is the largest market in the world. It accounted for 31% of total lactic acid consumption in 2013; China surpassed Western Europe to become the second largest lactic acid market, with growth in consumption mainly benefiting from increasing demand in the food and beverage processing industry and also robust export demand. According to Grand View Research, Inc., the global lactic acid market size is expected to reach USD 8.77 billion by 2025 (www.grandviewresearch.com/.../global-lactic-acid-and-poly-lactic-acid-market). Increasing use of cosmetics and personal care products due to the development of advanced products coupled with increased focus on formulation enhancement for targeted consumer groups is expected to drive the market. The global lactic acid market was valued at approximately USD 2.9 billion in 2018 and is expected to generate around USD 10.06 billion by 2025 (https://globenewswire.com/.../ Global-Lactic-Acid-Market-Will-Reach-USD-10-06-Bil). The market is expected to be driven by growing product usage in the end-user industries, such as food, chemicals, cosmetics, and pharmaceuticals, in the coming years. The high product demand in the food industry as a preservative and antimicrobial agent is the primary growth driver expected to stimulate the growth of the lactic acid market worldwide. Lactic acid prevents the growth of bacteria and other microorganisms and helps to increase the shelf life of food items. The increasing manufacturing of lactate ester is expected to positively affect the global lactic acid market in the future. The world's top lactic acid manufacturers are PURAC, Cargill, and Henan Jindan Lactic Acid Technology Co. These three companies produced 505,000 tons in 2013. Cargill is mainly supplying lactic acid products to its subsidiary—NatureWorks for the production of PLA. The most important application of lactic acid is PLA. It is seeing incremental application in biomedical, automotive, electronics, and other areas since the smooth realization of industrial production in the 1990s. Extensive developmental efforts are being made in several countries. The leading PLA producers are located in the United States, the Netherlands, Germany, and other developed countries. Lactic acid is used for producing implants, dialysis solution, pills, controlled drug release systems, and surgical sutures in the pharmaceutical industry. The product is also used for producing cosmetics and hygiene products in the personal and oral care industry because of its rejuvenating, antimicrobial, and moisturizing properties. Lactic acid is also used in the production of biocompatible and biodegradable PLA polymers, oxygenated chemicals, and solvents (www.grandviewresearch.com). In the food industry, the lactic acid is used to regulate the pH and inhibit residual bacteria, in processing of several products including sweets, soft drinks, bread, and beer. Lactic acid is not only an important ingredient in fermented foods such as yogurt, canned vegetables, and



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butter but also used in pickled vegetables and olives as a preservative and as an acidulant (www.grandviewresearch.com). The consumption of pharmaceutical grade creams and other medicines is increasing among consumers. This is expected to augment the lactic acid market in next few years. Furthermore, the pharmaceutical industry acts as a major asset to the European economy as it is one of the top performing sectors of the region. So, the growth of the industry in Europe is expected to increase the demand for lactic acid.

6.1.3  Propane-1,3-diol (1,3-PDO) 1,3-PDO has the formula CH2(CH2OH)2. This three-carbon diol is a colorless viscous liquid that is miscible with water. Propane-1,3-diol

An important use of 1,3-PDO is in the production of the polyester, polytrimethylene terephthalate (PTT). PTT is produced by the condensation reaction between 1,3-PDO and ­benzene-1,4-dicarboxylic acid (often called terephthalic acid). Catalysts generally used for the reaction are titanium alkoxylates such as tetrabutyl titanate (IV) (www.essentialchemicalindustry.org/...applications/biotechnology-in-the-chemical-ind). The structure of PTT is very similar to the well-known polyester—polyethylene terephthalate (PET), which is produced from ethane-1,2-diol and benzene-1,4-dicarboxylic acid.

Polyethylene terephthalate (PET)

Polytrimethylene terephthalate (PTT)

But the extra methylene groups in PTT gives the polymer pronounced kinks in the chain and therefore different properties to PET. PTT has better stretch recovery properties to PET and can be easily dyed. It is used in the fiber form in textiles, clothes, and carpets and also as a thermoplastic in car parts and electrical and electronic systems. 1,3-PDO is also used in the production of cosmetics, laminates, adhesives, paints, and inks and also as a replacement for ethane-1,2-diol as an engine coolant and as a solvent (www. essentialchemicalindustry.org).

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Dupant is manufacturing biobased 1,3-PDO through a proprietary fermentation process using glucose from plants instead of petroleum feedstocks (www.duponttateandlyle.com/ our_process). From “cradle to gate” (extraction and production prior to delivery to customers), biobased 1,3-PDO produces 56% less GHG emissions and consumes 42% less nonrenewable energy than petroleum-based 1,3-PDO. Compared with propylene glycol (PEG), biobased 1,3-PDO produces 42% less GHG emissions and uses 38% less nonrenewable energy from cradle to gate. Compared with butanediol (BDO), biobased 1,3-PDO produces 52% less GHG emissions and uses 32% less nonrenewable energy from cradle to gate. 1,3-PDO is one of the oldest known fermentation products. It was identified in 1881 by August Freund, in a glycerol fermentation mixed culture containing Clostridium pasteurianum as an active organism. 1,3-PDO can be produced by fermentation from glycerol by several bacteria such as Klebsiella pneumoniae, Citrobacter freundii, and Clostridium pasteurianum (Saha, 2003a,b). “Efforts were made to produce 1,3-PDO from glucose by using two methods: fermentations of glucose to glycerol and glycerol to 1,3-PDO by using a two stage process with two different organisms and the genes responsible for converting glucose to glycerol and glycerol to 1,3-PDO can be combined in one organism” (www.kieferdistillery.us). It is a versatile intermediate compound used in the synthesis of heterocycles. Because of the presence of two hydroxyl groups at 1 and 3 positions, it is used in the production of polymers, such as polyesters and polyurethanes (Biebl et al., 1999). Polymers based on 1,3-PDO have better wash fastness. The production of PTT has caused a significant increase in the demand of 1,3-PDO because of its superior stretching and stretch recovery characteristics (Kurian, 2005). PPT is a biodegradable polymer, which finds application in textile, carpets, and upholstery manufacturing (Witt et al., 1994). 1,3-PDO was traditionally considered as a “specialty chemical” but is now undergoing a transition into a “commodity chemical” (Sheldon et  al., 2007). The technological breakthroughs in the preparation of 1,3-PDO have resulted in reduced market prices. The market for 1,3-PDO is growing ­rapidly (Kraus, 2008). The development of economic production processes for 1,3-PDO is a success story for the establishment of a new market for a bulk chemical. The compound and its favorable properties have been known for a long time; also the fermentation of glycerol to 1,3-PDO had been described more than 120 years ago. Nonetheless, the product remained a specialty chemical until recently, when two new processes were introduced, providing 1,3-PDO at a competitive price. One of the processes is in the field of white biotechnology based on microbial fermentation, converting a renewable carbon source into a bulk chemical. (www.eurekaselect.com)

1,3-PDO is produced from maize. The maize is cooked and then ground for releasing the starch. The starch is subjected to hydrolysis for producing glucose. This is fed to a genetically modified Escherichia coli, which ferments the glucose into 1,3-PDO. The global 1,3-PDO market is estimated to reach $621.2 million by 2021 (https://globenewswire.com/.../2015/.../Global-1-3-Propanediol-PDO-Market-2015-20) is highest in United States. The production and consumption of 1,3-PDO are highest in the United States. DuPont Tate and Lyle Bio Products Company, LLC a joint venture betweenDuPontandTateandLyle is the largest manufacturer of 1,3-PDO and has its plant at London, Tennessee, in the United States with a production capacity of 63,500 tons. Europe, Middle East, and Africa are expected to grow at a highest growth rate; this can be attributed to the increasing use of biobased materials in Europe.



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1,3-PDO is used to produce PTT, polyurethane, cosmetic, personal care and cleaning products, and others (engine coolants, deicing fluid, heat transfer fluid, unsaturated polyester resins, and others). PTT production represents the largest application in terms of volume consumed in 2013. More than 90.0% of the 1,3-PDO produced is used to produce PTT fibers. The application of 1,3-PDO is continuously growing. 1,3-PDO is also used in food and beverages, adhesives and sealants, paints and coatings, etc. The 1,3-PDO market is currently dominated by DuPont Tate and Lyle and is expected to see entry of new manufacturers in the near future. Few Chinese companies are currently producing 1,3-PDO in small amounts. The Asia-Pacific region is attracting new producers because of its domestic demand and reduced raw material and labor costs.

6.1.4  Amino acids Amino acids are the most important macromolecules for the functions of humans and animals. These acids contain both amino and carboxyl functional groups and are the building blocks of proteins. In most of the living organisms, 20 l-amino acids are generally found. Nine amino acids are important for human and animal nutrition, and L-lysine is one of them. Amino acids are organic compounds consisting of amine and carboxylic acid as functional groups. The main elements are carbon, hydrogen, oxygen, and nitrogen. They can be segmented as alpha, delta, beta, and gamma structurally. Amino acids are generally used in nutritional supplements, food technology, and fertilizers due to their biological importance. In industry, they are used for the production of biodegradable plastics, chiral catalysts, and drugs. The industrially produced amino acids are chiral, and the two isomers (d and l) have different properties in biologically induced reactions with the exception of aminoethanoic acid (glycine). However, chemical synthesis produces equimolar amounts of d- and l-forms, and additional expensive steps are needed for producing a pure stereoisomer. But with biotechnological processes, pure optically active amino acids can be obtained. Amino acids are produced from the fermentation of sugar to which small quantities of nitrogen-containing compounds (e.g., ammonia or urea) have been added. The global demand for amino acids is expected to see strong growth driven by consumption in major developing markets. More growth opportunities will turn up in the next few years as compared with the last few years, suggesting rapid pace of change (www.animalhealthmedia.com/amino-acids-market-reach-11-million-metric-tons-value). According to research and market report, the market is expected to reach a volume of 11 million tons in 2023 (www.researchandmarkets.com/reports/4514538/amino-acids-market-global-industry-trends). Animal feed is the largest and also the fastest, g ­ rowing ­application for amino acids globally. Asia-Pacific is the world’s largest market for amino acids, in terms of both volume and value. South America is projected to record the growth rates at par with Asia-Pacific, mainly driven by growth in the animal feed sector. Increasing health consciousness among the consumers has increased the demand for food products rich in nutrients. This has prompted the producers to introduce protein-rich foods and beverages in the market, resulting in an increase in the demand for amino acids. Amino acids are also used to produce supplements that are consumed by athletes and also the aged population. Amino acids are used in several applications and perform several functions in animals and humans. The technology is rapidly advancing, and the understanding of the functions and properties of amino acids is increasing. So, the commercial use of amino acids is also

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i­ncreasing. New production technology and large-scale production of amino acids are making it more economical and thus increasing its user base and usage rate. Animal feed and human food formed a major part of the amino acid market. The use of amino acids in feed and food applications is also expected to grow steadily because the population and the standard of living of people are increasing. Commercially, amino acids can be used in animal feed, as nutritional additives, and for flavoring. It is also used in cosmetics, medical, research, therapeutic, and industrial applications. Amino acids are being used as a nutritional protein for fortifying animal feeds and as food supplements for humans. It is also being used in medicine for synthesizing any number of polypeptides. Their uses as food additives and animal feed would continue to grow as there are no substitutes for amino acids. A few companies in this industry are Ajinomoto, RSP amino acids, Biaffin, AnaSpec, ChemPep Inc., IRIS Biotech, PepTech Corporation, and Synthetech. Ajinomoto is one of the leading producers of amino acids. This company is offering amino acids for research, product development, and process development (www.prweb.com/releases/2013/2/prweb10411583.htm). It has developed and used innovative processes for producing these acids. Major producers are focusing on research and development for developing new market segments of amino acids. The two acids produced on the largest scale are L-glutamic acid and L-lysine.

6.1.5 Lysine Lysine is an organic compound having a chemical formula C6H14N2O2. Lysine is biologically active in its L-stereoisomer form and is one of the essential amino acids. This is not synthesized biologically in our body. Lysine is generally used as a supplement in human food and animal feed. L-lysine is commercially produced as L-lysine monohydrochloride with purity higher than 98.5 wt.%, corresponding to 78.8 wt.% of free lysine (www.chemengonline.com). About 1 million tonnes of L-lysine is produced annually with China producing 35% (www.

L-lysine

essentialchemicalindustry.org/materials-and-applications/biotechnology-in-the-chemical-industry.html). It is an essential amino acid, meaning that most vertebrates cannot synthesize it (Anastassiadis, 2007). It is often lacking in livestock diets; therefore, L-lysine is mostly used in animal feed. Table 6.8 shows uses of L-lysine. Lysine is injected in meat for providing necessary nutrients. Use of lysine as a protein synthesizer in animal feed is a main application driving the overall demand. This compound is used by the cattle breeding industry for improving the protein content in chicken, egg, and beef. There has been a great demand for processed meat having high protein content. This is propelling the usage of lysine in animal husbandry and cattle breeding. The global lysine market is expected to reach USD 6.96 billion by 2020 (www.marketwatch.com/.../ lysine-market-to-witness-a-steady-growth-on-account-).



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TABLE 6.8  Uses of L-lysine. L-lysine is an essential amino acid required for the human nutrition It is used as supplementary for cereal proteins Protein quality of certain foods like wheat (based foods) is improved by addition of L-lysine that results in the improved growth and tissue synthesis It is used as a nutraceutical

Market demand of lysine is expected to see a continuous growth on account of its increasing use in the production of animal feed. Its use as an herbal supplement is also driving the market demand in the recent years. Busy lifestyle and increasing consumer awareness have created a trend of consumption of protein supplements. These supplements are known to improve muscle growth and play an important role in the absorption of calcium. Lysine plays an important role in diet for people recovering from injuries. Their intake for improving body enzymes and antibodies is expected to provide significant opportunities in the coming years. Government regulations and safety related to this organic compound offer a main challenge for the growth of industry. This compound is considered as GRAS by US Food and Drug Administration. There are no significant regulatory standards for the use of lysine as an herbal supplement or for medicinal uses. The culture media used in the batch and fed-batch fermentation mode are prepared by mixing process water, glucose, and nutrients. The fermentation is conducted in fed-batch mode and under aerobic conditions. In the batch mode, the microorganism seed is introduced into the fermenters, which have been filled previously with the fermentation medium. After consumption of glucose, the batch phase completes, and the fed-batch phase gets started. During the fed-batch phase, glucose and nutrients are continuously fed until the desired L-lysine concentration is obtained. At the end of the fermentation, the broth is sent to a buffer tank and subjected to the downstream process steps. The fermentation broth is sent to an ultrafiltration system to remove cell debris and other suspended solids. Afterward, the liquor from ultrafiltration is fed to an ion exchanger, where L-lysine is selectively adsorbed. The adsorbed L-lysine is eluted from the ion-exchange resins by washing with an aqueous solution of ammonia (www.chemengonline.com). The L-lysine is eluted from the ion-exchange columns and is mixed with mother liquor from the product filtration step and concentrated by evaporation. The concentrated lysine solution is acidified with hydrochloric acid, and free L-lysine is converted to L-lysine hydrochloride. The L-lysine hydrochloride solution is then sent to the crystallizer to filter lysine. The mother liquor is recycled to the evaporator, and the wet cake is taken to the dryers. Final dry L-lysine hydrochloride of 98.5 wt.% purity is obtained and sent to a packaging line before being stored in bags. Fig.  6.1 shows the production of L-lysine HCl via a conventional fermentation process (www.chemengonline.com/l-lysine-hcl-production-glucose). The total capital investment estimated for construction of a plant producing 100,000 metric tons per year of L-lysine hydrochloride in the United States is about $350 million. Worldwide production of white meat (for example, poultry and pork) has significantly increased over the past forty years. This has led to a much higher demand for L-lysine. (www.chemengonline.com/l-lysine-hcl-production-glucose/)

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Nutrients Glucose syrup Process water

Process water 1

Water vapor

Ammonia

HCI ST

Glucose syrup

2

6

5

3

L-lysine HCI to packaging and storage

9

Ammonia CHW

Effluent to waste treatment

4

Air

7 8

Mother liquor

CHW

Solids to waste

10

CW

11

ST

12

1. Medium vessels 2. Fermenters 3. Buffer tank 4. Ultrafiltration unit 5. Ion-exchange unit 6. Evaporator 7. Crystallizer 8. Product filter 9. Dryer 10. Chiller 11. Cooling tower 12. Steam boiler CW Cooling water ST Steam CHW Chilled water

FIG. 6.1  Production of L-lysine HCl via a conventional fermentation process (www.chemengonline.com/l-lysinehcl-production-glucose/). Reproduced with permission from Intratec Solutions.

Fig. 6.2 illustrates the growth in L-lysine production over the past several decades. In recent years, a “single state” fermentation process is being used for the commercial production of lysine. This process has replaced the “two-stage” fermentation process of lysine production in which E. coli was used to produce diaminopimelic acid (DAP) in the first stage and Enterobacter aerogenes was used for the formation of lysine from diaminopimelic acid through decarboxylation by an enzyme called diaminopimelic decarboxylase (DAPdecarboxylasc) in the second stage. The “single-stage” process of fermentation of lysine involves mutants of Corynebacterium, Brevibacterium, etc. These bacteria are grown in a synthetic medium containing carbohydrates (glucose), an inorganic nitrogen source, small concentration of homoserine or methionine, and small concentration of biotin. The process of fermentation is completed within 48–70h, and the yield of the amino acid reaches up to 30g/L. According to Global L-Lysine Market 2019 Industry Research Report (https://www. marketwatch.com/press-release/l-lysine-market-2019-top-leading-countries-companiesconsumption-drivers-trends-forces-analysis-revenue-challenges-and-global-forecast-2025-

Thousands of metric tons

2500 2000 1500 1000 500 0 1970

1980

1990 2000 Year

2010

2020

FIG.  6.2  Growth in L-lysine production over the past several decades (www.chemengonline.com/l-lysine-hclproduction-glucose/). Reproduced with permission from Intratec Solutions.



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2019-03-22), global L-lysine market size will increase to 3970 Million USD by 2025, from 3070 Million USD in 2018.

6.1.6  l-Glutamic acid

L-Glutamic acid (2-aminopentanedoic acid)

The history of the first amino acid production dates back to 1908 when Dr. K. Ikeda, a chemist in Japan, isolated glutamic acid from kelp, a marine alga, after acid hydrolysis and fractionation. He also observed that glutamic acid, after neutralization with sodium hydroxide, developed a delicious taste (www.biologydiscussion.com/industrial-microbiology-2/ glutamic-acid-history-production-and-uses-with-diagram). Glutamic acid is a nonessential amino acid naturally occurring in the L-form. It is very important for proper functioning of cells and for disposing excess nitrogen from human body. Glutamate acts as a neurotransmitter in the nervous system. Glutamic acid commonly occurs in almost every protein compound. It is found in abundance in cereals, meats, certain algae, and soy protein. Glutamic acid is also known as proteinogenic amino acid, and its salts are known as glutamates. These are mainly used in food to enhance the flavor. Corynebacterium glutamicum is widely used for glutamic acid production (Hermann, 2003). L-Glutamic acid is used in food, beverage, pharmaceutical,health and personal care products, and agriculture/animal feed/poultry. Glutamic acid is mainly used to enhance the flavor in the processed food industry. Glutamic acid is mostly used in the production of monosodium glutamate (MSG), which is commonly known as the “seasoning salt.” It is extensively used as food additive in the food industry. MSG is condiment and flavor-enhancing agent, it finds its greatest use as a common ingredient in convenient foodstuffs. The sodium salt is used rather than the acid as it is the glutamate ion that produces flavor and the salt is more soluble in water than the parent acid. L-Glutamic acid is used as nutritional supplement in food such as processed cheeses, fruits, meats, soups, wines, seasonings, yogurts, canned shrimps, and crabs. L-Glutamic acid can be used to enhance flavor in soft drink and wine. In addition, glutamic acid is used in milk products for improving the taste. Glutamic acid can be used in cosmetics and personal care products. Glutamic acid can be used as a baby product, bath product, cleansing hair product, eye makeup, shaving preparation, and hair and skin care products. In cosmetic and personal care products, glutamic acid functions mainly as a hair-conditioning agent and a skin-conditioning agent. In agricultural and animal feed and poultry, glutamic acid can be used as a nutritional supplement. Glutamic acid is used in animal feeds for the growth of poultry. In pharmaceutical industry, glutamic acid is used to cure metabolic disorder and also used as a nerve stimulant. The global glutamic acid market is expected to expand significantly in the near future because of increasing application of processed food coupled with growth in the animal feed industry (www.transparencymarketresearch.com/glutamic-acid-market.htm).

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The animal feed industry is expected to experience substantial growth because of rising meat consumption, increase in population, high disposable income, and growing demand for better quality of meat food. Increase in consumption of meat products would speed up the growth of the glutamic acid industry. Increasing trend among consumers to opt for healthy and nutritional food is projected to increase the demand for glutamic acid in the coming years. Additionally, the preference of natural food additives over synthetic food additives to eliminate health concerns would positively impact the demand for glutamic acid. Increase in consumption of dairy products is likely to increase the demand for glutamic acid. About 1.7 million tonnes of L-glutamic acid are produced by fermentation every year, the majority is produced in Asia. One company in China is producing 33% of the world's output (www.essentialchemicalindustry.org/.../biotechnology-in-the-chemical-industry.html). Commercial production of glutamic acid by fermentation provides 90% of world’s total demand, and the remaining 10% is met through chemical methods. For the fermentation, the microorganisms are cultivated in fermentors as large as 500m3. The raw materials used include carbohydrates (glucose, molasses, sucrose, etc.), peptone, inorganic salts, and biotin. Biotin concentration in the fermentation medium significantly affects the yield of glutamic acid. Fermentation takes 2–4 days to complete. After the completion of the fermentation, the broth contains ammonium salt of glutamic acid. Mutants of C. glutamicum or genetically modified E. coli are efficient producers of L-glutamic acid. C. glutamicum possesses several characteristics: the bacterium is gram positive, nonsporulating, and nonmotile. It needs biotin for growth and shows little activity of α-ketoglutaric acid dehydrogenase and increased activity of glutamate dehydrogenase. Mutants of C. glutamicum secrete L-glutamic acid in large quantities even in the presence of high concentration of biotin. For example, a lysozyme-sensitive mutant of C. glutamicum is able to convert 40% of the added carbon source to L-glutamic acid even in the presence of 100 mg per liter of biotin. In a typical downstream process, the bacterial cells are removed, and the broth is passed through a basic anion-exchange resin. Glutamic acid anions get bound to the resin, and ammonia is released. This ammonia can be recovered using the distillation process and reused in the fermentation. Elution is performed with sodium hydroxide to directly form MSG in the solution and to regenerate the basic anion exchanger. From the elute, MSG may be crystallized directly followed by further conditioning steps like decolorization and serving to yield MSG. α-Ketoglutaric acid serves as the precursor of glutamic acid, and the conversion of the α-ketoglutaric acid to glutamic acid takes place in the presence of enzyme glutamic acid dehydrogenase. The production of glutamic acid can be increased several fold if penicillin is added in the medium. As stated earlier, glutamic acid is mostly used in the production of MSG, which is commonly known as the “seasoning salt.” The world production of glutamic acid is in the tune of 800,000 tonnes per year. MSG is condiment and flavor-enhancing agent and is mostly used as a common ingredient in convenient foods. L-Glutamic acid accounted for more than 40% of total market volume in 2014. Lysine, threonine, methionine, and tryptophan are mostly consumed feed additives for livestock production. Tryptophan is expected to be the fastest growing amino acid. There has been a strong demand in Japan and East Asia for L-glutamic acid as a seasoning since MSG was found to present umami taste in 1907. The discovery of glutamate fermentation by C. glutamicum in 1956 allowed abundant and low-cost production of the amino acid,



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TABLE 6.9  Companies producing glutamic acid. Sichuan Tongsheng Company Ltd. Bachem AG, Suzhou Yuanfang Chemical Co. Ltd. Luojiang Chenming Biological Products Co. Changzhou Huatong Biochemical Company Ltd.

creating a large market. The discovery also prompted researchers to develop fermentative production processes for other L-amino acids, such as lysine. Currently, the amino acid fermentation industry is so huge that more than 5 million tonnes of amino acids are produced annually all over the world, and this number is continuously growing. Some of the global market players operating in glutamic acid market are presented in Table 6.9 (www.grandviewresearch.com/industry-analysis/glutamic-acid-market).

6.2  Food additives and food supplements The basic food components in agricultural raw materials are carbohydrates, proteins, and lipids (fats and oils). These can be produced from alternate sources by using the fermentation process or plant tissue culture. Glutamic acid and lysine, which are the protein building blocks, are produced in large amounts by fermentation and are used as animal feed. Single-cell protein (SCP) is receiving a lot of attention as a way for improving the nutrition in developing countries. Before the prices of oil increase in the 1970s, petroleum hydrocarbons were considered a desirable substrate. In the United States, Amoco and Phillips Petroleum developed processes using yeast that were successful for producing yeast-based flavors but were not quite economical for producing commodity protein ingredients. In the United Kingdom, ICI and Rank Hovis McDougall developed a process that was based on a Fusarium fungus and switched to a conventional substrate. It was approved for sale in 1985 and is presently being marketed in Europe as Quorn mycoprotein by Zeneca’s Marlow Foods subsidiary (cdnet.stic.gov.tw). Quornis a meat substitute product available in 19 countries. Among food biotechnology applications, the production of basic food ingredients from unconventional sources is expected to have the enormous environmental implications due to the involvement of huge volumes. Developments in the near future are not likely due to the relatively low cost of traditional ingredients such as soy protein and the comparatively high cost of alternate substrates such as petroleum hydrocarbons. Food additives include acidulants, leavening agents, gums, emulsifiers, vitamins, minerals, preservatives, flavors, and colors. The preferences of consumers for natural products give biotechnology-derived additives a benefit over the chemical ones, if their cost is competitive. Few biotechnology-derived products from nonrecombinant sources include xanthan gum from Xanthomonas campestris and citric acid from Aspergillus niger. One of the most important potential applications is in the production of natural flavors, for example, vanilla by using plant tissue culture, but this has yet not been commercialized (www.sustentabilidad.uai.edu.ar). Another important category is preservatives obtained by fermentation. Most conventional food preservatives are chemically synthesized fatty acids or other organic acids that reduce

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6.  Application of biotechnology in chemical industry

pH of food and inhibit different types of microorganisms. One trend is the development of preservatives produced by fermentation such as “Upgrade” developed by Stauffer Chemical and now being produced in the United States by the Quest unit of ICI, with the same active ingredient (propionic acid) found in calcium propionate obtained chemically. Other preservatives produced by fermentation are Delvocid (pimaricin), or nisin. Delvocid is produced from Streptomyces natalensis by the Dutch company Gist-brocades. Nisin, is produced from Streptococcus lactis by the Australian company, Burns Philp. These preservatives have special properties or applications. Bacteriocins like nisin are especially important because these can be produced by lactic acid bacteria and are effective against particularly challenging pathogens like Listeria monocytogenes. The environmental advantages of producing food additives by fermentation or using enzymes instead of organic synthesis resemble with those of other speciality chemicals. The processing steps are reduced, and the use of organic solvents is also reduced. In the case of preservatives obtained by fermentation, there is an even more desirable effect when the fermentation broth is added to the final product. The most desirable situation involves the use of bacteriocin-producing cultures in situ for fermented foods (such as sausage or sauerkraut), where they utilize unstable carbohydrates, naturally preserve the final product, and contribute nutritive value of their own. Processing aids, including enzymes, are generally used in small amounts for their functional effect during production but are not an important part of the final product (cdnet.stic.gov.tw). Food and Beverages is the largest market for Industrial Enzymes with an estimated share of 26% that is equivalent to US id="dq0030".4 billion in 2017, followed by Biofuels and Detergents with 18% (US$969.3 million) and 14% (US$754.4 million) respectively in the same year. Industrial Enzymes in Biofuels is expected the fastest growing segment. (www.prnewswire.com/.../global-industrial-enzymes-market-2018-2024-key-busin)

Demand for industrial enzymes is showing rapid growth because of the increase in demand for food and beverages. This is ascribed to changing lifestyles and increase in income in a wide segment of the population. Governments in different countries have given incentives for using biofuels as cleaner alternatives to fossil fuel–based energy options, which has induced the market for industrial enzymes. Developments in different areas of biotechnology, especially protein engineering, have provided further push to the demand for industrial enzymes. Food and beverages account for about a quarter of the worldwide market for industrial enzymes, although demand for them in the biofuel industry is likely to result in the fastest growth in the coming years (www.researchandmarkets.com). Applications of enzymes in the food industry are diverse (Table  6.10) (Kirk et  al., 2002; Schafer et al., 2002; Bhoopathy, 1994. Muir, 1996; Farkye, 1995; Novozymes, 2011; Kuraishi et al., 2001; Bajpai, 2018d; www.biology.uoc.gr). Enzymes are playing an important role in the food industry in both traditional and novel products. The old processes of brewing and cheese making depended on enzyme activity at different stages of production. The first important breakthrough for microbial enzymes in the food industry came in the early 1960s with the introduction of glucoamylases, which were able to break starch into glucose. In the recent years, almost all glucose production is conducted using enzymatic hydrolysis. By using the enzymatic process, steam cost is reduced by 30%. Ash and by-products are reduced by 50% and 90%, respectively. The starch processing industry has now grown to be one of the largest markets for enzymes. The use of enzymes such as rennet in cheese making and barley amylases in brewing is as old as the food and beverage industry



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TABLE 6.10  Enzymes used in food industry. Food (including dairy) Protease Milk clotting Infant formulas (low allergenic) Flavor Lipase Cheese flavor Lactase Lactose removal (milk) Pectin methyl esterase Firming fruit-based products Pectinase Fruit-based products Transglutaminase Modify viscoelastic properties Baking Amylase Bread softness and volume Flour adjustment Xylanase Dough conditioning Lipase Dough stability and conditioning (in situ emulsifier) Phospholipase Dough stability and conditioning (in situ emulsifier) Glucose oxidase Dough strengthening Lipoxygenase Dough strengthening Bread whitening Protease Biscuits, cookies Transglutaminase Continued

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TABLE 6.10  Enzymes used in food industry—cont’d Laminated dough strengths Beverage Pectinase Depectinization Mashing Amylase Juice treatment Low-calorie beer Glucanase Mashing Acetolactate decarboxylase Maturation (beer) Laccase Clarification (juice) Flavor (beer) Cork stopper treatment Based on Kirk et al. (2002); Schafer et al. (2002); Bhoopathy (1994); Muir (1996); Farkye (1995); Novozymes (2011); Kuraishi et al. (2001); Bajpai (2018d).

itself. The production of the amylase enzyme represents the first example of the production of industrial enzyme for application in the food industry. The amount and type of enzymes used in the food and beverage industry have increased significantly in the last decade. Starch processing involves the conversion of corn or another grain into dextrose and other syrups by a hydrolysis reaction. This was earlier done using acid at high temperature and pressure, but dextrose yields were limited to about 80 per cent, and the process was hazardous and expensive and produced large quantities of salt as a by-product. The initial change to enzymatic hydrolysis in the 1960s increased dextrose yields and eliminated the drawbacks of the acid process. In the 1970s, development of immobilised glucose isomerase enzymes enabled the production of high-fructose corn syrup. In the 1980s, thermostable alpha-amylases contributed to increased yields, and in the 1990s, recombinant thermostable amylases contributed to reduced costs. (www.oecd.org)

Enzymatic hydrolysis is used for producing syrups by using liquefaction, saccharification, and isomerization processes. Baking industry is another important market for food processing industry. Supplementary enzymes are mixed to the dough for ensuring high quality of bread in terms of volume and a uniform crumb structure. Special enzymes are able to increase the shelf life of bread by preserving its freshness for a long time (Novozymes, 2011). Another application is in the dairy industry. It is used to coagulate the milk as the first step in the making of cheese. Both microbial enzymes and enzymes from animal sources are used. In several large breweries, industrial enzymes are added for controlling the brewing process and produce good beer of consistent quality.



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In food processing, animal or vegetable food proteins having improved functional and nutritional properties are produced by using enzymatic hydrolysis of proteins. In the juice and wine industries, enzymes are used for the extraction of plant material for breaking down the cell walls. Improved color and aroma of extracts and clear and higher juice yields are obtained.

6.2.1  Starch modification, production of sweetener, and glucose syrups Modified starches and syrups of different compositions and physical properties are obtained and used in different types of food products. By selecting the appropriate enzymes and the proper reaction conditions, valuable enzyme products can be produced for meeting virtually any specific requirement in the food industry (Novozymes, 2011). Several nonfood products obtained by fermentation are obtained from enzymatically modified starch products. For example, enzymatically hydrolyzed starches are used in the production of alcohol, ascorbic acid, polyols, enzymes, lysine, and penicillin. The following steps are involved in the conversion of starch: • Liquefaction • Saccharification • Isomerization Industrial enzymes are being used in the starch industry for a long time. Special types of syrups that could not be produced using the traditional chemical hydrolysis were the first products produced using the enzymes (Novozymes, 2011). Several valuable products are obtained from starch. Extensive work was conducted to develop the application processes. Enzymes are the ideal catalysts for the starch industry as these can work under mild conditions, show high specificity and reaction efficiency, and show a high degree of purification. The saccharifying enzyme—glucoamylase—completely breaks down starch to glucose. The immobilized glucose isomerase was developed in 1973, which made the industrial production of high-fructose syrup possible. These sweeteners are added in soft drinks, candies, baking, jams, and jellies and several other foods. The environmental benefits are presented in Table 6.11. Glucose syrups are obtained by complete hydrolysis of the starch. This process breaks the bonds that link the glucose units in the starch chain. The method and extent of hydrolysis affect the final composition of carbohydrates and therefore several functional properties of starch syrups. The degree of hydrolysis is generally defined as the dextrose equivalent. Originally, the acid conversion process was used for producing glucose syrups. Now, due to their specificity, enzymes are generally used for controlling the hydrolysis. With this method, tailor-made glucose syrups with well-defined sugar profile are produced. The sugars are analyzed using high-performance liquid chromatography (HPLC) and gel permeation chromatography (GPC) for obtaining information on the molecular weight distribution and overall carbohydrate composition of the glucose syrups. Modern enzyme technology is used extensively in the corn wet-milling sector. The enzymatic steps are discussed in the succeeding text. Corn starch, wheat, tapioca, and potato are used as raw material. Corn starch is most extensively used. The native starch is only slowly hydrolyzed using alpha-amylase enzyme.

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TABLE 6.11  Environmental benefits of using glucoamylase enzymes for the conversion of starch to glucose. Reduced use of strong acids and bases Reduced energy consumption Reduced generation of greenhouse gases Less corrosive waste Safer production environment for workers

Therefore, a suspension containing 30%–40% dry matter should first be gelatinized and liquefied for making the starch susceptible to further enzymatic attack. This is obtained by adding a stable alpha-amylase enzyme to the starch suspension (genet.univ-tours.fr). The liquefaction process uses stirred tank reactors, continuous stirred tank reactors, or jet cookers. Most plants producing sweeteners conduct starch liquefaction in a single-dose, jet-cooking process. Thermostable alpha-amylase is added to the starch slurry before it is pumped through a jet cooker. Here, live steam is injected to increase the temperature to 105°C and the subsequent passage of the slurry through a series of holding tubes provides the retention time of 5 minutes required to fully gelatinize the starch. The temperature of the partially liquefied starch is then reduced to 90–100°C by flashing, and the enzyme is allowed to further react at this temperature for one to two hours until the required DE is obtained. The enzyme hydrolyzes the ­alpha-1,4-glycosidic bonds in the gelatinized starch; the viscosity of the gel rapidly decreases and maltodextrins are produced. The process is stopped at this point, and the solution purified and dried. Maltodextrins (DE 15–25) are commercially valuable for their rheological properties. They are used as bland-tasting functional ingredients in the food industry as fillers, stabilizers, thickeners, pastes, and glues in dry soup mixes, infant foods, sauces, gravy mixes, etc. (www.novozymes.com)

When maltodextrins are saccharified by further hydrolysis using glucoamylase or fungal alpha-amylase enzyme, several types of sweeteners can be produced. These have DE in the range of 40–45 for maltose, 50–55 for high maltose, and 55–70 for high conversion syrup. By using beta-amylase, glucoamylase, and debranching enzymes, intermediate level conversion syrups having maltose contents of about 80% can be produced. A high yield of 95%–97% glucose can be produced from most starchy materials. The debranching enzyme mostly used is pullulanase (alpha-dextrin endo-1,6-alpha-glucosidase). Glucose can be converted to fructose in a reversible reaction. Under industrial conditions, the equilibrium point is reached when the level of fructose is 50%. The conversion is generally stopped when the yield of fructose reaches about 45% for avoiding a lengthy reaction time. The isomerization reaction in the reactor is efficient, fast, and economical when an immobilized enzyme is used.

6.2.2 Baking Fungal alpha-amylase enzymes and enzymes from malt have been used for making bread for decades. Several new enzymes are now available for the baking industry due to advances in the area of biotechnology. The use of enzymes is expected to increase because the consumers are demanding natural products that are free of chemical additives. The dough for bread, rolls, buns, and similar products consists of flour, yeast, water, salt, and other ingredients such as sugar and fat. Flour consists of gluten, starch, nonstarch polysaccharides, lipids, and



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minerals in trace amounts. As soon as the dough is made, the yeast starts working on the fermentable sugars and converts them into alcohol and carbon dioxide, which makes the dough rise. The major component of wheat flour is starch. Amylases are able to degrade starch and produce small dextrins for the yeast to act upon. There is also another type of amylase that modifies starch during baking and gives a substantial antistaling effect. Gluten is a combination of proteins that forms a large network when the dough is formed. This network holds the gas in during dough proofing and baking. Therefore, the strength of this network is very important for the quality of all bread raised using yeast. Enzymes such as hemicellulases, xylanases, oxidases, and lipases can directly or indirectly improve the strength of the gluten network and also improve the quality of the finished bread. Another area is baking where enzymes have been used, in the form of barley malt to standardise the starch degrading activity of wheat flour. Since the 1970s, fungal enzymes have partly replaced malt for this purpose. Different amylases with a specific “intermediate” temperature optimum came into use to retard bread staling in the 1980s. This application has increased in the 1990s with the developments of recombinant products. Combinations of glucose oxidase and other enzymes are being used to replace potassium bromate as an oxidant in flour for bread making due to concern about possible carcinogenicity of bromate. Recombinant and non-recombinant hemicellulases are being used to improve the processing and softness of whole grain and high fibre breads. Most recently, recombinant lipases have been used to replace or supplement the fat and emulsifiers used to give bread its volume and softness although all these enzymes are available from non recombinant sources. (cdnet.stic.gov.tw; genet.univ-tours.fr)

6.2.3  Dairy products The application of enzymes for the processing of milk and particularly rennet for the production of cheese has been used for a long time. Rennet is an enzymatic coagulant. It is a protease with a milk clotting role and is indispensable in the production of cheese. The rennet contains c­ hymosin, and in these days, there are several industrially produced chymosin products or similar type of proteases available as substitutes. Proteases are also used to modifying the functional properties of cheese, to accelerate the cheese ripening process, and to modify milk proteins for reducing allergenic properties of some dairy products. Protein is not the only possible allergen in milk. Many adults are not able to drink milk. Cow’s milk contains 5% lactose, and for breaking it down, the enzyme lactase is required. This enzyme is high in humans at birth, but in the adulthood, only low levels are found in certain sections of the world's population during adulthood. Lactase is used to hydrolyze lactose for increasing digestibility or for improving the solubility or sweetness of several dairy products. Finally, lipases are used mainly in ripening of cheese. Chymosin has been one of the recombinant enzyme used in food applications with the largest impact. Chymosin is the milk clotting enzyme used to make cheese. It was earlier extracted from calf stomachs, but the gene for the enzyme was cloned in microorganisms so that it could be produced by fermentation. Pfizer started producing chymosin using E. coli bacteria in 1990, Gist-brocades used Kluyveromyces lactis yeast in 1992, and Genencor International used Aspergillus niger fungus in 1993. (Maryanski, 1995)

6.2.4 Brewing Beer is traditionally produced using the mashing process. Barley malt is crushed and mixed with hot water in large circular vessels, called mash copper. Few adjuncts are also added to

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the mash. After mashing, the mash is filtered, and the resulting liquid, known as sweet wort, is then run off to the copper, where it is boiled with hops. The hopped wort is cooled and taken to the fermenter, and yeast is added. After fermentation, the green beer is matured before final filtration and bottling. The conventional source of enzymes used for the conversion of cereals into beer is barley malt, one of the major ingredients in brewing. If very little enzyme is present in the mash, there will be unwanted results listed in the succeeding text: • • • • • •

Extract yield will be quite less. Separation of wort will take a long time. Fermentation process will be very slow. Very little alcohol will be produced. Filtration rate of beer will be reduced. Flavor and stability of the beer will be of poor quality.

Industrial enzymes are used to supplement the malt’s own enzymes for preventing these problems. In addition, industrial enzymes can be used for producing low carbohydrate beer to ensure better adjunct liquefaction, to reduce the beer maturation time, and to produce beer from inexpensive raw materials.

6.2.5  Distilling potable alcohol Fermented alcoholic beverage production from raw materials containing starch has been practiced for long time. Before the 1960s, the enzymatic degradation of starch to fermentable sugars was obtained by adding malt or koji. Koji is used as a source of enzyme for alcohol production in Japan and China. Today, in several countries malt has been completely replaced in distilling operations by industrial enzymes. This offers many advantages. A few liters of enzyme preparation can be used to replace 100kg of malt. Because of this enzyme handling and storage becomes much easier. When switching to commercial enzymes, savings of 20– 30% can be expected on raw material costs. Moreover, since industrial enzymes have a uniform standardized activity, distilling becomes more predictable with a better chance of obtaining a good yield from each batch of fermentation. The quality of malt, on the other hand, can vary from year to year and from batch to batch, as can koji. Microbial amylase enzymes are available with activities covering a broad pH and temperature range, and so suitable for the low pH values found in the mash. The commercial enzymes have replaced malt in all but the most conservative parts of the distilling industry. The selection of raw material differs around the world. In the alcohol industry, starch is usually hydrolyzed by enzymes in two stages – liquefaction and saccharification. The yeast can then transform the smaller molecules – mainly glucose – into alcohol. (www. novozymes.com)

6.3  Feed additives Animal producers are facing increasing economic pressures that are demanding more effective utilization of feedstuffs of low grade. Moreover, consumer consciousness and new legislation need that any increase in animal production cannot be obtained with drugs promoting growth or other chemical substances. One interesting approach to this problem is to add hydrolytic enzymes to the animal diets for helping in the digestion and absorption of the nutrients that are poorly available or for removing antinutritional factors from the diet (Bajpai, 2018d; Aehle, 2004; Selle and Ravindran, 2007; Bedford and Schulze, 1998; Bedford and Partridge, 2011;



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Easter et al., 1993). Exogenous enzyme products in high-forage diets fed to growing cattle have been examined by several researchers (Beauchemin and Rode, 1996; Beauchemin et al., 1995, 1997, 1999; McAllister et al., 1999; Michal et al., 1996; Pritchard et al., 1996; Wang et al., 1999; ZoBell et al., 2000). There are certain concerns raised by this practice as these enzymes can survive at the processing temperatures and even the digestive tract of the animals. In livestock and poultry production, animal feed is the largest cost item. This accounts for 60%–70% of total expenditure. Several producers add enzyme additives to the feed to reduce the cost enabling them to produce more meat per animal or to produce the same amount of meat faster and cheaper. The feed enzymes market was estimated at USD 842.9 million in 2016. (www.marketsandmarkets.com). The global animal feed enzyme market is expected to reach USD 2.67 billion by 2025 according to a new study by Grand View Research (www. grandviewresearch.com/press-release/global-animal-feed-enzymes-market). Some types of feed ingredients are not completely digested by livestock. But by the addition of enzymes to the feed, the digestibility of the feed ingredients can be improved. Enzymes have now become a successful tool and also well proven. Enzymes allow feed producers to extend the range of raw materials used in feed and also to improve the efficacy of the current formulations (Bajpai, 2018d). “Enzymes are added to the feed either directly or as a premix together with vitamins, minerals, and other feed additives. In premixes, the coating of the enzyme granulate protects the enzyme from deactivation by other feed additives such as choline chloride. The coating has another function in the feed mill – to protect the enzyme from the heat treatments sometimes used to destroy Salmonella and other unwanted microorganisms in feed. Enzyme products in a liquid formulation are developed for those cases where the degree of heat treatment (conditioning) for the feed is high enough to cause an unacceptable loss of activity. Thereby addition can be performed accurately after the conditioning with insignificant loss of activity” (www.novozymes.com). A diverse range of enzymes are now available for degrading substances such as phytate, ­beta-glucan, starch, protein, pectin-like polysaccharides, xylan, raffinose, and stachyose (Table 6.12). Cellulose and hemicellulose can also be degraded. As shown by several feed trials conducted so far, the major advantages of adding enzymes to the feed are shown in Table 6.13. Table 6.14 shows the prerequisite of enzymes used in animal nutrition.

6.3.1  Phytase enzymes In the pig and poultry diets, about 50%–80% of the total phosphorus is present in the form of phytate or phytic acid. The phosphorus that is bound to phytate is largely not available to monogastric animals because they lack the enzyme that is required for breaking down the phytase. There are two important reasons to include phytase to the feed. - One is to reduce the adverse environmental impact of phosphorus from animal manure in those areas having intensive livestock production. Soil microorganisms degrade the phytate or phytic acid in manure. This leads to high levels of free phosphate in the soil and finally in surface water. Research has shown that optimization of phosphorus intake and digestion with phytase reduces the release of phosphorus by about 30%. According to Novozymes, the amount of phosphorus released into the environment is expected to be reduced by 2.5 million tons a year worldwide if phytases were included in all the feed for monogastric animals.

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TABLE 6.12  Type of feed enzymes. Enzyme

Substrate

Phytases

Phytic acid

β-Glucanases

β-Glucan

Xylanases

Arabinoxylans

α-Galactosidases

Oligosaccharides

Proteases

Proteins

Amylase

Starch

Lipases

Lipids

Mannanases, cellulases, hemicellulases, pectinases

Cell wall matrix (fiber components)

Based on Ravindran, V., 2013. Feed enzymes: science, practice and metabolic realities. J. Appl. Poult. Sci., 22, 628–636.

TABLE 6.13  Advantages of supplementing feed with enzymes. Growth of the animals is faster Feed conversion ratio is better Production is more uniform Health status is better Better environment for chickens because of reductions in “sticky droppings”

TABLE 6.14  Prerequisite of enzymes used in animal nutrition. Should be able to act under acidic pH condition of stomach Should be able to resist low pH Should be able to resist proteolytic action of pepsin Must be able to act in other parts of digestive tract

- The other reason is based on the fact that phytate can produce complexes with proteins and inorganic cations like calcium, magnesium, iron, and zinc. The use of phytase releases the bound phosphorus and other important nutrients for providing a higher nutritional value to the feed.

6.3.2  Nonstarch polysaccharides (NSP) degrading enzymes Cereals such as barley, wheat, and rye are added into animal feeds for providing energy. But much of the energy remains unavailable to monogastrics due to the presence of NSP that interferes with digestion. It prevents access of the animal’s own digestive enzymes to



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the n ­ utrients present in the cereals, and also, NSP can get solubilized in the gut and create problems of high gut viscosity, which further interferes with the digestion. The addition of selected carbohydrase enzymes will break down NSP, releasing the nutrients (energy and protein), and also reduce the viscosity of the gut contents (Ao et al., 2010; Montoya et al., 2011; Novozymes, 2011). The carbohydrase enzymes include xylanases, glucanases, and amylases. They act in the stomach to break down and degrade carbohydrates such as fiber, starch, and nonstarch polysaccharides into simple sugars that provide energy for use by the animal. The overall effect is better use of feed and a more “healthy” digestive system for monogastric animals. One of the most common carbohydrases is xylanase. This enzyme attacks the arabinoxylan structure of corn or wheat and allows the animal to absorb its components as an energy source. This limits the need for additional fat or energy in the final diet.

6.3.3 Proteases Protease enzymes are able to hydrolyze the proteins that are not much digestible in animal feeds and break them down into more usable peptides. So these enzymes are very important for digestion of protein. Improving the digestibility of dietary protein using a good protease enzyme can reduce the cost of feed by allowing the use of lower crude protein feedstuffs with lesser quality amino acids, effectively reducing protein and digestible amino acid levels required from the feedstuffs by up to 10% (www.allaboutfeed.net). Proteases break down anti-nutritional factors associated with various proteins. Proteases improve the digestion of proteins and increase amino acid availability, which helps to release the valuable nutrients. The result is improved animal growth and performance and very small negative effects of undigested protein in the hindgut. Raw ingredients with low digestibility of amino acid respond greatest to an exogenous protease. This is why its greatest value is when alternative ingredients are used in the diet. Proteases help producers manage the nutritional risks associated with feedstuff quality and allow them to best utilize all available feed ingredients. Proteases are not limited to diets with alternative ingredients. Animals consuming a traditional corn-soybean meal diet cannot utilize 100 percent of the protein fraction. Therefore, adding a protease enzyme to a corn-soybean meal diet will enhance amino acid digestibility and animal performance. (www. wattagnet.com)

The advantages of enzymes are becoming better realized as more research and development continue. For the animal, enzymes optimize gut health, produce uniform growth, and improve overall health. For the producer, they decrease the cost of feed and improve the profitability. Each type of enzyme has its own specific function and therefore do not interfere with one another (www.wattagnet.com).

6.3.4  Mode(s) of action of enzymes Different feed enzymes have different modes of action (Table 6.15). In spite of their increasing acceptance as feed additives, the exact mode of action of feed enzymes is not very much clear. The general opinion is that one or more of the mechanisms are responsible for the observed benefits (academic.oup.com). German multinational BASF is widening its collaboration with a German biotechnology company to develop feed enzymes for monogastric food animals (https://animalpharm.agribusinessintelligence.informa.com/AP003053/BASF-collaborates-on-biotech-feed-additives).

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TABLE 6.15  Mode of action of different feed enzymes. Degradation of specific bonds in ingredients that do not get generally hydrolyzed by endogenous digestive enzymes Disruption of endosperm integrity and the release of nutrients that are bound to or entrapped by the cell wall Degradation of antinutritional factors that limit the digestion of nutrients directly, increase intestinal digest viscosity indirectly, or both Shifting of digestion to more efficient digestion sites Reductions in endogenous secretions and protein losses from the gut resulting in reduced maintenance requirements Reduction in the weight of the intestinal tract and changes in the morphology of intestine Changes in the profile of microflora in the small intestine. As enzymes affect the amounts and form of substrate present within the gut, their use has a direct effect on the bacteria that make up the microfloral populations Augmentation of endogenous digestive enzymes, which are either not enough or are absent in the bird, resulting in better digestion. This will be particularly true for newly hatched chicks with immature digestive systems Based on Ravindran, V., 2013. Feed enzymes: science, practice and metabolic realities. J. Appl. Poult. Sci., 22, 628–636; japr.oxfordjournals.org

6.4 Agrochemicals Agrochemicals closely resemble to basic chemicals and specialty chemicals. A feature that distinguishes the agricultural chemicals is that one end-use customer industry—farming— clearly controls the demand patterns. The business consists of two main segments: fertilizers and crop protection. These segments have both commodity and specialty segments. Some other businesses, like construction and utilities, are also using agricultural chemicals, as many institutional segments are doing. It is distinctly possible that some undercounting takes place in this business segment. Furthermore, the value of seeds and traits based on biotechnology are not included in crop protection.

6.4.1 Fertilizers Fertilizers are added to soil for replacing or supplementing important nutrients for promoting the growth of plant particularly crop growth. These contain three basic elements—nitrogen, phosphorus, and potassium. Phosphorus is present in phosphate rock, and potassium is present in potash. Fertilizers mainly serve the farm sector. The prices are largely driven by the cost of the raw material, and major economic factors include the following: • • • •

Increasing overseas demand A high degree of seasonality Volatility in farm incomes Potentially reduced demand arising from genetically modified crops

The natural gas resources are increasing; therefore, the nitrogenous fertilizers are experiencing renewed competition in the recent years (www.massey.ac.nz).



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Biofertilizers and inoculants are currently drawing significant attention (Gavrilescu and Chisti, 2005). These are cheap and safer alternative to chemical fertilizers that provide nitrogen, phosphorus, potassium, sulfur, and other inorganic nutrients needed for the growth of crop (Subba Rao, 1982). The first-generation biological fertilizers included nitrogen fixing rhizobium bacteria, which are present in the root nodules of legumes. These bacteria fix nitrogen and provide the assimilable nitrogen to the plants. Microbial inoculants can be used to complement traditional fertilizers, by increasing their absorption by plants. Increased use of biofertilizers contributes significantly to reduce pollution, energy, and resource consumption associated with the use of traditional fertilizers (www.massey.ac.nz). In 2016, the global biofertilizer market size was estimated at USD 787.8 million (www. grandviewresearch.com/industry-analysis/biofertilizers-industry). The increasing use of microbial fertilizers proves potential for sustainable farming method and food safety. There is a great concern regarding food safety, which is expected to drive the industry growth over the next few years. Some biofertilizers and soil conditioners that are presently used in agriculture are shown in Table 6.16. Nonleguminous plants are being engineered with symbiotic rhizobial root nodules so that like the legumes they can be grown without the requirement for additional nitrogen fertilizers. Furthermore, the research on biofertilizer is focusing to increase the consistency and reliability of performance of products, developing stable formulations and effective delivery systems, proving the efficacy under different field conditions, and explaining the m ­ echanisms of action. Work is in progress to produce mycorrhizal soil inoculants for increasing the efficacy of plant root systems (www.massey.ac.nz). TABLE 6.16  Biofertilizers and soil conditioners used in agriculture. Rhizobium spp N2 fixation Cyanobacteria N2 fixation Azospirillum spp. N2 fixation Mycorrhizae Nutrient acquisition Penicillium bilaii Phosphorous solubilization Directed compost Soil fertility Earthworm Humus formation Based on Pimentel, D., 2002. Encyclopedia of Pest Management. Dekker, New York.

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6.4.2  Crop protection Crop protection products help to control weeds, pests, and diseases. These products include fungicides, herbicides, insecticides, miticides, and pesticides. These also control disinfectants, rodenticides, and other products used for controlling germs. The farm sector is the major end-use market, but other sectors—household, hospital, other institutional, electric utilities, telecommunications, and industrial applications—are also important. Main economic factors include the requirement for increasing agricultural productivity, sustainable development, growing population, high regulatory barriers, higher costs for product development, cost cutting, globalization, and consolidation (www.americanchemistry.com). The business is affected by the increased use of GMOs (genetically modified organisms) and other biotechnology innovations. The use of GM crops has increased rapidly over the past two decades. In 2012, 88 percent of the corn, 94 percent of the cotton, and 93 percent of the soybeans planted in the United States were varieties produced through genetic engineering. (www.usda.gov/wps/portal/usda/usdahome?navid=BIOTECH)

Pesticides are microscopic biological agent and are used for controlling pests, including weeds. Pesticides are used for controlling insects, protection of crop, management of weeds, insect control, and treatment of seeds and for controlling algae in swimming pools and ­preservation of wood and textiles (Waxman, 1998). Biopesticides are “biological pesticides,” a green chemical technology in agriculture application (Table 6.17). These products are derived from microorganisms and are used to control insects, weeds, and rodent pests. These types of pesticides are obtained from biobased resources like animals, plants, bacteria, and certain minerals. Regulatory positions have been affected by public perceptions. Packaging, handling, storage, and application methods are quite similar to those for traditional pesticides. Biopesticides fall into three major classes (Table 6.18). Botanical origin biopesticides and microbial origin biopesticides are described in Tables 6.19 and 6.20, respectively. Table 6.21 shows advantages of using biopesticides. In 2014, there were more than 430 registered biopesticide active ingredients and 1320 active product registrations. Biopesticides have had some amazing successes, but there were ­certain issues about their effectiveness (Auld and Morin, 1995). Biopesticides usually are highly specific, do not leave any toxic residues, reduce the risk of resistance development in the target species, and produce a reduced environmental impact in comparison with the traditional chemical pesticides. Biofungicides have been used in phylloplane and rhizosphere TABLE 6.17  Biopesticides. European Union Form of pesticide based on microorganisms or natural products United States Environmental Protection Agency Naturally occurring substances that control pests (biochemical pesticides), microorganisms that control pests (microbial pesticides), and pesticidal substances produced by plants containing added genetic material (plantincorporated protectants) Based on foreverest.cn



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TABLE 6.18  Types of biopesticides. Biochemical pesticides Naturally occurring substances that control pests by nontoxic mechanisms. On the contrary, conventional pesticides are usually synthetic materials that directly kill or inactivate the pest. Biochemical pesticides include substances that interfere with mating, such as insect sex pheromones, and also several scented plant extracts that attract insect pests to traps. As it is sometimes difficult to determine whether a substance is meeting the criteria for classification as a biochemical pesticide, EPA has constituted a special committee for making such decisions Microbial pesticides This group consists of microorganisms. These pesticides can control several different kinds of pests although each separate active ingredient is relatively specific for its target pest(s). For instance, there are fungi that control certain weeds and other fungi that kill specific insects Bacillus thuringiensis (Bt) is the most widely used microbial pesticides. This bacterium produces a different mix of proteins and specifically kills one or a few related species of insect larvae. While in contrast some Bt ingredients control moth larvae found on plants, other Bt ingredients are specific for larvae of flies and mosquitoes. The target insect species are determined by whether the certain type of Bt produces a protein that can bind to a larval gut receptor, thus causing the insect larvae to starve Plant-incorporated protectants Plants produce these pesticidal substances from genetic material that has been added to the plant. Scientists can take the gene for the Bt pesticidal protein and introduce the gene into the plant's own genetic material. Then, the plant produces the substance that destroys the pest. The protein and its genetic material are regulated by EPA Based on www.epa.gov

TABLE 6.19  Biopesticides of botanical origin. Derisom from Pongamia glabra Margosom from Azadirachta indica Anosom from Annona squamosa Based on www.agrilife.in

for suppressing fungal infection in plants. Bacillus and Pseudomonas species have been used as seed dressings for controlling certain soilborne plant diseases (Pimentel, 2002; Johnsson et al., 1998; Gavrilescu and Chisti, 2005). Table 6.22 presents few commercial biopesticide products being marketed for use against soilborne plant pathogens (www.massey.ac.nz). There are different types of biopesticides (Hall and Menn, 1999; Koul and Dhaliwal, 2002). Pest pathogenic bacteria, fungi, viruses, and parasitic nematodes are being evaluated for controlling several pests in addition to biologically produced chemicals. Both spore-forming and nonsporulating bacterial entomopathogens are being used or evaluated for controlling pests. Pseudomonadaceae and the Enterobacteriaceae families are nonspore forming species. These are being used as potential biocontrol agents. The spore formers Bacillus popilliae and Bacillus thuringiensis (Bt) are popular insecticides. In the United States, before a pesticide can be marketed and used, the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) requires that EPA examines the proposed pesticide to make sure that its use will not create any annoying risk human health and the environment.

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TABLE 6.20  Biopesticides from microorganisms. Fungicides Pseudomonas fluorescens Bacillus subtilis Fusarium proliferatum Trichoderma viride Trichoderma harzianum Insecticides Bacillus thuringiensis var. kurstaki Metarhizium anisopliae Beauveria bassiana Verticillium lecanii Ha-NPVS L-NPV Larvicides Bacillus thuringiensis Based on www.agrilife.in

TABLE 6.21  Advantages of using biopesticides. Biopesticides are generally less toxic in comparison with conventional pesticides Biopesticides are specific and affect the target pest and closely related organisms, contrary to broad spectrum, conventional pesticides Biopesticides are usually effective in small doses and decompose rapidly, resulting in reduced exposures and mostly avoiding the problems caused by traditional pesticides When used as a component of Integrated Pest Management programs, biopesticides can reduce the use of traditional pesticides, while crop yields remain high Based on www.epa.gov

Biochemical pesticides control pests by nontoxic mechanisms. These are natural substances such as plant extracts, fatty acids, or pheromones. On the other hand, traditional pesticides are usually synthetic materials that directly kill or inactivate the pest. Biochemical pesticides include substances, such as insect sex pheromones and several scented plant extracts. Insect sex pheromones interfere with mating, and the scented plant extracts attract insect pests to traps. As it is sometimes difficult to determine whether a substance is meeting the criteria for classification as a biochemical pesticide, a special committee may be formed for making decisions for determining whether a pesticide meets the criteria for a biochemical pesticide (Burges, 1998; Burges et al., 2001; Sarwar et al., 2012, 2013).



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TABLE 6.22  Few commercial biocontrol products for use against soilborne crop diseases. Ampelomyces quisqualis M-10 AQ 10 biofungicide Biofungicide Powdery mildew, cucurbits, grapes, ornamentals, strawberries, tomatoes Ecogen Inc., USA Candida oleophila I-182 Aspire Botrytis, Penicillium Citrus, pome fruit Ecogen Inc., USA Fusarium oxysporum (nonpathogenic) Biofox C Fusarium oxysporum Basil, carnation, cyclamen, tomato SIAPA, Italy Trichoderma harzianum and T. polysorum Binab T Wilt and root rot pathogens, wood decay pathogens Fruit, vegetables, flowers, ornamentals, turf BioInnovation, Sweden Coniothyrium minitans Contans Sclerotinia sclerotiorum and S. minor Canola, sunflower, peanut, soybean, lettuce, bean, tomato Prophyta, Biologiscare, Planzenschutz, Malchow/Poel, Germany Fusarium oxysporum (nonpathogenic) Fusaclean Fusarium oxysporum Basil, carnation, tomato, cyclamen, gerbera, Fusarium oxysporum Basil, carnation, tomato, cyclamen, gerbera, Natural Plant Protection Natural Plant Protection, Nogueres, France Pythium oligandrum Polygandron Pythium ultimum Sugar beet Plant Protection Institute, Slovak Republic Continued

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TABLE 6.22  Few commercial biocontrol products for use against soilborne crop diseases—cont’d Trichoderma harzianum and T. viride Promote Pythium, Rhizoctonia, Fusarium Greenhouse nursery transplant seedlings; tree and shrub transplants JH Biotech, USA Trichoderma harzianum RootShield, Bio-Trek T-22G, Planter Box Pythium, Rhizoctonia, Fusarium, Sclerotinia homoeocarpa Corn, cotton, cucumber, bean, ornamentals, potato, soybean, cabbage, tomato, turf Bioworks, USA Phlebia gigantean Rotstop Heterobasidium annosum Trees Kemira Agro Oy, Finland Gliocladium virens GL-21 SoilGard (formerly GlioGard) Damping-off and root pathogens, Pythium, Rhizoctonia Ornamentals and food crops grown in greenhouses, nurseries, homes, interiorscapes Thermo Trilogy, USA Trichoderma harzianum Trichodex Botrytis cinerea, Colletotrichum, Monilinia laxa, Plasmopara viticola, Rhizopus stolonifer, Sclerotinia scelrotiorum Cucumber, grape, nectarine, soybean, strawberry, sunflower, tomato Makhteshim Chemical Works, Israel Trichoderma harzianum and T. viride Trichopel, Trichoject Armillaria, Botryosphaeria, Fusarium, Nectria, Phytophthora, Pythium, Rhizoctonia Agrimm Technologies, New Zealand Based on Pimentel (2002)

Microbial pesticides are able to control several different kinds of pests, although each separate active ingredient is relatively specific for its target pest, for instance, there are fungi which are able to control certain weeds, and other fungi that kill specific insects. The most popular microbial pesticides are different types of the bacterium Bacillus thuringiensis, or Bt, which can control certain insects in cabbage, potato and other crops. The Bt produces a protein which is detrimental to specific insect pest. Certain other microbial pesticides act by out-competing pest organisms. Each strain of this bacterium produces a different mixture of proteins, and specifically kills one or a few related species of insect larvae. Whereas some Bts control moth larvae found on plants, other are specific for larvae of flies and mosquitoes. The target insect species are determined by whether the particular Bt produces a protein which can



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bind to a larval gut receptor, thus causing the insect larvae to starve. Microbial pesticides should be continuously ­monitored for ensuring that these do not become capable of harming non target organisms, including humans. (www.ijeart.com; Pal and Kumar, 2013;Crickmore, 2006; Pigott and Ellar, 2007; Perez-Guerrero et al., 2012)

Bacterial biopesticides are most widely used and inexpensive in comparison with the other methods of pest bioregulation. Insects can be infected with various bacterial species, but Bacillus sp. are most widely used as pesticides. Bt has developed several molecular mechanisms for producing pesticidal toxins; most of these are coded for by many cry genes (Schnepf et al., 1998). Since the discovery as a microbial insecticide, Bt has been widely used to control insect pests important in agriculture, forestry and health. Its principal characteristic is the synthesis, during sporulation of a crystalline inclusion containing proteins known as endotoxins or Cry proteins, which have insecticidal properties. To date, over one hundred Bt based bioinsecticides have been developed, which are mostly used against lepidopteran, dipteran and coleopteran larvae. In addition, the genes that code for the insecticidal crystal proteins have been successfully transferred into different crop plants, which have led to significant economic benefits. Because of their high specificity and the safety in the environment, Bt and Cry proteins are efficient, safe and sustainable alternatives to chemical pesticides for the control of insect pests. The toxicity of the Cry proteins have traditionally been explained by the formation of transmembrane pores or ion channels that lead to osmotic cell lysis. In addition to this, Cry toxin monomers also seem to promote cell death in insect cells through a mechanism involving an adenylyl cyclase/PKA signalling pathway. However, despite of this entomopathogenic potential, controversy has arisen regarding the pathogenic lifestyle of Bt. Some reports claim that Bt requires the co-operation of commensal bacteria within the insect gut to be fully pathogenic. (www.ijeart.com; Roh et al., 2007; de Maagd et al., 2003; Kiliç and Akay, 2008).

Biopesticides used for controlling crop diseases have already established themselves on a several crops, for instance, biopesticides already play an important role in controlling of downy mildew diseases. Their advantages include the ability to use under moderate to severe disease pressure, and the ability to use as a tank mix or in a rotational program with other registered fungicides. Because some market studies estimate that as much as 20% of global fungicide sales are directed at downy mildew diseases, the integration of biofungicides into production has substantial advantages in terms of extending the useful life of other fungicides, particularly those in the reduced risk category. A major growth area for biopesticides is in the area of seed treatments and soil amendments. Fungicidal and biofungicidal seed treatments are used for controlling soil borne fungal pathogens that cause seed rots, damping-off, root rot and seedling blights. These can also be used for controlling internal seed borne fungal pathogens and also fungal pathogens that are on the surface of the seed. Several biofungicidal products also show the ability to stimulate plant host defense and other physiological processes which can make treated crops more resistant to several biotic and abiotic stresses. (www.ijeart.com; Francis et al., 2011; Matthews et al., 2014)

The impact of biopesticides on the water quality and environment is less and are ecofriendly option to chemical insecticides. These can be also used where pests have developed resistance (Moazami, 2007; Sarwar, 2012, 2013, 2014, 2015). Since 1920s, Bt has been used as an insecticide spray and is mostly used in organic farming. Bt is the source of the genes used for genetically modifying several food crops so that they produce their own toxins to discourage several insect pests. The toxin is fatal to many insects, including Lepidoptera (butterflies, moths, and skippers), Diptera (flies), and Coleoptera (­beetles), though several types of Bt strains are available for making its use more specific (www.britannica.com). Japanese scientist in 1901 discovered Bt. He investigated the reduction in silkworm moth populations, which he attributed to the rod-shaped, gram-positive bacterium. A German scientist rediscovered Bt in 1911. A solution of crystallized Bt was found to be very effective against certain crop pests, which included the following:

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• Corn borer • Corn rootworm • Corn earworm • Bollworms The product was first used on a commercial scale as an insecticide spray in 1958 in the United States, and many different strains of the bacterium are used for controlling several agricultural insect pests and their larvae. Bt is used in crops, as a spray, or, sometimes, in granular form. Also, Bt can be applied to the crops directly by using genetic engineering. Bt crops are engineered for producing a protein toxic to specific insects and are applied in areas where the infestations of the targeted pests is high. United States Environmental Protection Agency (EPA) first approved the use of this technology in 1995. Since then, the production of Bt corn, cotton, potatoes, and rice has increased significantly in several countries, although plantings usually fluctuate depending on the level of pest infestation. Susceptible insects must ingest Bt crystals for getting affected. Chemical insecticides target the nervous system, but Bt acts by producing a protein that blocks the digestive system of the insect, effectively starving it. Bt acts fast. Infected insect stops feeding within hours of ingestion and dies, from starvation or a rupture of the digestive system, within days. Bt strain is found to be effective against a narrow range of insects whether used in spray form or through genetic engineering. Commonly used strain of Bt are kurstaki, or Btk. These target only certain species of caterpillars. Since the late 1970s, Bt strains (e.g., israelensis, or Bti) have been developed, which control certain types of fly larvae, including those of mosquitoes, black flies, and fungus gnats. Other strains include san diego and tenebrionis. These are found effective on certain leaf beetles, like the Colorado potato beetle and elm leaf beetle (www.britannica.com). Most insecticides usually target a broad spectrum of species, including both pests and beneficial insects, but Bt is toxic to a narrow range of insects. Bt does not have any harmful effect on the natural enemies of insects. It also does not harm the honey bees and other pollinators crucial to agroecological systems. Bt is used for integrated pest management by organic farmers. It is able to integrate well with other natural controls. The use of insect-resistant Bt plants may reduce the use of chemical insecticides, which are very toxic and costly. Use of traditional pesticides recommended for controlling the European corn borer, for instance, reduced by ~30% after Bt corn, was introduced. Although Bt is harmful to certain insects, its use as an insecticide or consumed with GMO food crops is nontoxic to mammals because they are lacking the digestive enzymes required to activate the Bt protein. But any introduction of new genetic material is potentially a source for allergens. Certain strains of Bt have not been approved for human consumption. Bt is susceptible to degradation by sunlight, which is one of the disadvantages. Most of the formulations are found to remain on foliage less than a week following application. Few new strains developed for leaf beetle control become ineffective in just 1 day. Few additives, such as sticking or wetting agents, are found useful in a Bt application. These additives improve the efficacy by covering foliage more thoroughly and to resist washing off. As Bt crops kill a narrow spectrum of the insects that attack them, additional insecticides are generally needed for protecting the plants by other pests. Furthermore, the potential for insects in developing a resistance to the toxin as a result of repeated exposure is of great concern in the extensive cultivation of Bt crops. This resistance would make useless one of the most eco-friendly insecticides in use today. Certain moth and cotton pest populations have already developed



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resistance. “Risk management strategies include the planting of refuges, such as a plot of non-Bt corn near a field planted in Bt corn, for maintaining a local population of insects which remain susceptible to Bt toxin. There is some degree of uncertainty about the impact of Bt proteins on the environment. According to the EPA, more research is required to study Bt protein accumulation in soils, the risks posed to nontarget organisms, and the likelihood of gene flow from Bt crops to wild relatives. A 2003 study from the Ohio State University, in which wild sunflowers were experimentally cross pollinated with genetically modified Bt sunflowers, suggests that modified genes in cultivated crops may drift into closely related populations and increase the hardiness of these plants, including potential weed species” (www.britannica.com). Almost 90% of the microbial biopesticides presently available are obtained from Bt (Kumar and Singh, 2015). At present, biopesticides are composed of a small share of the total crop protection market worldwide, with a value of about $3 billion, accounting for about 5% of the total crop protection market (Marrone, 2014; Olson, 2015). More than 200 products are available in the United States, in comparison with 60 similar products in the European Union (EU) market (Kumar and Singh, 2015). Although the use of biopesticide on a global scale is increasing by about 10% every year (Kumar and Singh, 2015), it looks that the global market should increase further in the future if these pesticides are to play a visible role in replacing the chemical pesticides and reducing our dependence on them. Biopesticides are examined in the EU by the same regulations used for the assessment of synthetic active substances, and this situation needed many new provisions in the existing legislation and also the preparation of new guidelines facilitating the registration of prospective biopesticide products (Czaja et al., 2015). Presently, there are few biopesticide active substances registered in the EU in comparison with the United States, India, Brazil, or China. The relatively low level of biopesticide research in the EU is related to the greater complexity of EU-based biopesticide regulations (Balog et al., 2017). Growth of biopesticides surpassed that of traditional pesticides, with compounded annual growth rates of more than 15% (Marrone, 2014). Biopesticides are expected to equalize with chemical pesticides, in terms of market size, between the late 2040s and the early 2050s, but main uncertainties in the uptake rates, particularly in Africa and Southeast Asia, account for a major portion of the flexibility in these projections (Olson, 2015). Biopesticides are considered safer than traditional pesticides. Biopesticides are by their nature less harmful and are more specific to the target pests. Furthermore, biopesticides are effective in small amounts, and decompose rapidly, without leaving problematic residues; so, the use of conventional pesticides can be reduced. The development of the biopesticide market in the future is related to research on biological control agents. Many researchers have done some preliminary research in this area but complete and systematic reports are scanty. Therefore, it is important to strengthen the collaboration of enterprises and research institutes in this area. Biopesticides cannot as yet completely replace chemical pesticides, so the agricultural sector must benefit from the co-existence of biopesticides with chemical pesticides. Increasing practical application of research results is expected to facilitate large scale industrial development. The pipeline of new chemistry has dropped substantially in recent decades, as regulations have become stricter, with products being withdrawn from the market as they do not meet the strict regulations. So, a more limited choice of chemical solutions remains focused on several pests in few staple crops. These consequences, which have always been apparent in the pesticide market, are now more evident than ever. (www.mdpi.com)

Several new substances have been explored as promising compounds for use as biopesticides (Table 6.23), but more research is needed to assess their effectiveness on specific pest problems under different cropping systems.

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TABLE 6.23  Substances with pest control properties. Strains of the fungus Talaromyces flavus SAY-Y-94-01 Anthracnose caused by Glomerella cingulata and Colletotrichum acutatum Extract of the species Clitoria ternatea (butterfly pea) Helicoverpa spp. Products of the fungus Trichoderma harzianum Fusarium root rot Bacillus thuringiensis var. tenebrionis strain Xd3 (Btt-Xd3) Alder leaf beetle (Agelastica alni) Alkaloid compound oxymatrine Spodoptera litura Helicoverpa armigera, Aphis gossypii Fermentation products of the bacterium Lactobacillus casei strain LPT-111 angular leaf spot caused by Xanthomonas fragariae Stilbenes isolated from grapevine extracts Spodoptera littoralis Olive mill waste Various pests Based on Damalas and Koutroubas (2018); Ishikawa (2013); Mensah et al. (2014); Kirk and Schafer (2015); Eski et al. (2017); Rao and Kumari (2016); Dubois et al. (2017); Pavela et al. (2017); El-Abbassi et al. (2017)

6.5 Biocolorants Biocolorants are produced by industrial biotechnology, particularly for food, pharmaceutical, or cosmetic applications. These substances can be produced by chemical synthesis and also industrial biotechnology. The production costs are comparable. Biocolorants produced using biotechnology show an important benefit because consumers do not like the synthetic substances (biotechsupportbase.com). Color is an important feature of food, which determines its appeal to the consumers. Biocolorants are mostly obtained from pigments such as anthocyanidin and carotenoids, but there are certain biocolorants, which are not pigments in any state such as light-emitting luciferin. The reasons for adding color to food are shown in Table 6.24 (Rymbai et al., 2011; FNB, 1971). The chemistry of natural colors has become an important part of any commodity (Clydesdale, 1993). The availability and use of natural colorants are increasing to a great extent as a result of consumer preference and also legislative action, which has continued the delisting of approved artificial dyes (Dweck, 2009; Garcia and Cruz-Remes, 1993). The consumer preference for natural colorants is mostly due to the reason that these are healthy and are of good quality. Furthermore, the taste of synthetic colorants is not good, and synthetic colorants are harmful to human beings, because these cause allergenic and intolerance reactions (Blenford, 1995). So, there has been a great interest in the development of natural food colorants (Francis, 1987; Lauro, 1991). The use of food colorants is advantageous for food manufacturers and also



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TABLE 6.24  Possible reasons for the use of colorants in food substances. To replace color in the food, which is lost during processing To enhance color of the food already present To minimize batch-to-batch variations To color otherwise uncolored food To supplement food with nutrients To maintain the original food appearance even after processing and during storage To assure the color uniformity for avoiding seasonal variations in color tone To intensify normal color of food and thus to maintain its quality To protect the flavor and light susceptible vitamins making a light-screen support To increase acceptability of food as an appetizing item Rymbai et al. (2011) and FNB (1971).

consumers in determining the acceptability of processed food (Spears, 1988; Griffiths, 2005). Approximately 43 colorants have been authorized by European Union (EU) as food additives, whereas, in the United States, approximately 30 color additives are approved (Wissgott and Bortlik, 1996). In Europe and the United States, most of the listed color additives are obtained from natural sources (Mapari et al., 2005). In these days, natural colorants are becoming the major alternatives to synthetic colorants (Roy et al., 2008; sphinxsai.com). Several biocolorants exist in nature, but quite a small number of them are available from natural extracts (Chattopadhyay et al., 2008). So, biotechnology can be a solution to provide coloring compounds that are difficult to synthesize by conventional methods. Over the last few years, efforts have been made to produce biocolorants through biotechnology. For this purpose, plants and microorganisms are more suitable because cultural techniques and processing are well understood (sphinxsai.com; Aberoumand, 2011). Table 6.25 shows naturally derived colors from plants sources, and Table 6.26 shows naturally derived colors from microorganism. Table 6.27 lists bacteria, fungi, yeast, algae, and actinomycetes producing pigments, and Fig. 6.3 shows structure of food-grade pigments. TABLE 6.25  Naturally derived colors from plants sources. Plant sources

Pigments

Color/appearance

Turmeric

Curcumin

Bright lemon yellow

Tomatoes

Lycopene

Orange red

Black grape skin, elderberries, black carrots, red cabbage

Anthocyanin

Pink/red to mauve

Red table beet root

Betanin

Pink to red

Turmeric

Curcumin

Bright lemon Yellow

Marigold and alfalfa

Lutein

Golden yellow

Palm oil

Natural mixed carotenes

Golden yellow to orange

Bixa orellana

Bixin/norbixin

Orange

Paprika/Capsicum annuum

Capsanthin/casorubin

Reddy orange

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TABLE 6.26  Naturally derived colors from microorganisms. Microorganisms

Pigments

Color/appearance

Serratia marcescens

Prodigiosin

Red

Staphylococcus aureus

Zeaxanthin

Golden yellow

Blakeslea trispora

Lycopene β-carotene

Red yellow-orange

Flavobacterium spp.

Zeaxanthin

Yellow

Dunaliella salina

β-Carotene

Cream

Pseudomonas aeruginosa

Pyocyanin blue

Green

Rhodotorula sp. Rhodotorula glutinis

Torularhodin

Orange red

Monascus roseus

Canthaxanthin

Orange pink

Based on Heer and Sharma (2017); Dufosse (2009); Venil and Lakshmanaperumalsamy (2009).

TABLE 6.27  Microorganisms producing pigments. Microorganism(s)

Pigments/molecule

Color/appearance

Agrobacterium aurantiacum

Astaxanthin

Pink red

Paracoccus carotinifaciens

Astaxanthin

Pink red

Bradyrhizobium sp.

Canthaxanthin

Dark red

Flavobacterium sp., Paracoccus zeaxanthinifaciens

Zeaxanthin

Yellow

Bacteria

Achromobacter

Creamy

Bacillus

Brown

Brevibacterium sp.

Orange yellow

Corynebacterium michiganense

Grayish to creamish

Corynebacterium insidiosum

Indigoidine

Blue

Rugamonas rubra, Streptoverticillium rubrireticuli, Vibrio gaogenes, Alteromonas rubra

Prodigiosin

Red Bluish red

Rhodococcus maris Xanthophyllomyces dendrorhous

Astaxanthin

Pink red

Haloferax alexandrinus

Canthaxanthin

Dark red

Staphylococcus aureus

Staphyloxanthin, zeaxanthin

Golden yellow

Chromobacterium violaceum

Violacein

Purple

Serratia marcescens, Serratia rubidaea

Prodigiosin

Red

Pseudomonas aeruginosa

Pyocyanin

Blue green

Xanthomonas oryzae

Xanthomonadin

Yellow

β-Carotene

Red

Algae Dunaliella salina



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TABLE 6.27  Microorganisms producing pigments—cont’d Microorganism(s)

Pigments/molecule

Chlorococcum

Lutein

Haematococcus

Canthaxanthin

Color/appearance

Fungi Aspergillus sp.

Orange red

Aspergillus glaucus

Dark red

Blakeslea trispora

β-Carotene

Cream

Helminthosporium catenarium

Red

Helminthosporium avenae

Bronze

Penicillium cyclopium

Orange

Penicillium nalgeovensis

Yellow

Fusarium sporotrichioides

Lycopene Red

Haematococcus pluvialis Monascus sp.

Monascorubramin, Rubropunctatin

Red orange

Monascus purpureus

Monascin Ankaflavin

Red yellow

Monascus roseus

Canthaxanthin

Orange pink

Monascus sp.

Ankaflavin

Yellow

Penicillium oxalicum

Anthraquinone

Red

Blakeslea trispora

Lycopene

Red

Cordyceps unilateralis

Naphtoquinone

Deep blood red

Ashbya gossypii

Riboflavin

Yellow

Mucor circinelloides, Neurospora crassa, and Phycomyces blakesleeanus

β-Carotene

Yellow orange Red

Penicillium purpurogenum, Paecilomyces sinclairii Paecilomyces farinosus

Anthraquinone

Red

Yeast Cryptococcus sp.

Red

Saccharomyces neoformans var. nigricans

Melanin black

Phaffia rhodozyma

Astaxanthin

Pink red

Rhodotorula sp., Rhodotorula glutinis

Torularhodin

Orange red

Yarrowia lipolytica

–

Brown

Streptoverticillium rubrireticuli

Prodigiosin

Red

Streptomyces echinoruber

Rubrolone

Red

Actinomycetes

Reproduced with permission from Malik, K., Tokkas, J., Goyal, S., 2012. Microbial pigments: a review. Int. J. Microb. Res. Technol. 1 (4), 361–365.

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FIG. 6.3  Structure of food-grade pigments. Reproduced with permission from Venil, C.K., Zakaria, Z.A., Ahmad, W.A., 2013. Bacterial pigments and their applications. Process Biochem. 48 (7), 1065–1079.



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β-Carotene (or provitamin A) is produced by organic synthesis and also by extraction from roots and also by fermentation with the fungus Blakeslea trispora. Zeaxanthin and astaxanthin are optically active hydroxycarotenoids. These are important as animal and human food, and are mostly used in the animal feed industry and also for fish. Astaxanthin is a pink pigment, it is added to the feed of sea-farm raised salmon for obtaining the beautiful pink salmon meat. Salmons get the pigment from their natural diet. Astaxanthin has been mostly produced by a chemical method, using a complex synthesis route. This route uses a combination of chemical and enantio-selective bioconversion steps. In the recent years there has been an increasing interest in the production of this pigment with yeast Xanthophyllomyces rhodozyma. The synthetic variant has been criticized because it differs slightly from natural astaxanthin. Phycocyanin which is a blue pigment is produced in Japan with Spirulina sp. Also the orange red food/drink pigment – monascin – is produced with the fungus Monascus purpureus using a fermentation process. (biotechsupportbase.com)

A bacteria, Bradyrhizobium sp., produces canthaxanthin (4,4′-diketo-β-carotene), and the carotenoid gene cluster was fully sequenced (Lorquin et al., 1997; Hannibal et al., 2000). This was also found in Halobacterium (Asker and Ohta, 1999). Flavobacterium sp. was found to produce zeaxanthin @ 190mg/L, with a cell concentration of 16-mg/g dried cellular mass (Shepherd et al., 1976). The nutrient medium contained glucose or sucrose and amino acids— methionine, cystine or cysteine, and pyridoxine—and bivalent metal ions. The alga Haematococcus lacustris is used for the production of astaxanthin in fermentors on a commercial scale (Yuan et al., 1997). Besides, echineone and canthaxanthin are also identified in Haematococcus cultures. Astaxanthin production needed high level of oxygen and high C/N ratio, but for cell growth, low C/N ratio is needed (Yamane et al., 1997; Chumpolkulwong et al., 1997a). Use of ethanol during the second stage increased the production of astaxanthin 2.2 times, while in contrast compactin-resistant mutants of Haematococcus pluvialis showed two times increase in yield (Chumpolkulwong et al., 1997b). Compactin inhibits HMGR which strongly blocks cholesterol formation. Many species of Dunaliella bardawil and Dunaliella salina produce β-carotene as their major carotenoid (Phillips et al., 1995). The fungus Blakeslea trispora is known to produce β-carotene (Chattopadhyay et al., 2008). The cell growth and β-­carotene production are increased in medium containing surfactants such as Span or Triton, except Triton X-100 (Kim et  al., 1997). Phycomyces blakesleeanus is another interesting fungus for β-­carotene production (Ootaki et al., 1996). In Blakeslea trispora, sexual stimulation of carotene biosynthesis remains important for increasing yield up to 35mg/g (Mehta et al., 1997). Many strains of Monascus are being used for commercial production of red and/or yellow pigments (Fabre et al., 1993).The red yeast, Xanthophyllomyces dendrorhous synthesizes astaxanthin and zeaxanthin as its main carotenoids (Andrews et al., 1976; Roy et al., 2008). Commercial production of carotenoids has been obtained in the case of astaxanthin, by red yeast fermentation (Chattopadhyay et al., 2008). Yeasts of the genus Rhodotorula including species R. glutinis, R. gracilis, R. rubra, and R. graminis can produce carotenoids (Chattopadhyay et al., 2008; Sakaki et al., 2000; Simova et al., 2004; Tinoi et al., 2005). The Czech Republic's Ascolor Biotech has been awarded patents of compounds from new strains of fungi that produce a red colorant for use in the food and cosmetic industries (Chattopadhyay et al., 2008). Penicillium oxalicum var. Armeniaca CCM 8242, obtained from soil, produces anthraquinone type of chromophore. Arpink red (red colorant) was recommended as 100mg/kg in meat products are in nonalcoholic drinks, 200mg/kg in alcoholic drinks, 150mg/kg in milk products including ice creams, and 300mg/kg in confectionery items (Sardaryan et al., 2004).

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Microbial pigments are produced either by solid substrate fermentation (SSF) or by submerged fermentation. In SSF, the microbial pigment biomass is formed on the surface of a solid (Tuli et al., 2014). This SSF method has several advantages including savings in waste water and higher yield of the metabolites. Contrarily, microorganisms are cultivated in liquid medium aerobically with suitable agitation to get homogenous growth of cells and media components in submerged fermentation method. The effect of several process parameters such as carbon source, nitrogen source, temperature, pH, and aeration rate for pigment production has been studied. But because of the high cost of using synthetic medium, there is a requirement for developing new low-cost and extraction process for the production of pigments. Efforts are underway to use the agro-industrial waste for large-scale production of microbial pigments. Research has been conducted on the production of carotenoids from agro-industrial waste such as whey, apple pomace, spent grain, and crushed pasta. So, such agro-industrial waste utilization procedures reduce the production cost and also act as an effective waste management tool (Araujo et al., 2010; Grossart et al., 2009; Cho et al., 2002; Vasanthabharathi et al., 2011; Lampila et al., 1985). The most common method for the production of plant pigments is cell culture. With this method, continuous production and uniform quality of pigment are produced. Biocolors have wide applications as colorants in various industries (Table 6.28). Table 6.29 shows application of bacterial pigments, Table 6.30 shows production of food colors from plants using biotechnological methods, and Table 6.31 shows important features of some biocolors. The benefits and limitations of biocolorants are presented in Tables 6.32 and 6.33. TABLE 6.28  Application of biocolors. Pharmaceutical industry Dairy industry Textile industry Nutritional supplements Printing industry Food colorants Based on Parmar, M., Phutela, U.G., 2015. Biocolors: the new generation additives. Int. J. Curr. Microbiol. App. Sci. 4 (7), 688–694.

TABLE 6.29  Application of bacterial pigments. Bacteria

Pigment (color)

Application

Agrobacterium aurantiacum, Paracoccus carotinifaciens, Xanthophyllomyces dendrorhous

Astaxanthin (pink red)

Feed supplement

Rhodococcus maris

Beta-carotene (bluish red)

Used to treat various disorders such as erythropoietic protoporphyria, reduces the risk of breast cancer

Bradyrhizobium sp., Haloferax alexandrines

Canthaxanthin (dark red)

Colorant in food, beverage, and pharmaceutical preparations

Corynebacterium insidiosum

Indigoidine (blue)

Protection from oxidative stress



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TABLE 6.29  Application of bacterial pigments—cont’d Bacteria

Pigment (color)

Application

Rugamonas rubra, Streptoverticillium rubrireticuli, Vibrio gaogenes, Alteromonas rubra, Serratia marcescens, Serratia rubidaea

Prodigiosin (red)

Anticancer, immunosuppressant, antifungal, algicidal; dyeing (textile, candles, paper, and ink)

Pseudomonas aeruginosa

Pyocyanin (blue green)

Oxidative metabolism, reducing local inflammation

Chromobacterium violaceum, Janthinobacterium lividum

Violacein (purple)

Pharmaceutical (antioxidant, immunomodulatory, antitumoral, and antiparasitic activities); dyeing (textiles), cosmetics (lotion)

Flavobacterium sp., Paracoccus zeaxanthinifaciens, Staphylococcus aureus

Zeaxanthin (yellow)

Used to treat different disorders, mainly with affecting the eyes

Xanthomonas oryzae

Xanthomonadin (yellow)

Chemotaxonomic and diagnostic markers

Reproduced with permission from Venil, C.K., Zakaria, Z.A., Ahmad, W.A., 2013. Bacterial pigments and their applications. Process Biochem. 48 (7), 1065–1079.

TABLE 6.30  Production of food colors from plants using biotechnological methods. Plant source

Method

Biocolorants (food grade)

Perilla frutescens

Cell culture

Anthocyanin

Vitis vinifera Aralia cordata Aralia cordata Fragaria anansa

Cell culture

Anthocyanin

Daucus carota

Cell culture

Anthocyanin

Crocus sativus

Somatic embryogenesis

Crocin

Beta vulgaris

Cell cultures Root cultures

Betalain, betacyanin, betaxanthins (portulaxanthin-II and vulgaxanthin-I), muscaauri-VII, dopaxanthin, and indicaxanthin

Kakegawa et al., 1995; Zhang et al., 1997; Suvarnalatha et al., 1994a; Zhong et al., 1991; Vogelien et al., 1990; Leathers et al., 1992; Kino-Oka and Tone, 1996; Chattopadhyay et al., 2008; Siva and Krishnamurthy, 2005.

TABLE 6.31  Important features of some biocolors. β-carotene Dunaliella salina, Euglena, Blakeslea trispora (yellow to orange depending upon color formulations). It is sparingly oil soluble and comprises all the transisomers and possesses provitamin A activity Astaxanthins Haematococcus pluvialis (Orange pink to red) Continued

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TABLE 6.31  Important features of some biocolors—cont’d Astaxanthin belong to the carotenoid family. Astaxanthin can protect against chemically induced cancers and is very strong antioxidant Phycobiliproteins Algae belonging to Rhodophyta and Chlorophyta (red and blue) These have good long-term stability when stored and refrigerated (2–5°C) as ammonium sulfate precipitates. These are relatively stable at room temperature and neutral pH Monascus pigments Monascus purpureus and M. anka (yellow, orange, and red) Pigment production and quality are good when the organism is provided with carbon source such as maltose, fructose, and glucose and yeast extracts as nitrogen source. Pigments are stable to pH change in temperature Based on Parmar, M., Phutela, U.G., 2015. Biocolors: the new generation additives. Int. J. Curr. Microbiol. App. Sci. 4 (7), 688–694.

TABLE 6.32  Benefits of biocolorants. Environmentally friendly and can be recycled after use Less toxic, less polluting, less health hazardous, noncarcinogenic, and nonpoisonous Prevent chronic diseases such as prostate cancer They are harmonizing colors, gentle, soft and subtle and create a restful effect. Most of them are water soluble (anthocyanins), which facilitates their incorporation into aqueous food systems Possess potent antioxidant and improve visual acuity properties Possess antineoplastic, radiation protective, vasotonic, vasoprotective, anti-inflammatory, and chemo- and hepatoprotective activities Based on Siva and Krishnamurthy (2005); Clinton (1998); Tsai et al. (2002); Wang (1997); Mazza and Miniati (1993).

TABLE 6.33  Limitations of biocolorants. Cost of dyeing with natural dyes is considerably higher than synthetic dyes due to tedious extraction procedures of coloring component from the raw material, low color value, and longer time Some of the natural dyes are fugitive and need a mordant for the enhancement of their fastness properties, while some of the metallic mordents are hazardous Difficulty in the collection of plants, lack of standardization, lack of availability of precise technical knowledge of extracting, and dyeing technique and species availability Sensitivity to temperature, oxygen, light, and pH so instable during processing They can also be decolorized or degraded during storage Based on Rymbai, H., Sharma, R., Srivastav, M., 2011. Biocolorants and its implications in health and food industry—a review. Int. J. PharmTech Res. 3 (4), 2228–2244, ISSN: 0974-4304.



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The major markets of food-grade biocolorants are in the United States, EU, and Japan and emerging markets are in China, India, and South Korea. Food colorants have been studied for biosafety before its promotion and are controlled by several regulatory bodies around the world, and regulation varies in different countries (Hallagan et  al., 1995). “The legislation specifies which colorant may be used - the source(s) of the colorant, the purity of the colorant, to which foods the colorant may be added, and at what level the colorant may be added to a specific food” (sphinxsai.com). Most of the food grade biocolorants approved by FDA or EU are also approved by other agencies. In India, Rule 26 of The Prevention of Food Adulteration Act (PFA) allows 11 colors for food use: Lactoflavin, Caramel, Annato, Saffron, Curcumin, etc., also approved by EU and FDA. (Chattopadhyay et al., 2008; sphinxsai.com) Under FDA regulations, a colorant added to a food product cannot be considered natural, no matter what the source is. Unless the colorant is natural to the food product itself, for example; strawberry juice or red beet color is used to make the ice cream a pink hue for strawberry ice cream, it would not be considered as naturally colored, as the colorant from strawberry or beet are not a natural component of ice cream. FDA considered only few colorants as food additives. (FDA/IFIC, 1993; www.academicjournals.org)

The preference of the consumers for natural colorants over synthetic ones started with the green movement of the 1960s and shows no trend of decreasing. This may result from a perceived uneasiness with the safety of the colorants on the part of the consumer, but another important factor is that most governments allow more flexibility and leniency in the use of natural colorants. Production of colors by fermentation has several benefits: • • • • •

Cheaper production Easier extraction Higher yield No lack of raw materials No seasonal variations

There is a lot of interest using bacteria as a possible alternate source of colorants used in foods, textile, pharmaindustry, etc. In this direction, biotechnology is expected to play an important role for large-scale fermentation of biocolorants.

6.6  Flavors and aroma compounds Flavors and fragrances are playing a predominant role in our daily lives. They are present in food and in cosmetics. Also, there is an increase in demand for natural ingredients instead of chemicals. Flavoring compounds are significantly used in food, perfumery, and pharma industries (Abraham and Berger, 1994; Chang et  al., 1995; Gatfied, 1988; Guenther, 1966; Hagedorn and Kaphammer, 1994; Molinari et  al., 1995; Tripathi et  al., 1997; Van Rensburg et  al., 1997). Generally, the main sources of natural flavor compounds are plants. Some of these are also chemically synthesized (www.ftb.com.hr). Plant cell culture is a viable method for producing flavors and aromas (Table 6.34). This approach is based on the distinctive biochemical and genetic capability and the totipotency (ability of a cell to produce unlike cells and so to develop a new organism or part) of plant cells (Harlander, 1994; Sahai, 1994; Scragg, 1997). Each cell of a plant culture consists

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TABLE 6.34  Flavors from plant cell culture. 2,3-Butanedione, (E,Z)-2,6-nonadienal and (E,Z)-2,6-nonadien-1-ol

Agastache rugosa

Apple aroma

Malus sylvestris

Cinnamic acid

Nicotiana tabacum

Caryophyllene

Lindera strychnifolia

Basmati flavor

Oryza sativa

Cocoa flavor

Theobroma cacao

Flavanol

Polygonum hydropiper

Garlic

Allium sativum

Monoterpenes

Perilla frutescens

Onion

Allium cepa

Triterpenoid

Glycyrrhiza glabra glandulifera

Vanillin

Vanilla planifolia

Based on Longo and Sanromán (2006); Kim et al. (2001); Drawert et al. (1984); Suvarnalatha et al. (1994a); Townsley (1972); Nakao et al. (1999); Ohsumi et al. (1993); Nabeta et al. (1983); Prince et al. (1997); Ayabe et al. (1990); Dornenburg and Knorr (1996).

the genetic information important for producing several chemical components, which constitute natural flavor. Feeding intermediates of the biosynthetic pathway can increase the production of flavor metabolites by precursor biotransformation. Some researchers presented the benefits of plant cell culture technique over traditional agricultural production (Rao and Ravishankar, 2002; Mulabagal and Tsay, 2004; www.readbag.com). Developments in the area of biotechnology are enabling the production of natural flavors to be performed in an economic and efficient manner. Enzymes are being used in the biotransformation, but whole cells of microorganisms show great promise for biotransformation because the microorganisms can be grown and used in the fermenters easily (Ravid et  al., 1997; Agrawal and Joseph, 2000). Biotechnological products are classified as natural; this is encouraging to use biotransformation systems (de Carvalho et al., 2000). Market study shows that consumers like natural products whereas the artificial products have a negative impact (Sharpell, 1985; Tyrrel, 1990; Kashi et al., 2008). A lot of interest is being shown for natural products. This is pushing the aroma industry to develop new techniques for obtaining natural aroma compounds. Another method for this natural synthesis is based on bioconversion. It is well documented that the production of aroma compounds for the food industry by the use of enzymes or microorganisms offers several benefits over traditional methods. Furthermore, the use of solid-state fermentation (SSF) produces higher yields or better product characteristics in comparison with submerged fermentation with reduced costs (www.ftb.com.hr). Use of biotechnological processes for the synthesis of flavor compounds are playing an important role in the food industry. This is due to the scientific advances in biological processes, using microbes or enzymes as an alternative to chemical synthesis, combined with the developments in analytical methods such as HPLC, GC, IR or mass spectrometry. (Christen and Lopezmunguia, 1994)



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Enzymatic biocatalysts offer several possibilities for producing food flavor. Their specificity allows the production of certain difficult to synthesize chemicals; their stereoselectivity is an important benefit for the food industry where a specific optical conformation may be linked with flavor properties. Enzymes can be used directly as food additives, not only for producing or liberating flavor from precursors but also for correcting bad flavors caused by certain compounds, either occurring naturally or produced during processing (Bigelis, 1992). Whitaker (1990) has discussed the prospective of enzymes in food technology. Enzymes used in flavors can be endogenous, can be inherent to the food, or can come from microbes added to foods or coming from contamination (Christen and Lopezmunguia, 1994). Most of the natural flavor/fragrance chemicals originate from plants and animals. But the quality and the supply of these conventional chemicals are limited. Innovative ways for synthesizing flavor and fragrance chemicals include fermentation and plant tissue culture routes (biotechsupportbase.com). Microorganisms are being used for producing aroma chemicals (Table 6.35). The ability to produce aroma chemicals by fermentation may supplement and improve the quality of plant based flavor/fragrance chemicals. Microbial biotransformation/biosynthesis of flavor and fragrance chemicals offer the advantages of producing optically active isomers which often have significant differences in flavor and fragrance quality and sensory intensity. (biotechsupportbase.com)

“Several enzymes can directly produce flavour molecules by hydrolysis of larger progenitors. Also, developments on biocatalysis in unconventional media have made possible the use of hydrolytic enzymes to catalyze the synthesis of several valuable compounds. This approach can be used for the production of food aromas, as is the case of ester synthesis by lipases in low water-content media” (www.ftb.com.hr; Dordick, 1989). The enzymes used for the production of aroma compounds are presented in Table 6.36. Many microorganisms are able to synthesize potentially valuable flavor compounds and enzymes used in flavor manufacturing. But yields are generally low, which impedes industrial use. In the last decades, there has been an increasing trend toward the use of SSF methods TABLE 6.35  Food aroma compounds produced by microorganisms. Diacetyl Lactones Esters Pyrazines Alcohols Terpenes Benzaldehyde Methyl ketone Vanillin Based on Longo, M.A., Sanromán, M.A., 2006. Production of food aroma compounds. Food Technol. Biotechnol. 44 (3) 335–353.

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TABLE 6.36  Enzymes used for the production of aroma compounds. Lipolytic enzymes Proteases Glucosidases Glutaminases Based on Longo, M.A., Sanromán, M.A., 2006. Production of food aroma compounds. Food Technol. Biotechnol. 44 (3) 335–353.

for producing various bulk chemicals and enzymes (www.ftb.com.hr). Several studies have been performed on SSF production of aroma compounds by microorganisms (Table 6.37). Commercial processes have been developed for producing or biotransforming natural precursors into important flavor/fragrance chemicals through microbial metabolic pathways. These include the following: (1) Production of Tuberose lactone (a new GRAS chemical) via hydroxylation of unsaturated fatty acids and limited β-oxidation of the hydroxylated fatty acids (2) Production of chirally active (R)-styrallyl acetate by regioselective reduction of acetophenone to styrallyl alcohol and subsequent esterification (3) de novo synthesis of chirally pure (+)-jasmonic acid and subsequent esterification to methyl jasmonate. (biotechsupportbase.com) Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the characteristic aroma component of the vanilla pod. It is used in a wide range of flavors for foods, confectionery, and beverages; as a fragrance ingredient in perfumes and cosmetics; and also for pharmaceuticals. Vanillin is produced through chemical synthesis from guaiacol and lignin. The increasing demand of customers for natural flavors has created interest in the production of vanillin from natural sources by biotransformation, which can then be considered as a natural aroma chemical. The production of vanillin from eugenol was developed in 1991. This process was based on a strain of Pseudomonas sp. HR199, which degrades eugenol via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillic acid, and protocatechuic acid (biotechsupportbase.com). Other methods were based on the biotransformation of ferulic acid to vanillin by Pycnoporus cinnabarinus or Amycolatopsis sp. strain HR167. Amycolatopsis sp. strain HR167 is used for the production of vanillin. To use eugenol as an inexpensive resource for providing ferulic acid for this biotransformation, the bacterium Ralstonia eutropha H16 was genetically modified for converting eugenol to ferulic acid (biotechsupportbase.com). Haarmann and Reimer (H&R) are leaders in the flavour field. They started production of natural vanillin. They have developed a fermentation process in which ‘eugenol’ the natural aroma chemical obtained from several plant extracts, including clove oil is used. Eugenol is converted to ferulic acid by oxidation with microorganisms and then ferulic acid is converted to vanillin by using oxidative degradation. The German company BASF started microbial synthesis of 4-decalactone, a peach aroma. It is based on a fermentation process with yeast Yarrowia lipolytica, whereas 12-0H-19 octadecenic acid is released from ricinus oil and subsequently converted to the desired 4-decalactone.

TABLE 6.37  Solid-state fermentation applications in food aroma production. Substrates

Microorganisms

Product Aroma compounds

Semisolid maize

Pediococcus pentosaceus Lactobacillus acidophilus

Butter flavor

Cassava bagasse and giant palm bran

Kluyveromyces marxianus

Fruity aroma

Cassava bagasse, apple pomace, amaranth, and soybean

Ceratocystis fimbriata

Fruity aroma

Pregelatinized rice

Neurospora sp.

Fruity aroma

Miso

Zygosaccharomyces rouxii

FIEMF

Coffee husk

Ceratocystis fimbriata

Pineapple aroma

Soybeans

Bacillus subtilis

Pyrazine

Rice koji

Aspergillus oryzae

Volatile compounds

Tropical agroindustrial substrates

Rhizopus oryzae

Volatile compounds

Coconut coir pith

Aspergillus niger

13-glucosidase

Cranberry pomace

Lentinus edodes

p-glucosidase

Wheat straw

Neurospora crassa

3-glucosidase

Sugar beet pulp

Aspergillus niger

Feruloyl esterase

Wheat bran, maize bran, rice bran, and sugarcane bagasse

Aspergillus flavipes Phanerochaete sp. Trametes sp.

Feruloyl esterase

Corn cobs

Sporotrichum thermophile

Feruloyl esterase and p-coumaroyl esterase

Wheat bran, rice husk, saw dust, coconut oil cake

Vibrio costicola

L-glutaminase

Olive cake and sugar cane bagasse

Rhizopus rhizopodiformis

Lipase

Enzymes

Rhizomucor pusillus Babassu oil cake

Penicillium restrictum

Lipase

Barley bran, triturated nut

Yarrowia lipolytica

Lipase

Coconut oil cake

Candida rugosa

Lipase

Coconut oil cake, groundnut and sesame, bombay rawa, soya beans, wheat rawa

Aspergillus sp.

Lipase

Gingelly oil cake

Aspergillus niger

Lipase

GY P medium

Aspergillus oryzae

Lipase

Peanut press cake

Neurospora sitophila Rhizopus oligosporus

Lipase

Soy cake

Penicillium simplicissimum

Lipase

Vegetable oil refinery residue

Penicillium citrinum

Lipase

Wheat bran

Penicillium candidum

Lipase

Wheat straw

Penicillium pinophilum

Phenolic acid esterase

Green gram husk

Bacillus circulans

Protease

Soy cake

Bacillus subtilis

Protease

Wheat bran, rice husk, rice bran, spent brewing grain, coconut oil cake, palm kernel cake, sesame oil cake, jackfruit seed powder, and olive oil cake

Aspergillus oryzae

Protease

Based on Longo and Sanromán (2006); Escamilla-Hurtado et al. (2005); Medeiros et al. (2001); Bramorski et al. (1998); Pastore et al. (1994); Sugawara et al. (1994); Soares et al. (2000); Besson et al. (1997); Larroche et al. (1999); Ito et al. (1990); Christen et al. (2000); Muniswaran et al. (1994); Zheng and Shetty (2000); Macris et al. (1987); Asther et al. (2002); Topakas et al. (2004); Chandrasekaran (1997); Cordova et al. (1998); Gombert et al. (1999); Domínguez et al. (2003); Benjamin and Pandey (1997); Adinarayana et al. (2004); Kamini et al. (1998); Ohnishi et al. (1994); Beuchat (1982); Di Luccio et al. (2004); Miranda et al. (1999); Ortiz-Vazquez et al. (1993); Castanares et al. (1992); Prakasham et al. (2005); Soares et al. (2005); Sandhya et al. (2005).

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Unilever in England produces the butter aroma, R-d-dodecanolide, starting from 5­ -ketododecane acid with the use of baker’s yeast. Butyric acid and its ethyl esters have been produced by fermentation since a long time and are used in cheese aroma and fruit aroma. Lee and Frost (1998) produced vanillin from glucose via the shikimate pathway using genetically modified E. coli in a fed-batch fermentation process. Strain E. coli KL7 with plasmid pKL5.26A or pKL5.97A was used for producing vanillic acid from glucose, which was recovered from the medium and reduced to vanillin by enzyme aryl aldehyde dehydrogenase isolated from Neurospora crassa. Vanillin can be obtained by fermentation using suitable microbes in the stationary growth phase. The microbial route of ferulic acid is recognized as one of the most attracting options for producing natural vanillin. Different types of bacteria are able to metabolize ferulic acid as the carbon source, producing vanillin, vanillic acid and protocatechuic acid as catabolic intermediates. Vanillin is used as fragrance in food preparations, intermediate in the productions of herbicides, antifoaming agents or drugs, ingredient of household products such as air fresheners and floor polishes, and, because of its antimicrobial and antioxidant properties, also as food preservative. (Bajpai, 2013b, 2018; Converti et al., 2003; Torre et al., 2004a,b; De Faveri et al., 2007;Davidson and Naidu, 2000; Gould, 1996; Priefert et al., 2001; Serra et al., 2005; Walton et al., 2003; Burri et al., 1989)

The price of the natural vanillin in comparison with the synthetic vanillin is very high. Therefore, flavor industry is focusing on natural sources (Davidson and Naidu, 2000; Yoon et al., 2005; Gurujeyalakshmi and Mahadevan, 1987). Pseudomonas fluorescens was found to produce vanillic acid from ferulic acid (Andreoni et al., 1995; Barghini et al., 1998), with the formation of vanillin as an intermediate (Narbad and Gasson, 1998). Good yield of vanillin was obtained from ferulic acid by Amycolatopsis sp. (Gurujeyalakshmi and Mahadevan, 1987; Rabenhost and Hopp, 1997) and Streptomyces setonii (Müller et  al., 1998; Gunnarsson and Palmqvist, 2006). The process development is not easy because of the slow growth of actinomycetes and high viscosity of fermentation broth; so, the development of new recombinant strains of fast-growing bacteria able to overproduce vanillin appears to be attractive. The process of producing vanillin from several agro by-products had been examined using different microorganisms. Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533 were used in a two-step process using sugar beet pulp (Lesage-Meessen et al., 1999; Bonnina et al., 2001), maize bran (Lesage-Meessen et al., 2002), rice bran oil (Zheng et al., 2007), and wheat bran (Thibault et al., 1998). Wheat bran and corn cob were found good substrate for vanillin production by E. coli JM 109/pBB1 (Di Gioia et al., 2007; Torres et al., 2009). After saffron, vanillin is the second most popular flavoring agent and is greatly used in several applications, e.g., as a food additive and as a masking agent in several pharmaceutical formulations. It is a valuable product for several other applications, such as metal plating and the production of other flavoring agents, herbicides, ripening agents, antifoaming agents, and personal and home-use products (such as in deodorants, air fresheners, and floor-polishing agents). Three types of vanillin, are available on the market. These are natural, biotechnological, and chemical/synthetic. But, only natural vanillins are considered as food grade additives by most food safety control authorities worldwide. (Banerjee and Chattopadhyay, 2019)

Increasing demand for natural flavoring agents over those derived from nonrenewable sources is expected to boost the market growth of biovanillin over the next few years. Biovanillin is derived from the ferulic acid that is present in crops such as wheat bran and rice bran and also tea leaves. Therefore, this product appears to be a key substitute for synthetic



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vanillin in the near future. In addition, the growth of key end users of mainly pharmaceutical and food and beverage industries is considered to be another major driving factor of the biovanillin market demand. Biovanillin improves the aroma, flavor, shelf life, and taste of various food and beverage products, keeping the nutritional value of foods unchanged. Therefore, it can be used for food and beverage processing applications particularly for manufacturing dairy products. Growing consumer preferences toward the use of highly nutritional and biobased food products particularly in developed countries such as the United States is expected to create a significant demand for biovanillin in the next few years. In pharmaceutical industry, biovanillin is used as a flavoring ingredient in the processing of several medicines for removing odor and bad taste. It is also used as a main intermediate during processing of pharmaceuticals such as therapeutic drugs for cancer treatment and in cosmetic lotions. Increase in health awareness among consumers in developing countries such as China and India is expected to increase consumption of pharmaceuticals in the future. This scenario, in turn, is expected to positively affect the market dynamics of biovanillin in the coming years.

6.7 Solvents Chemical processes use solvents as major elements for several chemical transformations. The role of solvents is to keep the reagents together in chemical reactions and is extensively used in several purification methods such as recrystallization, liquid-liquid extractions, or chromatographic separations. Solvents are components of several consumer and household products like degreasing cleaners, paints, varnishes, or building materials. Several sectors and industries are consuming solvents. The exposure to organic solvents is not an issue that affects the chemist and people involved in chemistry activities, but rather an important question of global concern because these are dangerous to health and environment. Table  6.38 shows the use of solvents in different sectors (Calvo-Flores et  al., 2018; Clark et  al., 2015). Several different classes of organic chemicals are used as solvents (Table 6.39). Most of the organic solvents are toxic, volatile, potentially flammable and explosive. National Institute for Occupational Safety and Health (NIOSH) has identified some solvents as reproductive hazards like benzene, carbon tetrachloride, and trichloroethylene as carcinogens, 2-ethoxyethanol, 2-methoxyethanol, and methyl chloride as reproductive hazards, whereas n-hexane, tetrachloroethylene, and toluene are tagged as neurotoxic. Also the World Health Organization (WHO), the US Environmental protection Agency (EPA) and the European Union (EU) have compiled information about risks, effects and limitations of the use of organic solvents in certain activities or installations. (pacaglogistics.com) Based on Green Chemistry principles, the search of less hazardous, and more environmentally benign solvents is a field of interest for chemists and chemical engineers to perform safer, environmentally-friendly and more sustainable procedures. The most evident solution is the elimination of solvents as reaction media. During the last years, many reactions have been carried out without solvent. They are called solventless reactions or dry media reactions. It can be found on literature numerous examples of such reactions. These reactions are environmentally friendly, they used to have a high reaction rate and are quite easy to purify, but the absence of the solvent is not a widely applicable procedure for every type of chemical reactions described in literature. Therefore, finding alternative solvents to traditional ones is necessary to achieve less dangerous reactions and processes for human health, and more environmentally friendly but equally effective. This would also lead to safer manufacture and less toxic industrial and household products. Such solvents are called green solvents. (link.springer.com; Kidwai and Mothsra, 2006).

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TABLE 6.38  Use of solvents in different sectors. Dry cleaning

1%

Agrochemicals

2%

Extraction food products

2%

Industrial cleaning

4%

Polymer manufacturing

4%

Adhesives

6%

Cosmetics

6%

Housework

6%

Printing inks

6%

Other industrial issues

8%

Pharmaceuticals

9%

Paints and coatings

46%

Based on Calvo-Flores, F.G., Monteagudo-Arrebola, M.J., Dobado, J.A., Isac-García, 2018. Green and bio-based solvents. J. Top. Curr. Chem. 201 (376), 18; Clark, J.H., Farmer, T.J., Hunt, A.J., Sherwood, J., 2015. Int. J. Mol. Sci. 16 (8), 17101.

TABLE 6.39  Organic chemicals utilized as solvents. Aliphatic and aromatic hydrocarbons Halogenated compounds Ethers Esters Ketones Based on Calvo-Flores, F.G., Monteagudo-Arrebola, M.J., Dobado, J.A., IsacGarcía, 2018. Green and bio-based solvents. J. Top. Curr. Chem. 201 (376), 18.

Solvents are an important ingredient of chemical processes and products and are used in several industrial applications. These days, solvents are selected not only based on their physical properties but also based on their effect on the environment. Several consumers are now preferring environmentally friendly solvents or green substitutes over traditional products; they value environmental benefits and the safety of workers. The industries are entering into an era of environmentally friendly products and environmental awareness, and since biobased solvents provide excellent performance, both financially and environmentally, they will become important in the coming years (www.biobasedpress.eu). Most of the biobased solvents are produced from sugar, corn, or beet. They are selected basically as they have the ability to replace oil-based solvents and are as safe and as effective as the traditional options. Furthermore, green solvents do not produce toxic by-products and volatile organic compounds (VOC) during production. Because of their high boiling point, lower toxicity, and miscibility, several green solvents are becoming popular nowadays. These



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solvents are generally used for coatings, paints, printing inks, personal care products, cosmetics, sealants, and pharmaceuticals, and so, the demand for biobased solvents is increasing (www.biobasedpress.eu). Biobased solvents are obtained from renewable sources, mostly agricultural crops having high carbohydrate content, such as corn, wheat, or beets, or residual organic matter considered as waste for a long time, although these are not the exclusive sources of biobased solvents. There are three main methods to produce solvents from biomass (Table 6.40). The biobased solvents and industrial biodegradable solvents are listed in Tables 6.41 and 6.42. TABLE 6.40  Methods to produce solvents from biomass. Fermentation Chemical transformation of biomass derivatives Use of waste material from other process Based on Calvo-Flores, F.G., Monteagudo-Arrebola, M.J., Dobado, J.A., Isac-García, 2018. Green and bio-based solvents. J. Top. Curr. Chem. 201 (376), 18.

TABLE 6.41  Biobased solvents. Biobased alcohols Bioethanol Butan-1-ol Octan-2-ol Propane-1,3-diol (PDO) Butane-1,3-diol Glycerol Biobased esters and related compounds Ethyl acetate Lactic acid and lactates Ethers and related compounds Valerolactone and 2-methyl-THF from levulinic acid Valerolactone (GVL) 2-Methyl-THF Green solvents from carbohydrates Cyrene Dimethyl isosorbide (DMI) Biobased ionic liquids Biobased deep eutectic solvents Based on Calvo-Flores, F.G., Monteagudo-Arrebola, M.J., Dobado, J.A., IsacGarcía, 2018. Green and bio-based solvents. J. Top. Curr. Chem. 201 (376), 18.

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TABLE 6.42  Industrial biodegradable solvents. d-Limonene p-Cymene Methyl soyates Based on Calvo-Flores, F.G., Monteagudo-Arrebola, M.J., Dobado, J.A., IsacGarcía, 2018. Green and bio-based solvents. J. Top. Curr. Chem. 201 (376), 18.

Green solvents can include a variety of products, especially biobased ethanol and methyl soyate. While methyl soyate is quickly gaining market share, many manufacturers choose biobased ethanol at this point because of the number of applications it can be used for and because of its widespread commercialization, making it more affordable and convenient to purchase. Ethyl lactate is also a green solvent that is growing in popularity, used primarily as a replacement for petroleum solvents. Biobased products include cleaners, degreasers and solvents. A solvent is defined as a solid, liquid or gas that dissolves another solid, liquid or gas, resulting in a substance that is able to be dissolved. Common uses for solvents in the cleaning industry are spot removers and detergents. Biobased solvents contain derivatives including methyl soyate, or soybeans; ethyl lactate (formed from lactic acid and ethanol, made from processing corn); and d-Limonene (citrus rind oil). Products containing these solvents perform quite well compared to their traditional counterparts, and d-Limonene is particularly effective on tough degreasing jobs. (www. cleanlink.com).

Table 6.43 shows the advantages of using biobased solvents. Green solvents are produced from beat, sugars, corn, and other products. Biobased solvents can be used as a replacement for crude oil–based solvents and are effective and also safe. This is due to their higher boiling point, reduced toxicity, recyclability, low mixability, and other features which traditional oil–based solvents do not possess. Sustainable solvents are attracting a lot of attention. Many people are especially interested in the fact that green solvents do not release the same high numbers of volatile organic compounds (VOCs) as ­petroleum-based solvents during manufacturing. Sustainable solvent suppliers are seeing increased demand for biobased solvents for a number of uses, including coatings, paints, printing inks, cosmetics, personal care products, sealants, cases, pharmaceuticals. Paints and coating are the most popular use, accounting for 40% of the global green solvent market in 2014. TABLE 6.43  Advantages of using biobased solvents. Reduced toxicity Biodegradability Low volatile organic compounds (VOC) Worker safety Environmental friendliness www.cleanlink.com/cleanlinkminute/details.aspx



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As customer demand for more sustainable products in these industries grows, manufacturers are more interested in purchasing greener ingredients and components, including green solvents. Sustainable solvents are useful in manufacturing, construction, cosmetics manufacturing, and more industries. In 2014, the worldwide market for green solvents was 4.3 billion (USD), and it is expected to grow to over 8 billion by 2023 (www.acme-hardesty.com/demand-bio-based-solvents). In 2014, North America had a 30% share in the worldwide green solvent market, putting it ahead of other regions. However, the Asia-Pacific region saw the largest growing market for these solvents in 2014. China, Malaysia, India, and Thailand are seeing an increasing interest in greener agrochemical solutions, pharmaceuticals, cosmetics, and other applications, which will likely drive the green solvent market. End market and consumer demands for greener products and a wider range of products are the biggest factors for promoting market growth in green solvents. However, petroleum-­ based solvents are mass produced, low cost, and easily accessible. Feedstock costs and more complex manufacturing do affect the cost and availability of green solvents for now. As green solvents are more commercialized, though, it is expected more manufacturers will choose them over the less sustainable alternatives. Already, manufacturers and researchers are seeking ways to make water-based and green solvents more commercially available. These innovations will likely shape the market in the years to come. Several companies are commercializing the production of n-butanol and isobutanol. The biobased production of n-butanol is an old process; it dates back to the early 20th century (Garcia et al., 2011; Jong et al., 2010). n-Butanol is produced by fermentation and is produced along with acetone and ethanol, the processes being known as the acetone-butanol-ethanol (ABE) process. Biobased n-butanol production stopped in the 1980s because of the reduced cost of crude oil. The petrochemical routes also became competitive. But increase in the oil prices and the interest in the chemicals derived from renewable sources have renewed the interest. The higher energy content and compatibility with existing infrastructure makes butanol an interesting biofuel proposition for the future. But current production costs are high. This is leading producers to focus on the development of high priced chemical applications. n-Butanol is used in a wide range of polymers and plastics and is also used as a solvent in paints and chemical stabilizers. In 2006 n-butanol had a market size of 2.8 million tones. Biobased butanol production has been reestablished in China to supply its growing chemicals market. In 2008 Cathay Industrial Biotech began supplying butanol under their BioSol brand, production capacity is now 100,000 tonnes. (pacaglogistics.com)

Companies commercializing biobased butanol include Butamax Advanced Biofuels (www. butamax.com.), a joint venture between energy producer BP and the chemical producer DuPont, the UK companies Green Biologics (www.greenbiologics.com) and Solvert (www. solvertltd.co.uk.), and US technology companies Cobalt Technologies (www.cobalttech.com/ about-cobalt) and Gevo Development (www.gevo.com). Butamax and Gevo Development are focusing on the production of isobutanol. The production of isobutanol would create the opportunity for isobutylene production and a variety of downstream products. Driven by government regulations and concerns regarding environmental preservation and exhaustion of natural resources, the biobased solvent industry is facing an exponential increase in demand and a push toward the development of new green solutions. These ­solvents are low-cost alternative to conventional solvents and are effective (www.biobasedpress.eu).

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Circa Group is offering solutions for converting waste biomass into advanced biomaterials. This company introduced Cyrene—a biobased dipolar aprotic solvent, which can be also used as an alternative to dimethylformamide (DMF) and N-methyl-2-pyrrolidone (NMP) derived from petroleum (www.biobasedpress.eu/2018/07/bio-based-solvents-on-the-rise). The major benefit is the chemical structure of Cyrene, which provides polarity similar to NMP without the addition of an amide group that is linked with toxicity. Nitrogen, chlorine, and sulfur are not present in it. Thereby, pollution problems are reduced. It has a density of 1.25g/mL. Furthermore, Cyrene is a stable biobased solvent and it yields only water and carbon dioxide upon biodegradation or incineration. Cyrene is becoming a greener substitute in several industrial applications such as carbon cross-coupling reaction and graphene synthesis (www.biobasedpress.eu). A report from Wageningen Food & Biobased Research shows that there are many green solvents that can be utilized instead of the controversial polar aprotic solvents including NMP, DMF, and DMAc. This study aimed at finding substitutes for these polar aprotic solvents because these cause fertility issues in females in chemical plants. It was found that new materials with special properties and chemical formulas can replace the aprotic solvents. Furthermore, these new materials can increase feasibility and viability. As the raw materials for the biobased products are easily available, these can be produced in a large amount. Daan van Es at Wageningen Food and Biobased Researchhas identified several substitutes, but so far, they are not in production as substitutes for polar aprotic solvents (www.biobasedpress.eu). Green Biologics has developed SOLVONK4. It is a biobased solvent used for dry cleaning. It is produced from corn. It is an ultrapure and green solvent that cleans better than the conventional solvents and does not pose any adverse environmental impact or create any health problems. It is the only biobased solvent in the dry-cleaning industry (www.biobasedpress. eu/2018/07/bio-based-solvents-on-the-rise/). A “green” solvent must fulfill several criteria. It should be biobased, functionally nonVOC, nontoxic, biodegradable, and also recyclable. Ethanol is the oldest and most important biobased chemical solvent used (Anastas et al., 2013; Kosaric et al., 2011; Taherzadeh et al., 2013). It is generally used in detergents, cosmetics, lotions, shampoos, soaps, and other consumer products accounting for a small percentage of total demand for solvents. Also, acetone is most extensively used. Bioacetone is available in substantial quantities and is present naturally in several fruits and vegetables. The demand for bioglycerol is increasing in the last few years. The use of glycerol as a solvent has been limited, but a study has shown that it shows a vast potential to replace several solvents. Bioglycerol is available at a low cost and is a by-product of biodiesel. Biobased solvents appear to be effective even after taking into consideration operating and one-off conversion costs and regulations linked with the use of a new solvent. It is quite certain that green solvents would play a part in securing a greener future. Nowadays, there has been an interest for producing biobased chemicals, and several biobased solvents are easily available. These solvents are not always safe and nontoxic, but since they are renewable, the concerns about the use of finite resources such as oil and natural gas are reduced. Few examples of biobased solvents include bioethanol, limonene, and 2-methyl tetrahydrofuran (2-MeTHF). “Bio-derived solvents may be renewably derived replacements to established solvents or novel molecules such as dihydrolevoglucosenone, a biobased alternative for dipolar aprotic solvents. A broad range of bioderived solvents



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will be required to reduce dependence upon traditional fossil derived solvents” (learning. chem21.eu). One consideration to be taken into account in the case of some biobased solvents is their higher viscosity and boiling points, which may lead to potential issues with recycling and recovery because of higher energy demands (Ashcroft et al., 2015).

6.8  Speciality products Speciality chemicals are produced by a complex, interlinked industry and are sold on the basis of their performance or function, rather than their composition (www.amaindia.org/ images/...page/SpecialtyChemicals-Ebook-2017Mar03.pdf). They may be single chemical entities or formulations whose composition strongly affects the performance and processing of the customer’s product. Products and services in the specialty chemical industry require in-depth knowledge and continuous innovation. Thereby, they are facing strict market entry barriers and are commanding higher prices. Several renewable resources remain to be used efficiently. Flora and fauna of several of the ecosystems of the world have been examined to a limited extent for existence of novel compounds of potential value. For instance, microalgae are contributing significantly to primary photosynthetic productivity on Earth but are scarcely used commercially. Microalgae appear to be an attractive source for several value-­ added products, including carotenoids and omega-3 and omega-6 polyunsaturated fatty acids (PUFAs), natural colorants and pigments, biopolymers, and therapeutics (Borowitzka, 1999; Cohen, 1999; Belarbi et al., 2000, 2003; Lorenz and Cysewski, 2000; Banerjee et al., 2002; Mirón et al., 2002; Lebeau and Robert, 2003a,b; Lopez et al., 2004; León-Bañares et al., 2004). Algae contain different types of pigments (chlorophylls and carotenoids [carotenes and xanthophylls]) and phycobilins (phycocyanin and phycoerythrin). Microalgae are being used as aquaculture feeds, biofertilizers, and soil inoculants. Microalgae appear to be interesting for wastewater treatments, as they provide a tertiary treatment coupled with the production of potentially valuable biomass, which can be used for many purposes. They can be used to remove excess carbon dioxide from the atmosphere (Gòdia et al., 2002). Microalgae grow rapidly; produce more biofuel per hectare in comparison with the oil plants; can remove excess carbon dioxide as hydrocarbons; produce a fuel that do not contain sulfur; does not compete with food, fiber, or other uses; and does not involve demolition of natural resources. Lipids and fatty acids are present in microalgae as sources of energy, membrane components, storage products, and metabolites. When algae are cultivated under standard conditions, they show big differences in percentages of the major macronutrients by dry weight, generally 25%–40% of protein, 5%–30% of carbohydrates, and 10%–30% of oils and lipids. Algae containing very high oil content have been found, and several species have been reported as possible candidates for producing biodiesel (www.oilseedcrops.org; Nandi and Sengupta, 1998; Banerjee et al., 2002). Algal lipids are subjected to transesterification for producing biodiesel (Zhu, 2015; Huang et al., 2014; Gonçalves et al., 2013; Zhu and Ketola, 2012). There are many benefits of using microalgae for biodiesel production in comparison with the crop plants. The growth of algae requires less land space and can also be cultivated in wastewater (Bhatt et al., 2014; Chisti, 2012; Pittman et al., 2011; Wijffels, 2008; Brennan and Owende, 2010).

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Depending on the strain and growth conditions, up to 75% of algal dry mass can be hydrocarbons. The chemical nature of hydrocarbons varies with the producer strain and these compounds can be used as chemical. Some microalgae can be grown heterotrophically on organic substrates without light to produce various products. As with microalgae, sponges and other marine organisms are known to produce potentially useful chemicals, but have not been used effectively for various reasons. Natural sponge populations are insufficient or inaccessible for producing commercial quantities of metabolites of interest. Production techniques include aquaculture in the sea, the controlled environments of aquariums, and culture of sponge cells and primmorphs. Cultivation in the sea and aquariums are currently the only practicable and relatively inexpensive methods of producing significant quantities of sponge biomass. (Gavrilescu and Chisti, 2005; Dennis and Kolattukudy, 1991; Banerjee et al., 2002; Wen and Chen, 2003; Belarbi et al., 2003; Thakur and Müller, 2004)

Extremophiles are adapted to extreme conditions like high pressure, heat, and total darkness. These are attracting a lot of attention as possible sources of unusual specialty products. Few commercial biotechnology products have been obtained from extremophiles. Till now, few extremophiles have been used on a large scale in the field of biotechnology, but their potential is irrefutable in several applications. Few success stories are listed in the succeeding text: • • • •

Thermostable DNA polymerases used in the polymerase chain reaction (PCR) Various enzymes used in the production of biofuels Microorganisms used in the mining process Carotenoids used in the food and cosmetic industries

Other potential applications include the following (Eichler, 2001; Henkel, 1998; Coker, 2016; Elleuche et al., 2014; Ishino and Ishino, 2014; Barnard et al., 2010; Johnson, 2014; Oren, 2010; Coker and Brenchley, 2006; Herbert, 1992; Dopson et al., 2016): • Production of lactose-free milk • Production of anticancer and antifungal drugs and antibiotics • Production of electricity or the leaching of electrons for producing current that can be used or stored

6.8.1 Fermentation Microbial fermentation is used for the production of certain products on a large scale (Weiss and Edwards, 1980; Strohl, 1997; Leeper, 2000; Liese et al., 2000; Schreiber, 2000). Table 6.44 lists the established fermentation products. The global antibiotics market size is expected to reach USD 62.06 billion by 2025. Increasing prevalence of infectious diseases, particularly in developing countries, is expected to contribute to the market growth. More than 15% of the children below the age of 5 years have died due to pneumonia. WHO has reported that there were about 9.2 million deaths in 2015. This disease is mostly found in the South Asian and sub-Saharan regions. Presently, the antibiotic treatment is available only to one-third of the infected population, thus increasing the burden of the disease (www.grandviewresearch.com/press-release/global-antibiotic-market). Other important pharmaceutical products produced by microorganisms are cholesterol-­ lowering agents or statins, immunosuppressants, antitumor compounds, and enzyme inhibitors (Demain, 2000). The global biopharmaceuticals market accounted for $186,470 million in 2017 and would increase to $526,008 million by 2025 (www.alliedmarketresearch.com/ biopharmaceutical-market).



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TABLE 6.44  Few established fermentation products. Bioethanol Food processing enzymes Vitamin C Gluconic acid Antibiotics Feed enzymes Xanthan Aspartame l-Methionine Dextran Vitamin B12 12 Provitamin D2 Glutamic acid (MSG) Citric acid l-Lysine Lactic acid l-Threonine l-Hydroxyphenylalanine 6-Aminopoenicillanic acid Nicotinamide d-p-Hydroxyphenylglycine Vitamin F 7-Aminocephalosporinic acid Based on Gavrilescu, M., Chisti, Y., 2005. Biotechnology—a sustainable alternative for chemical industry. Biotechnol. Adv. 23, 471–499.

Biopharmaceuticals have a high-therapeutic value. These are produced using microorganisms and animal cells. These are also known as biotech drugs. Biotechnological processes are used to produce several of these drugs. In 2017, the global cholesterol-lowering industry was worth $19.2 billion. In 2022, it is expected to be worth $24.4 billion. Novel fermentation methods for established drugs and drug precursors are being developed continuously (Moody, 1987; Chisti, 1998, 1999; Gavrilescu and Roman, 1993, 1995, 1996, 1998; Roman and Gavrilescu, 1994; Sanchez and Demain, 2002). Cholesterol-reducing drug lovastatin is produced, which is also used for producing other semisynthetic statins (Chang et  al., 2002; Casas López et  al., 2003, 2004a,b, 2005; Vilches Ferrón et  al., 2005; tur-www1. massey.ac.nz).

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The global cholesterol-lowering industry was worth $19.2 billion in 2017. This would grow to 4.9% each year during the next 5 years and is expected to be worth $24.4 billion in 2022 (www. statinnation.net/blog/2017/10/4/cholesterol-lowering-industry-still-worth-more-than-19-billion-and-increasing). Several novel bioprocess strategies are used to significantly increase productivities and efficacy of several bioprocesses (Chisti and Moo-Young, 1996). Vitamins are important micronutrients needed in small quantities that cannot be produced by mammals. These are needed for metabolism of all living organisms and are produced by microbes or plants. Vitamins are being used as food additives, as medical therapeutic agents, as health aids, and also as technical aids, besides their in vivo nutritional-physiological roles as growth factors for microbes, plants, men, and animals. Nowadays, several processed foods, feeds, pharmaceuticals, cosmetics, and chemicals contain externally added vitamins or vitamin compounds. Most of the vitamins are produced on an industrial scale and used in foods, pharmaceuticals, and cosmetics in large amounts. Currently, few of the vitamins are generally produced using chemical process, whereas a few others are produced either by chemical process or through extraction process. These processes suffer from high cost of waste disposal and are energy intensive. Moreover, these processes have growing consumer consciousness with regard to safety of food additives. This is creating great interest in substituting these processes with biotechnological processes. These processes for the production of most of these compounds are being developed quickly, and few processes are already competing with the conventional processes (www.ftb.com.hr). A list of vitamins produced by biotechnological methods is presented in Table 6.45. Various methods such as mutation and screening, media optimization, and genetic engineering and enzymatic methods are being used for the production of vitamins. DSM TABLE 6.45  Vitamins produced by biotechnological methods. Fat-soluble vitamins Vitamin E (a-tocopherol) Freshwater microalgae Euglena gracilis Fermentative production from glucose Vitamin K2 Mutated strain of Bacillus subtilis Fermentation using soybean extract Water-soluble vitamins Ascorbic acid (vitamin C) 2,5-Diketo-d-gluconic acid reductase Cyanobacterium sp. Fermentative process to 2-keto-l-gulonic acid followed by chemical conversion to l-ascorbic acid Biotin Fermentation (Serratia marcescens) Multiple enzyme system (Bacillus sphaericus) Fermentative production from glucose by genetically engineered bacterium Conversion from diaminopimelic acid using the biotin biosynthetic enzyme system of mutant of Bacillus sphaericus



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TABLE 6.45  Vitamins produced by biotechnological methods—cont’d Riboflavin Fermentation (Eremothecium ashbyii, Ashbya gossypii, Bacillus sp., etc.) Fermentative production from glucose Vitamin B12 Fermentation (Propionibacterium shermanii and Pseudomonas denitrificans) Fermentative production from glucose Based on Survase et al. (2006); Takeyama et al. (1997); Sato et al. (2001); Boudrant (1990); Hancock and Viola (2002); Gloeckler et al. (1990); Horiuchi and Hiraga (1999); Marwaha et al. (1983).

Nutritional Products replaced the conventional process containing six steps for producing vitamin B2 (riboflavin) with a single-step fermentation process, which has a reduced ecological impact in comparison with the traditional production process. The bacterium Bacillus subtilis was used. Production was made possible by genetic engineering the bacterium for increasing the vitamin yield by 300,000-fold in comparison with that obtained with the wild-type strain. The single-step fermentation process reduced cost of production by 50% as compared with the traditional process. Microbial or microalgal processes for vitamin production have several benefits in comparison with the traditional processes. The products from chemical processes are often racemic mixtures, whereas fermentation process yields the desired enantiomeric compound. Moreover, advances in biochemistry and DNA technology along with genomic revolution have broadened the options available for using biotechnological process in vitamin production. These processes and products usually have a positive ecological impact and a positive appeal to people (tur-www1.massey.ac.nz; www.ftb.com.hr). Microorganisms producing vitamin B12 and the respective yields were published by Martens et al. (2002) (Table 6.46). TABLE 6.46  Microorganisms producing vitamin B12. Microorganism

Vitamin B12 (mg/L)

Nocardia rugosa

18.0

Rhizobium cobalaminogenum

16.5

Micromonospora sp.

11.5

Streptomyces olivaceus

6.0

Nocardia gardneri

4.5

Butyribacterium methylotrophicum

3.6

Pseudomonas sp.

3.2

Arthrobacter hyalinus

1.1

Propionibacterium freudenreichii

206.0

Rhodopseudomonas protamicus

135.0

Propionibacterium shermanii

60.0

Pseudomonas denitrificans

60.0

Based on Martens, J.H., Barg, H., Warren, M.J., Jahn, D., 2002. Microbial production of vitamin B12. Appl. Microbiol. Biotechnol. 58, 275–285.

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Biopharmaceuticals, mainly recombinant proteins, vaccines, and monoclonal antibodies, represent different class of drugs in comparison with small molecules such as a­ ntibiotics. Examples are tissue plasminogen activator (tPA), insulin, and recombinant hepatitis B ­vaccine. Selected biopharmaceuticals are listed in Table 6.47 (Gavrilescu and Chisti, 2005). The market for recombinant proteins is much larger when nonbiopharmaceutical products are included. A generics industry would emerge around some of the older biopharmaceutical products (Melmer, 2005). Better processes for producing biopharmaceuticals such as alpha-1-antitrypsin are being developed. Like many enzymes, several naturally occurring first-generation protein therapeutics such as insulin and tissue plasminogen activator (being produced by modern biotechnology processes) are being protein engineered to products that are potentially superior to their natural counterparts. For instance, several modifications of

TABLE 6.47  Few selected biopharmaceuticals. Insulin Diabetes Blood clotting factors Hemophilia Erythropoietin Anemia Colony-stimulating factor Neutropenia Monoclonal antibody Cancer Growth hormone Growth disorders Monoclonal antibody Various Plasminogen activator Thrombotic disorders Interleukin Cancer, immunology Growth factor Wound healing Therapeutic vaccines Various Other proteins Various Interferon alpha Cancer, hepatitis Interferon beta Multiple sclerosis, hepatitis Based on Gavrilescu, M., Chisti, Y., 2005. Biotechnology—a sustainable alternative for chemical industry. Biotechnol. Adv. 23, 471–499.



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streptokinase have been used to extend its half-life in circulation, improving plasminogen activation, and reducing or eliminating immunogenicity. Protein engineering is found to be successful in changing bioactivity, stability, ease of recovery and other properties of proteins. (Gavrilescu and Chisti, 2005; Galler, 2000; Banerjee et al., 2002; Rouf et al., 1996; Tamer and Chisti, 2001; Nosoh and Sekiguchi, 1990; Sassenfeld, 1990; El Hawrani et al., 1994; Nygren et al., 1994)

6.8.2 Enzymes Enzymes are being used in the chemical industry as catalysts for several reasons. The worldwide enzyme market was estimated at $7,082 million in 2017 and is expected to reach $10,519 million in 2024 (www.prnewswire.com/news-releases/global-enzymesmarket-expected-to-reach-10-519-million-by-2024-898959866.html). Table  6.48 shows few examples of industrial enzymes and their application in industry. TABLE 6.48  A few examples of enzymes in industry. Proteases Detergents, food, pharmaceutical, chemical synthesis Carbohydrases Food, feed, pulp and paper, sugar, textiles, detergents Lipases Food, effluent treatment, detergents, fine chemicals, biodiesel Phospholipases Bread making, egg yolk industry (emulsification for different applications), and refinement of vegetable oils (degumming) Pectinases Food, beverage Cellulases Pulp, textile, feed, detergents Amylases Food Zymase Conversion of carbohydrates into ethanol in alcoholic beverages Invertase Food industry Lactase Food industry Rennin Coagulation of milk to make cheese Bajpai (2018).

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Several years of evolution have provided enzymes with unique capabilities of facilitating life reactions in ways that are sustainable. In comparison with the traditional catalysis, enzymes are highly specific and function under temperatures, pressures, and pHs that are compatible with life (Scheper, 1999; Bommarius, 2004; Abramovicz, 1990; Roberts et al., 1999). Dissimilar to several processes of traditional synthetic chemistry, enzymes need nontoxic and noncorrosive conditions. In the detergent, food, and starch processing industries, about 75% of the enzymes are used. These are mainly hydrolytic enzymes, for example, amylases, proteases, lipases, and cellulases. Specialty enzymes account for about 10% of the enzyme market and are finding increasing applications in the development of new drugs, medical diagnostics, and several other analytical applications. Of the commercial enzymes, about 60% are products of modern biotechnology. In addition to their increasing analytical and diagnostics applications, new applications are being developed for enzymes in the production, degradation, and biotransformation of chemicals, foods and feeds, agricultural products, and textiles (Gavrilescu and Chisti, 2005). A few examples for bulk enzymes are presented in the succeeding text: In the animal feed, amylase and protease enzymes are being added for improving digestibility by supplementing the animals’ own enzymes (Bajpai, 2018). Several animal feed derived from plants contains antinutritional factors, which interfere with digestion or absorption of nutrients. Addition of enzymes such as β-glucanases and arabinoxylanase to feed cereals breaks down nonstarch polysaccharide (NSP) antinutritional factors, helping in digestion and absorption of nutrients. Phytic acid present in plant matter reduces dietary absorption of essential minerals such as iron and zinc. Phytic acid finally appears in animal manure as highly polluting phosphorus. By the addition of phytases to feed, digestion of phytic acid is facilitated. “Phytases are digestive enzymes which release plant phosphorus from phytic acid. Phytase was available in sufficient amounts only after it was produced in recombinant microorganisms. Monogastric animals lack sufficient phytases to release the phosphorus. Addition of extra phytases to the diet increases phytate breakdown and subsequent utilization of plant phosphorus. If more phosphorus is available naturally, then less of this substance has to be added to the diet. This greatly reduces feed costs. If phosphorus in the diet is used more efficiently, then less of this substance is excreted. This reduces the impact of livestock production on the environment” (www.christa.bg; Gavrilescu and Chisti, 2005). Isomalto-oligosaccharides (IMOs), a new class of sugars, are produced using glucosyl transferases. These oligosaccharides find applications in food industry as nondigestible carbohydrate bulking agent. They repress the decay of tooth associated with consumption of sugars and prevent baked goods going stale. IMOs are becoming popular as a low-calorie, high-fiber carbohydrate in protein bars. IMOs are found in some fermented foods and natural sweeteners, but this is mostly produced in a lab and then produced on a large scale. Manufacturers are producing IMOs using enzyme-catalyzed hydrolysis on starch obtained from cereal crops, pulses, rice, tapioca, and potatoes. IMOs are generally 91% oligosaccharides, 2% glucose, and 7% various high-molecular-weight molecules. IMOs are 60%–70% as sweet as sucrose, less viscous than maltose, and are partially digestible, containing ~2.0–2.4 calories per gram (www.tigerfitness.com/blogs/supplements/isomalto-oligosaccharides-guide). Cellulases are complexes of enzymes that break down cellulose in a synergistic manner. These enzymes are used for the conversion of lignocellulosics and used for the production of ethanol and single-cell protein (SCP), for bleaching of pulp, for treatment of waste p ­ apers,



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and for fruit juice extraction (Bajpai, 2018). The biological degradation of cellulose has been studied. Several cellulolytic enzymes are produced by fungi and bacteria. Cellulases are being intensely researched. Cellulases produced by Trichoderma fungi are used for stone washing jeans. By changing the relative proportions of the enzymes in the cellulase complex, different effects on the textile fibers can be produced. Extremophile enzymes are extremely stable, and the application of these enzymes as biocatalysts is attractive because they are stable and active under conditions that were earlier regarded as incompatible with biological materials. Extremophilic enzymes are finding more and more application due to their ability to tolerate high temperatures and other conditions (Eichler, 2001). Extremozymes have a great potential in several industrial processes, including agricultural, chemical, and pharmaceutical applications. Several consumer products are benefiting from the addition of extremozymes. The toolbox to select and make such enzymes available is increasing. Less than 10% of the organism in a defined environment can be cultivated, and so, further improvement of gene expression technologies will increase the exploration of microbial diversity. It is possible to construct gene expression libraries from different sources. If such libraries are screened with rapid detection techniques several new extremozymes will be discovered in the coming years. These extremozymes will be used in novel biocatalytic processes which are rapid, more accurate specific and eco friendly. Concurrent developments of protein engineering and directed evolution technologies will result in tailoring and improving biocatalytic traits which will increase the application of enzymes from extremophiles in industry. (www.biotechnology.uwc.ac.za; www.scialert.net)

Use of enzymes in nonaqueous media has created new possibilities for producing several important chemicals. These are modified fats and oils, structured lipids, and flavor esters (Sharma et al., 2001; Krishna, 2002). These reactions present new possibilities for the production of several important chemicals using reactions that are not possible in aqueous media. Use of enzymes in nonaqueous media is finding use in the following areas: • • • •

Organic synthesis Synthesis or resolution Modification of fats and oils Synthesis of sugar-based polymers

Lipases are used in esterification reactions for producing several useful products such as emulsifiers, surfactants, wax esters, chiral molecules, biopolymers, modified fats and oils, structured lipids, and flavor esters. The interest in using lipases for performing reactions in both macro- and microaqueous systems is picking up significantly. Enzymes have been explored for pharmaceutical applications (Choi et  al., 2015; Anbu et  al., 2015). Bornscheuer et  al. (2012) reviewed the biocatalytic routes for pharmaceutical manufacturing showing the advantages of enzymes over the conventional process. One successful example in the use of enzymes in the pharmaceutical industry is the antidiabetic compound, sitagliptin (Desai, 2011; Savile et al., 2010). Sitagliptin is used for type II diabetes that is sold under the trade name Januvia by Merck (Desai, 2011). Enzymes are being used for producing β-lactam antibiotics such as semisynthetic penicillins and cephalosporins (Volpato et al., 2010). The semisynthetic penicillins are replacing natural penicillins, and about 85% of penicillins sold for medicinal use are semisynthetic.

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6-Aminopenicillanic acid (APA) is obtained by the hydrolysis of the amide bond of the naturally occurring penicillin with penicillin amidase, which does not open the β-lactam ring unlike chemical hydrolysis. Synthesis of complex chiral pharmaceutical intermediates is the important applications in biocatalysis. Esterases, lipases, proteases, and ketoreductases are mostly used in the preparation of chiral alcohols, carboxylic acids, amines, or epoxides (Zheng and Xu, 2011). Kinetic resolution of racemic amines is commonly used in the synthesis of chiral amines. BASF is using acylation of a primary amine moiety by lipase for the resolution of chiral primary amines in a scale of several thousand ton (Sheldon, 2008; Liu and Kokare, 2017). Atorvastatin, the active ingredient of Lipitor, can be produced using enzyme. The process is based on three enzymes such as a ketone reductase, a glucose dehydrogenase, and a halohydrin dehalogenase. Several iterative rounds of DNA shuffling for these three enzymes resulted in a 14-fold reduction in reaction time, a 25-fold reduction in enzyme use, a 7-fold increase in substrate loading, and a 50% improvement in isolated yield (Ma et al., 2010; Liu and Kokare, 2017). Therapeutic enzymes have several uses such as oncolytics, thrombolytics, or anticoagulants and as replacements for metabolic deficiencies (Liu and Kokare, 2017). Enzymes are being used for treating cancer, cardiac problems, cystic fibrosis, dermal ulcers, inflammation, digestive disorders, etc. (Moon et al., 2003; Lee-Huang et al., 1999). - Proteolytic enzymes are found to be good anti-inflammatory agents. - Collagenase enzyme hydrolyzes native collagen and spares hydrolysis of other proteins. This enzyme has been used to treat dermal ulcers and burns. - Papain produces significant reduction of obstetrical inflammation and edema in dental surgery. - Deoxyribonuclease is used to treat chronic bronchitis. It acts as a mucolytic agent. - Trypsin and chymotrypsin are used in the treatment of athletic injuries and postoperative hand trauma. - Hyaluronidase shows hydrolytic activity on chondroitin sulfate and can help in the regeneration of damaged nerve tissue. - Lysozyme hydrolyzes the chitins and mucopeptides of cell walls of bacteria. Therefore, it is used generally in combination with standard antibiotics as antibacterial agent. Lysozyme shows activity against HIV, as the RNase A and urinary RNase U present selectively degrade viral RNA showing the possibility for the treatment of HIV infection. Few examples of the use of enzyme therapeutics in cancer are presented in the succeeding text (Gurung et al., 2013; Ensor et al., 2002): - Arginine-degrading enzyme (PEGylated arginine deaminase) can impede human melanoma and hepatocellular carcinomas. - Another PEGylated enzyme, Oncaspar1 (pegaspargase), has shown good results for the treatment of children newly diagnosed with acute lymphoblastic leukemia. Further application of enzymes as therapeutic agents in cancer is described by antibody-­ directed enzyme prodrug therapy (ADEPT). A monoclonal antibody carries an enzyme



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s­ pecific to cancer cells where the enzyme activates a prodrug and destroys the cancerous cells but not normal cells. This strategy is being used for the discovery and development of cancer therapeutics based on tumor-targeted enzymes, which activate prodrugs. Certain enzymes such as L-asparaginase have been found to be useful for the treatment of cancer. l-Asparaginase, by reducing asparagine concentration, hampers the growth of cancerous cells. It has been found useful in treating lymphoblastic leukemia and certain forms of lymphomas. Genetic engineering basically involves taking the relevant gene from the microorganism that naturally produces a particular enzyme (donor) and inserting it into another microorganism which will produce the enzyme more efficiently. The first step is to cleave the DNA of the donor cell into fragments using restriction enzymes. The DNA fragments with the code for the desired enzyme are then placed, with the help of ligases, in a natural vector called a plasmid that can be transferred to the host bacterium or fungus. In recombinant DNA technology, restriction enzymes recognize specific base sequences in double helical DNA and bring out cleavage of both strands of the duplex in regions of defined sequence. Restriction enzymes cleave foreign DNA molecules. The term restriction endonuclease comes from the observation that certain bacteria can block virus infections by specifically destroying the incoming viral DNA. Such bacteria are known as restricting hosts, since they restrict the expression of foreign DNA. Certain nicks in duplex DNA can be sealed by an enzyme-DNA ligase which generates a phosphodiester bond between a 5′-phosphoryl group and a directly adjacent 3′-hydroxyl, using either ATP or NAD+ as an external energy source. (Liu and Kokare, 2017; Adrio and Demain, 2014)

Use of enzymes is not limited to specialty chemicals. Enzymes are used for the conversion of corn starch to high-fructose corn syrup that is a major sweetening agent in foods and beverages. About USD 1 billion worth of high-fructose corn syrup is produced annually (Gavrilescu and Chisti, 2005). Enzymatic processes for producing commodity chemicals such as acrylamide have been developed. Acrylamide has been traditionally produced from acrylonitrile by using following two chemical processes: • Hydrolysis with sulfuric acid • Hydrolysis catalyzed with copper Mitsubishi Rayon Co., Ltd. (www.mrc.co.jp) started to produce acrylonitrile from acrylamide using immobilized bacterial enzyme nitrile hydratase using the process developed in 1985 (Vandamme and Bienfait, 2004). This process is low cost and eco-friendly and produces a quality product. New production facilities based on this process are being built globally (OECD, 2001). About 100,000 tons of acrylamide is produced annually by using this process (Vandamme and Bienfait, 2004). Comparison of the traditional and biotechnology-based production of acrylamide shows that higher single-pass conversion is obtained in the bioprocess. Also, mild reaction conditions are required, and a higher final concentration of the product is obtained. The bioprocess uses about 20% lesser energy and produces much less carbon dioxide in comparison with the traditional process. Whereas enzyme have clear advantages over their chemical alternatives, much research is required to make them cost-competitive for use in the broader chemical industry. A report entitled New Biocatalysts: Essential Tools for a Sustainable 21st Century Chemical Industry (www.eere.energy.gov/biomass/pdfs/biocatalysis_roadmap.pdf) identified the following major objectives for biocatalysts for a sustainable chemical industry:

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- developing biocatalysts that are better, faster, less expensive and more versatile than comparable chemical catalysts - development of biocatalysts that can catalyze an increased range of reactions, have higher temperature stability and improved solvent compatibility - developing molecular modeling and other tools to permit rapid design of new enzyme catalysts. Progress is underway in all of the above areas to provide the chemical industry with diverse new useful biocatalysts. Newer ways of using enzymes and cells in bioreactors are being established. (Gavrilescu and Chisti, 2005; Drioli and Giorno, 1999; Park and Chang, 2000)

6.9  Personal care products Consumers are demanding for household and personal care products that have higher content of renewables and better environmental health outcomes. This demand is creating opportunity for growth of industrial biotechnology. Federal policy is expected to play an important role in helping the biotechnology companies to commercialize new technologies for meeting the market demand. Consumers like products that improve the health of the environment specifically within the home. Biobased consumer item products made with renewable biomass content by using industrial biotechnology can meet this demand if consumers are aware about their potential contribution to a healthier environment. The personal care and household product markets present several opportunities (www.teknoscienze.com/tks_article/ industrial-biotechnology-applications-for-personal-care-and-household-products/). Industry researchers and biotech companies are actively exploring the potential consumer demand for biobased products (http://vitalabactive.com/actives.html). The consumer knowledge of biobased products is still low. In Iowa State in 2011, a study found that 44% of those surveyed did not know much about biobased cleaning products. Furthermore, about one-third of respondents were not sure whether they are using such products in their own home (https://www.teknoscienze.com/tks_article/ industrial-biotechnology-applications-for-personal-care-and-household-products/). Biotechnology has an impact on cosmetics in several ways. Cosmetic companies are using biotechnology for discovering, developing, and producing components of cosmetics and to evaluate these components on the skin, particularly how they affect the changes associated with aging. Consumer safety and testing reproducibility should be guaranteed by cosmetic producers, because they are subjected to very strict manufacturing and inspections of production environment. Furthermore, both the industry and the consumers are now aware that biosustainable ingredients are good for the environment. Fewer resources such as water, soil, and or electricity should be used. The ingredients should not be exposed to pesticides and pollutants to provide higher safety levels for the consumers. During the last decade, cosmetic companies have invested excessively in molecular, genomic, and proteomic research into what causes skin cells to age, with the hope of determining ways to interfere with that process. When examining the success of both cosmetic and cosmeceutical products, we should consider the proper integration of skin structure and functional aspects with the type of the formulation, its effectiveness that is defined by the objective of the product, and its safety.



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Antiaging active ingredients can be classified into moisturizing, antioxidant, and extracellular matrix boosters according to their functions or effects. Active ingredients may differ in their manufacturing origin and also in their mechanism of action (www.mdpi.com). The global beauty market (cosmetics and toiletries or personal care products [PCP]) in the last two decades has grown by 4.5% a year on average with annual growth rates ranging from about 3% to 5.5% (Barbalova, 2011; Sunar et al., 2016). The global cosmetic industry is worth tens of billions of USD, and the industry is continuously seeking new products with ingredients having specific actions for which enzymes are the most preferred option for enhancement of personal care products. Enzymes have recently been started to be used for cosmetic application in developing PCPs for wide acceptability as they have good consumer appeal and improved performance. But these have always been poorly evaluated for their functionality in cosmetic science. Proteolytic enzymes like bromelain, papain, etc. have been used in PCPs for skin peeling and smoothing for several years, but, the general problem associated with such use is the irritation caused by some enzymes on the skin surfaces due to their proteolytic activities. The area where the topical applications of enzymes are widely explored and have shown substantial benefits is in skin protection, with enzymes having excellent stability. The enzymes used for skin protection can capture free radicals caused by environmental pollution, microorganisms, sunlight, radiations etc. The trend on use of enzymes in PCPs shows ample variability in terms of enzymes used from different types of classes for their specific function and roles. Studies of enzyme formulations suitable for topical use have also shown that such dosage forms are relatively easy to handle. However, the choice of base, surface active agent, etc., is important to provide for a stable formulation, and proper vehicle selection is also crucial for the proper activity. Another approach to cosmetics and skin care product development is to increase the effectiveness of existing ingredients that might improve skin functioning. Many new topical ingredients (from mushrooms to salmon caviar to sea urchin spines to green algae to knotweed) have been placed in complex antiaging formulations Nanoparticles are revolutionizing many areas of chemistry, physics, and possibly cosmetic formulation. The long term effects of nanoparticles are not known currently. Yet, nanoparticles could be the next frontier in cosmetic dermatology. Nanoparticles have great potential to create topical cosmeceutical formulations that behave in ways that enable better penetration of active skin ingredients. In the future nanoparticle therapy, nanoemulsions, polymeric nanoparticle spheres, and nanoliposomes may be used for improving the appearance of the skin. Nanotechnology may allow ingredients to show new skin effects, improving effectiveness of cosmetics and skin care product. (Zappelli et al., 2016; Draelos, 2012; Sonneville-Aubrun et al., 2004; Tadros et al., 2004;Sunar et al., 2016)

Table 6.49 shows the use of enzymes in cosmetics.

TABLE 6.49  Use of enzymes in cosmetics. Proteases

Peeling/antiaging/antiwrinkle

Lipases

Anticellulitis

Hyaluronidase

Moisturizing agent

Tyrosinase

Tanning agent

Superoxide dismutase

Antifree radicals

Peroxidase

Antifree radicals

Alkaline phosphatase

Antiwrinkle

Based on Bajpai, P., 2018. Industrial Enzymes—An Update, first ed. ISBN: 978-87-403-2129-6, Pages: 118.

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6.9.1  Superoxide dismutase Superoxide dismutase (SOD) is an antioxidant. It catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. It is naturally found in the body. This enzyme is being used in different fields. It plays an effective role in catalyzing superoxide free radicals. SOD is popular and the most effective enzyme being used in skin care products. It is also present in barley grass, brussels sprouts, broccoli, wheatgrass, cabbage, and most green plants. Therefore, they are an important antioxidant defense in almost all cells exposed to oxygen, such as skin cells. Superoxide dismutase overcomes the adverse effect of superoxide and protects the cell from superoxide toxicity. Superoxide is the most common free radical in the body and has the fastest turnover number of any known enzyme. It is used as an antiaging ingredient and antioxidant in cosmetics and personal care products due to its ability to reduce free radical damage in the skin. It prevents wrinkles, fine lines, and age spots; helps with wound healing; softens scar tissue; protects against ultraviolet rays; and reduces several other signs of aging (Sunar et al., 2016; www.truthinaging.com). Mann and Leilin discovered this enzyme as a blue/green protein in 1938. It was later characterized as an enzyme and named as superoxide dismutase by McCord and Fridovitch in 1969.

6.9.2 Peroxidase There are two different types of hydroxyl free radical scavenging enzymes. These enzymes belong to the oxidoreductase class of enzymes and are known as peroxidases and catalases. Plants contain heme-containing peroxidases, which are nonspecific peroxidases and can act on several substrates including hydrogen peroxide. In animals, similar nonspecific enzymes are as follows: • Lactoperoxidase (thiocyanate ion oxidation) • Myeloperoxidase (phagocytosis) • Thyroid peroxidase (iodine ion oxidation) The most studied enzyme is the horseradish peroxidase, which is obtained from the roots of horseradish. These free radical scavenging enzymes are been used largely in PCPs. For example, fennel seed extracts containing peroxidase are being used in cosmetics due to their high lipid peroxidation activities and low odor. The pale yellow/green liquid extract contains nonirritating and nonsensitizing activity and shows higher activity than tocopherol. Lignin peroxidase obtained from a fungus is a novel skin-lightening active agent. It is being studied to develop as an ingredient in products for treating pigmentation problems. From these discoveries, the development of lignin peroxidase as a skin-lightening agent resulted (US Patent and Trademark Office Patent Application 20060051305). Lignin peroxidase is an extracellular enzyme and is produced during submerged fermentation of Phanerochaete chrysosporium, which is a white-rot fungus (Woo et al., 2004). It is then purified from the fermented liquid broth. The safety of lignin peroxidase as a skin-lightening active ingredient has been shown in preclinical trials. It is nonmutagenic and nonirritating to the eyes, and the potential for skin irritation is very low. The trade name of lignin peroxidase is Melanozyme. It identifies eumelanin in the epidermis and breaks down the pigment without having any effect on melanin biosynthesis or blocking tyrosinase. Melanozyme is a glycoprotein and is



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presently proprietary and is available only in a new skin-lightening product known as Elure. It is active at pH 2–4.5 (Sunar et al., 2016).

6.9.3 Tyrosinase Tyrosinase is an oxidase and is the rate-limiting enzyme for controlling the production of melanins. This enzyme is involved in two distinct reactions of melanin synthesis (Hideya et al., 2007; Kumar et al., 2011): • The hydroxylation of a monophenol • The conversion of an o-diphenol to the corresponding o-quinone o-Quinone undergoes several reactions to form melanin. “The melanin synthesis in melanocytic cells is regulated by tyrosinase enzyme. This is, a membrane-bound copper containing glycoprotein, and is the critical rate-limiting enzyme. Tyrosinase is produced by melanocytic cells, and following its synthesis and subsequent processing in the endoplasmic reticulum and Golgi, it is send to specialized organelles. These are, termed melanosomes, wherein the pigment is synthesized and deposited. In the hair and skin, the melanosomes are transferred from melanocytes to neighboring keratinocytes and are distributed in those tissues for producing visible color” (Sunar et al., 2016; Hideya et al., 2007). The cosmetic industry has worked with substances involved in natural melanin formation during the past years. Not similar to the melanoidin process, a natural tan is induced, and also, protection against ultraviolet radiation is provided. The tyrosinase enzyme converts the tyrosine, which is an amino acid, into dihydroxyphenylalanine (DOPA) and into its quinoid form, the DOPA quinone, which is the base for the formation of both types of melanin—eumelanin (dark brown) and pheomelanin (reddish yellow). The combination of both types is responsible for the skin tone, which varies from skin to skin. The tyrosinase is induced by the α-melanocytes stimulating hormone and controlled by ultraviolet radiation. Other tyrosinase stimulators are the β-endorphins. Endorphin-related substances are found in vegetable extracts, and along with synthetic acetyl tyrosine, they are able to induce the ultraviolet-independent formation of melanin. Additional ultraviolet radiation will accelerate and stimulate the melanin formation process after the product has been used. New developments focus on additional tyrosinase activators and adequate transport systems for integrating the substances into the skin (Lautenschläger, 2007). Zymo-tan complex, which is a tanning activator, consists of tyrosine amino acids (precursors of melanine) and tyrosinase. Tyrosinase enzyme is able to catalyze the reaction forming the melanin in the presence of solar radiation. This enzyme is found in many plants and has been also isolated from yeast, milk, and leucocytes.

6.9.4 Proteases Protease enzymes hydrolyze the protein bonds of amino acids. Proteases are playing an important role in industrial biotechnology, particularly in detergents, foods, pharmaceuticals, and PCPs (Gupta and Khare, 2007; Kalpana Devi et al., 2008). Proteolytic enzymes are important for many physiological processes: • Digestion of food proteins • Protein turnover

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Cell division Blood clotting cascade Signal transduction Processing of polypeptide hormones, etc.

Proteases are used abundantly in the pharmaceutical industry for the preparation of medicines, such as ointments for debridement of wounds. They are also used in cleaning the dentures and for cleaning contact lens (Ogunbiyi et al., 1986). Proteases used in the detergent and food industries are produced in huge quantities and are used in crude form, whereas those used in medicine are produced in small quantity but are extensively purified before use (Bholay and Patil, 2012).

6.9.5 Lipases Lipases are present in all types of living organisms. These enzymes exert their activity on the carboxyl ester bonds of triacylglycerols and other substrates. Their natural substrates are insoluble lipid compounds which are susceptible to aggregation in aqueous solution. Among the lipases from higher eukaryotes, porcine pancreatic lipase has been used for several years as a technical enzyme. Active lipases can mostly be found in cosmetics for cleansing (anticellulite treatment) or overall body slimming, where they are responsible for the mild loosening and removal of dirt and/or small flakes of dead corneous skin and/or assist in breaking down fat deposits, often in combination with proteases. Further applications have been mentioned for nose cleansing, makeup beauty masks, and hair care. Based on the broad variety of compounds obtained from fats and carboxylic acids in cosmetic products, lipases and their hydrolytic, esterifying, and acylating activities show huge potential for implementation in the production of cosmetic ingredients. (Lotti and Alberghina, 2007; Sunar et al., 2016)

Immobilized lipases are used for the preparation of water-soluble retinol derivatives and are commercially very important in cosmetics and pharmaceuticals such as skin care ­products. Lipases are used as ingredients of topical antiobese creams or as oral administration and in hair waving preparation (Gurung et al., 2013).

6.9.6 Hyaluronidase Hyaluronidase (HA) enzymes catalyze the hydrolysis of certain complex carbohydrates such as hyaluronic acid and chondroitin sulfates. The enzymes are found in mammalian tissues (testis being the richest mammalian source), insects, leeches, snake venom, and bacteria. HA is gaining much importance in cosmetics for its popularity in cosmetic facial augmentation. HA is a naturally occurring glycosaminoglycan disaccharide present in skin and synovial joint structural properties. These varying properties may inform clinicians as to which HA filler would be most suitable for a specific clinical use. For instance, a more highly crosslinked HA filler would likely be strong in its ability to hold its form, making it suitable for the correction of deep wrinkles. Furthermore, a more monophasic filler can cleanly retain its form and clinically have a smoother appearance. Hyaluronidase is FDA (US Food and Drug Administration) approved as a temporary dispersion agent for injectable fluids, particularly local anesthetics during retrobulbar blocks. It has been used medically for more than 60 years (Silverstein et al., 2012).



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Biotech and pharmaceutical companies are making enormous investments in new technologies to drive product innovation. In the last 10 years, we greatly increased investment in scientific research and new technologies, driven by the assumption that “defeating” skin aging with beauty products will become increasingly possible. Biomimetics, 3-D bioprinting, and plant tissue culture techniques will all be areas of research of the beauty industry in the future. All of these developments should finally benefit consumers by resulting in the next generation of safer and more effective products (www.mdpi.com).

6.10  Soaps and detergents Soaps are mostly produced from oils and fats, which are obtained from plants and animals. Biotechnology as such is not used in processing of soaps and detergents, but enzymes are present in most washing detergents. Biotechnology-based cleaning agents based on enzymes are extensively used in industries. The biotechnology-based cleaning agents are cheaper and do not have much adverse effect on the environment (ETBPP, 1998). These cleaning agents have specific cleaning action and can be used at reduced temperatures also. The effluents produced have reduced COD and are not corrosive. Enzyme-based cleaners are becoming very popular in the food industry in comparison with caustic or acid cleaning agents (D'Souza and Mawson, 2005). Four classes of enzymes are generally used in detergents (Table 6.50). Enzymes in detergent industry are key to cleaning. These enzymes possess better cleaning properties in comparison with synthetic detergents (Gavrilescu and Chisti, 2005). They are able to work at low washing temperatures and are also eco-friendly (Kumar et al., 1998). The enzymatic activity in the detergents is not lost after the stain is removed. The enzyme-based detergents keep the color bright and also improve the fabric quality. TABLE 6.50  Four classes of enzymes are generally used in detergents. Proteases Most widely used enzymes in the detergent industry remove protein stains such as grass, blood, egg, and human sweat, which have a tendency to adhere strongly to textile fibers Amylases Used to remove residues of starch-based foods like potatoes, spaghetti, custards, gravies, and chocolate Lipases Decompose fatty material. Lipase is capable of removing fatty stains such as fats, butter, salad oil, and sauces and the tough stains on collars and cuffs Cellulases Modify the structure of cellulose fiber on cotton and cotton blends. When it is added to a detergent, it results in color brightening, softening, and soil removal, indeed a sustainable alternative to chem Based on Ito, S., Kobayashi, T., Ara, K., Ozaki, K., Kawai, S., Hatada, Y., 1998. Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures. Extremophiles 2 (3), 185–190.

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The enzyme-based detergents are used in small quantity in comparison with synthetic chemicals. They are active at very low temperature and are eco-friendly and completely biodegradable. Removal of proteins using the nonenzymatic detergents can result in permanent stains because of oxidation and denaturation caused by bleaching and drying. Protease enzymes hydrolyze the proteins and produce free amino acids or more soluble polypeptides. The use of surfactants and enzymes in combination remove the hard stains from the fibers. One of the major application of enzymes is in laundry and dishwashing detergents (Schafer et al., 2002; Gerhartz, 1990; Bajpai and Tyagi, 2007; Novozymes, 2011; Kirk et al., 2002). This is the largest application both in terms of volume and value. Enzymes can work at moderate temperature and pH, which characterize modern laundering conditions, and in laundering, dishwashing, and industrial cleaning, they offer many benefits (Table 6.51). In developed countries, the use of enzymes in detergent is very common. About half of the detergents currently available contain enzymes. Detergents amount to 25%–30 % of the total sales of industrial enzymes (link.springer.com). Use of enzymes in detergents started in the early 1930s. The pancreatic enzymes in presoak solutions were used. Otto Rohm from Germany first patented the use of pancreatic enzymes in 1913. The enzymes were obtained from the pancreases of slaughtered animals. These included proteases (trypsin and chymotrypsin), carboxypeptidases, α-amylases, lactases, sucrases, maltases, and lipases. Thus, the trend was already laid in 1913 for the use of enzymes in detergents. These days, enzymes are continuously being used in detergent formulations for laundry, automatic dishwashing, or cleaning of industrial equipment in the food industry. Soils and stains are removed by mechanical and enzymatic action. Surfactants, polymers, etc. are also used. Different types of surfactants help the wash liquor wet fabrics by reducing the surface tension at the interface and help in removing different kinds of soilings. Moreover, anionic surfactants and polymers increase the repulsive force between the original enzymatically degraded soil and the fabric and thus help in preventing the redeposition of soil. Builders chelate, precipitate, or ion-exchange calcium and magnesium ions, for providing alkalinity and buffering capacity, and inhibit corrosion. Enzymes present in heavy duty detergents degrade and thus help to solubilize substrate soils attached to fabrics or hard surfaces (e.g., dishes). (www.novozymes.com).

TABLE 6.51  Advantages of using enzymes in detergents. A better cleaning performance in general Rejuvenation of cotton fabric through the action of cellulases on fibers Reduced energy consumption by enabling lower washing temperatures Reduced water consumption through more effective soil release Minimal environmental impact since they are readily biodegradable Environmentally friendlier wash water effluents (in particular, phosphate free and less alkaline) Furthermore, the fact that enzymes are renewable resources also makes them attractive to use from an environmental point of view Based on Bajpai, P., 2018. Industrial Enzymes—An Update, first ed. ISBN: 978-87-403-2129-6, Pages: 118.



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TABLE 6.52  Enzymes used in detergents (laundry and dishwash). Protease

Protease enzyme removes the protein-based stains like blood, milk, grass, egg, and minced meat

Amylase

Amylase hydrolyses starch-based products like cereals, pasta, potatoes, and rice

Lipase

Lipase hydrolyses fatty stains such as lipstick, frying fats, butter, salad oil, and sauces and the tough stains on collars and cuffs containing residues of human sebum

Cellulase

Cellulase imparts biofinishing where it improves the general cleanness and whiteness of laundry

Mannanase

Mannanan stain removal (reappearing stains)

Based on Kirk, O., Borchert, T.V., Fuglsang, C.C., 2002. Industrial enzyme applications. Curr. Opin. Biotechnol. 13, 345–351.

Proteases are the major component in detergent enzymes. But other hydrolases are introduced for providing several advantages, such as the effective removal of specific stains (Table 6.52). New and better variety of the conventional detergent enzymes has been developed. These new second- and third-generation enzymes (proteases and amylases) are optimized for meeting the requirements for performance in detergents, the composition of which is also continuously changed. The compatibility of enzymes with detergents is especially addressed, but their ability to work at reduced temperature has also been among the recently reported improvements. For saving energy, the temperature in household laundering and automated dishwashers has been reduced nowadays. This usually results in problems with effective cleaning and stain removal that enzymes can help to overcome. Examples of second-generation detergent enzymes include the development of novel amylases having improved activity at reduced temperatures and alkaline pH while maintaining the necessary stability under detergent conditions. These enzymes were developed by using the microbial screening and rational protein engineering methods (Bisgaard-Frantzen et al., 1999). Proteases that show activity at low temperatures not only have been isolated from nature but also have been evolved in the laboratory using a directed evolution strategy (Wintrode et  al., 2000). Moreover, from a starting material of 26 subtilisin proteases, Ness et al. (1999) used DNA shuffling to isolate new proteases with better properties. The improvements included characteristics very relevant for detergent proteases (i.e., improved activity and stability at alkaline pH). New enzyme mannanase was introduced into detergent jointly by Procter & Gamble and Novozymes (McCoy, 2001). This enzyme removes several food stains containing guar gum, which is used in food products. Guar gum is used as stabilizer and thickening agent. Cellulases also clean indirectly by gently hydrolyzing certain glycosidic bonds in cotton fibers. Thus, particulate soils attached to microfibrils are removed. Another desirable effect of cellulases is to achieve greater softness and improved color brightness of worn cotton surfaces. Several detergent brands are based on a blend of two or more, even up to eight different enzyme products. One of the driving forces behind the development of new enzymes or the modification of existing ones for detergents is to make enzymes more tolerant to other ingredients, for example builders, surfactants, and bleaching chemicals, and to alkaline solutions. The trend towards lower wash temperatures, at least in Europe, has also increased the requirement for additional and more effective enzymes. Starch and fat stains are relatively easy to remove in hot water, but the additional cleaning power provided by enzymes is required in cooler water. The most widely used detergent enzymes are hydrolases, which remove soils consisting of proteins, lipids, and polysaccharides. Currently, research is being conducted with a view to extending the types of enzymes used in detergents. Many problem stains come from a range of modern food products such as chocolate ice cream, baby food, desserts, dressings and sauces. (www.novozymes.com)

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For removing these stains and classical soilings such as blood, grass, egg, and animal and vegetable fat, several different types of hydrolases are added to detergents. The major enzymes are proteases, cellulases, lipases, amylases, mannanases, and pectinases. Historically, proteases were the first enzymes to be used widely in laundering to increase the efficacy of detergents. Cellulases perform cleaning by maintaining, or even rejuvenating, the appearance of washed cotton-based garments through selective reactions not available earlier when washing clothes. Certain type of lipase enzymes can act as a substitute to current surfactant technology targeting greasy lipid stains. Thus, lipases are an important part of enzyme solutions used for replacing surfactants (www.wesrch.com). Often, multienzyme systems can replace up to 25% of a surfactant system in laundry detergent without have any effect on cleaning. This leads to a more sustainable detergent that allows cleaning at low temperatures. Mannanase and pectinase enzymes are used to remove stains that are difficult to remove such as salad dressing, ketchup, mayonnaise, ice cream, frozen desserts, milkshakes, body lotions, and toothpaste and also tangerines, banana, tomatoes, and fruit containing products such as marmalades, juices, drinking yogurts, and lowfat dairy products. The distinct benefits of enzymes make them universally acceptable for meeting the needs of consumer. Because the enzymes are catalyst, they are needed in small amounts in the formulations. This is of especially important at a time where detergent producers are compactifying their products (www.novozymes.com). In many parts of the world, laundry detergent bars are used for removing strongly colored and stubborn stains from sebum, blood, food soils, cocoa, and grass. After several years of very little performance improvement for laundry bars, a new solution that involves the use of enzymes has been developed. A specially formulated protease enzyme enables the producer to produce products that stand out from nonenzymatic laundry detergent bars, offering effective washing. Stain removal and washing by hand are very time-consuming and physically demanding domestic activities. With the addition of proteases, washing is reduced by at least one rinse and needs much less scrubbing. Laundry bars consisting of the enzyme may be formulated in such a way that these are milder to the hands than old type of bars without enzymes (www.novozymes.com). Most of the energy that is spent during a household machine wash is used for heating the water. Therefore, for energy saving and thus helping to reduce carbon dioxide emissions, the most effective measure is to reduce washing temperatures. Increased use of enzymes combined with a selection of appropriate other ingredients, including surfactants and bleaching systems particularly selected to work at reduced temperatures, has enabled the producers to produce cold water detergents (www.novozymes.com). Modern dishwashing detergents face increasing consumer demands for efficient cleaning of tableware. Enzymes are major ingredients for efficiently removing difficult and dried-on soils from dishes and leaving glassware shiny. Enzymes clean well under mild conditions and thereby assist to reduce clouding of glassware. In addition, enzymes also enable environmentally friendly detergents. Phosphates have been used in dishwashing detergents to get dishes clean, but they harm the aquatic environment and are increasingly being banned in detergents around the world. The combination of modifying detergent compositions and using multienzyme solutions enables the detergent manufacturers to replace phosphates without compromising the cleaning performance. For removal of starch soils, amylases are used; and proteases are used for removal of protein soil. (www.aocs.org).



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There is a movement in the market toward enzymes in hand dishwash. Amylases are being used for cleaning dishes from starch-containing soils. The amylase removes stubborn starch without scrubbing. An alkaline extracellular enzyme obtained from Bacillus strains is used in detergents. Other enzymes such as proteases, α-amylases and cellulases are produced on large scale and have been added in detergents (Ito et al., 1998). The dishwashing detergents contain different chemicals which are chelating agents, surfactants, polycarboxylates (PCA), phosphonates, active chlorine compounds etc. These substances have high environmental risks, particularly via sewage biosolids, poor biodegradability, increased organic load to sewage works, toxicity. (CEEP-phosphates in dishwasher detergents, 2007; www.academicjournals.org)

Enzymes are added in detergents for increasing the cleaning ability of detergents. Enzymes can be used instead of chlorine bleach to remove stains from the cloth. The enzyme protease was produced from alkaliphilic Bacillus clausii KSM-K16 and strain KP-43 and Bacillus sp. strain KSM-KP43 and has been included into laundry detergents. Subtilisin-like serine proteases have been used in laundry and dishwashing detergents (Saeki et al., 2007). These enzymes belong to family A of subtilase superfamily. Several alkaliphilic Bacillus produce alkaline cellulase. These are used as an additive to improve the efficacy of detergents. Properties of some cellulase enzymes fulfilled the necessary requirements for enzymes to be used in laundry detergents (Ito, 1997; www.academicjournals.org). Among other enzymes used in some detergents are Guardzyme, which is a peroxidase. This enzyme inhibits the transfer of dye. Carezyme removes the fuzz that builds up on cotton clothes. Enzymes used in detergents should be effective at reduced dose, compatible with several detergent components, and active at wide range of temperatures (Kumar et al., 1998; www.academicjournals.org). A variety of detergent formulations such as anionic or nonionic surfactants and the powdered lipases are used. Lipases are used to remove oil stains, and proteases are used to remove protein stains; cellulases are used for preventing pilling of cotton (Kirk et al., 2002). These enzymes are produced to a great extent by the use of genetically modified microorganisms. In detergent formulations, less than 1% enzyme by volume is present, but the enzymes contribute ~8% to the cost of the detergent. Use of enzyme reduces severity of washing. Their use can produce several benefits. Clothes washed with enzyme-containing detergents tend to be much cleaner in comparison with clothes washed with phosphate-containing detergents. In comparison with traditional detergents, enzyme-containing detergents may be formulated with less phosphate, to significantly reduce the release of this eutrophication agent to the environment. Washing detergents containing enzymes are more eco-friendly. Henkel Company (www. henkel.com) has used natural enzymes in detergent formulations since the 1970s. Enzymes produced using genetically engineered enzymes have been added to detergents since the late 1980s (Maurer and Kottwitz, 1999; Maurer, 2010). For instance, Bacillus lentus alkaline protease (BLAP) has been developed. This enzyme is expected to reduce environmental pollution associated with detergents by more than 65% and is used in washing detergents. This is a genetically modified enzyme and has been produced since 1995. Microbial proteases have several other uses also (Kumar et al., 1998).

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6.11  Bioplastics and other biopolymers Plastics are the biggest consumers of fossil fuel outside energy and transport. Concern over persistence of petrochemical plastics in the environment is a renewing interest in polymers obtained from biological sources (www.plastics.fi; Gavrilescu and Chisti, 2005; Kim et  al., 2000; Babel and Steinbüchel, 2001; Stevens, 2002). The term bioplastics encompasses numerous different plastics. Bioplastics are biobased and biodegradable and can be used in short-life, disposable products; however, they can also be used for durable applications. It should be remembered that biobased plastics are not always biodegradable, and biodegradable plastics are not always biobased. The goal in nondisposable applications of bioplastics is not to achieve biodegradability, but to produce items from sustainable resources. There are a number of bioplastics that are either commercial or in very active development. Bioplastics are a large family of materials. The term bioplastics is generally used to discuss two entirely different classes of polymer:

6.11.1  Biobased polymers These are produced in whole or in part from renewable raw materials, for instance, sugar, starch, vegetable oils, cellulose, and also food residues. The concept is related to the “origin of the carbon building block.” There is a requirement to switch from petroleum-based resources to renewable ones for controlling greenhouse gas emissions.

6.11.2  Biodegradable polymers These polymers possess some degree of inherent biodegradability. These can be decomposed by the activity of bacteria or fungi and produce natural metabolic products. In this case, the concept is related to the “end of life and disposal” of polymeric materials, and the focus is on waste management methods. The benefits of bioplastics are the main reason for the dynamic development of industry. It is growing regularly, at a rate of about 20%–100% per year. A survey performed by the University of Applied Sciences and Arts Hanover (Germany) on behalf of European Bioplastics reports that, between 2011 and 2016, worldwide production capacities are expected to grow fivefold to about 6 million tonnes. Because of their ability to improve economic growth and ecologic footprints, bioplastic materials and products would be a welcome supplement to any product portfolio of the company. The European Commission is recognizing the great importance of the bioplastics sector. It has been identified as an important pillar of the bioeconomy (tur-www1.massey.ac.nz). Bioplastics are not a single plastic but a family of materials, which can vary significantly from one another. There are three groups in the bioplastics family; each group has its own characteristics (Table 6.53). Bioplastics can be processed into a several type of products using traditional plastic processing techniques. The process parameters of the processing equipment need to be adjusted according to the individual specification of each polymer. Several types of converters are now transforming bioplastic materials to products. Driven by a growing need for sustainable solutions, the types of available bioplastics are increasing continuously. Every day, new bioplastic



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TABLE 6.53  Types of bioplastics. Biobased or partly biobased, nonbiodegradable plastics, for instance, PE, PET (drop-in solutions), or technical polymers such as polyamides Plastics that are biobased and biodegradable. This includes polylactic acid, cellulose, and polyhydroxyalkanoates Plastics that are obtained from fossil resources and are biodegradable, such as polybutyrate adipate terephthalate or polybutylene succinate Based on www.plastics.fi

TABLE 6.54  Market drivers. Internal market drivers Advanced technical properties and functionalities Possibility of cost reduction through economies of scale Sustainable and new recycling option External market drivers High consumer acceptance Change in climate Increase in the price of fossil materials Dependence on fossil raw material Based on www.sari-energy.org

TABLE 6.55  Bioplastic opportunities. Reduced emissions of greenhouse gases Reduced use of nonrenewable raw materials Composting/anaerobic digestion—most of the bioplastics can be managed using these methods and thereby offer opportunities for diverting waste from landfill Agricultural film disposal—starch mulch was found to offer environmental benefits over linear low-density polyethylene, although it would need to be left on the land after use for realizing these benefits Renewable conventional plastic disposal—these do not need to be differentiated from conventional plastics and as such do not affect the UK waste system Bioplastics are anticipated to offer jobs in the agricultural sector. By 2020, 20,000 new jobs worldwide are expected to be created Based on randd.defra.gov.uk

materials, compounds, and master batches are produced, and more and more production facilities go onstream (tur-www1.massey.ac.nz). The market drivers are presented in Table 6.54. Bioplastic opportunities are presented in Table 6.55. Political support is also expected to play a conclusive role in the coming years in addition to the market drivers mentioned here. A strong regulatory framework that does not constrict

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industry is a prerequisite for a flourishing bioeconomy in European countries and strong and flourishing development of the bioplastics sector (tur-www1.massey.ac.nz). Bioplastics are driving the evolution of plastics and are substantially contributing to a sustainable society. Bioplastics are saving fossil resources and carbon dioxide emissions. Moreover, they are offering additional end-of-life options where suitable. This makes bioplastic materials and products an attractive option for industries and consumers who are aiming to reduce their impact on the environment. Interest in bioplastics is increasing continuously, and the market is characterized by high and stable growth rates of ~ 20%–100% every year (scientiaresearchlibrary.com). The production facilities are increasing, and the capacities are growing; therefore, supply options for bioplastic materials and products will increase significantly in the coming years. Europe is a major center for the industry; it is occupying the top position in the field of research and development and is the huge market of the industry globally. Regarding the actual production of bioplastics, a supportive framework at European Union and Member State level is required for ensuring a balanced worldwide development (tur-www1.massey.ac.nz). Feedstocks that are used for producing bioplastics are, in fact, biomaterials that are obtained from biomass. The feedstocks used are sugar and starch bioproducts obtained through fermentation and chemical processes, such as alcohols, acids, starch, and xanthium gum, and obtained from feedstocks including corn, sugarcane, sugar beets, rice, potatoes, sorghum grain, and wood; oil- and lipid-based bioproducts obtained through chemical processes, such as fatty acids, oils, alkyd resins and glycerin, and obtained from feedstocks including soybeans, castor oil, rapeseed, and other oilseeds; cellulose derivatives and plastics, such as cellulose nitrate, cellulose acetate (cellophane) and triacetate, alkali cellulose, and regenerated cellulose, obtained from wood pulp and cotton linters; protein (wheat gluten, chitin, soy protein, zein, and silk); and, finally biomass (randd.defra.gov.uk). Several of these are used to produce bioplastics such as biopolyethylene, polylactic acid, polyhydroxyalkanoate, epoxy resins, alkyd resins, and regenerated cellulosics. Algae are found to be a good feedstock for plastic production because of its high yield, and also, it can grow in different environments. Algae bioplastics mainly evolved as a by-product of algae biofuel production, where companies were exploring alternate sources of biobased fuels. The use of algae also opens up the possibility of using carbon, neutralizing greenhouse gas emissions from factories or power plants. On the heels of conventional methods of using feedstocks of corn and potatoes as plastics, algae-based plastics have been a new trend in the era of bioplastics. While algae-based plastics are in their early stages, once they are into commercialization, they will be used in different industries (www.plastic.org.il). Current biomass conversion technology generally starts with starches obtained from biomass, sugars, and oils that are then converted to major building block chemicals through biological or chemical conversions and eventually converted to biobased chemicals and polymers. There are two main processes for manufacturing bioplastics: Direct extraction from biomass yields a series of natural polymer materials; alternately, the renewable resources/biomass substrates can be converted to biomonomers by fermentation or hydrolysis and then further converted to bioplastics using chemical s­ynthesis. Biomonomers can also be transformed to bioplastics like polyhydroxyalkanoates using microorganisms. Vegetable oils are offering another important carbon platform to polyols (precursors for polyurethanes and polyesters) and other functional monomers/macromers.



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Bioplastics are ­generally used in similar applications as petropolymers (i.e., petroleum-based plastics) (au.nec.com; www.icis.com) Process conversion issues to consider for bioplastics are as follows: several of the early bioplastics lack the same thermal and mechanical performance of their analogous petropolymers. Bioplastic grade innovation has provided better thermal and mechanical performance, but the nature of bioplastics must still be considered, and rarely can a bioplastic be substituted directly for a petropolymer in a conversion process. For extrusion and other molding processes, bioplastics may need a change in screw design because bioplastics tend to be more shear sensitive than conventional petropolymers, but a new screw does not always solve all the problems related to conversion. Rather, bioplastics may need new designs for extrusion dies and new molds for injection or blow molding tooling, as the dies and molds designed for conventional petropolymers do not always fit bioplastic rheological characteristics. Power is a major cost for extrusion and molding processes, but in case of bioplastics, there can be other costs, also as additional compounding of compatibilizing agents and other polymers. Bioplastics are found to be more sensitive to variation in heat cycling, dissipation, cooling, and overall heat history in comparison with traditional petropolymers (www.bioplasticsmagazine.com). Conversion speed should be more closely monitored, as the aforementioned issues will have an effect on the conversion performance and the quality of final product. Several methods are used for conversion: • Injection molding • Extrusion • Thermoforming • Blow molding • Transfer molding • Reaction injection molding • Compression molding Each method has its benefits and drawbacks and is better suited for specific applications. Several durable biobased plastics, with different biobased content (starch-polyolefin blends, polyethylene, polypropylene, polyethyleneisosorbide terephthalate, polytrimethylene terephthalate, polyurethanes, polyvinyl acetate, polyamides, alkyd resins, epoxy resins, and thermosetting polyesters), are being commercialized and even more are currently being developed. The growth prospects of the biobased durable plastics in the next decades are much greater as compared with biobased biodegradable plastics. Few companies have developed and commercialized biobased polyols for the production of polyurethane. The application possibilities of these biobased polyols rapidly increase because of improved functionalization techniques. Biobased polymers not only replace existing polymers in several applications but also provide new combinations of properties for new applications. In an unusual innovation, NEC Corporation of Japan announced the development of a new durable bioplastic produced from nonedible plant resources. The bioplastic is produced by bonding cellulose fibers from different types of plant stems, with cardanol—a primary component of cashew nut shells— which obtains a level of durability that is appropriate for electronic equipment and has a high biomass composition ratio of more than 70%. The durable plastic market is anticipating new materials produced from renewable feedstock. Experts say that there are many properties for durable plastics that cannot be met by compostables. Bioplastics represent just 1% of the 230

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tonnes of plastics used worldwide. The increasing demand for biobased, semidurable, and durable products for household products is driving development of building blocks for existing plastics and also new materials from renewable raw materials. Applications of durable bioplastics include the following: • Automotive • Electronics/electrical • Durable biomedical materials • Consumer goods • Building and construction • Textiles • Coatings The use of bioplastics in the field of construction is considered to be a more sustainable activity when compared with commercial PVC because bioplastics are using less carbon sources and produce fewer greenhouse gas emissions. The occurrence of biodegradable plastics such as polyhydroxyalkanoic acids in bacteria has been reported since the 1920s. The Japan Institute of Physics and Chemical Research genetically engineered a microorganism to produce up to 96% of its dry weight as biodegradable plastic (Lenz, 1995). Several different plastic and nonplastic biopolymers are currently available. Although they are expensive, their production and use are environmentally sustainable. Substantial efforts are being made in developing process for improved production of polyhydroxyalkanoates and other biodegradable, renewable biopolymers (Tamer et al., 1998a,b; Grothe et al., 1999; Grothe and Chisti, 2000; Babel and Steinbüchel, 2001; Stevens, 2002; Salehizadeh and Van Loosdrecht, 2004). Biopolymers with improved properties and microorganisms for producing them are being produced. More effective fermentation and product recovery processes have been explored (Tamer et al., 1998a,b; Grothe et al., 1999; Grothe and Chisti, 2000; Salehizadeh and Van Loosdrecht, 2004). The use of mixed cultures and inexpensive substrates can significantly reduce the production cost of polyhydroxyalkanoates (Salehizadeh and Van Loosdrecht, 2004; www.plastics.fi). In addition to bioplastics, several nonplastic biopolymers are available for use as thickeners, gelling agents, lubricants, and other applications (Paul et al., 1986; Sutherland, 1994; GarcíaOchoa et al., 2000; Laws et al., 2001). In 1999, about 30,000 tons of the polysaccharide biopolymer xanthan valued at USD 408 million was produced (Demain, 2000; www.plastics.fi). Biobased natural polymers consist of starch, cellulose, chitin, chitosan, and several other polysaccharides and proteins. These polymers occur naturally and offer a diverse range of properties and applications. Examples are starch, cellulose, chitin and chitosan, pullulan, alginates, carrageenan, xanthan, dextrans, pectin, glucans, gellan, collagen, gelatin, soy protein, whey protein, zein, casein, and gluten. Many starch-based products are being produced for several applications. “Applications of thermoplastic starch polymers include films for packing materials. shopping, bread, and fishing bait bags, overwraps, flushable sanitary product and special mulch films. Potential future applications could include loose-fill packaging and injection-molded products such as ‘take-away’ food containers. Starch and modified starches have a broad range of applications both in the food and non-food sectors” (Huber and BeMiller, 2010; Daniel et al., 2007; paperity.org). Conversion of acrylonitrile to acrylic acid for the production of anionic polyacrylamides is an example of large-scale biotransformation showing environmental and commercial



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­advantages. Ciba Specialty Chemicals (www.cibasc.com) is producing a variety of polymers based on acrylamide and acrylic acid using biotechnological processes. The traditional method for producing acrylic acid was an unsound, multistage process using large amount of energy. This process needed higher concentrations of toxic acrylonitrile, operated at high temperature and produced hazardous emissions. Biotransformation route used by Ciba has the following advantages: • • • • •

A simple, one-step cost-effective process providing a better quality product Production at ambient temperature and atmospheric pressure Low concentration of hazardous acrylonitrile during the production Generation of fewer byproducts Near quantitative conversion

Another example is the Mitsubishi Rayon’s bioprocess for producing acrylamide. This is then polymerized to the conventional plastic polyacrylamide. In the United Kingdom, Baxenden Company (www.baxchem.co.uk) is producing polyesters, acrylic polymers, and emulsions and other specialty chemicals using biocatalytic processes, which have reduced the reaction temperature to 60°C in comparison with 200°C for equivalent chemical processing (Gavrilescu and Chisti, 2005; www.plastics.fi).

6.12 Biofuels 6.12.1 Bioethanol There are several disadvantages of fossil fuel–derived transportation fuels. These are as follows: • Release of greenhouse gases • Pollution • Exhaustion of resources • Unbalanced supply–demand relations These disadvantages are significantly reduced or even absent with the use of biofuels. Of all the biofuels, ethanol is being produced on a large scale. Ethanol has several advantages. These are listed in the succeeding text: • Biodegradable. • Greenhouse emissions are lesser in comparison with fossil fuel. • Can replace harmful fuel additives. • Produces employment for farmers and refinery workers. • Can be easily applied in present-day internal combustion engine vehicles as it is possible to blend with gasoline. Ethanol is already commonly used in a 10% ethanol/90% gasoline blend. Adapted internal combustion engine vehicles can use a mixture of 85% ethanol/15% gasoline (E85) or even 95% ethanol (E95). Addition of ethanol increases octane and reduces carbon monooxide, volatile organic carbon, and particulate emissions of gasoline. Furthermore, via onboard reforming to hydrogen, ethanol can be used in future fuel cell vehicles. These vehicles have approximately doubled the current internal combustion engine vehicle fuel efficiency (Bajpai, 2013a).

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Ethanol is being produced and used in all corners of the world. The issues over petroleum supplies and global warming are continuously growing; many countries are exploring ethanol and renewable fuels as a way to counter dependence on oil and ecological impacts. Global production peaked in 2017 after a dip in 2011 and 2012. The United States is the world's largest producer of ethanol, having produced nearly 16 billion gallons in 2017 alone. Together, the U.S. and Brazil produce 85% of the world's ethanol. The vast majority of U.S. ethanol is produced from corn, while Brazil primarily uses sugarcane. (https://afdc.energy.gov/data/10331)

It is expected that, by the end of the year 2020, the world production would exceed 120,000 million mark. China, India, Thailand, and other countries are expeditiously expanding their ethanol industries. This is actually leading to a growing international trade for the renewable fuel. Whereas the large amount of ethanol is utilized in the country in which it is produced, some countries are exporting ethanol to countries such as the United States and Japan. “High spot market prices for ethanol and the rapid elimination of MTBE by gasoline refiners has led to record imports into the United States in the last few years. More than 500 million gallons of ethanol entered through American ports, paid the necessary duties, and competed effectively in the marketplace. The increased trade of ethanol around the world is helping to open up new markets for all sources of ethanol” (Bajpai, 2013a). The sustainable production of bioethanol needs very good planning and development programs for assuring that several environmental, social, and economic concerns related to its use are addressed properly. Ethanol production can be made competitive when it is produced from low-cost biomass. Several countries in the world are developing new technologies for ethanol production from biomass; the conversion of lignocellulosic materials appears to be a very good option (Bajpai, 2013a). Ethanol is “a wonderfully clean burning fuel that can be produced from farm crops, agricultural wastes, even garbage”—Alexander Graham Bell, 1917. Over the last decade, a lot of interest has been shown in fuel ethanol because of increasing environmental issues, of higher price of crude oil, and, by the prohibition in few regions, of the gasoline additive, methyl tert-butyl ether, which can be interchanged directly with ethanol (Kirk et al., 2002). Therefore, extensive efforts have been made for developing improved enzymes that would allow the use of inexpensive and partially used substrates such as lignocellulosics, for making ethanol more competitive with fossil fuels. The enzyme cost needed to convert lignocellulosics into a suitable fermentation substrate is a main issue, and the recent study has focused on developing enzymes having high activity and stability and also their production in an efficient manner. The Department of Energy in the United States has launched a program for supporting these developments, induced by the emphasis on the reduction of pollution and the requirement to fulfill the Kyoto protocol. The processes for the production of bioethanol vary significantly depending on the substrate involved, but few important stages in the process remain the same, even though they are performed in different conditions of pressure and temperature, and they use different microorganisms. These stages include the following (Olsson et al., 2005): • Hydrolysis • Fermentation • Distillation Hydrolysis is performed chemically or enzymatically. Two types of process technology are presently being used. These are mainly called first-generation technology and second-­ generation technology (Bajpai, 2013a).



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Sugars and starch-rich crops such as grain and corn are used for the production of ethanol in the first-generation technology. Sugars are converted to ethanol directly. However, starches are first converted to fermentable sugars by using enzymes from malt or molds. This technology is well established. However, the prices of the raw material are the main problem (Bajpai, 2013a). The ethics about using food products mater is also an issue. Lignocellulosic materials are used in the production of second-generation ethanol. These are wood, straw, and agricultural residues. These materials are often available as wastes and are not costly. However, the technology is more advanced in comparison with first-generation technology (Fan et al., 1987; Badger, 2002). The lignocellulosic raw material consists of lignin, cellulose, and hemicelluloses (Bajpai, 2013a). Table 6.56 shows first-generation and second-generation feedstocks for bioethanol production (Bajpai, 2013a). Table 6.57 shows enzymes involved in biofuel production. Fig. 6.4 shows stages of bioethanol fuel production. Fig. 6.5 shows different types of forest biomass, and Fig. 6.6 shows schematic of a biochemical cellulosic ethanol production process (Achinas and Euverink, 2016). TABLE 6.56  First-generation and second-generation feedstocks for bioethanol. First-generation feedstocks Sugar beet Sweet sorghum Sugar cane Maize Wheat Barley Rye Grain Sorghum Triticale Cassava Potato Second-generation feedstocks Corn stover Wheat straw Sugarcane bagasse Municipal solid waste Third-generation feedstocks Algae Based on Walker, 2010; Bajpai, 2019. Third Generation Biofuels. Springer Nature America Inc.

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TABLE 6.57  Enzymes involved in biofuel production. Bioethanol First-generation ethanol Alpha-amylase Beta-amylase Glucoamylase Second-generation ethanol Endoglucanases Cellobiohydrolases Beta-glucosidases Biodiesel Lipase

FIG. 6.4  Stages of bioethanol fuel production. Reproduced with permission from Achinas, S., Euverink, G.J.W., 2016. Consolidated briefing of biochemical ethanol production from lignocellulosic biomass. Electron. J. Biotechnol. 23, 44–53.

Enzymes are playing a very important role in the production of biofuels, which are the fuel of the future. Cellulase, xylanase, and amylase enzymes act on cellulosics and starchy substrates and produce a mixture of carbohydrates that can be converted into ethanol after fermentation with suitable microbes (secure.palgrave-journals.com) Ethanol produced from cellulose releases 87% less emission than burning petrol. On the other hand, ethanol from cereals releases 28% less emission. Ethanol produced from cellulose contains 16 times the energy required to produce it, petrol only 5 times whereas ethanol from corn only 1.3 times. The main issue is how to break the bonds of this molecule for converting it into fermentable sugars. Actually, lignocellulosics are the type of raw material



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FIG.  6.5  Different types of forest biomass. Reproduced with permission from Achinas, S., Euverink, G.J.W., 2016. Consolidated briefing of biochemical ethanol production from lignocellulosic biomass. Electron. J. Biotechnol. 23, 44–53.

Biomass handling

Biomass pretreatment

Enzyme production

Cellulose hydrolysis

Ethanol

Glucose fermentation

Ethanol recovery

Pentose fermentation

Lignin utilization

FIG. 6.6  Schematic of a biochemical cellulosic ethanol production process. Reproduced with permission from Achinas, S., Euverink, G.J.W., 2016. Consolidated briefing of biochemical ethanol production from lignocellulosic biomass. Electron. J. Biotechnol. 23, 44–53.

that is the most difficult to process. Lignin binds cellulose, hemicellulose, pectin, and protein in lignocellulosics. Lignin withstands attack by microbes and imparts strength to the plant. Pretreatment is needed for opening the biomass by degrading the lignocellulosic structure and releasing the polysaccharides (Bajpai, 2016). After pretreatment, treatment with cellulose and hemicellulase enzymes is conducted. The cellulose fraction releases hexose sugars, and the hemicellulose fraction releases pentose sugars. “Out of carbohydrate monomers in lignocellulosic materials, xylose is second most abundant after glucose. Glucose is easily fermented into ethanol, but another fermentation process is required for xylose – for example using special microorganisms. The second generation holds great advantages with the

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f­ ermentation of biomass in form of agricultural waste materials but there are some challenges such as efficient pre-treatment and fermentation technologies together with environmentally friendly process technology” (Bajpai, 2013a). Production of ethanol from cellulose holds great promise because of the following reasons: • Abundance • Widespread availability • Low cost of cellulosic materials Substantial investment has been made to develop the commercial processes using the biochemical and thermochemical conversion technologies for ethanol. Johnson et al. (2010) have published an excellent review on the current status of commercial production of ethanol from lignocellulosics raw material. In United States, in 2016 the production of a record 15.25 billion gallons of ethanol supported 74420 direct jobs in renewable fuel production and agriculture as well as 264 756 indirect and induced jobs across all sectors of the economy. (ethanolrfa.org)

United States has targeted 136,260 million liters per year (ML/yr) of renewable fuels production by 2022. This target can be achieved with most of this renewable fuel coming from lignocellulosics (wood, corn stover, switch grass, wheat straw, and energy crops). Several demonstration-scale cellulosic ethanol plants are being constructed as part of the government’s objective for making cellulosic ethanol cost competitive. These plants cover different types of raw materials, conversion technologies, and plant configurations for identifying viable technologies and processes for commercialization. The demonstration plants, which are sized at 10% of a commercial scale biorefinery, would become operational soon. Several commercial scale plants are in the planning stages (www.becaamec.co.nz). Demonstration and commercial plants include: Abengoa –Alico, Alltech, American Energy Enterprises (AEE), Bluefire Ethanol, Coskata, Flambeau River Papers, Park Falls, Wisconsin, Fulcrum-Bioenergy, Sierra Biofuels Plant, ICM, Mascoma, The Wisconsin Rapids, Pacific Ethanol, Red Shield Environmental (RSE), The BioGasol process, Poet, Pure Energy & Raven BioFuels, Range Fuels, Verenium, Virent. Several efforts are being made in North America to commercially produce ethanol from wood and other cellulosic materials as a primary product. (Bajpai, 2013a) National Renewable Energy Laboratory and its partners say that the research conducted in this area is an important step toward realizing the potential of biorefineries (www.ethanol.org/documents/6-05_ Cellulosic_Ethanol.pdf). Biorefineries, analogous to today’s oil refineries, will use plant and waste materials to produce an array of fuels and chemicals – not just ethanol. Biorefineries will extend the value-added chain beyond the production of renewable fuel only. Progress towards a commercially viable biorefinery depends on the development of real-world processes for biomass conversion. With these new technologies for the production of cellulosic ethanol, its promise becomes closer to reality with each passing day. (Bajpai, 2013a) Cellulosic ethanol is on track to be cost competitive with corn-based ethanol, a development that could drive the fuel’s production, according to an industry survey conducted by Bloomberg New Energy Finance. The survey focused on 11 major producers in the cellulosic ethanol industry, all of which use a method known as enzymatic hydrolysis to break down and convert the complex sugars in non-food crop matter, and a fermentation stage for converting the material into ethanol. Cellulosic ethanol cost 94 cents a liter to produce in 2012, about 40 percent more than ethanol made from corn. That price gap will close slowly according to the prediction made by cellulosic ethanol producers. Project capital costs, feedstock and enzymes used in the production process are still the largest costs of running a cellulosic ethanol plant. But the operating costs are ­reduced due



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to developments in technology. For instance, enzyme costs for a liter of cellulosic ethanol dropped 72 percent between 2008 and 2012 because of technological improvements. (www.environmentalleader.com)

The conversion of the woody biomass into fermentable sugars involves the use of expensive technologies. These methods involve pretreatment with special type of enzymes, which means that second-generation biofuels cannot be produced profitably on a commercial scale. Therefore, third-generation biofuels produced from microalgae appear to be a viable alternative energy resource lacking the main disadvantages associated with first- and second-generation biofuels. “Microalgae are able to produce 15–300 times more oil for biodiesel production compared to the traditional crops on an area basis. Furthermore, compared with conventional crop plants which are usually harvested once or twice a year, microalgae have a very short harvesting cycle allowing multiple or continuous harvests with substantially higher yields” (Bajpai, 2019). Algal biomass does not compete with agricultural food and feed production (Demirbas, 2007a,b). Microalgae are photosynthetic microorganisms and need light, carbon dioxide, and nutrients such as nitrogen, phosphorus, and potassium for its growth and for producing large amount of carbohydrates, which can be further processed into biofuels and other valuable coproducts (Brennan and Owende, 2010; Nigam and Singh, 2011; repositorium.sdum.uminho.pt). In algal biomass, the lignin content is zero, and hemicelluloses content is very low. This results in an increased hydrolysis and/or fermentation efficiency (Saqib et al., 2013; Bajpai, 2019). Algae have also applications in many other areas such as animal feed, human nutrition, pharmaceuticals, nutraceuticals, cosmetics, pollution control, biofertilizer, and wastewater treatment (Choi et al., 2012; Tamer et al., 2006; Crutzen et al., 2007; Thomas, 2002; Hsueh et al., 2007). The cultivation of microalgae also can complement approaches such as bioremediation of wastewaters and carbon dioxide sequestration, thereby addressing the serious environmental concerns (Bajpai, 2019).

6.12.2  Other biofuels made by assistance from enzymes Biofuels include products made via sustainable processing; substantiated by reducing the need for energy from fossil fuel, obtaining better production efficiencies and reducing environmental impact. Biodiesel is an example of such a product having combustion properties like petro-diesel. Biogas is a renewable energy source resulting from biomass – mainly waste products from industrial or agricultural production. A biorefinery is a facility that integrates biomass conversion processes and equipment to produce fuels, power, and value-added chemicals from biomass. Enzymatic catalysis is needed as the way to a sustainable, selective and mild production technique. (www.novozymes.com) Biodiesel is methyl or ethyl esters of fatty acids made from renewable biological resources: vegetable oils or animal fat. The esters are typically made by catalytic reactions of free fatty acids (FFA) or triglycerides with alcohols, preferably methanol or ethanol. The overall reaction is a sequence of consecutive and reversible reactions, in which diglycerides and monoglycerides are formed as intermediate compounds. The complete stoichiometric reaction requires 1 mol of triglycerides and 3 mol of alcohol. The reaction is reversible and therefore excess alcohol is used to shift the equilibrium to the products’ side. Methanol and ethanol are frequently used in the process. Transesterification as an industrial process is generally carried out by heating an excess of the alcohol under different reaction conditions in the presence of an acid or a base, or by heterogeneous catalysts such as metal oxides or carbonates, or by a lipase enzyme. The biodiesel yield in the transesterification process is affected by process parameters like moisture, content of free fatty acids (FFAs), reaction time, reaction temperature, catalyst type and molar ratio of alcohol to oil. (www.novozymes.com; Monisha et al., 2013; Nieves-Soto et al., 2012; Balat and Balat, 2008)

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6.13  Processing of oil and fats The use of enzymes in the oil and fat industry is providing many solutions to the industry problems and the key to produce novel oils and fats (scialert.net). Processing enzymes offer several advantages in the oil and fat industry. These are (www. danisco.com) as follows: • Increase in the yield • Increasing the extraction of oil • Reduction in energy Table 6.58 show the enzymes used in processing of fats and oils. Lipid modification and synthesis catalyzed by lipases has now developed from a simple lab-scale concept to an industrial scale. Lipase enzymes act on carboxylic ester bonds and hydrolyze triglycerides into diglycerides, monoglycerides, fatty acids, and glycerol. Furthermore, lipases are able to catalyze esterification, interesterification, and transesterification reactions, thus offering several strategies for synthesizing desired lipid products. A few products produced commercially using lipase enzymes are listed in the succeeding text (www.enzymeinnovation.com): • • • •

Human milk fat substitutes Cocoa butter equivalents Diacylglycerol cooking oil Polyunsaturated fatty acid concentrates

The transesterification in organic solvents catalyzed by lipases is a developing industrial application (Nakajima et al., 2000). Lipase enzymes are highly specific and are able to catalyze reactions (the industrial hydrolysis of fats and oils or the manufacture of fatty acid amides) under mild reaction conditions; they can be so used for producing chemicals of high value for food and industrial uses at competing production costs. For instance, cocoa butter fat used for chocolate production generally is in short supply, and the price can vary greatly. But transesterification catalyzed by lipase enzymes of inexpensive oils can be used, for instance, for producing cocoa butter from middle fraction of palm. So, enzyme technology based on lipase enzymes involving mixed hydrolysis and synthesis reactions are generally used on a commercial scale for upgrading some of the less desirable fats to cocoa butter substitutes (Undurraga et al., 2001). TABLE 6.58  Enzymes used in the processing of fats and oils. Lipase Transesterification Phospholipase Degumming Lysolecithin production Based on Kirk, O., Borchert, T.V., Fuglsang, C.C., 2002. Industrial enzyme applications. Curr. Opin. Biotechnol. 13, 345–351.



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In one application, immobilized Rhizomucor miehei lipase was used for the transesterification reaction, which is able to replace the palmitic acid in palm oil with stearic acid. Likewise, interesterification of butter fat catalyzed by lipase enzymes was used for reducing the longchain saturated fatty acids. A corresponding increase in C18:0 and C18:1 acid at position 2 of the selected triacylglycerol was also observed (Pabai et al., 1995). Lipase enzymes have been also used for enriching polyunsatured fatty acids (PUFAs) from lipids of animals and plants. Free PUFAs and their mono- and diglycerides are later used for producing several types of pharmaceuticals such as anti-inflammatories and thrombolytics (Jaeger and Reetz, 1998; Belarbi et al., 2000). Due to their metabolic effects, PUFAs are largely being used as pharmaceuticals, nutraceuticals, and food additives (Belarbi et al., 2000). Several of the PUFAs are needed for normal synthesis of lipid membranes and prostaglandins. Microbial lipase enzymes are used for obtaining PUFAs from lipids of animals and plants like tuna oil, menhaden oil, and borage oil. Furthermore, the flavor development for dairy products (cheese, butter, margarine, bakery products, alcoholic beverages, milk chocolate, and sweets) is obtained by selectively hydrolyzing fat triglycerides for releasing free fatty acids that act as flavor precursors (Jaeger and Reetz, 1998). Immobilized Rhizomucor miehei lipase in organic solvent was found to catalyze the reactions of enzymatic interesterification for the production of vegetable oils like sunflower oil, corn oil, soybean oil, peanut oil, and olive oil and containing omega-3 PUFAs. Lipases are important for hydrolyzing lipids for obtaining fatty acids and glycerol, both of which have important applications in industry. For example, fatty acids are used in soap production, and glycerol is used as a feedstock for pharmaceutical industries (Hoq, 1985; scialert.net). The European industry association Euro Fed Lipid, which represents the oils & fats industry in Europe, has awarded Novozymes the prestigious European Lipid Technology Award 2008. The award recognizes Novozymes’ enzymatic interesterification process. According to Hans Christian Holm, Novozymes’ Global Marketing Manager for Oils & Fats, the advantage of the enzymatic process over the traditional method is that the enzymatic process does not form trans fatty acids, which are suspected of increasing the risks for cardiovascular diseases. Trans fatty acids are typically found in products such as margarine, cookies, and potato chips, forming during a production process that aims to achieve the correct melting temperature for the end product; for example, so that chocolate cookies melt in the hand and not in the mouth. Traditionally, baking fats and margarines have either contained hardened fats with trans fatty acids, or had them removed through a chemical interesterification process, that gives poorer-quality oil while also being problematic in terms of both safety and the environment. Using enzymatic interesterification in the production of oils and fats removes the trans fatty acids, reduces the negative environmental impacts, and gives a more natural, better tasting product that contains healthier oils. (www.novozymes.com)

According to Novozymes, “it has taken a few years to convince oil producers for using the new technology because it requires changes to the production process. But, the final argument, has been the increasing focus in recent years on trans fatty acids and their adverse effects on health” (www.novozymes.com). Enzymatic interesterification is quite effective to control the melting characteristics of edible fats and oils (Christensen et al., 2001). Chemicals are not used in the process, and there is no production of trans fatty acids. The technology was not used extensively until recently because of the high cost of the enzyme, but now, this is a cost-effective technique to both chemical interesterification and hydrogenation as washing or bleaching of the interesterified fat is not needed. Furthermore, the low-temperature enzymatic process does not produce any

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side products. The capital investment is low as the enzymatic process needs only one simple column or tank as special equipment. A specific melting profile of the fat is obtained by passing the oil once through the column-containing enzyme. Different from both hydrogenation and chemical interesterification, the enzymatic process does not need any chemicals. The enzyme is immobilized in the column during the production process, so the only handling of the enzyme is when it is changed after the production of several hundreds of tons of fat (www.novozymes.com; genet.univ-tours.fr). Phospholipids are removed from vegetable oils using a highly selective microbial phospholipases (Clauson, 2001). This process is termed “degumming.” This is another example where the use of an enzymatic step shows savings in both energy and water for the benefit of the industry and the environment. “Enzymatic degumming is a physical refining process in which phospholipases converts nonhydratable phosphatides into fully hydratable lysolecithin. In industrial degumming this facilitates gum removal. In most physical refining methods, a fundamental criterion should be that the crude oil is degummed as effectively as possible. Using different phospolipases a variety of products, for instance lyso-­phospholipids, free fatty acids, diacylglycerols, choline phosphate, and phosphatidates are produced. Chemical refining uses large amounts of caustic soda as a main refining agent. The enzymatic degumming process has shows several benefits. An overall higher yield is obtained because the gums contain up to 25% less residual oil, and because no soapstock is produced, no oil is lost. Moreover, enzymatic degumming works with crude oil and also water-degummed” (www.novozymes. com; www.biology.uoc.gr).

6.14 Bioremediation Several pollutants like polychlorinated biphenyl compounds (PCBs), heavy metals, hydrocarbons, dyes, pesticides, esters, petroleum products, and nitrogen-containing chemicals are found in the environment. These pollutants get generated from different industrial and agricultural resources. These are very toxic and are carcinogenic. The accumulations of these chemicals are harmful to the environment and also to the flora and fauna living in the environment (Wasilkowski et al., 2012). To remove and degrade these pollutants is becoming a problem. Earlier, wastes generated from different industries and agricultural resources were treated by incineration process (high temperature) and using ultraviolet rays. However, these methods are not very effective because of their complex nature, higher cost, and formation of resistant derivatives. Bioremediation provides a method for the degradation of these chemicals (Dua et al., 2002; Vidali, 2001; Dzionek et al., 2016; Sharma et al., 2018). Biotechnology is playing an important role in maintaining a cleaner environment. This role is expected to increase considerably as methods are developed and used for bioremediation of different types of industrial effluents (tur-www1.massey.ac.nz) Bioremediation deals with the use of living organisms such as microorganisms for removing contaminants, toxins, and pollutants from water and soil. It can be used for cleaning up environmental problems such as an oil spill or contaminated groundwater (www.investopedia.com). Bioremediation is used for recovering brown fields for developing and for preparing contaminated industrial effluents before being discharged into waterways. Bioremediation is



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also applied to contaminated wastewater, surface water, or groundwaters and also soils, sediments, and air where there has been either deliberate or incidental release of pollutants that create a risk to the health of animals, humans, or ecosystem (www.thebalance.com). Bioremediation stimulates the growth of certain microorganisms that use contaminants such as oil, solvents, and pesticides as a source of food and energy. These microorganisms consume the contaminants and convert them into water and harmless gases such as carbon dioxide. Effective bioremediation requires a combination of the appropriate temperature, nutrients, and food, or else, it may take a long time to clean up the contaminants. If conditions are not suitable for bioremediation, they can be improved by adding some amendments like molasses, vegetable oil, or simply air. These amendments help in creating optimum conditions for microorganisms to grow well and complete the bioremediation process. Bioremediation can be performed at the site of the contamination itself or at a location away from the site. This may be important if the climate is too cold for microbial activity or the soil is too dense for the nutrients to get spread uniformly. This may also require digging up the soil and cleaning it above ground, which will increase the process cost. The process of bioremediation may take from a few months to several years. The amount of time needed depends on the following variables (www.investopedia.com): • • • •

Size of the contaminated area Concentration of contaminants Temperature and soil density In situ or ex situ process

Bioremediation shows several benefits over other cleanup methods (Table 6.59). In 2012, bioremediation was used to clean up more than 100 superfund sites in the United States (www.investopedia.com). Bioremediation technique most appropriate for a specific site is determined by many factors. These are as follows: • Site conditions • Indigenous population of microbes • Type, quantity, and toxicity of contaminant chemicals present Few treatment techniques involve the addition of nutrients for stimulating or enhancing the activity of indigenous microorganisms. Optimization of environmental conditions increases the growth of microorganisms and increases the population of microorganisms resulting in better degradation of hazardous substances. But if the biological activity required TABLE 6.59  Advantages of bioremediation. Uses natural processes, so it is a relatively green method that causes less damage to ecosystems Usually takes place underground as amendments and microorganisms can be pumped underground for cleaning up the contaminants in groundwater and soil, so it does not cause disruption to nearby communities Creates few harmful by-products Bioremediations is cheaper in comparison with most cleanup methods Based on www.investopedia.com/terms/b/bioremediation.asp

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for degrading a certain type of contaminant is not present at the site, suitable microorganisms from other locations can be added and nurtured. Other techniques being demonstrated are phytoremediation, or the use of plants for cleaning up contaminated soils and groundwater, and fungal remediation, which uses white-rot fungi for degrading contaminants (www.hawaii.edu). Bioremediation applications are classified as in situ or ex situ: - In situ applications treat contaminated soil or water in the location in which they are found. These techniques are usually less costly, produce less dust and debris, and generate less contaminants in comparison with ex situ techniques as no excavation processes are needed. - Ex situ bioremediation needs excavation or pumping of contaminated soil or groundwater, respectively, before starting the treatment. These techniques are usually easier to control, faster, and able to treat a diverse range of contaminants and soil types in comparison with in situ methods There is a lack of understanding of processes and control, which is presently limiting the use of bioremediation process. Suitable engineering techniques need to be understood properly for broadening the applicability of this technique. Furthermore, reliable cost data are required to command full acceptance and extensive use by technical and regulatory entities (www.hawaii.edu). Bioremediation takes benefit of the metabolic processes of various microorganisms for degradation or sequestering and concentration of various contaminants. For instance, bioremediation of soil might be conducted under anaerobic or aerobic conditions. The metabolic pathways of fungi or bacteria for degradation of aromatic compounds, pesticides, or hydrocarbons are optimized. Phytoremediation uses plants and generally has been proposed for bioaccumulation of metals, although there are several other types of phytoremediation. The bioremediation technique is becoming very popular in the 21st century. Genetically engineered microorganisms carry recombinant proteins that can accelerate the breakdown of explosives such as TNT, but these are relatively uncommon because of regulatory constraints related to their generation and control. Other methods of enzyme optimization that do not include gene cloning methods might be used to indigenous microbes for increasing their pre-existing characteristics. Bioremediation is found to be very effective on a small scale. For instance, the 1986 Chernobyl nuclear disaster was far too catastrophic to be positively affected by bioremediation efforts and is basically beyond repair. One example of bioremediation is to add nutrients to the soil for increasing bacterial degradation of contaminants on a brown field site. Bioremediation was used largely to fight the destructive effects of the Exxon Valdez oil spill in 1989 and BP’s Deepwater Horizon oil spill in 2010. In both oil spills, microbes were used for consuming petroleum hydrocarbons and played an important role in reducing the ecological impact. Bioremediation technique is a good choice for pollution abatement, but it does not work for all. For instance, bioremediation may not be successful at sites having high concentrations of chemicals that are toxic to most microbes. These chemicals include certain metals such as cadmium or lead and salts such as sodium chloride. Bioremediation can also be conducted on a personal level. Anyone can do composting, and it is an excellent method to recycle garden and yard waste nutrients for producing a soil conditioner. Nutrients are added



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to the soil for increasing the degradation of contaminants and increase the bioremediation rate on the brown field site (www.thebalance.com). Use of activated sludge method for treating municipal wastewater was probably the first main application of biotechnology in bioremediation. Activated sludge treatment remains a workhorse technology to control pollution of aquatic environment. Likewise, aerobic stabilization of solid organic waste by composting is being used for a long time. Both of these techniques have undergone significant improvement. There are many microbes and enzymes that are used in various bioremediation applications, for example, effective removal of nitrate and phosphate contamination from wastewater and fast and highly specific detection of several pollutants by the use of biosensors (tur-www1.massey. ac.nz; Pletsch et al., 1999; Macek et al., 2000; Gavrilescu, 2004b; Jördening and Winter, 2004; Khin and Annachhatre, 2004; Liu and Tay, 2004; Baeumner, 2003; Wolfbeis, 2004; Baeumner, 2003; Wolfbeis, 2004). Different microbes have been isolated, selected, mutated, and genetically engineered for effective bioremediation for degrading recalcitrant pollutants, obtaining increased rates of degradation of target compounds, and assuring better survival and colonization in target polluted niches (Renner, 1997; Pieper and Reineke, 2000). Microorganisms and enzymes have been the major focus of the effort for increasing bioremediation, but the use of higher plants in phytoremediation is significantly developing (Macek et al., 2000; Glick, 2003). High importance is being given to using ecologically integrated mixed bioremediation systems. Bioremediation processes have been established for the treatment of contaminated soil and groundwater (both in situ and ex situ). Bioremediation offers substantial cost and environmental advantages as compared with alternate techniques. Bioremediation techniques offer the industry significant new tools for increasing profitability and sustainability (Lee and de Mora, 1999; Jördening and Winter, 2004; Khan et al., 2004; Gavrilescu and Chisti, 2005). Use of microorganisms and their enzymes for the removal of pollutants is a successful, safe, and less costly technique. Developments in molecular and recombinant DNA technology and genetic machinery of the plants and microorganisms can be changed to increase the bioremediation (Karigar and Rao, 2011; Vallero, 2016; Kumar et al., 2016). There are many different types of enzymes involved in bioremediation (Kadri et al., 2017) (Table  6.60). Different fields of bioremediation such as microbial and enzymatic including phytoremediation and their strategies are shown in Fig. 6.7(Sharma et al., 2018). Bioremediation is found useful in reducing emissions of vapors of organic compounds especially from gaseous effluents that are low in VOCs (Cohen, 2001; Jorio and Heitz, 1999; Burges et al., 2001; Moo-Young and Chisti, 1994; Deshusses, 1997). VOC emissions are usually produced in processes involving drying of products (Lewandowski and DeFilippi, 1997; Hunter and Oyama, 2000; Penciu and Gavrilescu, 2004; tur-www1.massey.ac.nz). There are few biotechnological processes available for the removal of VOCs from gases (Table 6.61). Biofilters are found to be quite effective in removing VOCs. Concept of biofiltration is quite simple. The contaminated air is passed through a media populated by microorganisms that biologically degrade the contaminants. Degradation of contaminants takes place when the microbes metabolize the carbon-based contaminant (VOC) molecules to their primary components, generally carbon dioxide, water, and other harmless substances. Clean air is

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TABLE 6.60  The disadvantages of bioremediation. Limited to biodegradable compounds. All compounds are not susceptible to fast and complete degradation There are certain issues that the biodegradation products may be more toxic or persistent than the parent compound Biological processes are highly specific. Important site factors needed for success include the presence of metabolically capable populations of microorganisms, appropriate growth conditions, and the presence of suitable nutrients Difficult to extrapolate from bench- and pilot-scale studies to full-scale operations There is a need to develop technologies suitable for sites with complex contaminants that are not evenly dispersed in the environment Often takes longer time than other treatment options Skilled man power is needed Evaluation of performance of bioremediation is difficult as there is no accepted definition of clean Based on Abatenh, E., Gizav, B., Tsegaye, Z., Wassie, M., 2017. Application of microorganisms in bioremediation-review. J. Environ. Microbiol. 1 (1), 2–7.

Bioremediation

Microbial bioremediation

Phytoremediation

Recombinant DNA technology

Post translational modification of enzymes

Enzymatic bioremediation

Discovery of new enzymes involved in bioremediation Oxidoreductase

Modification of metabolic pathway

Lyases

Peroxidases

Hydrolase

Dehalogenases

FIG. 6.7  An overview of combinatory methodologies for bioremediation. Reproduced with permission from Sharma, B., Dangi, A.K., Shukla, P., 2018. Contemporary enzyme based technologies for bioremediation: a review Environ. Manag. 210, 10–22.



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TABLE 6.61  Microbial enzymes involved in bioremediation. Microbial oxidoreductases Oxygenase Monooxygenase Incorporation of oxygen atom to substrate and utilize substrate as reducing agent. Desulfurization, dehalogenation, denitrification, ammonification, and hydroxylation of substrate Dioxygenase Introduction of two oxygen atom to the substrate results in intradiol cleaving and extradiol cleaving with the formation of aliphatic product Microbial laccases Oxidation, decarboxylation, and demethylation of substrate Microbial peroxidases Lignin peroxidase Oxidation of substrate in the presence of cosubstrate H2O2 and mediator like veratryl alcohol Manganese peroxidase In the presence of Mn2+ and H2O2, the cosubstrate catalyzes oxidation of Mn2+ to Mn3+ that results in an Mn3+ chelate oxalate, which in turn oxidizes the phenolic substrates Versatile peroxidase The enzyme catalyzes the electron transfer from an oxidizable substrate, with the formation and reduction of compound I and compound II intermediates Hydrolytic enzymes Lipase The hydrolysis of triacylglycerols to glycerols and free fatty acids Cellulase Hydrolyses the substrate to simple carbohydrates. Protease Enzymes that hydrolyze peptide bonds in aqueous environment Based on Chakraborty et al. (2014); Shraddha et al. (2011); Bansal and Kanwar (2013); Mehta et al. (2017); Bhardwaj et al. (2017); Singh (2014); Santillan et al. (2016); Nagata et al. (2015).

then released to the atmosphere from the filter. This process does not produce any additional waste; transfer of pollutants to another media does not take place, and so, there is no additional environmental problem. This results in saving of disposal costs. Other advantages include low system maintenance and low operating costs (Wani et al., 1997; www.dtic.mil; tur-www1.massey.ac.nz). Despite these benefits, biofiltration technology did not gain much importance in the United States until after the implementation of the Clean Air Act (CAA) amendments in 1990. During the 1970s and 1980s, biofiltration technology gained research and development momentum

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in Netherlands and Germany, whereas, in the United States and Europe, this technique did not become popular (Devinny et al., 1999). Portions of the CAA focus on controlling and removing VOCs from contaminated air and these regulations, along with a new found public interest in air pollution control issues, created a renewed interest in the technology in the United States that has carried through to today (Wani et al., 1997; www.dtic.mil). BIP Ltd. in the United Kingdom installed a trickling biofilter to remove and degrade VOCs in its gaseous effluent (Bio-Wise Case Study 7, Department of Trade and Industry, Oxfordshire, UK, 2001). The biofilter obtained compliance with emission legislation in a safe manner and saved the company up to £100,000 annually on running costs in comparison with the alternative technology of incineration. The capital expenditure of installing the biofilter was about £500,000 lesser in comparison with the incineration alternative (Gavrilescu and Chisti, 2005). Several enzymes are found to be involved in the biodegradation of toxic organic pollutants (Table  6.62). Bioremediation is a cost-effective and eco-friendly biotechnology powered by microbial enzymes. Table 6.63 shows enzymes used in the bioremediation of different harmful chemical compounds. TABLE 6.62  Biotechnological processes for removing VOCs from gases. The gaseous effluent is scrubbed with an aqueous medium with or without suspended microorganisms, to absorb the VOCs in the scrub liquid where they are degraded by microbial action. The VOC containing liquid leaving the scrubber may be recycled through a separate aerated slurry suspension bioreactor where most of the degradation takes place VOC containing liquid effluent is passed over a trickle bed of immobilized microorganisms to achieve degradation of the dissolved pollutants. Another method that is used frequently is direct biofiltration of the effluent gas through a porous bed of soil, or other particulate matter, that supports the VOC degrading microbial community. The moisture content in the bed is controlled by spraying with water and humidification of the gaseous effluent entering the bed Based on Moo-Young and Chisti (1994); Deshusses (1997); Jorio and Heitz (1999); Burges et al. (2001); Cohen (2001).

TABLE 6.63  Enzymes used in the bioremediation of different harmful chemical compounds. Enzymes

Microorganisms

Chromium reductase

Pseudomonas, Chromium Bacillus, Enterobacter, Deinococcus, Shewanella, Agrobacterium, Escherichia, Thermus

Pollutants

Mechanism Chromium transform Cr (VI) into Cr (III) in the presence of oxygen via a two-step reaction; first, Cr (VI) accepts one electron from one molecule of NADH to generate Cr (V), and then, Cr (V) accepts two electrons to form Cr (III) (Thatoi et al., 2014)

Atrazine dechlorinase, triazine hydrolase

Nocardioides sp. C190, Pseudomonas, Rhodococcus erythropolis

Triazine herbicides

Metabolized atrazine into chloride, ammonia, and carbon dioxide with the help of enzymes (Scott et al., 2010)

Alkane hydroxylases (monooxygenase and dioxygenase)

Arthrobacter, Burkholderia, Mycobacterium, Pseudomonas, Sphingomonas, Rhodococcus

Hydrocarbon (aromatic and aliphatic)

Hydrocarbon degradation starts with the oxidation of a terminal methyl group into a primary alcohol, which is further oxidized into aldehyde and finally converted into fatty acid (Das and Chandran, 2010)



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TABLE 6.63  Enzymes used in the bioremediation of different harmful chemical compounds—cont’d Enzymes

Microorganisms

Pollutants

Mechanism

Laccase

Laccase Trametes versicolor, Pleurotus ostreatus

Polychlorinated biphenyls (PCBs)

PCBs metabolized into its constituent elements and laccase are able to dechlorinate chlorophenols by oligomerization of the substrate (Dodor et al., 2004)

Carboxylesterases

Pseudomonas aeruginosa PA1

Malathion and parathion

Carboxylesterase transforms malathion into malathion monocarboxylic acid and dicarboxylic acid (Qiao et al., 2003; Singh et al., 2012)

Peroxidases

Phanerochaete chrysosporium

TNT (2,4,6-trinitrotoluene) nitroaromatic compounds

2-Amino-4,6-dinitrotoluene (2amDNT) and 4-amino-2,6-dinitrotoluene (4amDNT) is the initial, intermediate products of TNT biodegradation, which is further mineralized into simpler compounds (Cameron et al., 2000)

Phytase

Aspergillus niger NCIM 563

Organophosphate

It can release inorganic phosphorus by the degradation of organophosphate containing compounds (Shah et al., 2017)

Laccase

Pycnoporus sanguineus

Dyes such as bromophenol blue, malachite green

Laccase catalyzes the oxidation of phenolic compounds and the reduction of one dioxygen (O0) molecule to two molecules of water (O2−) (Mayer and Staples, 2002)

Horseradish peroxidase (HRP)

Horseradish (Armoracia Chlorophenol, phenol rusticana) Chlorophenol, phenol

Horseradish peroxidase enzyme degrades the phenolic compounds by both meta- and orthopathway. Phenol is converted into aldehyde and pyruvate after degradation by meta pathway (Flock et al., 1999; Bilal et al., 2017)

Reproduced with permission from Sharma, B., Dangi, A.K., Shukla, P., 2018. Contemporary enzyme based technologies for bioremediation: a review Environ. Manag. 210, 10–22.

6.15  Bioprocessing of pulp and paper To produce paper from wood, the following steps are required: • Wood processing • Pulping • Bleaching • Stock preparation • Paper making In the pulping process, the wood fibers are separated from each other, which are then reformed into a sheet. The wood fibers are glued together by lignin, and separation of these fibers is called chemical pulping. The chemical pulp is subjected to bleaching for removing lignin to increase the brightness of the paper. The bleaching results in the production of

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numerous toxic derivatives of lignin that are hazardous to the environment. To minimize the action of bleaching, biotechnological approach can be adopted in paper industry. By the use of biotechnology and improved silviculture practices, trees and other nonfossil biogenic resources used in papermaking can be tailored for matching the properties needed in cellulose fibers for various product applications (Buschle-Diller and Ren, 2002; Bajpai, 2018c). This will largely increase paper yield from trees, improve product quality, and reduce energy consumption and chemicals used in manufacturing paper. Producing optimal fibers for papermaking by the use of genetic engineering technique is a major objective in the long term that needs an improved understanding of fiber biosynthesis in plants. Moreover, the use of genetically engineered microbes and enzymes can remove several environmentally harmful practices used in pulp making. Some of these developments are discussed here (tur-www1. massey.ac.nz).

6.15.1 Biodebarking The first step in wood processing is the removal of tree bark. In this step, considerable amount of energy is required. Severe debarking is needed to produce mechanical and chemical pulps of better quality as the presence of even a small quantity of bark residue darkens the product (Bajpai, 2018a,c). Complete debarking also results in the loss of raw material because of long treatment in the mechanical drums. Cambium is a border between wood and bark. This consists of only one layer of cells that produces xylem cells toward the inside of the stem and phloem cells toward the outside. In most of the woods, the cambium contains a high content of pectins, and the lignin is either absent or present in low amount. The pectin content in cambium cells varies in different wood species, but it can be as high as 40% of the dry weight. In the phloem, pectic and hemicellulosic components are very high. Pectinases are reported to be main enzymes in the debarking process, but xylanase enzymes can also play a role due to the high hemicellulosic components in the phloem of the cambium. Enzymatic treatments cause substantial reduction in energy requirement during debarking (Bajpai, 2006, 2015, 2009, 2013c, 2018a; Hakala and Pursula, 2007). The energy used in debarking is reduced by 80% after pretreatment with pectinases. The enzyme treatment also results in significant savings in raw material. Enzymes can increase existing debarking capacity, which results in saving of capital costs, and also can be used when debarking becomes difficult.

6.15.2 Biobleaching Enzymes are being used in pulp bleaching during the last two decades (Bajpai, 1999, 2004, 2006, 2018a,c; Bajpai and Bajpai, 1997a,b; Paice and Zhang, 2005; Viikari et  al., 2002, 2006, 2009. Hemicellulase enzymes particularly xylanase enzymes are being used on a commercial scale for pulp bleaching. In the pulp and paper industry, “This technology has become one of the solutions considered by the to give an innovative, environmentally, and economically acceptable answer to the pressures exerted on chlorine bleaching by regulatory authorities in Western countries and by more demanding, environmentally minded consumers” (Bajpai, 1999). The use of xylanases results in several advantages in pulp bleaching. These include the following:



• • • • •

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Reduction of AOX discharges, mainly by reducing the usage of chlorine Debottlenecking mills limited by chlorine dioxide generator capacity Eliminating use of chlorine gas for mills using high chlorine dioxide substitution Increasing the brightness especially for mills using ECF and TCF bleaching Reducing cost of bleaching chemicals, especially for mills using high dose of peroxide or chlorine dioxide

These advantages are realized over the long term when the enzymes are selected and used in proper way in the mill. The use of oxidative enzymes from white-rot fungi is able to attack lignin directly (Bajpai, 2018c). These enzymes show the following advantages: • Highly specific toward lignin • No damage or loss of cellulose • Larger chemical savings in comparison with xylanases But the process has not been commercialzed yet. It is being researched in many labs all over the world. Certain white-rot fungi are able to delignify kraft pulps, increasing their brightness and their response to brightening with chemicals. Treatment with fungi is very slow, but the enzymes manganese peroxidases and laccases can also delignify pulps. The enzymatic processes are easier to use in comparison with the fungal treatments and can be optimized easily. Research and development work on laccases and manganese peroxidases is continuing.

6.15.3 Deinking The combination of xylanases and cellulases is being examined for the deinking of waste paper. This method is found to be effective and economical on a commercial scale. Cellulase and xylanase enzymes show a very good effect on the enzymatic deinking of old newsprint (ONP). The deinking efficiency and the fiber modification are improved (Wang and Kim, 2005). The most important effect of enzymatic deinking is that the dewatering and dispersion steps and also the later flotation and washing stages may not be needed. This results in saving of capital costs in construction of deinking plants. Furthermore, electrical energy consumption for dewatering and dispersion is reduced. The bleach chemical requirements are generally lesser for enzymatic deinking in comparison with conventional chemical deinking. Reduction in chemical consumption would result in reduced waste treatment cost. The impact on the environment would be also reduced. Reduction in bleaching costs and reduction in pollution can also be expected, as enzyme pulps are easier to bleach and need less chemicals as compared with pulps deinked by chemical methods. Enzyme pulp also shows fast drainage, better physical properties, improved brightness, and lower residual ink in comparison with conventionally deinked recycled pulps. Faster drainage results in faster machine speed. This results in substantial energy savings and thereby the overall cost. Furthermore, the use of recycled fiber reduces the requirement for virgin pulp. This results in substantial savings on the energy needed for pulping, bleaching, refining, etc. This will finally result in reduced pollution problems (Bajpai, 2018c).

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6.15.4  Production of dissolving pulp Different types of cellulosic materials such as acetate, cellophanes, and rayons are produced from dissolving pulp. Highly purified cellulose is derivatized and solubilized for producing these products. The presence of hemicellulosic contaminants results in color and haze in the product. Also, insolubles are produced that hinder the production process. Their extraction from the pulps needs high dose of sodium hydroxide and proper pulping conditions, the latter restricted to kraft (acid pretreated) and sulfite pulping. The use of xylanases for purifying cellulose has been attempted. It is quite difficult to achieve complete enzymatic hydrolysis of the hemicellulose in the pulp. Even using high dose of enzyme, only a small amount of xylan can be solubilized. The large amount of the xylan in pulps is inaccessible. This limits the potential of this application, despite that xylanase use may reduce the chemical doses needed during extraction stage or facilitate xylan extraction from kraft pulps. Extensive work has been conducted on the use of xylanases in bleaching of dissolving pulp. A process has been developed for bleaching of dissolving pulp using an enzymatic pretreatment with xylanase enzyme (Bajpai et al., 2005; Bajpai, 2018a,c). A mild prehydrolysis is conducted for retaining relatively higher pentosans in the unbleached pulp. The higher pentosans results in higher unbleached pulp yield at the same kappa number. Results of bleaching of enzyme pulp in a CEHED sequence showed reduction in requirement of bleach chemical, along with higher brightness of the final bleached pulp, desired levels of pentosans, and a significant increase in bleached pulp yield. Enzyme pulp showed higher rayon yield and better reactivity (Bajpai, 2009).

6.15.5  Fiber modification Fiber modification is aimed at the following: • Increased beatability of chemical pulps • Reduced energy consumption in the production of thermomechanical pulps • Improvement of fiber properties Xylanase enzymes have been found to produce pulp fibers, which resemble in properties with those of slightly beaten pulps. Studies performed with blend of xylanases and cellulases with different types of kraft and mixed pulps showed appreciable reduction in refining energy and no negative effect on the strength properties of pulps. Refining with enzymes is now a commercial reality and is economically attractive (Bajpai, 2006). Enzymes can be easily integrated into the present production processes and do not create any problem in the normal operation. The major benefits of enzymes include the following: • Reduced electrical energy for refining • Reduced steam consumption • Reduced backwater consistency These advantages can be converted into the following: • • • •

Faster machine speed Better machine runnability Reduction in retention aids Improved formation of paper



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• Debottlenecking of refiner capacity • Possibility of using difficult to refine pulp Other benefits of enzymatic refining include easier operation of backwater clarification, reduced pitch problem, better biodegradability of machine effluent, and reduction in environmental problems. Drainage becomes faster on the paper machine due to limited hydrolysis of the fibers in recycled paper. A blend of xylanases and cellulases at lower concentrations significantly increases the freeness of recycled fibers without having any impact on the yield. The lower the initial freeness, the higher the gain following treatment. Freeness shows a fast initial increase with over half of the observed effect taking place in the first 30min. A small amount of enzyme is needed. The initial effects are largely advantageous, but increasing the reaction time with high dose of enzyme is harmful. When OCC fibers were treated with commercial xylanases, large improvements in drainage rate were obtained when xylanase was used on fiber after beating (Bobu et  al., 2003). Considerable improvement in relative bonded area and wet fiber flexibility were found with xylanases used before beating. Xylanases actually induce swelling of the fibers that has a strong effect on any later processing (Bajpai, 2009, 2015).

6.15.6  Removal of shives Shives are small bundles of fibers that do not get separated into individual fibers during the pulping process. “Shives are darker than the pulp. shive count is the most important quality criteria for bleached kraft pulp. By treating the brownstock pulp with xyanases, mills can significantly increase the degree of shive removal in the later bleaching stages. Xylanases for shive removal were sold by Iogen corporation, Canada under the trade name Shivex. This enzyme increased the degree of shive removal by 50% in the later bleaching stages. At a given bleached brightness, Shivex treatment resulted in a lower shive count. Xylanases, help to remove shives from the pulp beyond the associated gain in the brightness. Removal of shives and ease of pulp bleaching by the use of xylanases help in reducing the energy consumption” (Bajpai, 2009, 2015).

6.15.7  Retting of flax fibers Xylanases have been used in processing plant-fiber sources such as flax and hemp. At present, fiber liberation is affected by retting, that is, the removal of binding material present in plant tissues using enzymes produced in situ by micro-organisms, Pectinases are believed to play the main role in this process, but xylanases may also be involved. The replacement of slow natural retting by treatment with artificial mixtures of enzymes could become a new fiber-liberation technology. (Bajpai, 2009)

6.15.8  Biological solutions to processing problems 6.15.8.1  Slime control The control of slime is the most worrying problem in the paper industry (Smook, 1992). Microorganisms producing slime secrete extracellular polysaccharides, which stick to the process machinery (Bajpai, 1999, 2018c). The formation of slime depends on the e­ nvironmental

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c­onditions of the mill. The remedy varies with the type and nature of slime deposit. Conventional methods generally use mixtures of different biocides. This results in high ­processing/­treatment costs. The effluent also gets toxic. Therefore, alternate control methods are in demand. Such methods are the use of “enzymes, biodispersants, bacteriophages, competing organisms, and biological complex formers. Enzymes represent a clean and sustainable technology. Hence, use of enzymes for slime control is expected to bring important benefits for the pulp and paper industry. Enzymes are produced using renewable raw materials and are nontoxic, biodegradable. Many enzymatic products are already in industrial use around the world and many additional products are currently being designed and tested for a wide variety of specific applications. Use of enzymes in combination with biodispersants appears to be a promising method for slime control. Nevertheless, whatever the nature of the new applications, the eventual changeover to biocide-free methods will be possible only when the new technologies become beneficial and realistic replacements. Results showed that addition of bacteriophage to mill whitewater was an effective technique for slime control. Simultaneous addition of bacteriophage with conventional biocides also was found to be effective. Practical application of this technique on a commercial scale awaits completion of fundamental studies in several key areas. Unlike conventional biocides, bacteriophages will not impair the activity of the sludge used in waste treatment systems”(Bajpai, 2018c; Van Haute, 2000). 6.15.8.2  Stickies control Stickies are naturally present in the recycled fiber and are gluey, hydrophobic, flexible organic substances (Smook, 1992). Stickies consist of different types of materials including adhesives, styrene-butadiene latex, vinyl acetate, rubber, and hot melts. The nature of stickies is variable, and there is a great variability in the composition of recycled fiber. This makes them very strenous to control. Stickies cause quality and runnability problems. A new method for controlling stickies has been developed that makes use of esterases for breaking them down into smaller and less tacky particles (Jones, 2005). Several mixtures of esterase-­ type enzymes have been examined for finding the one that has the ability to break down the ­stickies. The size of the sticky is reduced by breaking the ester bonds. It gets broken into smaller components. A major benefit of this strategy is that, once broken down, the possibility of the particles reagglomerating further in the process is curtailed to a great extent. Another significant effect on the stickies is the enzymatic modification of the stickies surface, which results in less gluey stickies (Bajpai, 2018c). “These enzymes have been proven to reduce downtime, decrease cleaning chemical costs, and increase machine-clothing life better than historical stickies control technologies. On treatment of old newsprints and old magazines with esterase-­type enzyme in a mill trial, a dramatic reduction in the size of the sticky particles is observed. The stickies content of all the sizes and the total stickies were much less on enzyme treatment as compared to those without enzyme (pretrial results). The bigger size stickies are totally absent in the recycled fiber treated with enzyme. When the recycled fiber from MOW was treated with esterase enzyme in a mill trial, the total stickies content came down appreciably (Jones and Fitzhenry, 2003). Without enzyme treatment of MOW, it was not possible to increase the recycled fiber content in the final furnish beyond 50%. Even then, the total stickies were more than 250ppm. However, on treating the recycled fiber from MOW with enzyme, total stickies content could be reduced to around 100 ppm, that too with higher content (60%) of recycled fiber. In another mill using MOW, the recovered fiber was treated



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with enzyme to reduce the percentage of high brightness virgin pulp in the final furnish without compromising on brightness of the finished stock. With addition of 35% high brightness pulp, the normal brightness gain from coarse screen to finish stock was only 12–14 points when no enzyme treatment was given to the recovered fiber. However, after esterase enzyme treatment of MOW-recovered fiber, it became possible to get a brightness gain of more than 15 points even when the high brightness pulp content was only 15–20%” (Jones and Fitzhenry, 2003;Bajpai, 2018c). 6.15.8.3  Enzymatic modification of starch for surface sizing Enzymes are being used in the paper industry for starch modification for coating and surface sizing (Smook, 1992; Bajpai, 2018c). Starch imparts several favorable properties to paper. These include stiffness, strength properties, and erasability. When the enzymatic modification of starch is controlled in a proper manner, uniform quality of starch paste is obtained, and the quality surface-sized papers can be produced. Enzyme used for modification of starch is alpha-amylase. The enzymatically modified starch is suitable for surface sizing of writing and printing paper (Bajpai et al., 2005; de Souza and de Oliveira Magalhaes, 2010). It meets all the quality requirements. No capital investment is required to change over from oxidized starch to in situ enzymatic modification of raw starch. This process can be applied to starches of different types. The operating conditions in terms of reaction time and enzyme charge can vary (Bajpai et al., 2005; de Souza and de Oliveira Magalhaes, 2010). 6.15.8.4  Bioremediation of pulp and paper mill effluents The pulp and paper industry is a highly polluting industry. Even with the use of most efficient methods, about 60m3 of water is needed to produce a ton of paper. This generates huge volumes of wastewater. Of the different waste streams, effluents generated from bleach plant are most toxic because of several organochlorine compounds produced during pulp bleaching. Several different types of organochlorine compounds have been identified (Bajpai, 2001, 2018c; Bajpai and Bajpai, 1997a,b). The organochlorine compounds in wastewater are divided into high-molecular-weight and low-molecular-weight compounds. The low-­molecular-weight compounds contribute to mutagenicity and bioaccumulation because they are hydrophobic and are able to enter the cell membranes. The low-molecular-weight compounds are found to bioaccumulate in the aquatic food chain. The toxic effects of organochlorine compounds include carcinogenicity, mutagenicity, and acute and chronic toxicity. Strict legislation has been imposed to limit these toxic compounds in effluents. Furthermore, during the bleaching process, highly oxidized, chromophoric lignin/chlorolignin derivatives are generated, which produces dark color in the effluent. Several methods have been studied for reducing the generation of pollutants (Bajpai and Bajpai, 1997a,b). These are internal process modification and external treatment. “Internal process modification is one of the options used by the pulp and paper industry for reducing the generation of pollutants at the source. Physicochemical methods are not economically viable. Biological methods have the potential to eliminate/reduce the problems associated with physicochemical methods. In secondary treatment processes, activated sludge is the most commonly used process. Aerated lagoons are efficient in removing BOD, COD, and AOX. Anaerobic contact reactors, anaerobic filters, and fluidized bed reactors are suitable in reducing organic pollutants. White-rot fungi are found to be very effective for efficient degradation of the refractory material” (Bajpai, 2018c).

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High removal of toxic pollutants is obtained when physicochemical and biological methods are combined. The confirmation of the reported results in their application in the real situation and economic evaluations are very important in the adoption of the process. Various methods have been studied for detoxification and decolorization of effluents from bleached kraft mills. These include physicochemical and biotechnological methods. Physicochemical treatments incur high cost, and reliability is also questionable. Biotechnological methods have the potential to eliminate/reduce the problems associated with physicochemical methods. These methods may be aerobic as well as anaerobic bacterial treatment, fungal treatment, or enzymatic treatment. The bacterial processes are not very effective owing to the limitation that they cannot degrade the high molecular-weight chlorolignin compounds and enzymatic processes are not cost effective. Among the biological methods tried so far, fungal treatment technology using white-rot fungi appears to be the most promising in this regard. One of the drawbacks associated with the fungal treatment has been the requirement of an easily metabolizable cosubstrate such as glucose for the growth and development of ligninolytic activity. To make the fungal treatment method economically feasible, there is a need to reduce the requirement of cosubstrate or identify a cheaper cosubstrate. Hence, efforts should be made to identify the strains that show good decolorization with less or no cosubstrate and can utilize industrial waste as a cosubstrate. (Bajpai, 2009, 2018c)

6.16  Organic synthesis Biocatalysis is now a well established technology within the chemical industry. This field has been the focus of vigourous scientific research. The accelerated reaction rates, along with the unique stereo-, regio-, and chemoselectivity (highly specific action) and mild reaction conditions offered by enzymes, make them highly attractive as catalysts for organic synthesis. Additionally, improved production techniques are making enzymes inexpensive and more widely available. Enzymes work at a broad pH and temperature range, and often also in organic solvents. Many enzymes have been found to catalyze a variety of reactions that can be dramatically different from the reaction and substrate with which the enzyme is associated in nature. (www. novozymes.com)

Enzymes are being used in industrial chemical synthesis over the traditional methods (Bajpai, 2018). Enzymes are highly beneficial, that is, chiral, positional, and functional group specific. High selectivity is very beneficial in chemical synthesis. Several benefits are obtained. These are reduced or no by-product formation and reduced environmental problems, and the separation is very easy. Apart from this, mild operational conditions and high catalytic efficiency are benefits of enzyme-catalyzed commercial applications (link.springer.com). Chemical synthesis is an area where the use of enzyme catalysis has long been seen as having great promise. Inspite of that, the chemical industry has been slow to implement enzyme-based processes and the use of enzymes in the chemical industry is still low in comparison with other industries. At present, however, very significant growth in this area is being seen and enzyme based processes are now, finally, being widely introduced for the production of a diversity of different chemicals; one major example is in the production of single-enantiomer intermediates used in the manufacture of drugs and agrochemicals. This market is characterized by a very high degree of fragmentation, as very few enzymes have applicability in a broad range of different processes. (Kirk et al., 2002)



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The class of enzymes most widely applied to organic synthesis is the hydrolases. Members of the hydrolase family that have been used extensively include lipases, esterases, and proteases. Nowadays, lipases are widely used for organic reactions. Enantiopure alcohols and amides, nitrilases for the production of enantiopure carboxylic acids, and acylases for the production of new semisynthetic penicillins are produced. Several companies are presently at an early stage in the use of enzymatic catalysis; several developments are expected in this area in the coming years. Lipases catalyze several regioselective and stereoselective transformations. Use for lipases includes kinetic resolution of racemic alcohols, acids, esters, or amines and also the desymmetrization of prochiral compounds. They are used in regioselective esterification or transesterification of polyfunctional compounds, for example, in the chemoenzymatic synthesis of nucleoside. Nonconventional processes, such as aldol reactions or Michael addition, have been obtained using lipase enzymes (Schmidt et al., 2001; Kazlauskas, 1994; Berglund and Hutt, 2000; Rajendra et al., 2016 Ghanem and Aboul-Enein, 2004; Bornscheuer and Kazlauskas, 2004; Kirk et al., 2002; scialert.net; Ferrero and Gotor, 2000; Gotor-Fernandez and Gotor, 2007; imb.usal.es; Garca-Urdiales et al., 2005). Most of lipase enzymes are used currently as catalysts in organic chemistry are derived from microorganisms. These enzymes work at hydrophilic-lipophilic interface and can withstand organic solvents in the reaction mixtures (scialert.net). Berglund and Hutt (2000) have reported the use of lipases in the synthesis of enantiopure compounds. For example, lipase enzymes from Pseudomonas are extensively used, particularly for the production of chiral chemicals that serve as basic building blocks in the production of pharmaceuticals, pesticides, and insecticides. These enzymes show clear differences in regioselectivity and enantioselectivity, in spite of a high amino acid sequence homology (scialert.net). Lipase enzymes have been used in the production of biodegradable compounds. Trimethylolpropane esters were synthesized as lubricants. Lipase enzymes can catalyze ester syntheses and also transesterification reactions in organic solvent systems. This has opened up the possibility of the production of biodegradable polyesters catalyzed by enzymes. Aromatic polyesters can also be produced by lipase biocatalysis (scialert.net; Bailey and Ollis, 1986). Lipase enzymes are mostly used, especially, in the formation of a diverse range of optically active alcohols, acids, esters, and lactones (Jaeger and Reetz, 1998; Hasan et al., 2006). Lipases are used for the production of (S,R)-2,3-p-ethoxyphenylglycyclic acid, an intermediate for diltiazem (Gentile et al., 1992). Oxidoreductases, such as polyphenol oxidase, is involved in the synthesis of 3,4-­dihydroxylphenyl alanine, a chemical used in the treatment of Parkinson’s disease (Faber, 1997). Oligosaccharides and polysaccharides play important roles in cellular recognition and communication processes. These are produced industrially using high regio- and stereoselectivity of glycosyltransferases (Ginsburg and Robbins, 1984). Lyases are utilized in organic synthesis of cyanohydrins from ketones, acrylamide from acrylonitrile, and malic acid from fumaric acid (Faber, 1997; Zaks, 2001). Nitto Chemical Industry Co. of Japan is producing acrylamide at a scale of more than 40,000 tons per year (Zaks, 2001). This process is mediated by nitrile hydratase.

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6.17  Transgenic plants In transgenic plants, one or more genes from another species have been introduced into the genome, using recombinant DNA techniques for creating plants having new characteristics. By using genetic modification (GM) technology, the genes for specific traits between species using laboratory methods can be transferred. In the United States, GM crops were first introduced in the mid-1990s. Currently, most of the GM crops grown in the United States are engineered for insect resistance or herbicide tolerance. The three largest acreage GM crops are cotton, corn, and soybeans. Genetic engineering (GE) technique is used for modifying microorganisms, plants, and animals according to the desired requirements. In actuality, genetic engineering facilitates the transfer of desired properties into other plants that is not possible through traditional plant breeding methods. The use of biotechnology has led to opportunities and novel possibilities for enhancing the qualitative and quantitative characteristics of organisms (Yamaguchi and Blumwald, 2005; Sun, 2008; docplayer.net; extension.colostate. edu; ir.kib.ac.cn:8080). In 2002, more than USD 20 billion in crop value was associated with biotech commercial crop varieties. This will increase rapidly as transgenic plants are put to use for biopharming, or production of pharmaceuticals in plants. Potentially, oil crops can be engineered to produce less toxic and biodegradable industrial lubricant oils, to reduce dependence of the lubricants sector on petroleum derived products. High euricic acid canola oils have found applications as industrial lubricants. The levels of adoption of transgenic crops in the United States were 40% for corn, 81% for soybeans, 73% for cotton and 70% for canola in 2003. Transgenic plants are potentially versatile chemical factories. (Gavrilescu and Chisti, 2005; tur-www1.massey.ac.nz; Runge and Ryan, 2004; Hood and Jilka, 1999; Giri and Narasu, 2000; Larrick and Thomas, 2001; Jaworski and Cahoon, 2003; Wheeler et al., 2003; Mascia and Flavell, 2004)

Biotechnology for crop improvement is becoming a sustainable strategy for fighting deficiencies in food by improving the composition of proteins, carbohydrates, lipids, and vitamins (Zimmermann and Hurrell, 2002; Sun, 2008). Since the last three decades, the main emphasis of agricultural biotechnology can be found on traits for improvement in crops related to virus resistance, nutritional quality, herbicide and insect resistance, shelf life, and biofuel production. These traits involve several genes; therefore, improvement in crop using genetic engineering is not so simple. Lack of basic knowledge of the molecular biology and genetics of the plant species makes this even more demanding. Transgenic plants are being developed using different genetic engineering methods but with several legal, political, and social problems (Ashraf and Akram, 2009). For instance, the World Health Organization has identified the following three main issues with genetically engineered crops, particularly GM food crops (docplayer.net): Generation of allergenic foods Incorporation of modified food genes into the human body Crossing of transgenic plants with non-transgenic conventional plants

These factors may present a threat to food safety. In spite of all these barriers, different countries including the United States, Canada, Brazil, China, Canada, and Argentina are now allowing transgenic crop production (James, 2008). Substantial improvement in yield has been obtained by the use of transgenic approach in several crops—soybean, brassica, wheat, rice, tobacco, etc.—but still, there is an urgent requirement of producing high yielding and quality transgenics (tur-www1.massey.ac.nz).



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Several crops have been engineered to enhance the resistance to a multitude of stresses (herbicides, insecticides, viruses, and a combination of biotic and abiotic stresses) in different types of crops including mustard, tomato, maize, rice, and potato. Besides the use of GE in agriculture, it is being widely used for modifying the plants for increased production of hormones, vaccines, etc. Vaccines against certain diseases are available in the market, but most of these are very expensive. Developing countries are not able to afford the control of disease through these costly vaccines. Alternately, attempts are being made for producing edible vaccines, which are inexpensive and have several benefits in comparison with the vaccines commercially available. Transgenic plants produced for this application can express recombinant proteins including bacteria and antigens and antibodies. Certain food plants such as banana, tomato, rice, and carrot have been used for producing vaccines against cholera, HIV, hepatitis B, etc. Therefore, the up- and downregulation of desired genes that are used for the modification of plants have a significant role in improving the genetic crops. Ahmad et al. (2012) have discussed the role of genetic engineering in producing transgenic lines/cultivars of different crops with improved properties: • • • •

Nutrient quality Biofuel production Increased production of vaccines and antibodies Increased resistance against insects, herbicides, diseases, and abiotic stresses and also the safety measures for their commercialization

Plant scientists, by using several genetic engineering methods, are attempting to increase crop production by the development of high yielding crops, disease-resistant crops, and crops with high nutritional value and biofuel production. “GM plants have been produced for their increased tolerance to herbicides and pests. Some others have been developed to provide nutritionally rich food and biofuel production. Healthier oils, vegetables and fruits with low calorie sugars and enriched with vitamins are under development. Golden rice is a genetically modified crop. It is rich in provitamin A (β-carotene) and iron. Many parts of the world experience inadequate levels of essential vitamins and minerals such as vitamin A and iron” (Ahmad et al., 2012). Golden rice appears to be a promising crop to solve this problem. Golden rice is being evaluated these days in Vietnam, the Philippines, and India for its ability to produce high amount of vitamin A and iron. In India, high-protein potatoes have also been developed by transferring a gene from an amaranth plant. Despite the disagreement by several countries on transgenic crops, substantial economic advantages have been obtained using agricultural biotechnology (docplayer.net). According to Brookes and Barfoot (2017), “the generation of GM crops has enabled using 393 million kg less pesticides by the growers. This effect has a substantial role in reducing greenhouse gas emission which in 2009 was equivalent to removing 7.8 million cars from the roads. Due to the present growing trend of transgenic crops, it is assumed that available transgenic crops in the future could boost crop yield, and the food produced from such crops will be nutritionally rich. Another achievement of plant biologists is that plants are being used for the production of biopharmaceuticals. Valuable proteins are expressed in transgenic plants that can be extracted and processed, which have many advantages over industrial ­proteins. Though plant-based vaccines have shown promising results, the oral tolerance to plant vaccines is a very important problem that needs in depth research. The genetic engineered plants being used need strict safety evaluation. The plant biotechnologists should

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keep in mind that the transformants that they are going to develop should be safe enough. Apart from the success stories in many cases, many concerns are yet to be mitigated before plant based vaccines become a real boom.” HIV and malaria are most dangerous and very complex diseases. Vaccines from plants have been found promising in controlling these diseases, but since these studies have been conducted on a limited scale, so for their effective universal use, up-scaling of these studies is very important. Though several vaccines for many diseases are provided by the WHO, there are many diseases for which the vaccines have to be purchased locally. For instance, hepatitis-B/DTP combination vaccines are to be purchased from the local market and the cost of the vaccines is too high. As a result, many children are deprived of vaccination and hence at the risk of this preventable disease. To abate this problem transgenic plants may provide an excellent expression system and the vaccines can be given directly to people in the form of edible vegetables, fruits etc. Plants such as banana, tomato, potato, spinach, tobacco, rice, corn, etc. are being used to fight diseases such as cholera, measles, hepatitis-B, Norwalk virus and rabies virus by inducting immunization edible vaccines. Edible plant vaccines are very safe and also cost-effective. (Ahmad et al., 2012)

In comparison with their conventional counterparts, transgenic plants offer several benefits (Table 6.64): “In 2003, global acreage planted with biotech crops already amounted to 167 million acres in 18 countries, representing a 15% increase in acreage over 2003. Major transgenic crops cultivated include soybean, maize, cotton, canola, squash and papaya. Dozens of other transgenic crops are expected to enter commerce over the next few years” (Gavrilescu and Chisti, 2005). Some of the major commercial players in plant biotechnology are listed in Table 6.65

TABLE 6.64  Advantages of transgenic plants. Higher yields Reduced need for fertilizers and pesticides Ability to better tolerate adverse environments and pests Improved nutrition and other functionalities Ability to produce products that a crop is not able to produce naturally Lower cost of production Based on Bohnert and Jensen (1996); Murphy (1996); Hirsch and Sussman (1999); Jaworski and Cahoon (2003); Mascia and Flavell (2004).

TABLE 6.65  Major commercial players in plant biotechnology. Syngenta, Monsanto Bayer CropScience DuPont/Pioneer Hi-Bred Dow AgroSciences BASF Based on Gavrilescu, M., Chisti, Y., 2005. Biotechnology—a sustainable alternative for chemical industry. Biotechnol. Adv. 23, 471–499.

References 171

Doubtlessly, there is a continuous increase in the use of genetically modified organisms for food or other important commodities. GM foods are environmentally friendly and are not dangerous to human health, profitable for farmers, and also well regulated; however, several people are still of the firm opinion that GM foods can be harmful to human and animal health, because they have not been properly examined. Also, what types of long-term effects GM foods can cause is not known. Critics say that transferring new genes into a food can change the chemical composition of that food, which may trigger the human body to respond differently to that food, thus developing allergies or causing long-term toxicity. Moreover, many GM crops have antibiotic resistance genes that could be taken up by bacteria present in the body, thus increasing bacterial resistance against antibiotics. Therefore, every country needs to frame well-defined rules and regulations for the use of GM organisms, although several developed and some developing countries have already formulated specific regulations (Ahmad et al., 2012).

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Further reading Abatenh, E., Gizav, B., Tsegaye, Z., Wassie, M., 2017. Application of microorganisms in bioremediation-review. J. Environ. Microbiol. 1 (1), 27. Bajpai, P., 1997a. Enzymes in Pulp and Paper Processing. Miller Freeman, San Francisco, CA137. Bajpai, P., 1997b. Microbial xylanolytic enzyme systems—properties and applications. Adv. Appl. Microbiol. 43, 141194. Bajpai, P., 2005. Surface sizing. In: Emerging Technologies in Sizing. PIRA International, UK, pp. 135. (Chapter 8). Bajpai, P., 2011. Durable Bioplastics. PIRA International, UK.



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Bajpai, P., 2012. Environmentally Benign Approaches for Pulp Bleaching, 2nd ed. Elsevier Science B.V, Netherland. 416. Bajpai, P., 2018b. Biermann's Handbook of Pulp and Paper Volume 2: Paper and Board Making. Elsevier, USA. Deloitte, 2014. Opportunities for the Fermentation-Based Chemical Industry. An Analysis of the Market Potential and Competitiveness of North-West Europe, UK. Gavrilescu, M., 2004a. Cleaner production as a tool for sustainable development. Environ. Eng. Manag. J. 3, 4570. Joshi, V.K., Attri, D., Bala, A., Bhushan, S., 2003. Microbial pigments. Indian J. Biotechnol. 2, 362369. Liu, X., Xiao, G., Chen, W., Xu, Y., Wu, J., 2004. Quantification and purification of mulberry anthocyanins with macroporous resins. J. Biomed. Biotechnol. 5, 326331. Malik, K., Tokkas, J., Goyal, S., 2012. Microbial pigments: a review. Int. J. Microb. Res. Technol. 1 (4), 361365. Maurer, H.W., 2001a. Surface sizing of paper. In: Maurer, H.W. (Ed.), Starch and Starch Products in Surface Sizing and Paper Coating. Tappi Press, Atlanta, GA, pp. 83. Maurer, H.W., 2001b. Enzyme conversion of starch for paper sizing and coating. In: Maurer, H.W. (Ed.), Starch and Starch Products in Surface Sizing and Paper Coating. Tappi Press, Atlanta, GA, pp. 65. Mortensen, A., 2006. Carotenoids and other pigments as natural Colorants. Pure Appl. Chem. 78 (8), 14771491. Office of Technology Assessment (OTA), 1984. Commercial Biotechnology: An International Analysis. OTA-BA-218. U.S. Government Printing Office, Washington, DC. Parmar, M., Phutela, U.G., 2015. Biocolors: the new generation additives. Int. J. Curr. Microbiol. App. Sci. 4 (7), 688694. Ravindran, V., 2013. Feed enzymes: science, practice and metabolic realities. J. Appl. Poult. Sci. 22, 628636. Scheper, T., 2004. Molecular biotechnology of fungal beta-lactam antibiotics and related peptide synthetases. Adv. Biochem. Eng. Biotechnol. 88, 284. Scott, C., Pandey, G., Hartley, C.J., Jackson, C.J., Cheesman, M.J., Taylor, M.C., Pandey, R., Khurana, J.L., Teese, M., Coppin, C.W., Weir, K.M., Jain, R.K., Lal, R., Russell, R.J., Oakeshott, J.G., 2008. The enzymatic basis for pesticide bioremediation. Indian J. Microbiol. 48 (1), 6579. https://doi.org/10.1007/s12088-008-0007-4. Suvarnalatha, G., Rajendran, L., Ravishankar, G.A., Venkataraman, L.V., 1994b. Elicitation of anthocyanin production in cell cultures of carrot (Daucus carota L.) by using elicitors and abiotic stress. Biotechnol. Lett. 16, 12751280. Svensson, G., 2006. Alternative Enzymatic Conversion of Surface Sizing Starch at Nymölla Mill. Department of Chemical Engineering. Available at www.chemeng.lth.se/exjobb/E256.pdf. Teknoscienze, www.teknoscienze.com/tks_article/industrial-biotechnology-applications-for-personal-care-and-household-products/. Venil, C.K., Zakaria, Z.A., Ahmad, W.A., 2013. Bacterial pigments and their applications. Process Biochem. 48 (7), 10651079. https://doi.org/10.1016/j.procbio.2013.06.006. Vitalab, Vitalab—Green Biotechnology for Sustainable Production of Cosmetic Active Ingredients. Available online: http://vitalabactive.com/actives.html. Wijffels, R.H., Barbosa, M.J., 2010. An outlook on microalgal biofuels. Science 329, 796799. https://doi.org/10.1126/ science.1189003.

C H A P T E R

7 The economical and ecological advantages of industrial biotechnology Industrial biotechnology (IB)—also known as white biotechnology—uses enzymes and microorganisms for making biobased products in various sectors such as food and feed, chemicals, detergents, paper and pulp, textiles, and bioenergy (OECD Report, 2001; Okkerse and Van Bekkum, 1999; Philp, 2011; Soetaert and Vandamme, 2006; Singh,2014). This process works by transforming organic waste or agricultural products into other substances, in the same manner as crude oil is used as a substrate in the production of chemicals. In this manner, industrial biotechnology saves energy in production processes and lead to substantial reductions in greenhouse gas emissions, which helps in fighting global warming. This may also result in improved performance and sustainability for industry and higher value products (http://industrialbiotech-europe.eu/about-industrial-biotechnology/benefits-industrial-biotechnology/). Biobased chemicals can produce substantial energy savings in comparison with fossil fuel counterparts. These technologies are undergoing innovation. Several cases show potential for further improvement in the environmental and the economic profile of these materials (www. industrialbiotech-europe.eu). Industrial biotechnology uses enzymes, which can work under mild conditions such as low temperatures. Enzymes accelerate the reaction, so industrial biotechnology can enhance the resource efficiency of processing raw materials in terms of energy, time, and water. Enzymes are eco-friendly and provide an alternative to several toxic chemicals. So, they show substantial environmental and economic benefits in comparison with many other systems. Industrial biotechnology significantly improves the environmental and economic performance of chemical production. Biotechnological routes are able to replace many chemical production steps. This can lead to reduced material inputs, generation of lesser by-products and wastes, and also reduced energy consumption (www.industrialbiotech-europe.eu). “Industrial biotechnology adds value to agricultural products and builds new industrial production schemes targeted towards an overall greater degree of sustainability. The environment benefits because biotechnological processes are efficient users of (often renewable) raw materials, creating little end-of-the-pipe waste which itself can be often used as input into a further biological process” (https://www.europabio.org/.../ industrial_or_white_biotechnology_research_for_eur).

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At the same time, shifting from chemicals to biological processes can result in substantial reduction in carbon dioxide emissions, water, and energy consumption. Industrial biotechnology has a double impact: it improves the process efficiency and allows the use of renewable substrates. So, it is a major tool in the development of sustainable production processes. The impact of industrial biotechnology on the environment is shown in Table 7.1 (DSM, 2004). In parallel, the economy benefits as biotechnology enables the introduction of more effective, less energy-intensive processes. Fermentation and enzymatic processes are commonly used in the fine chemical sector for producing vitamins, pharmaceutical intermediates, and flavors. They are also making their first inroads into large volume segments such as polymers, bulk chemicals and biofuels, and several other industrial sectors. New polymers are finding multiple applications in the automotive and consumer industries. As industrial biotechnology moves from the fine chemicals into the commodity chemicals and finally into bulk products, prices of raw materials are becoming even a more important matter. The impact of industrial biotechnology on the economy is shown in Table 7.2 (DSM, 2004). As industrial biotechnology is making industry more sustainable, the benefits are expected to be seen across a range of critical society-based areas, job retention/creation, development of new technology platforms, and the reduction of dependence of society on valuable fossil TABLE 7.1  The impact of industrial biotechnology on the environment. Increased process efficiency and renewable feedstock Reduction of GHG emissions Reduction of emissions to air Reduction of emissions to water Resource usage Based on DSM, 2004. Industrial (White) biotechnology—an effective route to increase EU innovation and sustainable growth. Position document on industrial biotechnology in Europe and the Netherlands, http://www.sustentabilidad.uai.edu.ar/pdf/tec/industrial_white_biotech.pdf, 20 pp.

TABLE 7.2  The impact of industrial biotechnology on the economy. Cost reduction Raw material Process cost Investment Generation of additional revenue New products Value-added processes Based on DSM, 2004. Industrial (White) biotechnology—an effective route to increase EU innovation and sustainable growth. Position document on industrial biotechnology in Europe and the Netherlands, http://www.sustentabilidad.uai.edu.ar/pdf/tec/industrial_white_biotech.pdf, 20 pp.



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TABLE 7.3  The impact of industrial biotechnology on society. Employment New technologies to meet future challenges Creation of new jobs Responsibility Innovation Save valuable resources for future generation Based on DSM, 2004. Industrial (White) biotechnology—an effective route to increase EU innovation and sustainable growth. Position document on industrial biotechnology in Europe and the Netherlands, http://www.sustentabilidad.uai.edu.ar/pdf/tec/ industrial_white_biotech.pdf, 20 pp.

resources, thereby saving them for future generations. Industrial biotechnology can promote high level education and research by providing highly qualified employment and by developing R and D initiatives (www.europabio.org). “Together, these environmental and economic benefits will contribute towards a more sustainable society, with greater opportunities for job creation and retention, and a reduced dependence on fossil fuels” (www.europabio.org). The impact of industrial biotechnology on society is shown in Table 7.3 (DSM 2004). “Industrial biotechnology can not achieve its full potential without a coordinated effort on the part of all stakeholders. As a first step, a dialogue amongst stakeholders needs to be started to share the facts and information as well as to discuss the opportunities, including the concerns related to this technology. Important stakeholders range from industry to academia and public institutions. Stakeholders also include NGOs, the financial community, suppliers and industry users and observers from institutions such as the OECD” (DSM, 2004). Biobased products already available include biopolymer fibers, which are used in both construction and household applications, biodegradable plastics, biofuels, lubricants, and industrial enzymes such as those used in detergents or in paper and food processing. Biotechnological processes also constitute a key element in the production of some antibiotics, vitamins, amino acids, and other fine chemicals (http://industrialbiotech-europe.eu/ about-industrial-biotechnology/benefits-industrial-biotechnology/). Biotechnology already has improved the production of paper, textiles, plastics, chemicals, fuels, and pharmaceuticals, accelerating the production process and reducing water, energy, and raw material inputs and also the pollution. For instance, bleaching pulp for paper through biotech processes can reduce the use of chlorine-based chemicals by up to 15%, and energy use by a third or more (https://industrialbiotechnology.weebly.com/advantages.html; Bajpai, 2018a, b). Replacing petrochemicals with feedstocks made from organic material such as corn stalks can reduce demand for petrochemicals by 20%–80% and produce biodegradable plastics that can eliminate up to 80% of the plastics in community waste streams. Biotechnology also makes it easier to produce medicines such as riboflavin (vitamin B2), according to the BIO report. (https:\industrialbiotechnology.weebly.com\advantages.html) Researchers are ­examining bacteria, microorganisms, and other natural materials with DNA probes for identifying enzymes with certain capabilities, such as the ability to bleach paper or break down

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plant matter, which can thereby enable or accelerate industrially useful biochemical reactions. In some cases, researchers are genetically engineering enzymes for further improving their performance (www.heartland.org). In a Greenwire article, BIO Vice President Brent Erickson mentioned that the biotech processes are “more robust, adaptable, and inherently cleaner than old-line manufacturing methods.” These benefits, which are often coupled with substantial cost savings, should encourage industries to use biotechnologies (https://industrialbiotechnology.weebly.com/advantages.html). Gene-spliced crops could greatly do the following (https://www.bio.org/media/ press-release/industrial-biotechnology-offers-significant-societal-benefits-case-studies-find). • • • • • • • •

Improve nutrition and food security Reduce soil erosion Reduce the use of fertilizer and pesticide Need less water Produce more food from less land (thus saving habitats and wildlife) Increase shelf-life for foods (even without refrigeration) Eliminate allergens Reduce contamination from fungal mycotoxins

Although industrial or white biotechnology is a relatively new approach to product manufacturing, it can achieve substantial environmental and economic benefits over conventional manufacturing processes, according to a report released in a BIO 2003 press conference in Washington, D.C. The report, “White Biotechnology: Gateway to a More Sustainable Future,” presents industrial biotechnology case studies involving the companies BASF, DSM, Novozymes, Cargill Dow, and DuPont (https://www.bio.org/.../press-release/ industrial-biotechnology-offers-significant-societa). The document discusses how each company has used biotechnology to replace conventional manufacturing processes and includes an independent assessment as to the estimated savings it has created in terms of environmental impact and costs (Table 7.4). DSM, Cargill Dow, Dupont, BASF, Novozymes, and Genencor, in cooperation with the European and US biotechnology industry associations (EuropaBio and BIO) and the reputed and independent German Öko Institut, conducted an assessment of the potential impact of white biotechnology. Studies were combined with a market analysis by McKinsey and Company (2003) for estimating the impact on the three elements of sustainable development: people, planet, and profit (Fig. 2.1). The results confirmed an earlier study by the OECD that the “social, environmental and economic benefits of industrial biotechnology go hand-in-hand” (https://www.europabio.org/.../white_biotechnology-_gateway_to_a_more_sustainable). According to Brent Erickson, vice president for the Biotechnology Industry Organization's (BIO's) industrial and environmental section, positive contribution industrial biotechnology can offer in following three key areas of sustainability: • Society • The environment • The economy The United States and Europe have done much to encourage this burgeoning area of technology. Industrial biotechnology supplants traditional manufacturing processes by using



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TABLE 7.4  Companies using industrial biotechnology for improving manufacturing processes. BASF By using a biobased fermentation process, BASF is producing vitamin B2 in a single-step process, whereas in the traditional process, eight steps are involved. This process reduces carbon dioxide emissions by 30%, resource consumption by 60%, and waste by 95% DSM The traditional method for producing the antibiotic cephalexin involves a 10-step chemical process. By replacing this approach with a biotech approach (combination of a fermentation and enzymatic reaction), the material use and energy consumption have been reduced by 65% and the variable costs by 50% Novozymes The scouring process used in the textiles industry involves harsh chemical solutions. Novozymes has developed enzymes for the water intensive textile industry. These enzymes produce a 25% reduction in primary energy demand and a 60% reduction in water emissions. The enzymatic process reduces costs by 20% Cargill Dow The company produced NatureWorks, a new biobased polymer, for producing clothing, packaging materials, and electronic goods. This product needs 25%–55% less fossil resources DuPont The company produced Sorona, a new biobased polymer, which will incorporate the use of dextrose from corn as one of its major substrates, reducing the use of fossil inputs by 50%. Both the Cargill Dow and DuPont products were based on a process developed in collaboration with Genencor Based on https://www.bio.org/media/press-release/industrial-biotechnology-offers-significant-societal-benefits-case-studies-find.

e­ nzymes rather than chemicals, thus reducing pollution. Further, the technology is being used for developing new forms of energy production based on agricultural waste derived from corn stalks and rice, rather than oil or coal. According to Erickson “These new industrial biotech processes will allow us to use enzymes and renewable carbon instead of fossil fuels created by the dinosaurs to fuel our automobiles and our economies, while at the same time helping our environment.” There is a growing awareness in government and by the public that a “new holistic way of doing things is in order if economic growth is to continue without producing major negative impacts on our environment,” Erickson said. “Sustainable industrial development, as defined by the use of clean technologies to reduce pollution levels and resource consumption through the use of innovative technologies and industrial improvements, is that new way” (https:// www.bio.org/.../industrial-biotechnology-offers-significant-societal-benefits-case). The report urges the European countries to adopt policies to foster the development of industrial biotechnology by providing financial incentives and a supportive regulatory framework, benchmarking Europe against countries already using the technology, encouraging competitive feedstock prices, and building public awareness and support. Industrial biotechnology would provide new chances to the chemical industry by allowing easy access to building blocks and materials that were only accessible before through complex routes or not at all. White biotechnology will have a significant impact by using biomass as an alternative to fossil resources for the production of biochemicals like biofuels and biopolymers. The use of renewable raw materials as alternative substrates will reduce consumption of the limited fossil resources and reduced European dependence on imports. Consequently, this could contribute to meeting of the Kyoto protocol targets for reductions in carbon dioxide

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emissions due to a more favorable carbon dioxide balance. This technology may also boost the rural economy by providing new markets for agricultural crops and through the development of integrated biorefineries in farming areas. It should be guaranteed that raw materials can be bought at the cheapest price all over the world (www.europabio.org). White biotechnology processes are helping to make industrial manufacturing processes more eco-friendly. They are conducted in a contained environment and have the potential to produce high yields of specific products with low-energy use and reduced generation of waste. The potential of white biotechnology appears to be very promising. The vision of Europiabiofor industrial or white biotechnology in 2025 includes the following (https:// www.europabio.org/sites/default/files/industrial_or_white_biotechnology_research_for_ europe.pdf): • The development of a strategic research agenda and road map • The removal of technical, economic, regulatory, and implementation barriers • The involvement of the society in decision-making via stakeholder dialogue A renewed interest in the sustainability of industrial processes has also contributed to the attractiveness of biotechnology. All major facets of society and economic activity, including agriculture, environmental protection, and industry, are being challenged to demonstrate their sustainability. Industrial biotechnology can make a major contribution (https://www. europabio.org/sites/default/files/industrial_or_white_biotechnology_research_for_europe.pdf).

References Bajpai, P., 2018a. Biermann’s Handbook of Pulp and Paper: Volume 1: Raw Material and Pulp Making. Elsevier. Bajpai, P., 2018b. Biotechnology for Pulp and Paper Processing. Springer Nature. DSM (2004). Industrial (White) biotechnology—an effective route to increase EU innovation and sustainable growth. Position document on industrial biotechnology in Europe and the Netherlands, http://www.sustentabilidad.uai. edu.ar/pdf/tec/industrial_white_biotech.pdf, 20 pp. McKinsey and Company, 2003. In: Bachmann, R. (Ed.), McKinsey & Company, Industrial Biotech—New ValueCreation Opportunities. Presentation at the Bio-Conference, New York. OECD Report, 2001. The Application of Biotechnology to Industrial Sustainability. http://www1.oecd.org/publications/e-book/9301061e.pdf. Okkerse, H., Van Bekkum, H., 1999. From fossil to green. Green Chem., 107–114. Philp, J., 2011. OECD Outlook on Industrial Biotechnology. OECD Directorate for Science, Technology and Industry. DSTI/STP/BIO(2011)3. Singh, R.S., 2014. Industrial biotechnology: an overview. In: Singh, R.S., Pandey, A., Larroche, C. (Eds.), Advances in Industrial Biotechnology. IK International Publishing House Pvt. Ltd, India, pp. 1–35. Soetaert, W., Vandamme, E., 2006. The impact of industrial biotechnology. Biotechnol. J. 1, 756–769. https://doi. org/10.1002/biot.200600066.

Relevant websites http://industrialbiotech-europe.eu/about-industrial-biotechnology/benefits-industrial-biotechnology/. https://industrialbiotechnology.weebly.com/advantages.html. https://www.bio.org/media/press-release/industrial-biotechnology-offers-significant-societal-benefits-case-studies-find.



Relevant websites

201

https://www.europabio.org/.../industrial_or_white_biotechnology_-_research_for_eur. https://www.europabio.org/.../white_biotechnology-_gateway_to_a_more_sustainable. https://www.europabio.org/sites/default/files/industrial_or_white_biotechnology_research_for_europe.pdf. www.europabio.org. www.heartland.org. www.industrialbiotech-europe.eu.

C H A P T E R

8 Efforts made by different countries toward industrial biotechnology According to OECD (2009) in Europe ~75% of the future economic contribution of biotechnology and substantial environmental benefits is likely to come from applications obtained by using agricultural and industrial biotechnology. But these sectors are presently receiving less than 20% of all research investments made by the public and private sectors. So, there is an urgent requirement to strengthen research in the area of industrial and agricultural biotechnology by increasing the investment in research, by reducing regulatory burdens, and by encouraging private-public partnerships (ec.europa.eu). Today, in Europe, besides individual companies, research in biobased products mostly funded through several public sources: European Union level: The Seventh Framework Program for R and D, running from 2007 to 2013, is the major instrument for funding research in Europe. The Framework Programs for Research is having two major strategic objectives: - To strengthen the scientific and technological base of European industry - To encourage its international competitiveness, while promoting research that supports policies in EU Calls such as the knowledge-based bioeconomy 2011 within Seventh Framework Program could continue to be supported and their financing could be increased in Eighth Framework Program (Horizon 2020). Member State level: In the EU member states, the public research funds for industrial biotechnology are available to a limited extent. The dedicated research programs are running in few countries only. Some are funded through general research programs or supported through parallel programs like energy and agriculture (www.bio-economy.net). Regional and/or national research councils are providing the funding. “Further, many Member States are trying to coordinate their research through ERA-NET schemes. In this scheme, national and regional authorities identify the research areas of common interest, and launch collaborations via joint calls for projects. The national partners in

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these actions are program “owners” typically ministries or regional authorities defining research programs or program “managers” such as research councils or other research funding agencies managing research programs. The partners usually have long term collaboration with relevant national research institutes and other national and international professional organizations and governmental authorities. The scope, objectives and deliveries are defined by the partnership in the ERA-NET” (www.eurosfaire.prd.fr). “EU distinguishes itself by a fragmented approach across the different Member States. Every country is having its own programs and initiatives in different research areas, with little EU-level coordination. Research funding is playing an important role in stimulating academic and industrial collaboration, though the processes are not always easy and transparent, and the regulatory environment creates some problems for collaboration between actors in different countries. International collaboration through joint calls could be seen to be important to future functioning of research in the area of industrial biotechnology in Europe” (https://ec.europa.eu/docsroom/documents/11283/attachments/5/translations/.../native; ec.europa.eu). In the year 2011, European biotechnology firms amounted to 3593 (OECD Key Biotechnology Indicators—June 2013 http://www.oecd.org/innovation/inno/keybiotechnologyindicators. htm). The advantages that these companies are bringing to Europe and what is in the pipeline are presented in the succeeding text (www.chilebio.cl): - Industrial biotechnology reduces the environmental impact and also boosts production and creates more jobs for Europe. - Europe is a world leader in the area of industrial biotechnology. Europe is producing ~60% of the enzymes produced in the world. - Industrial biotechnology uses enzymes and microorganisms for making products that enhance the efficacy of detergents so that clothes are washed at reduced temperatures and the production of pulp and paper, chemicals, food, clothing, and bioenergy is done in an environmentally friendly way using less energy and water. Also there is reduced generation of waste. - Biobased industries use renewable raw materials and or use biobased processes in their production processes. Industrial biotechnology is the major enabler of biobased industries. The bioeconomy is worth about €2t and provides about 22 million jobs in European countries alone in agriculture, forestry, fisheries, food, chemicals, and biofuels sectors. - Industrial biotechnology avoids releasing 33 million tonnes of carbon dioxide, which is equivalent to more than seven coal-fired power plants or equivalent to the energy use of 2.5 million homes per year. - Biotechnology allows saving up to 30% of the electricity used on laundry by washing at 30°C rather than 60°C. - By driving in a car with advanced biofuels, carbon dioxide emissions is reduced by a minimum of 80%. Today, parts of automobiles are made from renewable feedstocks, such as biobased plastics for car parts, tires partially made from renewable rubber, and biofuels. - Enzymes are able to improve the intake of several nutrients, reducing phosphate output in manure. This provides an environmental advantage to farms.



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- Alternatives to antibiotics and overall improvement to animal nutrition are available. This makes the utilization of animal feed more effective (https://ec.europa.eu/ docsroom/documents/11283/attachments/5/translations/.../native; https://www. ey.com/.../EY-biotechnology-in-europe.../EY-biotechnology-in-europe.pdf). Major competitors of Europe in industrial biotechnology are the United States and Japan. United States is supporting industrial biotechnology in a big way and is spending about 10 times as much as member states of the EU on research in this area. In the last decade, in the United States, the development of the liquid biofuel and biobased chemical industries increased substantially as these industries are using enzymes, microorganisms, and renewable raw materials for manufacturing chemicals and fuels. The large increase in sales of liquid biofuels and biobased chemicals showcased the use of industrial biotechnology. A substantial part of the increase was accounted for by the biofuel industries, which were supported by tax incentives offered by government, mandatory use regulations, or both. Sales of biobased chemicals and liquid biofuels remain small as compared with conventional chemicals and liquid fuels. Innovation is key to the future competitivity and productive capacity of US firms and all innovation indicators increased in the last decade, including R and D expenses, patent and trademark activity, strategic alliances, and government grants. But operating income as a share of total net sales of biobased products was relatively flat during the period, mostly because of the significant increase in the prices of agricultural raw materials. Accounting for more than 50% of production costs for liquid biofuels, feedstocks along with the inability to attract enough investment are some of the most important issues to the successful development and use of industrial biotechnology. “The difficulty in attracting research and investment capital comes from the risk inherent in new technology, including the uncertainty of whether such technologies can be fully developed and adopted. Other important roadblocks identified by liquid fuel and chemical producers as affecting the use of industrial biotechnology include production costs and limits of technology” (www.bio.org). “Industrial biotechnology can potentially benefit the United States economy by allowing the substitution of liquid biofuels for conventional liquid fuels, potentially reducing crude petroleum imports, stimulating the development of rural economies because of increased agricultural feedstock consumption” (www.bio.org). Industrial biotechnology can provide a range of environmental advantages, including sustainable production, reduced greenhouse gas emissions, and reduced generation of waste and can also improve production efficiencies. The activities in industrial biotechnology in several countries also increased in the last decade. The United States was not alone in its industrial biotechnology growth. Like in the United States, other governments also use tax incentives and mandatory use regulations. Funds are being provided for R and D for supporting industrial biotechnology industries with the noteworthy examples of Brazil, China, and the EU (https://www.bio.org/articles/ industrial-biotechnology-development-and-adoption-us-chemical-and-biofuel-industries; https://www.usitc.gov/publications/332/pub4020.pdf). China and other countries like India are also developing in this area speedily and are becoming a major threat to European industrial biotechnology. The United States is ­encouraging

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industrial biotechnology as part of its governmental program and sanctioned a huge budget to facilitate further development and implementation of the use of industrial biotechnology. Main R and D programs are focusing on biofuels and biomaterials. The American vision of development till 2020 is structured around a coherent scheme aimed at becoming less dependent on energy. In the United States, federal agencies give importance to biobased products, which have been approved, and legislation is being changed speedily to coordinate federal programs encouraging the use of industrial biotechnology products. Budgets are allocated for research programs on enzyme and biomass technologies and also biobased products and bioenergy. Research and development expenses in biofuels increased 400 % from 2004 to 2007, reaching $152.5 million, which is three times the conventional R and D expenses. For biobased chemicals, the R and D expenses reached $3.4 billion—much larger than for biofuels (www.bio-economy.net). Japan is also taking a lot of interest in biotechnology considering that the old biotechnologies have played an important role in Japanese industry in the last decades. Earlier, Japan selected technologies and industries pertinent to its future and had instituted bureaucracies to support this redirection. But currently, it started mergers of government ministries to form super ministries in an attempt for providing more integrated and consistent approaches to change across government (ec.europa.eu). In China, biotechnology is strongly connected with foreign networks and getting benefited from the competence of those Chinese who came back from abroad. The government has developed framework of science and technology policy structured in three main programs and the government also invests in quasi-venture capital companies for supporting start-up firms and attracting private investment into life science through tax incentives, etc. Local governments are developing high tech parks and are attracting funds through matching investments and marketing campaigns aimed at foreign investors (ec.europa.eu). “The biotechnology sector of India is highly innovative and is on a strong growth trajectory. The sector, with its immense growth potential, will continue to play a significant role as an innovative manufacturing hub. The sector is one of the most significant sectors in enhancing India's global profile as well as contributing to the growth of the economy” (indiainbusiness.nic.in). “India is among the top 12 biotech destinations in the world and ranks third in the Asia-Pacific region. India has the second-highest number of United States Food and Drug Administration (USFDA)—approved plants, after the United States and is the largest producer of recombinant Hepatitis B vaccine. Out of the top 10 biotech companies in India (by revenue), seven have expertize in bio-pharmaceuticals and three specialize in agri-biotech” (www.ibef.org). “India has no dearth of talent in biotechnology, as a number of institutions, both government and autonomous, provide the necessary opportunities for the students seeking to obtain a degree in this sector. The Government of India has provided adequate scope to this sector by providing facilities for Research and Development in the field of biotechnology” (www.ibef.org). The biotech industry in India is holding about 2% share of the global biotech industry. There are 800 companies in the industry. These were valued at USD 11.6 billion in 2017. “The government has to invest US$ 5 billion to develop human capital, infrastructure and research initiatives if it is to realize the dream of growing the sector into a US$ 100 billion industry by 2025,” according to the Union Minister for Science and Technology.



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“Biopharma is the largest sector contributing about 62% of the total revenue followed by bioservices (18%), bioagri (15%), bioindustry (4%), and bioinformatics contributing (%)” (www.ibef.org). There is high demand of different biotech products. This is also opening up opportunity for the foreign companies to set up base in India. India is emerging as a leading destination for clinical trials, contract research, and manufacturing activities because of the growth in the bioservices sector. The biotech sector in India is attracting a lot of attention over the last two decades. Many companies in the world are aggressively joining hands with Indian companies because of its strong generic biotechnology potential. Some of the recent investments and developments in this sector are presented in Table 8.1. “A Network of Technology Centres and promotion of start-ups by Small Industries Development Bank of India (SIDBI) are among the steps taken by the Indian Government for promoting innovation and entrepreneurship in the agro industry proposed by the Ministry of Micro, Small and Medium Enterprises in a new scheme. Indian Government has taken many initiatives for improving the biotechnology sector in the country and also offer enough scope for research in this field. The Department of Biotechnology (DBT) along with other government funded institutions such as National Biotechnology Board (NBTB) and many other autonomous bodies representing the biotechnology sector, are working jointly in order to project India as a global hub for biotech research and business excellence” (www.ibef.org). Some of the recent major initiatives are presented in Table 8.2. The biotechnology industry in India has huge potential to emerge as a global key player. The country is offering several comparative benefits in terms of R and D facilities, knowledge, skills, and cost-effectiveness.

TABLE 8.1  Recent investments and developments in biotechnology sector in India. The flagship pharma and biotech event BioAsia 2017 of Telangana state government has attracted investments to the tune of Rs 3382 crore 54 MoUs worth Rs 5022 crore in the biotechnology sector was signed by 37 companies during the Vibrant Gujarat Global Summit 2017 Syngene International Ltd is setting up a drug discovery and development center in Bengaluru for Amgen Inc., a biotechnology company based in the United States. Based on www.ibef.org.

TABLE 8.2  Major initiatives taken in India. In 2017–18, DBT received Rs 2222.11 crore (US$ 333.31 million), an increase of 22%, to continue implementing the department’s national biotech strategy and target increasing the turnover from the sector to $100 billion by 2025 from $7 billion in 2016 The Telangana government also inked an MoU with PE firm Cerestra to explore a “Life Sciences Infrastructure Fund” with a corpus of Rs 1000 crore for establishing a sophisticated modular plug and play infrastructure for pharma biotech and medical devices industry Based on www.ibef.org.

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India is constituting about 8% of the total global generics market, by volume, showing an enormous untapped opportunity in the sector. Outsourcing to India is projected to spike up after the discovery and development of formulations. Hybrid seeds, including GM seeds, represent new business opportunities based on yield improvement (www.ibef.org). “India currently has a marginal share in the global market for industrial enzymes. Therefore, there is an opportunity in focused R and D and knowledge-based innovation in the field of industrial enzymes, which can innovatively replace polluting chemical processes into environment friendly processes that also deliver environmental sustainability. Another interesting field of study is the area of bio-markers and companion diagnostics, which will enable to optimize the benefits of biotech drugs” (https://www.ibef.org/industry/biotechnology-india.aspx).

Reference OECD, 2009. The Bioeconomy to 2030: Designing a Policy Agenda. Main Findings and Policy Conclusions. https:// www.oecd.org/futures/long-termtechnologicalsocietalchallenges/42837897.pdf.

Relevant websites www.chilebio.cl. https://ec.europa.eu/docsroom/documents/11283/attachments/5/translations/.../native. http://www.oecd.org/innovation/inno/keybiotechnologyindicators.htm. https://www.ey.com/.../EY-biotechnology-in-europe.../EY-biotechnology-in-europe.pdf. https://www.bio.org/articles/industrial-biotechnology-development-and-adoption-us-chemical-and-biofuel-industries. https://www.usitc.gov/publications/332/pub4020.pdf. https://ec.europa.eu/programmes/horizon2020/en/what-horizon-2020. www.bio-economy.net. www.eurosfaire.prd.fr.

C H A P T E R

9 Major challenges facing industrial biotechnology “Industrial biotechnology is the third wave of modern biotechnology, and it promises to be the biggest wave—bigger than biopharma and agricultural biotechnology” (Davison and Lievense, 2016). Grand View Research, Inc. reports that the global biotechnology market is expected to reach USD 727.1 billion by 2025 (https://www.grandviewresearch.com/ press-release/global-biotechnology-market). Certain major themes have emerged in the biotechnology market. This would drive the growth in this industry to a profitable extent. Industrial biotechnology is dominated by traditional products, such as organic acids, enzymes, ethanol, bulk antibiotics, and amino acids. The production of most of these products is now increased by the application of genetic engineering methods. Whereas biofuels are attracting a lot of interest among the public, press and policy makers, renewable chemicals represent another main opportunity for commercializing industrial biotechnology in existing markets, at reduced capital costs. Higher returns are also expected. Earlier attempts to lay a roadmap for the production of biochemicals from renewable sugars focused on those that would provide coproducts for integrated biorefineries producing biofuels and bioenergy as the main product. Several companies are exploring specialty chemicals as an entry point for building up the biobased economy (Erickson et  al., 2012). It appears industrial biotechnology is at a central point, with substantial and ongoing growth in technology contributing to several opportunities: • New products • New and more sustainable ways to make established products • Improvements to existing products and processes (https://www.aiche.org› Publications › CEP Magazine › CEP: June 2016) Now the important questions are as follows: When there will be a commercial breakout in industrial biotechnology like in biopharma and agricultural biotechnology? What advances in technology are required to make this possible? Commercialization is a challenge in all the fields, including industrial biotechnology, but it is also highly rewarding for, societal, corporate, and personal reasons (Singh et al. 2014). Biobased

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industrial fuels and chemicals offer potential benefits in customer acceptance (e.g., sustainability); product properties, particularly in the case of complex molecules that are otherwise difficult to produce; feedstock diversification; simpler, on purpose, lower-capital production; and reduced production costs. Rapid developments in biological tools are increasing the portfolio of accessible products and reducing technology risks (Davison and Lievense, 2016). There is now growing openness in industrial biotechnology to share insights and to form partnerships. The industrial biotech community must encourage these interactions. They are important to overcome blind spots and naiveté that otherwise exist in any isolated organization, especially in an emerging field, and to catalyze innovations in all stages of a project. They also allow for better risk management in R and D and across the supply chain. No institution (government, academia, corporate, or national laboratories) can do this alone. As an industrial biotech community, there is a need to eliminate barriers at institutional, regulatory, and career levels. Cross-training, communication, and advocacy must be promoted. There is a saying: “a rising tide lifts all the boats.” Directed toward the right commercial targets and with collaboration across the supply chain, technology can be that tide in industrial biotech field, in spite of the current macro environment challenges (Davison and Lievense, 2016; https://www.entrepreneur.com/article/308661). The commercialization of industrial biotechnology is not as fast as was expected. Originally, it was believed that production of bulk chemicals including polymeric materials, biofuels, and chemical agents using microorganisms or enzymes will provide environmentally friendly low-cost products, to partially replace petrochemicals products (Chen and Kazlauskas, 2011). But this does not appear to be so easy to materialize. The reasons are presented in Table 9.1. The European association for bioindustries has described the major challenges facing industrial biotechnology (Table 9.2). Different areas—biomass, bioprocess and bioproducts, and biofuels—have been covered (https://www.europabio.org/sites/default/files/industrial_or_white_biotechnology_research_for_europe.pdf). TABLE 9.1  Reasons for slow commercialization of biotechnology. The price of petroleum did not increase too much after 2008 financial crisis. Other alternative energy sources particularly shale gas natural gas hydrate and sand oil have been discovered in large amount and their uses are rapidly moving toward a very competitive price The depletion of petroleum seems to be a remote reality Agricultural feedstock for bioprocessing are becoming expensive Cellulose, which is a low-cost feedstock, cannot be used easily for microbial processes at least for the next 5–10 years Bioprocessing is still not as effective as the chemical processing is. This results in high cost of bioproducts Bioprocessing that requires large amount of fresh water has had increasing concerns in several areas facing water shortage The chemical industry is also becoming competitive in several ways including eco-friendliness, the use of renewable raw materials for producing chemicals that are usually obtained from petrochemicals The fast development of C1 chemical engineering products Funds are not available in large amounts in the area of industrial biotechnology Based on Chen, G.Q., 2012. New challenges and opportunities for industrial biotechnology. Microb. Cell Fact. 11, 111. doi: 10.1186/1475-2859-11-111.



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TABLE 9.2  Challenges facing industrial biotechnology. Biomass area Identifying competitive biomass substrates, which are suitable for EU requirements Conducting LCA and economic and EIA analysis for identifying optimal biomass substrates for the EU The development and optimization of viable processes for the conversion of biomass materials into substrates suitable for The creation of added value for the coproduct and by-products of bioprocesses, for improving the overall economics The development of bioprocesses based on other alternative substrates such as lignin or glycerol, for the chemical industry and energy industry The development of a closed loop fermentation cycle where the waste of one process can be recycled as input for another process Bioprocesses and bioproduct area The development of innovative bioproducts with new applications and properties. A major challenge will be the identification of new applications As it is sometimes difficult to replace existing products due to the higher price of the bioproduct, more efficient processes need to be developed, including improved enzymes and microbes or improved properties should be developed or identified for the bioproduct The development of new bioproducts showing better performance in the existing applications The development of novel processes, bioreactors, and operating strategies along with novel downstream processes The development of models for predicting cellular functioning under industrial conditions Biofuels area The development of improved enzymes and robust fermentation systems having the ability to convert lignocellulose directly and fermenting it to ethanol or other higher alcohols Making these technologies cost-effective The development of novel fermentation processes based on crude glycerol as a carbon source Use of waste fats and side streams of the edible oil processing industry as a substrate for producing biodiesel and bulk oleochemicals Analyzing the potential for producing biodiesel economically with biotechnological methods based on methanol or bioethanol Identification, evaluation, and production of other liquid fuels Based on https://www.europabio.org/sites/default/files/industrial_or_white_biotechnology_research_for_europe.pdf.

So it is important to develop such processes, which combine the benefits of chemical industry to supplement the weaknesses of industrial biotechnology as presented in Table 9.3. The newly emerging synthetic biological approaches may provide some clues for developing competitive technology for industrial biotechnology to produce “high volume and low price” products (Table 9.4).

TABLE 9.3  Comparison of industrial biotechnology and chemical technology. Items

Industrial biotechnology

Chemical technology

Reaction time

Slow: production takes days

Fast: production takes hours

Substrates

Agricultural products

Petroleum or its derivatives

Conversion of substrates to products

Low: for example, PHB/glucose ≈33 wt% PHA/fatty acids ≈60 wt%

High: for example, polyethylene/ ethylene ≈100%

Medium

Water

Mostly organic solvents

Consumption of water

A lot

Less

Reaction conditions

30°C–40°C, normal pressure

Generally >100°C, high pressures

Product concentration

Low: several mg to 100 g/L

Very high

Product recovery cost

Very high

Low to medium

Processing

Normally discontinuous one

Can be continuous

Sterilization

Necessary

No need

Production facility cost

Very high

Low to high (explosive proof)

Wastewater

Not toxic, easier to treat

Generally toxic, difficult to treat

Reproduced with permissions from Chen, G.Q., 2012. New challenges and opportunities for industrial biotechnology. Microb. Cell Fact. 11, 111. doi: 10.1186/1475-2859-11-111.

TABLE 9.4  Problems to be solved for making industrial biotechnology competitive to chemical technology. Problems

Weakness of Industrial biotechnology

Possible solutions

Microorganisms grow too slow

Slow: production takes days

Minimizing the microbial cells

Microbes cannot use mixed substrates

Agricultural products are mostly mixed substrates

Assembling pathways that can metabolize mixed substrates

Low conversion of substrates to products

Cell metabolism turn substrates into CO2, H2O, and by-products

Removing unnecessary pathways consuming substrates

High consumption on fresh H2O

Fresh H2O as medium, etc.

Utilization of seawater for cell growth

Microbial cells grow to very low density

Product concentration low: several milligrams to 100 g/L

Minimizing oxygen demand for aerobic cells and reducing Quorum sensing effects

Discontinuous processing

Contamination concerns

Developing continuous process

Sterilization costs high

High pressed steam

Contamination resisting strains grown in open systems

High-energy demand for intensive aeration

Aerobic microorganisms need a lot of oxygen for growth

Developing anaerobic bioprocesses

Difficulty to control the bioprocesses

Complicated cellular metabolisms

Artificial cells that contain only necessary metabolic pathways

One product by one microbial organism

Different organism has different strength

Development of a platform organism for many products

Organisms consume food related products

Food for fuels (chemicals)

Kitchen wastes or activated sludge as substrates

Production facility costly

Costly materials and sensors

The use of carbon steel facilities

Reproduced with permissions from Chen, G.Q., 2012. New challenges and opportunities for industrial biotechnology. Microb. Cell Fact. 11, 111. doi: 10.1186/1475-2859-11-111.



Further reading

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Simultaneously, bioprocessing should try to become as similar as the chemical industry, including the requirement for developing continuous and open fermentation processes, for example, producing biofuels and polyhydroxyalkanoates plastics (Johnson et al., 2009; Zhang et al., 2009; Tan et al., 2011). Combination of bio- and chemical processes can provide several benefits (Lee et al., 2011; Chen and Patel, 2012).

References Chen, G.Q., Kazlauskas, R., 2011. Chemical biotechnology in progress. Curr. Opin. Biotechnol. 22, 1–2. Chen, G.Q., Patel, M., 2012. Plastics derived from biological sources: present and future—a technical and an environmental review. Chem. Rev. 112, 2082–2099. Davison, B.H., Lievense, J.C., 2016. SBE supplement: commercializing industrial biotechnology—technology challenges and opportunities. Chem. Eng. Prog. 112, 35–42. Erickson, B., Nelson, J.E., Winters, P., 2012. Perspective on opportunities in industrial biotechnology in renewable chemicals. Biotechnol. J. 7 (2), 176–185. https://doi.org/10.1002/biot.201100069. Johnson, K., Jiang, Y., Kleerebezem, R., Muyzer, G., van Loosdrecht, M.C.M., 2009. Enrichment of a mixed bacterial culture with a high polyhydroxyalkanoate storage capacity. Biomacromolecules 10, 670–676. Lee, J.W., Kim, H.U., Choi, S., Yi, J., Lee, S.Y., 2011. Microbial production of building block chemicals and polymers. Curr. Opin. Biotechnol. 22, 758–767. Singh, R.S., Pandey, A., Larroche, C., 2014. Advances in Industrial Biotechnology. I. K. International Publishing House Pvt. Ltd, New Delhi, Bangalore, India. ISBN: 978-93-8332-76-3. Tan, D., Xue, Y.S., Gulsimay, A., Chen, G.Q., 2011. Unsterile and continuous production of polyhydroxybutyrate by halomonas TD01. Bioresour. Technol. 102, 8130–8136. Zhang, X.J., Luo, R.C., Wang, Z., Deng, Y., Chen, G.Q., 2009. Applications of (R)-3-hydroxyalkanoate methyl esters derived from microbial polyhydroxyalkanoates as novel biofuel. Biomacromolecules 10, 707–711.

Relevant websites https://www.aiche.org› Publications › CEP Magazine › CEP: June 2016. https://www.entrepreneur.com/article/30866. https://www.europabio.org/...biotech/.../white-biotechnology-gateway-more-sustainabl. https://www.europabio.org/sites/default/files/industrial_or_white_biotechnology_research_for_europe.pdf. https://www.grandviewresearch.com/press-release/global-biotechnology-market.

Further reading Chen, G.Q., 2012. New challenges and opportunities for industrial biotechnology. Microb. Cell Fact. 11, 111. https:// doi.org/10.1186/1475-2859-11-111.

C H A P T E R

10 Future perspectives “Industrial biotechnology is much larger than just a new source of liquid fuels. Significant benefits can be realized by switching significant production that is currently dependent on fossil resources to biological sources. A wide range of bio-based industrial products and technologies continues to penetrate diverse industrial markets. Ethanol and other oxygenated chemicals derived from fermentable sugars have served as key precursors in the marketplace to other industrial chemicals traditionally dependent on petroleum. In the long term, with advances in genetic engineering, large-scale fuel production from lignocellulosic plant materials are likely to become cost competitive with petroleum fuels. But already, biobased technologies such as enzyme catalysts are promising replacements for industrial chemical processes. And low-volume, high-value chemical production from biomass offers an opportunity to commercialize biotechnology applications in ready-made markets for existing chemicals, if quality, price and performance are equal to reference petrochemicals. In a few cases, biobased products may see a price premium for performance” (Erickson et al., 2012; www.ncbi.nlm. nih.gov). Technology push and industry and market pulls are driving the chance for commercializing industrial biotechnology in existing markets (OECD, 2001). Fast and extraordinary progress in the major technologies of the modern biological sciences, such as genetic engineering and synthetic biology, are driving bioproducts and processes to be more effective and profitable and are stimulating innovation in the chemical industry. While it is taking time for commercializing new technologies, the use of new biotech tools to conventional chemical processes is reducing the timeline. Bioproducts from biorefineries and renewable chemicals are growing swiftly and are gaining higher market share (Erickson et al., 2012). An encouraging fact is the extent of industry pull for products produced using industrial biotechnology. Industry is not only using the bioproducts but also generating more requirements for these and also the new products. For instance, the production of bioplastics from the laboratory to large-scale production is attracting new industry customers, especially in the consumer electronics and automotive industries. The development of highly efficient biorefineries integrating production of several biobased products may aid in reducing the costs and enable biobased products, which can compete more effectively with products from petroleum on price basis (Erickson et al., 2012).

Biotechnology in the Chemical Industry https://doi.org/10.1016/B978-0-12-818402-8.00010-0

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Industrial biotechnology is offering a clear value proposition. But several hurdles must be addressed for fully realizing the commercial potential of biobased products and chemicals in the next decade. Economical solutions will depend on the following factors: • • • •

Continuously conducting R and D Investment made by government and private sector Establishment of new supply chains for renewable and sustainable biomass substrates Market acceptance linked to compatibility with the existing infrastructure

Luckily, we seem to be on the path for commercializing these solutions and to build a biobased economy globally. This is quite good for the consumers, business, and our environment (Erickson et al., 2012). Industrial biotechnology is now moving rapidly as a combined result of international political desire, particularly in the case of biofuels, and significant progress in the area of molecular biology, which has provided the enabling technologies. The priorities of different geographical regions are different, but the common drivers are the need for energy independence and climate change mitigation. “Industrial biotechnology has now reached the centre of scientific and political attention. At no time in the past has there been a more pressing requirement for coherent, evidence-based, proportionate regulations and policy measures; they are at the heart of responsible development of industrial biotechnology” (https://www.oecd-ilibrary.org/science-and-technology/ future-prospects-for-industrial-biotechnology_9789264126633-en). Biotechnology is playing a significant role in the use of renewable resources and so is a research topic of strategic importance for traditional chemical companies (Woodley et al., 2013; Kashangura 2018; Tang and Zhao, 2009; Griffiths, 2001; Bachmann, 2003; OECD 2001, 2009). It is one of the key technologies for developing economically and ecologically successful new processes and products, since it allows petroleum-independent, carbon-neutral production of many substances and paves the way to “green chemistry.” The use of biotechnology in different industrial sectors has invariably led to both economic and environmental advantages listed in the succeeding text: • • • •

Processing is less expensive. Better product quality. Novel products. Eco-friendly processing in comparison with conventional methods.

Economic drivers are the major factor for increasing acceptance of bioprocessing and bioproducts, but sustainability considerations are also playing a very important role. In actual fact, the application of biotechnology has contributed to an uncoupling of economic growth from harmful ecological impact. Industrial biotechnology is in fact changing the way energy, chemicals, and other products are produced. By using engineered biocatalysis, biotechnology is allowing the use of renewable substrates, which were earlier unusable, and production of novel products. Functionally acceptable products, which generate less pollution and are persistent in comparison with conventional counterparts, are being developed. All this is being obtained with reduced ecological impact and increased sustainability. Biotechnology is now all set to transform industrial production to a basis that is more compatible with the biosphere.



10.  Future perspectives

217

For biotechnology to truly reach its full potential, the industry requires sound policy decisions, which support innovation and risk taking as well as a public that is well informed about how biotechnology is creating a healthier, greener, more productive, and more sustainable economy. The biotechnology industry is worth trillions of pounds and provides million jobs, and the objective now is to build on this momentum. Within the healthcare sector, biotechnology is already benefiting more than 350 million patients around the world through the use of medicines obtained using biotechnology for treating and preventing every day and chronic diseases. These medicines account for more than 20% of all marketed medicines, and in the near future, 50% of all these medicines will be obtained using biotechnology (www. europabio.org). Industrial biotechnology is helping to fight global warming as an alternative and safer form of global energy instead of dwindling and volatile fossil fuels. It has led to substantial reductions in greenhouse gas emissions with the aim to reduce a 2.5 billion tonnes of carbon dioxide equivalent per year by 2030. Agricultural biotechnology helps to reduce fuel use and carbon dioxide emissions while producing food containing fewer toxins. It also offers new, better, and adapted agricultural crops for reducing poverty and increasing food security for a growing population. Biotechnology is one of the most exciting sectors at the moment. “No other sector has the same promise of extraordinary rewards for investors as biotech stocks, to say nothing of the patients who will benefit from the new drugs and treatments that are developed” (www. stockring.com). “The future of biotechnology is strong. We envision a day when breakthrough drugs lead to a world without cancer, or AIDS or Alzheimer’s, a world where there is sustainable development that will tackle energy, food and environmental needs without compromising the Earth’s resources or its future” (https://ckscience.co.uk/candidate/biotechnology/nextsteps-in-your-biotech-career/the-future-of-biotechnology/; www.gene.com). White biotechnology can contribute to a more sustainable future for society as a whole. But white biotechnology cannot obtain its full potential without a coordinated effort on the part of all stakeholders either directly or indirectly for supporting the technology (Singh, 2014). We will be able to develop the true potential of this technology for creating a more comfortable and environmentally stable society only through cooperation. A dialogue among stakeholders should be started for sharing the facts and information and also to discuss the opportunities. The concerns related to this new technology must be also discussed (www. europabio.be). The application of white biotechnology in industry is not always smooth, as there are several issues, which get in way of its full-scale implementation. In Europe, there are three important issues concerning the progress of white biotechnology (www.europabio.be): - Development of a long-term strategy - Setting of favorable economic and regulatory framework conditions - Encouraging main technological capabilities Other countries have made more progress in addressing and breaking down these obstacles. In the United States, for instance, representatives from academia, industry, agriculture, and different governmental bodies worked jointly on a project called “Vision 2020” with an objective to promote usage of white biotechnology over the next decades. Lower prices of

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10.  Future perspectives

feedstock and the support of the agricultural community in the United States encourage the use of white biotechnology (www.europabio.be). Industrial biotechnology is moving rapidly in recent years due to the international political desire—particularly in the case of biofuels—and remarkable progress in molecular biology research, which has provided the enabling technologies. Different geographical regions have different priorities, but climate change mitigation and the wish for energy independence are the common drivers. Industrial biotechnology is now reaching the center of scientific and political attention. At no time in the past has there been a more acute requirement for coherent, evidence-based, proportionate regulations, and policy measures; they are at the heart of responsible development of industrial biotechnology (http://www.oecd.org/sti/emerging-tech/futureprospectsforindustrialbiotechnology.html). “The last six decades have seen enormous progress in the development of industrial biotechnology for chemical production and today many companies using such processes in an effective way to produce chemicals. Indeed today we can state that it is no longer a new technology but one that is taking a mainstream role in many research and development organizations in industry. Nevertheless education of the next generation of engineers and the continuing education of those already working in industry is also required. In such developments Europe has a particularly strong position and partly this is as a result of supportive funding for research from the European Commission and partly the result of excellent collaboration between industry and academia. This not only represents an exciting development but also helps all those in university to understand industrial needs and for industry to pick the very latest developments from academia. Such a synergistic relationship has served bioprocessing very well in the past six decades and there is little doubt it will continue to do so in the next decades as well” (Woodley et al., 2013). “The use of industrial biotechnology for the production of chemicals is well established in the pharmaceutical industry but is moving down the value chain toward bulk chemicals. Chemical engineers will have an essential role in the development of new processes where the need is for new design methods for effective implementation, just as much as new technology. Most interesting is that the design of these processes relies on an integrated approach of biocatalyst and process engineering” (Woodley et al., 2013). “Industrial Biotechnology is commonly being called the Third biotechnology wave, in which biowaste comes into value. The technology expands from the previous step of manufacture; the experimental tasks in laboratory; to the end of life of products. It provides a recirculating flow of feedstock that would otherwise be discarded. During the manufacturing process, waste and by-products are produced, including liquid, solid and gas streams, and may be revalorized industrially to biomass. Therefore, biomass can be employed as feedstock for other industrial production processes” (https://www.newfoodmagazine.com/article/27653/ sustainability-industrial-biotechnology/). Several interesting things are happening due to the speedy developments in biotechnology. The genome editing of living organisms, including microorganisms, plants, and animals, is quite interesting for several applications. With these developments, we could increase biobased chemical production, increase the food production, and maintain a better nutritional value, or we could even manufacture organs for transplant. Genetic engineering and synthetic biology are also advancing very swiftly. That has resulted in the production of several chemicals, fuels, and materials from renewable raw materials, rather than depending



10.  Future perspectives

219

on fossil resources. We are seeing some interesting developments in healthcare and also in the medical sector. New, highly complex natural compounds from biosources appear to be satisfactory for pharmaceutical applications. Stem cell therapy, ICT-integrated biotechnology, and several others will help in addressing the health challenges brought on by an aging population (www.weforum.org). “There will be larger number of biotechnology companies, both big and small, along with an increasing number of venture companies. Biotechnology will become as common as having a cellphone or going online. In small villages or even at home, biotechnology might be used, just like in Science Fiction novels. You might simply ask a machine to make some household chemicals you need, rather than go buy it at the supermarket. Biotech trash converters could do away with waste. Biotechnology could also help to tackle large national issues such as healthcare. Global healthcare spending, currently, is about 8 trillion US dollars. That price tag could be as high as we have to go, thanks to biotechnology. Even as the population grows, costs shouldn’t increase thanks to technologies such as efficient disease prevention and well being programs, precision medicine, genome editing, organ production, and stem-cell therapy. All of these will become rather routine. So by 2030, biotechnology will become a part of our life, from drugs, medicine and therapeutics to environmentally friendly chemicals, fuels and materials” (https://www.weforum.org/.../12/what-is-biotechnology-how-will-it-change-our-lives/). OECD (2009) sees several industrial biotechnological applications with a high probability of reaching the market by 2030 (Table 10.1). The biofuel area is likely to provide increasing opportunities because of the increasing price of oil and the growing policy support for fighting the climate change. Furthermore, this will go closely together with remarkable technological progress for producing more sustainable advanced biofuels at competitive prices. Strong growth is also expected for fine chemicals, particularly because of the increasing importance of chiral active pharmaceutical ingredients and to new simplified synthesis pathways through genetic engineering for complex molecules. Substantial growth in the biobased polymer area will result from the development of new polymers with improved properties, greater incentives for reducing costs through the use of renewable feedstocks, and increasing regulatory pressure for reducing carbon footprint (for instance, for applications in the packaging area). Furthermore, enzymes will be used more and more in applications, because of the improvements and benefits especially in the food, textile, and cosmetic industries in accordance with customer requirements and TABLE 10.1  Industrial biotechnological applications with a high probability of reaching the market by 2030. Improved enzymes for different applications in the chemical sector Improved microbes producing a large number of chemical products in single step, some of which build on genes identified through bioprospecting Biosensors for detecting environmental pollutants Biometrics for identification of people High-energy density biofuels obtained from sugary raw material and cellulosic sources Large market share for biomaterials like bioplastics, particularly in niche areas where they show some benefits Based on https://ec.europa.eu/docsroom/documents/11283/attachments/5/translations/.../native.

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10.  Future perspectives

stringent environmental regulations. Certain building block chemicals obtained from sugar substrates may potentially replace petrochemical building blocks (ec.europa.eu).

References Bachmann R (2003). Industrial Biotechnology—New Value-Creation Opportunities. McKinsey and Co., Presentation at the Bio-Conference, New York. Erickson, B., Nelson, J.E., Winters, P., 2012. Perspective on opportunities in industrial biotechnology in renewable chemicals. Biotechnol. J. 7 (2), 176–185. https://doi.org/10.1002/biot.201100069. Griffiths, M., 2001. The Application of Biotechnology to Industrial Sustainability, OECD-Report, 2001. http://www1. oecd.org/publications/e-book/9301061e.pdf. Kashangura, C., 2018. Industrial biotechnology: then, now and the future September 2018. In: Conference: 2nd Training Workshop on Industrial Biotechnology: Driving Value Addition and Beneficiation, 22–24 August 2017 at: Harare, Zimbabwe. OECD, 2001. The Application of Biotechnology to Industrial Sustainability. OECD, 2009. The Bioeconomy to 2030: Designing a Policy Agenda. Singh, R.S., Pandey, A., Larroche, C., 2014. Advances in Industrial Biotechnology. I. K. International Publishing House Pvt. Ltd., New Delhi, Bangalore, India. 978-93-8332-76-3. Tang, W.L., Zhao, H., 2009. Industrial Biotechnology: Tools and Applications. Biotechnol. J. 4, 1725–1739. Woodley, J.M., Breuer, M., Mink, D., 2013. A future perspective on the role of industrial biotechnology for chemicals production. Chem. Eng. Res. Des. 91 (10), 2029–2036.

Relevant websites ec.europa.eu. http://www.oecd.org/sti/emerging-tech/futureprospectsforindustrialbiotechnology.html. https://ckscience.co.uk/candidate/biotechnology/next-steps-in-your-biotech-career/the-future-of-biotechnology/. https://ec.europa.eu/docsroom/documents/11283/attachments/5/translations/.../native. https://www.newfoodmagazine.com/article/27653/sustainability-industrial-biotechnology. https://www.oecd-ilibrary.org/science-and-technology/future-prospects-for-industrial-biotechnology_9789264126633-en. https://www.weforum.org/.../12/what-is-biotechnology-how-will-it-change-our-lives/. www.europabio.be. www.europabio.org. www.gene.com. www.ncbi.nlm.nih.gov. www.stockring.com.

Index Note: Page numbers followed by f indicate figures and t indicate tables.

A Absolute specificity of enzymes, 52t Acetone-butanol-ethanol (ABE) process, 115 Acrylamide, 127 Actinomycetes, 98–99t Advanced biofuels, 204, 219–220 Aerated stirred tank batch fermenter, 40–42f, 42 Agriculture biofertilizers in, 86–87, 87t biotechnology in, 17, 217 byproducts of, 27 raw materials, food components in, 75 soil conditioners, 87t Agrochemicals, 86 crop protection, 88–95 fertilizers, 86–87 Air-lift fermenter, 40–42f, 43 Ajinomoto, 70 Alcohols distilling potable, 82 using fermentation, 59–60t Algae biomass, 149 bioplastics, 140 producing pigment, 98–99t, 101 Alkane hydroxylases, 158–159t Alkylpolyglucosides, 26–27 Alpha-acetolactate decarboxylase, 52 Alpha-amylase enzyme, 79–81 Amino acids, 69–70 L-glutamic acid, 73–75, 73f, 75t lysine, 70–73 growth, 72f hydrogen chloride production, 71, 72f structure, 70f uses of, 71t using fermentation, 59–60t, 74–75 6-Aminopenicillanic acid (APA), 125–126 Ammonia, 34 Amylase enzyme, 76–78 animal feed, 124 in detergents, 133t, 137 Angiotensin-converting enzyme (ACE) inhibitor, 10 Animal feed amino acids, 69–70



glutamic acid, 73 growth in, 73–74 livestock and poultry production, 83 L-lysine, 70–71 from plants, 124 Anionic polyacrylamides, 142–143 Antibiotics, 10 β-lactam, 125–126 semisynthetic, 10 using fermentation, 59–60t Antibody-directed enzyme prodrug therapy (ADEPT), 126–127 Archaebacteria, 52–53 Arginine-degrading enzyme, 126 Aroma compounds enzymes, 107, 108t from plant cell culture, 105, 106t produced by microorganisms, 107, 107t solid-state fermentation, 107–108, 109t Aspergillus niger, 43–44, 62–64, 75 Aspergillus oryzae, 46 Astaxanthin, 101 Atorvastatin, 126 Atrazine dechlorinase, 158–159t

B Bacillus lentus alkaline protease (BLAP), 137 Bacillus popilliae, 89 Bacillus subtilis, 120–121 Bacillus thuringiensis (Bt), 89, 89t, 93–95 Bacterial entomopathogens, 89 Bacterial pigments application, 102–103t production, 98–99t, 101 Baking, 77–78t, 78, 80–81 BASF company, 85, 108, 126, 198, 199t Basic chemicals.. See Commodity chemicals Batch-fed fermentation processes, 39 Batch fermentation lactic acid, 64–65 L-lysine, 71 Baxenden Company, 143 β-carotene, 101 Bioacetone, 116 Biobased chemicals, 7, 195, 205

221

222 Index Biobased polymer, 138, 219–220 Biobased product, 197, 203, 215 federal agency, 205–206 research in, 203 total net sales, 205 Biobased solvents, 112–113, 113–114t, 116 Biobased technology, 215 Biobleaching, 160–161 Biocatalysts, 6, 17, 58 Biochemical cellulosic ethanol production process, 147f Biochemical pesticides, 89t, 90 Biocolorants, 96–105 application, 102t benefits of, 104t features of, 103–104t food-grade, 100f, 105 in food substances, 97t limitations, 104t naturally derived colors from microorganisms, 98t from plants sources, 97t, 103t Biodebarking, 160 Biodegradability, 26–27 Biodegradable plastics, 9, 58–61, 197–198 Biodegradable polymers, 138–143 Bioenergy, 10 Bioethanol, 143–149, 147f enzymes in, 146t first-generation and second-generation feedstocks for, 145t production stages, 146f Biofertilizers, 86–87, 87t Biofiltration technology, 157–158 Biofuel, 10, 205–206, 209, 219–220 challenges facing industrial biotechnology, 211t enzymes in, 146t, 149–150 ethanol, 143–149, 145t, 146–147f Biogas, 59–60t, 149 Bioglycerol, 116 Biological process, 18 Biomass, 10, 218 agricultural waste materials, 146–148 algal, 149 biomaterials from, 140 challenges facing areas, 211t direct extraction from, 140–141 forest, 147f methods to produce solvents from, 113t microbial pigment, 102 significant impact, 199–200 solvents from, 113t total annual production, 28–29 Biomaterials, 205–206 from biomass, 140

Biopesticides, 88, 88t advantages, 90t botanical origin, 89t compounds, 95, 96t for controlling crop diseases, 93 on global scale, 95 from microorganisms, 90t products against soilborne crop diseases, 88–89, 91–92t types of, 89t water quality and environment impact, 93 Biopharmaceuticals, 18, 49–50, 118–119, 122, 122t, 169–170, 207 Bioplastics, 138–143 biodegradable, 9 market drivers, 139t opportunities, 139t production, 215 types of, 139t BioPreferred procurement program, 7–8 Bioprocess, 37–38 advantages, 38t challenges facing industrial biotechnology, 211t enzymatic process, 50–55 fermentation, 38–39 solid-state fermentation, 44–50, 46–50t submerged, 39–43, 44t surface, 43–44 Bioprocess engineering, 37 Bioprocessing techniques, pulp and paper industry, 159–160 biodebarking, 160 bioremediation, 165–166 bleaching, 160–161 dissolving pulp, 162 enzymatic deinking, 161 enzymatic modification of starch, 165 fiber modification, 162–163 flax fibers, 163 shive removal, 163 slime control, 163–164 stickies, 164–165 Bioproduct, 211t, 215 Bioreactor, 37, 39 fermentation process, 38 solid-state fermenters, 49t tray, 48–49 Biorefinery, 29–30, 215 Bioremediation, 152–158 advantages, 153t applications, 154 combinatory methodology, 155, 156f disadvantages, 156t enzymes in, 155, 156f, 157–159t

Index 223

microbial enzymes in, 155, 157t of pulp and paper mill effluents, 165–166 Biosensors, 155 Biosuccinium, 7 Biotech drugs, 119 Biotechnology, 4, 5t, 16, 197–198, 216 in agriculture, 17 application of, 5 in chemical production, 11t color code differentiating main areas of, 2, 2t environmental and economic benefits, 4, 5t healthcare sector, 217 industrial sectors (see White biotechnology) objective of, 38 products, 61 reasons for slow commercialization of, 210, 210t routes of, 195 sustainability, 15–20 vitamins produced by, 120, 120–121t Biotechnology Industry Organization's (BIO's), 198 Biotin, 74 Biotransformations, 51, 106 Ciba Specialty Chemicals, 142–143 ferulic acid to vanillin, 108 Biovanillin, 110–111 β-lactam antibiotics, 125–126 Blakeslea trispora, 97, 101 Bloomberg New Energy Finance, 148 Botanical origin biopesticides, 89t Bradyrhizobium sp., 101 Brewing, 81–82 British Petroleum Oil Spill, 25 Bubble column reactor, 40–42f Bulk chemicals, 58, 61, 210 amino acids, 69–70 citric acid, 61–64, 61f, 62–63t L-glutamic acid, 73–75 lactic acid, 64–67, 64f, 65–66t lysine, 70–73, 70f product produced using fermentation, 59–60t products in developmental stage, 60t propane-1,3-diol, 67–69, 67f Butamax Advanced Biofuels, 115

C Cambium, 160 Candida rugosa, 10 Captopril, 10 Carbochemistry, 26 Carbohydrate sources, 64 Carbon dioxide emissions, 25, 28, 136, 196, 204 Carbon-neutral production, 216 Carboxylesterases, 158–159t Cargill Dow, 198, 199t

Cellulases, 124–125, 135–136 in detergents, 133t Centrifugation, 39 Chemical industry, 1, 18, 31–32, 58 basic chemicals, 33–34 benefits of, 211 challenges, 33 consumer care products, 35 financial crisis, 31–32 life science chemicals, 35–36 products from, 33t specialty chemicals, 32, 34–35 subsegments, 33t, 36 Chemical pulping, 159–160 Chemical technology industrial biotechnology vs., 212t weakness and problems solutions, 212t Chernobyl nuclear disaster, 154–155 China industrial biotechnology, 206 Chlorine-based chemicals, 197–198 Chromium reductase, 158–159t Chymotrypsin, 126 Ciba Specialty Chemicals, 142–143 Circa Group, 116 Citric acid, 58, 61–64 microorganisms with produce, 63t structure, 61f uses of, 62t Clean Air Act (CAA), 157–158 Clean technology, 15–16, 18, 199 Coal burning, 25 Cocoa butter, 150 Collagenase enzyme hydrolyzes, 126 Commodity chemicals, 32–34, 59t Consumer chemicals, 35 Continuous fermentation processes, 39 Corn starch, 79–80 Corynebacterium glutamicum, 73–74 Cosmetics, enzymes in, 66, 128–129, 129t hyaluronidase, 132–133 lipases, 132 peroxidase, 130–131 proteases, 131–132 superoxide dismutase, 130 tyrosinase, 131 Crop protection, 36, 88–95 Crude oil, 27 Cry proteins, 93 Cyrene, 116 Czech Republic's Ascolor Biotech, 101

D Dairy products, 81 Debranching enzyme, 80

224 Index Degumming process, 152 Deinking process, 161 Delvocid, 76 Deoxyribonuclease, 126 Department of Biotechnology (DBT), 207 Detergent enzymes, 133–137, 133–135t Diaminopimelic acid (DAP), 72 Diaminopimelic decarboxylase (DAP-decarboxylasc), 72 Dihydroxyphenylalanine (DOPA), 131 Dimethylformamide (DMF), 116 Dissolving pulp, 162 Distilling potable alcohol, 82 Dry media reactions, 111 DSM company, 7–8, 198, 199t Dunaliella bardawil, 101 Dunaliella salina, 101 DuPont, 54, 61, 67–68, 115, 198, 199t

E Ecofriendly process, 15–16 Elure, 130–131 Elution, 74 Endotoxins, 93 Enterobacter aerogenes, 72 Environmental Protection Agency (EPA), 94 Enzymatic interesterification, 151–152 Enzymes, 8–9, 50–55, 195–196. See also specific enzymes advantages, 51t in animal nutrition, 84t aroma compounds, 107, 108t in biofuels, 146t, 149–150 in bioremediation, 155, 156f, 157–159t catalysts, 215 in cosmetics, 129t hyaluronidase, 132–133 lipases, 132 peroxidase, 130–131 proteases, 131–132 superoxide dismutase, 130 tyrosinase, 131 in detergents, 133–137, 133–135t fermentation broth and, 39 in food industry, 76–78, 77–78t hydrolysis, 10, 48, 76–79, 148, 162 limitations of, 52–53 nonaqueous medium, 125 in oil and fat industry, 150–152, 150t speciality products, 123–128, 123t specificity, 51, 52t starch modification, 165 therapeutic, 126–127 total market for, 55 types, 53–54t

ERA-NET schemes, 203–204 Escherichia coli, 68, 72, 74, 110 Ethanol biofuels, 143–149, 145t, 146–147f Ethyl lactate, 114 Eukaryotes, 52–53 European biotechnology, 203–204 advantages, 204 competitors, 205 European Union level, 203 major threats to, 205–206 Member States level, 203–204 European Common Agricultural Policy (CAP), 26 Extremophile enzymes, 118, 125

F Farm Security and Rural Investment Act, 7–8 Fat-soluble vitamins, 120–121t Fed-batch fermentation, L-lysine, 71 Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), 89 Feed additives, 82–85 Feed enzymes, 52, 82–83 advantages, 84t modes of action, 85, 86t nonstarch polysaccharides degrading enzymes, 84–85 phytase, 83–84 proteases, 85 type of, 83, 84t Feedstocks, 140 Fermentation, 6, 8–9, 29–30, 38–39, 58, 196 bulk chemicals product groups produced using, 59–60t chemical production, 9 food preservatives produced by, 75–76 glutamic acid, 74 lactic acid, 64–65 L-lysine, 71–72, 72f solid-state fermentation, 44–50, 46–50t speciality products, 118–123, 119t submerged, 39–43, 44t surface, 43–44 Fermentation broth, 39, 43–44, 71 Fertilizers, 86–87, 87t Fiber modification, 162–163 Fibers, 26 flax, 163 Filamentous fungi, 44–45, 48 Fixed bed bioreactor, 40–42f Flavobacterium sp., 101 Flavoring compounds, 105–111, 106t Flax fibers, 163 Fluidized bed bioreactor, 40–42f Fly larvae, types, 94 Food additives, 75–76 baking, 80–81

Index 225

brewing, 81–82 dairy products, 81 distilling potable alcohol, 82 environmental advantages, 76 glucose syrups, 79–80 starch modification, 79–80 Food and Drug Administration, 36 Food, Conservation, and Energy Act, 7–8 Food-grade biocolorants, 100f, 105 Food industry, enzymes in, 76–78, 77–78t Food substances, biocolorants in, 97t Food supplements, 75–82 Forest biomass, types, 147f Fossil fuels, 23–25, 24t cost of, 24, 27t, 28 safety concerns, 25 Fractionation technology, 29–30 Fungal alpha-amylase enzymes, 80–81 Fungi, producing pigment, 98–99t

G Gel permeation chromatography (GPC), 79 Generally Recognized As Safe (GRAS), 61–62, 71 Gene-spliced crops, 198 Genetically engineered microorganisms (GEMs), 17 Genetically modified Escherichia coli, 68, 74, 110 Genetic engineering, 51, 62–63, 127, 209, 215, 218–219 microbial strains, 64 substantial growth in, 219–220 Genetic modification (GM) technology, transgenic plants, 168–171 Genome editing, 218–219 Gevo Development, 115 Global L-Lysine Market 2019 Industry Research Report, 72–73 Global warming, 24–25 Glucoamylase enzymes, 79, 80t Glucose isomerase enzyme, 52 Glucose syrup, 79–80 Glucosyltransferase enzyme, 52 Glutamates, 73 L-Glutamic acid, 73–75 companies producing, 75t structure, 73f Grand View Research, Inc., 10, 62, 66, 83 Green Biologics, 116 Green chemistry, 1, 216 Greenhouse gas emissions, 23, 58, 195, 217 Green Revolution, 17 Green solvents, 111, 114–115 Group specificity of enzymes, 52t Guardzyme, 137 Guar gum, 135

H Haarmann and Reimer (H&R), 108 Haematococcus lacustris, 101 Haematococcus pluvialis, 101 High-performance liquid chromatography (HPLC), 79 Homolactic acid bacteria, 64 Horseradish peroxidase (HRP), 158–159t Hyaluronidase, 126 in cosmetics, 132–133 Hydrogen chloride (HCl) production, 71, 72f Hydrolases, 52 Hydrolytic enzymes, 10, 48, 76–79, 144, 148, 162 Hydroxyl free radical scavenging enzymes, 130

I Immobilized lipases, 132 India, industrial biotechnology, 206–208, 207t Industrial biodegradable solvents, 114t Industrial biotechnology (IB).. See White biotechnology Industrial enzymes, 50–51 application, 123 from biological systems, 50–51 breweries, 78 demand for, 76 in food industry, 76–78 global market for, 55, 208 hydrolases, 52 manufactured by three suppliers, 53 producing low carbohydrate beer, 82 starch industry, 79 using fermentation, 59–60t Industrialization of biology, 1, 2t Industrial Revolution, 25–26 International Renewable Energy Agency (IRENA), 28 Isobutanol production, 115 Isomalto-oligosaccharides (IMOs), 124

J Japan, industrial biotechnology, 206 Jungbunzlauer, 62

K

α-Ketoglutaric acid, 74 Kluyveromyces lactis, 81 Koji fermentation, 44–46

L Laccase enzyme, 52, 158–159t Lactic acid, 64–67 manufacturers companies, 66 purification, 64–65 speciality foods produced by fermentation, 65t structure, 64f uses of, 66t

226 Index Lactic acid bacteria (LAB), 64–65 L-asparaginase, 127 Life science chemicals, 35–36 Lignocellulosic materials, 10, 145 Linkage specificity of enzymes, 52t Lipase, 125 in cosmetics, 132 in detergents, 133t, 136–137 in oil and fat industry, 150–151 organic reactions, 167 Liquefaction process, 79–80 Liquid biofuels, 205 Listeria monocytogenes, 76 Lonza, 10 Lovastatin, 119 Lux Research, 7 Lyases, 167 Lyle Bio Products Company, 68 Lysine, 70–73 growth, 72f hydrogen chloride production, 71, 72f structure, 70f uses of, 71t Lysolecithin, 152 Lysozyme, 126

M Mannanase, 135–136 Mash copper, 81–82 Melanozyme, 130–131 Membrane technology, 19 Microalgae, 117, 120–121, 149 Microbial enzymes in bioremediation, 155, 157t in food industry, 76–78 Microbial fermentation, 62–63 Microbial inoculants, 87 Microbial pesticides, 89–90t, 90, 95 Microbial pigments, 102 Microbial process solid-state fermentation, 45 for vitamin production, 120–121 Microorganisms aroma compounds produced by, 107, 107t biopesticides from, 90t naturally derived colors from, 98t with produces citric acid, 63t producing pigment, 98–99t producing vitamin B12, 120–121, 121t types, 64 Ministry of Micro, Small and Medium Enterprises, 207 Mitsubishi Rayon Co., Ltd., 127, 143 Molecular biology, 218 Monoclonal antibody, 126–127

Monomers, 58–61 Monosodium glutamate (MSG), 73–75 Monsanto, 61 Mucor miehei, 151 Mycorrhizal soil inoculants, 87

N National Biotechnology Board (NBTB), 207 National Institute for Occupational Safety and Health (NIOSH), 111 Natural gas, 23–24 Neurospora crassa, 110 Nitto Chemical Industry Co., 167 N-methyl-2-pyrrolidone (NMP), 116 Nonleguminous plants, 87 Nonrenewable energy, 25. See also Renewable energy Nonstarch polysaccharides (NSP) degrading enzymes, 84–85 Novozymes company, 54, 151, 198, 199t Nozzle sparger, 43t

O Oil and fat industry, enzymes in, 150–152, 150t Oil-based diacids, 7 Oil crisis, 26 Oil reserves, 27 Oil sands, 25 Oil spills, 154–155 Old newsprint (ONP), 161 Oleochemistry, 26 Oncaspar1 (pegaspargase), 126 Organic acids, using fermentation, 59–60t Organic synthesis, 166–167 Organization for Economic Cooperation and Development (OECD) analysis, 7–8, 11, 16 Organization of the Petroleum Exporting Countries (OPEC), 26 Orifice sparger, 43t Oxidoreductases, 167

P Pancreatic enzymes, 134 Papain, 126 Pectinase enzymes, 136, 160 Penicillium oxalicum var. Armeniaca, 101 Performance chemicals.. See Speciality chemicals Peroxidase, 158–159t in cosmetics, 130–131 Personal care products, 66, 128–133 Personal hygiene products, 35 Pesticides, 88. See also Biopesticides biochemical, 89t microbial, 89–90t, 90, 95 substances with, 95, 96t

Index 227

Petrochemical, 9, 58 emergence of, 26 feedstocks, 26, 58, 61, 197–198 production, 34 Phanerochaete chrysosporium, 130–131 Phosphates, in laundry detergent, 6 Phospholipase, 152 Phycomyces blakesleeanus, 101 Phytase, feed enzymes, 83–84, 158–159t Phytic acid, 124 Phytoremediation, 153–155 Pigment bacterial, 98–99t, 101, 102–103t microbial, 102 microorganisms producing, 98–99t yeast, 97, 98–99t, 101 Plant biotechnology, commercial players in, 170t Plant-incorporated protectants, 89t Plants sources, biocolorants from, 97t, 103t Plasmids, 127 Plastic resins, 34 Polychlorinated biphenyl compounds (PCBs), 152 Polyethylene terephthalate (PET), 67, 67f Polylactic acid (PLA), 64–66 Polymers biobased, 138, 219–220 biodegradable, 138–143 using fermentation, 59–60t Polytrimethylene terephthalate (PTT), 67–69, 67f Polyunsatured fatty acids (PUFAs), 151 Porous sparger, 43t The Prevention of Food Adulteration Act (PFA), 105 Propane-1,3-diol (1,3-PDO), 67–69, 67f Protease enzymes animal feed, 124 in cosmetics, 131–132 in detergents, 133t, 135 feed additives, 85 production of cheese, 81 Protein engineering, 51, 76 Proteinogenic amino acid.. See L-Glutamic acid Proteolytic enzymes, 126, 131 Pseudomonas fluorescens, 110 Pulp and paper industry, 159–160 biodebarking, 160 bioremediation, 165–166 bleaching, 160–161 dissolving pulp, 162 enzymatic deinking, 161 enzymatic modification of starch, 165 fiber modification, 162–163 flax fibers, 163 shive removal, 163

slime control, 163–164 stickies, 164–165 Purification methods lactic acid, 64–65 solvents, 111 Pyrococcus furiosa, 52–53

Q Quornis, 75 Quorn mycoprotein, 75

R Raw materials, 2–4 agricultural, 75 renewable (see Renewable raw materials) Recombinant DNA technology, 4 Red biotechnology, 2 Red yeast fermentation, 101 Renewable chemicals, 215 Renewable energy, 23–24. See also Nonrenewable energy investment, 24–25 types of, 24t, 25 Renewable raw materials, 26, 28–30, 29t, 199–200 bulk chemicals, 61 fermentation medium, 39 Renewable resources, 18–19, 23–25, 27 based on biomass use, 28 chemical production from, 4 efficient use of, 8–9, 16, 117 enzymes from, 53 in Europe, 28 world market price, 27t Restricting hosts, 127 Rhizomucor miehei, 151 Rhodococcus, 10 Riboflavin (vitamin B2), 120–121, 197–198 Roche, 54

S Saccharifying enzyme, 79 Saccharomyces, 46 Seasoning salt, 73–74 Semisynthetic antibiotics, 10 Seventh Framework Program, 203 Shive removal, 163 Single-cell protein (SCP), 75, 124–125 Sitagliptin, 125 Slime control, 163–164 Small Industries Development Bank of India (SIDBI), 207 Soaps and detergents, 133–137 Soilborne crop diseases, 88–89, 91–92t Solar panel projects, 24

228 Index Solid-state fermentation (SSF), 44–45, 106–108 advantages, 46–47, 46t aroma compounds, 107–108, 109t Aspergillus oryzae, 46 bacterial, 48 disadvantages, 47t factors affecting enzyme production, 49–50, 50t filamentous fungi, 44–45, 48 microbial pigments, 102 products, 48t traditional, 48t types of, 48–49, 49t Solventless reactions, 111 Solvents, 111–117 biobased, 113t from biomass, 113t in different sectors, 112t industrial biodegradable, 114t organic chemicals as, 112t Sparger, 42–43, 43t Speciality chemicals, 32, 34–35, 117–128, 209 Speciality products, 117–128 applications, 118 enzymes, 123–128, 123t fermentation, 118–123, 119t Specificity of enzymes, 51, 52t SSF. See Solid-state fermentation (SSF) Stakeholders, white biotechnology, 197 Starch modification, 79–80 enzymes, 165 Stauffer Chemical, 75–76 Stereochemical specificity of enzymes, 52t Stickies control technology, 164–165 Streptococcus lactis, 76 Streptomyces natalensis, 76 Submerged fermentation, 39–43 advantages, 44t disadvantages, 44t Substantial energy savings, 195 Superoxide dismutase (SOD), 130 Surface fermentation, 43–44 Sustainability, biotechnology, 15–20 Sustainable chemistry, 18–19 Sustainable development, 15, 17, 199, 217 based on conviction, 16–17 concept of, 15 elements of, 19, 19f, 198 industrial goal, 15–16 Sustainable production process, 16–17 Sweetener production, 79–80 Sweet wort, 81–82 Synthetic biology, 7, 211, 215, 218–219

T Terephthalic acid, 67 Therapeutic enzymes, 126–127 Third wave in biotechnology, 5, 209, 218 Transgenic plants, 168–171, 170t Tray bioreactor, 48–49 Triazine hydrolase, 158–159t Trypsin, 126 Tryptophan, 74 Tyrosinase, 131

U Ultrafiltration, 71 United States Food and Drug Administration (USFDA), 206 United States, industrial biotechnology, 205–206

V Value-added products, 49 Vanillin (4-hydroxy-3-methoxybenzaldehyde), 108, 110 Vitamins microbial process for, 120–121 produced by biotechnological methods, 120, 120–121t using fermentation, 59–60t vitamin B2, 120–121 vitamin B12, 120–121, 121t Volatile organic compounds (VOC), 112–114, 155–157, 158t

W Wageningen Food & Biobased Research, 116 Water-soluble vitamins, 120–121t White biotechnology, 1–2, 4, 195, 217. See also Biotechnology application, 7–8, 217, 219, 219t areas of sustainability, 198 bioprocess in (see Bioprocess) challenges facing, 209–210, 211t chemical production, 218 vs. chemical technology, 212t in China, 206 clear value proposition, 216 community, 209–210 companies for improving manufacturing processes, 199t companies operating in, 19 drivers for, 2 economic impact, 196, 196t environmental impact, 11, 196, 196t enzymes and, 195 in Europe, 203–205 fermentation methods, 6 historical developments in, 3t

Index 229

impact of, 8–9 in India, 206–208, 207t in Japan, 206 products of, 9–11, 215 range of environmental advantages, 205 significant benefits, 199–200, 215 society impact, 197, 197t stakeholders, 197 traditional products, 209 United States, 205–206 value chain, 3f vision of, 8t, 200, 217–218 weakness and problems solutions, 212t Whole-cell processes, 6 Wood, 23 World Commission on Environment and Development, 15

X Xanthomonas campestris, 75 Xanthophyllomyces dendrorhous, 101 Xylanase enzymes fiber modification, 162 pulp bleaching, 160

Y Yeast, 10 baking, 80–81, 110 pigment, 97, 98–99t, 101 red yeast fermentation, 101 Saccharomyces, 46

Z Zymo-tan complex, 131