Valorization of Biomass to Bioproducts: Organic Acids and Biofuels [1 ed.] 0128228881, 9780128228883

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VALORIZATION OF BIOMASS TO BIOPRODUCTS

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VALORIZATION OF BIOMASS TO BIOPRODUCTS Organic Acids and Biofuels Edited by

VIJAI KUMAR GUPTA MARIA TUOHY PRAMOD RAMTEKE QUANG NGUYEN RAJEEV BHAT

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 Copyright © 2023 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. ISBN: 978-0-12-822888-3 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Susan Dennis Editorial Project Manager: Kathrine Esten Production Project Manager: Erragounta Saibabu Rao Cover Designer: Miles Hitchen Typeset by STRAIVE, India

Contents

Contributors

SECTION 1

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Organic acids

1. Propionic acid chemistry and production

3

Ahmed M. Abdel-Azeem, Fatma A. Abo Nouh, Sara A. Gezaf, Amira M.G. Darwish, and Mohamed Ahmed Abdel-Azeem 1. Introduction 2. Physicochemical properties 3. Chemical production of PA 4. Microbial production of PA 5. Fermentative producing pathways of PA 6. Biotechnological methods of PA production 7. Industrial applications of propionic acid References

2. Alpha linolenic acid

3 4 4 5 6 9 11 12

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Neelam Upadhyay, Priya Yawale, and E. Eswari 1. Audience 2. Introduction 3. Techniques adopted for extraction of ALA from bio-wastes 4. Extraction of ALA from potential bio-wastes 5. Unconventional sources for extraction of ALA 6. Conclusion and future prospective References

3. Citric acid

17 17 20 24 28 31 32

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Ramandeep Kaur and Kandi Sridhar 1. 2. 3. 4. 5. 6. 7. 8.

Introduction Biochemistry of citric acid formation Production of citric acid Citric acid fermentation methods Recovery and purification of citric acid Factors affecting citric acid production Applications of citric acid Citric acid global market scenario

37 37 40 43 44 47 52 53

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9. Concluding remarks and future directions Declaration of competing interests References

4. Applications of itaconic acid in biofuel production

55 57 57

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Ahmed M. Abdel-Azeem, Teroj A. Mohamed, Sara A. Gezaf, Fatma A. Abo Nouh, Amira M.G. Darwish, and Hebatallah H. Abo Nahas 1. Introduction 2. Physicochemical properties 3. Historical background 4. Biochemical pathways of IA production 5. IA biosynthesis and recombinant microorganisms 6. IA producing microorganisms and fermentation 7. Industrial applications of IA References

5. Lactic acid

63 64 65 65 67 68 71 73

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Praveen Kumar Dikkala, Barinderjeet Singh Toor, Pradeepa Roberts, Blessy Sagar, Kairam Narsaiah, Srinu Dhanavath, Zeba Usmani, Vijai Kumar Gupta, Rajeev Bhat, and Minaxi Sharma 1. Introduction 2. Biomass feedstocks 3. Enzymatic saccharification of biomass 4. Microbial fermentation 5. Product recovery 6. Technical challenges in LA production 7. Process advancements 8. Conclusions References

6. Valorization of biomass to levulinic acid

79 80 81 82 87 91 94 95 95

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Nazim Hussain, Muhammad Asim Raza Basra, and Aatika Sadia 1. 2. 3. 4. 5. 6. 7. 8.

Introduction Physical and chemical properties of levulinic acid Traditional production of levulinic acid Biomass conversions to levulinic acid Factors affecting levulinic acid yield Industrially important derivatives of levulinic acid Side products during biomass conversions: The humins Conclusion and future outlook

101 102 102 103 103 106 111 113

Contents

Acknowledgments Conflict of interest References

7. Production of pyruvic acid into value-added products using genetically modified microbes

114 114 114

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P. Jeevitha, J. Ranjitha, M. Anand, Shahid Mahboob, and S. Vijayalakshmi 1. Introduction 2. Production of pyruvate using microbial pathway 3. Optimization of media conditions for the production of pyruvate from different microbial strains 4. Various metabolic pathway production of pyruvic acid 5. Co-enzymes enhanced pyruvic acid production 6. Gene-modified microbial strains for pyruvic acid production—Genetic engineering 7. Other biological methods 8. Pyruvate recovery from fermentation processes 9. Conclusions References

SECTION 2

117 118 120 121 125 127 128 128 130 130

Biofuels and bio-oil

8. Microbial production of hydrocarbon and its derivatives using different kinds of microorganisms

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R. Shobana, J. Ranjitha, M. Anand, Shahid Mahboob, and S. Vijayalakshmi 1. Introduction 2. Genetically modified microbes used for the production of alkane/alkene derivatives 3. Using the Fatty acid carboxy-lyases metabolic pathway 4. Production of hydrocarbon and its derivatives using different microbes 5. Metabolic Pathway of polyketide synthase and fatty acids 6. Hydrocarbon derivatives titer, rate, and yield 7. Toxicity of the biosynthetic pathway 8. Future scope of the study 9. Conclusions References

9. Biomass valorization to biobutanol

137 138 140 141 142 142 143 144 146 146

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Tahir Mehmood, Fareeha Nadeem, Bisma Meer, Hajra Ashraf, Kushif Meer, and Shagufta Saeed 1. Introduction 2. History of biobutanol

151 154

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3. Global energy scenarios 4. Lignocellulosic biomass 5. Biomass pre-treatment 6. Acetone-butanol-ethanol (ABE) fermentation 7. Physiochemical properties of biobutanol 8. In-situ product recovery in ABE fermentation 9. Applications of biobutanol 10. Conclusions and future trends References

10. Bioethanol—A promising alternative fuel for sustainable future

155 155 158 161 164 167 172 173 173

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R. Reshmy, Eapen Philip, Rekha Unni, Sherely A. Paul, Raveendran Sindhu, Aravind Madhavan, Ranjna Sirohi, Ashok Pandey, and Parameswaran Binod 1. Introduction 2. Various biomass sources of bioethanol 3. Environmental impact 4. Advantages and disadvantages of bioethanol 5. Statistical analysis of bioethanol production in various countries 6. Conclusion and future prospectives Acknowledgments References

11. Hydrogen production from biomass gasification with carbon capture and storage

179 184 189 190 191 193 194 194

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Daya Shankar Pandey, Sanjay Mukherjee, and Faisal Asfand 1. Introduction 2. Production process 3. Biomass to hydrogen production technology 4. Product gas cleaning 5. Biomass to hydrogen with carbon capture and storage technologies 6. Conclusion References

12. Production of 2,3-butanediol from various microorganisms

197 199 203 214 216 219 220

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P. Jeevitha, J. Ranjitha, M. Anand, Shahid Mahboob, and S. Vijayalakshmi 1. 2. 3. 4. 5.

Introduction Genetically modified microbes in the production of 2,3 butanediol Microbial production of butanediol Several microorganisms are used for the butanediol production Chemical and physical properties butanediol

223 224 231 233 234

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6. Butanediol applications 7. Conclusion References

13. Enhanced anaerobic digestion: Recent advancements and future prospective

234 235 235

241

Neelamegam Annamalai, Sivaramasamy Elayaraja, Piotr Oleskowicz-Popiel, Daochen Zhu, and Channarong Rodkhum 1. Anaerobic digestion 2. Anaerobic system design and technology 3. Benefits and advantages of anaerobic digestion 4. Enhanced anaerobic digestion (EAD) 5. Physical pre-treatments 6. Chemical pre-treatments 7. Biological pre-treatments 8. Thermal pre-treatments 9. Addition of chemical and other materials 10. Full-scale application 11. Economic feasibility 12. Future perspectives Credit authorship contribution statement Acknowledgments References

14. Application of convective heat transfer process in reduction of consumption of alcohol during the biodiesel production process: A theoretical study

241 243 246 247 248 248 249 250 250 251 252 252 253 253 253

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A.V.S.L. Sai Bharadwaj 1. Introduction 2. Biodiesel production process 3. Heat transfer to solution mixture (feedstock + alcohol) 4. Conclusions References Index

257 258 259 260 260 261

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Contributors

Ahmed M. Abdel-Azeem Botany and Microbiology Department, Faculty of Science, Suez Canal University, Ismailia, Egypt Mohamed Ahmed Abdel-Azeem Pharmacognosy Department, Faculty of Pharmacy and Pharmaceutical Industries, University of Sinai, Arish, Egypt Hebatallah H. Abo Nahas Zoology Department, Faculty of Science, Suez Canal University, Ismailia, Egypt Fatma A. Abo Nouh Botany and Microbiology Department, Faculty of Science, Suez Canal University, Ismailia, Egypt M. Anand Department of Pharmacology, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India Neelamegam Annamalai Thayer School of Engineering, Dartmouth College, Hanover, NH, United States Faisal Asfand School of Computing and Engineering, University of Huddersfield, Huddersfield, United Kingdom Hajra Ashraf Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan Muhammad Asim Raza Basra Institute of Chemistry, University of the Punjab, New Campus, Lahore, Pakistan Rajeev Bhat ERA-Chair for Food (By-) Products Valorisation Technologies (VALORTECH), Estonian University of Life Sciences, Tartu, Estonia Parameswaran Binod Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Trivandrum, Kerala, India Amira M.G. Darwish Department of Food Technology, Arid Lands Cultivation Research Institute (ALCRI); Department of Food Science, Faculty of Agriculture, Saba Basha, Alexandria University, Alexandria, Egypt Srinu Dhanavath College of Food and Dairy Technology, TANUVAS, Chennai, India

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Praveen Kumar Dikkala Department of Food science and Technology, Punjab Agricultural University, Ludhiana, Punjab, India Sivaramasamy Elayaraja Centre of Excellence in Fish Infectious Diseases (CE FID), Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand E. Eswari Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India Sara A. Gezaf Botany Department, Faculty of Science, Al-Arish University, Arish, North Sinai, Egypt Vijai Kumar Gupta Biorefining and Advanced Materials Research Center; Center for Safe and Improved Food, SRUC, Kings’s Buildings, Edinburgh, United Kingdom Nazim Hussain Center for Applied Molecular Biology (CAMB), University of the Punjab, Lahore, Pakistan P. Jeevitha CO2 Research and Green Technologies Centre, Vellore Institute of Technology, Vellore, India Ramandeep Kaur Department of Food Technology, Eternal University, Baru Sahib, Himachal Pradesh, India Aravind Madhavan School of Biotechnology, Amrita VishwaVidyapeetham, Amritapuri, Kerala, India Shahid Mahboob Department of Zoology, Kingdom of Saudi Arabia, King Saud University, Riyadh, Saudi Arabia Bisma Meer Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan Kushif Meer Institute of Chemistry, University of the Punjab, Lahore, Pakistan Tahir Mehmood Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences; Centre for Applied Molecular Biology (CAMB), University of the Punjab, Lahore, Pakistan Teroj A. Mohamed Department of Dental Basic Sciences, College of Dentistry, University of Duhok, Duhok, Iraq Sanjay Mukherjee Infrastructure and Engineering, Energy Systems Catapult, Birmingham, United Kingdom Fareeha Nadeem Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan Kairam Narsaiah AS & EC Division, ICAR-CIPHET, Ludhiana, India

Contributors

Piotr Oleskowicz-Popiel Water Supply and Bioeconomy Division, Faculty of Environmental Engineering and Energy, Poznan University of Technology, Poznan, Poland Ashok Pandey The Center for Energy and Environmental Sustainability, Lucknow, Uttar Pradesh; Centre for Innovation and Translational Research, CSIR-Indian Institute for Toxicology Research (CSIR-IITR), Lucknow, India Daya Shankar Pandey School of Computing and Engineering, University of Huddersfield, Huddersfield, United Kingdom Sherely A. Paul Post Graduate and Research Department of Chemistry, Bishop Moore College, Mavelikara, Kerala, India Eapen Philip Post Graduate and Research Department of Chemistry, Bishop Moore College, Mavelikara, Kerala, India J. Ranjitha CO2 Research and Green Technologies Centre, Vellore Institute of Technology, Vellore, India R. Reshmy Post Graduate and Research Department of Chemistry, Bishop Moore College, Mavelikara; Department of Science and Humanities, Providence College of Engineering, Chengannur, Kerala, India Pradeepa Roberts Millet Processing and Incubation Centre, PJT Agricultural University, Hyderabad, India Channarong Rodkhum Centre of Excellence in Fish Infectious Diseases (CE FID), Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand Aatika Sadia Institute of Chemistry, University of the Punjab, New Campus, Lahore, Pakistan Shagufta Saeed Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan Blessy Sagar Department of Food process Engineering, Acharya N.G. Ranga Agricultural University, Bapatla, India A.V.S.L. Sai Bharadwaj Department of Chemical Engineering, Indian Institute of Technology-Madras, Chennai, India Minaxi Sharma Department of Applied Biology, University of Science and Technology, Baridua, Meghalaya, India

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R. Shobana CO2 Research and Green Technologies Centre, Vellore Institute of Technology, Vellore, India Raveendran Sindhu Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (CSIR-NIIST), Trivandrum; Department of Food Technology, TKM Institute of Technology, Kollam, Kerala, India Ranjna Sirohi The Center for Energy and Environmental Sustainability, Lucknow, Uttar Pradesh, India Kandi Sridhar Department of Food Science, Fu Jen Catholic University, New Taipei City, Taiwan Barinderjeet Singh Toor Department of Food science and Technology, Punjab Agricultural University, Ludhiana, Punjab, India Rekha Unni Department of Chemistry, Christian College, Chengannur, Kerala, India Neelam Upadhyay Dairy Technology Division; Krishi Vigyan Kendra, ICAR-National Dairy Research Institute, Karnal, Haryana, India Zeba Usmani Department of Applied Biology, University of Science and Technology, Baridua, Meghalaya, India S. Vijayalakshmi CO2 Research and Green Technologies Centre, Vellore Institute of Technology, Vellore, India Priya Yawale Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India Daochen Zhu Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, China

SECTION 1

Organic acids

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CHAPTER 1

Propionic acid chemistry and production Ahmed M. Abdel-Azeema, Fatma A. Abo Nouha, Sara A. Gezafb, Amira M.G. Darwishc,d, and Mohamed Ahmed Abdel-Azeeme a

Botany and Microbiology Department, Faculty of Science, Suez Canal University, Ismailia, Egypt Botany Department, Faculty of Science, Al-Arish University, Arish, North Sinai, Egypt c Department of Food Technology, Arid Lands Cultivation Research Institute (ALCRI), Alexandria, Egypt d Department of Food Science, Faculty of Agriculture, Saba Basha, Alexandria University, Alexandria, Egypt e Pharmacognosy Department, Faculty of Pharmacy and Pharmaceutical Industries, University of Sinai, Arish, Egypt b

1. Introduction Propionic acid (PA) is generally regarded as safe (GRAS) by the Food and Drug Administration (FDA). It is a C-3 platform chemical with applications in a wide variety of industries. PA is primarily used for its antimicrobial properties, with major markets as a food preservative and herbicide (Zidwick et al., 2013). PA and its derivatives are used in the agriculture, food, and pharmaceutical industries: e.g., PA is an important chemical intermediate in the synthesis of herbicides, perfumes, cellulose fibers, and pharmaceuticals. As a C-3 building block, it is used as a precursor for high-volume commodity chemicals such as propylene (Stowers et al., 2014). PA and its calcium, sodium, and potassium salts are widely used as preservatives in animal feed and human food (Liu et al., 2015). The most common way to produce PA is via nonsustainable petrochemical routes, e.g., the Reppe, Larson, and Fischer-Tropsch processes. However, because of growing concerns about greenhouse gas emissions and sustainability, the chemical industry is transitioning from traditional fossil resources to renewable ones (Bozell, 2008). Thus biotechnological production of PA from renewable bioresources has recently attracted increasing interest because of the problems associated with rising oil prices and the benefits of ecofriendly production (Zhang and Yang, 2009). Therefore the production of PA using novel biological methods must be reconsidered, to provide more sustainable production. Propionibacterium, which is classified as an anaerobic bacterium, is the most commonly used bacterium in PA production; it is gram-positive, nonmotile, non-spore forming, and rod-shaped (Zhu et al., 2010). Multiple feedstocks have been explored in PA fermentation, including whey permeate (Yang et al., 1994), hydrolyzed corn meal (Paik and Glatz, 1994), corn steep liquor (CSL) (Ozadali et al., 1996), glycerol (Dishisha et al., 2012), and Jerusalem artichoke tuber hydrolysate (Liang et al., 2012). In PA production from lignocellulosic biomass, enzymatically hydrolyzed aspen (Ramsay et al., 1998),

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corncob molasses (Liu et al., 2012), and sugarcane bagasse hydrolysate (Zhu et al., 2012) have been used as substrates. However, biobased PA is more expensive than that synthesized from chemical routes because of the low PA concentration and levels of productivity, caused partly by product inhibition. The produced byproducts, e.g., acetic and succinic acids, make downstream processing more complicated and costly in terms of product recovery and purification, but there is heightened interest in biologically synthesized PA (Guan et al., 2014). In 2014, the commercial price of chemically produced pure PA was $1 USD/kg, while biologically produced PA was $2 USD/kg (Liu et al., 2012) and this price varied depending on the purity of PA and the region of sale (Stowers et al., 2014). Global production of PA was estimated at 450,000 tonnes per year with a 2.7% annual growth and a price ranging between $2–$3 USD/kg. Four manufacturers supply 90% of the global propionate market: BASF covers approximately 31% of the market with plants in Germany and China; Dow Chemical Company supplies 25% of the global market with production in the United States; Eastman Chemical provides 20% of the market with production in the United States; and Perstorp in Sweden supplies 14% of the global market (Gonzalez-Garcia et al., 2017). The cost of synthetic propionic acid is 1000 USD/1000 kg, while 1000 kg of natural propionic acid produced by Propionibacterium bacteria costs 2000 USD (Piwowarek et al., 2021).

2. Physicochemical properties PA has physical properties that are intermediate between the smaller carboxylic, formic, and acetic acids and the larger fatty acids (Ahmadi et al., 2017). PA possesses the common properties of carboxylic acids and can form amide, ester, anhydride, and chloride compounds (Neronova et al., 1967). This acid is a colorless, corrosive, and naturally occurring organic liquid acid with a rancid, pungent, disagreeable, and sharp odor with a moderate cheese-like taste (Es¸ et al., 2017). It is water soluble, but the addition of salts leads to separation from water. It is also soluble in ether, alcohol, and chloroform and can react with alcohols, esters, and organic salts (Goldberg and Rokem, 2009). PA can be detected using paper or thin-layer chromatography (TLC) (Luck and Jager, 2012). Its quantitative determination can be represented using GC or HPLC (Rodriguez et al., 2014). Some of the physical and chemical properties of propionic acid are summarized in Table 1.

3. Chemical production of PA PA is chemically produced using various methods. The first was the Reppe process, which produces a wide range of aliphatic compounds using ethylene, CO, and steam, for the production of PA. Another common method is the Larson process, in which PA is produced from ethanol and CO using BF3 as catalyst (Wang et al., 2015). PA can also be obtained by oxidation of propionaldehyde as a byproduct of the Fischer-Tropsch process in the synthesis of fuel (Boyaval et al., 1987). In general, these

Propionic acid chemistry and production

Table 1 Physical and chemical properties of propionic acid. Chemical and physical properties

IUPAC name Other name Chemical formula CAS number Molar mass Melting point Boiling point Density Solubility in water Acidity Viscosity Median lethal dose (LD50)

Propionic acid Ethanecarboxylic acid C3H6O2 or CH3CH2COOH 79-09-4 74.08 g/mol 21°C 141°C 0.99 g/cm3 Miscible 4.87 10 mPa s 1370 mg kg 1 (white mice/oral administration)

pathways rely on nonrenewable petrochemical feedstock that causes environmental pollution, which is a globally serious issue. The primary advantage of chemical process production is the low cost of the chemical synthesis of PA, but there are many disadvantages of these methods, such as the use of complex catalysts, toxic reagents, high energy consumption, and the consequent pollution. Therefore microbial production of PA using agricultural and industrial wastes has attracted considerable attention, particularly due to its use of environmentally friendly processes (Zhuge et al., 2014).

4. Microbial production of PA PA is produced via fermentation by several gram-positive propionibacteria species, e.g., Propionibacterium acidipropionici, Propionibacterium thoenii, Propionibacterium jensenii, Propionibacterium shermanii, Propionibacterium beijingense, and Propionibacterium freudenreichii, and also other gram-positive (Clostridium spp.) and gram-negative bacteria (Veillonella spp., Fusobacterium spp., and Selenomonas ruminantium) (Feng et al., 2011; Zhu et al., 2012). First described by Albert Fitz (1878), Propionibacterium species can ferment sugars into PA as their main fermentation product. Later on, Swick and Wood (1960) described the set of reactions involved in the process of propionate production currently known as the Wood-Werkman cycle. Lignocellulose is a low-cost renewable source of fermentable sugars, mainly glucose, but also xylose and arabinose, which can be microbially converted to value-added products, such as biofuels and organic acids. Among the approaches employed to reduce the cost of biosynthesis of PA is identifying a low-cost renewable feedstock. Glycerol, an abundant renewable byproduct of the biodiesel industry, has been utilized in propionic acid fermentation (Barbirato et al., 1997; Himmi et al., 2000). Metabolic pathways leading to the production of PA can be classified into three categories: (1) primary

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fermentation pathways that catabolize different carbon sources to propionate and include the well-known acrylate and Wood-Werkman cycle pathways of native propionate producers; (2) catabolic pathways that can degrade a number of amino acids to propionic acid; (3) anabolic pathways associated with the production of biomass precursors from pyruvate or carbon dioxide that can be harnessed to produce propionate (GonzalezGarcia et al., 2017). Unfortunately, PA fermentation suffers from end-product inhibition and byproduct formation, e.g., acetic and succinic acids, which lower the yield and productivity of PA ( Jin and Yang, 1998). To overcome these limitations, various bioprocessing approaches have been applied (Wang et al., 2014, 2012), including metabolic engineering to enhance the producer strains, but genetic manipulation of propionibacteria has proven to be difficult (Ammar et al., 2014, 2013). PA fermentation is still not economically competitive with petrochemical methods and hence further improvements are needed.

5. Fermentative producing pathways of PA Three different biochemical pathways are known for the production of propionic acid: the propanediol pathway, the succinate pathway, and the acrylate pathway. Compared to the amino acid degradation and biosynthetic pathways, fermentative pathways provide energy and help to consume reduced cofactors that result from the catabolism of sugars (Louis et al., 2014).

5.1 Propanediol pathway Many different bacteria are known to produce 1,2-propanediol from fucose, rhamnose, or lactate. But in some bacteria 1,2-propanediol can be further metabolized to propionate or propanol, e.g., Salmonella typhimurium and Roseburia inulinivorans (Reichardt et al., 2014). Fucose is converted to 1,2-propanediol through L-fucose, L-fuculose, fuculose 1-phosphate, and L-lactaldehyde. The enzymes are L-fucose isomerase, L-fuculokinase, L-fuculose phosphate aldolase, and propanediol oxidoreductase. Then 1,2-propanediol is converted to propionaldehyde by propanediol dehydratase. Secondly, propionaldehyde is catabolized to propionyl-CoA and propanol by propionaldehyde dehydrogenase and propanol dehydrogenase, respectively. Finally, propionyl-CoA is converted to propionylphosphate and then to propionate by phosphotransacylase and propionate kinase (Scott et al., 2006). This pathway provides a source of ATP and carbon compounds that can be diverted to central metabolism via known pathways (Fig. 1).

5.2 Succinate pathway The succinate pathway, alias dicarboxylic acid pathway, follows glycolysis, which converts glucose and glycerol into pyruvate. Pyruvate is converted into oxaloacetate (OAA)

Propionic acid chemistry and production

Fig. 1 Propanediol pathway for L-fucose conversion to propionate via 1,2-propanediol.

by OAA transcarboxylase and propionate is generated through several intermediates, including malate, succinyl-CoA, and propionyl-CoA (Liu et al., 2016). Bacteroidetes, several Firmicutes, and some gram-negative bacteria use the succinate pathway via methylmalonyl-CoA for propionate production. While Bacteroidetes mainly utilize polysaccharides and peptides as substrates of this metabolic pathway, strains belonging to Firmicutes use organic acids to produce propionate (Marchandin et al., 2010). In these processes there are two key enzymes that catalyze multiple reactions: oxaloacetate transcarboxylase, which transfers the carboxyl group from methylmalonyl-CoA to pyruvate with the simultaneous formation of oxaloacetate and propionyl-CoA, and CoA transferase, which reversibly transfers the CoA part from propionyl-CoA to succinate to form succinyl-CoA together with the end product propionate (Parizzi et al., 2012) (Fig. 2).

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Fig. 2 Succinate pathway from glycerol to propionic acid proceeds from glycerol to dihydroxyacetone (DHA) to dihydroxyacetone phosphate (DHAP) to phosphoenolpyruvate (PEP) to pyruvate and oxaloacetate and from oxaloacetate to malate, fumarate, succinate, succinyl coenzyme A (CoA), methylmalonyl CoA, propionyl CoA, and propionate.

5.3 Acrylate pathway The third propionate-forming pathway is called the acrylate pathway (Fig. 3). The acrylate pathway for propionate production makes use of Clostridium propionicum (Kandasamy et al., 2013), Coprococcus catus (Flint et al., 2015), Clostridium homopropionicum (Seeliger et al., 2002), Megasphaera elsdenii (Prabhu et al., 2012), and Prevotella ruminicola (Hosseini et al., 2011). In this process lactate is converted to propionate (Flint et al., 2015). This reduction process allows the cell to balance the anaerobic oxidation of lactate to acetate and carbon dioxide, which appears to be the primary source of ATP generation. Key steps of the pathway are catalyzed by the following enzymes sequentially: propionyl-CoA transferase, lactyl-CoA dehydratase, and acrylyl-CoA reductase. Propionyl-CoA transferase typically catalyzes the interconversion of DL-lactate/ DL-lactyl-CoA and propionate/propionyl-CoA translocations. This CoA-transferase catalyzes the transfer of the CoA moiety from propionyl-CoA to lactate. Lactyl-CoA dehydratase converts lactyl-CoA to acrylyl-CoA. Propionyl-CoA is generated from acrylyl-CoA by acrylyl-CoA reductase (Wang et al., 2013).

Propionic acid chemistry and production

Fig. 3 Acrylate pathway for the metabolism of lactate into propionate.

6. Biotechnological methods of PA production Biobased PA processes are limited because of their low levels of productivity, yield, and final propionic acid concentration, caused by strong end-product inhibition. Currently, production of PA via petrochemical routes is more economical than applying biotechnological methods. For economical biotechnological production the process requires higher levels of productivity from the use of various technologies, including genetic engineering, which provides new tools for generating mutants with improved fermentation capabilities (Kiatpapan and Murooka, 2002; Zhang and Yang, 2009), co-culture (Sabra et al., 2013), and biocatalyst immobilization (Es et al., 2015).

6.1 Genetic and metabolic engineering for propionic acid production Propionibacteria metabolic-engineering mutants provide increased productivity and yield in terms of PA production as well as enhanced tolerance for high concentrations of the acid. Hence, advanced genetic engineering has become a routine technique to generate mutants with improved fermentation capacities, enhanced resistance to final product inhibition, and significant viability during long fermentation times. In 1990, the first endogenous plasmids from propionibacteria were characterized (Rehberger and Glatz, 1990). The number of studies is increasing on the synthesis of genetically engineered mutants with the capability to tolerate an acidic environment that could produce PA in higher concentrations (Suwannakham et al., 2006). Shuttle vectors represent the systems designed to generate genetically engineered propionibacteria, e.g., Propionibacterium-Escherichia coli, to improve the production yield; the undesired influence of pH on PA production could also be moderated. In one study, the shuttle vector was designed using an endogenous plasmid, pZGX01, isolated from P. acidipropionici ATCC 4875, a pUC18 plasmid from E. coli, and a chloramphenicol resistance-encoding gene

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(Zhuge et al., 2013). The innovative developments in genetic manipulation systems of propionibacteria have offered more efficient PA production (Kandasamy et al., 2013). The number of studies related to metabolic engineering for propionic acid production is continually increasing. Cloning the gene encoding phosphoenolpyruvate carboxylase (PPC) from E. coli to express in Propionibacterium freudenreichii could be an applicable approach to improving propionic acid productivity. PPC catalyzes the conversion of phosphoenolpyruvate to oxaloacetate with the fixation of one CO2, and its expression showed major effects on PA fermentation (Ammar et al., 2014). Compared to the wild type, the mutant bacteria grew more rapidly and consequently consumed the substrate more efficiently, resulting in higher propionic acid production.

6.2 Co-culture A co-culture is cell cultivation in which different cell populations are cultured together with a certain degree of interaction (Goers et al., 2014). Although PA fermentation processes are mainly focused on monoculture, recently the production of PA using co-culture systems has been well developed. The co-culture systems for PA production could act as a suitable alternative to the production of PA by monoculture with low productivity and yield (Sabra et al., 2013). The cultivation of PA-producing bacteria (PAB) in the presence of lactic acid bacteria (LAB) is a typical application for PA production based on strong commensalism (Smid and Lacroix, 2013). The lactic acid produced by LAB serves as a substrate that can be consumed by PAB to produce PA as well as acetic acid. In a study, Lactobacillus acidophilus and Propionibacterium shermanii resulted in a significant increase in PA acid yield in comparison with its production using P. shermanii as a monoculture (Liu and Moon, 1982). The co-culture also increased the growth rate of both microorganisms (from 0.24 to 0.37 h 1 for L. acidophilus and from 0.12 to 0.37 h 1 for P. shermanii) (Baer and Ryba, 1995). In a study by Sabra et al. (2013), they used a co-culture of Lactobacillus zeae (DSMZ 20178) and Veillonella criceti (DSMZ 20734); the highest productivity (0.33 g L 1 h 1) was achieved with free cells using flour hydrolysate as a carbon source.

6.3 Biocatalyst immobilization and bioreactor design Immobilization is considered to be one of the most useful and powerful tools applied to improve the overall process efficacy. It becomes necessary to use an immobilization technique in combination with bioreactor systems, such as stirred tank reactors, bedadsorption bioreactors, fibrous-bed bioreactors, and packed-bed bioreactors, in order to obtain higher volumetric productivity and reduce the demand for complex nitrogen sources. Although it is an expensive part of fermentation, it produces fewer byproducts, facilitates separation and purification of biocatalysts, and provides the recovery of biocatalysts for reuse (Es et al., 2015). However, the high cost of materials used for

Propionic acid chemistry and production

immobilization could limit the economic production of PA at an industrial scale (Feng et al., 2011). The number of investigations on whole-cell immobilization has dramatically increased (Dishisha et al., 2012). For instance, techniques such as physical adsorption, entrapment, and encapsulation have been introduced to immobilize different biocatalysts. However, entrapment techniques do not seem suitable for PA production due to low mechanical stability, especially when the production is performed in continuously stirred bioreactor systems (Es et al., 2016). In general, the fibrous-bed immobilized cell bioreactor can be regarded as a promising system, with the ability of significantly improving volumetric productivity, product yield, and final PA concentration. By using the fibrous-bed bioreactor as an effective immobilizing system, the high cost of PA production could be lowered, since the system could provide more efficient use of support materials (Zhu et al., 2012). Besides engineering, the use of immobilization materials has become an alternative solution to provide additional protection for propionibacteria against organic acids. Support materials, such as calcium alginate or calcium polygalacturonate beads, offer a certain protection (Coronado et al., 2001).

7. Industrial applications of propionic acid PA, which occurs naturally in grains, apples, strawberries, cheese, and human sweat, is utilized as a solvent (alkyl propionate esters) (Playne and Moo-Young, 1985; Boyaval and Corre, 1995), and is important in the industrialization of several chemicals, such as production of herbicides, pharmaceuticals, polymers (e.g., acrylonitrile cellulose fiber and modification of carbide slag), perfumes, animal feeds and grain, and food preservatives.

7.1 Herbicides (for the synthesis of sodium 2, 2-dichloropropionate) Modern agriculture makes use of a large group of herbicides to eliminate harmful organisms. However, these herbicides may also affect the beneficial activities of certain organisms that grow on the crops. Thus it is important to use biodegradable, target-specific agents; derivatives of PA are promising herbicides to avoid agricultural losses (Thompson and Baker, 2012). PA does not cause any health hazards during application when suitable formulations and respiratory protection are used, and it does not cause any threat to the environment, as it degrades first to formic acid and acetic acid, and then to carbon dioxide and water. It is less caustic and corrosive than formic acid, another common herbicide (Es¸ et al., 2017).

7.2 Chemical intermediate PA serves as a chemical intermediate in the manufacture of plasticizers, textiles, plastics, rubber auxiliaries, and dyes (Nelson et al., 2017).

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7.3 Preservative and safe food additive PA is used as a preservative due to its ability to prevent the growth of various fungi, along with its antibacterial properties. It is also used as a flavoring agent in different types of packaged foods, including cheese and baked goods like tortillas and bread. It is a generally recognized as safe (GRAS) food preservative (Ranaei et al., 2020).

7.4 Animal feed and grain PA is also used in animal food preservation and in grains such as corn, oats, wheat, sorghum, and barley. It has the ability to inhibit growth of various microorganisms like Aspergillus flavus, aerobic Bacillus spp., Salmonella spp., and yeast (Huang et al., 2011; Ranaei et al., 2020).

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Ozadali, F., Glatz, B.A., Glatz, C.E., 1996. Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production by Propionibacterium acidipropionici. Appl. Microbiol. Biotechnol. 44, 710–716. Paik, H.D., Glatz, B.A., 1994. Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici. Appl. Microbiol. Biotechnol. 42, 22–27. Parizzi, L.P., Grassi, M.C.B., Llerena, L.A., Carazzolle, M.F., Queiroz, V.L., Lunardi, I., Zeidler, A.F., Teixeira, P.J.P.L., Mieczkowski, P., Rincones, J., Pereira, G.A.G., 2012. The genome sequence of Propionibacterium acidipropionici provides insights into its biotechnological and industrial potential. BMC Genomics 13 (562), 1–20. Piwowarek, K, Lipi
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Wang, P., Jiao, Y., Liu, S., 2014. Novel fermentation process strengthening strategy for production of propionic acid and vitamin B12 by Propionibacterium freudenreichii. J. Ind. Microbiol. Biotechnol. 41 (12), 1811–1815. Wang, Z., Ammar, E.M., Zhang, A., Wang, L., Lin, M., Yang, S.T., 2015. Engineering Propionibacterium freudenreichii subsp. shermanii for enhanced propionic acid fermentation: effects of overexpressing propionyl-CoA:succinate CoA transferase. Metab. Eng. 27, 46–56. Yang, S.T., Zhu, H., Li, Y., Hong, G., 1994. Continuous propionate production from whey permeate using a novel fibrous bed bioreactor. Biotechnol. Bioeng. 43, 1124–1130. Zhang, A., Yang, S.T., 2009. Propionic acid production from glycerol by metabolically engineered Propionibacterium acidipropionici. Process Biochem. 44 (12), 1346–1351. Zhu, Y., Li, J., Tan, M., Liu, L., Jiang, L., Sun, J., Lee, P., Du, G., Chen, J., 2010. Optimization and scale-up of propionic acid production by propionic acid-tolerant Propionibacterium acidipropionici with glycerol as the carbon source. Bioresour. Technol. 101, 8902–8906. Zhu, L., Wei, P., Cai, J., Zhu, X., Wang, Z., Huang, L., Xu, Z., 2012. Improving the productivity of propionic acid with FBB immobilized cells of an adapted acid-tolerant Propionibacterium acidipropionici. Bioresour. Technol. 112, 248–253. Zhuge, X., Liu, L., Shin, H.D., Chen, R.R., Li, J., Du, G., Chen, J., 2013. Development of a Propionibacterium-Escherichia coli shuttle vector as a useful tool for metabolic engineering of Propionibacterium jensenii, an efficient producer of propionic acid. Appl. Environ. Microbiol. 79, 4595–4602. Zhuge, X., Liu, L., Shin, H.D., Li, J., Du, G., Chen, J., 2014. Improved propionic acid production from glycerol with metabolically engineered Propionibacterium jensenii by integrating fed-batch culture with a pH-shift control strategy. Bioresour. Technol. 152, 519–525. Zidwick, M.J., Chen, J.S., Rogers, P., 2013. Organic acid and solvent production: propionic and butyric acids and ethanol. In: The Prokaryotes. Springer, Berlin/Heidelberg, Germany, pp. 135–167.

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CHAPTER 2

Alpha linolenic acid Neelam Upadhyaya,b,*, Priya Yawalea,*, and E. Eswaria,* a

Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal, Haryana, India Krishi Vigyan Kendra, ICAR-National Dairy Research Institute, Karnal, Haryana, India

b

1. Audience This book chapter is intended for academicians, researchers, and students.

2. Introduction A massive amount of organic biodegradable solids, semi-solids, and/or fluid-like consistency waste is generated by the agro-food processing industry. It has been reported that beverage, dairy, fruit/vegetable production and processing, cereal processing and manufacturing, meat product processing and preservation, oil extraction and processing, fish processing and preservation industry generate approximately 26%, 21%, 14.8%, 12.9%, 8%, 3.9%, and 0.4% of waste, respectively (Baiano, 2014). The main objective of technologies dealing with waste management until the 1990s was the treatment of residues in effluent treatment plants for landfill, storage, and/or sorting. Thereafter, the concern of government bodies and socioeconomic apprehensions laid to research studies dealing with direct recycling of residues or wastes and/or extraction of valuable components from the waste for their valorization. This change in the trend is possibly due to the increase in emanation of greenhouse gases, the threat of exhaustion of natural resources, and the necessity for sustainable development (https://www.springer.com/ journal/12649, n.d.). In this context, the global sustainable development goals (SDGs) were formulated by world leaders and adopted by UN members in 2015. Four goals under SDG, i.e., 2, 3, 12 and 13, directly or indirectly focus on endorsing nutrition as well as plummeting waste through reprocessing and recycling. The present chapter aims at deliberating the valorization of bio-waste by extracting valuable alpha-linolenic acid (ALA) from a plethora of bio-waste sources. The chapter is divided into different sections and sub-sections and will sequentially discuss ALA, its importance in human health, the sources of ALA, and its extraction from bio-waste. ALA, or all-cis-9,12,15-octadecatrienoic acid, is an omega-3, plant-based, essential fatty acid (EFA) containing 18 carbon atoms and 3 double bonds. It can be represented ⁎

All authors contributed equally in the book chapter.

Valorization of Biomass to Bioproducts https://doi.org/10.1016/B978-0-12-822888-3.00005-0

Copyright © 2023 Elsevier Inc. All rights reserved.

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using delta and omega nomenclature. In terms of delta nomenclature, it is written as 18:3 Δ9,12,15 where 18 represents the number of carbon atoms in fatty acid (FA), 3 indicates the number of double bonds, Δ indicates that the counting of double bonds is being done from the carboxylic acid group, while 9, 12, and 15 are the locations of double bonds. In terms of omega nomenclature, it is written as 18:3 ω-3 or n-3 where 18 represents the number of carbon atoms in FA, 3 represents the number of double bonds, ω-3 or n-3 represents that the first double bond is at the 3rd carbon when numbering is done from the methyl end of the FA (Fig. 1). ALA is considered an EFA as it cannot be synthesized de novo by our body and is required to be taken from the diet on account of numerous important functions it performs in the human body. The common sources of ALA include walnuts, rapeseed (canola), several legumes, flaxseed, avocados, Spanish sage (chia seed), and garden cress oils. It is also present in green leafy vegetables where it is located in chloroplasts (Gebauer et al., 2006). Turpeinen and Merimaa (2011) reported that the amount of ALA present varies with the types of seed oil, such as perilla (58%), linseed (55%), sea buckthorn (32%), rapeseed (10%), and soya bean (8%). In general, omega-3 FAs are reported to promote brain health during pregnancy and early life of a new born baby, improve heart and mental health, reduce the symptoms of attentiondeficit hyperactivity disorder (ADHD) in children and metabolic syndrome, fight against inflammation, auto-immune disease, and age-related diseases like Alzheimer’s disease, prevent cancer, reduce asthma in children, reduce fat in the liver, improves bone and joint health, alleviate menstrual pain, improve sleep, and improve skin health (Hjalmarsdottir, 2018). These also help fight against anxiety and depression (Hjalmarsdottir, 2018; Grosso et al., 2014). ALA is a potential nutraceutical as it protects the brain from stroke, characterized by its pleiotropic effects in neuroprotection, vasodilation of brain arteries, and neuroplasticity. It may lower blood pressure by acting as a precursor for eicosanoids, which can generate prostaglandins and leukotrienes that may reduce vascular tone (Takeuchi et al., 2007). It has been reported that an increase in 1% ALA intake was associated with a 39% reduction in the risk of myocardial infarction (Turpeinen and Merimaa, 2011; Ascherio et al., 1996). The health benefits of ALA are given in Fig. 2. Further, Holman (1998) reported that ALA is the

Fig. 1 Delta and omega nomenclature of ALA.

Alpha linolenic acid

Fig. 2 Health benefits of ALA.

precursor of three other important FAs in the body, namely, eicosapentaenoic acid (EPA, C20:5), docosapentaenoic acid (DPA, C22:5), and docosahexaenoic acid (DHA, C22:6). This conversion is facilitated by the action of desaturase and elongase in the body (Fig. 3). However, conversion of ALA to EPA and DHA takes place at a lower rate; therefore, these FAs are generally termed as conditionally essential and are required to be taken through the diet (Blondeau et al., 2015). On an average, 107 kg per year per person (kg/p/y) of food waste are generated in developed countries, while 56 kg/p/y are generated in developing countries (Thi et al., 2015). Owing to the generation of a huge amount of waste and the beneficial health effects of ALA, a plethora of research is being initiated on the extraction of this high-value functional ingredient from a number of bio-waste generated by agro-food industries. Further, the global market for natural FAs is rising. It was reported to be US$13.5 billion in 2018 and is estimated to elevate to US$17.5 billion by 2023 (BCC Research, 2019). To counterbalance the waste issue, administer environmental sustainability and overcome the economic development model “take, make, and dispose,” the concept of circular economy has been introduced by the application of sustainable and profitable technologies to utilize by-products (Maina et al., 2017). The application of valorization strategies, relying on circular economy models, or bio-refinery approaches could also be used for the abstraction of ALA. The advantages and disadvantages of valorization of biomass for production and extraction of ALA are shown in Fig. 4, while extraction techniques and sources of waste for extraction of ALA are discussed in subsequent sections.

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Fig. 3 ALA as precursor of important fatty acids.

3. Techniques adopted for extraction of ALA from bio-wastes There is a rapid development in the field of extraction of crude polyunsaturated fatty acids (PUFAs) from bio-materials (Medina et al., 1998). The extraction of ALA and purification method mainly includes molecular distillation, low-temperature crystallization, column chromatography, silver ion complexation and urea inclusion, supercritical fluid extraction, ultrasonic extraction using solvent, imidazolium-based ionic liquids (ILs) having silver tetrafluoroborate extraction, etc. (Yan et al., 2014; Ni, 2017).

3.1 Molecular distillation Molecular distillation is liquid-liquid extraction which separates the liquids like FAs based on the difference in their molecular weight and mean free path, particularly at a temperature of lower boiling point (Martins et al., 2006). In spite of the simple operation of the technique, it provides high separation efficiency (Lutisˇan et al., 2002). However, the demands and requirements of the equipment are too high to be applied in mass industrialized production. This method of purifying ALA has been adopted by several workers. Chen et al. (2013) carried out purification of ALA from linseed oil by molecular distillation and reported a recovery rate of 76.20%, while its content increased from 53.36% to

Alpha linolenic acid

Fig. 4 Advantages and disadvantages of extraction of ALA.

80.27% under the optimized conditions. The ideal conditions for purification of ALA were optimized at distillation temperature, wiper speed, preheating temperature, and feeding intensity of 90°C, 235 r/min, 70°C, and 0.9 mL/min, respectively. Similarly, Huang et al. (2016) purified ALA after optimizing the procedure for Fructus perillae oil and revealed a final ALA mass fraction of 86.04%.

3.2 Low-temperature crystallization The low-temperature crystallization technique involves dissolving a mixture of fatty material in organic solvents like acetone or ethanol at a low temperature for obtaining purification and concentration of a specific FA (ALA in our case) by dissolving FAs at a low temperature so as to separate LC-PUFAs from short-chain FAs and unsaturated fatty acids (UFAs) from saturated fatty acids (SFAs). It is a simple operation and involves low energy cost, but yields ALA with lower purity (Gu et al., 2009). The ideal conditions in the purification process are reported to be 95.82% acetone as the extraction agent for a FA ratio of 6.56 at 12.35 pH, 45°C temperature, and 4.74 h of crystallization time for increasing ALA concentration from 46.0% to 80.3%. Yong et al. (2014) used the mixed FA, acetonitrile and acetone in the ratio of 1:6:8 (v/v/v), 10 h freezing crystallization time, solid-liquid separation at 18°C and 7000 r/min refrigerated centrifuge for 3 min as the optimal condition. The ALA content from rubber seed oil increased from 16.32% to 31.52% in addition to the total UFAs’ content, which reached 98.28%.

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3.3 Column chromatography Chromatography is a robust technique of analytical chemistry employed for separation of a particular chemical moiety from a mixture of compounds present in gaseous or liquid form in the mixture. Chromatography on silver nitrate-silica gel is a complex column chromatography and is commonly used to separate FAME. This method is based on the different polarities of PUFAs (Liu et al., 2014). The main advantages of this method are that it can be performed under mild conditions without involving high temperature or pressure and it yields high purity of FAME (Guil-Guerrero et al., 2003). The purity of fractionated ALA from pepper seed oil was obtained to be 97% using this method with silver-silica gel as a stationary phase (Zhang et al., 2005).

3.4 Silver ion complexation In the silver ion complexation method, a number of carbon double bonds in different FAs are used to form complex polarity with silver ions. In the presence of a higher number of double bonds, the complexation is stronger. Furthermore, it is used to achieve both separation and purification ( Jiang et al., 2008; Sun et al., 2011). The silver ion complexation method was used by Ryu et al. (1997) to purify ALA in perilla oil with 10 g silver nitrate/ 100 g silica gel and passing 2 to 3 g ALA-containing fatty acid mixture/100 g stationary phase (column). Subsequently, adopting the technique of gradient elution of 2%, 5%, and 7% acetone, a hexane solution each of 200 mL and a purity of ALA of more than 90% was obtained. Ge et al. (2017) obtained ALA from Phyllanthus emblica seed oil with 93.3% purity and 73.4% yield by silver ion complexation. The best conditions were 0°C complexation temperature, 2.29 mol/L silver nitrate concentrations, 38% methanol volume fraction, and 1.93 h complexation time. Furthermore, the recovery of silver nitrate was 93.8% and the recovery of Ag + showed good complexation effect.

3.5 Urea inclusion The urea inclusion method involves urea molecules in the crystallization process for combining with SFAs or monounsaturated fatty acids (MUFAs) to form a stable crystalline clathrate after precipitation. Gu et al. (2009) reported that inclusion is difficult to form with PUFAs due to the certain space configuration with multiple double bonds and bending carbon chains. It is characterized by simple equipment and process, low temperature and maintains the nutrients and active for extraction which affects the suitability for mass production. But the FAs with different carbon chain lengths and similar saturation cannot be separated easily (Sajilata et al., 2008). Gu et al. (2009) concentrated ALA (91.50%) from crude perilla oil by the gradient cooling urea inclusion method with optimized conditions of urea-to-FA ratio (3), solvent-to-FAs ratio (7), reaction temperature (348 K), and crystallization time (690 min). Lee et al. (2016) obtained ALA (81.75%) from Perilla frutescens var. japonica oil using 2 g urea treatment with cooling at 10°C for 24 h.

Alpha linolenic acid

Tartaric acid can assist in the inclusion of FAs with urea in this method. Under the ideal conditions of tartaric acid-to-urea ratio of 1:3 (n/n), urea-to-mixed FAs (MFAs) ratio of 2.5:1 (m/m), methanol-to-MFAs ratio of 10:1 (V/m), crystallization temperature of 8°C and crystallization time of 8 h, the purity and yield of ALA in the enriched product were 78.6% and 60.9%, respectively (Cheng et al., 2017).

3.6 Supercritical fluid extraction It is a relatively new separation method adopted in recent years with the basic principle of adjusting the temperature and pressure of supercritical fluid so as to make raw material components soluble in it for their efficient separation from the matrix. In this process, supercritical fluids, usually carbon dioxide (scCO2), are used as the extraction fluid for the extraction of an oil rich in EFAs owing to the non-toxic, environmentally benign, non-polluting, non-flammable, economical, and compatible nature of scCO2. Besides this, it is a fast, simple, efficient method and requires less organic solvents (Gouveia et al., 2007; Nisha et al., 2012). However, it is difficult to separate ALA from the UFAs having a carbon number equal to ALA (Teramoto et al., 1994). Li and Jia (2004) used ideal conditions for the separation of ALA from pine nutlet using the supercritical CO2 fluid extraction method; 35 MPa extraction pressure, 40°C extraction temperature, 34°C column temperature, and 90-min extraction time were used. The recovery rate of ALA was 34.9% from gas chromatography (GC) analysis. Pan et al. (2012) extracted oak silkworm pupal oil using supercritical CO2 fluid with the content of 77.29% of UFAs and 34.27% of ALA in the total oil. The optimal conditions used for extraction were at 28.03 MPa pressure, 1.83 h time, 35.31°C temperature, and a 20.26 L/h flow rate of CO2. In one pilot-scale scCO2 process, pressure and temperature were used in the range of 23 to 37 MPa and 52 to 73°C, respectively, with 4.5 kg of CO2. The highest yield of oil extraction was achieved in mango seed kernels (83 g/kg) using 37 MPa and 63°C. The EFA-rich lipid fraction containing 37 g/kg linoleic and 4g/kg α-linolenic acids, and a high oleic acid (155 g/kg) content was obtained using a low pressure (23 MPa) during extraction (Cero´n-Martı´nez et al., 2021).

3.7 Ultrasound based extraction using a solvent The acoustic cavitation force is applied for the extraction of a chemical compound using ultrasound. The ultrasounds cause compression and rarefaction in the system/solvent molecules having the desired compound to be extracted. This leads to the formation of bubbles which subsequently propagate in size, resulting in unstable sizes with temperature and pressure reaching their maximum and causing the bubble to collapse. For the extraction of oil from rapeseed flakes, the authors used pulsed ultrasound at a power of 400 W with a frequency of 12 kHz (Perrier et al., 2017). This involved the introduction of rapeseed flakes into a 229 mL volume of solvent (hexane, isopropanol, or ethanol)

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preheated to 50°C. Three liquid-to-solid ratios were studied (ratio 15: 15 g of solid and 229 mL of solvent; ratio 30: 7.5 g of solid and 229 mL of solvent; ratio 40: 5.7 g of solid; and 229 mL of solvent). Then, ultrasounds were applied for 5, 10, 20, or 30 min. The stirring speed for control was maintained at 140rpm. When the temperature exceeded 50°C, flakes were placed in an ice cube tray for maintaining the temperature during the treatment. Control experiments corresponded to solid/liquid extraction under agitation (140 rpm) and under the same conditions of temperature, time, and liquid/solid ratio but without ultrasound application. The authors indicated that isopropanol and hexane yielded similar results, i.e., an oil yield of 79 and 80%, respectively, under the optimum conditions of extraction of 20min (using ultrasound) followed by 2 h of additional solid/liquid extraction.

3.8 Imidazolium-based ionic liquids having silver tetrafluoroborate extraction The hydrophobic ILs with silver salts are also used as an extraction phase for the extraction of omega-3 polyunsaturated fatty acid methyl esters (PUFAMEs). The IL biphasic extraction equilibrium is mainly influenced by extraction time, organic solvents, IL structures, and AgBF4 concentrations. When extraction phase silver tetrafluoroborate (AgBF4) was used after dissolving in 1-hexyl-3-methylimidazolium [hmim] with anion [PF6], both distribution ratios of PUFAMEs and stability constants of PUFAME-Ag+ complexes increased significantly with an increase in the degree of unsaturation of the PUFAMEs. It is a more efficient extraction method for PUFAMES compared with aqueous AgNO3 extractions as it provides higher extraction capacities and less operation time (Li et al., 2009).

4. Extraction of ALA from potential bio-wastes Studies have revealed that some plant biomass is either rich in ALA content (like date palm waste, tomato peel, halophytes, and tropical fruit biomass) or is induced to produce ALA (such as micro algae). ALA from these is extracted using suitable techniques as described in the previous section.

4.1 Extraction of ALA from conventional sources 4.1.1 Mango seed kernel Tropical fruits represent one of the most important crops in the world. The continuously growing global market for the key tropical fruits is currently estimated at 84 million tons, of which approximately half of the production is lost or wasted throughout the whole processing chain (Villacı´s-Chiriboga et al., 2020). The kernels and peels of tropical fruit biomass are also reported to be used for the extraction of ALA as described hereunder. Worldwide, production of mango and area under cultivation ranks second which is only next to banana (FAO, 2020). A large amount of waste, i.e., close to 40%–60% of

Alpha linolenic acid

the whole fruit, is generated by the mango processing industry. The seed kernels of mango contribute half of this waste (Ballesteros-Vivas et al., 2019). Cero´n-Martı´nez et al. (2021) optimized protocol for extraction of lipid-rich fractions from mango seed kernel using a supercritical fluid extraction technique. The researchers reported that at a relatively lower pressure (23 MPa) and temperature (73°C), the per kg of extract showed a concentration of 172 g oleic acid, 46 g linoleic acid (LA), and 5 g ALA. The authors suggested that the application of Hansen solubility theory is effective in selective extraction of these FAs. According to this theory, the solubility of a specific component in supercritical carbon dioxide is improved on the addition of a cosolvent to it at a cosolvent volume fraction of 5% in a supercritical homogenous mixture. This improves extraction by enhancing separation factors and/or also utilizing less CO2 and decreasing working pressure. The details of the same can be found at the study by Hansen (2007); Tirado et al. (2019). 4.1.2 Tomato peel Tomato peel is a valuable source of pectin, polyphenols, and FAs. For extraction of ALA from tomato peels, these are first de-pectinized using a mixture of ammonium oxalate and oxalic acid. This is followed by analyzing the de-pectinized peels for polyphenols and FAs using chromatography, which is carried out by extracting lipid using Soxhlet apparatus with 70 and 96% of ethanol, and again, obtained residues are extracted in a mixture of chloroform and methanol (50:50, v/v) (Grassino et al., 2020). The extracted lipid is converted to FAMEs using trans-esterification with a methanolic solution of potassium hydroxide followed by GC analysis of FAMEs using a column temperature of 220°C at the rate of 7°C/min. These authors used helium as a carrier gas at a flow of 1.5 mL/min. The injector and detector temperature was set at 250 and 280°C, respectively, and the injection volume was 1 μL with a split ratio of 1:30. The ALA content of de-pectinized tomato peel extracts treated with 70% and 96% ethanol was found to be 2.02% and 2.37%, respectively. 4.1.3 Moth bean biomass Moth bean (Vigna aconitifolia L.) is consumed as pulses worldwide and its seed is either used in the form of split or whole grain. Milling of moth bean seed yields a substantial amount of by-product rich in valuable sources of macronutrients with remote chances of commercial value. The husk of moth bean contains valuable essential amino acids, unsaturated FAs, and minerals with less functional attributes. Kamani et al. (2020) extracted the lipid from the dried powder of husk and other parts of moth bean using the Soxhlet extractor and further dried it in oven at 60°C for 1 h to remove solvent. Later, the researchers prepared FAMEs by subjecting lipids to direct methylation for GC analysis. For the preparation of FAMEs, lipid (100 mg) was properly mixed with 4 mL of methanolic NaOH (0.5 N) followed by boiling for 5 min. The boiling step

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was repeated with 5 mL of BF3-Methanol for 2 min and 2 mL of heptane for 1 min. Further, on cooling, a saturated NaCl solution was added for allowing the heptane to float on the surface of the flask. The organic layer with methyl esters was then dried using anhydrous sodium sulfate and transferred to a glass vial for GC analysis. The ideal conditions used for GC analysis were 220°C column temperature for 10 min followed by an increase to 230°C at a rate of 10°C per min for 20 min. The injector and detector temperatures were maintained at 250°C and 260°C, respectively. Nitrogen was used as the carrier gas at a flow rate of 1 mL per min with a 1:20 split ratio. The major FAs were found to be palmitic acid (C16:0), oleic acid (C18:1), LA (C18:2), and linolenic acid (C18:3) in all fractions of moth bean biomass. The authors reported that linolenic acid was found to be high in whole moth bean (18.53%) followed by other fractions of moth bean, namely, protein-rich by-product (17.7%), cotyledon (16.97%), and husk (14.79%).

4.1.4 Olive leaves Olive (Olea europea) is cultivated majorly for the extraction of oil from its fruit. The leaves contribute to approximately 10% of the total weight of the yield of olives. Ghanem et al. (2019) studied olive leaves as by-products of the olive oil industry for bio-chemical analysis. For this, olive leaves were first washed using tap water followed by oven drying (40°C) and grinding into powdered form. The prepared powdered leaves (600 g) were sonicated using hexane (3  1 L) and the resultant extract was mixed and evaporated at 40°C to obtain a dark greenish black residue (12.26 g). Five grams of this hexane extract were dissolved in hot acetone and kept in the refrigerator overnight, followed by filtration. Saponification of acetone-soluble fraction was carried out using refluxing with 100 mL alcoholic potassium hydroxide (0.5 N) for 8 h, followed by concentration to about 25 mL and then dilution with cold distilled water. Mother liquor, produced after saponification, was rendered acidic with sulfuric acid. The FAs were extracted many times with ether, washed using distilled water, dehydrated over anhydrous sodium sulphate, and lastly, evaporated under vacuum (40°C) till dry. The fraction of FA was dissolved in dry methanol (30 mL) containing dry hydrochloric acid (4%–5%) and refluxed for 3 h in a boiling water bath. After that, it was diluted with distilled water and extracted with ether (3  25 mL) followed by washing using distilled water, drying over anhydrous sodium sulfate, filtered, and lastly, evaporated under vacuum (40°C). The prepared FAMEs were analyzed by GC/MS using ideal conditions that included 0.2 μL injection volume, helium as a carrier gas at a flow rate of 1.0 mL per min, and a split ratio of 1: 10. Using temperature 80°C for 1 min, increasing at 4°C per min to 300°C, and giving a 1-min hold, the injector, and detector were held at 240°C. Mass spectra were achieved using electron ionization (70 eV) with a spectral range of mass number/charge number (40–450). The authors revealed that ALA was the major FA in olive leaves with 49.45%.

Alpha linolenic acid

4.1.5 Bilberry seed Bilberry seed oil is known as an ideal source of ALA and LA. Bilberry waste obtained from the production of juice syrup that mainly comprises seeds and vegetal residues from the bilberry pulp and peel fractions was utilized for the ALA extraction. The lipid extraction from waste bilberry seeds was carried out by Cante et al. (2020). The extraction procedure involved the use of hydrofluorocarbon (HFC) as solvent under subcritical conditions of 35°C temperature and 10 bar pressure, with 1, 1, 1, 2-tetrafluoroethane (Norflurane, R134a). Norflurane-haloalkane refrigerant (boiling point 26.3°C at atmospheric pressure and 25°C at a pressure of 6.61 bar) is generally used as a propellant for inhalers and as an extinguishing agent. Percolation of liquid solvent through the solid matrix in the extraction reactor at 8–10 bar (30–45°C) enriched bilberry oil. Further, saturated solvent was fed into the expansion vessel to gasify at a pressure of 4–5 bars to release oil at the bottom. Norflurane was then recompressed and recycled in liquid form into the extraction chamber. This procedure compensated for the considerably lower solubilities of lipids in Norflurane through continuous solvent regeneration and recirculation. The resultant extracted lipid was transesterified using methanolic potassium hydroxide and analyzed for FAs using GC. Helium was used as the carrier gas (1.5 mL/min). The injection volume of 1 μL, run time of 30 min, and split ratio of 1:20 were used for analysis. The conditions used for GC were 180°C column temperature for 5 min, which was increased to 250°C. The temperatures for the injector and detector were 230°C and 300°C, respectively. The extracted bilberry oil showed FA profiles having a high content of UFAs such as ALA (34.98%) and LA (32.93%). A similar study on the extraction of bilberry oil was conducted using sc-CO2 and yielded 36.32% ALA and 36.12% LA in bilberry oil (Yang et al., 2011). 4.1.6 Salmo salar fish waste Fish by-products are used to produce fishmeal or fertilizers, with fish oil as a by-product. S. salar fish waste samples were grouped into heads and soft tissues. Ghaly et al. (2013) reported that fish wastes are a huge potential source of useful molecules, including bioactive peptides, enzymes, antimicrobial components, and PUFAs. Characterization of FAs composition was done through GC/mass spectroscopy (MS). Inguglia et al. (2020) used Salmo salar fish waste (head and soft tissues) for the extraction of ALA. The workers first homogenized the fish waste, followed by diluting with distilled water (1:1) and heating at 90°C for 1 h to coagulate proteins and increase oil release from samples. After that, filtration of the homogenate sample was done for obtaining a liquid fraction which was centrifuged at 10,000 g at 4°C to separate aqueous fraction from the liposoluble component corresponding to the fish oil. FAs were analyzed using the GC-MS method after extraction and hydrolysis of triacylglycerols. The optimum conditions used were 250°C column temperature for 8 min under isothermal conditions. Helium (carrier gas) was used at a flow rate of 1 mL/min and the injection volume of

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Table 1 Wastes sources for production/extraction of alpha linolenic acid. Source

Alpha linolenic acid yield (w/w)

Microalgae Biomass

1.15, 0.8, and 0.7 with ionic liquids, namely, [TMAm][Cl], [EMPyrro][Br], and [EMIM][Cl]a 0.13%–0.20%

Grape seed oil Blueberry pomace Blackberry waste Fig seeds

Rosehip seeds

5.4% 3% 0.30%

13.46%

Method used for extraction

Room temperature ionic liquids Supercritical fluid extraction Supercritical fluid extraction-CO2 Supercritical fluid extraction-CO2 Room temperature using n-hexane An automatic Soxhlet device using hexane

References

Motlagh et al. (2020) Souza et al. (2020) Campalani et al. (2020) Campalani et al. (2020) Nakilcio
gluTas¸ (2019) G€ uney (2020)

a [TMAm][Cl]: Tetramethyl ammonium chloride; [EMPyrro][Br]: 1-ethyl-1-methyl pyrrolidinium bromide; [EMIM] [Cl]: 1-ethyl-3-methyl imidazolium chloride.

the sample was 1 μl with a split ratio of 1:100. The conditions for MS included an ion source temperature of 260°C, MS transfer line temperature of 265°C, an injector temperature of 250°C, an ionization voltage of 70 eV, and a mass range scanned between 35 and 550 m/z. The results of the study were interesting and revealed that ALA was the most abundant omega-3 FA found in the fish waste oil representing 5.91% in head oil and 4.46% in soft tissue oil. Besides this, various waste sources used for extracting the ALA and method employed for its extraction are mentioned in Table 1.

5. Unconventional sources for extraction of ALA 5.1 Silkworm pupal oil The silkworm pupae, from which silk is obtained, are generally known as desilked silkworm pupae (DSP). These are used as fertilizer and fish or chicken feed. However, most of it possess a serious threat to the environment as products of putrefaction of this waste are toxic. DSP are reported to be rich in ALA (up to 40% of total FAs) as per the reports pertaining to the FA composition of neutral lipids (Shanker et al., 2006; Zhou and Han, 2006). Wang et al. (2010) optimized the process for enriching ALA from DSP oil. The researchers extracted crude FAs from DSP and enriched ALA using the urea clathration method, which is a phenomenon of isolating linear paraffins (including FAs) from a mixture of hydrocarbons via the formation of urea-n-paraffin-clathrates. The authors

Alpha linolenic acid

reported that the ratio of saturated urea/ethanol solution to crude mixed FA, clathration temperature, and clathration time were 2:1 (v/v), 4°C, and 2 h, respectively, for enriching ALA to a level of 31.87%.

5.2 Microalgae There are many methods used for extracting lipid from microalgal biomass, such as the Kochert method, Soxhlet method, liquid-liquid extraction, and supercritical fluid extraction. However, a few treatments are utilized to improve the extraction of valuable components from microbial lipid, namely microwave-assisted technique, ultrasonication, electro-flotation by current and autoclave, lyophilization, enzymatic disruption, and acid treatment ( Japar et al., 2017). Microalgae are oleaginous microorganisms which grow heterotrophically supported by volatile fatty acids (VFAs) as a carbon source and accumulate great amount of valuable products, such as omega-3 FAs and exopolysaccharides. Liu et al. (2013) produced omega-3 FAs from algae (Chlorella vulgaris and Auxenochlorella protothecoides) fermentation using VFA as a carbon source. Under dark conditions, some strains of microalgae utilize carbon sources (such as glucose, glycerol, ethanol, and volatile organic acids) and produce useful metabolites such as lipids and pigments. The biomass from 19 species of plants belonging to the 5 different families was collected and mixed; samples were dried at a temperature of 40°C in an oven for three days. After drying, the samples were milled finely in a grinder, sieved (50 μm mesh size) and stored ( 20°C) until analysis. Then, the dried mass was converted into FAME for analyzing the FA content. Finally, the obtained extract was filtered and stored at 20°C before GC-MS analysis. The ALA content in C. coronopifolia, P. australis, S. vera, H. marinum, Sporobolus sp. species was reported to be 39.9%, 37.9%, 35.8%, 33.8%, and 33.5%, respectively (Vizetto-Duarte et al., 2019). Sijil et al. (2019) concluded from their study that accumulation of ALA in microalgal biomass increased when it was subjected to the stress conditions of nitrogen depletion and low temperature (5°C). It was observed that 1.7- and 1.5-fold increase in its lipid content at low temperature occurred, which led to 1.8-fold increase in ALA fraction of total FAs. The other extraction method employed for extraction of lipids included placing frozen pelletized algae in tin foil cups and drying overnight at 65°C (Axelsson and Gentili, 2014). Othman et al. (2019) isolated ALA from freshwater microalgae, i.e., Acutodesmus obliquus (CN01), Chlorella sp. (Carolina-15-2069), and Chlorella culgaris (National Institute for Environmental Studies (NIES)-1269). The researchers extracted the lipid from an algae suspension (50 mL) produced using centrifugation (12,000  g and 25°C for 5 min) of freeze dried cells with a 6 mL mixture of methanol: chloroform (1:2 v/v). 100 μL of internal standard of heptadecanoic acid (C17) was incorporated into crude lipid at a concentration of 500 μg/mL followed by vortexing and centrifugation (1000  g and

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25°C for 5 min). Further, 1.25 mL of potassium chloride (0.1 M) was added to the supernatant collected in the other tube, mixed properly using vortexing (30 s) and centrifuged again. The lipid content was obtained by evaporation (2 h) of chloroform present in the bottom layer using a rotary evaporator. The FAMEs were prepared by the addition of 4 mL of methanolic hydrochloric acid (0.1 N) to the dried lipid and kept in water for 1 h at 100°C. After that, 4 mL of hexane was added to the cooled FAMEs followed by vigorous mixing to obtain the resultant FAMEs in two layers. The upper layer containing FAMEs was transferred to a new tube, and the bottom layer with methanolic hydrochloric acid was again extracted with 2 mL of hexane and 2 mL of distilled water. Later, the obtained supernatant was collected in a tube containing the first upper layer and then evaporated for 30 min in a rotary evaporator. The solution was then prepared by adding hexane (300 μL) to the dried FAMEs and transferred into a glass vial for GC analysis. The conditions followed for GC analysis included the initial temperature of 100°C for 1 min, increased to 200°C at a ramping rate of 25°C/min and held for 1 min, then further raised to 250°C at a rate of 4°C/min and held again for 7 min. Helium was used as the carrier gas with a 7.10 mL per min flow rate. The authors obtained the highest ALA of 38% in Acutodesmus obliquus CN01 as compared to C. vulgaris NIES-1269 and Chlorella sp. Carolina-15-2069, which had 35% and 17.9% ALA, respectively. In a study undertaken by Pleissner et al. (2016), restaurant food waste, namely, noodles, eggs, rice, bread, vegetables, sauce, and meat, was enzymatically digested using amylolytic and proteolytic enzymes. The subsequent hydrolysate was used as nutrients for heterotrophic microalgal Chlorella pyrenoidosa in continuous flow cultures using stirred and aerated Sartorius bioreactors at 6.5 pH, 28°C temperature, and 400–600 rpm speed. The inocula were grown for four days in a conical flask (250 mL capacity) containing 100 mL of diluted food waste hydrolysate with glucose 5 g/L, fructose 0.05 g/L, free amino nitrogen (FAN) 0.2 g/L, and phosphate 0.1 g/L. The hydrolysate was filtered using a 0.22 μm membrane filter. From this, 5% inoculum was used for fermentation with a dilution rate of 0.36 per day. Then, it was centrifuged at 7000  g for 20 min, and the obtained pellets were stored at 80°C and the supernatants were stored at 20°C. The glucose, fructose, FAN, and phosphate were determined by HPLC. The resultant nutrient concentration in feeds for the continuous flow cultures with a 0.36 per day dilution rate was 89.01 g/L glucose, 1.90 g/L fructose, 0.23 g/L FAN, and 0.27 g/L phosphate. Pleissner et al. (2016) found 29 mg ALA per g biomass of C. pyrenoidosa produced in continuous flow cultures with a dilution rate of 0.36 per day.

5.3 Crude tall oil Crude tall oil (CTO) is a by-product derived from kraft pulping of softwoods and paper mills. It is extracted from partially concentrated (20%–30% solids) black liquor and allowed to settle to separate into two layers. Among them, the resultant top layer

Alpha linolenic acid

containing tall oil soap was skimmed off. In general, CTO contains 30%–60% saponified FAs, 40%–60% resin acids, and 5%–10% unsaponifiables. In a study carried out by Islam et al. (2020) for the extraction of ALA from CTO, raw CTO was first treated with 1% sodium chloride for removing impurities such as sulfuric acid, sodium salts, dissolved lignin, residual pulping chemicals, etc. The CTO and sodium chloride (60:40) were mixed vigorously for 10 min in separating funnel. After separation, two layers were formed, out of which the bottom layer represented an aqueous salt solution, which was discarded, whereas the upper layer contained non-aqueous CTO, which was then further centrifuged (10,000 rpm for 10 min). The obtained CTO was subjected to fractional distillation (temperature 130–265°C and pressure of 10 mmHg) in order to separate tall oil fatty acids (TOFAs). Later, the separation of PUFAs from SFAs and MUFAs was done using urea complexation method in which ethanoic urea solution (ethanol:urea-96:4) was used with TOFAs at a ratio of 80:20. Further, a sample of PUFAs (5 g) and acetone (45 g) were mixed, crystallized at a temperature of 15°C for 24 h, centrifuged (4100 rpm for 7.5 min) and filtered, whereas acetone was recovered using evaporation. 200 μL of the obtained fraction was taken in a 2 mL vial and mixed with 800 μL of isopropyl alcohol. 20 μL of sample was taken from vial and injected into GC for analysis. The temperature was raised to 20°C per minute up to 240°C and held at this temperature. The total run time for GC was 24 min. This study revealed that the amount of ALA in CTO was 1%.

5.4 Molasses as medium for production of biomass with fatty acid The biomass of the photosynthetic bacterium Rhodopseudomonas faecalis PA2 accumulates ALA, LA, and dihomo-gamma-linolenic acid and also produces high levels of lipid, carotenoid, and protein (Saejung and Puensungnern 2020; Saejung and Ampornpat, 2019). In research carried out by this team, the bacterium was grown in a photo bioreactor (5 L) having molasses as a culture medium, which was used as a carbon source. The culture inoculum and nitrogen gas were added at the rates of 10% and 99%, respectively. The nitrogen gas was added via port to the headspace for developing anoxygenic condition in bioreactor and agitation speed used was 100 rpm. The illumination on the bioreactor was done by an external light source with 4000 lx light intensity. After the cultivation for 10 days, the bacterial mass was collected by centrifugation and washed with a sterile saline solution (0.85% w/v). The cell pellets were lyophilized using a freeze dryer. The lipid content and FA composition were analyzed by hydrolytic extraction and the GC method. The ALA content was found to be 0.183% using molasses as the medium.

6. Conclusion and future prospective Valorization of waste refers to converting the waste biomass into a valuable product through a number of interventions. This chapter explores the extraction of ALA from underexploited food sources, including bio-wastes like fruit pomace and seed, vegetable

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peel, fish waste, algae, etc. representing a source of valuable FAs and bioactive compounds. Scientific literature in the field of valorization has been given immense weightage over the last few years and it is increasing year by year, which indicates the growing interest toward the utilization of food waste for the extraction of valuable biomolecules. The application of these food wastes for ALA extraction is of growing public interest as this could lead to improved health of the general public due to its known health benefits. Besides this, ALA extracted from fruit and vegetable waste can make a substantial contribution to safeguarding the environment from these wastes demanding high oxygen for their disposal and also on account of being vegan source of ALA. Reiterating it, utilization of food waste for ALA extraction reduces not only the waste but also checks the environmental pollution. Further research is needed for exploring different waste sources, appropriate approaches, and techniques for improving the purity, yield, and reduce the extraction time of ALA. The extracted ALA could be analyzed through in-vitro and in-vivo study for accessing their health benefits in an array of food products.

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Liu, C.-H., Chang, C.-Y., Liao, Q., Zhu, X., Chang, J.-S., 2013. Photoheterotrophic growth of Chlorella vulgaris ESP6 on organic acids from dark hydrogen fermentation effluents. Bioresour. Technol. 145, 331–336. Liu, X.-x., Cai, N.-c., Ping, S., 2014. Research progress in extraction technology of α-linolenic acid from prickly ash seed oil. Sci. Technol. Food Ind. 08. Lutisˇan, J., Cvengrosˇ, J., Micov, M., 2002. Heat and mass transfer in the evaporating film of a molecular evaporator. Chem. Eng. J. 85 (2–3), 225–234. Maina, S., Kachrimanidou, V., Koutinas, A., 2017. From waste to bio-based products: a roadmap towards a circular and sustainable bioeconomy. Curr. Opin. Green Sustain. Chem. 8, 18–23. Martins, P.F., Ito, V.M., Batistella, C.B., Wolf, M.R., Maciel., 2006. Free fatty acid separation from vegetable oil deodorizer distillate using molecular distillation process. Sep. Purif. Technol. 48 (1), 78–84. Medina, A.R., Grima, E.M., Gimenez, A.G., Gonza´lez, M.I., 1998. Downstream processing of algal polyunsaturated fatty acids. Biotechnol. Adv. 16 (3), 517–580. Motlagh, R., Shiva, R.H., Biak, D.R.A., Hussain, S.A., Omar, R., Elgharbawy, A.A., 2020. COSMO-RS based prediction for alpha-linolenic acid (ALA) extraction from microalgae biomass using room temperature ionic liquids (RTILs). Mar. Drugs 18 (2), 108. Nakilcio
glu-Tas¸, E., 2019. Biochemical characterization of fig (Ficus carica L.) seeds. J. Agric. Sci. 25 (2), 232–237. Ni, R.X., 2017. Research progress on α-linolenic acid extraction and separation technology. Appl. Sci. Technol. 13, 76. Nisha, A., Udaya Sankar, K., Venkateswaran, G., 2012. Supercritical CO2 extraction of Mortierella alpina single cell oil: comparison with organic solvent extraction. Food Chem. 133 (1), 220–226. Othman, F.S., Jamaluddin, H., Ibrahim, Z., Hara, H., Yahya, N.A., Iwamoto, K., Mohamad, S.E., 2019. Production of α-linolenic acid by an oleaginous green algae acutodesmus obliquus isolated from Malaysia. J. Pure Appl. Microbiol. 13, 1297–1306. Pan, W.-J., Liao, A.-M., Zhang, J.-G., Dong, Z., Wei, Z.-J., 2012. Supercritical carbon dioxide extraction of the oak silkworm (Antheraea pernyi) pupal oil: process optimization and composition determination. Int. J. Mol. Sci. 13 (2), 2354–2367. Perrier, A., Delsart, C., Boussetta, N., Grimi, N., Citeau, M., Vorobiev, E., 2017. Effect of ultrasound and green solvents addition on the oil extraction efficiency from rapeseed flakes. Ultrason. Sonochem. 39, 58–65. Pleissner, D., Lau, K.Y., Lin, C.S.K., 2016. Utilization of food waste in continuous flow cultures of the heterotrophic microalga Chlorella pyrenoidosa for saturated and unsaturated fatty acids production. J. Clean. Prod., 1–8. Ryu, S.-N., Lee, J.-I., Jeong, B.-Y., Hur, H.-S., 1997. Method for separating and purifying α-linolenic acid from perilla oil. U.S. Patent 5,672,726, issued September 30,. Saejung, C., Ampornpat, W., 2019. Production and nutritional performance of carotenoid-producing photosynthetic bacterium Rhodopseudomonas faecalis PA2 grown in domestic wastewater intended for animal feed production. Waste Biomass Valorization 10 (2), 299–310. Saejung, C., Puensungnern, L., 2020. Evaluation of molasses-based medium as a low cost medium for carotenoids and fatty acid production by photosynthetic bacteria. Waste Biomass Valorization 11 (1), 143–152. Sajilata, M.G., Singhal, R.S., Kamat, M.Y., 2008. Fractionation of lipids and purification of γ-linolenic acid (GLA) from Spirulina platensis. Food Chem. 109 (3), 580–586. Shanker, K.S., Shireesha, K., Kanjilal, S., Kumar, S.V.L.N., Srinivas, C., Rao, J.V.K., Prasad, R.B.N., 2006. Isolation and characterization of neutral lipids of desilked eri silkworm pupae grown on castor and tapioca leaves. J. Agric. Food Chem. 54 (9), 3305–3309. Sijil, P.V., Sarada, R., Chauhan, V.S., 2019. Enhanced accumulation of alpha-linolenic acid rich lipids in indigenous freshwater microalga Desmodesmus sp.: the effect of low-temperature on nutrient replete, UV treated and nutrient stressed cultures. Bioresour. Technol. 273, 404–415. Souza, D., de Ca´ssia, R., Machado, B.A.S., de Abreu Barreto, G., Leal, I.L., dos Anjos, J.P., Umsza-Guez, M.A., 2020. Effect of experimental parameters on the extraction of grape seed oil obtained by low pressure and supercritical fluid extraction. Molecules 25 (7), 1634. Sun, L.-p., Ma, L., Ya-hua, W., Hui, X., 2011. Research progress of separation and purification techniques for α-linolenic acid. Packag. Food Machin. 2.

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Takeuchi, H., Sakurai, C., Noda, R., Sekine, S., Murano, Y., Wanaka, K., Kasai, M., Watanabe, S., Aoyama, T., Kondo, K., 2007. Antihypertensive effect and safety of dietary α-linolenic acid in subjects with high-normal blood pressure and mild hypertension. J. Oleo Sci. 56 (7), 347–360. Teramoto, M., Matsuyama, H., Ohnishi, N., Uwagawa, S., Nakai, K., 1994. Extraction of ethyl and methyl esters of polyunsaturated fatty acids with aqueous silver nitrate solutions. Ind. Eng. Chem. Res. 33 (2), 341–345. Thi, N.B., Dung, G.K., Lin, C.-Y., 2015. An overview of food waste management in developing countries: current status and future perspective. J. Environ. Manag. 157, 220–229. Tirado, D.F., Rousset, A., Calvo, L., 2019. The selective supercritical extraction of high-value fatty acids from Tetraselmis suecica using the Hansen solubility theory. Chem. Eng. 75. Turpeinen, A., Merimaa, P., 2011. Functional fats and spreads. In: Functional Foods. Woodhead Publishing, pp. 383–400. Villacı´s-Chiriboga, J., Elst, K., Van Camp, J., Vera, E., Ruales, J., 2020. Valorization of byproducts from tropical fruits: extraction methodologies, applications, environmental, and economic assessment: a review (part 1: general overview of the byproducts, traditional biorefinery practices, and possible applications). Compr. Rev. Food Sci. Food Saf. 19 (2), 405–447. Vizetto-Duarte, C., Figueiredo, F., Rodrigues, M.J., Polo, C., Resˇek, E., Custo´dio, L., 2019. Sustainable valorization of halophytes from the mediterranean area: a comprehensive evaluation of their fatty acid profile and implications for human and animal nutrition. Sustainability 11 (8), 2197. Wang, J., Wu, F., Liang, Y., Wang, M., 2010. Process optimization for the enrichment of α-linolenic acid from silkworm pupal oil using response surface methodology. Afr. J. Biotechnol. 9 (20). Yan, X.S., Gu, K.R., Ma, L., Zhang, B., Zhao, H., 2014. Research progress on α-linolenic acid purification technology. Food Oil 9, 9–13. Yang, B., Ahotupa, M., M€a€att€a, P., Kallio, H., 2011. Composition and antioxidative activities of supercritical CO2-extracted oils from seeds and soft parts of northern berries. Food Res. Int. 44 (7), 2009–2017. Yong, L.M., Wang, L.R., Liu, S.S., 2014. Separation process of α-linolenic acid from rubber seed oil by double solvent freezing crystallization. Food Sci. Technol. 39 (7), 233–237. Zhang, C., Kan, J.Q., Chen, Z.D., 2005. Study of purification α-linolenic acid with the silver nitrate-silica gel. Ion Exchange Adsorpt. 21 (1), 47–54. Zhou, J., Han, D., 2006. Proximate, amino acid and mineral composition of pupae of the silkworm Antheraea pernyi in China. J. Food Compos. Anal. 19 (8), 850–853.

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CHAPTER 3

Citric acid Ramandeep Kaura and Kandi Sridharb a

Department of Food Technology, Eternal University, Baru Sahib, Himachal Pradesh, India Department of Food Science, Fu Jen Catholic University, New Taipei City, Taiwan

b

1. Introduction Citric acid (CA) is a weak organic acid and natural preservative that has widespread applications in the food, pharmaceutical, and cosmetic industries (Luo et al., 2017). The consumption of CA is witnessing high annual growth due to advanced applications in functional industries (Singh Dhillon et al., 2011). Consequently, its production by different biotechnological approaches is a topic of utmost interest (Amato et al., 2020). Moreover, in order to cope with the declining prices and satisfy the increasing market demand for CA, there is a need to develop cost-effective, eco-friendly, and industrially feasible production technology (Singh Dhillon et al., 2011). Recently, numerous agricultural by-products and waste have been investigated for their potential as a substrate for CA production by fermentation methods. For example, fruit peels and pomace, molasses, wheat bran, cassava bagasse, as well as plant materials like wood, straw, and coffee husk (Vandenberghe et al., 2000) were used for the production of CA. Consequently, valorization of agri-waste to produce CA provides benefits by minimizing waste utilization as well as lower capital costs for carbon substrates. Along with this, the waste is renewable in nature and easily accessible for microbiological synthesis of CA (Morgunov et al., 2018). Over time, a wide variety of microbes have been documented, ranging from fungi and bacteria to yeasts to produce CA (Show et al., 2015). A white rot fungus, Aspergillus niger is the most commonly used choice of microbe for the CA production process as it is adaptable and suitable to grow on various substrates. Modern technologies for CA production are based on utilizing the mutant strains of A. niger. Moreover, many bacteria like Arthrobacter paraffinens, Bacillus licheniformis, and Corynebacterium sp. (Soccol et al., 2006) and yeast (Yarrowia lipolytica) have been known to yield CA from n-alkanes and carbohydrates (Rymowicz et al., 2010).

2. Biochemistry of citric acid formation The biochemistry of CA formation is a complex task due to the combined effect of different nutritional conditions in the medium (Max et al., 2010). From a technological point of view, understanding of biochemical reactions to produce CA is very meaningful Valorization of Biomass to Bioproducts https://doi.org/10.1016/B978-0-12-822888-3.00004-9

Copyright © 2023 Elsevier Inc. All rights reserved.

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for understanding of kinetics and reactor designs (Behera, 2020). Excessive CA can be produced by using A. niger through the glycolytic pathway under specific environmental conditions. In certain microbes like A. niger, CA can be produced as an overflow product due to faulty operation of the CA cycle (tricarboxylic acid cycle (TCA)) (Angumeenal et al., 2003). In brief, CA biosynthesis takes place through uptake of sugar substrate and then glycolytic catabolism of glucose to 2 mol of pyruvate and subsequently their conversion to oxaloacetate and acetyl-CoA. After condensation of these two precursors, CA excretes from the mitochondria and mycelia, respectively (Karaffa et al., 2001). Different conditions, such as carbon source concentration, dissolved oxygen, hydrogen ions, and suboptimal concentrations of trace metals or phosphate, synergistically may affect the production yield of CA (Max et al., 2010). For instance, the ability of A. niger to overproduce citrate through a glycolytic pathway has gained substantial interest because citrate is one of the best-renowned inhibitors of glycolysis. Under specific nutrient conditions, due to the accumulation of numerous positive factors of the phosphofructokinase gene 1 (PFK 1), such as ammonium ions, citrate inhibition is curbed (Arts et al., 1987). In the case of in vivo, citrate inhibition of PFK 1 to be antagonized by the ammonium ions (Habison et al., 1983) and the antagonism is functionally related to the wellknown effect of metal ions like manganese ions on CA accumulation. As documented by Habison et al. (1983), the breakdown of proteins under manganese deficiency leads to a high intracellular NH concentration, and this phenomenon is known as “ammonium pool.” It inhibits the vital phosphofructokinase enzyme, which converts glucose and fructose into pyruvate (Show et al., 2015). Consequently, there is a flux through glycolysis and CA formation (Papagianni, 2007). The high glucose and NH concentrations repress the formation of 2-oxoglutarate and eventually inhibit the catabolism of CA present within the TCA cycle. As observed in Fig. 1, at the outside of the cell, the way of reactions leading from sucrose to CA commences, where the membrane-bound invertase enzyme converts the sucrose to fructose and glucose to transport into the cell. A. niger contains numerous enzymes, and one of them has a 1000 times more affinity for glucose than fructose. But the citrate has been observed to be a non-competitive inhibitor of this enzyme (Steinb€ ock 2+ et al., 1994), which may be due to the chelation of the Mg (needed to bind ATP). However, under physiological conditions, it may be irrelevant where an excess of Mg2+ ions are present (Papagianni et al., 2005). The content of trehalose-6-phosphate is imperative in order to regulate the flux from glucose into glycolysis, as indicated by research experiments on mutants of A. niger (Arisan-Atac et al., 1996). The phosphorylation of fructose 6-phosphate by one of the two phosphofructokinase (PFK1 and PFK2) enzymes is the first and foremost reaction step of this pathway. The PFK1 phosphorylates the C1 position of the fructose 6-phosphate to make it fructose-1, 6-bisphosphate (Papagianni, 2007). The combination of activators and inhibitors content in the productive cytoplasm permits the accumulation of CA through glycolysis (Habison et al., 1983).

Citric acid

Fig. 1 Schematic representation of the metabolic process in CA production. CA, citric acid. (From Show, P.L., et al., 2015. Overview of citric acid production from Aspergillus niger. Front. Life Sci. 8(3), 271–283. https://doi.org/10.1080/21553769.2015.1033653.)

In order to use glucose and other carbohydrates for maintenance and biosynthesis, both the glycolytic and pentose phosphate pathway function as channels for Aspergillus species (Show et al., 2015). As documented previously, pyruvate kinase is an essential regulatory stage in CA synthesis; however, a research study exhibited that the enzyme in its pure form was only marginally affected by the levels of inhibitors. The catalytic breakdown of glucose through glycolysis produces two molecules of pyruvate. Two separate reactions subsequently convert the pyruvate into the precursors of citrate that are oxaloacetate and acetyl CoA. The study of Cleland and Johnson (1954) for the first time exhibited that in these reactions; A. niger uses an amount of carbon dioxide (CO2) for the production of oxaloacetate that equals the amount of carbon dioxide released throughout the acetyl CoA formation. It is imperative for producing high CA contents, because the only alternate to from oxaloacetate would be a complete cycle of the TCA cycle, which is associated with the loss of two molecules of CO2. If this occurs, only two-thirds of the carbon source would accumulate in the form of CA and the remaining at least a third would be wasted (Papagianni, 2007). However, if oil is used as a substrate for Y. lipolytica, the first step involves utilization of oil by microbes is its hydrolysis by extracellular lipases with the formation of glycerol and

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fatty acids, which appear in the medium in the phase of active growth. Lipase and the key enzymes of glycerol metabolism (glycerol kinase) and the glyoxylate cycle responsible for the metabolism of fatty acids (isocitrate lyase and malate synthase) are induced just at the beginning of the growth phase and remain active in the course of further cultivation (Morgunov et al., 2018). Glycerol enters into cellular metabolism by means of its phosphorylation through glycerol kinase (Makri et al., 2010). However, fatty acids enter yeast metabolism through the glyoxylate cycle, which functions actively because of the great quantities of acetyl-CoA (main product of lipid oxidation). Consequently, the produced high concentration of acetyl-CoA, suppress the pyruvic acid oxidation and thereby the functioning of the Kreb’s cycle. The key enzymes of the glyoxylate cycle, i.e., isocitrate lyase and malate synthase, catalyze the conversion of iso-citrate to succinic and glyoxylic acids with the formation of an end product, malate (Papagianni, 2007).

3. Production of citric acid Although CA fermentation is one of the primitive fermentations, its production has been growing over the years (Auta et al., 2014). On an industrial level, most of the CA production is performed by using the A. niger strain, while various other species belonging to the same genus are capable to produce CA (Torrado et al., 2011). The increased demand for CA collectively motivated the researchers to focus on more economical production methods (Auta et al., 2014). Various kinds of microorganisms (e.g., fungi, yeast, and bacteria) have been documented for their potential to produce CA and are thus discussed in the following sections.

3.1 Filamentous fungi The selection of an appropriate fungi strain for the production of CA is very critical because it plays a vital role in the process. On an industrial scale operation, fungus strains should have high sporulation, long-term stability, short fermentation time, resistance to other microbes, and good growth in the substrates, which produce the high yield of CA. The effect of methanol (presence and absence) on the production of CA at various moisture levels in the solid-state fermentation using pineapple, masomi, and mixed fruit waste as substrate for A. niger was determined. Results reported that the stimulating effect of the methanol was less at lower moisture content. In the presence of methanol, the highest content of CA was produced at 70% moisture content, while in the absence of methanol, maximum CA was produced at 60% moisture content. Based on the sugar consumed and in the presence of 4% methanol, the highest content of CA produced by the pineapple, masomi, and mixed fruit waste was 51.40%, 50%, and 46.50%, respectively, and metal ions showed no inhibitory effects (Kumar et al., 2003). In a submerged culture, bioproduction of CA by using A. niger (NRRL 599) on medium prepared after solubilization of sugars by orange peel autohydrolysis was studied. Under the best conditions, the

Citric acid

maximum yield of CA was 9.20 g/L, in the presence of 40 mL of methanol/kg of medium (Rivas et al., 2008). In their next study, as compared to submerged culture, the solid-state fermentation proved a versatile fermentation method and showed no requirement of any additional nutrients and/or treatments before sterilization. They obtained the highest concentration of CA (193.20 mg/g) by using Valencia orange (Citrus sinensis) as substrate for the same microbe in solid-state fermentation. In this process, 0.50
106 spores per g of dry orange was the initial inoculum concentration used, and maximum yield was obtained at 85 h of incubation (Torrado et al., 2011). Utilization of banana peels as a substrate at optimum moisture content gave a yield of 52.08 g/L (Abbas et al., 2016). Under optimal conditions (pH 4.30, 30°C temperature, 3% methanol, and 38 g/L glucose concentration, 50 h fermentation time), fermentation of pineapple waste with A. niger yielded 15.51 g/L of CA (Ayeni et al., 2019). Parkia biglobosa fruit pulp can be harnessed for citric production at an industrial level in lower amounts. The highest yield of CA produced by A. niger on this substrate was 1.15 g/L at pH 2 and at 0.54 mM reducing sugar concentration (Auta et al., 2014). When a basal medium of oat bran was used as a substrate for A. niger, it produced the maximum amount of CA of 62 g/kg (Rao and Reddy, 2013).

3.2 Yeast The various yeast species like Candida oleophilis, C. tropicalus, Y. lipolytica, C. guilermondi (Angumeenal et al., 2003), Debaromyces, Kloekera, Hansenula, Pichia, Saccharomyces, Torula, and Zygosaccharomyces can also produce CA by utilizing carbohydrates and n-alkanes (Weyda et al., 2014; Angumeenal et al., 2003). Among these, Y. lipolytica is a wellrenowned favorable yeast species for CA production (Auta et al., 2014). However, the major disadvantage of utilizing yeast fermentation is that it also produces abundant amounts of isoCA that is an unwanted by-product. Consequently, mutant microbial strains having low aconitase activity are needed (Show et al., 2015). The advantages of using yeast over the A. niger strain are shorter fermentation time and higher productivity. In addition, yeast strains are insensitive to substrate variation (especially molasses), more tolerant for contamination and metal ions, and capable of metabolizing the n-alkanes or high initial sugars (40–60 g/L). All of these advantages lead to remarkable reductions in costs related to substrate, waste treatment, and product recovery (Crolla and Kennedy, 2001). Various studies documented the higher yields of CA production, particularly by C. lipolytica, C. oleophila, and Y. lipolytica on substrates, such as n-paraffins, biodiesel production, glucose, and glycerol from olive mill wastewater-based media (Crolla and Kennedy, 2004; Rywi nska et al., 2010). The use of wild and mutant strains of Y. lipolytica resulted in industrially sufficient quantities of CA production. For example, Aurich et al. (2003) achieved a CA concentration of 198 g/L and a yield of 1.16 g/g, after 300 h with fed bath growth of a wild strain of Y. lipolytica (H181). In other studies, wild strains of Y. lipolytica W 29 and Y. lipolytica

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H222, cultivation on glucose resulted in 49 g/L (Papanikolaou et al., 2009) and 41 g/L (Moeller et al., 2007), respectively. In a study, medium containing glucose under cell growth limitation utilizing phosphor and sulfur resulted in 80–85 g/L of CA with a yield of 0.70–0.75 g/g when using a wild strain of Y. lipolytica VKM 2373 (Kamzolova and Morgunov, 2017). In their next study (Morgunov et al., 2018), 100–140 g/L of CA production was achieved by using rapeseed oil, raw glycerol, and ethanol (waste of the biodiesel industry) as a substrate for the mutant strain of Y. lipolytica NG 40/UV5. Y. lipolytica ACA-DS 50109 was cultivated on the glucose and olive mill waste water, resulted 28.90 g/L and yield of 0.53 g/g (Papanikolaou et al., 2008). This production was increased to 52 g/L and yielded 0.64 g/g by improving the process using the Y. lipolytica ACA-YC 5033 strain and also removed the harmful phenolic compounds from the olive mill waste water (Sarris et al., 2017). Y. lipolytica ACA-DC cultivated in nitrogen-limited aerobic cultures on raw glycerol (by-product produced during the bio-diesel production process) exhibited 62.50 g/L highest CA production and 0.56 g/g of yield after nitrogen depletion in the medium (Papanikolaou et al., 2008). In one study, 27 strains of Y. lipolytica and 5 strains from three other species of Y. clade (Candida bentonensis, Candida hispaniensis, and Aciculoconidium acyleatum) were tested for their potential to produce CA by using glycerol as a substrate under nitrogen-limited conditions. Results reported that non-Y. lipolytica strains could still grow on glycerol but none of them produced the CA. The highest CA-producing strain was the Y. lipolytica NRRL YB-423, with 21.60 g/L CA production and with a 54% yield. Moreover, the CA-to-isoCA ratio resulted from this yeast strain was 11.30 in the initial screen, whereas most of the strains reported a ratio of between 2 and 6 (Levinson et al., 2007). Due to the continuous increase in production of biodiesel, crude glycerol, obtained as a by-product in this process, can be used as a viable substrate for the production of CA and lipids (Souza et al., 2014). The optimized parameters for the production of CA, which resulted in 0.19 g/L CA production was the agitation of 184 rpm, temperature of 30°C, and 38.40 g/L of the crude glycerol (Souza et al., 2014).

3.3 Bacteria Besides yeast and fungi, bacteria are also capable to produce CA. Very few studies emphasizing the production of CA by utilizing the bacteria are very scarce. The most commonly used bacteria for the production of CA include Arthrobacter, Aerobacter, Bacillus, Brevibacterium, Corynebacterium, Klebsiella, Micrococcus, and Pseudomonas. Among all these, A. paraffinens, B. subtilis, B. flavum, and B. licheniformis are the most desirable microorganisms (Pometto et al., 2005). Based on the strain and composition of the medium, all these fermentations were performed under aerobic conditions for 2–5 days at 30–37°C. These studies opened a new horizon toward the production of CA by bacteria (Pometto et al., 2005).

Citric acid

4. Citric acid fermentation methods 4.1 Surface fermentation Surface fermentation (surface culture) is a process in which microorganisms grow on the surface of fermentation media. For example, A. niger is used for CA production by surface fermentation, it grows over the surface of the media as a thick floating mycelial mat (Singh Dhillon et al., 2011). It is an original CA industrial production method that consists of two phases. In the first phase, the development of fungus in the form of mycelial mat on the surface of the medium occurs, and in the second phase, CA is produced from carbohydrates by utilizing them. At the end of the second phase, recovery of CA is performed by washing of mycelial mats, and the impregnated CA is extracted (Max et al., 2010). Although submerged fermentation has acquired tremendous attention in recent years, but still there are small- and medium-scale industries that use surface fermentation (Bauweleers et al., 2014). The main advantages of this fermentation method include the requirement of less energy for aeration and agitation, thereby have lower installation and energy costs with no foam development. However, more labor requirements and sensitivity to changes in the composition of the media are the disadvantages of this system (Benghazi et al., 2014). Conventionally, this fermentation process was performed in fermentation chambers made up of high purity aluminum or polythene and/or special grade steel, but stainless steel is preferred because of its resistant to deformation with prolonged use (Bauweleers et al., 2014). This chamber needs to be significantly ventilated as sterile air passes through a bacteriological filter, continuously over the medium surface. This air serves many purposes, such as supplying the desired oxygen demand (owing to the highly aerobic nature of the process), controlling the temperature and humidity (through evaporative cooling), and removing carbon dioxide (which is an inhibitor to CA production at concentrations >10%). In surface cultures, contamination is an imperative concern and most commonly occurs due to the lactic bacteria, yeasts, Penicillia, and species of Aspergillus (Soccol et al., 2006). This fermentation method has the potential to compete with the promising and well-established method for large-scale production of CA. Consequently, it is an imperative requirement to design and develop same tray bioreactors like submerged fermentation to alleviate the abovementioned disadvantages (Singh Dhillon et al., 2011).

4.2 Submerged fermentation The submerged fermentation system is the most widely used batch fermentation system; however, its continuous system is also possible and is used in practice (Kishore et al., 2008). Submerged fermentation is developed after surface fermentation and has several advantages, such as higher yields and productivity, lower capital, labor costs, low maintenance costs, and lower chances of contamination. Additionally, submerged

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fermentation is less sensitive to media composition changes, thereby provides a broad range of substrates and better control. However, this system has more sophisticated installation, rigorous control, higher energy costs, and the formation of foam (Max et al., 2010).

4.3 Solid-state fermentation In this fermentation, microorganisms grow on solid materials without the presence of free liquid (Krishna, 2005). Moreover, this process refers to the cultivation of microorganisms under low-water activity conditions on a non-soluble material, which functions as both a physical support and a nutrient source (Rao and Reddy, 2013). This solid or support material is usually a natural compound made up of agricultural and agro-industrial waste and residues, urban waste, or a synthetic material (Pandey, 2003). The solid-state fermentation process, also known as “Koji” fermentation, originated in Japan, has been used for the production of industrial enzymes by using a tremendous amount of waste/agroindustrial waste (Kareem et al., 2010). A study documented the utilization of solid-state fermentation as an alternative to submerged fermentation (Kumar et al., 2003) due to higher product yield, lower energy requirements, lower risk of bacterial contamination, less energy concerns about disposal of generated waste, and less production of waste water (Shojaosadati and Babaeipour, 2002). The major benefit of this process is its ability to use inexpensive, extensively available agro-industrial waste as a substrate, and eco-friendlier in contrast to submerged fermentation. This process requires less water and has a lower operating cost, and no requirement of complex equipment. In this system, there is no requirement for pre-treatment as the system is less sensitive to the presence of trace elements in contrast to the submerged fermentation (Berovic et al., 2007). However, the limitation of this method is the incomplete utilization of the available nutrients due to poor heat and oxygen transfer in the substrate. Moreover, a limited pool of viable microbes is available, and strains having large phosphorus and nitrogen needs cannot be utilized (Show et al., 2015). To perform this process, many fermenters have been used, such as Erlenmeyer conical flasks, trays, rotating, and horizontal drum bioreactors.

5. Recovery and purification of citric acid Generally, CA as an end product contains several impurities that include mineral salts, organic acids, and proteins. The recovery of CA from fermented broth depends on the raw materials used in fermentation (Sawant et al., 2019). Therefore, a wide range of recovery methods were established, including fine filtration, precipitation, solvent extraction, adsorption and absorption (by ion exchange resins), and crystallization, as shown in Fig. 2. In general, these recovery processes may remove all solid impurities.

Citric acid

Fig. 2 Schematic representation of CA recovery and purification. CA, citric acid. (From Vandenberghe, L.P.S., et al., 2000. Solid-state fermentation for the synthesis of citric acid by Aspergillus niger. Bioresour. Technol. 74(2), 175–178. https://doi.org/10.1016/S0960-8524(99)00107-8.)

5.1 Precipitation Precipitation is the conventional method used in the CA processing industry by the addition of calcium salt in order to form the soluble tricalcium citrate tetrahydrate (Singh Dhillon et al., 2011). However, this method required the addition of sulfuric acid forming gypsum in order to remove micelles and suspended materials by filtration. Wang et al. (2020) recovered CA (6416 kg/h) by the calcium precipitation recovery method, in which calcium hydroxide (90°C, pH 7) was added and allowed to react for 2 h and then filtered. The residue is mixed with sulfuric acid for the extraction of CA from tricalcium. Another study produced CA from green olive processing wastewater by A. niger using the calcium citrate precipitation method (Papadaki and Mantzouridou, 2019). The findings concluded that the calcium citrate precipitation method could able to recover the 83 g/L of CA with a yield of 0.54 g/g. A systematic review highlighted the 100% yield recovery of CA at 50°C for 20 min (Li et al., 2016); however, studies reported many disadvantages on precipitation method, including high energy consumption, less recovery, multi-step process, generation of secondary pollution, and large waste generation (Singh Dhillon et al., 2011; Sun et al., 2017). The operation cost for a CA plant using precipitation as a recovery method was found to be $70 million with a selling price of $ 0.85/kg (Wang et al., 2020). Sun et al. (2017) used bipolar membrane electrodialysis (BMED) for the recovery of CA from fermented liquid and achieved a remarkable recovery in CA production. The authors selected this method due to its efficiency in the recovery of organic acids via splitting at the interface layer of a bipolar membrane. This study concluded that the BMED could be an alternative to the conventional method for the enhancement of the recovery of CA. Although there are a few limitations in the

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precipitation method, it is a well-established technique for the recovery of CA. Many separation techniques have been introduced recently in CA production to overcome possible disadvantages for CA recovery and purification.

5.2 Solvent extraction Although the precipitation method has been industrially well established, solvent extraction is an alternative to the purification and crystallization of CA. Generally, amine solvent, trioctylamine, and n-octanol (3:7, v/v) are used as the extractants for the extraction of CA, which reduced the environmental impact and eliminates the use of calcium hydroxide, calcium sulfate, and sulfuric acid (Show et al., 2015; Wang et al., 2020); however, other solvents, such as trioctylphosphine oxide, tributylphosphate, combination of trioctylphosphine oxide and tributylphosphate, quaternary ammonium salts, and ionic liquids can be used (Sprakel and Schuur, 2019). In the same study, the solvent systems for the extraction of carboxylic acids, including CA, were highlighted; for example, one study stated the three categories of solvents, including nitrogen-based extractants, phosphorous-based extractants, and ionic liquids (Sprakel and Schuur, 2019). In the same study, it was also explained the CA extraction mechanism using different solvent systems. At low pH, CA is able to form protonation that could highly interact with tertiary amines, and the extraction temperature was maintained at 27°C for 30 min (Wang et al., 2020) since the process was exothermic. Amine-based extractants reported high extraction efficiency and selectivity; and solutes could be used for the re-extraction. A study compared the different CA recovery methods (e.g., solvent extraction, precipitation, and ion exchange). Solvent extraction method recovered 6416 kg/h of CA with an annual operating cost of $54 million and further considered as the technically feasible method for extraction of CA due to its low production cost (Wang et al., 2020). The main advantages of solvent extraction are: low-cost production, eliminates by-product formation, consumes negligible amounts of mineral acids, and technical feasible method over the conventional method of CA recovery.

5.3 Ion exchange Ion exchange or adsorption resins may be used in the recovery of CA separation and purification. These resins (ion exchange resins and macro porous adsorption resins) collectively act as adsorbents, while other components pass through the elution bed (Gluszcz et al., 2004). The good physical and chemical properties of resins (stable, insoluble in acid, alkali, and organic solvents) led to their selection in the recovery of CA. Jianlong et al. (2000) studied the adsorption of CA from molasses integrated with in-situ product separation by ion-exchange resin adsorption. The authors connected the anion-exchange resin packed-column with a fermenter to separate CA from fermentation broth. The results concluded that the production of CA was increased by 1.60

Citric acid

times that of initial production (0.338 g/L). Gluszcz et al. (2004) investigated the properties of 18 types of ion-exchange resins for citric and lactic acid recovery. According to the study, weakly basic resins are recommended for the recovery of organic acids. Moreover, Amberlite IRA-67 was reported as the best resin for the separation of CA. Wang et al. (2020) also investigated the adsorption mechanism of CA using anion and cation resins. In the same study, the CA production rate by the ion exchange recovery method was recorded as 6416 kg/h with a $61 million annual operation cost and reported less environmental impacts. Overall, production and recovery of CA are involved with major production costs; however, precipitation and solvent extraction methods are frequently used recovery methods in organic acid production. There are several different technologies used for the separation of CA, including electrodialysis with bipolar membranes (Pinacci and Radaelli, 2002), biological electrodialysis (Luo et al., 2017), and electrodeionization (Widiasa et al., 2004). These methods could be prospective and cleaner technologies for the production of CA.

6. Factors affecting citric acid production Generally, CA production is strongly influenced by various factors (e.g., major nutrients) in the fermentation medium. Over the years, many techniques have been introduced to increase CA productivity by optimizing several fermentation conditions. It was shown that the strongly influenced factors including chemical and physical factors (Table 1). Monitoring and optimization of these factors are essential for attaining the high productivity of CA.

6.1 Carbon source The carbon source can play an important role in the production of CA. Therefore, several studies that centered to understand the role of different carbon sources on CA production. More recent study used different carbon sources (e.g., ethanol, glycerol, starch, glucose, sucrose, fructose, maltose, and mannitol) for the production of CA and concluded that glucose was preferred carbon source for two novel yeast strains (Hesham et al., 2020). A systematic review highlighted the role of mono and disaccharides as a preferred source in the recovery of high CA (Show et al., 2015). Mono and disaccharides are quickly available for the microorganisms in fermentation and help in the high recovery of CA. A study focused on the production of CA using glycerol as a carbon source (Da Silva et al., 2009). Another study optimized the different sugar sources (e.g., glucose, sucrose, and fructose) and concluded the high recovery (1.76 g/L) of CA using sucrose as a carbon source (Lee et al., 2015). These results could be attributed to the intracellular concentration of fructose-2, 6-diphosphate that activated the phosphofructokinase gene (PFK 1). In contrast, another study suggested that glucose as an effective carbon source is followed by galactose, maltose, dextrose, fructose, sucrose, lactose, and trehalose. It was also highlighted that the

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Table 1 Chemical and physical factors affecting CAa production. Sources

Examples

Chemical factors

Carbon

Nitrogen

Phosphorous

Trace elements Lower alcohols Other factors

Plant oils (Darvishi et al., 2009); crude glycerol (Souza et al., 2014); ethanol, glycerol, starch, glucose, sucrose, fructose, maltose, and mannitol (Hesham et al., 2020); mono and disaccharides (Show et al., 2015); glycerol (Da Silva et al., 2009); glucose, sucrose, and fructose (Lee et al., 2015); analytical glycerol, raw glycerol, cane molasses, glucose syrup, high fructose syrup, and high glucose syrup (Cavallo et al., 2020); sugarcane bagasse (Campanhol et al., 2019). Urea (Darvishi et al., 2009); corn steep liquor (Liu et al., 2015); ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium dihydrogen phosphate (Mostafa and Alamri, 2012); urea and yeast extract (Xie and West, 2009); diammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium chloride, ammonium ferrous sulfate, urea, ammonium phosphate monobasic, and ammonium sulfate (Hesham et al., 2020); intracellular ammonium ion (Ikram-Ul-Haq et al., 2005); ammonium nitrate (Pietkiewicz and Janczar, 2000). Monosodium phosphate, dipotassium phosphate, monopotassium phosphate, phosphoric acid, and disodium phosphate (Mostafa and Alamri, 2012); dipotassium phosphate (Francisco et al., 2020); phosphorous (Kim et al., 2006); potassium phosphate (Campanhol et al., 2019). Fe+3, Zn+2, and Mn+2 (Guilherme et al., 2008); Cu, Fe, and Zn (Alani et al., 2007); sodium, calcium, zinc, and copper (Lotfy et al., 2007). Ethanol and/or methanol (Campanhol et al., 2019); ethanol and methanol (Haq et al., 2003); methanol (Alani et al., 2007); ethanol and methanol (Yaykas¸lı et al., 2005). Almond, caster, maize, nigella, olive, peanut, soybean, and sunflower (Adham, 2002); lipids (Souza et al., 2014); plant oils (Darvishi et al., 2009).

Physical factors

pH Aeration a

3–5 (Karthikeyan and Sivakumar, 2010); 5 (Alani et al., 2007); 2 (Auta et al., 2014); 60 g/L IA yield. By overexpressing the glucoamylase gene in a genetically engineered A. terreus, the production rate increased to 77.6 g/L (Huang et al., 2014a). Starches from sago, sorghum, sweet potato, wheat, potato, and cassava have been successfully used to produce IA with varying degrees of success the use of these sugars as substrates for IA production contributes significantly to the production costs. The use of cheaper substrates, such as agro-wastes, could potentially bring down the cost of IA production (Alvira et al., 2010). Vassilev et al. (2013) produced 44 g/L IA from dried olive wastes and beet press mud using Aspergillus terreus CECT 20365, while a yield of 55g/L IA was obtained in a patented SSF method utilizing a mutant, A. terreus M8, on sugar solutions or starch hydrolysate adsorbed on sugarcane press mud (Tsai et al., 2001). MuralidharaRao et al. (2007) reported production of IA with a maximum yield of 24.5g/L from jatropha seed cake after five days of cultivation of a soil isolate A. terreus. The use of market refuge apple and banana extracts (90 g L 1) as substrates for fermentation by A. terreus mutant strains N45 and UNCS gave yields of 29–30 and 31–32 g IA L 1, respectively, in 6 days at 34°C and pH 3.0 (Reddy and Singh, 2002).

6.2 Fermentation of IA process requirements Fermentation of the IA process needs some requirements for reach to adequate fermentation (Table 1). IA fermentation is strictly aerobic. An adequate oxygen supply is essential because anaerobic conditions will irreversibly damage the biomass. Kuenz et al. (2012) reported that an interruption of the oxygen supply for 10 min has an influence on morphology and causes a dramatic decrease of IA productivity. IA production is strongly affected by several medium components, including Fe, Mn, Mg, Cu, Zn, P, and N. Many studies have been conducted on the influence and regulation of these substances during the production process (Roehr and Kubicek, 1996). The IA yield is significantly improved by the presence of nitrogen and trace amounts of Zn2+ and Fe2+ ions (Lockwood and Reeves, 1945), while phosphate ion should be limited once mycelial growth is established to prevent carbon diversion into further mycelial production. A wide range of fermentation times have been studied for IA fermentation ranging from 2 to 14 days (Bressler and Braun, 2000). The optimum production time has however been reported to be seven days by many authors (Kocabas et al., 2013). Hevekerl et al. (2014) studied the effect of pH on IA yield, and the highest concentration of itaconate achieved was 146 g/L at pH 3 with A. terreus DSM 23,081 strain. The initial pH has to be low for the microorganisms to achieve the IA production capability (Larsen and Eimhjellen, 1955). The reported temperatures for IA production also vary, but it is optimally kept at around 37°C (Willke and Vorlop, 2001). After mutagenesis, for example, at 40°C, an A. terreus strain was able to produce five-fold higher amounts of

Applications of itaconic acid in biofuel production

IA than the parent strain (Kariya and Fujiwara, 1994). As observed in various studies, different parameters influence the final production of IA: the used microorganisms, temperature, medium concentrations, the initial pH and the variation of pH values through the fermentation process, oxygen supply, and also the formation of side products (Kuenz and Krull, 2018). So that, the creation of competent microbial cell factories for IA production through genetic engineering should bring substantial benefits for the production levels (Huang et al., 2014b). To compete with conventional chemical methods, bio-based processes yielding chemical building-blocks should have a production rate of 50 g/L, 80% theoretical yield, and a volumetric capacity of about 3 g/L (Bozell and Petersen, 2010).

7. Industrial applications of IA Currently, IA has gained importance as a fully sustainable building block chemical for wide applications for the manufacture of various synthetic resins, coatings, and polymers. It has applications as super-absorbents, phosphate-free detergents and cleaners, and bioactive compounds, particularly in the pharmaceutical industry and agriculture (Fig. 3).

7.1 Polymeric hydrogels and nanohydrogels applications IA and its polymers are utilized in several applications as novel substitute monomers. Hydrogels are used in both medical biology (Stanojevic et al., 2006) and biotechnology

Fig. 3 Some industrial applications of IA.

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(El-Halah et al., 2019) due to their capability to absorb high amounts of liquid. The structure of hydrogels consists of cross-linked polymer chains and can absorb amounts of liquid when placed in aqueous solutions (Ahmed, 2015). IA, together with its various esters, can feasibly co-polymerize with acrylamide and facilitate the swelling parameters of formed hydrogels. The highest degree of swelling capability was achieved with acrylamide and monomethoxyethyl itaconate hydrogel synthesized with a molar ratio of 70/30 (Dannert et al., 2019). Amonpattaratkit et al. (2017) studied high and low molecular weight of gelatin-methacrylate and gelatin-IA cross-linkers percentages for hydrogel microstructure, the improvement of biodegradation, cross-linking behavior, and swelling ratio characteristics of hydrogels. According to this study, the low molecular weight hydrogel had a slightly higher swelling ratio and faster degradation, but with the increase of IA content, the swelling ratio increased in both hydrogels. Biodegradable acrylamide hydrogels that include IA and itaconate groups have essential application in waterdecontamination due to outstanding features such as low-price, precise handling, and reusability (Foungfung et al., 2011). Naturally, starch is occurring a biopolymer in high concentrations with several applications (Elgadir et al., 2012). A study by Soto et al. (2016a) starch-based superabsorbent materials with the natural compounds IA and maleic acid. The use of maleic acid has high water solubility, but its use as an adsorbent was difficult. Itaconate starch mono and diester show competent cross-linking and substitution degrees. Itaconate starch diester hydrogel separated the metal ions Pb2+, Cd2+, Ni2+, and Zn2+ in the concentrations of 11.24, 7.11, 5.10, and 8.44 mg/g and with a retention capacity of 145.03, 82.78, 56.74, 101.44 mg/g individually (Soto et al., 2016b). Hydrogel presented a competent and cheap Mn2+elimination method from polluted waters (Sharma and Tiwari, 2016). The renewable IA is used in the food industry, especially in active packaging like smart nanohydrogels and in the delivery of food preservatives (Trif et al., 2019). Bio-based lactic acid and its co-polymers present a valuable alternative building block in the production of synthetic polymers. The bulk polycondensation, a co-polymer was synthesized from lactic acid, butanediol, and IA (Mehti€ o et al., 2017). The co-polymerization of IA with different natural biopolymers usually improves the hydrogel structure and increases the degree of cross-linking (Ge et al., 2017). The essential challenges are that face hydrogels are resource recovery, regeneration, reusability, and recovery of hydrogels (Khan and Lo, 2016). In the anti-microbial field, hydrogels are useful biopolymers because of their high absorption capacity and controlled drug release (Yang et al., 2018). IA and its derivatives are novel co-monomers used in a variety of pH-sensitive microgels in anti-tumor drug delivery (Sun et al., 2015). IA is a remarkably hydrophilic anti-microbial agent, and it is capable of building hydrogen bonds with analogous groups. With the addition of different amounts of IA and cross-linking agents, drug release and drug-loading quantities can be efficiently controlled (Stanojevic et al., 2006). Also, due to pH tolerance of co-monomer is efficiently used for drug delivery in the

Applications of itaconic acid in biofuel production

gastrointestinal tract. The introduction of IA in polymeric chains improves the pH-sensitive and complexation characteristics of the hydrogel. The enhanced swelling behavior owed to the two carboxylic groups in its structure reacts to electrostatic repulsion at the appropriate pH and medium (Peppas et al., 2000). IA-based polymeric hydrogels accomplished the best action in inhibiting the Candida albicans fungus. So that IA shows a promising application in vaginal infections and wound treatment to protect against infections. Hydrogels polymerized with IA are non-toxic and 88% biodegradable under natural processes. A wide variety of microorganisms like fungi, bacteria, protozoa acted on the hydrogels under aerobic degradation (Sakthivel et al., 2018). The increase of IA and reduction of the crosslinking agent positively influenced the water and drug uptake of the investigated hydrogels (Stanojevic et al., 2006).

7.2 Other applications Currently, the annual production of methylacrylic acid is about 3.2 million tons (Choi et al., 2015). The polymerized esters of IA are used as plastics, adhesives, elastomers, and coatings. IA is also used as a co-monomer in polyacrylonitrile and styrene-butadience co-polymers. It can be used for the synthesis of 3-methyltetrahydrofuran, a potential biofuel (Corma et al., 2007). IA is used in the manufacture of emulsion paints, where it improves the adhesion of the paint. When IA is added at a level of 5% in acrylic resins, it imparts to the resins the ability to hold printing inks (van Balken, 1997). In plastics and coatings, a 1%–5% addition confers on the product benefits such as a light color, ease of painting and separation, water-fastness, and antiseptic properties. IA provides opportunities for selective enzymatic transformations to create useful polyfunctional building blocks (Ferraboschi et al., 1994). It has the potential for replacing the markets for malic anhydride and sodium tripolyphosphate which are used for making unsaturated polyester resin and detergent builders, respectively (Choi et al., 2015). IA plays an important role during inflammation and acts as an endogenously produced anti-microbial compound against pathogens (Cordes et al., 2015). It can inhibit microbial growth by interfering with enzymes of central metabolism, including the glyoxylate shunt, 2-methylcitrate cycle, and TCA cycle. In the pharmaceutical industry, IA is used as a hardening agent in organosiloxanes for use in contact lenses; in binders for use in diapers and feminine napkins, in the production of glass ionomer cement, a biocompatible cement used in dentistry (Okabe et al., 2009).

References Ahmed, E.M., 2015. Hydrogel: preparation, characterization, and applications: a review. J. Adv. Res. 6, 105–121. Alvira, P., Toma´s-Pejo´, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101, 4851–4861.

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CHAPTER 5

Lactic acid Praveen Kumar Dikkalaa, Barinderjeet Singh Toora, Pradeepa Robertsb, Blessy Sagarc, Kairam Narsaiahd, Srinu Dhanavathe, Zeba Usmanif, Vijai Kumar Guptag,h, Rajeev Bhati, and Minaxi Sharmaf a

Department of Food science and Technology, Punjab Agricultural University, Ludhiana, Punjab, India Millet Processing and Incubation Centre, PJT Agricultural University, Hyderabad, India c Department of Food process Engineering, Acharya N.G. Ranga Agricultural University, Bapatla, India d AS & EC Division, ICAR-CIPHET, Ludhiana, India e College of Food and Dairy Technology, TANUVAS, Chennai, India f Department of Applied Biology, University of Science and Technology, Baridua, Meghalaya, India g Biorefining and Advanced Materials Research Center, SRUC, Kings’s Buildings, Edinburgh, United Kingdom h Center for Safe and Improved Food, SRUC, Kings’s Buildings, Edinburgh, United Kingdom i ERA-Chair for Food (By-) Products Valorisation Technologies (VALORTECH), Estonian University of Life Sciences, Tartu, Estonia b

1. Introduction The economic value of the agricultural industry waste is generally less for collection and recovery for reuse, which affects waste utilization that could be valuable resource if appropriate bioprocessing techniques are utilized to produce a range of novel products (Ezejiofor et al., 2014). Agricultural residues are produced at a rate of 3.5 billion tons per year worldwide, mainly used as carbohydrate feedstock. These feedstocks are used to produce different value-added substances like amino acids, enzymes, organic acids, and vitamins ( John et al., 2007). The wastes generated from agricultural and food industries are the richest sources of fermentable sugars, which can be easily degraded by microbial strains and further they are converted into highly valued components (Ali and Zulkali, 2011; Usmani et al., 2020). Organic acids are the most common examples of valuable products obtained from fermentation technologies from different industrial wastes (Singhania et al., 2009). The purest form of glucose is the substrate for lactic acid (LA) production. The different waste substrates such as molasses, rice, wheat bran, wheat straw, and bread waste (Ghaffar et al., 2014) have generally been utilized in LA production. The by-products and processed food wastes like bran, seeds, pomace, and peel and are the richest source of different nutrient components such as starch, protein, minerals, and also dietary fibers that support the growth of LA strains to produce LA (NunezGaona et al., 2010; Saman et al., 2011; Kim et al., 2003). In the fermentation, the corn steep liquor is used as a nutrient source in the commercial application of the fermentation process. It is an inexpensive complex broth received from the wet milling process (milling of food grains) that consists of proteins, carbohydrates, minerals, and other organic acids (Nancib et al., 2001) can be a good platform for the production of LA using these Valorization of Biomass to Bioproducts https://doi.org/10.1016/B978-0-12-822888-3.00003-7

Copyright © 2023 Elsevier Inc. All rights reserved.

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nutrients by LA bacteria. The trends in the development of renewable resources and their relationship with sustainability in the environment play an important role in the recovery of different fermentation products such as LA and other bioactive ingredients.

2. Biomass feedstocks LA is produced by using different technologies, that is mainly depending on the cost of raw materials, plays an important role. The major and important sources of nutrients for LA bacteria growth and the production of valuable products are food industry wastes (Grewal et al., 2020). 1/3rd of the total food is wasted globally from the initial production to consumption of the food, which is around 1.3 billion tons per year. Various bioactive compounds can be extracted from agri-food waste, having huge applications in the food, pharmaceutical, and therapeutic industry (Sharma et al., 2021). The food industrial wastes include different carbohydrate polymers (cellulose, hemicellulose, lignin), organic acids, proteins, and lipids. The total sugars range from 35.5% to 69% and proteins range from 3.9% to 21.9% (Kiran et al., 2014). LA bacteria grow preferably in the carbohydrate environment because of that the different starchy biomass is gaining momentum in the agricultural feedstocks due to their abundance availability and low amounts of lignin content (Zhao et al., 2014). The proper utilization of agricultural and food-based feedstocks ensures greater environmental and economic benefits. Moreover, the lignocellulosic pre-treatment has many advantages; these by-products are mass utilized in the different biorefineries (Usmani et al., 2021). The complex composition leads to the generation of inhibitors after the pre-treatment process, which extended the lag phase (Kim, 2018). The low-cost agricultural residues which are extensively used in LA production are rice saccharificate, corn steep liquor, sugarcane juice, cassava fibrous waste, corn flour hydrolysate, residues from corn, cotton meal, whey protein hydrolysate, palm jaggery, and whey permeate (Fukushima et al., 2004; Coelho et al., 2011; Zhao et al., 2014). With amylolytic enzymes, the different types of microbial strains are used in hydrolyzing agricultural residues such as wheat, barley, and corn to produce LA. And, the corn steep by-product is utilized effectively in the LA production. The liquor from the corn steep consists of 85% of the nitrogen, containing proteins, peptides, and amino acids (Wee et al., 2005, 2006). In the bio-based chemical production lignocellulosic biomass is used, which is an abundant renewable resource of cellulose, lignin, and hemicellulose which are made up of pentose sugars (Kawaguchi et al., 2016). The use of lignocellulose plays a key role due to its wide availability, less cost, no competition, and renewability. The annual production of lignocellulosic biomass around the world is about 10 metric ton (Ahring et al., 2016). The valorization of lignocellulosic materials using biotechnological processes in the different sectors exhibits a variety of applications. However, lignocellulosic feedstocks are good substrates for LA production, but the presence of complex carbohydrates such as cellulose and hemicellulose limits their exploitation process.

Lactic acid

However, pre-treatment is required for LA production, which is a multi-step process. The pre-treatment steps for the degradation of lignocellulosic biomass are: 1. Saccharification—with the different hydrolytic enzymes, including cellulases, cellobioses, and xylanases. 2. Microbial fermentation—monomeric sugars are converted into LA. 3. Downstream processing—is the final step for the recovery of pure LA. The LA production with the fermentation process depends on many different factors, including the cost of raw materials, which plays an important role. Thus, lignocellulose biomass play a key role in the bio-economy (Ahring et al., 2016). However, the energy-rich crops such as switch grass, elephant grass, and other industrial residues are used in LA production. Hydrolysis is the major step which converts the polysaccharides into the potentially fermentable sugars. This is done through the process of acid or enzymatic catalysis. In the process of substrate conversion by enzymatic hydrolysis, this process requires low-energy input, which gives the high conversion efficiencies (Ballesteros, 2010).

3. Enzymatic saccharification of biomass Enzymatic hydrolysis of the biomass is one of the most critical steps of the LA production system. Generally, biomass is a mixture of starch, cellulose, hemicellulose, and lignin. It is very important to break down these molecules into their simpler forms so that the microorganisms can ferment the simple sugars into LA and achieve higher production efficiency (Hari Krishna and Chowdary, 2000). Although dilute acids (like sulfuric acid) have been used for this purpose, enzymatic hydrolysis has certain advantages, for instance, higher degree of hydrolysis, fewer inhibitory by-products generation, low toxicity, mild processing conditions, and less energy requirements. However, certain disadvantages include high cost of enzymes, longer hydrolysis period, and product inhibition (Mohammad and Keikhosro, 2007; Chen et al., 2008). Saccharification of cellulose and hemicellulose is carried out by cellulase and hemicellulase enzymes. For cellulose, a hydrolysis mixture of three cellulase enzymes is utilized which synergistically acts on the structure to yield simple sugars (Abdel-Rahman et al., 2011; Sarkar et al., 2012). These include: (a) Endo-β-1,4-glucanases—in the amorphous region, these enzymes randomly hydrolyses the β-1,4 glycosidic bonds and yield oligosaccharides. (b) Cellobiohydrolases (Exo-β-1,4-glucanases)—these enzymes act on both the reducing and non-reducing ends of the polymer and yield glucose and/or cellobiose. They can also carry out their biological activity in the crystalline region of a polymer. (c) β-Glucosidases—these enzymes hydrolyse the cellobiose molecules into glucose units and complete the hydrolysis process. They have an important role in relieving the system from end product inhibition. In contrast to cellulose, hemicellulose is a heteropolysaccharide containing various fractions, including xylan as the major one. Xylan possesses a complex chemical structure and

81

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requires multiple enzyme systems to achieve the desired degree of hydrolysis. Enzymes such as endo-1,4-β-xylanase, β-glucuronidase, acetylxylan esterase, β-xylosidase, and α-L-arabinofuranosidase have successfully been utilized to degrade the xylan molecule (Carvalheiro et al., 2008). Moreover, α-mannosidase and β-mannanase are required for cleaving the backbone of glucomannan molecules (Kumar et al., 2008). Although hemicellulose is more complex, the lower degree of crystallinity makes this polymer more bio-accessible to enzymatic hydrolysis than cellulose (Keshwani and Cheng, 2009). Microbial species like Bacillus, Thermomonospora, Erwinia, Trichoderma, Clostridium, Phanerochaete, Cellulomonas, Streptomyces, Fusarium, and Penicillium, are certain examples who can produce cellulase and hemicellulase enzymes for the hydrolysis of biomass. Trichoderma reesei and Trichoderma viride are most widely studied for the production of cellulase enzymes, which is ascribed to the beneficial properties of the enzymes produced, including high stability and resistance to chemical inhibitors. However, the low activity of β-glucosidase enzyme is major limitation of Trichoderma. Furthermore, Aspergillus has also been utilized due to its ability to efficiently produce β-glucosidase. Several investigations have utilized Trichoderma and Aspergillus’s mixed cultures to obtain the desired degree of hydrolysis (Sarkar et al., 2012) (Fig. 1).

4. Microbial fermentation During the fermentation process, LA bacteria produces a number of bioactive components, such as bacteriocins, biogenic amines, exopolysaccharides, proteolytically released by peptide groups. The LA is synthesized by two industrial processes either by the chemical process or microbial fermentation. The fermentation process with the different microbial strains gives the pure LA, whereas LA chemical synthesis gives the racemic

Fig. 1 Value-added products of enzymatic hydrolysis of lignocellulosic biomass.

Lactic acid

mixture (Randhawa et al., 2012). L (+)-LA existence has high purity, which provides poly-LA with a high melting point and crystallinity (Oh et al., 2005). LA is utilized in the polymerization process to form the poly-LA with a great interest because of its biodegradable nature. The production of poly-LA with the use of starch as a potential substrate. Different types of poly-LA-based products are replacing the petroleum based products (Ilmen et al., 2007). The preference of feedstock always depends on price, availability, recovery, and purification of LA. Lignocellulosic biomass is low-cost substrate, extensively available, renewable source of carbon that does not have any food value, so it can be used as a conventional source. In the process of fermentation Kluyveromyces, Rhizopus, and Escherichia strains were used to produce latic acid by Maas et al. (2008). LA production always depend upon the strain and also the conditions during the process (temperature, pH, initial substrate concentration, concentration of nitrogenous nutrients). For L. delbrueckii, the optimum temperature was 45–60°C with a pH of 5.0–6.5 and for L. bulgaricus, the temperature was 43°C with a pH of 6.0–7.0 as suggested by Zhou et al. (2003). The time for the fermentation process is about 24–48 h under optimum conditions. The LA yield depends upon the initial starch or sugar content (Zhou et al., 2003). In the batch distillation process, the hydrolysis of methyl lactate enhances the production of LA (Edreder et al., 2010). The process of fermentation always depend upon the microbial growth, substrate nature, and broth viscosity. The developments in the fermentation process can enhance the fermentation of LA production. Advance fermentation techniques include batch, fed-batch, repeated, and continuous fermentations are discussed in brief.

4.1 Batch fermentation It is the most commonly used method. Except for the neutralizing agents (to control pH), the carbon substrates and other components are not included in this process. Different kinetic studies showed that with the increase in glucose concentration, LA concentration also increased (Yun et al., 2003; Kadam et al., 2006). The highest concentration of LA was observed when batch fermented with Lb. paracasei subsp. paracasei CB2121 (Moon et al., 2012). The risk of contamination is less in this process, which yields more LA when compared with the another fermentation process (Hofvendahl and Hahn-Hagerdal, 2000). The batch fermentation process has some disadvantages, including low percent of cell concentrations with less amounts of nutrients and low productivity.

4.2 Fed-batch fermentation This is better fermentation method that maintains the substrate concentration at a low level of feeding when compared with batch and continuous fermentation. Continuous or sequential substrate utilization takes place in this process of fermentation (Ding and Tan, 2006). It is more beneficial when high substrate concentrations affect the growth

83

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of cells and productivity (Lee et al., 1999; Roukas and Kotzekidou, 1998). As like other fermentation processes, in the fed batch fermentation is also influenced by many factors (substrate time and concentration, fermentation broth, substrate feeding method). It is very difficult to perform the trail and error tests with so many open-ended variables, which play an important role in the fed-batch fermentation process (Wu et al., 2011). For the production of LA, different innovative fed-batch fermentation processes have been developed and discussed, as shown in Table 1.

4.3 Repeated fermentation In this type, both the fed-batch and batch fermentations are involved in repeating the cycles using inoculated cells (Zhao et al., 2010a). The different methods that are used include bacteria cell recycling, centrifugation, filteration process, or mycelia pellet precipitation process. The rate of LA production could be increased with the repeated microbial fermentation process. Zhao et al. (2010b) showed that, in a 2–6 open and repeated batch fermentation process, the maximum L-LA was produced with Bacillus strains. The purity of LA was about 99.8%, obtained from this process. The study by Yu et al. (2007) revealed that small mycelial pellets of R. oryzae NRRL 395 for 9 cycles over 14 days, got 2.02 g/L/h of L-LA. In an airlift bioreactor, LA productivity is 1.9 fold higher than the other methods of fermentation.

4.4 Conventional continuous fermentation As LA production is always associated with cell growth, the cells always receive the energy for their growth from LA pathways. With product dilution in the fermentation broth and medium, the end products are separated in this method (Wee and Ryu, 2009; Yu et al., 2015). In chemosata fermentation, the method of feeding with fresh medium and withdrawing the fermentation broth takes place at a time to maintain the concentration of components in the fermentation broth. In chemostat fermentation, the substrates, cells, and products concentration in the fermentation broth can be maintained at constant levels during certain periods. In this process, the dilution rate adjusts the growth rate that would maintain the equilibrium (Bustos et al., 2007). The dilution rate plays a key role in productivity improvement. Productivity is decreased during the lag phases in the continuous fermentation process. It has less frequency than the batch fermentation process (Ohara et al., 1992; Gassem et al., 1997). Carbon sources, efflux products, and some other cells are unutilized in the fermenter, these reduces the concentration of the LA by increasing the dilution rate in the LA production (Zhang et al., 2011).

4.5 Fermentation using different substrate In the batch fermentation process, different fermentation methods are used, such as simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF). In this process, a mixed culture or open fermentation process is used to

Table 1 Different studies of LA production by batch fermentation. Lactic acid Isomer and optical purity (%)

Strain

Concentration (g/L)

Yield (X/S) (g/g)

Production (P/S) (g/L/h)

Broken rice

Lb. delbrueckii

79.0

0.81

3.58

D (96.1)

Corn starch

Lb. plantarum NCIMB 8826 (engineered)

73.2

0.85

3.86

D (99.6)

Corn stover

Lb. rhamnosus and Lb. brevis (mixed culture)

21.0

0.70

0.58

ND

Glucose

Lb. paracasei subsp. paracasei CHB2121

192

0.96

3.99

L (96.6)

Glucose

Bacillus sp. Na-2

106

0.94

3.53

L (99.5)

Fermentation substrate

Fermentation method

Reference

SSF with glucoamylase performed in 5-L fermenter with 2.5-L basal medium at 40°C, 150 rpm, pH controlled at 6.0 with Ca(OH)2 Performed in a 2-L bioreactor with a 700-mL working volume, at 37°C, 100 rpm, pH controlled at 5.5 by NH3 solution SSF with cellulases Performed in 250-mL flasks containing 100-mL media at 37°C, shaking at 100 rpm, initial pH 5 with CaCO3 Performed in a 2.5-L jar fermenter with a working volume of 1.5-L at 38°C and 200 rpm, pH controlled at 6.5 by addition of NaOH Two-stage aeration method Open fermentation performed in a 5-L bioreactor

Nakano et al. (2012)

Okano et al. (2009)

Cui et al. (2011)

Moon et al. (2012)

Qin et al. (2010) Continued

Table 1 Different studies of LA production by batch fermentation—cont’d Lactic acid

Fermentation substrate

Strain

Concentration (g/L)

Yield (X/S) (g/g)

Production (P/S) (g/L/h)

Isomer and optical purity (%)

Glucose

Rhizopus oryzae GY18

115

0.81

1.6

L (98.5)

Jerusalem artichoke tuber extract

Lb. paracasei KCTC13169

92.5

0.98

1.2

L (93.2)

Sucrose

H. halophilus JCM 21694

65.8

0.83

1.1

L (98.8)

Sucrose

Escherichia coli (engineered)

85.0

0.85

1.0

D (98.3)

Sucrose

Rhizopus oryzae GY18

80.1

0.89

1.67

L (98.5)

Fermentation method

containing 4-L unsterilized fermentation medium at 50° C, pH controlled at 6.0 by NaOH Performed in 500-mL flask at 35°C and CaCO3 as a neutralizing agent Performed in 5 L jar fermenter containing 2-L medium at 37°C, at 150 rpm, pH controlled at 6.0 with NaOH. Performed in a 5-L jar fermenter with 2.5-L fermentation medium at 30° C, 250 rpm, pH-controlled at 9.0 by NaOH. Performed in 15-L fermenter with 10-L medium at 37°C, 200 rpm, and pH 7.0 controlled by of a 3.5 M Ca(OH)2 slurry Performed in a 500 mL-flask at 35°C and CaCO3 as a neutralizing agent

Reference

Guo et al. (2010) Choi et al. (2012)

Calabia et al. (2011)

Wang et al. (2012)

Guo et al. (2010)

Lactic acid

increase the LA production from various substrates as shown in the Table 1. The SSF method has many advantages, those helps in enhancing the productivity of LA. The advantages in this process includes single reaction vessel, less enzyme loading, quick processing time, reduced end product inhibition by hydrolysis. In SSF fermentation method, Lb. delbrueckii strains are used for broken rice, in which starch is broken down by hydrolysis to glucose and then to LA (Nakano et al., 2012). In SSF method of fermentation 0.97 g/g of LA and in SHF 0.81 g/g of LA is produced from the recycled sludge with Lactobacillus rhamnosus ATCC7469 (Marques et al., 2008). They concluded that the SHF fermentation process is a prolonged process to separate enzymatic hydrolysis, which can reduce productivity. The mixed strains of Lactobacillus brevis, and L. rhamnosus used in the consumption of cellulose and hemicellulose derived sugars from corn straw (Cui et al., 2011). The saccharification and fermentation processes improved the LA production yield by 0.70 g/g with NaOH treatment with corn straw with mixed fermentation process. Thus, improving the production of LA by 18.6 and 29.6% from L. rhamnosus and L. brevis, respectively. Wang et al. (2012, 2014) utilized Actinobacillus succinogenes and metabolically engineered Escherichia coli for the production of LA and reported improved production efficiency of these strains. Different fermentation modes using different microbial strains can be shown in Table 2. Several studies found an increase in the productivity of LA by the open fermentation process by using complex natural microbes (Sakai et al., 2004; Ennahar et al., 2003). Different conditions and methods used for fermentation processes show the effect on microbial count and its structure which consequently produce LA. For example, maintenance of pH in the fermentation broth reduced the activity of the bacteria with rice bran powder. In this process, the pH is maintained at 5, with strain Lb. delbrueckii IFO 3202 that produces 28 g/L of D-LA from 100 g/L of rice bran with a yield rate of 0.78 g/g with an optical purity of 95% (Tanaka et al., 2006). With the different fermentation methods, the yield rate, concentration and productivity has been enhanced. The pH during the process of fermentation partly reduces the production and recovery of LA that has resulted in an increase in cell concentration by 50 fold with dialysis membranes (Savoie et al., 2007). During the process of fermentation, the acids are removed selectively during the process of electrodialysis that reduces the productivity of LA. Some of the challenges that can be achieved by reverse electro enhanced dialysis can be achieved higher yield of LA and biomass and fouling effect can be reduced with the periodic charge reversal (Prado-Rubio et al., 2011). Such methods are used for the better fermentation of LA and its characteristics with high purities and yields and concentrations in addition to the eco-friendly LA is obtained instead of lactate salts.

5. Product recovery As the demand of LA is increasing day by day in the fields of medicine and pharmaceuticals, the product recovery is an important aspect in the production process. Chemically,

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Table 2 LA production in different fermentation modes by different producers. Fermentation mode

Concentration (g/L)

Yield (X/S) (g/g)

Productivity (g/L/h)

K. marxianus Lb. helveticus Lb. bulgaricus Lb. helveticus and K. marxianus (mixed culture) Lb. bulgaricus and K. marxianus (mixed culture) Lb. helveticus and Lb. bulgaricus (mixed culture) Lb. helveticus and Lb. bulgaricus and K. marxianus (mixed culture) Lb. casei NRRL B-441

Batch Batch Batch Batch

8.8 10.1 9.6 15.5

0.24 0.23 0.30 0.45

4.3 5.1 4.8 10.0

Batch

16.2

0.41

10.5

Batch

14.6

0.35

Lb. casei SU No. 22 and Lb. lactis WS 1042 (mixed culture) Lb. bulgaricus ATCC 8001, PTCC 1332 Lb. helveticus R211

Strains

Reference

Plessas et al. (2008)

9.4

Batch

19.8

0.47

12.8

Batch

96.0

0.93

2.2

Batch

22.5

0.48

0.93

Fed-batch

46.0

0.77

1.91

Batch

24.6

0.81

–

Fakhravar et al. (2012)

Continuous

38.0

–

19–22

Schepers et al. (2006)

B€ uy€ ukkileci and Harsa (2004) Roukas and Kotzekidou (1998)

Lactic acid

LA is a mixture of D- and L-forms (racemic mixture) (Zhou et al., 2003). Separation and purification of LA play an important role to meet the standards of commercial applications. Racemic mixture is impossible to control both the physical and chemical properties of the final product. Some industries require pure form of enantiomers for LA production with specifications ( Juodeikiene et al., 2015). The fermentation process of LA production is more attractive than the chemical process. It is relevant, when LA monomer for polymer applied in different food and pharmaceutical industries. With the fermentation, there are many advantages, including low substrate cost, low temperatures, low energy consumption with high purity. It is very easy to create products with tailor-made characteristics (Wang et al., 2015). Product recovery is a vital step in LA production, which is always associated with the separation, purification of LA from fermentation broth. In the conventional process of LA production, purification plays an important role in the purity of LA extraction. The different alternatives for this industrial procedure are being started. The LA purification involves different types of techniques such as separation by ion exchange method, reactive extraction, distillation, electrodialysis, and membrane technology (Gonzalez et al., 2008).

5.1 Separation and purification of LA In the process of extraction of LA, the separation and purification methods are same. Various methods of separation and purification used for LA production are shown in Table 3. The fermentation broth is primarily treated with calcium carbonate for neutralization in the traditional process of separation. The calcium lactate with broth is filtered, treated with carbon, and it is acidified with sulfuric acid to produce LA which is insoluble in the calcium sulfate (Datta and Henry, 2006). In the process of pure LA extraction, different steps are involved, such as hydrolysis, esterification, and distillation. The major disadvantage in this process is that it produces a large amount of calcium sulfate by-product with high sulfuric acid consumption (Qin et al., 2010). The current advancements in methods of LA separation techniques include adsorption, reactive distillation, electrodialysis, ultrafiltration, and nanofiltration. The LA purification method doesn’t yield the salt waste (Kumar et al., 2006; Datta and Henry, 2006; Ha´bova´ et al., 2004; Madzingaidzo et al., 2002; Li and Shahbazi, 2006). These processes of separation are more costly and energy efficient in comparison with the traditional chemical separation process. Equilibrium-based processes, membrane separation techniques, solid-liquid separation processes, and reaction-separation processes are the major techniques involved in the LA separation process (Ramaswamy et al., 2013; Huang et al., 2008).

5.2 Purification with the downstream process Economic and ecological implications play an important role in the final LA purification process. LA recovery and purification plays represent around 20%–50% of the operating

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Valorization of biomass to bioproducts

Table 3 Various separation technologies used in lactic acid production. Process of separation

Principle

Equilibrium-based processes

Absorption Distillation Solvent extraction

Ion exchange resins used in the absorption process Volatility of the compounds and heat plays an important role in this process It is based on the solubility of solute in two solvents

Affinity-based separation

Adsorption Ion exchange Simulated moving bed chromatography

Ion exchange resins are used in the absorption process The absorption of product with the ion exchange resins The mobile phase changes counter current to the stationary phase

Membrane separation

Electrodialysis Filtration

With the electric potential difference, the ions from the solution are separated with the ion exchange membrane In this process, depending on size of pore, pressure, size the particles, or molecules are retained in the membrane

Solid-liquid separation

Conventional filtration Crystallization and precipitation

Depending upon the size of pore, flow, and pressure the particles are retained in the membrane In this process, the product is concentrated by the process of evaporation and further it is crystallized

Reaction-separation systems for process intensification

Separation by reaction membrane method Fermentation by extraction Process of reactive distillation Reactive absorption

Bioreactors-membrane In situ extraction of product Chemical reaction. After the process of distillation. It is used when the product is having low volatility Product extraction process with in situ with the resins (ion exchange)

costs. The LA recovery is the conventional process (extraction by the precipitation process). First, LA is precipitated with calcium hydroxide, during downstream processing. Then the filtration process further recovers this. Finally, sulfuric acid is utilized to release LA that generates large amounts of CaSO4 (Ramaswamy et al., 2013). The diluted product is purified sequentially with the activated carbon and evaporated further, by the process of crystallization. The extraction of LA by precipitation has some disadvantages, the cost is high due to the reagents that are applied during the process, more filtration steps are required and more solid waste is generated (calcium sulphate sludge), which leads to

Lactic acid

environmental problems (Lee et al., 2017; Wasewar et al., 2002, 2003; Datta et al., 1995). This process usually reaches up to 50% of the LA production (Wasewar et al., 2002, 2003). In LA purification, the elimination of residues plays an important role. Residual ions, sugar, cells, and broth components play a vital role at the end of the process. As the LA process is a non-volatile process, it has a higher affinity toward the molecules of water. The distillation and evaporation processes are inefficient energetically (Yankov et al., 2004). In this aspect, research is required for the alternative separation, purification of LA through fermentation, which reduces the cost of production and its impact on the environment, which is essential to increase the process competitiveness. There are different techniques for the separation process, as shown in Table 4. Neutralization and acidification steps are crucial unit operations used during the pure form of LA production. Due to fermentation, acidity increases, leading to low pH and high concentrations in fermenting chamber. It impedes the cellular viability through the highly protonated form of LA, thereby incidence of intracellular reserves is packed with LA as it intersects through membranes (Grabar et al., 2006; Abdel-Rahman and Sonomoto, 2016). Hence, in neutralization, excess calcium carbonate or calcium hydroxide is added to obstruct the excess acid produced during fermentation. The neutralizer added retains the pH and forms a calcium salt of the acid, i.e., calcium lactate (Datta and Henry, 2006). Other than neutralizers, even acid tolerant strains can also be used. Further, using standard techniques such as electrodialysis, solvent extraction, adsorption and membrane bioreactors (Abdel-Rahman and Sonomoto, 2016) can also be used for in-situ separation of LA so that microbial growth is also hindered. Acidification is generally done with sulfuric acid to precipitate calcium sulphate or gypsum, which revokes separation and then evaporates to produce LA. Further researchers proposed certain neutralizers such as Ca(OH)2, NH4OH, and NaOH, addition of ethanol during acidification of ammonium lactate (Kwak et al., 2012) and purification and recovery using base of alkaline and precipitation with sulfuric acid.

6. Technical challenges in LA production Conventional methods of LA production by fermentation, yield a pure quality of LA, involve several purification steps to reach the final outcome. The primary challenge starts in the form of raw material collection, and it is generally obtained from refined sugars, starch, etc., and this consequently shares 40%–70% of LA production (Tejayadi and Cheryan, 1995). As a substitute affordable feedstock, like lignocellulosic biomass, is also recommended for reliable production. Meanwhile, care should be taken while preliminary processing when this type of material is required to produce LA, as these mixed sugars and undesirable compounds which reduce the production efficiency. This problem can be confronted by pre-treatment and hydrolysis of complex lignocellulosic biomass (Abdel-Rahman et al., 2011). But certain consequences arises due to pre-treatments

91

Table 4 Different separation processes in downstream fermentation for lactic acid recovery. Separation method

Feedstock

Concentration

Yield

Purity

Reference

Evaporation process by the short-path method Hybrid short-path evaporation

Synthetic

–

–

7.13  0.80

Fermentation

–

–

89.7

Nano and micro filtrations

Fermentation

937

–

99.7

Fermentation and separation integration system Reactive distillation

Fermentation

183.4

97

–

Komesu et al. (2015) Komesu et al. (2017) Pleissner et al. (2016) Wang et al. (2014)

Synthetic

347.68

99.94  2.17

–

Esterification (Vapor permeation)

Fermentation

–

95

–

Membrane integration separation process Nanofiltration with cross flow FPA 53 and CR 5550 (Amberlite resins)

Fermentation Fermentation Fermentation

– – –

76 – 90

99.5 85.6 –

Molecular Distillation process Membrane- integrated hybrid reactor system (three phase)

Fermentation Fermentation

– 250

74.09 96

95.6 95

Komesu et al. (2015) Khunnonkwao et al. (2012) Lee et al. (2017) Sikder et al. (2012) Pleissner et al. (2017) Yu et al. (2015) Pal and Dey (2013)

Lactic acid

which lead to the inception of other by-products such as phenolic compounds released during lignin degradation. This kind of inhibitory by-products formation remains as impediment but certain productive approaches, for instance usage of fabricated feedstocks that produce less affective inhibitory compounds, and use of designed strains and inhibitor tolerant, etc. can meet some challenges. Different detoxification methods such as activated charcoal treatment, ion exchange method, reduction agents addition, microbial, or enzymatic detoxification have been suggested to decrease the inhibitory compounds (Zhao et al., 2013). These methods also intensify the processing efficiency and enhance the production cost. Different modifications should require on pre-treatment strategies such as isolation of new inhibitor tolerant strains, detoxification of huge concern about the development of the dexterous process in the process of LA from the lignocellulosic biomaterials. Several other challenges in the form of substrate inhibitory effects can be addressed by using powerful strains with high substrate tolerance, but these again decline the LA production after considerable time, which may be due to the formation of undesirable products, and this can be trounced by isolating novel strains and modifying cultivation conditions and inherently modifying genetic strains (Zhang and Vadlani, 2015). Furthermore, LA deposition backlogs the fermentation process, which affects the production economically. Therefore, cell recycling by membrane filtration, cell immobilization, washing and sterilization of the fermenter also uplifts the LA production (Abdel-Rahman et al., 2013). In addition, purification of LA needs to clean up the broth loaded with finest impurities such as residual sugars, color, nutrients, and other organic acids as part of cell mass. Hence, these impurities should be deterged before further processing. Separation and purification are the methods followed during LA production, and separation by precipitation is the most known method. The raw material and the culture added to it instigates the fermentation process, but it would be more difficult to separate the LA from the broth. Hence, technical challenges need to overcome during these steps. The process of LA, though, seems to be complex, but the technological innovations thrash critical problems and produce pure products. The principal issue is the procedure followed for LA separation in conventional technique, which is incompetent to produce a high value of end product owing to certain imperfections in the process (Komesu et al., 2017; de Oliveira et al., 2018). Consequently, the innovative methods adopted for better product purification exaggerate product cost, and it turns out to be more expensive production. The optimization of production aspects should furnish a better economic outcome. Hence, from raw material to end product evolution, critical attention is necessary for such kinds of products. Though advanced techniques yield high-quality products, still certain drawbacks impedes the production on a large scale. Extensive research should be intended on technologies that can produce genuine products on a large scale and enhance production yield but not shoot up economic terms.

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Valorization of biomass to bioproducts

7. Process advancements Every technology has certain advantages and disadvantages, and the trouble shooting of the problems definitely adds more benefits to the process. Similarly, though traditional techniques involved in LA production are inaccurate in meeting global trends, the innovative technologies augmented the yield and showcased high productivity (Ismail et al., 2018). Novel technologies and their approaches involved in LA production to enhance the yield and purity are briefly described in Table 5. Table 5 Novel approaches in LA production. Novel technology

Principal

Problem with earlier technique

Co-culture technique

Two or more strains with a specific degree of interaction produce the desired product.

Due to homo fermentative strains utilization of by-products such as acetic acid and ethanol were produced adding extra costs in the separation and purification phase in LA production.

Genetic and metabolic engineering

These focusses on heterologous gene expression or pathway redirection

Immobilized bioreactor design

Technique of physically or chemically fixing biocatalysts in a specific region to carry out catalysis of a particular reaction Enhancement of production by increasing cell density

The low acid tolerance of strains and their ability to utilize a limited amount of substances is one of the common problems. Due to continuous usage biocatalysts activity comes down

Cell recycle fermentation

Industrial production relatively lowers due to end product inhibition

Novel approach

Using of homo fermentative strains along with hetero fermentative strains reduces the production of by-products such as ethanol and acetic acid finally reducing separation and purification costs to some extent. Increases acid tolerance and re-direct pathways to consume substrate more efficiently Immobilization is avoiding activity loss of valuable biocatalyst and allowing its repeated use.

As the production of LA is based on cell growth and final biomass the novel process enhances cell density and productivity.

Lactic acid

Table 5 Novel approaches in LA production—cont’d Novel technology

Principal

Simultaneous saccharification and fermentation

Process aims in turning polysaccharides into soluble sugars form by hydrolysis in order to facilitate the use of substrate more efficiently

Problem with earlier technique

In traditional process hydrolysis process occurs separately

Novel approach

This method provides single-step enzymatic hydrolysis and microbial fermentation.

8. Conclusions With different innovative techniques, sustainable and conventional development of LA production is required. It is a well-known organic acid, widely used in different food processing, medicinal, and chemical industries. The derivative poly-LA (PLA) is an important biodegradable polymer and is gaining tremendous attention in the food packaging industry. LA purity and yield can be increased by using different innovative techniques with engineered microbial strains (fermentation and biotechnological processes). The raw materials which are used in the recent years to produce LA play an important role in the LA production. For the commercial production of LA, the use of agri-food waste, mainly lignocellulosic biomass, is a considerable approach which further supports environmental health. There is a need to improve LA production with fermentation and food-biotechnological processes to improve the purity and yield using improved or engineered LA strains. Biotechnological utilization of agricultural and food industrial wastes to produce LA solves the environmental issues and makes the process cheaper and more productive. For the efficient production of LA, there is still need to further research to address the importance of different factors such as type of: agri-industrial waste, involvement of microbial strains, fermentation process, downstream processing, etc.

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CHAPTER 6

Valorization of biomass to levulinic acid Nazim Hussaina, Muhammad Asim Raza Basrab, and Aatika Sadiab a Center for Applied Molecular Biology (CAMB), University of the Punjab, Lahore, Pakistan Institute of Chemistry, University of the Punjab, New Campus, Lahore, Pakistan

b

1. Introduction Levulinic acid, IUPAC name: 4-oxopentanoic acid (C5H8O3), is recognized as a gamma keto-acid containing 5 carbons. Due to its keto and acid functional groups, levulinic acid exhibits multi-functionality and is classified as a top industrially important platform chemical by the US Department of Energy (Chen, 2015). It is derived from biomass that includes organic matter of plant and animal origins. It has gained importance as an economic biofuel material. Biomass contains chemical energy acquired from the sun and stored in the form of biochemicals such as hexoses and pentoses. Green chemistry utilizes this form of renewable energy so that these natural biomolecules are processed and converted to various platform molecules (Tukacs et al., 2012). The platform molecules derived from levulinic acid are sought as potential substitutes for the traditional non-renewable energy resources, especially petrochemicals. Biomass provides raw material in the manufacture of hydrocarbon fuel chemicals as compared to other renewable energy resources such as wind, water, tidal, solar, and thermal sources, which only provide potential energy, respectively (Braden et al., 2011). The surplus natural production of biomass, the cost-effective, and eco-friendly synthesis of levulinic acid and the vast variety of derivatives produced are the key factors that make it an economical and favorable platform chemical. The lignocellulose-derived levulinic acid is a chief contributor to liquid hydrocarbon fuels that are utilized by the transportation sector. Levulinic acid has been utilized industrially as fuels, plasticizers, and polymers. While its use is not limited to the industries, the levulinic acid derivatives are also utilized as cosmetic stabilizers and humectants, pesticides, and herbicides. It is evident that levulinic acid, due to its poor solubility in hydrocarbon liquids, requires to be converted to its derivatives to become a petrochemical fuel. However, it requires energy to start and maintain the derivatization process of levulinic acid. The valeric fuels are reported to overcome this issue by first hydrogenating levulinic acid to give n-pentanol and n-pentanoic acid, also called valeric acid. By forming a 10-carbon straight chain ester, the valeric acid and pentanol produce potent biodiesel. Many other

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chemicals derived from levulinic acid are synthesized for the improvization of petrochemicals. Such platform chemicals chiefly constitute γ-valero lactone along with methylated tetrahydrofurans and ethyl levulinate ester. These chemicals are easy to blend with the traditional fuels and improve the efficiency of the fuels (Pileidis and Titirici, 2016). The utilization of levulinic acid is not limited to the fuel and transportation industries. Many derivatives of levulinic acid are used in the manufacture of polymers, varnishes, and resins. Therefore, levulinic acid is a renowned and distinguished platform chemical as it serves as a multi-dimensional use. Above all, the preservation of non-renewable energy resources and consumption of renewable resources with eco-friendly conversion processes, the reduction of pollution as a consequence of reduced combustion of fossil fuels, the vast types of derivatives and their respective applications, and multi-dimensional uses make levulinic acid a cornerstone platform chemical in green chemistry.

2. Physical and chemical properties of levulinic acid Levulinic acid occurs in solid and liquid forms. The general appearance of levulinic acid is that of a white crystalline solid. The liquid form is yellow to brown in color, it may be found congealing into the solid crystalline form. Levulinic acid has a specific caramel-like odor. The presence of the hydroxyl and keto functional groups in the molecule makes it fairly soluble in water as well as alcoholic solvents (Table 1).

3. Traditional production of levulinic acid A traditional method to produce high purity, analytical grade levulinic acid is by the hydrolysis of maleic anhydride. In this process, the production of diethyl ester of maleic anhydride is followed by acylation of the activated double bond and the formation of acetyl succinate. The selective decarboxylation of this acetyl succinate in the vicinity of a basic or acidic catalyst produces levulinic acid and gives a prominent yield of up Table 1 Physical and chemical properties of levulinic acid (Antonetti et al., 2016). Physical properties

Chemical properties

Molar mass Melting point Boiling point Relative density Appearance

pKa Solubility

4.64 H2O, alcohol, diethyl ether, polar organic solvents, and oils.

Heat of formation Heat of vaporization


2417 kJ/mol

116.11 g/mol 33.0°C 245.5°C 1.340 g/cm3 at 20°C White crystalline solid

74.4 kJ/mol

Valorization of biomass to levulinic acid

to 94% (Moens, 2002; van der Waal and de Jong, 2016). However, this process is quite expensive. The transformation of biomass into a platform chemical like levulinic acid diminishes its cost of manufacture. Lignocellulose-based biomass materials are inexpensive feedstock with less than 5% of the cost as compared to that of maleic anhydride. Although maleic anhydride is a predominant feedstock for the production of high quality, analytical grade levulinic acid, it is not economical to use expensive raw materials. Therefore, the use of fuels derived from lignocellulosic materials can considerably alleviate the market price.

4. Biomass conversions to levulinic acid Biomass is generally composed of organic matter from animal and plant origins. The natural production of biomass exceeds 150 billion tonnes C/year approximately (Corma et al., 2007). Natural biomass production makes it a popular choice for fuel production since the raw material is constantly being replenished naturally by solar energy (Field et al., 1998). The biomass conversions of levulinic acid provide environmentally friendly substitutes of traditional industrial chemicals and fuels. Levulinic acid and its derivatives such as γ-valerolactone, δ-Amino levulinic acid, α-angelica lactone, and tetrahydrofuran have vast range of applications in industries (Kang et al., 2018). Hence, levulinic acid and its derivatives are economical in production, safe for the environment, and provide practical solutions to the problem of rapidly diminishing non-renewable energy resources. The lignocellulosic biomass chiefly comprises cellulose fibers, lignin, and hemicellulose, which are categorized as residues from crops and forests and the municipal and paper wastes. The cellulose part of lignocellulosic biomass consists of polysaccharides and disaccharides, chiefly featuring glucose combined with glucose to give long chains and with fructose, respectively. While the hemicellulose and lignin content of biomass play the role of a barrier that protects the cellulose contents from detriment, the hexoses from hemicellulose can also be processed to form levulinic acid (Kumar et al., 2008). Various catalytic conversion routes can be adopted to synthesize levulinic acid originating from the lignocellulose-derived biomass. The conversion of glucose and fructose to levulinic acid requires a series of acid catalyzed hydrolytic reactions (Fig. 1). However, the levulinic acid is shallow in yield due to its reactivity (Ya’aini et al., 2012). The biomass conversion routes for levulinic acid include homogenous and heterogenous acid catalyzed hydrolysis, solvolysis with supercritical and ionic liquids, hydrolysis of furfuryl alcohol, and from maleic anhydride, respectively.

5. Factors affecting levulinic acid yield The presence of two different functional groups in the molecule makes levulinic acid a versatile reaction intermediate. During its synthesis, various different routes and methods

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Fig. 1 Generalized scheme of synthesis and applications of levulinic acid.

can be adopted to obtain a better yield. The yield of levulinic acid depends on several factors such as type of raw material used, pre-treatment methods including temperature, residence time, concentrations of solvents, and acids along with the kinetic factors of reaction such as the type of catalyst used.

5.1 Selection and pretreatment of raw materials The raw material used for the production of levulinic acid is usually plant-based such as paper, crops, and forestry residues, including wheat straw, bagasse, starch, sorghum grains, pulp residues, and water hyacinth. The best theoretical mole percent yields are given by using bagasse (82.7%), wheat straw (68.8%), and pulp residues (70%–80%) as raw materials (Rackemann and Doherty, 2011). The pre-treatment of the raw material is done by separating unwanted material, followed by crushing and grinding of the raw material. The depolymerizing is done by chemical treatments of the raw material, which exposes cellulose and makes the glucose residues accessible and easier to be converted to levulinic acid products. The temperature required for this process is more than 200°C for better yield. The better homogenizing yields a higher quantity of levulinic acid.

5.2 Homogenous catalysis by mineral acids Mineral acids such as HCl, H2SO4, and HBr are extensively utilized as catalysts in the synthesis of levulinic acid from lignocellulosic-derived biomass. The maximum yield of levulinic acid is obtained by using HBr, followed by HCl and H2SO4, respectively. HNO3 does not give a better yield owing to the formic acid formed as the main product, which is undesired (Hayes et al., 2006). This method is widely applied in the synthesis of levulinic acid on an industrial scale as there is no requirement of neutralization of the products as the acid catalyst and water are recycled to be used for further synthesis of levulinic acid. The use of dilute mineral acids and controlled hydrolysis under medium heat and pressure are some of the key factors in the synthesis of levulinic acid.

Valorization of biomass to levulinic acid

The commercial synthesis of levulinic acid is based on degradation of sugar polymers like lignocellulose by the use of strong acid as catalysts. The raw material in the form of lignocellulose is treated with mineral acid and processed under high pressure in the presence of steam. The products are filtered to separate the solid by-products. The levulinic acid formed is extracted from the acid by a suitable solvent and the acid is recycled for further use. Hence the acid catalyzed synthesis of levulinic acid is cost-effective. The levulinic acid thus produced is purified by evaporation of solvent followed by distillation.

5.3 Heterogenous catalysis The biomass conversion to levulinic acid is carried out by using solid catalysts such as amberlite, nafion SAC-13, and clay catalyst. The yields of levulinic acid formed using solid catalysts are much lower as compared to those using homogenous acid catalysts. However, this process is quite efficient for the manufacture of hydroxymethyl furfural in the absence of water by using solid catalysts like amberlyst-15, ion exchange resins, and methyl isobutyl ketone, exhibiting mole percent of 100%, 97%, and 73%, respectively (Shimizu et al., 2009). Heterogenous catalysts exhibit the highest potential for the levulinic acid synthesis attributed to their simple yet energy-efficient separation, recyclable nature, lesser corrosion, and debris disposal issues. In this regard, additional advanced studies are required for use of heterogenous catalysts so that the efficient synthesis of platform chemicals may be brought to use on an industrial scale.

5.4 Hybrid catalysts A relatively new technique uses hybrid catalysts such as the catalysts composed of Bronsted-acid zeolites like LZY zeolite, supported with metal halides as Lewis acid. Such catalysts, for example, CrCl3 supported by HY zeolite (SiO2/Al2O3 ¼ 30) or other similar hybrid catalysts are used for the manufacture of levulinic acid from biomass for their synchronous reactions (Ya’aini et al., 2012). These hybrid catalysts are efficient in the synthesis of levulinic acid by catalyzing two or more reactions at the same time, producing a levulinic acid yield of up to 66.1%.

5.5 Hydrolysis of furfuryl alcohol Apart from the hexose sugars, the pentoses can also be utilized as precursors for the synthesis of levulinic acid through furfural formation. This type of conversion involves additional steps involving the formation of a furfural intermediate. The acid catalyzed hydrolysis of pentoses gives up to 50 mol% of furfural, which is then reduced to furfuryl alcohol by catalytic reduction. The acid hydrolysis of furfuryl alcohol produces levulinic acid with a yield of up to 93 mol% (Otsuka et al., 1973). Levulinic acid produced by this process is of high purity.

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5.6 Solvolysis of biomass While hydrolysis utilizes water molecules for the breakdown and processing of the reactants, another phenomenon called solvolysis utilizes solvents other than water to selectively drive the reaction in the desired direction. Solvolysis is also used in biphasic reactions aiming to isolate certain products from the reaction mixture prevention the subsequent conversions and repolymerization reactions. Much greater yields have been achieved by using solvolysis, for example, hydroxymethyl furfural gave an 87% yield from fructose by using dimethyl sulfoxide and dichloromethane instead of water (Chheda et al., 2007).

5.7 Solvolysis by supercritical liquids Supercritical fluids are substances that are being utilized above their critical temperature and critical pressure. Acidic and basic characteristics are simultaneously displayed by the supercritical liquids. Solvents such as water, acetone, and carbon dioxide are some of the substances that are utilized in their supercritical states for the production of furfural from biomass. Supercritical acetone is reported to give more than 75 mol% yield of furfural from fructose source materials while that of carbon dioxide gives approximately 90% yield of furfural (Sangarunlert et al., 2007).

5.8 Solvolysis through ionic liquids Ionic liquids act as solvents and catalysts simultaneously as these are the salts in their molten state at room temperature. The ionic liquids are thermally stable, non-volatile, and non-inflammable. Many ionic liquids have been used to synthesize hydroxymethyl furfural from biomass, such as N,N-methylacetamide, 1-ethyl-3-methylimidazolium hydrogen sulphate, and 1-H-3-methylimidazolium chloride with 92%, 88%, and 92%, respectively, at temperatures ranging from 90°C to 100°C (Lima et al., 2009). Ionic liquids are advantageous like heterogenous catalysts in that they can be extracted for recycling much easily from the reaction mixture and they retain higher extent of activity. Ionic liquids are also advantageous for their capability to process the reaction at lower temperatures, of the order of less than 100°C.

6. Industrially important derivatives of levulinic acid Levulinic acid has versatile applications due to the large number of various types of derivatives produced by it. Levulinic acid derivatives find applications as anti-freeze agents, fuels and fuel additives for the transport industry, as building blocks of various types of polymers, in medicine; and in the agriculture industry as herbicides and pesticides. There are several derivatives of levulinic acid that are industrially important and have a broad range of applications. Some of the levulinic acid derivatives are discussed as follows.

Valorization of biomass to levulinic acid

6.1 Hydroxymethyl furfural Hydroxymethyl furfural is also called as 5-hydroxymethyl furfural. Its IUPAC name is 5-(Hydroxymethyl) furan-2-carbaldehyde. It is freely soluble in the polar, organic solvents but sparingly soluble in petroleum ether, while in carbon tetrachloride its solubility is very less. It is formed as an intermediate in the synthesis of levulinic acid from hexoses or pentoses. In the fresh natural food materials, hydroxymethyl furfural is absent but it is formed as a result of decomposition by heating. Hydroxymethyl furfural is extensively used as a platform molecule. It has a huge potential as a biomass-derived commodity chemical and is utilized on an industrial scale for the synthesis of various commercial amines, acids, alcohols, and aldehydes. It is also utilized in the manufacture of 2,5dimethylfuran which is a promising fuel. It is also used in the manufacture of petrochemical fuels as carbon-neutral feedstock (Yan et al., 2014).

6.2 δ-Aminolevulinic acid The δ-Aminolevulinic acid is a simplest delta-amino acid and 4-oxo dicarboxylic acid. IUPAC name of δ-Aminolevulinic acid is 5-amino-4-oxopentanoic acid. It is a substitution derivative of levulinic acid and is produced by the replacement of the hydroxyl functional group with that of the amino group. It is approved by the United States Food and Drug Authority (FDA) to be used as a cancer/tumor locating agent. It is used as photodynamic therapy of cancer of various types, including gynecological cancers, bladder cancers, and malignant gliomas. δ-Aminolevulinic acid is also used as a biodegradable herbicide in the agrochemical industry. Another important aspect is that an intermediate in the synthesis of δ-aminolevulinic acid, β-acetylacrylic acid can potentially be used in the manufacturing of new acrylate polymers. In this way, the δ-aminolevulinic acid is utilized in the acrylate polymer industry (Pileidis and Titirici, 2016).

6.3 γ-Valerolactone Gamma-valerolactone or γ-valerolactone is an important derivative of levulinic acid. IUPAC name of γ-valerolactone is 5-methyloxolan-2-one. The reduction of levulinic acid is done by catalytic hydrogenation. This reduction reaction of levulinic acid is followed by dehydration to give γ-valerolactone. The γ-valerolactonecan potentially be utilized as a solvent and has the potential to replace ethyl acetate. It can also serve as a building block of polyesters and also as a precursor for the pyrrolidone isomers. Many catalysts and synthesis routes for the synthesis of γ-valerolactone as feedstock are known. Several processing routes originating from levulinic acid with catalysts such as Pd/SiO4 and RuSn4 have been reported. These aforementioned catalysts exhibit a prominent selectivity of 96% and a conversion of 88%. The greater extents of selectivity and conversions lead to smaller recycle flow and lesser α-angelica lactone formation, which is an important factor, since α-angelica lactone gives rise to coke formation and hence

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catalysts become deactivated. Both of these catalysts can work at a temperature of 180°C for a duration of 10 h. γ-valerolactone is distilled from the mixture as it boils at a lower temperature as compared to that of levulinic acid. The unconverted levulinic acid is then recycled as such from the stream of reaction mixture without further processing (Wettstein et al., 2012).

6.4 Sodium and calcium salts of levulinic acids Like all carboxylic acids, levulinic acid also forms salts with alkali and alkaline earth metals. Sodium levulinate, IUPAC name sodium4-oxopentanoate-based formulations are used as skin conditioners. Some sodium levulinate-based formulations are utilized as humectants, which absorb moisture from the skin. The calcium salt of levulinic acid, IUPAC name calcium4-oxopentanoate is utilized as a mineral supplement (Sharath et al., 2019; Thompson et al., 2008).

6.5 Methyl and ethyl levulinate Methyl levulinate, IUPAC name methyl 4-oxopentanoate, and ethyl levulinate, IUPAC name ethyl 4-oxopentanoate are two important esters of levulinic acid. They have extensive applications in industry from catalytic as well as processing perspectives. There are several advantages of using methyl levulinate as predominantly because its physical state is liquid at room temperature. The lower boiling point of methyl levulinate permits gasphase reactions. It does not act as a weak organic acid and hence is less corrosive. It resists conversion into angelica lactone and hence has more stable catalytic processes (Christensen et al., 2011).These characteristics of methyl levulinate make it a versatile biomass-derived platform chemical. Nonetheless, on the other hand, ethyl levulinate is also a viable biomass-based fuel. The levulinate esters are quite efficiently extracted from the levulinate and mostly formed directly in the manufacturing process, starting from hexoses and pentoses. To keep the fuel products entirely based on biomass, both precursors, including levulinic acid and the alcohols utilized in this process, are to be extracted from biomass. Nonetheless, the biomass-based schemes produce methanol, ethanol, and butanol from biomass. Until now, industrial production schemes for the fuels constituting methyl and ethyl esters of levulinic acid are known to have satisfactory properties as gasoline. The slightly less caloric value per kg of fuel is their only drawback. The levulinic acid esters also find application as fruity fragrances and flavor-enhancing ingredients.

6.6 Pentanoic acid Pentanoic acid, or valeric acid, is an important derivative of levulinic acid. The pentanoic acid is capable to deliver components of both gasoline and diesel fuel to the vehicle and hence is extremely compatible with transportation. The pentanoic acid and its esters have potential

Valorization of biomass to levulinic acid

to outperform the traditional non-renewable fuels (Lange et al., 2010). These biomass-based fuels emerge as an excellent opportunity to conserve non-renewable energy resources. The undeviating synthesis of pentanoic acid from levulinic acid is complicated due to the reason that under the conditions of reaction required for the entire hydrogenation of ketone, the levulinic acid is actively converted to the α-angelica lactone. This reactive molecule either hydrogenates to the stable γ-valerolactone or is rapidly polymerized; therefore, the catalyst becomes denatured. Hence, as a consequence, a direct chemical conversion route of levulinic acid to pentanoic acid is not found. However, the synthesis of pentanoic acid by indirect routes via γ-valerolactone has been reported with satisfactory yield and selectivity. The synthesis of γ-valerolactone and straight conversion of γ-valerolactone to the required pentanoic acid is completed through the formation of pentenoic acid as an intermediate by the use of Pd/Niobium-Silica as a catalyst at a temperature of 300°C.

6.7 Diphenolic acid Diphenolic acid, IUPAC name 4,4-bis(4-hydroxyphenyl)pentanoic acid, is a potential derivative of levulinic acid. It is of particular interest because it can replace the use of bisphenol-A in the polycarbonates manufacture. The resin polycarbonates are extensively used in industry. Hence, the diphenolic acid finds application as a precursor of polycarbonate resins. The scratch-resistant finishes in coatings and decorations are also applications of diphenolic acids. This compound is synthesized by the condensation of methyl levulinate with two equivalents of phenol. In this process, the catalyst used is strong HCl. The selectivity of the two consecutive alkylation reactions on the two phenol rings works as the chief parameter for the synthesis of diphenolic acid. The use of concentrated HCl along with the mercapto acetic acid catalyst system provides greater selectivity. However, a high selectivity of 97.1% is obtained on use of strong HCl and greater phenol-levulinic acid ratio (Guo et al., 2008).

6.8 α-Angelica lactone The α-Angelica lactone, IUPAC name: 5-methyl-3H-furan-2-one, is a butanolide. The dehydration of levulinic acid occurs on prolonged heating. A water molecule is removed, and α-angelica lactone is formed. It is an important building block constituent of the polymer industry. The polymers of α-angelica lactone include caprolactone polymers and co-polymers with styrene (Pei et al., 2020).

6.9 2-Methyl tetrahydrofuran 2-Methyl tetrahydrofuran, IUPAC name: 2-methyloxolane, is a potential derivative of levulinic acid. It is less dense than water, has ether-like odor and appears as a colorless mobile liquid. It is a highly flammable liquid. It is often used as an eco-friendly and

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high-boiling point substitute in place of tetrahydrofuran. Its lesser volatility makes it stable and appropriate option for a biomass-based fuel. The US Department of Energy approves use of 2-methyl tetrahydrofuran as a fuel additive. 2-Methyltetrahydrofuran can be mixed into approximately 50% of gasoline fuel to increase the vehicle’s performance and also reduce the emissions of pollutant gases. 2-methyl tetrahydrofuran also finds application as a solvent for biphasic and other chemical reactions of Grignard reagents. It is also used as a component in alternative fuel cells for the formulation of electrolytes for lithium electrodes (Pace et al., 2012).

6.10 Methyl vinyl ketone and methyl ethyl ketone The methyl vinyl ketone, IUPAC name but-3-en-2-one, and methyl ethyl ketone, IUPAC name butan-2one, are derived from levulinic acid. Methyl vinyl ketone is a reactive compound. It is a highly flammable, toxic chemical if inhaled. However, it can be used by dissolving it in water or other organic solvents. It is particularly useful as an alkylating agent and as a building block of polymers in organic synthesis reactions. Methyl vinyl ketone is also used as precursor of insecticides and fungicides. On the other hand, methyl ethyl ketone is a common solvent. It is utilized in the manufacture of textiles, plastics, lacquers, varnishes, glues, and as a cleaning agent. It is very similar to acetone in properties except that it boils at a higher temperature. Methyl ethyl ketone dissolves plastics, including polystyrene. Hence, it is greatly utilized as a plastic welding agent in the industry.

6.11 Succinic acid Succinic acid, IUPAC name: butanedioic acid is an α,ω-dicarboxylic acid composed of four carbons. It is extensively found in nature as well as a biomass conversion product of levulinic acid. Succinic acid is extensively used as a biomass converted building block in the polymer industry for the manufacture of various polyesters and polybutylene succinate polymers (Siracusa and Lotti, 2018). Apart from polymers, succinic acid is utilized in the manufacture of resins and plastics through their esterification. As a linear aliphatic ester, the succinate ester readily forms plastics and resins. Hence, the industrial applications of succinic acid and succinate ester are vast. It finds extensive use as a feedstock chemical for the production of important industrial chemicals like 1,4-butanediol, adipic acid, tetrahydrofuran, etc., which are applied in agriculture, food, medicine, plastics, textiles, and cosmetics.

6.12 Adipic acid Adipic acid, IUPAC name: hexanedioic acid, is an α,ω-dicarboxylic acid and is a sixcarbon derivative of levulinic acid. It is a white crystalline solid and is practically insoluble in water. It is an extremely important chemical from an industrial perspective as it is used as a main precursor of nylon. Adipic acid forms nylon-66 by a polycondensation reaction

Valorization of biomass to levulinic acid

with hexamethylene diamine. Other than the nylon-66 polymer, it is also used in the manufacture of polyurethane, while the esters of adipic acid are used as plasticizers in the manufacture of polyvinyl chloride (PVC). The biomass-derived adipic acid is especially important as it is involved in the synthesis of modern materials through renewable resources (Marckwordt et al., 2019). Apart from application in the polymer industry, adipic acid is also utilized in medicine for controlled release formulations of hydrophilic drugs and flavoring agents in the food industry.

6.13 Ketals based on levulinic acid The levulinic acid-derived ketals, such as ethyl levulinate ethylene glycol ketal, IUPAC name ethyl 3-(2-methyl-1,3-dioxolan-2-yl)propanoate, have been characterized as bio-surfactants. The levulinate-derived ketal surfactants have applications as detergents such as sodium dodecyl sulfate (Freitas et al., 2015). This is a novel type of ketal produced by the combination of alcohols containing two or more hydroxyl groups, such as glycerol, with the keto functional group of the levulinic acid in the vicinity of a homogenous and strong mineral acid such as HCl. This new class of ketals has outstanding functions. These ketals have the potential to replace the pre-existing surfactants, solvents of petrochemical origin, and the plasticizing agents. A wide range of biomass-derived esters, alcohols, and amines can be utilized for the production of ketal esters of levulinic acid by using standard reaction conditions and catalysts. The ketals formed from levulinic acids find application as detergents on industrial and domestic levels. The production of detergents from biomass conversion is a noticeable achievement of modern times as it is derived from renewable sources and is a promising substitute of the traditional detergents being used. The biomass-derived ketals also present preservation of aquatic organisms as these generate less toxic waste and are eco-friendly alternatives for the traditional detergents.

7. Side products during biomass conversions: The humins Humins are undesired side products formed during the levulinic acid synthesis from biomass. The generation of humins is regarded as inevitable during levulinic acid and hydroxymethyl furfural production due to the thermodynamic nature of the reaction by homogenous acid catalysts. The generation of humins is greatly affected by the initial feedstock concentration as compared to that of the required product. This can be ascertained from the order of the reaction. Since a high order of reaction usually depicts a higher impact of the concentration of the feedstock on the rate of reaction. For instance, in the process of cellulose conversion, the reaction order of hydroxymethyl furfural and glucose in the general reaction is 0.88 and 1.09, respectively. Both these values are lesser than their values of 1.23 and 1.30 in the unwanted side reactions, respectively. Likewise, in the hexose-based reaction, the order of reaction for the hydroxymethyl furfural

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production (1.09) was lower than that for humins (1.30) (Girisuta et al., 2006). It is inferred that the formation of humins exhibits second-order kinetics and that the initial carbohydrate concentration is related to the order of reaction. Although the order of reaction for the humin formation depends on reaction systems, the generally accepted notion is that high starting concentrations of the feedstock give rise to a high yield of humins. The humins have been generally regarded as unwanted side-products due to the following reasons. • The humins are accumulated on the inside of the reactor. Hence, this accumulation results in obstruction of the reactor along with a decrease in the efficiency of heat transfer and an increase in pressure. The increased pressure might cause safety problems; hence, to avoid the safety problems, the humin formation should be avoided. • The carbon content of cellulose is wasted by the formed humin side-products. Due to humin formation, the productivity of valuable platform chemicals decreases in the biorefineries. The yield of humins is generally 20% to 50% which comprises a carbon content of 55% to 65%. This indicates that about 30 to 80% of the hexose carbon is not selectively utilized for the production of a desired chemical. Nonetheless, the relative high formation of humins cannot be ignored in the pilot or industrial level plants. About 25% to 30 wt% of the carbohydrate material is wasted as a humin by-product. The fructose forms approximately 20 mol% humins through reactions catalyzed by oxalic acid in the small-scale production of hydroxymethyl furfural. It is worth noticing that the yield of humins is greatly influenced by the type of the feedstock used, and it is generally found that the order of humins yields is greatest in fructose, followed by xylose and then glucose, respectively. • The effectiveness of the solid acid catalyst is decreased by the formation of humins. Since by the humins may deactivate the solid-state acid catalysts via blockage in their acidic active sites. • Levulinic acid, hydroxymethyl furfural, and carbohydrates can be potentially adsorbed by the humins. These active products, such as levulinic acid, formic acid, and carbohydrate intermediates, are grafted onto the humin matrix via condensation reactions. The humins can also adsorb the homogenous acid catalysts such as H2SO4 as after removing humins by filtration, only about 90.5% of the H2SO4 catalyst remains in the aqueous solution (Kang and Yu, 2016). Therefore, washing with water is needed for the absolute recovery of targeted chemicals, such as H2SO4 catalyst and levulinic acid. In other cases, a large proportion of the final product and catalyst might be wasted owing to the adsorption phenomena of humins.

7.1 Insight into humin structure The humins in the solid states are in fact unusual hydrocarbons which are produced via acid-catalyzed hydrolytic reactions of biomass. Certain humins identified as water

Valorization of biomass to levulinic acid

soluble, in the biomass conversion reaction mixtures that are able to precipitate continuously. The molecular weights of these soluble humins (300–500 g/mol) obtained from the hexoses are about 2 to 3 times higher than those of hexoses (180 g/mol) and 2 to 5 times higher than that of the intermediate hydroxymethyl furfural (126 g/mol). The soluble humins are hence oligomeric comprising 16 to 27 carbons. A carbon content of 65 wt% is assumed for the humins. Of the precipitated humins, approximately 20 wt% may be obtained by using acetone for separation. The framework of insoluble humins is identical to that of the acetone soluble fraction, which indicates that the soluble fraction can be considered as smaller oligomers featuring insoluble humins with a lesser density (van Zandvoort et al., 2013).

7.2 Inhibition of humin generation To inhibit the generation of humins in biomass conversion processes, many methods have been developed. One of these desirable ways is the adjustment of the reaction parameters. Lowering the biomass concentrations, increased acid concentrations along with decreased reaction temperatures are usually able to inhibit the formation of humins in the biomass conversion reactions. A highly concentrated acid (e.g., 0.2 M H2SO4), for instance, can enhance the selectivity of levulinic acid generation from the hydroxymethyl furfural in the process of rehydration catalyzed by acid. Replacing the medium of reaction solution from water to other specialized media is also regarded as a considerable option. These specialized media comprise certain ionic liquids, organic solvents, and biphasic solvent systems, including aqueous-organic solvent systems or ionic liquidsorganic solvent systems. Protection of the activated functional groups present in the main intermediates such as glucose and hydroxymethyl furfural can also be utilized to suppress the generation of humins. The alcohols are considered highly efficient humin-suppressive agents. The larger quantities of methanol present in the reaction mixture cause the generation of methoxymethyl-2-furaldehyde intermediates and methyl glucosides. This leads to a very low yield of humins but high yields of levulinic acid and methyl levulinate. Hence, the formation of humins crucially affects the acid catalyzed biomass conversions of the lignocellulose derived biomass. To prevent the formation of humins in biorefineries, it is important to develop efficient and simple technologies (Patil and Lund, 2011).

8. Conclusion and future outlook Levulinic acid exhibits a phenomenal degree of derivatization and is therefore considered a high priority platform chemical. A variety of hydrocarbons, petrochemicals, polymer precursors, and other highly valuable chemicals can be synthesized by the naturally sustained lignocellulose-derived levulinic acid. The maximized generation of levulinic acid from biomass is hurdled by humin-side products which compromise the synthesis of energy-rich and versatile compounds from it. Using concentrated mineral acid catalysts

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and optimization of media systems can enhance the selectivity and yield of levulinic acid. However, the maximum yield generation of levulinic acid in biorefineries by determination of economic conditions such as affordable feedstock, cheap solvent, and catalyst systems along with easily manageable physical parameters still remains a valuable direction for advanced research.

Acknowledgments All the authors listed are thankful to their respective universities and institutes for providing the literature services.

Conflict of interest The authors of this work have no conflicting interests to declare.

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Kang, S., Yu, J., 2016. An intensified reaction technology for high levulinic acid concentration from lignocellulosic biomass. Biomass Bioenergy 95, 214–220. Kumar, R., Singh, S., Singh, O.V., 2008. Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives. J. Ind. Microbiol. Biotechnol. 35 (5), 377–391. Lange, J.-P., Price, R., Ayoub, P.M., Louis, J., Petrus, L., Clarke, L., Gosselink, H., 2010. Valeric biofuels: a platform of cellulosic transportation fuels. Angew. Chem. Int. Ed. 49 (26), 4479–4483. https://doi.org/ 10.1002/anie.201000655. Lima, S., Neves, P., Antunes, M.M., Pillinger, M., Ignatyev, N., Valente, A.A., 2009. Conversion of mono/ di/polysaccharides into furan compounds using 1-alkyl-3-methylimidazolium ionic liquids. Appl. Catal. A Gen. 363 (1–2), 93–99. Marckwordt, A., El Ouahabi, F., Amani, H., Tin, S., Kalevaru, N.V., Kamer, P.C., Wohlrab, S., de Vries, J.G., 2019. Nylon intermediates from bio-based levulinic acid. Angew. Chem. 131 (11), 3524–3528. Moens, L., 2002. Sugar cane as a renewable feedstock for the chemical industry: challenges and opportunities. In: Advances in the Chemistry and Processing of Beet and Cane Sugar: Proceedings of the 2002 Sugar Processing Research Conference, New Orleans, Louisiana, USA, 10–13 March 2002. Sugar Processing Research, Institute, Inc, pp. 26–41. Otsuka, M., Hirose, Y., Kinoshita, T., Masawa, T., 1973. Manufacture of levulinic acid. Google Patents. Pace, V., Hoyos, P., Castoldi, L., de Marı´a, P.D., Alca´ntara, A.R., 2012. 2-Methyltetrahydrofuran (2MeTHF): a biomass-derived solvent with broad application in organic chemistry. ChemSusChem 5 (8), 1369–1379. https://doi.org/10.1002/cssc.201100780. Patil, S.K., Lund, C.R., 2011. Formation and growth of humins via aldol addition and condensation during acid-catalyzed conversion of 5-hydroxymethylfurfural. Energy Fuel 25 (10), 4745–4755. Pei, M., Peng, X., Shen, Y., Yang, Y., Guo, Y., Zheng, Q., Xie, H., Sun, H., 2020. Synthesis of watersoluble, fully biobased cellulose levulinate esters through the reaction of cellulose and alpha-angelica lactone in a DBU/CO2/DMSO solvent system. Green Chem. 22 (3), 707–717. https://doi.org/10.1039/ C9GC03149A. Pileidis, F.D., Titirici, M.-M., 2016. Levulinic acid biorefineries: new challenges for efficient utilization of biomass. ChemSusChem 9 (6), 562–582. https://doi.org/10.1002/cssc.201501405. Rackemann, D.W., Doherty, W.O., 2011. The conversion of lignocellulosics to levulinic acid. Biofuels Bioprod. Biorefin. 5 (2), 198–214. https://doi.org/10.1002/bbb.267. Sangarunlert, W., Piumsomboon, P., Ngamprasertsith, S., 2007. Furfural production by acid hydrolysis and supercritical carbon dioxide extraction from rice husk. Korean J. Chem. Eng. 24 (6), 936–941. Sharath, B.O., Tiwari, R., Mal, S.S., Dutta, S., 2019. Straightforward synthesis of calcium levulinate from biomass-derived levulinic acid and calcium carbonate in egg-shells. Mater. Today Proc. 17, 77–84. https://doi.org/10.1016/j.matpr.2019.06.403. Shimizu, K.-i., Uozumi, R., Satsuma, A., 2009. Enhanced production of hydroxymethylfurfural from fructose with solid acid catalysts by simple water removal methods. Catal. Commun. 10 (14), 1849–1853. Siracusa, V., Lotti, N., 2018. Biobased plastics for food packaging. In: Reference Module in Food Science. Elsevier, https://doi.org/10.1016/B978-0-08-100596-5.22413-X. Thompson, R.L., Carpenter, C.E., Martini, S., Broadbent, J.R., 2008. Control of listeria monocytogenes in ready-to-eat meats containing sodium levulinate, sodium lactate, or a combination of sodium lactate and sodium diacetate. J. Food Sci. 73 (5), M239–M244. https://doi.org/10.1111/j.1750-3841.2008. 00786.x. Tukacs, J.M., Kira´ly, D., Stra´di, A., Novodarszki, G., Eke, Z., Dibo´, G., Kegl, T., Mika, L.T., 2012. Efficient catalytic hydrogenation of levulinic acid: a key step in biomass conversion. Green Chem. 14 (7), 2057–2065. https://doi.org/10.1039/C2GC35503E. van der Waal, J.C., de Jong, E., 2016. Avantium chemicals: the high potential for the levulinic product tree. In: Industrial Biorenewables, pp. 97–120, https://doi.org/10.1002/9781118843796.ch4. van Zandvoort, I., Wang, Y., Rasrendra, C.B., van Eck, E.R., Bruijnincx, P.C., Heeres, H.J., Weckhuysen, B.M., 2013. Formation, molecular structure, and morphology of humins in biomass conversion: influence of feedstock and processing conditions. ChemSusChem 6 (9), 1745–1758.

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Wettstein, S.G., Alonso, D.M., Chong, Y., Dumesic, J.A., 2012. Production of levulinic acid and gammavalerolactone (GVL) from cellulose using GVL as a solvent in biphasic systems. Energy Environ. Sci. 5 (8), 8199–8203. https://doi.org/10.1039/C2EE22111J. Ya’aini, N., NAS, A., Asmadi, M., 2012. Optimization of levulinic acid from lignocellulosic biomass using a new hybrid catalyst. Bioresour. Technol. 116, 58–65. https://doi.org/10.1016/j.biortech.2012.03.097. Yan, L., Greenwood, A.A., Hossain, A., Yang, B., 2014. A comprehensive mechanistic kinetic model for dilute acid hydrolysis of switchgrass cellulose to glucose, 5-HMF and levulinic acid. RSC Adv. 4 (45), 23492–23504. https://doi.org/10.1039/C4RA01631A.

CHAPTER 7

Production of pyruvic acid into value-added products using genetically modified microbes P. Jeevithaa, J. Ranjithaa, M. Anandb, Shahid Mahboobc, and S. Vijayalakshmia a

CO2 Research and Green Technologies Centre, Vellore Institute of Technology, Vellore, India Department of Pharmacology, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India c Department of Zoology, Kingdom of Saudi Arabia, King Saud University, Riyadh, Saudi Arabia b

1. Introduction Pyruvate is the three-carbon compound formed at the neutral pH. Other names of pyruvates are acetylformic acid, 2-oxopropanoic acid, and 2-ketopropionic acid. Pyruvate is formed biochemically after the glycolysis and tricarboxylic acid pathway (TCA) (Fig. 1). Both carboxylic acid and keto groups are present in pyruvate, and it’s the important precursor for many polymers, food additives, chemicals, and mainly in pharmaceuticals. L-DOPA is biochemically synthesized with the help of pyruvate (Park et al., 1998), (R)phenylacetylcarbinol (Rosche et al., 2001), N-acetyl-D-neuraminic acid (Neu5Ac) (Zhang et al., 2010), butanol (Reisse et al., 2016), propionate is synthesized enzymatically and it’s the starting material for propionate (Stine et al., 2016). Escherichia coli is the major cell factory because it contains a large amount of pyruvate and it is used in the major metabolic pathway for the production of many commercial products (Zhang et al., 2016). The major subjects for previous reviews are the production of microbial pyruvate (Li et al., 2001a,b; Xu et al., 2008). Pyruvate has been produced by the metabolic pathway, and it has several health benefits when compared to others. For example, oxidative stress causes human neuroblastoma cells and it is protected by pyruvate (Mazzio and Soliman, 2003; Wang et al., 2007). Pyruvate protects rat cortical neurons (Nakamichi et al., 2005), zinc toxicity was protected by pyruvate with the help of retinal cells (Yoo et al., 2004), pyruvate improves brain metabolism, which prevents hemorrhagic shock (Mongan et al., 2001), and ischemia-reperfusion injury of the brain was protected by pyruvate (Ryou et al., 2012). In myocardial function, pyruvate plays a major role in the ejection and maintenance of heart rate (Hasenfuss et al., 2002; Hermann et al., 2004). In this study, 6 g per day of pyruvate supplementation was given for 6 weeks and the person with mild physical activity leads to decrease in the fat mass. Physical exercise leads to increased triacylglycerol Valorization of Biomass to Bioproducts https://doi.org/10.1016/B978-0-12-822888-3.00012-8

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Fig. 1 Conversion of lactic acid from pyruvic acid.

and cholesterol very low-density lipoprotein (VLDL) and HDL cholesterol level were decreased (Koh-Banerjee et al., 2005). For food supplementation, magnesium and calcium pyruvate are used (EFSA, 2009). In the environment pyruvate is converted to hydrogen peroxide by a detoxification process, and this stimulates the ammoniaoxidizing archaea’s growth (Kim et al., 2016).

2. Production of pyruvate using microbial pathway The major central metabolite is pyruvate, microorganisms have been reportedly present in small amounts in a variety of environmental circumstances. Pyruvate is formed biochemically through glycolysis process, and the overall reaction is represented: Glucose + 2NAD+ + 2Pi + 2ADP ! 2Pyruvate + 2NADH + 2ATP + 2H+ … (1) Glucose production is 0.966 g/g in the maximum theoretical yield of pyruvate and in this equation is balanced and this process requires nicotinamide adenine dinucleotide (NAD) and adenosine diphosphate (ADP) and again it’s regenerated. Glucose is the major energy/carbon sources are used for some cellular materials. In Eq. (1), pyruvate accumulates in a high concentration without any carbon by-products. ADP and NAD formation rate is affected and this suggests the pyruvate formation in the Eq. (1). These carbon by-products rapidly increased the availability of ADP and NAD. For the formation of pyruvate, several key enzymes are involved and the catabolisms are listed in the Table 1. For the generation of pyruvate, several significant processes were carried out, and they produce glucose with the help of fungi, and they require thiamine as a co-factor, and the result states that no pyruvate is formed in the excess amount of thiamine, and in the absence of thiamine, 3 g/L of pyruvate is produced (Kitahara and Fukui, 1951a,b,c,d, 1955). In lipoic acid, auxotroph pyruvate can be observed. Researchers came to

Production of pyruvic acid into value-added products

Table 1 Production of pyruvate from different organisms. Strains

Nitrogen source

Carbon source

Pyruvate (g/L)

Yield (g/g)

Nocardia fumifera

(NH4)2SO4

Gluconate

23

0.46

E. coli W1485lip2

Polypeton

Glucose

25.5

0.51

E. coli TBLA-1

Polypepton

Glucose

30

0.60

E. coli AJ 12631

Polypepton

Glucose

29.2

0.58

Actinetobacter sp., 80-M Pseudomonas sp., TB-135

Polypeptone

1,2Propanediol 1,2Propanediol

8.1

0.41

14

0.35

–

Glucose

36.9

0.37

Corn steep liquid (NH4)2SO4, NH4NO3

Acetamide

1.5

0.15

Citrus peel

11.5

–

Saccharomyces cerevisiae IFO 0005 Candida liploytica AJ 4546 Debaryomyces coudertii IFO 1381

NH4NO3

References

Uchio and Hirose (1975) Yokota et al. (1994a) Yokota et al. (1994c) Tomita and Yokota (1993) Izumi et al. (1982a,b) Shigene and Nakahara (1991) Yonehara and Yomoto (1987) Uchio et al. (1974) Yanai et al. (1994)

recognize that pyruvate might accumulate in microorganisms with a reduced ability to decarboxylate pyruvate oxidatively (low pyruvate dehydrogenase activity) or that were auxotrophic for thiamine or lipoic acid as a result of greater understanding of enzyme kinetics mechanisms. These auxotrophs result in the lipoic acid and thiamine co-factor is responsible for the pyruvate dehydrogenase multi-enzyme complex: Lipoic acid transfers the acetyl group through an amide linkage to a singly lysyl residue of E2 transacetylase (aceF gene in E. coli) and thiamine binds to E1 decarboxylase (aceE gene in E. coli). To isolate the pyruvate-accumulating microorganisms, vitamin auxotrophy has been used. For example, Acinetobacter contains thiamine are isolated which converts 20 g/L of 1,2-propanediol produce 12 g/L of pyruvate (Izumi et al., 1982a,b), Schizophyllum strains convert glucose to pyruvate of about 19 g/L in 5 days and a maximum yield of 0.38 g/g (Takao and Tanida, 1982), and Debaryomyces coudertii is a citrus fruit which contains pectin and the citrus peels generate pyruvate at a range of 9.7 g/L of pyruvate in 48 h (Moriguchi, 1982). Eighteen different strains of yeast were screened to produce pyruvate with the help of glycerol or glucose, one yeast which contains Yarrowia lipolytica of thiamine-auxotrophic which generates 61 g/L of glycerol is produced from pyruvate at a time of 78 h and the maximum yield obtained was 0.71 g/g (Morgunov et al., 2004).

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Lipoic acid and thiamine enterobacter auxotrophs are exposed to some mutagens and after the exposure the mutagen N-nitrosoguanidine which generates 4.7 g/L pyruvate from 20 g/L glucose after 72 h, in the fermentation process, E. coli lipoic acid auxotrophs produce 25 g/L of pyruvate and 50 g/L glucose in a controlled fermenter for 40 h (Yokota et al., 1994b). One hundred and thirty-two strains were studied from that Trichosporon cutaneum was isolated, which generates 35 g/L of pyruvate is produced from glucose at a maximum yield of 0.43 g/g (Wang et al., 2002). To produce pyruvate selected strains are used and they used oxythiamine and analog of thiamine as the several strains of yeast to produce pyruvate. Candida glabrata (Torulopsis glabrata) is isolated auxotrophic for biotin, nicotinate, thiamine, and pyridoxine, which generates 57 g/L of pyruvate was produced from glucose, and 40 g/L of peptone was produced in 59 h with a maximum yield of 0.57 g/g. C. glabarata produces pyruvate with the help of mutagen and they generate valine/isoleucine and arginine and they accumulate at a range of 60 g/L of pyruvate was produced from glucose in 43 h and a maximum yield of 0.60 g/g. Natural strains are used to accumulate pyruvate and the majorly used strains are C. glabrata, which are mainly used in the production of pyruvate (Li et al., 2016). Blastobotrys adeninivorans is the recently used strain, which generates 43 g/L of pyruvate when produced from glucose at 192 h has a maximum yield of 0.77 g/g (Kamzolova and Morgunov, 2016).

3. Optimization of media conditions for the production of pyruvate from different microbial strains Most of the strains lost their ability to synthesize the organic compound. The enzyme activity is important for the accumulation of pyruvate by the strains, and the production of pyruvate can be improved by the optimization of media. For example, thiamine and nicotinate are the key factors for the optimization of nitrogen, and they used fed-batch process 68 g/L of pyruvate from glucose was achieved from C. glabrata and required 63 h, and a maximum yield of 0.49 g/g. 57 g/L of pyruvate is from the glucose was achieved from C. glabrata and requires 55 h and a maximum yield of 0.50 g/g of the nitrogen nutrients (Li et al., 2000), vitamin was subsequently optimized by statistical method, and they generate 69 g/L of pyruvate was generated from glucose, which required 56 h 0.62 g/g was obtained by a batch process (Li et al., 2001a,b). Thiamine was present majorly in yeast, and it is not suitable for the optimization of media (Wang et al., 2002). Most of the complex compounds such as vitamins are mainly used in the optimization of media and these play a major role in the aeration of the formation of pyruvate and they produce the NAD for the optimization of strains. C. glabrata genome analysis says that yeast is mainly used in the transport of glucose and that require multi-vitamins for the transport of organic acids and also for the accumulation of pyruvate (Xu et al., 2013, 2016). The formation of pyruvate has been optimized and isolated from the several strains. 86 g/L of

Production of pyruvic acid into value-added products

pyruvate was achieved by C. glabrata and a maximum yield of 0.70 g/g and nitrogen source can be obtained from ammonium chloride and the major source present is urea are some of the examples (Yang et al., 2014). If the glucose concentration was increased, productivity of pyruvate also increased and the ammonia was produced from the reduced futile cycling and ammonia was used as a nitrogen source and they generated NADPH. Similarly, pyruvate production rate was decreased, and the concentration of the baseneutralized and NaCl capacity was increased in the production strain of C. glabrata (Liu et al., 2007). Under continuous culture, the strains were supplemented with some amount of salt, and the mutant strains produced 94 g/L of pyruvate in 82 h and the concentration was compared to the parent strain. Proline was supplemented to C. glabrata, which protects the high osmotic pressure with the help of growth, and 60 to 74 g/L of pyruvate was produced in an increased amount (Xu et al., 2010). Most of the strains are naturally tolerant to osmotic pressure and salt, organic acids can be produced microbially for the production of halophilic microbes (Calabia et al., 2011; Yokaryo and Tokiwa, 2014; Yin et al., 2015). One halophilic, alkaliphilic 63 g/L of pyruvate was generated. Halomonas was continuously unsterilized at aerobic conditions for 48 h with ionic strength (Kawata et al., 2016). In the last three decades, pyruvate titer was increased by optimization/isolation and the yield titer value was increased. In the last 20 years, genetic methods have been developed for the microbial production of pyruvate, which has been improved by microbial metabolism.

4. Various metabolic pathway production of pyruvic acid Commonly, most of the strains are metabolic engineered with the help of genetic techniques to purposefully optimize the pathways of cells and regulatory circuits in order to influence the creation of an end product. Most of the strains are constructed with the help of genetic techniques and predictive algorithms for the production of pyruvate from the simple isolation techniques of the pyruvate-containing strains. When compared to other products, the production of pyruvate enhances the yield of final concentration and also the productivity and substrate. In biochemical pathways like glycolysis, pyruvate is the end product formed from the direct conversion of glycerol or glucose (Fig. 1). These accumulates the product formed from this pathway, and this involves in the elimination or restricting for further production of other products, which increases the glycolysis rate. Metabolic engineering of pathways will provide NADPH and other biochemical molecules to satisfy the demands of other biosynthetic pathways. Increasing the rate of glycolysis was one of the first metabolic engineering approaches for pyruvate generation. Researchers have known for a long time that adding 2,4dinitrophenol to the medium diminishes a cell’s energy charge and raises the consumption of glucose rate in E. coli (Dietzler et al., 1975). Using genetic tools, alterations in the adenosine triphosphate (ATP)operon allowed for a more direct uncoupling of respiration

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in E. coli, resulting in a doubling of glycolytic flux ( Jensen and Michelsen, 1992). Essentially, cells increase the rate at which ATP is produced by glycolysis to compensate for the diminished ATP generation via proton motive force. Glycolysis be linked directly to the formation of pyruvate. Many researchers used gene manipulation tools for the introduction of ATP operon, which will affect the formation of pyruvate. Introduction of atpA mutated gene in E. coli lipoic acid strain resulting in the formation of 31 g/L of pyruvate is produced from glucose at a time of 32 h and the yield is 0.64 g/g in the batch culture of strains (Yokota et al., 1994b, 1997). In the atpA medium, the pyruvate formation rate was increased from 0.8 to 1.2 g/L h and the biomass yield was decreased from 0.26 to 0.14 g/g. It continues to develop more advanced techniques for regulating the intracellular ATP content. An example is the construction of the INH1 gene from S. cerevisiae a copperinducible F0F1-ATPase inhibitor (Zhou et al., 2009). C. glabrata overproduces the pyruvate, which will increase the pyruvate production by 23% to 1.69 g/L h. Because increasing glycolytic flow has a limited effect on pyruvate yield, scientists have turned their attention to different ways to deliver more substrate to the product. Pyruvate is the primary catabolic pathway, and the enzyme is the pyruvate dehydrogenase complex, which was an early target for deletion. Unlike previous efforts that used naturally or an auxotrophy lower the activity of enzyme to low enzyme activity to manage pyruvate dehydrogenase, growing a pyruvate dehydrogenase mutant usually required supplying a supplementary carbon source such as ethanol or acetate to give cells with a source of acetyl CoA. Growing E. coli mutants defective in pyruvate dehydrogenase components (lpd, aceE, and aceF genes) with a supplement of acetone resulted in a significant pyruvate output from glucose (Tomar et al., 2003). In 35 h, the best-performing E. coli, aceF ppc strain produced 35 g/L pyruvate from acetate and glucose, yielding 0.78 g/g glucose (Tomar et al., 2003). The generation of acetate and lactate in aceE and aceF strains also means that pyruvate oxidase is active under specific situations (poxB) and lactate dehydrogenase (ldhA), despite aerobic settings, are significant conduits for pyruvate metabolism and thus possible future gene knockout targets. The relevance of pyruvate oxidase in E. coli without pyruvate dehydrogenase was proven using 13C-flux analysis, which revealed that in the functional pyruvate dehydrogenase is absent, pyruvate oxidase, as well as the Entner-Doudoroff and anaplerotic pathways, are all elevated (Li et al., 2006). Other experiments with E. coli, aceEF pflBpoxB pps ldhA and a specified media revealed a link between acetate intake, quantifiable CO2 consumption, and cell growth (Zeli
c et al., 2003). These researchers produced 62 g/L pyruvate in 30 h with a yield of 0.55 g/g by carefully controlling both the glucose and acetate feeds (glucose concentration and using online measurements of CO2 evolution rate, respectively). Using electrodialysis to extract pyruvate in combination with a repeated (fed)-batch fermentation process using the same E. coli strain minimized product inhibition and permitted an average productivity of 0.82 g/g and a productivity of 3.9 g/L h for four cycles of products began to decline at 40 h (Zeli
c et al., 2004a,b). Growth and pyruvate formation

Production of pyruvic acid into value-added products

were adequately represented by an unstructured model combining inhibition of pyruvate product and growth formation, but the higher glucose consumption rate of pyruvate and maintenance than envisaged was not taken into consideration (Zeli
c et al., 2004a,b). The characteristics of products and substrate concentration changes in the acetate feeding were better predicted using a neural network technique (Zeli
c et al., 2006). Pyruvate dehydrogenase will reduce the activity and other components of pyruvate dehydrogenase will be eliminated, and they will also use metabolic engineering methods to disturb the enzyme activity. In this study, they used E. coli, sucA pflB ackA frdBC poxB adhE and atpFH which generates 2 g/L of pyruvate from the glucose; they used carbon as the sole source for a period of 43 h, and the yield obtained was 0.76 g/g (Causey et al., 2003). This pyruvate strategy combined a variety of features (1) prevent the formation of ethanol (adhE), acetate (poxB, ackA) and lactate (ldhA gene deletion); (2) they reduce the both reductive (frdABC) TCA cycle and oxidative (sucA); and (3). The glycolysis rate is increased by uncoupling with the respiration (atpFH). The process was performed under low levels of oxygen (5% saturation) that tended to increase the NADH pool, an inhibitor of the PDHase and the component of dihydrolipoamide dehydrogenase (Hansen and Henning, 1966) and citrate synthase (Weitzman, 1966). Because of these operating conditions, pyruvate yield was in a critical condition. In pyruvate accumulation pathways, the importance of pyruvate oxidase (poxB). Evidently, the primary difference between this strategy and those involving the gene deletion process in the pyruvate dehydrogenase complex is that an acetate requirement is obviated by the preservation of certain pyruvate dehydrogenase operations. New and exciting approaches to metabolic engineering suggest rather than deletion of genes or silencing of genes is used. Silencing the aceE gene, for example, resulted in 26g/L pyruvate from glucose in 72 h when paired with the deletion or silencing of other genes (Akita et al., 2016). PDC negative strain cannot grow in ethanol or acetate and they require a low amount of acetate for their feed and they produce 135 g/L of pyruvate is produced in a batch process at a time of 100h and the yield obtained was 0.54 g/g (Van Maris et al., 2004). In a related study, a negative PDC strain have the ability to grow on glucose condition without the help of ethanol or acetate, and after 1000 generations, it was isolated and it gave low concentration of ethanol and high concentration of glucose (Wang et al., 2012). The utilization of lignocellulosic hydrolysates as an affordable supply of carbohydrates has sparked an interest in cells that can metabolize both glucose and xylose at the same time. PDC1 and GPD1 (coding for glycerol-3P dehydrogenase) were disrupted, and a xylose transporter and numerous metabolic genes were introduced into Kluyveromyces marxianus, resulting in 29 g/L pyruvate in 36 h from a sugar combination (Zhang et al., 2017). A recent work (Kawai et al., 2014) used the bacteria Sphingomonas with an ldh deletion to produce pyruvate from alginate. In just 3 days, the strain produced over 3 g/L pyruvate, demonstrating the importance of oxygen availability. L-Glutamate and L-lysine are the amino acids produced by the industrial bacteria called Corynebacterium glutamicum. Strains with gene deletion of aceE were shown to generate

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2 g/L pyruvate in one investigation focused on the formation of L-valine, which is produced in four enzymatic steps from pyruvate (Blombach et al., 2007). The deletion of pyruvate oxidase (poxB or pqo gene) improved L-valine production while also introducing an acetate growth requirement, suggesting that the combination of acetate kinase, pyruvate oxidase, and phosphotransacetylase in C. glutamicum can bypass pyruvate dehydrogenase to allow acetyl CoA generation (Blombach et al., 2008). Lactate dehydrogenase (ldhA) and two transaminase genes were also knocked out (avtA and alaT), and replacing native acetohydroxyacid synthase with inhibited variation (C-T ilvN) resulted in 46 g/L pyruvate from acetate and glucose 105 h, yielding 0.48 g/g glucose (Wieschalka et al., 2012). C. glabrata and S. cerevisiae are the varieties of yeasts, and C. glutamicum, pyruvate carboxylase is present, but it is not present in E. coli.

4.1 Lactic acid to pyruvic acid conversion Pyruvate is the simplest form of alpha-keto acids which contains carboxylic acids and the functional group present is ketone. Pyruvate is the key intermediate, and it’s a conjugate base. In the glycolysis pathway, pyruvate and lactate are the end products, and this product is the starting compound for other glucose metabolism. In this pathway, pyruvate is converted to lactate by the enzyme lactate dehydrogenase.

4.2 Succinic acid to pyruvic acid conversion Succinic acid is classified as a dicarboxylic acid. Succinic acid has several multiple roles in the biological pathway, and it is converted to fumarate, and the enzyme involved is succinate dehydrogenase. Nitrogen gives a high amount of succinic acid with the help of glutamate. In this, glutamate is an oxidative agent and aspartate is a reducing agent in the formation of succinic acid. In the citric acid cycle, pyruvic acid is converted to acetyl CoA and it undergoes several chemical reactions which form succinate (Fig. 2).

4.3 Acetic acid from pyruvic acid conversion In the fermentation of yeast, acetic acid is formed. It is a volatile acid present in wine. The mechanism of acetic acid is not fully formed, and it can be formed only by two possible pathways. In the first step, acetyl CoA is formed from the pyruvic acid with the help of hydrolysis reaction and takes place in anaerobic conditions because these compounds

Fig. 2 Conversion of succinic acid from pyruvic acid.

Production of pyruvic acid into value-added products

Fig. 3 Conversion of acetic acid from pyruvic acid.

found in the mitochondria and its functions in the absence of oxygen. In second step, acetaldehyde was oxidized previously and converted from pyruvic acid with the help of NADP+ co-enzyme, and the resulting NADPH is used in the fatty acid synthesis. This pathway favors the formation of acetic acid from acetyl CoA and the enzyme involved in this formation is acetyl-coA synthetase (Fig. 3).

4.4 2,3-Butanediol, acetoin, and butanedione from pyruvic ACID conversion In the fermentation of yeast acetoin, 2,3-butanediol and -butanedione are formed. 2,3butanediol attached to thiamine pyrophosphate (TPP) and undergoes a decarboxylation reaction gives TPP-C2 also called active acetaldehyde. Acetaldehyde is condensed to produce acetoin in the pathway (1). Thiamine pyrophosphate C2 complex binds to pyruvic acid and gives to α-acetolactic acid. In the non-oxidative decarboxylation method, acetoin is formed in pathway (2). Butanedione is formed by oxidative decarboxylation of α-acetolactic acid. Acetoin is produced by the reduction of butanedione (3). This is a reversible reaction (Fig. 4).

5. Co-enzymes enhanced pyruvic acid production By using co-factor engineering, NAD/NADH availability can be altered for the production of pyruvate, and it is another method for pyruvate production. In the glycolysis process, glucose is converted to pyruvate, producing 2 mol NADH is produced per molecule of glucose (Eq. 1). Pyruvate dehydrogenase is the component of dihydrolipoamide dehydrogenase which is inhibited by NADH (Hansen and Henning, 1966). In this process, pyruvate is produced in a direct method in a redox state. In the presence of pyruvate dehydrogenase activity, pyruvate catabolism is suppressed by elevated levels of NADH.

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Fig. 4 Conversion of 2,3-butanediol, -acetoin, and -butanedione from pyruvic acid.

The glycolysis rate is increased with the help of elevated levels of NAD and NADH and is enhanced by the oxidation process. Based on the genetic background of strains, pyruvate is formed by co-factor engineering and metabolic engineering of the strains. When compared to wile-type strains, 70% of the increased rate of glucose uptake in E. coli of Streptococcus pneumoniae (nox gene) is seen in the NADH oxidase and it is produced in the water (Vemuri et al., 2006). In E. coli, poxB aceEF ldhA pps pflB are produced in the acetate conditions and no improvement is seen in the glycolysis rate and NOX expressing gene was produced in increased amount (Zhu et al., 2008). 12% increased glucose uptake was seen in NADH oxidase of arcA and atpFh are the additional knockout genes and 8% of pyruvate is formed in a specific rate (Zhu et al., 2008). In C. glabrata, mitochondrial endogenous alternative oxidase (AOX1) or the overexpression of water-forming NADH oxidase (nox) produce pyruvate at an increased rate and the productivity was 15%–30% ranging from 1.63 g/L h and 0.79 g/g (Qin et al., 2011). E. coli transhydrogenase (udhA) and Lactococcus lactis (noxE gene) are produced from the expression of water-forming NADH oxidase, evolved in PDc-knockout of Saccharomyces cerevisiae for the increased production of pyruvate ranges from 75 g/L in 120 h with a maximum yield of 0.63 g/g (Wang et al., 2012). In acetaldehyde medium, the formation of pyruvate is enhanced in this medium. In C. glabrata, ethanol is produced in a reduced amount. A high level of alcohol is produced in the dehydrogenase activity. This leads to the increased production of pyruvate, yielding and concentration was attained to elevated levels of concentration of NAD in the acetaldehyde-reducing strains (Liu et al., 2007). NADH concentration is decreased and this is the major goal for the pyruvate production. E. coli of Mycobacterium vaccae (fdh) more amount of NADH is generated by expressing the formate dehydrogenase and the strains having pyruvate dehydrogenase activity (Ojima et al., 2012). The media is supplemented with peptone, glucose, and

Production of pyruvic acid into value-added products

formate by the enzyme formate dehydrogenase, which leads to increased production of pyruvate ranging from 6.8 to 9.0 g/L in 24 h with a maximum yield of 0.48 g/g of glucose.

6. Gene-modified microbial strains for pyruvic acid production—Genetic engineering For example, low levels of oxygen favors the uptake of glucose in C. glabrata and high levels of oxygen favor the yield of pyruvate. This two-stage process at low-to-high oxygen levels improves the overall formation of the oxygenation (Hua et al., 2001; Li et al., 2002). The cost of yield is very high, which compromises the light levels of oxygenation, which shows the complexity of both productivity and yield maximization. As NADH is produced by the conversion of glucose to pyruvate (Eq. 1), high levels of oxygen are needed for the formation of pyruvate. In glycolysis, high concentrations of NAD are required and reduced by-products in strains lacking the knockout are prevented by oxygenation. For example, E. coli is formed from lactate and pyruvate formate lyase activity can be discouraged by high levels of oxygen in the generation of pyruvate (Ojima et al., 2012). Most of the strains contain the pyruvate dehydrogenase as the functional compound, and pyruvate is generated in low oxygen conditions, which initiates the accumulation of NADH (Causey et al., 2004). The formation of pyruvate will impact the environmental conditions and pH. At pH 6, pyruvate is produced with the help of lipoic acid and the production reaches its maximum level. Pyruvate dehydrogenase shows the greatest level at a pH of 7 and generated at 32°C (Tomar et al., 2003). Based on the genetic background and optimal conditions of the strains, they are used for the accumulation of pyruvate. Nutrient-limited conditions are also used by processes to monitor the growth rates and provide carbon to the target product. Limited amount of nutrients are supplied to the microbes to prevent the growth rate and it can be metabolized maximally by the nutrients. With the help of acetate, phosphorous, glucose, and nitrogen, they are used for the steady-state and also for the production of pyruvate by E. coli aceEF poxB ldhA pflB pps and glycolytic rates are compared (Zhu et al., 2008). Under acetate-limited conditions, the maximum yield (0.70 g/g) and specific productivity (1.11 g/g h) were achieved for the culture of the fed-batch process, and the constant growth rate was 0.15 h1. ArcA and atpFH knockouts are the additional E. coli strains that are used for this process, and they produce 90 g/L of pyruvate at 44 h and a maximum yield of 0.68 g/g of glucose (Zhu et al., 2008). The primary carbon source are similar with acetate by fed-batch process ad they produce 40 g/L in a less period of 40 h and the yield obtained was 0.62 g/g of glycerol (Zhu et al., 2010). A small amount of glucose was added to the fed-batch process to improve the yield of glycerol and the productivity was 0.95 g/g.

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The reaction was catabolized by NADPH generation and biomass used for the synthesis of glycolysis with the help of glycerol (Zhu et al., 2010).

7. Other biological methods In non-growing cells, biotransformation takes place; pyruvate is formed by the biochemical process. In the fermentation process, biotransformation takes place, and they require free salt and other impurities. They are required for the production of purified products, and they use many biocatalysts for several process. Several biocatalysts are produced, and they generate pyruvate and have several advantages for the production of pyruvate involves several cycles. Pyruvate is produced from the reduction of more compounds, and several materials are used as starting materials, and they generate NADH in cells, which helps in the maintenance of energy. 0.5 M L-lactate is converted to pyruvate in 12 h with a concentration range of 6 g/L (dry cells) and they used Acinetobacter sp. (Ma et al., 2003). Pichia pastoris and Hansenula polymorpha are the yeast cells, which express the catalase and glycolate oxidase, which oxidizes the 0.5 M L-lactate is converted to pyruvate after 12 cycles of reduction, and 98% is mainly converted to lactate (Eisenberg et al., 1997). Above 0.5 M L-lactate concentration shows that they inhibit the substrate of P. pastoris and oxygen is recombined and glycolate oxidase is used as a substrate for the conversion process (Gough et al., 2005). Enzyme activities are prolonged at 5°C and are operated by glycolate oxidase/catalase for the conversion process. Pseudomonas stutzeri is used for the conversion of D/L-lactate into pyruvate, which generates 23 g/L of pyruvate at a maximum yield of 0.89 g/g of pyruvate (Hao et al., 2007). 48 g/L of pyruvate is generated at P. stutzeri at a maximum yield of 0.98 g/g after 29 h and the temperature is 30°C at a pH of 7–8 (Gao et al., 2012). In E. coli, 14.6 g/L of pyruvate is formed in 30 h by the conversion of 50 g/L D/L-alanine and in E. coli, they deleted some native alanine and pyruvate transporters for L-amino acid deaminase (Hossain et al., 2016). For the production of pyruvate, glycerol or glucose are mainly used as the carbon sources. For the production of commodities and chemicals, lignin is used as a major untapped feedstock. Several research studies have been demonstrated for the production of pyruvate by aromatic catabolic pathways, which replace the ortho-cleavage and metacleavage pathways of Pseudomonas putida, which significantly increases the production of pyruvate from benzoate and p-coumarate in aceEF knockout strains ( Johnson and Beckham, 2015).

8. Pyruvate recovery from fermentation processes Pyruvate recovery from the fermentation broth: variety of separation methods can be investigated. Pyruvate recovery is the general procedure for the preparation of organic

Production of pyruvic acid into value-added products

acids. It is used to precipitate calcium salt, which is poorly soluble and acidified using H2SO4 which makes free acid and gypsum. It is not a favorable technique which utilizes high volumes of acid and produces poorly soluble calcium sulfate in the waste stream. The advisable method is to extract the product selectively from the fermentation broth with an immiscible solvent and regenerate the impurity-free solvent for reuse. Reactive extraction is a suitable method for modification. In this, an extract is involved in the extraction system (Hong et al., 2001). The additional chemical introduced into the system has the ability to undergo reversible complex formation with the product. Phosphoryl-containing extractants like trioctyl phosphine oxide (TOPO) or tributyl phosphate (TBP), as well as aliphatic amines like tri-n-octylamine, are common organic acid extractants. The chemical characteristics of the diluent and extractant of the functional groups (i.e., the solvent) influence the extraction equilibria, and hence the selectivity of the intended desired extraction. A diluent composition change a temperature shift, or pH shift can all be used to regenerate the extractant and diluent. For carboxylic acids, aliphatic amines with a suitable diluent are frequently used because they give high distribution coefficients at pH values close to or below the target carboxylic acid’s pKA (Wasewar et al., 2002). Pyruvate extraction equilibria in diluent/extractant mixtures have been compiled in a variety of studies. Diluent and cyclic alcohol gives high extraction efficiency. Alamine 336 is used as an extractant for tertiary amine and is the alternative precipitation method for extraction of liquid-liquid. Senol (2006) explained the highest separation factor for yield was provided by 1-dodecanol/Alamine among aliphatic alcohols (Senol, 2013). In the first-order reaction, the TOA/1-octanol system reveals the kinetics and equilibrium studies (Marti and Gurkan, 2015). To achieve the recovery of >98% pyruvate the mixing extracts of phosphate along with extract of amine in decanol are the recent studies (Pal et al., 2016). TBP extraction equilibria with various diluents indicate that toluene and n-heptane produce the best results (Pal and Keshav, 2014). Pyruvate and extractant react with a 1:1 M ratio, found in most of the system. Selecting a method that limits co-extraction of other organic acids as well as water must be done carefully. A comparative study was carried out for the selective pyruvate recovery from the fermentation broth or from the solution containing a mixture of acids. pH swing extraction using the pKA differences between pyruvate (2.49) and lactate (3.86) was examined with various diluents in a study. The same pH swing extraction with trimethylamine gives 97% pyruvic acid recovery and the amine can be removed by a simple distillation method (Ma et al., 2006). Salt-assisted solvent extraction, also known as repulsive extraction, is a comparable recovery approach that can be used to recover a variety of biochemicals obtained by fermentation (Birajdar et al., 2015; Zhigang et al., 2001). The process includes decreasing the solubility of the target product selectively. In fermentation broth, the use of acetone to reduce the pyruvate solubility and combining extraction of acetone by evaporation

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(to concentrate the mixture), microfiltration, and crystallization resulted in pyruvate purity of 97% and recovery of 75% (Ingle et al., 2015). Electrodialysis is another general method for the recovery of organic acids. Cationexchange and anion-exchange membranes are supplied with alternation current. The fact that there is no problem with organic solvent impurity is the greatest advantage of this method. This method is also used for pyruvate recovery (Zeli et al., 2005). Using the ultrafiltration technique, the cells were removed prior to the electrodialysis. It can work in two modes, both monopolar and dipolar. In monopolar mode, sodium pyruvate is formed, and in dipolar mode, hydroxide and pyruvic acid are formed in the fermenter to control the pH. As a result, the fermentation and electrodialysis processes can be fully combined. Co-separation of other medium components and membrane fouling are still obstacles to overcome before electrodialysis may be entirely compatible with fermentation. Adsorption is another approach for recovering pyruvic acid, and a weakly basic sorbent and primary amine can be employed to absorb the pyruvic acid at its pKA (2.49) (Huang et al., 2008). In comparison to extraction or electrodialysis, ion exchange has a limited capacity.

9. Conclusions Pyruvate is the major key metabolite involved in the glycolysis and tricarboxylic acid cycle. This pathway is the major building block component of many fatty acids and amino acids. Several bacteria, fungi, and yeast are involved in the production of pyruvate. Several enzymes are involved in the production of pyruvate, and vitamins act as co-factors in the production of pyruvic acid. Most of the researchers used glucose as the major carbon source, and it produces pentoses or glycerol and stress conditions are given to the strain, such as pH, temperature, and high osmolarity to find the production.

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SECTION 2

Biofuels and bio-oil

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CHAPTER 8

Microbial production of hydrocarbon and its derivatives using different kinds of microorganisms R. Shobanaa, J. Ranjithaa, M. Anandb, Shahid Mahboobc, and S. Vijayalakshmia a

CO2 Research and Green Technologies Centre, Vellore Institute of Technology, Vellore, India Department of Pharmacology, Saveetha Dental College and Hospital, Saveetha Institute of Medical and Technical Sciences, Saveetha University, Chennai, India c Department of Zoology, Kingdom of Saudi Arabia, King Saud University, Riyadh, Saudi Arabia b

1. Introduction Concerns about the environment and the exhaustion of remnant fuels contain sparked attention in generating biofuels and bio-based compounds with a lower ecological footstep than existing fossil fuel manufacture ( Jang et al., 2012; Peralta-Yahya et al., 2012; Yan and Liao, 2009). Metabolic engineering was used largely to enhance the titer and productivity of commercial fermentation systems until the early 2000s (Nielsen, 2014). Incorporation of artificial environmental science and biological systems into the ground of metabolic trade has outcome within significant progress in the field to date (Church et al., 2014; Dai and Nielsen, 2015). Gratitude to these improved technologies, engineered cell factories can now generate a wider range of chemicals that can be utilized as medicines, compound building blocks, and fuels (Nielsen and Keasling, 2016). Furthermore, using enormous biological statistics sets given by systems environmental science methodologies, we have gained a better understanding of complicated cellular networks, and these insights have resulted in innovative tools and procedures that may be utilized to design cell factories. Alkanes and alkenes are a family of hydrocarbons that are widely utilized in liquid transportation fuels and polymers. However, breaking crude oil is required to get alkanes and alkenes with the desired characteristics. The process’s intricacy might make it harder to get particular compounds and raise expenses (Kissin, 2001). A lot of organisms spontaneously synthesize alkanes and alkenes to defend themselves from environmental challenges (Howard and Blomquist, 2005; Jetter and Kunst, 2008; Schirmer et al., 2010), but their production levels and structures make them unsuitable for use as drop-by fuels. Numerous processes and enzymes are occupied in the production of alkane and alkene as of normal sources encompass been discovered (Fig. 1) along with preface of biosynthetic pathways into heterologous microbial hosts has enabled the synthesis of a variety of alkanes and alkenes with different structures. Many Valorization of Biomass to Bioproducts https://doi.org/10.1016/B978-0-12-822888-3.00007-4

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Fig. 1 Biosynthetic pathway and enzymes of alkene and alkane.

attempts to generate alkanes and alkenes in engineered microbial strains have been attempted, and current progress indicates assurance for future industrialized invention (Nielsen and Keasling, 2016). Conversely, the titer, rate, and yield (TRY) of alkenes along with alkanes generated through hosts of heterogonous is currently insufficient toward fulfilling commercial needs; moreover numerous hurdles must be solved before these compounds are able to produce through microbial fermentation. In this paper, we examine the research on alkane/alkene biosynthesis and the related biosynthesis pathway of fatty acids, as well as how to overcome the TRY problem to create effective cell factories.

2. Genetically modified microbes used for the production of alkane/alkene derivatives 2.1 Fatty aldehydes decarbonylation Fatty aldehydes are the most common route to alkanes/alkenes (Kunjapur and Prather, 2015). Aldehyde decarbonylases (ADs) have been identified in a variety of species, as well as flora, cyanobacteria, and insects (Bernard et al., 2012; Qiu et al., 2012), with they may renovate fatty aldehydes to alkanes/alkenes while simultaneously producing carbon dioxide (CO2), carbon monoxide (CO), or formate (Marsh and Waugh, 2013). To minimize water desertion and defend themselves against ecological stressors, certain plants produce

Microbial production of hydrocarbon and its derivatives using different kinds of microorganisms

very long-chain (VLC) alkanes (Bourdenx et al., 2011). Alkane synthesis in heterologous microbial systems has been demonstrated using the Arabidopsis thaliana aldehyde decarbonylase CER1 (Bernard et al., 2012; Choi and Lee, 2013). The cuticular layer of insects is made up of a combination of alkanes and alkenes, and it serves as a barrier beside both ecological and communication pheromone assaults. The P450 enzyme of Pest CYP4G1 and CYP4G1-Drosophila co-expression by means of P450 reductase in cytochrome (CPR) allowed Saccharomyces cerevisiae to produce C23, C25, and C27 alkanes (Qiu et al., 2012). Many cyanobacterial strains have been shown to produce alkanes, other than the purpose used for alkane manufacture in cyanobacteria are unknown. The cyanobacteria of ADs were first described as generating CO and alkenes/alkanes (Schirmer et al., 2010), except isotope tracer studies eventually revealed the true co-product as formate. As a result, aldehyde deformylating oxygenase (ADO) was given to the cyanobacteria AD (Li et al., 2012). The act of fatty acid reductase (FAR) or fatty acyl carrier protein (ACP) reductase (AAR) on free fatty acids provides two pathways for the synthesis of alkanes/ alkenes through Ads (Fig. 1). Plant mutant and overexpression studies revealed Arabidopsis CER1 to be an alkane biosynthesis enzyme. While CER1 was uttered only in mold, however, no alkanes were identified. Only by co-expressing Arabidopsis CER1–CER3 in yeast was the synthesis of alkanes VLC with lengths of the chain is C27–C31 verified. Moreover, CYTB5s co-expression or/and LACS1, which act as electron transport mechanisms and acylCoA synthetase on long-chain, respectively, increased alkane titer, resulting in 86 g/mg of DW being converted to nonacosane (C29) (Bernard et al., 2012). Despite the fact that VLC alkanes are produced from CER1 in mold along with flora, the enzyme was revealed to create C8–C14 alkanes in an E. coli engineered species to generate fatty acids short-chain. The strain, which also included numerous extra manufacturing strategies, was formed by means of: (a) obstructing the—pathway of oxidation through the fadE gene is deleted, it encodes acyl-CoA dehydrogenase; (b) the arrangement of shortchain FFAs increased through establish customized thioesterase (TE), TesA among an mutation of L109P and fadR gene of deleting, it encodes acyl-CoA dehydrogenase; (c) fadD gene of expression, which codes on behalf of fatty acyl-CoA synthetase, in support of effective conversion of fatty acyl-CoAs from FFAs, and (d) expression of CER1 as of Arabidopsis thaliana and AAR as of Clostridium acetobutylicum to generate A. thaliana fatty acyl-CoAs as of short-chain alkanes (Choi and Lee, 2013). The AAR activity in cyanobacteria was evaluated using acyl-CoA and acyl-ACP as two substrates, in the occurrence of NADPH in previous work. AAR converted both substrates to fatty aldehydes, although acyl-ACP was the favored one (Schirmer et al., 2010). The particular enzyme is widely utilized to reconstruct alkane/alkene biosynthetic pathways and combining ADO and AAR engineered strains of expression leads to the C15 and C17 alkane synthesis mainly (Buijs et al., 2015; Coursolle et al., 2015; Schirmer et al., 2010; Song et al., 2016; Wang et al., 2013; Zhou Yongjin et al., 2016). The S. cerevisiae of alkane titer, on the other hand, has not yet approached the levels seen in other hosts with AAR-ADO

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co-expression. It was recently discovered that the existence of aldehyde dehydrogenase activity in yeast contributes to this. The S. cerevisiae alkane titer was increased to 23 g/g of DW when HFD1, the gene encoding hexadecenal dehydrogenase (HFD), was deleted (Buijs et al., 2015). Furthermore, removing competing pathways (pox1 and adh5), increasing aldehyde supply (expression of Mycobacterium marinum of carboxylic acid reductase), and increasing ADO expression (Synechoccocus elongatus and Nostoc punctiforme of ADOs) with an S. cerevisiae sp. engineered (Yan and Liao, 2009), accomplishes an constant higher alkane titration (0.83 mg/L), however, it is still not similar to titers obtained with an E. coli strain (300 mg/L) (Schirmer et al., 2010). Fatty acids have long been regarded as ideal substrates for the production of alkanes/alkenes due to their abundance in cells. When the Photorhabdus luminescens of the FAR complex (Howard et al., 2013), M. marinum of CAR (Akhtar et al., 2013), and Oryza sativa of fatty acid-dioxygenase (DOX) (Foo et al., 2016) were uttered along with AD, they were able to produce alkanes/alkenes. The FAR compound is prearranged through the luxC, luxE, and luxD gene operons, with which it exchanges various sequence lengths of fatty aldehydes of fatty acids. When uttered jointly among N. punctiforme in E. coli of ADO, it consequence during a varied series of alkane/alkene chain lengths at a titer of 5.1 mg/L balanced to using the N. punctiforme from the pathway of AAR-ADO (Howard et al., 2013). Furthermore, FatB1 expression, a Cinnamomum camphora C14 fatty acyl-ACP at TE explicit, enhanced C13 alkane and C14 fatty acid synthesis in E. coli. Co-expression of Prochlorococcus marinus of CAR and AD produces 2 mg/L of alkanes/alkenes C11–C17 in E. coli (Akhtar et al., 2013) and adding up of NADPH and ATP converts FAs to their equivalent fatty aldehydes. While a fatty acid translating enzyme, O. sativa DOX has the benefit of utilizing co factor of dioxygen, whereas CAR and enzymes of AAR need NADPH, with its substrate series is smaller cyanobacterial AAR (C16–C18) than the (C12–C18).

3. Using the Fatty acid carboxy-lyases metabolic pathway The alkene terminals, often known as olefins, are significant chemicals in the chemical industry because they are used to make detergents, lubricants, and polyethylene. OleTJE, UndA, and UndB are three kinds of enzymes concerned in the undeviating enzymatic alteration of fatty acids to alkene ends, and the expression heterogonous of this enzyme allows for the synthesis of terminal alkenes in designed microbial species (Rude et al., 2011; Rui et al., 2015, 2014). It is a Cytochrome P450 enzyme of OleTJE from Jeotglicoccus sp. ATCC 8456, and it belongs to the CYP152 family. To a certain extent of decarbonylation, which is accomplished by means of the CYP4G group of enzymes from pest, the family of CYP152 OleTJE produces alkenes by a decarboxylation mechanism. Although the CYP152 family members have been reported to employ solely H2O2 as an electron and oxygen donor, alternative co-factor systems of biocatalysis, such as Fdr/Fdx, CamAB, and RhFRed, have recently been used to accomplish

Microbial production of hydrocarbon and its derivatives using different kinds of microorganisms

H2O2-independent alkene biosynthesis (Dennig et al., 2015; Liu et al., 2014). UndA and UndB, aldehyde decarboxylases as of Pseudomonas species, were freshly identified, and together they produced terminal alkenes while uttered in E. coli (Rui et al., 2015, 2014). The UndA was discovered to be a non-heme oxidase of Fe that transformed 1-undecene from lauric acid (LA) when Fe2+ was present. It also has a limited substrate range, since it can only convert “C-1” terminal alkenes from fatty acids of standard chain (C10–C14) (Rui et al., 2014). Due to sequence homology, UndB was initially categorized as a fatty acid desaturase, but it was subsequently discovered to be a decarboxylation of aldehyde. UndB has a larger substrate variety than UndA, spanning from C6 to C16; however, like UndA, it prefer fatty acids of C10–C14. The homologue UndB, exhibited Pmen 4370 the uppermost undecanoic acid (C11) conversion rate co-expressing of TE-E. coli, UcFatB2 in E.coli strains (Rui et al., 2015), whereas OleT had the highest overall titration of alkane/alkene in strains of E. coli designed (OleT: 96.8 mg/L, UndA: 6.1 mg/L, UndB: 54 mg/L) (Liu et al., 2014; Rui et al., 2015, 2014).

4. Production of hydrocarbon and its derivatives using different microbes Two study groups discovered alkene long-chain production in Sarcina lutea about half a century ago, but they were unable to fully understand the biochemical and genetic data (Albro and Dittmer, 1969; Albro and Huston, 1964; Tornabene et al., 1967). During a topical investigation of Micrococcus luteus biosynthesis of long-chain alkene, the three genes encoding were identified Ole-ABCD concerned in alkane biosynthesis (Mlut 13230–13250). Mlut 13230 contains a preserved energetic position remains seen in enzyme biosynthesis of fatty acid, and its capacity to translate unsaturated monoketones from acyl-CoA in vitro indicates its activity as an OleA homologue (Beller et al., 2010). The Mlut 13250 along with 13240 contain sequence similarity to oleD, and in Stenotrophomonas maltophilia, oleABCD is combination of oleB and oleC outcome in the manufacturing of 40 g/L on alkenes long chain, and oleABCD of heterologous expression as of M. lutea outcome in the manufacture of 40.1 g/L of alkenes long-chain. Individual alkenes, long-chain 3, 6, 9, 12, 15, 19, 22, 25, 28-hentriacontanonaene, are produced in strains of Shewanella oneidensis, and the chemical was interestingly discovered in several bacteria isolated from cold settings. When the oleABCD homologue was removed from the MR-1 strain, alkane production was eliminated, showing that the composite enzyme is implicated in the biosynthesis of alkanes (Sukovich et al., 2010b). Based on a comprehensive sequence homology investigation, OleABCD enzymes were characterized as a combination of thiolase (OleA)/-hydrolase (OleB) and dehydrogenase/reductase (OleD) of short-chain enzymes based on comprehensive sequence homology study (Sukovich et al., 2010a). OleABCD is thought to be begun by OleA, which generates a -keto acid by non-decarboxylative Claisen condensation, and

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then OleD creates a -hydroxy acid via reduction of NADPH-dependent (Frias et al., 2011; Sukovich et al., 2010a). The OleC consumes ATP to convert alkene from-hydroxy acid after the process (Frias et al., 2010). Despite the fact that OleB’s function is unknown, a combination of oleB along with oleC in certain species suggests a connection to OleC’s activity or that it serves as a scaffolding or Ole complex in regulatory function (Wackett and Wilmot, 2015).

5. Metabolic Pathway of polyketide synthase and fatty acids The mechanics of the polyketide and fatty acid biosynthesis routes are extremely similar, and both are regarded promising for biofuel generation. PKSs are made up of acyltransferases (AT), ACPs, and 3-keto-acyl synthases (KS), as well as dehydratases (DHs), enoyl reductases (ERs), and -keto reductases (KRs). The reaction begins by Acyl substrates, while the length of the chain is via the elongation module made by malonyl-CoA, AT, ATP, and KS. After extension, the -hydroxyl group is transferred into -keto grouping, and the domain of TE catalyses decarboxylation with dehydration to liberate the product of alkenes. The C19 alkenes (1, 14-nonadecadiene and 1-nonadecene) synthesized by the Synechococcus strain. PCC 7002 with an end of double bond, and the Curacin A PKS progression was utilized to seem to be for similar proteins that might be occupied in production of alkane. The CurM is the preceding component of Curacin A PKS, which in Lyngbya majuscule, a marine cyanobacterium, generates the terminal double bond. A single enzyme with 45% amino acid similarity to CurM was discovered and designated olefin synthase based on the sequence alignment results (Ols). The consignment domains (LD), KS, ACP2, AT, ACP1, KR, sulfotransferase (ST), as well as TE are among the conserved PKS domains found in Ols. To validate the function of Ols in the biosynthesis of alkene long-chain, a mutant strain of Ols deletion and an overexpression of Ols gene species were produced, and it was observed that alkene biosynthesis was abolished by Ols deletion while high Ols expression enlarged the production of alkene by 4.2 mg/L/OD730 (Mendez-Perez et al., 2011). In one more work, modified Enediyne antibiotic was produced by Streptomyces globisporus C-1027 (37.5 mg/L) and the 1,3,5,7,9,11,13-pentadecaheptaene (PDH, 129.3 mg/L) after co-expression PKS, SgcE, and TE from enediyne, Streptomyces of SgcE10. A SgcESgcE10 make was also commencing interested in E. coli, which outcome in PDH synthesis. PDH, a cell culture take-out product, may be transformed to pentadecane at a titer of 141 mg per liter via a chemical hydrogenation method (Liu et al., 2015).

6. Hydrocarbon derivatives titer, rate, and yield While the initial discovery of alkane synthesis in E. coli (Schirmer et al., 2010), there has been a surge in interest in genetically engineering microbes to create efficient cell

Microbial production of hydrocarbon and its derivatives using different kinds of microorganisms

factories for alkene/alkane production in trade. Even though considerable understanding of S. cerevisiae and E. coli metabolism, the primary cell industrial unit of two was raised area, the productivity of alkenes/alkanes produced through these microbes falls well short of what is necessary for commercial manufacturing. Microbial biofuel production is thought to be commercially feasible at a cost of goods sold (COGS) of about $0.6 per liter. However, producing hydrocarbons at this price is difficult. In S. cerevisiae, for example, the manufacturing of pentadecane (C15) necessitates the use of NADPH and ATP, which reduces glucose yield. However, comprehensive metabolic modeling was used to calculate the theoretical capitulate for the synthesis of several hydrocarbons in mold, including alkanes/alkenes, and it was discovered that as to, while the molar acquiesce is lesser aside from ethanol, the energy capitulate is merely 6%–10% inferior (Caspeta and Nielsen, 2013). Moreover, a techno-economic research revealed that alkanes/ alkenes may be generated more cost-effectively than ethanol if 90% of the utmost theoretical capitulate is able to be attained (Caspeta and Nielsen, 2013). One reason for this is that hydrocarbon separation costs are expected to be cheaper than the very luxurious distillation process utilized to make ethanol. However, it is apparent that in order to achieve industrialized practicality, microorganisms in terms of TRY have to be optimized, and we will examine the current state of designing cell industrial units for increasing TRY of alkanes/alkenes in the next section.

7. Toxicity of the biosynthetic pathway When high chemical productivity is required in microorganisms, toxicity is a critical factor to consider. In microorganisms, alkanes/alkenes are hazardous chemicals that alter membrane cell reliability and purpose, causing growth reserve or even cell death (Sikkema et al., 1995). Various methods have been used to alleviate chemical toxicity in microbial hosts ( Jin et al., 2014). Conversely, every method only addresses a portion of the problem, necessitating further basic study into alkane toxicity. To plan cellular responses to toxicants, one method is to integrate toxicology with genomes, dubbed toxicogenomics by others. Transporters have recently materialized as candidates for trade in regulating to decrease the toxicity level of chemicals, based on mechanistic knowledge of cellular activities (Dunlop, 2011; Mukhopadhyay, 2015). The cells were treated with C9–C12 alkanes to explore alkane/alkene associated carriers in S. cerevisiae, and transcriptome data revealed numerous increased plasma membrane efflux pumps. In excess of expression of Snq2p as well as Pdr5p enhanced the tolerance of alkanes by lowering intracellularly decane with undecane attentiveness (Ling et al., 2013), while more phrase of Snq2p along with Pdr5p enhanced alkanes acceptance via dropping intracellularly decane with undecane attentiveness. ABC transporters in Yarrowia lipolytica were investigated in another investigation to find effective efflux pumps. For the susceptibility experiment, diverse chain measurement lengthwise (C8–C12) alkanes were provided, and the phrase

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of transporters of ABC2 and ABC3 enhanced easiness to alkanes of C10 and C11 (Chen et al., 2013). Among the four ABC transporters studied, an essential was shown to be the best efflux pump, and the rationale for increased alkane tolerance is made clear through a drop in intracellular alkane levels. The efflux pumps, on the other hand, it will not develop C8 and C9 alkane acceptance, and in excess of expression of efflux pumps of Snq2p and Pdr5p in S. cerevisiae only raised acceptance to alkane C10 and C11 other than not to alkane C9 (Ling et al., 2013). As a result, it is proposed to extend the tolerance ranges of efflux pumps using a variety of techniques such as heading for progression and enzyme production based on configuration-based studies (Chen et al., 2013; Ling et al., 2013).

8. Future scope of the study Alkanes/alkenes are scientifically important compounds that can be utilized as chemical starting materials or as liquid transportation fuels. Despite the fact that many species spontaneously manufacture alkanes/alkenes, industrial demand for these compounds is now met only by fossil fuels. Microorganisms that can perform alkane/alkene biosynthesis may be able to produce these essential compounds in the future in a sustainable and environmentally friendly manner. In heterologous microbial hosts, fatty acid biosynthesis supplies various pathways to recreate alkane/alkene production. Microbial engineering for alkane/alkene synthesis has demonstrated that microbes can be engineered to produce a wide spectrum of alkanes/alkenes. However, the chemicals’ TRY levels are much below those required in the industry, and additional research is required to create well-organized cell factory. Intracellular biosensors might be useful in this situation since they permit for high-throughput transmission and strain selection. Alkane/alkene biosensors have been developed using microorganisms that decompose alkanes/alkenes. Alkane biosensors are made up of three parts: a transcriptional regulator that responds to alkane, a supporter that is made active via a regulator, and a reporting protein (e.g., GFP—green fluorescent protein). Due to restricted detection range or correct operation in a heterologous host, previous biosensors have difficulty being used in heterologous hosts (Sticher et al., 1997; Zhang et al., 2012). However, a novel biosensor based on a chimeric alkane response element (cARE) (Wu et al., 2015; Yan and Liao, 2009) shows tremendous potential. This was reassembled using an older biosensor along with allowing in-situ uncovering of mutually alkane of standard and long chain in E. coli using fluorescence-activated cell sorting (FACS) (Qiu et al., 2012; Yan and Liao, 2009). Using a biosensor like this will allow for quicker viewing of better strains. Despite the fact that alkanes/alkenes can be produced as of sugar in a development comparable to ethanol first-generation manufacture, our technology financial examination revealed to with the feedstock of biomass would permit additional cost reductions (Caspeta and Nielsen, 2013), as well as a significant reduction in greenhouse gas emissions, even more

Microbial production of hydrocarbon and its derivatives using different kinds of microorganisms

than secondary bioethanol. As a result, reducing the cost of pre-treatment for lignocellulosic biomass and engineering microbes to use diverse carbon sources should be explored. Finally, novel enzymes have been discovered in diverse natural alkane/alkene routes that can be utilized to recreate alkane/alkene biosynthetic pathways in heterologous hosts of microbes. By means of diverse combinations of these enzymes, researchers were able to boost output while also allowing for a greater variety of complex forms. Table 1 shows the titration of engineered strains in alkane/alkene, with the AAR-AD route having the greatest titer between the pathways of biosynthetic has been illustrated to date. Dissimilar substrate specificities and activity were observed in the enzymes. UndB, for example, has a wider substrate variety apart from UndA (Rui et al., 2015), while CAR from P. marinus synthesizes numerous aldehydes, whereas it generates two cyanobacteria AAR merely (Akhtar et al., 2013; Schirmer et al., 2010). Furthermore, Table 1 Production of alkanes/alkenes through biologically engineered microbes.

1

Ols

2 3

UndB AAR-AD

S. globisporus E. coli Synechoccus sp. PCC7002 E. coli E. coli E. coli

Alkene (15:7) Alkene (C15) Alkenes (C19, C19:2)

E. coli E. coli S. cerevisiae

Alkenes (C5–C17) Alkanes (C15, C17) Alkanes (C9, C12, C13, C14), Alkene (C13) Alkanes (C15, C17) Alkanes (C15, C17), Alkene (C17) Alkanes (C13, C15, C17)

S. cerevisiae

Alkanes (C27–C31)

S. cerevisiae

Alkanes (C13, C15, C17), Alkenes (C15, C17) Alkanes (C15, C17), Alkene (C17)

4 5

OleABCD CAR-AD

Synechocystis strain. PCC6803 E. coli E. coli

6 7

DOX-AD FAR-AD

S. cerevisiae E. coli

8

OleT

S. cerevisiae E. coli

9

UndA

E. coli

Alkenes (27:3, 27:2, 29:2, 29:3) Alkenes (C15, C17), Alkanes (C11, C13) Alkanes (C14, C16) Alkanes (C13, isoC13, C15, C16, C17), Alkenes (C13, C15, C16, C17) Alkenes (C11–C19) Alkenes (C11, C13, C15, C15:2, C17:2) Alkenes (C9–C13)

129.3 mg/L 140 mg/L 4.2 mg/L/ OD730 55 mg/L 300 mg/L 580.8 mg/L 7.7 mg/L 255.6 mg/L 22 μg/g of DW 86 μg/g of DW 0.82 mg/L 26 mg/L 40.1 μg/L 2.1 mg/L 73. 6 μg/L 5.1 mg/L 3.7 mg/L 97.6 mg/L 6.2 mg/L

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compared to OleT and UndA, UndB had a higher fatty acid conversion rate for alkene synthesis (Rui et al., 2015). As a result, it is predicted that the identification of novel enzymes for alkane biosynthesis will help to enhance alkane/alkene production. Furthermore, biosensors are likely to speed up the enzyme screening and selection process, as well as the assessment of various ways to increase the titer, rate, and yield of cell factories manufacturing alkanes/alkene and therefore minimize strain progress moment. Engineered biosensors have also been used to regulate gene expression while avoiding the buildup of hazardous intermediates in the cell (Dahl et al., 2013) and to optimize specific reactions in biosynthetic pathways using concurrent monitoring (Rogers and Church, 2016). Alkane/alkene biosensors will be achievable advances in finding bottleneck stages sooner as well as establishing efficient cell factories by overcoming present strain engineering difficulties.

9. Conclusions Alkenes/alkanes are important scientific substances that can be used as chemical starting materials or liquid transportation fuels. Even though the reality is that lots of microbial strains produce alkanes/alkenes on their own, fossil fuels are presently the primary sources of these chemicals for industrial use. Microbial species are capable of alkane/alkene biosynthesis and could be able to generate these important chemicals in a sustainable and ecologically acceptable approach in the outlook. As a consequence, it’s expected that discovering new alkane biosynthesis enzymes will aid in increasing alkane/alkene output. Moreover, the speed up of biosensors are expected to improve the enzyme screening and selection process, along with the evaluation of various methods for increasing the titer, rate, and yield of cell factories producing alkanes/alkenes and thereby reducing microbial strain improvement time. The biosensors of have also been utilized to optimize particular processes in biosynthetic pathways via contemporaneous supervision and to control gene expression although keeping away from the accumulation of dangerous intermediates in the cell. Alkane/alkene biosensors will enable faster detection of bottleneck phases and the establishment of proficient cell factories by addressing current microbial species engineering challenges.

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CHAPTER 9

Biomass valorization to biobutanol Tahir Mehmooda,b, Fareeha Nadeema, Bisma Meerc, Hajra Ashrafc, Kushif Meerd, and Shagufta Saeeda a Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan Centre for Applied Molecular Biology (CAMB), University of the Punjab, Lahore, Pakistan c Department of Biotechnology, Quaid-i-Azam University, Islamabad, Pakistan d Institute of Chemistry, University of the Punjab, Lahore, Pakistan b

1. Introduction The demand for energy production is high because of a rampant increase in the human population. The escalating trend of energy consumption has led to dependence on nonrenewable energy sources, mostly fossil fuels, i.e., coal, gas, and petroleum, which would shortly exhaust the current global supplies. Thus, they will not be capable of persuading the future needs for everlasting production and consumption of energy. Various environmental concerns have been taken into consideration by the utilization of fossil fuels, such as global warming due to the emission of greenhouse gases during fossil fuel combustion in forming energy. Therefore, another source is needed, which is quick as well as readily available and greener in nature. In order to have little miniature alternatives, the world is moving toward biofuel production by biomass valorization to solve energy disasters. The familiar biofuel, i.e., biodiesel, is identified traditionally to have explosive perspective in contrast to fossil fuels, besides to its renewability for ceaseless applications (Leong et al., 2018). Biomass can be defined as a living material that is derived from animals, plants, and microorganisms (fungi and algae) that are present in a variety of environments across the surface of the earth. Biomass can be categorized into two components in the case of terrestrial plants: non-lignocellulosic and lignocellulosic materials (Ravindra, 2015). Non-lignocellulosic material mainly constitutes structural components (proteins, sugar, and lipids), low molecular weight cytoplasmic components, inorganic molecules, as well as ions and a soluble fraction of cellulose and lignin. Lignocellulosic material constitutes non-starch along with fibrous structural parts forming lignin, cellulose, and hemicellulose (Braghiroli and Passarini, 2020). Butanol is particularly interesting among the potentially usable renewable energies due to its less emission of pollutants, low vapor pressure, and safety for handling and storage (Kattela et al., 2019; Baral and Shah, 2016). Butanol, also known as biobutanol,

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can be formed from the same feedstock such as corn grain, alternative biomass, and ethanol (Motghare and Wasewar, 2019). Biobutanol is an alcohol that can be made from the fermentation of sugars from biomass, i.e., food waste, cassava starch, inulin, palm mill effluent, corn stover, algal biomass, cane molasses, cheese whey, and sugarcane bagasse by utilizing aerobic as well as anaerobic bacteria (Abo et al., 2019). This process is regarded as acetone-butanol-ethanol (ABE) fermentation (Gao et al., 2015).

1.1 Generations of feedstock Biofuel is a fuel obtained from waste biomass, algae biomass, and plant material. Since biomass is sustainable, biofuel is regarded as a renewable source of energy as compared to non-renewable sources such as coal, petroleum, and natural gas (The Three Different Types of Biofuel and their Uses, 2020). Biofuel obtained from natural sources could aid in lessening greenhouse gases in the environment and also aid in continuing the carbon balance in the atmosphere. Biofuels are categorized into two groups: primary and secondary biofuels. Primary biofuels are referred to as the natural, unprocessed sources utilized for the production of heat and electricity, e.g., firewood, crop residue, forest materials, animal waste, and plants. In contrast, biofuels that are derived from biomass that is processed referred to as secondary biofuels. Secondary biofuels are further divided into three groups: first generation, second generation, third-generation biofuels, and fourth-generation biofuels, primarily rely on the feedstock utilized (Pattanaik et al., 2019) shown in Fig. 1. 1.1.1 First-generation biofuels Biofuels that are directly formed from food crops such as sugarcane, rapeseed, soybean, corn are indicated as first-generation biofuels as they are edible biomasses and it also minimizes the need of arable land for the production of food (Pattanaik et al., 2019). Their extra masses are used in the production of biofuel (bioethanol and biodiesel) but with time demand of biofuel increases. It leads toward food shortage ( Jang et al., 2012). 1.1.2 Second-generation biofuels While the main focus of second-generation biofuels is lignocellulosic biomass (LCB) in the form of non-edible plants or their parts, crop residues, and forest waste. Disposal of such waste is complicated. As there is a tremendous increase in energy demand, this type of biomass is generally used for obtaining energy ( Jang et al., 2012). Some other benefits of second-generation biofuel are as follows: It is comparatively cheap, non-food-based, and uses biomass derived from waste material (Pattanaik et al., 2019). 1.1.3 Third-generation biofuels Third-generation biofuels raised by the utilization of various oleaginous microorganisms, i.e., microalgae, fungi, and bacteria have preceded the rise of thirdgeneration biofuels. The utilization of microalgae biomass feedstock has benefited over

Biomass valorization to biobutanol

Fig. 1 Generations of feedstock.

other species of microbes because of its higher growth capabilities, high oil/lipid carbohydrates, lipid, oil and protein content, biomass productivity, and ability to gather large cell lipid content (20%–77%) (Leong et al., 2018). The biomass obtained from this source has an advantage over the other two sources because it does not demand any fertile and cultivated land. Moreover, its lipid content is very high as compared to others. Due to a lack of technology, a high amount of biofuel production from this biomass could not be achieved. But in upcoming years, it will be in great demand (Srirangan et al., 2012) 1.1.4 Fourth-generation biofuels Through genetic modification of microalgae, their quality and productivity can be enhanced and used as a fourth-generation biofuel. Lipid, protein, carbohydrates, and nucleic acid content are very high in microalgae. The lipid may be polar or in the form of neutral lipid in microalgae. Polar lipids determine the shape of the cell membrane and give it strength. Neutral lipids are involved in the storage process. Among other content, lipid provides more incredible energy approximately 37.6 kJ 1. Fourth-generation biomass becomes efficient due to its high lipid content (Shokravi et al., 2019).

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2. History of biobutanol Usage of biofuels was widespread in the 19th century. Their invention is not novel. In 1912, Rudolf Diesel ran his engine with peanut oil (D€ urre, 2007). Later Nikolaus August Otto and Henry Ford ran their engines with ethanol. It is reported that David Ramey used biobutanol as a fuel instead of gasoline to run his car, although more than 9% of biobutanol was consumed. But at the same time, the emission of hydrocarbons was reduced significantly. Biobutanol production is not among the latest inventions; in 1862, during Vibrion butyrique fermentation, Louis Pasteur felt the presence of biobutanol as a by-product (Pasteur, 1862). It was not produced from the pure medium but from the medium of Clostridium acetobutylicum and Clostridium butyricum (Sauer et al., 1993). Albert Fitz achieved the production of the first pure culture of Bacillus butylicus by fermentation of glycerol and sucrose to butanol. Butyrate, CO2, and hydrogen were carried out by Bacillus butylicus. This formed its clostridial forms, which were highly sensitive to butanol. By increasing butanol concentration, it undergoes severe damage. This laid the foundation for the scientist to further isolate solvent-forming bacteria. In 1905, Scherzinger reported acetone formation during the fermentation process (Fitz, 1878). In the 20th century, scientists paid great attention toward the synthesis of biobutanol as natural rubber production had fallen, scientists made great efforts to produce synthetic rubber. It was found that butanol or isoamyl alcohol was involved in the production of butadiene or isoprene rubber. Between 1912 and 1914, Chaim Weizmann was the first to perform biological screening of microbes during fermentation. Isolation of C. acetobutylicum resulted in the production of more acetone and butanol. In World War I, British army wanted to produce smokeless explosive material. In their production, a large amount of acetone was required (Qureshi and Blaschek, 2001). Weizmann helped the army to build setup that produced enormous acetone. Later, Great Britain also adopted this mechanism. When the US troops joined Britain’s army in the war, they built another production house for acetone. This house was closed within one year. At the end of World War I, a large amount of butanol had been obtained as a by-product of acetone. Also, a joint project started by the US and Britain armies for the production of acetone was again opened. But this time, butanol production started there (Gao and Rehmann, 2014). In 1936, after the expiry of Weizmann potent, a large setup was design for fermentation by an anaerobic bacterium. Fermentation by molasses started, isolation of new microorganisms occurred. An enormous increase in the production of acetone and butanol by patented microorganisms has been witnessed (Li et al., 2014). During World War II, acetone utility had been increased tremendously, so large amount of acetone was produced from molasses fermentation. Not only Great Britain and the US, but some other countries like Australia, Africa, and India are involved in its production. While after the end of World War II fermentation process become slow. As farmer interest toward

Biomass valorization to biobutanol

molasses increased, which result in increased molasses price, making acetone, and butanol production expensive (Galadima and Muraza, 2015).

3. Global energy scenarios Demands of global energy are generally fulfilled by non-renewable sources such as natural gas, oil, and coal. But continuous use of these resources caused their depletion. Moreover, the high rate of fossil fuels and environmental issues related to them force researchers to adopt another way to fulfil increasing energy demand. For this purpose, biofuels are used as an alternative way to fulfil these demands (Rathour et al., 2018). In 2019, production of biofuels in all developing regions increased globally, such as in Brazil, China, Indonesia, etc. Biofuels are environment-friendly fuels and obtained generally from animal fats and plant oils, so they prove a good and reliable alternative fuel. They are predominantly obtained from biomass (Mofijur et al., 2015). Biomass has been ranked fourth as an energy source around the globe. It is estimated that it fulfils approximately 14% of the world’s energy needs. In addition, biomass is a clean, renewable energy source. Ethanol is used as a biofuel, and it is used primarily in transportation. World production of ethanol and biodiesel in 2011 was around 545 and 147 million barrels, respectively. The US globally shares 61% of ethanol production. Brazil shares 26%. In biodiesel production, the EU shares 44%, US 16%, and Brazil 11%. These shares were estimated in 2011 (To and Grafton, 2015). Four carbon alcohols which are produced by fermentation of biomass are biobutanol. Energy obtained from biobutanol is the same amount as from gasoline. In America and Europe, fleet testing of biobutanol has started. As the numbers of cars and vehicles are increasing rapidly on the roads. It is expected that in 2050, there will be more than 2 billion cars on the road. This demand can be fulfilled by biobutanol. In the global market, China consumes the maximum amount of biobutanol as it has the largest market of paint and adhesive tapes. Biobutanol is a quality standard fuel, and in the future, it can be used in automobiles with traditional fuels up to 12.5% biobutanol share same feedstock of ethanol. Globally, biobutanol tends to replace conventional fuels such as ethanol, diesel, and gasoline (Motghare and Wasewar, 2019). The overall production method of biobutanol from biomass is shown in Fig. 2.

4. Lignocellulosic biomass LCB is an ample and sustainable resource from plants mainly constituted polysaccharides, i.e., cellulose, hemicellulose, and lignin (an aromatic polymer). LCB is the most potent source to form second-generation biofuels, bio-sourced materials, and chemicals without embracing global food security. A major restriction in the

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Fig. 2 Steps for biobutanol production from the valorization of biomass.

valorization of LCB is that it’s resistant to enzymatic hydrolysis due to heterogenous multi-scale structure of the cell wall of plants (Zoghlami and Pae¨s, 2019). Hemicellulose, cellulose, and lignin are not evenly distributed within the cell walls. The quantity and structure of components of the plant cell wall alter according to the maturity of the plant cell wall, species, and tissues. Commonly, LCB constitutes 35%–50% cellulose, 20%–35% hemicellulose, and 10%–25% lignin. Ash, proteins, and oils make up the remaining fraction (Zoghlami and Pae¨s, 2019; Isikgor and Becer, 2015).

4.1 Cellulose Cellulose is the superabundant lignocellulosic polymer, with 40–60% in weight, constituting of ß-D-glucopyranose units associated by (1,4) glycosidic bonds, with the basic repeating unit named as cellobiose (Sharma et al., 2019). Cellulose chains are made up of 500–1400 D-glucose units that are ordered together to form microfibrils that are packed together to generate cellulose fibrils (Robak and Balcerek, 2018). Cellulose fibrils are implanted in a lignocellulosic matrix due to this it is resistant to enzymatic hydrolysis. The content of cellulose was correlated positively with glucose release. A significant role in LCB recalcitrance is played by the number of glucose units in the polymer referred to as the degree of polymerization. But the exact role is still not understandable. Variations in the degree of polymerization always go along with the alterations in structural parameters such as porosity and crystallinity. It is reported that a decrease in the degree of polymerization of cotton linters by γ-irradiation had a little impact on the rate of saccharification. It is indicated that the degree of polymerization of cellulose was correlated negatively to the cellulose hydrolysis. It is predicted that long chains of cellulose constitute more hydrogen bonds, challenging to hydrolyze. In contrast, shorter chains of cellulose constitute a weaker hydrogen bond system and, therefore, aid in enzyme accessibility (Meng et al., 2015).

Biomass valorization to biobutanol

4.2 Hemicelluloses and acetyl groups A heterogenous group of biopolymers with 20%–35% biomass weight is called hemicelluloses (Chandel et al., 2018). It constitutes several monosaccharide subunits to form xylans, mannans, xyloglucans, and glucomannans. The degree of polymerization of hemicellulose is in the range of 100–200 units. It is very less in contrast to cellulose, but it can indicate a high degree of less or more complex substitutions. Hemicellulose physical strength is little, with an amorphous nature. It is hydrolyzed readily by dilute bases or acids along with hemicellulase enzymes. Hemicelluloses function as a physical barrier, hindering the enzymes’ accessibility (de Oliveira Santos et al., 2018). It has been indicated that the removal of hemicelluloses by steam explosion or dilute acid pretreatment could enhance cellulose conversion by modifying enzyme accessibility to cellulose. It is determined that when pre-treated pine cellulose is removed, it modifies the area accessible for enzymes and fibers porosity. The effect of hemicelluloses on the recalcitrance of LCB is still not clearly understood as some lignin is also separated with hemicelluloses. Several studies have suggested that removal of hemicelluloses was more efficient in contrast to the removal of lignin for modifying the rate of enzymatic hydrolysis, whereas others reported that the removal of lignin was much more important (Kruyeniski et al., 2019). LCB can be acetylated extensively with acetyl groups. Acetyl groups may hinder the accessibility of cellulose by interrupting with enzyme recognition. It also might predict the productive binding formation between the catalytic domain of cellulases and cellulose through enhancing the cellulose chain diameter or altering its hydrophobicity. It is suggested that the reduction in the acetyl content modified the effectiveness of the enzyme. While several studies on switchgrass, wood, bagasse, and wheat straw indicated that the impact of deacetylation is more remarkable on the digestibility of hemicellulose than on digestibility of cellulose. It is also observed that the effect of acetyl groups depends on biomass crystallinity, lignin, and cellulose content (Zoghlami and Pae¨s, 2019; Zhu et al., 2008).

4.3 Lignin The second most abundant polymer in LCB is lignin, after cellulose, with 15%–40% dry weight (Ragauskas et al., 2014). It is a very composite amorphous, heteropolymer of phenylpropanoid building units (Sinapyl alcohol, p-coumaryl, and coniferyl). It is responsible for structural rigidity and hydrophobicity. It attaches hemicelluloses to cellulose in the cell wall. It is familiar that lignin performs a negative role in cellulose conversion governed by various factors such as lignin composition/structure (especially S and G units’ content and hydroxyl groups), total lignin content (Santos et al., 2012). First of all, lignin can hinder the accessibility of polysaccharides physically; it performs a function as a physical barrier that inhibits enzyme access to cellulose. It can also adsorb cellulases irreversibly and other enzymes during enzymatic hydrolysis due to its hydrophobic structural attributes

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constituting polyaromatic structures, hydrogen bonding, and methoxy groups. Previous studies reported that lipid content was correlated negatively with enzymatic digestibility in wheat straw, in poplar, in miscanthus and in transgenic rice. Lignin removal commonly disrupts the carbohydrate-lignin matrix, enhances the porosity, and decreases the adsorption sites that are non-productive for enzymes. It has been suggested that compounds that are derived from lignin (phenolic hydroxyl groups) are responsible for reversible inhibition of cellulases. Inhibiting free phenolic hydroxyl groups by chemical reactions, i.e., hydroxypropylation, crucially decreased by 65%–91% the inhibitory effect of lignin. Studies suggest that the lignin S/G ratio is prime as an independent recalcitrance factor. However, the correlation between recalcitrance and the S/G ratio is still not clear. It is observed a positive correlation between hydrolysis yields for woody chips, miscanthus, and S/G ratio to cellulase because of G that have branched structure showed higher binding capacity over S with a low degree of polymerization and linear structure. While others indicated a negative correlation between enzymatic hydrolysis and S/G ratio in woody chips, in genetically engineered poplar, in pre-treated wheat straw and in pre-treated miscanthus. Comprehensively, lignin contributes firmly to LCB recalcitrance affected by its structure and chemical composition, restricting the enzymes’ accessibility to cellulose (Zoghlami and Pae¨s, 2019).

4.4 Non-lignocellulosic biomass The biomass whose primary components constitute proteins, lipids, starch, minerals, and inorganic material is referred to as non-LCB and includes manure, sewage sludge, and algae. Non-LCB poses a severe threat to the environment because of more content of heteroatoms and heavy metals such as phosphorus, nitrogen. Water system polluted by these heavy metals, gather in food chains and result in serious health concerns (Anukam and Berghel, 2020).

5. Biomass pre-treatment Pre-treatment for the production of biobutanol includes physical, chemical, and biological treatments, which are explained further.

5.1 Physical pre-treatment Pre-treatment of LCB is necessary for the conversion of LCB into useful products. It’s required to convert large-sized particles into small ones, which results in an increase in surface area and in decrease in crystallinity and polymerization (Rajendran et al., 2018). Physical methods are more eco-friendly than other chemical methods and rarely produce any harmful material (Shirkavand et al., 2016). However, the major drawback of this method is the high energy requirement. Softwoods require lower energy consumption

Biomass valorization to biobutanol

as compared to hardwoods and the estimated amount required for switchgrass and corn stover is 27.6 and 11 kWh/metric ton, while poplar chips and pine require 118.5 and 85.4 kWh/metric ton (Rajendran et al., 2018). The common pre-treatment methods are the following: 5.1.1 Milling Milling is used for reduction in size and crystallinity of LCB. It reduces the size of LCB to approximately 0.2 mm. There are different types of milling depending upon the operating equipment, i.e., vibratory milling, rod milling, colloid milling, two-roll milling, wet disk milling, and hammer milling. Change in size of particle depends on time, type of milling method adopted, and biomass used (Marousˇek, 2012). However, the major drawback of using milling is the cost of equipment and the high amount of energy. Though milling does not produce any harmful or inhibitory compounds, it can be used as a preliminary pre-treatment for many biomasses. 5.1.2 Microwave-assisted size reduction Microwave irradiation is one of the non-conventional methods used for LCB pre-treatment. It has several advantages, including least inhibition formation, easy operation, maximum heating capacity in a short period of time and energy-efficient. This treatment is operated at 150–250°C in a closed bioreactor (Intanakul et al., 2003). 5.1.3 Ultrasonication Ultrasonication pre-treatment is the application of ultrasonic radiation through the principle of cavitation. This cavitation produces shear stress which breakdown the complex structure of LCB into required compounds like lignin, cellulose, and hemicellulose (Xie et al., 2016). Studies revealed that the ultrasonication pre-treatment scales down the biomass hydrolysis time, making this method more favorable for biofuel production. However, the major drawback of this method is that it requires highly energy-intensive and detailed optimization of the procedure before operating (Shi et al., 2013).

5.2 Chemical pre-treatment Chemical pre-treatment includes further subtypes: 5.2.1 Ozone pre-treatment Ozonolysis of LBM started in the 80s, but during the last few years, it has become frequently used for LCB pre-treatment (Kumar et al., 2009). Pre-treated biomass has simpler sugar, which is used for the production of biofuel, biogas, etc. During ozonolysis, short-chain carboxylic acids are produced which act as inhibitory compounds, but they are removed after washing. Ozonolysis of biomass depends upon several factors, including ozone concentration, particle size, ozone airflow, moisture content, pH, and reactor

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design (Travaini et al., 2016). It was indicated that the measure of devoured ozone dictates the reactivity of plant substrates pre-treated with ozone, and the pace of ozone utilization relies upon the substance of water is an example. Ideal estimations of dampness (2FSP) and ozone utilization (2–3 mol O3/C9PPU lignin) were found. 5.2.2 Hot water treatment Liquid hot water treatment is an eco-friendly pre-treatment used for the breakdown of hemicellulose. It does not only reduce the time for the production of biofuel by making reducing sugar available for enzymes but also minimizes the formation of the inhibitory product (Zhuang et al., 2016). At a higher temperature, usually at 200°C LHWT dissolves all hemicellulose without using chemicals and removes lignin within the time duration of 2 min (Sreenath et al., 1999). 5.2.3 Organosolvent process An organic or aqueous organic solvent is used to degrade or remove lignin or a small amount of hemicellulose. In this method, solvent alone in a combination of catalyst, i.e., salicylic acid, oxalic acid, is used to minimize the operational time. A number of organic solvents used are ester, ketone, ether, etc. This method can be used in combination with acid hydrolysis, which proved more beneficial as more than 70% of lignin removed at a lower temperature (Palonen et al., 2004).

5.3 Biological pre-treatments Biological pre-treatments are commonly non-toxic, have low vitality prerequisites, and do not create inhibitors for downstream cycles. Bacteria can be utilized to hydrolyze cellulose or to depolymerize and eliminate lignin. Conditions for natural pre-treatments are affected by physical, (for example, temperature, size of the particles), chemical (for example, pH), and organic (strain of microbes or parasites) conditions. Other than the creation of bioethanol from starch, biogases can likewise be delivered relying upon the states of the pre-treatment, and the microbes/parasites utilized (Sindhu et al., 2016). Biological treatment is not only used for lignin removal but also used for the removal of selective compounds, i.e., anti-microbial substances. Little environmental conditions, lesser energy requirements, and no need for chemicals make it more attractive for lignocellulosic pre-treatment; however, this requires more time than other physical and chemical treatments (Berlin et al., 2006). 5.3.1 Detoxification To increase the rate of fermentation, the detoxification method is generally used. In this method, inhibitors have been removed from lignocellulosic hydrolysate, and microbes get easy access to available sugars (Alriksson et al., 2005). For the fermentation process, different approaches have been used that are:

Biomass valorization to biobutanol

Table 1 Different pre-treatment method for biomass valorization. Microorganism

Biomass

Pre-treatment

References

Clostridium beijerinckii

Corn fiber, corn stover Rice bran, defatted rice bran Empty fruit bunch

Acidic pre-treatment

Ezeji et al. (2007) Lee et al. (2009)

C. beijerinckii NCIMB 8052

Clostridium acetobutylicum

C. acetobutylicum SE-1

Corncob

Clostridium thermocellum and Clostridium saccharoperbutylacetonicum C. acetobutylicum B 527

Rice straw

C. acetobutylicum

C. acetobutylicum

C. saccharoperbutylacetonicum N1-4

Pineapple peel waste rice straw, sugarcane bagasse rice straw, sugarcane bagasse Switchgrass

Acidic treatment, enzymatic hydrolysis Acidic and enzymatic hydrolysis Wet disk milling with enzymatic hydrolysis Alkaline pretreatment Acid hydrolysis – Detoxification Alkaline and enzymatic pretreatment Alkaline and enzymatic pretreatment Acidic pre-treatment

Noomtim and Cheirsilp (2011) Zhang et al. (2013) Kiyoshi et al. (2015) Khedkar et al. (2017) Tsai et al. (2020) Tsai et al. (2020) Wang et al. (2019)

1. Liquid extraction (supercritical CO2, tri-alkylamine, ethyl acetate) 2. Solid-liquid extraction (activated carbon treatment, ion-exchange, and lignin) 3. Alkaline treatment (NaOH, Ca(OH)2, and NH4OH) 4. Enzyme treatment (peroxidase and lactase) 5. Microbial treatment ( J€ onsson et al., 2013) Coniochaeta ligniaria, a fungal strain and thermophilic bacteria, Ureibacillus thermosphaericus, have the ability to remove furfural, phenolic compounds, and 5-hydroxymethylfurfural (HMF). Organic acids, furfural aldehydic compounds, and phenolic substances have been removed easily from detoxification methods (Olofsson et al., 2008). Table 1 shows different pre-treatment methods for biomass.

6. Acetone-butanol-ethanol (ABE) fermentation Even though the formation of ethanol from renewable sources has gained greater attention, the butanol and the acetone-butanol-ethanol (ABE) production processes have also been examined as having more inherent. Biobutanol, named after biodiesel and

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bioethanol, has been severely examined having more potential. Due to this, scientists have shown an interest in biobutanol as a suitable renewable fuel (Abdi et al., 2016). Several types of sugar can be used by bacterial species and result in ABE fermentation, utilizing an anaerobic fermentation process. Initially, the production of biobutanol was done through ABE fermentation and further proceeded by contributing to the production of ethanol using yeast, making the ABE process the second-largest industrial process. The method of ABE fermentation resulted in the formation of acetone, butanol, and ethanol. These substances contributed to the ratio of 3:1:6. Among these products, a considerable amount of biobutanol is formed (Pfromm et al., 2010). Several aspects, such as low substrate cost, eco-friendly nature, economic stability, and availability, make biobutanol a leading biofuel. In contrast to ethanol, the calorific value of biobutanol is higher with a lower freezing point. The process of end-product inhibition influences ABE fermentation for ABE products. This may be due to the existence of a solvent system in it. To rectify this, much care is needed for solvent recovery ( Jones and Woods, 1986). Production of biobutanol occurs by ABE fermentation which is biphasic: first butyric acid and acetic acid are formed in the acidogenesis phase, then re-assimilation of acids occurred to yield the solvents, i.e., acetone, butanol, and ethanol (Terracciano and Kashket, 1986). The batch fermentation process works on a simple principle, but the fermentation process can be repressed at once by supplying the substrates to communities of microbes. Conditions in the process vary continuously due to cell metabolism, and there is no control and as such steady production is not achieved. Improper mixing can also stimulate settlement layers, making the technique inefficient (Patinvoh and Taherzadeh, 2019). The most studied fermentation is batch fermentation due to little risk of contamination and simple operation (Lee et al., 2008), and readers can approach various original studies of batch fermentation of LCB to form biobutanol (Ezeji and Blaschek, 2008). Low productivity can be the result of low density, so biofilm reactors and absorbed substrate fermentation have been applied to cope with this issue in the batch process (He and Chen, 2013; Qureshi et al., 2005a). The most beneficial mode to solve substrate inhibition is fed-batch by slowly adding substrate, thus keeping the concentration of substrate below toxic levels (Li et al., 2011). Fed-batch processes are a combination of both continuous and batch processes; the bioreactor with the inoculum is fed with a little amount of the feedstock at the commencement of the process, and that’s why there is periodic or continuous feeding without fermentation broth removal (Zabed et al., 2017). This operational model with the recycling of cells is mostly utilized due to the possibility of achieving higher volumetric productivity; feeding optimization is crucial for improved product yield (Sanchez and Cardona, 2008). Various studies have examined the optimization and feasibility of the fed-batch process for potent production of biofuel, better productivity, higher product yield, and shorter retention time were reported in contrast to batch fermentation (Patinvoh and Taherzadeh, 2019).

Biomass valorization to biobutanol

Continuous batch allows the substrate to continuously feed into the reactor, and an equal quantity is separated to achieve a constant working volume. A little amount of substrate is provided at the initial stage, and then the loading rate is enhanced gradually to attain the target, the balance between feed as well as the discharge is maintained for enough retention time to achieve steady state condition (Brethauer and Wyman, 2010) with low operational cost and high productivity (Mears et al., 2017). This process can be utilized for fermentation of lignocellulosic hydrolysate constituting inhibitors; it will reduce the inhibitory impact on the cells since the addition of the feed at low concentration (Patinvoh and Taherzadeh, 2019). Chemostat (continuous fermentation) has benefited over fed-batch, and batch modes such as its productivity improved (Li et al., 2011). To improve the performance of chemostat cell recycling, multi-stage, immobilized cell, and bleeding techniques have been applied (Birgen et al., 2019).

6.1 Substrates and biomass for ABE fermentation Under batch conditions, ABE biosynthesis at an industrial scale was carried out by ABE fermentation. Clostridium species such as C. acetobutylicum, Clostridium saccharoperbutylacetonicum, and Clostridium saccharobutylicum are the most commonly utilized bacterial strains for the production of biobutanol fermentation. Other strains like Escherichia coli, Saccharomyces species, Bacillus subtilis, and Pseudomonas species can also be used but need genetic modifications for the biosynthesis of products. Another important characteristic of ABE fermentation is the inhibition of the produced product due to the presence of biobutanol as biobutanol retards the growth of microorganisms. Substrates utilized for ABE fermentation are mostly biomass from the plant. Improvement in ABE fermentation occurred if LCBs (low-cost substrates) were used. Besides bacterial species, there is a chance for algal species to be utilized as well as algal culture, cultivation does not need much handling and is relatively low cost (Chauhan, 2010).

6.2 Phases of ABE fermentation The ABE fermentation process comprises two stages: an acidogenic stage that involves mainly the formation of acids (butyrate and acetate) and a solventogenic stage, where these acids are reused to form acetone, butanol, and ethanol. Biobutanol formation is suitable at low pH, but there can be a failure of process if pH lessens beyond 4.5 before the formation of acids (Lee et al., 2008). Microbial cells can be retarded at low concentrations of biobutanol, i.e., 5 g/L and enhanced stress level on the cells occurs by high concentrations between 7 and 25 g/L (Patinvoh and Taherzadeh, 2019). Fig. 3 shows the schematic presentation of ABE fermentation. Major issues retarding industrial application of ABE fermentation from lignocellulosic hydrolysates constitute feedback inhibition, low yield of biobutanol, inhibitor presence in the hydrolysates, cost of the bioreactor, high cost of recovery of biobutanol as well as the cost of wastewater

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Fig. 3 Schematic representation of microbial pathways of acid and solvent formation in ABE fermentation in Clostridia species. ABE, acetone, butanol, and ethanol (Dharmaraja et al., 2020).

treatment. These issues can be resolved through different strategies of optimization, such as microbial modification of strains, detoxification, and increased bioreactor and product recovery systems (Green, 2011).

7. Physiochemical properties of biobutanol Biofuels, i.e., biobutanol that arises from various feedstocks, have different characteristics in contrast to biofuels that originate from organic sources. Biobutanol has specific properties in comparison to other fuels; therefore, properties of biobutanol are discussed here (da Silva Trindade and dos Santos, 2017; Anandharaj et al., 2020). Molecular formula of butanol is C4H9OH, and its molecular weight is 74.11. Density is defined as the weight of the unit volume of fuel. Viscosity is referred to as the resistance between adjacent layers of a fluid that retards the sliding over one another (da Silva Trindade and dos Santos, 2017).

Biomass valorization to biobutanol

A cetane number is basically an ignition quality indicator of the fuel. This determines whether the fuel has a shorter or longer ignition delay during the period of combustion. A melting point predicts the temperature at which a solid transforms into a liquid state, and it can be visualized by using standard conditions. A flash point is defined as the fuel vapor with the lowest possible temperature that can be flammable directly if in contact with fire (Bharathiraja et al., 2019). Some physicochemical properties of biobutanol are shown in Table 2. Table 2 Physiochemical properties of biobutanol (da Silva Trindade and dos Santos, 2017; Bharathiraja et al., 2019; CABI, 2022; Yasin et al., 2019; Bankar et al., 2013; Jin et al., 2011). Properties

Biobutanol

Molecular formula Molecular weight Taste Odor Color Melting point (°C) Optical rotation at 20°C/D (water) Specific gravity Ignition temperature(°C) Autoignition temperature (°C) Flash point (°C) Boiling point (°C) Relative density (water: 1.0) Vapor pressure (kPa at 20°C) Critical pressure (hPa) Relative vapor density (air: 1.0) Critical temperature (°C) Explosive limits (vol% in air) Density at 20°C (g/mL) Viscosity (10 3 Pa s) Solubility in 100 g of water Dipole moment (polarity) Energy density (MJ L) Octanol/water partition coefficient (as log Po/w)a Energy content/value (BTU/gal) Motor octane number Air-fuel ratio Research octane number Heat of vaporization (MJ/kg) Liquid heat capacity (Cp) at STP (kJ/k-mol. K) Cetane number Saturation pressure (kPa) at 38°C Flammability limits (%vol.) Stoichiometric ratio

C4H9OH 74.11 Banana, fuel taste Rancid, sweet Colorless liquid 89.3 9.8 0.810–0.812 35–37 343–345 25–29 117–118 0.81 0.5 48.4 2.6 287 1.4–11.3 0.8098 2.593 Immiscible 1.66 27–29.2 0.88 110,000 78 11.2 96 0.43 178 25 2.27 1.4–11.2 11.21

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7.1 Striking features of biobutanol in contrast to ethanol From the analysis of the characteristics of biobutanol, it is possible to prove that it has some fascinating features which specify that it can conquer some disadvantages caused by lower carbon alcohols used as fuel additives or fuel, like low lubricity, problems of ignition in cold weather, corrosion, and higher consumption of fuel (da Silva Trindade and dos Santos, 2017; Brassat et al., 2011). The striking features of biobutanol that make it efficient over other fuels are as follows: • It shows higher viscosity and lubricity. It is stated that with the increase in the number of carbon atoms in a molecule, the viscosity of alcohol also increases. Biobutanol inherently shields some components of the engine that have a direct association with fuel, i.e., injectors, fuel pumps, and fuel rails against wear problems (da Silva Trindade and dos Santos, 2017). The main benefit of utilizing biobutanol in fuel is its positive impact on decreasing the viscosity of fuel (Pexa et al., 2016). • A higher heating value is observed in biobutanol. Generally, with the increase in carbon content, there is a rise in the low heating value of alcohol. As biobutanol is a 4 carbon sugar, so its energy density in volume is 50% more in contrast to ethanol, i.e., 2 carbon sugar. It determines that lower consumption of fuel and better mileage are predicted when an engine is using biobutanol as compared to ethanol (Rakopoulos et al., 2010). In contrast to ethanol, biobutanol is better for utilizing in diesel engine due to its good solubility, more heating value and no corrosion to pipelines that are existing (No, 2016). • Biobutanol with gasoline shows good indissolubility because alcohols with higher carbon numbers are easily blended with gasoline as they are increasingly less polar because of longer non-polar chains of hydrocarbons and their affinity with water is also less (Fletcher, 2015). While ethanol is a lower carbon alcohol, it is more soluble and more polar in water than hydrocarbons with non-polar nature. It may be a dare then starts some more problems regarding distribution that must inhibit water contamination. It also lets down the upper limit of blending in petroleum fuels despite the use of co-solvent (Sarathy et al., 2014). • Engine running on biobutanol showed fewer ignition problems (less issue of cold start) in contrast to the engine running on ethanol by considering that air to fuel relation is the same. It occurs due to the heat of vaporization of biobutanol is less than half that of ethanol ( Jin et al., 2011; Yilmaz et al., 2014). • Biobutanol has little capability to vaporize because the volatility of alcohol reduces with greater carbon content (Patakova et al., 2011). Besides its low volatility, biobutanol has a flash point higher than that of ethanol. These two properties determine that it is potentially safe to use butanol at high temperatures and when kept in view of transportation (da Silva Trindade and dos Santos, 2017).

Biomass valorization to biobutanol

8. In-situ product recovery in ABE fermentation The goal of in-situ product recovery (ISPR) is to separate the product from the locality of the cell immediately after it is produced. This should usher in enhanced productivity and overall titers to reduce the cost of wastewater treatment and to impede fermentation. More research is needed to prove stability, scalability, reduced energy consumption, long-term robustness, and to enhance recovery of the products. Since 2012, there has been an increase trend in considering on ISPR from ABE fermentation. After this, more attention was paid to the separation capability of the technique rather than product improvements (Outram et al., 2017b). Various separation techniques can be utilized for the recovery of biofuel from fermentation broth to increase the production of biobutanol. But if biobutanol concentration is more than 10 g/L in a fermentation broth, it will proceed to retardation of growth of microbes and also influence yield (Motghare and Wasewar, 2019). Various separation techniques for ABE fermentation products recovery from fermentation broth are as follows: distillation, gas stripping, liquid-liquid extraction, membrane reactor, perstraction, pervaporation, adsorption, reverse osmosis (RO), mutation, genetic engineering, and ionic liquid (IL) (green method) as shown in Fig. 4 (Bharathiraja et al., 2019; Kujawska et al., 2015).

8.1 Distillation Distillation is one of the common separation techniques in which separation performs due to a difference in volatilities of each separated component. When a mixture constituting components of different volatilities is brought to the boiling, the composition of the vapor

Fig. 4 Recovery methods of biobutanol.

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that is released will vary in contrast to the solvents in the boiling liquid. There are various types of distillation, i.e., steam distillation, batch distillation, continuous flash distillation, and fractional distillation. An aqueous ABE mixture is a complex system in which organic azeotropic-water mixtures can be produced during the process of distillation. Although this technique is the most familiar but it possesses several disadvantages, such as low selectivity, high cost of investment, and high energy consumption (Kujawska et al., 2015; Berk, 2009). The less quantity of ABE fermenters in ABE indicates that there is an increasing demand of energy involved in conventional distillation separation from batch fermentation (Outram et al., 2017a). For biobutanol recovery, distillation is also not suitable because butanol has a boiling point higher than water. Therefore, other techniques such as adsorption, gas stripping, pervaporation, RO, membrane protraction have been formed to improve the performance of recovery as well as lower its cost (Liu et al., 2013).

8.2 Gas stripping Recovery of biobutanol from the fermentation broth can be made by nitrogen gas stripping (Lodi and Pellegrini, 2016). To enhance the production of biobutanol, anti-foam is introduced inside the fermentation broth because of this inhibition rate of the microbes reduced (Ezeji et al., 2013). For gas stripping, H2, N2, and CO2 gases can be utilized as stripping agents and improve the yield of the product. It is regarded as an efficient method that predicts maximum separation efficiency of biobutanol in contrast to other products and also decreases the operational cost of the process (Motghare and Wasewar, 2019; Xue et al., 2012). It has several benefits than other methods of recovery because it is not difficult to operate, scale-up, does not need expensive chemicals or equipment; it does not extract reaction intermediates as well as nutrients from fermentation broth and not provide any harm to the cell. In this method, the fermentation broth is in contact with an inert gas, catching the solvents and then moved through the condenser. Therefore, components of strip condense, while recycling of gas occurred to the section of stripping. Gas stripping results in the utilization of concentrated solutions of sugar in the fermenter, resulting in higher utilization of sugar and a decrease in inhibition of biobutanol. The biobutanol concentration in the recovered stream is more in contrast to the fermentation broth. Therefore, gas stripping also excludes a huge amount of water with biobutanol and needs a high energy input (Lodi and Pellegrini, 2016; Ezeji et al., 2003).

8.3 Liquid-liquid extraction To extract solvents from the fermentation broth, liquid-liquid extraction can be utilized. In this technique, organic extractant, i.e., water-insoluble, is mixed with fermentation broth. In contrast to fermentation broth (aqueous phase), biobutanol is more soluble in the extractant (organic phase). As fermentation broth and extractant are immiscible, the extractant can be separated easily after the extraction of biobutanol from the

Biomass valorization to biobutanol

fermentation broth. Despite these, this process also includes some issues such as the production of an emulsion, the toxicity of the extractant, and extraction solvent losing. Researchers widely utilized oleyl alcohol as a good extractant with low toxicity (Liu et al., 2013). It is suggested that liquid-liquid extraction is an excellent alternative of product disposal with the saving of a considerable amount of energy in contrast to distillation for the recovery of biobutanol from very dilute broths (Vane, 2008; Qureshi et al., 2005b; Salemme et al., 2016; Gonza´lez-Pen˜as et al., 2020).

8.4 Membrane reactor Another process for the removal of biobutanol is the immobilization of microbes in the membrane or utilizing membrane reactors. By this technique, productivity can be increased. If continuous ABE fermentation proceeded by immobilized cells of C. acetobutylicum with a carrier, i.e., fibrous, 4.6 g/L/h productivity was achieved. But if fermentation of biobutanol was done by immobilized Clostridium beijerinckii cells with various carriers such as clay brick, the productivity escalated to 15.8 g/L/h. Though this technique increased the productivity of biobutanol but the frequent problem in the industrial stage is the cells’ leakage from the matrices, enhanced mass transfer resistance, and poor mechanical strength (Liu et al., 2013). Membrane separation is regarded as one of the most efficient approaches due to several benefits, i.e., easy to operate, greater freedom in fermentation broth treatment at operation conditions, smaller footprint, no formation of additional waste, and achieving concentrated solutions of organics in a single step in water (Groot et al., 1990; Qureshi et al., 1992; Grobben et al., 1993; Volkov et al., 2020).

8.5 Perstraction In the perstraction method, the membrane is utilized along with oleyl alcohol as an extractant. Fermentation was done in batch mode that constituted 5 g/L extract of yeast, 60 g/L whey permeate and ABE fermentation proceeded for 67 h. At this stage, broth circulation through the membrane was started by utilizing a peristaltic pump (at a rate of 24 L/h). A continuous feed of whey permeates the medium, i.e., concentrated was commenced at a rate at which the concentration of lactose in the reactor was maintained between 30 and 60 g/L. The extractant’s volume was approximately 950 ml, and it was kept anaerobic utilizing nitrogen gas which is oxygen-free (Qureshi et al., 1992). Recovery of ABE by perstraction was quicker in contrast to its formation in the reactor, and the highest concentrate of ABE in oleyl alcohol was 9.75 Gl. It is proven that ABE recovery from oleyl alcohol would be more economical as compared to recovery from fermentation broth. It is said that a new membrane can be formed that can offer a higher flux of ABE (Qureshi and Maddox, 2005).

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8.6 Pervaporation Pervaporation is also referred to as pervaporative separation. It is the process for the separation of butanol in which through the partial vaporization, mixtures of substances are recovered with the aid of a porous or non-porous membrane from the fermentation broth (Strathmann, 2001). By utilizing this method in the fed-batch fermentation process, the concentration of product enhanced from 0.34 g/Lh to 0.5 h/Lh and 24.2 g/L to 32.8 g/Lh by C. acetobutylicum. It is predicted that the pervaporation process is to separate water-biobutanol mixture by utilizing ceramic composite membrane/polydimethylsiloxane. It is reported that biobutanol separation from the fermentation broth occurs and 36 extractants are applied (Motghare and Wasewar, 2019). Among various separation techniques, pervaporation is regarded to be safe and economically advantageous for the removal of products (Van Hecke et al., 2018). The benefit of pervaporation constitutes high efficiency of separation, low consumption of energy, compact operation space, low operating temperature, high selectivity, and reasonable performance-to-cost ratio (Abdehagh et al., 2014; Arregoitia-Sarabia et al., 2020).

8.7 Adsorption Production of biobutanol through the ABE fermentation process can be separated by the process of adsorption utilizing hydrophobic adsorbents. It is reported that six types of adsorbents were applied and it was concluded that activated carbon (AC) F-400 is the most efficient adsorbent for the recovery of biobutanol. Selective recovery of biobutanol predicted by utilizing different adsorbents, such as KA-I, XD-41, H-511 resin, among this KA-I resin proved a potent resin because of the maximum separation of biobutanol that occurred by this resin. Adsorption of biobutanol is performed by utilizing silicalite material that has hydrophobic characteristics with a zeolite like structure. But on a large scale, the low capability of adsorption may enhance the economy of this method (Motghare and Wasewar, 2019). Adsorption has been suggested as an efficient recovery method that could be successfully applied for energy-efficient separation of biobutanol from fermentation broth in industrial applications (Nielsen and Prather, 2009; Raganati et al., 2020).

8.8 Reverse osmosis Membrane-based technique frequently applied in the removal of salt from water and formation of portable water is referred to as RO. In this technique, feed solution separated by semi-permeable membranes into two streams: solution constitutes retained compounds and salts (concentrate) and purified water (permeate) (Perez-Gonzalez et al., 2012). For the recovery of biobutanol by using RO, polyamide membranes are regarded as a good material with the rejection range of 85%. It is reported that a 98% rate of rejection and the optimal rejection of BuOH in ferment liquor done at 20%–25% recoveries

Biomass valorization to biobutanol

(Garcia III et al., 1986). It also showed that to separate pure biobutanol from the solution containing biobutanol, the first step is to separate a fermentation broth; nanofiltration is proceeded (Ito et al., 2015). After this, filter solution is passed to the RO module. Retenate constitutes a two-phase system enriched in biobutanol. The last stage is the recovery of the biobutanol-rich phase. This process allows getting biobutanol-water mixture constituting 80% of BuOH (Kujawska et al., 2015).

8.9 Genetic improvement Several cellulosic feedstocks are recently being utilized for the production of biobutanol, such as corn stover, wheat, cassava bagasse, miscanthus, and barley straw. Besides this, costly edible feedstocks like corn starch and glucose also used for the production of biobutanol. Generally, biomass could be utilized as a substrate for anaerobic digestion by the C. acetobutylicum (Heap et al., 2007; Ni and Sun, 2009) bacterium to form biobutanol through ABE fermentation. Several biosolvents such as ethanol, acetone, butanol have been produced from spore-producing microbes and strict anaerobes named Clostridium by anaerobic fermentation of various oligosaccharides, monosaccharides, and hydrolysate. Accumulation of butanol, i.e., butanol toxicity, is the major issue during fermentation of biobutanol that restricts the yield of butanol and enhances the production as well as recovery costs. During the production of biobutanol in anaerobic fermentation, if butanol produce >7.4 g/L then it suppresses the growth of Clostridium and fermentation process would be stopped simultaneously. These issues resolved by genetic engineering. A genetic transformation is a fascinating and important method that can modify the production of biobutanol utilizing Clostridium. Considering the prime issue with Clostridia, knock out or insertion or overexpression of heterogenic genes can be performed to modify the quality of the strain. Researchers have formed tolerant strains of solventogenic Clostridia toward the production of biobutanol and also have limited the formation of spores. Keeping in view, the problems associated with Clostridia strains, researchers moved toward E. coli, which is well manageable and speedily growing. Along with the E. coli strain, another strain also introduced for the production of biobutanol that is resistant to high concentrations of biobutanol, i.e., Saccharomyces cerevisiae. To utilize these microbes for the production of biobutanol, genetic tools have been recruited to manipulate their metabolism. Genetic engineering can modify the quality of strain, produce yield, and decrease toxicity, which will concurrently modify and simplify the production of biobutanol at the industrial scale (Xue and Cheng, 2019).

8.10 Green method (ionic liquid) Various methods have been suggested to separate biobutanol from the fermentation broth but liquid-liquid extraction would be expensive. So, ILs can be utilized as a novel extractant and could be a potential solution in replace of toxic solvents and conventional

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volatiles. Currently, research has empathized on ILs as environmentally friendly, designer green solvents, non-volatile for the separation industry. Biobutanol recovery by IL could be used in combustion systems driven by gasoline due to magnificent blending characteristics with lesser emissions. Therefore, ILs that have currently attained unique identification in industry and science may be regarded as highly significant for the recovery of biobutanol. Imidazolium-based ILs, phosphonium-based ILs, non-fluorinated taskspecific ionic liquids (TSIL) and ammonium-based ILs are applied as an extractant for the recovery of biobutanol from the fermentation broth, and this technique is the most potent process because it has a higher separation efficiency than previous methods (Motghare and Wasewar, 2019; Garcia-Chavez et al., 2012).

9. Applications of biobutanol Biobutanol is attaining much attention in the biofuel research field due to its better potential for sustainable biofuel production (Mahapatra and Kumar, 2017). Some of the industrial applications of biobutanol are also discussed as follows: Biobutanol is used as a solvent for rubber formation, quick-drying lacquer to reveal an excellent surface finish, and also in the industry of dying, such as the printing ink industry. It is utilized as a precursor for the formation of butyl acetate, acrylic esters, butyl amines, and glycol ethers. It also acted as an eluent in paper and thin-layer chromatography. Biobutanol is a supplement in cleaners and polishes utilized for industrial and domestic cleaning. It is an extractant in pharmaceuticals for natural substrates, i.e., vitamins, antibiotics, hormones, and it also acts as an extractant for drugs. It is a de-icing fluid in the textile industry for solubilization and gasoline in cold regions (Liu et al., 2013; Mahapatra and Kumar, 2017; Gottumukkala et al., 2017; Visioli et al., 2014). Biobutanol has applications in different fields as shown in Fig. 5.

Fig. 5 Applications of biobutanol.

Biomass valorization to biobutanol

10. Conclusions and future trends This chapter summarizes the potential and significance of biomass waste valorization into a useful product like biobutanol. The valorization of largely available, low-cost, and renewable waste (biomass) could offer vital benefits in reply to growing fossil fuel demands and its manufacturing expenses, along with enhancing environmental distress. Biobutanol is considered a next-generation biofuel because of its specialty to intermingle with gasoline up to 95%. Biobutanol can be useful in pharmaceutics, rubber formation, chemical synthesis, and dying and textile industry. Although ABE fermentation has a lengthy history of virtually hundred years, the present curiosity and progress by innovative plants scheduled or previously being constructed in the France, Austria, America, Brazil, China, and England show that the biological biobutanol fabrication through the ABE procedure has an enlightened future. The current research on the biochemistry, physiology, and genetics of C. acetobutylicum, it is evident that the biological effectiveness of solvent making is enhanced regularly by metabolic engineering, improved downstream handling and consuming alternate and renewable substrates as a substitute in the place of sugar.

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CHAPTER 10

Bioethanol—A promising alternative fuel for sustainable future R. Reshmya,b, Eapen Philipa, Rekha Unnic, Sherely A. Paula, Raveendran Sindhud,e, Aravind Madhavanf, Ranjna Sirohig, Ashok Pandeyg,h, and Parameswaran Binodd a

Post Graduate and Research Department of Chemistry, Bishop Moore College, Mavelikara, Kerala, India Department of Science and Humanities, Providence College of Engineering, Chengannur, Kerala, India c Department of Chemistry, Christian College, Chengannur, Kerala, India d Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology (CSIRNIIST), Trivandrum, Kerala, India e Department of Food Technology, TKM Institute of Technology, Kollam, Kerala, India f School of Biotechnology, Amrita VishwaVidyapeetham, Amritapuri, Kerala, India g The Center for Energy and Environmental Sustainability, Lucknow, Uttar Pradesh, India h Centre for Innovation and Translational Research, CSIR-Indian Institute for Toxicology Research (CSIR-IITR), Lucknow, India b

1. Introduction The repercussion of extensive use of petroleum products has led to crop up an astonishing demand of bioethanol in the global market. Today, environmental defilement is a comprehensive issue due to the rapid rise in industrialization, urbanization, and human resource utilization (Rameshprabu et al., 2015). Research studies on the emergence of renewable feedstocks for the production of biofuels are the only solution to control the reducing reserves and existing fossil fuel issues (Saı¨dane-Bchir et al., 2016). Biofuels have been implemented as exceptional combustion-substitute fuels to reduce the amount of fossil fuel burned to release CO2 from renewable energy sources. Nowadays, a part of the energy produced in developed countries is from renewable agricultural waste feedstocks, which allows for the creation of bioenergy through environmentally friendly and economically viable routes (Naik et al., 2010). Biological resources including agricultural wastes, trees, algae, wood processing residues, food and municipal wastes, etc., could be utilized as a source for biofuels because of this reason biofuels are one of the most attractive renewable energies for future (Nigam and Singh, 2011; Vohra et al., 2014; Anubhuti and Prakash, 2015; Deshavath et al., 2019). Subsequently, there is a persistent value in plant or organic waste fuel production and utilization as it reduces adverse environmental effects of greenhouse gas (GHG) emissions by decreasing fossil fuel consumption (Manmai et al., 2020). Depending on the current fossil fuel usage rate, it is estimated that the fossil fuel reserve will diminish in the near future. Also, the rising prices of fossil-based fuels and hazardous gas emissions hinder their use and push us to find other resources. More than 80% of the global gross carbon emissions, 40% of all nitrogen emissions, Valorization of Biomass to Bioproducts https://doi.org/10.1016/B978-0-12-822888-3.00006-2

Copyright © 2023 Elsevier Inc. All rights reserved.

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and 14% of total GHG emissions are contributed by the use of fossil fuels in automobiles (Demirbas, 2011). Around 8 kg of CO2 is emitted during the combustion of one gallon of gasoline fuel. In 2011, worldwide CO2 emissions exceeded about 34 billion tons, generating an additional pollution control expense of $1.2 trillion. It is particularly the need of the hour to focus on the development of low-cost and eco-friendly biofuels as substitutes to fossil fuel. Bioethanol is a green and comfortable fuel based on non-petroleum that has been considered a hopeful alternative to deal with the rising energy crisis. Since bioethanol has a lower energy content compared to conventional fuels and the greater octane number, that has resulted in improving the efficiency of the gasoline-bioethanol blends (Demirbas and Demirbas, 2007; van Dam et al., 2008). In addition, bioethanol has a higher oxygen content compared to gasoline. This high oxygen concentration and higher vaporization heat of bioethanol results in a safe combustion. Bioethanol combustion thus decreases emissions of harmful pollutants relative to gasoline fuel combustion. Bioethanol is expected to be capable of reducing up to 90% of gas emissions when combined with 95% of petroleum fuel. Thus, bioethanol is a revolutionary product for the development of low-emission sustainable energy. The selection of appropriate microalgae, the exploitation of viable feedstocks, and the implementation of novel methodologies are some of the challenges in bioethanol production. Numerous developments and research studies have been undertaken in recent years to establish viable commercial routes for the production of bioethanol (Ibeto et al., 2011; Carrillo-Nieves et al., 2019). This chapter discusses in detail the developments of bioethanol production using biomass waste materials as feedstocks, processing technology, advantages, and the current industrial status of solid waste material bioethanol production.

1.1 Biofuels and its generations Bioethanol (C2H5OH) is an alternative fuel developed using various conversion technologies from numerous biomass feedstocks. This bio-based fuel can be utilized as an alternative for petroleum-based fuels because of its sustainable nature, less particulate emissions, and highly oxygenated combustion (Nair et al., 2016). Municipal waste and wastes produced by agricultural activities, forestry, etc., are used for the development of biofuels. They are usually categorized as first (1G), second (2G), third (3G), and fourth generation (4G) biofuels, depending on the sources and production technologies (Acheampong et al., 2017). Different types of biofuels and their sources are depicted in Fig. 1. Biofuels’ structure doesn’t change from generation to generation. As biofuels of the 1G are fuels obtained from edible food crops, namely, starch, sugar, vegetable oils, and animal fats, it varies with food utilization and thus has a harmful effect on the agricultural sector (Naik et al., 2010). It would also increase carbon emissions, water and land

Bioethanol—A promising alternative fuel for sustainable future

Fig. 1 Sources of different generations of biofuels.

overuse, traditional agricultural conditions, and demands for fast development. While in a foreseeable future, the popularity of 1G feedstock utilizations for biofuel is decreasing, and thus improved alternatives are being developed (Sims et al., 2010). Biofuels of the 2G are derived from non-edible crops like lignocellulosic biowastes, whereas biofuels of the 3G utilized algae as feedstock. Non-food biomass, including forest residues, sugarcane bagasse, cereal straw, and organic components of municipal solid waste, are the major feedstocks for 2G biofuels (Binod et al., 2010; Talebnia et al., 2010). Hence, building a green and healthy world plays a vital role. Many 2G biofuels tend to be particularly effective, given their benefits and costs for the production of biofuels (Hill et al., 2006). If biofuel technologies of 2G are fully commercialized, they are likely to benefit from policies designed to reward national priorities such as environmental sustainability or supply protection over several alternatives of 1G. Nanotechnology techniques are to be carried out to increase the overall device performance in both process and strain engineering stages. The significance of algae as a biofuel feedstock is that it can be directly integrated into carbon sources releasing carbon from power plants or factories, and converted into usable fuels. So there is zero release of carbon dioxide from these environments. However, the algae-derived biofuel is easily vulnerable to degradation due to high insaturation and high volatility compared with sources of lignocellulosic feedstock. In this area, comprehensive research is underway into engineering algae in an attempt to increase the development of biofuels as well as studying algal biodiversity in order to discover extremely competent fuel-producing organisms. The 4G biofuels consist of solar

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photobiological and electro fuels. New artificial biological technologies can be used to produce solar fuel and to discover inexhaustible, inexpensive raw materials through biological systems. While these 4G fuels seem to offer an essential advancement in the development of biofuel at an infant stage (Abdullah et al., 2019). To date, bioethanol constitutes up to 75% of the overall consumption of biofuels. With a revenue of $58 billion per annum, bioethanol dominates the market. The United States of America (USA) and Brazil are currently the major producers of bioethanol from agro-wastes like corn and sugarcane. India is the world’s second largest producer of sugarcane, yet it only contributes 2% of global production of bioethanol. Traditional processing strategies require being incorporated into streamlined processes to evade excessive development of inhibitory sugar derivatives and achieve high bioethanol titers in order to generate bioethanol from lignocellulosic feedstocks, which is commercially viable, renewable, and compatible with gasoline-based fuels (Chandrasekhar et al., 2015).

1.2 Processing technologies Thorough research has centered on the utilization of agricultural biomass as a source of biofuels, materials, and chemicals due to increased understanding of economic changes and environmental issues, becoming one of the most promising options for replacing fossil resources. The bioethanol production process as a whole is shown in Fig. 2. 1.2.1 Preparation of feedstock The method of preparation of feedstock is typically the primary step in the pre-treatment of biomass and aims to minimize the dimension of the content. Due to significant energy usage during the milling process, this may not be always feasible. The corresponding pretreatment process can also have a harmful impact, as in the case of agricultural residues or wood wastes, such as straw (Talebnia et al., 2010). The energy utilization throughout the physical phase is rigorously linked to the reduced particle size and the type of feedstocks used (Tao et al., 2014). 1.2.2 Pre-treatments methods Lignocellulosic materials offer a potential feedstock alternative for biofuel production considering their ratio of energy efficiency, availability, low cost, and higher yields of ethanol (Balat, 2011; Nair et al., 2016; Carrillo-Nieves et al., 2019). The pre-treatment of lignocellulosic biomass is one of the main rate-limiting phases. The complex cellulose structure in association with lignin and hemicelluloses limits the opportunities of simple pre-treatment processes. Still, most of the available methods of pre-treatments, like alkali or acid treatments, involve a pre-hydrolysis stage of neutralization. In addition, the degradation of hexose and pentose sugars and lignin during acid pre-treatments occurred, and the resulted products act as fermentation inhibitors. Sulfuric acid is also causing

Bioethanol—A promising alternative fuel for sustainable future

Fig. 2 Schematic representation of different stages of bioethanol production.

corrosion in the reactor and sulfur toxicity due to its high concentration. Therefore, treatments using other acids such as dilute phosphoric acid are preferred. In addition, the selection of an effective pre-treatment process and extensive screening are required in the case of raw materials, such as municipal solid wastes (Lang et al., 2001). A pretreatment stage for biomass waste, such as residues of food waste from coffee, is generally not required. But, an increased bioethanol yield after pre-treatment of coffee extract residue at lower temperatures confirmed the importance of the pre-treatment process (B€ orjesson, 2009). 1.2.3 Saccharification and hydrolysis Although the hydrolysis process differs between substrates based on lignocelluloses or starch, it is mostly conducted using acids or enzymes. Pullulanase, amylase, glucoamylase, and isoamylase are the usual enzymes for starch substrates, while cellulase and glucosidases are the main enzymes for lignocellulosic substrates. Researchers reported that endproduct accumulations usually decrease enzyme activity and ultimately result in process inhibition. Cellobiohydrolases and endoglucanases, for example, result in the accumulation of cellobiose (Halder et al., 2019), thus affecting the yield of hydrolysis. Similarly, the range of substrate components also includes the supply of anti-microbial promoters like tetracycline or cycloheximide to avoid microbial contamination during the hydrolysis process (Nair et al., 2016).

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1.2.4 Fermentation and bioethanol production The overall fermentation process, using traditional microorganisms, is another significant challenge to bioethanol production from waste biomass. For different biotechnological processes and industries, such as breweries, Saccharomyces cerevisiae is considered a competitive microorganism. However, its restrictions on the use of lignocellulosic hydrolysate materials, primarily pentoses, have proved to be a major obstacle to the acquisition of higher ethanol yields from different biomass waste. The creation of pentose-fermenting S. cerevisiae resulted in gene modifications by recombinant DNA technologies. S. cerevisiae with elevated cellulolytic activity. Genetically engineered S. cerevisiae strains are currently essential for applying in food wastes and other lignocellulosic feedstocks for a better fermentation yield (Oreb et al., 2012). In bioethanol fermentation, many bacteria and fungi, such as the recombinant Escherichia coli, Zymomonas mobilis, and filamentous fungi, have also been used. However, the use of pentose sugars to increase the production of ethanol is not optimally effective and poses significant challenges for utilizing biomass in this environment. Fermentation inhibitors that form during pre-treatments are present in the waste stream like volatile acids and antimicrobial agents, and pose a serious challenge to the growth of microorganisms (Balat, 2011; Nair et al., 2016; Carrillo-Nieves et al., 2019).

2. Various biomass sources of bioethanol 2.1 Bioethanol from lignocellulosic biomass/cellulose It is possible to create cellulosic ethanol from parts of plants that are usually referred to as lignocellulosic biomass. As major ingredients, these plant parts consist of lignin, hemicellulose and cellulose, and also contain oils, free sugars, pectin, starches, minerals, and proteins as minor ingredients. Cellulose is the largest component and comprises between 35 and 50% of basic plant matters. Hemicellulose is the second largest fraction (15%–30%) of basic plant matter and the rest is mainly lignin. As a minor ingredient, hemicellulose consists of five sugars, namely, glucose, xylose, arabinose, mannose, and lactose. Lignin is a composite structure made up of phenyl propene units that act to hold the fibrous cellulose together with hemicellulose. Lignin plays a critical role as biomass recalcitrance in the production of bioethanol by promoting the biological breakdown of cellulosic biomass for the release of sugars. The percent composition of different biomass sources is shown in Table 1. Three pathways are involved in the biological conversion of lignocellulose to bioethanol that involves catalytic hydrolysis of cellulose to produce fermentable glucose by (1) concentrated acid treatments, (2) supercritical deconstruction of biomass into water-based cellulose, and (3) enzymatic hydrolysis, followed by fermentation of ethanol-released sugars (Lambert et al., 1990).

Bioethanol—A promising alternative fuel for sustainable future

Table 1 Composition of different biomass sources. Sources

Cellulose

Mannan

Galactan

Arabinan

Lignin

Sugarcane bagasse Corn stover Wheat straw Monterey pine Hybrid poplar Eucalyptus Switchgrass Sweet sorghum

39.01 37.61 32.64 41.7 39.23 48.07 30.97 34.01

0.35 0.38 0.31 10.7 1.81 1.23 0.29 0.2

0.46 0.87 0.75 2.4 0.88 0.74 0.92 0.52

2.06 2.42 2.35 1.6 0.89 0.3 2.75 1.65

23.09 18.59 16.85 25.9 25.18 26.91 17.56 16.09

Concentrated acids, such as 72% sulfuric acid, are commonly used to submerge cellulose fibers in cellulose crystallinity to break down hydrogen bonds, resulting in disaggregation to form amorphous cellulose fibers that can be easily broken down into glucose (Balat, 2011). Moreover, this process requires ambient pressure and temperature for reducing energy requirements for the hydrolysis. The dried biomass avoids aggressive heat release during concentrated acid pre-treatments. The acid recovery requires operations such as evaporation and ion exchange that makes the overall process costly during commercial implementation (Deshavath et al., 2019). Supercritical deconstruction using high-energy water molecules can lead to rapid disintegration of biomass components particularly cellulose to glucose (Lachos-Perez et al., 2017). Supercritical water oxidation or gasification processes are utilized for the treatment of lignocellulosic feedstocks. Also, these conditions accelerate the hydrolysis of cellulose. These approaches are ultra-clean because of the absence of corrosive acids, flammable solvents, or harmful reagents. Another route for the conversion of lignocelluloses to bioethanol is by utilizing cellulase enzymes, namely, Trichoderma reesei (T. reesei). While using these enzymes, one must consider several factors like cost, the nature of culture, and environmental conditions. In this process, the vigorously agitated glucose/sophorose mixture in an aerated vessel can be directly fed into the enzymatic hydrolysis (Tao et al., 2014). After hydrolysis, the content was fermented using a fermentative bacterium for 36 h using 0.25% corn steep liquor as nutrients . Finally, bioethanol was separated by distillation coupled with molecular sieve dehydration. In thermal routes, cellulosic materials can be gasified using high-temperature steam to syngas. This syngas can be utilized to produce methanol followed by a catalytic reaction to form ethanol (Shen et al., 2017). Many methods have been used over the years to resolve biomass temerity, among that a few have only been proficient to provide the high yields of bioethanol. A yield analysis of bioethanol from 1 kg of biomass is illustrated in Fig. 3.

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Fig. 3 Schematic illustration of yield analysis of bioethanol from lignocelluloses.

2.2 Bioethanol production from starch and sugar Sugar and starch are other renowned sources for bioethanol production. Classical yeast such as Saccharomyces cerevisiae is utilized as a microorganism in commercial scale bioethanol manufacture from these sources because of its easy processibility (Fig. 4). The yeast strains need to tolerate elevated ethanol levels for the optimum conversion of sugar to bioethanol. Only hexose sugars like fructose or glucose and disaccharides like maltose or sucrose can be converted by classical yeast. Under anaerobic conditions, yeast can preliminarily convert sugar to bioethanol through the Embden-Meyerhof strategy, and glucose to bioethanol conversion is effectively carried out at temperatures of 28–35°C and pH ranges of 3.5–6.0. During fermentation, a low partial oxygen pressure (7–13 Pa) must be established. Yeast needs less amounts of oxygen for survival due to fat and lipid synthesis. Higher partial oxygen pressure values would lead to increased cell growth instead of the development of bioethanol, termed as the “Pasteur effect”.

Bioethanol—A promising alternative fuel for sustainable future

Fig. 4 Flow chart of bioethanol production from starch or sugar feedstocks within the concept of biorefinery.

During fermentation, the emitted carbon dioxide (CO2) provides a gaseous atmosphere in the fermentation vessel and can be collected for further use, filtered, and compressed. An optimum condition is necessary for the yeast to achieve the maximum output of bioethanol and the highest possible conversion of sugar. The nutrients needed for the yeast on combining with the sugar components are provided by many feedstocks during industrial bioethanol production. They can be given as salts of potassium and ammonium or as a low-cost complex medium, namely, steeped corn liquor. The propagation of yeast may be carried out in a semi-continuous or continuous mode. These methods chosen depend on the bioethanol fermentation’s output mode itself (Chandrasekhar et al., 2015). The key goal of fermentation is the conversion of almost all sugar into bioethanol. The final step is the upturn of bioethanol from the fermentation vessel after completing the overall process. This is achieved by adding a mixture of columns of distillation and rectification (Balat, 2011). To extract the pure form of bioethanol, the entire fermentation broth should be heated up to 78°C. The evaporation takes place in conjunction with their vapor liquid balance, leading to higher bioethanol content in the vapor.

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2.3 Bioethanol production from microalgal biomass One of the recently developed sources of bioethanol is microalgae due to their efficiency and compatible nature. On comparing to land-based feedstocks, algae are obtainable 5–10 times more due to photosynthetic efficiencies. There is no lignin in the algae, which is a physical barrier to enzymatic hydrolysis and cannot be eliminated by pre-treatment. The pre-treatments and saccharification stages of the bioethanol production depend on the character of the microalgae. Microalgae are intensively grown in an artificial environment, either in an open pond or in enclosed tubes called photobioreactors. They are also grown in a medium of growth rich in nutrients and CO2. The cultivated microalgae are processed to produce bioethanol in the same manner as other lipid-based feedstocks (Fig. 5). The carbohydrates in the cells may also be fermented to improve the yield of bioethanol (Saı¨dane-Bchir et al., 2016). As a source of biomass, algae have gained significant attention. Different algal cultivation systems include (a) open ponds; (b) flat PBRs; (c) fermenter cultivation;

Fig. 5 Flow chart of production of bioethanol from microalgal biomass.

Bioethanol—A promising alternative fuel for sustainable future

(d) biofilm-based PBRs; (e) tubular PBRs; and (f ) cultivation of algal biomass using waste water. The key carbohydrate reserve types in microalgae are starch and glycogen. The main factors affecting bioethanol production are temperature, nutritional techniques, light intensity, and saline tension. Nitrogen limitation is one of the most powerful strategies in which degradation of N-based macromolecules in microalgae leads to the accumulation of carbohydrates and lipids (Ibeto et al., 2011; Halder et al., 2019). A number of attempts have been made to produce less costly photobioreactors for algae cultivation for the development of bioethanol. A closed photobioreactor may be a technology that eliminates some of the key problems associated with the production systems of open ponds. The higher yield compared to open systems is one of the big advantages of closed reactors (Guo et al., 2015). It minimizes the possibility of risks of external emissions and contamination. For classified algal strains, these systems are more suitable as the closed form enables regulation of likely emissions. Due to high machinery and substrate prices, only a few of these bioreactors can technically be used for large-scale algae cultivation. Fouling was also reported (Humbird et al., 2011). While the problem of pollution is smaller than open ponds, maximum regulation has not been observed. One of the most effective outdoor microalgal mass cultures is the tubular photobioreactor as their exposure to solar radiation covers a wide surface area. Difficult temperature regulation, a certain degree of wall growth, and a limited degree of hydrodynamic stress are the problems with this method. Algal biofilms can play a key role in mitigating the key limitations of other algae development and harvesting systems (Demirbas, 2011). Biofilm formation occurs due to the concentration on immersed surfaces of cations, organic molecules, and proteins relative to the main aqueous environment, providing a suitable space for microalgae growth (Shen et al., 2017). Another alternative solution is an electrolytic process that is used without adding any chemicals to extract algae. Initiatives in the electric field advise algae to step out of the solution. Water electrolysis creates hydrogen that sits next to the microalgal flocks and transfers them to the outside. There are numerous advantages to the use of electrochemical processes, along with ecological compatibility, energy efficiency, adaptability, low cost, and protection. Ultrasound also has a very powerful influence on algae flocculation, but application aspects are lower than other methods, with a severe concentration growth of 20 times the amount of food. For maximum output of bioethanol, the harvested algal biomass is processed using dehydration and extraction procedures. Dehydration is carried out using a suitable drying process (Kim et al., 2014).

3. Environmental impact Global bioenergy production occurs at different levels, such as solid, liquid, and gaseous types of biofuels. Solid biofuels are generally the most commonly obtainable and low-cost fuel with the most efficient recovery of feedstock energy. But, they are heavy,

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inconvenient to treat, low density, and relevant to solid fuel burners only. Liquid biofuels are transport-friendly, and be capable of replacing petrol and diesel. But, they are less energy efficient; require rigid feedstock, complex conversion technologies, and production costs. Gaseous biofuels can be generated by using reported techniques from organic wastes and residues, but there are problems with fuel processing and by-product disposal. In 2012, global biofuel use exceeded 0.55–1020 J/year, accounting for 10% of global energy use and 80% of total renewable energy productions (Haberl et al., 2013). In particular, bioethanol is extensively produced to supplement the fast-depleting reserves of petroleum. By 2050, bioethanol is projected to be the leading fuel for driving passenger cars and heavy vehicles. Significant economic, social, and environmental impacts have been produced by the intensive production and use of biofuels. Socioeconomic impacts include property rights, economic viability, food security, local growth, energy security, conditions of employment, and policies. The environmental impacts include emissions of GHGs, ecosystems, land use, protection of soils, and water supplies. The results differ according to the uniqueness of the supply chains of biofuels, the management of the production environment, and the factors of the processing regions. Generally, the development and use of biofuels enhances the sustainability of the nation’s resources, decreases traditional pollution and GHG emissions, stimulates research studies, creates jobs, and enhances agricultural productivity. Biomass feedstock processing, on the other hand, involves water, land, fertilizer, and other resources and generates additional demand of water resources and enhances food costs. However, these adverse effects can be reduced by proper preparation and advancements in technology (Guo et al., 2015).

4. Advantages and disadvantages of bioethanol Biofuels provide significant benefits over petroleum-based fuels: (1) they are readily accessible from popular sources of biomass, (2) they serve as a carbon dioxide cycle in combustion, (3) eco-friendly potentials, (4) environmental and consumer friendly, and (5) are biodegradable. Biofuels will lead to the improvement of GHG emissions, led to a renewable and stable source of energy and, in developing countries, improve agricultural incomes for rural poor people. Due to improved land availability, favorable climate conditions for crops, and lower production costs, several countries have competitive advantages in bioethanol production. Other environmental concerns and socioeconomic implications could affect the ability of developing countries to take advantage of increased biofuel production. Large-scale bioethanol production is a breakthrough for some developing countries to cut short their dependency on importing oil. In developing countries, there is an increasing movement toward the use of modern technology and the effective conversion of bioenergy using a variety of biomasses that are compatible on a cost basis with fossil fuels. Thermal processes

Bioethanol—A promising alternative fuel for sustainable future

such as gasification, pyrolysis, liquefaction, supercritical water liquefaction, supercritical fluid extraction, and biochemical are used to create biofuels from bio-based materials (Vohra et al., 2014; Busˇic et al., 2018). Thermo-chemical biomass reforming is concerned with catalytic as well as non-catalytic pyrolysis processes and gasification, and aims to maximize the production of powerfully exploitable liquid and gaseous goods. Renewable liquid biofuels for transport have recently attracted enormous attention in different countries across the globe due to their recyclability, sustainability, GHG emission reduction, and biodegradability. It was used in France and Germany by the then emerging internal combustion engine industry (ICEs) as early as 1894. Brazil has made use of bioethanol as a fuel since 1925. Bioethanol production at that time was 70 times greater than the production and consumption of petrol. In Europe and the United States, the use of bioethanol for fuel was common until the early 1900s. The potential of bioethanol was widely ignored until the oil crisis of the 1970s, because production was more costly than petroleum-based fuels, particularly after World War II. There has been an increasing trend in using bioethanol as an alternative fuel for transportation since the 1980s. In order to ensure that “sustainable” bioethanol is generated with regard to the benefits of GHGs, the following requirements must be met: (1) bioethanol plants should use biomass rather than fossil fuels; (2) on carbon-rich soils such as peat soils used as natural grasslands, annual feed crop cultivation should be avoided; (3) by-products should be used effectively to enhance their energy and GHG advantages; and (4) efficient fertilization techniques should keep nitrous oxide emissions to a minimum and the industrial nitrogen fertilizer used should be produced in nitrous oxide gas cleaning system plants. The benefits of GHG are an significant advantage of crop-based bioethanol (Nigam and Singh, 2011). The drawbacks of bioethanol comprise its lower density compared to gasoline, less luminosity of the flame, low vapor strength, corrosiveness, and water miscibility (Ibeto et al., 2011).

5. Statistical analysis of bioethanol production in various countries In Brazil, the USA, and European countries, bioethanol has already been implemented on a wide scale. Output has increased dramatically as many nations are trying to reduce oil imports, boost rural economies, and enhance their air quality. In 2007, the global production of bioethanol arrived at about 51,000 million liters, with the USA and Brazil becoming the foremost producers, together accounting for about 70%. On average, fuel bioethanol accounts for 73% of the bioethanol produced worldwide, 17% for beverage bioethanol, and 10% for industrial bioethanol. Bioethanol production accounted for approximately 4% of the 1300 billion liters of gasoline consumed worldwide in 2007. Moreover, bioethanol production is constantly growing globally, with the potential of reaching 120,000 million liters per annum by 2025. More than 94% of the world’s biofuel production is currently accounted for by

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bioethanol. Approximately 60% of the global production of bioethanol comes from sugarcane, which in Brazil is predominant, and 40% from other crops. Almost all bioethanol is currently produced in the USA by fermentation of corn glucose or sucrose in Brazil. By 2020, the European Commission aims to gradually replace 20% of traditional fossil fuels with renewable fuels in the transport sector, with a sporadic target of 5.75% in 2010. Most Member States have implemented this indicative goal within their national biofuel goals. The set quota has already been reached by some Member States, such as Sweden, Finland, or Germany. The quantity of biofuels produced in the EU generally remains low on comparing to the total amount of mineral-based transport fuel sold. Around 0.3% of all EU petrol and diesel fuel was sold in 2003. In Serbia, bioethanol production is currently focused on molasses (50%) and cereals (50%). The projected demand for bioethanol as a transportation fuel in EU countries was approximately 6 billion liters in 2006 and 12.7 billion liters in 2010. This is disproportionate to the existing EU production capacity of approximately 2 billion liters per annum. The feedstocks used for bioethanol in Europe are mainly wheat, sugar beet, maize, and wine industry waste. It is anticipated that the production of the biofuels needed to comply with the Directive would require between 4% and 13% of the total agricultural land in the EU. Today, most developing countries depend on fossil oil, and steady rises in global fuel prices have been economically affecting these nation’s developments. As per UN Energy, in the absence of access to energy, no nation in recent times has significantly reduced poverty. The processing of bioethanol, however, poses challenges as well as job opportunities. There are three major factors to consider, such as increased food costs, the effect of increased cultivation on the environment, and climate change. There is no question, therefore, that it is important to closely track these threats, but it is also important to be conscious that food prices rely on many factors other than the demand for biofuel crops. When it comes to price rises in relation to bioethanol, maize is the most discussed crop. However, it should be noted that only about 8% of all the maize at present produced is utilized for bioethanol on a global scale. The environmental effects of increased agricultural production, which can contribute to a rise in fertilizer and water use, are another area of concern. In fact, this may contribute to an increased release of nutrients into the marine ecosystem and GHG emissions. Finally, the growing demand for bioethanol can result in changes in the use of land. Natural vegetation like rainforests may well be affected by growing agricultural crops, a trend that may possibly lead to affect biodiversity, and a decline in the land’s carbon-binding ability. The stated concerns indicate the need for sustainable bioethanol development, which includes the conservation of high biodiversity and carbon stock lands and the advancement of agricultural practices and novel bioethanol feedstocks. The annual production of bioethanol is continuously growing and the worldwide production and consumption of bioethanol is probable to grow to almost 134.5 billion liters by 2024 (Busˇic et al., 2018). The percentages of bioethanol production and consumption by 2024 in different countries are shown in Fig. 6 (Busˇic et al., 2018).

Bioethanol—A promising alternative fuel for sustainable future

Fig. 6 Prophecy of the world bioethanol: (A) Production and (B) Consumption by 2024.

6. Conclusion and future prospectives The development of an alternative and safe source of fuel is a key concern for all nations around the world. There are great prospects for bioethanol extracted from various agroindustrial wastes to fulfill the aim of designing a continuous energy supply method. Major investments in technology, infrastructure, and building capacity are required in

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most developing countries to enhance the productivity and sustainability of the agriculture sector. This will allow the dedication of agricultural land to enhance food production, as a result, in the future, more feedstock for secondary ethanol production. Since the agricultural sector is different in each of the developing countries, more comprehensive, residue-specific studies are required by each country to assess the economic viability of collecting and pre-processing forestry and agricultural residues for 2G biofuel production. Recently, applications of nanotechnology have enhanced the efficiency of the pre-treatment system, which helps to improve reliable, cost-reducing, and environmentally friendly processes. Bioethanol can be utilized as an alternative to resolve the petroleum-based fuel disadvantages. The industrial scale production of bioethanol can be recommended from lignocellulosic biowastes because of its wide availability and processibility. However, in order to improve efficiency to meet market demand, this type of industry still faces some challenges. In the near future itself, comprehensive research studies and the developments of innovative technologies are required to solve bioethanol production problems and increase the quality of production. Depending on the feasibility of conversion technologies, 2G and 3G of bioethanol could offer numerous advantages, like less GHG emissions, significantly zero competition for food, soil preservation, carbon sequestration, and improved habitat. The development of more efficient pre-treatment technologies, the development and implementation of stable production of microorganisms in industrial fermentation processes, and the introduction into the economy of bioethanol production systems of the optimal components achieved, and the creation of a biorefinery concept. To conclude, different biomasses have immense potential to satisfy future energy demands in the modern world. They are valuable sources of energy in order to overcome our unsustainable dependency on petroleum-based fuels that are proven to cause global climate change and environmental pollution.

Acknowledgments Reshmy R and Raveendran Sindhu acknowledge DST for sanctioning projects under DST WOS-B Scheme (SR/WOS-B/587/2016 and SR/WOS-B/740/2016).

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CHAPTER 11

Hydrogen production from biomass gasification with carbon capture and storage Daya Shankar Pandeya, Sanjay Mukherjeeb, and Faisal Asfanda a

School of Computing and Engineering, University of Huddersfield, Huddersfield, United Kingdom Infrastructure and Engineering, Energy Systems Catapult, Birmingham, United Kingdom

b

1. Introduction The International Energy Outlook predicts that the global energy demand will increase by about 48% by 2040 (IEO, 2016). Most of the developing country’s economy are dependent on fossil fuels, such as coal, oil, and gas, which are the major anthropogenic contributors to carbon emissions. However, the international community is committed to decarbonize the planet and several countries have set a goal to reduce their dependency on fossil fuels. In addition, as fossil fuel reserves are depleting, an alternative clean and sustainable energy source needs to be explored. Therefore, exploitation of an integrated biomass conversion and valorization technologies to produce biofuels, bio-hydrogen, biopower, renewable chemicals, and materials are needed. Biomass-driven technology could create a new domestic bio-based industry while mitigating the carbon footprint originating from fossil fuels. The European Union set-out an ambitious but achievable plan that by 2030 up to one quarter of the total transport fuel demand should be met by clean and efficient biofuels to curb greenhouse gas (GHG) emissions from fossil fuels and its impact on global climate change. As per the EU biofuel policy, it is anticipated that biofuel and hydrogen production will be a major contributor to the European economy with the twin benefits of a large potential for job creation and a reduction in carbon dioxide (CO2) emissions. As a result, the goal of limiting CO2 emissions and associated environmental problems can be achieved by using alternative low-carbon fuels, such as green and blue hydrogen. Hydrogen fuel can also provide a sustainable, accessible, and clean energy source to meet the future energy needs. Therefore, sustainable, energy efficient, and innovative technologies are needed to produce alternate fuels and hydrogen from wide range of biomass feedstocks while adhering to the societal, economic, and environmental norms. The combustion of hydrogen does not produce oxides of carbon, sulfur, and polyaromatic hydrocarbons because it does not contain carbon and sulfur molecules. When

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hydrogen is used as an energy source, it only produces water vapor as a by-product. During the combustion process of hydrogen, the chemical energy stored in the HdH bond is released and the hydrogen atom combines with oxygen to form water as the combustion product. Given that combustion of hydrogen does not contribute to GHGs, it has a huge potential to reduce the CO2 emissions produced by combustion of fossil fuel precursors. Hydrogen as a fuel has high fuel quality such as high flammability limit, high flame speed, ignition quality, and requires less ignition energy. Although, less ignition energy requirement is advantage from combustion point of view as ignition can easily be initiated but it poses a safety concern. Hydrogen can be produced by using a wide variety of renewable energy resources including waste heat from industries, biomass, wind energy, solar energy, tidal energy, wave energy, and geothermal energy. Although the amount of hydrogen is negligible on the Earth in its free form, it can be produced by means of electrochemical, thermochemical, or biochemical processes. The most common production technologies include electrolysis of water, steam reforming of natural gas, and coal/biomass valorization.

1.1 Prospects of hydrogen Future energy security can only be guaranteed by considering a more agile and diverse energy portfolio with an increased share of renewable energy source. Hydrogen fuel can provide a cost effective and environmentally friendly solution to improving our future energy needs. Hydrogen offers flexibility to be used in a variety of ways in combination with current technologies to efficiently produce, move, store, and deliver energy. Moreover, hydrogen can be used as an alternative fuel in the transportation sector, for producing electricity or domestic heating. For domestic use, hydrogen could be blended into existing natural gas networks as it can be transported through the existing natural gas pipes and infrastructure, if it is mixed with natural gas at a ratio of up to 20%. However, for higher percentage of hydrogen or in the case of pure hydrogen gas, the existing gas networks would need to be modified/ changed to make them compatible. The direct use of hydrogen fuel in hydrogen boilers or in fuel cells has a higher potential in commercial settings, dwelling and can play a pivotal role in minimizing carbon emissions in population dense cities. The use of hydrogen in the transport sector will reduce dependence on oil. Hydrogen fuel has the highest gravimetric energy density compared to any known fuel. In addition, it is compatible with both electrochemical and combustion processes. Hydrogen fuel cells are not only environmentally friendly but also more efficient compared to internal combustion engines. In hydrogen fuel cells, the chemical energy stored in the HdH bond is converted into electrical power without relying on the thermodynamic power cycles, thus, allowing higher efficiency. However, the competitiveness of hydrogen driven vehicles will depend on hydrogen fuel cost and adequate number of refueling stations that

Hydrogen production from biomass gasification

cover a wider area. Absence of refueling stations can limit its share in the transport industry. In contrast, marine and aviation industries have limited options available to reduce their carbon emissions and therefore hydrogen fuel can represent a potential alternative. Hydrogen can also be used as an energy storage medium particularly for storing renewable energy. This will allow reduction in energy losses compared to other energy storage types like batteries and thermal energy storage which lose the energy stored in them over time. Hydrogen can be stored as a pressurized gas or liquid, which means it will never dissipate until it is used. In addition, hydrogen fuel can be used in gas fired power plants to increase power system flexibility.

1.2 Hydrogen market Hydrogen economy is gaining a global acceptance and the rigorous progression in fuel cell technology has proven that hydrogen can play a vital role in replacing internal combustion engines and other conventional fossil fuel driven powertrain devices. Hydorgen is a widely used feedstock in oil refining and has become an integral part of the energy industry (Balat and Kırtay, 2010). In addition, hydrogen is used for the synthesis of ammonia to make fertilizer to be applied in the agriculture sector. This has given rise to a global business in the production and supply of hydrogen. Hydrogen market is continuously growing and the annual production of hydrogen in its pure form is estimated to be 70 million tons which shows an increase of three-fold compared to 1975 (IEA, 2019). The contribution of hydrogen-led economy is worth around $40 billion in the global economy. Fig. 1 shows its main applications in ammonia production (51%) and oil refining (31%), about 10 and 8% hydrogen is being used in methanol production and other reduction processes, respectively (Arregi et al., 2018).

2. Production process The common building blocks for hydrogen production are water and hydrocarbons such as methane (CH4) and carbon monoxide (CO). Hydrogen gas is produced through three 8% 51%

31%

10% Others

Oil refining

Methanol

Ammonia

Fig. 1 Global consumption of hydrogen.

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main routes, electrochemical, biological, and thermochemical processes. Hydrogen can sustainably be produced from water via hydrolysis using electricity, provided energy used in the process coming from renewable resources. Alternatively, hydrogen can also be produced via thermochemical process where hydrocarbons react with steam (steam methane reforming and water-gas-shift reaction). Using biomass as a source of hydrocarbons ensure renewable and CO2 neutral production of hydrogen. Although, all the above mentioned routes allow low or zero-carbon hydrogen production, thermochemical route is the most adopted technique because of its technological maturity compared to other processes. Although hydrogen gas can be produced using renewable energy resources, yet at present, 95% of hydrogen production technologies are based on fossil-derived resources (natural gas, coal, and oil) (Thomas et al., 2018), which means that hydrogen production is also responsible for annual carbon emissions. Fig. 2 shows the share of different sources in the production of hydrogen gas, and it can be noticed that the current source of hydrogen gas using renewable energy is very low. To address the issue of climate change, small-, and large-scale CO2 neutral hydrogen production facilities need to be promoted. Renewable energy sources, particularly biomass feedstocks, have a great potential to be used to provide a clean route for hydrogen production. Fig. 3 summarizes the various energy sources, different conversion routes to hydrogen production, and the applications of hydrogen fuel in a wider context.

2.1 Steam reforming of hydrocarbons Hydrogen is widely produced on the industrial scale using the steam reforming of methane. This is the most economical technology to produce hydrogen on a large-scale. In this process, the methane gas reacts with steam over a nickel catalyst at a temperature of 900–1000°C and a pressure of 20–30 bar. One mole of methane gas can produce three moles of hydrogen gas when it reacts with steam. Carbon monoxide is also produced during the steam reforming of methane which further reacts with steam to produce extra 18%

4%

48%

30% Natural gas

Oil

Coal

Electrolysis

Fig. 2 Current sources of hydrogen (Thomas et al., 2018).

Hydrogen production from biomass gasification

Fig. 3 Routes for hydrogen production with potential applications (Thomas et al., 2018).

hydrogen via water-gas shift reaction. The final product is mostly hydrogen, a comparatively smaller amount of CO2, traces of CO and unreacted methane gas.

2.2 Electrolysis of water Electrolysis of water is another way to produce hydrogen by splitting water into hydrogen and oxygen using electricity. An electrochemical cell consists of two electrodes, linked by the flow of current, and placed in an ionically conducting electrolyte. An alkaline medium is used to increase the electrical conductivity. During electrolysis of water, the reduction reaction at the cathode produces hydrogen and the oxidation reaction at the anode produces oxygen.

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Hydrogen produced using electrolysis is not cost effective and therefore it is only preferred for small-scale hydrogen production for a particular application. To reduce the cost of hydrogen production and increase productivity, high temperature driven chemical reactions that split water to produce hydrogen are adopted. The high temperatures can be generated by solar concentrators or nuclear reactors which can help in reducing the electricity consumption and consequently reduce the overall cost. Photo-electrolysis is another method which uses sunlight for water splitting to produce hydrogen gas. The method includes conversion of solar radiations into electricity using photovoltaic cells and the electricity is then used in separate electrolysers to generate hydrogen from water. Hydrogen can also be produced when sunlight (photons) absorption creates electron-hole pairs that electrochemically split water molecules. The electricity used in the electrolysis process can also be obtained from renewable energy sources, such as solar thermal power, wind energy, geothermal energy, etc. In addition, the surplus electricity during the off-peak time could be used to produce hydrogen energy using electrolysis, which can then be used as a source of energy during the peak time.

2.3 Biomass to hydrogen Any organic material that is derived from plants or animals is known as biomass. Hydrogen gas can be produced from biomass using several approaches, including biophotolysis, indirect biophotolysis, photo-fermentations, dark fermentations, and thermochemical conversion. These technologies are not fully commercialized yet and research is ongoing to improve the production efficiency of these processes. Gasification and pyrolysis of biomass are attractive thermochemical route to produce sustainable hydrogen. The thermal processing techniques of pyrolysis and gasification of biomass is similar to fossil fuels in which heat is used in a limited oxygen environment. Biomass is partially oxidized at high temperatures (
1000 K) to produce a gaseous mixture together with carbon residue that is subsequently reduced to form hydrogen, methane, carbon dioxide, and carbon monoxide. The methane gas produced in the process can also be converted into hydrogen by steam-methane reforming. Hydrogen is then purified and separated from other volatile gases present in the syngas. The proportion of hydrogen in the syngas is increased using a water-gas shift (WGS) reaction. Processing of biomass via thermochemical processes has several advantages over chemical (hydrolysis) or biological conversion (anaerobic digestion and fermentation technologies) routes such as shorter conversion time, pathogens elimination and is quite adaptable to different kinds of biomass and waste. Nevertheless, the gasification of biomass has a downside of tar formation which impedes the production of hydrogen by steam reforming. However, appropriate design of gasifier, suitable catalyt additives, and controlled operating parameters can lower the risk of tar formation. This chapter focuses on carbon neutral hydrogen production from biomass (renewable resources)

Hydrogen production from biomass gasification

Fig. 4 A process layout for hydrogen production from biomass gasification with carbon capture and storage.

via gasification route. Fig. 4 provides a generic process layout of hydrogen production via biomass gasification along with carbon capture and storage.

3. Biomass to hydrogen production technology This section provides an overview of thermochemical technologies for hydrogen production with focus on exploiting biomass-based fluidized-bed gasification system.

3.1 Pyrolysis Pyrolysis (also called devolatilization) is a thermochemical process involving heating lignocellulosic or waste feedstock materials at a moderate temperature (normally in the range of 300–600°C) in the reducing environment during which the unstable carbonaceous species breakdown into pyrolytic gases (mainly CO, CO2, CH4, H2, and other light hydrocarbons) and condensable liquid bio-oil (mainly oxygenates, aromatics, water, products of low degree of polymerization, etc.) and solid by-products (unconverted

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carbon residue called biochar and ash). The yield of the desired products is controlled by the operating parameters such as temperature and residence time. For example, low temperature and long residence time (slow pyrolysis) result in higher char yield whereas the liquid bio-oil production is favored at higher temperatures with a relatively short residence time (fast pyrolysis) (Bridgwater, 2012; Pandey, 2016). The produced bio-oil from biomass can substitute fossil-derived oil or diesel in power generating devices can help in decarbonizing the energy sector. The calorific value of the bio-oil is normally around 17–20 MJ/kg about 60% of diesel on a volume basis (Pandey et al., 2019). Despite extensive on-going research activities pertaining to improve the quality and usability of biomass derived bio-oil as an alternative fuel in power generation, its application is still limited. Some of the problems related to the utilization of bio-oil as a fuel are low volatility index, high viscosity, and its corrosive nature due to impurities. Therefore, it is imperative that the produced bio-oil must be upgraded before it can be used as an alternative transportation fuel or utilizing them to produce chemicals such as adhesives for wood, fertilizers, resins, acetic acids, and chemicals destined for industrial applications. The pyrolytic gases can be directly utilized for process heating. Since the hydrogen content increases sharply with an increase in pyrolysis temperature, it can be used in internal combustion engines for power generation. Finally, the solid residue (biochar and ash) can either serve as a supplementary fuel in the combustion process or as a soil amendment. In addition, considering that biochar has a high surface area, it could be used in water purification, catalyst, or adsorbent.

3.2 Gasification Gasification is one of the oldest thermochemical conversion technologies, which converts solid feedstocks into clean combustible gas called product gas (synthesis gas) by partial combustion. The application of gasification technology has been recognized as a promising method to provide an alternative approach for resource recovery from waste and biomass, viz. waste to energy, hydrogen-rich syngas, and chemical building blocks. Furthermore, gasification processes provide flexibility to produce fuels, heat, and power-based on a clean biomass/waste-derived product gas while being compliant with the latest emission standards (Pandey et al., 2016). In particular, the produced gas can be supplied in steam turbines, internal combustion engines and in fuel cells, for the generation of heat and electricity. The amount of external oxidizing medium (oxygen) fed into the gasifier is always kept below the stoichiometric amount of oxygen needed for a complete combustion which prevents the fuel from being completely oxidized. The vital difference between combustion and gasification is that the former releases the heat by breaking chemical bonds while the later relocates/repacks the energy into chemical bonds in gaseous products. The composition of the evolved product gas varies and depends on the feedstock

Hydrogen production from biomass gasification

composition, gasifying medium (oxidation agent), and process parameters. Typically, a gasifier temperature is over 650°C. Air, steam, and/or oxygen are mainly used as the gasifying media. Air is commonly used as a gasifying medium since it is inexpensive; however, the calorific value of the gas is lower compared to gas produced from oxygen or steam gasification due to dilution with N2. The air gasification process increases the burden of downstream gas cleaning equipment. Additionally, feedstock properties such as shape, size, moisture content and chemical composition, significantly affect the composition of product gas and impurities. For example, the LHV of the product gas can be in the range of 4–7 MJ/m3 when gasified with air whereas it can be as high as 10–15 MJ/m3 when the same feedstock was gasified in a pure oxygen environment. Moreover, the later one required a very high investment cost for air separation units (ASUs) to produce oxygen in and is favorable only for large-scale applications. Oxidation with steam produces a better LHV in the range of 15–20 MJ/m3 which is directly correlated with the higher share of hydrogen in the product gas due to the improved water gas shift reaction (Pandey, 2016). The National Renewable Energy Laboratory revealed that biomass and waste gasification process represent only 0.33% of the total worldwide gasification capacity compared to fossil fuel derived feedstocks (approximately 99.67%) such as peat and coke (NREL, 2010). Fig. 5 shows the existing gasification capacity and expected growth rate worldwide. Gasification process yields a mixture of gases CO, H2, CH4, CO2, H2O, N2, and contaminants, such as particulates, tar, alkali metals, hydrochloric acid (HCl), sulfurcontaining compounds (H2S, COS), and nitrogen containing compounds (NH3, HCN). The low to medium calorific value of the produced gases (4 and 6 MJ/m3) can either be

Fig. 5 Worldwide gasification capacity and growth, input from Gasification Technology Centre database, 2014 (Higman, 2014).

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burned directly or used as a fuel for gas engines and turbines after the necessary gas cleaning steps. The main components are also called “syngas”. Furthermore, the main components of the evolved gases (CO and H2) can be used as a building block for further processing and production of chemicals or cleaner fuels (e.g., methanol, ethanol) and Fisher-Tropsch synthesized gasoline. The gasification process involves drying, devolatilization, partial oxidation, and reduction (gasification). These processes occur simultaneously in a real gasifier. Gasification process involves a sequence of endothermic and exothermic reactions. The gasification process can for example be either direct (autothermal) or indirect (allothermal). The amount of heat required for converting a unit mass of solid fuel into gaseous products at standard temperature and pressure is called the heat of gasification. The heat of gasification is the sum of the heat required to raise the temperature of the solid fuel and gasification medium to the gasification temperature, and the energy needed for the endothermic gasification reactions (Pandey, 2016). Tð gasif

ΔH gasif ¼



  mfuel C p,fuel T + magent C p,agent T dT + ΔH r

(1)

T0

A brief description of different processes involved in gasification is provided here: Heating and drying of the biomass: This phenomenon takes place when fuel is fed into the gasifier, it uses heat transferred from the heating zone to remove the free and the chemically bound water. The required heat for the drying process is met by the exothermic reactions occurring within the gasifier reactor. Typically, the moisture content of biomass ranges from 5 to 35% but freshly harvested biomass can have a moisture content as high as 60%. Therefore, drying of the feedstock is usually desirable and sometimes essential before gasification to ensure satisfactory gasifier operation and improve product gas. The process of drying takes place in a temperature range between 150°C and 200°C. X     Cn Hm Op fuel,ar + Heat ! Cx Hy Oz fuel,dry + moisture (2) Pyrolysis (devolatilization): After the drying process, the feedstock is heated up between 200°C and 600°C in an oxygen-free environment. During the devolatilization process, the feedstock releases light permanent gases (non-condensable gases), primary tar (condensable gases), and char due to its thermal decomposition. Tar is a black and sticky material formed during the pyrolysis process that potentially gives rise to system malfunction if condensation occurs (Horvat et al., 2016). In other words, tar is a mixture of organic molecules including acids, aldehydes, ketones, alcohols, phenols, larger polycyclic aromatic hydrocarbons, and particulate matter whose amount in the product gas depends on the operating conditions (Pandey, 2016). The composition of the volatiles depends on the original feedstock, temperature, pressure, atmosphere, and heating rate. Presence of tars in the product gas composition can cause the malfunction of reforming

Hydrogen production from biomass gasification

catalysts and sulfur removal systems, thus an effective cleaning system is essential for maintaining the system’s performance. Char may also contain ash from the biomass which generally contains inorganic mineral and alkali metals. Light permanent gases are mixture of H2, CO, CO2, and CH4 but it also contains a small amount of C2H6, C2H4, C3H8, and C3H6. The condensed primary tar is also called bio-oil and is composed of oxygenated hydrocarbons (levoglucosan, acetic acid, and alcohols). At elevated temperature (>800°C), primary tar is further cracked to permanent gases (CO, CO2, H2, CH4, steam) and secondary tar. The secondary tars comprised a mixture of phenols, cresol, benzene and methyl- and hydroxyl-derivates. Cracking of secondary to ternary tars produces methyl-derivate of aromatic components (toluene and xylene) and polycyclic components like naphthalene (Horvat et al., 2016). X X X Cx Hy Oz + Heat ! Ca Hb Oc + Cj Hk Ol + C (3) liquid

gas

solid

Partial combustion: It is the only exothermic process in gasification. The heat that drives the gasification process comes from combusting the products of the pyrolysis process, either tar and gases or char. Partial combustion of tar or char provides heat for drying, devolatilization, endothermic gasification reactions, and maintaining the temperature in the gasifier. Gasification (reduction): During gasification endothermic reactions between gasgas and solid-gas phases take place in a reducing environment. Instead of burning the char and the other volatiles produced in the pyrolysis process, they react with oxygen, steam, and CO2 to produce partially oxidized compounds called product gas or synthesis gas. The final product gas predominantly contains H2, CO, CO2, CH4, and water. The gasification reactions generally occur in excess of 750°C. Steam reforming reaction produces carbon monoxide and hydrogen whereas carbon monoxide is consumed in water-gas shift reaction. The water-gas shift reaction where carbon monoxide reacts with steam to produce hydrogen and carbon dioxide. Since hydrogen is characterized by higher energy content this reaction is highly aimed. Furthermore, the steam-methane reforming reaction plays an important role to increase the hydrogen and carbon monoxide content in the product gas. Steam-methane reforming reaction is highly endothermic, and it is generally favored at higher temperatures. The efficiency of the gasification process is measured through an index called cold gas efficiency (CGE) on a dry basis and it is described in Eq. (4). CGE ¼

m_ Syngas LHV Syngas % m_ fuel LHV fuel

where m_ Syngas : Mass flowrate of the product gas on a dry basis exiting the gasifier (kg/s) LHVSyngas: LHV of the product gas on a dry basis exiting the gasifier (MJ/kg) m_ fuel : Mass flowrate of the initial fuel input (kg/s)

(4)

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LHVSyngas: LHV of the initial fuel input (MJ/kg) Table 1 lists prominent chemical reactions involved in the gasification process. The endothermic gasification reactions listed here are all equilibrium-based reactions. It is worth mentioning that the water gas and Boudouard reactions are the key reactions which help to improve the concentration of CO and H2 in the product gas at a higher temperature. Moreover, the product gas composition is driven by the chemical kinetics of reaction and catalytic effects, if any. For example, reactions involved in the devolatilization process are several orders of magnitude faster than char gasification with CO2 and H2O. This is Table 1 Typical gasification reactions at 25°C (Basu, 2010). Carbon reactions

R1 R2 R3 R4

(Boudouard) (Water-gas or steam) (Hydrogasification) (Partial oxidation)

C + CO2 $ 2 CO C + H2O $ CO + H2 C + 2 H2 $ CH4 C + 0.5 O2 ! CO

+172 kJ/mol +131 kJ/mol 74.8 kJ/mol 111 kJ/mol

C + O2 ! CO2 CO + 0.5 O2 ! CO2 CH4 + 2 O2 $ CO2 + 2 H2O H2 + 0.5 O2 ! H2O

394 kJ/mol 284 kJ/mol 803 kJ/mol 242 kJ/mol

CO + H2O $ CO2 + H2

41.2 kJ/mol

2 CO + 2 H2 ! CH4 + CO2 CO + 3 H2 $ CH4 + H2O CO2 + 4 H2 ! CH4 + 2 H2O

247 kJ/mol 206 kJ/mol 165 kJ/mol

CH4 + H2O $ CO + 3 H2 CH4 + 0.5 O2 ! CO + 2 H2

+206 kJ/mol 36 kJ/mol

Oxidation reactions

R5 R6 R7 R8 Water-gas shift reaction

R9 Methanation reactions

R10 R11 R12 Steam-reforming reactions

R13 R14 Pyrolysis

Biomass ! permanent gases + tar + char + steam

R15 Tar reactions

R16 R17 R18 R19 R20

(Partial oxidation) (Dry reforming) (Steam reforming) (Hydrogenation) (Thermal cracking)

CnHm + (n/2) O2 ! n CO + (m/2) H2 CnHm + n CO2 ! 2n CO + (m/2) H2 CnHm + n H2O ! n CO + (m/2 + n) H2 CnHm + (2n-m/2) H2 ! n CH4 CnHm ! (n-m/4) C + (m/4) CH4

Hydrogen production from biomass gasification

relevant for biomasses with high moisture and volatile matter content, once oxygen is fed into the gasifier it predominantly reacts with the volatile gaseous species before reacting with relatively stable char compounds. The reaction rate of char gasification reaction is generally slow. In addition, char conversion is also restricted by attrition and elutriation of chars as well as the available reaction time. The reactivity of the char particles is kinetically limited and depends on the composition of the original feedstock and hardly reaches an equilibrium condition. The condensate (tars) produced during the devolatilization process is thermally cracked at a higher temperature. A schematic of these processes is illustrated in Fig. 6.

3.3 Type of gasifier reactors The type of gasification system can be classified based on different parameters such as gasifying agents used (air, oxygen or steam), design of the reactor (fixed, fluidized, or entrained flow), direct or indirect, etc. Moreover, the key difference among different types of gasifier is how feedstock and oxidizing agents move inside the reactor. The selection of gasifier type predominantly depends on the characteristics of fuel. Generally, all types of feedstock require some sort of pre-treatment/preparation owing to their heterogeneous nature. Fuel requirement and an appropriate design of the gasifier for different capacities are presented in Table 2. A typical gasification unit consists of a biomass handling and feeding system, gasifier reactor, gas clean-up system, and ash or solid residue removal system.

Fig. 6 Schematic presentation of gasification process. (Adapted from Gómez-Barea, A., Leckner, B., 2010. Modeling of biomass gasification in fluidized bed. Prog. Energy Combust. Sci. 36 (4), 444–509.)

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Table 2 Selection of gasifier design (Basu, 2010). Gasifier type

Fixed bed

Fluidized bed

Entrained flow

Size (mm) Moisture content (ar) Ash content (db) Ash melting point (°C) Bulk density (kg/m3) Application area Nature of ash produced Problem areas

20–100 500 10 kWth–10 MWth Dry Tar production

5–100 MWth Dry C Conversion

50 MWth Slagging Raw-gas cooling

3.3.1 Moving/fixed-bed gasifiers This type of gasifier has been used for a long time and the technology is quite simple and robust. In this type of gasifier, cyclone units are not required for unconverted carbon and ash separation as they are removed from the bottom of the reactor. The thermal capacity of the fixed-bed gasifier is in the range of 10 kWth–10 MWth. The thermal capacity of this type of reactor is limited mainly due to problems associated with achieving a homogenous bed. Another big challenge involved with the fixed-bed reactor is that the quality of product gas and the amount of tar in the product gas show large deviations due to a significant variation in temperature along the bed. Fixed-bed gasifiers are mainly categorized into updraft, downdraft, and cross-draft gasifiers according to where the gasifying medium is introduced. Updraft gasifier The updraft gasifier is the oldest and simplest design. In an updraft gasifier (countercurrent gasifier), the feedstock is fed into the gasifier from the top and moves down in a counter-current fashion to the product gas flow (Fig. 7A). The feedstock passes through drying, devolatilization, reduction, and oxidation zones. Warmed air enters from the bottom of the reactor and meets the hot ashes and any unconverted carbon (char) descending from the top. The temperature in the bed is higher than the ignition temperature of carbon initiating exothermic reactions (R4 and R5). The hot gases (mixture of CO and CO2) move up in the reduction zone where char from the pyrolysis zone is gasified (R1 and R2). High char combustion, internal heat exchange, and low exit temperature of the product gas results in relatively high gasification efficiency. Due to the upward movement of hot gases, the feedstock is dried in the upper section allows fuel with higher moisture, up to 60% to be used. The CH4 content in the product gas is relatively high indicating that the temperature in the devolatilization zone is not high enough to reform the CH4. However, the presence of high amounts of tar in the product gas is a major drawback because the temperature in the devolatilization zone is not high enough to crack the tar which is ultimately carried away by the upward moving product

Hydrogen production from biomass gasification

Fig. 7 Schematic of fixed-bed gasifiers (A) updraft, (B) downdraft and (C) cross-draft. (Source: Knoef, H., Ahrenfeldt, J., Eds., 2012. Handbook Biomass Gasification. BTG Biomass Technology Group, Enschede.)

gas. Therefore, the product gas from such a type of gasifier is only suitable for direct combustion in Kiln/furnace otherwise extensive cleaning is required. Downstream gas cleaning equipment is necessary for the use of the product gas in gas turbines and engines, which includes the removal of particulates, tar, and trace impurities associated with fuel bound nitrogen sulfur and chlorine (Basu, 2010; Pandey, 2016). Downdraft gasifier Downdraft (co-current) reactors are designed in a way that the gasifying agent is injected at a certain height below the top (Fig. 7B). Both the product gas and solid (char and ash) move down along with the gasifying medium. Therefore, a part of volatile gases and char get partially combusted before the gasification zone. The hot gases flow downward through the remaining hot char bed where gasification takes place. Since the product gas passes through the hot bed, tars are easily cracked, and their production rate is kept very low but the product gas contains more entrained particles. Furthermore, the tar content in the product gas is dependent on load. Since producer gas from downdraft gasifiers is tar free (low tar), it can be used in an internal combustion engine/gas turbine. Nevertheless, it requires that the feedstock should have a relatively low moisture content (