Microbial Organic Acids Production: Utilizing Waste Feedstocks [1 ed.] 3110792567, 9783110792560, 9783110792584

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Microbial Organic Acids Production

Also of interest Microbial Nanotechnology. Advances in Agriculture, Industry and Health Sectors Kumar, Singh (Eds.),  ISBN ----, e-ISBN ----

Green Pulp and Paper Industry. Biotechnology for Ecofriendly Processing Kumar, Pathak, Dutt (Eds.),  ISBN ----, e-ISBN ----

Industrial Biotechnology. Plant Systems, Resources and Products Yadav, Kumar, Sehrawat (Eds.),  ISBN ----, e-ISBN ----

BioProducts. Green Materials for an Emerging Circular and Sustainable Economy Vijayendran (Ed.),  ISBN ----, e-ISBN ----

Chemistry for Biomass Utilization Alén,  ISBN ----, e-ISBN ----

Microbial Organic Acids Production Utilizing Waste Feedstocks

Edited by Amit Kumar and Vikas Kumar

Editors Amit Kumar Sharda University School of Engineering and Technology 32-34 Knowledge Park III, Plot no. 32-34 Uttar Pradesh-201310 Greater Noida India E-mail: [email protected] https://orcid.org/0000-0002-5354-420X Vikas Kumar Department of Bio-Sciences and Technology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207, Haryana India and Department of Microbiology International Medical School University of International Business (UIB), Almaty Kazakhstan E-mail: [email protected] https://orcid.org/0000-0002-6044-3239

ISBN 978-3-11-079256-0 e-ISBN (PDF) 978-3-11-079258-4 e-ISBN (EPUB) 978-3-11-079263-8 Library of Congress Control Number: 2023949515 Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available on the internet at http://dnb.dnb.de © 2024 Walter de Gruyter GmbH, Berlin/Boston Cover image: kontrast-fotodesign / E+ / Getty Images Typesetting: TNQ Technologies Pvt. Ltd. Printing and binding: CPI books GmbH, Leck www.degruyter.com

About the editors Dr. Amit Kumar currently works as an Assistant Professor at the Department of Biotechnology, Sharda School of Engineering and Technology, Sharda University, Greater Noida, India. Previously, he worked as an Assistant Professor at Department of Biotechnology, College of Natural and Computational Sciences, Debre Markos University, Ethiopia. He completed his Doctorate in Biotechnology from the Indian Institute of Technology Roorkee, India. He is extensively involved in research on microbial fermentation, industrial enzymes, pulp and paper biotechnology, biofuels production, and environmental biotechnology. He has published several research and review articles in various reputed international journals. He has also co-edited four books and published several book chapters. He has guided several graduate, post-graduate, and doctoral students. Dr. Vikas Kumar, currently, is serving as an Associate Professor in the Department of Microbiology, International Medical School, UIB, Almaty, Kazakhstan. Prior to this, he was Assistant Professor in the Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana (Ambala), Haryana, India during 2015-2022. He received his doctorate from Department of Microbiology, Kurukshetra University Kurukshetra, Haryana (India). He completed his M.Phil. in Microbiology from Chaudhary Charan Singh University, Meerut, India in 2011. During 2019, he also visited CINVESTAV, Irapuato, Mexico as a Postdoc fellow on the project funded by CONACYT. He has more than eight years of teaching experience in the field of microbiology and biotechnology. He has authored over 100 refereed research/review papers and 05 scientific books in Journals/Publishers of national/international reputes. He has guided several M.Sc., M. Tech and Ph.D students for their research work. He is acting as an associate editor and reviewer for various reputed journals. As a researcher, his research interest includes areas like development of biological control agents, plant pathology, mycology and antimicrobial activity of natural and chemical compounds.

https://doi.org/10.1515/9783110792584-201

Contents About the editors V List of contributing authors

XIII

Mansha Ghai, Nivedita Agnihotri, Vikas Kumar, Rajesh Agnihotri, Amit Kumar and Komal Sahu 1 1 Global organic acids production and their industrial applications 1 1.1 Introduction 2 1.2 Citric acid 5 1.3 Succinic acid 6 1.4 Lactic acid 7 1.5 Fumaric acid 8 1.6 Gluconic acid 9 1.7 Itaconic acid 10 1.8 Alpha-ketoglutaric acid 10 1.9 Malic acid 11 1.10 Butyric acid 12 1.11 Conclusions 12 References Birhan Aynalem, Himani Negi, Yigrem Alemu, Nirmala Sehrawat, and Amit Kumar 2 Citric acid: fermentative production using organic wastes as 21 feedstocks 21 2.1 Introduction 22 2.2 Microbial sources and fermentative production of citric acid 26 2.3 Organic wastes as potential resource for citric acids production 26 2.3.1 Citric acid production using fruit wastes and peels 28 2.3.2 Citric acid production using agricultural wastes 29 2.4 Recovery and purification of citric acid 30 2.5 Applications of citric acid 30 2.6 Conclusions 31 References Sushmita Chauhan, Shreya Mitra, Mukesh Yadav and Amit Kumar 3 Microbial production of lactic acid using organic wastes as low-cost 35 substrates 35 3.1 Introduction 37 3.2 Lactic acid production using organic wastes 37 3.2.1 Corn stover and corncob for lactic acid production 41 3.2.2 Rice-washing drainage and rice straw for lactic acid production 42 3.2.3 Spent coffee grounds (SCG) for lactic acid production

VIII

3.2.4 3.2.5 3.2.6 3.2.7 3.2.8 3.2.9 3.2.10 3.3 3.3.1 3.3.2 3.3.3 3.4

Contents

Paper sludge for lactic acid production 42 43 Sugarcane bagasse and molasses for lactic acid production 43 Lactic acid production using wheat straw 43 Production of lactic acid using corn steep liquor Production of lactic acid using organic fraction of municipal solid wastes 44 (OFMSW) 44 Production of lactic acid using agave bagasse 44 Production of lactic acid using cheese whey 45 Applications of lactic acid 45 Applications of lactic acid in food industry 45 Applications of lactic acid in pharmaceutical industry 46 Polylactic acid (PLA) 46 Conclusions 46 References

Mayank Raj, Tamanna Devi, Vikas Kumar, Prabhakar Mishra, Sushil Kumar Upadhyay, Mukesh Yadav, Anil Kr Sharma, Nirmala Sehrawat, Sunil Kumar and Manoj Singh 4 Succinic acid: applications and microbial production using organic wastes as 51 low cost substrates 52 4.1 Introduction 53 4.2 Strategies and methods of succinic acid production 54 4.2.1 Production of succinic acid by bacteria 55 4.2.2 Production of succinic acid by fungi 56 4.3 Optimization of various parameters for succinic acid production 56 4.3.1 pH 56 4.3.2 CO2 concentrations 57 4.3.3 Redox potential 57 4.4 Metabolic engineering of succinic acid 4.5 Production of succinic acid utilizing low cost of raw materials (agro-industrial 58 waste) 58 4.5.1 From cane molasses 58 4.5.2 Fermentative production of succinic acid from straw hydrolysate 59 4.5.3 From vine shoots and surplus grape must 59 4.5.4 Conversion of crop stalk wastes into succinic acid production 59 4.5.5 Production of succinic acid from pineapple peel waste 60 4.6 Recovery and purification of succinic acid 60 4.6.1 Filtration/ultra-filtration 61 4.6.2 Crystallization 61 4.6.3 Selective extraction 61 4.6.4 Ion exchange and sorption 62 4.6.5 Electro dialysis

Contents

4.7 4.8

Applications of succinic acid 62 Conclusion and future prospective 64 References

IX

63

Masrat Mohmad, Nivedita Agnihotri and Vikas Kumar 5 Fumaric acid: fermentative production, applications and future 69 perspectives 69 5.1 Introduction 70 5.2 General properties of fumaric acid 71 5.3 Fermentative production of fumaric acid 5.3.1 Fermentation as a green and economic alternative for fumaric acid 72 production 73 5.3.2 Potential fumaric acid producing microorganisms 74 5.3.3 Optimum conditions for fumaric acid production 76 5.4 Biochemical aspects of fumaric acid 77 5.5 Key strategies to accelerate fumaric acid production 78 5.5.1 Gene modification in microbial strains 78 5.5.2 Mutagenesis of Rhizopus spp. to improve fumaric acid production 79 5.5.3 Morphology control 80 5.6 Auxiliary substrates for fumaric acid production 81 5.6.1 Xylose 81 5.6.2 Glycerol (propane-1,2,3-triol) 82 5.6.3 Waste apples 83 5.6.4 Waste water from brewery 83 5.6.5 Food waste 83 5.7 Post-processing and purification of fumaric acid (downstream process) 84 5.8 An outlook towards the application aspects of fumaric acid 85 5.8.1 Applications in food industries 85 5.8.2 Role in dairy and poultry 86 5.8.3 Hotspot compound for resin industry 5.8.4 Fumaric acid and green chemistry (green approach for Beckmann 88 rearrangement) 88 5.8.5 Medicinal applications 89 5.9 Conclusion and future perspectives 90 References Monika Chopra, Vikas Kumar, Manoj Singh and Neeraj K. Aggarwal 6 An overview about the approaches used in the production of alpha95 ketoglutaric acid with their applications 96 6.1 Introduction 96 6.2 Production of α-ketogulatric acid

X

6.2.1 6.2.2 6.3 6.4 6.5 6.6 6.7 6.8

Contents

Synthesis of alpha ketoglutarate from succinate and succinate 97 semialdehyde Synthesis of α-ketoglutarate from iso-citrate in a glycerol medium with 97 Mn 99 Alpha-ketoglutaric acid as a product of bacterial oxidation of glucose 100 Alpha-ketoglutaric acid production by Yarrowia lipolytica yeasts Utilization of Proteus mirabilis L-amino acid deaminase for the single step 101 production of alpha ketoglutaric acid from glutamic acid 102 Substrates used for the production of alpha-ketoglutaric acid 103 Significance of alpha-ketoglutaric acid 104 Future prospects and advancements 105 References

Mayank Raj, Manoj Singh, Vikas Kumar, Tamanna Devi, Sushil Kumar Upadhyay, Prabhakar Mishra, Sunil Kumar, Mukesh Yadav, Nirmala Sehrawat and Mamta Kumari 7 Gluconic acid: strategies for microbial production using organic waste and 111 applications 112 7.1 Introduction 113 7.2 Physico-chemical properties of gluconic acid 113 7.3 Strategies for gluconic acid production 115 7.3.1 Gluconic acid production through bacteria 116 7.3.2 Gluconic acid production through fungi 117 7.3.3 Fermentative production of gluconic acid using wastes 119 7.4 Application in industry and biomedicine 120 7.5 Conclusions 121 References Meena Sindhu, Shikha Mehta, Shubham Kumar, Baljeet Singh Saharan, Kamla Malik, Monika Kayasth and Sushil Nagar 8 Itaconic acid: microbial production using organic wastes as cost-effective 125 substrates 125 8.1 Introduction 126 8.2 Application of IA 127 8.3 Microbial production of IA 128 8.3.1 Submerged fermentation (SmF) 129 8.3.2 Solid state fermentation (SSF) 130 8.4 Low cost organic waste as substrate for IA production 131 8.5 Microorganisms involved in production of IA using organic waste 131 8.5.1 Aspergillus strains 133 8.5.2 Ustilago strains 134 8.5.3 Other natural producers

Contents

8.6 8.6.1 8.6.2 8.7 8.7.1 8.7.2 8.8

Extraction of IA 134 134 Biomass removal 136 Product Isolation and purification Problems associated with IA fermentation from agrowaste 138 Effect of substrate and microbial strains 140 Effect of metal ions and other inhibitors 141 Conclusion and future perspectives 142 References

XI

137

Mukesh Yadav, Nirmala Sehrawat, Sunil Kumar, Anil Kumar Sharma, Manoj Singh and Amit Kumar 149 9 Malic acid: fermentative production and applications 149 9.1 Introduction 150 9.1.1 Strategies for malic acid production 9.1.2 Microbial malic acid production: developments and advances in bioprocess 151 technology 9.1.3 Microbial organic acids production (malic acid): an environment friendly 155 approach 156 9.1.4 Major substrates to produce microbial malic acid 156 9.1.5 Applications of malic acid, climate change and industrial perspective 157 9.2 Conclusions and future perspectives 158 References Urvasha Patyal, Vikas Kumar, Manoj Singh, Amit Kumar, Anil K. Sharma, Syed Fahad Ali and Sheikh Mudasir Syed 10 Butyric acid: fermentation production using organic waste as low-cost 163 feedstocks 163 10.1 Introduction 164 10.2 Butyric acid features and applications 165 10.3 Butyric acid production 167 10.3.1 Metabolic Pathway 167 10.3.2 Efficient production of butyric acid with low-cost feedstock 170 10.4 Product recovery 170 10.4.1 Acidification 171 10.4.2 Ion exchange 171 10.4.3 Solvent extraction 171 10.4.4 Distillation 171 10.4.5 Esterification 172 10.4.6 Membrane methods 172 10.5 Conclusion and future perspective 172 References

XII

Contents

Sachin Kumar, Priya Panwar, Nirmala Sehrawat, Sushil Kumar Upadhyay, Anil Kumar Sharma, Manoj Singh and Mukesh Yadav 11 Oxalic acid: recent developments for cost-effective microbial 177 production 177 11.1 Introduction 178 11.2 Various strategies/methods for production of oxalic acid 179 11.2.1 Carbohydrate oxidation 179 11.2.2 Synthesis from carbon monoxide 179 11.2.3 Synthesis from alkali formate 180 11.3 Microbial production of oxalic acid 180 11.3.1 Production of oxalic acid from bacterial sources 180 11.3.2 Production of fungal oxalic acid 11.4 Production of oxalic acid by utilization of cost effective raw substrates or agro185 industrial wastes 186 11.5 Potential advantages of producing oxalic acid from microbial sources 187 11.6 Various applications of oxalic acid 189 11.7 Conclusions and future perspectives 190 References Index

195

List of contributing authors Neeraj K. Aggarwal Department of Microbiology Kurukshetra University Kurukshetra, 136119 India Nivedita Agnihotri Department of Chemistry Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana India https://orcid.org/0000-0002-3588-5852 Rajesh Agnihotri Department of Applied Sciences University Institute of Engineering & Technology Kurukshetra University Kurukshetra 136119 Haryana India Yigrem Alemsu Department of Biotechnology Collage of Natural and Computational Sciences Debre Markos University Debre Markos Ethiopia Syed Fahad Ali Department of Pharmacology International Medical School UIB Almaty Kazakhstan Birhan Aynalem Department of Biotechnology Collage of Natural and Computational Sciences Debre Markos University Debre Markos Ethiopia

https://doi.org/10.1515/9783110792584-202

Sushmita Chauhan Department of Biotechnology School of Engineering and Technology Sharda University Greater Noida India Monika Chopra Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana Ambala, 133207 India Tamanna Devi Department of Bio-Sciences & Technology MMEC Maharishi Markandeshwar, Deemed to be University Mullana-Ambala Haryana India Mansha Ghai Department of Chemistry University of Wisconsin Madison 53706 USA Monika Kayasth Department of Microbiology College of Basic Science & Humanities Chaudhary Charan Singh Haryana Agricultural University Hisar, 125004 India Amit Kumar Department of Biotechnology School of Engineering and Technology Sharda University Greater Noida India E-mail: [email protected]

XIV

List of contributing authors

Sachin Kumar Department of Bioinformatics Janta Vedic College Baraut-Baghpat Uttar Pradesh 250611 India Shubham Kumar Department of Microbiology College of Basic Science & Humanities Chaudhary Charan Singh Haryana Agricultural University Hisar, 125004 India Sunil Kumar Department of Microbiology Faculty of Biomedical Sciences Kampala International University Western Campus Ishaka Uganda Vikas Kumar Department of Bio-Sciences and Technology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207, Haryana India and Department of Microbiology International Medical School UIB, Almaty Kazakhstan E-mail: [email protected] https://orcid.org/0000-0002-6044-3239 Mamta Kumari Department of Biotechnology M.S. Ramaiah Institute of Technology Bengaluru, Karnataka India Kamla Malik Department of Microbiology College of Basic Science & Humanities Chaudhary Charan Singh Haryana Agricultural University Hisar, 125004 India

Shikha Mehta Department of Microbiology College of Basic Science & Humanities Chaudhary Charan Singh Haryana Agricultural University Hisar, 125004 India Prabhakar Mishra Department of Biotechnology School of Applied Sciences REVA University Bengaluru, Karnataka India Shreya Mitra Department of Biotechnology School of Engineering and Technology Sharda University Greater Noida India Masrat Mohmad Department of Chemistry Maharishi Markandeshwar (Deemed to be University) Mullana Ambala 133207 India Sushil Nagar Department of Biochemistry College of Basic Science & Humanities Chaudhary Charan Singh Haryana Agricultural University Hisar, 125004 India Himani Negi Department of Biotechnology School of Engineering and Technology Sharda University Greater Noida India Priya Panwar Department of Biotechnology M.M.E.C. Maharishi Markandeshwar (Deemed to be University) Mullana-Ambala 133207 India

List of contributing authors

XV

Urvasha Patyal Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) MMEC Mullana, Ambala Haryana India

Anil Kr Sharma Department of Bio-Sciences & Technology MMEC Maharishi Markandeshwar, Deemed to be University Mullana-Ambala Haryana India https://orcid.org/0000-0002-9768-1644

Mayank Raj Department of Bio-Sciences & Technology MMEC Maharishi Markandeshwar, Deemed to be University Mullana-Ambala Haryana India

Anil Kumar Sharma Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana-Ambala India

Baljeet Singh Saharan Department of Microbiology College of Basic Science & Humanities Chaudhary Charan Singh Haryana Agricultural University Hisar, 125004 India Komal Sahu Department of Bio-Sciences and Technology Maharishi Markandeshwar (Deemed to be University) Mullana, Ambala 133207 Haryana India Nirmala Sehrawat Department of Bio-Sciences & Technology MMEC Maharishi Markandeshwar, Deemed to be University Mullana-Ambala Haryana India Anil K. Sharma Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) MMEC Mullana, Ambala Haryana India

Meena Sindhu Department of Microbiology College of Basic Science & Humanities Chaudhary Charan Singh Haryana Agricultural University Hisar, 125004 India E-mail: [email protected] https://orcid.org/0000-0002-8755-4705 Manoj Singh Department of Bio-Sciences & Technology MMEC Maharishi Markandeshwar Deemed to be University Mullana-Ambala Haryana 133207 India E-mail: [email protected] https://orcid.org/0000-0002-9257-927X Sheikh Mudasir Syed Department of Genral Surgery International Medical School UIB Almaty Kazakhstan

XVI

List of contributing authors

Sushil Kumar Upadhyay Department of Bio-Sciences & Technology MMEC Maharishi Markandeshwar, Deemed to be University Mullana-Ambala Haryana India

Mukesh Yadav Department of Biotechnology Maharishi Markandeshwar (Deemed to be University) Mullana-Ambala India E-mail: [email protected] https://orcid.org/0000-0001-8526-3747

Mansha Ghai, Nivedita Agnihotri, Vikas Kumar*, Rajesh Agnihotri, Amit Kumar and Komal Sahu

1 Global organic acids production and their industrial applications Abstract: Organic acids are key to the biological, physical, and chemical functions of the life. These acids naturally occur in animals, foods, and microorganisms. Their molecular configurations drive several physical characteristics imperative to well-being. Organic acids are applied in the pharmaceutical, cosmetic, cleaning and food industries. For decades, natural and chemical production of organic acids has thrived, however microbial fermentation has been considered environmentally sustainable approach. Various low-cost substrates are employed as substrate during microbial fermentation. The organic acids production from microbial origin account for the majority of the acids produced on a large industrial basis. Numerous organic acids from bacterial and fungal origin have significance and their biological production offers clear benefits as compared to chemical synthesis in terms of cost. The article illustrates a brief description of the various organic acids in a systematic way along with a survey on the relative production methods. Keywords: organic acids; production; fermentation; microorganisms; metabolism

1.1 Introduction Organic acids represent a class of bulk chemicals that have been extensively used in pharmaceutical, cosmetics, detergent, polymer, food, and textile sectors. Biological or chemical processes can be used to precisely produce organic acids. Growing

*Corresponding author: Vikas Kumar, Department of Bio-Sciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, Haryana, India; and Department of Microbiology, International Medical School, UIB, Almaty, Kazakhstan, E-mail: [email protected]. https:// orcid.org/0000-0002-6044-3239 Mansha Ghai, Department of Chemistry, University of Wisconsin, Madison 53706, USA Nivedita Agnihotri, Department of Chemistry, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, Haryana, India. https://orcid.org/0000-0002-3588-5852 Rajesh Agnihotri, Department of Applied Sciences, University Institute of Engineering & Technology, Kurukshetra University, Kurukshetra 136119, Haryana, India Amit Kumar, Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida 201310, Uttar Pradesh, India Komal Sahu, Department of Bio-Sciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, Haryana, India As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: M. Ghai, N. Agnihotri, V. Kumar, R. Agnihotri, A. Kumar and K. Sahu “Global organic acids production and their industrial applications” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0157 | https://doi.org/10.1515/9783110792584-001

2

1 Global organic acids production

environmental pollution concerns among the general population have a significant impact on the creation of technology that makes it easier to remove dangerous substances. A growing amount of attention is being paid to organic acid generation using biological means due to its environmentally friendly and sustainable characteristics [1]. In biological means, microbial processing has long been used to conventionally produce organic acids. The market for microbe-assisted organic acid production has expanded due to the rise in demand for more environmentally friendly bio-based processes for the manufacture of fuel, biochemicals, and materials [2, 3]. Due to the expanding global market for organic acids, the microbial synthesis of these substances has emerged as a rapidly growing field. One report on the organic acid industry estimates that by 2026, the global organic acid market would have grown to US$12.54 billion which was at US$6.55 billion upto 2015 [4]. Citric acid, with an average annual growth rate (AAGR) of 3.5–4.0 %, is the highly produced organic acid on an annual basis (1.6 million tons), followed by acetic and lactic acids with 0.19 and 0.15 million tons, respectively [5]. Co-cultivation technique is anticipated to be suitable for the industrial manufacture of some bulk chemicals given the idea of the efficient use of wastes and low-value raw materials. Numerous studies have reported and analyzed in-depth the use of less expensive, renewable substrates from a variety of industries, including agricultural (sugars, molasses, plant oils, oil wastes, starchy substances, lactic whey), distillery wastes, animal fat, and oil industries [6]. A high yield of product at the lowest cost is achieved by using low-cost materials in the majority of biotechnological product processes. Many relatively affordable and plentiful substrates from many industrial sectors are currently usable as carbon sources. Utilizing economically more affordable substrates is enabling industry to produce organic acids more effectively. An overview of the most recent advancements in the manufacture of organic acids, the substrates, and their future are given in this review.

1.2 Citric acid Citric acid (C6H8O7·H2O), discovered by Swedish–German chemist, Carl Wilhelm Scheele in 1874, plays a pivotal role in our daily life. It is a weak organic acid (pH 3.0–6.0) and commonly occurred metabolite of plants, animals and some bacteria (Table 1.1). It naturally occurs in the juice of citrus fruits (lemon and lime juice), pineapples, tomatoes, broccoli, and berries [7, 8]. Pure form of citric acid is a tricarboxylic acid, an organic acid containing three carboxyl (–COOH) functional groups, with a molecular weight of 210.14 g mol−1. Physical characteristics of citric acid include colorless, odorless, a sour taste and readily soluble in water [9]. It occurs as a solid form at room temperature and decomposes at higher temperatures. It is also a biodegradable, environmentally friendly, economical, safe, and versatile chemical. Citric acid has widespread use in preparing medicinal citrates, confectionery, soft drinks, and effervescent salts. In foods and beverages, it is used as a preservative to increase shelf life as well as balance acidity. Additionally, citric acid is commonly used in cosmetics and in several skin products to

1.2 Citric acid

3

Table .: Molecular formula, molecular weight, and molecular structure of organic acids. Organic acid

Number of carbon

Chemical formula

Lactic acid

-Carbon acid

CHO

.

Fumaric acid

-Carbon acid

CHO

.

Malic acid

CHO

.

Butyric acid

CHO

.

Succinic acid

CHO

.

CHO

.

CHO

.

CHO CHO

. .

Itaconic acid

-Carbon acid

Alpha-ketoglutaric acid

Gluconic acid Citric acid

-Carbon acid

Molecular weight

Molecular structures

exfoliate the skin. It is present in hairspray, body spray, and deodorant. It can be derived naturally from citrus fruits or synthetically (by microbial fermentation and chemical reactions). Upon discovery, the prominent method adopted in the 19th Century by England was commercial production through extracting the citrus juices from fruit, until 1919 when synthetic production was discovered. As demand for the acid increased, innovative production methods had to advance to fulfill its need. Fermentation accounts for the majority of global citric acid production. Industrial development using Aspergillus niger, a common black mold for fermentation is mainly used for citric acid production. This further led to the experimentation on the production of this acid from several microorganisms. Notably, various mutants from

4

1 Global organic acids production

Table .: Microbial production of citric acid using organic substrates. Substrate Apple pomace Brewery waste Yam bean Kiwifruit peel

Yield (%)

Reference

 . – >

[] [] [] []

Aspergillus genus, such as A. aculeatus, A. carbonarius, A. wentii, and A. foetidus [10] were also found suitable for citric acid production in significant amounts. In addition to fungi, yeast such as Saccaromicopsis lipolytica, Candida tropicalis, Candida oleophils, Candida guilliermondi, Candida parapsilosis, C. citroforman, and Yarrowia lipolytica [11–16] were strong citric acid producing candidates. Currently, beverage and food industries account for more than 60 % of citric acid application [17]. Another method for citric acid production was proposed by Adam and Grimaux in 1880, using glycerol as a starting material and using pure chemical reactions. However, this procedure was not economically viable in comparison to its substitute i.e., fermentation. Yeasts such as Y. lipolytica at one time were a prominent citric acid producer from n-alkanes (C9–C23) in a concentration of 100 g L−1 through batch or fed-batch fermentation; however, changes in the world oil market caused the demand to decrease for this substrate [18]. Thus, out of all the microorganisms studied, A. niger prevailed in the conversion of organic material to citric acid because it is easily manageable, has high yields, and can be improved through mutagenesis and strain selection. Fermentation by fungi utilizes several inexpensive and easily available carbon substrates, like molasses, corn-cobs, and oranges. The main substrates used are molasses, particularly beet or cane which contain high concentrations of sucrose. Notably, the various types of organic acids such as aconitic, glutaric, lactic acids, malonic, malic, and succinic are found in molasses. These acids have the ability to combine with calcium to create insoluble salts that improve the recovery of citric acid crystals. Some of the substrates which are used in citric acid production are highlighted in Table 1.2 with their respective percentage yield. The fermentation process is affected by variety and concentration of carbon source (glucose syrups, sugar beet molasses and sugarcane), pH, phosphate, nitrogen limitation in cultural media, aeration, trace elements, and microorganisms [23]. However, plants using submerged fermentation can use a variety of substrates such as hydrolyzed starch, refined or raw sugar, purified and condensed beet, or cane juice, etc. It had also been proven that beet molasses typically produce more citric acid than cane molasses [24]. An imperative presence of citric acid is displayed in the Krebs or tricarboxylic acid (TCA) cycle in aerobic metabolism to break down carbohydrates. Many aerobic and anaerobic bacteria are produced from the byproducts of glucose metabolism. Citric acid is the primary intermediary in the metabolism of carbohydrates and also acts as one of the

1.3 Succinic acid

5

building block in many aerobic and anaerobic microbes. It is produced as an excess product in some microbes, such as A. niger, under specific circumstances as a result of the TCA cycle’s malfunctioning. TCA is a transitional cycle that involves the final steps in converting sugars, proteins, and lipids into carbon dioxide and water while simultaneously releasing energy for processes like growth, movement, luminescence, etc.

1.3 Succinic acid Succinic acid (C4H6O4) is a dicarboxylic acid initially purified from amber in 1546 by Georgius Agricola. It is considered as a weak organic acid (molecular weight 118.09 g mol−1) that has chemical and biological significance. It serves as a micronutrient that serves a critical role in energy metabolism. The physical characteristics include water soluble, colorless, odorless, white crystals and slightly dissolved in ethanol [25]. It provides economic returns with its diverse applications in food, beverages, pharmaceuticals, polymers, paints, cosmetics, and inks [26]. It is applied as a precursor to generate bulk chemicals, including resins, polymers, and others [27]. This acid is an intermediate in the Kreb’s cycle and synthesized in all living organisms. Upon discovery, succinic acid was primarily produced by liquified petroleum gas [28]. However, due to high costs and environmental concerns, researchers examined alternative sources and methods, such as fermentation for its production. The mostly used method to synthesize succinic acid is fermenting petrol-derived substrates (benzene and butane). However, this process is found to be costly and environmentally unfriendly, driving research approaches toward fermentation technologies by utilizing renewable resources. Bio-based manufacturing expenses of succinic acid are influenced by acid yield in fermentation, costs of raw materials and recovery method [29]. The prominent succinic acid producing microorganisms are bacteria and fungi. Important fungi such as A. niger, A. fumigatus, Byssochlamys nivea, Lnetinus degener, Penicillium viniferum and Paecilomyces varioti had been studied to produce succinic acid [30]. Some bacteria, such as Corynebacterium glutamicium and Enterococcus faecalis had also been examined for succinic acid production. Additionally, some strains of Escherichia coli are also known to Table .: Microbial production of succinic acid using organic wastes. Substrate

Microorganism

Whey Corn steep liquor Wood hydrolyzate Plant hydrolyzate Wheat

A. succiniciproducens A. succinogenes M. succiniciproducens M. succiniciproducens MBELE A. succinogenes

Yield (%)

Reference

 %  %  %  %  %

[] [] [] [] []

6

1 Global organic acids production

have a succinic acid producing ability [31]. High yield of succinic acid is also observed in case of Actinobacillus succinogenes [32], Anaerobiospirillum succiniciproducens [33], and Mannheimia succiniciproducens [34]. Each of the aforementioned bacteria can be cultivated from substrates, such as whey, wood hydrolyzate, galactose, glucose, and glycerol. Some substrates commonly used in the production of succinic acid have been mentioned in the table below (Table 1.3) with microorganisms.

1.4 Lactic acid Lactic acid (C3H6O3), discovered in 1780 by Karl Willhelm Scheele in sour milk, plays a vital role in the chemical and biological functions of life. It is a weak organic acid (molecular weight 90.08 g mol−1) which act as an essential energy source in glucose production. Lactic acid is soluble in water (colorless solution) or occurred as white powder in solid form, has a sour taste and is nearly odorless [40]. The presence of carbonyl (CO) and hydroxyl (OH) groups generates versatility of the acid. In general, lactic acid is used in an industrial fashion as a food preservatives, dairy products, as well as cosmetics. Similar to citric acid, lactic acid can be produced both naturally and synthetically. It initially was extracted from dairy products until, in 1881, Frémy introduced industrial production method using fermentation. This process is controlled by several important factors including the microbes, substrates and methods used in fermentation. To reduce the costs of production, several methods have been employed, the three main methods of fermentation include batch, fed-batch, and continuous batch [41]. Batch and fed batch produce significant yields; however, continuous fermentation is the most productive [42]. Microbial lactic acid producers are classified as per microbes used i.e., bacteria, fungi, yeast, cyanobacteria, and algae. Bacterial production poses several challenges, such as forming both L- and D-lactic acid or racemic DL-lactic acid [43], low yield due to byproduct, requirement of a nutritionally rich substrate, a high risk of cell lysis, and necessity of mixed bacterial strains for development of phage-resistance strains [44]. The four prominent bacterial genera used in lactic acid production are: lactic acid bacteria (LAB), Bacillus strains [45], E. coli and C. glutamicum. LABs are Gram-positive bacteria that produce lactic acid through anaerobic respiration and cannot grow at a pH below 4. They typically inhabit plants, meat and dairy products. LABs are classified into two groups: hetero-fermentative that produce by products such as acetic acid, ethanol and carbon dioxide in addition to lactic acid; and homo-fermentative that produce more than 95 % lactic acid from glucose [46]. LABs require complex nutrient substrates, such as amino acids, peptides, nucleotides, and vitamins, to flourish which dampens their ability to recover as well as drive production costs up [47]. Bacillus strains, such as B. coagulans [48], B. stearothermophilus [49], B. licheniformis [50], B. subtilis, and Bacillus sp. [51] had been examined to produce lactic acid. These bacterial strains could uniquely reduce the production costs of lactic acid through reducing input costs. Additionally, certain strains

1.5 Fumaric acid

7

Table .: Microbial production of lactic acid using organic wastes. Substrate used

Microorganisms

Apple pomace Banana wastes Cellulose Food wastes Paper sludge Sugarcane bagasse Wheat straw Wastewater sludge

Lactobacillus rhamnosus ATCC  (CECT) Lacticaseibacillus casei B. coagulans D L. manihotivorans LMG B. coagulans strains D Lactococcus lactis IO- L. brevis CHCC  and L. pentosus CHCC  Lh. paracasei strain LA

Yield (g/g)

Reference

. . . . . . . .

[] [] [] [] [] [] [] []

of fungi of the Rhizopus genus, namely R. oryzae were also studied to produce lactic acid [52]. Fungi are potentially more beneficial than LABs due to their analytic characteristics, low requirement for nutrients, lower cost and fungal biomass as a useful fermentation byproduct [53]. However, a limitation to these fungi is the unwanted products, such as ethanol and fumaric acid [54]. Several substrates, such as food waste, carbohydrates, dairy wastes, glycerol, industrial waste, and microalgae were reported to produce lactic acid (Table 1.4). However, certain substrates exhibited additional production costs. Refined materials significantly reduce the production purification cost, but persists high production costs. Conventional batch fermentation of whey displayed a considerable lag period in the formation of lactic acid, requiring a larger fermentor and higher operating expenses [55]. The cost effective materials include starchy (corn, potato, rice, wheat starch) and cellulosic (wood, cellulose) materials are inexpensive and available in great quantities around the world [56]. Chemical synthesis is another way to make lactic acid. The primary chemical method used for lactic acid production (L-lactic acid and D-lactic acid) is the hydrolysis of lactonitrile by strong acids. Other methods for producing lactic acid chemically include the oxidation of propylene glycol and the oxidation of propylene by nitric acid. However, all chemical synthesis methods with the exception of lactonitrile hydrolysis are not technically or commercially feasible. The fermentation process uses inexpensive raw resources such starchy waste, molasses, and other materials, which is a key benefit over chemical synthesis as a biological mechanism [64].

1.5 Fumaric acid Fumaric acid (C4H4O4) (Table 1.1), is a organic acid, first time isolated from the plant, Fumaria officinalis. The acid is a butenedioic acid (C–C double bond in E conformation), with a molecular weight of 116.07 g mol−1 and is known as a colorless crystalline solid with combustible nature [65]. The acid is an intermediate metabolite in the citric acid cycle,

8

1 Global organic acids production

Table .: Microbial production of fumaric acid using organic wastes. Substrates

Microorganisms

Corn straw Corn starch Diary manure Molasses Xylose Wood chips hydrolyzate Crude glycerol Brewery waste

R. oryzae ME-F R. oryzae R. oryzae R. nigricans R. nigricans R. arrhizus R. arrhizus RH-- R. oryzae 

Yield (g/g)

Reference

. . . . . . . –

[] [] [] [] [] [] [] []

making it imperative to all life. It plays an essential role as an acidulant due to its nontoxic nature and is used to prepare L-malic acid. The acid is prominently produced from petrochemical processes. It serves as a primary material for producing acids such as maleic acid, malic acid, succinic acid and L-aspartic acid [66]. Currently, with petroleum prices on the rise and growing environmental concerns, researchers have turned to alternatives. In the 1940s, fermentation was an operational method, but was replaced by the then cost-effective process of petrochemical production of fumaric acid. A limitation of petrochemical processes is that the conversion proceeds through an equilibrium, resulting in by-products from maleic and fumaric acids, making yields below equilibrium yields [67]. This limitation prompts the motivation for studying enzymatic conversion. In 1911, Flix Ehrlich discovered Rhizopus nigricans strains to produce fumaric acid, leading to Foster and Waksman screening of 41 strains of eight genera to produce fumaric acid [68]. These strains were identified as: Rhizopus, Mucor, Cunninghamella and Circinella species. The different species of Rhizopus i.e., R. nigricans, R. arrhizus, R. oryzae, and R. formosa produces fumaric acid under both aerobic and anaerobic conditions [69–71]. Various substrates used for the production of fumaric acid have been mentioned in the table (Table 1.5) with the microbes used in production. In-depth economic study reveals that fermentation may become a financially feasible alternative because of its reduced raw material costs, which could offset the greater yields of petrochemical manufacture from maleic anhydride [72].

1.6 Gluconic acid Gluconic acid (C6H12O7), one of the sugar acids, discovered in 1870 by Hlasiwetz and Habermann, is a carboxylic acid formed by oxidizing the first carbon of glucose. It is primarily found in plants, honey and wine [87]. It is a solid white mild acid, having a molecular weight of 196.16 g mol−1 and soluble in water [88]. It is mainly used in pharmaceuticals, cosmetics and food products like additives or buffer salts. Shortly after its

1.7 Itaconic acid

9

discovery, Boutroux discovered that acetic acid bacteria, Acetobacter aceti, serve as a gluconic acid-producing strain in fermentation [89]. Afterward, several bacterial species, such as Acetobacter, Pseudomonas and Gluconobacter demonstrated gluconic acid production. Fungal species such as A. niger, A. foetidus, Penicillium chrysogenum, Penicillium notatum, Phanerochaete chrysosporium, Talaromyces flavus, A. foetidus, Candida tenuis and Tricholoma robustum [90–95] were studied to produce gluconic acid. Primarily the oxidation of glucose substrates is used for the majority of commercial production of gluconic acid. Chemical methods of oxidizing the substrates are limited due to their lack of specificity, producing non-significant yields and by-products [96]. However, during fermentation, these challenges are minimal, allowing fermentation to be the dominant technique for gluconic acid manufacturing. Currently, gluconic acid is commercially produced by fermentation through oxidation of glucose or glucose-containing raw-materials. However, hydroxylates of several raw materials, such as agro-industrial waste, corn starch hydrolyzate, Indian cane molasses, lactose, xylose, and cellobiose were used as alternative substrates for gluconic acid production [97].

1.7 Itaconic acid In 1837, Baup discovered itaconic acid (C5H6O4), which is a byproduct of the pyrolytic distillation of citric acid. It is a renewable carboxylic acid (one of the methyl hydrogen is substituted by a carboxyl group) with a molecular weight of 130.1 g mol−1 [98]. It is an olefinic compound and a fatty acid metabolite exhibited both in fungi and humans. Some physical characteristics include – dry and white solid, dissolves in water, alcohol and acetone and is hygroscopic (absorbs moisture). It is used in the polymer industry, wastewater treatment and ion-exchange chromatography [99]. Since itaconic acid production was first introduced, there has been little interest in it, and the method is still the same. When itaconic acid was named by the US Department of Energy (DOE) as one of the 12 components having the highest potential to be produced by industrial biotechnology, however, views were inverted. Itaconic acid production is also similar to that of citric acid. However, large scale production is through aerobic fermentation using Aspergillus terreus and low cost carbohydrates from beet or cane. The acid is derived biosynthetically and can serve as a nontoxic substitute for many petroleum-derived monomers to produce polymers. With a yield of around 80 %, itaconic acid is produced industrially using five primary steps: fermentation, filtering, crystallization, decolorization, and drying [100]. Utilizing A. terreus poses certain limitations due to a regular oxygen supply being required, resulting in a high concentration of NADH, inhibiting activities of the key enzymes. Additionally, growth inhibition of A. terreus due to itaconic acid, a low carbon flux and absence of metabolite transporters serve as paths for improvement [101]. Further, itaconic acidproducing agents include – Ustilago maydis, candida sp., Pseudozyma antarctica, A. niger, Y. lipolytica, Saccharomyces cerevisiae, C. glutamicium and E. coli [102–108]. Glucose is the

10

1 Global organic acids production

consolidated carbon substrate for high yields of itaconic acid [109]. However, lower-cost carbon sources can be substituted for glucose, such as corn starch [110]. Finally, glycerol can act as a potential substrate for itaconic acid production from E. coli [111].

1.8 Alpha-ketoglutaric acid Alpha-ketoglutaric acid is a keto and oxo acid, containing both carboxylic and ketone groups in its structure. Its conjugate base, alpha-ketoglutarate (AKG) is a ratedetermining intermediate in the tri-carboxylic acid cycle, making it vital for cellular energy metabolism [112]. AKG supplementation is enough in the mature human stage but insufficient in the senescent stage [113]. Since AKG from the TCA cycle cannot be used in cellular metabolism, it must be given as a pure food supplement to carry out amino acid synthesis. The upper small intestine is known to absorb AKG much better than the distal parts. AKG serves as an indispensable source of glutamine and glutamate for cellular metabolism, which in turn increases protein synthesis, prevents muscle protein breakdown and serves as a vital source of metabolic fuel for gastrointestinal tract cells. In the liver, glutamine plays a crucial role in the inter-organ transport of nitrogen and carbon and acts as a precursor for the processes of ureagenesis, gluconeogenesis, and acute phase protein synthesis [114]. In general, alpha-ketoglutaric acid is used in the food industry (dietary supplement), chemical industry (component of infusion solutions), agriculture, and medicine (improve nitrogen balance in patients with burns). Diethyl succinate and diethyl oxalate are used in the chemical production of the alpha-ketoglutaric acid mostly. It is a multi-step, time-consuming, low yielding, and overall dangerous process due to the frequent use of hazardous substrates such as cyanides, toluene, and sodium metal [115, 116]. The process is also not selective, further limiting acid production. These disadvantages to chemical synthesis prompt research into biotechnological methods to convert carbon sources into organic compounds, such as alpha-ketoglutaric acid. One such method is microbial fermentation, through which studies discovered that microorganisms such as Arthrobacter paraffineus, Bacillus spp., Corynebacterium glutamicum, Pseudomonas fluorescens, Serratia marcescens, Candida spp., Pichia spp. and Y. lipolytics [117–122], have the potential to synthesize large amounts of this acid. Several substrates such as ethanol and rapeseed oil have been used as carbon sources for the production of alpha keto-glutaric acid.

1.9 Malic acid Malic acid (C4H6O4) is a 2-hydroxyldicarboxylic acid that has similarity with succinic acid with one of the hydrogens attached to a carbon replaced by a carboxyl group. Physical characteristics include – dry, white or colorless crystals, faint arduous odor, high solubility in methanol, ethanol, acetone and other polar solvent. It is typically derived from

1.10 Butyric acid

11

fruit, particularly apples, bananas and plums [123]. It plays an important role as a food acidity regulator and a fundamental metabolite [124]. It is denoted as a building block material for the synthesis of homo and heteropolymers. The acid was initially discovered by Carl Willhelm Scheele in apples in 1785. It can be synthesized by both chemical and biological methods. The majority of malic acid is synthesized from chemical method by maleic anhydride, an intermediate chemical used for production of polyester resins. It was initially synthesized by S. cerevisiae as a microbial product in 1924 [125]. Common substrates for chemical production are hydrocarbons and hydrocarbon mixtures. However, the present day resources include n-butane and benzene [126]. Both of these substrates are obtained from fossil resources, such as natural gas and petroleum, proven to be catastrophic to the environment. Current research highlights industrial production of malic acid by microbial and natural methods. Microbial production is advantageous to chemical production due to its use on a broader scale of renewable substrates. Some of these substrates include soy and sugar cane molasses, sweet potato, Jerusalem artichoke, and waste materials. Species of Aspergillus, Rhizopus and Ustilago are prominent natural producing microorganisms of malic acid [127–132]. Glucose is the most researched substrate for malic acid production, however, due to its high expenses; it could not be a suitable substrate for production. Cost-effective substrates for malic acid production are industrial waste or lignocellulose-derived materials. The highest yield of malic acid come from beech wood hydrolyzate (0.97 g/g) and corn straw hydrolyzate (0.96 g/ g).

1.10 Butyric acid The short-chain fatty acid, butyric acid (also known as butanoic acid), is hypothesized to have a number of advantageous effects on the digestive system. Enteric cells may quickly absorb butyric anion and utilize it as their primary energy source. Due to its role in many other processes, such as immunoregulation and anti-inflammatory activities, butyric acid is a key regulator of colonocyte proliferation and death, gastrointestinal tract motility and bacterial microflora composition. Only chemical synthesis employing petrochemical feedstocks in the “oxo” process is used to produce butyric acid. Notably, the worldwide market of butyric acid is approximately 80 scales to approximately 80,000 metric tons per year [133]. Petrochemical stock, particularly propylene, is prevalent due to its cost-effective nature and abundance in the production of butyric acid [134]. However, the environmental concerns attributable to the production of various toxic compounds such as sulfide, cyanide, and chloride pose a great consequence to chemical synthesis versus biosynthesis [135]. Fermentation production is a contemporary method of butyric acid biosynthesis. Various species of genera related to Clostridium, Butyrivibrio, Butyribacterium, and Sarcina are actively involved in the acid’s production [136–138]. pH, carbon supply, H2 partial pressure, reducing agents (NADH), acetate and butyrate acid content, yield, and productivity are all factors that affect fermentation [139–141]. Metabolic engineering and

12

1 Global organic acids production

Table .: Microbial production of butyric acid. Microorganism Clostridium acetobutylicium C. tyrobutyricum C. tyrobutyricum C. tyrobutyricum C. tyrobutyricum C. tyrobutyricum

Substrate Glucose Jerusalem artichoke Wheat straw Glucose and xylose Oilseed rape straw Sugarcane molasses

Yield −

.–. g L . g L− – % . g L− and . g L− . g L− . g L−

Reference [] [] [] [] [] []

process development techniques centralized around the use of low-cost biomass feedstocks for butyric acid production is gaining attention [142]. The type of substrate used dramatically impacts butyric acid yield. In Candida thermobutricium, the BA/AA−1 ratio decreased from 10.5 g/ g to close to 1.1 g/ g with pyruvate as the substrate [143]. Butyric acid can be produced by Clostridium bacteria from a variety of substrates such as glucose, xylose, lactose, and glycerol. Feedstocks are typically less expensive than sugars, but before fermentation, they must be pretreated and subjected to enzymatic hydrolysis. The table below summarizes substrate used and their butyric acid production (Table 1.6).

1.11 Conclusions This review delves into the intricacies surrounding each organic acid and their productions. Organic wastes have been utilized as substrates for the production of organic acids by microorganisms. Contemporary scientific findings serve as evidence for the costeffectiveness of biological organic acid production through biomass substrates. Environmental concerns make it ever so important to transition away from petroleum-based sources into unique renewable substrates. Organic acid application across several industries makes them imperative to global economies. Acknowledgments: The authors would like to thank the editors Amit Kumar and Vikas Kumar for their guidance and review of this article before its publication.

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and bioengineering: production, isolation and purification of industrial products. Amsterdam: Elsevier; 2017:543–56 pp. Litchfield JH. Lactic acid, microbially produced. In: Encyclopedia of microbiology, 3rd ed. Cambridge: Academic Press; 2009:362–72 pp. Budhavaram NK, Fan Z. Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulan strains. Bioresour Technol 2009;100:5966–72. Mozzi F. Lactic acid bacteria. In: Encyclopedia of food and health. Cambridge: Academic Press; 2016:501–8 pp. Singh SK, Ahmed SU, Pandey A. Metabolic engineering approaches for lactic acid production. Process Biochem 2006;41:991–1000. Ou MS, Mohammed N, Ingram LO, Shanmugam KT. Thermophilic Bacillus coagulans requires less cellulases for simultaneous saccharification and fermentation of cellulose to products than mesophilic microbial biocatalysts. Appl Biochem Biotechnol 2009;155:76–82. Danner H, Neureiter M, Madzingaidzo L, Gartner M, Braun R. Bacillus stearothermophilus for thermophilic production of L-lactic acid. Appl Biochem Biotechnol 1998;70:895–903. Wang Q, Zhao X, Chamu J, Shanmugam KT. Isolation, characterization and evolution of a new thermophilic Bacillus licheniformis for lactic acid production in mineral salts medium. Bioresour Technol 2011;102: 8152–8. Bai DM, Li SZ, Liu ZL, Cui ZF. Enhanced L-(+)-lactic acid production by an adapted strain of Rhizopus oryzae using corncob hydrolysate. Appl Biochem Biotechnol 2008;144:79–85. Bulut S, Elibol M, Ozer D. Effect of different carbon sources on L(+)-lactic acid production by Rhizopus oryzae. Biochem Eng J 2004;21:33–7. Magnuson JK, Lasure LL. Organic acid production by filamentous fungi. In: Tkacz JS, Lange L, editors. Advances in fungal biotechnology for industry, agriculture, and medicine. Boston: Springer; 2004:307–40 pp. Zayed G, Winter J. Batch and continuous production of lactic acid from salt whey using free and immobilized cultures of lactobacilli. Appl Microbiol Biotechnol 1995;44:362–6. Åkerberg C, Zacchi G. An economic evaluation of the fermentative production of lactic acid from wheat flour. Bioresour Technol 2000;75:119–26. Gullon B, Yanez R, Alonso JL, Parajo JC. L-Lactic acid production from apple pomace by sequential hydrolysis and fermentation. Bioresour Technol 2008;99:308–19. Chan-Blanco Y, Bonilla-Leiva AR, Velazquez AC. Using banana to generate lactic acid through batch process fermentation. Appl Microbiol Biotechnol 2003;63:147–52. Ou MS, Ingram LO, Shanmugam KT. L(+)-Lactic acid production from non-food carbohydrates by thermotolerant Bacillus coagulans. J Ind Microbiol Biotechnol 2011;38:599–605. Ohkouchi Y, Inoue Y. Direct production of L(+)-lactic acid from starch and food wastes using Lactobacillus manihotivorans LMG18011. Bioresour Technol 2006;97:1554–62. Laopaiboon P, Thani A, Leelavatcharamas V, Laopaiboon L. Acid hydrolysis of sugarcane bagasse for lactic acid production. Bioresour Technol 2010;101:1036–43. Garde A, Jonsson G, Schmidt AS, Ahring BK. Lactic acid production from wheat straw hemicellulose hydrolysate by Lactobacillus pentosus and Lactobacillus brevis. Bioresour Technol 2002;81:217–23. Nakasaki K, Adachi T. Effects of intermittent addition of cellulase for production of L-lactic acid from wastewater sludge by simultaneous saccharification and fermentation. Biotechnol Bioeng 2003;82: 263–70. Anuradha R, Suresh AK, Venkatesh KV. Simultaneous saccharification and fermentation of starch to lactic acid. Process Biochem 1999;35:367–75. Goldberg I, Rokem JS. Organic and fatty acid production, microbial. In: Encyclopedia of microbiology, 3rd ed. Cambridge: Academic Press; 2009:421–42 pp. Yang ST, Zhang K, Zhang B, Huang H. Fumaric acid. In: Comprehensive biotechnology, 2nd ed. Cambridge: Academic Press; 2011.

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Birhan Aynalem, Himani Negi, Yigrem Alemu, Nirmala Sehrawat and Amit Kumar*

2 Citric acid: fermentative production using organic wastes as feedstocks Abstract: Citric acid is the most important organic acid produced in tonnage and is used extensively in the pharmaceutical, chemical and food industries due to its low cost and high efficiency compared to other acidulates. Citric acid is produced by fungi, bacteria and yeasts under solid-state and submerged state fermentations. Aspergillus niger is one of the most dominant producer of citric acid. Different fruit wastes and agricultural residues are employed as surplus resources for microbial production of citric acid. In this review, the microbial sources and different organic wastes involved in citric acid production have been discussed. Furthermore, the recovery, purification and application of citric acid in different human utilities have also been reviewed. Keywords: citric acids; organic wastes; submerged fermentation; solid-state fermentation; microorganisms

2.1 Introduction Organic acids are economically important natural products produced by chemical synthesis, extraction from acidulated fruits and microbial fermentation from the different substrates. Organic acids are playing significant role in food and beverage production and preservation, production of animal feed, soap, pharmaceuticals, industrial solvents, perfumes, oils and gas stimulators [1]. Particularly, citric acid, tartaric acid and malic acid enhances the flavor, shelf life and antioxidative properties of the vegetable foods and alcoholic beverages [2]. Food and beverage acidification is common in traditional practice to reduce microbial contaminants especially, bacteria. The large scale production of organic acids is mainly a concern of microbial origin and number of

*Corresponding author: Amit Kumar, Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, India, E-mail: [email protected] Birhan Aynalem and Yigrem Alemu, Department of Biotechnology, Collage of Natural and Computational Sciences, Debre Markos University, Debre Markos, Ethiopia Himani Negi, Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, India Nirmala Sehrawat, Department of Biotechnology, MMEC, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, Haryana, India As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: B. Aynalem, H. Negi, Y. Alemu, N. Sehrawat and A. Kumar “Citric acid: fermentative production using organic wastes as feedstocks” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0158 | https://doi.org/10.1515/9783110792584-002

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2 Citric acid production using organic wastes

Figure 2.1: Molecular formula, molecular structure, model and molecular weight of citric acid.

identified microbial organic acids are exceeding beyond hundreds worldwide [3]. Solidstate fermentation (SSF) and submerged state fermentation (SmF) are ecofriendly and high yielding processes to produce different types of organic acids [4, 5]. Citric acid is 6-carbon organic acid that originated from the different fruits such as limes, lemons, oranges, pineapples and grapes with broad applications in food, beverage, cosmetics, pharmaceutical and detergent producing industries in the form of additive or preservative components (Figure 2.1) [6]. It is a natural compound involved in detoxification, preservation, balancing the energy level and improving healthy dietary system/processes in physiological function [5]. The global demand of citric acid consumption has increased with high market opportunities. The annual sales of citric observed more than 3.6 billion USD in 2020 [7]. However, fermentative citric acid production requires selection of effective microbial species or strains and availability of fermentable feedstocks. SSF is effective process to convert organic wastes into value-added citric acid with low energy requirement, high yield, little microbial contamination and less waste regeneration [8]. Therefore, this review summarized the sources of microbes involved in citric acid production, sources of feedstock employed for citric acid production, suitable inducers for fermentative citric acid production, citric acid recovery, purification process and applications of citric acid in different human utilities.

2.2 Microbial sources and fermentative production of citric acid Microorganisms are morphologically, ecologically and physiologically divers in nature and playing both useful and harmful effect to the human being [9]. Different industries are using microbes and their products as important inputs to enhance the productivity because of their adaptation of harsh conditions, utilization of different substrates and production of value added products to generate revenue [10].

2.2 Microbial sources and fermentative production of citric acid

23

Fungal species showed high importance in the organic acid production and takes a considerable share compared to other microbial categories in fermentative degradation of agricultural residues, industrial and domestic wastes [11]. The genus Aspergillus is the main fermentative producers of citric acid in the word (Tables 2.1–2.3). Specially, Aspergillus niger produces the most essential enzymes in citric acid production including cell bounded invertase that hydrolyze sucrose into glucose and fructose, hexokinase with more affinity to glucose than fructose and glucokinase, which is non-competitive to both substrates [12]. In high concentration of glucose, glucose oxidase is more potent enzyme that stimulates A. niger to oxidize glucose to gluconic acid under highly aerated condition and low concentration of other nutrients. Citric acid is produced under surface fermentation or liquid surface culture [16], and SmF or SSF [17]. However, its production can be influenced by moisture content, particle size and type of the substrate, sugar level of the substrate, incubation period, type and concentration of inducers used [12]. In addition, carbon concentration, dissolved oxygen, hydrogen ions, suboptimal concentration of phosphate, trace metal, nitrogen and manganese affects the production of citric acid in fermentative processes [12]. Particularly, shortage of nitrogen, manganese and phosphate influences the protein degradation and increase concentration of ammonium ions in fermenting medium. For the mass production of citric acid, the deactivation of aconitase and/or isocitrate dehydrogenase is involved during production of intermediate biomass formation in Krebs cycle [14, 18], which is series of eight reactions that produce acetate and carbon dioxide.

Table .: Microbial sources of citric acid. Microbial category

Genus

Species

Fungi

Aspergillus

Yeasts

Penicillium Trichoderma Talaromyces Absidia Acremonium Candida

A. niger, A. flavus, A. awamori, A. nidulans, A. phoenics, A. wenti P. citrinum, P. janthinellu, P. leteum, P. restrictum T. viride Talaromyces sp. Absidia sp. Acremonium sp. C. lipolytica, C. tropicalis, C. guilliermondii, C. oleophils, C. parapsilosis, C. citroformans P. kluyveri Y. lipolytica B. licheniformis, B. subtilis A. paraffinens B. flavum Corybebacterium sp.

Bacteria

Pichia Yarrowia Bacillus Arthrobacter Brevibacterium Corybebacterium

Reference [, , ]

[] [] [] []

24

2 Citric acid production using organic wastes

Table .: Microbial production of citric acid using organic wastes. Citric acid producer

Substrate

Type of Fermentation fermentation conditions

A. niger

Beet molasses, corn steep liquor

SmF

A. niger FUO

Cassava peel

SmF

Y. lipolytica NG/UV

Biodiesel waste SmF having glycerol, aspen waste having glucose

A. niger CECT-

Orange peel waste

SSF

A. niger NRRL

Apple pomace

SSF

A. niger

Cassava bagasse

SSF

A. niger DS

Sugarcane bagasse

SSF

A. niger IBO-MNB

Oil palm empty fruit bunches

SSF

A. niger ATCC

Sugarcane bagasse

SSF

A. niger NRRL

Corn husk

SmF

A. niger NRRL

Corn cob

SmF

Temperature: . °C, pH: ., incubation time:  h, aeration rate:  mL/min Temperature:  ±  °C, pH: ., incubation time:  h, agitation:  rpm Temperature:  ± . ° C, pH: ., incubation time: six days, agitation:  rpm, pO:  % (air saturation) Temperature:  °C, incubation time:  h, moisture saturation:  % Temperature:  °C, pH: ., incubation time:  days, initial moisture content:  % Temperature:  °C, incubation time:  h, moisture content:  %, air humidity  % Temperature:  °C, incubation time: six days, moisture content:  % Temperature:  °C, pH: .–., incubation time: six days, moisture content:  % Temperature: – °C, pH: –., incubation time: – days, moisture content: – % Temperature:  °C, pH: ., incubation time:  h, agitation:  rpm Temperature:  °C, pH: ., incubation time:  h, agitation:  rpm

Citric acid yield

Reference

. %

[]

. g/L

[]

– g/L

[]

 mg/g dry orange peel

[]

 ± . g/kg dry substrate

[]

 g/kg dry substrate

[]

. % (w/w)

[]

. g/kg dry substrate

[]

. g/kg dry substrate

[]

 ±  g/kg dry substrate

[]

 ± . g/kg dry substrate

[]

2.2 Microbial sources and fermentative production of citric acid

25

Table .: (continued) Citric acid producer

Substrate

Type of Fermentation fermentation conditions

A. niger KS-

Pineapple waste SSF

A. niger ACM

Pineapple waste SSF

A. niger BC

Apple pomace

SSF

A. niger MTCC Peels of banana SSF 

Temperature:  °C, incubation time:  days, initial moisture content:  % Temperature:  °C, pH: ., incubation time: seven days, initial moisture content:  % Temperature:  °C, incubation time: five days, aeration rate: . l/min, moisture content:  % Temperature:  °C, incubation time:  h, moisture content:  %, pH: .

Citric acid yield

Reference

. g/kg dry substrate

[]

 g/kg dry substrate

[]

 g/kg dry substrate

[]

 g/kg dry substrate

[]

Table .: Fermentative citric acid production improvement using inducers. Fermentative microbes

Substrates

Fermentation inducers

A. niger NRRL

Apple pomace

A. niger NRRL

Apple pomace

A. niger CGMCC  A. niger DS A. niger (NRRL, )

Residual sugar Sugarcane bagasse Corn cobs Apple pomace Orange pulp Apple peels Grape pomace Grape pomace Grape pomace Banana peels Apple peels

 % Methanol  % Ethanol  % Methanol  % Ethanol HGT-overexpression Sucrose (moistening agent) NaOH + Rapidase Pomaliq No inducer  % Methanol  % Methanol  % Methanol  % Methanol  % Methanol  % Methanol +  % glucose  % Methanol +  % sucrose

A. niger LPB BC A. niger DTO: -E A. niger ATCC  A. niger ATCC  A. niger ATCC  A. niger EU. A. niger DTO: -E

Citric acid production

Reference

 g/kg  g/kg . g/kg . g/kg . g/L  g/kg . g/kg  g/kg  g/kg  g/kg  g/kg  g/kg  g/kg  g/kg  g/kg

[] [] [] [] [] [] [] []

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2 Citric acid production using organic wastes

2.3 Organic wastes as potential resource for citric acids production Appropriate utilization of organic wastes is environmental friendly, inexpensive and safe approach to convert surplus resources into value added products. Most of the organic wastes are rich in carbon, nitrogen, energy, different minerals, electron acceptors and growth factors that support the growth of microorganism [18, 19, 27]. A variety of organic wastes are reported as the important substrates of citric acid production through microbial fermentation [28]. The organic wastes that composed of carbohydrates, proteins, fatty acids, minerals and different growth factors could be suitable for cultivation of different fermentative microbes and satisfy their requirements of carbon, nitrogen and electron acceptor [1].

2.3.1 Citric acid production using fruit wastes and peels Fruit wastes and peels are used as substrates for citric acid production through microbial fermentation. In food industry during the manufacturing of jellies and jams, fruits have a large quantity of solid by-products generated as wastes. These wastes can be discarded in landfill sites that create environmental pollution otherwise could be used as cheap substrate to produce citric acid through microbial fermentation. It could reduce the cost of citric acid production and environmental problems [29]. Fruit wastes like banana peel, orange peel, sweet lime peel, apple pomace, pineapple waste, jackfruit waste, grape pomace and kiwi fruit peel are successfully used as substrate for the citric acid production [30]. The availability and presence of high quantity of cellulose, starch, carbohydrates, fiber and other nutrient contents food wastes are attractive raw material for microbial production of organic acid [31]. Hang et al. [32] demonstrated that strain of A. niger NRRL567 yielded 90 g of citric/kg apple pomace under SSF at 30 °C of incubation temperature for five days induced by methanol at a concentration of 4 % v/w. Methanol concentration has enhancing impact on the production of citric acid [32]. Fungal sources of citric acid production perform best at temperature ranged 27–30 °C however, lower than 27 °C might be decreased the citric acid production [33]. A. niger BC1 produced 124 g of citric acid from the 1 kg of dry-weight apple pomace using multi-layer packed bed solid-state bioreactor at optimized conditions of 30 °C incubation temperature, five days of incubation period, 78 % of moisture content [34]. Orange peel contains soluble and insoluble carbohydrates. Monosaccharide including glucose and fructose are the soluble sugars available in orange peel while cellulose, hemicellulose and pectin are the insoluble polysaccahrides. Orange peel consists 16.9 % of soluble sugar, 3.75 % starch, 3.5 % ash, 1.95 % lipids, and 6.5 % proteins. The fiber comprises 9.21 % of cellulose, 10.5 % hemicellulose, 0.84 % lignin, and 42.5 % of

2.3 Organic wastes as potential resource for citric acids production

27

pectin [35]. A. niger CECT-2090 (ATCC 9142, NRRL 599) was used to produce citric acid and yielded 193 g of citric acid per gram of dry orange peel under optimum conditions at 85 h incubation period, 30 °C temperature and 70 % of moisture saturation in SSF [36]. Metals like Fe, Zn, Cu, Mn and Co are essential for the growth of A. niger, which can increase the production of citric acid [37]. A. niger van Tiegh 1867 yields 640 g of citric acid/kg of orange peel reinforce with corn molasses with appropriate conditions of submerged fermentation conditions. A 72 h of incubation time and 65 % w/v of moisture content with bed loading of 20 % were found to be optimum for citric acid production. In the medium the final sugar concentration was adjusted to 14 % using molasses and 3.5 % of methanol that supplemented to improve the citric acid production [38]. Pineapple is major vegetable grown in the tropical regions of the world including India. Pineapple peel contains proteins, fats and carbohydrates and is suitable raw material for microbial growth [39]. Pineapple juice is the third most preferred juice after apple and orange juices [40]. Aspergillus foetidus ACM3996 produced 9.9 g of citric acid per 100 g of the dry weight of pineapple waste under SSF. The incubation time, temperature and initial moisture of four days, 30 °C, and 65 %, respectively, are found to be optimum for maximization of citric acid production. The supplementation of 3 % methanol increased citric acid production up to 16.1 g/100 g of dried pineapple waste [41]. Kareem et al. [39] investigated the citric acid production by A. niger KS-7 and observed 60.61 g/kg of pineapple peel and media supplemented with glucose, sucrose and nitrogen source under SSF at a 65 % of moisture content, 30 °C of temperature and five days of incubation period. Banana is cultivated in tropical regions and the harvesting of fruit generates a huge quantity of biomass residue. Global production of banana is approximated 48.9 million ton, of which India contributes 10.4 million tons [20]. Priscilla et al. 2020 [42] investigated citric acid production by A. niger UABN 210 using banana peel as substrate under SSF. A. niger UABN 210 produced 82 g of citric acid per kg of dry weight banana peel supplemented with nitrogen source of ammonium sulphate and potassium hydrogen phosphate. A 96 h of incubation time, temperature of 30 °C, and moisture content of 60 % were found suitable for maximum production of citric acid. 1 % v/v methanol was supplemented in medium as inducer. A. niger MTCC282 was reported for the production of citric acid using koji fermentation. Under optimized conditions, 70 % of moisture content, 28 °C temperature, pH 3, 108 spores/ml inoculum and 72 h of incubation maximized the production of citric acid [42]. However, increasing moisture content lowers the porosity of the substrate and reduces the citric acid production [8]. Artrococarpus heterophyllus (Jackfruit) is a large tree cultivated in tropical regions and it is a native fruit of south India. It contains carpel fiber, which consists of sugar, amino acids and minerals. It was treated chemically to extract the present reducing sugar and carpel fiber used as a carbon source for the production of citric acid [30]. Hydrolysis of carpel fiber was performed with 14.6 mol/L HCL followed by batch fermentation. A. niger NRRL322 produced 73 g/L of citric acid using batch fermentation at 48 h of incubation time and 3.092 g/ml of biomass concentration [43].

28

2 Citric acid production using organic wastes

Among different strains of A. niger, wild type AN2 produced the maximum amount of citric acid (14.32 mg/mL) from a grape pomace concentration of 250 g/L under SmF [44]. Hang et al. [45] reported the yield of 100 g/kg of kiwi fruit peel under SSF by using A. niger NRRL-567. The incubation temperature of 30 °C, moisture level of 65 % and the addition of 2 % methanol were found optimum for the citric acid production [45]. The yield of citric acid is based on the amount of sugar consumed. A. niger 360 yielded the maximum (278.5 g/kg) amount of citric acid per dry peel of pomegranate under SSF using non-aseptic conditions. The strain produced more citric acid when it was cultivated on wet peel wastes as compared to dried peel wastes [46].

2.3.2 Citric acid production using agricultural wastes Agricultural residues such as sugarcane bagasse, sugarcane molasses, wheat bran, wheat straw, rice husk, rice straw, soybean hulls, corn husk, oil palm empty fruit bunches and date waste contains high amount of carbohydrates that are resources to produce citric acid. The protective cover of rice grain is known as rice husk. The milling process separates it from rice grain. Rice husks consist of comparatively high levels of lignocellulose [47]. It can be used as a substrate in fermentation medium for the production of citric acid. Pretreatment process is required to separation lignin from the rice husks before utilizing it as a substrate in fermentation because lignin may prevent cellulose and hemicellulose from being hydrolyzed into reducing sugars [48]. The abundant availability of rice husks is a major benefit as rice is the most widely cultivated grain worldwide. It is a cost-effective carbon source to produce citric acid using strain A. niger under SmF. A yield of 19.12 g/L of citric acid was observed at 400 rpm of agitation, pH 2.34 and 22.34 % (w/v) of substrate concentration in fifth day of fermentation [49]. Thousands of tons of sugarcane have been cultivated worldwide and its processing generates a huge amount of sugarcane bagasse [50]. It is burnt and used as fuel. Sugarcane bagasse contains 43–52 % w/w fiber, 50 % cellulose, 25 % hemicellulose and 25 % lignin, 46–52 % water and 2–6 % soluble solids [51]. Investigations showed high production of citric acid from the sugarcane bagasse using as a substrate in SSF. Khosravi-Darani et al. [50] reported a yield of 94.5 g of citric acid per kg of sugarcane bagasse fermenting by A. niger ATCC 9142 at six days of incubation time and temperature of 37 °C with 75 % of moisture content during SSF. Urea pretreated sugarcane bagasse has played an important role in increasing the productivity of citric acid. Under optimum conditions, per kg of urea-pretreated dry bagasse produced 137.6 g of citric acid that is corresponds to a yield of 96 % (based on sugars consumed). This gives the productivity of 26.45 g/kg per day [50]. Ram horns also called fibrous proteins that are commonly produced worldwide. Every year 600 tons of ram horns are discarded by the slaughterhouse in turkey. Ram horn waste can lead to large problems for the environment [52]. Ram horn contains α-keratin, which is rich in cysteine and contains other amino acids [53]. A. niger NRRL 330 yielded 78 g/L of citric acid from Ram horn hydrolysate (RHH) media in submerged

2.4 Recovery and purification of citric acid

29

culture. This yield was obtained at 8 % RHH concentration at 30 °C for six days [54]. In 2020–2021, with an annual production of 362.1 million metric tons soybean (Glycine max) is considered one of the most cultivated crops in the world [55]. India is ranked fifth globally for soybeans production [56]. During de-hulling process, soybean hulls are generated that accounts 5–8 % of the total mass of the soybeans. Soybean hulls is a biomass with high polysaccharides content, consist of cellulose, hemicellulose and low lignin [57]. Citric acid pretreatment and enzymatic hydrolysis of raw soybean hulls optimized to release fermentable sugars [58]. Sugar processing industries produce large amounts of waste known as molasses [30]. Molasses is a rich source of sugar that can be utilized by microorganisms to produce citric acid [59]. It needs the treatment of molasses with ammonium oxalate followed by treatment with di-ammonium phosphate to make it potential substrate for citric acid production. This is mandatory to reduce inhibitors of fermentation process due to the presence of organic and inorganic compounds [43]. A. niger yielded 72.3 g/L of citric acid using 20 % (v/v) of molasses during the fermentation [60]. Molasses have been tested for citric acid production by A. niger under SmF. Medium with molasses as a carbon source gave a yield of 12.6 mg/ml with 5 % sucrose at an optimum incubation temperature of 30 °C, 200 rpm of aeration, and initial pH of 6.5 with methanol as inducer [61]. Sweet corn processing generates a large volume of solid waste, termed as corn-husk. It contains carbohydrates i.e. cellulose and hemicelluloses that are used as carbon source for microorganisms [62]. The pretreated corn-husk with 0.5 mol/L NaOH (20 g/100 mL) fermented with A. niger NRRL 2001 and yielded 259 ± 10 g of citric acid per kg of dry matter of corn husk after 120 h of fermentation at 30 °C [63].

2.4 Recovery and purification of citric acid There are various methods for citric acid production. However, around the world over 90 % of the citric acid produced through fermentation. The production of citric acid could be carried out by different fermentation processes with high possibility of undesirable by-products production or contaminates like microbial parts (mycelium), organic acids other than citric acid, proteins, salts, substrate residues, and other impurities produced during citric acid production. Recovery of citric acid from fermented media needs precipitation, extraction and purification. Different impurities like oxalic acid are removed by uprising of pH to 3.0 with calcium hydroxide at temperature of 72–75 °C [28]. The precipitated calcium oxalate can be removed by filtration or centrifugation process. The citric acid in the fermented broth converts into calcium citrate and precipitated by the addition of calcium oxide at 90 °C at pH of 7. The precipitated calcium citrate is recovered by centrifugation of filtration [64].

30

2 Citric acid production using organic wastes

2.5 Applications of citric acid Citric acid is a versatile and inoffensive alimentary additive. It is listed as ‘generally regarded as safe’ (GRAS) and approved by the Joint FAO/WHO Expert Committee on Food Additives [24]. Citric acid finds applications in food, pharmaceuticals, chemical, and cosmetic industries. Because of pleasant taste, high water solubility, good chelating and buffering properties, citric acid has been extensively used in food and pharmaceutical industries. It is act as a buffering agent in various processes including cosmetics and toiletries. It is also considered a reactive intermediate compound for chemical synthesis. Moreover, the carboxyl and hydroxyl groups of citric acid are involved in the synthesis of different complex molecules and industrially important reactive products [6, 24]. Citric acid is the leading organic acid for its wide range of application in human welfare and has been briefly discussed in previous literature reviewed by Lende et al. [64]. The monohydrates of citric acid are generally employed as pH controlling agent, flavoring and preservative of candies, cookies, biscuits, jams, jellies, snacks, prompt foods and sauces. In pharmaceutical industries, citric acid served as antioxidant preventives of effervescent, vitamins, pH correctors, blood, iron citrate tablets and ointments [65]. Citric acid acts as a good cleaning agent in various industries. It is used for operational cleaning of iron and copper oxides, leather processing, printing inks, floor cement, textiles, photographic reagent and plaster. Citric acid has also been used passivation process of stainless steel to replace nitric acid. It is utilized along acid dyes for home dyeing as odorless agent instead of white vinegar. Moreover, citric acid might be used as active ingredients for the preparation of antiviral tissues [5, 66].

2.6 Conclusions Citric acid have important applications in various industries. This review has been focused on fermentative production of citric acid using organic wastes as carbon source. The recovery process of citric acid and its applications in food and other sectors have also been discussed. Fruit wastes and agricultural residues are generated in large amount all over the world and create disposal problems. These organic wastes are employed as lowcost surplus substrate for citric acid production under SSF and SmF. The microbial production of citric acid has been improved by optimizing the cultural conditions. Moreover, the citric acid production has been enhanced with the addition of inducers such as ethanol, methanol, sucrose and glucose in the production medium. Acknowledgement: The authors would like to thank the editors Amit Kumar and Vikas Kumar for their guidance and review of this article before its publication.

References

31

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Sushmita Chauhan, Shreya Mitra, Mukesh Yadav and Amit Kumar*

3 Microbial production of lactic acid using organic wastes as low-cost substrates Abstract: Lactic acid is a natural organic acid with diverse of applications in food, pharmaceutical, cosmetics, and chemical industry. Recently, the demand of lactic acid has been grown due to its utilization for polylactic acid production. Microbial production of lactic acid production is preferable due to optical purity of product, utilization of low cost substrates, and low energy requirement. Lignocellulosic biomass and other organic wastes are considered potential raw materials for cost-effective production of lactic acid. The raw materials are either hydrolyzed by enzymes or dilute acids to release the reducing sugars that are fermented in to lactic acid. This review has been focussed on microbial production of lactic acid using different organic wastes as low cost substrate. Keywords: lactic acid, lactic acid bacteria, organic wastes, Rhizopus, submerged fermentation

3.1 Introduction Lactic acid (2-hydroxypropanoic acid) is a naturally occurring organic acid that is applicable to several industries. The market of lactic acid is rapidly expanding due its demand in food, pharmaceutical, textile, leather, and chemical industries. The demand of lactic acid is predicted around 1960.1 kilotons by 2025 with an estimated annual growth rate of 18.7% that represents about 9.8 billion USD in the international market [1, 2]. Lactic acid is produced through chemical synthesis or microbial fermentation. The chemical synthesis from hydrocarbon-based sources generates a racemic mixture of D/L (±) lactic acid. Lactic acid production through microbial fermentation has gained more attention due to utilization of renewable carbohydrate biomass, low production temperature and low energy consumption. Moreover, optically pure L (+) or D (−) lactic is produced by microbial fermentation. Presently, almost all lactic acid is produced globally, comes through the microbial fermentation route due to recent developments in bioconversion technology [3, 4].

*Corresponding author: Amit Kumar, Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, India, E-mail: [email protected] Sushmita Chauhan and Shreya Mitra, Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, India Mukesh Yadav, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), MullanaAmbala, Haryana, India As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: S. Chauhan, S. Mitra, M. Yadav and A. Kumar “Microbial production of lactic acid using organic wastes as low-cost substrates” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0159 | https://doi.org/10.1515/9783110792584-003

36

3 Microbial production of lactic acid

Several strategies have been employed to minimize the production cost of lactic acid. The lactic acid production can be improved by screening of hyperproducer microbial strain from natural habitat or optimizing the cultural parameters. Another strategy to improve the lactic acid production is microbial strain improvement by genetic modification. Moreover, the utilization of low-cost substrates such as lignocellulosic biomass and food wastes appear to be an attractive approach to minimize the production cost of lactic acid. The utilization of low-cost substrates such as lignocellulosic and food wastes appear an attractive approach due to their wide availability, low cost, and renewability. Lignocellulosic materials such as wheat bran, rice bran, corn stover, corn cob, paper sludge, sugarcane bagasse, corn steep liquor, poplar hydrolysate, sugarcane pressmud, etc. have been evaluated as low-cost substrate for the production of lactic acids [5]. A microbial strain is selected based on the yield, productivity, and purity of lactic acid. In microbial fermentation process, another important parameter during selection of the right microorganism is the nutritional requirement. The microbial strain able to utilize hexose and pentose for lactic acid production are preferable [6, 7]. Most of the lactic acid is produced by either acetic acid bacteria or filamentous fungi namely Rhizopus spp. using fermentable sugars [8] (Figure 3.1). Different species of Rhizopus are able to produce lactic acid anaerobically using glucose. Pyruvic acid act as a precursor molecule during lactic acid production by the microorganisms. NAD-dependent L-lactate dehydrogenase and NAD-dependent D-lactate dehydrogenase are the enzymes that convert pyruvic acid into lactic acid. The type of enzyme involved in the production of lactic acid determines the stereospecificity of the product [9]. Metabolic engineering of microbes have also been used to improve the production of lactic acid. Saccharomyces cerevisiae has been genetically engineered for hyper-production of lactic acid due its higher resistance against low pH [10, 11].

Figure 3.1: Microbial sources for lactic acid production [2, 4, 5, 10–12].

3.2 Lactic acid production using organic wastes

37

3.2 Lactic acid production using organic wastes Chemical or microbial processes are used for production of lactic acid. Mostly microbial fermentation is preferred because it utilizes renewable organic wastes to produce optically pure lactic acid [13]. A variety of organic wastes has been utilized for the production of lactic acid (Table 3.1). Lignocellulosic biomass is an attractive carbon sources for the production of lactic acid. It contains both cellulose and hemicelluloses. Cellulose is a homopolymer, hydrolysis of cellulose generates D-glucose whereas hemicellulose is a heteropolymer. Hydrolysis of hemicellulose produces various reducing sugars such as glucose, xylose, galactose, arabinose and mannose. Cellulases and xylanases are used for the hydrolysis of cellulose and hemicellulose respectively and converts them into hexose and pentose sugars [6, 7]. For the conversion of lignocellulosic material into lactic acid by microbial fermentation, these three steps must be followed: (1) pretreatment of lignocellulosic material, (2) hydrolysis of pretreated lignocellulosic material to release fermentable sugars (acid, enzyme alkali hydrolysis), (3) fermentation process for lactic acid production at controlled pH and temperature [14].

3.2.1 Corn stover and corncob for lactic acid production After corn is harvested stalks, leaf and husk are remained in the field that is known as corn stover. It contains about 11.1% lignin, 27.1% hemicellulose, 32.6% cellulose, 24.5% pentosans, and 33.0% α-cellulose [33]. It contains high amount of carbohydrate, and considered as of the one of best lignocellulosic feedstock for lactic acid production. A corncob is a hard, cylindrical kernel that supports the corn kernel. It contains cellulose (70%), hemicellulose (22%), and lignin (8%). Corncob has higher cellulose content compared to corn stover, and has lower lignin content [34]. Harvesting and processing of corn produces a number of by-products such as corn stover, corncob, corn germ, concentrated soluble grains and processing effluents. Among these by-products the most important source of lactic acid production are corn stover. The use of these byproducts makes a significant contribution to increasing the efficiency of corn industry, generating revenue for rural communities and benefiting the environment [35]. L. pentosus is a facultative hetero-fermentative species. Lactic acid is produced as the only product from hexose via Embden–Meryerhof–Parnas (EMP) process in anaerobic conditions, and produces lactic acid and acetic acid from pentoses by using phosphoketolase (PK) pathway for conversion. It produces 2 mol of lactic acid per mol from hexose and 1 mol of lactic acid per mol from pentose under anaerobic conditions [6]. Corn stover was pretreated with NaOH followed by washing. Enzymatic hydrolysis was carried out at 37 °C and pH 6.0 with cellulase dosage of 30 FPU/g was found to produce 1.28 g/L/h at 12% (w/w) of lactic acid by Pediococcus acidilactic PA204 in 5 L bioreactor. This bacterium has been reported to ferment xylose for the production of lactic acid [36].

–

–

Corncob molasses (by-product during xylitol production) Sophora flavescens residue & food waste

Corn stover

Alkaline pretreatment

Enzymatic hydrolysis

Enzymatic hydrolysis Enzymatic Corn stover Ground stover was treated with % NaOH at hydrolysis  °C for  h Enzymatic Corn steep liquor, oak -.% HSO soaking -Steam explosion at hydrolysis wood chips  °C for  min Poplar hydrolysate – –

Rice washing drainage having rice bran Recycled paper sludge Neutralized with HCl

Ground to  mm and Enzymatic pretreated with % NaOH hydrolysis at  ±  °C – –

Enzymatic hydrolysis

Aqueous ammonia

Corn stover

Type of hydrolysis

Pretreatment

Substrate

Temperature:  °C, pH: ., agitation:  rpm, N was sparged at . vvm to maintain anaerobic conditions Temperature:  °C, pH: ., incubation . g/g of substrate time:  h, agitation:  rpm L. brevis ATCC, L. plantrum ATCC L. brevis ATCC, L. plantrum ATCC

. g/g of substrate with productivity . g/L h . g/g of substrate

[]

 g/L or . g/g of carbohydrates . g/L or . g/g of stover

Temperature:  °C, pH: ., agitation:  rpm

[]

 g/L or . g/g of sugars

[]

[]

[]

[]

[]

. g/L or . g/g total sugars

[]

[]

 g/L

Temperature:  °C, agitation:  rpm, flushed with pure N to create anaerobic conditions Temperature:  °C, static, pH: – (maintained by CaCO), incubation time:  h Temperature:  °C, agitation:  rpm, solid to liquid ratio was :, flushed with pure N to create anaerobic conditions Temperature:  °C, pH: ., incubation time: days, intermittent N sparging for anaerobic conditions Temperature:  °C, pH: ., incubation time:  h Temperature:  °C, pH: ., incubation time:  h, agitation:  rpm

Reference

. g/L

Lactic acid yield

Fermentation conditions

Lactobacillus sp. RKY

L. rhamnosus ATCC B. coagulans LA 

L. rhamnosus M-

L. casei CICC

Bacillus sp. XZL

L. pentosus

Fermentation microorganism

Table .: Microbial production of lactic acid using organic wastes.

38 3 Microbial production of lactic acid

– Enzymatic hydrolysis Enzymatic hydrolysis –

Acid hydrolysis Acid hydrolysis Acid hydrolysis

–

–

–

–

–

–

–

Corncobs Corn stover Paper sludge

Biosludges

Sugarcane pressmud

Rice bran

Wheat bran Corn steep liquor

Sugarcane bagasse, corn steep liquor

Bacillus sp.  C

L. rhamnosus LA--

L. rhamnosus (NBRC )

L. casei subsp. casei CFTRI

L. rhamnosus CECT-

B. coagulans D B. coagulans P-B

L. rhamnosus SF

L. rhamnosus LA--

Bacillus sp. NL

% HSO soaking for  h, Enzymatic and processed at  °C hydrolysis for  min – Acid & enzymatic

Corn stover

White rice bran

Fermentation microorganism

Type of hydrolysis

Pretreatment

Substrate

Table .: (continued)

Batch: . g/L Fed-batch: . g/L

Lactic acid yield

Productivity-batch: . kg m− h− Fed-batch: . kg m− h− Temperature:  °C, agitation:  rpm, Corncobs: . g/L incubation time – h Corn stover: . g/L Temperature:  or  °C, feeding fre- D strain:  g/L quency: , residence time:  days, half of P-B strain: . g/L reaction mixture was added every  days Temperature:  °C, pH: ., agitation:  g/kg biosludge with  rpm productivity of . g/ Lh Temperature:  °C, moisture content:  g/kg of substrate %, incubation time  h, static conditions Temperature:  °C, pH: ., incubation  g/L time  h, N sparged to maintain anaerobic conditions Temperature:  °C, pH: ., incubation . g/g of substrate time  h, agitation:  rpm with productivity of . g/L h Temperature:  °C, pH: ., incubation ./g of sugars with % time  h, agitation:  rpm of theoretical yield

Temperature:  °C, pH: ., rotation speed: . Hz

Temperature:  °C, pH: ., agitation:  rpm without aeration

Fermentation conditions

[]

[]

[]

[]

[]

[]

[]

[]

[]

Reference

3.2 Lactic acid production using organic wastes

39

Rice bran

Sugarcane bagasse

Office waste paper

–

Rice & wheat bran

Type of hydrolysis

Enzymatic hydrolysis – Enzymatic hydrolysis Ground to obtain particle Acid size of less than  mm hydrolysis – Enzymatic hydrolysis

Pretreatment

Substrate

Table .: (continued)

L. delbrueckii subsp. delbrueckii IFO

L. lactic IO-

R. oryzae NRRL 

Lactobacillus sp. RKY

Fermentation microorganism Temperature:  °C, pH: ., incubation time  h, agitation:  rpm Temperature:  °C, pH: ., incubation time  h, agitation:  rpm Temperature:  °C, pH: ., incubation time  h, agitation:  rpm Temperature:  °C, pH: .–., incubation time  h, agitation:  rpm

Fermentation conditions

[]

[]

. g/L  kg m− from  kg m− of rice bran

[]

[]

Reference

 g/L with productivity of . g/L h . g/L

Lactic acid yield

40 3 Microbial production of lactic acid

3.2 Lactic acid production using organic wastes

41

L. brevis ferments xylose (hetero-fermentation pathway) 1 mol xylose is converted into 1 mol of acetate [18]. L. brevis produced 3.1 g/L of lactate in monoculture. The mixed culture of L. brevis and L. rhamnosus resulted in 0.70 g/g of yield and 0.58 g of productivity using NaOH pretreated corn stover as substrate under simultaneous saccharification and fermentation [37]. B. coagulans LA204 was reported to produce lactic acid under nonsterile condition in a 5 L fermenter at a cellulase dosage of 30FPU per gram stover and 10 g/L yeast extract under simultaneous saccharification and fermentation. The lactic acid titer, yield and average productivity were found 97.59 g/L, 0.68 g/g and 1.63 g/L/h, respectively, at fermentation pH 6.0 and temperature of 50 °C [18]. Kluyveromyces marxianus YKX071, capable to ferment glucose and xylose was studied for lactic acid production at 42 °C. Fed-batch fermentation was performed having 180 g/L of corncob that resulted in 103 g per liter of lactic acid [38].

3.2.2 Rice-washing drainage and rice straw for lactic acid production In rice mills, large amounts of drainage from rice washing are released and treated using costly procedures. This rice washing drainage contains large number of solids composed of starch, protein, and vitamins. It is considered as carbon and a nutrition source for microorganisms. Bacteria able to produce lactic acid was isolated from the rice washing water. A minimum amount of 35 g/L of lactic acid was obtained from rice wastewater and bran mixture. Strain M−23 had capacity of about 83 g lactic acid/L that was maximum compared to other four strains. This strain was Gram-positive curve rod, oxidase positive that produced lactic acid with optical purity of 95% and it was identified as L. rhamnosus JCM1136. At pH 6.5 high lactic acid concentration was achieved (58 g/L) with 48 h of cultivation [16]. World rice cultivation feeds about half of the world’s population and produces more than one billion tons of straw residue per year. Traditionally, rice straw is burnt in the field to recycle nutrients to the soil and avoiding biopathogens for subsequent crops. Most of the national and international organizations have banned open burning of rice straw to prevent biomass loss, carbon dioxide emissions, and smog. In the 1990s, developed countries such as USA and European Union introduced bans. After that, counties such as China, India, Pakistan, Australia, and Southeast Asia also have imposed bans [39]. Straw has many properties such as it contain large number of cellulose and hemicellulose, easily hydrolyzed to reducing sugars that can be converted into lactic acid [40]. L. casei was used for the production of lactic acid using from rice straw under solid-state fermentation. Hydrolysis of pretreated rice straw was performed by cellulase and hydrolysate having reducing sugars was fermented by L. caseii to produce 3.467 g of lactic acid per 5 g pretreated rice straw after 5 days of duration. The temperature of 37 °C, pH 6.5 and moisture content of 72% was maintained [41]. Wheat straw pretreated with 1-ethyl3-methylimidazolium-acetate [EMIM][OAc] has been used to produce lactic acid from L. plantarum SKL-22. A maximum amount of 37.16 g/L of lactic acid was obtained in a single pot bioprocess [42].

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3 Microbial production of lactic acid

3.2.3 Spent coffee grounds (SCG) for lactic acid production According to a report of International Coffee Organization, world’s coffee consumption was more than 9.7 billion kg in 2017–2018. The preparation of grounds and instant coffee produces a large amount of coffee grounds. Large amount of coffee residues is released which causes various issues [43]. SCG consist of 8.6–15.3% cellulose, 31.7–41.7% hemicellulose, 22.2–33.6% lignin, and 21.3–40.7% other materials [44]. Alkali pretreatment was optimized and enzymatic hydrolysis of SCG was carried to release of fermentable sugars. SCG hydrolysate containing sugars was fermented by L. brevis ATCC8287 and L. parabucuneli ATCC49374 to produce lactic acid. The maximum yield of lactic acid was observed 101.2 g per 1000 g of SCG by L. parabuchnri ATCC49374 [14]. Hudeckova et al. [45] also studied the utilization of SCG as substrate for lactic acid production. Dilute acid pretreatment and enzymatic hydrolysis was performed to obtained SCG hydrolysate. Among several microbial strains tested, L. rhamnosus CCM1825 produced maximum amount of lactic acid (25.69 ± 1.45 g/L) with yield of 98% despite of the inhibitory effects of furfural and phenolic compounds in the fermentation medium [45].

3.2.4 Paper sludge for lactic acid production The wastewater treatment facilities of recycled fiber plants generates large amount of paper sludge that causes major environmental pollution problem. The generated paper sludge contains organic cellulose, lignin, and other sugar of 30.6%, 8% 12.1% and 41.6% respectively. The inorganic materials such as Al, Si, Ca, Cu, Fe, Mg and other are also found in the recycled sludge [17, 46]. Due to high polysaccharides content, the paper sludge can be valorize into useful products. This large amount of polysaccharides in sludge needs to be broken down into fermentable sugars [47]. Acid hydrolysis or enzymatic hydrolysis can be used for breakdown of polysaccharides. Enzymatic hydrolysis is considered most suitable method of hydrolysis due its mild reactions conditions and environment-friendly nature. Marques et al. [17] evaluated the performance of recycled paper sludge as substrate for lactic acid production. Maximum lactic acid production was obtained with simultaneous hydrolysis and fermentation of sludge based medium supplemented with MRS components and CaCO3. L. rhamnosus ATCC7469 gave 73 g/L−1 of lactic acid that was equivalent to productivity of 2.9 g/L/h with 0.97 g of lactic acid per g of carbohydrates in the substrate [17]. R. oryzae MTCC5384 was also employed for lactic acid production from paper sludge. After 144 h of simultaneous saccharification and fermentation, 27 g/L of lactic production was achieved using mixture of commercially available and manufactured mixed cellulase with a ratio of 50:50. The temperature of 40 °C, pH six and feed concentration 75 g/L was maintained [46]. B. coagulan strains 36D1 and P4-102B were also used for production of lactic acid. CaCO3 was also added as a buffering agent to avoid pH regulation. A maximum of 80 g/L and 47 g/L of lactic acid was produced by 36D1 and P4-102B strains respectively [23].

3.2 Lactic acid production using organic wastes

43

3.2.5 Sugarcane bagasse and molasses for lactic acid production Globally, sugarcane is cultivated on 20.1 million hectares of land and the processing of sugarcane generates large amount of residues [48]. Brazil, India, China, Pakistan and Thailand are the largest producers of sugarcane, with total output of 1870 million tonnes in 2020 [49]. The processing of each tonne of sugarcane produces 740 kg of juice and 260 kg of moist bagasse [50]. González-Leos et al. [51] studied lactic acid production using sugarcane bagasse as substrate. L. pentosus CECT4023T produced 0.82 g of lactic acid per g of sugarcane bagasse [51]. B. coagulans NCIM5648 was used to produce lactic acid using sugarcane baggases after alkali pretreatment. The NaOH pretreatment of sugarcane bagasse was carried out for 120 min at 80 °C. The productivity, yield, and titer of 2.86 g/L/h, 0.92 g/g, and 68.7 g/L respectively were achieved [52]. Sugarcane molasses is considered a suitable and low-cost source for microbial growth. Sugarcane molasses was fermented by Bacillus amyloliquefaciens J2V2AA to produce 178 g/L/24 h of lactic acid [53].

3.2.6 Lactic acid production using wheat straw With annual production of approximately 529 million tonnes worldwide, wheat straw is considered potential crop residue for the production of value-added products [54]. L. pentosus CECT4023T was been reported to ferment wheat straw hydrolysate to produce lactic acid. When aerobic conditions were changes to anaerobic, especially strictly anaerobic lactic acid production was increased. Therefore, the lower oxygen concentration resulted in the higher yield of lactic acid (0.55 g/g) after 120 h of fermentation from enzymatically hydrolyzed wheat [55].

3.2.7 Production of lactic acid using corn steep liquor Corn milling produce large amount of corn waste. Corn steep wastewater contains large amount of sugars and nutrients such as nitrogen, amino acids, trace elements, and vitamins that encourages microbes growth during fermentation processes [56, 57]. About half of the nitrogen is present in the form of free amino acids. The rest are small peptides while proteins are rarely intact. Vitamins and trace elements are also present in large amount [58]. A high temperature tolerant strain of Enterococcus faecium WH51-1 was selected for lactic acid production. Under optimized conditions a maximum amount of 44.6 g/L of lactic was produced using 60 g/L of corn steep liquor with 10% of inoculum size. Sodium hydroxide or calcium carbonate was used as a neutralizing agent [59]. De Lima et al. [60] studied the production of lactic acid by the fermentation of corn steep liquor and yeast autolysate using Lactobacillus LMI8. The optimal amounts of corn steep liquor (16.48 g/L) and autolytic yeast (4.93 g/L) resulted in lactic acid production of 41.52 g/L [60].

44

3 Microbial production of lactic acid

3.2.8 Production of lactic acid using organic fraction of municipal solid wastes (OFMSW) The world has projected the generation of 2.2 billion tons of municipal solid waste (MSW) per year by 2025 around the world. Improper handling of MSW results in degradation of environment by polluting water, air, and soil. Organic fraction is a major component of MSW that can be valorize into value added products. It has been utilized as substrate for the production of lactic acid [61]. A mixture of D- and L-lactic acid is produced in OFMSW due to natural lactic acid producers that reduces the enantiomeric purity of final product. Therefore, monopolar electrodialysis was carried out to remove unfavorable D- and Lform of lactic acid OFMSW hydrolysate. The OFMSW hydrolysate was fermented by B. Coagulans A166 to produce 61.1 g/L of lactic acid with a yield of 0.94 g/g. The optical purity of final product was estimated 98.7% [62].

3.2.9 Production of lactic acid using agave bagasse Agave is native of Mexico and Central America and propagated in different countries. It is cultivated for food, fiber, animal feed, and alcoholic beverages. The processing of these products generates large amount of residues that have potential to be converted into biofuels and fine chemicals [63]. Agave bagasse have been tested for the production of lactic acid by L. rhamnosus. Agave bagasse was acid hydrolyzed to release the fermentable sugars. The acid hydrolysates were fermented to produce 90.1 mg/g and 67.9 mg/g of lactic acid after HClhydrolysis fermentation and H2SO4-hydrolysis fermentation after 20 h of duration [64].

3.2.10 Production of lactic acid using cheese whey A large volume of whey containing milk sugar is produced by dairy industry worldwide. This whey are can be used for several purposes including lactic acid production. Patel and Parikh [65] performed lactic acid production by L. delbrukii MTCC911 using whey as substrate. Under optimized conditions 29.6 g/L of lactic acid was obtained with an incubation duration of 72 h [65]. The lactic acid production by L. helveticus using whey as feedstock was studied under batch fermentation and a maximum amount of lactic acid (53 g/L) was achieved at optimum temperature and pH of 42 °C and 5.9 respectively [66].

3.3 Applications of lactic acid Lactic acid is an extensively used in a wide range of industries for various purposes. Lactic acid finds several applications in food industry, biomedicines, cosmetic preparation, and chemical industry (Figure 3.2). Lactic acid is polymerized into polylactic acid, a biodegradable and biocompatible polymer [67].

3.3 Applications of lactic acid

45

Figure 3.2: Applications of lactic acid.

3.3.1 Applications of lactic acid in food industry Lactic acid is used as preservative, flavoring agent, and pH regulator in food industry. It also controls the pathogens and improves the shelf life of foods. It prevents bacterial growth to control the food spoilage. Sodium and potassium salts of lactic acid are used for the improvement of shelf life of meat, poultry, and fish. Lactic acid is also used as an acidulant in pickled vegetables, baked goods, salad, and beverages due its mild acidic taste. Lactic acid is used confectionary due to its low inversion rate, ease of handling, and ability to produce clear candies [8, 68].

3.3.2 Applications of lactic acid in pharmaceutical industry Lactic acid is used as an electrolyte in various parenteral or intravenous solutions. It has also been used for preparation of minerals, tablets, prostheses, controlled drug delivery system, surgical sutures, and preparation of dialysis solution for dialysis processes [8].

3.3.3 Polylactic acid (PLA) PLA is a biodegradable polymer with good mechanical properties and it have shown packaging applications. It has been used making of salad containers, cups, candy wraps, and bottles for packaging material in food industry. PLA has been employed for curtains, towels, and apparels manufacturing. It has also been used for the production of flexible film applications such as shopping bags or mulch films [69].

46

3 Microbial production of lactic acid

3.4 Conclusions Organic wastes are attractive sources of sugars that are metabolized by microorganisms to produce lactic acid. Lignocellulosic materials contains cellulose and hemicelluloses that are hydrolyzed into fermentable sugars. Some of organic wastes such as sugarcane pessmud, sugarcane molasses, rice washing drainage, and milk whey have reducing sugars. Therefore, pretreatment and hydrolysis step is not required and they can be fermented directly into lactic acid. Several organic wastes such as corncob, corn stover, corn steep liquor, food waste, rice straw, rice bran, rice washing drainage, recycled paper sludge, office waste paper, sugarcane pressmud, sugarcane bagasse, wheat bran, wheat straw, cheese whey, OFMSW, agave bagasse etc. have employed for the production of lactic acid. The utilization of low cost substrate for lactic acid production decreases overall cost of the product.

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Mayank Raj, Tamanna Devi, Vikas Kumar, Prabhakar Mishra, Sushil Kumar Upadhyay, Mukesh Yadav, Anil Kr Sharma, Nirmala Sehrawat, Sunil Kumar and Manoj Singh*

4 Succinic acid: applications and microbial production using organic wastes as low cost substrates Abstract: Succinic acid is a valuable organic acid with a high commercial value that may be employed in a variety of sectors including food, cosmetics, and chemistry. Through bacterial fermentation, succinic acid can be easily produced. This paper includes a broad body of literature assessment spanning the previous two decades on the evaluation of succinic acid (SA) production procedures in to further drive research toward membranebased sustainable and affordable production. The best natural method of SA producer is through Actinobacillus succinogenes. The process of microbial fermentation is used to produce bio-succinic acid utilizing agro-industrial waste. There are different methods under metabolic engineering which are being frequently used for bio-based succinic acid production using representative microorganisms, such as Mannheimia succiniciproducens, Pichia kudriavzevii, Saccharomyces cerevisiae, Actinobacillus succinogenes, Corynebacterium glutamicum, Basfia succiniciproducens, and Escherichia coli. This review summarizes the evolution of microbial production, fermentative methods, various organic substrates and the effects of efforts to recover and refine components for a wide range of applications in the perspective of biologically produced succinic acid for commercialization state. Keywords: Actinobacillus succinogenes; Aspergillus niger; bio-based; metabolic engineering; succinic acid

*Corresponding author: Manoj Singh, Department of Bio-Sciences & Technology, MMEC, Maharishi Markandeshwar, Deemed to be University, Mullana-Ambala, Haryana 133207, India, E-mail: [email protected]. https://orcid.org/0000-0002-9257-927X Mayank Raj, Tamanna Devi, Sushil Kumar Upadhyay, Mukesh Yadav, Anil Kr Sharma and Nirmala Sehrawat, Department of Bio-Sciences & Technology, MMEC, Maharishi Markandeshwar, Deemed to be University, Mullana-Ambala, Haryana, India. https://orcid.org/0000-0001-8526-3747 (M. Yadav). https:// orcid.org/0000-0002-9768-1644 (A.K. Sharma) Vikas Kumar, Department of Bio-Sciences & Technology, MMEC, Maharishi Markandeshwar, Deemed to be University, Mullana-Ambala, Haryana, India; and Department of Microbiology, International Medical School, UIB, Almaty, Kazakhstan. https://orcid.org/0000-0002-6044-3239 Prabhakar Mishra, Department of Biotechnology, School of Applied Sciences, REVA University, Bengaluru, Karnataka, India Sunil Kumar, Department of Microbiology, Faculty of Biomedical Sciences, Kampala International University, Western Campus, Ishaka, Uganda As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: M. Raj, T. Devi, V. Kumar, P. Mishra, S. K. Upadhyay, M. Yadav, A. K. Sharma, N. Sehrawat, S. Kumar and M. Singh “Succinic acid: applications and microbial production using organic wastes as low cost substrates” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0160 | https://doi.org/10.1515/9783110792584-004

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4 Succinic acid: applications and microbial production using organic wastes

4.1 Introduction Organic acids are chemical substances that occur naturally as regular elements of plant or animal tissues. For many years, organic acids have been used in the food items, chemical, pharmaceutical industries, and agriculture. The presence of carbon, hydrogen and oxygen elements distinguishes organic acids. Succinic acid, citric acid, itaconic acid, lactic acid, gluconic acid, acetic acid, lactobionic acid, propionic acid, and fumaric acid, are the most typical organic acids synthesized by microbial action. To address these shortcomings, microbial production of organic acids was developed, which provides a sustainable and environmentally friendly alternative to chemical processes for future use. Different microbes have been identified as organic acid producers that can be used to produce concerned organic acid in a cost-effective and environmentally friendly manner [1]. SA is an organic acid having a broad spectrum of industrial uses. Succinic acid (also called as butanedioic acid, 1,2-ethanedicarboxylic acid, or amber acid), is a four-carbon dicarboxylic acid that is a tricarboxylic acid cycle intermediate and is often found in atmospheric aerosol samples and C4H6O4 is its chemical formula [2]. Before the invention of fermentation technologies, succinic acid was produced by catalytic hydrogenation of maleic anhydride, a fossil-based chemical. SA is also frequently found in atmospheric aerosol particle samples [3]. Succinic acid has been as one of the 12 sugar-based building blocks because it can subsequently be transformed into new and valuable compounds, and it has been listed as one of the greatest value-added chemicals from biomass by the US Department of Energy [4]. Amber has a long history of usage in Europe as a natural antibacterial and general medicinal. Georgius Agricola was the first to isolate succinic acid from amber by dry distillation, and he recognized three amber fractions: succinic acid, amber oil, and amber colophony in 1546 [5]. Since then, succinic acid has been reported to be used in the food industries, agriculture, and pharmaceutical. SA has long been used in food additives, cosmetics, detergents, toners, pharmaceutical intermediates, pigments, soldering fluxes, and cement additives. Previously, succinic acid had a small market share [6]. It is also utilized as a beginning point in the production of a lot of chemicals, including N-methylpyrrolidinone, adipic acid, 2-pyrrolidinone, maleic anhydride, 1,4-butanediol, succinate salts, g-butyrolactone, and tetrahydrofuran, which are widely employed in the pharmaceutical industry. Butanedioic acid, another major organic acid, is likewise employed as a growth regulator for plants. Among the most recent application of succinic acid is used in the production of Bionelle, a novel biodegradable plastic that is a succinic acid and 1, 4-butanediol ester [7]. Succinic acid production has been discovered in a wide range of microorganisms, including fungi (such as Yarrowia lipolytica, Saccharomyces cerevisiae, Paecilomyces varioti, and Byssochlamys nivea) and bacteria (such as Pichia kudriavzevii, Mannheimia succiniciproducens, Actinobacillus succinogenes, Corynebacterium glutamicum, Basfia succiniciproducens, and Escherichia coli). Recognizing the significance of succinic acid, the authors provide an

4.2 Strategies and methods of succinic acid production

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in-depth examination of succinic acid synthesis strategies, methods and applications in the chapter. Prospects and outlook are also discussed in combining current research and worldwide demand [8].

4.2 Strategies and methods of succinic acid production Succinic acid may be generated in a variety of ways, and the market for succinic acid is predicted to increase by 6.8 % between 2018 and 2023 [9]. Succinic acid was developed from petroleum basis using chemical methods, resulting in higher efficiency costs and limited application areas. Now, succinic acid is created through various chemical techniques such as paraffin oxidation, catalytic hydrogenation, and electro reduction of maleic anhydride [10]. A manganese or calcium catalyst is used in paraffin oxidation technology to create a variety of dicarboxylic acids, distillation, crystallization, and drying techniques to purify generated succinic acid. The purity of the succinic acid produced is insufficient. Catalytic hydrogenation is a maturation process that uses heterogeneous or homogeneous catalysts to create succinic acid with high yield and purity; nevertheless, it is a costly technique that might lead to environmental problems [11]. A homogeneous catalyst is utilized in the same phase since all of the reactants reside in the same matter state. Heterogeneous catalysts could be utilized in a different phase

Figure 4.1: Array of intermediate and end products stemming from succinic acid.

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4 Succinic acid: applications and microbial production using organic wastes

than the reactants, as well as in a distinct matter of state. The hydrogenation of fossilderived maleic acid produces succinic acid and become undesirable due to the high cost and non-renewability of fossil-derived products [12]. Figure 4.1 depicts variable chemical derivatives of succinic acid.

4.2.1 Production of succinic acid by bacteria Many bacteria, including Actinobacillus succinogenes, Anaerobiospirillum succiniciproducens Mannheimia succiniciproducens and Escherichia coli may produce succinic acid. Ruminant rumen is an abundant resource of anaerobic bacteria such as Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, and Bacteroides fragilis which, under anaerobic conditions and in the presence of CO2 creates a mixture of volatile organic acids coupled with SA [13, 14]. In certain cases, a combined culture (fungus and bacterium) of Rhizophus sp. and Enterococcus faecalis produces a high titer of SA (yield, 0.95 g/g, and productivity, 2.2 g/L$h) in a two-step procedure in which the fungus synthesizes fumarate, which further get transferred into succinic acid by the bacteria [15]. Since SA must cross two boundaries (the mitochondrial and cytoplasmic membranes) in order to be excreted in later circumstances. The bacteria are a better option for SA fermentation than fungi or yeast. A. succinogenes, a facultative anaerobic microbe can grow in a variety of carbon sources, including refined sugars, sugarcane or beetroot molasses, starch, fruit juice, corn, sweet sorghum, wheat, barley, fruit and vegetable waste (FVW), citrus peel waste, and lignocellulosic biomass such as cellulose, lignin, and hemicelluloses obtained from agricultural [16]. The production of succinic acid using various organic wastes as substrate involving fermentation strategies have been tabulated in Table 4.1. Table .: Succinic acid production using different carbon sources, microorganisms and methodology. Microorganism

Substrate

Strategy of fermentation

Ref.

A. succinogenes M. succiniciproducens B. succiniciproducen C. crenatum C. glutamincum C. acetoacidophilum Synechocystis sp. PCC  M. succiniciproducens MBELE A. succinogenes Z A. succinogenes (ATCC)

Glucose Whey Sulphite liquor Wheat bran hydrolysate Glucose Glucose NaHCO and CO Glucose Xylose Galactose

Repeat-batch Batch Batch Batch Fed-batch with membrane for cell recycling Anaerobic, fed-batch Anaerobic, batch Anaerobic, continuous fermentation Anaerobic, continuous fermentation Anaerobic, batch

[] [] [] [] [] [] [] [] [] []

4.2 Strategies and methods of succinic acid production

55

When metabolizing glucose, A. succinogenes uses the oxidative pentose phosphate pathway and glycolysis to create phosphoenolpyruvate (PEP). The resultant intermediate PEP is the junction of the succinate-producing C4 pathway and the byproduct-producing C3 pathway, which uses a variety of carbon sources to make byproducts such formate, acetate, and ethanol. To increase the level of succinic acid, NADH, a cofactor is required for the C4 route to produce SA. SA is a major intermediate in the tricarboxylic acid cycle (e.g. A. succinogenes). Using genetic engineering strategies to upgrade metabolic processes, screening naturally strong producing strains, and process optimization can all boost SA production during industrial manufacturing [26]. Several factors such as including the removal of genes associated with by-product formation, pathways involved directly in SA synthesis, improvements in substrate transfer, and the development of effective cofactor metabolism, can be used in genetic manipulation of model strains to increase SA yield. A higher SA yield can also result from optimizing physical parameters like temperature, stirring rate, pH, and duration as well as media elements like concentration of substrate, CO2 expelling rate, and cofactor availability [27]. To create SA with fewer byproducts, a variety of sugars were employed in minimal cost medium with metabolically modified E. coli AFP184. Co-producing long-chain alcohol and SA using lignocellulosic carbon sources has demonstrated significant economic and environmental benefits as because the CO2 generated during alcohol synthesis might be used as a precursor material for SA fermentation. Microbiologically synthesized SA also uses glycerol waste as a substrate [28].

4.2.2 Production of succinic acid by fungi In the past of microbial synthesis of organic acids, Aspergillus niger is well known as a reliable commercial powerhouse for bio-based succinic acid production. It possesses a number of traits that considerably aid in the improvement of biological processes for the manufacture of organic acids that includes effective organic acid secretion, strong feedstock utilization, and high stress tolerance to pH and heavy metals [29]. The best alternative form of manufacturing other organic acids such itaconic acid, malic acid, and lactic acid has therefore been considered through A. niger [30]. In contrast, A. niger attempts to synthesize succinic acid that have met with little success. However, genetic modifications to these processes did not lead to increase carbon fixation for the formation of succinic acid [31]. On the other hand, it is challenging to reroute the carbon to fix from other primary organic acids toward succinic acid using route engineering because the methods of efficient organic acid excretion in A. niger are not fully understood. A further black, Aspergillus carbonarius, that has an organic acid profile quite similar to that of A. niger have been used to discover the limitations behind mechanism to synthesize C4-dicarboxylic acid [32]. Following the discovery and over expression of the biological membrane protein AcDCT (C4-dicarboxylate transporter) in Aspergillus carbonarius, C4-dicarboxylic acid production, including malic acid and succinic acid, increased

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significantly [33]. A. niger was the subject of another recent study that discovered effective malic acid synthesis through the over expression of several genes connected to the cytosolic rTCA. These results show that by maintaining carbon fixation to succinate in the cytosolic route, A. niger could enhance succinic acid production. In order to test this theory, a soluble NADH-dependent fumarate reductase (FRD) was earlier expressed in a naturally occurring malic acid-producing strain of Aspergillus saccharolyticus to A. niger. This process successfully increased succinic acid production [34].

4.3 Optimization of various parameters for succinic acid production 4.3.1 pH pH is amongst the most important variables influencing the formation of succinic acid. The pH controls CO2 availability by affecting its solubility, which in turn controls the enzyme action that produces succinic acid [35]. Although A. succinogenes may grow at a pH range of 6.0–7.4 but 7.0 is the ideal pH for its growth. Low pH values negatively impacts the cell development. In reality, the medium will get more acidic as the fermentation progresses due to the generation of organic acids such as acetic, formic and succinic acids. Therefore, using neutralizing agents is essential in maintaining the pH within the proper range for sustained growth and fermentation [36]. MgCO3 was found to be the most efficient pH controlling reagent for A. succinogenes. According to research, there was no discernible variation in succinic acid synthesis between pH levels 6.0 and 7.2. As a result, MgCO3 could be used in stirred bioreactors for neutralization of pH. Succinic acid synthesis did not improve by adding certain neutralizing agents like NaOH and KOH. Additionally, when pH was controlled with only one neutralizing agent, substantial cell flocculation was seen because of a higher metal ion concentration. Alternative inexpensive alkaline neutralizers including Na2CO3, NaHCO3, and NH3H2O. However, this could be possible only under low carbon source concentrations initially. However, relatively low carbon source ratios made this possible. In order to substitute the costly MgCO3 for industrial succinic acid production, Li et al. created combined alkalis Mg (OH)2 and NaOH as a pH controller [37].

4.3.2 CO2 concentrations According to previous research on rumen bacteria, higher CO2 concentrations were necessary for the commencement and optimal growth of those species of rumen bacteria that produce SA as one of their main end products [38]. According to Xi et al., optimization model analysis on cell cultures for CO2 fixation to produce SA, the accessible CO2 in the

4.4 Metabolic engineering of succinic acid

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fermentation broth is made up of HCO3−, CO32− and CO2 and is regulated by the composition of the media, CO2 partial pressures and temperatures. Under CO2 limiting conditions, succinic acid formation decreased in contrast to higher CO2 concentration [39]. The quantities of dissolved CO2 in the fermentation broth greatly control the metabolic transit of carbon and the action of phosphoenolpyruvate kinase (PEPK), thereby encouraging the flow of carbon towards the SA generation of the TCA cycle. As a result, A. succinogenes produces higher succinate and much less ethanol, acetate, and formate in response to high CO2 concentrations [40]. Theoretically, SA can be synthesized in the presence of CO2 in following way: Glucose + 2CO2 + 4H+ → 2Succinate + 2H2 O

4.3.3 Redox potential Few researches that looked at how redox potential affected A. succinogenes and its ability to produce SA were originally explained. These were supplying hydrogen, using carbon sources with varying levels of oxidation and supplying electrons through electricity [41]. According to Li et al. employing carbon sources with various degrees of oxidation, varied amounts of NADH accessibility and the NADH/NAD+ proportion can be obtained. Redox potential regulation may result in a shortened fermentation cycle, increased SA production and optimal metabolite distribution. A. succinogenes NJ113 appeared to produce fermentative SA at a redox potential of 350 mV, when redox potential values in the range of 100–450 mV were tested [42].

4.4 Metabolic engineering of succinic acid There are many methodologies for SA metabolism in traditional industry where biocatalysts have been effectively. As an illustration, E. coli has undergone substantial engineering to optimize SA biosynthesis by enhanced substrate transport, higher carbon flux and elimination of by-product synthesis [43]. Number of other hosts, such as S. cerevisiae and Corynebacterium glutamicum have also used with similar tactics. Succinate dehydrogenase, which catalyses the conversion of SA to fumarate, must be knocked out or inactivate hosts that encode a classical tricarboxylic acid (TCA) cycle in order to stop the oxidative pathway TCA cycle [44]. Additionally, these modified strains frequently use dual-phase aeration (aerobic/anaerobic) to generate high titer fermentations. An appealing approach for different framework has been provided for the development and application of anaerobic strains with high native permeability for SA, such as A. succinogenes strain 130 Z and A. succinogenes in particular. They are facultative anaerobes that can form biofilms as well as has the ability to ferment variety of carbon sources into SA as a major byproduct. Thus, they produce highest and recorded amount of

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SA yield [45]. Prior research on the organisms has pinpointed its essential metabolic and succinic acid biosynthesis pathways and enhanced fermentation engineering techniques by utilizing its distinctive partial TCA cycle [46]. A. succinogenes mutants that lack pyruvate-formate lyase (PFL) completely lack the ability to synthesize formate. Moreover, till date, there have been very few reports of impactful metabolic engineering techniques with A. succinogenes, because of the organism’s limited adaptability; as a result, mechanisms governing flux to SA are still not fully understood. Recognition and transformation of additional strain which aims to offer improved succinic acid production are under research [47].

4.5 Production of succinic acid utilizing low cost of raw materials (agro-industrial waste) Currently, maleic anhydride is used as the primary raw material in the industrial process that produces succinic acid, which costs $1.03/kg. Succinic acid manufacturing from petrochemical feed stocks is very expensive and has significant pollution issues. More work is being done nowadays to produce succinic acid through microbial fermentation utilizing renewable polysaccharides [48].

4.5.1 From cane molasses A byproduct of the sugarcane industry, cane molasses contains water, roughly 50 % (w/w) total sugars, suspended dispersions or colloidal, heavy metals, micronutrients and nitrogen-containing compounds, etc. After the pretreatment of molasses, nitrogen sources and initial sugar concentration were optimized by A. succinogenes. A succinic acid concentration of 50.6 gL−1 with an accumulation of 79.5 % is acquired in anaerobic bottles fermentation and the sugar utilization. The succinic acid content and the productivity in the fed batch fermentation in bioreactor reaches up to 55.2 gL−1 [49].

4.5.2 Fermentative production of succinic acid from straw hydrolysate A. succinogenes CGMCC 1593S a succinic acid producing bacteria has been studied that uses maize straw, corn core, rice straw, and wheat straw as carbon source. The amount of sugar content around 45 gL−1 was used for batch fermentation process with a variety of hydrolysate-based medium in anaerobic fermentation bottles. Corn straw hydrolysate and corn core hydrolysate produced 33.7 gL−1 and 32.1 gL−1 succinic acid respectively after 48 h of fermentation [50].

4.5 Production of succinic acid utilizing low cost of raw materials (agro-industrial waste)

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4.5.3 From vine shoots and surplus grape must A. succinogenes and Basfia succiniproducens were evaluated as potential feed stocks for the manufacture of succinic acid from vine shoots and extra grape must. Vine shoots hydrolyzed by enzymes and acid produced 35–40 g/L of total sugars. From this hydrolysate, both types of bacteria produced 18–21 g/L of succinic acid in 120 h. A. succinogenes was found to be very effective strain using grape must as a substrate than B. succiniproducens. A. succinogenes. That does not require any additional nutrients other than yeast extract as a source of organic nitrogen and vitamins. A. succinogenes produced 88.9 g/L of succinic acid after 96 h of ideal conditions [51].

4.5.4 Conversion of crop stalk wastes into succinic acid production The effect of various carbon sources involving A. succinogenes BE-1’s as microbial source for generation of succinic acid using waste feedstock were examined for batch fermentations. Crop stalk wastes, such as cotton and corn stalks were used as carbohydrate-rich feedstock that were enzymatic ally converted for yielding maximum glucose concentrations of around 65–80 %. The generation of succinic acid for the anaerobic batch culture using cotton stalk hydrolysates was 15.8 gL−1, with a good yield of 1.23 g glucose. Glucose and xylose were used simultaneously, but cellubiose wasn’t used until the other two sugars had been used up completely [52].

4.5.5 Production of succinic acid from pineapple peel waste Pineapple has long been one of the most profitable crops in 48 tropical countries. The core region of pineapple being used as waste as it contains 17.2 % 63 (w/v) cellulose, 12.3 % (w/v) hemicellulose, and 1.8 % (w/v) lignin, whereas the peel section contains 64 22.9 % (w/v) cellulose, 13.9 % (w/v) hemicellulose, and 5.1 % (w/v) lignin 65 [53]. Major sugars identified in pineapple peel were glucose, xylose, fructose, arabinose, and galactose in varying amounts depending on hydrolysis. In other reports, pineapple waste from a canning plant has been used as a substrate for succinic acid fermentation. The sugar content of the liquid waste was 40.23 g/L sucrose, 88.26 g/L glucose, and 40.27 g/L fructose. Under aerobic conditions, E. coli AFP 184 produced 6.26 g/L of succinic acid from liquid pineapple wastes [54]. Ferone et al. did another investigation on the liquid waste from a pineapple juice manufacturing factory. As carbon sources for succinic acid fermentation using pineapple juice approximately contain 34 g/L glucose, 12 g/L fructose, and 10 g/L sucrose. A. succinogenes generated 38 g/L of succinic acid after 400 h of anaerobic fermentation. Dessie et al. studied the feasibility of solid fruit and vegetable as substrates for succinic acid bioproduction using different material [55].

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4.6 Recovery and purification of succinic acid Succinic acid recovery and purification from fermentation broth may be a laborious and multi-step process. Knowledge of purification processes and the required purity of the finished product both had a role in the inputs chosen for the biosynthesis of succinic acid [56]. Each of the current succinic acid firms created and licensed their own extraction and purification systems due to the costs connected with the procedures. Several articles have been written about the recovery of succinic acid from various microbiological sources [57]. In Figure 4.2 various methods for succinic acid retrieval in form of schematic flow charts has been described. There are often no rigid requirements for succinic acid purification. The process of purification frequently entails several distinct steps, including the neutralization of salts, ion exchange, ion exchange, filtering, electro dialysis, acidification, and crystallization. Converting salt to acid, eliminating cellular and protein-like impurities, and refining the free acid to the necessary purity are the main technological hurdles encountered during purification [58].

4.6.1 Filtration/ultra-filtration In order to remove succinic acid from fermentation broth, ultra-filtration is frequently being used. Unwanted proteins and cell debris are taken out of the fermentation broth

Figure 4.2: Recovery and purification process for succinic acid.

4.6 Recovery and purification of succinic acid

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using a bypass cross-flow hollow-fiber ultra-filtering system. The succinic acid fermentation broth was cleaned using ultra filtration as the first operational component of a succinic acid bio-refinery. Optical analysis of the fermentation broth following these two treatments showed that the ultra-filtered broth is significantly clearer than the centrifuged soup. Several colorants were accumulated on the surface of the membrane after ultra-filtration. This implies that ultra-filtration involves chemical adsorption between both the membrane and small molecules in the broth in addition to physical filtration, which is governed by pore diameter and particle size in the broth. Therefore, super filtration might produce a broth that is cleaner [59].

4.6.2 Crystallization The simultaneous fermentation and crystallization of the calcium salt accompanying the insertion of calcium hydroxide to the aqueous fermentation broth is the basis for the recovery of succinic acid. Additionally, these crystals are recovered by a straightforward filtration process, and protein as well as other debris is eliminated through washing. The crystal splits into soluble succinic acid solution and insoluble calcium sulfate molecules when strong sulfuric acid is introduced to it. Filtration is used to recover succinic acid, and then acid and base ion converters are used to further refine it [60].

4.6.3 Selective extraction In addition, succinic acid is purified using this method. The two most popular kinds of selective extraction are amine-based extraction and acid–base pair extraction. The two main extraction techniques are the creation of ion pairs and hydrogen bonds. Aminebased extraction is a feasible and economical method for isolating succinic acid from fermentation broth because it advances quickly and operates at normal temperatures and pressures [61].

4.6.4 Ion exchange and sorption A well-known technique for succinic acid’s ultimate purification is ion exchange. This method has been used to extract succinic acid in a number of studies. This paper aims to provide a comprehensive and organized overview of the application of adsorbent and ion exchange techniques in the food industry. This includes a thorough treatment of the various kinetic model, thermodynamics, and equilibrium models that are frequently used to describe adsorption and ion exchange events in various systems, as well as the characterization of the primary adsorbent and ion exchange components permitted for food application, their preparation, and a background of this technology [62].

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4.6.5 Electro dialysis It is one of the greatest methods for recovering succinic acid. By moving ions via semipermeable membranes while being affected by a direct electric current, this is a rapid approach to alter the composition and/or amount of electrolytes in a solution. Electro dialysis is an engineering technology with more commercialization potential and environmentally acceptable succinic acid purification procedures, as per Zeikus et al. [63] and Glassner and Datta [64]. This process for making succinic acid is affordable and simple to scale up for commercial use. Due to the membranes’ inability to handle divalent cations, this method cannot acidify and purify fermentations that have been neutralized with magnesium or calcium hydroxide.

4.7 Applications of succinic acid Succinic acid is a fascinating core component for the industry because it may be used to make a wide range of derivatives with diverse applications in all facets of life [65]. Succinic acid was made from petroleum to supply a comparatively small market for use in food and medicine. The construction of bio-based succinic acid manufacturing techniques was motivated by the possible use of succinic acid as a starting feedstock for the synthesis of industrial chemicals and consumer goods with large markets [66]. Additionally, succinic acid and its derivatives have been used in the leather industry to improve water proofing and wet strength, as well as in metallurgy to improve the foamy floating of certain ores. Succinic acid manufacture has gained popularity as a green feedstock for the production of synthetic resins and biodegradable polymers like polybutylene succinate (PBS) and polyamides [67]. It also functions in paints as an emulsion dispersing agent. Succinic acid’s most recent use was to make bionelle, a brandnew biodegradable polymer that is an ester of succinic acid and 1, 4-butanediol (BDO). 1, 4-Butanediol is the most popular bio-based succinic acid. A flexible industrial chemical is 1, 4-butanediol (BDO). It can also be combined with succinic acid to create polybutylene succinate (PBS) and poly (butylene succinate-co-butylene terephthalate), two biodegradable plastics with a wide range of uses. It can be used as a solvent starting material for a variety of other significant industrial chemicals, including tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP), gamma-butyrolactone (GBL) [68]. The production of the succinic acid in recent use is particularly associated with aqueous media catalysis, the creation of catalysts for the transformation of succinic acid to 1, 4-butanediol (BDO), gamma-butyrolactone (GBL), and tetrahydrofuran (THF) as the subject of their interest and active research. Additionally, catalysts for the transformation of succinic acid into pyrrolidones have been carefully studied [69]. The majority of the time, 1, 4-butanediol synthesis involves high-value rare metal-containing catalysts. A California-based startup called Genomatica realized this and directly altered E. coli to produce 1, 4-butanediol

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(BDO). 1, 4-Butanediol (BDO) is much less acidic than succinic acid, necessitates a distinct recovery process, and has a comparatively lower demand during fermentation. Given that it does not require expensive hydrogenation catalysts that are susceptible to impurities, it might have a reduced purity requirement [70]. Contrary to the strong theoretical results seen for anaerobic manufacture of succinic acid, the biological generation of 1, 4-butanediol (BDO) has a higher need for biological reducing equivalents. The present bio-based succinic acid producers are actively developing technology for the creation of other potential succinic acid-based products [71]. Succinic acid and its derivatives have also been used to improve water repellency and wet strength in the leather industry, as well as in metallurgy to improve the froth floating of certain ores. Succinic acid synthesis has grown in popularity as a green feedstock for the manufacturing of synthetic resins and biodegradable polymers such as PBS and polyamides. It is also used in paints as an emulsion coalescing agent [72]. The most recent application of succinic acid is in the creation of bionelle, a novel biodegradable plastic that is an ester of succinic acid and BDO. Showa Highpolymer Co., Ltd. manufactured this (Tokyo, Japan). Succinic acid possible applications based on commodity and specialist chemicals are depicted.

4.8 Conclusion and future prospective Succinic acid has evolved as a particularly intriguing bio-based product that has given its applications and status as a component in many different products. This is crucial for the success of bio-refineries in the future. There are numerous different fermentation processes that have been developed for succinic acid production. Through metabolic pathway investigation and optimization, efforts to increase and enhance the efficiency of succinic acid production have so far been highly successful. However, chemical synthesis continues to be more competitive economically. A range of engineering techniques and computer analytical tools can be utilised to further examine metabolic systems and reactions, such as significant enzymes, intracellular balance, and production limitations. The utilization of low-cost raw materials, direct CO2 consumption, microbial co-cultivation, and process optimization are only a few of the factors that have a major impact on manufacturing cost reduction. Further, it can be utilized as starting materials in multiple applications, including food where it can be used primarily as an acidity regulator in the food and beverage industry and it also add flavors to the food. It has a wide role in chemical, cosmetic, and pharmaceutical industries owing to their various functional properties. Acknowledgment: Authors (MR, TD, VK, PM, SKU, MY,AKS, NS, SK, MS) acknowledge the help and support by Head, Department of Bio-sciences and Technology, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, India.

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Masrat Mohmad, Nivedita Agnihotri* and Vikas Kumar

5 Fumaric acid: fermentative production, applications and future perspectives Abstract: The rising prices of petroleum-based chemicals and the growing apprehension about food safety and dairy supplements have reignited interest in fermentation process to produce fumaric acid. This article reviews the main issues associated with industrial production of fumaric acid. Different approaches such as strain modulation, morphological control, selection of substrate and fermentative separation have been addressed and discussed followed by their potential towards production of fumaric acid at industrial scale is highlighted. The employment of biodegradable wastes as substrates for the microorganisms involved in fumaric acid synthesis has opened an economic and green route for production of the later on a commercial scale. Additionally, the commercial potential and technological approaches to the augmented fumaric acid derivatives have been discussed. Conclusion of the current review reveals future possibilities for microbial fumaric acid synthesis. Keywords: downstream process; fermentation; fumarates; fumaric acid; genetic engineering; Rhizopus oryzae.

5.1 Introduction Fumaric acid (FA), a naturally occurring tetra-carbon unsaturated organic acid, is also known by the names lichenic acid, tumaric acid, allomaleic acid and boletic acid. The acid was isolated for the first time from a pink-coloured flower bearing plant, Fumaria officinalis, a herb belonging to the family Papaveraceae. It flourishes during the months of April to October in the northern hemisphere, and the plant is considered to be the prime fount of fumaric acid [1–5]. A meagerly soluble white solid in aqueous medium, fumaric acid is a trans-isomer of maleic acid. Chemically it is (E)-2-butenedioic acid or trans-1,2-ethylenedicarboxylic acid (Figure 5.1) bearing a double bond at α, β positions and two carboxylic groups trans to each other. Owing to the presence of two carboxylic groups and olefinic bond between α and β carbon atoms, FA is amenable to chemical

*Corresponding author: Nivedita Agnihotri, Department of Chemistry, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, India, E-mail: [email protected]. https:// orcid.org/0000-0002-3588-5852 Masrat Mohmad, Department of Chemistry, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, India Vikas Kumar, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala 133207, India. https://orcid.org/0000-0002-6044-3239 As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: M. Mohmad, N. Agnihotri and V. Kumar “Fumaric acid: fermentative production, applications and future perspectives” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0161 | https://doi.org/10.1515/9783110792584-005

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modifications and has, therefore, been reappraised with pioneering outlooks in its production and utility domain [6]. In the year 2004, the U.S. Department of Energy (DOE) recognized fumaric acid as one of the “top 12” building-block chemicals due to its application as a significant intermediate in variable fields of chemical food, resin synthesis industries and other areas [7, 8]. Fumaric acid is an indispensable speciality organic acid that has a wide range of applications in numerous industries. Since 1946, fumaric acid has been used as a food and beverage acidulant. Currently, it is an ingredient of a wide variety of foods, such as maize and wheat tortillas, chilled biscuit doughs, rye breads, gelling agents and beverages. Fumaric acid, according to research, enhances food quality while lowering food expenditures. It is also added to the feed to increase its efficacy. Methane emissions are significantly reduced (by up to 70%) as a result of its use as a supplement in cattle feed. Fumaric acid is mostly used to manufacture a variety of resins, all of which have promising prospects for future market expansion. In addition to this, fumaric acid esters show promising medicinal properties [3, 9]. Furthermore, the emerging novel experimental evidence on the efficiency and safety of using FA and its ester derivatives [10] in manifold fields has opened new avenues for this dexterous scaffold chemical. Keeping in view, the aforestated remarkable applications and characteristics of fumaric acid, attentiveness ought to be given to its effective manufacturing. Due to rapid rise in petroleum prices, the use of maleic anhydride (a petroleum derivative) for the production of fumaric acid being non-economical and cost-ineffective is avoided. Out of newer and economic strategies employed for production, fermentation [11, 12] has received an outstanding and eye-catching attention and has been extensively studied as an operation for its production. The well-known capacity of production of organic acids by fungi qualifies them as successful candidates to be employed in fumaric acid production techniques. The current article emphasizes on the development of fermentation as an advantageous manufacturing process for acid’s production. Various factors governing ideal manufacturing of FA, metabolic routes for fumaric acid formation, application aspects of the acid and its future prospects will be described after outlining fermentation as a process for fumaric acid synthesis.

5.2 General properties of fumaric acid Chemically known by the names trans-1, 2-ethylenedicarboxylic acid and (E)2-butanedioic acid, fumaric acid has the molecular formula C4H4O4 with molecular

Figure 5.1: Structure of trans-1, 2-ethylenedicarboxylic acid.

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mass 116.07 g mol−1 and consists of two acid carboxyl groups and an olefinic bond. It is colourless, tasteless pure white crystalline solid with a melting point 560 K and density 1.635 g cm−3. When carefully heated to 200 °C, the acid creates traces of maleic anhydride as the compound like fumaric anhydride does not exist. In cold water, fumaric acid is only slightly soluble having solubility less than 10 g l−1. The solubility of different fumarate salts in water varies. Calcium fumarate dissolves poorly in water with a meagre 21.1 g l−1 solubility at 30 °C, while sodium fumarate has a greater solubility of 220 g l−1 and is substantially more soluble at the same temperature (Table 5.1). It is also soluble in organic solvents [10]. The associated physical and chemical properties of the acid are as given in Table 5.1.

5.3 Fermentative production of fumaric acid Fumaric acid is manufactured chemically from maleic anhydride, which in turn is prepared from butane or benzene by oxidation, catalyzed by vanadyl pyrophosphate [3, 12, 13] (Figure 5.2). However, due to rapid rise in petroleum prices, the use of maleic anhydride (a petroleum derivative) for the production of fumaric acid is avoided being Table .: Some of the general properties of fumaric acid []. S. No.

Property

Assigned value

. . . . . . . .

Molecular formula Molar mass Density Crystal geometry Electric dipole moment Melting point Boiling point Solubility

. . . . . . .

Thermal capacity Standard enthalpy of catalytic hydrogenation Calorific value Heat of sublimation Heat of formation Gibbs-free energy pKa values

CHO . g mol− . g cm− Monoclinic, prismatic .  K  K Ethyl alcohol (%) = . (. K) Trichloromethane = . g l− ( K) Tetrachloromethane = . mg ml− ( K) Diethyl ether = . mg ml− at  K Propan--one =  g ml− (. K) -Butenenitrile = . mg ml− ( K) Water = . mg ml− ( K) . kJ mol− −. kJ mol− −. kJ mol− . kJ mol− −. kJ mol− −. kJ mol− . and .

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Figure 5.2: Schematic representation of fumaric acid synthesis in petrochemical industries.

non-economical and cost-ineffective. In addition to this, the process is accompanied by the evolution of toxic and green-house gases like CO and CO2, further hindering its utilization [12]. Owing to the stated difficulties associated with production of fumaric acid, there has been considerable interest in developing and designing the costeffective and ecofriendly strategies for its production.

5.3.1 Fermentation as a green and economic alternative for fumaric acid production Dating back to 1940s, fermentation was used as a key methodology to produce fumaric acid in the US [14]. However, this methodology was discontinued and substituted by synthetic and chemical-based routes using maleic anhydride as the basis for its production. Due to the ever increasing cost of petroleum-based chemicals and the escalating interest about food safety and dairy supplements, interest in fermentative production of fumaric acid has been rejuvenated and fortified [15]. Overview of the steps involved in the fermentative process can be shown with the help of schemes (Figure 5.3).

Figure 5.3: Fermentative production of fumaric acid.

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5.3.2 Potential fumaric acid producing microorganisms In the world of microorganisms, fungi are well known for their potential of producing organic acids and have hence been employed for FA production as well. Keeping in view the fact, researchers, time to time, have done thorough investigations for the purpose of identification of the best species for the same. During 90s, scientists studied fumaric acid producing capability of Rhizopus nigricans, R. oryzae and R. formosa [16–18, 48]. Similarly, Jiménez-Quero and Pollet [19] selected eight different genera of the order Mucorales and tested 41 strains to identify high yielding fumarate strains. In continuation, they concluded with Rhizopus, Circinella, Cunning hamella and Mucor as powerful fumaric acid producing genera of which only a few had the capacity to synthesize the acid in considerable amount. After an assiduous and long research, it was observed that the species that could be considered for the production of fumaric acid at industrial scale comes under the genus Rhizopus including R. arrhizus, R. nigricans,, R. formosa and R. oryzae. The literature regarding Rhizopus species assisted fumaric acid production can be tabulated as follows (Table 5.2). During 1970s and 1980s, Ling et al., reported R. arrhizus as the key producer of fumaric acid with highest product concentration being 121 g l−1 [27]. However, at the same time, it is noteworthy to mention that the aforesaid species came out with a limited yield (0.37 g l−1) and was also less economic in terms of the nutrient requirements of the strain. In contrast to this, using R. oryzae, the nutrient requirements are met at lower costs and hence 1990s onwards, R. oryzae was commonly used for fumaric acid production with highest productivity of 4.25 g l−1h−1 was achieved in this way. It is important to note that all the strains of R. oryzae are not potent fumaric acid producers and keeping the same in consideration, Abbe et al. [28] (2003) categorized R. oryzae strains into two – type I producing only FA with little or no lactic acid and type II producing lactic acid in major proportion while fumaric acid is produced in negligible amount by this strain. Thus it can be inferred that among various species of genus Table .: List of fungi involved in fumaric acid production with type of fermenter and substrate. Fungi

Type of fermenter

Substrate

Rhizopus oryzae

Bubble column Air lift Stirred tank Shake flask Fluidized bed Shake flask Shake flask Stirred tank Shake flask

CHO CHO Manure from dairy Starch from corn Molasses CHO Xylose (CHO) CHO Cassava

Rhizopus nigricans

Rhizopus formosa

Production (g l−)

References

. .    . .  .

[] [] [] [] [] [] [] [] []

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Rhizopus, R. oryzae and R. arrhizus can be highlighted as potent fumaric acid producers. Two species of Aspergillus (A. niger and A. flavus) [29] also were noticed to possess the capacity of producing fumaric acid as a fermentation product in low concentrations. However, these species have so far not been used for the commercial production.

5.3.3 Optimum conditions for fumaric acid production The maintenance and setting of optimum conditions is an essential criterion to increase the productivity and yield, both being an important consideration of any industrial process. Similarly, fumaric acid production by aerobic microorganisms (Rhizopus) following the process of fermentation is governed by the variables comprising pH maintenance by neutralizing agents and oxygen control. 5.3.3.1 pH regulation and role of neutralizing agents pH plays a substantial part in regulation of overall activities of microorganisms and, hence, an important condition to be taken into consideration during the fermentative production of FA [30]. In 2011, Roa Engel [31] reported that in Rhizopus-controlled FA production, pH declines from 5.0 to 2.0 during initial 20 hours in absence of any neutralizing agent. The highly acidic environment created had an inhibitory impact on the growth of microorganisms and, hence, on the overall fumaric acid production. In order to maintain ambient pH for the survival of microorganisms, use of neutralizing agents became inevitable. In this context, a number of neutralizing agents have been put to use, which includes bicarbonates, carbonates and hydroxides of sodium and calcium. However, from an exhaustive research, it has been found that calcium carbonate is maximum effective and extensively used neutralizing agent in organic acid producing industries. This property of calcium carbonate has been attributed to the lesser dissolution of calcium fumarate formed during process, the later switching all the involved equilibria during the course of metabolism to production of fumaric acid, hence lowering down byproducts concentration. Additionally, during course of FA production, CaCO3 serves as a source for CO2 production for formation of oxaloacetic acid (first substance formed during Kreb’s cycle) along with its role as a neutralizer [32–34]. Owing to the lower solubility of calcium fumarate (2.1%), its use during the process has been found to be less economic. Another flaw associated with the lower solubility of calcium fumarate is that the product gets precipitated leading to the formation of highly viscous mass, which may result in stoppage of the process due to failure in oxygen transfer. Also, to recuperate calcium fumarate from broth culture, mineral acids like HCl and H2SO4 are required resulting in the formation of sulphate and chloride salts of calcium, which may act as the sources of environmental pollution during fumaric acid recovery [9]. An attempt has been made to replace CaCO3 with

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some other neutralizing agents, which include NaHCO3, Na2CO3 and (NH4)2CO3; however, in all the cases, the production was low in comparison to using CaCO3 in the process [35–37]. This is observed that the use of sodium and ammonium salts for fumaric acid production generates sodium fumarate and ammonium fumarate, respectively, both having higher solubility than calcium fumarate. The resultant fermentation broth can be employed to produce L-maleic acid and L-aspartic acid without performing the recovery step [35]. Another advantage of using the mentioned salts is that the Rhizopus cells can be regenerated to be used again. The waste mycelia can be utilized as animal feed. The above said merits of the non-calcium salts during the fermentation process, thus, qualify their use for FA production [38, 39]. As is evident from the performed studies, fumaric acid production by the process of fermentation is best achieved at neutral pH values resulting in the consumption of higher amounts of alkalis, hence adding to the overall costs of production. Therefore, the cost effectiveness of the manufacturing process would have been increased by conducting the later at a lower pH value. However, FA production by Rhizopus species is hindered by a lower pH. Various strategies have been analyzed in order to enhance acid production at lower pH [40]. In 2011, Rao Engel et al. while studying a fermentation process mediated by R. oryzae switched off pH control during the late hours (90 h) and allowed the pH to reach to a value of 3.6 during the process [41]. Under these set of conditions, the fermentation process carried out for a time duration of 160 h resulted in a production of 20 g l−1 concentration of fumaric acid. Similarly, by regulating CO2 volume percentage at 10%, they obtained 19.84 g l−1 concentration of FA at low pH of 3.5. Although a sound research has been carried out regarding the effect of pH on the active role of Rhizopus species during fermentative production of FA, yet mechanism of acid tolerance and the impact of lower pH conditions is undifferentiated, and at a stage of infancy, an additional study is demanded in this direction. 5.3.3.2 Effect of oxygen concentration on Rhizopus species mediated fumaric acid production Being aerobic, Rhizopus species need a steady supply of oxygen during fermentation. A significant impact is observed on rate of production of FA and byproducts upon varying the concentration of oxygen. Studies have been carried out under oxygen deficient as well as in oxygen rich environment. It has been observed that under limited supply of oxygen, production of ethanol may increase during the course of metabolic flow. At the same time under oxygen-rich conditions, surplus oxygen may activate Kreb’s cycle leading to an increased cellular growth and metabolism. Furthermore, during the production of fumaric acid, the influence of dissolved oxygen (DO) has been examined thoroughly. A fermentation approach was developed [20] that limited the DO levels between 30 and 80% to promote the fumaric acid synthesis. By employing this methodology, they were able to procure R. arrhizus (NRRL 1526) that produced 130 g l−1 fumaric acid in a

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time period of 142 h. However, this procedure resulted in the production of pretty good byproducts comprising succinic acid, L-maleic acid and α-ketoglutaric acid. In 2010, Fu and co-workers proposed a two-stage dissolved oxygen control technique by putting into use a 5-L stirred reactor with DO concentration maintained at 80% which in turn was switched to 30% following 18 h of cultivation. The said study was carried out on R. oryzae (ME-F512) under various constant dissolved oxygen concentrations. During this course of time, the productivity of fumaric acid (0.7 g l−1) displayed an increment of 37, 21 and 9% in contrast with fumaric acid strength maintained consistent, respectively, at 80, 60 and 30%. Same time, a decline in concentration of ethanol (7.4 g l−1) by 66% was observed in comparison to the strength of DO maintained at 30% (15.2 g l−1) [31, 42]. Zhou (1999) [43] developed a method of pressure pulsation to improve the oxygen transfer from gaseous to liquid-phase during FA synthesis in 2-L reactor fermenter with a stirred tank. After 35 h of fermentation, yields (mass and volumetric) were determined by periodically closing and opening up of the valve on exit to raise or lower the pressure inside the fermentor. It was observed that mass yield and fumarate productivity in terms of volume was 70.1% and 0.99 g l−1 h−1, respectively, greater than those without pressure pulsation. The investigations and studies related to the role of DO in fumaric acid production have been carried out exhaustively at laboratory level. Therefore, a vent ought to be given to carry such investigations at pilot scale [44]. Additionally, a relationship between the two has to be explored and the emphasis on the production of FA at low-cost with high yield has to be layed down.

5.4 Biochemical aspects of fumaric acid Apart from being an illustrious intermediate of Kreb’s cycle occurring in the mitochondria of cells, Rhizopus species utilizes fumaric acid during a diverse range of pathways. During the fixation of carbon dioxide by C1 + C3 mechanistic pathways in the reductive phase of tricarboxylic acid (TCA) cycle, fumaric acid is a dominant product. Enzyme activity and C13 NMR studies performed by Keanly et al. and Peleg et al. further supported this mechanism [45, 46]. An enhanced rate of fixation of CO2 during the reductive phase of TCA cycle would aid in the paramount production of fumaric acid, theoretical yield being 200%, which means for every mole of glucose consumed, 2 mol of fumaric acid are produced. However, due to the release of energy as ATP or GTP along with the elimination of reducing powers (NADH2 and FADH2) during oxidative part of Kreb’s cycle, the experimental yields are relatively lower [45, 47, 48]. The energy currencies and reducing powers produced are essential for maintenance of cells and transport of the acid. Furthermore, it has been recently (2012) proposed by Yu and co-workers [49] that in addition to Embden–Meyerhof–Parnas (EMP) pathway and Kreb’s cycle (oxidative as well as reductive) both of which constitute the central carbon metabolism, alternate pathways may be involved in FA production. During their studies on R. oryzae, both

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wild and mutant strains, they examined that in both the strains other pathways comprising fatty acid and amino acid metabolism were involved during the course of synthesis of fumaric acid. It was further observed that the ratio of unsaturated to the saturated fatty acids was higher in mutant strains. For example, the level of palmitoleic acid and oleic acid was found to be higher in case of mutant strain and at the same time the ratio of saturated fatty acids like stearic acid, heptadecanoic acid, palmitic acid and arachidic acid was found to be more in parental strains. The primary step of fumaric acid production involves conversion of pyruvic acid to oxaloacetic acid by the addition of carbon dioxide in presence of ATP. This step is catalyzed by the enzyme pyruvate carboxylase. The four carbon compound, oxaloacetic acid, thus formed is converted into malic acid by malate dehydrogenase and malic acid is then converted into FA by fumarase. The functioning of different enzymes during fumaric acid synthesis was further explored in several FA producing fungal strains where process of conversion of fumaric acid to L-malic acid is irreversibly catalyzed by the fumarase present both in cytoplasm as well as mitochondrion of S. cerevisiae [50]; however, in R. oryzae, hydration of fumaric acid is reversibly catalyzed by fumarase. Earlier it was taken into consideration that the fumarase of the aforementioned fungi are coded by the same gene and difference in the process and mechanism of the reactions catalyzed by the enzyme results due to changes after the process of translation. Further investigation and extensive studies in this direction have revealed that in case of R. oryzae, there exists two separate genes for the purpose of encoding of the two fumarases (mitochondrial and cytoplasmic one) [51, 52]. The cytosolic fumarase catalyzes the conversion of fumaric acid to L-malic acid while the mitochondrial fumarase catalyzes the reverse process. Ding et al., have reported that the concentration of nitrogen has an evident impact on the functioning of cytosolic fumarase [53]. They observed an increase in the activity of cytoplasmic fumarase by 300% with decreasing urea concentration from 2 to 0.1 g l−1, thereby enhancing FA production from 14.4 g l−1 to 40.3 g l−1. Time to time studies performed in this direction have revealed that one of the essential factors governing the accumulation of fumaric acid is the ratio of nitrogen to oxygen in the cells.

5.5 Key strategies to accelerate fumaric acid production Aerobic fermentation employing Rhizopus spp. is put into application for the manufacturing of fumaric acid from inexhaustible resources. However, it has been found that the existing fermentation procedures suffer from several shortcomings in terms of yield, formation of byproducts and technological hurdles as well. In order to overcome the obstacles faced and for the production of sufficient amounts of FA on an industrial scale, following key strategies can be followed [10].

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5.5.1 Gene modification in microbial strains Building genetically modified strains has been suggested as a method of producing fumaric acid increasing its production. Saccharomyces cerevisiae was created as a mutant by Kacliková et al. in 1992 that lacked mitochondrial fumarase. Fumaric acid gathered during growth on glucose medium [54]. Under ideal circumstances, however, just 12% glucose was converted into FA with less than 0.5 g l−1 concentration of resulting substance. To investigate pyruvate carboxylase effects on fumarate synthesis, Wu et al. [55] (2011) estimated the effect of pyruvate carboxylase in Pichia pastoris. The yields of oxaloacetate and L-malic acid increased despite carbon being transferred to the fumarate pathway. By over-expressing endogenous pyruvate carboxylase and heterologous, fumarase and malate dehydrogenase genes from R. oryzae in S. cerevisiae, the biosynthesis pathway for FA was rebuilt by Xu and co-workers in 2012 with 3.18 g l−1 concentration of product with glucose as a medium [44]. Other genetically altered species include Rhizopus spp. Three different lengths of ldhA gene fragments were introduced into R. oryzae by Skory with intention of raising lactic acid concentration [56]. Moreover, the mutant with the greatest ldhA fragment displayed maximum lactic acid generation (roughly seven percent increased). A uracil auxotrophic isolate of Rhizopus spp. is typically used for genetic modification, but this isolate is difficult to be procured. It is noteworthy to mention that even if the concentration of FA achieved by genetically modified strains is fewer than those obtained by the process of fermentation from the Rhizopus spp. yet genetic engineering has been taken into consideration as a tool because of its cost effectiveness [36].

5.5.2 Mutagenesis of Rhizopus spp. to improve fumaric acid production Random mutagenesis has been employed in several researches to modify and enhance various R. oryzae strains. High energy radiation with two stages (one UV radiation and second X-rays) with selection dependent upon colony diameter is the most popularly used method. Thus R. Oryzae, RUR709 was created from the mutant strain of R. oryzae KTC 6946 using irradiation technique, which increased final 45.45 percent productivity and 88.81 percent fumaric acid content [57]. To ensure the survival of cells lacking the alcohol dehydrogenase gene, the commonly used irradiation techniques could be supplemented specific mutagens addition like nitrosoguanidine, in combination with a selection pressure consisting of the presence of alkyl alcohol. MU-UN-8 strain was procured, yielding 21.15 percent FA, larger than wild type strain [58]. Among the mutagens used, N+ is used satisfactorily on R. oryzae ME-F12, resulting in an increase of 28.22% in the final FA concentration. Furthermore, PCR mutagenesis has also been employed to bring an enhancement in fumaric acid production from R. oryzae strain [59].

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5.5.3 Morphology control One of the major challenges for maintaining the productivity and yield on a consistent level is to control the fungal morphology [60]. The morphology exhibited by fungi is controlled by variable parameters comprising pH, quantity of nitrogen, agitation and mass of inoculum. Rhizopus species can develop in clumps, pellets or filamentous mycelium morphologies [61, 62]. The various fungal morphologies and their impact on the overall acid production can be summarized as follows: 5.5.3.1 Filamentous form The filamentous form of mycelium is acknowledged to be economic and highest fumaric acid producing morphology, though facing certain limitations [63]. Significant enhancement in the viscosity of broth has been observed, thus causing an overall hindrance in the smooth running of various operations. For increased fumaric acid production, the need exists for development of the methodologies and strategies that would lead to an overall flourishment of the mentioned morphology while causing reduction in the production of biomass. A decrease in biomass production and at the same time enhancement in the fumaric acid production is achieved by subjecting the cells to nitrogen and phosphorus stress conditions. 5.5.3.2 Clump morphology The clump morphology exhibited by certain strains of fumaric acid producing fungi suffers from several flaws, which restricts its use in the acid production. Zhang and coworkers have reported that clump morphology is marked by the presence of large sized clump, the core of which is devoid of nutrients and the aerobic conditions, hence switching on the formation of ethanol and at the same time leading to an overall increase in the formation of byproducts while perishing the inner cell mass [64]. 5.5.3.3 Pellet morphology The recommended pellet morphology for the industrial process prevents a significant rise in viscosity, but the pellet’s size must be tuned to allow for practical mass transfer and prevent clump associated issues [64]. Fungal morphology can be controlled and modified by optimizing the required conditions including concentration of spores in the inoculum, utilization of different nitrogen sources and providing a solid support to get fungal strains of preferable morphology [64, 65]. The controlling factors are described as under. (i) Strength of spores in the inoculum

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The overall morphology of fumaric acid producing strains is affected by the initial concentration of the spores in the inocula as was suggested out of the in depth study by Zhang et al. in the year 2012 [64]. The mycelia of dispersed morphology were obtained at high concentration (106 spores l−1) of the spore while at the lower spore concentrations (102 spores l−1), small pellet morphology of the strains got revealed. (ii) Variable nitrogen sources The source of nitrogen employed for the growth of fungi has an inevitable impact on morphology of the later. In case of Rhizopus arrhizus, metabolism is highly influenced by the nitrogen containing compound, ammonium sulphate {(NH4)2SO4}. An appreciable increase in the rate of metabolism is experienced at higher ammonium sulphate concentrations. Additionally, alternate nitrogen sources that can act as a substitute to the ones used in industries have been searched out. For example, it has been noticed that incorporating soyabean meal as a nitrogen source, duration of the final growth phase (lag phase) gets minimized, enhancing the overall cell growth and at the same time resulting in the formation of the desired dispersed mycelium [63, 64]. (iii) Alteration of surface area The preferential and desired changes in the fungal morphology can also be achieved by the employing the solid support particularly the one having higher surface area. Surfaces comprising micro-nanoparticles are the best options in this regard because using such surfaces, fungal strains get an optimum area to flourish reducing the chances of formation of clumps and mega pellets, the latter being hindrances in an ideal fungal functioning process. The micro-nanoparticle surfaces interestingly supplement a few micro-nutrients including Zn+2, Mn+2 and Fe+2 ions. Ferric oxide (Fe3O4) has been found to enhance the growth and hence production of FA in R. oryzae 1526. 95.7% germination rate of the strain was observed at a concentration of 1 g l−1. However, micronanoparticles based on MnO2 do not have any significant positive impact on germination (less than 10%) of the spores of the fungi studied [65].

5.6 Auxiliary substrates for fumaric acid production Substrate expenses in an industrial process could account for 30–40% of the entire cost of manufacturing [66]. Constitution of the culture medium is determined as per needs of the microorganism. During initial stages of the process development, intricate media are utilized, which later are followed by their optimization and simplification accordingly. Molasses and starch, the biodegradable raw materials such as molasses and starch are used intermittently in fermentation process. However, use of such materials may be accompanied by elimination of compounds like phenolics and terpenes possessing antimicrobial properties; therefore, prior complementation and optimization is

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necessary. Complementation involves the adjustment of carbon–nitrogen ratio and to check the enhanced activity of fumarase enzyme [53]. The vast study on renewable materials used as substrates during the process of fermentation brings together the substrates as follows:

5.6.1 Xylose Along with cellulose and lignin, xylose makes up the majority of agricultural waste and wood lignocellulosic biomass. Together with mannose, it is one of the most prevalent monosaccharides and can be found in hemicellulose fraction of corn stalk, wheat straw, sugar-cane and numerous wood species like Pinus radiate etc. [67]. Owing to the abundance of this simple sugar, it becomes quite mandatory to come up with new processes based on this carbon source. A comprehensive investigation has revealed that the Rhizopus species of the wild type face certain hurdles while growing in a xylose rich medium, so this is essential to select several strains subjected to mutation and possessing the capability to thrive on xylose rich medium. Out of various species of Rhizopus, R. arrhizus RH 7-13-9# has been successfully grown on xylose rich medium during the production of fumaric acid. The species has been found to be a potential fumaric acid producer coming up with a yield of 73%. Furthermore, studies reveal that while adjusting the fermentation conditions, combining xylose and cellulose also resulted in higher production of fumaric acid in contrast to the fermentation process employing only xylose as a substrate [30]. The transcriptome analysis of R. oryzae [68] showed that it responds differently to the presence of xylose or glucose and experiences oxidative stress during xylose fermentation. As a result of this stress, the requirement for carbon rises, making the overall growth and fumaric acid production a challenging process.

5.6.2 Glycerol (propane-1,2,3-triol) Glycerol (propane-1,2,3-triol), which is regarded as a C3 building block in bio-refinery framework, is the primary byproduct of the biodiesel production process. Glycerol is produced from oil biomass at a rate of 10%. The production of glycerol has expanded enormously as a result of the recent rapid expansion of biodiesel industry, taken up as a waste if not in sufficient demand from the other glycerol-based processes. Many industries including the cosmetics and automotive sectors can be benefitted greatly from the usage of pure glycerol as a raw material; however, it must be processed before applying commercially being impure possessing lower concentration of triol, hence directing a need of its refinement and purification. Furthermore, it has been found that the process of purification of glycerol is economically infeasible, turning out glycerol as

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a waste that needs to be disposed of. Therefore, owing to the cost-ineffectiveness associated with the purification of glycerol, the concept of employing crude glycerol as a raw material in biotechnological processes sounds intriguing. Numerous biological processes can convert it into high and medium-value compounds including succinic acid, fumaric acid or diols [69]. It has been reported that glycerol does not serve as an ideal carbon source for all microbes and even those which utilize it demand additional carbon source as a supplement. For this reason, co-fermentation techniques employing glycerol as an additional carbon source have been investigated. In continuation with this, in-depth studies have been carried out and genetic engineering has been used as a tool to address the issue. Fumaric acid production was accomplished by removing three fumarases from a well-known succinic acid producer (E. coli strain) using glycerol as substrate. FA production was attained during the process, obtaining acetate as a byproduct during process. A decrease in the production of acetate was achieved by overexpressing PEP carboxylase, resulting in the production of 41.5 g l−1 of FA in a feedbatch process [70]. The process of adaptive evolution has been employed for the modification of optimum fumaric acid producing fungi R. oryzae so that the later can use glycerol as a source of carbon. In this context, novel fumaric acid producing strain G80 has been developed in which unique pathway for production of FA from glycerol is followed. In an optimized-feed batch process, while following this mechanistic pathway 25.5 g l−1 of FA concentration has been achieved [71].

5.6.3 Waste apples While apple pressing and its juice preparation process, roughly 12 MM tonnes [72] of residues per annum is usually generated, the solid-waste comprising apple pomace that accounts to the 20–35% of the fruit weight. Apple pomace ultrafiltration sludge is also formed during further processing of the juice produced. The high percentage of sugar in the wastes produced during the process qualifies their use as suitable raw materials for the process of fermentation, hence contributing to overall decrease in raw material expenses. In 2017, Syzmaneska et al. reported [72] that 25.2 g l−1 of FA was generated from apple pomace ultrafiltration sludge by idealising fermentation conditions and modifying sugar and solid content. A change in morphology including use of small-sized pellets further enhanced the acid production. Solid-state fermentation is preferred in cases where apple pomace is used. Das and coworkers [73] have reported that while carrying out R. oryzae 1526 mediated solid-state fermentation, 52 g FA per kg of the solid was produced. Furthermore, an analysis done via electron microscopy has revealed that the apple pomace is used by the fungus to enhance the growth of hyphae.

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5.6.4 Waste water from brewery The process of sub-merged fermentation has been carried out by using brewery waste waters as the substrates. Following the suitable fermentation conditions mediated by R. oryzae 1526, 31.3 g l−1 productivity was achieved supporting the fact that brewery waste waters are the less expensive and promising raw material for fumaric acid production [74].

5.6.5 Food waste A significant issue in contemporary culture is the huge output of urban waste and litter. Dumping and storing of all this garbage securely poses an environmental danger due to the generation of huge quantity of waste annually. As per the reports of FAO, 1.3 MM tons of garbage is discarded every year of which food waste forms an essential part. The wastes produced during the process could be in solid or liquid form both but the most commonly encountered food wastes are rich in water content, proteins and carbohydrates, therefore, making their application suitable for the process of fermentation. Using R. arrhizus RH-07-13 and the proper strategy to improve food wastes as a raw material, a higher yield of 32.68 g l−1 was obtained from liquid fraction of the wastes in contrast to glucose as substrate [75].

5.7 Post-processing and purification of fumaric acid (downstream process) The downstream operations necessary to retrieve and refine product are the major cost drivers and stumbling blocks to the commercialization of microbial fumaric acid and pro-drugs in addition to that of fermentation expenses. A number of separation processes such as solvent-extraction, ion-exchange adsorption, electro-dialysis and precipitation are satisfactorily used in the past for the post-processing and purification of fermented organic acids involving citric acid, succinic acid and lactic acid. However, strategies to recover have not been developed at an advanced scale. Out of the mentioned methods, precipitation is the most commonly used method for the recovery of fumaric acid [36]. The property of lower solubility and high polarity of fumaric acid in water is used for its separation from other acids, salts, carbohydrates and nitrogen compounds. Lower dissolution of the acid enables its precipitation from the broth, which can be polished by the process of adsorption using active carbon resulting in the formation of monomer of the acid (polymer-grade) [76]. Following this, acetone can be used for desorption of the acid with a high recovery yield of 93%, which can be further increased up to 98% by the subsequent water sweeping process [77, 78].

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Ion-exchange resins like IRA-900 [78] can be used to perform adsorption during separation and purification of fumaric acid, and it has been observed that during the process, the intermittent removal of acid from the broth and combination of bioprocess with the separation-unit enhanced the fumaric acid yield by 25% and even higher rate of productivity. This is due to the fact that fumaric acid plays the role of self-inhibitor during the course of its production by fungi. Thus, it is evident that hybrid strategies combining more than one process act as an important tool for the purpose of increasing fumaric acid production and have been widely used in industrial processes. In cases, where the separation of fumaric acid is cumbersome and is hindered by the byproducts like acetic acid, highly sensitive chromatographic techniques incorporating ionexchange resins in simulated moving-beds is extended to the process by which high purity and recovery (both 99%) have been achieved [79].

5.8 An outlook towards the application aspects of fumaric acid FA, a versatile chemical intermediate is used in almost all industrial chemistry fields. In the recent past, fumaric acid derivatives, especially the esters (FAEs), have found diverse range of applications in bio-medicine including sclerosis and psoriasis treatment and a support material for tissue engineering [32, 63]. A thorough go through of the literature has revealed the usability of fumaric acid as a bactericidal agent and in food industry as well [9, 44]. The percentage wise utility of fumaric acid in different fields along with its worldwide percentage consumption is expressed, in Figures 5.4 [5] and 5.5 [15], respectively.

Figure 5.4: Percentage consumption of fumaric acid in different products [5].

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Figure 5.5: Percentage consumption of fumaric acid throughout the world [15].

The detailed description of the numerous applications of fumaric acid in various industries is as in the succeeding discussion.

5.8.1 Applications in food industries Being a cheap and affordable organic-grade food acid, fumaric acid has been in use since 1946. The characteristic feature of fumaric acid to govern the growth of microbes, to maintain pH and to intensify flavours has made its successful use as a food-grade acid in food and farming industry. Studies have shown that due to the hydrophobic nature, its savour influence and sourness is pertinacious and long-standing. Owing to its low molecular mass, the buffering capacity of fumaric acid is appreciable at pH 3 in contrast to other food acids. A small amount of it is added to food and nutrition products possessing pH more than 4.5 [80]. Due to its least expensive and non-toxic nature, 33% fumaric acid consumption occurs in the preparation of food articles and beverages. In addition, fumaric acid is involved in the preparation of edible products such as L-maleic acid and L-aspartic acid, which are in turn used in the production of artificial sweeteners and many food articles. Thus, a mega scale production of fumaric acid is qualified [9, 32, 81].

5.8.2 Role in dairy and poultry Domesticated animals including emit enormous quantity of CH4 as a part of their natural digestion process. Furthermore, these animals’ stored manures also contribute to the formation of methane (CH4) gas. Methane gas is thought to make up about 5% of the atmosphere while the others like carbon dioxide, nitrous oxide and fluorinated gases account for 9% of all green house gases on a global scale. Enteric fermentation and manure management are popular in the United States and provide, respectively, 23

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and 9% of the total methane emissions [82]. The rising consciousness of global warming has forced development of methane curtailment techniques, which include alterations in the way animals are fed. Several in vitro investigations have demonstrated the incorporation of fumaric acid in ruminant diets leading to reduction in enteric methane emissions. In the year 1999, Iwamoto and co-workers [83] discovered that incorporation of 30 mM of fumarate in ruminal inoculum in the batch cultures led to a 10% reduction in CH4 production. During the same year, Valdes and co-workers [84] investigated the impact of addition of fumaric acid salt, the sodium fumarate of concentration 0–10 mM to a diet comprising hay (50%) and barley (30%), declining production of methane by 5–6%. The results were further confirmed by employing Rumen Simulation Technique. Kanda et al. in 2001 [85] performed an in vivo study wherein they incorporated 20 × 103 mg per kg of fumaric acid in the form of dry matter having an approximate concentration of 18 mM to cattle diet feed comprising sorghum silage and marked 23% fall in methane gas production. In batch cultures, Caro and Ranilla (2003) observed that upon the addition of fumaric acid to concentrate feeds in the concentration range 0–10 mM, declined CH4 production by 5% [86]. In addition to this, a number of studies reported by Skinner and co-workers (1999) proved a positive impact in the feeding efficacy of the breed broilers and in particular the laying hens upon the addition of variable concentrations of fumaric acid into their feeds. An increase in overall weight of the broilers was observed without affecting the rate of feed consumption [87]. In the year 2011, Luckstadt and Mellor [88] observed that fumaric acid can be successfully tested as a substitute to primitive growth promoters and revealed a considerable amendment in feed-to-gain ratio, providing a novel option for the broiler-farmers to achieve a lesser ratio by amalgamation of FA in poultry feeds.

5.8.3 Hotspot compound for resin industry The resin industry extensively uses FA for manufacturing resins of different chemical nature. Of the total 90,000 tons of fumaric acid produced annually, resin industry accounts for 56% of its consumption where 35% is utilized in the paper resin industry, 15% in unsaturated polyester resins industry and 6% in alkyl resins (Figure 5.6) [5]. The presence of two carboxylic groups and carbon–carbon olefinic bond qualifies the application of fumaric acid for the processes of esterification and polymerization. Fumaric acid plays a potential role in one of the hotspot reactions, the polymerization. This characteristic feature of FA has been employed for the synthesis of unsaturated polyester resins at commercial scale, which are produced by the condensation reaction between FA and polyhydric alcohols. On varying the number and type or proportion of the polyhydric alcohol, numerous polyesters can be synthesized (Figure 5.7). The resins synthesized from fumaric acid are long-lasting, hard and more corrosion

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Figure 5.6: Percentage consumption of fumaric acid in different sectors of resin industry [5].

Figure 5.7: Synthesis of PBF from fumaric acid.

resistant in comparison to the resins synthesized from maleic anhydride, the starting material for fumaric acid production [13]. A study carried out by Shao and co-workers [89] reveals that fumaric acid-based polymers are biodegradable and environmental friendly. In addition to this FA acidbased resins being unsaturated, its macromers possess the property to cross-link between themselves or else with the cross-linking agent to form novel type of polymerbased products. One of the important linear fumaric acid-based polyester is polypropylene fumarate (PPF) [90], which leads to the formation of fumaric acid by disruption of the ester linkages. The unsaturated sites present in the copolymer backbone can be utilized in the subsequent cross-linking reactions. In addition to this, PPF can be copolymerized with polyethylene glycol (PEG) to produce another popular group of synthetic biopolymers, the hydrogels which find numerous applications like in tissue engineering and drug delivery [91].

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5.8.4 Fumaric acid and green chemistry (green approach for Beckmann rearrangement) The use of organic solvents during the course of divergent syntheses is associated with several deleterious effects to the environment and hence to the mankind. In the recent past, attempts have been made by the researchers to perform these reactions in a solvent-free manner and also to replace the conventional acid catalysts with the environment friendly ones. Many reactions of economic importance have been modified to make them more green and ecofriendly. One such reaction is the Beckmann rearrangement, an organic synthesis involving use of organic solvents during the course of reaction. Additionally, the reaction is associated with the use of a varied number of acid catalysts including chlorosulfonic, sulfamic, p-toluene sulfonic and trifluoromethane sulfonic acids. However, these acid catalysts are associated with drastic effects and their use needs to be avoided. To overcome the problems associated with the reaction route followed earlier, Rohokale et al. [90] had worked out the role of fumaric acid as one of the efficient promoters of the Beckmann rearrangement under solvent-free conditions using thermal and microwave irradiations. Similarly, many reactions of commercial importance including esterification and aldol earlier involving the use of non-green catalysts have been modified by substituting these with FA [91].

5.8.5 Medicinal applications For decades, mixtures of dimethyl fumarate (DMF) and FAE salts have been employed as oral treatment of psoriasis [92] and also DMF coated tablets have been found to have

Figure 5.8: Applications of fumaric acid.

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less gastrointestinal side effects. Furthermore, under in vivo conditions, one of the esters of DMF that gets hydrolyzed to yield monomethyl fumarate has been found to be biologically active, thus possessing anti-carcinogenic behaviour. Fumaric acid esters are also satisfactorily applied to treat a variety of skin disorders including cutaneous sarcoidosis, necrobiosis lipoidica, diabeticorum and disseminated granuloma annulate [93]. The schematic presentation of numerous applications of fumaric acid is given in Figure 5.8.

5.9 Conclusion and future perspectives Owing to the expanding range of the uses of fumaric acid and derivatives, microbial production of the acid, first investigated around the turn of 20th century, is still an intriguing subject of study. A noticeable enhancement in the fumaric acid production has been achieved by ascertaining certain potent FA microbial strains and by employing more effective bio-processes. Nowadays, researchers focus on the designing of cost-effective procedures in terms of energy consumption and required raw materials. Although an exhaustive research and in-depth studies have been carried out on fermentation as a production process for fumaric acid, yet it is not fully competent with the chemical methods in terms of economy. Hence, more investigation ought to be done in this direction. The three most crucial factors to be taken into account while designing a fermentation process are product concentration, yield and productivity. However, studies frequently find that fermentation of fumaric acid is primarily concerned with the product concentration and a little thought is given to the productivity and yield. Since fumaric acid has a low solubility and can be isolated from broth at low concentrations as previously mentioned, we believe that product concentration is no longer an issue and a hurdle in the way of microbial fumaric acid’s commercialization production. Low product yields from the fermentation process, however, are always followed by a growth in byproduct creation like succinic acid, L-malic acid and ethanol, consequently raising the difficulty, materials needed and expense of recovering the downstream a procedure. Low productivity processes also use more energy and have lower average capacities. Therefore, yield and productivity are key elements in the production of microbial FA. Strategies such as strain modification, control of pH and DO should be optimized to improve the parameters. Furthermore, process optimization tactics ought to take into account the viability of mega-scale fermentations in addition to concentrating on boosting production at the laboratory size. Keeping in view the utility domain of FA derivatives like L-malic and L-aspartic acids, their synthesis needs further focus. L-Malic acid and L-aspartic acid are every time 1.5 to 2 times more expensive in general the cost of fumaric acid. Direct application of microbiological sodium fumarate or ammonium fumarate as substrates to produce the latter is a sustainable and economic approach. Thus, in the near future, production of L-malic acid or L-aspartic acid would be highly advantageous economically as

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compared to fumaric acid. In addition, commercial bioprocesses for manufacture of L-malic acid and L-aspartic acid would present substantial chances for the advancement of biotechnologies for the synthesis of fumaric acid and microbial fumaric acid might in future, will be more appealing economically. To sum up, additional research into metabolism, namely acid transport, pellet formation and environmental tolerance, is necessary to comprehend fumaric acid accumulation by the mechanisms over a variety of Rhizopus spp. Building up this fundamental understanding will guide future techniques for process optimization.

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Monika Chopra, Vikas Kumar*, Manoj Singh and Neeraj K. Aggarwal

6 An overview about the approaches used in the production of alpha-ketoglutaric acid with their applications Abstract: Alpha ketoglutaric acid is a biological compound found naturally in the human body. It plays an important role in the cell metabolism and has a role in various metabolic pathways including Kreb’s cycle, protein metabolism and so on. Keto glutaric acid is chemically prepared from succinic acid and oxalic acid. It is a direct precursor of glutamic acid and triazines. It can be produced by oxidative decarboxylation of isocitrate by isocitrate dehydrogenase. The yeast Yarrowia lipolytica is used as a prospective producer of alpha ketoglutaric acid from ethanol. The capability to synthesize Keto glutaric acid has so far been investigated for many microorganisms such as Pseudomonas fluoroscens, Bacillus subtilis etc. P. fluoroscens have the ability to synthesize a huge amount of alpha ketoglutaric acid in a glycerol medium supplemented with manganese (Mn). The Mangnese has a significant impact on glycerol metabolism resulting in the buildup of alpha ketoglutaric acid. The metabolism of succinate may result in the production of alpha ketoglutarate. Despite its importance in TCA cycle, alpha ketoglutaric acid buildup as an intermediate product of bacterial glucose oxidation. Along with chemical synthesis and microbial fermentation, enzymatic transformation can also be used to produce alpha ketoglutaric acid. Biodiesel waste is considered as cheap and renewable carbon source for the development of alpha ketoglutaric acid. Alpha ketoglutarate is used for kidney disease, intestinal and stomach disorders and many other conditions. It also plays an important role in the food industry as food and nutrient enhancers. The review is covering all the aspects related with the Alpha ketoglutaric acid production, utilization and product recovery. Keywords: alpha ketoglutaric acid; glycerol; Kreb’s cycle; succinate; Yarrowia lipolytica.

*Corresponding author: Vikas Kumar, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, 133207, India, E-mail: [email protected]. https://orcid.org/0000-0002-6044-3239 Monika Chopra and Manoj Singh, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, 133207, India Neeraj K. Aggarwal, Department of Microbiology, Kurukshetra University, Kurukshetra, 136119, India As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: M. Chopra, V. Kumar, M. Singh and N. K. Aggarwal “An overview about the approaches used in the production of alpha-ketoglutaric acid with their applications” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0162 | https://doi.org/10.1515/9783110792584-006

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6.1 Introduction Alpha ketoglutaric acid (AKG) is a low molecular weight chemical substance which is categorized asketo-acid or oxo-acid which includes carboxyl groups in addition to ketones and is commonly observed in animals [1]. The keto acids that contain a carbonyl group inthe alpha position are crucial intermediates in the amino acid metabolism and Kreb’s cycle.Additionally, it is anticipated that demand for this metabolite will increase in the upcoming years due to the compounds’s broad application in a number of industries including food, chemical, agriculture, medicine etc [1]. On the other hand, Alpha ketoglutarate may react with ammonia and transformed to glutamate and the glutamate, in turn reacts with ammonia to produce glutamine [2]. Alpha ketoglutarate could also provide energy for intestinal cell functions by providing a lot of ATP in the TCA cycle. Furthermore, It has a protective effects against oxidative stress in intestinal mucosal cells and helps to maintain cell redox homeostasis [3]. Apart from this, It also provide antioxidative protection through enzymatic and non-enzymatic oxidative decarboxylation [4]. According to the emerging evidences, alpha ketoglutarate has shown a critical role in systemic, intestinal and gut bacterial metabolism [5]. The majority of alpha ketoglutaric acid is produced through the chemical synthesis of diethyl succinate and diethyl oxalate. This technique, however, is multi-step, low yielding and requires hazardous compounds such as cyanides, toluene, and sodium metal, all of which generate toxic wastes [6]. It has a role in a variety of metabolic pathways, including the Krebs cycle, amino acid and protein metabolism and so on [7]. Ketoglutaric acid is made chemically from succinic acid and oxalic acid diethyl esters as raw materials all over the world whereas microbiological production relies on bacteria like Pseudomonas fluorescens and Serratia marcescens, as well as yeasts like Torulopsis glabrata and Yarrowia lipolytica [8]. It has been shown that thiamine auxotrophic yeasts like T. glabrata, a multi-vitamin yeast can over synthesize keto acids [9]. Ketoglutaric acid is a direct precursor of glutamic acid and triazines. Pyridasine is made from ketoglutaric acid and hydrazine derivatives and has excellent insecticide, antiviral and antibacterial properties [7].

6.2 Production of α-ketogulatric acid α-ketogulatric acid is now manufactured using a multi-step chemical process. The chemical production of α-ketogulatric acid is multi-step process. However, flaws such as low yield, low purity, residual cyanides, and other harmful waste are difficult to overcome. Biotechnological approaches have been investigated for decades to overcome these difficulties as appealing alternative means of α-ketoglutaric acid synthesis. Over production of α-ketoglutaric acid have been detected in a variety of bacteria and yeasts [10]. The production of α-ketoglutaric acid on a large scale is thought to bring up new possibilities for its use in the food industry, medicine, agriculture and other fields [11]. Toluene, chloroform, diethyl ether, absolute alcohol and sodium metal are commonly

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used in the chemical synthesis of KGA from diethyl succinate and oxalate ester; it is a multi-stage process involving explosive and toxic compounds such as toluene, chloroform, diethyl ether, absolute ethanol and sodium metal [7]. Pseudomonas sp., Bacterium ketoglutaricum, Aerobacter aerogenes and Serratia marcescens, were found to be the most active makers of α-ketoglutaric acd from carbohydrates [12]. A high concentration of carbon source in the medium and a low nitrogen concentration are required for the over synthesis of α-ketoglutaric acid [13].

6.2.1 Synthesis of alpha ketoglutarate from succinate and succinate semialdehyde Alpha ketogultarate may be produced as a result of succinate metabolism. Prevotella bryantii was the first bacteria to be identified producing alpha ketoglutarate from succinate approximately 50 years ago [14]. Despite the fact that the Tricarboxylic acid cycle is commonly thought of as a highly oxidative mechanism that creates reactive oxygen species (ROS), it can also function in reductive manner. Unlike the traditional Tricarboxylic acid cycle, which oxidizes acetyl CoA into two molecules of CO2, as carbondioxide is fixed via the reductive TCA cycle, a variety of organic compounds are produced [15]. Because most enzymatic processes in the TCA cycle are reversible, so it is possible to operate reductive TCA with the help of additional enzymes. Additional enzymes such as citrate lyase (CL) and fumarate reductase are required for a complete rTCA cycle to occur (FRD). While citrate lyase catalysis the conversion of citrate to acetyl CoA and oxaloacetate, the latter catalyses the conversion of fumarate to succinate, making it essential for the growth of decarboxylic acid [16]. The other distinctive enzyme in the rTCA cycle is 2-oxoglutarate synthase. The production of AKG and CoA from succinyl CoA and CO2 is mediated by this enzyme. As a result, the rTCA cycle is another metabolic network used by microbial systems to generate this crucial keto-acid [17]. The synthesis of AKG can be supplemented by succinate semialdehyde (SSA) which is produced by the oxidation of succinate, SSA dehydrogenase (SSADH) is responsible for this mechanism [18]. The transformation of SSA to Alpha ketoglutarate with the addition of CO2 is catalysed by AKG decarboxylase. The enzyme gamma-aminobutyrate (GABA) transaminase catalyses the synthesis of SSA from GABA, which promotes the formation of alpha ketoglutarate [19]. When glycerol serves as the only carbon source, it has been shown to improve the production of this 5 carbon keto acid when manganese is added to a medium [17]. (Figure 6.1).

6.2.2 Synthesis of α-ketoglutarate from iso-citrate in a glycerol medium with Mn A significant derivative of the biodiesel industry is glycerol, and it presents a technological difficulty to transform it into a useful product [20]. P. fluoroscens have the

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Figure 6.1: Pathways for the production of alpha-ketoglutaric acid from succinate.

ability to synthesize copious amount of alpha ketoglutarate in a glycerol medium supplemented with manganese (Mn). Increased activity of iso-citrate dehydrogenase (ICDH)-(NAD)P dependent and amino-transaminases were responsible for the increased synthesis of this keto acid [19]. Oxaloacetate, a crucial metabolite in the production of ketoglutarate can be produced effectively by two isoforms of pyruvate carboxylase (PC) that are present in cells that have been exposed to manganese [21]. Additionally, the enhanced activity of phosphoenol pyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) ensured the effectiveness of KG producing metabolic system by delivering pyruvate and ATP from the oxaloacetate produced by PC [19]. The complete cells treated to Mn transform 90% of industrial glycerol to ketoglutarate. The cost effective conversion of glycerol to ketoglutarate can be optimized by using this manganese triggered metablic network [17]. To fuel the synthesis of KG, oxaloacetate, an important product in a range of metabolic pathways may go through a number of changes. Aspartate transaminase (AST) may recognize to convert glutamate and oxaloacetate into KG and aspartate very easily. Cells isolated from cultures that received manganese supplements showed an increase in this enzyme’s activity. When the enzymes (PC/AST) were incubated with pyruvate, HCO3 and glutamate, the excised activity band produce KG [22]. Manganese supplementation is relatively simple procedure for activating a variety of metabolic networks involved in the production of ketoglutarate. Mangnese significantly affects the metabolism of glycerol, which causes a buildup of ketoglutarate [19]. (Figure 6.2).

6.3 Alpha-ketoglutaric acid as a product of bacterial oxidation of glucose

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Figure 6.2: Synthesis of alpha ketoglutarate from iso-citrate in a Glycerol medium.

6.3 Alpha-ketoglutaric acid as a product of bacterial oxidation of glucose Despite its importance in Kreb’s TCA cycle, alpha ketoglutaric acid buildup as an intermediate product of bacterial glucose oxidation [23]. Lockwood and Stodola conducted the first research in 1946. They discovered that this acid is produced when P. fluorescens NRRL B-6 2- ketoglutonic acid fermentation progresses beyond the maximal accumulation, and that when all of the 2-ketoglutonic acid has disappeared, roughly 25% alpha ketoglutaric acid is present for the initial glucose provided [24]. Recently, Koepsell, Stodola and Sharpe found a greater yield, around 41% for glucose provided, in shaken Pseudomonas cultures [25]. Masuo et al. validated the accumulation of this acid, as well as tiny amounts pyruvic and oxaloacetic acid in a Pseudomonas strain. Katagri, Tochikura and lmai discovered that a strain of the Escherichia coli group produces a significant amount of this acid. Masuo and Wakizaka have identified a novel bacterium that produces a high yield of alpha ketoglutaric acid, with yield of 50–60% of the acid for initial glucose given in shaken cultures in numerous experiments [26]. According to Bergey’s manual of determinative bacteriology (6th edition), this bacterium belongs to the genus Bacterium and it has been given the name Bacterium alpha ketoglutaricum n. sp. These discoveries are important not only for the industrial production of alpha ketoglutaric acid but also for the research of the bacterial oxidation of glucose process [23]. Apart from Pseudomonas and E. coli, Serratia marcescens, Bacillus megatherium, B. natto (a variant of B. subtilis), Bacterium succinicum and Gluconoacetobacter cerinum were found to produce significant amount of alpha ketoglutaric

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acid during glucose fermentation in shaken culture [27]. The effect of environment on acid accumulation was studied in these bacteria, particularly Serretia marcescens, which appeared to have some use for the industrial manufacture of alpha ketoglutaric acid [28].

6.4 Alpha-ketoglutaric acid production by Yarrowia lipolytica yeasts The yeast Y. lipolytica differs physiologically from Sacchromyces cerevisea. It belongs to the hemiascomycetes class and the dipodascacea family taxonomically. Candida, Endomycopsis and Sacchromycopsis lipolytica were some of the previous names for it [29]. Y. lipolytica is an oblicate aerobe that can only utilize a few carbon sources that are widely examined in yeasts classifications. At the same time, it has the ability to metabolise hydrophobic substances like n-alkanes, oils, lipids, and fatty acids which is unusual among yeasts [30]. Y. lipolytica strains have been extracted from dairy products (cheese, yoghurt, and kefir) beef, chicken and shrimp salads as well as polluted settings with hydrocarbons and oils. The ability of this yeasts to utilize n-paraffin sparked initial interest in it in the late 1960s and it has since been utilized to manufacture single-cell proteins from crude oils containing long chain hydrocarbons [31]. When strains of Y. lipolytica were grown on these substrates, it was shown that they can create rather substantial amounts of organic acids such as citric acid and alpha-keto glutaric acid [32]. Y. lipolyticahas been the most thoroughly studied non-conventional yeast because of its several benefits such as broad substrate spectrum, intense secretary ability, greater product output, waste reduction and an existing efficient system for genetic engineering transformation [33]. Furthermore, the food and drug administration (USA) considers Y. lipolytica to be non-pathogenic, and several methods based on this bacterium are designated as generallyrecognized as Safe (GRAS) [34]. Y. lipolytica strains cultivated on n-alkanes, ethanol, glycerol and vegetable oils results in high production of alpha ketoglutaric acid [35]. Tsugawa and Lozinov’s groups were the first to mention Y. lipolytica ability to release and generate ketoglutaric acid in the late 1960s [11]. Y. lipolytica Aj5004 synthesized roughly 46 g/L ketoglutarate in 72 h using 8% (w/v) n-paraffin as carbon source, according to a selection of various microorganisms for ketoglutarate production cultured on n-paraffin [36]. Optimization of growth conditions and the development of new appropriate strains were two other ways for increasing ketoglutaric acid output with n-paraffins [37]. The generation of 185 g/L was demonstrated with 10% n-paraffin after 240 h for the diploid strain D 1804. Further research revealed that ethanol can also be used as a substrate for alpha ketoglutaric synthesis [11]. For efficient ketoglutaric creation with ethanol, other approaches were used, such as determining optimal culture conditions, such as thiamine content, dissolved oxygen, nitrogen and initial pH. Environmental

6.5 Utilization of Proteus mirabilis L-amino acid deaminase

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impacts such as the identification of major enzyme activities involved in ketoglutaric acid production and ethanol metabolism were also investigated [38]. The results show that a low thiamine concentration (3 μg/L), an acidic pH (4.5), and nitrogen excess, as well as a low oxygen concentration (5%) are critical for alpha ketoglutaric acid overproduction on ethanol containing medium [38]. Otto et al. (2012) recently demonstrated that reducing the amount of by-products improves ketoglutaric acid output. It was studied whether a gene dose-dependent mechanism could regulate the concentration of organic acids (main products of KGA and PA as a major by-product, fumarate, malate and succinic acid as minor by-products) [11]. During culture of these strains on raw glycerol as carbon source in bioreactors, over expression of the genes FUM1 and PYC1 resulted in significantly elevated enzyme activity [39]. Kamzolova et al. published a follow-up study on the generation of ketoglutaric acid from ethanol. They discovered that the formation of ketoglutaric acid from ethanol requires an increased number of zinc and iron ions [35]. With all prior study, the impact of thiamine deficiency, abundant carbon and nitrogen sources as well as a low ph on intensive keto glutaric acid production is revealed. The Y. lipolytica strain VKM-Y 2412 produced upto 172 g/L KGA under ideal circumstances with a mass yield coefficient of 0.70 g [40].

6.5 Utilization of Proteus mirabilis L-amino acid deaminase for the single step production of alpha ketoglutaric acid from glutamic acid The production of alpha ketoglutaric acid can also be accomplished through enzymatic transformation in addition to chemical synthesis and microbial fermentation. Alpha keto acids can be theoretically produced by three different types of enzymes: amino acid dehydrogenase (ADH), amino acid transferase (ATs) and amino acid deaminase (AAD) [41]. ADH requires a cofactor recycling mechanism since they depend on either NADH or NADPH to provide reducing equivalents. To overcome the unfavourable balance in the case of ATs, an enzyme system containing two amino acids is required. The coupling mechanisms and cofactors are not required for AAD and therefore, it is considered as the best choice for the production of alpha keto acids from cheap L-amino acids [42]. Enzymatic transformation has potential benefits including less environmental contamination and lower production costs as compared to the chemical route and microbial fermentation [43]. The enzymatic production of alpha Keto glutaric acid by L-AAD hasn’t been reported yet, though. Natural L-amino acids are stereospecifically oxidatively deaminated by L-amino acid deaminases (EC 1.4.3.2) to produce the corresponding alpha keto acids and ammonia [44]. Several bacterial sources, including Proteus, Provindecia, and Morganella, have been found to contain the L-AAD [45]. P. mirabilis KCTC 2566 has been shown to contain two different kinds of l-amino acid deaminase. One is very active toward a variety of aliphatic and aromatic amino

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acids, typically l-phenylalanine, whereas the other operates on a relatively small range of basic amino acids, notably histidine, arginine, and glutamic acid. Both varieties of P. mirabilis’ L-AAD are membrane-bound [46]. Between residues 7 and 29 amino acid residueS, the second kind of L-AAD from P. mirabilis KCTC 2566 has one transmembrane region. The second variant of P. mirabilis KCTC 2566 L-AAD, which is more active on basic l-amino acids, was created in this study by eliminating one N-terminal transmembrane region (from nucleotides 21 to 87), and switching out the low-usage codons with the favoured codons observed in E. coli [47]. When expressed in E. coli BL 21, this engineered N-LAAD developed into inclusion bodies, therefore solubilization and refolding operations were carried out to produce the soluble and active N-LAAD. The biochemical properties of the refolded N-LAAD were also evaluated, and they were contrasted with the characteristics of the full-length L-AAD. Finally, to achieve the highest bioconversion rate, the conversion of glutamic acid into alpha-keto gultaric acid was enhanced [46].

6.6 Substrates used for the production of alphaketoglutaric acid Biodiesel waste is a cheap and renewable carbon source for the development of microbial technology of alpha ketoglutaric acid production [48]. The yeast Y. lipolytica, which is generally recognized as safe (GRAS) is a promising microbe for such technique [49]. Biodiesel output in the globe now stands at 41 million tons per year, upto 4.5% per year [50]. The production of alpha ketoglutaric acid from glycerol containing biodiesel waste using yeast Y. lipolytica has recently gained popularity [51]. The method developed in the early 1950s was the first literature studies revealing the feasibility of AKG biosynthesis and isolation from the actual post fermentation broth. Classic separation methods such as filtration, centrifugation, acidification with a strong mineral acid, solvent extraction, evaporation and crystallization were the primary processes of the proposed solution [1]. The method for the separation and concentration of alpha ketoglutarate based mainly on the classical methods i.e. ion exchange, acidification and organic solvent extraction. But, these techniques have several limitations such as the need to use additional chemicals and generate nuisance waste. Many studies have found that electrically driven membrane approaches such as classical electrodialysis are effective [52]. Membrane electrodialysis and bipolar membrane electrodialysis (EDBM) is a chemical that can be used to separate organic acids from post fermentation broth [53]. In the recent years, the usage of EDBM has attracted a lot of attention from academic as well as industry since it provided an effective and cost-effective solution in obtaining valuable and ecofriendly raw material [54]. Glucose, lard oil, salicylic acid, hydrocarbons and corn steep liquor are some of the raw materials required to produce alpha ketoglutaric acid [55].

6.7 Significance of alpha-ketoglutaric acid

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6.7 Significance of alpha-ketoglutaric acid –

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Medical importance of alpha ketoglutaric acid:- Alpha ketoglutaric acids are commonly utilized todiagnose a variety of disorders. For instance, increased levels of keto acids in serum as well as in urine could indicate maple syrup urine disease (MSUD), Phenylketonuria (PKU) and tyrosinosis [56]. As a result, in vivo assessment of alpha ketoglutaric acids results in the examination of various diseases. The analogues of alpha ketoglutaric acid have been found to be effective substitutes for particular amino acids, which is crucial in the patients with kidney problems. Because alpha keto acids lack amino groups, replacing amino groups in the diet with the corresponding alpha ketoglutaric acids helps the kidney as well as the liver in reducing urea formation [57]. Importance of alpha ketoglutaric acid in animal nutrition:- Alpha ketoglutaric acids are employed as nutrient supplements in animal feed, particularly in the poultry industry to address environmental and animal growth concerns [58]. It has been stated that excess nitrogen excretion in chicken manure as a result of high amino acids content meal is a significant issue that negatively affects the environment and the farmers [59]. To limit, nitrogen accumulation in the manure, farmers can start supplying alpha ketoglutaric acids in the chicken’s diet rather than amino acids [60]. It has been shown that alpha ketoglutaric acid can reduce the amount of nitrogen consumed in food while still delivering sufficient nutrients to prevent weight loss [61]. Early studies found that all amino acids accept lysine and threonine, each alpha ketoglutaric acids of amino acids can be employed as modifications for the meals of rats. Chow and Walser investigated the efficacy of alpha keto acids in another study. They discovered that when compared to high amino acid supplement, rats can grow at the same rates with just reduced doses of alpha ketoglutaric acid [62]. Importance of alpha ketogultaric acid in food industry:- Food flavours are quite important in today’s world because in daily diets, improved foods have gained enormous popularity. In the smoke created during the pyrolysis of various compounds like ketones, aldehydes, acids and esters have been discovered [63]. On the other hand, only ketones and aldehydes are thought to be useful in the formation of colour, fragrance and texture in smoked meals [64]. Several researches have found that alpha keto acids have beenemployed in flavor enhancers as well as in nutrient enrichers. Alpha ketoglutaricacids have a considerable influence in cheese taste and flavor development [65]. They demonstrated that one pathway for producing savoury chemicals in cheese is the degradation of numerous amino acids in aminotransferase reactions. Because of sulphur metabolism, Brevibacterium linens, which were cultured on low-fat cheddar cheese, boosted consumer approval. By virtue of its proteolytic actions, B. linens aids in ripening of cheese [66]. Volatile sulphur compounds, alpha keto-acids and fatty acids are formed as a result of proteolysis and they play a key role in flavor production [67].

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Importance of alpha ketoglutaric acid in cyanide antidote:- Cyanide is xa neurotoxic present in fire smoke, medications as well as diets that can cause quick death by rupturing cellular adenosine triphosphate (ATP) and inhibiting the electron transport chain’s mitochondrial oxidase enzyme [68]. The function of alpha ketoglutaric acids as a potential cyanide antidote was investigated by Satpute et al. They used potassium cyanide to treat different quantities of rat pheochromocytoma cells (PC12) for 4 h in both in existence and absence of 0.5 Mm alpha-keto acid and 0.25 Mm N-acetyl Cysteine (NAC). Alpha ketoglutaric acid and NAC efficiently addressed the unfavorable effect of cyanide on antioxidant enzymes, which play a significant part in oxidative pressure, in the PC12 cells in the treatment group [69] (Figure 6.3).

6.8 Future prospects and advancements Overall, the physiological significance of alpha ketoglutaric acid is multi-directional and not all metabolic pathways have been well investigated. Alpha ketogluraic acid can be produced chemically but biotechnological approaches are used as an alternative way to overcome the difficulties faced by the other process [10]. Succinate metabolism results in the generation of alpha ketoglutarate [14]. A variety of raw materials can be used for the production of alpha ketoglutaric acid but biodiesel waste is recognized as the cheap and renewable carbon source for development of microbial technology for the production [48]. The action of mechanism of alpha ketoglutaric acid impact on the skeletal system are likely complex and include glutamate receptor activation, proline mediated bone

Figure 6.3: Applications of alpha ketoglutarte in various metabolic pathways.

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collagen synthesis and possibly anti-catabolic and anabolic actions of 17b-oestradiol [70]. Furthermore, alpha ketoglutarate’s beneficial effects on chest function and internal organ protection in pre-mature and low-birth-weight neonates should be expected [71]. The current findings could have significant clinical consequences, prompting further research into the use of alpha keto glutarate in the prevention and treatment of metabolic bone diseases in human and animals [72]. As a result, more research is needed to better understand the function of alpha ketoglutarate, define its mechanism and investigate its potential applications in human society and other fields. Some exciting discoveries in the field of ageing revealed that TORC1 is involved in the wide range of human disorders including diabetes, obesity, heart disease and cancer [73]. Ageing is a common risk factor for various diseases and it has been discovered that link between cellular senescence, diseases and organism ageing is mediated through the TOR pathway [74]. As a result, the inhibition of TOR function by alpha ketoglutarate metabolism suggests that alpha ketoglutarate may play a key role in tumor suppression [75].

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Mayank Raj, Manoj Singh*, Vikas Kumar, Tamanna Devi, Sushil Kumar Upadhyay, Prabhakar Mishra, Sunil Kumar, Mukesh Yadav, Nirmala Sehrawat and Mamta Kumari

7 Gluconic acid: strategies for microbial production using organic waste and applications Abstract: Gluconic acid is one of the most important natural acids which are moderately generated from glucose via a straightforward oxidation reaction process. Through the use of microorganisms like Gluconobacter (bacteria) and fungi, the reaction is facilitated through various enzymes such enzyme glucose oxidase and glucose dehydrogenase. The typical widespread, fermentation procedure is characterized by the use of Aspergillus niger (fungi). The primary gluconic acid derivatives, such as sodium gluconate, are widely used in the agricultural and food industries. Gluconic acid has several uses in the pharmaceutical, food, beverage, textile, cement, metal chelating agent, leather, and dairy sectors. Commercial production of gluconic acid made by fungi is well-established. Therefore, fermentation procedures and effective microorganisms are employed to produce gluconic acid with a higher yield and higher quality. These processes are also more economical and effectively convert inexpensive substrates into carbon sources. Production of gluconic acid has been reported with corn starch, grapes must, banana must, egg shells, and potato pulp using both solid state and submerged fermentation. This article provides a thorough analytical analysis for the gluconic acid production through microbial fermentation and its uses in agriculture and food. Additionally, this contemporary paper thoroughly examines the literature from recent years on the growth of gluconic acid production for the global market.

*Corresponding author: Manoj Singh, Department of Biotechnology, MMEC, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, Haryana, India, E-mail: [email protected]. https:// orcid.org/0000-0002-9257-927X Mayank Raj, Vikas Kumar, Tamanna Devi, Sushil Kumar Upadhyay, Mukesh Yadav and Nirmala Sehrawat, Department of Bio-sciences and Technology, MMEC, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, Haryana, India. https://orcid.org/0000-0002-6044-3239 (V. Kumar) Prabhakar Mishra, Department of Biotechnology, School of Applied Sciences, REVA University, Bengaluru, Karnataka, India Sunil Kumar, Department of Microbiology, Faculty of Biomedical Sciences, Kampala International University, Western Campus, Ishaka, Uganda Mamta Kumari, Department of Biotechnology, M.S. Ramaiah Institute of Technology, Bengaluru, Karnataka, India As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: M. Raj, M. Singh, V. Kumar, T. Devi, S. K. Upadhyay, P. Mishra, S. Kumar, M. Yadav, N. Sehrawat and M. Kumari “Gluconic acid: strategies for microbial production using organic waste and applications” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0163 | https://doi.org/10.1515/9783110792584-007

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Keywords: Aspergillus niger; bioprocess technology; fermentation; gluconic acid; microbial production.

7.1 Introduction Gluconic acid (GA) is a naturally occurring organic acid that is present in both micro- and macro-organisms, including human beings. The poly-hydroxycarboxylic acid, also known as gluconic acid, has a wide range of uses in the pharmaceutical, food, leather, and other sectors since it is a nontoxic, soft, nonvolatile organic acid. The gluconate ion is formed by gluconic acid in aqueous solution at neutral pH. The “gluconates” are considered to be gluconic acid salts [1]. Three commercially available processes can be used to make gluconic acid: electrolytic addition of oxygen to a glucose solution, chemical oxidation of glucose with a hypochlorite solution containing a known amount of bromide and fermentation, which involves the growth of specific microbes in a production medium that includes glucose as a substrate and other ingredients [2]. The utilization of microbial fermentation is a crucial striking strategy for addressing issues with chemical synthesis, such as expected negative effects, and for further optimizing the bioprocess for the synthesis of gluconic acid. GA can be produced by a wide range of microorganisms, including yeast, bacteria, and especially filamentous fungi. Different species of fungi from various genera include Gonatobotrys, Aspergillus, Gliocadium, Penicillium, and Scopulariopsis having previous experience of producing GA. While various bacterial species, such as Enterobacter, Gluconobacter oxydans P. fluorescens and the species of Pseudomonas, Scopulariopsis, Morexella, Acetobacter, Acetobacter methanolicus, Pullularia, Tetracoccus, and Gluconobacter participate in a specific pathway for the oxidation of glucose with glucose dehydrogenase to produce GA [3]. Fungal gluconic acid synthesis is a basic dehydrogenation reaction in which the enzyme glucose oxidase (also known as β-D-glucose: oxygen 1-oxidoreductase) catalyses the conversion of glucose to gluconic acid. The development of glucose to glucono-δ-lactone with the exclusion of the water molecule is catalyzed by the enzyme glucose oxidase. Honey and fruit juices naturally contain gluconic acid. Its core ester, glucono-δ-lactone, and releases an initially pleasant taste that eventually becomes somewhat acidic. It is mostly used in baked goods but also has applications in dairy and meat products [4]. Additionally, it discovered various modifications that reduced the absorption of fat in doughnuts and cones. It has a wide range of applications and has been employed in the concrete and food sectors as a food additive, a bottle washing agent, and a retardant agent. A. niger produces gluconic acid by an aerobic fermentation with a high oxygen requirement. Even though living things are capable of producing an enormous amount of organic acids, only a tiny subset primarily those derived from fungi are made in large quantities for commercial use [5]. Citric acid, gluconic acid, itaconic acid, and lactic acid are a few examples of these acids. Citric acid and gluconic acid, the two most popular fungi-derived organic acids, are produced commercially from glucose or sucrose using certain Aspergillus niger strains. Rhizopus oryzae, a Zygomycetae fungus, and Aspergillus terreus strains can be used to produce lactic acid, which can then be

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converted to taonic acid. Other fungal acids, such as oxalic, fumaric, and mallic acid, are being synthesized utilizing more practical chemical conversion processes as a result of the low market demand. Several more organic acids have also been researched in order to create fresh methodologies. The production processes of several international companies are currently focused on the industrial production of gluconic acid, its salts (especially sodium gluconate), and gluconolatone from glucose or maltose using interrupted batch, or repeated batch and fed batch submerged fermentations using A. niger and glucose oxidizing enzymes [6].

7.2 Physico-chemical properties of gluconic acid The powder form of anhydrous gluconic acid is white, flavorless, and crystalline. It is challenging to create free D-gluconic acid in crystalline form. The anhydrous material may crystallize below 30 °C, according to Milsom and Meers [7], and a monohydrate has been reported to crystallize at 3 °C with a distinctive crystalline structure and a melting point of 85 ° C. Gluconic acid has a melting point that ranges from 120 to 131 °C. The spread in the melting point range results from the formation of intermolecular anhydrides, which lower the melting point. It is insoluble in ether and the majority of other organic solvents, but is easily soluble in water and very marginally soluble in alcohol. A commercial mixture of 50 % gluconic acid has a pH of 1.82 at 20 °C. At 25 °C, gluconic acid has a density of 1.23 g/cm3. Solution density rises almost linearly as concentration increases in this concentration zone. Lactones are produced when gluconic acid is heated above 50 °C or stored at room temperature over a desiccant. Above 200 °C, pyrolysis takes place [8]. It is known that gluconic acid has two lactones: 1, 4-lactone, also known as glucono-g-lactone, and 1, 5-lactone, also known as glucono-d-lactone. These can be found in both the solid and liquid states of aqueous solutions, where they are in equilibrium with the free acid and one another. The inner ester of gluconic acid is glucono-d-lactone. It gives off a sweet flavor at first, followed by a little acidic flavor. It is well known that sodium gluconate aqueous solutions exhibit strong temperature resistance to oxidation and reduction [9]. It works well as both a plasticizer and a set retarder. It degrades quickly (98 % after 48 h), because it contains certain lactone structures. The concentrated gluconic acid solution has antibacterial properties (neutral cyclic ester). Sodium gluconate has a strong sequester capacity. Its chelating ability is superior to EDTA, nitrilotriacetic acid, and other chelators at alkaline pH [10]. Additionally, sodium gluconate reduces the bitterness of food.

7.3 Strategies for gluconic acid production Only the oxidation of raw materials containing glucose or glucose-containing molecules is used for the majority of development of D-gluconic acid and its salts for commercial purposes. Chemical, electrolytic, catalytic, or biological oxidation processes are all

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possible [11]. Despite the fact that there are many different oxidizing agents, chemical methods have the drawback of limited specificity even when reaction conditions are carefully monitored and optimized. In contrast to fermentation methods, this leads to unsatisfactory yields and undesirable byproducts, making it more challenging to isolate and purify the product. Therefore, one of the most effective and widely used processes for producing gluconic acid has been fermentation. For the generation of gluconic acid, bioconversion has benefits over fermentation [12]. Gluconic acid is a useful organic acid that has industrial applications. It has found use in a variety of sectors, including both food and non-food businesses. Various techniques for gluconic acid synthesis, extraction, and manufacturing have been described in Figure 7.1. GA can be produced by two methods mainly a microbe mediated method and plant based method. Microbe mediated production is done by both bacteria and fungi which is enzyme mediated bioconversion. It has been documented that several optimization procedures use submerged fermentation with a specific chemical medium to create gluconic acid. The GA is produced by allowing the fungal spores to germinate and mycelia development to take place in such a fermentation medium. Additionally, there are publications that describe solid-state fermentation techniques for gluconic acid production employing agricultural waste products or agricultural crops [13]. The fermentation process uses fungus mycelia, is the focus of most research on the production of gluconic acid. Currently, the specialised fermentation method of A. niger, was developed in 1952 by Blom et al. [14], and improved by Ziffer et al. in 1971 [15], is the main method used in industrial fermentation to produce GA. A mycelia inoculum generated in a medium with low glucose and high nitrogen content is transferred into a medium with high glucose and low nitrogen content during the process. For fed-batch production of GA, intermittent glucose feedings, and the neutralizing agent sodium hydroxide are required. A temperature of about 34 °C and a pH range of 6.0–6.5 are maintained. This procedure has a very high productivity since it converts glucose at a rate of 15 g/L/h. A. niger and G. oxydans are the main organisms used nowadays for the extensive biological production of gluconic acid. One of the most popular production techniques makes use of the fungus A. niger [16]. No matter whether fungi or bacteria are utilised, the main goal is to manufacture products like sodium gluconate, calcium gluconate, etc. Since the end product of this reaction is acidic, neutralization using neutralizing agents is crucial. Otherwise, the acid deactivates such as glucose oxidase stops the generation of gluconic acid. Both sodium and calcium gluconate are produced, although they do so in quite different ways. The pH control and glucose concentration (both start and final) are two of the several fermentation procedures and settings. The sodium gluconate method is far superior because of employing safe glucose concentrations of up to 350 g/L. The automatic addition of NaOH solution regulates pH [17]. Water quickly dissolves sodium gluconate (39.6 % at 30 °C). To regulate the pH during the synthesis of calcium gluconate, calcium carbonate slurry is added. However, compared to sodium gluconate generation, calcium gluconate manufacturing has some drawbacks. At 30 °C, calcium gluconate is only 4 % soluble in water. Super saturation happens at high glucose concentrations, above 15 %. Additionally, if it goes

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Figure 7.1: Schematic presentation of strategies for large scale production of gluconic acid utilizing waste feedstock.

over the limit, calcium salt precipitates on the mycelia and prevents the transport of oxygen [18]. To prevent the Lobry du Bruynevan Ekenstein reaction, which modifies the structure of glucose and causes a yield drop of roughly 30 %, separately from the glucose solution, the neutralizing agent should also be sterilized.

7.3.1 Gluconic acid production through bacteria Pseudomonas savastanoi and acetic acid bacteria were the first cultures found to make gluconic acid. Contrary to fungi, bacteria carry out the reaction by GDH (glucose dehydrogenase) (EC 1.1.99.17), It converts glucose to gluconic acid, which is then converted to 2-ketogluconate by gluconic acid dehydrogenase. By use of 2-ketogluconate dehydrogenase, the last oxidation step to 2,5- diketogluconic acid is carried out. High glucose concentrations (>15 mM) stimulate all three enzymes, which are located in the cell membranes [19]. PQQ (Pyrroloquinoline quinine) serves as a coenzyme for the extracellular protein known as GDH. Additionally, there is an internal enzyme called a NADP-dependent GDH that plays a smaller role in the synthesis of gluconic acid than the external enzyme does. Produced GA is transferred to the cell where it is further catabolized through processes along the pentose phosphate pathway. The pentose phosphate

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pathway is suppressed and gluconic acid accumulates when the amount of glucose in the medium exceeds by 15 mM [9]. The obligate aerobic bacteria G. oxydans has two different routes to oxidize glucose. The pentose phosphate pathway is followed by oxidation in the first step after initial phosphorylation. The second mechanism is known as “direct glucose oxidation,” and it produces gluconic acid and ketogluconic acid. Three membrane-bound NADP-independent dehydrogenases in G. oxydans unique pathway for oxidizing glucose convert D-glucose into 2,5-diketogluconic acid. Acetobacter diazotrophicus, acidotolerant acetic acid bacteria, produced gluconic acid at high rates. The organism’s glucose oxidation was less susceptible to low pH levels than that of G. oxydans [20]. It appeared that the direct oxidative and phosphorylative routes of glucose metabolism were both active. A. diazotrophicus included a PQQ-dependent GDH in addition to a pyridine nucleotide (strictly NAD)-dependent GDH, which was principally in charge of gluconic acid synthesis. Since the oxidation occurs with the secondary processes that result in oxogluconic acids, bacterial gluconic acid synthesis has encountered limited success on an industrial scale. It has been utilised to take advantage of the capabilities of Pseudomonas and Gluconobacter spp. to create gluconic acid and gluconolactone [21]. Additionally, gluconic acid is created using A. methanolicus to stimulate the glucose conversion. The main benefits of employing this facultatively methylotrophic bacterium as a catalyst are that, unlike other bacterial fermentation processes, the gluconic acid generated is a metabolic dead-end result, and the organism employs methanol, a cheap raw material, as a substrate. Additionally, since glucose is not consumed or absorbed throughout the process, the maximum potential yield coefficient is attained. In 1930, Currie and Carter submitted a patent application [22] that described a process in which a medium containing 200 g/L of glucose along with the air was carried upward via the packing of a tower filled with wood shavings or coke that had been inoculated with G. oxydans, together with other nutrients and a neutralizing agent. Working with group of scientist Parabrimidius ovalis, Tsao and Kempe [23] discovered the particular strain that had a 99 % yield in the conversion of glucose to gluconic acid and the rate was inversely proportional to the efficiency of aeration.

7.3.2 Gluconic acid production through fungi Due to its vigorous growth, high output, and simplicity in separating GA products, A. niger makes an excellent option for fermentation [24]. Glucose oxidase (GOD, EC 1.1.3.4) is the enzyme in charge of catalyzing the conversion of glucose to GA. As demonstrated in both Aspergillus sp. and Penicillium sp. GOD is a flavoprotein that is mostly found in cell walls and extracellular fluid and whose activity accounts for almost 80 % of the total enzymatic activity. All the enzymes necessary for converting glucose into gluconic acid, including lactonase, GOD, mutarotase, and, catalase are produced by A. niger [25]. The GOD-catalyzed oxidation reaction is a highly oxygen-intensive aerobic fermentation method. The biochemical metabolism of gluconic acid synthesis from A. niger, which

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includes the complete oxidation process. H2O2 is produced as a byproduct of the dehydrogenation reaction that transforms D-glucose into D-glucono-lactone. In the presence of catalase, H2O2 is then broken down into O2 and H2O [26]. A. niger generates the enzyme mutarotase, which helps to speed up the conversion. The GOD in A. niger is undertaking self-reduction by losing 2 hydrogen’s during the conversion of glucose. Hydrogen peroxide is produced as a byproduct of the reaction when the enzyme’s reduced form is oxidized forward by molecular oxygen. Catalase, which is produced by A. niger, reacts with hydrogen peroxide to release water and oxygen [27]. Lactonase acid in the hydrolysis of glucono-d lactone to gluconic acid. Because the cleavage of lactone happens quickly at a pH approaching neutral, which is caused by the addition of calcium carbonate or sodium hydroxide, the reaction can happen spontaneously [28]. It is advised to remove any lactone that has accumulated in the medium because it slows down the oxidation of glucose and the creation of gluconic acid and its salt. According to some accounts, A. niger also contains the enzyme glucono lactonase, which accelerates the conversion of glucono-d-lactone to gluconic acid. The GOD activity is closely related to the production of gluconic acid. It is advised to remove any lactone that has accumulated in the medium because it slows down the oxidation of glucose and the creation of gluconic acid and its salt [29]. GOD activity is intimately connected to gluconic acid synthesis. Depending on the purpose, fermentation broths containing sodium gluconate or calcium gluconate are generated by adding solutions of sodium hydroxide or calcium carbonate for neutralization. The concentration of weak organic acids produced by A. niger, such as citric acid, gluconic acid, and oxalic acid, is dependent on the medium’s pH [30].

7.3.3 Fermentative production of gluconic acid using wastes The simplest and most effective approach in terms of economics for producing gluconic acid from glucose is fermentation. It also has a number of drawbacks, including challenges with by product formation control, waste water disposal, and the separation of microorganisms and product [31]. There are some issues and drawbacks associated with the current fermentative gluconic acid and gluconate production, which in addition to the material cost generates significant amounts of sewage materials in the millions of gallons of industrial and biological wastes, including biomass and leftover mother liquid [32]. Having to recycle, further purify, or dispose of the sewage raises the cost of production. Traditional fermentation techniques also call for a lot of resources, a wellorganized infrastructure, as well as sewage disposal facilities. Continuous reductions in manufacturing costs and increases in production efficiency are required due to the rising demand for ecologically safe gluconate for use in industrial and pharmaceutical applications [33]. Process synthesis and analysis are the two main components of process design. Process synthesis entails choosing and organizing a set of unit operations (process steps) that can produce the desired product at a reasonable cost and quality, while

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process analysis involves assessing and contrasting various process synthesis solutions [34]. Three steps are necessary for creation of novel multi-step biotechnological methods: 1. Developing and presenting an effective biological system (microorganism, biocatalyst). 2. Productivity improvement for bioreactors by analytical media modification and process-specific fermentation technology adaptation (process development and fermentation technology). 3. Downstream method (cell separation by centrifugation or ultrafiltration, separation, evaporation and drying). Gluconobacter and A. niger are production strains, particularly A. niger and Gluconobacter suboxidans, are primarily used to produce gluconic acid in discontinuously operating fermentation processes. Fermentation, cell separation by centrifugation or ultrafiltration, product separation and purification, evaporation, and drying are all part of this multi-step process. A. niger can block pipes and is not suitable for continuous development since chemostat culture would not allow for simultaneous cell growth and gluconic acid production. However, it has been discovered that Gluconobacter produces a sizable amount of keto-acids during synthesis, which makes gluconic acid processing and isolation are more difficult [35]. According to Figure 7.2, fermenter containing sodium gluconate can be purified in various ways depending on the kind of product. In order to obtain technical grade sodium gluconate (98 %) purity, mycelium from the fermentation was removed by filtration, then decolorized using a granular activated-carbon column and concentrated under vacuum dryer to 40–50 % of total solids, using NaOH to get

Figure 7.2: Schematic presentation of typical conventional fermentative method for downstream processing and purification of gluconic acid. Reprinted with permission from Ref. [35].

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sodium gluconate, and dried using spray or drum dryer. Thus, the calcium salt of gluconic acid crystallizes and is removed from the fermentation broth to get pure gluconic acid. These crystals are then retrieved and refined further. Again, calcium gluconate is precipitated in hyper saturated solutions, and sulfuric acid is stoichiometrically added to remove the calcium. Another technique for creating pure gluconic acid is ion exchange, which involves sending calcium gluconate via a cation exchanger where calcium ions are absorbed [36]. Some studies have been conducted on the bioconversion of waste paper to gluconic acid by the filamentous fungus R. oryzae, with the production rate being limited by either xylose generated from hemicellulose or unknown chemicals originating from paper pulp. The gluconic acid yield and production rate utilizing waste paper hydrolysate were compared to those obtained with a glucose medium in a flask and bioreactor using the filamentous fungus A. niger [37]. Potato pulp, the industrial byproduct of potato processing for starch manufacturing, can be utilised to make biochemicals. The method for gluconic acid production from potato pulp is environment friendly, sustainable, and extremely efficient. Cellulase cocktails made from Penicillum oxalicum and Trichoderma reesei cellulases were combined with commercial pectinase to hydrolyze hydrothermally treated potato pulp into fermentable sugars. Glucan in potato pulp could be transformed into glucose. The overall glucose to gluconic acid conversion yield was 94.9 %. Finally, this procedure yielded 546.48 g of gluconic acid per kg of dry potato pulp. To achieve higher hydrolysis efficiency with the potato pulp, different enzyme systems and their mixes were utilised, and the hydrolysates were fermented by Gluconobacter oxidans to create gluconic acid [38].

7.4 Application in industry and biomedicine In the creation of foods, medications, and cosmetics, glutamic acid and its esters and salts are frequently employed. It is a component of leavening agents for preleavened items and is utilised in meat, dairy products, and especially baked goods. It serves as an ingredient in flavors (for example in sherbets). It is also used to doughnuts and cones to lessen the absorption of fat [39]. Additionally, it is employed in food pickling. D-glucono-δ-lactone can be found in a variety of foods, including beef, yoghurt, cottage cheese, bread, and bean curd. In bottle washing formulas, sodium gluconate acts as a detergent and aids in both the prevention of scale formation and its removal from glass [40]. Gluconic acid compounds have acidity-regulating actions such as raising, sequestering, hardness, and flavour enhancement. GA avoids clouding by binding various metals potentially present at trace amounts in drinks, such as Ca and Fe in fruit juices, in addition to improving the sensory characteristics of food goods by imparting a bitter but pleasant taste. GA is also employed as a preservative in pickled foods, and glucono-δ-lactone is utilised in cured meat-based sausages. Furthermore, certain food processing industries use GA derivatives as industrial cleaning agents. As a result, alkaline sodium gluconate solutions are used to

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clean glassware, but GA is preferable for metal (steel, alloyed) components. The dairy industry, for example, employs GA derivatives to avoid calcium salt precipitation in processing equipment and glass storage vessels. Furthermore, the bakery sector employs glucono-δ-lactone as an acidifier and chemical baker’s yeast to reduce fatty compound absorption [41]. Due to its sequestering ability over a wide pH range, it is utilised in the textile industry to avoid the iron coating. Desiring polyester and polyamide fabrics also uses it. Sodium gluconate is used in metallurgy to remove metal carbonate precipitates without producing corrosion, wash painted walls, and alkaline de-rust metal [42]. It serves as a cement ingredient that regulates the setting time. Additionally, it aids in the production of frost and fracture resistance and boosts the cement’s strength and water resistance. The treatment of water and the paper industry are some more applications [4]. Supplements containing to prevent anemia and a calcium shortage, the calcium and iron salts of gluconic acid are utilised. Additionally, calcium gluconate is used in animal feeding. In iron therapy, iron gluconate and iron phosphogluconate are employed. Zinc gluconate is used to treat zinc deficiency symptoms such delayed sexual maturation, mental drowsiness, skin abnormalities, and infection susceptibility [43]. Additionally, it is a component of medicines used to treat wounds and the common cold. Ramachandran et al. [3] described the ability of GA to sequester iron over a broad pH range is utilised in the textile sector to prevent the deposition of iron and to resize polyester and polyamide fabrics. Additionally, in metallurgy gluconic acid is utilised to remove metal carbonate precipitates without producing corrosion, wash painted walls, and perform alkaline degusting. In the pharmaceutical business, Calcium gluconate is utilized as a calcium source to treat calcium shortage through oral or intravenous delivery. Common colds, wound healing, and a number of illnesses caused by zinc deficiency, including delayed mental drowsiness, sexual development, skin changes, and susceptibility to infections, are all treated with zinc gluconate [44].

7.5 Conclusions Gluconic acid has numerous applications in the field of culinary, pharmaceutical, textile, and building sectors. Traditionally, some fungal species and acetic acid bacteria have been used in technical processes to produce GA. Wastes with a high sugar content have been used as raw materials for GA production with improved techniques due to the need to increase the efficiency and profitability of current biotechnological processes and the rising volumes of agro-industrial residues produced worldwide. The possibility of culminating in the development of a small, compact, flexible, and energy-saving plant capable of offering green production while maintaining an attractive profit margin. A conventional oxidation process for the manufacturing of gluconic acid is required that can be accomplished via electrochemical, biochemical, or a mixture of the two ways, although the fermentation process employing fungus and bacteria is the most widely used and economically proven approach. Future advancements in genetic engineering

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and the use of novel molecular biology techniques may enhance the great prospects in this field. Further the use of waste raw materials for large scale production of gluconic acid needs to be investigated and techniques to be developed. Acknowledgments: Authors (MR, MS, VK, PM, SKU, SK, MY, NS, MK) acknowledge the help and support by Head, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, India.

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Meena Sindhu*, Shikha Mehta, Shubham Kumar, Baljeet Singh Saharan, Kamla Malik, Monika Kayasth and Sushil Nagar

8 Itaconic acid: microbial production using organic wastes as cost-effective substrates Abstract: Itaconic acid is one of industrially important organic acid having wide application in environmental protection, food and textile industries. Microorganisms mainly fungi have vast potential to be exploited for itaconic acid production. But low yield and higher cost of production are major drawback creating a settle back for industrial production. This problem can be solved by using low cost organic waste as substrate. This review summarizes recent research on production of itaconic acid using organic wastes, microorganisms involved, extraction, application and problem faced during utilization of agro-industrial wastes. Keywords: itaconic acid; fungi; microorganism; organic waste; industry

8.1 Introduction Microorganisms are an important cell factory, which can be used for synthesis of industrially important valuable products [1]. Microorganisms are easy to grow, can be easily manipulated using genetic modification and can be used for product formation with in short span. Synthesis of value added chemical using microbes is green alternative as compare to production using petroleum based products. It will help in solving the problem of fossil based depletion and will also help in environmental protection [2]. One of industrially important organic acid is itaconic acid (IA) which can be synthesised using microorganisms mainly fungi. Structure of IA consists of double bond and two carboxylic acid groups, which provides it property to be used as monomer for synthesis of polymers such as papers, resins, plastics and super-adsorbent polymers [3, 4]. It is an alternative for petroleum-based monomer for production of important polymers [5, 6]. Demand for IA is increasing in world market as more policies are focused towards usage of natural products for sustainable development. Global market demands for IA

*Corresponding author: Meena Sindhu, Department of Microbiology, College of Basic Science & Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, 125004, India, E-mail: [email protected]. https://orcid.org/0000-0002-8755-4705 Shikha Mehta, Shubham Kumar, Baljeet Singh Saharan, Kamla Malik and Monika Kayasth, Department of Microbiology, College of Basic Science & Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, 125004, India Sushil Nagar, Department of Biochemistry, College of Basic Science & Humanities, Chaudhary Charan Singh Haryana Agricultural University, Hisar, 125004, India As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: M. Sindhu, S. Mehta, S. Kumar, B. S. Saharan, K. Malik, M. Kayasth and S. Nagar “Itaconic acid: microbial production using organic wastes as cost-effective substrates” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0164 | https://doi.org/10.1515/ 9783110792584-008

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valued at $96 million in 2019, which has been increased to $116.6 million till 2026 having CAGR of 2.8 % (Global IA Market). Presently, IA is mainly synthesized using microorganisms such as bacteria, fungi and actinomycetes. Among all the microbes, most investigated fungi are Aspergillus terreus strain, one of the prevalent hosts for bio-production of IA, reaching titres up to 129 g/L [7]. In industry, the main problem faced with IA is lesser production and higher cost of production, therefore, it is not able to replace petroleum synthesised chemicals. So, to make it economically competitive, there is need to find inexpensive material such as agricultural and industrial waste for production of industrially important organic acids [2] to reduce production cost. Agriculture wastes include agriculture processing residues such as straw, baggase and other processing residues and industrial wastes include residue after processing, waste water etc. Need of the hour is to find such microbes, which can efficiently utilize agricultural and industrial wastes for production of IA. Such strains can be obtained using biotechnological and other bioengineering approaches [8]. Polymer from IA can be used as superabsorbent for removal of heavy metals from water [9] and can be used in pesticides, anti-microbial applications, synthetic resins and pharmaceuticals [10, 11].

8.2 Application of IA Being highly reactive in nature, IA has ability to undergo polymerization along with other polymers as acrylonitrile and can undergo esterification and acetylation [12]. There are various applications of IA as mentioned in Figure 8.1:

Figure 8.1: Application of IA.

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1. Water purification: Contaminants of water cause damage to soil, surface water, ground water, which ultimately lead to negative impact on soil, ecological and environmental health [13]. Film made by IAs had improved characteristics, like enhanced tensile strength, impermeable to oxygen, heat tolerance, so can be mixed with various polymers such as chitosan, guar gum and nanoparticles used for water purification [14]. Mixing of IA with guar gum increased extraction of pollutants, like coal particles (81.36 %) and kaolin (88.15 %), reducing biological oxygen demand, alkalinity and hardness with respect to commercial flocculants [15]. Sharma et al. in their study developed a hydrogel chitin-cl-poly (IA-co-acrylamide)/ zirconium tungstate an efficient photocatalyst-absorbing nanocomposite for removal of sulphon black dye [16]. Similar nano-hydrogel was developed by Sharma et al. for removal of pesticide atrazine from water having adsorption efficiency of 204.08 mg/g [17]. Heavy metal present in water such as cadmium, zinc, mercury and arsenic is very toxic for aquatic animals. Hydrogel composed of IA mixed with poly acrylic acid was manufactured having detection limit 3 ppb of Cd (II). 2. Antimicrobial polymers: Polymer synthesised using IA have anti-microbial property against Gram + ve bacteria, so can be used for treatment of drug resistant bacterial infections and biofilm removal. Prabakaran et al. 2020 in their study integrated IA with ricinoleic acid and α, ω-aliphatic diols and found it effective against bacteria [18]. Some polymers have anti-cancerous activity. 3. Food packaging: Polymer for food packaging should have antimicrobial and antioxidative properties along with biodegradable nature. Incorporation of IA with food packaging help in improving its bioactivity, so it can be used as food preservative. IA was incorporated into gelatin films along with dopamine as an active ingredient, and biofilms were found to have anti-microbial activity against Staphylococcus aureus (0.5 mg/mL), Pseudomonas aeruginosa (2–4 mg/mL) and E. coli (8 mg/mL).

8.3 Microbial production of IA Many microorganisms such as bacteria, fungi and yeast have been reported to produce IA, among them major producer are fungi such as A. terreus and filamentous fungi Ustilago maydis [19, 20]. Major yeast species include Candida sp., Rhodotorula sp. and Pseudozyma antarctica [21, 22]. A list of low cost substrates along with microorganism used is mentioned in Table 8.1. Most of these fungi are pathogenic, during IA production; pH is decreased, which enhances its pathogenic characteristics. These fungi can be used for decomposition of agricultural wastes by producing different hydrolytic enzymes, which can be further converted into value added products using solid state fermentation. Yeast is more preferred for production as they can produce acid in shorter time period as compare to fungi [23, 24]. Metabolic performance of the microbial culture also affected by cell morphology resulting in lower yield. Production of IA can be further enhanced by optimizing cultural conditions such as temperature, pH, aeration and media composition

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Table .: List of low cost substrates used for IA production. Substrate

Microorganism

Corn starch

Aspergillus niveus MG Palm kernel cake Aspergillus terreus (ATCC ) Aspergillus niger (ATCC ) Rayon waste material Escherichia coli Lactobacillus paracasei Aspergillus niger Sweet potato peel Aspergillus niger (ATCC ) Aspergillus terreus (ATCC ) Potato waste Aspergillus terreus strain C Wheat chaff Aspergillus terreus DSM  Purified glycerol and Aspergillus niveus algal biomass hydrolysate Wheat A. terreus ATCC bran hydrolysate  Rice husks Aspergillus terreus (ATCC )

A. terreus NRRL  Food waste (potatoes, Aspergillus terreus rice & noodles) BD strain

Production yield (g/L)

Optimum conditions

. ± .

 h incubation time and pH  pH .; substrate concentration of  g; inoculum size of  % at  ±  °C on day 

. .

References [] []

. IA, . lactic – acid and . chitosan

[]

. + .

–

[]

[]

. ± .

 rpm; aeration rate . vvm;  °C; pH . pH .;  °C;  FPU/ gBiomass  h;  °C;  rpm

. ± .

–

[]

.

 °C; pH .; inoculation of  ×  CFU mL− cellular concentration;  rpm pH .;  °C;  rpm

[]

 % biomass loading;  % enzyme concentration; pH .;  °C

[]

. + . . .

Wheat straw

.

[] []

[]

[25, 26]. Nutrient limitation is also one of important factor affecting IA production. In filamentous fungi, phosphate is one of the limiting factors for IA production along with carbon and nitrogen [27]. IA can be produced using solid state as well as submerged fermentation. Brief process for industrial production of IA is mentioned in Figure 8.2.

8.3.1 Submerged fermentation (SmF) In industry, IA is produced using submerged fermentation majorly. In this method, due to shaking conditions, there is sufficient amount of nutrients and dissolved oxygen. Lack of

129

8.3 Microbial production of IA

Figure 8.2: Industrial production of IA.

oxygen leads to negative impact on IA production as mycelia got damaged due to oxygen deficiency. IA production is enhanced under deficient nitrogen conditions. There is need to explore such microbes having ability to produce higher amount of IA without any feedback inhibition. Moreover, microbe should have ability to tolerate stress condition such as high temperature, pH and shear stress.

8.3.2 Solid state fermentation (SSF) Low cost agricultural and industrial wastes are potent substrate for production of IA using solid state fermentation. Fermentation media should be simple, so that it can be optimized to enhance production. Other parameters such as temperature, aeration and Table .: Organic wastes used as substrate for IA production along with their production yield. Substrate

Microorganism

Production yield

A. oryzae . mg of IA U. maydis (MTCC . g/kg No-) Oil palm empty Aspergillus terreus K . g/L fruit bunch Bamboo Aspergillus terreus . g/L residues AtYSZ- Corn cobs A. oryzae . mg of IA/g of biomass

Corn cob Orange pulp

Optimum condition

References

pH ;  % humidity after  h pH ; moisture  %;  °C; incubation period of  days  °C; pH .; aeration rate .

[] []

 °C;  rpm after  days; pH .  days

[]

[]

[]

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8 Itaconic acid: microbial production

moisture should also be maintained. If moisture is below optimum during solid state fermentation, nutrients will not be available to microbes and if moisture is high, it will negatively affect the aggregation of substrate. High temperature is required for spore germination and production of IA. SSF provides better yield as no foam is produced. Moreover, chances of contamination are very less [37]. IA production using solid state fermentation of some readily available organic wastes have been listed in Table 8.2.

8.4 Low cost organic waste as substrate for IA production IA was produced by synthetic chemical processes, such as citric acid pyrolysis, heating to decarboxylate aconitic acid, carboxylating acetylene, oxidising isoprene, oxidising mesityl oxide etc. The main drawbacks of these techniques, however, include being timeconsuming, unsustainable, and impractical for industrial production with environment detrimental effect [42]. In contrast, it was discovered that the biological pathway was more advantageous in terms of production rate, yield, sustainability, ease of use, power consumption along with other economic considerations [43, 44]. As per Van Dien’s findings, bio-based manufacturing techniques should be efficient enough to outperform traditional fossil-based production techniques for the commercial manufacture of important chemicals. A bio-based manufacturing process must produce at least a final titre of 50 g/L, 80 % of the theoretical yield, with a production rate of 2 g/L/h in order to compete successfully in the commercial market [45]. Therefore biological production via low organic waste as substrate could be a more efficient and cheaper method. The selection of low cost substrate is another obstacle as it should have following characteristics: i. Comparatively cheaper ii. Throughout availability in whole year iii. Interchangeable compatibility iv. Specificity and/or compatibility towards the microbial strain used v. Low or no pre-processing vi. Should not be sources of animal or human food to avoid ethical issues Considering these points at priority, many substrates have been used over the time by the researchers for the production of IA. These substrates majorly include glucose, mannose, galactose and D-xylose. These substrates have been found yielding maximum IA as compared to other substrates which includes glycerol, beech wood, wheat flour, corn starch etc. Lists of substrates that have been found efficient for the production of IA have been listed in Table 8.3.

131

8.5 Microorganisms involved in production of IA using organic waste

Table .: Different substrates along with microorganism used and amount of IA produced. Microorganism

Substrate used

Method of fermentation

A. terreus DSM  A. terreus NRRL  A. terreus NRRL  A. terreus DSM 

Glucose D-Xylose Glucose Galactose Mannose Glycerol Beech wood Glycerol Bleached eucalyptus pulp Wheat flour Corn starch Sweet potatoes Corn stover hydrolysate Pretreated rice husk Food waste extract

Fed batch Submerged Biofermentor Shake flask

U. vetiveriae TZ A. terreus A. terreus DSM  A. terreus NRRL  A. niveus MG

Aspergillus terreus M A. terreus ATCC  A. terreus m. N

Submerged Batch bioreactor Stirred tank reactor Submerged Submerged

Submerged Submerged Submerged

Amount of IA produced (g/L)

References

 . . . . . . . . . . . . . –

[] [] [] [] [] [] [] [] []

[] [] []

8.5 Microorganisms involved in production of IA using organic waste 8.5.1 Aspergillus strains Itaconic, gluconic, kojic and citric acids are among the organic acids produced industrially by the Aspergillus species of filamentous fungi [55, 56]. There has been much research into and use of the biotechnology for IA production using Aspergillus strains, particularly A. terreus, at both the laboratory and industrial scale employing a variety of substrates and processing conditions. Due to A. terreus’ superior synthesis capabilities, it has been producing IA through glucose fermentation since 1955 [44, 57]. After two days of cultivation, Hevekerl et al. got a total of 146 g/L IA by shifting the pH later studying the impact of pH on production efficiency [58]. According to Kuenz et al. it was discovered to be 68 % higher than the already reported concentration of 90 g/L regardless of pH optimization [27]. Using the A. terreus DSM 2308, Krull et al. created a procedure that depends on timely and precise pH control could produce the maximum of 160 g/L IA titer [46]. There have also been further studies on the effects of media formulations and/or composition, sources of nitrogen, supply of oxygen, fermentation methods, and recovery procedure on A. terreus’s IA yield [12, 27, 36, 43, 59–61]. The most effective substrate with regard to strain growth, production, and simplicity of microbe metabolism is glucose, although it is not the most economical. Another high-yielding but costly substrate is sucrose [19, 43]. Numerous investigations were carried out to identify low-cost yet

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8 Itaconic acid: microbial production

effective substrates to lower the cost of fermentation. According to Juy and Lucca, A. terreus MJL05 produced IA with glycerol as the only source for carbon, and with further medium optimization, they obtained final titer of 27.6 g/L [62]. Saha et al. have tested 100 A. terreus strains and attempted to ferment the biomass constituent’s hexose viz; mannose and galactose and pentose viz; xylose and arabinose sugars [63]. Although the yields from these monosaccharides were relatively large, they were shown to produce less IA than glucose [64]. Because they don’t need additional hydrolysis stages like polysaccharides do, monosaccharides are more desirable sources of carbon for fermentation [41]. Starch is the finest polysaccharide substrate because it may be hydrolyzed by enzymes or acids in the bioreactor to produce glucose. IA has been produced utilising a variety of sources of starch, but utilising foods-based substrates for industrial chemical production raises ethical concerns and causes friction with the food sector [65, 66]. The fermentation of several raw starchy substances (flour made from cassava, wheat and potato, sweet potato, commercial maize starch and potato flour) using A. terreus NRRL 1960 was investigated by Petruccioli et al. [67]. The highest yield of IA was obtained by using starch waste of potato, as per a study based on the starch-rich waste fermentation from industries (rice, potato and maize). Increased yield was achieved by further deionizing potato starch [11]. In a different investigation, A. terreus SKR10 was used to ferment hydrolysate of amylase and nitric acid from corn starch, yielding, respectively, 31 and 28.5 g/L IA [54]. In a recent work, IA synthesis via an enzymatic hydrolysate of synthetic food residue i.e., potato, rice and noodles was examined using a newly obtained thermo tolerant strain of A. terreus BD. The resulting titer of IA and its yield production derived from starchy food waste was found to be 41.1 g/L and 0.27 g/L, respectively which were in stark contrast to the yield of pure glucose i.e., 44.7 g/L and 30 g/g, respectively [7]. This strain demonstrated excellent IA production at high temperature (45 °C). IA can be produced by microbial fermentation using solid-state or submerged fermentation, both of which use lignocellulosic biomass as an affordable carbon source. The complex and tenacious character of biomass substrate, however, necessitate substantial pretreatment [41]. When fermentation inhibitors are present in biomass hydrolysates, A. terreus is very sensitive to them. Tippkotter et al. found that pretreatment of organosolv beech wood hydrolysate (for removal of phenolic and ionic compounds) is necessary for the growth of A. terreus NRRL 1960 [68]. Due to the unavailability of lignin and other inhibitory substances, Kerssemakers et al. employed Eucalyptus cellulose pulp obtained from paper industry as feedstock produced a significant quantity of IA [69]. In other research, agricultural biomass for producing IA was used, including bran, husk, straw, stalk, maize stover, corn cob, bamboo residues, sugarcane baggase etc. [41]. Other Aspergillus strains besides A. terreus have also been identified as having the capacity to naturally biosynthesize IA. Simultaneous saccharification and fermentation can be done using Aspergillus oryzae as this strain provides excellent supply of hydrolytic enzymes along with IA synthesis [70]. Using an isolated fungal strain, Aspergillus japonicas, Ramakrishnan et al. performed solid-state fermentation on the rind of Citrullus lanatus [71]. In a different investigation, the soil-isolated Aspergillus niveus MG 183,809 was employed

8.5 Microorganisms involved in production of IA using organic waste

133

to produce IA using economical substrates (corn starch, wheat flour and sweet potato), with corn starch yielding the highest titre of 15.6 g/L [28]. A. niveus was also used to produce IA using glycerol from the biodiesel industry and ultrasonically hydrolyzed algal biomass, yielding an overall titer of 31.5 g/L [33]. Aspergillus flavus which was isolated and grown from soil sample, later evaluated by Sudarkodi et al. for its ability to produce IA, and under ideal conditions, it produced 8.8 g/L of the acid [72]. Despite having numerous positive traits, A. terreus has a number of negative ones as well. A. terreus produces a large number of byproducts and has a long fermentation period thus making the process more expensive. The fungus grows in a controlled manner in the best medium for producing IA. Additionally, the growth is slowed down by the shear stress. As a result, the fermentation continues to be unsuitable for the standard conditions [73]. However, the titer values compared to different native Aspergillus spp. are generally quite low. Due to increased medium viscosity and sensitivity to harsh circumstances, the filamentous shape of these fungi additionally adds to the handling challenges [73]. Additionally, filamentous fungi have a propensity to aggregate into globules when disturbed, which prevents oxygen from reaching the cells at the centre of the globular mass.

8.5.2 Ustilago strains A genus well known for parasitic smut fungi called Ustilago infects grasses, plants and agricultural products. Due to their capacity to create high-value compounds like IA, many species of this genus are economically significant [74]. A total of 15 g/L of IA was produced as the metabolic byproduct in the submerged cultivation of fungus, and Ustilago maydis’ synthesis of IA was first documented in 1955 [75]. U. maydis has been a greatest choice for large-scale fermentation devoiding the issues related with filamentous fungus due to its ability for efficient IA production and morphology like that of non-filamentous yeast [76, 77]. In order to produce IA from glucose through submerged fermentation, Rao et al. employed a strain of U. maydis and investigated the influence of various circumstances on yield [78]. At 34 °C along with pH 3.0 and 4 % (w/v) glucose concentration, they were able to achieve the highest yield i.e., 29 g/L in 5 days. In a membrane bioreactor, “reverseflow diafiltration” was used by Carstensen et al. to recover the product from continuous fermentation for the production of IA using U. maydis MB215 [79]. There have been studies done in the past that involved U. maydis fermenting biomass substrates to make IA. William et al. found that Pseudozyma antartica NRRLY-7808 strain under limiting nitrogen conditions produced IA from carbohydrates and other sugars [22]. Klement et al. during accessing the effect of pretreatment of biomass found that production of IA demands nitrogen-limited conditions, while product inhibition occurs at IA levels higher than 25 g/L [77]. Among the U. maydis strains tested by Maassen et al. U. maydis MB215 produced the most IA [80]. With glucose and xylose substrate in identical circumstances, it produced similar results. A maximal rate of 0.74 g/L/h was used to create 44.5 g/L of IA in a medium containing 200 g/L of glucose and 75 mM of ammonium chloride. The IA

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8 Itaconic acid: microbial production

production by other Ustilago spp., including Ustilago cynodontis 2217 (3.4 g/L), Ustilago xerochloae 2221 (0.05 g/L), and Ustilago vetiveriae 2220 (0.05 g/L) along with U. maydis strains, was reported by Geiser et al. after screening 68 Ustilaginaceae strains [74]. In a different investigation, itaconic synthesis utilising glycerol as a substrate was reported for U. vetiveriae RK 075 and U. xerochloae UMa702. After 21 repetitions, the U. vetiveriae began producing IA. According to Zambanini et al. the finest single colony (U. vetiveriae TZ1) was isolated and generated 34.7 g/L of IA using glycerol [50]. According to a study, Ustilago rabenhorstiana is a potent natural generator of IA that can grow on various monosaccharide substrates and is unaffected by weak acids in the media. From glucose media, a maximum titre of 50.3 g/L was produced [81].

8.5.3 Other natural producers There are also other fungi that can naturally make IA, according to research. According to Tabuchi et al. out of 140 isolated yeast strains, Candida sp. produced IA with a 35 % yield when phosphate availability was limited [82]. Another naturally occurring producer that produced a considerable quantity of IA (30 g/L) in nitrogen-limited conditions was Pseudozyma antarctica NRRL Y-7808 which is a non-pathogenic Ustilaginomycetes closely related to Ustilago sp [83]. IA productivity has also been noted in Rhodotorula sp., Helicobasidium mompa and other species [43, 84].

8.6 Extraction of IA IA must be separated from the fermenting culture prior to decolonization and drying, frequently through filtration and crystallization. With an average yield of roughly 80 %, IA is mostly recovered during crystallization. Alternative processes to crystallization include liquid–liquid extraction, precipitation, adsorption and membrane separation.

8.6.1 Biomass removal Substrates are transformed into IA through fermentation, and the resulting broth is made up of biomass, various organic acids, their minor constituents, and fermentation medium leftovers. Typically, the concentration as well as purification of the acid to the required level comes after the removal of the biomass in the downstream process. IA synthesis depends on the initial concentration of acid (recovered from the broth). High final concentrations and reduced throughput reduce net operating and equipment expenses [85]. The most used purification methods for IA are summarized in Table 8.4.

Procedure

Advantages

Cooling followed by evaporation in acidic environment A quick and easy method yields a pure product, but it requires The crude crystals are solubilized and treated with activated carbon at expensive machinery and plenty of energy  °C Precipitation Precipitation using calcium and lead salts Cost of the reagent is low and/or it can be recycled contributing towards simplicity and feasibility of the practical Liquid–liquid Solvents like long-chain alkanes, esters and alcohols are used Compared to water, organic solvents have a lower differential extraction coefficient Reactive extraction Using diluents and extractants like organophosphorus compounds, High productivity helps maintain pH without adding basic solutertiary or the quaternary amines tions, increases substrate to product conversion, and can improve the energy efficiency of continuous processes Membrane Diafiltration with reverse flow. By utilising a concentration gradient Reverse flow direction along with wash solution prevents perforseparation and permeable membranes mance loss and maintain a constant volume;  % pure IA is recovered; minimizes hydro-mechanical stress that leads to membrane wear and the risk of oxygen limitations; produce a product stream through hydrophilic cultra-filtration with the hollow-fiber membrane submerged in the bioreactor Adsorption Using various kinds of synthetic ion exchange resins, alumina, acti- In contrast to extraction, it is more selective and lacks solvent vated carbon, silica, and other materials emission low energy requirement in comparison to membrane separation and crystallization Electrodialysis IA is recovered using ED and univalent electrolytes, which transform It is possible to use ED in fermentation while simultaneously the acid to create a disodium salt. The solute recovery yield for removing IA. IA can be made from Itaconate salt using electrodiItaconate anions passing through the anion membrane is  % alysis with bipolar membranes (EDBM), which can also be used to regulate the fermenter’s pH

Crystallization

Method

Table .: Methods for purification of IA.

[]

[]

[]

[]

[]

[]

[]

References

8.6 Extraction of IA

135

136

8 Itaconic acid: microbial production

8.6.2 Product Isolation and purification 8.6.2.1 Crystallization and precipitation IA generated by fermentation is typically recovered through crystallisation. It has been found when two fermentation broths comprising glucose and digested sago starch were used to create IA by crystallisation. The yields of the hydrolysates of sago starch was nearly double than glucose. Advancement in research strategies has asserted that industrial IA synthesis by crystallisation has an overall product yield of 80 % [86]. Although it is expensive and energy-intensive, it is quick and efficient. However, a pure product is produced. Calcium and lead salts precipitation can also be used for IA recovery [4]. Calcium hydroxide is used in a different IA precipitation technique. This process produces calcium itaconate, which is recovered by filtration. The calcium itaconate remains less soluble compared to the acid it precipitates. Calcium itaconate and sulfuric acid can be used to regenerate IA, which is then further refined with activated carbon and crystallisation. However, this recovery method produces a sizable amount of waste calcium sulphate sludge. Precipitation is feasible only when chemical costs are low enough or when recycling is practical, as in the case of CaSO4, that requires costly calcination to CaO and SO3 [89]. 8.6.2.2 Solvent extraction Due to IA’s weak distribution coefficient, which means that it is only slightly more soluble in organic solvents than in water, esters, long-chain alcohols and alkanes are useless as solvents for IA purification [87, 89]. In order to purify IA, tertiary or quaternary amines or compounds containing organophosphorus could change the distribution coefficient. Reactive extraction (RE) is the name given to this technique. The developed acid-extractant mixture has a high affinity towards the organic phase (solvents referred to as diluents). This compound’s acid could be extracted, and the extractant recycled and employed in a second extraction (back-extraction) are both options. When employed with specific extractants and diluents, RE has been examined and shown to be more successful at organic acids recovery [87]. Aliphatic amines and organophosphates have been studied as solvents for extracting IA from the aqueous phase for their thermal stability and simplicity of recovery by simple distillation [90–93]. 8.6.2.3 Electrodialysis Ions in solution can be simultaneously removed and absorbed by electrodialysis (ED), which is an exciting method for IA purification [94]. IA is separated from various residues in the fermented broth by the exchange of itaconate ions when an electric field

8.7 Problems associated with IA fermentation from agrowaste

137

is applied to the anion exchanger membranes. The non-fermented sugars can be used by recycling the dialysate that results. ED works in a low-temperature setting and doesn’t require heating or hazardous chemicals [89]. Monad elements (univalent) electrolytes are employed in ED to convert IA into di-sodium salt and recover around 98 % of IA; however, the main drawbacks of ED include the high price of the membrane used, membrane fouling, and the lack of ability to separate charged nutrients and inorganic ions [95]. 8.6.2.4 Membrane filtration In situ product recovery (ISPR), a desirable technology for developing a financially viable and petrochemically advantageous biotechnological process for IA production, comprises the combination of fermentation with large-scale reactive extraction or recovery and downstream units [87]. Reverse-flow diafiltration (RFD), a new ISPR approach developed by Carstensen et al. employing a membrane bioreactor [67]. A ten-fold increase in production was achieved by using RFD for IA manufacturing from U. maydis and A. terreus fermentation systems in conjunction with pertraction-based IA purification [96]. There always needs to be a recycling phase because a single crystallisation stage cannot recover all of the products in a stream. As a polishing process, crystallisation is used to crystallise previously concentrated solutions, which reduces the cost, size and energy requirements [97]. Because adsorbent is expensive, adsorption has a comparatively high capital cost for generating a lot of compounds. However, it contrasts favourably with extraction because it is more selective and doesn’t produce solvent emissions. It also contrasts favourably with membrane and crystallisation processes in terms of energy consumption and can be applied to fermenters for improved substrate transformation by-product sequestration.

8.7 Problems associated with IA fermentation from agrowaste IA is an unsaturated dicarboxylic acid that serves as building block for the synthesis of several valorized products such as surfactants, lubricants, detergents, resins, poly IA etc. [8]. Due to depletion of fossil fuels at an alarming rate, attention is being given to the generation of bio-based IA which is environment friendly as well as acts as cleanest alternative to petroleum based products which are non-sustainable [98]. However, bioproduction of IA still faces several challenges/bottlenecks such as high production cost, inability to use raw materials and low production efficiency (Figure 8.3) [99].

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8 Itaconic acid: microbial production

produc on of Inhibitory substances during pretreatment and hydrolysis

High viscosity and low oxygen transfer due to mycelial growth

Problems associated with IA produc on from agro waste

Inhibi on by metal ions and nutrients such as phosphates

Inhibi on of microbial strains due to fluctua ons in pH

Figure 8.3: Bottlenecks associated with IA production.

8.7.1 Effect of substrate and microbial strains Glucose and sucrose are the prominent substrates/carbon sources for the production of IA by fermentation using A. terreus [100, 101]. One constraint with the use of pure monosaccharides and disaccharides is the expensiveness of these substrates which ultimately leads to high production cost of IA so exploration towards cheaper carbon sources is essential to make the process cost effective. Several renewable carbon sources have been identified to replace glucose and sucrose for IA production and these include food waste, lignocellulosic wastes, glycerol, soft wood etc. [41, 99]. Soft wood derived sugars such as mannose, glucose and galactose serve as low cost source of sugars for its production [63]. Food waste extract, pretreated rice husk, starchy food waste, corn cobs, wheat bran etc. can also serve as economically feasible raw materials in terms of economy [86]. Starch has been found as one among the best alternative to glucose because of its low cost, high purity as well as stability. Delidovich et al. reported yield of IA upto 0.35–0.37 g/g starch by using corn starch as substrate saccharified with enzyme or acid [10]. IA has been synthesized directly by [28] with the maximum yield approximately 16 g/L of corn starch, sweet potato and wheat flour using A. niveus strain. Starches from sorghum, potato, sweet potato, wheat etc. are generally used for the production of IA but there are several problems associated with their uses as these are food based substrates so leads to food scarcity and high production cost issues and creates conflicts with food applications [19]. In this regard, lignocellulosic materials (agricultural or forest residues) can be a good source for IA production as these are widely distributed, sustainable and renewable. The

8.7 Problems associated with IA fermentation from agrowaste

139

use of agro-wastes can also lower down the production cost which was major constraint by using food based substrates. Lignocellulosics are generally pretreated in order to remove lignin as well as to produce hydrolysates in order to generate simple sugars which can then be utilized by the microbes. Several methods have been employed for pretreatment like physical, chemical, biological and combined pretreatment. Choice of reasonable pretreatment method can help to improve the digestibility and assist in enzymatic hydrolysis in an economical way [102, 103]. Detoxified phosphoric acid hydrolysate of rice bran was used to synthesize IA and 1.9 g/L IA was produced with a yield of 49 mg/g of sugar [56]. Forest residues can also serve as potential substrate for IA production. U. maydis can utilize cellulose and hemicellulose in the hydrolysate of beech wood [77]. Several issues have been faced by the use of these complex substrates for IA production. The yield of the product from the recalcitrant substrates is generally lower than the sugars. Moreover, the production time increases as these are firstly pretreated to obtain hydrolysates. In addition to this, the chemicals which are used for pretreatment negatively affect the production process [19]. The pretreatment of lignocellulosic substrates may produce several inhibitors like acetic acid, furfurals, phenols, metal ions, formic acid, hydroxymethylfurfural etc. which may inhibit the microbial growth and ultimately IA production. Acetic acid produced during its production can strongly suppress the growth and metabolism of Aspergillus strain [32]. The impurities in the end product may increase the cost of the process in the form of expensive downstream processing as several unwanted acids are also produced along with IA. A number of microorganisms have been employed for the genesis of IA. Aspergillus species such as A. terreus, Aspergillus itaconicus and Ustilago strains have been used for its production [82] and out of these A. terreus has been found as the main strain used for industrial production of IA. As discussed earlier that food waste and lignocellulosic substrates can be used to produce simple sugars such as monosaccharides and disaccharides but the product yield is low because several inhibitory intermediates may produce due to pretreatment and these may either alter or inhibit the activity of A. terreus and lower the IA production. An important step to overcome this issue is to produce the mutant strains with high tolerance and productivity [98]. Several breeding strategies have been employed to produce high yielding strains such as protoplast fusion, physico-chemical mutagenesis and genetic engineering. Strain engineering is an economically beneficial and effective method to enhance the IA production along with increasing the tolerance ability of the microbes [99]. Several microorganisms have been genetically engineered such as Aspergillus niger, Escherichia coli, Pseudomonas putida, U. maydis, U. cynodontis and many other strains [104, 105]. Many microorganisms which do not have the ability to produce IA can be made efficient IA producers by incorporation of relevant genes and their ability to utilize various substrates such as cellobiose, glycerol, xylose, glucose, acetates etc. can be increased. Table 8.5 shows several microorganisms that have been engineered to produce IA.

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Table .: Microorganisms engineered for enhanced IA production. Microrganism

Substrate utilized Gene expression

Strain

Escherichia coli Candida lignohabitans Ustilago maydis Aspergillus niger Pseudomonas putida Aspergillus terreus

Acetate Glucose Glucose Glucose Pretreated lignin Lignocellulose

WCIAG . g/L CBS  – g/L K  g/L  g/L KT  . g/L  g/L

cadA- A. terreus cadA- A. terreus mttA- A. terreus cadA, mttA, mfsA- A. terreus cadA- A. terreus pfkA- A. niger

IA yield References [] [] [] [] [] []

Mutagenesis and protoplast fusion are another technique which can help in improving the ability of strains to produce more IA. Mutagenesis using nitrosoguanidine, colchicine and sodium azide has been found to enhance the productivity of A. terreus SKR10 and IA yield can reach up to 46–50 g/L from acid and enzymatically hydrolyzed corn starch [54]. A. terreus strain has also been mutagenized by plasma mutagenesis and found to produce approximately 20 g/L IA using corn straw as a substrate hydrolyzed with steam explosion pretreatment whereas in wild type strain, yield (0.54 g/L) was significantly low [58]. Protoplast fusion technique has also been reported to increase the IA production. When A. terreus T730 and citric acid producing A. niger Ni-5K strains are fused together, the yield of IA can reach upto 41 g/L [111]. The members of Ustilaginaceae family are the efficient microorganisms for the generation of IA from waste [50, 76]. Ustilaginaceae are the plant pathogenic fungi but most of them have been found to produce IA such as Ustilago maydis, U. vetiveriae, Ustilago cynodonti and U. rabenhorstiana [75, 81, 112]. U. maydis possess both mycelial as well as yeast-like morphology in cultivation. Yeast like morphology is preferred over mycelial form for IA production as mycelium formation during fermentation makes the medium viscous which results in low oxygen availability and thus low yields of IA are obtained [113]. Nitrogen sources also regulate the production of IA by U. maydis. Nitrogen source in limited amount promotes the yield of IA but on the other hand it shifts the U. maydis from yeast like form to mycelial form which ultimately inhibits IA production [80]. This problem can be resolved by maintaining C/N ratio and acidity/alkalinity of the medium. One of the solution to this is co-culturing of the strains i.e. U. maydis can be co-cultured along with Trichoderma reesei as U. maydis has been found to show tolerance to lignocellulosic substrates and T. reesei is capable to degrade lignocellulose by producing cellulase and yield of IA can be improved [98].

8.7.2 Effect of metal ions and other inhibitors Several minerals and nutrients also affect the IA production. It has been observed that yield of IA is considerably increased by the presence of nitrogen source along with Fe and

8.8 Conclusion and future perspectives

141

Zn ions [114]. Studies have indicated that hydrolysates of various agro-residues pretreated with dilute acid contain acetic acid, furfurals, hydroxymethylfurfurals along with calcium, magnesium, potassium, sodium, copper, manganese etc. which could partially inhibit the growth and interfere with the metabolic activity of Aspergillus strains thus inhibiting the IA production [36]. Several strategies have been explored to alleviate the problems related with the use of lignocellulosic materials. These include removal of metal ions by ion exchange method, neutralization of acids produced during the process by acid base neutralization technique; denaturation of proteins by heating etc. but it will again increase cost of production [56]. The already present phosphorus in potato starch can increase the growth cycle of A. terreus and hence decrease the yield of the IA [67]. Similarly, the already present nitrogen in plant-derived hydrolysate may reduce the IA production by low nitrogen demanding U. maydis [77]. One way to lower down the cost of manufacturing process is to use integrated technique i.e. integration of different substrates like industrial, agricultural or food wastes for IA production [115].

8.8 Conclusion and future perspectives IA production is gaining much attention because of its varied applications such as it acts as building block for synthesis of many polymers, can eradicate water impurities thereby help in waste water treatment as oxygen barrier film can be made from IA and chitosan or guar gum, in addition to this, IA based hydrogels can be effectively used for controlled drug delivery. But the production of IA from conventional substrates like mannose, galactose, glucose or food based substrates raises the production cost and leads to unavailability of food sources to increasing population. The most promising way to lower down the manufacturing cost of IA is to shift towards the use of low cost renewable raw materials which are non-food substrates such as lignocellulosic materials, forest residues or industrial wastes. However, the presence of harmful substances in the substrates or intermediates formed during the hydrolysis using chemicals may affect the growth and hamper the activity of microorganisms involved in the production; therefore, substrates should be tested for the presence of inhibitors such as manganese, phosphates, sulphates etc. and their pretreatment should be carried out to avoid the inhibition of the process. Enzymatic hydrolysis is one way to prevent the formation of byproducts and inhibitors. Further research could be carried out to determine the inhibition mechanism of IA synthesis by molecular or synthetic biology approaches. Another efficient technique to avoid inhibition and increase the yield of IA is to develop genetically engineered microorganisms. In addition, the IA yield can be enhanced by improving key metabolic enzymes, blocking pathways for inhibitory products and increasing the tolerance of microbial strains to pH fluctuations and carbon flux. This can provide a way to bring out breakthrough in the economical and sustainable production of IA.

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Acknowledgments: The authors would like to thank the editors Amit Kumar and Vikas Kumar for their guidance and review of this article before its publication.

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Mukesh Yadav*, Nirmala Sehrawat, Sunil Kumar, Anil Kumar Sharma, Manoj Singh and Amit Kumar

9 Malic acid: fermentative production and applications Abstract: Microbial metabolites have gained lot of industrial interest. These are currently employed in various industries including pharmaceuticals, chemical, textiles, food etc. Organic acids are among the important microbial products. Production of microbial organic acids present numerous advantages like agroindustrial waste may be utilized as substrate, low production cost, natural in origin and production is environment friendly. Malic acid is an organic acid (C4 dicarboxylic acid) that can be produced by microbes. It is also useful in industrial sectors as food, chemicals, and pharmaceuticals etc. Production/extraction of malic acid has been reported from fruits, egg shells, microbes, via chemical synthesis, bio-transformation and from renewable sources. Microbial production of malic acid seems very promising due to various advantages and the approach is environment-friendly. In recent years, researchers have focused on fermentative microbial production of malic acid and possibility of using agro-industrial waste as raw substrates. In current article, malic acid production along with applications has been discussed with recent advances in the area. Keywords: agro-industrial waste; bioprocess technology; fermentation; food industry; malic acid.

9.1 Introduction Microbes are routinely used for fermentative production of various metabolites having industrial applications. Microbes are now well known for their primary and secondary metabolites of industrial importance. Microbes have been considered as important alternative sources of many chemical compounds. Microbes provide several

*Corresponding author: Mukesh Yadav, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, India, E-mail: [email protected]. https:// orcid.org/0000-0001-8526-3747 Nirmala Sehrawat, Anil Kumar Sharma and Manoj Singh, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, India Sunil Kumar, Department of Microbiology, Faculty of Bio-Medical Sciences, Kampala International University, Kampala, Uganda Amit Kumar, Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, U.P., India As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: M. Yadav, N. Sehrawat, S. Kumar, A. K. Sharma, M. Singh and A. Kumar “Malic acid: fermentative production and applications” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0165 | https://doi.org/10.1515/ 9783110792584-009

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advantages over chemical processes including fast growth, independent to season, energy efficient and as an environment friendly approach. Microbes have also been reported for organic acids productions that have wide industrial applications. Microbes have capability to produce the organic acids. Several microbial organic acids are in use among various industrial aspects and the demand of microbes originated organic acids are increasing. Malic acid is also an important intermediate of cellular TCA (tri-carboxylic acid) cycle [1, 2]. Therefore, the microorganisms and plants are able to accumulate malic acid naturally [1, 2]. Initially, malic acid was recognized in form of fruit acid and was isolated from the fruit i.e., apples [2, 3]. Extraction of malic acid has also been reported from fruits and eggshells [4]. Though, the extraction of malic acid from fruits and egg shells was reportedly expensive and laborious [4, 5]. Malic acid has numerous applications in various industries (i.e., food & beverage industries) in form of acidulant as well as a flavor enhancer [1, 2]. It is also useful for metal cleaning, pharmaceutical, textile and agriculture industry. Moreover, it has been found useful for PMA (Polymalic acid) synthesis in chemical industries [1]. Malic acid is regarded as the promising element or chemical base to produce biodegradable polymers [1, 6] and that can be produced with the help of renewable sources [7]. Synthesis of this useful acid can be possible chemically by hydration of fumaric acid by applying high pressure and temperature that produce combination of L-malate and D-malate (Racemic mixture) as a result of chemical reaction [1, 8]. Beside this, malic acid can also be produced by enzymatic process either by utilizing fumarase enzyme in purified form or microbes as a whole cell having fumarase enzyme activity that can catalyze the conversion of fumaric acid into L-malate [1, 9]. Due to increasing demand of malic acid, microbes mediated production of this useful acid may contribute major percentage of this acid to the demanding industries in a cost effective manner. New and efficient microbial sources for malic acid production need to be identified characterized and require extensive research for bioprocess technology development particularly for utilizing the agricultural waste or feed stock waste for malic acid production by fermentation.

9.1.1 Strategies for malic acid production Malic acid is a valuable organic acid of industrial importance. It has found applications in various industries including both food and non-food industries (Figure 9.1). Synthesis, extraction and production of malic acid using various methods have been reported (Figure 9.1). Earlier, malic acid extraction was reported from apple juice, fruits and egg shells [1, 4]. But extraction of malic acid from these natural sources has been found laborious and therefore, expensive. West [4] has discussed the various strategies for malic acid production. Currently, the petroleum-based method is the in-progress method of malic acid synthesis for industrial purpose [4]. Hydration of maleic anhydride under higher pressure and temperature leading to synthesis of a mixture

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Figure 9.1: Schematic presentation showing various strategies for production of malic acid and potential applications in various industries.

containing both L-malic acid and D-malic acid (Racemic mixture) that further utilized for malic acid production at commercial level. The produced isomers by this method require chiral resolution [1, 4, 6]. Alternatively, synthesis of malic acid is possible by enzymatic process either by using fumarate hydratase or by using yeast cells i.e., Saccharomyces cerevisiae harboring the over-expressed fumarate hydratase gene for catalytic transformation of fumarate to malate [1, 4, 5, 10]. Recently, acidic hydrolysis of polymalic acid (PMA) has been practiced to produce malic acid. The PMA can be synthesized by various suitable microbes using fermentation approach of product formation. Currently, the researchers are working on possibilities of cost effective and environment friendly approaches for malic acid production using cheaper biomass and also for fermentative production of microbial malic acid using agricultural and industrial waste products as efficient substrate at lower cost.

9.1.2 Microbial malic acid production: developments and advances in bioprocess technology Production of malic acid from fungal sources and bacterial species has been reported. Filamentous fungi have been found to produce higher amount of this beneficial acid. Yeasts are also promising sources to produce malic acid via fermentation process as investigated in earlier studies. Further, yeasts and filamentous fungi have been genetically engineered for modifications in the metabolic pathways to obtain higher yield of malic acid. Bacterial sources may be the better choice for production of malic acid for industries due to their advantages including fast growth and easy genetic manipulations. General strategy for production and down stream processing has been shown in Figure 9.2.

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Figure 9.2: General strategy for fermentative production of microbial malic acid.

Fermentative production of microbial malic acid is currently limited due to low yield of product, titer, and also low productivity due to the inhibition of end-product formation. Zou et al. [11] reported new method of malic acid production by fermentation of polymalic acid or PMA and its hydrolysis in successive step. Polymalic acid producing microbial strain i.e. Aureobasidium pullulans ZX−10 was isolated and investigated. The selected microbial strain produced PMA in the form of chief fermentation product. Authors reported a titer equivalent to value of 87.6 g/L for malic acid with higher efficiency obtained with fermentation (free-cell) carried out in stirred-tank bioreactor. Further, immobilized cells were investigated by fed-batch fermentations in a fibrous-bed bioreactor and authors achieved a productivity of 0.74 g/L h along with maximal titer of the product as 144.2 g/L in this study. The PMA produced by microbial fermentation was purified using specified resins (anionexchange) having purity of near about 100% along with great recovery rate of 84%. The produced PMA was further utilized for malic acid production in pure form by hydrolysis with sulfuric acid (2 M) at temperature of 85 °C. Authors suggested the described process as an efficient road for production of PMA as well as malic acid [11]. The method

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was economic and seems promising for the industrial application. Yeast strains also seem promising for microbial production malic acid. The yeast Saccharomyces cerevisiae has been reported as efficient microbial source for malic acid production. Metabolic engineering of this yeast has been suggested important for production of higher level of malic acid [12]. The effect of 03 genetic modifications at individual and combined form was investigated on an earlier specifically engineered S. cerevisiae strain. The three modifications at genetic level included: (a) Over-expression of native enzyme pyruvate carboxylase coded by PYC2 gene, (b) Higher expression of an allele of malate dehydrogenase (MDH3) gene, and its retargeting towards the cytosol, and (c) Expression of malate transporter gene SpMAE1 of the Schizosaccharomyces pombe to perform specific function. Although, the modifications at genetic level either single or double resulted in improved production of malate, but instantaneous incorporation of all three modifications is necessary to achieve highest malate yields and titers [12]. Further, the authors observed that genetically engineered strain in batch cultures (glucose-grown) resulted in significant production of malate titers (59 g L−1). The yeast S. cerevisiae was also investigated for production of malate using metabolic engineering as promising approach. With the over expression of fumarase, improved malate titers of approximate 6 g L−1 has been reported by researchers [13]. The attainment of improved titre was due to increase in activities of malate dehydrogenase (Mdh) enzyme. Further, authors found that enhanced expression of Mdh2 (cytosolic isozyme) is responsible for increased malate titers i.e., 12 g L−1 of malate [14]. Engineered Aspergillus niger has been reported for malic acid production but byproduct formation i.e., citric acid has remained a major concern. Xu et al. [15] have reported the improved production of malic acid by engineering of Aspergillus niger. This fungal species was engineered to over express specific genes and inhibiting the citric acid accumulation. Due to disruption of cexA gene, the strain stopped accumulation of citric acid but also showed low production of malic acid. Therefore, the genes including pkiA (pyruvate kinase); hxkA (hexokinase); mstC (glucose transporter) and pfkA (6-phosphofructo-2-kinase) were over-expressed in experimental strain S895 and lead to construction of strain S1149. The constructed strain S1149 produced higher titer of malic acid (201.13 g per litre) as a result of fermentation in fed-batch culture system [15]. Moreover, the byproduct i.e., citric acid was eliminated completely. In the study, authors constructed a new engineered strain with significantly high industrial potential for malic acid production [15]. Wei et al. [7] have discussed the malic acid production from native microbial strains and from mutant as well as genetically engineered strains. Majority of studies have been carried out on Aspergillus sp. and Penicillium sp. due to their capacity of higher malic acid production [7]. Both, Penicillium sp. and Aspergillus sp. are now well known for their potential to produce malic acid. Schizophyllum commune [16] and Zygosaccharomyces rouxii [17] has also been reported for ability of malic acid production. Initially, Aspergillus flavus was reported for malic acid production [18] along with optimal process for higher malic acid production [19]. But, this fungal

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species produced aflatoxins during fermentation process therefore, it was not investigated further to produce malic acid at large-scale [19, 20]. Aspergillus oryzae was later investigated and found capable of malic acid production [21]. Then the same fugal species (A. oryzae) was examined later by researchers for the production of malic acid by using renewable sources of carbon i.e., xylose and glycerol [22]. Aspergillus niger is among the important fungal sources of industrial metabolites. Various strains of A. niger have been studied and found efficient as well as promising source of malic acid for fermentative production. Strains of A. niger have been investigated for their potential to produce malic acid using thin stillage in another study where authors reported significant malic acid titers [23]. A. niger ATCC 12,486 was also assessed for the potential of producing malic acid by considering crude glycerol as feedstock and the investigated strain showed production of malic acid (23 g/L) at 25 °C after 192 h [24]. Various strains of Penicillium species have also been investigated as potent source of malic acid [25, 26]. The higher titer of malic acid along with good yields has been reported for Penicillium sp. [25, 26]. Wang et al. [25] have reported production of higher level of calcium malate by using Penicillium sclerotiorum K302. Authors achieved a significant titer (92.0 g/L) and yield of glucose (0.88 g/g). Authors obtained significant productivity rate (1.23 g/L/h) after fermentation of 72 h. Also, yeasts including Saccharomyces cerevisiae, Zygosaccharomyces rouxii, and Aureobasidium pullulans have been recognized as potent microbial sources of malic acid [17, 27–29]. Liu et al. [30] have reviewed L-malate production as a result of biocatalysis process and using microbial fermentations. Fermentative production of microbial malic acid seems to be hope of industries relying on malic acid for their final product. Authors discussed the various types of microorganisms used for malic acid production along with the substrates for carrying fermentations, biosynthetic pathways involved in malic acid production, and strategies for metabolic engineering. Kövilein et al. [2] also discussed production of malic acid using microbial sources. Authors discussed about the native strains, genetically modified bacteria and genetically modified fungal strains capable of production of malic acid. Also, Aspergillus sp., Rhizopus delemar, and Ustilago trichophora TZ1 have been discussed as fungal sources for production of this important acid. Authors further discussed about genetic engineering for specific genes to enhance production of malic acid by genetically engineered microbial strains. Use of Saccharomyces cerevisiae and Schizosaccharomyces pombe has been reviewed in genetic engineering of fungal strains. Similarly, genetic modification of bacterial strains has also been discussed [2]. Recently, enhanced L-malic acid production from A. oryzae DSM 1863 by repeating batch culture system has been reported by Schmitt et al. [31]. Authors investigated the process characteristics at shake flask level and compared the repeated-batch, fed-batch, and also batch cultivation systems for improved malic acid production by using selected strain of Aspergillus oryzae. Authors obtained higher production (around 178 g/L) in repeated-batch cultivation system with productivity of 0.90 ± 0.05 g/L/h. Also, regular addition of calcium carbonate was found to positively

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affect the malic acid production and pH stability. Further, the nitrogen supplementation was also found to play crucial role in lengthened production of malic acid using similar cultivation system. Enhanced production of malic acid in investigated cultivations was observed if product is removed in routine process because accumulation of product can limit its production from selected microbial strains. These findings were very impactful and provided the important insights to produce L-malic acid.

9.1.3 Microbial organic acids production (malic acid): an environment friendly approach Demands of various chemical and energy are increasing continuously to meet the requirements of increasing global population. Scientists are looking for the alternate solutions and sources to satisfy the increasing demands with sustainable environment friendly approaches. Large number of industries depends on the petroleum and energy industries. To meet the increasing chemicals and energy demand in near future for coming generations, alternative to petrochemicals are required along with reducing the impact of the chemical industry on environment [32]. Microbes mediated conversion or translation of renewable feed-stocks has enormous potential for production of fuels and chemicals at industrial scale in cleaner as well as sustainable way [32]. Organic acids play important role in industrial sectors. Organic acids also serve as basic chemical structure for synthesis of various chemical compounds [4]. Fermentation based microbial organic acids production seems very capable and efficient approach to produce chemicals to be used as basic building blocks and that have potential to replace the petro-chemically derived equivalents [32]. Several organic acids are produced by microbes. The industrial biotechnology approach covering suitable microbial sources, microbial fermentations, advancements in bioprocess technology, and cost effective downstream processing will be the keys in future for sustainable production of microbial organic acids in environment friendly way. Microbial fermentations and organic acids production using agricultural and industrial wastes as raw substrates (non-food) may help to tackle the climate changes. Malic acid has found important place in chemical industry to its various applications including basic chemical structure for synthesis purposes. Recent findings have supported the microbial production of malic acid using bacteria as well as fungal sources including suitable yeast strains. The biosynthesis pathways associated with production of L-malic acid in microbes are to some extent apparent. Three metabolisms related pathways such as oxidative pathway, glyoxylate cycle and non-oxidative pathway have also been recognized to synthesize L-malic acid from glucose [1]. Strain improvements, genetic engineering and metabolic engineering of microbes allow controlling the biosynthetic pathways to obtain higher titer of malic acid. Moreover, agricultural feedstock can be utilized as potential substrate to produce malic acid by fermentation in a cost effective and environment friendly way. Microbes have been found useful in producing the

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industrial metabolites and the microbial practices have been found environment friendly and cost effective due to use of raw substrates. Microbes are currently cultured at large scale for production of industrial metabolites including enzymes [33–38].

9.1.4 Major substrates to produce microbial malic acid Majority of investigations focused on using glucose as a key substrate to produce microbial malic acid. One major drawback for using glucose is that it is edible and its pure form is comparatively costly. Therefore, suitable alternate substrates will be required for industrial production of microbial malic acid. A suitable microbe along with specific substrate will be the important criteria to achieve the cost effective production of malic acid. The substrates should be readily available and should be cheaper. Several alternate substrates have also been investigated time to time for production of microbial malic acid. Inexpensive waste products or by products from industries and material derived from lignocelluloses may be utilized for low cost microbial malic acid production [2]. Kövilein et al. [2] have reviewed various alternate substrates that were previously investigated to produce L-malic acid i.e., pure/crude glycerol [2, 22, 29, 39, 40], fraction of beech wood hemicelluloses, hydrolysate product of beech wood cellulose, corn straw & Miscanthus cellulose [41, 42], Syngas [43] and thin stillage [4, 23]. Also, hydrolysate of corncob, cornfibre, Jerusalem artichoke tuber, soybean hull, sweet potato, and wheat straw has been studied for polymalic (PMA) acid production [44–48]. Besides these, soy molasses, sugarcane molasses and waste xylose mother liquors have also been reported for polymalic (PMA) acid production [44, 49, 50].

9.1.5 Applications of malic acid, climate change and industrial perspective Organic acids of microbial origin represent good alternate to meet continuously increasing industrial demands along with potential to tackle the climate change [1, 4]. Among various important organic acids, Malic acid is an essential acid having applicability in diverse sectors. Various strategies reported earlier to produce malic acid production includes extraction from specific natural sources, one-step fermentation based production, enzyme mediated transformation of fumaric acid using the purified fumarase into L-malate, fumarase expessing microbial cells mediated transformation of fumaric acid and also the acid hydrolysis of polymalic acid [1, 4, 5, 9, 10]. It can also be generated by chemical process for example, hydration reaction of fumaric acid with application of high pressure along with high temperature which yields racemic assortment of isomeric forms L-malate and D-malate [1, 8]. Recently, the findings suggested production of malic acid can be done by acidic hydrolysis of polymalic acid

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(PMA) and this acid is a metabolic product of selective microorganisms including Aureobasidium pullulans [47, 48]. Production of malic acid by suitable microbial sources has potential to cover the industrial demand of malic acid. Higher malic acid producing microbial strains are required to meet the industrial requirement and to reduce the burden of petrochemical industry. The mechanism involved in L-malic acid biosynthesis and secretion further needs to be elaborated in detail to target its higher production [1]. New and efficient sources for malic acid production should be screened and process should be developed for its higher production using cheap raw substrates. Recently, production of L-malic acid by acidic hydrolysis of polymalic acid has been investigated [1, 11, 51]. The polymalic acid (PMA) was obtained in this study from PMA-producing fungi [1, 11, 51]. L-Malic acid has found applications in different industries including beverages, food, chemical, pharmaceutical, and medical industries [1, 4]. In food and beverages, this acid is applicable to enhance flavor and as an acidulant [1, 4]. It has been found that malic acid exert less bitter taste if used as an acidulant as compared to citric acid [1, 6]. It also finds application in cleaning of metal, finishing of textile, agriculture industrial sectors and pharmaceutical industries [1, 6, 26]. It has been recognized as important and basic building block useful to produce several chemical compounds [4] as polymalic acid (PMA) and polymers [1, 4, 6]. Malate has been accepted as potential base molecule for production of significant number of chemicals in a sustainable way [32, 52]. Its extraction from traditional sources i.e., from eggshells, fruits and apple juice is comparatively expensive as well as laborious [1, 4]. On the annual basis, the global market of malic acid ranges from 40,000 to 60,000 metric tons accompanied with a significant increase in growth rate annually [4, 53]. Increasing pressure on natural resources has adversely impacted the environment and resulted in gradual changes in global climate. Green approaches are need of current time to tackle the climate change and using the resources in sustainable way. Fermentative production of microbial malic acid using low value biomass should be investigated more rapidly. New and efficient microbial strains should be isolated and screened for their capability to produce malic acid in a cost effective way. Cost effective production may lead to reduced pricing of malic acid as compared to malic acid produced from petroleum-based products without harming the environment and to meet the glaobal demand of malic acid.

9.2 Conclusions and future perspectives Malic acid is highly useful organic acid having great potential for industrial sector. It has been found useful in various industries including food, pharmaceutical, agricultural, metal cleaning, and others. It also serves as important basic material for synthesis of other useful chemicals. Different strategies have been followed to produce malic acid. Microorganisms can be used for production of malic acid. Recent trends

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showed that production of malic acid from microbes is very promising. The strategies for microbial production of malic acid may be cost effective due to use of cheaper substrates. Use of microbial sources for malic acid production has several advantages over chemical methods and process based on petro-chemicals. Moreover, approach of microbial production of malic acid is environment friendly and may help to tackle the climate change. Research on malic acid production from microbes has increased in recent years. New and efficient sources of malic acid are required along with extensive research on process development of acid production as well as down stream processing. Raw substrates need to be explored as substrates for microbial fermentations and malic acid production. The microbial malic acid may be the solution of increasing demand of malic acid in a cost effective way but this area need to be investigated with elaborated investigations. Acknowledgment: Authors (MY, NS, AKS, MS) acknowledge the help and support by Head, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, India.

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33. Singh RS, Yadav M. Single-step purification and characterization of recombinant aspartase of Aeromonas media NFB-5. Appl Biochem Biotechnol 2012;167:991–1001. 34. Singh RS, Yadav M. Enhanced production of recombinant aspartase of Aeromonas media NFB-5 in a stirred tank reactor. Bioresour Technol 2013;145:217–23. 35. Singh RS, Singh RP, Yadav M. Molecular and biochemical characterization of a new endoinulinase producing bacterial strain of Bacillus safensis AS-08. Biologia 2013;68:1028–33. 36. Yadav M, Sehrawat N, Sharma AK, Kumar V, Kumar A. Naringinase: microbial sources, production and applications in food processing industry. J Microbiol Biotechnol Food Sci 2018; 8:717–20. 37. Mahto RB, Yadav M, Sasmal S, Bhunia B. Optimization of process parameters for production of pectinase using Bacillus subtilis MF447840.1. Recent Pat Biotechnol 2019;13:69–73. 38. Mahto RB, Yadav M, Muthuraj M, Sharma AK, Bhunia B. Biochemical properties and application of a novel pectinase from a mutant strain of Bacillus subtilis. Biomass Conv Bioref 2022. https://doi. org/10.1007/s13399-021-02225-y. 39. Zambanini T, KleinebergW, Sarikaya E, Buescher JM, Meurer G, Wierckx N, et al. Enhanced malic acid production from glycerol with high-cell density Ustilago trichophora TZ1 cultivations. Biotechnol Biofuels 2016;9:135. 40. Iyyappan J, Baskar G, Bharathiraja B, Saravanathamizhan R. Malic acid production from biodiesel derived crude glycerol using morphologically controlled Aspergillus niger in batch fermentation. Bioresour Technol 2018;269:393–9. 41. Dörsam S, Fesseler J, Gorte O, Hahn T, Zibek S, Syldatk C, et al. Sustainable carbon sources for microbial organic acid production with filamentous fungi. Biotechnol Biofuels 2017;10:242. 42. Li X, Liu Y, Yang Y, Zhang H, Wang H, Wu Y, et al. High levels of malic acid production by the bioconversion of corn straw hydrolyte using an isolated Rhizopus delemar strain. Biotechnol Bioproc Eng 2014;19:478–92. 43. Oswald F, Dörsam S, Veith N, Zwick M, Neumann A, Ochsenreither K. Sequential mixed cultures: from syngas to malic acid. Front Microbiol 2016;7:1–12. 44. Cheng C, Zhou Y, Lin M, Wei P, Yang ST. Polymalic acid fermentation by Aureobasidium pullulans for malic acid production from soybean hull and soy molasses: fermentation kinetics and economic analysis. Bioresour Technol 2017;223:166–74. 45. Zan Z, Zou X. Efficient production of polymalic acid from raw sweet potato hydrolysatewith immobilized cells of Aureobasidium pullulans CCTCC M2012223 in aerobic fibrous bed bioreactor. J Chem Technol Biotechnol 2013;88:1822–7. 46. Xia J, Xu J, Liu X, Xu J, Wang X, Li X. Economic co-production of poly(malic acid) and pullulan from Jerusalem artichoke tuber by Aureobasidium pullulans HA-4D. BMC Biotechnol 2017;17:20. 47. Leathers TD, Manitchotpisit P. Production of poly(β-L-malic acid) (PMA) from agricultural biomass substrates by Aureobasidium pullulans. Biotechnol Lett 2013;35:83–9. 48. Zou X, Yang J, Tian X, Guo M, Li Z, Li Y. Production of polymalic acid and malic acid from xylose and corncob hydrolysate by a novel Aureobasidium pullulans YJ 6–11 strain. Process Biochem 2016;51: 16–23. 49. Xia J, Xu J, Hu L, Liu X. Enhanced poly (L-malic acid) production from pretreated cane molasses by Aureobasidium pullulans in fed-batch fermentation. Prep Biochem Biotechnol 2016;46:798–802. 50. Feng J, Li T, Zhang X, Chen J, Zhao T, Zou X. Efficient production of polymalic acid from xylose mother liquor, an environmental waste from the xylitol industry, by a T-DNA-based mutant of Aureobasidium pullulans. Appl Microbiol Biotechnol 2019;103:6519–27. 51. Zhang X, Wang X, Shanmugam KT, Ingram LO. L-malate production by metabolically engineered Escherichia coli. Appl Environ Microbiol 2011;77:427–34.

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Urvasha Patyal, Vikas Kumar*, Manoj Singh, Amit Kumar, Anil K. Sharma, Syed Fahad Ali and Sheikh Mudasir Syed

10 Butyric acid: fermentation production using organic waste as low-cost feedstocks Abstract: Butyric acid is an important chemical which has many applications in the chemical, food, and pharmaceutical industries. Butyraldehyde, which is derived from propylene, is now converted into butyrate by petrochemical processes known as oxo synthesis. Because of its poor productivity and low butyrate concentration in the fermentation broth, biotechnological production of butyric acid is not economically viable. Typically, a sizable amount of the overall production expenses goes toward the cost of the fermentation substrate. If the fermentation process can use minimal biomass as the feedstock, a cost-competitive production of butyric acid from the fermentation technique would be generated with a strong market prospect. Organic wastes are recommended as a source of butyric acid fermentation feedstock because they are inexpensive, can be generated in huge numbers, and are biodegradable. With a focus on the low-cost feedstock, the many uses of butyric acid are discussed, with its present production status. As a result, this paper explores several butyric acid fermentation-related problems and offers ideas for potential solutions. Keywords: butyric acid; feedstock; fermentation; metabolism; pharmaceutical.

10.1 Introduction Butyric acid also referred to as butanoic acid, is a straight-chain triglyceride found in animal fats and plant oils in the form of esters. Butyrate is created as a by-product of an obligatory anaerobic bacteria via the fermentation process [1]. Prokaryotes and eukaryotes both employ fermentation as one of their earliest metabolic processes. Butyric acid is used in the chemistry, polymers, fabrics, foodstuff, and pharmaceutical industries,

*Corresponding author: Vikas Kumar, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), MMEC, Mullana, Ambala, Haryana, India; and Department of Microbiology, International Medical School, UIB, Almaty, Kazakhstan, E-mail: [email protected]. https://orcid.org/0000-0002-6044-3239 Urvasha Patyal, Manoj Singh and Anil K. Sharma, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), MMEC, Mullana, Ambala, Haryana, India. https://orcid.org/0000-0002-9257-927X (M. Singh) Amit Kumar, Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, India Syed Fahad Ali, Department of Pharmacology, International Medical School, UIB, Almaty, Kazakhstan Sheikh Mudasir Syed, Department of Genral surgery, International Medical School, UIB, Almaty, Kazakhstan As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: U. Patyal, V. Kumar, M. Singh, A. Kumar, A. K. Sharma, S. F. Ali and S. M. Syed “Butyric acid: fermentation production using organic waste as low-cost feedstocks” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0166 | https://doi.org/10.1515/ 9783110792584-010

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and it is now generated from fossil resources. Furthermore, as customers seek organic components in their food, pharmaceutical, and aesthetic goods, demand for naturally and economically generated butyric acid is increasing [2]. Unfortunately, because of the high expense of the procedure, there is a considerable gap between demand and supply for various biologically generated chemicals, including butyric acid. Feedstock expenses can be minimised by employing industrial or agricultural by-product streams, and fermentation costs can be cut by creating systems with high cell density and high product titre. Clostridium species create butyric acid, which, like other acids (acetic acid, lactic acid, propionic acid), is harmful to bacteria after a certain concentration [3]. As a result, product titres are often low, making downstream costly. In situ elimination of butyric acid can be developed to lower these expenses. Although in situ removal techniques for butyric acid are not yet widely used in industry, it is important area where work must be done to make the process commercially feasible. Commercial butyrate manufacturing is now limited to chemical processing, having a business world of 80,000 metric tonnes produced a year at a price of $1.8/kg [4]. However, the production from sustainable sources will become an inescapable trend as demand from the pharmaceutical and food industries, which favour organically produced products, develops [5, 6]. For the fermentation biosynthesis of butyrate, research has centered on strain generation via biochemical technology and process innovation employing low-cost biomass feedstocks [2, 7, 8]. In the economy, the low-cost feedstock is a prevalent word, with many comparing it to “trash”. Organic wastes are recommended as a source of butyric acid fermentation feedstock because they are inexpensive, can be generated in huge numbers, and are biodegradable [9]. Food trash, paper waste, effluent, plant leftovers, and animal and human bodily waste are all examples of organic waste. If low-grade biomass can be used as a feedstock in the fermentation process, cost-competitive butyric acid production with a large market potential can be achieved [10].

10.2 Butyric acid features and applications Butyric acid has the molecular formula CH3CH2CH2–COOH and is a carboxylic acid with different physical properties. Butyrates are butyric acid’s salts and esters. It smells bad, tastes harsh, and leaves behind a slightly sweet aftertaste (much like ether) [11]. This translucent, fatty liquid may be split from an aqueous solution using ions and is readily soluble in water, alcohol, and ether. While alkaline potassium permanganate can oxidise to acetic acid and carbon dioxide, sulfuric acid and potassium dichromate can only oxidise to carbon dioxide. Butyric acid has its own importance in different industries (Figure 10.1) and is used in the production of synthetic flavouring esters, as a food ingredient, in the production of coatings, and the demineralisation of hides [12]. Perfume, flavourings, medicines, and disinfectants are all synthesized with it [13, 14]. It’s a major flavouring ingredient in a

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Figure 10.1: Importance of butyric acid in different industries.

variety of foods, including beer, and it’s also prevalent in cosmetics and detergents [15]. When used according to acceptable manufacturing or feeding methods, it is considered a safe food ingredient.

10.3 Butyric acid production In 1861, Pasteur found the fermentation that produced butyric acid, and he named the bacteria “anaerobic” due to air elimination. In the 1880s, Fitz, Gruber and Grimbert investigated the fermentation process of various strains like Clostridia, but they failed to discriminate between species that produced butyric acid and those that produced butanol. In 1923, Lefranc and his partners made the initial and only reasonable attempt to synthesise the acid for commercial use by fermentation. Butyric acid-producing bacteria include Clostridium, Butyrivibrio, Butyribacterium, Sarcina, Eubacterium, Fusobacterium, and Megasphaera. Butyrate-producing bacteria include Bacteroides melaninogenicus, Treponema phagedenic, and Peptococcusasacelarolyticus [16]. The most often utilised microbes are Clostridium and Butyribacterium (Table 10.2). Clostridia are the bacteria of choice for commercial butyric acid synthesis because they are stable and prolific [17]. The most typical bacteria employed in butyric acid fermentation are Clostridium [18]. These bacteria, which also include Clostridium populate, Clostridium beijerinckii and other Clostridia strains, generate butyrate as the primary end product and acetate, carbon dioxide, and hydrogen as the primary byproducts [19]. These strains are strictly anaerobes, chemoorganotrophic, Gram-positive, and spore-forming. A suspension of spores in sterilized solution at very high temperature i.e. 400 °C or a sterile glycerol stock at −70 °C are both viable alternatives for long-term preservation after lyophilization and shortterm storage in a medium that contains just a modest amount of simple sugars. A combination of nitrogen and carbon dioxide, and a pH 4.5–7.0 are the ideal culture conditions at 35–38 °C. Other frequently utilised carbon sources include starch, potato byproducts, corn meal, wheat flour, cellulose, and xylose, as well as degraded corn fibre. Lactose from whey and sucrose from molasses are other common sources of carbon. To produce

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Table .: Micro-organisms, carbon source and culture design for the synthesis of butyric acid []. Microorganisms

Source of carbon

Culture design

C. tyrobutyricum

Glucose Xylose, cane molasses Glycerol Paper and wheat straw Starches, whey lactos and xylose Cheese whey Cheese whey and lactose Glucose/glycerol Glucose, lactate Glucose CO

Continuous culture fed batch, fed batch bioreactor Batch and fed-batch Chemostat and batch Batch

[]

Batch Mixed culture with Bacillus cereus Chemostat Batch Batch Continuous

[]

C. butyricum

C. beijerinckii C. acetobutylicum C. thermobutyricum Butyribacterium methylotropicum

References

[]

[] [] []

butyric acid, various species of microorganisms use various sources, necessitating the use of various culture designs (Table 10.1). The efficiency, productivity, and yield of traditional butyric acid synthesis via batch fermentation are all inadequate. Because of the challenging product recovery and expensive purification, downstream processing is cost-prohibitive due to the low product concentration [20]. As a result, various researches have been conducted to increase cell concentration, overall product concentration, rate constants, and total reactor performance employed different operational modes, immobilised cell reactors, and fermentation. Because traditional fermentation materials are highly priced and frequently make a contribution up to half of the finished product expense, latest techniques have focused on using inexpensive biomass feedstocks, such as food manufacturing by-products and plant waste, for butyric acid fermenation. Batch, fed-batch [25] and continuous [25, 27] fermentation techniques have been used often to make the acid due to their respective efficiency. Typically, butyric acid was produced using Clostridium tyrobutyricum in containers or bioreactors using standard free-cell batch fermentation with a final titer of less than 25 g/L, output of less than 1 g/L, and yield of less than 0.4 g/g from glucose. Using limited glucose introduction in fed-batch process, it is possible to overcome the strong substrate inhibition that occurs when cells are cultivated in batch system at elevated glucose levels of >100 g/L. As a consequence, hydrolyzed wheat flour has a high final acid content of 62.8 g/L, a yield of 0.45 g/g, and an economic output of 1.25 g/L [3]. Finally, a fed batch fermenter with coupled cells may produce 6.78 g/L of butyric acid and 86.9 g/L of butyric acid [25, 28] in a fed batch process. Process of Continuous fermentation could enhance output; however, the product concentration was only marginally higher.

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Immobilization of cells in bioreactors can considerably enhance cell density and consequently fermentation product. Immobilized cell fermentation may also generate greater product output and titre since it uses less carbon source to produce cells and has improved cell resistance to product impediment. Pack-bed and fluidized-bed bioreactors are conventional rendered immobile bioreactors [25, 29]. In membrane bioreactors, cell retention through microfiltration is also typical. The Fed batch, a fermenter with packed column that has a permeable fibrous network for entrapment, is greatly analysed for fermentation process in comparison to free-cell fermentation, with appreciable increases in final butyrate titre, output, and production.

10.3.1 Metabolic Pathway Butyrate, acetate, CO2, H2, and lactate are all potential end products of the acidogenic Clostridium metabolic process. The Embden–Meyerhof–Parnas (EMP) pathway [30] converts glucose to pyruvate once it is transported into the cytoplasm (Figure 10.2). From 1 mol of glucose, 2 mol ofATP and NADH are generated [30]. The pentose–phosphate route, which combines phosphorylation, isomerization, and epimerization, metabolises pentoses like xylose. Fructose 6-phosphate and glyceraldehyde-3-phosphate are made from the phosphorylated intermediates that follow. When 3 mol of pentose is changed into pyruvate, 5 mol of adenosine triphosphate and nicotinamide adenine dinucleotide are produced [31]. Pyruvate is largely degraded by pyruvate–ferredoxin oxidoreductase to generate acetyl-CoA, carbon dioxide, and reduced Fd under typical circumstances. Reduced Fd electrons can be used to produce H2 by depleting protons, or they can be transferred to NAD (P). Acetyl-CoA is a critical step in the major metabolic pathway of acidogenic Clostridia and may be found at the junction that separates the acetateproducing branch from the butyrate-formation branch. The formation of acetic and butyric acids follows two similar processes. Every route comprises of 2 reactions: the first produces an acyl-phosphate from the appropriate acyl-CoA, and the second produces an acid from the acyl-phosphate while simultaneously phosphorylating ADP. An acyltransferase and a kinase catalyse the two sequential events, although the similar reactions of the parallel routes are catalysed by different enzymes. Both acids’ creation methods entail the production of ATP. Acetate synthesis produces 4 mol of ATP from 1 mol of sugar glucose, but butyrate production produces only 3 mol of ATP. The following is a diagram showing theoretical glucose fermentation with perfect butyrate conversion:

10.3.2 Efficient production of butyric acid with low-cost feedstock Butyric acid manufacturing from flow resources has recently become a more enticing option to petroleum-based chemistry due to concerns about environmental contamination brought on by the petroleum sector and customer’s interests in meals, aesthetics, and

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10 Butyric acid: fermentation production

Figure 10.2: Metabolic pathway of butyric acid synthesis.

healthcare for microbially based substances. It is first important to swap out the substrates with inexpensive feedstocks to form butyric acid production a feasible truth.Different hexose and pentose sugars can be fermented by the stringent anaerobe Tyrobutyricum, a Gram-positive, spore-producing organism. It’s been successfully investigated to manufacture butyrate from a range of substrates and biomass feedstocks utilising batch, FBB, repeated FBB, continuous, and cell-recycled production (example, corn fibre, cane molasses, brown algae, corn meal, wheat flour, sweet sorghum, wheat straw, waste paper, and oilseed rape straw). Cell immobilisation [32] and in situ product removal [33] can significantly enhance butyric acid free-cell fermentation, which often suffers from decreased yield, Reactor efficiency and output intensity due to mass transfer and product inhibition [4, 32, 33]. When affordable substrates furthermore, organic matter materials made from industrial waste and agricultural residues have been promoted as affordable substrates for the effective manufacture of a variety of biofuels and

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chemicals, with few biomass products even achieving greater amounts of fermentation output than glucose at the very last butyric acid proportion [4, 11]. Therefore, future commercial manufacturing of butyrate using Clostridium make use of biomass feedstocks. Different types of inexpensive substrates can be by different type of bacteria to produce butyric acid (Table 10.2). It was determined how quickly Clostridium tyrobutyricum ATCC 25,755 immobilised in a fibrous-bed bioreactor at pH 6.0 and 37 °C produced butyrate from glucose and xylose. In comparison to xylose fermentation, fermentation using glucose resulted in increased cell biomass output and reactor productivity. When xylose served as the substrate, acetate production was similarly decreased. However, both glucose and xylose fermentations produced butyrate yields of 0.43 g/g and comparable butyrate concentrations of around 40 g/L. With the addition of corn steep liquor, butyrate fermentation of maize fibre hydrolysate produced a high butyrate production of 0.47 g/g carbon source consumed and reactor productivity of 2.91 g/L. This study showed that butyrate can be produced effectively from low-value by-products of corn refining [19].The antagonists present in lignocellulosic hydrolysates have the potential to drastically lower the productivity and cell growth of industrial strains even though lignocellulosic biomass is a renewable source for biological fermentation. The ATCC 25,755/groESL strain performed better when butyric acid was fermented utilising hydrolysates of maize cob, corn straw, rice straw, wheat straw, soybean hull, and soybean straw, respectively. When maize straw and rice straw hydrolysates, which demonstrated cytotoxic effect to C. tyrobutyricum, were used as the substrates, the quantity of butyric acid generated in batch fermentation increased by 26.5% and 19.4%, respectively, in comparison to the wild-type strain. The synthesis of butyric acid also rose to 0.31 g/L/h because to the decreased lag phase (compared to 0.20–0.21 g/L/h for the wild-type strain) [35]. Clostridium tyrobutyricum ATCC 25,755 performed butyric acid fermentation using either glucose or brown algae as a carbon source. In order to examine several fermentation Table .: Substrates for cost-efficient production of butyric acid []. Cost effective organic feedstock

Sources

Timber biomass Grasses Plants Agriculture wastes Algae Used or off-specification oils Tires Incompatible plastics Organic municipal solid wastes Industrial waste and chemical

Bark, sawdust, crowns of trees, branches, and scrap wood Switchgrass, yard chips Foliage, shrub trimmings Manure, rotten substances, and crop remains Cyanobacteria, seaweeds Automobile oils, waste food oils, and commercial lubricants Vehicles, aircraft, and mechanical components Food wrappers, bubble wrap, and auto materials Cardboards, foodstuff, and newspaper Effluent from paper mills, materials used in chemical plants or refineries, and off-spec materials

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10 Butyric acid: fermentation production

modes (batch, fed-batch, and semi-continuous) at pH 6 and 37 °C, a model medium using glucose as a carbon source was first used. Brown algae, Laminaria japonica and Undaria pinnatifida, were used as the substrates for the butyric acid fermentation, and the established conditions were then applied to the fermentation. After being autoclaved for 60 min in a 1.5% H2SO4 acid solution with 100 g/L of L. japonica, the resultant butyrate concentration was around 11 g/L [36].

10.4 Product recovery Product segregation is by far the most power utilizing process for the synthesis of large chemical products and biofuels, according to energy budget analyses and economic evaluations. Because the substance and liquid have a significant variation in volatility, distillation may be utilised with compounds like ethanol, acetone, and butanol [37]. Acetic, propionic, and butyric acids, on the other hand, do not have this property. Most acidogenic fermentation works best when the pH is between 6.0 and 7.5, which implies the acids are ionised and hence entirely non-volatile [38]. Before adequate quantities of free unionised acids may be formed, the pH must be reduced to about the pKa values of the acids. The fermentation medium contains lignocellulosic substrates, microbiological biomass, inorganic ions, colloidal particles, soluble NH3 and CO2, and other biological non-selected troglodytes which are normally found in amounts less than 100 g L−1 [39]. There are a variety of product isolation procedures that may be used in conjunction with fermentation (Figure 10.3). Many published techniques address the possibility of product removal in situ or online. Since the by-products are separated as soon as they are created from the cells and the fermentation liquid, these procedures provide a number of advantages. Product losses, which are mostly brought about by contact with cells and medium portions can be minimised.

10.4.1 Acidification The initial prerequisite for butyric acid extraction is to devise a method for lowering the pH of the medium to 4.8 or less, which is lower than the recommended fermentation pH of

Figure 10.3: Product recovery techniques for butyric acid.

10.4 Product recovery

171

roughly pH 7.0 [5]. A multiple fermenting system can be constructed with the first phase held at pH 6.0–7.0 to allow for bacterial growth and substrate transformation, and the next phase following lacking alkali neutralisation to enable for direct acidification [40].

10.4.2 Ion exchange Particles in the fermentation media rapidly reduce anion-exchange resin capability [41]. Other ions are also coated onto the resin (HCO3−, Cl−, SO42−, S2−, NO3−, HPO42−) potentially reducing vital nutrients for microbial development and contending with acid anions for exchange sites. As an adsorber, Kawabata compared cross-linked poly(4-vinyl pyridine) to the Amberlite resins, as well as the adsorbent materials [42].

10.4.3 Solvent extraction Before butyric acid fermentation and extraction are combined, a variety of methods must be explored. In solvent extraction, the organic solvent needs to be refreshed after being saturated with the product [43]. The process can be utilised when the organic phase is needed for subsequent phases [44]. Reactive extraction is considered more efficient because the mobile phase generally contains a carrier for the acid to be recovered or reacted. 12 pH has a significant impact on the extraction procedure’s efficiency as well as the distribution coefficient.

10.4.4 Distillation Evaporation followed by distillation or liquid–liquid extraction with an entrainer were two early techniques for acid recovery [45]. If the solvent’s acid-to-water ratio is greater than the aqueous inflow, solvent extraction and distillation are favoured over entrainer distillation. If the solvent utilised is not capable of absorbing impurities, the process of solvent extraction can be utilised as a purification method [46].

10.4.5 Esterification Separating product acids has been proposed by esterifying acids with methanol or ethanol. This method makes use of esters’ lesser water solubility as compared to their corresponding acids, as well as their lower boiling temperatures. There are three methods for ester formation: (1) adsorption of organic acids followed by catalytic conversion to esters via ester formation; (2) extraction of the acids into an organic solvent phase followed by chemical esterification in that phase with added alcohol; and (3) enzymic esterification in dilute solutions by suitable microorganisms [47].

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10 Butyric acid: fermentation production

10.4.6 Membrane methods Researchers are interested in the use of membranes to separate acids from water because emulsification issues during the extraction technique of acids from fermentation mixtures have sparked their attention [48]. The use of ultrafiltration membranes in acid extraction has previously been recommended as a way to minimize clogging issues [49]. Water-splitting electrodialysis membranes and coupled-transport water repellent membranes are indeed being developed using short-chain lipids membranes [50].

10.5 Conclusion and future perspective Butyric acid fermentation, in comparison to other carboxylic acids currently produced by fermentation necessitates additional positive improvements in ultimate product density, output, and yield attained mostly by cellular engineering via biosynthetic pathways, as well as manufacturing technology via increased cell concentration. However, various challenges must be overcome before microbial based butyric acid can compete in applications that now employ petroleum-derived butyric acid. More study in this area is required to create a butyric acid microbe that produces close to 100%. Structured cellular metabolic research, such as gene expression, enzymatic modulation, and molecule correlations, can give the data needed to develop metabolic engineering strategies. In order to create a variant with sufficient regulatory and metabolic pathways, biotechnological tools like catalysts, transcriptional regulatory elements, and ribosome receptors must also be altered. A centralised metabolic pathway utilising Clostridium cellulovorans could be a possible way to produce butyric acid using microbes by converting cellulose to that acid, but more research is required to improve production, titre, and output. Finally, biorefinery using food material waste, which is generally high in starch and protein, merits more research as a low-cost feedstock for butyric acid production.

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Sachin Kumar, Priya Panwar, Nirmala Sehrawat, Sushil Kumar Upadhyay, Anil Kumar Sharma, Manoj Singh and Mukesh Yadav*

11 Oxalic acid: recent developments for costeffective microbial production Abstract: Organic acids are the important compounds that have found numerous applications in various industries. Oxalic acid is one of the important organic acids with different industrial applications. Different microbes have been reported as important sources of various organic acids. Majority of studies have been carried on fungal sources for oxalic acid production. Aspergillus sp. has been found efficient oxalic acid producer. Microbial productions of metabolites including organic acids are considered cost effective and eco-friendly approach over chemical synthesis. Fermentative production of microbial oxalic acid seems to be a good alternative as compared to chemical methods. Microbial production of oxalic acid still requires the extensive and elaborated research for its commercial production from efficient microbes using cost effective substrates. The present text summarizes the production of oxalic acid, its applications and recent developments in the direction of fermentative production of microbial oxalic acid. Keywords: fermentation; metabolic engineering; microbial production; oxalic acid.

11.1 Introduction Organic acids are abundant in nature and naturally present in plants, animals and various microbes [1]. Production of organic acids from microbes is a promising approach for obtaining building-block chemicals from renewable carbon sources [1]. Microbes provides several obvious advantages over animal and plants for production of useful and industrial metabolites. Although, not all organic acids are produced commercially solely through microbial fermentations but still microbes seems promising as more research have focused on microbial production of organic acids. Production of organic acids from microorganisms seems to be a

*Corresponding author: Mukesh Yadav, Department of Biotechnology, M.M.E.C., Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala 133207, India, E-mail: [email protected] Sachin Kumar, Department of Bioinformatics, Janta Vedic College, Baraut-Baghpat, Uttar Pradesh 250611, India Priya Panwar, Nirmala Sehrawat, Sushil Kumar Upadhyay, Anil Kumar Sharma and Manoj Singh, Department of Biotechnology, M.M.E.C., Maharishi Markandeshwar (Deemed to be University), MullanaAmbala 133207, India As per De Gruyter’s policy this article has previously been published in the journal Physical Sciences Reviews. Please cite as: S. Kumar, P. Panwar, N. Sehrawat, S. K. Upadhyay, A. K. Sharma, M. Singh and M. Yadav “Oxalic acid: recent developments for costeffective microbial production” Physical Sciences Reviews [Online] 2023. DOI: 10.1515/psr-2022-0167 | https://doi.org/10.1515/ 9783110792584-011

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viable method for obtaining chemical building blocks based on renewable carbon sources [2]. Various organic acids including citric acid, succinic acid, lactic acid, fumaric acid, acetic acid, propionic acid etc. have been reported for their microbial production [3]. Different organic acids are used in food, chemical, agricultural and pharmaceutical industries. The cost effective production of various organic acids will open up new markets and prospects for the chemical industry [1]. Organic acids are used as main building blocks for a wide range of polymer and solvent manufacturing methods used in the chemical industries. Also, some organic acids have found their applications in food industries due to their organoleptic qualities as well as flavour and antioxidant activity [3]. Organic acids that have a molecular weight of approximately 300 g/mol are termed LMWOA (Low molecular weight organic acids), characterized by the presence of one or more carboxylic groups. Oxalic acid is the most prevalent LMWOA [4]. Oxalic acid is widely distributed in nature and found in plants, animals and also microbes [5]. Oxalic acid has been proposed to play several roles in different types of organisms. In fungi, oxalic acid has been found to play role in pathogenicity, mineral weathering, nutrients acquisition, wood degradation and metal tolerance. In bacteria, oxalic acid play role in pathogenicity, mineral weathering and metal tolerance while in plants heavy metal detoxification, calcium regulation, ion homeostasis and plant defence are its main function. In animals, oxalic acid has been suggested to play role in stimulation of Na+, Cl− and H2O uptake, production of H2O2 to increase phagocytosis [6, 7]. Among the major applications, oxalic acid has found applications in agriculture sector to be used as nematicide, preservative [8]. In other industries, it is used for metal recovery [9–13]. Oxalic acid is used as a stain remover and mordant in the textile industry and can also be used to remove calcium from water in water treatment applications. Oxalic acid has been used to eliminate ticks from the environment and to prevent the spread of varroosis, a condition brought on by the parasite mite Varroa destructor, during honey bee production. The main uses of oxalic acid have been roughly divided into four categories: metal treatment, textile treatment, bleaching agents and chemical synthesis [5, 14]. Oxalic acid and oxalate salts have occasionally been used as anticoagulants in blood samples drawn for chemical analysis [15]. The market for oxalic acid is anticipated to develop sharply in near future due to continuous increasing demand of oxalic acid for a variety of applications [16]. Production of oxalic acid from microbes may ensure its cost effective production for the demanding industries in an environment friendly approach. The present review provides a concise overview of production strategies of oxalic acid including microbial production of oxalic acid and its applications in various industries.

11.2 Various strategies/methods for production of oxalic acid In general, two processes are used for production of oxalic acid i.e., chemical processes and biological processes. In chemical processing, ethylene glycol, propylene, CO, ethanol, and

11.2 Various strategies/methods for production of oxalic acid

179

carbohydrates are used in the commercial production of oxalic acid [5, 17]. Oxalic acid is produced by a variety of chemical processes that have been employed by different industries. Some of strategies used for production of oxalic acid are summarized as follows.

11.2.1 Carbohydrate oxidation Oxidation of carbohydrate using strong nitric acid is the oldest method of chemical production. The ability to collect and recycle the nitrogen oxides produced during the reaction allowed this technology to attain industrial importance until around 1940. Sugar, glucose, fructose, corn, wheat, recovered starch, potato, corncobs, tapioca and molasses are some of the raw ingredients used in this procedure. Various countries have adopted this method for oxalic acid production [5, 18, 19]. Sawdust has also been explored to produce oxalic acid using a mixture of strong nitric acid and concentrated sulfuric acid with coal fly ash acting as a catalyst [20]. Oxalic acid dihydrate can be synthesized in a single step by reacting ethylene glycol with nitrogen tetroxide, oxygen, or nitric acid and sulfuric acid [21]. This technique is also used for oxalic acid production in some countries [5].

11.2.2 Synthesis from carbon monoxide Oxalic acid can be produced by reaction between carbon monoxide and alcohol under pressure with the help of platinum or palladium salt and also ferric or cupric chloride salt. In this method, the alcohol and carbon monoxide are first combined to create an oxalic acid diester, followed by subsequent hydrolysis to generate oxalic acid and restore the original alcohol [5]. Carbon monoxide based chemical synthesis of oxalic acid is also carried out in two steps with a recycling of the caustic soda [22].

11.2.3 Synthesis from alkali formate Oxalic acid can be synthesized using sodium hydroxide. Under pressure, the sodium hydroxide is heated and absorbed by carbon monoxide which leads to production of sodium formate. The sodium formate is then evaporated and heated once again to produce sodium oxalate, sodium carbonate, and hydrogen gas. To precipitate calcium oxalate and regenerate sodium hydroxide at the same time, sodium oxalate is treated with calcium hydroxide and water. Sulfuric acid is used to treat calcium oxalate to create calcium sulphate and oxalic acid [23]. Alternative methods include treating sodium oxalate with nitric acid to produce oxalic acid and sodium nitrate [24]. Oxalic acid can be produced by other methods also. Among all the commercially used chemical procedures, biomass oxidation, fomate coupling, carbon mono oxide to oxalic acid through reverse gas shift and oxalate acidification are among the most sustainable methods [5].

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11.3 Microbial production of oxalic acid Oxalic acid is naturally present in different organism including plants, animal, bacteria and fungi. Numerous studies have been carried out to examine the ability of microbes to produce oxalic acid.

11.3.1 Production of oxalic acid from bacterial sources Fermentative production of oxalic acid using bacteria for commercial purpose requires extensive research. Very few studies have been reported on bacterial production of oxalic acid [25, 26]. Hamel et al. [25], have reported the production of oxalic acid in an aluminium stressed bacteria Pseudomonas fluorescens. Oxalic acid was produced as a significant metabolite in response to aluminium stress. As compared to the control cells, the aluminium stressed cells produced eight times more oxalic acid in the inner membrane fraction. Additionally, it was found that the cell-free extract can produce oxalate from glyoxylate [25]. The oxalic acid biosynthetic enzyme was identified, cloned and partially characterized from the animal bacterial pathogen Burkholderia mallei [26] and the discovered gene was reported as oxalate biosynthetic component (obc)1 [26]. In another study Nakata and He [27] reported that oxalic acid biosynthesis is encoded by an operon in Burkholderia glumae. Authors reported oxalate biosynthetic component (obc)A locus and obcB locus required for expression of oxalic acid [27]. The research in oxalic acid production from bacteria is in its initial stage and requires pace for developing and establishing its commercial production. Cost effective production of oxalic acid using agro-industrial waste is the demand of current time. More elaborative research required to find new bacterial sources, efficient production of oxalic acid in a cost effective manner.

11.3.2 Production of fungal oxalic acid Fungi are well known for production of various metabolites with industrial applications. Currently, various fungal sources are employed at large scale for production of industrial products useful for food, pharmaceutical, chemical and other industries. Fungal sources have also been investigated for organic acid production. Though, limited literature is available on oxalic acid production from fungal sources but still it seems promising. Initially, the oxalic acid production from fungi was reported by Hamlet and Plowright in 1877 [28]. Various fungal strains have been reported to produce oxalic acid including Aspergillus niger, Botrytis cinerea, Sclerotinia sclerotiorum, Sclerotium rolfsii, Clarireedia jacksonii, Cristulariella pyramidalis, Sclerotinia minor, Penicillium islandicum, Pleurotus ostreatus, Rhizoctonia solani and others [7, 10, 29–34]. Due to varied physiologies and ecologies among

181

11.3 Microbial production of oxalic acid

different fungi, the role of oxalic acid in fungal metabolism seems to be important [6]. Oxalic acid has been found to be involved in mineral weathering, nutrient acquisition, metal tolerance, pathogenicity and other functions of fungi [6, 7, 28, 30, 34]. For the synthesis of oxalic acid, three different pathways in fungi have been reported [28, 32, 35]. A pyruvate can be used as precursor to make oxalic acid (OA), pyruvate is transformed into either acetyl-CoA by the pyruvate dehydrogenase multi-enzyme complex or oxaloacetate by the pyruvate carboxylase (PYC) (EC 6.4.1.1) before entering the tricarboxylic Acid (TCA) or glyoxylate (GLOX) cycles. Oxaloacetate produced from either of three different sources i.e., within the cytoplasm, from TCA cycle, or from GLOX cycle is hydrolysed to oxalate by oxaloacetate acetyl hydrolase (OAH) (EC 3.7.1.1). Hence three pathways are termed as cytoplasmic pathway, TCA pathway and GLOX pathway. The enzyme glyoxylate dehydrogenase (GLOXDH) (EC 1.2.1.17) can convert glyoxylate from the GLOX cycle directly into oxalate [6, 28]. Synthesis and quantity of oxalic acid produced by the fungi have been found to be influenced by a variety of environmental conditions including mineral type, pH and the source and availability of carbon or nitrogen [29, 34, 36–38]. Various studies carried on fungal production of oxalic acid have been summarized in Table 11.1. Table .: Production of oxalic acid from various fungal sources and various production aspects. Fungal Source

Major objective of research

Sclerotium rolfsii Sr Oxalic acid production

Media/Substrate

Optimized Tsao medium pH  buffered  days Sclerotinia minor Oxalic acid production as Modified Richard’s Virulence factor to develop solution plus  mM bioherbicide sodium succinate pH . Cristulariella Oxalic acid production on Media with glucose pyramidalis selected culture media and fructose buffered with citrate buffer Sclerotium rolfsii Sacc. Influence of media liquid basal salts composition medium (LSM) with modifications Clarireedia jacksonii Effect of pH on oxalic acid PDB, amended with and Sclerotinia production as a pathoge- cell wall sclerotiorum nicity factor components Aspergillus Effect of deletion of Synthetic medium carbonarius glucose oxidase on with glucose as carorganic acid production in bon source Aspergillus carbonarius A. niger CGMCC Comparison of organic Potato dextrose No., NJDL- acid production between  broth, initial pH . isolates of fungi phosphate solublizer

Fermentation type

Reference

shake flask

[]

shake flask;  days;  °C;  rpm

[]

Flask; pH .;  days

[]

Shake flask; initial pH .

[]

Submerged

[]

Shake flask;  days; pH .

[]

Shake flask;  °C

[]

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11 Microbial oxalic acid: recent developments

Table .: (continued) Fungal Source

Major objective of research

Penicillium oxalicum CGMCC No., NJDL- A. niger

Impact of glucose and sucrose a sole carbon source on OA production Aspergillus niger Effect of temperature and nutrient feed, pH A. niger  Optimisation of fermentation with milk whey as carbon source A. niger (MW) Kinetic modelling, fermentation study was fitted into the Monod, Leudeking-Piret and Andrews kinetic models A. niger Optimization of variables for oxalic acid production by using RSM A. niger Production of oxalic acid by A. niger A. niger

A. niger

A. niger

A. niger

A. niger  A. niger 

Influence of sucrose and milk whey as carbon source Appraisal of ANN and RSM in modelling and process variable optimization Application of RSM to optimize variable for OA production Nutritional effects on oxalic acid production and solubilization of gypsum by A. niger Oxalic acid production by A. niger

A. nigerWU-L

Metabolic engineering of A. niger to hyper produce oxalic acid

A. nigerWU-L

Metabolic engineering of A. niger to hyper produce oxalic acid

Media/Substrate

Fermentation type

Glucose

Shake flask;  days

[]

Glucose,  °C pH . Milk whey

Fed batch system;  days Stirred tank;  rpm;  days

[]

Cassava whey

Submerged fermentation system; pH .; maintained  days

[]

Algal biomass and sucrose mixture

Submerged fermentation system; pH .;  °C;  days Submerged fermentation;  days

[]

Chlorella vulgaris grown with an industrial effluent Milk Whey

Reference

[]

[]

Shake flask

[]

Cashew apple juice

Surface fermentation; pH .

[]

Cashew apple juice

Submerged fermentation; pH .

[]

Czapek Dox liquid medium

 mL flask; static  days

[]

Whey permeate lactose as carbon source OAP medium with initial pH ., maintained above . OAP medium with initial pH ., maintained above .

Batch fermentation;  days

[]

Shake flask;  days

[]

Shake flask;  days

[]

11.3 Microbial production of oxalic acid

183

Table .: (continued) Fungal Source

Major objective of research

Media/Substrate

Fermentation type

Reference

A. niger methanol resistant strain

Optimization of inoculum size and substrate concentration Effect of condition, immobilized mycelia in polyurethane foam (bioparticle) or free mycelium cells Effect of pH, carbon and nitrogen source

Corncob pH .

Semi solid-state Fermentation;  °C;  days Submerged; pH .;  days

[]

pH maintained –;  days

[]

Submerged fermentation

[]

fed-batch run in − stirred tank reactor

[]

Submerged culture;  °C and  rpm with an aeration rate of . vvm Stirred;  rpm;  days

[]

A. niger NCIM 

A. niger NCIM 

A. niger

A. niger

A. niger F

A. niger 

Glucose

Lactose, ammonium dihydrogen phosphate Effect of span  detergent Post refining fatty on OA production from acids Post refining fatty acids High-yield production of Strasser medium, oxalic acid for metal pH . maintained leaching Oxalic acid production and Chemical media nematocidal activity

Production of oxalic acid using whey permeate

Whey permeate pH . Room Temp.

[]

[]

Production of oxalic acid from Cristulariella pyramidalis has been investigated [31]. Authors compared growth of C. pyramidalis and also oxalic acid production using various carbon sources. Mannose resulted in higher oxalic acid production (34.9 mg/40 mL) and also supported higher growth of mycelia measured in terms of dry weight [31]. Dry weight of 95.5 mg/flask was obtained with mannose as compared to other carbon sources [31]. Strasser in 1994 [55] reported that lactose permeate as a carbon source resulted in increased production of oxalic acid [55]. Similarly, Handayani [42] reported the effect of temperature, nutrient feed and pH on oxalic acid production by A. niger [42]. Potential of two microbial strains i.e., Penicillium oxalicum NJDL-03 and A. niger NJDL-12 for the production of organic acids have been investigated [40]. Both strains were found to produce oxalic acid also. A. niger NJDL-03 was found to produce more organic acid as well as oxalic acid and therefore, revealed significantly higher ability of solubilizing P minerals [40]. The pH was found to affect the activities of microbial strains. Production capacity of oxalic acid by Clarireedia jacksonii and Sclerotinia sclerotiorum was also found to be affected by pH [34].

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Production of oxalic acid from A. niger F22 has been reported by Lee et al. [10]. Authors have optimized the fermentation parameters for production of oxalic acid by A. niger in form of submerged culture in 5-L jar fermenters. Authors evaluated the nematicidal activity of culture filtrate and found to be proportional to the content of oxalic acid. Further, authors scaled up the oxalic acid production to a 500-L pilot vessel. Fungi seem to be promising source of oxalic acid that needs to be explored for production of oxalic acid in a cost effective manner. In majority of studies, fungal sources have been reported for oxalic acid production. Bacterial and fungal sources need to be investigated more extensively. The oxalic acid producing potent microbial strains are needed to be isolated, screened and identified for higher production of oxalic acid. The already reported fungal strains should be investigated for optimization of oxalic acid production. 11.3.2.1 Optimization of various parameters for production of oxalic acid Since a long time, response surface methodology (RSM) and artificial neural network (ANN) has been applied in fermentation studies for optimization of microbial production of metabolites and other products. Emeko et al. [48] have compared the efficacies of ANN and RSM in modelling and optimizing of the process for oxalic acid production. It was found that cashew apple juice have potential to be used successfully as a major carbon source for oxalic acid production by A niger under surface fermentation [48]. Similarly, use of central composite design (CCD) and RSM has also been reported for oxalic acid production using cashew apple juice (CAJ). Authors investigated the potential of cashew apple juice (CAJ) to be used as carbon source for production of oxalic acid via fermentation process [37]. The investigated variables included CAJ concentration, time, pH, NaNO3 concentration and methanol concentration. Effects and interactions of these variables on oxalic acid production were determined in a central composite design (CCD) and RSM was used to model and optimize the process. Authors reported a maximum oxalic acid concentration (122.68 g/L) with the optimum levels of variables [37]. A. niger was used in the study for oxalic acid production using CAJ [37]. Oxalic acid production from A. niger (MW188538) has also been investigated by Chioma et al. [44]. Authors studied the growth kinetics along with the modelling of oxalic acid production. The oxalic acid was produced from cassava whey by using A. niger (MW188538) in a batch fermentation system. The production kinetics of oxalic acid in a batch fermentation system was fitted into the Monod, Leudeking–Piret and Andrews kinetic models [44]. Results revealed production of oxalic acid by A. niger and a significant growth rate was obtained using the cassava whey as a sole carbon source. The Leudeking–Piret and Monod models described a healthy utilization of the cassava whey and yeast extract by the microbial strain as evident from the substrate consumption rates [44]. The results showed the utility of the modelling in bioprocess technology and production of microbial acid. Recently, Amaro et al. [29] investigated six fungal species (A. niger FS1, Penicillium islandicum FS41, Pleurotus ostreatus PO1, Rhizoctonia solani Rhiz555, Sclerotium rolfsii Sr25 and Sclerotinia sclerotiorum Scl134) and 03 culture media for maximal production of

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oxalic acid. The three media included potato dextrose broth, Strasser media and Tsao media. The fungal strain S. rolfsii Sr25 and Tsao culture media resulted in efficient oxalic acid production. Tsao media was optimized using RSM and significant level of oxalic acid was obtained [29]. 11.3.2.2 Metabolic engineering Attempts have been made to engineer metabolic pathways for increased production of oxalic acid from fungal sources [39, 51, 52]. Yang et al. [39] have suggested Aspergillus carbonarius as potent microbial source for organic acid production. At pH 5.5 the A. carbonarius was found to accumulate high amount of gluconic acid while the lower pH resulted in citric acid production. Authors identified and removed the glucose oxidase gene from A. carbonarius to shift the carbon flux towards other organic acids. The genetically engineered strain (glucose oxidase deficient mutants) showed higher production of citric acid, malic acid and oxalic acid [39]. A. niger has been studied extensively for the production of organic acids. Under specific cultivation conditions, A. niger has been found to accumulate oxalic acid [51]. The oxalic acid biosynthesis pathway in A. niger involves the cytoplasmic Mn-dependent oxaloacetate acetyl hydrolyase (OAH), which converts oxaloacetate (arising from the action of pyruvate carboxylase on pyruvate generated from glycolysis pathway) to oxalate and acetate [57]. Metabolic engineering of A. niger WU-2223L for oxalic acid production has been reported by Kobayashi et al. [51]. The transformants overexpressing the oahA gene (coding for oxaloacetate hydrolase; OAH) were constructed in A. niger WU-2223L [51]. The oahA-overexpressing transformant (EOAH-1) produced higher amount of oxalic acid as compared to A. niger WU-2223L. Authors reported that A. niger WU-2223L and EOAH-1 revealed 15.6 and 28.9 g/L oxalic acid production, respectively, during the 12-day cultivation period [51]. A. niger also have a cyanide (CN)-insensitive respiration pathway which involve alternative oxidase (EC 1.10.3.11; AOX), supports continuous glycolysis because it reoxidizes NADH without producing ATP and hence aox gene encoding AOX have been used as a tool for metabolic engineering to generate efficient oxalic acid (OA)-producers by genetic engineering of A. niger using aoxA gene [52].

11.4 Production of oxalic acid by utilization of cost effective raw substrates or agro-industrial wastes Oxalic acid can be derived from agro-industrial waste biomass through various methods including the oxidation of sugars, alkali heating of plant matter, or fermentation of sugars. Oxalic acid is produced predominantly from the sugary components of the plant matter [17]. Microbes provide several advantages for production of

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metabolites of industrial importance. One of the advantages is the utilization of raw or waste material from different industries as cost effective substrate. Microbes have been investigated for organic acid production and also for utilizing the agro-industrial waste in form of medium or substrate. Various scientists have assessed the potential of raw materials to be used as cost effective substrates for production of microbial oxalic acid. Milk whey has been used as a substrate for the production of oxalic acid using A. niger [43, 47], while post refining fatty acids have also been reported [54]. The potential of cashew apple juice (CAJ) to be used as carbon source for production of oxalic acid from A. niger has been investigated and promising results were found [37]. The investigated variables included CAJ concentration, time, pH, NaNO3 concentration and methanol concentration. The study showed that CAJ may be used as potential carbon source for the fermentative production of oxalic acid using A. niger. Enhanced production of oxalic acid has been investigated in semi-solid state fermentation of methanol-resistant A. niger using corncob [53]. Semi-solid state fermentation of corncob was developed for the application of environment friendly and low-cost biomass waste to organic acid production. Oxalic acid production was found to be dependent of inoculum size and initial substrate concentration [53]. Hilt et al. [50] have also reported production of oxalic acid by A. niger. Authors reportedly used the whey permeate as a substrate for production of oxalic acid. Whey permeate has been suggested as desired substrate due to its high lactose content. Further, it is considered as a by-product of the dairy industry. Authors compared two strains of A. niger, i.e., A. niger ATCC 9029 and A. niger ATCC 6275 [50]. Chioma and Agwa [46] have investigated oxalic acid production by A. niger using Chlorella vulgaris as an potent feedstock. The Chlorella vulgaris was cultured and algal formulation was inoculated with A. niger spores. Production of oxalic acid by A. niger was assessed by gas chromatography-mass Spectrophotometry [46]. Results suggested that lagal formulations may be used as cheaper substrates for oxalic acid production. In a study, Chioma and Agwa [45] used response surface methodology for optimal production of oxalic acid by A. niger. Authors investigated three media formulations including algal biomass medium, sucrose medium and combined algal biomass with sucrose medium [45]. The growth kinetics and modelling of oxalic acid production from cassava whey by A. niger (MW188538) has been studied in a batch fermentation system where cassava whey as carbon source was used, a strong utilization of the Cassava whey is reported [44].

11.5 Potential advantages of producing oxalic acid from microbial sources Microbes are well known for production of industrial metabolites having industrial applications. Microbes provide various obvious advantages over animal sources, plant sources and chemical synthesis. Microbes are now well known for their potential to

11.6 Various applications of oxalic acid

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produce organic acids. Oxalic acid is among the important industrial organic acids. Currently, oxalic acid is synthesized chiefly via chemical methods. Microbes have been found to produce oxalic acid through fermentative approach. Further, oxalic acid can be produced in an eco-friendly manner through microbes mediated fermentation. Microbes are easy to grow and can be cultured at large scale. Also, waste materials like agricultural waste, industry effluents, wood waste and other raw materials can be utilized by microbes for production of metabolites and organic acids. Microbial production of oxalic acid seems promising. New potent microbes are required for oxalic acid production. New microbes need to identified, characterized along process development for higher production of oxalic acid using cost effective substrates. This process is appealing due to its environmental friendliness. Various microorganisms have been researched for the production of oxalic acid but due to its ease of handling, ability to use a variety of cheap raw materials, and high yield, Aspergillus continues to be the most productive microbe for the microbial synthesis of oxalic acid [48]. As mentioned above several waste materials have been proved to be a good substrate for the microbial production of oxalic acid, hence microbial production of oxalic acid may be an advantageous alternative.

11.6 Various applications of oxalic acid Organic acids have found various applications in different industries. Oxalic acid has potential to be used in various industries. The major potential applications of oxalic acid and related industrial sectors have been shown in Figure 11.1. Naturally, oxalic acid has significant role in the soil environment, mineral solubilisation and also fungal pathogenicity [7, 28, 58, 59]. Oxalic acid producing fungal strain (A. niger) has been recommended as a phosphate solubilizing fungus (PSF) in agricultural soil for enhancing the phosphate release [40]. The oxalic acid producing fungi seems promising and having biotechnological potential for phosphate solubilisation [29]. Betiku et al. [37] have briefed the applications of oxalic acid. According to authors, oxalic acid has various applications in food industry, pharmaceutical industry, waste water treatment and also hydrometallurgy. In food industry, oxalic acid serves as anti-browning agent for some fruits [37]. Oxalic acid has potential applications in metallurgy. The iron leaching from China clay with oxalic acid has been reported by Mandal and Banerjee [60]. It has been observed that oxalic acid is a sustainable chemical that enables effective metal recovery from industrial materials via dissolution. As a crucial response in a prospective clean iron-making process, the dissolution process has recently received interest [13]. One of the numerous techniques for recovering valuable components from industrial wastes is acid leaching. Application of oxalic acid in form of an efficient leaching agent of aluminium from industrial waste has been reported [12]. The uses of oxalic acid as and in bleaching agent, textile treatment (mordant for the printing and dyeing of wool and cotton), metal treatment, for preparation of slats, chemical synthesis and others have been discussed [5].

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Figure 11.1: General presentation showing major industries and potential applications of oxalic acid.

The primary phosphorous (P) source for the production of fertilizers is rock phosphate (RP) and solubilization of RP by microbial activities is promising in producing phosphate fertilizers [29]. Oxalic acid has shown significant solubilization of RP. Oxalic acid has potential for extraction of phosphate from rock phosphate (RP). By solubilizing RP in pure oxalic acid solutions, reaction parameters have been determined. It was found that the concentration of oxalic acid was the primary influence on reaction kinetics [61]. Nematicidal activity of oxalic acid has also been reported. Culture filtrate of Aspergillus sp. was found extremely effective against Meloidogyne incognita with noticeable second-stage juvenile mortality and egg hatching suppression [9]. Oxalic acid has revealed significant nematicidal activity against the root-knot nematode Meloidogyne incognita [10]. Mutinelli et al. [11] assessed the efficacy of oxalic acid to control varroosis, and found a mean efficacy of 95%. Therefore, oxalic acid has potential for use by beekeepers as a miticide against the parasitic varroa mite [11]. Oxalic acid has been investigated for potent application in horticulture. Anwar et al. [62] have investigated application of low dosage on strawberries and found that oxalic acid treatments at low doses increased the levels of nitrogen, phosphorus and potassium in leaf petioles. Foliar application of oxalic acid improved strawberry plant growth and fruit quality in an eco-friendly, economical and safer manner [62]. Oxalic acid has been reported to reduce the fruit softening and exo-PG enzyme activity while enhancing the activities of antioxidative enzymes [8]. Hossain et al. [63] investigated the effects of oxalic acid and 1-methylcyclopropane (1-MCP) treatments in combination with low-density polyethylene (LDPE) and high-density polyethylene (HDPE) on the post-harvest storage of litchi fruits. Authors proposed the combined treatment of 1-MCP and HDPE as a sustainable strategy to preserve red pericarp, improve quality and increase fruit shelf life of litchi [63]. Oxalic acid application in pre-treatment of maize residues for ethanol production has also been reported [64]. Also, pre-treatment of Typha latifolia with oxalic acid for

11.7 Conclusions and future perspectives

189

production of reducing sugar has been studied [65]. Sar et al. [66], investigated the effects of phosphoric acid and oxalic acid as possible substitutes for sulfuric acid in the organosolv pretreatment of oat husk. These acids can be used in place of sulfuric acid in solvent acidification since they have been shown to be as effective at producing highquality lignin and glucan [66]. Oxalic acid is also considered very useful in organic synthesis [14]. Oxalic acid serves as starting compound for a variety of high value products e.g., glyoxylic acid, glycolic acid, glyoxal, glycol aldehyde and ethylene glycol [17]. Oxalic acid is a potent new sustainable chemical for producing ingredients for the cosmetics, polymer and pharmaceutical industry [17]. Recently, Schuler et al. [17] have discussed the sustainable production of oxalic acid from CO2 and biomass. The complexity of fermentation processes has posed a challenge however, biotechnologists are still researching to fully comprehend microbial fermentation, through kinetic modelling and metabolic engineering [44, 52]. Thus, the developed models may be useful for controlling the microbial growth, substrate consumption kinetics and production of oxalic acid at a large-scale fermentation/industrial scale in the near future. The applications of oxalic acid may increase in future and therefore, more production of oxalic acid will be desirable to satisfy the increasing demand. Microbes may provide an efficient alternate to produce the oxalic acid in a cost effective manner by utilizing cheap and raw substrates. As already discussed, microbes are widely used for production of industrial products and also for organic acids [67–71]. New efficient microbial sources of oxalic acid should be identified and characterized. Process should be developed for higher production of oxalic acid from microbes. Fermentative production of microbial oxalic acid at commercial scale requires extensive as well as elaborated research on all aspects related to commercial production of microbial oxalic acid.

11.7 Conclusions and future perspectives Organic acids are important compounds that have been found useful in various industries due to wide range of applications. Oxalic acid is one of the important organic acids that have great potential for industrial sector. It has been found useful in various industries including agriculture sector, food industries, pharmaceuticals, chemical, metal industries, textile industries, fertilizer industry and others. Currently, chemical methods are used to produce oxalic acid. Time to time, researchers have investigated the potential of microbes for fermentative production of oxalic acid because microbes may provide several advantages over chemical methods. Also, the fermentative production of microbial oxalic acid may be cost effective due to possibility of using cheaper substrates such as raw substrates, agricultural waste, industrial wastes etc. The approach of microbial production of oxalic acid is environment friendly over chemical methods and

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therefore, may help to tackle the climate change. The studies showed that oxalic acid production from microbes seems promising. Majority of research has been carried on fungal sources of oxalic acid particularly Aspergillus sp. Currently, new and efficient sources of oxalic acid are required along with extensive research on process development for production as well as downstream processing. The microbial oxalic acid may be the solution of increasing demand in a cost effective way but this area need to be investigated extensively with elaborated investigations. Acknowledgements: Authors (MY, NS, AKS, MS, PP, SKU) acknowledge the help and support by Head, Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala, India.

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Index 2-ketoglutonic acid 99 2-oxoglutarate synthase 97 A. niger 55 acid catalysts 88 acidification 60, 171 acid tolerance 75, 91 acidulant 8, 70 aconitase 23 aconitic 4 additives 52 ADP 167 adsorbent 61 aerobic conditions 79 agave bagasse 44 agricultural and industrial wastes 126, 129 agro-industrial waste 180 alkali 171 alpha ketoglutaric acid 10, 95, 96, 104 alternative oxidase 185 aluminium stress 180 amino acid dehydrogenase 101 aminotransaminases 98 aminotransferase 103 anaerobic 59 animal feed 21 animal nutrition 103 anion-exchange 171 anti-cancerous 127 anti-carcinogenic 89 antibacterial 52 antimicrobial 127 antioxidative 21, 96, 127 arginine 102 artificial neural network 184 artrococarpus hete 27 aspartate transaminase 98 aspergillus foetidus 27 aspergillus niger 3, 21, 117 ATP 96, 98, 104, 167 auxotrophic 78 B. coagulan 43 Bacillus subtilis 95 bacteria 163, 165, 169 bactericidal 84 balancing 22 batch 166

https://doi.org/10.1515/9783110792584-012

beckmann rearrangement 88, 94 beech wood cellulose 156 bergey’s manual 99 bio-based 5 bio-medicine 84 bio-refineries 63 biocatalyst 118 bioconversion 114 biodegradable 2, 52, 69, 80, 87, 163, 164 biodegradable polymer 46 biodiesel waste 95, 102 biofilms 127 biomass 79, 81, 93, 163, 164, 166, 168, 169, 170 bioprocess technology 184 bioreactors 58, 166, 167 biosynthetic pathways 78, 154 biotechnological approaches 96 broad substrate spectrum 100 butyric acid 11, 163, 164, 165, 167, 172 byproducts 81, 82, 89, 114, 165 C4H4O4 70 C13 NMR 76 calcium fumarate 71 calcium gluconate 117 calcium oxalate 179 candida 127, 134, 145, 146 carbohydrate biomass 35 carbon source 167, 169 carboxyl groups 69, 96 carrier 171 cashew apple juice 184 catalyst 179 cell 164, 166, 167, 168, 169, 172 cellular engineering 172 cellulases 37 cellulose 81, 165, 172 CH4 production 86 cheaper substrates 189 chelating ability 113 chemical synthesis 95, 96, 97, 101 chitosan 127, 141 citric acid 2, 4, 21, 22, 23, 26, 28, 29, 30 clostridium 164, 165, 167, 169, 172 clumps 79, 80 co-fermentation 82 CoA 97

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Index

coffee grounds 42 continuous 166, 168, 170 corn steep liquor 44 corn stover 37 cornfibre 156 corynebacterium 9 cost effective manner 184 cristulariella pyramidalis 183 crop stalk wastes 59 crystallization 60, 136, 137 cyanide antidote 104 cyanide 96 cytotoxic 169 D-malate 156 deaminases 101 dehydrogenase 95 deodorant 3 department of energy 70, 90 derivatives 63, 119 diagnose 103 diethyl oxalate 10 diethyl succinate 10, 96, 97 dilute acid pretreatment 42 direct precursor 95, 96 distillation 170, 171 DO levels 75 domestic wastes 23 downstream 83, 89, 118 ecofriendly 102 electrodialysis 102, 136, 172 electrons 167 embden–meryerhof–parnas 37 emulsification 172 enzymatic hydrolysis 42 enzyme activity 76 escherichia coli 54 esterification 86, 88, 171 esters 96, 103 ethanol production 188 ethylene glycol 178 evaporation 171 extraction 170, 171, 172 FAO 83 fatty acids 100, 103 fed batch 58 fed-batch culture 153 fed-batch fermentation 4 feed batch 82 feedstock 163, 164

fermentable sugars 42 fermentation 3, 11, 55, 69, 70, 72, 74, 75, 77, 78, 80, 81, 82, 83, 85, 89, 90, 91, 92, 94, 95, 99–102, 116, 150 fermentative approach 187 fermentative production 72, 74, 75, 180 ferric oxide 80 filamentous fungi 36, 151 filtration 29 flavoring agent 45 fluidized-bed 167 food industry 45, 95, 96, 103, 187 food packaging 127 food wastes 36 fossil-based 130 fruit wastes 26, 30 fumarase 77, 78, 81, 92, 153 fumarate 97, 101 fumaric acid (FA) 7, 8, 69 fumaric anhydride 71 fungal metabolism 181 gas chromatography 186 GDH 115 generally recognized as safe (GRAS) 102 genetic engineering transformation 100 global climate 157 global warming 86 glucokinase 23 gluconates 112 gluconic acid 8, 185 glucono-δ-lactone 112 gluconobacter oxydans 116 glutamine 10, 96 glycerol 4, 6, 7, 10, 12, 95, 97, 100, 101, 102, 130, 132, 134, 138, 139, 143, 144 glyoxylate cycle 155 glyoxylate dehydrogenase 181 grape must 59 heavy metal 127 hemicellulose 139 hetero-fermentative 6 high energy radiation 78 histidine 102 hydrocarbons 100, 102 hydrogenation 54 hydrolysates 169 hydrolysis 117 hydrophobic 100 hydroxymethylfurfural 139 immobilization 167, 168

Index

industrial metabolites 156, 177 industrial wastes 155 inexhaustible resources 77 infection 120 ingredient 164 inoculum size 186 intermolecular 113 intracellular 63 invertase 23 IRA-900 84, 93 itaconate ions 136 itaconic acid 9, 125, 147 jerusalem artichoke tuber 156 juvenile mortality 188 keto-acid 96, 97 ketoglutarate 95, 96, 97, 98, 100, 102, 104 ketogultaric acid 103 kluyveromyces marxianus 41 kojic 131 kreb’s cycle 74, 75, 76, 95, 96 KTC 6946 78 L-amino acids 101 L-malate 154, 156 L-maleic acid 75, 76, 85 L. brevis 41 lactic acid 6, 35 lactose permeate 183 large-scale fermentation 189 lignin 132, 139, 146 lignocellulose 11 lignocellulosic 55, 132, 138, 139, 140, 141, 143, 146 lignocellulosic biomass 36 limitations 63 lipids 100 low cost substrates 35 low-cost biomass 12 maleic acid 8 maleic anhydride 8, 11 malic acid 10, 150 mannose 81 manufacture 63 manure 103 maple syrup urine disease 103 medicine 62 membranes 102, 172 metabolic engineering 153 metabolic process 167 metabolite 57 metal tolerance 178

197

metallurgy 120 methodologies 57 methylotrophic 116 microbial acid 184 microbial contamination 22 microbial fermentation 1, 3, 10, 21, 26, 35, 155, 189 microbial oxalic acid 186 microbial production 184 microbial synthesis 2 microorganisms 22, 112, 125, 131, 166, 171 monosaccharides 132, 138 MSW 44 mutagenesis 139, 140 mycelia 75, 80, 115 N-LAAD 102 n-paraffins 100 N-terminal transmembrane region 102 NADPH 101 neurotoxic 104 neutralization 114 neutralizing agent 56, 74, 91 nitrogen excretion 103 nitrogen source 80, 93 nitrosoguanidine 78 non-volatile 170 olefinic bond 69, 71, 86 optimization 56, 100 orange peel 26 organic acids 2, 5, 6, 7, 12, 52, 70, 73, 83, 94, 112, 150 organic wastes 37, 125, 130 organophosphorus 136 organosolv 132 output 166, 167, 168, 169, 172 over production 96, 101 oxalate biosynthetic component 180 oxalic acid 178 oxaloacetate hydrolase 185 oxaloacetic acid 77 oxidation of glucose 99 oxidative pathway 155 oxidative stress 81 oxygen control 74, 76, 92 pack-bed 167 palladium salt 179 papaveraceae 69 PCR 78 pellets 79, 80, 82 penicillium oxalicum 183 penicillium sclerotiorum 154

198

Index

pentose 167, 168 petrochemical industry 157 petroleum based 125, 137 pharmaceutical 1, 21, 22, 30, 35, 189 phenolics 80 phosphoenol 98 Phosphoenolpyruvate kinase 57 phosphorus stress 79 physical parameters 55 pigments 52 pilot scale 76 Pinus radiate 81 plant growth 188 polyethylene glycol (PEG) 87 polyhydric alcohols 86 polymalic acid 152 polymerization 86 polysaccharides 58, 132 pomace 82 PQQ 115 precipitation 134, 136 prevotella bryantii 97 product 163, 165, 166, 167, 168, 170, 171, 172 production 58, 120 properties 63 pseudomonas fluoroscens 95, 97 pseudozyma antarctica 127, 133 psoriasis 84, 88, 94 purification 60, 166, 171 pyruvate 167 R. arrhizus 73, 75, 81 R. formosa 73 R. nigricans 73 R. oryzae 73, 77, 78, 81, 82 ram horns 28 raw material 81, 82, 83 recycle 117 recycled fiber 42 reducing powers 76 renewable carbon source 95, 102, 104 renewable feed-stocks 155 renewable substrates 2, 11, 12 resins 70, 84, 86, 87 response surface methodology 184 reverse-flow diafiltration 133 rhizopus spp. 77, 78, 90 rhodotorula 127, 134 rice straw 41 rice wastewater 41

saccharomyces cerevisiae 36, 78, 92, 100, 151 saturated fatty acids 77 savour 85 schizosaccharomyces pombe 153 secondary metabolites 149 segregation 170 semipermeable 62 simultaneous saccharification and fermentation 41 sodium gluconate 113 solid state 127, 129, 143 solid support 80 solid-state and submerged 21 solid-state fermentation 41, 82 solvent 171 starches 138 stirred-tank bioreactor 152 straw, baggase 126 submerged culture 184 submerged fermentation 113, 128, 132, 133 submerged state fermentation 22 substrates 168, 169, 170 succinate metabolism 104 succinate semialdehyde 97 succinic acid 5, 52, 95, 96, 101 sugar-cane 81 sugarcane bagasse 43 sugarcane molasses 28, 43 sulphur metabolism 103 supplements 120 sustainable 164 sweet corn 29 synthetic 63 TCA cycle 95, 96, 97, 99 technique 119 textile treatment 178 TOR 105 TORC1 105 Torulopsis glabrata 96 transcriptional 172 transformation 62 translation 77 tricarboxylic acid (TCA) cycle 76 tricarboxylic acid 4, 181 trichoderma reesei 140 triol 81 tumor suppression 105 tyrobutyricum 168 tyrosinosis 103 ultra filtration 61, 118

Index

ustilaginaceae 134, 140, 145 ustilago 127, 133, 134, 139, 140, 143, 144, 145, 147 value added products 22, 26 vanadyl pyrophosphate 71 vine shoots 59 viscosity 79 waste 119, 163, 164, 166, 168, 172

water purification 127 wheat straw 43 xylanase 37 xylose 81, 91, 93, 154, 165, 167, 169 yarrowia lipolytica 95, 100 yield 119

199