Multifunctional Microbial Biosurfactants 9783031312304, 3031312309

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Pankaj Kumar Ramesh Chandra Dubey   Editors

Multifunctional Microbial Biosurfactants

Multifunctional Microbial Biosurfactants

Pankaj Kumar • Ramesh Chandra Dubey Editors

Multifunctional Microbial Biosurfactants

Editors Pankaj Kumar Department of Botany and Microbiology H.N.B. Garhwal University (A Central University) Srinagar Garhwal, Uttarakhand, India

Ramesh Chandra Dubey Department of Botany and Microbiology Gurukul Kangri Vishawavidyalaya Haridwar, Uttarakhand, India

ISBN 978-3-031-31229-8 ISBN 978-3-031-31230-4 https://doi.org/10.1007/978-3-031-31230-4

(eBook)

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Mātṛ Devo Bhav. Pitṛ Devo Bhav. Ācārya Devo Bhav. (Tattirīya Upaniṣad 1.11.2) (See God in Mother, Father and Teachers)

Preface

The book entitled ‘Multifunctional Microbial Biosurfactants’ encompasses 22 chapters on various types of microbial biosurfactants and their applications in different area. The chapters also provide an overview of the different production process of biosurfactants and its future aspect. Therefore, this book will be beneficial for postgraduate students, research scholars and scientists working in various areas of biosurfactants. Biosurfactants are attractive amphiphilic surface-active molecules derived from microorganisms (bacteria, fungi, actinomycetes and cyanobacteria) and differ in their structural and physico-chemical properties depending on the organism. Different techniques are used to isolate these surface-active agents. Biosurfactants are environment-friendly and an alternative of synthetic surfactants, which are highly selective, biodegradable and impart limited toxicity. Biosurfactants are usually applied as emulsifiers and reducers of surface tension in different fields, mainly in the oil and detergent industries. As compared to chemical surfactants, biological surfactants have better surfactant properties and are essential for the remediation of soil and sea water. In recent times, the bio-pesticides have gained attention in the management of fungi, pest and insects and have long been boosted as potential alternative of chemical pesticide. Lipopeptide and rhamnolipid biosurfactants are low in toxicity to the ecosystem and highly biodegradable in nature and promising surface-active compound that could be used as bio-pesticides. The biosurfactant producing PGPRs are important to raise the disease-free crop and help in counteracting the problem of food security. Besides, the biosurfactants are the best alternative for the biological control of mosquito. The two vital features promoting the applications in markets are economic growth and cost-effectiveness. Biosurfactants are safer for the environment, less poisonous and easily decomposable than the chemical surfactants. It has numerous applications in the food industry, healthcare and cosmetic industries. Biosurfactants are used for oil clean-up, soil remediation, pesticide manufacturing, plant growth promoters, drug delivery, medicine, agriculture and environmental safety. Cosmeceuticals are cosmetic products having some specific therapeutic effects. vii

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The role of biosurfactants in cosmetic and detergents industry and their applications have also been covered in this book. Biosurfactants also exhibit antioxidant, antimicrobial, anti-ageing, cytotoxicity and anti-inflammatory antiviral activities. It kills herpesvirus, retrovirus and coronavirus by interacting with viral membrane and carbon atoms in surfactin’s acyl chain. It is predicted that biosurfactants might be the potential inhibitors of SARSCoV-2. Biosurfactants-mediated nanoparticles have exhibited multipurpose roles in biomedicine, particularly as antibacterial, antifungal, antibiofilm, anticancer, wound healing, anti-inflammatory, mosquitocidal and dermal drug delivery agents without showing toxicity to the normal cells. We extend our heartiest thanks to all contributors for providing an insight into these important areas of research and development. We also thank Dr. Sofia Costa and the entire team of SPRINGER-NATURE for publishing this book. We are indebted to our teachers, parents and family members because this tedious journey could not be completed without their blessing and support. Srinagar Garhwal, Uttarakhand, India Haridwar, Uttarakhand, India

Pankaj Kumar Ramesh Chandra Dubey

Contents

Screening Methods for Biosurfactant-Producing Microorganisms . . . . . Sumeyra Gurkok and Murat Ozdal

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Purification Assessment and Assay of Biosurfactant Efficacy . . . . . . . . . Abiram Karanam Rathankumar, Kongkona Saikia, Suganyadevi Palanisamy, Rathi Muthaiyan Ahalliya, and Mariadhas Valan Arasu

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Methods of Screening and Applications of Biosurfactants Produced by Actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O. Archana and Lokesh Ravi

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Methods of Screening and Applications of Biosurfactants Produced by Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. R. Shree Kumari and Lokesh Ravi

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Fungal Biosurfactants and Its Applications . . . . . . . . . . . . . . . . . . . . . . Hoda Nouri, Hamid Moghimi, and Elham Lashani

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Production of Biosurfactant by Bacteria from Extreme Environments: Biotechnological Potential and Applications . . . . . . . . . . . . . . . . . . . . . . 129 Mariana Amaral Azevedo, Letícia Portugal do Nascimento, Maria dos Remédios Vieira-Neta, and Iolanda Cristina Silveira Duarte Microbial Biosurfactants: An Eco-Friendly Perspective Environmental Remediation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 Bruna Gabryella Andrade de Lima, Renata Raianny da Silva, Italo José Batista Durval, Leonie Asfora Sarubbo, and Juliana Moura de Luna Lipopeptide and Rhamnolipid Biosurfactant as Biopesticides . . . . . . . . . 171 S. Nalini, S. Sathiyamurthi, T. Stalin Dhas, and M. Revathi

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Biosurfactants for Formulation of Sustainable Agrochemicals . . . . . . . . 189 Maria da Gloria C. Silva, Fabiola Carolina G. de Almeida, Anderson O. de Medeiros, and Leonie A. Sarubbo Biosurfactants: Role in Plant Growth Promotion and Disease Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Sumit Kumar, Pankaj Kumar, and Ramesh Chandra Dubey Rhamnolipids from Pseudomonas aeruginosa in the Cleaning of Polluted Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Arelis Abalos-Rodríguez, Odalys Rodríguez-Gámez, and Yaima Barrios-San Martin Mosquitocidal Activity of Biosurfactants . . . . . . . . . . . . . . . . . . . . . . . . 251 R. Parthasarathi, S. Harini, P. Poonguzhali, K. Akash, and N. Kavinilavu Biosurfactants as Promising Surface-Active Agents: Current Understanding and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Harmanjit Kaur, Pankaj Kumar, Amandeep Cheema, Simranjeet Kaur, Sandeep Singh, and Ramesh Chandra Dubey Role of Biosurfactants in Enzyme Production . . . . . . . . . . . . . . . . . . . . . 307 Rengasamy Sathya, Mariadhas Valan Arasu, Naif Abdullah Al-Dhabi, and P. Vijayaraghavan Microbial Biosurfactants in Food Processing Industry . . . . . . . . . . . . . . 329 Muhammad Bilal Sadiq, Muhammad RehanKhan, R. Z. Sayyed, and Imran Ahmad Biosurfactants in Cosmetic Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Suganyadevi Palanisamy, Rathi Muthaiyan Ahalliya, Abiram Karanam Rathankumar, Kongkona Saikia, Mariadhas Valan Arasu, Varshini Rajapandian, and Manokiruthika Vellingiri Applications of Microbial Biosurfactants in Detergents . . . . . . . . . . . . . 363 Murat Ozdal, Sumeyra Gurkok, and Volkan Yildirim Application of Biosurfactant in Petroleum . . . . . . . . . . . . . . . . . . . . . . . 383 Eduardo J. Gudiña, Jéssica Correia, and José A. Teixeira Biosurfactants in Medical Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 Kongkona Saikia, Abiram Karanam Rathankumar, Suganyadevi Palanisamy, Rathi Muthaiyan Ahalliya, and Mariadhas Valan Arasu Biosurfactants: An Antiviral Perspective . . . . . . . . . . . . . . . . . . . . . . . . 431 Sethuramalingam Balakrishnan, Marimuthu Ragavan Rameshkumar, Avoodaiappan Nivedha, Krishnan Sundar, Narasingam Arunagirinathan, and Mariadhas Valan Arasu

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Biosurfactants-mediated Nanoparticles as Next-Generation Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455 Ameer Khusro, Chirom Aarti, and Mariadhas Valan Arasu Production Cost of Traditional Surfactants and Biosurfactants . . . . . . . 495 Rathi Muthaiyan Ahalliya, Femil Selta Daniel Raja, Kavitha Rangasamy, Vijayachitra Arumugam, Suganyadevi Palanisamy, Kongkona Saikia, Abiram Karanam Rathankumar, Naif Abdullah Al-Dhabi, and Mariadhas Valan Arasu

About the Editors

Pankaj Kumar completed his master’s and doctorate degrees at the Gurukula Kangri Vishwavidyalaya, Haridwar, Uttarakhand (India). He has more than ten years of teaching and research experience. His research interest and expertise are rhizosphere microbiology, biosurfactants, and biofertilizers etc. He has published several research papers in national and international reputed journals. He has edited two books Rhizosphere Engineering (2022), Macrophomina Phaseolina: Ecobiology, Pathology and Management (2023) published by Academic Press (Elsevier), along with Prof R.C. Dubey. He is also a coauthor of An Objective Compendium on Food Science (Brillion publishing, India, 2022). He is life member of the Association of Microbiology of India (AMI), New Delhi and Indian Science Congress, Kolkata (West Bengal) and serving as reviewer and editorial board member of several national and international reputed journals. Currently, Dr. Pankaj is associated with the Department of Botany and Microbiology, H.N.B. Garhwal University (A Central University), Srinagar Garhwal, Uttarakhand, India.

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About the Editors

Ramesh Chandra Dubey is a Professor, the Director (Research and Development Cell), Dean-Research, Dean-Faculty of Medical Science and Health, and the former Head of the Department of Botany and Microbiology, Gurukula Kangri Vishwavidyalaya, Haridwar, Uttarakhand (India). He obtained his M.Sc. and Ph.D. degrees from the internationally renowned Banaras Hindu University, Varanasi (India). He has more than 35 years of teaching and research experience in the field of Agricultural Microbiology and Biotechnology. He has published more than 208 research papers in the national and international journals of repute. His Google citation index is above 5138 till date. He has authored 7 books and co-edited 10 books under different subjects such as agriculture, microbiology and biotechnology. In 2022, Dr. Dubey published a book, Vedic Microbiology with Motilal Banarsidass International, New Delhi (India). Dr. Dubey is a Life Member and Fellow of the Indian Botanical Society, Indian Phytopathological Society and the International Society for Conservation of Natural Resources. He also previously served as the Chairman of the Institutional Animal Ethical Committee (IAEC) for 6 years (2015–2021) and the Councillor of the Indian Botanical Society for 3 years (2011–2013).

Screening Methods for Biosurfactant-Producing Microorganisms Sumeyra Gurkok and Murat Ozdal

Abbreviations (NH4)2SO4 BATH BTB CMC CTAB EU FC FTIR GC-MS H2SO4 HCl HIC HPLC LC-MS MATH NaCl NaOH nm NMR OD SDS TBA TLC ZMA

Ammonium sulfate Bacterial adhesion to hydrocarbon Bromothymol blue Critical Micelle concentration Cetyltrimethylammonium bromide Emulsification activity Foaming capacity Fourier transform infrared spectroscopy Gas chromatography-mass spectrometry Sulfuric acid Hydrochloric acid Hydrophobic interaction chromatography High-performance liquid chromatography Liquid chromatography-mass spectrometry Microbial adhesion to hydrocarbon Sodium chloride Sodium hydroxide Nanometer Nuclear magnetic resonance spectroscopy Optical density Sodium dodecyl sulfate Tributyrin agar Thin layer chromatography Zobell marine agar

S. Gurkok (✉) · M. Ozdal Department of Biology, Science Faculty, Ataturk University, Erzurum, Turkey © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_1

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1 Introduction Surfactants are amphiphilic chemical compounds containing hydrophilic and hydrophobic moieties that partition at physical interfaces and reduce the surface and/or interfacial tension between different phases (Santos et al. 2016). Biosurfactants, on the other hand, are attractive surface-active molecules derived from mostly microorganisms. Biosurfactants have many advantages over synthetic surfactants in terms of structural diversity, lower toxicity, lower critical micelle concentration (CMC), stability, and biodegradability (Edwards et al. 2003; Jahan et al. 2020). Therefore, they are in great demand in different industries with emulsification, foam formation, detergent, and oil dispersion activities. The biggest obstacle that limits the use of biosurfactants in different industrial sectors instead of their synthetic counterparts is that they are produced in low quantities and at high cost. Majority of the chemical surfactants are obtained from petrochemical industry and can therefore be produced with high efficiency and low cost. However, this mode of production is widely considered as unsustainable and contrary to sustainable green economy strategies, and also damages the ecosystem due to toxicity and bio-incompatibility issues. They are also not preferred because of consumers’ tendency toward natural products and sustainable production systems. On the other hand, biosurfactants avoid the concerns related to the use of petrochemical sources. Cost-effective production is extremely important in increasing the application areas of biosurfactants instead of synthetic surfactants. Using cheap substrates and waste materials in production and optimizing the production process are frequently applied strategies (Ozdal et al. 2017; Rastogi and Kumar 2021). However, the isolation of strains that efficiently produce novel biosurfactants with diverse properties is the critical step to overcome the economic constraints of biosurfactant production. The production of biosurfactant has gained importance recently, and in parallel with this, the discovery of novel biosurfactant-producing organisms is kept in the foreground. The process for microbial biosurfactant production begins with sampling, and areas contaminated with hydrocarbons are among the most suitable environments for sampling. Following sampling, various screening methods are used for the detection and isolation of promising microbial strains able to produce high yields of biosurfactants. While majority of the screening methods rely directly on the surface and/or interface activity of the cell culture supernatant. Others are dependent on the microbial cell surface hydrophobicity, which is an indication of biosurfactant synthesis. An ideal screening assay should (1) be able to detect the promising microorganisms, (2) even if it is a qualitative method, it should provide an idea about the production yield and ensure the selection of the most effective microorganisms, (3) be economical and easy to implement, (4) allow screening of a large number of candidates simultaneously, and (5) give fast results and save time. Microbial biosurfactants have a great structural diversity and can be classified according to different criteria, such as their microbial origin, molecular weight, biochemical structure, and mechanism of action. According to their molecular

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weights, they are generally divided into two large groups as high-molecular-weight and low-molecular-weight biosurfactants. Protein, lipoprotein, lipopolysaccharide, polysaccharide, and biopolymers are classified as high-molecular-weight biosurfactants. Lipopeptide, glycolipid, phospholipid, and fatty acids have been grouped as low-molecular-weight biosurfactants (Rosenberg and Ron 1999). Biosurfactants are also classified according to their biochemical structure and the microorganisms that produce them. The five main biosurfactant classes are: (1) glycolipids, (2) phospholipids and fatty acids, (3) lipopeptide/lipoproteins, (4) polymeric surfactants, and (5) particulate surfactants (Parra et al. 1989). Since it is not possible to screen all types of biosurfactants with a single method, it is necessary to use various screening methods in combination for effective, reliable, and accurate screening and isolation (Ariech and Guechi 2015; Gurkok 2022). Based on this, the current chapter provides commonly used methods involved in screening for biosurfactant-producing microorganisms.

2 Sampling for the Isolation of Biosurfactant-Producing Microorganisms Biosurfactants can be obtained by sampling from a wide variety of environments as seen in Table 1. Although the isolation of biosurfactant-producing microorganisms can be performed by sampling from various undisturbed and contaminated areas (Bodour et al. 2003; Gurkok 2022), hydrocarbon contaminated sites are mostly preferred. Hydrocarbon contamination often results from leakage of crude oil from storage facilities or tanks, spills during transportation of petroleum products, and deliberate discharge of petroleum derivatives and by-products into soil or water. Environments contaminated with hydrocarbons for such various reasons have proven to be good sources for biosurfactant-producing microorganisms in many studies (Shoeb et al. 2015; Joy et al. 2017; Patowary et al. 2017; Astuti et al. 2019; Balakrishnan et al. 2022). Biosurfactants produced by extremophiles are also of interest for different biotechnological purposes, and therefore, extreme habitats are also preferred as sampling areas (Cameotra and Makkar 1998; Schultz and Rosado 2020). Numerous studies have shown that hot environments, such as deserts, volcanoes, and hot springs (Zarinviarsagh et al. 2017) and cold environments, such as alpines, glaciers, permafrost, ice caves, deep and polar oceans (Perfumo et al. 2018), extreme pH (Arulazhagan et al. 2017), and saline (Sarafin et al. 2014) environments can also be used for sampling.

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Table 1 Biosurfactant-producing strains, sampling areas, and screening methods Strain Pseudomonas mendocina ADY2b

Bacillus sp., Streptomyces sp., Microbacterium sp., Micrococcus sp., Rhodococcus., Pseudomonas, Arthrobacter sp., Staphylococcus sp. B. subtilis, P. aeruginosa, B. tequilensis, B. safensis

Serratia sp., Paenibacillus sp., Citrobacter sp. B. subtilis

Pseudoxanthomonas sp.

Geotrichum candidum, Galactomyces pseudocandidum, Candida tropicalis

Franconibacter sp. C. parapsilosis

Aspergillus terrus, A. fumigatus Brevibacterium casei Halomonas elongata

B. halotolerance Pseudomonas sp.

S. marcescens

Sampling area Hydrocarboncontaminated soil of Chennai Harbor Oil batteries, Chauvin, Alberta

Screening method Drop collapse, E24, hemolysis, oil spreading

References Balakrishnan et al. (2022)

CTAB, drop collapse, oil spreading, emulsification, hemolysis

Rani et al. (2020)

Potwar oil fields, Pakistan

Drop collapse, E24, emulsification assay, hemolysis, tilted glass slide, oil spreading Drop collapse, E24, oil spreading, surface tension Bath, drop collapse, E24, oil spreading, surface tension

Sohail and Jamil (2020)

E24, hemolysis, interfacial tension, oil spreading

Astuti et al. (2019)

CTAB, E24, hydrocarbon overlay agar plate, hemolysis, oil spreading, ParafilmM, phenol sulfuric acid, phenol red test, surface tension CTAB, drop-collapse, E24, oil spreading, Parafilm M Drop collapse, E24, hemolysis, oil spreading

Eldin et al. (2019)

Soil, Amapaense Amazon Brackish water of Chilika Lake, Odisha, India Petroleum reservoir, South Sumatra Rhizosphere soil, Egypt

Soil from Lakwa oil field, Assam Contaminated dairy products, India Crude oil sludge, Malaysia Textile wastewater Khewra slat mines, Pakistan Oil fields, CNPC, China Motor oil-contaminated soil, Tunisia Hydrocarboncontaminated site in Melaka, Malaysia

Drop collapse, E24, oil spreading, parafilm test, surface tension E24, oil spreading, surface tension CTAB, drop collapse, E24, hemolysis Oil spreading E24, oil spreading, surface tension E24, surface tension

Oliveira et al. (2021) Nayarisseri et al. (2018)

Sharma et al. (2022) Garg and Chatterjee (2018) Othman et al. (2022) Carolin et al. (2021) Fariq and Yasmin (2020) Wang et al. (2022) Chebbi et al. (2017) Almansoory et al. (2019)

(continued)

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Table 1 (continued) Strain S. quinivorans, Psychrobacter arcticus Janthinobacterium svalbardensis

Ochrobactrum intermedium

Kocuria marina

Sampling area Pony Lake, Ross Island, Antarctica Cotton glacier, Transantarctic Mountains, Antarctica GheynarjeNir hot spring Ardebil, Iran Condenser pond of Kovalam, India

Screening method E24, oil spreading E24, oil spreading

References Trudgeon et al. (2020) Trudgeon et al. (2020)

Drop collapse, E24, hemolysis, oil spreading

Zarinviarsagh et al. (2017)

Drop collapse, E24, hemolysis, oil spreading

Sarafin et al. (2014)

3 Methods for Screening Biosurfactant-Producing Microorganisms Biosurfactants are structurally heterogeneous amphiphilic molecules derived from mostly microorganisms. As a result of the heterogeneity, diverse approaches for screening prospective biosurfactant-producing microorganisms have been wellestablished, devised, and implemented as shown in Table 1. Some of the screening methods including, bacterial adherence to hydrocarbon, hydrophobic interaction chromatography, salt aggregation test, and replica plate tests are based on measuring cell surface hydrophobicity, which is directly related to biosurfactant production (Pruthi and Cameotra 1997). In most screening methods, crude oil, hexane, n-hexadecane, xylene, and sunflower oil are used as hydrocarbons. In the following screening methods, controls are not individually specified for each method, but typically, 1% (w/v) SDS or Triton X-100 is used as the positive control, and distilled water or a buffer is applied as the negative control.

3.1

Bacterial Adhesion to Hydrocarbons Assay

The test known as Bacterial Adhesion to Hydrocarbon (BATH) or Microbial Adhesion to Hydrocarbon (MATH) was first used for measuring cell-surface hydrophobicity by Rosenberg et al. (1980). According to this method, biosurfactant producer microorganisms attach to hydrocarbons due to their hydrophobic cell surfaces. By measuring the hydrophobicity of the cell, this method offers a simple and rapid spectrophotometric test for prescreening for biosurfactant-producing microorganisms. Detection of biosurfactant-producing bacterial strains of Acinetobacter calcoaceticus, Bacillus pumilus, B. laterosporus, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aceticus was achieved rapidly

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by testing cell surface hydrophobicity, which has a direct correlation with biosurfactant production (Pruthi and Cameotra 1997). Using this method, numerous microorganisms producing various types of biosurfactants including lipopeptides, phospholipids, glycolipids, fatty acids, and polymeric biosurfactants have been identified (Nayarisseri et al. 2018). In this assay, after 18 to 24 h of incubation in liquid medium, cells are harvested by centrifugation. Cell pellets are washed several times with phosphate buffer and suspended in the same buffer to reach an optical density of about 0.5 at 600 nm. In a test tube, 2 mL of cell suspension and 100 μL of a hydrocarbon, such as octane, hexane, xylene, hexadecane, or crude oil, are mixed and vortexed briefly for 2 to 3 min. The mixture is left for approximately 1 h to allow separation of aqueous phase and hydrocarbon phase. The OD of the aqueous solution is then determined at 600 nm to estimate the reduction in turbidity. The formula below is used to calculate the percentage of cell adhesion to the hydrocarbon. Cell adhesion ð%Þ = ½1ðOD600 aqueous solution =OD600 starting cell solution Þ × 100:

3.2

Bromothymol Blue (BTB) Assay

BTB assay is a quantitative colorimetric assay used to screen the strains producing lipopeptide-containing biosurfactants by mixing a solution of BTB (0.2 mM) in phosphate buffer, pH 7.2 with an equal volume of cell-free culture medium. Color change is determined spectrophotometrically at 410 and 616 nm. This method can be used for both culture broth and purified lipopeptide-containing biosurfactants, such as surfactin, iturin, and fengycin. Color changes are determined as yellow for iturin, light green for fengycin, and green for surfactin (Ong and Wu 2018). The advantages of this method are that it is suitable for rapid, simple, quantitative analysis, and screening of lipopeptide type biosurfactant-producing species. In addition, similar to this test, cetylpyridinium chloride-bromothymol blue (Yang et al. 2015) and polydiacetylene (Zhu et al. 2014) methods were also used for surfactin determination. Recently, Kubicki et al. (2020) have reported a useful colorimetric method with the potential to detect biosurfactant in culture supernatants. Victoria Pure Blue BO, a hydrophobic blue dye, can also be used in the comparative assessment of biosurfactant quantification in supernatants of bacterial cultures (Kubicki et al. 2020). The quantity of dye released is measured spectrophotometrically at 625 nm.

Screening Methods for Biosurfactant-Producing Microorganisms

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CTAB Agar (Blue Agar) Plate Assay

Siegmund and Wagner (1991) described this method for the detection of rhamnolipids. The blue agar plate assay is another name for the CTAB (cetyltrimethylammonium bromide) agar plate assay. This method is used for screening of extracellular glycolipids or anionic surfactants, which have a polar head and a nonpolar tail. Microorganisms that produce glycolipid-type biosurfactant, such as P. aeruginosa, form a clear halo around the colony as shown in Fig. 1. For screening, microbial isolates are spot-inoculated on mineral salt agar medium supplemented with 0.005 g/L methylene blue and 0.2 g/L CTAB and grown for 1 to 2 days. Formation of the clear blue zone surrounding the streaks on dark blue agar plates is attributed to the secretion of anionic biosurfactant. CTAB assay is a semiquantitative approach because the size of the zone is related to the amount of biosurfactant released. CTAB screening assay provides fast, simple, and accurate results, but CTAB itself has been shown to inhibit the development of several bacteria. Furthermore, its specificity for anionic biosurfactants like glycolipids limits its use in screening microorganisms producing other types of biosurfactants. Therefore, this approach has often been used to evaluate producer of glycolipid biosurfactants (Eldin et al. 2019; Rani et al. 2020).

3.4

Drop-Collapse Assay

Drop-collapse assay works on the idea that biosurfactant destabilize or collapse the liquid droplets on hydrocarbon surface. In drop-collapse screening assay, described by Jain et al. (1991), drops of culture supernatant are deposited on a surface coated Fig. 1 Clear halo formation by P. aeruginosa (+) on mineral salt agar plates supplemented with 0.005 g/ L methylene blue and 0.2 g/ L CTAB. No zone formation is observed in microorganisms that cannot produce biosurfactant (-)

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with oil. In the lack of biosurfactant, the hydrophobic surface repels the polar water molecules, keeping the drop stable on oil-coated surface. Drops from the culture supernatant of biosurfactant-producing colonies collapse and spread as a result of the decrease in the interfacial tension between the oil-coated surface and the liquid droplet. The consistency of drops is connected with surface tension but not with emulsifying activity, and it is dependent on biosurfactant concentration. Bodour and Miller-Maier (1998) developed the drop-collapse assay on 96-well microtiter plate cover. Two microliters of oil is placed on 96-well microplate lid and left to equilibrate at room temperature for 24 h. A drop of supernatant is applied to the surface coated with oil and monitored after 1 min by the use of a magnifying glass. In the absence of biosurfactant, the drops remained stable and rounded; but in its presence, they spread and became flat. Drop-collapse assay is a reliable technique often used in screening as it offers a quick and simple approach to evaluate large numbers of samples simultaneously, does not require the use of special equipment, and only a minimal volume of sample is required for examination (Sohail and Jamil 2020; Rani et al. 2020; Oliveira et al. 2021; Balakrishnan et al. 2022).

3.5

Emulsification Assay

Emulsification assay precedes the most commonly used methods for screening biosurfactant-producing colonies (Nayarisseri et al. 2018; Rani et al. 2020; Sohail and Jamil 2020; Carolin et al. 2021). Assay, described by Rosenberg et al. (1979), evaluates the emulsification of a hydrocarbon by spectrophotometric measurements. Culture supernatant, suspended in Tris buffer (pH 8), is mixed with equal volume of crude oil and vortexed for 1 min. The emulsion is allowed to stand for about 20 min, and the optical density in the aqueous phase is then measured in the spectrophotometer at 400 nm. The formula below is used to calculate emulsification activity (EU/mL). EU=mL = 0:01 OD400 × Dilution Factor

3.6

Emulsification Index Assay (E24)

Emulsification index approach described by Cooper and Goldenberg (1987) is another frequently used method for screening of the biosurfactant producers (Zarinviarsagh et al. 2017; Chebbi et al. 2017; Almansoory et al. 2019; Trudgeon et al. 2020; Fariq and Yasmin 2020). It is simple to implement and requires minimal specialist instruments. Emulsifying activity is measured by calculating the emulsification index (E24) for a crude oil.

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In E24 examination, culture supernatant is mixed with equal volume of hydrocarbon in a test tube, vortexed thoroughly for at least 2 to 3 min, and allowed to stand for 24 h at room temperature. Emulsification index is calculated by using the formula as given below: Emulsification index ðE24 Þ = ðHeight of the emulsion layer=Total heightÞ × 100:

3.7

Foam Test

Biosurfactants have foaming properties due to their amphiphilic (hydrophobic and hydrophilic groups) nature (Gurkok and Ozdal 2021a). The foaming properties of biosurfactants can be used to screen for biosurfactant producers. Foaming is related to the reduction of surface tension by surfactants. Foam, in the presence of surfactant, reduces the surface tension between an aqueous solution and air, resulting in the mixing of the two different phases and, consequently, the formation of bubbles. The foaming capacity is determined by transferring 10 mL of the cell-free culture broth into a 50 mL graduated measuring cylinder and vigorously shaking or vortexing for 1 to 2 min (El-Sheshtawy and Doheim 2014). To calculate the foaming capacity, the foaming height and the total height are measured. The foaming capacity is determined according to the following equation. Foaming capacity = ðHeight of foam=Total heightÞ × 100: This method is an easy and simple test to screen biosurfactant production (Hamzah et al. 2020).

3.8

Hemolysis Test

The basic idea behind the hemolysis test invented by Mulligan et al. (1984) is that biosurfactants can lyse erythrocytes. In this assay, isolates to be screened are streaked on blood agar plates containing 5% blood and incubated for 2 days at 25 °C. Formation of hemolysis halos surrounding the colonies due to the blood cells lysis indicates the production of biosurfactant as shown in Fig. 2. The fairly easy to implement hemolysis assay is often used as a preliminary screening for biosurfactant producers (Astuti et al. 2019; Sohail and Jamil 2020; Rani et al. 2020; Balakrishnan et al. 2022), but the method has some limitations, such as giving false-positive and false-negative results. In the absence of biosurfactant, lytic enzymes also cause the formation of a transparent hemolysis zone; in the presence of some biosurfactants, clear zone formation may not be

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Fig. 2 Hemolysis zone formation by P. aeruginosa (+) after incubation on blood agar plates with 5% blood at 25 °C for 2 days

observed because they do not have hemolytic activity (Schulz et al. 1991). Therefore, this test needs to be validated by other screening methods.

3.9

Hydrocarbon Overlay Method

Hydrocarbon overlay assay uses Zobell Marine Agar (ZMA) coated with a hydrocarbon, such as benzene, toluene, and hexadecane, for qualitative screening of biosurfactants producers. Isolates are spot-inoculated on ZMA plates and incubated at 25 °C for 3 to 5 days. The formation of an emulsified halo surrounding the colonies suggests the production of biosurfactants (Nayarisseri et al. 2018; Eldin et al. 2019).

3.10

Hydrophobic Interaction Chromatography (HIC)

Hydrophobic interaction chromatography (HIC) allows the rapid and reliable screening for biosurfactant producer isolates depending on the hydrophobicity of the cell surface, which has a direct relationship with biosurfactant production (Smyth et al. 1978). Since, HIC is a chromatographic separation technique that separates molecules based on differences in their surface hydrophobicity, it can simultaneously purify these isolates in addition to screening. The column resin with hydrophobic moieties like butyl, phenyl or octyl, is stabilized with a buffer containing salt to increase hydrophobic interaction. Cells are suspended in this buffer, and suspension is applied to the column. Non-retained cells are eluted whereas organisms having hydrophobic cell surface are retained by

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the column. The initial cell suspension is compared to the elution by cell count or spectrophotometer measurement to determine the hydrophobic index as the percentage of retained bacteria.

3.11

Lipase Assay

The principle of using the lipase assay to screen biosurfactant-producing bacteria is that there is a correlation between production of lipases and biosurfactants (Colla et al. 2010). Lipase production is the characteristic feature of the biosurfactantproducing organisms (Kalyani and Sireesha 2014). Qualitative lipase assay is performed on tributyrin agar (TBA) plate (Gurkok and Ozdal 2021b). Isolate is streaked on TBA plate, and lipolytic clear zone formation is monitored after 3 to 5 days of incubation at 25 °C (Fig. 3). This assay is used as prescreening approach and should be confirmed by additional screening methods (Kalyani and Sireesha 2014; Chittepu 2019).

3.12

Oil-Displacement (Oil-Spreading) Assay

Oil-spreading test also known as oil-displacement test has been described by Morikawa et al. (2000) and is widely used for screening of biosurfactant-producing microorganisms (Zarinviarsagh et al. 2017; Garg and Chatterjee 2018; Trudgeon et al. 2020; Othman et al. 2022; Wang et al. 2022; Balakrishnan et al. 2022). It is a reliable, fast, and simple method and requires only a small amount of sample, and therefore, it is one of the most commonly used tests. Fig. 3 Lipolytic zone formation by B. cereus on TBA plate after 3 days at 25 °C

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Fig. 4 Oil-spreading assay of P. aeruginosa cell-free culture supernatant by using diesel motor oil

Fig. 5 Parafilm M test by P. aeruginosa cell-free culture supernatant deposited on parafilm after 1 min (a) and 5 min (b). SDS is the positive control; H2O is the negative control

In this screening method, 1 mL of oil is deposited on the surface of 20 to 30 mL distilled water in a Petri plate to create a fine coating of crude oil. Ten microliter of culture supernatant is deposited in the center of the oil layer surface. In the presence of biosurfactant, a distinct zone surrounding the supernatant is observed as shown in Fig. 4. There is a linear correlation between the size of the zone and the amount of biosurfactant.

3.13

Parafilm M Test

Parafilm M test is fairly simple and rapid test requiring modest amount of sample. This approach is often used for qualitative preliminary screening of biosurfactant producer organisms in conjunction with other procedures (Eldin et al. 2019; Sharma et al. 2022; Othman et al. 2022). A droplet of supernatant is deposited with a micropipette on parafilm M, which is used as a hydrophobic surface. After a minute, the shape of the droplet on the parafilm is monitored. The droplet spreads on the surface when biosurfactant is present; otherwise, it remains dome-shaped (Fig. 5).

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13

Penetration Assay

Penetration test, developed by Maczek et al. (2007), is another assay appropriate for high throughput screening of biosurfactant producers (Chittepu 2019). This test is based on the color change that occurs when two insoluble phases come into contact. The wells of 96-well microplates are filled with 200 μL of hydrophobic paste containing oil and silica gel. Hydrophobic paste is covered by 10 μL of crude oil. Ninety microliter of supernatant stained with 10 μL safranin is deposited to the wells, and color change is monitored. Oil cover is destroyed in the presence of biosurfactant, and silica gel reaches to the hydrophilic phase within 10 to 15 min, and the color of the supernatant shifts from bright red to hazy white. The color of the supernatant still gets cloudy but stays red in the lack of biosurfactant.

3.15

Replica Plate Assay

Rosenberg (1981) developed the replica plate experiment on the idea that cell producing biosurfactant binds to hydrophobic polystyrene because of its hydrophobic cell surface. This method allows both screening and isolation of biosurfactantproducing colonies. Cells to be examined are incubated on agar medium. A sterile and flat polystyrene disc is pressed on colonies, and the replicas of the colonies are formed. The disc is rinsed with water to eliminate the loosely attached colonies. Biosurfactant-producers due to their hydrophobic surfaces have affinity to polystyrene and firmly attach to disc. Remaining securely adhered colonies are fixed with methanol and dyed for visibility. The positive colonies can easily be obtained by isolation from the original plate.

3.16

Salt Aggregation Assay

Salt aggregation assay invented by Lindahl et al. (1981) is also a screening approach based on cell surface hydrophobicity of biosurfactant-producing cells. Salt aggregation testing, which requires no special equipment, provides an easy way to screen for bacteria-producing biosurfactants (Pruthi and Cameotra 1997; Walter et al. 2010). Depending on their cell surface hydrophobicity, various cell types precipitate at different salt concentrations. Cells that produce biosurfactants have more hydrophobic cell surfaces and precipitate at lower salt concentrations. Due to its great solubility, ammonium sulfate ((NH4)2SO4) is widely employed in salt aggregation assays. In the assay, an overnight culture is centrifuged, and the harvested cell pellets are dissolved in phosphate buffer. Increasing quantities of (NH4)2SO4 (0.01–4.0 M) are

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arranged in the same buffer. Equal amounts of cell suspension and salt solutions are combined at room temperature on a glass depression slide. The aggregation result, which produces a clear solution and white aggregates, is then evaluated on a black background.

3.17

Surface/Interfacial Tension Analysis

Determination of surface or interfacial activity of a cell culture supernatant is the easiest way of screening for biosurfactant-producing microorganisms. The surface tension is defined as the surface force between a liquid and air, while interfacial tension is the force between two liquids (Bodour and Miller-Maier 1998). Surface/ interfacial activity is measured directly by the use of a digital tensiometer coupled with a Du Noüy platinum ring (Du Noüy 1925). Du Noüy platinum ring is positioned beneath the supernatant surface. The force needed to move the ring from the aqueous phase to the air is determined and used to determine surface tension using the equation below: ST = ½ðF-P0 Þ=4Πr × 1000 Where, F is the force measured, P0 is the force measured prior to removing the ring, and r is the ring radius. The surface/interfacial tension can also be measured by the stalagmometric method (Dilmohamud et al. 2005), pendant drop shape method (Nierderhauser and Bartell 1950; Tadros 2008), and axisymmetric drop shape method (Van der Vegt et al. 1991). Although surface/interfacial tension analysis requires specialized equipment, it is a frequently used method due to ease of application and to get fast and accurate results (Astuti et al. 2019; Eldin et al. 2019; Oliveira et al. 2021).

3.18

Tilted Glass Slide Assay

In the tilted glass slide assay described by Persson and Molin (1987), a single colony to be screened is transferred to a drop of 0.9% NaCl placed at one end of a glass slide. The slide is tilted to the opposite side and the movement of a water droplet along its surface was monitored. In the presence of biosurfactant, the water runs across the surface. Tilting glass slide assay is a relatively less used method compared to the others (Sohail and Jamil 2020).

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Determination of the Biochemical Composition of Biosurfactants

The biochemical composition of biosurfactants can be determined by total protein, total carbohydrate, and total lipid content analysis.

3.19.1

Phenol-Sulfuric Acid Test

Phenol sulfuric acid test applied for the screening of glycolipid surfactants was developed by Dubois et al. (1956). One milliliter of culture supernatant is mixed with 1 mL of 5% phenol. To this mixture, 3 to 5 mL of concentrated sulfuric acid (H2SO4) was added drop by drop. The presence of glycolipid biosurfactant is indicated by a color shift from yellow to orange. This method is generally used to quantify rhamnolipids by using a rhamnose standard curve (Ozdal et al. 2017; Eldin et al. 2019).

3.19.2

Biuret Test

In the presence of peptides, the copper (II) ion forms mauve in a basic solution and albumin is used as a standard. The biuret test is used to detect the presence of lipopeptide biosurfactants, such as lichenysin, fengycin, iturin, and pumilacidin produced by Bacillus genus (Kumar and Ngueagni 2021). Two mL of crude extract solution is heated to 70 °C and 10 drops of 1 M NaOH solution are added. Next, 1% CuSO4 is slowly added to the mixture to observe the violet or pink color change and measurements are made at 540 nm (Patel and Patel 2020).

3.19.3

Phosphate Test

This test is used to detect the presence of phospholipid biosurfactants. After adding 10 drops of 6 M HNO3 to 2 mL of crude extract solution, it is heated to 70 °C. Five percent ammonium molybdate is added dropwise to the mixture. The formation of a yellow precipitate after the formation of a yellow color indicates the presence of phospholipids (Patel and Patel 2020). In addition to these techniques, new screening approaches continue to be developed. The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to screen for glycolipid-type biosurfactant-producing organisms is an example to recent approaches (Sato et al. 2019). With the discovery of novel biosurfactants, new screening systems will undoubtedly be put into use in the near future.

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4 Analytical Methods for Compound Detection of Biosurfactants Characterization is generally performed with the purified biosurfactants obtained after extraction and purification steps. Some of the characterization methods used to analyze the biosurfactant properties are listed in the Table 2.

4.1

Extraction of Crude Biosurfactant

The main purpose of the extraction is to obtain crude biosurfactants that are free from other culture media components. The most common biosurfactant recovery method is acid precipitation followed by extraction with organic solvents (chloroformmethanol, ethyl acetate). As it is relevantly cheap method, acid precipitation is generally preferred. Concentrated HCl is widely added to culture supernatant for acidification until pH reaches 2.0 and the mixture is left overnight at 4 °C for precipitation. The biosurfactant is then extracted with chloroform:methanol (2:1 v/ v) or ethyl acetate using a separatory funnel (Abdelli et al. 2019; Ratna and Kumar 2022). Different methods, such as ammonium sulfate precipitation, acetone

Table 2 Biosurfactant-producing microorganisms and analytical methods used for their characterization Microorganism B. safensis Ochrobactrum anthropi, Citrobacter freundii Candida tropicalis B. velezensis Metschnikowiahurdharensis B. altitudinis Gordonia sp. P. putida P. aeruginosa P. aeruginosa

Recovery process Acid precipitation+ ethyl acetate Acid precipitation + chloroform: methanol (2:1) Chloroform: methanol (2: 1) Ammonium sulfate Ethyl acetate Acid precipitation + ethyl acetate Acid precipitation + chloroform: methanol (2:1) Acid precipitation + chloroform: methanol (2:1) Acid precipitation + chloroform: methanol (2:1) Acid precipitation + chloroform: methanol (2:1)

Analytical method TLC, LC-MS, HPLC TLC, FTIR TLC, FTIR, NMR LC-MS, GC-MS TLC, HPLC, GC-MS TLC, FTIR, LC-MS, GC-MS TLC, FTIR, HPLC, GC-MS TLC, FTIR, NMR, GC-MS, LC-MS TLC, FTIR, LC-MS TLC, GC-MS, HPLC TLC, FTIR, NMR, GC-MS, LC-MS

References Abdelli et al. (2019) Ibrahim (2018) Almeida et al. (2021) Meena et al. (2021) Kumari et al. (2021) Goswami and Deka (2019) Zargar et al. (2022) Mishra et al. (2020) Hrůzová et al. (2020) Ratna and Kumar (2022)

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precipitation, centrifugation, crystallization, adsorption, and foam fractionation are the others methods for extraction of biosurfactants.

4.2

Chromatographic and Spectroscopic Methods

Thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), Fourier transform infrared spectroscopy (FTIR), and nuclear magnetic resonance spectroscopy (NMR) are recently preferred approaches used for detection of biosurfactants because of their automation, high sensitivity, and accuracy (Table 2). These methods have both advantages and disadvantages and can be chosen according to the properties of biosurfactants, including stability, solubility, molecular size, and charge. TLC is a straightforward technique for determining the presence of substances such as lipids, peptides, and sugars. The purified biosurfactant (approximately 0.1 g) is dissolved in methanol or chloroform then aliquots (20 μL) are applied to silica gel TLC plate. The TLC plate is run with a mobile phase of chloroform:methanol:water (65:25:4) (Abdelli et al. 2019). Molish’s reagents, iodine vapor, and 1% ninhydrin solution are sprayed on dry plate for staining of sugars, lipids, and free amino groups, respectively (Ibrahim 2018). HPLC is a special type of column chromatography where it can separate the mixture of surfactant compounds, identify, quantify, and purify the biosurfactant components separately. HPLC applications have been reported for the purification, characterization, and quantification of biosurfactants (Twigg et al. 2021). FTIR is a useful tool for rapid analysis and determination of functional groups of biosurfactants. This method determines hydroxyl, ester, and carboxylic groups in biosurfactants according to their IR absorption bands (Eslami et al. 2020; Sen et al. 2021). GC-MS is widely used for structural analysis of biosurfactants. Frequently, it is used for quantitative or qualitative analysis of fatty acid structures. When combined with MS, information about the molecular mass and elemental composition, functional groups, and molecular geometry of each separated compound can be obtained. GC or GC-MS analysis is also used for the analysis of fatty acid chain derivatives (Biniarz et al. 2017). LC-MS provides an excellent measurement for rapid, inexpensive, and quantitative measurements of organic molecules in a wide variety of applications. In general, LC-MS analyzes the hydrophilic (water-loving) part of the biosurfactant compound, while GC-MS identifies the hydrophobic (water-repellent) part (Jimoh and Lin 2019). NMR gives information about the bond structures and functional groups in lipids and carbohydrates. NMR is a suitable technique to accurately correlate the chemical structure and position of the presence of biosurfactant compounds in a sample (Kim

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et al. 2018). It is a suitable method for the chemical structure determination of the novel biosurfactants.

5 Future Directions The vast majority of biosurfactants have been detected from microbial sources in a culture-dependent manner, leaving mostly unexplored supply of uncultured microorganisms producing possibly new biosurfactant structures. Recent progresses in understanding biosurfactants at the genome level have made screening of microorganisms much easier. Parallel to these developments, modern technologies rather than traditional culture-dependent approaches have begun to be considered and applied for the discovery of new microorganisms. Advanced techniques, such as metagenomic and meta transcriptomic analysis, have been used to investigate the potential of microorganisms for biosurfactant production (Jackson et al. 2015; Thies et al. 2016; Williams and Trindade 2017; Williams et al. 2019). A new gene related to biosurfactant production and hydrocarbon degradation has been identified by da Araújo et al. (2020). They extracted environmental DNA from soil samples and constructed a metagenomic library. They identified a biosurfactantpositive clone by functional screening and an open reading frame with high similarity to sequences encoding a hypothetical protein. They purified the protein and observed biosurfactant activity. Also, they observed elevated hydrocarbon degradation in the E. coli cells transformed with the gene encoding this protein. With these promising methods, environmental DNA samples as well as microorganisms can be genetically examined and rapidly screened without the need for culturing microorganisms, and enable the identification of novel biosurfactants with different and desired properties in the future (Perfumo et al. 2018).

6 Conclusion The main barriers to large-scale biosurfactant production are still high production costs and low yields. In addition to using inexpensive substrates and waste or by-products in production and optimizing the production process, the isolation of strains that efficiently produce novel biosurfactants with diverse properties is a critical step to overcome the economic constraints of biosurfactant production. Currently, biosurfactants such as rhamnolipid, sophorolipid, and surfactin are commercially produced. It is necessary to find new biosurfactants with suitable structural diversity for specific purposes in different industries. One of the approaches to find new biosurfactants is the application of different screening methods. The shift to more precise, efficient screening methods is seen as the key to the discovery of new biosurfactants. In order to accelerate the discovery of new biosurfactants, technological devices should also be utilized. Although there are many screening methods,

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with the discovery of novel biosurfactants with various chemical structures and physicochemical properties, it is clear that more advanced methods will be needed in the future to screen the producer of microorganisms.

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Purification Assessment and Assay of Biosurfactant Efficacy Abiram Karanam Rathankumar, Kongkona Saikia, Suganyadevi Palanisamy, Rathi Muthaiyan Ahalliya, and Mariadhas Valan Arasu

Abbreviations CAGR CaLB CMC DEAE ELS FAMEs FID FTIR GC HCl HPLC kDa NMR pI rf RID SPE

Compound annual growth rate Candida antarctica lipase B Critical micelle concentration Diethylaminoethyl Evaporative light scattering Fatty acid methyl esters Flame ionized detector Fourier’s transform infrared spectroscopy Gas chromatography Hydrochloric acid High-pressure liquid chromatography Kilodaltons Nuclear magnetic resonance Isoelectric point Retention value Refractive index Solid phase extraction

A. K. Rathankumar (✉) Department of Biotechnology, FoE, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India e-mail: [email protected] K. Saikia · S. Palanisamy · R. M. Ahalliya Department of Biochemistry, FASCM, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India M. V. Arasu Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_2

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TLC UPLC

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Thin layer chromatography Ultra-performance liquid chromatography

1 Introduction Surfactants or surface-active compounds are the compounds with the ability to reduce the surface tension between two phases by forming micelles (Rathankumar et al. 2021b). Chemically, surfactants are amphiphilic in nature and act as antimicrobial agents by disrupting their cellular membranes. Surfactants exist as monomer and form micelles after reaching a particular concentration called critical micelle concentration (CMC). The monomers of surfactants at CMC value aggregate to form micelle tubes or micelle bilayer or vesicles (Rathankumar et al. 2021b). The surfactants increase the solubility of hydrophobic compounds in hydrophilic matrix or vice versa with the increase in concentration till it reaches CMC value. Due to these properties, surfactants have a wider application in various industries as cleaning agents, soap and detergent, emulsifying agent, paint and pharmaceutical industries (Rathankumar et al. 2021b). With its wide application, surfactants had a potential market of 41.3 billion USD in 2019 and is expected to raise to 58.5 billion USD by the end of 2027 with a compound annual growth rate (CAGR) of 5.3%. In addition, extensive utilization of surfactant during COVID-19 pandemic further boosted its market value (Allied market research). The surfactants are generally synthesized using animal fats, petrochemicals products, and plant fats. However, persistence to degradation along with toxicity of synthetic surfactant has a huge impact on the environment. Alternatively, biosurfactants and bio-based surfactants, with their origin from biological compounds, are slowly replacing the synthetic surfactants. The current market for the biosurfactants was over 1.75 billion USD in 2020 and was expected to grow by 5.5% CAGR over 2021 to 2027 (Allied market research). Owing to this potential market, several strategies have been developed for the production of biosurfactants from microorganisms and other biological sources. Being a secondary metabolite, biosurfactants are generally produced along with several other metabolites, which require extensive application of downstream process for the purification. However, due to structural variability and diverse chemical properties, purification of these compounds is tedious in nature. Hence, the current chapter focuses on various downstream processes utilized for the purification of biosurfactants.

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2 Properties of Biosurfactants and Its Applications Compared to synthetic surfactants, biosurfactants can be synthesized using board spectrum of substrates, making them industrially feasible for the production. Most of biosurfactants have the ability to reduce the surface tension of water from 72 mN/m to a minimum of 26 mN/m (rhamnolipids) and exhibit low CMC value than synthetic surfactants (Jiang et al. 2020). Depending on the origin of the biosurfactants, the properties of biosurfactant vary greatly. The biosurfactants synthesized by thermophilic bacteria were reported to show higher tolerance to extreme temperatures. For instance, lichenysin from Bacillus sp. exhibit thermal resistance up to 50°C (McInerney et al. 1990). Similarly, the biosurfactants produced by alkaliphiles exhibit high tolerance to basic pH (Khalikova et al. 2019). Being a secondary metabolite, most of the biosurfactants act as a food source (carbon source) for the cellular metabolism, making them easily degradable. Due to their antimicrobial nature, biosurfactants are reported to be less toxic than synthetic surfactants and were proved to be safe for medical, food, and cosmetic applications (Bjerk et al. 2021). The unique properties of biosurfactants resulted in several advantages when compared to synthetic surfactants and are widely utilized in various industries for their biodegradability, high bioavailability, less toxicity, and high foaming capacity (Bjerk et al. 2021). Figure 1 shows the properties of biosurfactants. Most of the Environmental properties Biodegradation

Lowtoxicity

Environmental friendly Low cytotoxicity

Biological properties Antimicrobial

Antibiofilm

CMC Anti corrosive Biosurfactant (Monomer)

Biosurfactant (Micelle form) Emulsification agent

Wetting agent

Functional Properties Detergent

Foaming agent

Dispersion agent

Fig. 1 Properties of biosurfactants

Antioxidant

Reduces Interfacial tension Reduces surface tension

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biosurfactants are multifunctional due to their emulsifying, antimicrobial, antifouling, stabilizing, wetting, and moisturizing properties (Rathankumar et al. 2021b). Recently, biosurfactants were also utilized as soil washing agents or soil flushing agents for the removal of contaminants from environment (Rathankumar et al. 2021b).

3 Types and Production of Biosurfactants Due to the diversity of biosurfactants, these compounds are broadly classified into three types based on their charge associated with hydrophilic group as anionic surfactants, cationic surfactants, and nonionic surfactants (Rath and Srivastava 2021) (Fig. 2). Anionic surfactants are negatively charged in their hydrophilic moiety and produce lot of foam when mixed, making them a common ingredient in soap and detergents. Whereas, cationic surfactants are positively charged which make them useful as antimicrobial agents. On the other hand, the nonionic surfactants have the net charge as zero and are considered as good emulsifying agents. In general, nonionic surfactants and anionic surfactants were utilized in combination to enhance the emulsification activity. Conventionally, biosurfactants are classified into four major groups based on their chemical diversity as glycolipids, lipoproteins or lipopeptides, phospholipids, and polymeric surfactants (Drakontis and Amin 2020). Glycolipids are the class of surfactants consisting of one or more carbohydrates as hydrophilic head and one or more fatty acid as hydrophobic tail. Rhamnolipids, trehalolipids, and sophorolipids are the best examples of glycolipids. Whereas, the biosurfactants with peptide or protein linked with fatty acid by ester or ether linkages are categorized under lipoproteins or lipopeptides. Most of the lipopeptides (surfactin, lichenysin) are antimicrobial agents and are generally used as therapeutical agents. On the other hand, phospholipid consists of phosphate group as head and fatty acid as tail joined by alcohol residue. Phospholipids are often used as medication for the replacement of pulmonary surfactants (Katyal and Singh

Fig. 2 Classification of surfactants based on physical and chemical properties

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1984). Polymeric biosurfactants are the surfactants that are produced by microorganisms as an extracellular polymer. Polymeric biosurfactants are good emulsifying agents, and best-reported polymeric biosurfactants include lipomanan, liposan, biodispersan, and emulsan. With respect to molecular weight, biosurfactants are categorized into lowmolecular-weight biosurfactants and high-molecular-weight biosurfactants (Elsoud and Ahmed 2021). Low-molecular-weight biosurfactants are effective in reducing the surface tension, and most of the glycolipids and lipopeptides come under this category. Polysaccharides, some lipoproteins, and polymeric particles are categorized into high-molecular-weight biosurfactants and are reported to be good emulsifying agents (Sharma et al. 2021).

4 Production of Biosurfactants Even though biosurfactants created an increasing acceptance in market, these biosurfactants failed to compensate the market demand of synthetic surfactants. High production cost, low yield, and involvement of several downstream processes for product recovery are considered to be main reasons in the difficulty for the production of biosurfactants (Webera et al. 2012). However, with the advancement in microbiology and enzyme technology, several strategies were reported for the production of biosurfactants with industrial feasibility. Among several biosurfactants, rhamnolipids production using Pseudomonas aeruginosa is a wellestablished industrially feasible fermentation process (Zhao et al. 2018). Conventionally plant oil, glycerol or soyabean oil were used as a substrate for the rhamnolipids synthesis using Pseudomonas aeruginosa (Li et al. 2019). With more than 20,000 publications and 6000 patents on the synthesis of rhamnolipids, several substrates were exploited as a starting material for the rhamnolipids synthesis. Similar to rhamnolipids, sophorolipids by Candida bombicola (Silveira et al. 2018), surfactin by Bacillus sp. (Hoffmann et al. 2021), and trehalose lipids by Rhodococcus sp. (Marqués et al. 2009), were reported to be commercially successful processes for biosurfactant production by fermentation technology. However, utilization of facultative pathogenic bacteria and production of biosurfactants along with other secondary metabolites limit their commercialization (Rathankumar et al. 2021a). The selection of carbon source was considered as a key step in the production of biosurfactants, which accounts for more than 60% of the production cost. Further, optimization of the conditions of temperature, pH, agitation speed, and incubation time must be performed for achieving a maximum yield of biosurfactants (Asha et al. 2018). Figure 3 shows an overview of biosurfactants production. As an alternative, enzymatic synthesis of biosurfactants under controlled environment stood as a promising strategy. Rathankumar et al. (2021a) exploited the CaLB (Candida antarctica lipase B) for the production of rhamnolipids by esterification of rhamnoside to fatty acid, as an alternative strategy for the production highly

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Isolation of microorganism

Screening of microorganism

Media Optimization Culturing of microorganism

Production of seed culture

Media formulation

Fermentation

Filtration/centrifugation

Extraction of biosurfactants

Purification of biosurfactants

Fig. 3 Flow chart of biosurfactant production using microorganisms

purified mono-rhamnolipids. Similarly, Saikia et al. (2020) utilized CaLB for the synthesis of 5-hydroxymethylfurfural (HMF) esters.

5 Purification of Biosurfactants Design of downstream process is often considered as crucial process in any industrial process, as it contributes immensely to the total cost of the product (Webera et al. 2012). Being extracellular in nature, the purification of biosurfactants is often dependent on the biochemical properties of biosurfactants (Thavasi and Banat 2019). A simple solvent extraction to complex multiple units operation have been reported for the extraction and purification of biosurfactants. The commonly utilize purification methods include solvent extraction, acid precipitation, centrifugation, and foam fractionization (Weber and Zeiner 2014). Further, the choice of purification method also depends on the production of primary and other secondary metabolites, cellular debris, microbial contamination, and extraction yield (Weber and Zeiner 2014). Among these, extraction yield and presence of metabolites were considered to play a major role in the selection of purification strategy, as other interference can be removed by using simple unit operations such as centrifugation and filtration. For example, in case of rhamnolipids, acid precipitation was considered as a commonly used extraction and purification strategy (Dardouri et al. 2021). Table 1 shows some of the commonly used strategies for the purification of biosurfactants.

Mode of purification Batch mode

Chloroform Ethyl acetate Ethanol Methanol Acetone or ethanol

Organic solvent extraction

Solvent precipitation

Ammonium sulfate Zinc sulfate

Salt precipitation

Strategy Acid precipitation

Commonly used chemical/ instrument Hydrochloric acid Sulfuric acid

Addition of ice cold acetone or ethanol to the fermentative broth

Lipopeptides Lipoproteins

Simple and easy process

Efficient for crude biosurfactant and partial purification

Rhamnolipids Trehalolipids Liposan

Solubilization of the broth with organic solvents

Applicable for selected biosurfactants. Utilization of organic solvents. Against green principles Less extraction yields.

Utilization of huge quantities of organic solvents

Effective in the extraction of lipoproteins and lipopeptides

Disadvantage Often requires other unit operation to achieve high purification. Effective only for positively charged hydrophilic (anionic) biosurfactants Contamination with cellular proteins are high

Advantage Low cost, efficient extraction of crude biosurfactant from fermented broth

Emulsion Surfactin

Commonly used for Rhamnolipids Surfactin Sophorolipids

Salting out the biosurfactant by adding salt to the fermentation broth

Purification principle Reduction of fermented broth pH to 2.0 using acid and allow the surfactant to precipitate

Table 1 Various reported strategies for the purification of biosurfactants

(continued)

Shah et al. (2016), Meena et al. (2021) Varjani and Upasani (2017) RodríguezLópez et al. (2020), Adetunji and Olaniran (2019)

References Dardouri et al. (2021) , Cazals et al. (2020)

Purification Assessment and Assay of Biosurfactant Efficacy 31

Mode of purification Continuous

Commonly used chemical/ instrument Electrolysis chamber

Fractionation column

Ultra filtration unit (membrane)

Column with stationary phase for the adsorption of biosurfactants

Strategy Isoelectric focusing

Foam fractionation

Ultrafiltration

Adsorption and desorption chromatography

Table 1 (continued)

Biosurfactants are adsorbed on to the surface of carrier matrix such as silica and are desorbed by eluting with a solvent or combination of solvents

The gas is sparged into vessel containing broth for the creation of foam, which is collected at the top of the column Micelle-formed biosurfactants are trapped in polymeric membrane

Purification principle Separation of biosurfactants by applying voltage of 10 V to 30 V

Fast and one-step recovery method with high efficiency

Cheaper and reusable

Glycolipids Lipoprotein

Advantage Simple and cheaper process for the extraction of biosurfactants based on charge Continuous process for the extraction of biosurfactants from the reactor

Glycolipids, lipopeptides

Glycoproteins

Commonly used for Ionic surfactants

Require more than two ultrafiltration units to achieve highest purity. Maintenance of membrane and membrane fouling reduces the efficiency Not highly specific

Disadvantage Presence of other charged particles reduces the purity of extracted biosurfactants Yield of the biosurfactant is relatively less

Dubey et al. (2005)

Satpute et al. (2010), Darton et al. (2004) De Andrade et al. (2016)

References Gidudu and Chirwa (2021)

32 A. K. Rathankumar et al.

Biosurfactants are purified based on the retention value

Separation based on the ability of charged biosurfactants to bind with counter charged support

Silica gel coated plate

Ion exchange column

Preparative TLC chromatography

Ion exchange chromatography

All the biosurfactants

All kinds of biosurfactants

Reusable and fast recovery with maximum purity

Conventional method for identification and purification of biosurfactants Purify only limited quantities of biosurfactants Time-consuming process and involves solvents Fouling of column and high cost association for elution buffer Flaih et al. (2016)

Meena et al. (2021)

Purification Assessment and Assay of Biosurfactant Efficacy 33

34

5.1

A. K. Rathankumar et al.

Acid Precipitation

Acid precipitation is a commonly used strategy for the purification of anionic biosurfactants such as rhamnolipids (Dardouri et al. 2021). In general, the ionic surfactant in solution interacts with other counter reactive ions and form precipitates. Some anionic surfactants, such as carboxylates surfactants react with positivity charged particles like magnesium ions or chloride ions to form hydrophobic solid colloidal particles. In acid precipitation method, the cell debris-free fermentation broth containing anionic surfactants can be precipitated by reducing the pH to 2.0 using acid. Commonly, hydrochloric acid or sulfuric acid with concentration ranges from 1 N to 6 N was used for precipitation, and the reaction was reported to be carried out overnight at 4°C to increase the precipitation yield (Cazals et al. 2020; Shen et al. 2019; Felix et al. 2019). The resultant precipitants can be recovered by either centrifugation or filtration process. After washing, the precipitants were resuspended either in buffer or water by adjusting the pH to neutral using sodium hydroxide. Being a complex biochemical broth, the presence of other anionic particles may affect the purity of biosurfactant by precipitating along with the biosurfactants during the acid precipitation process. To overcome this problem, the acid precipitation process is often coupled with organic solvent extraction. Chloroform, methanol, ethyl acetate, or combination of chloroform and methanol are commonly used organic solvents for the extraction of biosurfactants (Shen et al. 2019). Anionic biosurfactants, such as rhamnolipids, are reported to be extracted using 2 N HCl by incubating overnight at 4°C and extracted using chloroform and methanol in 2:1, v/v ratio. On the other hand, Vanjara and Dixit (1996) reported simple cationic surfactant recovery method by precipitating cetylpyridinium chloride using potassium iodide.

5.2

Salt Precipitation

Lipoproteins and other polymeric biosurfactants were reported to be extracted by precipitating the protein or peptide moiety of the biosurfactants using salting out method. Salts such as ammonium sulfate and zinc sulfates are the widely reported salts used for the salting out of surfactants (Shah et al. 2016; Varjani and Upasani 2017). The ammonium sulfate-based extraction of biosurfactants was first reported by Rosenberg et al. (1979) for the extraction of bio-emulsifier from Arthrobacter. Depending on the type and concentration of biosurfactants, various concentrations of ammonium sulfate were reported for the extraction of high-molecular-weight biosurfactants. Meena et al. (2021) extracted surfactin from B. Velezensis KLP2016 culture broth by saturating with ammonium sulfate and incubating for overnight at 4°C. Timmis et al. (2010) reported a purification strategy of lipoprotein by saturating the cell debris-free fermentation broth with ammonium sulfate and the

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resultant precipitant was further purified using dialysis to remove smaller molecule contaminants.

5.3 5.3.1

Organic Solvent Extraction Acetone and Ethanol Precipitation

Apart from acid precipitation and salt precipitation, acetone- and ethanol-based precipitation of biosurfactants is a commonly reported strategy for the extraction of protein- or peptide-based biosurfactants. The ability of ice cold acetone to precipitate protein is exploited for the purification process. Rodríguez-López et al. (2020) reported the precipitation of biosurfactants using acetone and incubating overnight at -80°C. The resultant precipitants were vacuum filtered and washed with cold acetone. Saranraij et al. (2022) reviewed various biosurfactants recovery strategies, and reported utilizing three volumes of chilled acetone for the extraction of biosurfactant by incubating for 10 h at 4°C. Similar to acetone, ethanol was also reported to be used to precipitate biosurfactants. Reduction of dielectric potential of water, followed by increased electrostatic interaction among the protein or peptide part of surfactants and counter charged region results in aggregation of surfactants in the presence of ethanol. Adetunji and Olaniran (2019) precipitated bio-emulsifiers produced by Acinetobacter using cold ethanol in 1:3 ratio and incubating overnight at -20°C. Similarly, de Veras Souza Maia et al. (2022) extracted biosurfactants produced by Bacillus sp. from cassava wastewater using ethanol precipitation method.

5.3.2

Other Organic Solvents

Apart from acetone and ethanol precipitation, ethyl acetate, chloroform, methanol, n-hexane, and butanol were reported to be commonly used solvents for the extraction of biosurfactants. Most of the organic solvent extractions are performed in combination with precipitation or chromatography methods. However, few reports utilized ethyl acetate as an organic phase for the separation of surfactants (Thavasi and Banat 2019). Saranraij et al. (2022) reviewed the application of chloroform and methanol in 2:1, v/v ratio for the biosurfactant extraction by liquid–liquid extraction using separating funnel.

5.4

Foam Fractionation

Foam fractionation process depends on the ability of the biosurfactants to selectively adsorb on the surface of bubble, which is formed during fermentation process. The

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foam fractionation-based biosurfactant recovery is considered as low cost with high efficiency and can be utilized in continuous process. In general, in foam fractionation, the gas is sparged into the vertical column fermenter or vessel containing culture broth at the bottom for the formation of foam. The raising stable air bubble in the liquid pool generates foam, which consist of surfactants. The surfactant adsorbed foams are collected at the top of the column. The collected foam is called foamate and is disrupted to collect surfactant-rich solution. By fabricating a simple fractionation column to a bioreactor, biosurfactants can be extracted. The extraction yields of the biosurfactants were reported to be enhanced by increasing the residual time of foam in the fractionation column, which allows the liquid in the bubble to drain by gravitational force (Satpute et al. 2010). The requirement of additional vessel for the storage of foam and requirement of longer duration for the destabilization of bubbles are reported to be major limitations. Darton et al. (2004) designed a continuous multistaged foam fractionation by introducing rotating device for the disruption of bubble. As per the design, the foam-containing biosurfactants enter the column from fermentation broth by bubbling gas. In the column, the bubbles are destroyed by a rotator and gives rise to foamate. The surfactant-rich foamate is collected and subjected to further purification. Chen et al. (2006) designed a foam fractionation column with rotator for the continuous extraction of biosurfactant produced by Bacillus sp. In some reports, foam fractionation is utilized in combination with other purification strategies to improve the purification efficiency. De Rienzo et al. (2016) extracted rhamnolipids by using foam fractionation process, and the resultant foamate was subjected to acid precipitation using hydrochloric acid for further purification.

5.5

Isoelectric Focusing

Isoelectric focusing is reported to be a simple purification strategy of biosurfactants. In isoelectric focusing, the biosurfactants are purified based on their isoelectric point (pI). At isoelectric point, the net charge of the biosurfactant is zero, and under this condition, a reduction in the electrostatic repulsion between neighboring molecules takes place. The reduction in the repulsive forces is reported to induce precipitation. Isoelectric focusing-based biosurfactants separation or purification was reported as early as the 1980s. Katyal and Singh (1984) reported the application of isoelectric focusing method for the separation of monkey lung and rat lung pulmonary surfactants using isoelectric focusing in the pH range of 4.64 to 5.53. Similarly, Gidudu and Chirwa (2021) designed an electrokinetic system for the extraction and recovery of biosurfactants. As per the report, the electrokinetic system was constructed with three compartments consisting of anode, medium, and cathode compartments. Graphite electrodes were positioned in anode and cathode compartments, and the electrode compartment was filled with deionized water. The interface of electrode and medium with fermented broth was sealed by cellulose filtration membrane. Upon subjecting to a voltage of 10 to 30 V, biosurfactant was separated based on

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the net charge of the compound (Gidudu and Chirwa 2021). In spite of achieving high purification efficiency using this strategy, very limited reporting on the commercial application was observed.

5.6

Ultrafiltration

Ultrafiltration is a unit operation and is utilized for the concentration or separation of biomolecules depending on their molecular weights. Ultrafiltration is a membrane filtration process with the application of membrane which ranges from 1 nm to 0.05 μm and hence comes between nanofiltration and microfiltration. This relative low temperature- and low pressure-driven mechanical unit operation is commonly performed using membrane filter or filter papers made of polymeric material (ploy ethersulfone, polysulfone, polyacrylonitrile, or polyvinylidene fluoride) or biological membrane. The ability to retain a biomolecule of particular molecular weight (molecular weight cut-off) is the main characteristic feature of ultrafiltration. As ultrafiltration does not require any chemical additives, products with high quality with minimized possibility of denaturation, deactivation, or degradation can be recovered. Due to the formation of micelles at the concentration above or equivalent to that of CMC, biosurfactants can be retained by using high molecular weight cut-off rather than smaller molecular weight cut off. De Andrade et al. (2016) utilized series of two ultrafiltration units for the separation of surfactin from Bacillus sp. fermented broth. As per the report, the membrane filtered fermented broth (0.45 μm) was subjected to two ultrafiltration units. Using first ultrafiltration unit with PES 100 kDa cut off, surfactant and proteins (large molecules) were separated as retentate and small protein, and salts and other biomolecules were removed as permeate. The resultant retentate was subjected to 75% ethanol to disrupt the micelles and subjected to 50 kDa ultrafiltration, which resulted in 78% recovery of surfactants in permeate. Similarly, Baxter et al. (2014) extracted bio-based surfaceactive compounds from urban refuse waste using a 5 kDa polysulfone membrane with molecular weight cut-off. The system was operated in tangential flow at 7 bar inlet pressure and 4.5 bar outlet pressure. Application of ultrafiltration units were not only limited to anionic surfactants but were also reported to be utilized for the purification of cationic surfactants (Klimonda and Kowalska 2019). It is clear from above the reports that due to the high efficiency and industrially feasibility, ultrafiltration has been a popular choice of downstream unit operation for the purification of biosurfactants.

5.7

Chromatographic Techniques

On utilizing starting materials such as distillery wastewater and biorefinery wastewater, the conventional downstream processes, like solvent extraction,

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centrifugation, precipitation/crystallization, and foam fractionation, are not ideal as they impart color in the final product. As an alternative, chromatographic techniques were utilized. Chromatographic techniques are commonly used either for purification or separation of the biomolecules based on their biochemical properties. In this context, several publications have been reported for the separation or purification of biosurfactants using various chromatographic techniques, which include preparative thin layer chromatography (TLC), ion exchange chromatography, and adsorption and desorption chromatography.

5.7.1

Preparative TLC Chromatography

Preparative TLC chromatography is a conventional method of purification, separation, and identification of biosurfactants based on the retention value (rf) in large scale (milligram to grams) than the traditional TLC. As compared to the traditional TLC, the preparative TLC consists of thicker silica coating to accommodate more samples. Similar to traditional TLC, the crude fermentation broth (sample) is loaded on the silica-coated plate and allowed to run in the presence of solvent system (mobile phase) for the separation of the biosurfactants. The separated biosurfactants (bands) can be visualized under UV after chromogenic reagent (ninhydrin) or acid treatment (orcinol reagent). After the confirmation, the separated bands can be scrapped out and extracted from silica by using solvent. Glycolipids (rhamnolipids) and lipoproteins (surfactin) are the commonly separated biosurfactants utilizing TLC method. Meena et al. (2021) utilized TLC for the purification and identification of surfactin in the presence of chloroform: methanol: water (39:15:3, v/v/v) solvent system based on the Rf value (0.94).

5.7.2

Adsorption and Desorption Chromatography

Adsorption and desorption chromatography is a technique in which the biosurfactant is adsorbed on to the surface of carrier matrix, such as silica, and is desorbed by eluting with a solvent or combination of solvents. The adsorption and desorption chromatography consists of three major steps, which include (1) loading of sample on the chromatography column and equilibration with buffer solution, (2) washing the column to remove any unbound contaminants, such as proteins, sugars, and cell debris, and (3) desorption of biosurfactants from the column using elution buffer or solvents. Hydrophobic polymeric XAD resins are the commonly reported carrier matrix for the adsorption of biosurfactants. However, to enhance the adsorption efficiency, various adsorbents, such as silica gel, activated alumina, wood-based activated carbon, and zeolite, were commonly screened. Dubey et al. (2005) screened various absorbents for the adsorption of biosurfactant produced by Pseudomonas aeruginosa strain BS2 from distillery wastewater and reported that wood-based activated carbon adsorbed 99.5% of biosurfactants under optimized conditions.

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Further, on eluting with acetone approximately 89% of biosurfactants was reported to be recovered. Weber and Zeiner (2014) reviewed adsorption desorption strategies for rhamnolipids purification and reported low loading capacity as a major disadvantage. Further it was reported that to absorb 1 g of rhamnolipids, approximately 20 g of resin is required, which is considered as commercially not feasible. On the other hand, the adsorbent, silica gel, is commonly utilized adsorbent, and the chromatography using silica gel is popularly called as “silica gel column chromatography.” Being highly porous in nature, silica gel has been universally utilized as an adsorbent for the separation of compounds with functional groups such as alcohol, phenol, amines, etc. Barale et al. (2022) reported an application of silica gel chromatography for the separation of lipopeptide using silica gel and eluting with linear gradient of methanol and chloroform. In continuous production of biosurfactants, more than one chromatographic strategy along with adsorption desorption chromatography were reported to be utilized for the purification (Webera et al. 2012; Weber and Zeiner 2014).

5.7.3

Ion Exchange Chromatography

Ion exchange chromatography is utilized as a single unit operation or in combination with adsorption–desorption chromatography for the purification of biosurfactants. Ion exchange chromatography works by separating the molecules based on their affinity toward the ion exchanger. Depending on the charge of stationary phase, the ion exchange chromatography is categorized as cation exchange chromatography and anionic exchange chromatography. The negatively charged anionic surfactants, such as rhamnolipids, are purified using anionic exchange chromatography. The application of ion exchange chromatography for purification of biosurfactants is a well-established technique from the early 1990s. Reiling et al. (1986) utilized a combination of adsorption–desorption chromatography and anionic exchange chromatography for the purification of rhamnolipids to achieve 90% pure rhamnolipids with an overall recovery of 60% (Reiling et al. 1986). On the other hand, Flaih AbdAl-Hussan et al. (2016) utilized anion exchange (DEAE-cellulose) followed by gel chromatography (Sepharose-6B) as a downstream process for purification of biosurfactants from Bacillus sp. fermented broth. Commercial resins, such as activated carbon, were also reported to have efficiency for the separation of surfactin. The work also studied the effect of particle size and reported that adsorption efficiency gradually increases with the reduction of particle size. Further, it was reported that temperature and pH play a major role in the adsorption.

5.8

Solid Phase Extraction

Solid phase extraction (SPE) is a simple technique, utilized for separation or purification of compound of interest before subjecting to other chromatographic

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analysis. The SPE technique is rapid with basic principle of transfer of compound of interest from liquid phase to the adjacent solid phase (stationery phase). The SPS column in general packed with either polar sorbent, such as silica or alumina, or nonpolar sorbent, such as silica phases or polymers. Most of the SPE columns are generally packed with nonpolar octadecylsilyl-derivatized silica and were reported to be used for the adsorption of anionic surfactants. The working step of the SPE is similar to that of other chromatographic techniques, and the process starts by loading centrifuged (6500 rpm for 10 min) or filtered fermented broth into the activated column. For activation, the column is washed with methanol and followed by water. The samples loaded can be eluted using solvent system and used either for further purification or characterization (Mohanty et al. 2013; Ismail and Baek 2020). Bodour et al. (2004) reported the utilization of C8 sorbent sandwiched between 27 mm polyethylene frits for the removal of secondary metabolites (pigment and impurities) from fermented broth of Flavobacterium sp. for the extraction of novel biosurfactants flavolipids.

6 Purification Assessment and Assay of Biosurfactants Efficacy 6.1

Purification Efficiency Determination

During the biosurfactants purification, the efficiency of the purification strategy is assessed by exploiting the physical and chemical properties of the biosurfactants. Conventional analysis of surface tension, emulsification index, oil drop method, or drop-collapse method cannot be used while assessing the purity of the biosurfactants, as after achieving CMC concentration, the biosurfactants, even in the presence of impurities, will exhibit most of the surfactant properties. Alternatively, spectroscopic techniques and chromatographic techniques are reported to be most utilized for assessing the purity of biosurfactants. Simple assessment strategies, such as thin layer chromatography to high end chromatographic techniques, such as UPLC are reported to be utilized depending on the application of biosurfactants (Table 2). Among several strategies, utilization of chromatographic techniques for the identification and determination of purity of biosurfactants is commonly reported.

6.2

Spectrophotometric or Colorimetric Assays

UV–Vis spectrophotometric or colorimetric assays are simple assessment methods for biosurfactants designed by the exploiting the ability of biosurfactants to develop color. Most of the reagents utilized in colorimetric methods utilized for the detection

Purification Assessment and Assay of Biosurfactant Efficacy

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Table 2 Assessment of biosurfactants after purification by different techniques Purity assessment of biosurfactants Surfactin

S. no. 1

Technique HPLC

2

Spectrophotometer

HPLC-purified rhamnolipids

3

LC-MS

Column-purified biosurfactants

4

FTIR

5

LC-MS

6

GC-MS

7

GC-MS and FTIR

Acid precipitated rhamnolipids

8

TLC

Acid precipitated rhamnolipids

9

UPLC-HRMS

Acid precipitated and solvent extracted

TLC and silica column-purified biosurfactants

Method C18 reverse phase column with photodiode assay detector. Acetonitrile, ammonium acetate (10 mM) in 40:60 (v/v) as mobile phase Microtiter plate coated with Victoria pure blue BO used for the colorimetric determination of biosurfactants at 625 nm Biosurfactants subjected to LC-MS with C18 column. Acetonitrile and water gradient (10–90)% with 0.01% formic acid was used as mobile phase. ESI-MS operated at positive ion mode and 150–2000 m/z as scanning range Scan range of 500 to 4000 cm-1 LC-MS with C18 column. Chloroform and methanol in 3: 1 v/v was used as mobile phase. 100 to 1200 m/z as scanning range 260°C as injection temperature, oven temperature increase from 40° to 260°C at 6°C/min. MS source temperature as 230°C and MS quad temperatures as 150°C FTIR spectra of biosurfactants was analyzed by preparing KBR pellet method. GC-MS using elite -5 MS column TLC plate subjected to iodine fumes and visualized under shorter and longer wavelength UV radiation UPLC coupled with HRMS. Gradient mobile phase water (0.1% formic acid) and acetonitrile

Reference Meena et al. (2021)

Kubicki et al. (2020)

Patowary et al. (2017)

Zargar et al. (2022)

Rath and Srivastava (2021)

Sohail and Jamil (2020)

Moro et al. (2018)

(continued)

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Table 2 (continued) Purity assessment of biosurfactants Silica gel-purified biosurfactants

S. no. 10

Technique GC-MS

11

NMR

12

TLC

C18 silica column

13

TLC

Acid precipitated biosurfactants

14

LC MS MS

Ultrafiltration with two cut-off

15

FTIR and NMR

16

UPLC-MS

HPLC-purified biosurfactants Dialysis membrane and TLC fractions of rhamnolipids

17

GC-MS

18

FTIR NMR

Acid precipitation followed by dialysis for 48 h Silica gel chromatography

Method GC-MS with VF-5MS column Sample dissolved in CDCl3 and subjected to 13CNMR The sample was loaded on silica and visualize using UV radiation Sample was loaded on activated TLC plate (120°C for 30 min) and using mobile phase consisting of chloroform, methanol, and water (70:26:4), the spots were identified under UV radiation The sample subjected to LC-MS/MS consisting of C18 column. Acetonitrile and water with 0.1% formic acid used as mobile phase. UV detector was used for the detection at 214, 254, and 280 nm Subjected to FTIR and NMR for the assessment UPLC with C18 column and multilinear gradient of water with 0.1% trifluoroacetic acid and acetonitrile with 0.1% trifluoroacetic acid was used. ESI was run in both positive and negative ionization mode Alditol-acetate-derivatized biosurfactant was subjected to GC MS The purified sample was subjected to FTIR for the presence of biosurfactant’s functional groups Samples were dissolved in deuterochloroform and subjected to NMR studies

Reference Sharma et al. (2015)

Balan et al. (2019) Rani et al. (2020)

Kourmentza et al. (2021)

Felix et al. (2019) Sharma et al. (2014)

Jain et al. (2013) Cruz et al. (2018)

(continued)

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Table 2 (continued) S. no. 19

Technique Spectrophotometer

20

TLC

Purity assessment of biosurfactants Acid precipitated biosurfactants followed by silica gel chromatography Solvent extraction

Method Orcinol reagent in the presence of sulfuric acid and the absorbance was recorded at 421 nm TLC using chloroform: methanol: acetic acid: water (25:15:4:2 v/v/v/v) as mobile phase and α-naphthol and sulfuric acid as spraying agent

Reference Patowary et al. (2017)

Saikia et al. (2012)

of glycolipids target either the carbohydrates or lipid moiety of the biosurfactants. Carbohydrate moiety in the glycolipids reacts with orcinol in the presence sulfuric acid at 50 °C and develops color, which is quantified at 625 nm using spectrophotometer. Similarly, anthrone reagent was reported to be used as a reagent for the quantification of glycolipids. In all these assays, the results are compared with calibration graph plotted with reference (commercial) biosurfactants by varying the concentration. Although the spectrophotometric methods are simple and quick, the accuracy in quantification and in assessing the purity of the biosurfactants vary due to the presence of glycolipid congener. Alternative to conventional reagent and substrate reaction methods, Kubicki et al. (2020) developed a simple assessment method for high-pressure liquid chromatography (HPLC)-purified rhamnolipids by coating the microtiter plate with Victoria pure blue dye for spectrophotometric analysis at 625 nm.

6.3

Thin Layer Chromatography

Thin layer chromatography (TLC) is considered as a basic conformation technique for testing the purity of the biosurfactants. In TLC technique, depending on the retention value of the compound and the spraying agent, the compounds can be determined. In general, the purified biosurfactants are loaded on the silica coated glass plate and allowed to separate using mobile phase. Using destructive and non-destructive techniques, the purity of the biosurfactants can be determined. In TLC technique, the mobile phase that cannot be used in HPLC or LC due to the interference can be used as combination or single mobile phase. The solvent with solubility in water and non volatile nature is preferred as a mobile phase. After running the TLC, the spots on the TLC can be detected either using destructive technique or non-destructive techniques. In non-destructive technique, the sample can be scrapped out from the plate and reused for further analysis. Iodine, water or UV radiation are commonly utilized in non-destructive technique. On the other

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hand, destructive techniques reagents such as orcinol, iodine, and resorcinol in combination with acids are reported to be used for the identification of functional groups present in the biosurfactants. Ibrahim (2018) checked the efficiency of purified biosurfactants by subjecting them to TLC with chloroform methanol and acetic acid in 65:15:2, v/v/v ratio as mobile phase. The carbohydrate, protein, and lipid moiety of the biosurfactants were determined by using Molisch’s reagent, 1% ninhydrin, and iodine vapors, respectively. Similarly, Moro et al. (2018) utilized 0.5% ninhydrin for protein moiety, anisaldehyde reagent and anthrone reagent for carbohydrate, and iodine vapors for lipid moiety for the identification of acid precipitated lipoprotein using TLC. On the other hand, Sohail and Jamil (2020) utilized TLC for assessing the purity of acid precipitated biosurfactants, by subjecting the iodine vapor, and visualized the bands in shorter and longer wavelength of UV.

6.4

High-Pressure Liquid Chromatography (HPLC)

HPLC is a chromatography technique involved in separation of compounds through a column (stationary phase) in the presence of liquid mobile phase. The basic components in HPLC include sample injector, pump, column, and detector. Through the sample injector, the biosurfactants is introduced into the column, which migrates according to the noncovalent interaction. The detectors, including electrochemical (EC), evaporative light scattering (ELS), and refractive index (RID), are commonly reported detectors utilized for the detection of biosurfactants. After purification of crude biosurfactants, the purification efficiency was reported to be test by subjecting to HPLC coupled with evaporative light scattering detector (ELSD) or RID and presence of single chromatogram confirms the high purity. On the other hand, presence of multiple chromatograms reflects partial purification, and the fractions of the respective peaks are collected for further analysis. Further, HPLC coupled with MS was also reported to be used for the assessment of biosurfactants purity. Patowary et al. (2017) utilized LC with C18 column coupled with ESI-MS for the assessment of column-purified biosurfactants from P. aeruginosa PG1. Similarly, Zargar et al. (2022) utilized LC-MC for the purity assessment of biosurfactants produced by Gordonia sp.

6.5

Ultra-Performance Liquid Chromatography (UPLC)

As an enhancement of HPLC, UPLC was introduced and has the ability to operate at high pressure with more than 15,000 psi, and the particle size of the column is less than 2 μm. Similar to HPLC, the samples are injected into the system via inlet and get separated in the column based on the polarity. In general, UPLC is often connected with mass spectrophotometer (MS) as an analyzer, which further aids in

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identification of biosurfactants (mass (m)/charge (z) ratio) with reference to the existing database. Most of the lipopeptides or lipoproteins are separated and quantified using UPLC couple with MS. Some of the researchers reported the efficiency of UPLC couple with MS/MS in assessing the purity of biosurfactants (Moro et al. 2018).

6.6

Gas Chromatography

Similar to HPLC, utilization of gas chromatography either with MS or FID detector is reported to be commonly used for the detection of biosurfactants. Contrary to HPLC, the biosurfactants, before subjecting to GC, are derivatized to fatty acid methyl esters (FAMEs) to reduce their polarity. Commonly used derivatization reactions include esterification, acetylation, alkylation, and silylation. With respect to biosurfactants, conversion of fatty acid residue into methyl ester is a widely reported technique. In GC, the derivatized sample is converted into gaseous phase and further into ions in the ionizing chamber and transferred to mass spectrometer (MS) analyzed or flame ionized detector (FID). In GC coupled with mass detector, the fractions separated in the GC are transferred to MS for mass to charge ratio analysis. With the reference with the mass to charge ratio database, the software predicts the basic information about the compound. Further, coupling of GC to MS/MS not only aid in detection but also helps in structural determination. In GC MS/MS, the single congener is fragmented, and the resultant daughter ions are analyzed.

6.7

Infrared Spectrophotometry

Infrared spectrophotometer is an analytic instrument utilized to identify the presence of biosurfactants and other impurities by detecting the absorption and emission of energy by the biosurfactants on exposing to IR. The third generation IR spectrophotometer (FTIR) is utilized for the determination of functional groups on the surface of compounds. Absorption of IR results in occurrence of atomic vibration in the molecules, and the transmitted signals are quantified for Fourier transformation of energy signals. Using this technique, the functional group on compounds can be determined. Biosurfactants are characterized by the presence of at least one ester bond, and detection of the ester bonds confirms the presence of surfactants. In general, standard biosurfactant along with the sample is reported to be used as background reference. Variation in the purified sample spectra from that of reference standard biosurfactants indicates the presence of contaminants or impurities. The disadvantage in utilizing FTIR for the analysis includes the presence of biosurfactant congeners produced during fermentation or presence on unutilized nutrient media components, which leads to false-positive error on subjecting to FTIR. Patowary

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et al. (2017) assessed the column-purified biosurfactants and identified bands at 3376 cm-1 (-OH- free stretch), 2928 cm-1 (C-H stretching), 1732 cm-1 (carbonyl stretching band), 1648 cm-1 (COO- group), and 1038 cm-1 (C-O-C) as major peaks that represent the chemical structure of rhamnolipids.

6.8

Nuclear Magnetic Resonance (NMR)

NMR is a common technique used for elucidating the structure of the compound and as a quality control to find the purity and content of the compound. NMR works on the principle of quantifying the change on intrinsic property of nuclei that is nuclear spin, on subjecting the compound (Biosurfactant) to external magnetic field in the presence of radiofrequency radiation. The change in nuclear spin to higher energy level by the adsorption of radiofrequency totally depends on the strength of external magnetic field applied and magnetogyric ratio. The NMR is a highly sensitive instrument, which provides information about the biosurfactants functional group and also differentiates structural isomers. For the analysis, the biosurfactants samples are dissolved in solvents such as deuterated DMSO or chloroform solution and subjected to series of 1H, 13CNMR, and Correlated Spectroscopy (COSY) analysis. Sharma et al. (2015) utilized NMR (13CNMR) study for the structural identification of silica gel-purified rhamnolipids.

7 Conclusion and Future Prospective The utilization of biosurfactants has grown in the recent years in order to achieve a sustainable bio-based green economy. For the commercial applications of biosurfactants, the purity of the compounds is necessary. Thus, in order to achieve highly pure commercial grade biosurfactants, assessment of the purity of biosurfactants is required, which influences the production costs and also their applications. Further, with the increasing demands of biosurfactants, there also arises a need to develop new rapid, simple, and robust biosurfactants assays. Simple TLC and spectrophotometric methods have the demerit of being not specific due to the occurrence of false-positive results in the presence of impurity, and they have limited commercial application. Even though several reports claimed various techniques for the assays, most of them involve sophisticated instruments, which are associated with high cost. Further, very limited data are reported for the assessment of biosurfactants purification exclusively as most of the techniques are meant for the estimation of properties such as structure or to elucidate the mass of biosurfactants. In addition, for the utilization of these techniques in large scale, more research along with the vigilant choice is required. Most of the researches aim for the chemical or structural analysis of biosurfactants after purification rather than giving importance to the purification yield and purification efficiency. Further, the assessment of

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purified biosurfactants is limited and is restricted to chromatography techniques especially to TLC and HPLC. Considering all these limitation, there is a major research gap, and more research and development have to be channeled into the design and development of simple and rapid techniques to assess the purity of biosurfactants. Apart from considering the purification techniques and further assessing the purity, the overall output of biosurfactants can also be influenced by (1) screening and identification of microbial and enzyme source for production; (2) optimization of the bioconversion process; and (3) expanding the market of biosurfactants in order to achieve a global economy. Finally, public awareness on the necessity of eco-friendly bioproducts and technology can also aid in escalating the biosurfactant market and boost the research on bio-based products, which will also positively influence the downstream processing of these components.

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Meena KR, Dhiman R, Singh K, Kumar S, Sharma A, Kanwar SS (2021) Purification and identification of a surfactin biosurfactant and engine oil degradation by Bacillus velezensis KLP2016. Microb Cell Fact 20(1):1–12 Mohanty S, Jasmine J, Mukherji S (2013) Practical considerations and challenges involved in surfactant enhanced bioremediation of oil. Bio Med Res Int Moro GV, Almeida RT, Napp AP, Porto C, Pilau EJ, Lüdtke DS (2018) Identification and ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry characterization of biosurfactants, including a new surfactin, isolated from oil-contaminated environments. J Microbial Biotechnol 11(4):759–769 Patowary K, Patowary R, Kalita MC, Deka S (2017) Characterization of biosurfactant produced during degradation of hydrocarbons using crude oil as sole source of carbon. Front Microbiol 8: 279 Rani M, Weadge JT, Jabaji S (2020) Isolation and characterization of biosurfactant-producing bacteria from oil well batteries with antimicrobial activities against food-borne and plant pathogens. Front Microbiol 11:64 Rath S, Srivastava RK (2021) Biosurfactants production and their commercial importance. EnvironAgric Microbiol Appl Sustain:197–218 Rathankumar AK, Saikia K, Ribeiro MH, Cheng CK, Purushothaman M, Kumar VV (2021a) Application of statistical modeling for the production of highly pure rhamnolipids using magnetic biocatalysts: evaluating its efficiency as a bioremediation agent. J Hazard Mater 412:125323 Rathankumar AK, Saikia K, Senthil KP, Varjani S, Kalita S, Bharadwaj N (2021b) Surfactant aided mycoremediation of soil contaminated with polycyclic aromatic hydrocarbon (PAHs): progress, limitation and countermeasures. J Chem Technol Biotechnol 97(2):391–408 Reiling H, Thanei-Wyss U, Guerra-Santos L, Hirt R, Käppeli O, Fiechter A (1986) Pilot plant production of rhamnolipid biosurfactant by Pseudomonas aeruginosa. Appl Environ Microbiol 51(5):985–989 Rodríguez-López L, Rincón-Fontán M, Vecino X, Cruz J, Moldes A (2020) Extraction, separation and characterization of lipopeptides and phospholipids from corn steep water. Sep Purif Technol 248:117076 Rosenberg E, Zuckerberg A, Rubinovitz C, Gutnick D (1979) Emulsifier of Arthrobacter RAG-1: isolation and emulsifying properties. Appl Environ Microbiol 37(3):402–408 Saikia RR, Deka S, Deka M, Banat IM (2012) Isolation of biosurfactant-producing Pseudomonas aeruginosa RS29 from oil-contaminated soil and evaluation of different nitrogen sources in biosurfactant production. Ann Microbiol 62(2):753–763 Saikia K, Rathankumar AK, Vaithyanathan VK, Cabana H, Vaidyanathan VK (2020) Preparation of highly diffusible porous cross-linked lipase B from Candida Antarctica conjugates: advances in mass transfer and application in transesterification of 5-Hydroxymethylfurfural. Int J Biol Macromol 170:583–592 Saranraij P, Sivasakthivelan P, Hamzah KJ, Hasan MS, Al-Tawaha ARM (2022) Microbial fermentation technology for biosurfactants production. Microbial surfactants: volume 2: applications in food and agriculture, pp 25–43 Satpute SK, Banpurkar AG, Dhakephalkar PK, Banat IM, Chopade BA (2010) Methods for investigating biosurfactants and bioemulsifiers: a review. Crit Rev Biotechnol 30(2):127–144 Shah MUH, Sivapragasam M, Moniruzzaman M, Yusup SB (2016) A comparison of recovery methods of rhamnolipids produced by Pseudomonas aeruginosa. Procedia Eng 148:494–500 Sharma D, Saharan BS, Chauhan N, Bansal A, Procha S (2014) Production and structural characterization of Lactobacillus helveticus derived biosurfactant. Sci World J Sharma D, Saharan BS, Chauhan N, Procha S, Lal S (2015) Isolation and functional characterization of novel biosurfactant produced by Enterococcus faecium. Springerplus 4(1):1–14 Sharma J, Sundar D, Srivastava P (2021) Biosurfactants: potential agents for controlling cellular communication, motility, and antagonism. Front Mol Biosci:893

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Methods of Screening and Applications of Biosurfactants Produced by Actinomycetes O. Archana and Lokesh Ravi

1 Introduction Biosurfactants are composites, which have ability to generate surface-active and emulsifying activities. They are also amphiphilic composites, containing of two parts, hydrophilic component (water loving, polar) and nonhydrophilic component (water hating nonpolar, hydrophobic). Biosurfactants are produced by microorganisms as secondary metabolites, that is why these are also named as microbial surfactants (Hamzah et al. 2013). Biosurfactants are made up of an ample variety of chemical compounds, like polysaccharides, protein composites, phosphorylated lipids, and many more. These are involved in enlarging the surface area, bacterial pathogenesis, biofilm formation, etc. (Rodrigues et al. 2006). These are enchanting advertence over the last few years on account of various benefits on top of chemical surfactants having less toxicity, inclusive biodegradability, and ecological acceptability (Gandhimathi et al. 2009). Class—Actinobacteria Subclass—Actinobacteridae Order—Actinomycetales Actinomycetes are Gram-positive bacteria with a special characteristic of having filamentous hyphae, and these hyphae do not usually incur fragmentation and produce asexual spore. They are rich in guanine and cytosine content, and they show morphology of fungi that is why these organisms are named as actinomycetes. They have the capability to break down large quantity and bulky organic composites into simplest constituents. Bacterial pool showed a large array of metabolic process for managing with the degradation of oil constituents and even surface-active agent

O. Archana Department of Botany, St Joseph’s University, Bengaluru, India L. Ravi (✉) Department of Food Technology, Ramaiah University of Applied Sciences, Bengaluru, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_3

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production and emulsifiers. These agents are tiny surfactant molecules having amphipathic features which increase the bacterial growth and bioremediation rate (Atuanya et al. 2017). Aerial mycelial color of actinomycetes is gray color, substrate mycelium will be in yellow–green color, they do not produce any pigment (melanin), and they could ferment mannitol, sucrose, and glucose but are not able to ferment xylose (Carrillo et al. 1996). Biosurfactants are generally divided into less molecular weight composites like glycolipids or lipopeptide and dense molecular weight composites like polysaccharides, proteins, lipopolysaccharides, and lipoproteins (Ron and Rosenberg 2001).

2 Type of Biosurfactants 2.1

Polymeric Biosurfactants

The greatest ubiquitous polymeric biosurfactants generated by actinomycetes are large-amphiphilic lipoglycans namely, lipomannan, phosphatidylinositol mannosides, lipoarabinomannan, and its antecedents. In comparison with cell wall of actinomycetes, peptidoglycan, arabinogalactan, etc. are noncovalently bound to the cell membrane, albeit phosphatidylinositol mannides are constructurally associated to lipomannan and other anchor components. And these polymeric glycolipids have been purified by Mycobacterium sp., Rhodococcus sp., Dietzia maris, Turicella otitidis, Tsukamurella paurometaba, and many more. Lipoarabinomannans are familiar to cause immune-repressive activities in diseases like leprosy, and this disease occurred by the action of Mycobacterium leprae (Kugler et al. 2015).

2.2

Macrocyclic Glycosides

Actinobacteria produces biosurfactants like macrocyclic glycosides, and macrocyclic dilactones are renowned and are frequently familiar to show bio-activity on various organisms. Nocardia brasiliensis generated an aliphatic macrolide antibiotic known as Brasilinolide, which shows antimicrobial activities (Tanaka et al. 1997). Elaiophylin and its derivatives were purified from several Streptomyces spp. and from high producer strains. This compound shows bio-active features against worms of intestine, including antimicrobial, immunosuppressant, and antitumor pursuits (Haydock et al. 2004). Fluvirucin is obtained from numerous Streptomyces sp., Actinomadura sp., S. mutabilis, and Microtetraspora sp. The various fluvirucins concerned similar lactam ring component but vary in glycosylation. They showed antiviral activities against influenza A and also exhibited antifungal features against Candida sp. (Chen et al. 2021).

Methods of Screening and Applications of Biosurfactants Produced. . .

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Lipopeptides

Lipopeptides are grouped into cyclic and noncyclic lipopeptide. Cyclic lipopeptides are very familiar lipopeptides. These are constituted by varieties of peptide chain and spheroid amino acids and are associated to form a fatty acid single chain. Daptomycin possesses 13 amino acids, which were secreted by S. roseosporus, and this is cyclic because of the existence of ester bond in between carboxyl terminus and hydroxyl swarm of either hydroxyl asparagine or threonine. Lipophilic tail mixture-enzymatic deacylation and chemical reacylation along with decanoyl fatty acid group lead to the daptomycin production (Debono et al. 1987). In 2003, the US FDA accepted Daptomycin as first antibiotic of its kind, marketed as Cubicin. This has shown vigorous activity against many gram-positive bacteria (Miao et al. 2005). Actinoplanes sp. produces depsipeptide ramoplanin, possessing 17 amino acids, out of which, 16 are cyclic sections which show antibacterial activities, especially against gram-positive bacteria. Ramoplanin members are differentiated from their acyl amides that are made from various diunsaturated fatty acids which are associated with the distal hydroxyl-asparagine (De la Cruz et al. 2017). Streptosporangium amethystogens produces linear lipopeptide. These lipopeptides are defined to shield against leucopenia infections caused during cancer therapies by patronizing cells of bone marrow (Christopherson et al. 2006).

2.4

Phenazine Ester

These are a sporadic alkaloid esters group. Streptomyces sp. has been delineated to generate these phenazine ester compounds having desoxypyranosequinovose, which are esterified at either Carbon 3 or 4 to the phenazine carboxyl end. Phenazine– quinovose ester has been noted to show antimicrobial activity (Kugler et al. 2015).

2.5

Amide Glycosides

Many biosurfactants with nucleoside fatty amide glycosides structures are generated by actinomycetes. Streptomyces sp. produced a glycoprotein known as tunicamycin, which shows antibacterial features. Streptavidin and Corynetoxin have been defined as a tunicamycin-based surfactants. Later, this amide glycoside was generated by C. rathayi, and this microorganism can replicate inside the sheep galls, spreading the poisonous metabolite (Chen et al. 2021). Some of the common biosurfactants produced by Actinomycetes are summarized in Table 1.

Elaiophylin

Compound name Brasilinolide

Chemical structure

Table 1 Name of the biosurfactants and their structure

1025.3

Molecular weight (g/mol) 1167.4

Streptomyces sp.

Name of the organism Nocardia brasiliensis

Haydock et al. (2004)

References Tanaka et al. (1997)

54 O. Archana and L. Ravi

Fluvirucin

456.7

Streptomyces sp., Actinomadura sp., Microtetraspora sp., and Saccharothrix mutabilis

(continued)

Chen et al. (2021)

Methods of Screening and Applications of Biosurfactants Produced. . . 55

Compound name Daptomycin

Chemical structure

Table 1 (continued) Molecular weight (g/mol) 1620.7 Name of the organism Streptomyces roseosporus

References Debono et al. (1987)

56 O. Archana and L. Ravi

Phenazine

Ramoplanin

180.20

2254.1

Streptomyces sp.

Actinoplanes sp.

(continued)

Kugler et al. (2015)

De la Cruz et al. (2017)

Methods of Screening and Applications of Biosurfactants Produced. . . 57

Compound name Streptavidin

Chemical structure

Table 1 (continued) Molecular weight (g/mol) 720.7 Name of the organism Streptomyces sp.

References Kugler et al. (2015)

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Corynetoxin

636.6

Corynebacterium rathayi

Kugler et al. (2015)

Methods of Screening and Applications of Biosurfactants Produced. . .

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3 Methods of Screening 3.1

Hemolytic Activity

Hemolytic activity in blood agar plate is the preliminary method to detect the biosurfactant’s production. Because of their amphiphilic character, biosurfactants can foment destruction of red blood cells at a particular concentration. 1. Screen purified strains on blood agar plate, having 5% blood 2. Incubate plate at 28 °C for 2 days (48 h) 3. The appearance of cleared zone around the colony is significant for microorganism-producing biosurfactants (Carrillo et al. 1996).

3.2

Drop-Collapse Test

Drop-collapse method is a qualitative method used to screen biosurfactants, and this test is performed in 96-microwell plate. (a) Add 2 μl of mineral or crude oil into 96-microwell plate. (b) And then, equilibrate plate for 60 min at 37 °C. (c) After 1 h equilibration of plate, add 5 μl of supernatant of the sample to be examined into the 96-microwell plate (d) If the oil drops on the supernatant became flat or collapsed after unifying indicates the biosurfactants producing actinomycetes, if the oil drops endure beaded were considered as negative for the test (Plaza et al. 2006).

3.3

Oil Displacement Test

This test works on biosurfactant’s properties that change the contact angle at the interface of oil–water. The biosurfactant displaced the oil surface pressure. 1. Add 15 μl of weathered crude oil in to the petri plate containing distilled water 40 μl. Consequently, a thin layer of oil will form. 2. Immediately after the occurrence of oil membrane thin layer, add sample to be examined at the center of thin layer. 3. If the clear zone occurred, then it is positive for the test, if not it is negative for the test and indicates absence of biosurfactants, clear of zone diameter can be measured for the further studies (Morikawa et al. 1993).

Methods of Screening and Applications of Biosurfactants Produced. . .

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61

Blue Agar Plate

This is a semiqualitative method, also known as CTAB agar plate method, utilized to determine extracellular glycolipids or for other biosurfactants. This method was introduced by Wagner and Siegmund. This mineral salt agar media is augmented with 2% of glucose (carbon source), 0.05% of cetyltrimethylammonium bromide (cationic surfactant), and 0.02% of methylene blue. If the actinomycetes produce anionic surfactants, they will form a dark halo blue color around the bacterial colonies. (a) Make 4 mm diameter wells on the methylene blue agar media with the help of cork borer. (b) Load 30 μl of culture that is needs to be examined. (c) Incubate plates at 37 °C for 48–72 h (2–3 days). (d) Look for dark blue halo formation around the bacterial colonies (Hathwar et al. 2015; Walter et al. 2010).

3.5

Du-Nouy-Ring Method

This a direct method used to measure the interface or supernatant surface activity of culture. The surface tension reduces with increasing concentration of biosurfactants. This test is entrenched on computing the force needed for segregating a wiring from surface. The force of detachment is mainly depended on surface tension (directly proportional). Surface or interface activity can also be measured by using commercially available automated tensiometer. Microorganisms which generate better biosurfactants are capable to decrease the interface tension of growth media by ≥20 mN/m (Walter et al. 2010). Note: The ring must be clean (without any contamination), and this is generally done by utilizing platinum ring, which should be sterilized.

3.6

Penetration Assay

Penetration assay depends on the contacting of two immiscible phases, which results in color change. (a) Take clean 96-well microplate, into that add hydrophobic paste (150 μl), containing of 10 μl silica gel and oil. (b) Add 10 μl of red stain to 90 μl supernatant of the culture to be examined. (c) Place, stained supernatant on hydrophobic paste surface. (d) If actinomycetes produce biosurfactants, then hydrophilic liquid will shatter via oil film barrier into the paste. Due to entry of silica gel into the hydrophilic phase from hydrophobic phase, color of upper phase will become cloudy white from red within 15 min (Walter et al. 2010).

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3.7

Crystalline Anthracene Solubility

This test works on the principle of crystalline, hydrophobic anthracene solubility. If the actinomycetes produce biosurfactants, they will solubilize the anthracene. (a) After adding anthracene compound into the supernatant, incubate a supernatant in shaker incubator at 25 °C for 1 day. (b) Measure the concentration of dissolved hydrophobic anthracene compound at 354 nm with the help of spectrophotometer. (c) And correlates the biosurfactant production (Walter et al. 2010).

3.8 (a) (b) (c) (d)

Parafilm-M Test Mix 2 ml of cell-free supernatant with 10 μl of bromophenol blue indicator. With the help of micropipette, put this mixture as a drop on parafilm-M. Look for the shape of drop on the surface. If the drop becomes flat, then it is positive for the test (presence of biosurfactant). If drop remains in shape of dome, then it is considered as negative for the test (absence of biosurfactants) (Alt et al. 2014).

3.9

Phenol-Sulfuric Acid Method

(a) Add 1 ml of 5% phenol to 1 ml of cell-free supernatant. (b) To the above mixture, add 2 to 5 ml of conc. H2SO4 drop by drop, until typical color is obtained. (c) Appearance of color denotes the glycolipids presence (Alt et al. 2014).

3.10

Microplate Assay

Microplate assay is performed to detect the biosurfactant’s presence. (a) Take 100 μl cell-free extract in a 48-well microplate. (b) And then, observe the plate against the backing sheet having grids. (c) If the optical distortion of background grids is observed, then the assay is considered as positive for the determination of biosurfactants, if not, it is negative (absent) for the biosurfactants (Walter et al. 2010).

Methods of Screening and Applications of Biosurfactants Produced. . .

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Orcinol Assay

The biosurfactant nature was decided by utilizing this test. (a) Mix 100 μl cell-free supernatant with 900 μl of orcinol reagent. (b) Incubate at 80 °C for half an hour. (c) Appearance of blue–green color indicates the presence of glycolipid in the supernatant (Roy et al. 2015).

4 Case Studies on Biosurfactants In a study, the sponge-correlated marine Actinomycetes N. alba MSA 10 was isolated and examined for the secretion of biosurfactant. Oil displacement, lipase production, drop-collapsing method, hemolytic activity, and activity of emulsification methods were utilized to affirm the biosurfactant’s occurrence. Ethyl acetate, diethyl ether, and dichloromethane solvents were utilized to extract those compounds showing activity, utilizing thin layer chromatography (TLC), and the compound was recognized as lipopeptide. Biosurfactants have been proving as an optimistic representative for hydrocarbon bioremediation and oil-polluted marine environment; hence, marine environment quests for the biosurfactants’ producers will have broader applications in bioremediation and industrial process, traversing the sedentary marine organism chemical environment (Gandhimathi et al. 2009). In a study, the biosurfactant-secreting actinomycetes were purified from the Raniganj Coal Mine and Rourkela Steel Plant region, and the strain was recognized as A. nocardiopsis A17, possessing more foam-exhibiting features. This showed protease, gelatinase, amylase, and lipase activity. Biosurfactants were distinguished by using Fourier Transform Infra-Red spectroscopy and liquid chromatography– mass spectrometry. From the experimental observation, researchers found that highest biosurfactant secretion was at the 2% of glycerol, 0.1% of yeast extract, monopotassium phosphate 0.05%w/v, at 28 °C temperature, and pH 6.8. Due to their minor toxicity and biodegradability activities, utilization of these biosurfactants increases as dashing excipients in medicine dosage formulation and in make-up kits (Chakraborty et al. 2015). In other study, T. spumae and T. pseudospumae nonpathogenic actinomycetes were cultured in sunflower oil; these actinomycetes were able to secrete extracellular trehalose. Trehalose lipid biosurfactant crude extract was refined utilizing silica gel chromatography, and with the aid of multidimensional nuclear magnetic resonance (NMR), 1-α-glucopyranosyl-1-α-glucopyranoside biosurfactant was recognized. And these actinomycetes detain contingent for the ecologically friendly surfactants production (Kugler et al. 2014). Some of the common types of biosurfactants reported from Actinomycetes sources are tabulated in Table 2.

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Table 2 Type of biosurfactants produced by actinomycetes Biosurfactant type Trehalose lipid Lipopeptide

Biosurfactant-producing actinomycetes Tsukamurella spumae and Tsukamurella pseudospumae Streptomyces sp. MN394821

Lipopeptide

Nesterenkonia sp.

Lipopeptide (Surfactin) Macrocyclic glycosides Macrocyclic glycosides

Micromonospora marina

Macrocyclic glycosides

Lipopeptide (daptomycin) Lipopeptide

Nocardia brasiliensis Streptomyces sp.

Streptomyces sp. Actinomadura sp. Microtetraspora sp. and Saccharothrix mutabilis Streptomyces roseosporus

Functions Surfactant production

Reference Kugler et al. (2014)

Exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus Effective emulsifier, and as a shielding agent against Staphylococcus aureus Anticancer agent against breast cancer Antibacterial and antifungal activities Intestinal worms including antimicrobial, immunosuppressant, and antitumor activities Showed antifungal activity against Candida sp. And exhibited antiviral activities against influenza A

Arifiyanto et al. (2020)

Antibiotic

Debono et al. (1987) De la Cruz et al. (2017) Christopherson et al. (2006)

Actinoplanes sp.

Antimicrobial activity

Lipopeptide

Streptosporangium amethystogens

Phenazine ester Amide glycosides

Streptomyces sp.

Protective against contagion in patients and against leucopenia caused by cancer therapies by patronizing cells of bone marrow Antimicrobial activity

Lipopeptide Lipopeptide Lipopeptide Lipopeptide

Glycolipid derivatives

Streptomyces sp. Corynebacterium rathayi Streptomyces coelicoflavus Rhodococcus sp. Streptomyces angustmyceticus CGSB11 Streptomyces althioticus RG3 and Streptomyces californicus RG8 Brachybacterium paraconglomeratum MSA21

Antibacterial activities

Antibacterial activity Bioremediation Bioremediation Antimicrobial and antifouling activities Antibiotic activities

Kiran et al. (2017) Ramalingam et al. (2019) Tanaka et al. (1997) Haydock et al. (2004)

Tanaka et al. (1997)

Kugler et al. (2015) Kugler et al. (2015) Alt et al. (2014) White et al. (2013) Jimenez et al. (2020) Hamed et al. (2021) Kiran et al. (2014) (continued)

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Table 2 (continued) Biosurfactant type Rhamnolipids (Dokdolipids A-C)

Biosurfactant-producing actinomycetes Actinoalloteichus hymeniacidonis

Glycolipids (rhamnolipids)

Nocardioides sp.

Functions Showed cytotoxicity against large intestine of a male dukes C colorectal cancer cell and other cancer cell lines Bioremediation

Reference Choi et al. (2019)

VasilevaTonkova and Gesheva (2005)

5 Applications 5.1

Environmental Remediation Industries

In most circumstances, environmental pollution brought out by industrial activity is because of fortuitous or intentional deliverance of inorganic and organic composites into the environment. Such composites show issues for remediation, as they get easefully bound to soil particles. Biosurfactant applications in the inorganic composite remediation, like, removal of chelating agents, heavy metals, and ions in a washing step eased by the interplay between the amphiphiles and the metal ions. The organic pollutants show specific issues for land remediation as they are often hydrophobic composites and rapidly become soil-bound residues. The hydrophobic composites sorption mechanism from water to soil is a separation method to the interior of soil humic material. Once bound, hydrophobic constituents are tough to eliminate, and in this state, their accessibility for biodegradation is highly reduced. The binding process becomes more laborious with reinforcement (Jain et al. 1992). The biosurfactant’s application to this issue concerns to organic pollutant solubilization; they occur as nonaqueous phase liquids or solids. The synthetic surfactant’s addition may provide further pollutants to the nature. Hence, the very less environmentally polluting biosurfactants may have a lead role. In a study where they examined two degradation phases for a model, first phase with oil system possessed 10% soil. The second phase was quantitatively the most significant, and depends on the glycolipids characteristic production of Rhodococcus erythropolis to lesser the interfacial tension between the aqueous phase and oil; it was observed that naphthalene was more readily degraded as contrasted with less water-soluble hydrocarbons including other aromatics. In further study, utilizing similar model, isolated glycolipids were added that enhanced the hydrocarbons degradation efficiency. The effect went along with enhanced biomass concentration (Oberbremer and Miiller-Hurtig 1989). Rhodococcus erythropolis can produce bioflocculants which effectively cause broad range flocculation of solid suspension. Glycolipids like glucose and trehalose– corynomycolates are significant in the growth media flocculating activity of R. erythropolis S-1. They predicted that the flocculant in the culture broth forms

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micelles constituted by several proteins and by above mentioned lipids. Those materials may help in elimination of suspended solids in treatment of waste water (Kurane et al. 1995).

5.2

Biosurfactants in Food Industry

Biosurfactants are utilized for many food processing applications. These are emulsion stabilizing agents, assimilated in food products to keep the firmness, texture, fat globules solubility, enhance fragrance, foaming, and dispersing features. These are utilized to avoid the fat globules clumps, maintain aerated system, enhance the starch shelf life possessing materials, alter wheat dough rheological features, and enhance consistency of fat containing foods. In ice cream and bakery articulation, biosurfactants play a role by avoiding consistency, delay staling, solubilizing aroma of oils and even used as stabilizers of fat and antispattering agents while oil and fats cooking. Enhancement in stability of dough, consistency, volume, and shelf life of bakery items is gotten by the addition of rhamnolipid biosurfactant. These are recommended for utilization of rhamnolipids to enhance the features of butter cream, croissants, and frozen sweet foods. L-rhamnose has noticeable capability as a precursor for flavoring. It is already utilized commercially as a precursor of goodquality flavor constituents like furaneol (Kiran et al. 2017).

5.3

Degradation of Engine Oil

Nocardiopsis VITSISB, a marine bacterium, was found to secrete rhamnolipid and even strong engine oil degrader with reference to oceanic conditions like pH, temperature, and salinity. The biosurfactant possessing high emulsification activity, lesser surface tension, higher viscosity, and lower density is reliably utilized in oil spill biodegradation in ocean than synthetic biosurfactants. Biosurfactants, secreted by Nocardiopsis VITSISB, provided a special benefit on renewable sources; so this feature of biosurfactant may put back chemical surfactants. Nocardiopsis VITSISB immobilized culture which can play lead role as an oil spill cleanup in ocean with reference to reusability and low cost (Roy et al. 2015).

5.4

Silver Nanoparticle Synthesis

The biosurfactants make an appearance as an encouraging nanoparticle stabilizing agents. A glycolipid biosurfactant secreted by Dendrilla nigra (marine sponge)-associated B. casei MSA19 showed the emulsification index, which was invariably greater as compared with synthetic surfactants like Tween 20 and 80, etc.

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The concentration of extracted surfactant was appeared to be 18 g/L. and the nanoscale silver is synthesized in reverse micelles by utilizing glycolipid as a stabilizer. Synthesized silver nanoparticles were stable, uniform for 60 days because they deduce the aggregates formation due to its electrostatic force of attraction. Hence, biosurfactants’ conciliated synthesized nanoparticles might be treated as “Nanoparticle green stabilizer” (Kiran et al. 2010).

5.5

Manufacturing of Antifoul

Biofouling is the unsolicited agglomeration of micro- and macro-organisms on submerged surfaces, this process starts instantly after water-submersion and takes hours to months to build. Marine biofouling creates threats to many industries like aquaculture, powerplants, shipping, and many more (Pereira et al. 2020). The biosurfactants secreted by Streptomyces althioticus RG3 deduced the bacterial cell density. Streptomyces aculeatus PTM-029 produces Napyradiomycin which is responsible for showing greater antibacterial activity, microfouling inhibition, and the most capable anti-microfouling activity (Hamed et al. 2021).

5.6

Medical Field

Dokdolipids A-C, three hydroxylated rhamnolipids, were purified from the Actinoalloteichus hymeniacidonis, and to study the cytotoxicity, purified biosurfactants were tested on six cancer cell lines, and these biosurfactants were able to show cell toxicity on all six cancer cell lines with inhibition concentration ranging from 13.7 to 41.5 μM (Choi et al. 2019). Succinyl trehalose lipids are of two types, secreted by Rhodococcus erythropolis SD-74, and were able to foment differentiation of cell instead of proliferation in the human promyelocytic leukemia cell line HL60. Additionally, they found to inhibit protein kinase C (PKC) HL60 cells functions. PKC inhibitors are anticipated to be anticancer agents (Isoda et al. 1997). Rhodococcus erythropolis SD-74-produced Succinoyl trehalose lipids also exhibited antiviral features. Certain mycolic acids are correlated with virulence in Nocardia strains. Isolated Rhodococcus erythropolis SD-74 glycolipid was able to affect lesions in mice (Lang and Philp 1998).

5.7

Oil Industry

Biosurfactants have various activities in the oil industry. Biosurfactants have fascinated engrossed majorly on two fields: increased oil retrieval and cleaning. In oil

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fields, crude oil is found in porous rock in correlation with brines. Traditional water flooding operations rescue only about 40% of the oil because of capillary forces, which avoid oil or water confederate from passing via narrow pores By decreasing the oil or water interfacial tension to very less range; increased oil recovery may be anticipated. Rhodococcus strain H13-A-produced trehalose glycolipid biosurfactant has lesser surface tensions in the range between 0.02 and 0.00005 mNM-1, and this biosurfactant meliorated the heavy crude oil displacement from rock cores by 20% (Jardim Pacheco et al. 2010). In a study, where 2 tons of culture medium possessing biosurfactants was utilized to clean oil sludge from a tank bottom and recovered almost 90% oil from the sludge during the process, and can be convenient in future for oil recovery (Banat et al. 1991). In another study, K. rosea ABR6 actinobacteria was used to study the applications of lipopeptide (biosurfactant) in oil sludge retrieval and lubrication. The biosurfactants secreted by this organism had the ability to piercing into various phases to decrease the surface tension. These properties could be employed in oil recovery, lubrication, and crude oil facilities in pipeline. Besides, 7% of crude oil was recuperated from petroleum sludge by using these biosurfactant. Furthermore, crude oil speed of transfer in pipeline was improved (Akbari et al. 2021).

5.8

Cosmetics

In the cosmetics industry, biosurfactants are utilized as emulsifiers; antimicrobial, wetting, and foaming agents; solubilizers; cleaners; and conciliator for enzymes reactions. Hydrophobic surfactants’ nature would make them more fitted to applications in face creams, pastes, sticks, and films (Lang and Philp 1998). The sophorolipids are utilized in lactone type, containing large parts of diacetyl lactones; it acts as an agent for stimulating skin dermal fibroblast cell metabolism and more specifically for invigorating collagen neo synthesis. The isolated lactone sophorolipid product is of significance in the dermis antisenescence formulation, repair, and restructuring products due to its effect on the stimulation of dermis cells by supporting new collagen fibers synthesis (Kugler et al. 2015).

5.9

Microbial Foams in Activated Sludge Plants

The most strenuous microbiological issues demonstrated when treating unwanted water by the activated sludge methods are filamentous bulking and pattern microbial foam formation. They can overflow to the secondary sedimentation tank, and usually they create maintenance tough and dangerous (Lang and Philp 1998). Rhodococcus was used to detect the microbial foams; hence, they have been incriminated in foaming circumstances (filamentous growth). Biosurfactants

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secreted by Rhodococcus strain, formed foams, which were same as those of generated in full scale activated sludge plants. So, four different strains of Rhodococcus rhodochrous were studied to test the ability to generate viscous foams. These strains varied in morphology of colony. The strains which have rough colony, generated viscous foam when aerated and the cells were observed in large quantity in the foams and showed high cell surface hydrophobicity. The strain which has mucoid strain produced a large amount of bubbles which looks like a soap bubble but which were not tenacious. Strain which contains smooth colony generated a smaller quantity of bubbles of the liquid culture surface (Sunairi et al. 1997).

6 Conclusion The enthusiastic biosurfactant’s properties have resulted in a broad spectrum of applications in the industrial and medical field. These are essential as antimicrobial agent, immune-modulatory substances, adhesive agents, adjuvants for antigens, etc., and their applications remain restricted and might be because of its cost and yield. Additional explorations on human cells and natural microbiota are needed to substantiate their uses in various biomedical fields.

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Methods of Screening and Applications of Biosurfactants Produced by Cyanobacteria G. R. Shree Kumari and Lokesh Ravi

Abbreviations 13C μ 16S rRNA 1H BuOH CMC CMD CTAB EPE EPS EtO H 2O HPLC IC50 L LPS Lux

Carbon 13 Micrometer 16S ribosomal ribonucleic acid, where S (Svedberg) unit of measurement (sedimentation rate) Hydrogen Butanol Critical micelle concentration Critical micelle dilution Cetyltrimethylammonium bromide Exopolysaccharides Extracellular polymeric substances Ethyl acetate Water High-performance liquid chromatography Half-maximal inhibitory concentration Liter Lipopolysaccharide SI derived unit of illuminance, measuring luminous flux per unit area

G. R. Shree Kumari Department of Biomedical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, India L. Ravi (✉) Department of Botany, St Joseph’s University, Bengaluru, India Department of Food Technology, Ramaiah University of Applied Sciences, Bengaluru, India Department of Biomedical Sciences, School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_4

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MeOH MFC-7 ml mm NaCl Nm NMR o C RPSs v/v W w/v

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Methanol Michigan Cancer Foundation-7, human breast cancer cell line with estrogen Milliliter Millimeter Sodium chloride Newton-meter Nuclear magnetic resonance Degree Celsius Cyanobacterial released polysaccharides Volume/volume percentage Watt Weight per volume

1 Introduction A phylum comprising of photosynthetic bacteria that live in aquatic habitats and moist soils is known as cyanobacteria. These are discovered to play a role in producing gaseous oxygen as a byproduct of photosynthesis (Rashmi et al. 2021). Usually, these are established to be in colonies, forming spheres or filaments or some live singly. They are commonly confused with the algae as these share traits with bacteria and algae; these are popularly identified as blue–green algae (Nayarisseri et al. 2018). Chlorophyll a, a green pigment, is the only type of chlorophyll that is seen in these photosynthesizing bacteria. In addition, they also contain pigments, such as carotenoids, phycobilin, and also a bluish pigment, phycocyanin (used for photosynthesis) (Flow and Modelling 2016). Cytotoxins produced by these photosynthesizing bacteria can induce illness to exposed animals well as human populations (Satpute et al. 2010). Almost all species of these photosynthesizing bacteria are buoyant and float on the water face and form floating mats. The accumulation of these algae is nominated as “blooms.” These blooms discolor the water and produce unwelcome odor as well as taste (Gudiña et al. 2016). They show some impact on the marine population, precisely the populations of fish, as well as reduce the quality of water. The corruption of the cyanobacterial blooms depletes the levels of oxygen and triggers the elimination of fish. These photosynthesizing bacteria may also produce biosurfactants that are surface active low-molecularweight compounds (Kaya et al. 2006). In general, there are different types of biosurfactants produced by these photosynthetic bacteria; some of the biosurfactants are lipopeptide or lipoproteins, glycolipids, phospholipids, etc. (Rashmi et al. 2021). Some of the examples for cyanobacteria are Oscillatoria, Microcystis, Anabaena, Nostoc, Spirulina, etc. These photosynthesizing bacteria are involved in the growth of other plants and play a significant role in their development. These bacteria possess the unique ability

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to convert the nitrogen present in the atmosphere into simpler nitrogen compounds like nitrates, nitrites and ammonia. That is why they are called bio-azeotropes. These bio-azeotropes facilitate in the cycling of nitrogen in the environment. Plants obtain the nitrogen fixed by these organisms from the soil. Nitrogen is one of the major essential elements for the growth of plants; hence, these organisms play a vital role in their survival. The nitrogen can also be obtained from the soil by the addition of bio fertilizers (Singh et al. 2016). Organisms like Anabaena and Nostoc that fix nitrogen have specialized nitrogen fixing cells in their filaments called “heterocysts.” These cells are larger with thick walls which creates an anaerobic environment as nitrogen fixation is inhibited by the presence of oxygen (Bodour et al. 2003). Bio-azeotropes have formed symbiotic associations with both higher and lower plants. With fungi they form a stable association called a lichen (Singh et al. 2016). Leguminous plants provide housing and shelter to the bacteria in special cells in the root called nodules. In return, the bacteria provide fixed nitrogen to the plants. This is one of the main principles of crop rotation. Rice is commonly grown along with the aquatic fern Azolla as it contains nitrogen fixing cyanobacteria in its leaves. This provides a major advantage in its cultivation as it provides a natural and cheap source of nitrogen in the paddy fields (Bento et al. 2005). Spirulina, a cyanobacterium, is consumed all over the world. It is considered a super food due to high protein contents. It is a very important dietary supplement; in tropical countries, it is eaten quite regularly. In the United States, it is dried and sold in the form of tablets as a health food. It is cheap and easy cultivation makes it an ideal source of protein (Singh et al. 2016). However, there are cyanobacteria that are harmful to humans, animals, and the environment. Eutrophication leads to the uncontrolled growth and leads to formation of algal blooms. These blooms are toxic to the aquatic organisms and if eaten can cause harm to plants and animals. It is responsible for the poisoning of cattle (Bodour et al. 2003). A species of Lyngbya causes irritation to the skin and is locally called “swimmer’s itch” (Bento et al. 2005).

1.1

Scientific Classification

Domain: Bacteria Phylum: Cyanobacteria Class: Cyanophyceae Order: Chroococcales Family: Cyanobacteriaceae Genus: Oscillatoria Species: princeps

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Characteristics of Cyanobacteria

The development or the blooming of cyanobacteria is greatly influenced by the availability of light. An emulsifier like Phormidium J-1 acts as a flocculent on the suspended clay particles in the water. This clears the water and increases the amount of light reaching the mat of cyanobacteria. The photosynthesizing bacteria possess some key characteristics that are unshared to these cyanobacteria as they thrive and survive extreme conditions (Rahman et al. 2019). These photosynthesizing bacteria possess some key characteristics that are unshared to these cyanobacteria as they thrive and survive extreme conditions. Cell hydrophobicity, which is regulated by the sediment of adjunct of cells, is put forward in the benthic forms of cyanobacteria that are ordinarily resolved by extracellular polymeric substances (EPS) (de Paniagua-Michel et al. 2014). These EPS are polysaccharides bearing nonsugar components. A majority of photosynthetic bacteria or cyanobacteria are distinguish an anionic nature; numerous species contain two different uronic acids, a trait infrequently encountered in the polymers released by strains related to other microbial groups (de Paniagua-Michel et al. 2014). These photosynthetic bacteria release polysaccharides, also identified as Cyanobacterial Released Polysaccharides (RPSs), and consist of one or two pentoses, sugars that are generally absent in other polysaccharides of prokaryotic origin. A majority of the RPSs that are synthesized by the cyanobacteria (approximately about 80% of the polymers) are pretty complex as these compose six or more monosaccharides (de Paniagua-Michel et al. 2014).

2 Biosurfactants Produced by Cyanobacteria Although cyanobacteria pose a significant potential in the field of biosurfactant production, the organism is yet to be exploited and demands more research for applications in the industrial sector. Few of the biosurfactants recently isolated as well as identified from cyanobacteria are tabulated in Table 1.

3 Methods of Screening Biosurfactants, such as glycolipids, lipopeptides, lipoproteins, lipopolysaccharides, or phospholipids, are structurally widely varied groups of biomolecules. As a result, majority of techniques used for broad screening of strains that produce biosurfactants are based on the physical actions of surfactants. Furthermore, it is possible to examine how strains might obstruct hydrophobic surfaces. Furthermore, some screening techniques, namely the colorimetric CTAB agar test, are only

–

Lipopeptides

Somocystinamide A

Chemical structure

Compound name Columbamide F (1), G (2), & H (3)

Table 1 Biosurfactants produced by cyanobacteria

759.2

Molecular weight (g/mol) 338.4

Cyanobacterium sp. Lyngbya majuscula

Name of the organism Moorea bouillonii

(continued)

Abdelghany et al. (2018) Mehjabin et al. (2020)

References Mehjabin et al. (2020)

Methods of Screening and Applications of Biosurfactants Produced. . . 77

Exopolysaccharides

–

840.12426

–

–

Phospholipids

Apratoxins A

Molecular weight (g/mol) 364.5

Chemical structure

Compound name 2-Acyloxymethyl phosphonate (AOEP) 1

Table 1 (continued)

Cyanobacterium sp.

Corynebacterium lepus Cyanobacterium sp.

Name of the organism Aphanizomenon flos-aquae

Sukla et al. (2019)

Samuel et al. (2015) Mehjabin et al. (2020)

References Mehjabin et al. (2020)

78 G. R. Shree Kumari and L. Ravi

Corynomicolic acids

496.8

Corynebacterium insidiosum

Ibrahim et al. (2018)

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effective with a minimal subset of biosurfactants. Screening techniques produce quantitative and qualitative data. Qualitative procedures are typically sufficient for preparatory screening of isolates (Walter et al. 2010). Surface/Interfacial Activity Most of the screening techniques for microorganisms that produce biosurfactants are based on the surface or interfacial activity. Several techniques have been devised for measuring this attributes (Alizadeh-Sani et al. 2018). Measures of Direct Face/Interfacial Pressure The simplest webbing system and one that is definitely relevant for a primary webbing of microorganisms that produce biosurfactants is the direct dimension of the interfacial or face exertion of the culture supernatant (Huy et al. 1999). It strongly suggests the development of biosurfactants. Various methods can be employed to determine the face or interfacial pressure of a liquid. The range of dimensions is still restricted (Vujosevic 1989). As more surfactant is added, the face pressure drops until the CMC is obtained. However, there are limitations on the measuring range. An increase in attention cannot be seen if the biosurfactant attention is higher than the CMC. Two communities that have distinctly different attention to biosurfactants may therefore exhibit the same face pressure. Periodic lacing can solve this issue up until a significant rise in face pressure is noticed (Singh et al. 2016). Critical micelle dilution or CMD is the dilution of the supernatant that corresponds to the attention of the biosurfactant. Likewise, elements like pH and ionic strength have a huge impact on the measurements. Additionally, industrial canvases might have an impact on the dimension as carbon sources. The following styles are recognized for use in webbing. They can all be used to gauge a liquid face and interfacial pressure. In particular, the Du-NouyRing method is used most frequently and with considerable ease (Gustafsson et al. 2009). The biosurfactant capabilities of well-known natural product structures, such as the cytotoxic apratoxins, 1-acyloxyethyl phosphonate, exopolysaccharides (EPS), and Somocystinamide A from cyanobacteria, have been extensively studied. Because of their high bioactivities and structural variety, the secondary metabolites produced by cyanobacteria make them ideal candidates for new biosurfactants (Kaya et al. 2006). A majority of studies follow Jakia Jerin Mehjabin et al. (2020) method to identify, characterize and isolate biosurfactants from cyanobacteria.

3.1

The Process of Screening and Isolating Cyanobacteria According to Mehjabin et al. (2020)

Step 1: Identification Identification of cyanobacteria is done by its morphological appearance and on the basis of 16S rRNA gene sequencing analysis, which helps in understanding the phylogenetic studies (Mehjabin et al. 2020).

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Step 2: Extraction The sample containing cyanobacteria is extracted with MeOH (Mehjabin et al. 2020). Step 3: A solvent partition is run by using H2O, EtOAc, and BuOH (Mehjabin et al. 2020). Step 4: The Ethyl acetate chemical profiling of the fraction by liquid chromatography–mass spectrometry (LC-MS) exhibits the existence of several familiar biosurfactant compounds, such as apratoxins A and C, lyngbyabellin A, and supplemental possibly new chlorinated compounds (Mehjabin et al. 2020). Step 5: Ethyl acetate fraction to normal-phase silica gel column chromatography is subjected and divided into 12 fractions (Mehjabin et al. 2020). Step 6: Run an oil displacement assay The silica gel fractions of 100% ethyl acetate, ethyl acetate/methanol in the ratio of 90:10 (v/v), and hexane/ethyl acetate in the ratio 50:50 (v/v) exhibit at a greater value for the broadness of the clear zone (140 mm) in comparison with other fractions in an oil displacement assay (Mehjabin et al. 2020). Step 7: Based on their molecular profile and bioactivity, the above-mentioned fractions are selected for new halogenated compound isolation (Mehjabin et al. 2020). Step 8: Comparison between the compounds obtained by NMR chemical shift data: The compounds are determined based on the compounds eluted with 50:50 (v/v) mixture of hexane/ethyl acetate which determines for the presence of columbamides F (1) and G (2) with di- and trichlorinated patterns (Mehjabin et al. 2020). From 100% EtOAc fraction, columbamide H (3) was purified along with known compounds columbamide D and Apratoxin A13. All the above compounds were established by contrasting their respective 13C and 1H Nuclear Magnetic Resonance chemical shift (Mehjabin et al. 2020). Step 9: To determine the absolute configuration acid hydrolysis and Marfey’s examination of the N, O-dimethylserinol moiety and Ohrui’s method followed by chiral-phase HPLC were executed (Mehjabin et al. 2020). Step 10: By using oil displacement assay for the isolated compounds from cyanobacteria, biosurfactant activities are measured (Mehjabin et al. 2020). With the help of utilizing MCF-7 breast cancer cells, the cytotoxicity of the respective compounds is assessed (Mehjabin et al. 2020).

3.2

Methods of Cultivation of Cyanobacteria

The culturing of cyanobacteria can be followed through some steps of cultivation as follows (Priatni et al. 2016): Step 1: Take 1 ml stock culture and cultivate primary into 5 ml of medium, continue to 10 ml and later into 20 ml of medium. Step 2: The cultivation should be done at 28 °C for 3 days.

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Step 3: The cultivation of marine cyanobacteria is then gradually increased to 100 ml of medium. Step 4: It is recommended to cultivate these cyanobacteria in bottles connected to an aeration pump which has a pump output of about 70 l per minute. These bottles with aeration pump are exposed to 2 × 10 W white lamp which is approximately equal to 500 to 2000 lux at 25 °C. It is suggested to carry out the experiment for about 21 to 15 days for better culture.

3.3

Production of Biosurfactants on Industrial Scale (Large Scale)

For the synthesis of biosurfactants, various fermentation strategies are inculcated. Into the bargain immobilized cultivation, genetic engineering is accompanied for the enhancement of biosurfactant production. Soap stock, oil waste and other vegetablebased oil from refineries and food industries are used in the production as raw materials. Vegetable oil yields higher amount of biosurfactant among the other carbon sources. For the production of biosurfactants, we use methods like shake flask method, fed-batch method, batch cultivation, etc. Continuous integrated enzymatic or microbial processes are involved. Certain substances like glucose and plant oil limit the growth and can be used in batch cultivation, whereas glycerol is used in fed-batch cultivation as a growth limiting substance. In continuous cultivation mode, glucose and hydrocarbons are used (De Carvalho and Fernandes 2010).

4 Bio-Activity of Biosurfactants Biosurfactants lower the interfacial tension or surface tension between a lipid and a solid or between two lipids. These are often organic compounds which contain hydrophobic groups at their tails also known as amphiphilic and hydrophilic groups at their heads. Therefore, these contain both water-soluble and a water-insoluble component. The water insoluble component may be oil soluble. These biosurfactants may serve as wetting agents, forming agents, dispersants, and detergents. These biosurfactants generally tend to diffuse in water and adsorb at interfaces between air and water. EPS that are mass-produced by cyanobacteria are another property of biosurfactant producing cyanobacteria. These are a desperate group of surface-active molecules or also identified as chemical compounds which are usually synthesized by other microorganism such as cyanobacteria. Both in mixtures of hydrocarbon and aqueous solutions biosurfactants reduce critical micelle dilution (CMD) and surface tension. Production of polymeric biosurfactant has been reported. In nature, glycolipids and phospholipids are the best-studied and most frequently isolated groups of biosurfactants. These biosurfactants are eco-friendly and have potential industrial and environmental applications (Samuel et al. 2015).

Methods of Screening and Applications of Biosurfactants Produced. . .

4.1

83

Surfactants of Cyanobacterium phormidium

The manufacture of an emulsifying agent is found to be correlated with the alteration in cell surface hydrophobicity of Cyanobacterium phormidium. Fatty acid esters, carbohydrate, and proteins are detected in the emuicyan that was partially purified. The cells resulted in becoming hydrophilic and were identified to be detached from phenyl Sepharose beads or hexadecane in addition to emuicyan to adhere the hydrophobic cells (de Paniagua-Michel et al. 2014).

4.2

Cyanobacteria Producing Exopolysaccharide

A very notable character that is predominantly detected in marine cyanobacteria living in hypersaline environmental conditions is that it produces a coating of mucilaginous envelop around it. Therefore, production of EPS is noted surrounding the media in a majority of these cyanobacterial strains. Uronic acids, with one or two acidic sugars and six to eight monosaccharides, constitute EPS. The detection of other chemical groups like pyruvyl, acetyl, and/or sulfate groups was also proposed. The EPS from these photosynthesizing bacteria are assuring candidates for various industrial applicants (de Paniagua-Michel et al. 2014).

4.3

Biosurfactant as Cytotoxin

An encouraging source of added-value compounds is present in the marine environment. A few new biosurfactants have been reported from these compounds, namely somocystinamide A, fellutamides, rakicidin, and apratoxin. Somocystinamide A, which is a lipopeptide, obtained from a cyanobacterium Lyngbya majuscula cells with IC50 values ranging from 1.3 μM to 970 Nm depending on the cancer model (Gudiña et al. 2016). Biosurfactants of somocystinamide A exhibit remarkable cytotoxicity in odds to leukemia, breast, lung, and prostate cancer. It is considered as a tumor cell proliferation and a pluripotent inhibitor of angiogenesis, the development of new blood vessels (Gudiña et al. 2016).

5 Parameters Influencing Activity and Production of Biosurfactants Few parameters that influence the activity and biosurfactant production are temperature, pH, ion concentration, and salinity (Samuel et al. 2015).

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1. Temperature: A variable criterion which depends on the type of medium and the strain cultivated in that particular medium. For example, EPS produced by Nostoc sp. at 37.5 °C show the maximum biosurfactant activity. 2. pH: It is another vital factor that is considered, but the properties dependent in respect of its respective strain and also its working condition is also taken into count. Generally, at neutral pH, the biosurfactant activity is very high. 3. Salinity: Salts like NaCl effecting on the production of biosurfactant have a highly minute change that has been absorbed up to 10% (w/v) NaCl.

6 Applications Biosurfactants offer numerous advantages over the chemical surfactants. These are environment or eco-friendly which can be produced provided a natural condition, and mostly less toxic (Elis acirc ngela et al. 2015). • These are cheaper as they have the ability to produce from industrial waste and by-products when compared to chemical surfactants (Mehjabin 2020). • The biosurfactants produced by a few marine microorganisms like cyanobacteria are employed as new weaponry to resist against human pathogens as these biosurfactants exhibit antimicrobial and anti-inflammatory activity in opposition to different human pathogens (Mandal and Mandal 2011). • Biosurfactants produced by cyanobacteria exhibit anticancerous and antimicrobial activity against different human pathogens (de Paniagua-Michel et al. 2014). • Some Nostoc sp. have been reported to produce polymeric biosurfactants (Satpute et al. 2010). • EPS that elongate from the germplasm in a few marine Cyanobacterium sp. issued as biosurfactant in the field of cyanobacterial clean-up as a new variegated arena called bio-adsorbent (Rashmi et al. 2021). • The marine Cyanobacterium sp. are also used in some rare fields like crude studies. • Cyanobacteria are applied in the treatment of effluent of oil industries with the help of EPS covering the cells or biosurfactant activity (Kaya et al. 2006). • Phormidium sp., a marine cyanobacteria, removes 45% of hexadecane and 37% of diesel oil also helping in bioremediation and enhancement of diesel oil (Wang et al. 2021). • Besides cyanobacteria being a promising source of galactolipids, it is not so explored upon. These compounds are found to be useful in bakery products and cancer preventive measures (Rahman et al. 2002). • They also show anti-inflammatory activity, a recent study reported a surfactinmediated reduction of proinflammatory mediators in lipopolysaccharide (LPS)induced macrophage stimulation (Anestopoulo et al. 2020).

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7 Conclusion Cyanobacteria have effectively been proven as a good source of production of biosurfactants. About eight biosurfactants have been extracted and reported from these cyanobacteria till date. Biosurfactants produced by cyanobacteria possess an outstanding functional property which forms basic foundation for various industries. Further studies on cyanobacteria in biosurfactant production could lead to discovery of novel bioactive biosurfactants with potential applications in industries and environment. The current review of literature proves cyanobacteria as a potent source for biosurfactants.

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Huy NQ, Jin S, Amada K, Haruki M, Huu NB, Hang DT, Ha DTC, Imanaka T, Morikawa M, Kanaya S (1999) Characterization of petroleum-degrading bacteria from oil-contaminated sites in Vietnam. J Biosci Bioeng 88(1):100–102. https://doi.org/10.1016/S1389-1723(99)80184-4 Ibrahim H, Wefky S, Abo Elela G, El Sersy N, EBD HA (2018) Bio-surfactants: a package of environmental and industrial benefits. Int J Adv Multidiscip Res 05(12):4275–4291 Kaya K, Morrison LF, Codd GA, Metcalf JS, Sano T, Takagi H, Kubo T (2006) A novel biosurfactant, 2-acyloxyethylphosphonate, isolated from waterblooms of Aphanizomenon flosaquae. Molecules 11(7):539–548. https://doi.org/10.3390/11070539 Mandal MD, Mandal S (2011) Honey: its medicinal property and antibacterial activity. Asian Pac J Trop Biomed 1(2):154–160. https://doi.org/10.1016/S2221-1691(11)60016-6 Mehjabin JJ (2020) Studies on new secondary metabolites from the marine cyanobacterium Moorea bouillonii collected in Sabah, Malaysia. https://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/ 79624/1/Mehjabin_Jakia_summary.pdf Mehjabin JJ, Wei L, Petitbois JG, Umezawa T, Matsuda F, Vairappan CS, Morikawa M, Okino T (2020) Biosurfactants from marine Cyanobacteriacollected in Sabah, Malaysia. J Nat Prod 83(6):1925–1930. https://doi.org/10.1021/acs.jnatprod.0c00164 Nayarisseri A, Singh P, Singh SK (2018) Screening, isolation and characterization of biosurfactant producing Bacillus subtilis strain ANSKLAB03. J Bioinform 14(6):304–314. https://doi.org/10. 6026/97320630014304 Priatni S, Budiwati TA, Ratnaningrum D, Kosasih W, Andryani R, Susanti H, Susilaningsih D (2016) Antidiabetic screening of some Indonesian marine cyanobacteria collection. Biodiversitas 17(2):642–646. https://doi.org/10.13057/biodiv/d170236 Rahman KSM, Rahman TJ, McClean S, Marchant R, Banat IM (2002) Rhamnolipid biosurfactant production by strains of Pseudomonas aeruginosa using low-cost raw materials. Biotechnol Prog 18(6):1277–1281. https://doi.org/10.1021/bp020071x Rahman PKSM, Mayat A, Harvey JGH, Randhawa KS, Relph LE, Armstrong MC (2019) Biosurfactants and bioemulsifiers from marine algae. In: Sukla L, Subudhi E, Pradhan D (eds) The role of microalgae in wastewater treatment. Springer, Singapore, pp 169–188. https://doi. org/10.1007/978-981-13-1586-2_13 Rashmi V, Darshana A, Bhuvaneshwari T, Saha SK, Uma L, Prabaharan D (2021) Uranium adsorption and oil emulsification by extracellular polysaccharide (EPS) of a halophilic unicellular marine cyanobacterium Synechococcus elongatus BDU130911. Curr Opin Green Sustain Chem:100051. https://doi.org/10.1016/j.crgsc.2020.100051 Samuel CSJ, Jagannathan A, Barath M, Shreenidhi KS (2015) A review: bioflocculant and biosurfactant activity of exo- polysaccharides produced by marine cyanobacteria- Nostoc. Int J Innov Res Sci:12747–12754. https://doi.org/10.15680/IJIRSET.2015.0412100 Satpute SK, Banat IM, Dhakephalkar PK, Banpurkar AG, Chopade BA (2010) Biosurfactants, bioemulsifiers and exopolysaccharides from marine microorganisms. Biotechnol Adv 28(4): 436–450. https://doi.org/10.1016/j.biotechadv.2010.02.006 Singh JS, Kumar A, Rai AN, Singh DP (2016) Cyanobacteria: a precious bio-resource in agriculture, ecosystem, and environmental sustainability. Front Microbiol 7:1–19. https://doi.org/10. 3389/fmicb.2016.00529 Sukla LB, Subudhi E, Pradhan D (2019) The role of microalgae in wastewater treatment. In: The role of microalgae in wastewater treatment. https://doi.org/10.1007/978-981-13-15862 Vujosevic N (1989) Izracun prehodne vrtilne hitrosti radialnih drsnih lezajev. Stroj Vestn-J Mech E 35(1–3):37–41 Walter V, Syldatk C, Hausmann R (2010) Screening concepts for the isolation of biosurfactant producing microorganisms. Adv Exp Med Biol 672:1–13. https://doi.org/10.1007/978-1-44195979-9_1 Wang BW, Huang CH, Liu LC, Cheng FJ, Wei YL, Lin YM, Wang YF, Wei CT, Chen Y, Chen YJ, Huang WC (2021) Pim1 kinase inhibitors exert anti-cancer activity against HER2-positive breast cancer cells through downregulation of HER2. Front Pharmacol 12:1–11. https://doi. org/10.3389/fphar.2021.614673

Fungal Biosurfactants and Its Applications Hoda Nouri, Hamid Moghimi, and Elham Lashani

1 Introduction Biosurfactant’s replacement with synthetic surfactants is at an enormous pace today. Rapid industrialization adversely affects the environment and environmental control legislation, and social pressure has led to a tremendous increase in research and development for eco-friendly product replacements (Santos et al. 2016; Vieira et al. 2021). Biosurfactants are structurally and functionally diverse and unique amphipathic biomolecules that reduce surface tension properties like synthetic surfactants. However, most currently produced surfactants are petroleum-derivative and have high commercial and industrial importance (Sanches et al. 2021; Santos et al. 2016; Vieira et al. 2021). Biosurfactants have drawn the scientific community’s interest due to their advantages over synthetic surfactants. These compounds have exhibited more excellent biodegradability, low toxicity, and more and unique structural diversity and broad capabilities. Other advantages of biosurfactants are their tolerance to environmental fluctuations like temperature, pH, and salinity, and industrial production by using renewable sources or wastes (Rawat et al. 2020; Sanches et al. 2021). Most researches related to microbially derived surface-active substances, and biosurfactants, describe the acquisition of these compounds from bacteria, yeasts, and fungi. The variety of metabolic and physiological capabilities of these microorganisms will result in a wide range of molecular structures and properties (Shakeri et al. 2021; Singh et al. 2021). Although several studies have demonstrated the potential of fungi as biosurfactant producers, most of them have focused on bacteria. However, there is little information on biosurfactant production by fungi as

H. Nouri · H. Moghimi (✉) · E. Lashani Department of Microbial Biotechnology, School of Biology, College of Science, University of Tehran, Tehran, Iran e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_5

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Fig. 1 Comparison of preferences of fungal over bacterial biosurfactants

compared to bacteria. As shown in Fig. 1, they posse unique features and advantageous properties that make them attractive for researchers and industry (Bhardwaj et al. 2013; Luft et al. 2020). In order to gain detailed insights into the biosurfactant production of fungi, a comparison between these two groups was made: 1. Although bacteria stand out as one of the main producers of biosurfactants, they are not offered in the pharmaceutical and food industries because of possible pathogenicity. In contrast, yeasts are classified as GRAS (generally regarded as safe) so they do not have pathogenic natures (Santos et al. 2016; Vieira et al. 2021). 2. Bacterial response to environmental fluctuation is another reason in terms of the management of environmental contaminants. Low pH, low moisture, and nutrient are harsh conditions that affect bacterial survival and function. However, fungi have various extraordinary strategies to counteract these nonfavorable conditions (Espinosa-Ortiz et al. 2022; Singh et al. 2021). 3. Tension-active characteristics of fungi to thrive in extreme habitats may result in the production of novel metabolites with specific surface activities and molecular structures and properties which are not often present in surfactants of bacterial origin. 4. Biosurfactants of fungal origin show a higher yield in comparison to bacterial. This feature may relate to the cell wall structure of these organisms (Bhardwaj et al. 2013). 5. There are many expectations of fungi in the secretion of essential enzymes to break down compounds of great complexity into simpler forms. In addition to

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strong metabolic capability, peculiar bio-degradative actions by extracellular nonspecific and nonstereoselective enzymes make these organisms promising from a practical viewpoint (Espinosa-Ortiz et al. 2022). 6. Hyphal network structures enable fungi to explore and penetrate trapped contaminations in the soil more professionally than bacteria. So, fungi are ubiquitous, and inaccessible pollutants will break down and be available to use by other organisms in that ecosystem (Sánchez 2020). As mentioned above, fungi have attracted attention not only because of their unique structure of surfactants but also for metabolic activity in the utilization of recalcitrant and complex structures that bacteria cannot catabolize (Luft et al. 2020). Various environmental, commercial, and industrial importance confirmed the essentiality of biosurfactant replacement with a synthetic one. However, there are economical and operational barriers to this procedure that many tools and techniques deal with. These limitations cause synthetic surfactants to be preferred in the industry; so solutions are vital for biosurfactant replacement. It should be noted that profit-making biosurfactant production depends on the ratio between cost and benefit in each industry (Sanches et al. 2021; Shakeri et al. 2021). In the following section, we tried to summarize limitations and solutions for cost-effective production of fungal biosurfactants: • Raw materials: using raw materials is one of the strategies for reducing production costs related to the substrates. Pure feed-stocks are expensive, and most of the product cost is related to this sector. From a financial point of view, agroindustrial wastes and renewable substrates, such as corn-steep liquor, cheese whey, and molasses, are low-cost substrates for manufacture. Fungi with the high enzymatic capability to use agricultural substrates are preferred over bacteria (Rane et al. 2021; Vieira et al. 2021). • Low yields: there are restrictive factors that limit the rate and yield of production. Understanding these factors is vital for overcoming and improving the yield. Inoculum size, substrate, culture medium component concentration, and environmental factors influence the final yield and should be optimized by using statistical procedures. Fungi possess a higher yield than bacteria (Vieira et al. 2021). • Microbial strain: hyperproducing microorganism is the main criteria in any profitable biotechnological industry. High-yielding naturally productive or recombinant isolates are applied in the biosurfactant production process. In this way, metabolic engineering techniques and mutation help to obtain superior strains (Rane et al. 2021; Sanches et al. 2021). • Optimization of culture conditions: The medium formulation and an optimized and efficient production process are essential since they collaborate with the reduction of production costs and higher quantity of the biosurfactants. In the case of fungi, there is an essential need to develop cost minimization strategies in medium and process engineering. The main parts that gain more attention are cultural, environmental and operational parameters. Cultural sectors focused on nutritional requirements include carbon and nitrogen sources, phosphorous, iron, magnesium, and other ion concentrations. Environmental and operational

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parameters are significant factors for regulating and controlling biosurfactant production. The most important factors are pH, temperature, stirring speed, aeration, dilution rate, and cultivation time. Medium engineering and statistical analysis can be applied to heighten biosurfactant production from fungi (Luft et al. 2020; Rane et al. 2021; Sanches et al. 2021; Shakeri et al. 2021). • Downstream processes and purification: Biosurfactant’s application determines the degree of purification. In some industries like pharmaceuticals and cosmetics, some standards determine the purification quality. The high cost of these quality controls is due to acceptable purification processes because of using low volume in manufacturers (Sanches et al. 2021; Shakeri et al. 2021). Outstanding participation of biosurfactants is in environmental remediation. Biosurfactants can be widely used in the biodegradation of toxic contaminants, enhanced oil recovery, elimination of heavy metals, or remediation of industrial effluents. Depending on biosurfactant’s application in environmental protection, nonmicrobial contamination is the only constraint. Since a large volume of biosurfactants is required in these situations, high cost and low yield will limit their wide range of applications. Consequently, recovery procedures are still a matter of concern. Different strategies are applied based on the chemical class of surfactants, their specified structures, degree of charge, solubility and stability, and other chemical and physical parameters. Recovery methods of fungal biosurfactants need further studies due to the unexplored and less-known fungal-derived biosurfactant structures (Daccò et al. 2020; Rane et al. 2021). Consequently, from the above-mentioned details, recovery efficiency and production costs are the most critical barriers to the industrial scale-up of biosurfactants. As fungi have unique features that can counter economic problems in biosurfactant production, the current chapter may provide a comprehensive overview of fugal biosurfactant production in detail. In the first step, the latest finding on the biodiversity of biosurfactant-producing fungi and their chemical structure was collected. In the second step, particular focus was placed on optimizing involved parameters, their inherent hurdles, and various downstream industrial developments. Finally, the application of fungal biosurfactants and the way from the laboratory to market were compiled to propose future avenues to fill existing market gaps.

2 Biodiversity of Biosurfactant-Producing Fungi and Classification of Fungal Biosurfactants Fungi constitute an essential part of the Earth’s biosphere; approximately 148,000 species of fungi have been described by scientists, but the global biodiversity of the fungus kingdom is not fully understood. As per 2017 estimate suggested, there might be between 2.2 and 3.8 million fungal species. Recent estimates based on nextgeneration sequencing (NGS) methods suggested that as many as 5.1 million fungal species exist. Understanding the biodiversity of fungi is very important because of

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their biotechnological and environmental importance (Naranjo-Ortiz and Gabaldón 2019). Fungal biosurfactants and their enzymes have caused those fungi to be prominent as primary decomposers for recycling some nutrients and elements to food and elements cycles. Moreover, ecological relationships with other microorganisms, such as mutualism, provide an appropriate environment for remediation and support nutritional requirements in harsh conditions (Chambergo and Valencia 2016). Fungal biosurfactants are mainly categorized according to a chemical and functional structure, molecular mass, and producing fungi (yeasts or filamentous fungi). Regarding the chemical and functional structure, biosurfactants are divided into two moieties with amphiphilic structures. Hydrophobic tail and hydrophilic head are connected via linking biochemical functional groups, such as esters (O–C=O), amides (N–C=O), and ethers (C–O–C). This structure confers the ability to make micro-emulsions between two hydrophobic and hydrophilic phases (Henkel and Hausmann 2019; Banat et al. 2021). Based on molecular mass, biosurfactants are categorized into low and high molecular weight. Low-molecular-weight biosurfactants with reducing surface and interfacial tension properties are included glycolipid, lipopeptide, lipoprotein, fatty acid, and phospholipid. In contrast, polymeric substances and particulate surfactants are considered high-molecular-weight biosurfactants with emulsion stabilizing property (Banat et al. 2021). Bacterial biosurfactants are produced mainly by different Pseudomonas and Bacillus strains and are mostly glycolipids, glycoproteins, and lipopeptides in structure. In comparison, fungal biosurfactants include only 19% of biosurfactants but have the broadest chemical structural diversity. Some types of biosurfactants, including sophorolipids, cellobiose lipids, lipid polyols, mannosylerythritol lipids, xylolipids, and hydrophobins, are produced only by fungi. The high diversity in the structure of fungal biosurfactants has led to their wide applications (Sunde et al. 2017). The different types of fungal biosurfactants are related to their molecular weight and chemical structure. Based on molecular weight, fungal biosurfactants are divided into two groups, including low molecular weight (0.5 and 1.5 kDa) (Mulligan 2005) and high molecular weight (bioemulsifiers) that can reach up to 500 kDa (Uzoigwe et al. 2015). Accordingly, different groups of fungal biosurfactants are shown in Table 1. Regarding chemical and functional structures, different groups of biosurfactants are briefly described.

2.1

Glycolipid

Glycolipids are composed of hydroxyl and long-chain aliphatic acids combined with carbohydrates, such as glucose, rhamnose, glucuronic acid, galactose, mannose, and galactose sulfate. Different types of glycolipids include rhamnolipids, trehalose

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Table 1 Low- and high-molecular-weight fungal biosurfactant classes Biosurfactant categories Low molecular weight

Biosurfactant group Glycolipids

Biosurfactant class Sophorolipids

Liposan

Biosurfactantproducing fungi Torulopsis bombicola, Torulopsis petrophilum, Torulopsis apicola Aspergillus sp. MSF1, Fusarium sacchari, Fusarium verticillioides Kurtzmanomyces sp. Penicillium spiculisporum Saccharomyces cerevisiae Candida lipolytica

Yasan

Yarrowia lipolytica

Rhamnolipids

Surfactin

High molecular weight

Polymeric substances

Spiculisporic acid Mannoprotein

References Cooper and Paddock (1983), Hommel et al. (1994), Cavalero and Cooper (2003) Seghal Kiran et al. (2010), Goswami et al. (2014), Borah et al. (2016) Kakugawa et al. (2002) Gautam et al. (2014) Cameron et al. (1988) Cirigliano and Carman (1985) Amaral et al. (2006)

lipids, glycoglycerolipid, diglycosyldiglycerides, sophorolipids, and mannosylerythritol lipids (Liepins et al. 2021). Among different types of microbial biosurfactants, sophorolipids are produced by Starmerella bombicola, Pseudohyphozyma bogoriensis, C. batistae, C. floricola, C. apicola, C. riodocensis, C. stellate, C. kuoi, Candida albicans O-13-1, C. rugosa, and C. tropicalis, Cyberlindnera samutprakarnensis, Lachancea thermotolerans, and Candida apicola (Daverey and Pakshirajan 2009; Santos et al. 2016). Mannosylerythritol lipids are another glycolipid biosurfactant produced by fungi (Table 1). Candida antarctica, Schizonella melanogramma, Geotrichum candidum, Candida sp. SY16, M. aphidis, and S. scitamineum are some fungal examples that produce mannosylerythritol lipids (Das et al. 2008). Furthermore, glycolipid production was observed in Sordaria macrospora, Rhynchosporium secalis, Komagataella phaffii, Gliocladium catenulatum, Rhodotorula glutinis, and Rhodotorulagraminis. Different species can produce mannosyl and glycosyl lipids, including Fusarium sp., and Simplicillium lamellicola, U. maydis, and C. humicola can produce both cellobiose lipids and mannosylerythritol lipids. Ustilago zeae, Pseudozyma fusiformata, and Sympodiomycopsis paphiopedili can produce cellobiose lipids, and the ability to biosynthesize cybersan was observed in Cyberlindnera saturnus (Kasai et al. 2005; Sakaki et al. 2001; Zibek and Soberón-Chávez 2022).

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Lipopeptide/Lipoprotein Biosurfactant

Lipopeptide is one of the most important groups of biosurfactants that contain two main regions, including a short linear peptide sequence and hydrophobic acyl tail (s) containing hydrocarbon. In the peptide region, the anionic and cationic residues are present, and sometimes nonproteinaceous amino acids are also available (Mondal et al. 2017). Because of various functional properties and structural diversity, different lipopeptide domains are used (Inès and Dhouha 2015). Cyclic lipopeptides are synthesized with the help of nonribosomal peptide synthetase (NRPS) (Strano et al. 2015). The lipoprotein is a biosurfactant type generally composed of many cyclic lipopeptides associated with a fatty acid. In addition to reducing surface tension, this biosurfactant has an antibacterial effect (Cawoy et al. 2014; Sharma 2016). Most lipopeptides are produced by bacteria (Kumar and Ngueagni 2021). However, Candida lipolytica is a fungus that can produces a lipopeptide biosurfactant. One of the lipopeptide types is surfactin, and its production was reported in Kurtzmanomyces sp. In addition, lipopeptide production was seen in Trametes versicolor, Penicillium chrysogenum SNP5, and Fusarium sp. BS8 (Gautam et al. 2014; Lourenço et al. 2018; Qazi et al. 2014).

2.3

Fatty Acid Biosurfactant

Some microorganisms can biodegrade hydrocarbon and produce extracellular freefatty acid biosurfactants when they are grown on alkanes. These saturated fatty acids act as biosurfactants and contain alkyl branches and hydroxyl groups or are in the range of C12 to C14 (Rahman and Gakpe 2008). Talaromyces trachyspermus and Penicillium spiculisporum (spiculisporic acid) can produce fatty acid biosurfactants (Santos et al. 2016).

2.4

Particulate Biosurfactants

This particulate biosurfactant plays a crucial role in saturated hydrocarbon adsorption by partitioning them into vesicles and creating a micro-emulsion. This process facilitates hydrocarbon uptake (Kamyabi et al. 2017). Bacteria generally produce this kind of biosurfactant.

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Polymeric Surfactants

This biosurfactant has a complex and variable composition and generally comprises hetero-polysaccharides (Table 1). Water surface tension is reduced from 72 to 30 mN/m by this high-molecular-weight surfactant (≥1000 Da) (Marcelino et al. 2020). Polymeric surfactant biosynthesis was seen in Candida lipolytica and Candida utilis (Santos et al. 2016). A type of glycoprotein is produced by Aspergillus niger (Bjerk et al. 2021). Mannoprotein and liposan are other polymeric biosurfactants biosynthesized in Saccharomyces cerevisiae and Candida lipolytica, respectively. In addition, Yarrowia lipolytica is a yeast that biosynthesizes liposan and yasan biosurfactant (Cirigliano and Carman 1985;Amaral et al. 2006;Walencka et al. 2007;Saikia et al. 2021). Fungi are a great source of metabolites with a broad spectrum of applications, such as cosmetics, biocatalysts, pharmaceuticals, agrichemicals, biosurfactants, nutritional supplements, bio-emulsifiers, biomaterials, and enzymes (Chambergo and Valencia 2016). There are nine different fungal phyla: Opisthosporidia, Mucoromycota, Chytridiomycota, Blastocladiomycota, Neocallimastigomycota, Zygomycota, Glomerulomycota, Ascomycota, and Basidiomycota, and most of the known species (65%) belong to the Ascomycota phylum (Naranjo-Ortiz and Gabaldón 2019). Among different phyla of fungi, the members of Ascomycota and Basidiomycota phyla are well-known. For biosurfactant production, more genera are related to the Ascomycota phylum, and the abundance of the genera belonging to the Basidiomycota is less than that of the Ascomycota. Different taxa such as Candida, Aspergillus, Ustilago, and Fusarium have drawn much attention for their potential as biosurfactant-producing fungi. Species belonging to the genus Candida, such as Candida bombicola and Candida lipolytica, are well-known yeast species for producing biosurfactants (Saranraj et al. 2021). According to Table 2, most biosurfactant-producing species belong to the lineages of Saccharomycotina and Pezizomycotina. These two taxa, placed in the Ascomycota and Basidiomycota phyla, respectively, can produce some surfactants such as glycolipid, polymeric surfactant, glycopeptide, and fatty acid. Some genera of these taxa, classified as yeasts, are known as GRAS microorganisms. Genera such as Candida, Saccharomyces, and Yarrowia are among the yeasts that can produce critical biotechnological products such as surfactants. From the Mucoromycotina phylum, Mucor is the only genus in which the ability to produce biosurfactants has been identified. Little studies have been conducted on species of other fungal phyla in case of metabolite production. Different fungal species and their biosynthesized biosurfactants are shown in Table 2.

Pezizomycotina

Mucoromycotina

Pezizomycotina

Ascomycota

Mucoromycota

Ascomycota

Biosurfactant type Sophorolipids

Fatty acids

Lineage Saccharomycotina

Fungal division (phylum) Ascomycota

Table 2 Biodiversity of biosurfactant-producing fungi

Mostly filamentous

Saprobes

Mostly filamentous

Main characteristic Mostly yeasts

Candida bogorensis Torulopsis petrophilum Candida (Torulopsis) apicola Metschnikowia churdharensis f.a Aureobasidium thailandense LB01 Mucor hiemalis UCP 0039 Cunninghamella echinulata UCP 1299 Fusarium sp. Fusarium fujikuroi

Biosurfactantproducing fungi Candida lipolytica Candida bombicola

Al-Kashef et al. (2018) Reis et al. (2018)

Andrade et al. (2018)

Ferreira et al. (2020)

Meneses et al. (2017)

Kumari et al. (2021)

(continued)

Hommel et al. (1994), Weber et al. (1992)

Cooper and Paddock (1983)

Deshpande and Daniels (1995), Casas and Garcia-Ochoa (1999), Cavalero and Cooper (2003) Tulloch et al. (1968)

References Sarubbo et al. (1999)

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Saccharomycotina

Ustilagomycotina

Ascomycota

Basidiomycota

Ustilagomycotina

Pucciniomycotina

Pezizomycotina

Ascomycota

Polymeric Surfactants Mannosylerythritol lipids

Pezizomycotina

Ustilaginomycotina

Basidiomycota

Ascomycota

Lineage

Fungal division (phylum)

Lipopeptide

Biosurfactant type

Table 2 (continued)

Unicellular and filamentous Saprotrophic yeast-like or dimorphic Unicellular and filamentous

Mostly yeasts

Mostly filamentous

Mostly filamentous

Unicellular and filamentous Extremotolerant black fungi

Main characteristic Extremotolerant black fungi

Penicillium chrysogenum SNP5 Fusarium SP BS-8 Beauveria bassiana Curvularia lunata IM 2901 Candida antarctica Candida sp. SY16 Pseudozyma aphidis Kurtzmanomyces sp. l-11 Pseudozyma parantarctica Pseudozyma fusifornata

Biosurfactantproducing fungi Aspergillus niger Penicillium citrinum Ustilago maydis

Morita et al. (2007b)

Morita et al. (2007b)

Kakugawa et al. (2002)

Rau et al. (2005)

Kim et al. (2006)

Kitamoto et al. (1992)

Paraszkiewicz et al. (2002)

Abdel-Aziz et al. (2020)

Qazi et al. (2014)

Gautam et al. (2014)

Hewald et al. (2005)

References Laine et al. (1972) Camargo-de-Morais et al. (2003)

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Pezizomycotina

Ascomycota

Not determined

Pucciniomycotina

Basidiomycota

Saprobes, occasionally mycoparasites or plant pathogens Mostly yeast Saprobes, plant pathogens

Mucoromycotina

Saccharomycotina

Pezizomycotina

Mucoromycota

Ascomycota

Extremotolerant black fungi

Extremotolerant black fungi Unicellular

Pezizomycotina

Lipids

Mostly yeasts

Mostly yeasts

Saccharomycotina

Ascomycota

Glycoprotein

Saccharomycotina

Ascomycota

A complex of protein, carbohydrate, and lipid

Yarrowia lipolytica Xylaria regalis

Pseudozyma tsukubaensis Pseudozyma rugulosa Candida lipolytica Yarrowia lipolytica NCIM 3589 Yarrowia lipolytica IMUFRJ 50682 Candida lipolytica ATCC 8662 Saccharomyces cerevisiae Aspergillus ustus MSF3 Rhodotorula glutinis Exophiala dermatitidis SK80 Mucor circinelloides

Adnan et al. (2018)

Fontes et al. (2010)

Hasani zadeh et al. (2018)

Chiewpattanakul et al. (2010)

Yoon and Rhee (1983)

Kiran et al. (2009)

Cameron et al. (1988)

(continued)

Cirigliano and Carman (1984), Cirigliano and Carman (1985)

Amaral et al. (2006)

Zinjarde et al. (1997)

Sarubbo et al. (2007), Sarubbo et al. (2001)

Morita et al. (2006)

Morita et al. (2007b)

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Biosurfactant type

Table 2 (continued)

Fungal division (phylum)

Lineage

Mostly filamentous

Main characteristic Extremotolerant black fungi

Biosurfactantproducing fungi Penicillium sp. Aspergillus niger Fusarium oxysporum LM5634 References Luna-Velasco et al. (2007) Silva et al. (2021) Sanches et al. (2018)

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3 Metabolic Pathways of Biosurfactant Production Biosurfactant production is dependent on cultural conditions and media composition. By producing a surfactant, microorganisms can solubilize insoluble-and harddegrading carbon sources and uptake them. Therefore, these factors define which metabolic pathway should be used by microorganisms (Bjerk et al. 2021; Ribeiro et al. 2020b). Although many investigations have been performed on microbial biosurfactants, their molecular pathway is not entirely revealed. Mannosylerythritol lipids (MEL) are one type of biosurfactants produced by different fungal species, such as Ustilago maydis, Schizonella melanogramma, Geotrichum candidum, and Candida antarctica. Two types of MEL are produced as secondary metabolites by Ustilago maydis namely, ustilagic acid and ustilipids. These glycoproteins were encoded by emt1 and cyp1 (Das et al. 2008). Hydrophobins are another low-molecular-weight surfactant produced by Trichoderma reesei. Two genes are involved in hydrophobin biosynthesis, namely hfb1 and hfb2 (Askolin et al. 2001). Figure 2 shows three metabolic pathways of biosynthesis of fungal biosurfactants. In the synthesis pathway of sophorolipids (SL), cytochrome P450 Cyp52M1 oxidized fatty acid and produced hydroxyl fatty acid. Then, glucose was added to a fatty acid by glucose transferase I, II UgtA1, and UgtB1 and acetylated by acetyltransferase (Fig. 2). The acidic form of SL was changed by lactone esterase and transferred across the membrane by the SL transporter (Zibek and SoberónChávez 2022). Some genes and enzymes are proposed to be involved in the SL biosynthesis pathway, including mfe-2 (which has a role in β-oxidation), three cytochrome P450 monooxygenases, and glyceraldehyde-3-phosphate dehydrogenase (Van Bogaert et al. 2008; Van Bogaert et al. 2009a; Van Bogaert et al. 2009b;Van Bogaert et al. 2011). In addition to U. maydis, the mannosylerythritol lipids (MEL) biosynthesis cluster was investigated in other species, such as P. hubeiensis, P. tsukubaensis, M. antarcticus, P. aphidis, and M. aphidis (Hewald et al. 2006; Konishi et al. 2013; Lorenz et al. 2014; Saika et al. 2014; Günther et al. 2015). Erythritol-mannosyltransferase (emt1), mannosylerythritol-acyl-transferases (mac1 and mac2), and mannosylerythritol-acetyltransferase (mat1) are three enzymes, and mannosylerythritol-major-facilitator protein (mmf1) is a transporter encoded by a gene cluster. In the MEL pathway production, lipase is produced and degraded triglyceride to fatty acids and glycerol when a triglyceride is used as a carbon source. Moreover, induction of MEL cluster genes happens when triglyceride is present (Hewald et al. 2006; Morita et al. 2007a; Zibek and Soberón-Chávez 2022). Fatty acid interacted with coenzyme A and converted to a short-chain fatty acid by ß-oxidation (Kitamoto et al. 1998; Freitag et al. 2014). Mannose and erythritol are affected by glycosyltransferase Emt1, and mannosylerythritol is produced and used as a substrate for two acyltransferases, Mac1 and Mac2 (Hewald et al. 2005; Hewald et al. 2006; Morita et al. 2010). These two enzymes added two fatty acids to

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Fig. 2 Schematic overview of metabolic pathways of fungal biosurfactant production. (a) sophorolipids (SL) production, (b) cellobiose lipids (CL) production, and (c) mannosylerythritol lipids (MEL) production

mannosylerythritol and formed MEL-D. After acetylation by acetyltransferase, Mat1, MEL-A, B or C are made depending on the position of acetyl groups. Exportation of MEL occurred by Mmf1 transporter. By deletion of mac1, mac2,

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and emt1, biosynthesis of MEL was inhibited indicating the prominent roles of these genes in biosurfactant production in M. antarcticus and U. maydis (Hewald et al. 2006; Morita et al. 2010). Twelve genes are involved in cellobiose lipids (CL) synthesis as a gene cluster. Palmitic acid is made by the de-novo pathway and was affected by cytochrome 450 monooxygenases Cyp1 and Cyp2 to produce 15, 16-dihydroxy palmitic acid. By glycosyltransferase activity, two glucose are added to dihydroxy palmitic acid and CL-A is produced. After acetylation by acetyltransferase Uat2, fatty acid is coupled to acetylate CL-A by the acyltransferase Uat1 to form CL-B. After hydroxylation by hydroxylase Ahd 1, this glycolipid is exported by transporter Atr1 (Teichmann et al. 2007; Teichmann et al. 2011a; Teichmann et al. 2011b). Other biosurfactant metabolic pathways are not extensively investigated in fungi.

4 Production, Optimization, and Downstream of Fungal Biosurfactants The fungal biosurfactant production depends on the production process yields maximization. Much research is going on for the successful establishment of industrial-scale production of biosurfactants (da Silva et al. 2021; Luft et al. 2020). As depicted in Fig. 3, different routes must be optimized to secure the cost aspects of biosurfactant production.

4.1

Commercial Production and Optimization of Fungal Biosurfactant

Selection and improvement of appropriate fungal species, medium and environmental factor optimization, and large-scale and process parameters optimization are the most critical factors determining the quality and quantity of the produced biosurfactant (da Silva et al. 2021; Luft et al. 2020). In the following section, we tried to discuss the routes mentioned earlier to achieve the commercial production objective.

4.2

Strain Improvement

The continuous demand to decrease production costs of biosurfactants has attracted more attempts to induce the overproduction of these compounds. As microbial production is regulated by the genetic background of the producer strain, enhanced yields will be achievable by screening more potent fungi or strain engineering

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Fig. 3 Strategies for improved fungal biosurfactant production

(da Silva et al. 2021; Deepika et al. 2017; Koglin et al. 2010). Lourenço et al. (2018) investigated biosurfactant biosynthesis by Trametes versicolor as the first study on white-rot fungi. After culturing this fungus on two-phase olive mill waste, the maximum biosurfactant production was 373.6 ± 19.4 mg in a 100 g culture medium. Protein and lipid exist in the structure of this biosurfactant (Lourenço et al. 2018). In addition, to yield more surfactants, this strain engineering can alter the scaffolds of biosurfactants and provide novel structural variants that expand their specified function. As diverse metabolic pathways are involved in producing these biomolecules, the knowledge about the biosynthetic pathways and regulation mechanisms require more attention. Random mutagenesis and genetic and metabolic engineering are the primary tools for gaining the desired strain. In order to design a superior stain, advanced analytics like genomics, transcriptomics, proteomics, and metabolomics can provide detailed insights into metabolic pathways in biosurfactant synthesis. Moreover, metabolic flux analysis can provide a comprehensive view of strain metabolism in a systems biology context. Consequently, this complete overview can help identify and eliminate bottlenecks and barriers in synthetic pathways

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and flow fluxes through the desired routes (Koglin et al. 2010; Deepika et al. 2017;da Silva et al. 2021). Due to the prominent role of microbial strain in generating particular compounds, rational design strategies to obtain strains that withstand the extreme environmental conditions enhance metabolic and substrate usage spectrum, eliminating competitive by-product pathways, and constructing streamlined base strain are some engineering projects to generate arrays of modified strains (da Silva et al. 2021; Koglin et al. 2010).

4.3

Optimization of Medium and Environmental Factors

Medium engineering and environmental optimization can provide higher yields, reduced overall production costs, and more diverse biosurfactant structures with favorable characteristics. It should be noticed that developing a culture medium for lab scale might not be good enough either for large scale or for other fungal strains. So, it is challenging to introduce a universal guideline for the medium optimization of fungal biosurfactants. Therefore, scale-up process, each fungal strain, and each class of biosurfactant production need to be optimized in a stepwise manner and a case-by-case study (Amaral et al. 2010; Sarubbo et al. 2022). Some general features should be considered in each medium and environmental optimization procedure. These factors are summarized as below.

4.4

Medium Optimization

Nutritional parameters include a carbon source, nitrogen source, carbon to nitrogen ratio (C:N), and minerals. These essential factors impact fungal biosurfactant’s physiological and economical characteristics, function and structure. While these critical factors directly influence overall production yields, the final production cost could be minimized by using the appropriate source, optimized concentration, and proportion of factors in the media (Nurfarahin et al. 2018). The carbon sources influence the growth of the fungal strain as well as the structure and yield of the target biosurfactant by applying different metabolic pathways. The main carbon sources are carbohydrates (glucose, sucrose, and glycerol), lipid-containing substrates or by-products (refined vegetable oils, wastewater from the oil industry), hydrocarbons, and agroindustrial wastes. By using waste as feedstock, we can cut the price in addition to overcome waste disposal difficulties (Olasanmi and Thring 2018;da Silva et al. 2021;Sarubbo et al. 2022). Pele et al. (2019) studied the production of biosurfactant on corn-steep liquor and crude glycerol as carbon sources by Rhizopus arrhizus UCP 1607. Optimum concentrations of crude glycerol and corn-steep liquors were 3% and 5%, respectively. Biosurfactant produced by this fungus has anionic glycoprotein structure moiety,

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and the yield of its production was 1.74 g/L. The water surface tension of this biosurfactant decreased from 72 to 28.8 mN/m, and the result of hydrophobic substrate dispersion in water was 53.4 cm2 oil displacement area. The emulsifying capacity of this biosurfactant was reported at 58.3%, 50%, and 79.4% for kerosene, motor oil, and burnt motor oil, respectively (Pele et al. 2019). Casas and GarciaOchoa (1999) reported the maximum yield of sophorolipids (120 g/L) by Torulopsis bombicola when glucose and soybean oil were used as a carbon source at the concentration of 80 and 40 g/L, respectively. In another study, Bednarski et al. (2004) showed that using soap stock as a supplemented carbon source in 5% v/v leads to 13.4 g/L sophorolipids production by Candida antarctica. Feeding of specific precursors may alter biosurfactant structure in addition to influencing the yield. Carbon sources influence both hydrophilic and hydrophobic moieties of biosurfactants. Hydrophilic substrates like carbohydrates regulate the cell growth and synthesis of the hydrophilic moiety, but hydrophobic substrates will control the hydrophobic portion of the biosurfactant. Therefore, optimal growth and maximized biosurfactant production must be considered in each procedure (Deepika et al. 2017; Luft et al. 2020). In a study, the ability of Candida glabrata UCP1002 in the production of biosurfactant and n-hexadecane emulsification was studied in a medium containing glucose, yeast extract, and cotton seed oil. The maximum emulsifying capacity was observed in 5%, 0.3%, and 7.5% glucose, yeast extract, and cotton seed oil, respectively. Also, the surface tension of this biomaterial was reported at 31 mN/m (Luna et al. 2009). The nitrogen sources with inorganic and organic origins contributed to biosurfactant production. Ammonium nitrates and sulfates are examples of the former, and yeast extract, urea, peptone and corn-steep liquor are the examples of the latter. Medium composition, producing microorganisms, and biosurfactant structure will determine a suitable nitrogen source (da Silva et al. 2021; Sarubbo et al. 2022). The proportion of carbon to nitrogen is a fundamental factor that induces the production of biosurfactants. The high C:N ratio and limited nitrogen source are required to redirect microbial metabolism to biosurfactant production. In contrast, extra nitrogen directs the cell metabolism towards fungal growth and biomass production. Different nitrogen sources were used to production of biosurfactants by fungi. Yeast extract, urea, and ammonium nitrate are introduced as suitable nitrogen sources for producing mannosylerythritol lipid and sophorolipids (Casas and Garcia-Ochoa 1999; Rufino et al. 2007; Sarubbo et al. 2007). Consequently, depletion of the nitrogen source followed by excess carbon and nitrogen sources are needed to alter the growth phase to the biosurfactant production phase (Nurfarahin et al. 2018; da Silva et al. 2021). The presence and proportion of minerals like phosphorus, magnesium, sulfur, iron, potassium, and some trace elements influence the medium formulation and biosurfactant production (Nurfarahin et al. 2018; da Silva et al. 2021).

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Environmental Factor Optimization

Cultural condition plays a crucial role in the overall output of the biosurfactant because changes may influence fungal performance. These conditions are dependent on fungal species and other medium components. Advanced techniques and tools are necessary for process monitoring and control (Amaral et al. 2010). Changes in pH and H+ ions can affect enzyme activity and cell permeability. An optimum pH range should be regulated for the generation of biosurfactant compounds. Wide pH ranges were reported for maximum yields of biosurfactants production, but in most studies, the pH range is from 5 to 7.8 (Casas and GarciaOchoa 1999; Felse et al. 2007; Cavalero and Cooper 2003). Also, temperature variation may interfere with microbial function and should be constant during the process (Sarubbo et al. 2022). The best temperature for biosurfactant production by various fungi is 30°C (Cavalero and Cooper 2003; Konishi et al. 2008). The volume of inoculum is a significant factor, especially in large-scale production, due to reaching higher yields in less time. An appropriate inoculum will result in no depletion of required nutrients throughout the process, reducing fungal activity and biosurfactant production rate. Incubation time is a strain-specific characteristic and depends on the growth phase of biosurfactant production. The maximum productivity in the exponential or stationary phase will determine different time intervals ranging from hours to several days (Bhardwaj et al. 2013; Shakeri et al. 2021). Various fungi produce biosurfactants at different incubation times. For example, the favorable incubation time by Aspergillus ustus was reported at 5 days, while this time for Candida bombicola was observed at 7, 8, and 11 days after incubation (Casas and Garcia-Ochoa 1999; Cavalero and Cooper 2003; Felse et al. 2007). Aeration or agitation speeds are critical factors for mass and oxygen distribution in the medium and provide the desired oxygen level for biosurfactant production. Therefore, higher aeration will provide higher yields in addition to more surfactant properties. Rotational speed in lab-scale production facilitates aeration, although, on a large scale, it is a vital factor that should be considered (Amaral et al. 2010; Deepika et al. 2017). In a study conducted by Ferreira et al. (2020), the effects of culturing on the shaker and static conditions are compared, and the results showed a difference in water surface tension in two static (40 mN/m) and aerobic (32 mN/m) conditions, while emulsification index for both was 96%. The first produced compound is a bio-emulsifier, and the other is a biosurfactant (Ferreira et al. 2020). Natural inducers like lipophilic compounds and triglycerides composition may induce biosurfactants production as they are applied in anabolic pathways to produce biosurfactants (Cavalero and Cooper 2003).

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Production Optimization via Statistical Procedures

Different fungal strains produce diverse biosurfactant structures during the cultivation process. Different medium and environmental requirements will result in novel biosurfactants, and the process must be optimized in each experimental case (Deepika et al. 2017). To address the cost parts of biosurfactant production, the first strategy is adopting waste substrate in medium engineering procedures. Low-priced raw materials, high product yields, appropriate quality and functionality of the produced biosurfactant, and minimum complexity in downstream processing are some constraints that should be employed in nutrient selection for medium formulation (Koglin et al. 2010; Olasanmi and Thring 2018). Due to the significant influence of maintaining fungal strain at the optimal condition, determining the most critical process parameters can lead to the maximization of biosurfactant production. Statistical analyses can be used to achieve this objective. In the first step, statistical techniques like the Plackett–Burman design are used to screen and select the most significant factors that affect fungal-based biosurfactant, as many cultural conditions and variables must be examined in biosurfactant-producing fungi and their novel biosurfactant structures. Using this factorial design will reduce the number of laboratory experiments. After selecting the main practical factors, optimization and evaluation of the interaction between selected variables are crucial in the second step. One of the most appropriate approaches could be Response Surface Methodology (RSM), which is a statistical model-based optimization approach. In this tool, by using reduced assays, a mathematical model will be developed, and the relationship and interaction between variables will be determined over a short period. After validation of models by lab experiments, this reliable model that formulated fermentation media can be applied in transferring lab scale optimization to a high yield and low-cost, large-scale production procedure (Campos-Takaki et al. 2010; Deepika et al. 2017; Bertrand et al. 2018). As shown in Table 3, different optimization designs and factors are considered to gain cost-effective medium formulation.

4.7

Large-Scale and Process Parameters Optimization

Lab scale optimization is the first step toward the large-scale production of each metabolite. There are some inconsistencies between results obtained between these two scales of production because oxygen, nutrient transfer and limitations are different based on the experimental scale. Few attempts have been made to produce fungal-based biosurfactants on a large scale. Both submerged and solid-state fermentation were used; each has some advantages over the other (Velioglu and Urek 2015; Costa et al. 2018). In the case of submerged fermentation, batch culture has been applied. As aeration is a limiting factor in biosurfactant production, a greater biosurfactant yield will be obtained where the oxygen and nutrient supply is sufficient. In

Groundnut oil refinery residue and corn-steep liquor

Waste soybean oil and cornsteep liquor

Hemicellulosic sugarcane bagasse hydrolysate

Sugarcane molasses, cornsteep liquor, waste frying oil

Crude glycerol, a biodiesel coproduct, and clarified cashew apple juice (CCAJ)

Molasses, residual frying

Candida sphaerica UCP 0995

Saccharomyces cerevisiae URM 6670

Scheffersomyces stipitis NRRL Y-7124

Candida tropicalis UCP0996

Yarrowia lipolytica

Candida tropicalis UCP0996

Sugarcane molasses, cornsteep liquor, waste frying oil concentrations, and inoculum size

Agitation speed, aeration rate, and culture time

Substrate composition, nitrogen supplements, extraction media, and pH

New isolate from soil contaminated grease waste

Grease waste and wheat bran/ waste cooking oil and wheat bran

Penicillium chrysogenum SNP5

Medium engineering

Aeration rate/ sucrose concentration

Strain improvement or screening

Aureobasidium pullulans LB 83

Microorganism

Renewable substrates

Central composite rotational design (CCRD) and response surface methodology (RSM)

Central composite rotatable design (CCRD)

One factor at a time

A central composite face-centered design (CCFD)

Statistical analysis

2 and 50 L bioreactors

4 L bioreactor

Solid-state fermentation

Stirred tank reactor

Fermentation scale

Different organic solvents for extraction

Filtration, centrifugation, and solvents extraction

Acid precipitation, solvent extraction, and evaporation

Solvent extraction, centrifugation, and saturated NaCl and MgSO4

Centrifugation, vacuum filtration, and solvent extraction

Filtration and centrifugation

Downstream processing

Glycolipid

4.11 g/L

7. 9 g/L in crude glycerol /6.9 g/L in CCAJ

4.19 g/L/ 5.87 g/L in 2 L and 7.36 g/L in 50 L

5.84 ± 0.17 g/ L

Glycolipid

Glycolipid

10.0 g/L

Biosurfactant (productivity or yield)

Glycolipid

Lipopeptide

Polyol lipid

Biosurfactant structure

Table 3 Survey of optimization of fungal biosurfactant production from the substrate to the application

68.0% on CCAJ and 70.2% on crude glycerol

70 ± 3.4%

90%

45% with oil / 23% with diesel

Emulsifying index

30.4 mN/m

18.0 mN/m on CCAJ and 22.0 mN/m on crude glycerol

29.98 mN/m

52 ± 2.9 mN/ m

26.64 ± 0.06 mN/ m

25.22 mN/m

Surface tension reduction

Almeida et al. (2021)

Fontes et al. (2012)

Almeida et al. (2017)

Franco Marcelino et al. (2017)

Ribeiro et al. (2020c)

Mendes da Silva et al. (2021, b)

Gautam et al. (2014)

Brumano et al. (2017)

References

(continued)

Motor oil adsorbed in marine stones

Oil dispersant

Larvicidal effect

Food formulations

Removing oil from soil

Antimicrobial activity and enhanced oil recovery

Application

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Animal fat and corn-steep liquor

Banana stalks powder

Candida lipolytica UCP0988

Aspergillus niger

Cassava wastewater

Sunflower acid oil

Pseudozyma tsukubaensis

Starmerella bombicola

Fusarium sp. BS-8

Sugarcane molasses, frying oil waste, and corn-steep liquor

oil, and cornsteep liquor

Starmerella bombicola (previously Candida bombicola)

Microorganism

Renewable substrates

Table 3 (continued)

pH, temperature, NaCl,

Inoculum size, pH, incubation time, temperature, sucrose, and yeast extract

New isolate from oil-contaminated soil

New isolate from soil

Temperature, pH, substrate concentration, and incubation time

pH, temperature, salt concentrations

Medium engineering

Mutation with EtBr

Strain improvement or screening

RSM under CCD

One factor at a time

Statistical analysis

Submerged fermentation (batch/continuous fermenter

5-L bioreactor

Solid-state fermentation

1.2-, 3.0-, and 50-L bioreactors

Fermentation scale

Adsorption chromatography

Foam fractionation and ultrafiltration

Optimization of solvent for extraction

Acid precipitation and centrifugation

Cold acetone precipitation, centrifugation, and evaporation

Acid precipitation and solvent extraction

Downstream processing

Sophorolipid

Mannosylerythritol lipids

Lipopeptide

Glycolipid

Biosurfactant structure

5.18 g/L

41.6 g/L at shake flask and 51.5 g/L at the fermenter

0.02 g/L/h/ 1.26 g/L

5.25 g/L

2.3 g/L in native strain/ 3.3 g/L in mutated strain

12.5 g/L in the flask-scale/ 19.5 g/L in the 1.2-L reactor/ 61 g/L in the 3-L reactor/ 221.9 g/L in the 50-L reactor

Biosurfactant (productivity or yield)

70%

57%/62.3%

47.0 ± 0.9% in the flask-scale/ 32.0 ± 0.9% in the 1.2-L reactor/10.0 ± 1.1% in the 3-L reactor/ 58.0 ± 1.1% in the 50-L reactor

Emulsifying index

31.7 mN/m

35.5 mN/m

26 mN/m

32 mN/m

29 mN/m in the flaskscale, 33 mN/m in the 1.2-L reactor, 31 mN/m in the 3-L reactor, and 30 mN/m in the 50-L reactor

Surface tension reduction

Remove spilled oil,

Microbial enhanced oil recovery (MEOR)

Removal of petroleum products and heavy metals

Additives for food products

Application

Jadhav et al. (2019)

De Andrade et al. (2017)

Qazi et al. (2014); Qazi et al. (2013)

Asgher et al. (2020)

Santos et al. (2017a)

Pinto et al. (2022)

References

108 H. Nouri et al.

Diesel oil

Rice-bran

Trichosporon asahii

Fusarium proliferatum

Ceriporia lacerata CHZJU

Canola waste frying oil

Two-phase olive mill waste

Corn-steep liquor and soybean oil waste

Candida utilis UFPEDA1009

Cyberlindnera saturnus SBPN27

Trametesversi color

Aspergillus flavus AF612

Cunninghamella echinulata UCP1297

New isolate from soil

New isolate from the rice-bran oil industry

New isolate from petroleum hydrocarbon contaminated soil

New marine yeast isolate

Temperatures, diesel concentrations and pH

Two-phase olive mill waste, wheat bran, and olive stones

corn-steep liquor, and soybean oil waste

Plackett– Burman design and response surface methodologycentral composite design (CCD)

D-optimal mixture design

Factorial design CCRD 22

3-L laboratory fermentor

Solid-state fermentation

Solvent extraction

Ultrasonication and column chromatography

Silica gel column chromatography followed by dialysis

Reverse phase (RP)-C18 silica gel (230–400 13 mesh) column chromatography

Acid precipitation or Solvent extraction

Mannosylerythritol lipids

Enamide

129.64 ± 5.67 g/L in optimized condition/ 81.10 g/L in nonoptimized condition

24.22 ± 0.23 g/L

Fatty acid

Sophorolipid

2.13 g/L

373.6 ± 19.4 mg/ 100 g by acid precipitation or 125.5 ± 10.0 mg/ 100 g by solvent extraction

1.6 g/g

Glycolipid

Lipoprotein

Glycolipid

78.5% on hydrocarbons, 76.1% on soybean oil, 77.4% on vacuum pump oil

Coconut oil = refined oil (95%), kerosene (90%), n-dodecane (43%)

89 ± 0.7%

81%

31.11 mN/m

36.6 mN/m

30 ± 0.6 mN/ m

28 mN/m

34.5 ± 0.3 mN/m

20 mN/m

Hydrocarbon degradation

Food applications/ antioxidant activity

Antimicrobial activity

Antifungal activity

such as diesel and kerosene, from marine sand

Niu et al. (2017)

Bhardwaj et al. (2015)

Chandran and Das (2010)

Ribeiro et al. (2020a)

Balan et al. (2019)

Lourenço et al. (2018)

Ishaq et al. (2015)

De Souza et al. (2018)

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Erlenmeyer flasks, as compared to fermenters, agitation, temperature, and contamination control are significant factors. Since batch culture is a closed system, better control and fewer contamination problems have occurred during the bioprocess. In addition, due to the remarkable homogeneity of the culture medium by agitation, oxygen and nutrients are maintained at the optimal value. In contrast, the intensity of aeration and agitation should have remained in an improved range because over-mixing promotes excessive foaming, leading to difficulties in process control. Foam formation trapped biomasses and caused washing out of the cells from media. Moreover, variation in the supply of oxygen and cell growth decreases the final yield of the process (Amaral et al. 2010; Almeida et al. 2017; Brumano et al. 2017). Solid-state fermentation is another large-scale process for biosurfactant production. As fungi prefer to grow on solid substrates like agroindustrial wastes, the application of these wastes as feedstock has an economic effect and ecological importance. Using these substrates will solve these challenges as much agroindustrial waste is produced and accumulated in nature. There are some advantages to this system over submerged production. Low-cost feedstock, ecological benefits, nonfoaming conditions, and low energy and water demand are the most apparent benefits of this fermentation process. As the complex substrates are used for cultivation in this process, efficient extraction techniques for eliminating impurities in the recovery process are essential. In addition, temperature, moisture, mass transfer, and control are other limiting factors that should be considered. Finally, designing a suitable fermentation system for solid-state cultivation scale needs special attention because monitoring and controlling the parameters is a significant challenge (Velioglu and Urek 2015; Costa et al. 2018). Currently, sophorolipids are one of the most promising fungal biosurfactants, and many efforts have been made for their commercial production on a large scale. Li et al. (2020) tried to produce sophorolipids in a semicontinuous fermentation system. The process lasted 300 h, and the maximum biosurfactant titer was 477 g/L (Li et al. 2020). Wang et al. (2020) reported the high volumetric productivity (2.43 g/L/h) of sophorolipids produced when a simple in situ separation method coupled with the fermenter using food waste (Wang et al. 2020). Consequently, it is clear that, besides optimizing the culture condition on a large scale, special attention should be directed towards the operation and overcoming the limitations in the biosurfactant production by fungi. A summary of fungal biosurfactants production optimization is presented in Table 3.

4.8

Biosurfactant Extraction and Recovery

Biosurfactant extraction is one of the main cost-related steps in its production process. It should be noted that integration to the market and competition in substitution with synthetic surfactant depends on the cost of refining steps. As the recovery and purification represent 60% to 80% of the total cost, the wanted degree

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of purity based on biosurfactant industrial application should be considered (Costa et al. 2018; Sarubbo et al. 2022; Silva et al. 2018). Based on structural diversity as well as the desired rate of purification, different protocols are applied. However, in general, intra or extracellular location of the biosurfactant, water or organic solvent solubility, ionic charge, and the physicochemical properties of the biomolecules influence and present more challenges in recovery procedures (Costa et al. 2018). As inexpensive techniques and procedures are the major priority for feasible industrial application, we focused on the recovery and purification methods of fungal biosurfactants in the following section (Table 3). According to the location of the biosurfactant, intracellular or cell-bound products need to be released at the beginning step. Cell lysis methods, in addition to sonication, are used for this purpose. In the case of extracellular biosurfactants, separation of mycelium from the fermentation broth is essential. Filtration is the first step followed by centrifugation for more efficient separation. These cell-free extracts will provide a suitable broth for the next extraction course. The selection of extraction and purification methods depends on the balance between cost, application, efficiency and safety (Bertrand et al. 2018; Luft et al. 2020). Precipitation is the most reported technique. It is done by using hydrochloric acid or ammonium sulfate. In the acidification method, the pH of cell-free broth was reduced to 2 to 3 for pellet formation. Ammonium sulfate protocol is the same with subsequent purification by dialysis. In solvent extraction methodology, based on the hydrophobicity of the biosurfactants, different solvents like methanol, chloroform, dichloromethane, ethyl acetate, ethanol, butanol, etc. can be used. This method favors the reuse of solvents besides a high yield of product. While environmental hazards and costly procedures are the drawbacks (Silva et al. 2018; Luft et al. 2020). Integrated production and recovery of biosurfactants are facilitated by gravity separation, foam fractionation, and membrane separation. Recycling media and microbial cells are the advantage of these methods. Due to foam formation in fed-batch or submerged fermentation of fungal biosurfactants, extraction of adsorbed biosurfactants in foams not only solve the mass and heat transfer but also avoid the accumulation of biosurfactants and reduce the risk of contamination (Luft et al. 2020; da Silva et al. 2021). Sequential strategies are developed based on the final products required purity. Adsorption-desorption on polystyrene resins or activated carbon, ion exchange chromatography, Hydrophobic interaction chromatography (HIC), high-pressure liquid chromatography (HPLC), gel filtration, and ultrafiltration are more sophisticated methods for the separation of biosurfactants from the culture medium (Luft et al. 2020; Sarubbo et al. 2022). Crystallization and lyophilization are helpful techniques for preserving the purified biosurfactants long-term. These traditional strategies should be optimized for each biosurfactant from lab to the industrial scale. Consequently, in addition to medium optimization, bioprocess optimization should be implemented to explore the best extraction factors affecting the final downstream process and extraction yield (da Silva et al. 2021; Olasanmi and Thring 2018).

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Biosurfactant Characterization

Purification is a vital step not only for biosurfactant application but also for its characterization. Unwanted contaminants may cause perturbation in the characterization of the biosurfactant. Due to less information about fungal biosurfactants, a group of techniques is required. Thin layer chromatography (TLC), highperformance thin-layer chromatography (HPTLC), HPLC, and Fourier-transform infrared spectroscopy (FTIR) are initial and routine methods that elucidate purity grade and functional groups of biosurfactant (Bhardwaj et al. 2013; Sanches et al. 2021; Sarubbo et al. 2022). For more accurate and detailed identification, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), protonor carbon nuclear magnetic resonance (1H-NMR or 13C-NMR), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LCESI-MS/MS) are applied. These high throughput techniques provide highresolution identification of different structures in a low-time course (Bhardwaj et al. 2013; Sanches et al. 2021; Sarubbo et al. 2022).

5 Biosurfactant Application Biosurfactants were used in various industries, such as foods, agriculture, pharmaceutics and medicine, oil, and bioremediation (Table 4). The main market of biosurfactants is in the recovery of petroleum and remediation of oil-polluted regions (Rodrigues et al. 2006). Biosurfactants have been applied for petroleum and hydrocarbon bioremediation by reducing the interfacial tension between oil/water and oil/rock and emulsion formation at the oil-water interface, which allows oil removal (Pacwa-Płociniczak et al. 2011). Among the other essential applications of biosurfactants, we can mention their use in medicine. Some biosurfactants are considered as antimicrobial compounds and lead to the death of pathogenic microbes through the destruction of the cell wall (Saikia et al. 2021). Cybersan, a glycolipid biosurfactant produced by Cyberlindnera saturnus SBPN-27, showed bactericidal activity against pathogens, with no toxicity on 3T3 fibroblast cells (Balan et al. 2019). Sophorolipids biosynthesized by Starmerella bombicola can effectively inhibit Pseudomonas aeruginosa and Enterococcus faecalis (Saikia et al. 2021). Some biosurfactant has antifungal activity. For example, a Sophorolipid produced by Rhodotorula babjevae has a great inhibitory activity on a broad range of fungi, including Fusarium verticillioides, Corynespora cassiicola, Colletotrichum gloeosporioides, Trichophyton rubrum, and Fusarium oxysporum (Sen et al. 2017). Ustilagic acid produced by U. maydis was used to inhibit a phytopathogenic fungus, Botrytis cinerea (Teichmann et al. 2007). The antifungal activity also was seen in cellobiolipids produced by different species,

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Table 4 Some applications of fungal biosurfactants Industry Food

Microorganism S. cerevisiae URM 6670

Application A replacement for egg yolk

Candida bombicola

Emulsifiers in mayonnaiselike sauces Bioemulsifier in mayonnaise Salad dressings

Candida utilis Candida utilis Environment bioremediation

Glycolipid

– Carbohydrate– protein–lipid Glycolipid

Candida sphaerica UCP 0995

Oil removal

Cunninghamella echinulata

Enhanced oil spill recovery (diesel and kerosene) Degradation of motor oil and oil spills Cassava wastewater Degradation of motor oil and cleaning oil spills Olive oil mill wastewater Dispersant in oil spills Skin-moisturizing damaged by UVA irradiation Emulsification and wetting property Antimicrobial action of toothpaste

–

Inhibition of biofilm formation toothpaste

Candida tropicalis UCP0996 Pseudozyma tsukubaensis Candida lipolytica UCP 0988

Cosmetics

Biosurfactant structure Glycolipid

Aureobasidium thailandense LB01 Candida bombicola URM 3718 Pseudozyma sp.

Starmerella bombicola Starmerella bombicola URM 3718 (previously Candida bombicola) Starmerella bombicola URM 3718 (previously Candida bombicola)

Reference Ribeiro et al. (2020c) Pinto et al. (2022) Campos et al. (2015) Campos et al. (2019) Mendes da Silva Santos et al. (2021) De Souza et al. (2018)

Glycolipid

Almeida et al. (2021)

Mannosylerythritol lipid Glycolipid

De Andrade et al. (2017) Santos et al. (2017b)

Lauric acid ester

Meneses et al. 2017) Freitas et al. (2016) Bae et al. (2019)

Sophorolipid Mannosylerythritol lipid (glycolipid)

Sophorolipid

Jadhav et al. (2019)

–

Resende et al. (2019)

–

Resende et al. (2019)

(continued)

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Table 4 (continued) Industry Petroleum

Microorganism Fusarium sp. BS-8 Penicillium chrysogenum SNP5 Trichosporon asahii

Microbially enhanced oil recovery (MEOR) Mining

Medicine

Starmerella bombicola (previously Candida bombicola) Starmerella bombicola (previously Candida bombicola) Candida tropicalis UCP 0996

Application Enhanced oil recovery Enhanced recovery of oil Emulsification of diesel oil Biodegradation of the oil spillages Removal of nickel from contaminated lipid wastes As (91%)

Biosurfactant structure Lipopeptide Lipopeptide Sophorolipid

Glycolipoprotein

Reference Qazi et al. (2014) Gautam et al. (2014) Chandran and Das (2010) Kiran et al. (2009)

Sophorolipid

Felse et al. (2007)

Sophorolipid (SL18)

Arab and Mulligan (2018)

Zn and Cu

Lipopeptide

Starmerella bombicola CGMCC 1576 Candida tropicalis

Cd (83.6%) Pb (44.8%)

Sophorolipid

da Rocha Junior et al. (2019) Qi et al. (2018)

Removal of Zn, Cu and Pb

Glycolipid

Fusarium proliferatum Penicillium chrysogenum SNP5 Cyberlindnera saturnus SBPN-27 Scheffersomyces stipitis NRRL Y-7124

Antioxidant activity Antimicrobial activity Antimicrobial activity Larvicidal

Enamide

Wickerhamiella domercqiae Ustilago maydis FBD12

Anticancer activity Antimicrobial activity

Glycolipid

Ustilago maydis FBD12

Antioxidant capacity

Glycolipid

Aspergillus ustus MSF3

Antimicrobial activity

Glycolipoprotein

Lipopeptide Cybersan Glycolipid

Glycolipid

da Rocha Junior et al. (2019) Bhardwaj et al. (2015) Gautam et al. (2014) Balan et al. (2019) Franco Marcelino et al. (2017) Chen et al. (2006) CortesSanchez et al. (2011) CortesSanchez et al. (2011) Kiran et al. (2009) (continued)

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Table 4 (continued) Industry

Microorganism Ustilago maydis

Cleaning

Fusarium oxysporum

Application Antifungal activity Emulsifiers

Biosurfactant structure Glycolipid Fatty acid

Reference Teichmann et al. (2007) Santhappan and Pandian (2017)

such as Pseudozyma fusiformata, Ustilago zeae, Sympodiomycopsis paphiopedili, and U. maydis (Desai and Banat 1997; Golubev et al. 2001; Golubev et al. 2004). In addition to the antimicrobial effect, biosurfactants can also have an antiviral effect by interacting with the virus lipid membrane and the diacetate-ethyl ester form of the sophorolipid biosurfactant could be used for inhibiting human immunodeficiency virus 1 (HIV-1) (Shah et al. 2005). Another application of biosurfactants in medicine is using them as anticancer agents. Wickerhamiella domercqiae produced a sophorolipid with anticancer activity (Chen et al. 2006). Biosurfactants are also applied as antiadhesive agents to hinder pathogen attachment to the surfaces and lead to biofilm formation inhibition. Mannoprotein produced by Saccharomyces cerevisiae associated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide can significantly inhibit Staphylococcus aureus and S. epidermidis biofilm formation. Also, Spiculisporic acid of Penicillium spiculisporum can be used in drug delivery (Walencka et al. 2007). Biosurfactants can be used for metal desorption of heavy metal polluted regions by interacting with metal ions and incorporating them in the micelle or absorption in the interface layer. Members of the genus Candida can effectively be used for this purpose (Santos et al. 2016). Another use of biosurfactant is its application in food industries which Candida utilis produces a bio-emulsifier used in processed salad dressings and mayonnaise (Campos et al. 2015). Saccharomyces cerevisiae produced a glycoprotein (manoprotein) which confers stability to water/oil emulsions in ice cream, mayonnaise, and cookies (Shepherd et al. 1995; Torabizadeh et al. 1996).

6 Biosurfactant: From Lab to Market The transition from lab-scale production of biological-oriented biosurfactants to industrial scale needs an optimization scenario. This scenario is focused on upstream processing, fermentation bioprocess and downstream processing. As fungi biosurfactants are less known on a large scale as compared to bacterial ones, more consideration is needed to emphasize all the phases to achieve our goal in marketing. As the high production cost is the major factor restraining the growth of the global biosurfactants market compared to chemical surfactants counterparts, many efforts

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are made to make biosurfactants production more economically feasible. For this purpose, initiation is from fungal species and metabolic production pathways toward process engineering, using cost-effective substrates or waste and raw materials, and optimization of culture and variables and completion is by high yield extraction process (Luft et al. 2020; Srivastava et al. 2021). The use of waste materials is strongly preferred because, in addition to being cheap and available, it also reduces environmental pollution. Glycerol, vegetable and animal fat, starchy effluents, molasses, corn-steep liquor, soap stock, cassava flour wastewater, oil distillery waste, and oily effluents are the waste material that could be applied in case of microbial surfactant production. Industrial waste with a high amount of carbohydrates or lipids is ideal for use as substrate (Santos et al. 2016). The cost of waste or the raw material is not the only determining factor, and the other factors, such as availability, stability, purity, variability, and size of the particles, texture, liquid, and solid forms in the case of raw materials, should be taken into consideration (Singh et al. 2019; Elsoud and Ahmed 2021). Choosing the best microbial strain with a high production yield and optimization of culture conditions to minimize by-products are the other critical elements for producing commercial surfactants. Moreover, genetic modification is another way to maximize the yield and productivity of biosurfactants by improving the enzymatic activity of microorganisms. In this regard, enzymatic specificity, mode of action, and catalytic properties are targeted for these genetic modifications (Srivastava et al. 2021). A workflow of upstream, fermentation, and downstream processing of fungal biosurfactant production is presented in Fig. 4. To analyze and forecast the biosurfactants market, different factors should be noticed, e.g., raw material suppliers, application of biosurfactants, and manufacturer region. Considering that the SARS-CoV-2 has spread all over the world; without surfactants, it would not be possible to provide essential standards of cleanliness and hygiene. So, concerning this pandemic, more opportunities are provided in biosurfactant production as a detergent (Vieira et al. 2021). Biosurfactant global market from 2017 to 2022 faces a considerable growth from 4.20 billion dollars to a predicted 5.52 billion dollars, and it is anticipated that it will reach about 7.9 billion dollars by 2026. Europe, Asia, and North America are leaders in the biosurfactant industry. Although the progress of Asia in the global biosurfactant market is significant, the largest market belongs to European countries. In Europe, consumer awareness of synthetic surfactant hazard, and in the Latin America, plenty of agricultural waste as feedstock provide a suitable platform to build commercial production of biosurfactants successful in the near future (Sanches et al. 2021). Biosurfactants as multifunctional biomolecules with various applications have shown remarkable growth in recent decades. However, the proportion between the needed financial investment and the yield of the final product limited the growth of this industry over the world from an economic point (Santos et al. 2016). Evonik (Germany) is a pioneer company in large-scale biosurfactant production in 2016 with a sophorolipid structure. Their product was to the sector of home care cleaning products and detergents. In an ongoing progress, this company and Unilever

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Fig. 4 A workflow of upstream, fermentation, and downstream processing of fungal biosurfactant production for the successful establishment of the cost-effective procedure from lab to market of biosurfactants

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introduced a rhamnolipid-containing detergent in 2019 that was well-accepted by the consumers. The growing interest of companies in biosurfactant production is observed in Stepan Company (USA), which is famous in chemical manufacture for producing Nat Sur Fact’s rhamnolipid. This biosurfactant is derived from a renewable source and vegetable oil, with a high yield in the lab and large scale. In 2014, TeeGene Biotech (United Kingdom) was developed to meet the demands of cosmetics, biopharmaceuticals, and waste management. Jenil Company (USA) offers a diverse array of natural rhamnolipids that provide the desired properties. In agriculture, these are used as a growth enhancer, in bioremediation for oil recovery and as sanitizers and cleaning solutions with their antimicrobial property (Vieira et al. 2021). Five companies named Ecover, Saraya, Urumqui Unite, MG Intobio, and BASF Cognis supply 90% of biosurfactants’ needs (Luft et al. 2020). In 2009, Ecovar (Belgium) made a breakthrough by introducing an eco-surfactant as a surface cleaning product. This biosurfactant is produced from sugar fermentation by unique yeast isolated from bumblebee hives. Saraya (Japan) focused on biosurfactant production because of environmental concerns. Their categories are health-related, environmental protection, and sanitation products. Urumqui Unite biotechnology company (China) was established in 2006 by producing rhamnolipid biosurfactants focused on oil pollution remediation and microbial-enhanced oil recovery. MG Intobio (South Korea) factory produces sophorolipids in soap formulation by using yeast as a producer microorganism. BASF Cognis (Germany) applied coconut oil as a substrate for producing cleaning agents, cosmetic products, and detergents. It should be noted that fungal-based biosurfactants are among the most famous companies in marketing. Consequently, biosurfactants of fungi origin will have more opportunities to contribute to industries in the near future. As mentioned above, the growing commercial interest in biosurfactant production as well as the environmental problems caused by the use of chemical surfactants lead to their price reaching more than 3 USD/kg, while the use of biosurfactants with a price of 3 to 10 USD/kg does not cause chemical surfactant difficulties (Dhanarajan and Sen 2014).

7 Conclusions The microbial biosurfactant production with sustainable and eco-compatible properties creates a considerable attention wave across the industries. Due to advantages of fungal surfactants over bacterial ones, many efforts are done to overcome the economic issues and low process productivity challenges. There is little information about fungal biosurfactants in the literature, and we tried to introduce fungal biosurfactants, their improvement procedures, and their applications in this chapter book. Due to the origin of a biosurfactant dictates its structure and application, securing high-yield fungi is a crucial prerequisite to solving industrial bottlenecks. Fungal biosurfactants grow in various physiological conditions and produce new or

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high amounts of biosurfactants with novel and specialized characteristics compared to bacteria. Due to their ability to produce a broad range of hydrolytic enzymes, fungi can use many hardly biodegradable materials as substrates and produce biotechnologically essential metabolites, such as biosurfactants. This process helps remove organic pollutants and leads to biosynthesis, a cost-effective biotechnologically valuable compounds. In addition, using metabolic engineering, the ability of microorganisms in biosurfactant production has been improved and new surfactant or high yield of products will be achieved. Considering various applications of fungal biosurfactants and the great potential of these microorganisms, a systematic study is required to improve knowledge about biosynthesis, optimization, and purification technologies. Without a doubt, future research should be devoted to scale-up as well as industrial low-cost, high-yield biosurfactants.

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Production of Biosurfactant by Bacteria from Extreme Environments: Biotechnological Potential and Applications Mariana Amaral Azevedo, Letícia Portugal do Nascimento, Maria dos Remédios Vieira-Neta, and Iolanda Cristina Silveira Duarte

1 Introduction Biosurfactants are extracellular secondary metabolites synthesized by fungi, bacteria, yeasts, and algae (Arifiyanto et al. 2020), with diverse applicability due to unique characteristics including promoting superficial tension reduction, emulsification, solubilization, foam formation, and wettability (Bujak et al. 2015). Despite the difficulties to establish the exact physiological roles and biochemical aspects of all kinds of biosurfactants (Markande et al. 2021), it is well known that their properties are a function of important media parameters, such as pH, temperature, salt concentration, presence of contaminants, and the ratio between carbon and nitrogen sources (Barakat et al. 2017). The use of biosurfactants is part of a wider scenario concerning the growing environmental awareness, the advent of more sustainable practices, and the industrial search for bioproducts to minimize hazardous aspects associated with synthetic alternatives (Salwan and Sharma 2020). Chemical surfactants are considered potential contaminants because they may accumulate in natural systems throughout time, usually transported to water bodies or leached to the soil. In high concentrations, these substances imply risks to the local microbiota, plants, vertebrates, and invertebrates (Ivanković and Hrenović 2010), since they may affect primary productivity in water bodies, increasing the permeability of cell membranes and the concentration of soluble and insoluble pollutants (Yuan et al. 2014). Furthermore, when applied to cosmetics and detergents, chemical surfactants may cause unwanted side effects concerning changes over skin microbiome, irritations, and allergic reactions (Bujak et al. 2015). Compared to chemical surfactants, biosurfactants present significant advantages, such as

M. A. Azevedo (✉) · L. P. do Nascimento · M. dos Remédios Vieira-Neta · I. C. S. Duarte Department of Biology, Laboratory of Applied Microbiology, Federal University of São Carlos (UFSCar), Sorocaba, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_6

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selectivity, biodegradability, biocompatibility, bioavailability, and ecological acceptability (Ambaye et al. 2021). In this context, the study of microorganisms from extreme environments has been a promising field in searching for novel biosurfactants with widespread applicability. Given their capacity to adapt and specialize, extremophiles are microorganisms with great biotechnological potential (Barakat et al. 2017; Giovanella et al. 2020), since they synthesize biosurfactants with unique properties and excellent stability in harsh conditions (Ibrahim 2018). This chapter provides an overview of the advances regarding microorganisms isolated from extreme environments and their biosurfactant production, focusing on biosurfactant applications and future challenges.

2 Biosurfactants: From the Definition to the Assessment Biosurfactants are surface-active molecules synthesized by different living organisms, such as fungi, yeasts, and bacteria, implying a wide range of molecular lengths, structures, conformations, and compositions (Manga et al. 2021). Despite the origin, all biosurfactants have at least one hydrophobic and one hydrophilic group, allowing its solubilization on both polar and nonpolar solvents. For this characteristic, molecules of biosurfactants are also said to be amphiphilic. When obtained from bacteria, biosurfactants are normally produced during the stationary phase of growth (Shah et al. 2016) and can be found attached to the cell membrane or secreted in the culture media (Vieira et al. 2021). Unlike synthetic surfactants, usually categorized due to their polarity, biosurfactants may be classified in terms of microbial production, chemical composition, molecular structure, and molecular weight (Vijayakumar and Saravanan 2015; Sarubbo et al. 2022). Low molecular weight biosurfactants, like glycolipids, lipopeptides, and phospholipids are more efficient in reducing water–oil interfacial tension and air–water surface tension, while high molecular biosurfactants, like lipoproteins and lipopolysaccharides, are mostly used to stabilize emulsions but do not lower the surface tension as much (Drakontis and Amin 2020; Manga et al. 2021; Sarubbo et al. 2022). In natural systems, microbial surfactant production is a sign of adaptation since it is associated with significant physiological roles such as the motility of microorganisms, biofilm formation, quorum sensing, cellular differentiation, and protection against toxic elements or other microorganisms (Van Hamme et al. 2006). Also, these compounds favor the nutrient-obtaining processes by increasing their availability within the substrates (Shah et al. 2016). Therefore, biosurfactants are essential to enable microbial growth and survivability in environments with harsh conditions. However, the amount of biosurfactant produced and its chemical properties depend on several factors. Besides the type of microorganism used, temperature, pH, fermentation time, the ratio between carbon and nitrogen sources, the presence of

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trace elements and oxygen levels are parameters that affect the synthesis of biosurfactants (Gurkok and Ozdal 2021). In addition to showing low toxicity, high biodegradability, and high ecological compatibility (Banat et al. 2010; Schultz and Rosado 2020), some properties of biosurfactants can be pointed out when highlighting the advantages of their biotechnological use in industrial, medical and environmental processes, like surface tension lowering, wettability, emulsification, dispersion and aggregation of solids and foaming (Drakontis and Amin 2020; Sarubbo et al. 2022). On the laboratory scale, high-performance liquid chromatography (HPLC), thinlayer chromatography (TLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and matrix-assisted laser desorption/ionization-time of flight mass spectroscopy (MALDI-TOF) are techniques frequently applied to characterize and identify biosurfactants (Ambaye et al. 2021), while hemolytic activity, drop collapsing test, oil spreading test, surface tension reduction, and emulsification index test are some methods commonly used to screen the efficiency of a biosurfactant (Shah et al. 2016).

3 Extremophile Microorganisms and Biosurfactant Synthesis With represents in all three domains of life, extremophiles are microorganisms that, through adjustments in their cellular machinery, are able to survive in environments in which physical–chemical properties are limiting to the majority of the living organisms (Basak et al. 2020). These limiting factors may be related to a single aspect or a combination of them, such as excess alkalinity or acidity, exposure to radiation, extreme temperatures, high salt concentrations, and the presence of toxic elements like potentially toxic metals (Salwan and Sharma 2020). Thus, these microorganisms may be found in natural environments, including hot and cold deserts, salty lakes, hot springs, glaciers, deep-sea and volcanic areas (Raddadi et al. 2015; Abbamondi et al. 2019; Trejos-Delgado et al. 2020; Moura et al. 2021; Zgonik et al. 2021), contaminated environments due to oil spills (Ibrahim 2018), or other sources of pollution. Extremophiles can be classified as extremophilic when extreme conditions are necessary to enable their growth, or extreme tolerant when they are able to tolerate adverse environmental conditions, but their optimal growth occurs in milder conditions (Salwan and Sharma 2020). Another type of classification is given in terms of their primary stressor (Basak et al. 2020) so that, according to the conditions for which they are adapted to survive or tolerate, they can be categorized into different groups including thermophiles, psychrophiles, halophiles, acidophiles, alkaliphiles, piezophiles, and radiophiles (Fig. 1).

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Fig. 1 Categories of extremophile microorganisms and the different extreme environments which they may inhabit

Natural environments distinguished by high temperatures like hot springs, volcanic sites, tectonically active faults, and deep-sea hydrothermal vents are inhabited by thermophiles and hyperthermophiles (Panda et al. 2019). Consequently, they are promising sites for the discovery of novel biosurfactants with significant characteristics. However, approximately 85% of the biosphere has a temperature of 5 °C or less. Most of these environments have a microbiota of extremophiles, called psychrophiles, which grow in a range between 0 and 20 °C (Hamdan 2018). The diversity of psychrophilic microorganisms is widespread in several matrices in both aquatic and terrestrial environments, such as snow, cryoconite holes, glaciers, lakes, deep sea, and cold soils (Perfumo et al. 2018). Saline and hypersaline environments comprise of a wide range of natural and artificial ambient, such as soils, subsurface salt deposits, continental waters, salt lakes, salt deserts, and solar salterns. Containing a varying concentration of NaCl, usually between 4% and 30%, natural saline environments may be harmful to microorganisms that are not adapted to such conditions (Zahran 1997) since high amount of salt negatively affect the stability of enzymes and proteins (Paul and Mormile 2017). Halomonas sp. and Bacillus sp. are species of halophilic bacteria commonly screened in studies concerning the assessment of extremophiles from saline environments (Keerthi et al. 2018; Joulak et al. 2019; Liu et al. 2019). Acidophiles are organisms that can grow in extremely low pH, such as hot springs and anthropic environments including mine drainage systems and sulfur mining

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areas (Johnson 1995; Ivanova et al. 2016; Arulazhagan et al. 2017). Usually, acidophiles are found with thermophilic microorganisms, since the environments with high temperatures have low pH (Elleuche et al. 2014). In the opposite way, alkaliphiles organisms are capable of growing in high pH environments, for example, in soda lakes that are usually extremely alkaline and saline and in contaminated soil or water areas (Tindall et al. 1984; Elazzazy et al. 2015; Dukhande and Ward 2016). Microorganisms that may be able to strengthen extreme barometric can be called piezophiles. At sea level, the pressure can reach 0.1 MPa, while in the depths of the ocean it approaches 38 MPa (Zhang et al. 2015). Gabani and Singh (2013) classified organisms that are capable of tolerating or growing in conditions with high levels of radiation, including ultraviolet, gamma, X-ray, and radio waves as radioresistant or radiophiles, respectively. In drilled oil areas and storage tanks, microorganisms presently use hydrophobic carbon sources and metabolites synthesized by other organisms for growth (Parthipan et al. 2018). Reservoirs can be considered extreme environments due to their high toxicity, hydrophobicity, and low water activity (Cai et al. 2015), and according to Hewelke et al. (2018), oil contamination in the soil increases water repellency causing the same characteristics mentioned above. Furthermore, microorganisms capable of growing in environments with low water activity are considered xerophilic or xerotolerant (Grant 2004). The primary and secondary metabolites produced by extremophile bacteria make them fundamental in the biotechnological development process. Of the intracellular or extracellular origin and produced by different pathways, they are characterized by their low molecular weight, less than 1 kDa, and the critical role they play in the metabolism of microorganisms (Pinu et al. 2017). When said to be primary, metabolites are synthesized uninterruptedly during the growth of the microorganism as they are essential to ensure cellular survival and processes, such as growth and reproduction (Pande and Kost 2017). When secondary, they are not necessary for the growth of the organism but provide adaptive advantages. As they are nonessential, they are produced in the stationary phase when substrates are depleted or when subjected to some stress condition. Among the secondary metabolites are antibiotics, pigments, toxins, enzyme inhibitors, and biosurfactants (Fouillaud and Dufossé 2022). Thus, extremozymes, compounds extremely stable to temperature and pH variations, and carotenoids (hydrophobic pigments precursors of vitamin A), growth regulators and antioxidants, are the examples of metabolites produced by extremophile microorganisms allowing their application in different processes, such as in industrial food, cosmetics, pharmaceuticals, and textiles (Kochhar et al. 2022). Furthermore, they can be used in the production of biofuels (Barnard et al. 2010) and in bioremediation processes (Ibrahim 2018). In this scenario, biosurfactants are characterized as one of the possible applications of secondary metabolites of extremophiles. Concerning bacterial production, Actinobacteria, Alcanivorax, Arthrobacter, Bacillus, Halomonas, Mycobacterium, Pseudoalteromonas,

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Pseudomonas, Rhodococcus, and Sphingomonas are the main genera known for the capacity to produce biosurfactants (Kashif et al. 2022). In the last few decades, several studies have reported the potential of extremophile bacteria species in producing biosurfactants (Table 1). Unlike mesophile submitted microorganism’s mechanisms which were not evolutionarily selected to survive in harsh environments, extremophile counterparts developed to overcome the barriers imposed by the environment, and consequently, they perform better when under those conditions and their biosurfactants show higher stability (Donio et al. 2013b; Salwan and Sharma 2020). Many extremophile bacteria are adapted to survive when submitted to multiple extreme conditions at the same time being called polyextremophiles (Urbieta et al. 2015). The biotechnological application of these microorganisms is made both as pure cultures or consortia improving the usage potentials of the biosurfactants produced. The isolation of bacteria may be achieved by cultivation-dependent or cultivation-independent methods (Basak et al. 2020). While the first usually involves the isolation of a certain microorganism on a general or selective media (McLain et al. 2016), the latter comprises a set of methods that do not rely on cultivation to study the organisms of interest (Cocolin et al. 2013). However, growing and maintaining pure cultures of extremophiles is not a simple process since standard cultivation methods are limited in terms of providing an exact replica of the original environment, which has all the essential aspects needed to favor the development of those microorganisms (Stewart 2012). Despite the specificities, both approaches are complementary as culture-independent tools favoring the identification of metabolites with biotechnological potential, and culture-dependent provide important information about microbial characteristics and phenotypes(Costa et al. 2020). Given the difficulties to isolate bacteria from extreme environments, genome sequencing methods have been used in an effort to assist the development of novel applications of biomolecules from extremophiles and to better comprehend the fundamental mechanisms associated with the bacterial capacity to adapt to nonstandard environments (Podar and Reysenbach 2006). These approaches commonly focus on strategies, such as recombinant DNA technology, mutagenesis, overexpression of extracellular peptides, substitution, replacement, modifications of amino acids and gene expression processes (Manga et al. 2021). However, it is important to mention that all known genes, processes, and metabolic pathways involved in the synthesis of biosurfactants are due to the observations made upon microorganisms that may be cultured on a bench scale, which comprises of only about 1% of the already mapped microbiota diversity (Dykhuizen 1998; Daniel 2005). Thus, metagenomics approaches may be considered a viable perspective to identify novel molecules produced by nonculturable microorganisms, from a biotechnological interest point of view (Berini et al. 2017; Araújo et al. 2020). Chakraborty et al. (2020), for instance, evaluating the composition of the microbial community from Lonar Lake, a hyperalkaline and hypersaline Soda Lake in India, used metagenome sequencing to map the prevalence of antibiotic and metal resistance genes. The DNA extracted from sediment samples revealed the presence of 29 bacterial phyla and 596 bacterial genera, with the predominance of

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Table 1 Biosurfactant-producing bacteria isolated from extreme environments Microorganism Thermophiles Bacillus axarquiensis Bacillus siamensis Bacillus subtilis Aeribacillus pallidus Streptomyces sp.

Source

Classification

References

Unkeshwar hot spring, India

Lipopeptide

Mehetre et al. (2019)

Sidoarjo mudflow region, Indonesia

Lipopeptide

Arifiyanto et al. (2020)

Byers peninsula, Antarctica

Glycolipid

Malavenda et al. (2015) Perfumo et al. (2018) Habib et al. (2020) Zhou et al. (2021) Kumar et al. (2022) Donio et al. (2013a) Sarafin et al. (2014)

Psychrophiles Rhodococcus spp. Pseudomonas sp. Idiomarina sp. Moesziomyces antarcticus Rhodococcus sp.

Lake Vanda, Antarctica

Glycolipid

Fellfield soils, Antarctica

Lipopeptide

Vibrio sp.

Cold-seep of the South China Sea

Phospholipid

Mucilaginibacter sp. Bacillus sp.

Proglacial water sample, Himalaya

Polysaccharide

Solar salt works, Thamaraikulam, India Kovalam solar salt works, India

Lipopeptide

Oil contaminated sites, Red Sea, Egypt Saline water conversion company Jeddah, Saudi Arabia.

Lipopeptide

Chott El-Djerid, Tunisia

Rhamnolipid

Hydrocarbons contaminated seawater, Tunisia Oil contaminated site, India

Lipopeptide Glycolipopeptide

Sea water, India

Rhamnolipid

Jeddah Region, Saudi Arabia

Lipopeptide

Sulfur blocks, Russia

N/A

Kocuria marina Halophiles Bacillus amyloliquefaciens Ochrobactrum halosaudia Pseudomonas aeruginosa Halomonas desertis Halomonas venusta Klebsiella sp. Alkaliphilic Pseudomonas aeruginosa Virgibacillus salarius Acidophilic Mycobacterium sp.

Lipopeptide

N/A

Barakat et al. (2017) Pugazhendi et al. (2017)

Neifar et al. (2019) Cheffi et al. (2021) Jain et al. (2013) Dukhande and Ward (2016) Elazzazy et al. (2015) Ivanova et al. (2016) (continued)

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Table 1 (continued) Microorganism Stenotrophomonas maltophilia Radiophile Ochrobactrum anthropi Citrobacter freundii Polyextremophile Franconibacter sp.

Source Mineral mining site, Saudi Arabia

Classification N/A

References Arulazhagan et al. (2017)

Used engine oil contaminated soil sample

Rhamnolipid

Ibrahim (2018)

Lakwa oil field, Assam, India

Glycolipid

Bacillus megaterium

Oil-contaminated water samples, Gulf of Suez, Egypt

Surfactin

Sharma et al. (2022) El-sheshtawy et al. (2022)

N/A: Information not available

Proteobacteria, Firmicutes, and Bacteriodetes. In the terms of functional metagenome analysis, the observed presence of genes related to antibiotic and metal resistance, especially As, Co, Cd, Cu, and Zn, is a sign of environmental contamination. Besides unveiling a huge potential in terms of extremophile diversity, the authors highlight those findings as essential to elaborate and further implementation of strategies to restore the Lonar Lake ecosystem. Araújo et al. (2020) used samples collected from the banks of the Jundiaí River (Natal, Brazil), identified as hypersaline, to create a metagenomic library. In that regard, a gene, MBSP1, was identified and related to the production and degradation of hydrocarbons by Haloferax lucentense. The ORF was subcloned into an expression vector, which was inserted into E. coli and the proteins were expressed in these clones. The emulsion index varied according to the substrate used, such as 56.7% for toluene and 49% for hexadecane. In addition, the droplet collapse and oil dispersion tests were positive, significantly reducing the interfacial tension. The stability in NaCl concentrations, 0 to 100 mg/mL, resulted in an increased emulsion index together with salinity, reaching 80% emulsion at the highest concentration, the biosurfactant was also stable with changes in temperature and pH, with approximately 60% emulsions.

4 Diversity of Microorganisms and Biosurfactants in Extreme Environments Studies concerning extremophile isolation and the characterization of their metabolites usually seek a group of species already known for their capacity to produce biosurfactants, and specific genes or just aim in exploring extreme environments in which the microbiota was not yet investigated in terms of taxonomic diversity. Hereafter, studies are discussed about the production of biosurfactants produced by bacteria isolated from different extreme environments.

Production of Biosurfactant by Bacteria from Extreme Environments:. . .

4.1

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Saline and Hypersaline Environments

With regard to biosurfactant synthesis, Donio et al. (2013b) described the isolation of halophilic microorganisms in water samples with 155% salinity collected from a solar salt works in Thamaraikulam, India. The isolates were cultured in nutrient agar plates containing from 5% to 20% of NaCl concentrations and were kept at 37 °C for 7 days. The colonies were further classified by the morphology pattern, gram staining, motility, and biochemical aspects. Among the isolates, a positive biosurfactant production was associated with a bacterium posteriorly identified as belonging to the genus Bacillus sp., being more than 90% similar to Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis. Besides being evaluated in terms of antibacterial, antifungal and antiviral activity, the secondary metabolite produced was also submitted to drop collapse test, oil spreading test, emulsification activity, and hemolytic activity. The biosurfactant extracted was a lipopeptide type and presented higher emulsification stability the higher the medium salinity, temperature, and pH. Furthermore, it was efficient in inhibiting pathogenic growth and reduced the mortality of shrimps infected with the White Spot Syndrome Virus (WSSV). Velmurugan et al. (2013) also explored the potential of the Thamaraikulam solar salt works area in terms of extremophiles, aiming at the characterization and analysis of the antimicrobial activity of biosurfactants produced by Halomonas salifodinae. Microorganisms were isolated from water samples with 230% salinity and were grown in halophilic agar plates at 37 °C until the first colonies appeared. The predominant colonies were assessed by morphology, physiology and biochemical aspects, being further identified through 16S rRNA sequencing. The biosurfactant was efficient in inhibiting bacterial growth and improved the immune system of shrimps infected with the WSSV, being a good candidate to replace commercial antibiotics and favor the development of antiviral drugs. Investigating water samples collected from 21 oil-contaminated sites in Shalateen, Red Sea, Egypt, known as the saltiest body of water in the world, Barakat et al. (2017) were able to isolate eight strains of haloalkaliphilic bacteria, cultivated in liquid minimal salt medium (MSM) in g/L (0.2 MgSO4, 1.0 K2HPO4, 1.0 NH4NO3, 0.05 FeCl3, 0.02 CaCl2, and 20.0 agar) with 1% (v/v) of sterile-filtered crude oil as the carbon source for 5 to 10 days, at 25 °C. Two of the strains, later identified as Bacillus amyloliquefaciens and Bacillus thuringiensis, produced biosurfactants with the highest emulsification index, 57 ± 2.85 and 56 ± 2.50, respectively, and were the only ones showing positive results toward hemolysis activity and surface tension reductions. The biosurfactants produced by those bacteria were characterized as lipopeptides and were considered thermally stable. The maximum production was observed at pH 11, 30 °C, and 15% (w/v) salinity. Moreover, evaluating bacterial growth under pH and salinity stress conditions, the results presented small changes over protein profile, denoting the capacity of the strains to adapt to extreme environments. In order to develop a bioremediation strategy to treat petroleum-contaminated sites under saline conditions, Neifar et al. (2019) evaluated the functional genomic

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analysis of a Halomonas desertis strain previously isolated from saline oil-contaminated sediments from Chott El-Djerid, a salty lake in Tunisia. The isolates were grown in a liquid mineral medium ONR7a (composition not described) supplemented with 1% of crude oil as the carbon source and 10% of NaCl. The production of the biosurfactant characterized as a rhamnolipid was directly affected by changes in pH and NaCl concentration. The maximum production of 0.42 g/L was obtained after 5 days of cultivation at 30 °C with initial pH of 10.0, 1% of inoculum 10% of NaCl, and 4% of crude oil, resulting in reduced surface tension to 31.2 mN/m and emulsification indexes of 69.4%, respectively. Furthermore, the biosurfactant was effective in degrading 92.2% of the alkanes and 89.0% of the polycyclic aromatic hydrocarbons.

4.2

High-Temperature Environments

The contributions of Arifiyanto et al. (2020) may be highlighted. The authors described the isolation process of thermophile bacteria from soil samples collected in the Sidoarjo mudflow region, the result of a mud erupting volcano in Indonesia with constant temperatures around 100 °C (Wibowo et al. 2018). The isolates were cultured with starch casein agar supplemented with griseofulvin and chloramphenicol 0.05 ppm at 70 °C for 1 to 3 weeks. Twelve isolates were obtained and characterized regarding biosurfactant production, focusing on antimicrobial activity. The best results concerning pathogen inhibiting were observed with the biosurfactant produced by a strain identified as a Streptomyces sp., capable of growing in temperatures greater than 70 °C. The biosurfactant was further classified as a lipopeptide and was considered suitable for being used as an anticancer agent and to favor the production of enzymes and antibiotics. The presence of thermophiles, however, is not restricted to natural environment; an example of an artificial environment is the photovoltaic panel (Moura et al. 2021). Tanner et al. (2019) reported that the microorganisms present are pigmented, and resistant to radiation and desiccation, which makes their metabolites exciting for biotechnology. Moura et al. (2021) reported the microbiota present in photovoltaic panels and, among them hydrocarbon degradation genes were found. The production of these metabolites should be studied considering stability at extreme temperatures.

4.3

Cold Environments

Malavenda et al. (2015) isolated 199 psychrotolerant bacteria from coastal sediments in the Arctic and Antarctic. Of these, 18 were selected due to stable emulsions (≥50%) or with a reduction in surface tension to CMC) S4 Rhamnolipid solution (< CMC) S5 Cell-free culture medium solution

95%

63%

57%

50% 21%

12% 0%

S1

S2

S3

S4

S5

Solutions

Fig. 5 Percentages of oil removal obtained by applying rhamnolipids from P. aeruginosa ORA9 during the washing of contaminated sea sand

21% removal quantified by applying the rhamnolipid solution at concentrations lower than the critical micelle concentration, CMC (Fig. 5). The 57% removal obtained by applying the cell-free culture medium (crude RL) is an important result, since it represents a significant decrease in the production costs of the rhamnolipid, due to the elimination of the purification and extraction stages, which make the procurement process more expensive. Torres et al. (2018) obtained 80% hydrocarbon (6500 mg kg-1) removal by using nonpurified rhamnolipids from Pseudomonas aeruginosa ATCC 9027, during the washing of contaminated soils. Recently, our research group evaluated crude oil degradation by applying rhamnolipid crude from Pseudomonas aeruginosa Y3-B1A at three different scales: laboratory (liquid medium), microcosm (soils), and field (soil plots). Cells were cultured in liquid medium by using a minimal mineral medium supplemented with crude rhamnolipids (1% m/v), and addition of crude rhamnolipids (crude) increases the biodegradation of hydrocarbons by 61.7% (Table 2). For the microcosms (Fig. 6), when applying crude rhamnolipids, percentage increases between 36 and 53% (Table 3) were observed for the different hydrocarbon fractions analysed. The addition of crude rhamnolipids was considered as a biostimulation treatment, as it was added to an autochthonous microbial population previously evaluated as a hydrocarbon degrader. The application of rhamnolipids in contaminated plots favored the degradation of TPH (36.7%) and aromatics (63.7%), while the biodegradation of resins and asphaltenes did not exceed 30%. The order of degradation of petroleum hydrocarbons is as follows: n-alkanes > branched alkanes > low molecular weight aromatics > monoaromatics > cyclic alkanes> polycyclic aromatics and > asphaltenes.

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Table 2 Effect of crude rhamnolipid addition from P. aeruginosa Y3-B1A on crude biodegradation by mixed culture Culture conditions Mixed culture (CM) Mixed culture + RL (CM + RL)

Biodegradation (%) 29.9a ± 7.0 78.0b ± 7.5

Increased (%) – 61.7 ± 3.9

Values with the same letter do not present statistically significant differences ( p < 0.05) Fig. 6 Biodegradation of hydrocarbons in microcosms applying the rhamnolipids of P. aeruginosa Y3-B1A

Table 3 Influence of rhamnolipids on hydrocarbon biodegradation in microcosm TPH 55.2 ± 1.5

O&F 3.3 ± 0.3

Saturated 35.2 ± 2.2

Aromatics 73.7 ± 3.3

Resins 5.8 ± 1.1

Asphaltenes 1.54 ± 1.7

TPH total petroleum hydrocarbons, O&F Oils and fats

5 Toxicity and Biodegradability of Rhamnolipids in Contaminant Removal Processes When introduced into the environment, surfactants can themselves be a source of pollution. The most described negative effect is the one caused when the biosurfactant is a carbon source that is more easily degraded than the pollutants. One of the most important mechanisms to explain the toxicity of biosurfactants is the alteration of the cell membrane due to the interaction with lipid components and the reaction of rhamnolipids with proteins. On the other hand, toxicity during the bioremediation process may be associated with the solubilization and/or emulsification of hydrocarbons that are toxic to the microbiota. To evaluate the toxicity of Pseudomonas aeruginosa Y3-B1A rhamnolipids, three concentrations were used, prepared from cell-free broth: ½ CMC, CMC, and 2CMC. The toxicity was evaluated with seeds of Solanum lycopersicum and Artemia

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salina (micro crustacean). The mean lethal concentration for Artemia salina was 163.4 mg/L, while for a germination of 50%, a rhamnolipid concentration of 190.6 mg/L was estimated (95% confidence). The median lethal concentration values for the two models were in the range of 100–1000 mg/L, and can be considered safe (Meyer et al. 1982). As in previous studies, rhamnolipids produced by pathogenic bacteria did not alter the toxicity levels of the sample (Dobler et al. 2020); which makes them more attractive for bioremediation strategies by biostimulation by the direct application of crude oil (Liu et al. 2018; Patowary et al. 2018; Dhanya 2021). Other authors have reported similar results using the same biological models (Oluwaseun et al. 2017; Nogueira et al. 2019; Dobler et al. 2020), in addition to Latuca sativa (lettuce) seeds and mammalian cells (OjedaMorales et al. 2016; Oluwaseun et al. 2017;). The low or absence of toxicity of biosurfactants is generally attributed to their polymeric structures, which gives them a lower polarity than that of synthetic surfactants and leads to the formation of micelles with less electrostatic charge, thus increasing compatibility with biological membranes (Nogueira et al. 2019). The application of rhamnolipids in the bioremediation of soils for biorecovery purposes requires the subsequent evaluation of the soil through toxicity tests that allow confirming their recovery (Franzetti et al. 2010b). In the research carried out, an acute toxicological test was carried out with Eisenia andrei (earthworm) 30 days after the rhamnolipids were applied to the plots. The results achieved showed that the addition of crude rhamnolipids from P. aeruginosa Y3-B1A did not cause toxic effects to the soil, since the earthworm mortality rate was 5%. These acute toxicity values qualify the soil as restored, taking into account that the earthworm is a widely used and accepted model for such purposes.

6 Conclusion Due to their physical-chemical and biological properties, rhamnolipids have multiple applications, in particular, the bioremediation and biodegradation of hydrocarbons and xenobiotic compounds and the removal of heavy metals in water, sediments, and soils. Cadmium, zinc, mercury, copper, iron, nickel, lead, and chromium are the most studied metals. However, the authors have recently demonstrated the feasibility of applying rhamnolipids in the removal of vanadium from oil sludge. The applications of rhamnolipids in the remediation of soils contaminated with hydrocarbons were directed solely to their use in the stimulation of biodegradation by microorganisms and direct extraction of hydrocarbons. However, the stability and efficiency of biosurfactants in the extraction of hydrocarbons in soils, as well as the percentages of elimination achieved in the remediation of impacted ecosystems, facilitates the application of these compounds in the assisted recovery of crude oil in oil wells.

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Mosquitocidal Activity of Biosurfactants R. Parthasarathi, S. Harini, P. Poonguzhali, K. Akash, and N. Kavinilavu

1 Introduction Vector-borne diseases are caused by etiological agents like parasites, bacteria, viruses, etc., that are transmitted to humans through the bite of the infected vectors, such as mosquitoes, sandflies, black flies, ticks, mites, bugs, etc. Mosquitoes are one of the most medically important vectors as they transmit deadly disease-causing agents like Dengue, Filariasis, Chikungunya Malaria, West Nile fever, Zika, Japanese encephalitis, etc. (Dhanasekaran and Thangaraj 2014). Because of the lack of effective vaccines and antiviral drugs for some of the major diseases, the effective ways to reduce this burden have often critically relied on mosquito control programs (Talaat et al. 2021). In order to control them, a large number of chemical compounds have been extensively used, which resulted in many negative effects on environment, such as contamination of food and water source, poisoning of nontarget organisms, concentration of the food chain and development of resistance, etc. (Dahmana et al. 2020). To overcome these problems caused by chemical pesticides, the best alternative is to use the biocontrol agents, which minimize these environmental problems.

R. Parthasarathi (✉) · S. Harini · K. Akash · N. Kavinilavu Department of Agricultural Microbiology, Faculty of Agriculture, Annamalai University, Annamalai Nagar, Tamilnadu, India P. Poonguzhali Department of Microbiology, M.R. Government Arts College, Mannargudi, Tamilnadu, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_12

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2 Biosurfactant Biosurfactants are amphipathic molecules having dual moieties of both hydrophilic, as well as hydrophobic moieties that accumulate at the interface between many phases like oil-water, liquid-solid, and air-water accomplishing the decrease in the surface tension (ST) and the interfacial tension between the phases of aqueous and other immiscible solutions. Surfactants produced from microbes as secondary metabolites are generally specified as biosurfactants that are secreted from microbial cell either extracellularly or intracellularly by attaching to the cell wall of microbes (Karlapudi et al. 2018; Kumar et al. 2018; Sharma et al. 2021). Biosurfactants are the surface-active biomolecules with emulsifying properties that have a known number of activities, including the solubilization to combat the microbial biofilms (Long et al. 2016; Rani et al. 2020; Srinivasan et al. 2021; Leonie et al. 2022). The biosurfactants have antimicrobial activities and insecticidal potential (such as Antifungal, Antibacterial, Antimycoplasma, Algicidal, Antiamoebial, Zoosporicidal, Antiviral); hence, these have medical applications.

2.1

Antimicrobial Action of Biosurfactants

The exploration for novel antimicrobials tends to impart a great challenge in the current scenario owing to the emergence of new trait of pathogens showing antibiotic resistance towards almost all antibiotic molecules available as of today (Hancock and Chapelle 1999). The advent of microbial metabolites serves as the reservoir of biomolecules renowned to be acknowledged in earlier studies exerting inventive structures having prominent biological activities (Donadio et al. 2002). In fact, certain biosurfactants prove to be the effectual therapeutic agents that are safe and are identified as the noteworthy substitute for replacing the synthetic drugs and antimicrobial agents (Cameotra and Makkar 2004). The underlying mechanism responsible for the biocontrol activity of biosurfactants is the alteration in the efflux pump of cell wall and it also involves the perturbations of cell surface on the pathogen. The finest biosurfactant exhibiting biological control activity is rhamnolipid; for example, P. aeruginosa PNA1 produces rhamnolipids. Interestingly, owing to biosurfactant synthesis, fluorescent Pseudomonads can hamper the fungal pathogens like Fusarium oxysporum (responsible for wilt disease in plants), Pythium ultimum (responsible for damping off and root rot in plant system), and Phytophthora cryptogea (responsible for rot disease in fruits and flowers). Apart from these, biosurfactants have also been involved in reducing the viability of Verticillium sp., stopping the growth of pathogenic fungus Rhizoctonia solani that produces a wide range of plant diseases, and inhibiting the growth of Phythium ultimum, which results in damping off and root rot in plants (Nalini et al. 2020).

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Fig. 1 Properties of Biosurfactant

The contribution of Haba et al. (2003) reveals the antimicrobial activity of rhamnolipid produced by Pseudomonas aeruginosa against various pathogenic Enterobacter aerogenes, Klebsiella pneumonia, Bacillus subtilis, Staphylococcus aureus and S. epidermis, Penicillium funiculosum and Fusarium solani with various minimum inhibitory concentration ranges from 0.5 μg/ml to 64 μg/ml. The antimicrobial activities of rhamnolipid against Proteus vulgaris, Streptococcus faecalis, Penicillum, and Alternaria were also observed by Besson et al. (1976). Likewise, Walia and Cameotra (2015) reported that, rhamnolipid biosurfactant produced by Pseudomonas aeruginosa had potential algicidal activity against harmful algal bloom (HAB) species, Heterosigma akashiwo. A medium containing rhamnolipid 0.4–3.0 mg L-1 can inhibit the algal growth. The rhamnolipid-producing P. aeruginosa LBI strain exhibits antimicrobial effect upon the pathogenic Staphylococcus aureus, Bacillus cereus and laboratory fungi, such as Micrococcus hiteus, Mucor michei, and Neurospora crassa (Nitschke and Pastore 2006). Another promising biosurfactant named flocculosin, which is a cellobiose lipid obtained from Pseudozyma flocculosa (yeast like fungus), is found to be very effective against Staphylococcus species, including methicillin-resistant Staphylococcus aureus (MRSA). Lankar et al. (2018) have evaluated biosurfactant from the strain Pseudomonas aeruginosa JS29 for biological control of anthracnose disease and have revealed that there is a significant reduction on the disease incidence by the application of biosurfactant. Khare and Arora (2021) reported Pseudomonas guariconensis LE3 from the rhizosphere of Lycopersicon esculentum that has been found to have biostimulant and biocontrol activities. The biosurfactants possess various other properties apart from antimicrobial activities; it is observed that the biosurfactants have been exploited in various applications (Fig. 1 and Table 1).

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Table 1 Application of biosurfactant Biosurfactant Types Rhamnolipids, Sophorolipids, and Lipopeptides

Industry Petroleum

Application Enhanced oil recovery, crude oil transport

Rhamnolipids, Sophorolipids, and Lipopeptides

Environmental

Bioremediation oil spill cleanup operations soil remediation and flushing treatment of wastewater heavy metal remediation Biofouling

Rhamnolipids, Sophorolipids, and Lipopeptides

Agriculture

Biocontrol plant protection Pest control

Glycolipid, lipopeptide, and polymeric surfactants

Food

Emulsification

Sophorolipids, Mannosylerythritol lipids, Rhamnolipids, Lipopeptides

Cosmetics

Beauty lotion

Function Of Biosurfactant Emulsification of oils, solubilization of solid surfaces, spreading, detergency, foaming,

Emulsification of oils, lowering of interfacial tension, dispersion of oils, solubilization of oils, wetting, mobilization, spreading, detergency, foaming, binding, desorption, and mobilization of heavy metals

Solubilization of flavoured oil Maintain consistency Wetting agent Spreading agent Foaming Thickening Emulsification Foaming agents Wetting agents Cleansers

References Nalini and Parthasarathi (2013), De Almeida et al. (2016), Fenibo et al. (2019), Rocha e Silva et al. (2019), Da et al. (2021), Leonie et al. (2022) Parthasarathi and Sivakumaar (2011), De Almeida et al. (2016), Kumar et al. (2015), Sarubbo et al. (2015), Carolin et al. (2021), Da et al. (2021), Melvin Joe et al. (2019), Leonie et al. (2022) Nalini and Parthasarathi (2014, 2018), Santos et al. (2016), Chaprao et al. (2018), Nalini et al. (2016, 2020) Santos et al. (2016); Ribeiro et al. (2020); Nalini et al. (2020)

Kumar et al. (2015), Santos et al. (2016), Bezerra et al. (continued)

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Table 1 (continued) Biosurfactant Types

Industry

Application

Rhamnolipids, Sophorolipids, and Mannosylerythritol lipids

Washing detergent

Detergent

Rhamnolipids

Nanotechnology

Nanoemulsion

Function Of Biosurfactant Moisturizing Skin toning Detergents for laundry Wetting Foaming agent Spreading agent Antifouling Emulsification

References (2018), Leonie et al. (2022) Chaprao et al. (2018), Nadaf et al. (2021), Nalini et al. (2019) Melvin Joe et al. (2011), Rocha e Silva et al. (2019)

3 Vector Control In the later part of nineteenth century, it was identified that several arthropods such as mosquitoes, sand fly, black flies, ticks, mites, and other insects play major roles in transmitting many of the diseases. For many of the vector-borne diseases transmitted by these vectors, there is no effective vaccine for its prevention; hence in order to control the disease transmission, vector control is the only best available effective method. Vector control mainly relies on following strategies such as 1. 2. 3. 4.

Chemical control method Biological control method Environmental management in vector control Genetic control method

3.1 3.1.1

Chemical Control Methods Insecticides

Insecticides are chemical compounds used primarily to control the insects. Insecticides are classified into four classes based on their chemistry and their mode of action in killing the vectors, such as Organochlorines, Organophosphates, Carbamates, and Pyrethroids. Chemical method of controlling vectors involves two techniques: (1) indoor residual spraying and, (2) use of insecticides treated nets (Pryce et al. 2022). Indoor residual spraying (IRS) involves spraying of insecticides on the surface of the house walls. The insecticide residues will be present on the sprayed surface for several months (CDC, malaria). IRS will not prevent the biting of mosquitoes rather

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it acts on the mosquitoes when it rests on the sprayed wall surface after blood meal. When the mosquitoes rest on the sprayed surface, they will be in contact with the insecticides sprayed on the wall through their feet and get killed.

3.2

Biological Control Methods

Biological control methods are the methods of controlling the mosquitoes by making use of living organisms or their products. Biocontrol agents, such as viruses, bacteria, fungus, protozoa, plants, fish, insects, parasitic worms, etc., are useful in mosquito control (Rozendaal 1997).

3.2.1

Insect Predators

Mosquito larvae and pupae serve as a natural food source for many of the predatory insects; these insects play an important role in decreasing the mosquito population in the natural environments (Louca et al. 2009). Predatory insects, such as dragon fly nymphs, Toxorhynchites species of mosquito larvae, cyclopoids, copepods and other small crustacean feeds on mosquito larvae. Similarly, the tadpole species Polypedates cruciger preys on the eggs of mosquitoes. Certain studies (Murugan et al. 2015; Priya et al. 2015) has suggested the predation of aquatic crustaceans such as Cyclops vernalis and Mesocyclops aspericornis preying on the mosquito larvae as the natural occurrence and this strategy could be intensively adopted to control the mosquito species, such as Anopheles stephensi, Culex and Aedes species intending to spread the vector-borne diseases like malaria and dengue.

3.2.2

Entomopathogenic Fungus

Coelomyces genus of fungus is known to be the first reported entomopathogenic fungus used in controlling the larvae of An. gambiae (Muspratt 1963). This entomopathogenic fungus when ingested by the mosquito larvae alters the physiology of the larvae and decreasing the blood feeding capacity (e.g. Beauveria bassiana in Stegomyia aegypti) and Metarhizium anisopliae in Aedes aegypti) (Paula et al. 2011; Darbro et al. 2012).

3.2.3

Larvivorous Fish

The release of larvivorous fish to the larval habitat plays a significant role in controlling the mosquito larvae (Wei et al. 2006). These larvirorous fishes were used worldwide in controlling the vectors of malaria and other mosquito species. Example of larvivores fishes is Gambusia affnis and Poecilia reticulate that are

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widely used in controlling the mosquito larvae in many countries (Chobu et al. 2015).

3.2.4

Protozoa

Microsporidia are the obligate parasite which do not possess mitochondria. Since, it can easily bind with host cells and relay energy from the host cells for their food source (Han and Weiss 2017), widely used microsporidia includes Nosema, Amblyospora, Thelohania, Vavraia and Parathelohania respectively.

3.2.5

Nematodes

Several species of nematodes are found to be effective in controlling the mosquito vectors. About 24 mermithid nematodes were described to have parasitic effects on mosquito vectors as wells as blackflies. Seven genera of mermithid nematodes that shows parasitic effects to mosquito vectors are Culicimermis, Empidomermis, Hydromermis, Octomyomermis, Perutilimermis, Romanomermis, strelkovimermis, Steinernema abbasi and Romanomermis iyengari (Poinar 2001; Liu et al. 2020; Elbrense et al. 2022). It has been reported in the tree holes that Octomyomermis muspratti has been isolated from Aedes and Culex. Also, other nematodes species known as Empidomer miscozii was isolated from the Anopheles vectors.

3.2.6

Bacteria

Microorganisms play a major role in killing the mosquito larvae. When mosquitos ingest the bacteria e.g. Bacillus thuringiensis (Bt), the alkaline pH (9 to 12) of the midgut region solubilizes the crystals. For Lepidoptera, the specific proteins are soluble at pH above 9.5. The effect of Cry toxin is greatly influenced by the pH of the midgut among insects. Some toxins are stimulated under neutral to acid pH conditions (Cry1b), whereas others are stimulated under alkaline conditions (CryII1A) (Canton et al. 2015). The most important step in stimulation of toxin is the cleavage of Cry toxins and its specificity in different insects. The active proteins of size 60–70 k Da is formed by the solubilization of toxin and upon the action of proteases on them. Post translational modification of the toxin fragments by the removal of amino terminus of the polypeptide chain makes them activate. Further the protoxins are stimulated by the influence of digestive enzymes in the mid gut leading to the subsequent binding of them to the specific receptors of microvillus; especially the gut region among the insects belonging to the order Lepidoptera involves the apical membranes of the columnar cells. Usually, cry gene encodes for Cry protein which exhibits distinct structures like bipyramidal, cuboid, rhomboid, oval, spherical, and sometimes the shape is not definite. The toxins so far identified from B. thuringiensis subs. Kurstaki HD1 Strain

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(Bt) includes Cry1Aa, Cry2Aa, Cry1Ab, and Cry1Ac Cry2Aa, respectively. The toxic Cyt and Cry proteins that are having a negative impact among the insects leading to its fatal effect are water soluble in nature and falling under the bacterial proteins category of δ-endotoxin (Sriwimol et al. 2015). Besides these, variety of toxins including α-exotoxin, β-exotoxin, hemolysins, enterotoxins, phospholipases, and chitinase has also been demonstrated from Bt. Furthermore, the bacterial Cyt and Cry proteins are considered as the pore forming protein, and have also been denoted as δ-endotoxin. These δ-endotoxins express either α-helix toxin form or α-helical and β-barrel toxin form. The α-helix toxin initiates pore formation in the host cell membrane, while the β-barrel toxins constituting the hairpin β-sheet monomer triggers the barrel formation in the host cell membrane (Mizuki et al. 1999a, 1999b; 2000, 2001; Mondal and Chattopadhyay 2020). The investigation of Vijayan and Balaraman (1991) demonstrated the production of secondary extracellular metabolites from 35 isolates of Actinobacteria showing larvicidal mode of action towards the mosquitoes belonging to the genus Culex and Anopheles. Previous studies have proved the mosquitocidial effect of the extracellular metabolites, such as tetranectin (Ando 1983), avermectins (Pampiglione et al. 1985), and flavonoids (Rao et al. 1995) obtained from Actinobacteria. Some insects, which were studied in vivo, have established their resistance for Cry1 toxins that displayed cross-resistance towards other Insecticidal Crystal Proteins (ICP). For instance, Plutellaxylostella (Tabashnik et al. 1994), Pectinophora gossypiella (Tabashnik et al. 2010, 2013; Tabashnik and Carrière 2017, 2019; Li et al. 2020; Fabrick et al. 2020), Spodopteraexigua (Moar et al. 1995) and Heliothis virescence (Gould et al. 1995). To avoid insect resistance development genetically modified plants ensuring insect-resistant characteristic is highly suitable (Shelton et al. 2000). World Health Organization has projected the statistical report insisting that more than 500 million clinical cases would emerge due to the mosquito species like Aedes aegypti and Culex quinquefasciatus by acting as the vector in transmission distinct disease-causing agents. Mosquitocidal exotoxin production from two different bacterial strains, such as Pseudomonas fluorescence (NCIM-2631) and Pseudomonas caryophilly (NCIM-5094), and its activity has been evaluated by Mahamuni et al. (2016) in which, Pseudomonas fluorescence (NCIM-2631) has synthesized 4 mg/ml of exotoxin, while Pseudomonas caryophilly (NCIM-5094) was found to excrete exotoxin 3 mg/ml. Examining these exotoxins against Aedes aegypti revealed 40% mortality rate for the metabolite from Pseudomonas fluorescens (NCIM-2631) and 20% mortality rate for the metabolite from Pseudomonas caryophilly (NCIM-5094), respectively (Mahamuni et al. 2016). However, Bacillus thuringensis sub sp. israelensis genes, such as cry4Aa, cry4Ba, cry11Aa and cyt1Aa confer four distinct ICPs (134, 128, 72 and 27 kDa) synthesis that exhibits larvicidal activity against mosquito (Neil et al. 1995; Bergamasco et al. 2013). These alpha-endotoxins differ in the specificity against different species of mosquitoes as well as the toxicity levels (Margalith and Ben-Dov 2000). The synergistic action of Cyt1Aa toxin with Cry toxins has been observed to elevate the larvicidal activity even the low toxic Cyt1Aa toxin under in vitro

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condition during their coexistence, which may be because of the difference in their mode of action (Wirth et al. 1997; Crickmore et al. 1998; Butko 2003). Most regularly used bacilli for larvicidal activity are Bacillus thuringiensis sub sp. israelensis (Bti) and B. sphaericus (Patil et al. 2012). Additional bacterial strains, such as Bacillus brevis (Khyami-Horani et al. 1999) and Bacillus subtilis (Das and Mukherjee 2006; Geetha et al. 2011), have been confirmed to produce larvicidal toxins. However, the intensified frequencies of resistance to Bacillus sp. have led researchers to look for an auxiliary biocontrol agent for larvae and pupae of mosquitoes.

3.3

Environmental Management of Vector Control

Environmental management aims to change the environment in order to minimize or control the vector multiplication, and also by reducing the interaction between pathogen and vector through altering, destroying, removing, or recycling the containers that provides habitat for vector breeding (Randell et al. 2010; Christian et al. 2021). The “Global Vector Control Response (GVCR) 2017–2030” was approved by the World Health Assembly in 2017, provides strategic guidance to countries and development partners for urgent strengthening of vector control as a fundamental approach to preventing disease and responding to outbreaks (WHO 2017).

3.4

Genetic Control of Vector

Genetic control of vector aims to suppress the target population or to introduce genetically modified harmless traits to the environment. The genetically modified mosquitoes are introduced to reduce the number of competent mosquitoes, which may result in the reduction of specific targeted pathogenic spread. This alternative approach has been adopted by Carter et al. (2014) to suppress the dengue virus by altering the expression of genomic RNA by targeting Bax group I intron.

4 Mosquitocidal Activities of Biosurfactant Typically, a wide range of harmful chemical components are imparted to eradicate the mosquitoes commercially in repellents in the form of mosquito coils and liquidators. Certain harmful chemicals that are employed in those repellants like N,N-diethyl-3-methylbenzamide (DEET), pyrethrum, methoprene, dimethylphthalate (DMP), temephos, allethrin and malathion, etc., results in various health issues and other related side effects upon human beings (Khater et al. 2019). Besides chemical, usage relying upon microbes for mosquitocidal activity has

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a higher impact, because the microbial metabolites like proteins or biosurfactants have been proved to be prompt in eradicating the distinct stages of mosquitoes inclusive of egg, larva, or its adult forms. The implementation of biosurfactant to eradicate the distinct stages of mosquitoes has been considered to be a potential eco-friendly approach as a finest solution for the commercially available chemical repellents and insecticides. The current scenario after the pandemic outbreak of COVID-19 urges and necessitates the vector control especially mosquitoes, which easily transmit many pathogens causing malaria, dengue, chikungunya, yellow fever, etc., owing to the emergence of various diseases among immuno-compromised human populations. There are different classes of biosurfactant (based on their chemical nature) produced from a variety of microbes in the earlier studies. Even though biosurfactant has been extensively studied for various applications in the field of medical, agriculture, petroleum, food, chemical and other cosmetic industries, the utilization of biosurfactant in mosquito control has been identified to be novel strategy.

4.1

Impact of Surfactin, Rhamnolipid and Other Biosurfactant on Mosquitoes

Nonetheless, most of the larvicidal properties of the biosurfactant research have been undertaken with surfactin, the lipopeptide type of biosurfactant produced by the Bacillus species (Medeiros et al. 2005; Das and Mukherjee 2006; Geetha et al. 2011, 2012; Parthipan et al. 2018) and the next line up in the hierarchy of frequent research of larvicidal property has been occupied by rhamnolipid, a glycolipid type of biosurfactant produced by Pseudomonas species (Prabakaran et al. 2015; Parthipan et al. 2018). Further, both the larvicidal and pupicidal activities of biosurfactants such as surfactin and rhamnolipid from the strains of Bacillus subtilis A1 and Pseudomonas stutzeri NA3 have been extensively assessed by Parthipan et al. (2018). The evaluation has focused and has proved the efficacy of the both the biosurfactants such surfactin and rhamnolipid in the concentration range of 2 to 10 ppm against Anopheles stephensi at different stages (larval and pupal stages) of its life cycle. The lipopeptide nature of the produced biosurfactants has been confirmed by FT-IR spectroscopy and Gas chromatography-Mass Spectrometry (GC-MS), which were effective against both the larval and pupal stages of A. stephensi. Furthermore, the insecticidal effect against the pupal and larval stages of mosquitoes due to the action of biosurfactant from Bacillus subtilis has also been extensively demonstrated (Ehrenberg) (Geetha et al. 2011; Manonmani et al. 2011). Few other reports insisting on the insecticidal property of the biosurfactant has also been proved earlier targeting the insect pest (Fazaeli et al. 2021) in which the biosurfactant from Staphylococcus epidermidis (Talaat et al. 2021) has exhibited a potential larvicidal activity against the insect pest Tribolium castaneum. Bacillus mojavensis has been involved in the

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production of lipopeptide type of biosurfactants such as surfactin and fengycin (Hmidet et al. 2017) exhibiting a potent bio-insecticidal activity against various pests (Blacutt et al. 2016) including mosquitoes (Salamun et al. 2022). The promising adulticide property of surfactin from B. subtilis sub sp. subtilis (VCRC B471) has been demonstrated during its low volume spray over the malaria causing adult mosquitoes belonging to Anopheles stephensi by Geetha et al. (2012). In contrary, the study of Randhawa and Rahman (2014) has reported the rhamnolipids from Pseudomonas sp. expressing a low toxicity against mosquitoes but has a high biodegradability and serves to be a prominent alternative for chemical surfactants owing to its physicochemical properties. Investigation of Jayasree et al. (2018) has evidenced the lipopeptide type of biosurfactant produced from Bacillus subtilis B50 exerting its larvicidal activity in which the crude form of biosurfactant has been examined ranging from 1 mg to 10 mg per 100 ml concentrations for larvicidal effect. It revealed that 1 mg to 4 mg concentration had an effectual mortality rate at the exposure duration of 72 h. Bacillus species produced necessary metabolites, which includes the cyclic lipopeptide antibiotics of surfactin, iturin, and fengycin. Among the bacilli, B. subtilis has the potential for biopharmaceutical and biotechnological operations (Cameotra and Makkar 2004; Nitschke and Pastore 2006). Yet another study of Fernandes et al. (2020) has revealed the larvicidal activity expressing 100% mortality at the lowest concentration biosurfactant of 6.25% produced by the yeast Wickerhamomyces anomalus CCMA 0358 against the mosquito species Aedes aegypti in 24 h of investigation. In fact, the efficacy of the biosurfactant against the mosquitoes has been generally expressed as lethal concentration (LC50). The study of Deepali et al. (2014) has established the larvicidal activity of the rhamnolipid type of biosurfactant extracted from Stenotrophomonas maltophilia expressing LC50 value of 100 mg per litre at 2 hours of exposure, endures in the reduction of the LC50 value corresponding to 30 mg per litre by increasing the time of exposure of biosurfactant to 24 h towards the test larvae belonging to Culex quinquefasciatus. On the contrary, the mortality rate of the test larvae of A. aegypti has been found to be constant at the biosurfactant concentration of 400 and 600 mg per litre during 18 h and 24 hours of exposure, respectively (Silva et al. 2015). Furthermore, biosurfactants extracted from Scheffersomyces stipitis was proved to exert larvicidal effect on A. aegypti, thereby eradicating the tropical disease vectors by implicating LC50 value of 600 mg per litre at 12 h of exposure (Franco Marcelino et al. 2017). Certain studies (Abd El-Salam et al. 2011; Revathi et al. 2013) have suggested the use of surfactin obtained from Bacillus subtilis as an effective biological control agent against the species Spodoptera littoralis as well as mosquitoes. Lipopeptide biosurfactants produced by Bacillus amyloliquefaciens have been identified to possess insecticidal property when tested against different mosquito species (Geetha et al. 2011; Yun et al. 2013; Ben Khedher et al. 2017), which makes a feasibility of trying alternatives to Cry toxins to overcome insect resistance.

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Key Factors for Mosquitocidal Activity

Although several studies spotlighted the applications of biosurfactant as biocontrol activities (Blacutt et al. 2016; Crouzet et al. 2020; Poonguzhali et al. 2021; Maity et al. 2022), yet only certain studies (Deepali et al. 2014; Geetha et al. 2011) have undertaken the effort to establish the effectiveness of biosurfactant against the mosquito at the pupal and larval stages. Initially, consideration of biosurfactant in the aspect of vector control has been originated owing to the reason that it could damage the cells and cuticles of insect pest with high specificity (Christophers 1960; Awada et al. 2005; Kim et al. 2011). In fact, such studies are still at the explorative state because the knowledge on maximum possible advantages and limitations of biosurfactant as vector control to attain its commercial importance has not been fully flourished. Biosurfactant is well known for its property to reduce the surface tension between two distinct phases and this property has been observed as one of the reasons behind the lethal effect among the pupae of mosquitoes. Perhaps, it has been noticed that as the biosurfactant is involved in reducing the surface tension of the water bodies where the pupae are present, they are not able to reach the surface of water bodies to fulfil the oxygen demand through respiration for its survival, finally resulting in the death of those larvae owing to drowning (Piper and Maxwell 1971; Fernandes et al. 2020). Similar report of adulticide property against mosquitoes from the microbial source has been reported by Yang et al. (2020). The mechanism underlying the adulticidal activity of biosurfactant against mosquitoes is due to the water penetration of cuticles or spiracle openings, resulting in the lethal effect upon mosquitoes (Silva et al. 2015). In fact, the siphon maintains the spiracle openings by not permitting the water flow as it loses its hydrostatic balance to reach the water surface and if the biosurfactant interludes in such region affecting its wettability or water flow, this may lead to the larval drowning, thus leading to its mortality.

4.3

Advantages, Limitations, and its Future Prospective

Undeniably, there are numerous advantages while employing the biosurfactants for eradication of the mosquitoes. As the biosurfactants are acquired from microbes, they could be mass multiplied in the laboratory or industries or the specific purpose even from the cheapest raw materials available at the locality. Unlike other synthetic repellents, these are not toxic for nontargeted host, due to which health issues faced by the human beings out of those synthetic repellents could be minimized. In addition, the biosurfactants are biodegradable, biocompatible, and eco-friendly in nature (Poonguzhali et al. 2018; Vandana and Singh 2018). On the other hand, the main limitation may be its production cost, selection of the strain, and low yield when compared with the huge demand. Yet another milestone that could be encountered during commercialization may be the resistant mosquito species. It is

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noteworthy that emerging of resistant species towards biosurfactant is not much easier as distinct classes of biosurfactants are accessible and are in existence. Indeed, the future prospective of the biosurfactant as vector (mosquito) control agent needs to travel a long way to attain its commercialization. Further research in this view necessitates exploring the potential high yielding strain with mosquitocidal properties.

5 Conclusion Mosquitoes being a predominating vector in the transmission of diseases in lieu of an emerging threat to world. Many of the currently available chemical agents for controlling vectors, especially mosquitoes, are incredibly toxic and nonbiodegradable and thus result in health hazards for living beings including humans, pets, and other beneficial insects. Hitherto, though different concepts of natural products have evolved in the current era, none have been consistent. Rather, the biosurfactants have known to exhibit a wide range of insecticidal activity towards mosquitoes and other insect pests belonging to the order Diptera and Depidoptera. Nevertheless, the nature of biosurfactants being eco-friendly, biodegradable, safe, and biocompatible, make it to be the best alternative and the sustainable for the target pest, especially to implement in the mosquito repellants in the prevailing situation for replacing other chemical repellants in market.

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Biosurfactants as Promising Surface-Active Agents: Current Understanding and Applications Harmanjit Kaur, Pankaj Kumar, Amandeep Cheema, Simranjeet Kaur, Sandeep Singh, and Ramesh Chandra Dubey

1 Introduction Surfactants are amphiphilic compounds made up of hydrophilic (water loving) and hydrophobic (water repelling) parts in the same molecule. They are being used since the period of Babylonians, 2800 years ago who exploited them in soaps (Geys et al. 2014). Nowadays, majority of surfactants are produced by chemical processes from petrochemical and oleochemical resources. Surfactants are classified depending on their origin as of microbial or synthetic type, produced either by microorganisms or derived from chemical synthesis, respectively. Surfactants acquired from microbes are known as biosurfactants (BSs). In 1960s, BSs were first obtained from microbes via hydrocarbon fermentation as extracellular compounds. BSs are surface-active compounds/ secondary metabolites of biological origin (bacteria, fungi and yeast), synthesized non-ribosomally by actively growing and/or resting microbial cells (Mulligan 2005). BSs form a distinct class of biomolecules with molecular weights ranging between 500 Da and 1000 kDa (van Hamme et al. 2006). They exhibit a distinctive characteristic feature of reducing interfacial tension between the two H. Kaur (✉) Department of Botany, University of Allahabad, Prayagraj, Uttar Pradesh, India P. Kumar (✉) Department of Botany and Microbiology, H.N.B. Garhwal University (A Central University), Srinagar Garhwal, Uttarakhand, India A. Cheema · S. Kaur Department of Agriculture, Sri Guru Granth Sahib World University, Fatehgarh, Punjab, India S. Singh Department of Botany, Kanya Maha Vidyalaya, Jalandhar, Punjab, India R. C. Dubey Department of Botany and Microbiology, Gurukula Kangri Vishwavidyalaya, Haridwar, Uttarakhand, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_13

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liquids (Sekhon et al. 2011) due to their ability to gather between them (Cunha et al. 2004). After produced by the microbe, they remain adherent to microbial cell surfaces or are oozed into the external medium (Femi-Ola et al. 2015). The former situation generally takes place when the microbes are cultured in water-insoluble substrates. In recent years, much interest has been focused toward BSs than their chemically synthesized ones because of their diversity, ecological recognition, low noxiousness, great selectivity, wide foaming attributes, efficacy at intense temperatures or pH conditions, consistency, and extensive applicability (Silva et al. 2014; Mnif and Ghribi 2015). These features allow BSs to be used in solubilization, emulsification, and separation, to lessen surface/interfacial tension and to support adsorption of bioactives via biological membranes (van Hamme et al. 2006; Nitschke and Silva 2018). Since BSs are synthesized from low-price renewable agro-origin raw materials as carbon sources and are easily decomposed, these are considered to be environment friendly (Chrzanowski et al. 2012a). Microbial species reported to produce BS compounds comprise specific unicellular eukaryotes and several Gram-negative and Gram-positive bacteria, for example, Burkholderia, Bacillus, Flavobacterium, Pseudomonas, etc. These microbes live in different environments; nevertheless, their isolation has been reported from mainly unpolluted and stable environments like natural soils and marine ecosystems (Thavasi et al. 2011). Additionally, several studies have revealed that contaminated environments, for example, those polluted with oil and wastewater treatment plants, produce large numbers and diverse BS-synthesizing microbes (Ndlovu et al. 2016). BSs generated by bacteria are of special interest because of their antibacterial, antifungal, and antiviral characteristics along with healing and biomedical perspectives (Inès and Dhouha 2015). BSs can be acquired from microbial activity (e.g., from Pseudomonas and Bacillus), via fermentation processes and enzyme-substrate reactions along with their extracellular production employing biocatalyst enzymes. Both hydrophilic and hydrophobic portions of BSs are synthesized via two independent pathways: either the portions can be substrate reliant or one can be produced de novo, whereas the other is stimulated by the substrate (Arima et al. 1968). Detailed understanding pertaining to the substrate dependency and biosynthetic pathway would improve the structure and, thus, properties of BSs via advances in metabolic and genetic engineering methods. A review by Banat et al. (2014) suggested various bacterial species used to generate a variety of BSs. Many researches have highlighted the impact of carbon source on the kind of BS produced by a particular microbial species (Singh et al. 2014). BSs have been reported to be synthesized from tropical agronomic crop remains (Onbasli and Aslim 2009), coffee-processing industry wastes, fruit-processing industry leftovers, and residue from oil-processing mills (Pandey et al. 2000). Several challenges are linked with the manufacturing of huge quantities of these compounds; specifically, problems are encountered while trying to culture isolated microbes under ordinary fermentation conditions. Other challenges associated with the production of BSs are that the kind, property, and amount of BS synthesized rely on the culture conditions (e.g., agitation rate and incubation temperature) along with macro- and micronutrients available to the BS-synthesizing microbe (Fakruddin 2012; Roy 2017). Therefore,

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high-throughput approaches for speedy and reliable selection of several promising BS-producing microbes are imperative for the discovery of new BSs and/or BS-producing microbial species. The physicochemical traits of BS compounds are essential for their functioning and also aid in screening for their presence in a culture medium. BSs are recognized for their outstanding surface activity, which serves many purposes. These consist of reducing the interfacial and surface tensions between diverse phases (liquid-solid, liquid-liquid, and liquid-air), which act as a factor in lowering critical micelle concentration (CMC) of these compounds and their tendency to form stable emulsions. The power to reduce surface and interfacial tension is due to adsorption of the BS to dissimilar phases. This causes higher interaction and amalgamation of different phases, which assist in solubilizing hydrophobic substrates (Uzoigwe et al. 2015). The screening techniques generally employed for BS production evaluation are hence based on their surface or interfacial property (Walter et al. 2010). Depending on their chemical composition, molecular weight, and microbial species of origin, BSs are categorized into different groups/classes. The lipid-soluble component of BS is typically a fatty acid hydrocarbon chain or one of its derivatives, but it can also be a protein or peptide with a significant amount of hydrophobic side chains. A peptide, a carbohydrate, or an ester group can make up the hydrophilic moiety (Nitschke and Costa 2007). The highest production of BSs is attributed to bacterial species followed by yeasts. Generally, BSs are grouped either as low(glycolipids and lipopeptides) or high-molecular-weight (polymeric BS) compounds, also known as bioemulsifiers (Cameotra et al. 2010). Some of the major classes of BSs include glycolipids, lipopeptides, lipoproteins, phospholipids, fatty acids, polymeric surfactants, and particulate surfactants (Thavasi et al. 2011; Kapadia and Yagnik 2013). Certain BS compounds are needed by the producing microbe for the formation of biofilms and solubilization of hydrocarbon compounds. Several of these compounds can also increase the motility of microbial cells. In addition, BSs display antiadhesive, anticarcinogenic, and antimicrobial traits (Liang et al. 2014). Therefore, they are multipurpose compounds and hence have several applications in agricultural, food, cosmetic, pharmaceutical, and oil industries (do Valle Gomes and Nitschke 2012; Dhasayan et al. 2014). They are also successfully exploited in environmental bioremediation (Mulligan et al. 2014). The majority of BS applications that have been commercialized are primarily propelled by low-cost manufacturing process and cost efficacy (Banat et al. 2010). This was made possible by the low purity standards required for such applications, which eliminated the downstream purification processing stages, which are responsible for nearly 60% of the total manufacturing costs (Sarubbo et al. 2015). The leading businesses in the global BS market are MG Intobio, AGAE Technologies, Jeneil Biotech, Ecover, Saraya, and Saraya, with prospective target markets in Europe, North America, and Asia-Pacific (Sajna et al. 2015). Jeneil BS Co. (Saukville, Wisconsin) made the most successful attempts to bring BS to an industrial level. Jeneil BS Co. (Saukville, Wisconsin), has developed a production method for BS based on rhamnolipids and the ability to conduct fermentation processes in batches up to 20,000 gallons (Rufino

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et al. 2014). With this background, the chapter aims to present an account of types, properties, and production of BSs. Moreover, their emerging potential commercial applications have also been highlighted and discussed in detail.

2 Properties of BSs Due to comparable characteristics of their chemically produced counterparts and the abundance of substrates, BSs are considered suitable for commercial applications (Vijayakumar and Saravanan 2015). Surface and interfacial activities, temperature and pH resistance, biodegradability, low toxicity, biocompatibility and digestibility, emulsion framing and emulsion breaking, and antiadhesive agents are some of the properties of microbial surfactants (Chakrabarti 2012; Vijayakumar and Saravanan 2015; Shakeri et al. 2021). The major features of each attribute of BSs are briefly discussed in the following subsections:

2.1

Surface and Interfacial Activities

Surfactant aids in lowering interfacial pressure and surface tension. Compared to common surfactants, BSs exhibit increased potency and productivity at a reduced mass and produce less surface tension. Bacillus subtilis produces surfactin, which has the ability to decrease surface tension of water to 25 mN m-1 and the interfacial strain between water and hexadecane to below 1 mN m-1 (Cooper et al. 1981). Rhamnolipids, which are synthesized by Pseudomonas aeruginosa, reduce the surface tension of water to 26 mN m-1 and the interfacial strain between water and hexadecane to below 1 mN m-1 (Chakrabarti 2012). Additionally, BSs have a Critical Micelle Concentration (CMC) that is little lower than chemically synthesized surfactants, indicating that less surfactant is essential for the maximum drop in surface strain (Roy 2017). While surface and interfacial tension (oil/water) are roughly at 1 and 30 ml m-1 in the order, CMC of BSs varies from 1 to 2000 mg L-1 (Joshi et al. 2016; Shakeri et al. 2021).

2.2

Temperature and pH Tolerance

BSs have the capacity to tolerate and function under conditions of high pH, temperature, or salinity. For instance, in pH range of 5–12, lipopeptides from Bacillus licheniformis JF-2 maintain their stability at temperatures around 75 °C for 140 hours. BSs can withstand salt concentrations of up to 10%, but chemically manufactured surfactants can only withstand 2% of salt (Joshi et al. 2016; Shakeri et al. 2021). According to McInerney et al. (1990), Bacillus licheniformis

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synthesized lichenysin was resistant to temperatures up to 50 °C, pH values ranging from 4.5 to 9.0, concentrations of NaCl up to 50, and Ca levels up to 25 g L-1. Arthrobacter protophormiae was found to produce another BS that was both thermo (30–100 °C) and pH resistant (2–12). As industrial operations often comprise temperature and pH extremes, it is crucial to distinguish innovative microbial items that are prepared to function in these situations (Roy 2017).

2.3

Biodegradability

BSs are apt for bioremediation and treatment of waste applications, since they are decomposed by bacteria and other microorganisms in soil or water, in contrary to synthetic chemical surfactants that have ecological problems (Gharaei-Fathabad 2011; Shakeri et al. 2021). For instance, Lee et al. (2008) explored how BS sophorolipid suppressed the blooms of the marine protoctist Cochlodinium with a removal potency of 90% in a thirty minute treatment, demonstrating biodegradable attribute of BS (Roy 2017; Swarnalatha and Rani 2019).

2.4

Low Toxicity

In comparison to synthetic allergen products, BSs are considered to be low- harmful or nonharmful compounds that can be employed in cosmetic, pharmaceutical, and food products (Fenibo et al. 2019; Shakeri et al. 2021). For example, sophorolipids from Candida bombicola, with a decreased toxicity profile, were useful in nutrient projects (Hatha et al. 2007; Roy 2017). Additionally, it has been reported that BSs exhibit greater EC50 values than synthetic dispersants (effectual concentration to eliminate 50% of test population) (Swarnalatha and Rani 2019).

2.5

Biocompatibility and Digestibility

The biocompatibility coupled with digestibility of BSs ensures their use in the food, oil, cosmetics, and pharmaceutical industries. Additionally, BSs can be easily acquired in large quantities and are produced using cheap basic ingredients that are also widely available as raw materials. For example, BSs that are more affordable and exhibit higher environmental compatibility can be produced using less expensive agro-industrial waste materials including bagasse, molasses, and plant material residues (Silva et al. 2014). Additionally, BSs are used in environmental control because of their high effectiveness in regulating industrial emulsions, controlling oil spills, detoxifying and biodegrading industrial effluents, and bioremediating polluted soil (Kosaric 2008).

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Emulsion Framing and Breaking

BSs exhibit emulsifying and de-emulsifying properties. An emulsion is a heterogeneous framework made up of immiscible fluids that are dispersed as beads, most of which are larger than 0.1 mm in diameter. Water-in-oil and oil-in-water emulsions are the two types of emulsions. They have a low level of stability that can be compensated by the addition of BSs, which can keep them stable for months or years (Hu and Ju 2001; Roy 2017).

2.7

Antiadhesive Agents

A biofilm is referred to as a collection of microorganisms or additional organic materials accumulated on a surface (Jadhav et al. 2011). The first step in the creation of biofilms is bacterial adherence to surfaces. Numerous factors, including the type of microbe, the electrical charges and hydrophobicity of the surface, ecological conditions, and the microorganisms’ capacity to create extracellular polymers that aid cells in adhering to surfaces, all have an impact on this process (Kachholz and Schlingmann 1987). The BSs can be used to alter the hydrophobicity of surfaces, which in turn affects how tightly microbes adhere to it. For instance, a surfactant produced by Streptococcus thermophilus prevents other thermophilic strains of Streptococcus from colonizing steel. Likewise, adhesion of Listeria monocytogenes to steel surfaces was also prevented by a BS from Pseudomonas fluorescens (Konishi et al. 2008; Roy 2017).

2.8

Biofilm Formation

By providing an environment that is conducive for bacterial adherence, BSs make use of the wettability trait. Bacterial cells congregate to form a common life form, called biofilm, that is encased in an extracellular matrix (ECM) (Dragoš and Kovács 2017). Exo-polymeric compounds, known as bioemulsifiers, aid in the production of biofilms in bacteria and the survival and defense of cells against predators, predatory circumstances, and most significantly water loss from the cell. The surface-attached community of different bacterial planktonic cell types is formed by multiple species that produce distinct signals. Surface area, smoothness, flow velocity, and nutrients are biofilm development elements that have an impact on the biofilm by creating an environment, which is conducive for bacterial growth and attachment (Karlapudi et al. 2018).

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3 Types of Biosurfactants Surfactants obtained from microbes can be classified using different approaches because of immense molecular and structural diversity. They are commonly categorized according to their origin, structural composition, molecular weight, net charge, or mode of action (Rosenberg and Ron 1999). Depending upon their molecular size, they can be classified as low-molecular-weight BSs, for example, glycolipids or high-molecular-weight surfactants, for instance, lipopolysaccharides including polymeric as well as particulate surfactants (Henkel and Hausmann 2019). Because of their amphiphilic behaviour, most of the microbial surfactants comprise of at least one hydrophobic moiety and one hydrophilic moiety. The hydrophilic moieties are made up of phosphates, carbohydrates such as mono-, oligo- or polysaccharides, carboxyl, hydroxyl group, simple esters, or sometimes proteins and peptides. On the other hand, hydrophobic moiety is made up of fatty acids (either unsaturated or saturated), hydroxyl fatty acids (with 8 or 18 carbon atoms), or fatty alcohols.

3.1 3.1.1

Low-Molecular-Weight BSs Glycolipids

Among low-molecular-weight BSs, glycolipids constitute the group that has been most thoroughly studied. They are made up of a hydrophobic moiety that contains long fatty acid chains and a hydrophilic moiety that contains saccharides of various sugars, including glucose, galactose, mannose, glucuronic acid, rhamnose, or sophorose (Paulino et al. 2016). Rhamnolipids, sophorolipids, mannosylerythritol lipids, trehalose lipids, cellobiose lipids, and glucose lipids are some of the further subgroups of glycolipid BSs (Inès and Dhouha 2015).

Rhamnolipids They were initially reported to be produced by Pseudomonas pyocyanea (now P. aeruginosa) cultured on glucose and were first described as “oily glycolipids” by Bergstrom et al. (1946). They are the most intensively studied BSs and their structure and features are well known due to development of several techniques for their detection and analysis. Rhamnolipids are composed of two units, that is, rhamnose (a glycon part) and lipid (an aglycon part). These two units are connected to each other via O-glycosidic bonds. The former part is hydrophilic in nature and consists of one or two α-l-rhamnose moieties linked to each other via α-1,2-glycosidic linkage. The latter part is hydrophobic in nature and consists of one, two, or three β-hydroxy fatty acid chains linked by ester bonds.

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Rhamnolipids were initially discovered in Pseudomonas aeruginosa (Jarvis and Johnson 1949) and even now, it is the predominant organism for production of rhamnolipids, mainly 3-[(6-Deoxy-α-L-mannopyranosyl)oxy] decanoic acid, 3-[3-(2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyloxy) decanoyloxy] decanoic acid, 3-[(2-O-α-L-Rhamnopyranosyl-α-L-rhamnopyranosyl)oxy] decanoic acid, and 3-[3-(α-L-Rhamnopyranosyloxy)decanoyloxy] decanoic acid (Thakur et al. 2021). Besides P. aeruginosa, many other species of Pseudomonas were also discovered for rhamnolipid production. Rhamnolipid production with 3-hydroxytetradecanoic acid by Burkholderia species has also been reported (Hörmann et al. 2010).

Sophorolipids These are low-molecular-weight microbial BSs consisting of sophorose sugar moiety covalently bonded to 17-hydroxyoleic acid. These are produced by a number of nonpathogenic yeast strains, predominantly by Candida torulopsis, C. apicola and Starmerella bombicola, formerly known as Candida bombicola (Henkel and Hausmann 2019). Sophorolipids exist in two states, that is, the lactonic and the acid state depending on the position of the hydroxyoleic acid to 4″sophorose either lactonically in former or presence of acetyl residue in the 6′ and/or 6″ positions in the latter (Nuñez et al. 2001).

Trehalolipids Trehalose lipids, also known as trehalolipids, are different from sophorolipids and rhamnolipids in terms of composition as well as activity. They consist of the nonreducing disaccharide trehalose as the alcoholic component of long-chain α-branched 3-hydroxy fatty acids. Trehalolipids were first reported by Anderson and Newman (1933) in the fat extracts of tubercle bacilli, but, in 1956, a pure form of trehalose lipid was effectively purified from Mycobacterium tuberculosis (Shao 2011). Diverse kinds of trehalolipids are synthesized by various microorganisms, such as Rhodococcus, Mycobacterium, Tsukamurella, Arthrobacter, and Nocardia (Franzetti et al. 2010).

Mannosylerythritol Lipids These are long-chain glycolipids containing 1-β-D- mannopyranosylerythritol or 4-O-β-D-mannopyranosyl-meso-erythritol as the hydrophilic head group attached to a fatty acid as the hydrophobic moiety (Arutchelvi et al. 2008). They are predominantly synthesized by resting cells of yeast species, such as Pseudozyma (Morita et al. 2015), Candida, Schizonella melanogramma (Coelho et al. 2020), and some species of Ustilago (Bölker et al. 2008).

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Cellobiose Lipids Cellobiose lipids are BSs that are composed of combination of diverse acylated low-molecular-weight D-glucolipids, glycosidically bonded to the terminal hydroxy-group of 15,16-dihydroxyhexadecanoic acid (ustilagic acid A) or a 2,15,16-trihydroxyhexadecanoic acid. They are produced as secondary metabolites by various microbes belonging to family Ustilaginaceae. They were first discovered by Haskins (1950) while working on metabolism of glucose and agricultural wastes by various fungal species (Haskins 1950). The major cellobiose lipid producers include Ustilago maydis, Kalmanozyma fusiformata, Anthracocystis flocculosa (formerly Pseudozyma flocculosa), Cryptococcus humicola, and Sporisorium graminicola (Kulakovskaya et al. 2007; Teichmann et al. 2011; Oraby et al. 2020).

3.1.2

Fatty Acids, Phospholipids, and Neutral Lipids

Several microorganisms like yeasts and bacteria secrete huge quantities of phospholipid surfactants and fatty acids and while growing on n-alkanes. Rehm and Reiff (1981) reviewed that biological oxidation of alkanes by microorganisms is responsible for the production of these BSs. These complex BSs typically consist of one or more alkyl residues or hydroxyl groups. Phospholipid vesicles are produced by Acinetobacter sp. HO1-N when cultivated on hexadecane as a source of carbon. The phospholipids produced in these vesicles exhibit surfactant properties similar to glycolipids during emulsification tests (Käppeli and Finnerty 1980). Lipophilic compounds, similar to phospholipids, have also been reported to be produced by Rhodococcus erythropolis when cultivated with n-alkanes as the single carbon source (Kretschmer et al. 1982).

3.1.3

Lipopeptides and Lipoproteins

These are the second biggest group of BSs, which are mostly made up of a fatty acid chain connected to a peptide chain (with 4–12 amino acids). They are extracellular products obtained from diverse Gram-positive (Streptomyces or Bacillus) and/or Gram-negative (primarily Pseudomonas sp.) bacteria. Surfactin was the first lipopeptide named and was obtained from Bacillus subtilis by Arima et al. (1968). Surfactin stands out for its superior surface qualities among the many other lipopeptides that have been found since then. It is a cyclic lipopeptide comprising of various 3-hydroxy fatty acids and seven amino acids. Other known cyclopeptides are lichenysins, polymyxins, iturins, viscosins, fengycins, putisolvins, and amphisins (Sharma 2016).

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High-Molecular-Weight BSs Polymeric BSs

These are high-molecular-weight BSs containing different proteins, polysaccharides, lipoproteins, and lipopolysaccharides produced by various species of bacteria. Among these, emulsan, liposan, alasan, and lipomannan are the most studied polymeric BSs. Candida lipolytica produces a water-soluble extracellular emulsifier called liposan, which contains 83% carbohydrates and 17% proteins (Cirigliano and Carman 1985). Alasan was first described by Navon-Venezia et al. (1995) from Acinetobacter radioresistens as a hetero-polysaccharide protein containing alanine. Cameron et al. (1988) reported the synthesis of huge quantities of mannoprotein by S. cerevisiae, which exhibited remarkable emulsifier activity toward several organic solvents, alkanes, and oils.

3.2.2

Particulate BSs

Extracellular vesicles were reported to be produced by Acinetobacter sp. HO1-N consisting of phospholipids, proteins, and lipopolysaccharide with a thickness of 1.158 cg/cm3 along with 20–50 nm diameter (Käppeli and Finnerty 1979). These extracellular membrane vesicles play an important role in alkane uptake by cells by partitioning the hydrocarbons into micro-emulsion (Rosenberg et al. 1979). These vesicles contain five times higher phospholipids and 360-fold higher polysaccharides as compared to the outer membrane of the microorganism.

4 Production of BSs The microbial BSs are diverse in structure and functionality and are gaining popularity in industries due to their eco-friendly characteristics and biodegradability (Shekhar et al. 2015). These BSs exhibit various advantages over their chemical counterparts in terms of biodegradability, specificity, lower toxicity and ability to function under extreme conditions which makes them exceptional candidates for use in various industries including food, agriculture, water remediation, oil recovery and fields like detergent and cleaning industries (Sachdev and Cameotra 2013; Nitschke and Silva 2018; Geetha et al. 2018; Singh et al. 2019). Literature indicates that these BSs increase the solubility and availability of several water-immiscible substrates and are produced under different environmental conditions (Chrzanowski et al. 2012b). Various types of microbial species including Rhodococcus sp., Pseudomonas sp., Bacillus sp. and Candida sp. are widely used in the production of different types of BSs (Shekhar et al. 2015; Luna et al. 2016).

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In huge level industries, the integration of production and downstream processing is crucial for economical production of BSs. There are several aspects that affect the effectiveness of microbes for BS production. These include: (1) substrates, and (2) physicochemical parameters: community of microbes and growth environment in the bioreactor.

4.1

Substrates

The main obstacle in producing microbial BSs on a large scale is their greater manufacturing costs when compared to chemically synthesized ones. For instance, the cost of the carbon sources accounts for 50–60% of the production expenses via fermentation process. A number of cheap and renewable waste materials for production of BSs have been explored in the past decade (Banat et al. 2014; Satpute et al. 2017). Prominent among these are substrates like sugars, oil wastes, plant oils from agriculture and various other industries, such as distillery (molasses), oil industries, etc. (Fig. 1). The usage of these industrial by-products as carbon source can be an excellent way in reducing the cost of production as well as providing sustainability for industries. Cheaper substrates like soybean oil have been reported to act as a nutrient as well as source for isolation of BS-producing microorganisms (Lee et al. 2008). Soybean oil or chicken fat is usually used as carbon source for production of rhamnolipids (Nitschke et al. 2010). Corynebacterium aquaticum has been reported to produce emulsifying BS by utilizing agricultural waste (Karnwal 2021).

4.2

Physicochemical Parameters: Growth Conditions in the Bioreactor

These are extremely necessary to be optimized as any change in pH, temperature, agitation speed, or aeration will affect the yield and characteristics of the BSs. The optimum temperature for the production of most of the BSs ranges between 25 °C and 30 °C (Desai and Banat 1997). Zinjarde and Pant (2002) examined the impact of pH on BS production and demonstrated that pH of 8.0 is the best for better yield of BSs. Another important aspect in synthesis of BSs is exposure to air and agitation of the media that facilitates the transfer of oxygen from gas to aqueous phase (Md 2012). Production of BSs in a bioreactor/fermenter is a complex method, and choosing a suitable culture method is a crucial step depending upon the type of microbe, bioreactor and product (Fig. 1). The main culture methods are continuous cultures, batch cultures or fed batch cultures (Karnwal 2021). Each of these culture methods has their specificity in relation to biomass kinetics, substrates or final product. The

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Fig. 1 Microbial Biosurfactants: production and applications

continuous culturing of microbes is characterized by their cultivation in a culture with constant rate of growth under constant environmental conditions (Saikia et al. 2012). In continuous cultures, physiological parameters like temperature, pH, substrate concentrations, aeration, agitation and metabolic products remain constant throughout the cultivation process but under regular monitoring (Santos et al. 2016). Batch culturing is extensively used in the synthesis of microbial products such as drug or vitamin industry (Kebbouche-Gana et al. 2013). In this process, both substrates and microbes are supplied on batch basis into a bioreactor (Vanavil et al. 2013). The batch culturing is a simple way of producing BSs but is comparatively costlier than continuous culture method due to periodical cleaning and filling of bioreactors (Behrens et al. 2016). The fed batch culturing is a modified form of batch fermentation. In this, the microbes and substrates are fed into bioreactors for a brief period of time. The fermentation process is then interrupted once the broth volume reaches 1/fourth of the bioreactor volume (Rodriguez-Contreras et al. 2013). This allows the steady flow of substrates for achieving high concentrations and prevents the undesirable changes in substrate inhibition or changes during cellular

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metabolism. The advantage of this method is that it can be applied to large amounts of substrate and can be managed at a certain target level (Sarilmiser et al. 2015; Xu et al. 2020).

5 Commercial Applications of BSs In many industrial processes, including lubrication, wetting, softening, etc., BSs have the potential to replace synthetic surfactants. For commercial uses, glycolipids and lipopeptides are the most significant BSs. The petroleum sector (33%), cosmetics (15%), antimicrobial agent and medicine (12%), and bioremediation are the industries with the highest percentage of patents on BSs (Shete et al. 2006; Silva et al. 2014). A significant number of the patents are for sophorolipids (24%), surfactin (13%), and rhamnolipids (12%). Applications of BSs in various fields have been described as follows and have been tabulated in Table 1 and represented in Fig. 1.

5.1

Agriculture

BSs can reduce surface tension at air/water interfaces as well as interfacial tension at oil/water interactions (Satpute et al. 2010; Banat et al. 2010); therefore, they can be widely used in agriculture-related fields to boost the biodegradation of pollutants and enhance soil quality (Table 2). They can also be used to stimulate plant growth indirectly, since they have antibacterial properties and promote favourable plantmicrobe interactions. It has been found that soil-dwelling bacteria use these BSs as a carbon source, which allows for the organic elimination of BSs from rural soil (Kim et al. 2011). Following are some BSs applications related to agriculture:

Table 1 Multifunctional prospective of biosurfactants in agriculture Green surfactants from soil microbes

Function Biodegradation of contaminated agricultural soil Enhancement of plant microbes interaction Plant growth by elimination of phytopathogen Application in pesticides industry

References Phule and Singh (2013), Satpute et al. (2010) Nihorimbere et al. (2011) Singh et al. (2007), Krzyzanowska et al. (2012) Takenaka et al. (2007), Lima et al. (2011a)

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Table 2 Applications of biosurfactants in medical, laundry, cosmetics, and oil industries In Medical Industry Biosurfactant types Surfactins

Microorganisms Lactobacillus sp.

Applications Antibacterial

Lactobacillus surlactin Bacillus pumilus Treahalose lipid

Rhodococcus erythropolis

Pumilacidin

Bacillus pumilus

Bacillomycin D, Iturin, Iturin A, Surfactin

Bacillus subtilis

Antifungal

Lipopeptide, Surfactin Iturin, Mannosylerythritol lipids

Bacillus subtilis

Antimycoplasma

Bacillus subtilis

Immunological adjuvants

Candida antatica

Anticancer

In Laundary, cosmetic, and oil industries Rhamnolipids Pseudomonas aeruginosa Pseudomonas cepacia CCT6659 Lipopeptides

Bacillus licheniformis JF-2

Antiviral (HSV) and influenza virus

Reduce surface tension, cosmetics Cleaning of beaker walls contaminated with a layer of oil Tolerate extreme temperature

B. subtilis strain A1

Emulsification

Lichenysin

Arthrobacter protophormiae

Tolerate extreme temperature

Sophorolipid

Cochlodinium

Control bloom

References Fracchia et al. (2012), Emmanuel et al. (2019) Hultberg et al. (2008) Biniarz et al. (2017) Krishnaswamy et al. (2008) Biniarz et al. (2017) Fariq and Saeed (2016), Mnif and Ghribi (2016) Kumar et al. (2007) GharaeiFathabad (2011), Pervaiz et al. (2022) Krishnaswamy et al. (2008), Ligia et al. (2006) Chakrabarti (2012) Rocha e Silva et al. (2013) Joshi et al. (2016), Shakeri et al. (2021) Parthipan et al. (2017) Das and Mukherjee (2007), Roy (2017) Lee et al. (2008), Roy (2017), Swarnalatha and Rani (2019) (continued)

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Table 2 (continued) In Medical Industry Biosurfactant types

Microorganisms Candida bombicola

Applications Helpful in nourishment ventures

Nocardiopsis

In toothpaste preparation

Phospolipids

Acinetobacter venetianus

Oil removal

Trehalolipids

Mycobacterium tuberculosis, Rhodococcus erythropolis, Arthrobacter sp., Nocardia sp., Corynebacterium sp. Pseudoalteromona agarivorans

Enhancement of the bioavailability of hydrocarbons

Exopolysaccharide

5.1.1

Moisture retention, absorption of free radicals, potential applications in food and cosmetics industries

References Hatha et al. (2007), Roy (2017) Das et al. (2013), Bhattacharya et al. (2017) Bach et al. (2003), Silva et al. (2014) Franzetti et al. (2010)

Hao et al. (2019)

Improvement of Soil Quality

Pollutants, both organic and inorganic, that cause abiotic stress in crop plants, are a major hindrance to agricultural land productivity. Bioremediation is needed to improve the condition of soil that has been contaminated by hydrocarbon and heavy metals. Both of these can be successfully removed using microorganisms that produce BSs. Through BSs, a highly important phenomenon of desorption of hydrophobic pollutants that are tightly bound to soil particles is enlarged, which is essential for the bioremediation process. Additionally, BSs can speed up the decomposition of some chemical pesticides accumulated in agricultural soils (Sharma et al. 2009; Zhang et al. 2011).

5.1.2

Plant Pathogen Elimination

In order to achieve sustainable agriculture, a number of BSs from microorganisms show antimicrobial activity against plant infections. As a result, they are regarded as attractive bio-control molecules. Rhizobacterial BSs are recognized to have antagonistic characteristics (Nihorimbere et al. 2011). Chemical surfactants and BSs are used in agriculture for the biocontrol of bacteria, which in turn promotes plant development through antibiosis, parasitism, competition, hypo-virulence, and induced systemic resistance (Singh et al. 2007). For instance, Pectobacterium and

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Table 3 Nature and properties of various surfactants Surfactant Sodium dodecyl sulfate Tween 20 Linear alkylbenzene sulfonate Rhamnolipid

Nature Anionic Nonionic Anionic Anionic

CMC value, mg/l 2380 140 120 20

References Arsyah et al. (2018) Effendi et al. (2018) Arsyah et al. (2018) Helmy et al. (2019)

Dickeya species that cause soft rot have been biocontrolled by rhizospheric isolates of Bacillus and Pseudomonas that produce BSs (Krzyzanowska et al. 2012). Rhamnolipids are shown to suppress fungal plant diseases that produce zoospores and have developed resistance to chemical insecticides used commercially (Kim et al. 2011; Sha et al. 2011) Additionally, research has revealed that rhamnolipid has the ability to boost plant immunity, which is considered to be a different approach for lowering plant pathogen infection (Vatsa et al. 2010) (Table 3). Rhamnolipid has recently been discovered to be an insecticide, and researchers have isolated BS from Pseudomonas strain that exhibits insecticidal action against Myzus persicae (green peach aphid) (Kim et al. 2011).

5.1.3

Asset for Plant Microbe Interactions

It is crucial to interact with the plant in order for rhizobacteria to benefit the plants. Microbial characteristics, like motility, the ability to form biofilm on root surfaces, and the production of quorum sensing molecules, are required to make contact with the plant. A study found that the rhizobacteria need quorum sensing molecules like acyl homoserine lactone (AHL) to create antifungal substances on surfaces like roots (Nihorimbere et al. 2011). The addition of BS is also believed to hasten the composition process by fostering microbial growth. As a result, these green surfactants are crucial elements for microorganisms to establish advantageous interaction with the root system and boost plant growth. Additionally, the BSs that rhizobacteria secrete increase the bioavailability of substances that are hydrophobic in nature and may serve as nutrients. The wettability of the soil and the allocation of chemical fertilizers in the soil are improved by BSs produced by soil microorganisms, assisting in the promotion of plant growth (Sachdev and Cameotra 2013).

5.1.4

Molecular Means for Profiling of BS Producing Microbial Community from Agricultural Soils

The traditional techniques for testing microorganisms for the formation of BS are well known. The techniques used to purify BSs include high pressure liquid chromatography, thin layer chromatography, and phase separation technology (Baker and Chen 2010). Molecular techniques are being used in addition to conventional methods to detect BS-producing bacteria. The literature has extensive

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documentation of methods like cloning, PCR, sequencing, transposon mutagenesis and homology analysis. PCR-based methods focus on genes either involved in BS synthesis or regulation. The molecular approach is limited to a small number of bacterial strains, thus novel BS from uncultured microbes in the soil biosphere must be investigated using cutting-edge approaches, such as functional metagenomics. The following procedures can be used to characterize BS production in bacteria from a particular habitat molecularly, especially those from agricultural soil that has been contaminated with heavy metals, hydrocarbons, and crude oil (Sachdev and Cameotra 2013): • Soil samples can be used to directly isolate whole DNA, which can then be analysed using PCR to target and amplify particular sequences. • Cloning or genetic fingerprint analysis can be used to examine PCR products. A quick and easy electrophoretic examination of the PCR results creates a genetic fingerprint that allows for the analysis of the community’s genetic makeup. • Assessment of a community’s genetic diversity and individuals’ phylogenetic affiliation are made possible by the characterization of cloned sequences. • Similarly, sequencing the bands in a fingerprint profile can help identify certain populations or types of BS that predominate in a given niche. • Functional communities can be examined using DNA microarray technology, SIP (Sterilization in place), and FISH (Fluorescence in situ hybridization), which can also be used to determine the genetic makeup of BS-producing communities.

5.2

Medicine

Besides chemical substances alone, a variety of microorganisms can produce surface-active molecules. In contrast to artificial surfactants, microbes can synthesize unique BSs that are less harmful and more biodegradable. BSs play an important role for controlling various diseases which are caused by bacteria, viruses, etc. (Atipan et al. 2020). Some of the antimicrobial activities of BSs are discussed underneath:

5.2.1

Antibacterial Performance of BSs

Due to the capacity of molecules of lipopeptides to self-assemble and form a micelle aggregation inside a lipid membrane (which is crucial for their antibiotic activity) they are thought to be the most effective antibacterial agents (Biniarz et al. 2017). For instance, the biological and physical characteristics of surfactin include antibacterial, antifungal, anticancer, antiviral, anti-inflammatory, antimycoplasma, antiplatelet, and hemolytic activity (Seydlova and Svobodova 2008; Deleu et al. 2008). Numerous research studies are currently being carried out in order to produce novel BSs and antimicrobial compounds that may be used for biotechnological and medical applications as well as to battle diseases with resistance. The efforts

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frequently fall short of an understanding of the molecular mechanisms underlying the production of secondary metabolites because the primary approaches typically involve a number of cultivation conditions and experimental analysis before considering which kinds of secondary metabolites are produced by a microorganism (Hippolyte et al. 2018; Emmanuel et al. 2019; Giani et al. 2021). With the development of novel bioinformatics techniques, there are now more bacterial genome sequences that can be used to reconstruct biosynthetic gene clusters (BGCs) that may encode routes for producing particular metabolites (Ceniceros et al. 2017; Zampolli et al. 2018). Therefore, the examination of a strain genome could serve as the starting point for the search of novel antibacterial agents and BS molecules.

5.2.2

Antifungal Activity

Although there have been only a few studies against human pathogenic fungi, biosurfactants have long been claimed to have antifungal properties (Table 2). Numerous human mycoses and pathogenic yeasts, such as Candida spp., Colletotrichum gloeosporioides, Cryptococcus neoformans, Corynespora cassiicola, Fusarium spp., Fusarium oxysporum, Trichophyton rubrum, Rhizoctonia spp. and Trichosporon asahii, were significantly suppressed in vitro (Fariq and Saeed 2016; Mnif et al. 2018), signifying antifungal property of BSs.

5.2.3

Anticancer Activity

Few microbial extracellular glycolipids encourage cell differentiation rather than proliferation in the human pro-myelocytic leukaemia cell line. Additionally, MEL (mannosylerythritol lipid) treatment of PC 12 cells elevated acetylcholine esterase activity and interrupted the G1 phase of the cell cycle, resulting in excessive neurites development and incomplete cellular differentiation. These findings support the use of the microbiota and imply that MEL encourages neural development in PC 12 cells (Krishnaswamy et al. 2008). BSs are secondary metabolite substances produced by bacteria, fungi, and other microorganisms that have an impact on surface chemistry by reducing surface interphase tension and promoting the development of emulsions, gels, and foams (Twigg et al. 2021). Additionally, the pharmaceutical and biomedical industries have shown interest in using glycolipids due to their potential benefits, including compatibility with human skin, low toxicity, anticancer, woundhealing, and immune-modulatory effects (Adu et al. 2020).

5.2.4

Immunological Adjuvants

When provided along with a particular antigen, immunological adjuvants are chemicals that typically increase the intensity and persistence of the immune response in an organism that is specific to the antigen. An immunologic adjuvant

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attacked by the immune response can either be humoral or cellular, depending on the type of antigen. A powerful immunologic adjuvant, lipopeptide BS of bacterial origin possesses good immune-modulatory activity (Naughton et al. 2019). In order to produce high titers of antigen-specific antibodies, immunologic adjuvants are typically combined or blended with haptens that have limited immunogenicity. Prior to combining and binding with immunologic adjuvants, low-molecular-weight antigens are typically associated with appropriate carriers, such as poly-L-lysine (PPL), bovine albumin (BSA), keyhole limpet hemocyanin (KLH), etc. (Pervaiz et al. 2022).

5.2.5

Antiviral Activity

There are reports related to the antibiotic as well as inhibitory effects on growth of human immune-deficiency virus in leucocytes by BSs in literature (Fechtner et al. 2017). In addition, Krishnaswamy et al. (2008) noted that a female regulated, effective, and safe vaginal topical microbicide was required because of the rise of HIV in women. Sophorolipid surfactants from Candida bombicola and their structural counterparts including the sophorolipid diacetate ethyl ester, the most potent spermicidal and virucidal agent against human semen, have been found to have nonoxynol-9-like virucidal effects. Surfactin and enrofloxacin have been found to work synergistically to destroy mycoplasma in a manner that is around two orders of magnitude more effective than when the drugs are used separately. The antibacterial, antiviral, anticancer, hypo-cholesterolemic, antiadhesive, insecticide, apoptotic, and hemolytic action of surfactin are just a few of its intriguing qualities (Bjerk et al. 2021).

5.2.6

Gene Delivery

According to Gharaei-Fathabad (2011), for both fundamental sciences and therapeutic applications, gene therapy is an effective and secure way to introduce the foreign nucleotides into mammalian cells. It is believed that the lipofection method using cationic liposomes is a potential way to transfer foreign genes to the target cells while avoiding any unfavourable side effects (Banat et al. 2010; Parsad et al. 2015). Additionally, the ability to use surfactants as agents for enhancing stem fibroblast metabolism and immune-modulatory function has been made possible by the isolation of genes for protein molecules of BSs and cloning in bacteria (Fakruddin 2012).

5.3

Commercial Laundry Detergents

Surfactants are a crucial component in contemporary commercial laundry detergents and are hazardous to freshwater living beings almost universally. The search for

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environmentally acceptable, natural alternatives to chemical surfactants in laundry detergents has been triggered due to an increased awareness among the human population regarding dangers and environmental issues associated with chemical surfactants. When heated at high temperatures, the surface-active property of BSs like Cyclic Lipopeptide (CLP), which is stable throughout a wide pH range (7.0–12.0), is not lost (Mukherjee 2007). These show good emulsion-forming ability with vegetable oils and strong stability and compatibility with conventional laundry detergents, justifying their incorporation in the formulation of laundry detergents. Cleansing solid things with detergents involves getting rid of undesirable items from the surface. During this cleaning procedure, materials can be separated in a number of ways, such as solvent separation, chemical separation (addition of water and surfactants), and simple mechanical separation (e.g., washing and immersing the cloth in water). The substrate or the items to be cleaned, that is, the dirt and stains that would be eliminated during the washing process and the liquid bath, make up a washing system with detergent (Helmy et al. 2019). Some detergents have just one component like toilet soap. The other detergents may contain multiple ingredients. Surfactants, builders, fillers, and additives are the main components of multimaterial detergent compositions. Additional supplementary ingredients used in commercial formulae include water softeners, antideposition agents, bleaching agents, enzymes, dispersion agents, perfumes, and other small ingredients (Arsyah et al. 2018). When it comes to lowering the surface tension of liquids, BSs made from extracellular microorganisms are on par with their synthetic counterparts. Such BSs possess a hydrophilic head, which is usually a simple sugar or peptide and a hydrophobic tail, which is usually a lipid. Rhamnolipids are a class of BSs often made by Pseudomonas aeruginosa bacterial strains. Table 3 shows the critical micelles concentration (CMC) value for the rhamnolipid BSs employed in this investigation. Surface tension was measured at various concentrations to determine the CMC value (Arsyah et al. 2018). The surface tension below the CMC decreases with increasing BS concentration as the quantity of active agent at the interface increases. The surface tension of solution remains steady, because the interfacial BS concentration is greater than the CMC. Table 3 shows that rhamnolipids have a significantly lower CMC of 20 mg/L than other surfactants (Effendi et al. 2018).

5.4

Food-Processing Industry

BSs have been employed in a variety of food-processing applications, but they are most frequently utilized as an antiadhesive agent and a component in food formulations. Due to their ability to lower surface and interfacial tensions, they contribute to the formation and stability of emulsions as a part of food compositions. Finding alternative natural sources for BS compounds suitable for use in novel and cuttingedge preparations in the food and other industries has gained increased attention due

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to the desire to reduce reliance on plant emulsifiers derived from genetically modified (GM) crops such as GM modified soybean. According to Marcelino et al. (2019), Cutaneotrichosporon mucoides UFMGCM-Y6148 was the primary producer of sophorolipids, a type of glycolipid on a medium containing xylose and sugarcane bagasse. Guerfali et al. (2019) found that Rhodotorula babjevae Y-SL7 grew in a medium supplemented with various carbon sources, resulting in the formation of glycolipids with high emulsification and mild toxicity. Since this raises the product’s consumer appeal, it is advantageous for the constituents included to a formulation in a food application to have overall nutritional value. This nutritional value is correlated with the presence of polyunsaturated fatty acids (PUFAs), particularly the n-3 series [mainly alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA)], and n-6 PUFA series [linolenic acid (LA)]. The nutritional value of foods increases with a higher concentration of 18-carbon acids, because these elements are crucial for the human body (Burdge 2019; Gramlich et al. 2019).

5.4.1

Food Emulsifiers

In most food industry products, emulsification affects consistency, phase dispersion, texture, and the solubilization of smells (Radhakrishnan et al. 2011). The purpose of an emulsifier is to stabilize the emulsion by controlling clustering of globule and keeping aerated systems in place (Patino and Anchez 2008). An emulsion consists of a heterogeneous structure made up of droplets of minimum one immiscible liquid dispersed across another. Surfactants can improve the stability of these systems by lowering the interfacial tension (Ribeiro et al. 2020). Both BSs and bioemulsifiers are frequently used in the same sense. Those that lower surface tension at the air-water boundary are referred to as BSs, while those that cause emulsification are known as bioemulsifiers. Colloids and food emulsions are intricate systems made up of various food ingredients. For liquid emulsions, such as drinks, dressings, sauces, alcoholic emulsions, and others, to remain stable, amphiphiles with low molecular weight are essential. As a result, the majority of food colloids are composed of protein and hydrocolloid mixtures, both monomeric and polymeric. The use of artificial emulsifiers is continuously subjected to regulations and prohibitions by food organizations and health authorities around the world (Tao et al. 2019). In addition to their important role as surface-tension- and interfacial-tension-lowering agents, improving stabilization of emulsion formation, microbial BSs have other potential advantages in food, such as maintaining the consistency and shelf-life of products having starch, improving the texture of fat-based products, stabilizing aerated systems, agglomerating fat globules, and altering the rheological features of wheat dough (Campos et al. 2013). In addition to acting as fat stabilizers and antispattering agents, BSs have been found to boost consistency, delay staling, and solubilize flavour oils in bread and ice cream formulations (Marchan and Banat 2012). Particularly,

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rhamnolipids have been utilized to improve the consistency, volume, stability, and preservation of bakery items (Kralova and Sjeoblom 2009).

5.4.2

Antioxidant Agents

Adaptable BSs like mannosylerythritol lipids (MELs) are known for their flexible biochemical and interfacial characteristics. BSs demonstrate some potential as antioxidant agents. In vitro antioxidant activity of BSs has been reported by Takahashi et al. (2012) by using a free-radical-scavenging analysis method. The scientists came to the conclusion that MEL-C had the highest shielding and antioxidant features in cells and suggested its possible use as an ingredient in skin-care products designed to fight ageing. Analogous results were obtained with a BS that was isolated from B. subtilis RW-I, which exhibited antioxidant property to eliminate free radicals and could be used as a replacement for naturally occurring antioxidants (Yalcin and Cavusoglu 2010). A Klebsiella polysaccharide emulsifier has been shown to have a strong inhibitory effect on self-oxidation of soybean oil. By isolating the oil from its surroundings and encapsulating it, the emulsifier prevented soybean oil from oxidization. In France, this polysaccharide was being developed as a potential source of rhamnose for producing furaneol, a flavouring ingredient (Singh et al. 2007). Several research teams have recently looked into numerous BSs that have excellent potential antioxidant qualities from various sources. Some of the BS also showed antibacterial and antiproliferative properties in addition to their putative antioxidant action (Merghni et al. 2017; Basit et al. 2018; Ekramu et al. 2020). The antibacterial, antioxidant, and antiadhesive characteristics of the BS compounds obtained from Bacillus licheniformis VS16 and Bacillus subtilis VSG4 were evaluated by Giri et al. (2019). They found that compared to Bacillus licheniformis VS16, Bacillus subtilis VSG4 had higher antioxidant activity (Kumar et al. 2021).

5.5

Cosmetics Industry

Cosmetics are essential for human life, since many of them are used regularly by people, including soaps, shampoos, toothpastes, deodorants, skin care products, perfume, and makeup. Baby products, toothpaste, mascara, lipsticks, and denture cleansers, etc. typically contain BSs. They are also used as wetting agents, emulsifiers, solubilizers, and foaming agents (Gharaei-Fathabad 2011; Md 2012). To promote the transport of molecules via skin, BSs can be utilized as percutaneous absorption enhancers. Ionic surfactants increase transdermal absorption by denaturing keratin and disordering the lipid layer of the stratum corneum. On the other hand, enhancers may promote cosmetic penetration by inducing the stratum corneum to enlarge and/or leach off part of its structural components, lowering the diffusion resistance, and increasing the skin permeability. The organized layer of

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intercellular lipids and the low water content of skin both contribute to its limited permeability. Because of the abundance of cysteine residues in keratinized tissue proteins, the insoluble character of this protein may be caused by the protein’s strong disulfide bonds. The use of BSs as reducing agents leads to reduction in the number of disulfide bridges, which raises the hydration of the proteins, thereby increasing skin permeability. Azone is one of the best percutaneous absorption enhancers as well, as it permeates lipid bilayers and damages their structure (Haque and Talukder 2018). The fluidization of the intercellular lipid lamellar area of the stratum corneum by azone is one potential mode of action. Azone is a relatively nonpolar chemical, while dimethyl sulfoxide (DMSO), which penetrates the aqueous region and interacts with the polar heads of lipid molecules to produce a thick solvation shell, is a substantially dipolar solvent. As a result, both azone and DMSO increase lipid fluidity, lowering the lipid barrier’s resistance to drug diffusion. Hexamethylene lauramine and N, N di-substituted amino acid alcohol derivatives also increase the permeability of cosmetics. Due to their low cost, BSs are also replacing chemical surfactants in some cases. For instance, natural or synthetic ceramides can reduce the roughness of the skin surface but are quite expensive to produce (Sethi et al. 2016). Mannosylerythritol lipids (MELs), which have comparable qualities, provide a feasible alternative at a cheaper production cost (Yamamoto et al. 2012). MELs have also been linked to skin roughness, water retention, and hydration (Lin et al. 2011; Yamamoto et al. 2012). In addition, Takahashi et al. (2012) demonstrated that MEL-C had antioxidant and protective properties in human skin fibroblasts against H2O2-induced oxidative damage. Among all glycolipids, the maximum radical scavenging activity was demonstrated by MEL-C, which exhibited 50.3% scavenging activity at 10 mg/mL (Pillaiyar et al. 2017). Similar to hyperpigmentation, which results in freckles due to excessive melanin production (Pillaiyar et al. 2017), the exploitation of MELs as a component of skin-whitening formulations has been suggested to be effective in reducing production of melanocyte and enhancing skin colour (Yoo et al. 2019; Adu et al. 2020). Additionally, with encouraging results, Owen and Fan (2013) tested the usage of oligomer BSs in the composition of conditioning hair masks. Pseudomonas antarcticain was used as the source of the formulation by Kitagawa et al. (2011) to create a skin-nourishing cosmetic. Additionally, a study by Rikalović et al. (2015) suggested formulating a shampoo with 2% rhamnolipid dissolved in water and using rhamnolipid biosurfactant derived from Pseudomonas aeruginosa. The afore-mentioned bio-antibacterial surfactant properties preserved the scalp odour-free and shiny for three days. Similarly, Cox et al. (2013) created a gentle formulation ideal for shower gels by combining an anionic surfactant with a sophorolipid biosurfactant. This proprietary recipe had 40–98% water, 1–20% sophorolipid, 1–20% chemical anionic surfactant, 0–10% foam-booster surfactant, 0–2% extra electrolyte, and 0–10% extra detergent additives (Masaru et al. 2007). Numerous cosmetic formulations including at least one BS and one fatty acid were examined by Allef et al. (2014). In order to create various cosmetic formulations, such as conditioning antidandruff shampoo, body cleanser, moisturizing skin cleanser, shower gel, etc., rhamnolipids and sophorolipids were combined with 10% oleic oil. In a recent study, sodium dodecyl sulphate (SDS) was

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replaced with a BS derived from the bacterium genus Nocardiopsis for the manufacturing of toothpaste. These findings show that BSs could replace synthetic surfactants, since they are more environment friendly as well as less toxic (Das et al. 2013; Bhattacharya et al. 2017).

5.6

Petroleum and Oil Recovery

Petroleum is an essential source of energy and a key factor in a nation’s economic progress, especially in emerging nations. BSs are very popular in the petroleum sector due to availability of effective production techniques, economically cheaper, and because they have immense applications in biotechnology (Banat et al. 2010). Utilizing any kind of BS can enhance, optimize, or improve all processes, including oil searching and production, product handling, refining, transportation, and managing situations involving accidental pollution or discharge of oil (De Almeida et al. 2016). Phase separation, hydrophilicity, solubility, foaming, emulsification, de-emulsification, corrosion resistance method, and viscosity diminution of heavy crude oils are some of the characteristics of BSs that make them widely used in the petroleum industry (Kapadia and Yagnik 2013; Santos et al. 2013). BSs have been employed successfully in the petroleum industry for searching heavy oil due to advantages they have over their synthetically synthesized counterparts along the entire chain of petroleum processing (Silva et al. 2014).

5.6.1

Crude Oil Reservoir Extraction

According to Al-Sulaimani et al. (2012), a number of commercially used improved oil recovery technologies (thermal, chemical, physical, etc.) are both expensive and detrimental to the environment. Therefore, BSs are useful in this field, since these organic substances boost the mobilization of hydrocarbons, which in turn improves the crude oil recovery from reservoirs through a procedure known as microbialenhanced oil recovery (MEOR) (Perfumo et al. 2010). It is a type of tertiary oil revival in which residual oil is recovered using microbes or their metabolic by-products. Polymers and BSs are made by microorganisms, which lessen the capillary pressures that prevent oil from passing through rock pores (Sun et al. 2018). BSs also assist in emulsifying and dissolving oil film in rock. MEOR employs a number of methods, including induction of BS-producing bacteria into the reservoir followed by their spreading, addition of nutrients to reservoir to promote growth of microbes, and the continued production of BSs in reactors before their introduction into the reservoir (Al-Bahry et al. 2013). These procedures improve recovery of oil from an exhausted reservoir, prolonging the reservoir’s life. MEOR costs less than oil recovery via chemicals, because microbes can manufacture effective products from inexpensive substrates or raw materials (Sarafzadeh et al. 2013).

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Pipeline Transportation of Crude Oil

Crude oil is frequently transported over large distances from the extraction regions to the refineries. Operational challenges associated with the transport of extra-heavy and heavy crude oil restrict its commercial viability. According to Cerón-Camacho et al. (2013), the main issues are low flow ability caused by heavy crude oil’s high viscosity and asphaltene content, which causes inconveniences like the deposition of paraffins and/or asphaltenes along with decline in pressure that leads to pipeline plugging issues. Recently, a potential technique for creating a steady oil-in-water emulsion, which promotes oil mobility, has been devised. For this application, BS-based emulsifiers (bioemulsifiers) are especially appropriate. Unlike glycolipids and lipopeptides, bio-emulsifiers are high-molecular-weight surfactants possessing unique characteristics. These products have a high ability to stabilize oil-in-water emulsions in addition to being effective for lowering interfacial tensions (Silva et al. 2014). Bioemulsifiers produce a strong bond with oil droplets coupled with a strong obstacle that thwarts drop coalescence because of the numerous reactive groups that are present in the molecule (Perfumo et al. 2010). Petroleum sector applications for emulsan, the strongest bio-emulsifier, include the production of heavy oil-water emulsions to reduce viscosity during pipeline transmission (Silva et al. 2014).

5.6.3

Cleaning of Oil Storage Tanks

Daily huge volumes of crude oil are transferred to refineries and stored in tanks. These tanks need to be cleaned occasionally for maintenance. Nevertheless, heavy oil and waste parts gets accumulated at the base and on storage tank walls, which are extremely viscous and solidify into deposits, which are impossible to remove with standard pumping. These must be manually cleaned and washed with solvents to be removed, which is a risky, labour-requiring, expensive, and time-intense process that may also involve hot water spraying and solvent liquefaction (Perfumo et al. 2010; Matsui et al. 2012). By creating an oil-in-water emulsion, which makes waste pumping easier, microbial BSs offer a different cleaning method for reducing the oil deposits and sludge viscosity. Furthermore, when the emulsion breaks, this procedure enables the recovery of crude oil. For instance, Gordonia spactinomycete JE1058BS,was used by Matsui et al. (2012) to clean oil tank bottom sludge. The BS was more effective at dispersing than a chemical- or plant-derived surfactant and was equally effective for at least three weeks. To clean oil-contaminated vessels, the supernatant from P. aeruginosa SH 29 was examined by Diab and Din (2013). The oil was separated from the supernatant and washed off from the vessel walls and bottom after about fifteen minutes. According to the scientists, the BS in P. aeruginosa SH 29’s sterilized supernatant may be used directly to clean crude oil storage tanks and other vessels employed for its transit and storage. A BS isolated from Pseudomonas cepacia CCT6659 was examined by Rocha e Silva et al. (2013)

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for its ability to clean walls of beaker polluted with oil. They discovered 80% clearance rate, which suggests the BS might be used to clean storage tanks. According to theories, solubilization, emulsification, or mobilization, which increase the surface area in contact with the hydrocarbons, are the processes responsible for BS-enhanced oil removal and recovery (Joseph and Joseph 2009; Santos et al. 2016). The solubilization capacity of a surfactant refers to its ability to increase the solubility of hydrophobic components in an aqueous phase. This ability is greatly enhanced when micelles are formed due to hydrocarbon partitioning in the hydrophobic region of the micelles. Since BS concentration affects how soluble hydrocarbons are in water, higher BS concentrations are often required in this procedure. Conversely, mobilization involves both displacement and dispersion. The interfacial tension is decreased when hydrocarbon droplets are released from the porous material due to displacement. The capillary forces that lead to the creation of residual saturation can also be overcome by a substantial decrease in the interfacial tension between water and oil phases. It can also happen when trapped hydrocarbon is subjected to displacement. Therefore, displacements are more closely related to the interfacial tension between water and hydrophobic phases rather than emulsion formation. Contrarily, the process of dispersion involves the dispersion of hydrocarbons into aqueous phases as a result of the production of emulsions, and thus it is connected to both interfacial tension and surfactant concentration (Santos et al. 2016). The BS generated by Pseudomonas sp. was capable of removing 80–90% of the motor oil that had been absorbed into the sand (Silva et al. 2017; Mondal et al. 2022). Additionally, BSs are useful for bioaugmentation of soil ecosystems polluted with hydrocarbons. Five bacterial synthesized BSs were shown to be especially effective in the cleaning of oil tanks and oily sludge from refineries as their application led to 95% recovery of oil from oily sludge (Lima et al. 2011b). The moisture-retaining ability of BSs allows them to absorb oil from porous mediums. The Acinetobacter venetianus–derived BS removed 89% of the crude oil from porous limestone at 0.1 mg mL-1, and 98% at 0.5 mg mL-1 concentration (Bach et al. 2003; Silva et al. 2014).

6 Conclusions and Future Prospects BSs have numerous applications in various industrial processes, making them multifaceted compounds of the twenty-first century; thanks to their immense structural diversity resulting in a wide range of properties, which explains continuous interest of scientific community in this group of molecules. The investigations in the field of BSs are progressing at a fast rate, encompassing other fields, such as surface science, medicine, organic chemistry, molecular biology, etc., thereby increasing the production of BSs. Conventional chemical synthesis techniques of BSs raise concerns about their toxicological impacts on environment, resulting in switching to more sustainable synthesis alternatives. BSs produced from renewable resources are favoured as they use low-temperature, cost effective methods, are sustainable, and

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produce least waste. Due to biodegradable and low toxicity nature, BSs have huge potential in remediation technologies. In spite of the rising demand, the industrial production of BSs is still a challenge owing to raw material and processing costs coupled with low output. Production of BSs may become more profitable and economically feasible under optimal growth/production conditions using inexpensive renewable raw materials and cutting-edge multistep downstream processing methods. The economic viability of BS production may also be increased through the co-isolation of other by-products, such as enzymes. Additionally, mutant and recombinant hyperproducing microbial species with the capacity to thrive on a wide array of low-cost raw materials may boost BSs’ yield and may lead to the much expected crucial breakthrough in their successful production. To regulate production and boost product yields, a thorough investigation of the genes involved in BS synthesis is required. BSs with novel properties and structures need to be developed using molecular biotechnological techniques. Lastly, the structural modifications of BSs may expand the range of applications for these compounds.

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Role of Biosurfactants in Enzyme Production Rengasamy Sathya, Mariadhas Valan Arasu, Naif Abdullah Al-Dhabi, and P. Vijayaraghavan

1 Introduction Microbial biosurfactants are highly valued due to the presence of hydrophilic and hydrophobic zones within a molecule, which allows them to reside in mixed-part systems and modify the balance environment. The majority of cosmetic products are made by enzymatic conversion of hydrophobic molecules. It is important to have biosurfactants in antimicrobial features, biofilm infraction, healthcare, food industry, hydrocarbon decomposition, and quantum sensing (Gudina et al. 2015) (Table 1). Biosurfactants are capable of a wide range of surface activities including solubilization, wetting, foaming, dissolution, dispersion, and emulsification. The surfactant is closely associated with the phase mixture of water+air, oil+water, and oil+solid +water (Banat et al. 2000). Bodour et al. (2003) show that biosurfactants have an important evolutionary benefit by permitting microbes to thrive under desired conditions (Bodour et al. 2003). Microbes are usually accepted with a variety of approaches to increase their bioavailability and increase their contact with hydrophobic components like hydrocarbons. There are different ways of contacting, such as surfactant-mediated solubilization, biofilm contact, and direct contact with oil drops. Microbes degrade substrates using enzymes that are linked to biosurfactant molecules. A majority of the substrates have low water solubility and high

R. Sathya Department of Microbiology, PRIST Deemed To Be University, Thanjavur, Tamil Nadu, India M. V. Arasu (✉) · N. A. Al-Dhabi Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia P. Vijayaraghavan Bioprocess Engineering Division, Smykon Biotech, Nagercoil, Tamil Nadu, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_14

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Table 1 General features and application of biosurfactants S. No 1.

Features Biodegradability

2.

Little harmfulness

3.

Raw constituents availability

4

Physical factor

5.

Surface and interface action

6.

Biosurfactant applied in Laundry detergent

7.

Biopesticide and insecticidal activities

8.

Bio uptake by microbes

9.

Hydrophilization

10.

Penetration power in pesticides

11.

Rhamnolipid Biosurfactant

12.

Fengycins

Role of biosurfactant Biosurfactants are quickly degraded by microbes. Biosurfactant has less toxicity than synthetic derivatives. Biosurfactant can be produced from easily available raw materials (Carbon sources: carbohydrates/lipids, hydrocarbon). Generally, biosurfactants are not affected by physiological factors like pH, temperature, and ionic strength. Biosurfactants can reduce the surface tension of water (75–35 mN/m), interfacial tension water/hexadecane (40-1mN/M); surfactin has the ability to decrease the surface tension of water to 25 m N/M and interfacial tension of water/hexadecane ≤1mN/M. Biosurfactants are highly suitable and alternative for chemical surfactant. Biosurfactants in laundry that reduce the environmental hazardous. (e.g., Cyclic lipopeptides are active in a wide range of pH 7.- = 12.0) during high temperature withstand their surface active ability. Lipopeptides containing biosurfactants synthesized by several bacteria reveal insecticidal properties against Drosophila melanogaster and hopeful to use biopesticide. Microbes can easily adsorb the soil pollutants and reduce the distribution route length among the site of absorption. Biosurfactants used for hydrophilization of heavy soils to acquire routine wettability and to complete smooth circulation of fertilizer in the soil. They also prevent the caking of certain fertilizer during storage and promote spreading and penetration of the toxicants in pesticides Biosurfactants can reduce the coating of firm fertilizer during packing and encourage dispersion and diffusion of the toxicants in pesticides. The bacterium Pseudomonas exhibits better antimicrobial properties. Biosurfactant (Rhamnolipid) does not cause any adverse effect to the human and environments. Biosurfactants (fengycins) are also stated to hold antifungal properties and also applied for biocontrol agent of more plant diseases.

References Mohan et al. (2006) Desai and Banat (1997) Kosaric (2001)

Krishnaswamy et al. (2008) Mulligan (2005)

Das and Mukherjee (2007)

Mulligan (2005)

Makkar and Rockne (2003)

Kachholz and Schlingmann (1987)

(continued)

Role of Biosurfactants in Enzyme Production

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Table 1 (continued) S. No 13.

Features Antimicrobial activity

14.

Anti-cancer activity

15.

Anti-adhesive agents

16.

Immunological adjuvants

17.

Antiviral activity

18.

Gene delivery

19.

Food-processing sector

Role of biosurfactant Most of the biosurfactants have strong antimicrobial activity (Antibacterial, antifungal, and antivirus activity). The biosurfactants play a vital role in antiadhesive properties to many pathogens and can be used for the treatment of numerous diseases. The best example is marine B. circulans produced biosurfactant fight against both gram (+) and (-) pathogens, etc. The microbial extracellular glycolipids enhance cell differentiation instead of proliferation in human cell. Whereas the practice of microbial extracellular glycolipids as new substances for the cancer treatment. Biosurfactant can prevent the adhesion of pathogenic microbes to solid surfaces / infection sites. The biosurfactants from L. acidophilus and L. fermentum adsorbed on glass and reduced by 77% inhibit the adherence of uropathogens (e.g., Enterococcusfaecalis). Bacterial lipopeptides are potential immunological adjuvants and have less toxic effects, they are nonpyrogenic with mixed predictable antigens. This type of biosurfactant improves the humoral immune response to the body (Antigen iturin Al, herbicolin A). The best antiviral biosurfactants inhibit the HIV in leucocytes. Recent reports suggest the requirement of female controlled, efficacious, and safe vaginal topical microbicidal agents. The Sphorolipids (biosurfactant) from C. bombicoloa synthesizing diacetate ethyl ester is most powerful spermicidal and virucidal activity. Recently, the most high tech efficient technology and safe method has introduced exogenous nucleotides into mammalian cells. The wide range of application has been found in food-processing (food formulation ingredients, antiadhesive agents, stabilization, and emulsion). Biosurfactants are used to protect the mass of fat globules, stabilization of aeration system, food texture, improving shelf life and changing the rheological properties of wheat dough and enhance consistency and appearance of fat-based products.

References GharaeiFathabad (2011)

Krishnaswamy et al. (2008)

Rodrigues et al. (2006)

GharaeiFathabad (2011)

Krishnaswamy et al. (2008)

GharaeiFathabad (2011) Krishnaswamy et al. (2008)

(continued)

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Table 1 (continued) S. No 20.

Features Biosurfactant in cosmetic industries

21.

Biosurfactant in petroleum

22.

Microbial enhanced oil recovery

Role of biosurfactant Biosurfactants are used in various forms in cosmetic sectors like emulsifiers, enzyme action (mediator), solubilizer, foaming agents, cleanser, antimicrobial agent, acne pads, antidandruff, lens solutions, baby products, lipsticks, mascara, tooth paste, etc. The modern microbial technology is focusing novel group of microbial surfactant can be used in biocorrosion, degradation of hydrocarbon within enzymes for petroleum extraction, transportation, upgrading and refining, and petrochemical production. Microbial surfactants are highly used in oil recovery in modern years. The potent microbes B. subtilis, Torulopsisbombicola, P. aeruginosa highly stated to exploit crude oil and hydrocarbons as only carbon source castoff for oil spill cleanups.

References GharaeiFathabad (2011)

Perfumo et al. (2010)

Das and Mukherjee (2007)

solid-liquid ratios. There are a number of factors that affect substrate bioavailability, such as physical, chemical, environmental, and kinetic parameters (Johnsen et al. 2005). Biosurfactants have more potential features than chemical surfactants. It consists of numerous key features such as degradable, stable at extreme environmental conditions, the use of inexpensive and cheap raw materials for production, and eco-friendliness (Diaz De Rienzo et al. 2016). According to most reports, biosurfactants are widely used in the remediation of petroleum-contaminated environments (Ferradji et al. 2014). Microorganisms are widely used for the production of biosurfactants. Recently, biosurfactants have played a crucial role in research and development as well as product development in industry (Thakur et al. 2021). A biosurfactant is produced through metabolic processes or the outer surface chemistry of a cell. Generally, biosurfactant production is carried out by microorganisms in submerged fermentation with carbon sources and types of biosurfactants recovered from bacteria (Acinetobacter, Bacillus and Pseudomonas), fungi (Fusarium, Aspergillus, etc.), and yeast (Pseudozyma and Candida) (Fenibo et al. 2019). We need to extend our understanding of biosurfactant producers to include genetics, morphology, physiological, and biochemical analysis, as well as screening of virulent strains to minimize production costs. Using bio-based surfactants, we can treat polluted soil, heavy metals, skin conditions; improve oil restoration; preserve food; clean up municipal waste, yard waste; treat crop residues; and treat plant diseases (Gayathiri et al. 2022). The growing market value of biosurfactants is driving advancement and improvement in bioremediation through the use of biosurfactants and microbes. The biosurfactant has improved the bioremediation

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rate of hydrocarbons linked to various enzymes (Zahed et al. 2022). Hence, the study looks at biosurfactants and their beneficial role in enzyme synthesis.

2 Current Status of Biosurfactants Global surfactant value increased from 17 million metric tons in 2000 to a growth rate of 3–4 percent per year in the near future (Rahman and Gakpe 2008). Surfactants are applied in food, petroleum, medicine, pharmaceuticals, and cosmetics; however chemical mixtures cause severe health and environmental problems (Makkar and Rockne 2003). Petroleum-based surfactants are toxic to the environment and are non-biodegradable. Environmental researchers have recently been focusing on the development of surfactants derived from microbial sources. The use of microbebased biosurfactants is thus highly valued on a global scale due to their eco-friendly nature (Benincasa 2007). Currently, biosurfactants have more commercial applications. By using genetic engineering tools, the unique and super-active microbial strain has developed continuously and emerged as a biosurfactant. A highly renewable substrate is used to produce biosurfactants on an industrial scale. Biosurfactants are characterized by low toxicity, biodegradability, and good foaming, selectivity, specific activity, wide pH, temperature, salinity range, and good surface and interfacial activity. The biosurfactants are the original group of particles and contain the most powerful and versatile by-products. The recent microbial biotechnology permits the biodegradation of hydrocarbons with the help of enzymes (biocatalysts). Biosurfactants are synthesized enzymatically by microbes. In recent years, the naturally presenting surface active mixtures caused by microbes have attracted significant attention (Fakruddin 2012). Biosurfactants are in high demand around the world. Surfactant is projected to reach a market value of USD 3064 billion in 2016, and USD 3986 billion in 2021. By 2022, the value is projected to be $552 billion (Markets and Markets 2016). In 2016, the revenue generated from biosurfactant reached 18 billion dollars; and we are expected to reach 26 billion dollars in 2023, and the quantity of biosurfactant will reach 540 kilotons in 2024, with rhamnolipids holding 8% of total market value (Grand View Research). Numerous renewable and less expensive raw materials can be applied for the synthesis of biosurfactants.

3 Source of Biosurfactants Biosurfactant-synthesizing microbes are widely distributed in nature. Both land and water ecosystems contain microbial surfactants (Fig. 1). The areas are covered by water ecosystems, such as marine, seashore, and freshwater and groundwater. The land areas covered by soil, sediment, sludge, and extreme oil reservoirs can also withstand high levels of temperature, pH, and salinity (Van Hamme et al. 2006). The

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Sludge Extreme Environm ent (Oil reservoirs)

Sea water

Sources of Bio surfactant

Fresh water

Marine Regions

Land Ground water

( Soil, Sediment, Sludge)

Fig. 1 Sources of microbial biosurfactant on different environment

most common crude-oil-degrading bacteria can synthesize biosurfactants that influence the activities of specific enzymes that contribute to the degradation process. Bacillus, Acinetobacter, Pseudomonas aeruginosa and Candida antartica are among the microbes that synthesize various biosurfactants. These are typically classified based on their chemical composition and molecular mass. Bacillus, Pseudomonas, and Sphingomonas are the most prominent genera responsible for producing biosurfactants in soil and sediments. In the most dominant fungi, biosurfactants are produced at low levels of substrate concentration. Sophorolipids are a major class of lipids produced by C. lipolytica, which is surrounded by wellpacked lipopolysaccharide (Rufino et al. 2007). Biosurfactants can be obtained from the marine ecosystem containing Alcanivorax, Acinetobacter, Pseudoalteromonas, and Halomonas. The reports on the screened population of microbes from uncontaminated soil (2–3%) and contaminated soil samples (25%) (Bodour et al. 2003). In contrast, the enrichment procedure detected nearly (80%) of biosurfactant producing microbes (Rahman et al. 2002) (Fig. 2). Recently, it was stated that Pseudomonas aeruginosa, produced biosurfactant and was phylogenetically related to P. chlororaphis. In both species, glycolipid family members and water soluble

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• Pseudomonas aeruginosa, P.chlororaphis, Rhamnolipid is class of glycolipid synthesized by Pseudomonas

Soil source • The function of Rhamnolipids includes solubilization, modification of outer surface, enhancement of bacterial Rhamno movement, formation and distruption of biofilms, virulemce and antimicrobial properties. lipid

Surface active lipids

Glycolipid marine source

Lipopeptide Sediment sample

• Rhodococcus and CMN group: Corvnebacterium, Mycobacterium and Nocardia), Gordonia genus • The Rhodococcus, species are considered as top biosurfactant and active for the help of potential ozygenases, enzymes, The main function of surfactant is lower the surface tension at the air liquid interface.

• Alcanivorax borkumensis, Halomonas, Bacilus, Pseudomonas, Arthrobacter and Micrococcus species are involved good potent role of biosurfactant synthesis. • The role glycolipids can reduce the bioadhesion of pathogenic bacteria enabling their use as anti adhesive agent and for distruption of biofilm formation.

• B. licheniformis and B. mojavensis, Pseudomonas and Bacilli. The bacteria hold an superb capacity to join forces with membrane, lipopeptides, antimicrobials, enzyme synthesis. • The Lipopeptides are microbial surface active compounds synthesized bacteria, bacteria, fungi and yeast and reduce the surface and interfacial tension.

Fig. 2 Biosurfactant-producing microbes and their distribution in the environment

plus hydrophobic substrate are present at high concentrations during late exponential growth phases to increase cell density (Gunther et al. 2005). Rhamnolipids alter the outer hydrophobic properties of the cell membrane through the production of lipopolysaccharide compounds with concomitant increases of outer hydrophobicity (Al-Tahhan et al. 2000). As the biosurfactant is exposed on a small surface material, the initial cell concentration is very low at the interface between oil and water, and carbon and nitrogen inputs have increased the number of bacteria (Garciaa-Junco et al. 2001). Rhodococcus species are considered to be the most effective biosurfactant for degrading hydrocarbons, primarily because of their oxygenase enzymes and cellular characteristics enhancing biosurfactant activity. Rhodococcus synthesizes biosurfactant that has chemical and physiological functions related to a specific glycolipid (trehalose) linked to sugars and fatty acids. The most stimulating feature of different forms of biosurfactants such as non-ionic derivatives trehalosemycolates and anionic tetraesters compounds (Rapp and Gabriel-Jurgens 2003). It appears that Gordonia species can convert straight contact to oil droplet into dispersed droplet by producing different intervals of cell bound surfactant and extracellular enzyme action (emulsifying agent) along with active modification of cell hydrophobic and hydrophilic activities (Franzetti et al. 2008). For the sources of biosurfactant, marine microbes hold an excellent community. There is an enormous population of hydrocarbonoclastic bacteria to ornament the popularity of the overall microbial population (Yakimov et al. 2007). Alcanivorax borkumensis are considered to be a specialized and dominant hydrocarbon degrading bacteria in marine environments. The genetic group of glycolipid synthesizing producers is still understudied, but A. borkumensisSK2 genome sequencing evidence has proven interspecies identification and putative (ABO _ 0822 OprF/OmpA protein) protein components of A. ka53 have been detected (Schneiker et al.

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2006). Halomonas, Bacillus, Pseudomonas, Arthrobacter and Micrococcus species play significant roles in producing biosurfactants (Calvo et al. 2002). Several genera of bacteria such as Pseudomonas and Bacillus produce lipopeptides that are biosurfactants. The bacteria exhibit an excellent ability to cooperate with membranes, lipopeptides, antimicrobials, enzyme synthesis, and hydrophobic mixtures, which act as strong surfactants (Ongena and Jacques 2007). Species of B. subtilis are grown in a variety of environments and produce a variety of compounds like surfactin, lipopeptides and iturin. The carbon source (starch) was used with an additional surfactin lipoprotein synthesized hydrocarbon degrading bacillus (Mukherjee and Das 2005). Bacillus licheniformis and B. mojavensis both synthesized biosurfactant lipopeptides lichenysin lipopeptides that are effective in decreasing interfacial tension and vital for allowing mobilization of oil form to the bacteria under the extreme environmental conditions. Lipoprotein synthesizing bacteria are unlimited to hydrocarbon contact and confer a wide range of evolutionary benefits to bacteria that have suitable substrate accessibility and environmental conditions (Ahimou et al. 2000). High molar masses of biosurfactants have complex structures such as biopolymers, lipopolysaccharides, proteins, and polysaccharides, and low molar masses of glycolipids and lipoproteins (Ron and Rosenberg 2002). The exopolysaccharide family was derived by a number of bacteria (Sphingans, Sphingomonads). The Sphingomonas species are widely distributed in soils and accumulate biofilm, from which they recoup their cells (Johnsen and Karlson 2004). As another important bacteria, Halomonas eurihalina produces EPS with emulsifying activity (EPS synthesize hydrocarbons, proteins, and uronic acid). Uronic acid is responsible for detoxifying hydrocarbons quickly. The H. eurihalinahas initiated the growth of the other biosurfactant producers (Pseudomonas, Micrococcus, Arthrobacter, and Bacillus)-(Calvo et al. 2002). Plant and Rhizosphere associated microbes synthesize biosurfactant and play a major role in signaling, motility, and biofilm formation to ensure plant microbe interaction (Williams and Cámara 2017). Biosurfactant is produced by environmental isolates and plays an important role in the agricultural sector (Sachdev and Cameotra 2013).

4 Nature of Biosurfactant Biosurfactants are amphiphilic polymers synthesized in living surfaces, mostly produced on microbial surface or emitted extracellular components (hydrophilic/ hydrophobic) that converse the ability to present between liquid phases by lowering external and interfacial tightness at the external and border separately (Cunha et al. 2004). In chemical-based surfactants, the biosurfactant mechanisms are completely utilized (Singh et al. 2007). Biosurfactants are classified based on their chemical structure and microbial source. There are four main types of biosurfactants: phospholipids, glycolipids, lipopeptides, and polymers (Yin et al. 2009), the various fatty acid derivatives such as sugar alcohols and fatty acids. They are used for industrial,

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household, and pharmaceutical applications. A variety of prokaryotes and eukaryotes produce biosurfactants. The microbial surfactants are lipoidal in nature, and they are excreted by microbes based on their growth rate (hydrophobic substrates). For robotic producers (microbes), substrate characteristics and quantity are crucial.

5 Physiological Role of Microbial Biosurfactant Biosurfactants are produced by a wide range of microbes (synthesize the extracellular/surface attachment part of cells). By reducing the surface area, biosurfactants enable microbes to grow on (water/impermeable) substrate. Its most important physiological role is antimicrobial activity (antimicrobial activity against different microbes), potent stability under harsh environmental conditions, and virulence. The surface parts are usually restricted, resulting in an increase in biomass and extracellular compounds.

6 Factors Affecting Bio Surfactant Production Biosurfactant structure and emulsifying ability are determined by cultural factors like carbon, nitrogen, nutritional role, and physical and chemical factors (temperature, aeration, divalent cations, pH). Biosurfactant quality and quantity are influenced by raw material environments (crude oil, sucrose, glucose, diesel, and glycerol used as a carbon source for the production of biosurfactant). For the growth of protein and enzyme production, the nitrogen source (peptone, yeast extract, ammonium sulphate, nitrate, meat extract, and malt extract) is crucial. Nitrogen sources such as ammonium salt and urea are highly suitable for the production of biosurfactants by Arthrobacter paraffineus and Pseudomonas aeruginosa. Surfactant production is always affected by pH, aeration, temperature and agitation speed, which are essential factors. The production temperature ranges from 25 to 30 °C, and the optimal pH is 8.0 (Zinjarde and Pant 2002). Aeration and agitation are crucial to improving the production of biosurfactant and simplifying the transfer of oxygen from gas to liquid. Microbial emulsifiers are physiologically affected by the environment. The salt concentration in the water is ideal for the production of biosurfactants. Microbes cellular activities are highly dependent on salt concentration (Desai and Banat 1997). Biosurfactants are used in leather processing, textile, food, pharmaceutical, chemical and agriculture processes (Fig. 3).

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Other purpose (8%) Cleanser and detergents

Agriculture (2%)

(54%)

Efficient use of Bio surfactant

Leather, textile and paper

Chemical process industries (10%)

(20%)

Food industry (3%)

Cosmetics and pharmaceut icals

(3%) Fig. 3 Application and percentage value of biosurfactants

7 Type of Biosurfactants Rhamnolipids biosurfactants are produced by Pseudomonas aeruginosa with different types of trehalolipids synthesized by Mycobacterium, Nocordia, and Corneybacterium. Rhodococcuserythropolis synthesized trehalosedimycolate (a novel anionic lipid compound). Arthrobacter and Rhodococcus decreased the surface and interface tension in the culture broth (25–40; 1–5 mN/m), respectively. Sophorolipid biosurfactant is synthesized by yeast (Torulopsisbombicola, T. petropgilum, T. apicola). Lipopeptides and lipoprotein surfactin are produced by B. subtilis, B. licheniformis. Antibacterial properties of lipopeptide, iturin were derived from Bacillus subtilis, and both compounds were capable of working at wide pH ranges (Singh and Cameotra 2004). As one of the most dominant surfactants, lipopeptide surfactin is linked with seven amino acid rings and fatty acid chain with lactone linkages. Bacteria and yeast produce fatty acids, phospholipids, and surfactants (Fig. 4). Polymeric biosurfactants, such as emulsan, liposan, manoprotein, polysaccharide, and other proteins are produced by Acinetobacter calcoaceticus (Gautam and Tyagi 2006). The food and cosmetic industries pay close attention to polymeric surfactants. Polymeric biosurfactants include alasan, liposan (extracellular water soluble),

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Class of Biosurfactant Lipopeptide and Lipoprotein Glycolipids

Rhamnolipids

Fatty acids

Trehalolipids

Sopohorolipids

Phospholipids, Neutral lipids

Polymeric Biosurfactant

Fig. 4 Major class and their types of biosurfactants

emulsan (agent used to dissolve hydrocarbons in water), lipomanan, and many other polysaccharide protein composites (Hatha et al. 2007). The most important particulate biosurfactant is extracellular membrane vesicles, which form microemulsions and play a significant role in microorganism uptake of alkane. Cesicle-like particles produced by Acinetobacter species (20–50 nm diameter; density 1.158 cubic gcm) which form a complex layer (protein, phospholipid, and lipopolysaccharide) (Chakrabarti 2012). Bacteria such as Bacillus sp., Pseudomonas sp., Rhodococcus sp. and Arthrobacter sp. were used for the commercial production of biosurfactants.

8 Biosurfactant Role in Enzyme Production Biosurfactants are incorporated with attractive enzymes and play a major role in bioremediation and improve the benefits of cosmetics, renewable energy, food, textiles, leather, and petroleum. The biosurfactant is obtained from renewable resources and has better surface activity as compared to the chemical surfactant (Xu et al. 2011). Lipopeptide, a biosurfactant with the most versatile properties, is widely used for the inhibition of fibrin clot formation, oil recovery, antiviral, antifungal, antitumor, antimycoplasma, and insecticidal properties. Recently, biosurfactant- and enzyme-producing microbes began synthesizing nanoparticles (Kiran et al. 2011). Most biosurfactant-producing strains are best suited to mediate enzymes and their application. Hydrolase, amylase, proteases and lipases are potent biosurfactant synthesizing strains (Table 2). Enzymes are used in several industrial sectors, including the food, fermentation, textile, detergent, and paper industries. Strains have already been reported to produce biosurfactants at a good level in Casava wastewater. The LB1a and LB5a strains were selected for using a synthetic medium

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Table 2 Impact of biosurfactant on enzyme synthesis S. No 1.

2. 3.

4.

5.

6. 7. 8.

Association role of biosurfactant and enzymes Pseudomonas aueroginosa synthesis surfactant that initiate the production of four kinds of proteases (acid, alkaline, thiol, metalloproteases). Keratinolytic bacteria commonly synthesize lipase and biosurfactant involved for bioremediation. The molecular synthetic technology of surfactin generates nonribosomal peptides that (Srf A, B, and C only contain thioesterase). The rubiwettin surfactant synthesized by the protein (Rhl gene) of S. rubidaea strain 1122 43% similarity to RhlA of Burkholderiaglumae94%. RhlA synthesises 3- (3 hydroxyalkanoyloxy) alkonic acids from triggered hydroxyl fatty acids glycosyltransferase RhlB catalysis dTDP-1-rhamnose. Removal of alkane degradation by microbes with the help of alkane hydroxylases enzyme. Metagenomic studies proved enzymes involved in the pathway of biosurfactant synthesis. Yeast recombination systems have added more successful level for biosurfactant synthesis.

Reference Samanta et al. (2012) Rahayu et al. (2019) Tanovic et al. (2008)

Hausmann and Syldatk (2014) Tiso et al. (2017)

Van Beilen and Funhoff (2007) Sachdev and Cameotra (2013) Weihmann et al. (2019)

that contains cassava water highly influencing (amylase, protease) production. Bacillus species are highly valued for their ability to produce enzymes. Nearly half of the total enzyme market (1.6 billion dollars) is accounted for by Bacillus species. Food, fermentation, textiles, paper, and detergents are some of the industrial applications of enzymes. The possibility of obtaining enzymes and biosurfactants by fermentation of cassava substrate has influenced the economic viability (Cavalcante Barros et al. 2013). Lipase and biosurfactant are highly used in many fields of biotechnology. Biosurfactants and enzymes are used in a wide array of applications, such as hydrolysis of oils, wastewater treatment, detergent industries, biomedicine, cosmetics, oleochemistry (fatty acid derivatives), and biodegradability (Zarinviarsagh et al. 2017). The majority of keratinolytic bacteria synthesize both lipase and biosurfactant, which are highly beneficial to the bioremediation process (Rahayu et al. 2019).

9 Enzymatic Degradation Microbes are responsible for enzyme degradation. Degradation of PHA (poly hydroxyalkanoate) by enzymes plus biosurfactants demonstrated that naphthalene and phenanthrene decompose. Bacterial strains (Sphingomonas, Pseudomonas, Mycobacterium, Sphingomonads, Sphingobium, Burkholderiafungorum,

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Fig. 5 Microbes and their commercial product of biosurfactants

Novosphingobium, Sphingopyxis) are responsible for the degradation of PHA (Saxena et al. 2019). The enzymes that help decompose PHA include dehydrogenase, oxygenase, ligninolytic, laccase, manganese peroxidase, and lignin peroxidase (Zhao et al. 2018). Bacillus strains that produce lipopeptide biosurfactants are thermophilic. The situation exchange has been found to increase the yield of surfactant synthesis (Cameotra 2002). The rhamnolipids products are multifunctional glycolipid biosurfactants synthesized by Pseudomonas species. It is common practice to use biosurfactant as an additive for improving enzymatic activity, because the ideal properties are renewable resources, microbial origin, and less impact on the environment (Zhang et al. 2009). Biosurfactants (rhamnolipids) are able to improve the stability and activity of enzymes (cellulase), since they increase the release of reducing sugars and untangle the mechanism of enzymes to produce positive effects (Wang et al. 2011). The saponins are linked with a hydrophobic unit (triterpenoidaglycone) called sapogenin that increases enzymatic hydrolysis. During the hydrolysis of lignocellulosic feedstocks, rhamnolipids and saponins are very stimulating (Fiallos-Jurado et al. 2016). It has been reported that the robotic biosurfactant, Bacillus species mediated several enzymes and degraded hydrocarbons (De Franca et al. 2015). Biodegradative enzymes are essential for biosurfactant synthesis (Yong and Zhong 2010). Biosurfactant plays a vital role in enzyme synthesis (Fig. 5). Many alkane-degrading microorganisms produce alkane hydroxylase, an enzyme that converts alkane alkanols (Van Beilen and Funhoff 2007). The biosurfactant process involves the coproduction of enzymes and performs a dual role in bioprocessing, the most important and most viable submerged state of enzyme production linked with biosurfactant; the need of microbes to consume water immiscible substrate enhances the biosurfactant along with the lipase enzyme (Ramos-Sanchez et al. 2015). Aspergillus sp. produced lipase and biosurfactant in submerged fermentation and solid state fermentation. Both lipase and biosurfactant have been synthesized under submerged conditions with lipolytic and emulsifying activities. Bacillus subtilis isolated from a dump yard produced pectinase and biosurfactant (Colla et al. 2010; Kavuthodi et al. 2015).

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Integrated Bioprocess of Enzyme and Biosurfactants

The enzyme and biosurfactant are incorporated into the same bioprocess and utilize the same kinds of substrates. P. aeruginosa strains that produce biosurfactants have genetically modified desulphurization processes (Raheb and Hajipour 2011). Bio-origin enzymes are most important and are found in all living organisms. Proteases from microbes are receiving a lot of attention (66%) in the industrial market. Protease-producing microbes synthesize biosurfactants that bind to the four types of proteases (acidic, alkaline, thiol, metallo). Pseudomonas aueroginosa produces surfactants in the pH range of 7–12 and at temperatures between 35 and 80 °C (Samanta et al. 2012). Biosurfactant synthesis is connected to the biosynthetic pathway of enzymes. Apparently fatty acids/hydroxyl fatty acids are made from carbon in prime metabolism. N-acyl amino acids are derived from activated fatty acids and amino acids are catalysed by N-acyl aminoacid synthase in bacteria (Van Wagoner and Clardy 2006). Lipopeptide-producing organisms have potential application as producers of various pharmaceutically active compounds or biocontrol agents (Trimble et al. 2016). Polypeptide chain of proteins and lipopeptides surfactant linked by nonribosomal peptide synthetases NRPS (nonribosomal peptides), each segment adds amino acids (NRPS catalyses the incorporation of lipids, lactonization, and epimerization) to the peptide backbone (Samel et al. 2006). The proteolysis segment comprises three domains (condensation, adenylation, liable for aminoacid selectivity, epimerization, liable for aminoacid transformation) to the peptide backbone. Lipopeptide surfactants (surfactants) and enzymes (protein synthesis) are always interconnected. The molecular synthetic machinery of surfactins creates NRPS that contain multifunctional proteins (SrfA, B and C only contain thioesterase). The final product discharge and lactonization of the peptide is catalysed by the thioesterase domain of the SrfA (Tanovic et al. 2008). P. aeruginosa may have established the pathway for the biosynthesis of bacterial glycolipids through rhamnolipid surfactant synthesis. Rhamnolipids are synthesized by enzymes (glycosyltransferase, rhamnosyltransferase I&II). The gene of interest rhlA&B is ordered as bicistronic operon selected rhamnosyltransferase I (bicistronic operon: RhlA&B) genes (Wittgens et al. 2017). RhlAsynthesises 3- (3 hydroxyalkanoyloxy) alkonic acids from triggered hydroxyl fatty acids glycosyltransferase RhlB catalysis the density between the dTDP-1-rhamnose (Tiso et al. 2017).RhlC is located at the chromosomal location of the rhlB gene of P. aeruginosa, which encodes rhamnosyltransferase II. The rubiwettin surfactant synthesized by the protein (Rhl gene) of S. rubidaea strain 1122 43% similarity to RhlA of Burkholderia glumae 94% (Hausmann and Syldatk 2014). Production of biosurfactant is carried out by conservative cloning restriction ligation utilizing novel cloning methods (Andreou and Nakayama 2018). Yeast recombination has received much attention for successful biosurfactant synthesis (Weihmann et al. 2019). Palindromic sequences are an advanced genetic engineering tool that is used for improving biosurfactant and enzyme production by moving the

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implied genes into another microbial host with suitable features or by removing a few defective genes (Elshafie et al. 2015). As a crucial advance in molecular biology, the target DNA sequence of biosurfactant-producing strains has been applied for recombination process that improves the enzyme that induces the bioremediation process (Dasgupta et al. 2013). Immobilization devices are crucial to the growth improvement in continuous fermentation of biosurfactant by downstream processing (washing of cells). Foam induction is an important step in final washout and enhancing free and immobilized cells (Abouseoud et al. 2008). This is the most important step in bioprocesses using resting cells. Cells of Pseudomonas fluorescens immobilized in alginate are highly used in the production of rhamnolipids. Media pH is more variable in immobilized cells than in free cells. The immobilized rhamnolipid producers are easy to isolate the product from the culture during final cell recovery. The enzyme-based immobilization of Bacillus subtilis onto Fe-coated polymeric particles produced 20–43 times higher than planktonic cells in batch conditions. However, even slight changes in P. aeruginosa immobilized on Ca-alginate must be applied for biosurfactant production. Rhamnolipid synthesis under resting conditions was tested in P. nitroreducens with Ca-alginate beads and 51 g of L-rhamnolipid was recovered (Onwosi and Odibo 2013). Though biologically versatile and diverse, the microbes do have a large repertoire of genes with significant biotechnological applications (Sebastianes et al. 2013). Degradative enzymes are proteases, xylanases, lignases, and cellulases (Zheng et al. 2016). Secondary bioactive metabolites such as enzymes and also extracellular active compounds such as biosurfactants are normally synthesized by the microbes. Several endophytic fungi possess enzyme abilities (oxidase, hydrolases) and bioremediation potential (Trichoderma camerunense). Degradation of hydrocarbons is largely dependent on biodegradative enzymes. Microbes remove alkane degradation with the help of alkane hydroxylase enzymes (Van Beilen and Funhoff 2007). The alkane hydroxylase enzyme converts alkanes to alkanols. Alkane enzymes of various molecular weights (low, medium, and high) participate in the degradation process; for example, methane monooxygenase (low MW), non-hemo alkane monooxygenase (medium MW) and the high-molecularweight flavin-binding monooxygenase, and the key enzyme alcohol dehydrogenase play a vital role in degradation of hydrocarbon (Singh et al. 2012). A number of bacteria, such as Pseudomonas sp. P. aeruginosa PSA5, Ochrobacterium intermedium, and Stenotrophomonas nitritireducens, have been studied for hydrocarbon degradation due to the role of biosurfactant and enzyme synthesis (Jauhari et al. 2014). A biosurfactant and enzyme-synthesizing Bacillus strain is primarily responsible for the degradation of crude oil. Optimized conditions with vital factors (pH 7.0, 40 ° C, 25% sucrose, and 3% yeast extract) are used for biosurfactant synthesis. The maximum production of biosurfactant (4.85 g L-1) is influenced by nutrients and environmental conditions. Low molecular compounds are completely degraded, whereas C15-C19 degrades to 97% from the total hydrocarbon. It was determined that the biosurfactant collected from the degraded hydrocarbon medium is a

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lipopeptide. The enzyme production and biosurfactant synthesis strains have the potential to degrade hydrocarbons in polluted environments (Cavalcante Barros et al. 2013).

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Advanced Technology

The biosurfactants are used to improve the stability of microbubble technology and to diagnose disease, create molecular imaging, deliver drugs and genes, and improve water purification and sewage treatment technology (Zhang et al. 2011). A variety of biosurfactants are becoming a useful tool for biotechnology and benefiting mankind. A number of patents are available for biosurfactant microbial producers. The majority patent rights has received the strains Pseudomonas sp., Bacillus sp., Acinetobacter sp., Candida sp. and categorized microbe-based biomolecule (Shete et al. 2006). Microbial communities synthesizing enzymes and its role in metagenomic studies by culture independent analysis (Schloss and Handelsman 2003). These studies allow us to identify the operons and genes that encode the pathways that direct the synthesis of biosurfactants. In functional metagenomic studies, the genes that encode for enzymes and proteins involved in the pathway of biosurfactant synthesis were identified, which are normally located in chromosome regions. Metagenomics is useful to obtain a new biosurfactant (uncultured bacterium) from agricultural samples of microbial (bacterial) surfactants (Sachdev and Cameotra 2013). Glycolipids activate enzymes for various bioprocesses by altering their activity. Enzymes are antiadhesive molecules that protect the cell from pathogenic bacteria (inhibit bioadhesion) (Inès and Dhouha 2015). The rhamnolipid synthesis occurs in three major steps. The production of rhamnolipids is controlled by quorum sensing signals (Rahim et al. 2001; Wittgens et al. 2017). The catalytic enzymes RhlI and LasI are critical in the synthesis of autoinducer compounds once they have reached the threshold concentration and undergo binding to the regulatory proteins RhlR and LasI (Reis et al. 2011). The Rhamnolipis biosurfactant is cytotoxic against cervical, bladder, breast, and leukaemia (Rollauer et al. 2015). It encloses the outer membrane protein (OmpA) through its action as an antioxidant enzyme SOD (Superoxide dismutase, CAT (Catalase), and GR (glutathione reductase). OmpA is usually secreted by gram-negative bacteria in their cytoplasm. The barrel proteins encode 8–22 strands. The OmpA gene exhibits different functions, such as quick action of enzymes like proteases, lipases, and palmitoyltransferases (Noinaj et al. 2013). In the use of rhamnolipid, the cellulolytic and lignolytic synthesizing fungus Phanerochete chrysosporium and Azotobacter chrococcum (nitrogen fixing bacteria), the growth rate of green waste composted using Eiseniafetida was increased (Subramanian et al. 2010). During composting, rhamnolipid and tween 80 influence bacterial growth. Bacillus and Streptomyces provide better breakdown of organic material degradation (Parry et al. 2015).

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Conclusion

Globally, the biosurfactant industry is highly competitive and profitable. Most of the biosurfactant and enzyme synthesis (protease, amylase, lipase, cellulase, etc.) are integrated biological processes, both are carried out in a bioremediation process and eco-friendly. Production of dual products by microbes greatly improves the economic feasibility. The new technologies have been applied to the production of enzymes with biosurfactant activity, improving the efficiency of biomolecules at the same time. A significant role is played by biosurfactants and enzymes in hydrocarbon degradation. This enzymatic degradation process is completely safe, effective, and eco-friendly. A top priority is the development of new biosurfactants and enzymes that can be used to produce green enzymes rather than artificial products. The combination of rhamnolipids and enzymes enhances sanitation; the ratio of biosurfactant to enzymes is considered to be the best detergent configuration. Biosurfactant boosting improved organic matter decomposition, quick metabolization, and an increase in enzyme concentration and biomass that led to rapid organic matter degradation. Therefore, enzymes and biosurfactants can both be utilized in conjunction to control the bioremediation process and to produce more organic compounds and metabolites for industrial applications.

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Microbial Biosurfactants in Food Processing Industry Muhammad Bilal Sadiq, Muhammad RehanKhan, R. Z. Sayyed, and Imran Ahmad

1 Introduction Surfactants reduce the interfacial tension due to their amphiphilic nature (Farias et al. 2021). Most of the commercial surfactants are synthetic in nature and based on low-cost petrochemicals. Such surfactants are associated with toxicity, lack of biocompatibility, and environmental threats which limits their application despite low production cost (Rebello et al. 2014). Biosurfactants are derived from microorganisms as a product of their metabolism during fermentation (Farias et al. 2021). Due to their natural origin, low toxicity, sustainable production, and environmental safety, biosurfactants are considered as an effective alternate to synthetic surfactants (Banat et al. 2010; Naughton et al. 2019). Biosurfactants constitute 10% of the total surfactant production globally and are used in petroleum, food, cosmetic, pharmaceutical, and agriculture industries (Sarubbo et al. 2022). Currently, the production cost of biosurfactants is high in comparison to synthetic biosurfactants, which is a major hindrance in the application of natural surfactants. However, the use of sustainable substrate sources, such as agro-industrial waste as a feedstock can

M. B. Sadiq (*) Kauser Abdullah Malik School of Life Sciences, Forman Christian College (A Chartered University), Lahore, Pakistan e-mail: [email protected] M. RehanKhan Department of Agricultural Science, University of Naples Federico II, Portici, Italy R. Z. Sayyed Department of Microbiology, PSGVP Mandal’s, Arts, Science, and Commerce College, Shahada, Maharashtra, India I. Ahmad Food, Agriculture and Bio-innovation Lab, Chaplin School of Hospitality and Tourism Management, Florida International University (Biscayne Bay Campus), North Miami, FL, USA © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_15

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minimize the production cost and as well reduce the environmental burden associated with the agro-industrial waste disposal (Sarubbo et al. 2015). Biosurfactants have diverse applications such as in medical field (antibacterial, antitumor, etc.), agriculture filed to improve soil quality by removing heavy metals and ability to control the pest growth (Díaz De Rienzo et al. 2016; Sachdev and Cameotra 2013; Mnif and Ghribi 2016). Biosurfactants are produced by various microorganisms but yeasts are considered as better option for food applications due to lack of toxicity and generally recognized as safe (GRAS) status. Recently, the applications of biosurfactants are extending in food industry due to their stabilizing ability, safety, and preservation potential (Dikit et al. 2019; Gudiña and Rodrigues 2019). Biosurfactants are used to improve viscosity, control of microbial growth, fat replacer, and texture modification in various food formulations, such as baking products and salad dressings (Campos et al. 2015; Giri et al. 2017). Biosurfactants, due to their antioxidant and preservative ability, have potential to replace conventional food additives. This chapter summarizes the applications of biosurfactants in food sector and as a possible alternative to conventional food additives due to additional benefits of biosurfactants in food formulations.

2 Biosurfactant Classification Biosurfactants can be classified based on different criteria such as molecular weight, ionic charge, chemical structure, etc. (Mnif et al. 2018). Microbial biosurfactants are classified into lipopolysaccharides, lipoproteins, phospholipids, and glycolipids, based on the chemical configuration. Due to diverse applications in food and other industrial sectors glycolipids and lipopeptides are predominant biosurfactants (Souza et al. 2017). Glycolipids are comprised of lipid and polysaccharide fractions which determine the characteristics of the molecule. Different types of glycolipids are produced by different microbial sources. Based on carbohydrate fraction, glycolipids can be rhamnolipids, trehalose lipids, sophorolipids, etc. (Williams and Trindade 2017). Lipopeptides are comprised of lipid and peptide fractions arranged in linear or cyclic configurations. Surfactin is a classic example of lipopeptide, due to its diverse applications in food sector, such as surface and interface activity (Bezerra et al. 2019). The biosurfactant yield from bacteria is relatively low than the yeast (Ali et al. 2016). Moreover, some yeast has generally recognized status (GRAS) and is associated with advantage of lack of toxicity and pathogenicity (Nwaguma et al. 2019). Saccharomyces cerevisiae has a potential to produce secondary metabolites with surface active and emulsification characteristics. Genus Candida has a potential for industrial-scale production of biosurfactants as previous reports have presented various members of this genus with potential to produce bio-emulsifiers (Ribeiro et al. 2020b).

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3 Production of Biosurfactants by Using Food Waste/By-products Food processing industries are generating huge amount of organic waste such as peels, pomace, etc., which if not managed properly can pose serious environmental hazards (Sadiq et al. 2017). Although the biosurfactants are associated with various benefits over synthetic surfactants but high cost is the major limitation in their use in food and feed industries (Campos et al. 2013). The production cost of biosurfactants can be reduced by using organic waste from food industries as feedstock for biosurfactant-producing microorganisms. The process of fermentation by optimizing culture conditions and feedstock ratio can serve the purpose in obtaining the desired biosurfactant with high yield and low cost. Ground nut oil was reported as a substrate to Candida lipolytica to produce biosurfactant and a yield of 4.5 g/l was obtained (Rufino et al. 2008). Vegetable fat waste, ground nut oil refinery residue, and corn steep liquor were reported as feedstock for Candida strains to produce biosurfactants (Coimbra et al. 2009; de Gusmao et al. 2010). Restaurant waste oil was reported as a feedstock for the production of 20 g/l rhamnolipids, when fed to Pseudomonas aeruginosa in a bioreactor (Zhu et al. 2007). Apart from various applications of biosurfactants in food products, their production from food waste can serve as an effective solution for food industry to manage waste products and produce low-cost biosurfactants.

4 Applications of Biosurfactants in the Food Industry The food industry has placed strict measures to control microbial contamination throughout the supply chain to ensure the safety of the consumers. Recently, consumers have been interested in food products rich in natural additives and with lesser chemically synthesized or artificial compounds (due to their toxic effects) (Ribeiro et al. 2020a). As a result, new plant-based formulations have been developed to replace synthetic additives; however, their use in formulations has been restricted due to various food processing operations i.e., irradiation and microwave heating which could lead to the loss of essential properties that these plant-based compounds possess (Hasenhuettl 2019). Thus, novel additives with desirable properties (i.e., antimicrobial, antioxidant, anticancer, etc.) should be produced to replace synthetic and plant-based additives. Biosurfactants have many advantages over chemical surfactants, especially during food processing and the finishing quality of the products. Since biosurfactants are resistant to various food processing conditions (i.e., temperature, salinity, and acidity), these biomolecules can retain their original properties and can have a positive influence on the final quality of the product (Campos et al. 2013). For instance, yeast biosurfactants are highly thermos-stable showing thermal stability up to 250  C (Ribeiro et al. 2020b). Additionally, lesser toxic effects, high biodegradability, and the tendency for stabilization and

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Fig. 1 Applications of biosurfactants in food industry

emulsification make them highly desirable food additives, to be used in the food processing industry. However, another aspect of these biomolecules that have not been given much attention is their use in edible films/coatings to extend the shelf-life of food products because of their ability to scavenge free radicals and ability to restrict microbial growth (Adetunji et al. 2018). Different applications of biosurfactants in food industry are summarized in Fig. 1.

5 Biosurfactants as Emulsifiers and Stabilizers Emulsification is a phenomenon that is required for the development of texture and consistency in foods along with solubilization and phase dispersion (Campos et al. 2013). An emulsion system is stabilized by stabilizing the heterogeneous system and restricting the agglomeration of fat globules with the help of an emulsifier (McClements et al. 2017). An emulsion system has at least one immiscible liquid (discontinuous internal phase) dispersed in another (continuous outer phase) in droplet form. The stability of this system is very low, which can be enhanced by the addition of a surfactant that reduces the interfacial tension between the two phases, thereby forming electrostatic and steric barriers to inhibit particle coalescence (Fig. 2) (Santos et al. 2016). There is enough evidence that biosurfactants exhibit a variety of properties that could positively contribute to improving or maintaining the characteristics of food products by showing greater resistance to adverse food processing operations. On the other hand, the efficacy of biosurfactants as an emulsifier and stabilizer does not only depend on the fact that they should

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Fig. 2 Biosurfactants with amphiphilic nature provide strong steric barrier to coalescence

actively reduce the interfacial surface tension at the air-water interface to stabilize the emulsion systems, but also on the phenomenon of reducing surface energy between both phases and by forming electrostatic barriers among phases to impede the clustering of the emulsion particles (Campos et al. 2015). Biosurfactants have been utilized in the processing industry for various food products (i.e., emulsion systems, such as nanoemulsions (NE), mayonnaise, butter, fillings, salad dressings, etc.) and to improve the emulsifying properties of butter, frozen dessert, and croissants (Campos et al. 2015; Sharma 2016), however, the food industry has not made a wide-scale use of biosurfactants despite their many advantages over their synthetic counterparts.

6 Nanoemulsions (NE) A NE is a thermodynamically unstable colloidal dispersion of two immiscible liquids having a particle size 98% emulsification ratio) with kerosene oil even at pH 11; however, below pH 3, emulsification did not occur due to protonation or ionization of the carboxyl group present in the molecular structure of surfactin (Long et al. 2017). Thus, its use as an emulsifier is limited to products where a pH below 3 is used during processing. This phenomenon was also confirmed by Bai and McClements (2016), the authors observed droplet aggregation in NE (stabilized by using rhamnolipid as a surfactant) at highly acidic (2–4 pH) and highly alkaline (200–500 mMNaCl) conditions due to reduction in electrostatic repulsion among droplets during extreme conditions. Another study demonstrated the ability of rhamnolipid-stabilized NE to avoid breakage caused by demulsifying enzymes (i.e., subtilisin A and alcalase) (Onaizi 2021), which suggests that NE stabilized by biosurfactants can be used as a delivery system for bioactive components.

7 Bakery Products and Desserts Flour-based bakery products i.e., cookies, bread, and cake have gained tremendous attention in the past few decades as target foods for novel and healthier formulations to satisfy the special dietary needs of the consumers (Campos et al. 2013). Biosurfactants isolated from microbial origin are regarded as ‘Generally Recognized as Safe’ (GRAS) and have been suggested to be beneficial for foods. Biosurfactants obtained from Candida and Bacillus genus are commonly used as emulsifier for bakery products (Campos et al. 2015). The main aim of adding biosurfactants into bakery products is to improve the rheological profile of the products and reduce the usage of commercialized additives in these products. Although limited literature is available on the said application of biosurfactant in bakery products; however, there is some evidence that biosurfactant can act as a bio-emulsifier to improve the textural profile of the bread with a decrease in firmness and adhesiveness of the bread samples (Mnif et al. 2012). Another study isolated a biosurfactant from Candida bombicola URM 3718 and used as a replacement for vegetable oil for the development of cupcakes. There was no significant difference between the physicochemical properties of the dough made with or without biosurfactant with reduced trans-fats in muffins with biosurfactant suggesting their potential application in the baking industry (Silva et al. 2020). Durval et al. (2021) formulated cookies with biosurfactant obtained from Bacillus cereus UCP 1615. The authors observed an increase in the crispness of the cookies in relation to an increase in biosurfactant concentration and no negative effect was observed related to textural properties after

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biosurfactant addition. In several studies, it has been reported that a biosurfactant concentration of 0.7–0.75% (w/w) is effective in improving the textural properties i.e., greater springiness and cohesiveness values and lesser hardness and chewiness values of bakery items (Campos et al. 2019; Kiran et al. 2017). The biosurfactants can also be utilized as emulsifiers for frozen desserts (i.e., ice cream (compound emulsion)) with the main aim of improving their textural and quality parameters. These biomolecules can not only solubilize the aromatic compounds added to intensify the flavour but also improve the physical stability of the ice cream by controlling its consistency (Anjum et al. 2016). An important processing step during ice cream production is the partial coalescence, thus biosurfactant should form an easily penetrable layer to encourage partial penetration of fat crystals into the droplets of the liquid portion (McClements and Gumus 2016). There is some evidence that biosurfactants can be used along with some proteins that have adhesion and adsorption properties; however, their ratio should be balanced to achieve a synergistic action at the interface for stabilizing the food product (Campos et al. 2015). Another possible reason for adding biosurfactants for stabilizing frozen desserts as opposed to their natural counterparts (i.e., saponin, carboxy methyl cellulose, etc.) is that they don’t affect the physiochemical and sensory properties of the product as much (Khosrow Shahi et al. 2021).

8 Biosurfactants as Antioxidants and Antimicrobials to Extend the Food Shelf-life Lipid peroxidation leads to the production of off-flavours, rancidity, and toxic compounds which leads to the reduction in the overall shelf-life of the food product. Antioxidants are a group of food additives that are intentionally added to food products to retard or impede oxidation reactions causing lipid oxidation (e Silva et al. 2017). Similarly, secondary metabolites obtained from microbes have been highlighted as antimicrobials, since conventional antibiotics no longer serve the purpose because of antimicrobial resistance. Biosurfactants have the potential to replace both synthetic antioxidants and antimicrobials because of their effectiveness both as an antioxidant and antimicrobial; thus can be used as a coating or film to extend the shelf-life of food products (Rizvi et al. 2021). Generally, a biosurfactant displays its antioxidant potential due to the presence of unsaturated fatty acids in its molecule, on the other hand, the method of antioxidant capacity determination can be used to highlight whether a particular biosurfactant displays its antioxidant activity by reducing complexes or by scavenging radicals (Ribeiro et al. 2020b). For instance, biosurfactant obtained from Bacillus cereus UCP 1615 displayed better complex reduction activity (with total antioxidant capacity reaching >100% at 5 mg/ ml concentration) than radical scavenging activity against both ABTS and DPPH assays (4–10%) (Durval et al. 2021). However, another study reported an excellent DPPH radical scavenging activity of biosurfactant at a similar concentration (5 mg/

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Table 1 Applications of biosurfactants in the formulation of food packaging films and coatings Biosurfactant type and source SophorolipidStarmerellabombicola

Packaging type Polylactic acid film

Rhamnolipid-Pseudomonas aeruginosa

Chitosan coating

GlycoproteinDyadobacter fermentas

Gum ghatti and xanthan gum coating

Rhamnolipid-Lactobacillus rhamnosus

PVA film

Rhamnolipids

Hydroxypropyl methylcellulose, carrageenan, and nisin composite films

Application Displayed antimicrobial potential against Listeria, Staphylococcus, and Salmonella Displayed antiradical activity against reactive oxygen species. Mesophilic bacterial count for oranges treated with active coating was around 2 Log CFU/g which was significantly lower than control (6 Log CFU/g) The coating effectively reduced the activity of reactive oxygen species to avoid oxidation stress in Jamun fruit Antibacterial activity against Staphylococcus and Pseudomonas The growth of food-borne bacteria in chicken fillets and cheese slices was effectively inhibited by nano-active packaging as compared to control and active packaging

References Silveira et al. (2020) Adetunji et al. (2018)

Solanki et al. (2022) Salman et al. (2014) Niaz and Imran (2021)

ml) obtained from Lactobacillus species (74.6–77.3%) (Merghni et al. 2017), which could be due to the difference in the structure of the biosurfactant and its antioxidant mechanism. On the other hand, antimicrobial activity of a biosurfactant could be due to several reasons i.e., rupture or destabilization of the bacterial cell membrane, change in membrane permeability, alteration of vital function, etc. (Rizvi et al. 2021). For instance, several species of lactobacilli bacteria are known to produce biosurfactants with antimicrobial and anti-adhesive properties against food-borne pathogens Escherichia coli, Salmonella typhi, Staphylococcus aureus and Yersinia enterocolitica (Merghni et al. 2017; Madhu and Prapulla 2014). The above-mentioned characteristics of biosurfactants render them useful for their application as an active food coating or packaging film on the food product to extend their shelf-life. However, there are very few studies that have exploited this domain till now. For instance, active packaging made from nano-rhamnosomes significantly reduced the bacterial count (from 7.5 Log CFU/ml to 2 Log CFU/ml) as compared to control samples (Niaz and Imran 2021). Another study reported a decrease in weight loss, malondialdehyde content, and improved superoxide dismutase activity for the oranges coated with coatings containing 2% rhamnolipid (Table 1). Furthermore, the coating also exerted an antimicrobial action on the fruits thus reducing yeast, mould, and mesophilic bacterial count due to the presence of

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monorhamnolipid and dirhamnolipid which possess antimicrobial activity (Adetunji et al. 2018). Some studies related to active packaging based on biosurfactants and their applications are presented in Table 1.

9 Preventive Role of Biosurfactants in the Formation of Biofilms Microorganisms adhere to the surfaces and to each other which results in the formation of microbial aggregates, termed as biofilms. Formation of these biofilms, on the surfaces of food processing units, presents a major source of contamination and food-borne disease outbreak. To ensure the delivery of quality and safe food to consumers, it is of prime importance for food industries to prevent the microbial adherence to food contact surfaces. Biosurfactants due to surface-active nature can be an effective solution in removing the microbial adherence or preventing the microbial attachment to food contact surfaces. Biosurfactants obtained from Pseudomonas spp. were reported to reduce the adherence of Listeria cells to stainless steel surfaces in food processing premises (Meylheuc et al. 2006). Rhamnolipids and surfactin applied to polypropylene and stainless steel surfaces were found effective in reducing microbial adherence (Nitschke et al. 2009).

10

Conclusion and Future Recommendations

Biosurfactants, due to their additional benefits such as preservation potential, low toxicity, and stabilizing effect, have the potential to replace conventional food ingredients. Biosurfactants have extensive applications in food products including emulsion formulations, bakery products, and food packaging system. However, biosurfactants constitute only a limited proportion of total surfactants used globally, which is due to high product cost and low recovery. Alternative low-cost feedstock such as food and agriculture waste and by-products can lower the production cost of biosurfactants.

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Kiran GS, Priyadharsini S, Sajayan A, Priyadharsini GB, Poulose N, Selvin J (2017) Production of lipopeptidebiosurfactant by a marine Nesterenkonia sp. and its application in food industry. Front Microbiol 8:1138 Long X, He N, He Y, Jiang J, Wu T (2017) Biosurfactantsurfactin with pH-regulated emulsification activity for efficient oil separation when used as emulsifier. Bioresour Technol 241:200–206 Madhu AN, Prapulla SG (2014) Evaluation and functional characterization of a biosurfactant produced by Lactobacillus plantarum CFR 2194. Appl Biochem Biotechnol 172(4):1777–1789 McClements DJ (2012) Nanoemulsions versus microemulsions: terminology, differences, and similarities. Soft Matter 8(6):1719–1729 McClements DJ, Gumus CE (2016) Natural emulsifiers—biosurfactants, phospholipids, biopolymers, and colloidal particles: molecular and physicochemical basis of functional performance. Adv Colloid Interf Sci 234:3–26 McClements DJ, Bai L, Chung C (2017) Recent advances in the utilization of natural emulsifiers to form and stabilize emulsions. Annu Rev Food Sci Technol 8:205–236 Merghni A, Dallel I, Noumi E, Kadmi Y, Hentati H, Tobji S, Amor AB, Mastouri M (2017) Antioxidant and antiproliferative potential of biosurfactants isolated from Lactobacillus casei and their anti-biofilm effect in oral Staphylococcus aureus strains. Microb Pathog 104:84–89 Meylheuc T, Renault M, Bellon-Fontaine MN (2006) Adsorption of a biosurfactant on surfaces to enhance the disinfection of surfaces contaminated with Listeria monocytogenes. Int J Food Microbiol 109(1–2):71–78 Mnif I, Ghribi D (2016) Glycolipid biosurfactants: main properties and potential applications in agriculture and food industry. J Sci Food Agric 96(13):4310–4320 Mnif I, Besbes S, Ellouze R, Ellouze-Chaabouni S, Ghribi D (2012) Improvement of bread quality and bread shelf-life by Bacillus subtilis biosurfactant addition. Food Sci Biotechnol 21(4): 1105–1112 Mnif I, Ellouz-Chaabouni S, Ghribi D (2018) Glycolipid biosurfactants, main classes, functional properties and related potential applications in environmental biotechnology. J Polym Environ 26(5):2192–2206 Naughton PJ, Marchant R, Naughton V, Banat IM (2019) Microbial biosurfactants: current trends and applications in agricultural and biomedical industries. J Appl Microbiol 127(1):12–28 Niaz T, Imran M (2021) Diffusion kinetics of nisin from composite coatings reinforced with nanorhamnosomes. J Food Eng 288:110143 Nitschke M, Araújo LV, Costa SG, Pires RC, Zeraik AE, Fernandes AC, Freire DM, Contiero J (2009) Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces. Lett Appl Microbiol 49(2):241–247 Nwaguma IV, Chikere CB, Okpokwasili GC (2019) Effect of cultural conditions on biosurfactant production by Candida sp. isolated from the sap of Elaeisguineensis. Biotechnol J Int 23(3): 1–14 Onaizi SA (2021) Demulsification of crude oil/water nanoemulsions stabilized by rhamnolipid biosurfactant using enzymes and pH-swing. Sep Purif Technol 259:118060 Rebello S, Asok AK, Mundayoor S, Jisha MS (2014) Surfactants: toxicity, remediation and green surfactants. Environ Chem Lett 12(2):275–287 Ribeiro BG, Guerra JMC, Sarubbo LA (2020a) Potential food application of a biosurfactant produced by Saccharomyces cerevisiae URM 6670.Front. Bioeng Biotechnol 8:434 Ribeiro BG, Guerra JM, Sarubbo LA (2020b) Biosurfactants: production and application prospects in the food industry. Biotechnol Prog 36(5):e3030 Rizvi H, Verma JS, Ashish (2021) Biosurfactants for oil pollution remediation. In: Inamuddin Ahamed MI, Prasad R (eds) Microbial biosurfactants. Environmental and microbial biotechnology. Springer, Singapore, pp 197–212 Rufino RD, Sarubbo LA, Neto BB, Campos-Takaki GM (2008) Experimental design for the production of tensio-active agent by Candida lipolytica. J Ind Microbiol Biotechnol 35(8): 907–914

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Biosurfactants in Cosmetic Industry Suganyadevi Palanisamy, Rathi Muthaiyan Ahalliya, Abiram Karanam Rathankumar, Kongkona Saikia, Mariadhas Valan Arasu, Varshini Rajapandian, and Manokiruthika Vellingiri

1 Introduction Cosmetics are used by people to stay clean and enhance their beauty. Lipstick, nail paint, deodorant, perfume, hairspray, shampoo, shower gel, tattoos, hair adhesives, hair removal products, hair dyes, most soaps, some tooth whiteners, and some cleansing wipes are among these goods. It’s crucial to use cosmetics responsibly. Before using cosmetics, the U.S. Food and Drug Administration (FDA) urges you to do your research (Lima and Alegre 2009). Cosmetics are used by people to stay clean and enhance their beauty. Lipstick, nail paint, deodorant, perfume, hairspray, shampoo, shower gel, tattoos, hair adhesives, hair removal products, hair dyes, most soaps, some tooth whiteners, and some cleansing wipes are among these goods. It’s crucial to use cosmetics responsibly. Before using cosmetics, the U.S. FDA urges you to do your research. Cosmetics are any of a wide range of substances—aside from soap—applied to the human body in an effort to improve, preserve, or alter its look, as well as to clean,

S. Palanisamy (*) · R. M. Ahalliya · K. Saikia Department of Biochemistry, FASCM, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India e-mail: [email protected] A. K. Rathankumar Department of Biotechnology, FoE, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India M. Valan Arasu Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia V. Rajapandian · M. Vellingiri Department of Biochemistry, FASCM, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_16

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colour, condition, or protect the skin, hair, nails, lips, eyes, or teeth; see also fragrance and cosmetics. The earliest cosmetics that archaeologists are aware of were in use in Egypt in the fourth millennium BC, based on remnants of items that were probably used for eye makeup and the application of perfumed unguents. In the early days of Christianity, cosmetics were widely used throughout the Roman Empire. With the use of kohl, a substance made from lampblack or antimony, the eyelashes, brows, and eyelids were all darkened and lined (Tuleva et al. 2002). The cheeks were reddened with a variety of white powders, including rouge, to simulate or enhance fairness of skin. Bath oils were commonly utilized as well as a variety of aggregates. The perfumes that were in use at the time were made with natural resins and based on floral and herbal scents (Nitschke et al. 2004). In the fifth century AD, the rise of the modern empire resulted in the eradication of cosmetics and other cultural accoutrements from a large portion of Europe. When crusaders returned from the Middle East with these products, the usage of cosmetics and perfumes was renewed during the middle ages. Cosmetics experienced a significant revival throughout the Renaissance, with France (starting in the seventeenth century) and Italy (during the 15th and 16th centuries) emerging as the two primary centres. At first, only members of the aristocracy, the royal family, and their courtiers used makeup, but by the eighteenth century, virtually every demographic utilized it. Religious culture in the United States and Britain opposed the open use of cosmetics during the conservative Victorian era of the nineteenth century. Despite this, French women continued to wear makeup, and the country was at the forefront of scientific developments in the creation of cosmetics at the time. Any AngloAmerican prejudices against makeup that persisted after World War I were eliminated by the advent of new goods, production methods, packaging innovations, and marketing strategies, which led to an unparalleled level of accessibility to cosmetics (Bhattacharya et al. 2017).

2 Skin-care Preparations Skincare products are a well-known subcategory of cosmetics. Cleaning is the first stage in facial care, and soap and water are still one of the best tools. However, if the skin is sensitive to soap or heavy makeup needs to be removed, washing creams and lotions can be helpful. They contain oil as their major active component, which also serves as a solvent and is combined with water to form an emulsion (a mixture of liquids in which one is suspended as droplets in another). One of the first cosmetics, cold cream is often made by adding water to a mixture of organic fats, such as lard or almond oil (Takahashi et al. 2012). To aid in the oil’s dissolution in water, modern formulations however blend mineral oil with an emulsifier. Emollients and night creams are thicker, cold creams that should be massaged into the skin. In order to prevent the skin from drying out, they often leave a thick layer on the face overnight. People apply hand creams and lotions on their hands to prevent or reduce the dryness and roughness that results from contact with household detergents, the sun,

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wind, and arid environments. While the body’s natural repair mechanisms are taking place, they work similarly to facial treatments by primarily restoring lost water and producing an oil layer to prevent further moisture loss (Suzuki et al. 2011).

2.1

Makeup, Face Powder, and Rouge

The traditional foundation is called vanishing cream, which consists mostly of an oiland-water mixture with 15% stearic acid (a solid fatty acid); some of which is saponified (changed into a crystalline form) to produce the shine feature. These creams provide a smooth, adherent substrate for face powder, which when applied on top of foundation gives the appearance of peach skin without leaving a greasy aftertaste. A face powder needs a lot of components in order to be considered excellent. Magnesium stearate makes it stick, talc makes it easier to apply, chalk or kaolin makes it absorb moisture, zinc oxide and titanium dioxide help it coat the skin more fully, and various colours give it pigment (Cameotra and Singh 2009). The current equivalent of rouge, which is used to mix more colour into the face, is blusher. Rouge is used to emphasize the cheekbones and provides heightened colour. Women frequently carry compact face powder, rouge, and blusher in their handbags.

2.2

Cosmetics for the Eyes

The components of eye makeup, which are often seen as being necessary to a full maquillage (makeup application), include eyeliner and eyebrow pencils to define the edges of the eyelids, mascara to emphasize the eyelashes, and eye shadow for the lids in a range of colours. Because they are administered so closely to a region that is especially sensitive, the ingredients in eye cosmetics must be secure (Santos et al. 2016).

2.3

Lipstick

Since the mouth is a prominent feature along with the eyes and can have beautiful colours and textures, lipstick is practically a global cosmetic. When applied, the fatty base of lipstick spreads smoothly despite being hard in its own right. The colour is typically provided by pigment, which primarily comes in reds but can also be a white material called titanium dioxide that adds brightness and cover. Since lipstick is applied to a fragile surface and eventually swallowed, it must meet the highest safety criteria (Rodrigues et al. 2006).

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Other Cosmetics

Examples of hair preparations include shampoos without soap (soap leaves a film on the hair), which are actually perfumed detergents, gloss- and body-improving items like resin-based sprays, brilliantines and pomades, lotions with alcohol as an active ingredient, and conditioners for damaged hair. In preparations for permanent waves and hair straightening, a chemical called ammonium thioglycolate is used to loosen hair from its natural set. Hydrogen peroxide is used to bleach hair to a blond tint, whereas permanent or semi-permanent dyes are used to add colour to dull or mousy hair. The vast majority of cosmetics and toiletries contain fragrances as a component. Antiperspirants, mouthwashes, depilatories, nail polish, astringents, and bath crystals are additional items for grooming and cleanliness (Cameotra and Singh 2009).

3 Types of Cosmetics Cosmetics have experienced a renaissance during the past 100 years, driven by quick technological advancements and swift shifts in fashion. Cosmetics can be broken down into a variety of unique categories due to the enormous number of items available on the market. There are just ten different types of cosmetic formulations, despite the fact that there are literally hundreds of different types of cosmetic goods. Here is a brief description of each variety, along with information on its traits, method of production, and prospective applications. Even if your company currently employs a formulation chemist, you should still strive to master the art of producing

Fig. 1 Types of cosmetics

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each kind (Elshikh et al. 2016). Fig. 1 shows the types of cosmetics applied to the face. The basic categories for cosmetic formulas include.

3.1

Solution Cosmetics

These are the most basic varieties of cosmetic formulations, and a variety of goods, such as shampoos, body washes, hand sanitizers, and colognes, are created using them. They are soluble mixtures of homogeneous components. After adding the main diluent, which is often water, you just need to blend the remaining ingredients in your container to make them. By slightly warming the system, you can occasionally make them faster (Fakruddin 2012).

3.2

Creams/Emulsions

A cream or an emulsion is utilized since the bulk of cosmetic ingredients are incompatible. Emulsions are supposedly stable combinations of immiscible liquids that are mixed with one another. Products like hand moisturizers, cosmetics, hair conditioners, sunscreens, etc. are produced using them. Your formula must contain an emulsifier, an oil phase, and an aqueous phase in order to produce them. The oil and water phases must first be heated separately, then blended completely while still hot (together with the emulsifier), and finally allowed to cool in order to create the formulations. The result is a cream with minute particles scattered throughout the diluent phase. For further details on producing emulsions, see our article on emulsion HLB (Hahn et al. 2017).

3.3

Lotions

Lotions can be utilized in a variety of settings where ointments and pastes cannot since they are less oily. They can be applied to the skin quickly and easily because they are neither greasy nor sticky. Creams can be excessively heavy or greasy for some purposes; therefore, they are not always suitable. In this situation, lotion is used. Lotions are essentially thin creams. They are found in moisturizing cleansers, leave-in hair conditioners, and skin moisturizers. Creating them is similar to making cream because they are emulsions. Because you do not have to worry about the emulsion thickening enough as it cools, they are frequently simpler. Skin moisturizers and sunscreens are the two most common kinds of lotion.

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Suspensions

Another product format for delivering incompatible substances is suspensions. These are often transparent goods, unlike creams, containing visible particles like inorganic minerals (like titanium dioxide) or gelatine beads suspended throughout. Incompatible ingredients in other products are overcome or removed using this category of cosmetics. They can be found in shampoos, hand soaps, and sunscreens. To create them, a polymer or clay must be added to the mixture to give them an internal suspending structure. Beneficial ingredients include bentonite clays and carbomer.

3.5

Ointments/Pastes

These incredibly thick products are utilized for things like medicated skincare treatments and haircuts. Typically, they are oily and sticky and anhydrous (contain no water). Paste-making frequently calls for the use of petrolatum, lanolin, or dimethicone. You may make them easily by heating the raw materials and quickly blending them until they are spread.

3.6

Tablets and Capsules

The tablet is another product type that is frequently utilized to make colour cosmetics. Numerous cosmetics are created as a powder that is pressed and moulded into the proper shape before being put on the face with brushes and paint sticks. Typically, face powders like eye shadow, cheek shadow, and others are packaged in this way. These are solids that have been pressed into shape after being physically combined. To make these goods, specialized tools are required. Additionally, they are typically more expensive.

3.7

Powders

The most popular and convenient type of makeup and powders are one of the most popular product formats for cosmetics of colour. They first appeared in Mesopotamian and Egyptian antiquity, and their present-day equivalents are not all that dissimilar. Today, we employ fine powder made from raw ingredients to improve our appearance and treat a variety of skin conditions. Baby powder and foot powder are two other products that use powders. They are merely a mixture of basic components that have been ground into a fine powder. Talc, silicates, and starch

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are a few typical constituents. These goods require specialized machinery since the fine powder can be hazardous.

3.8

Gels

Another well-liked kind of cosmetic product is gels. These are ‘shear thinning’ materials, which are thick and frequently clear. In other words, they don’t become thin or flowable unless a force is applied that causes them to do so. Anyone who has tried to open a bottle of ketchup is familiar with the situation. Gels are a component of products including toothpaste, body cleansers, shave cream, and hair care items. They are produced using a gelling ingredient, such as an acrylic polymer, natural gum, or a cellulose thickener.

3.9

Sticks

Keeping our hands away from various cosmetics, such as underarm deodorant and lipstick, it is the ideal way to utilize them. Therefore, they are packaged in sturdy containers that may be opened to administer the product to the body by rubbing it against the skin. There are times when you have to produce a product that the customer would not necessarily want to touch, like underarm deodorant or lipstick. A stick product shape will be employed in these circumstances. When rubbed together, sticks, which are solid dosage forms, can release active substances. They are frequently fashioned from materials that are solid at room temperature when they are created. The components are mixed, heated until they liquefy, then either poured into a mould or used to create the completed item. They adopt the shape of their container as they cool.

3.10

Aerosols

Aerosols are less of a particular formulation type and more of a packaging product shape. If you had the correct can, propellant, and nozzle configuration, you could truly turn practically any cosmetic composition into an aerosol. Any cosmetic dispersed from a pressurized container is an aerosol. They are made up of a propellant and a concentration. The formula is first prepared like you would any other cosmetic, and then it is poured into the can. The can is sealed and the necessary propellant is used to pressurize it. The usage of aerosols in cosmetic products has decreased as a result of recent VOC limits.

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Other Products

• Primers, foundations, and concealers: The materials (powders or pastes) known as concealers are used to cover up skin imperfections such as pores, discolourations, pigment spots, bruises, and blemishes. On the other side, primers and foundations are used to prepare skin so that additional cosmetics will stick to it better and last longer. Makeup products can be distinguished by their area of application: • Lips—Lipsticks, lip gloss, lip liner, lip plumper, lip balm, lip conditioner, lip primer, lip boosters. • Eyes—Mascara, mascara primer, eye shadows, eye primers, eyelash glue, eyelash curler. • Eyebrows—Eyebrow pencils, creams, waxes, gels, and powders. • Nails—Nail polish, nail gloss. • Face—Face powders, foundations, primers, concealers, blush powder, bronzer, setting spray, contour powder/cream.

4 Role of Biosurfactant on Cosmetics Biosurfactants are organic substances that have a lot of promise for use in the creation of cosmetic goods due to their biodegradability and positive effects on human health. In reality, a lot of these biosurfactants can have a ‘prebiotic’ quality. In order to increase the bioavailability of organic contaminants, bacteria make biosurfactants (BSs), ‘green’ amphiphilic compounds. BSs, surface-active substances with the capacity to lower interfacial and surface tension, have been proven to be helpful for a variety of applications. They are employed in the food industry for stability, texture and flavour enhancement, and shelf-life extension. Cosmetics are a necessary part of everyone’s life. People use a wide range of cosmetic items on a daily basis, including soap, shampoo, toothpaste, deodorant, skincare, perfume, and makeup. The cosmetics industry has a number of negative environmental, social, and economic effects that are being addressed by looking for more effective manufacturing processes, reducing waste and emissions, and promoting personal hygiene. These efforts improve public health while also creating job opportunities. The search for natural ingredients in cosmetic goods is a contemporary consumer trend because many of these products offer comparable, superior, or additional benefits to those offered by chemical-based products. Given their biodegradability and effects on health, BSs are natural substances with a lot of promise for use in the formulation of cosmetic goods. In reality, a lot of these BSs can have a ‘prebiotic’ quality. This paper examines the state-of-the-art in BS research for cosmetic uses and goes into additional detail about upcoming difficulties for cosmetic applications.

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Surfactants can lower surface and interfacial tension in water-oil and oil-water systems because of their amphiphilic nature (Lima and Alegre 2009). Amphiphilic compounds called surfactants have a hydrophobic head and a hydrophobic tail. These compounds are used in numerous industrial processes that include emulsification, foaming, detergency, wetting, dispersion, or solubilization. BSs are those made from biological sources, whereas synthetic surfactants are those made from chemical sources (Bhattacharya et al. 2017). BSs are amphiphilic compounds produced by living surfaces, primarily on the hydrophobic and hydrophilic hydrophobic and extracellular moieties that are ejected from microbial cell surfaces, and have the ability to aggregate between fluid phases in order to reduce surface and interfacial tension (Bondi et al. 2015). BSs have a variety of physical and chemical properties, including as low toxicity, biodegradability, foaming ability, and stable activity at both high and low temperatures (Brown 1991; Lima and Alegre 2009; Bhattacharya et al. 2017; Bondi et al. 2015; Brown 1991). These BSs have a wide range of uses in industries like oil recovery, cosmetics, food, agriculture, and pharmaceuticals. BSs have both antibacterial and anticancer properties. Because they have the ability to disturb the biofilm, they are also utilized as anti-adhesive agents. It also displayed antiviral properties (Cameotra and Singh 2009). Additionally, they have shown promising results in the fields of cleaning products, food emulsifiers, pesticide dispersants, antifungal agents, environmental bioremediation, and enhanced oil recovery technologies (Das et al. 2013). BSs are a group of biomolecules with a very diverse structural composition. There are numerous methods for a general screening of BS-producing strains that take into account the physical impacts of surfactants. Alternately, it is possible to investigate how stresses can obstruct hydrophobic surfaces. Results from the screening techniques might be both qualitative and quantitative. The interfacial or surface activity is the basis for the screening techniques for bacteria that produce biosurfactants (Das and Kumar 2016). The potential to extract surfactants naturally present in some plants could also constitute an evolution of bio-based surfactants (no chemical transformation steps). For instance, lecithin, a co-product that is commonly used in cosmetics, is abundant in soybeans. Therefore, using this strategy would enable the fusion of “sustainable development” with ‘100% natural’. This kind of BS would satisfy the need for total naturalness, but it also needs to function above acceptable levels and be generated affordably (Hahn et al. 2017). Surfactants are essential while making cosmetics. They provide important functions, such as those of detergents, emulsifiers, and foaming agents, and are used nearly routinely in a variety of applications. These compounds of interest are, however, frequently derived from petrochemical sources and are not always biodegradable. Surfactants serve numerous purposes in personal care items such as hand soaps, body washes, cosmetics, lotions, and toothpaste. This includes serving as abrasive cleaners, foaming agents for bubbles and lather, or emulsifiers for stabilizing and maintaining the mixing of materials. The chemicals and components that are utilized to produce these items have come under the spotlight as consumer awareness of

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product sustainability and safety has grown. As a result, surfactants made from plants rather than petroleum were used in personal care products (particularly palm and to a lesser extent coconut) (Elshikh et al. 2016).

5 Cosmeceutical Applications of Biosurfactant 5.1

Anti-aging Skincare Products

Intrinsic and extrinsic ageing are the two types of ageing that occur in the human body. Free radicals are released by environmental factors like cigarettes, pollution, and UV light. These free radicals bond to the skin’s surface and cause chemical changes that hasten the ageing process (Bhattacharya et al. 2017). With intrinsic ageing, the collagen and elastin fibres enlarge, get more clumped and loosen, resulting in inelastic and brittle skin that eventually wrinkles and sags (Fakruddin 2012). (MEL) Mannosylerythritol lipid antioxidant-rich skincare products like antiaging face gels and lotions are used to decrease or postpone the signs of skin aging. To evaluate the antioxidant capacity of mannosylerythritol lipid (MEL) derivatives A, B, and C, we performed an experiment with fibroblasts NB1RGB cells using the 1,1-diphenyl-2-picryl hydrazine (DPPH) free-radical technique and a superoxide anion scavenging assay. MEL-C showed the best cellular oxidative stress protection (30.3% at 10 lgm/l of MEL-C) and the greatest antioxidant activity (50.3% at 10 g/l) (Fakruddin 2012). According to their findings, triacylated mannosylalditol lipid, also known as mannosylalditol lipid, can prevent cells from aging and is available in the forms MEL and 165 MML (Fakruddin 2012; Gudiña et al. 2015).

5.2

Sunscreen Products

UVA (ultraviolet A) radiation on skin can have negative consequences as a result of exposure to natural light. Skin protection against solar exposure is essential to reduce the risk of developing skin cancer, especially in people with light skin, as well as the immediate effects, such as sunburn, use of skin-protection compounds in sunscreen. In addition to being used as moisturizers in other products, sunscreen chemicals can be found in a variety of formulations, including lotion, cream, wipes, and lip balm (Elshikh et al. 2016). There are different types of BSs, such as surfactin BS, BS from an agro-industrial stream as an ingredient in sunscreen, and BS that could be used to increase the UV protection of mica minerals (UV light). Sunscreen using BS derived from an agricultural industry stream and BS that could be utilized to boost mica minerals’ UV protection (UV light). The solar protection factor (SPF) of several biocomposites is made from various mica minerals, either by themselves or in conjunction with an extract of a BS from the corn industry (Elshikh et al. 2016).

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The surfactin that B. subtilis generates may be used by the maize industry (Regos and Hitz 1974). A systematic procedure was used to determine the SPF ratings of water-based cosmetics, including mica, both with and without the BS extract. The absorbance was determined at wavelengths between 290 and 320 nm. Without adding BS, the SPF values for the mica minerals ranged from 0.2 to 11, and in some cases, doing so improved the micas’ SPF values by more than 2000% (Das et al. 2013; Cho et al. 2022; Ricon-Fontan et al. 2018).

5.3

Toothpaste

To remove the dental plaque and food particles from the mouth, promote oral hygiene, and get rid of bad breath, toothpastes are a necessary cosmetic that is frequently available and sold in sachets or flexible tubes (Walter et al. 2010). A ‘humectant’ is an ingredient that keeps toothpaste moist. Other ingredients in toothpaste include a binder (which holds the ingredients together), such as sodium alginate, baking soda, a foam-based substance that helps remove food debris from the teeth, such as sodium lauryl sulphate, fluoride, water, and an antibacterial agent (prevent gum disease). Few chemicals, like sodium lauryl sulphate, which is an ingredient in toothpaste, are bad for our bodies’ health (Rodrigues et al. 2006). Therefore, a lot of effort has gone into finding an alternative surfactant that is biocompatible and less hazardous. The production of industrial products is becoming more and more interested in bacterial BSs. Amphiphilic molecules called BSs, which are produced by both bacteria and fungus, have the ability to act on surfaces. Due to their superior environmental compatibility, non-toxicity, high biodegradability, strong foaming capacity, and particular activity at severe temperatures, pH levels, and salinities, they offer a unique advantage over chemical surfactants. Due to their environmental friendliness and ease of degradation, BSs are being used in the environment, food, petroleum, pharmaceutical, and other industries. So, in the current study, a formulation of dental paste based on BSs that are biocompatible and less toxic is produced, and its efficacy is established to produce a safe product. Sophorolipids (SLPs) BS; Nocardiopsis VITSISB, a marine actinobacterium, is the source of BS. It is utilized in the cosmetic composition of toothpaste, taking the place of the surfactant sodium lauryl sulphate, which is often found in commercial toothpaste. The spreadability, foaming, abrasiveness, brine shrimp hatchability, and cleansing ability of this BS toothpaste were all evaluated on a qualitative level. For the manufacture of toothpaste, the BS derived from actinobacteria can be a good substitute for commercial surfactants. According to the findings, BSs are more effective and less hazardous than chemical surfactants (Gudiña et al. 2015). The glycolipid BSs known as sophorolipids (SLPs) are made by Candida bombicola ATCC 22214 and are also utilized in toothpaste (Das et al. 2013). Therefore, as a potential replacement for chemical surfactants in various cosmetic formulations like shampoo, face wash, etc., the BS may be employed in the future due to its compatibility with the environment.

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Face Wash

Rhamnolipid: NatSurFact is a series of BS products based on rhamnolipid. A group of naturally occurring substances known as rhamnolipids have characteristics of surface-active (surfactant) chemicals. Vegetable oil, a renewable resource, is used to produce rhamnolipid through a natural fermentation process. When we use it to cleanse our face and hair, it is gentle. Conventional surfactants like sodium lauryl sulfate can be replaced by rhamnolipid surfactants (Cho et al. 2022).

5.5

Shampoo Rhamnolipid biosurfactant

In a recent work, a shampoo with 2% rhamnolipid dissolved in water was created using rhamnolipid BS from Pseudomonas aeruginosa. The aforementioned BS’s antibacterial properties prevented the scalp’s odour for three days while preserving its lustre Natsurfact.

5.6

Hair Conditioners

Hair conditioners are used to protect from UV radiation while also enhancing the feel, look, and manageability of hair. BS made of mannosylerythritol lipids (MELS): Pseudozyma produces MELS, which have hair care qualities like Repair the brittle hair. MELS are being considered as a new element for hair care products. They are a highly effective agent for both restoring damaged hair and producing smooth, flexible hair (Walter et al. 2010).

5.7

Skin Moisturizing

Creams are applied to the skin to lubricate, moisturize, and protect it. Transepidermal water loss is the name for the process whereby water continuously evaporates from the deeper layers of skin (TEWL). Any increase in the water content of the skin is thought to improve its quality since dry skin is stiff and brittle. Sophorolipid biosurfactants have the ability to store moisture. Bombicolu is treated with alkylene oxides to form the Sophorolipids that Torulopsis makes, which are long-chained alkyl-sophorolipids. These chemically altered substances have been shown to enhance the natural moisturizing component. The oleyl- sophorolipid displayed specific skin compatibility, an HLB Value of 7–8, and had exceptional moisturizing capabilities (Brown 1991; Morita et al. 2010).

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However, BSs, or surfactants derived from microorganisms, are the most environmentally friendly choice for personal care and cosmetics because they are highperforming natural chemicals.

5.8

Ingredients Present in Moisturizers

The ingredients present in moisturizers are as below:

5.9

Emollients

Emollients are substances that are water-insoluble but do not create an occlusive film, like oils and lipids. A substance’s molecular weight may frequently tell an emollient from an occlusive. They are frequently used because they can give skin a silky feel and make it smooth and supple. Since they are mostly made up of lipids that resemble the intercellular lipids of the skin, they may be able to replenish the lipid matrix that has been depleted. Ceramide, fatty acids and cholesterol found in moisturizers can help to restore lipid bilayers damaged by solvents, soaps, and extremely dry or cold temperatures by taking the place of the essential lipid components.

5.10

Humectants

Humectants are hygroscopic conditioning agents that, as a result of their chemical makeup, draw and bind water. The deeper dermis and epidermis are where they are most likely to get water from. The stratum corneum (SC) molecules’ capacity to absorb and retain water, which supplies moisture to the skin tissues and enhances skin hydration, is facilitated by a number of hydroxy (OH) activities. In addition to capturing water from the atmosphere, they can also suck water from the dermis into the epidermis.

5.11

Occlusives

By limiting excessive water loss from the surface of the skin, occlusive substances aid in keeping the SC wet. They do not completely occlude the passage of water, but they still allow it, which is necessary for the skin to operate normally. The extra water content has the result of hastening the recovery of the barrier. The majority of occlusive medications are incapable of adhering to water because they lack OH

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functional groups in their chemical makeup. However, because they can generate homogeneous hydrophobic coatings, they can effectively lock moisture into skin.

6 Exfoliants Glycerin, lactic acid, and urea are ingredients with exfoliating qualities.

6.1

Solvents

Since it is generally available and reasonably priced, water is the most important and frequently utilized raw material in the creation of moisturizing cream. Water is employed in skin creams as a solvent to dissolve other ingredients. Water activity, as an important parameter for self-preservation, can help to control microbial growth in cosmetic formulations (Nadarzynski et al. 2022).

6.2

Antioxidants

Two antioxidants, vitamins C (ascorbic acid) and E (tocopherol), are helpful for shielding the skin from oxidative stress brought on by pollutants and UV radiation. However, when they are exposed to sunlight, they become unstable. Additionally, these vitamins can be found in more enduring forms like vitamin E acetate and magnesium ascorbyl phosphate.

6.3

Preservatives

To prevent or eliminate the growth of microorganisms that are mistakenly introduced during production or usage, preservatives are utilized. Contaminants could also include pathogens and nonpathogens. The ideal preservative should be safe to use, stable in the product, have a broad spectrum of activity, and have no impact on the physical features of the product.

6.4

Fragrances

The aesthetic benefit of fragrances and colouring compounds outweighs their moisturizing capabilities in most applications (Table 1) (Mawazi et al. 2022) These

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Table 1 The common Ingredients present in moisturizers (Mawazi et al. 2022) S. No 1

Ingredients Emollients

2

Humectants

3

Occlusives

4 5

Exfoliants Thickeners

6 7

Buffers Preservatives

8

Lipids

Examples Fatty emollients (octyl stearate, jojoba oil, propylene glycol, castor oil, glyceryl stearate), dry emollients (isopropyl palmitate, decyloleate, isostearyl alcohol), protective emollients (isopropyl isostearate, diisopropyldilinoleate) and astringent emollients (octyloctanoate, cyclomethicone, isopropyl myristate, dimethicone) Alpha hydroxyl acids (Lactic acid and glycolic acid), glycerine (glycerol), sodium pyrrolidine carboxylic acid (PCA), allantoin, honey, panthenol, propylene glycol, butylene glycol, PEG, hyaluronic acid, aluminium lactate, sodium lactate, urea, gelatine, and sorbitol Hydrocarbons (mineral oil, petrolatum, caprylic/capric triglyceride, paraffin, squalene), fatty alcohols (stearyl alcohol, cetyl alcohol, lanolin), fatty acids (stearic acid, lanolin acid), polyhydric alcohols (Propylene glycol), vegetable waxes (Candelilla, carnauba), phospholipids (Lecithin), sterols (cholesterol) and wax esters (Lanolin, beeswax, stearyl stearate) Lactic acid, urea, malic acid Carbomer, sorbitol, oleic acid, xanthan gum, isostearic acid, stearic acid and glyceryl stearate NaOH, TEA, maleic acid and citric acid Potassium sorbate, rice bran oil, phenoxyethanol, disodium EDTA, propylparaben, methylparaben and vitamin C (L-Ascorbic Acid) γ-linoleic acid

9

Fragrance

Hazelnut fragrance

10

Emulsifiers

Cetearyl alcohol, sorbitanmonolaurate and cetyl alcohol

References Dixit (2001); Peters (2001); Bagajewicz et al. (2010); Tamura et al. (2020)

Sethi et al. (2016); Levi et al. (2010)

Dixit (2001); Peters (2001); Bagajewicz et al. (2010); Tamura et al. (2020)

Levi et al. (2010) Dederen et al. (2012)

Dederen et al. (2012) Dederen et al. (2012)

Xu et al. (2017); Dederen et al. (2012) Levi et al. (2010); Manzano et al. (1994) Levi et al. (2010); Ryu et al. (2015)

components include menthol, cinnamates, cinnamic acid, and benzoin resin. Although they may result in irritating dermatitis, colouring agents create subtle colours and other optical effects that lead to increased acceptance. Despite being marketed as ‘fragrance-free’ or ‘unscented’, some moisturizers contain fragrance

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elements in the form of masking agents that prevent the brain from detecting their odour.

6.5

Emulsifiers

The stability of moisturizers depends on the presence of emulsifiers. The most common ways to make moisturizers are as kinetically stabilized colloidal suspensions of two immiscible liquids or as emulsions of two immiscible liquids. Because of this, it is challenging to distinguish between the phases, and over the product’s shelf life, there is no change to the in-use experience. Emulsifiers can be anything from big polymeric fragments, surfactants, and aggregates of lamellar liquid crystal to microscopic monomeric surfactants (Table 1). BSs, unlike bio-based surfactants, do not involve chemical modification steps in their production process. These surfactants are produced by biotechnological means, either by microbial fermentation or by recovery from plants (extraction). It is possible to produce molecules with surfactant properties within microorganisms. The production of surfactants by microorganisms is based on a fermentation process which is very well mastered and is currently considered for the production of sustainable surfactants. The use of microorganisms also offers the possibility to modulate metabolic pathways, allowing the properties of BSs to be adjusted in cosmetics. Finally, the microbial pathway is also potentially beneficial because it could recover co-products and/or biological waste from industries by using them as a source of carbon for microorganisms. It is important to point out that this mode of production has not yet been proposed on an industrial scale, although some major players are interested in it. For example, Unilever launched in 2019 a dishwashing liquid containing rhamnolipids – BSs produced by bacteria. BSs have good surface and biological properties, are non-toxic, and are easily produced. Microorganisms from sustainable sources produce them. Due to their low toxicity, biological activity, biocompatibility, and biodegradability, they also have the potential for extensive application in personal care and cosmetic goods, which is advantageous for the products’ accessibility, efficacy, and efficiency. In several branches, the use of BSs rather than synthetic surfactants ensures both very high efficiency and compliance with ever-stricter environmental requirements. BS may eventually replace synthetic surfactants.

6.6

Enzymes in Cosmetic Industry

Enzyme is used in a variety of cosmetic processes. The use of enzymes will increase along with the rapid development of cosmetics industries. In the cosmetics sector, enzyme can be employed as an antioxidant as well as moisturizing agents, whitening

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agents and other functional additives. The biological enzyme that has been the subject of the greatest research and is employed the most in the cosmetics sector is superoxide dismutase. Superoxide dismutase or simply superoxide dismutase is the body’s first line of defence against free radicals. When the body takes in oxygen to support metabolism, superoxide anion-free radicals are created. The body will start a chain reaction that will kill human cells if free radicals are not eliminated. Free radicals are a significant factor in the development of many diseases as well as aging, according to modern medicine. A natural free radical killer is superoxide dismutase. Cosmetics containing superoxide dismutase have some anti-aging, anti-wrinkle, anti-inflammatory, antiradiation, sunscreen and whitening properties. Superoxide dismutase makes skin more delicate, making it ideal for people who work in front of computers and in the sun a lot because it efficiently blocks UV damage and inhibits the development of melanin, senile plaques, and acne on the face (Nwakanma et al. 2014). 1. Safety evaluation of enzymes 2. Enzymes are produced by organisms. Enzymes for industrial purpose derive from animals, plants and microorganisms. Enzymes used for cosmetics can be obtained from animal and plant tissues generally. 3. The following points of enzymes used in cosmetics 4. Enzymes used in cosmetics are mostly stable under 40  C. In the process of cosmetics manufacturing, enzymes should be added below 40 degrees, avoiding heating. 5. Enzymes used for cosmetics except pepsin, most of which are stable between PH 5.O and PH 9. is stable. Each enzyme has its own best PH, avoiding strong acid and alkali. 6. Pay attention to the question whether there is any inhibitor or activator in the formulation of cosmetics.

General Tips Follow these safety guidelines when using cosmetics products of any type: • • • •

Examine the label. Pay attention to all instructions and warnings. Hands should be washed before using the product. Avoid sharing cosmetics. Protect the containers from temperature extremes and keep them clean and securely closed while not in use. • If cosmetics change in colour or scent, discard them. • Use spray cans or aerosols in well-ventilated spaces. Use caution when using them around open flames or when smoking. It might cause a fire.

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Eye Make-up Tips

There are special safety guidelines for using cosmetics in the eye area. Be sure to keep these practices in mind: • Only use cosmetics that are intended for your eyes near your eyes. For instance, avoid using eyeliner over your lips. • Avoid putting saliva or water in your mascara. Germs could be added. • If you have an eye infection, throw away your eye makeup. The cosmetics might have gotten sick. • Avoid tinting or dying your eyelashes. The FDA has not approved any products for brows or eyelashes that are permanently stained or dyed.

6.8

Understanding Cosmetic Labels

It is crucial to be knowledgeable about the product you are utilizing. Make careful to read the full label, including the components list, cautions and usage guidelines. Additionally, be cautious of the terms listed below that you could find on the label: • Hypoallergenic: Don’t believe a product won’t trigger allergic responses. ‘Hypoallergenic’ is not defined by the FDA. • Natural or organic: The degree of safety is unrelated to the origin of the substances. Don’t automatically assume that these goods are safer than those created using components from other sources. What constitutes ‘organic’ cosmetics is defined by the U.S. Department of Agriculture (USDA). However, ‘natural’ has no official definition from the USDA or FDA. • Expiration Dates: Cosmetics are exempt from having an expiration date under the legislation. However, if you keep a cosmetic product improperly, such as in a warm or moist environment, it could become bad. By labelling the container with the date you open a cosmetic, you may find it useful to keep track of the age of your cosmetics.

6.9

Report Problems to FDA

Before being sold in stores, cosmetics are not required by law to receive FDA approval. FDA does keep track of consumer reports of unfavourable reactions to cosmetic items. Get in touch with FDA if you use a cosmetic product and then experience a rash, redness, burn or any other unexpected reaction. Contact FDA if you discover a problem with the cosmetic product itself, such as an offensive odour, a change in colour or a foreign object inside the product.

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7 Follow these Steps • • • •

Put an end to using the product. To learn how to solve the issue, contact your doctor via phone. Use either of these methods to inform FDA about issues: Contact MedWatch, FDA’s Safety Information and Adverse Event Reporting Program: • By Phone: 1-800-FDA-1088 • Online: file a voluntary report • Get in touch with the local consumer complaint coordinator.

8 Conclusion Like chemical surfactants, BS are excellent emulsifiers and maintain wetting and foaming properties, non -toxic in natures that are valued in several applications including the cosmetics industry. Unlike chemical surfactants, BSs are readily biodegradable contributing to environmental compatibility. Overall it may conclude that BS can act as a replacement candidate for chemical surfactant in cosmetic formulation.

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Applications of Microbial Biosurfactants in Detergents Murat Ozdal, Sumeyra Gurkok, and Volkan Yildirim

Abbreviations CAGR LAS SDS

Compound annual growth rate Alkylbenzenesulfonate Sodium dodecyl sulfate

1 Introduction Surfactants, consisting of a hydrophilic (water-loving) head and a hydrophobic (water-hating) tail, are amphiphilic compounds. They reduce the surface tension between two phases and disrupt the interface between water and oil. They have emulsifying, foam formation, detergent, and oil dispersion activities. Therefore, they are in great demand in various industries such as detergent, cosmetics, medicine, food, textile, paint, petroleum, and agriculture (Sarubbo et al. 2022). Most of the commercially available surface-active agents are chemical surfactants, which are generally produced from petroleum products (Jemil et al. 2016). However, synthetic surfactants pose a threat to the environment as they are generally disposable and about 60% of them are released into the aquatic environment (Pradhan and Bhattacharyya 2017). They damage the macro- and micro-biota of the aquatic and terrestrial environments (Rebello et al. 2020). Accordingly, environmentally conscious consumers prefer environmentally friendly surfactants of biological origin (Perfumo et al. 2018). Biosurfactants have recently gained popularity as a more environmentally friendly alternative to chemical surfactants. Biosurfactants, the natural equivalent M. Ozdal · S. Gurkok (✉) · V. Yildirim Department of Biology, Science Faculty, Ataturk University, Erzurum, Turkey © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_17

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to synthetic surfactants, have also emulsifying, foam formation, detergent, and oil dispersion activities, and are mostly derived from microorganisms. Biosurfactants have distinct advantages such as low toxicity, biodegradability, and production from renewable substrates. In addition, low concentrations of biosurfactants have greater surface and interfacial effectiveness and are active and stable under high temperature, extreme pH, and salinity conditions (Jemil et al. 2016; Schultz and Rosado 2020). However, due to economic concerns, chemically derived surfactants are still in widespread use over biosurfactants and they are used in large quantities in households and various industries. As a result of the extensive use of surfactants, the global surfactant market in 2019 showed a turnover of US$ 42.1 billion. The surfactant market is expected to reach $52.4 billion by 2025; the overall market is expected to surge at a compound annual growth rate (CAGR) of 5.0% (https://www. marketsandmarkets.com/Market-Reports/biosurfactants-market-493.html). On the other hand, the global biosurfactant market is expected to grow from $3.82 billion in 2021 to $5.31 billion in 2026, with a CAGR of approximately 7% (https://www. thebusinessresearchcompany.com/report/biosurfactants-global-market-report). One of the areas where surfactants are widely used is the detergent industry. They are used in the formulations of soap, detergent, and shampoo. They provide the removal of oil and dirt by disrupting the interface between water and oil with their surface tension-reducing properties (Khamis et al. 2022). In this way, surfactant additives in detergents make it easier to remove dirt and stains from the skin, fabrics, and household items. In this chapter, detergents and surfactants as the main components of detergents, the types of surfactants, their harms on humans and ecosystems will be discussed. Afterwards, as their biological equivalents, biosurfactants will be introduced and strategies for cost-effective production of biosurfactants in industrial quantities will also be discussed.

2 Detergents and Surfactants Detergents are materials with complex components that help remove dirt, stain, and microorganisms from contaminated surfaces. The use of detergent is a worldwide need that must be met in terms of health, hygiene care, and lifestyle. They are used in both industrial and domestic areas to clean living surfaces (human skin and hair) and inanimate surfaces (textiles, glass materials, floors, installations, and machines). Detergents are also used in pesticide preparation in agriculture and to disperse oil spills in bioremediation (Curkovic 2016; Farias et al. 2021). In general, detergents can be in different forms such as liquid, powder, tablet, and gel (Bouassida et al. 2018). There are different brands of commercial detergents, and most of them claim some special qualities about their products. Detergents have a specific formula for cleaning and consist of different ingredients depending on the usage of the final product. While hand soaps are made up of only a few ingredients,

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most detergents have more than one complex ingredient. For example, laundry detergents are mixtures containing more than 20 compounds (Gaubert et al. 2016). The typical components of detergent ingredients include enzymes, surfactants, bleaching agents, softening agents, stabilizers, viscosity builders, colour, and perfume (Gurkok 2019; Zhang et al. 2021). The most essential components in detergents are the surfactants, accounting for 15% to 50% of the entire detergent formulation. Most of the detergents on the market are products with active ingredients, usually in the form of surfactants derived from petroleum derivatives (Helmy et al. 2020). With their surface tension-reducing properties, surfactants disrupt the interface between water and oils, thus facilitating the removal of dirt and stains from contaminated items. These amphiphilic molecules are also used for their solubilizing, dispersing, foaming, wetting, and emulsifying properties (Gaubert et al. 2016). The low surface tension of the water facilitates the removal of oil and this helps to keep the oily dirt suspended in the water. Surfactants differ in their ability to remove different types of soils, activity on different surfaces, and response to water hardness. The hydrophilic head pulls the oil towards the water and the head stays in the water (Bouassida et al. 2018). Surfactants act in three different ways: (i) Roll-up mechanism, (ii) Emulsification, and (iii) Solubility (Mishra et al. 2009). With ‘roll up mechanism’, surfactants decrease the interfacial tension between ‘oil and solution’ and ‘cloth and solution’, lifting the soil off the cloth. With ‘emulsification mechanism’, they decrease the interfacial tension of the oil solution and facilitate oil emulsification. With the ‘solubilization mechanism’, the material spontaneously dissolves to produce a clear and stable solution after interacting with the surfactant’s micelles in the water (Bouassida et al. 2018). The surfactant used in a detergent is determined by several parameters, including fabric or glassware type, wash temperature, builder type, foam level, detergent form (liquid or powder), biodegradability, eco-toxicity, and manufacturing process. Surfactants are divided into 4 groups according to their polar head structure: anionic, nonionic, cationic, and amphoteric. Anionic surfactants have been widely used in detergents since ancient times. They include alkyl sulfonates, sodium laureth sulfate, ammonium laureth sulfate, sodium stearate, etc. Anionic surfactants have greater wetting, foaming, and emulsifying properties (Vecino et al. 2017). Among them, sodium dodecyl sulfate (SDS) and linear alkyl benzene sulfonate (LAS) are frequently used in laundry detergents, household, and personal cleaning items. Cationic surfactants are also applied in detergents, softeners, and conditioners, but they are not as effective as anionic surfactants. Because the surfaces of the hair and fabric are generally charged negatively, which causes the cationic surface-active agents to be easily adsorbed into surfaces, but removed with difficulty. Being adsorbed by this interaction changes the surface textures and makes them feel softer (Arai et al. 2001). In addition, cationic surfactants inhibit bacterial growth due to their bacteriostatic effects and can be used to develop cleaning agents, disinfectants, and antimicrobial products (St. Laurent et al. 2007). Amphoteric surfactants are typically used in body washes, shampoos, and handwashes. They are the least strong surfactants and are included in sensitive skin personal care products. In addition, effective foaming

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feature of amphoteric ones makes them preferred for hand washing. After anionic surfactants, charge-free nonionic surfactants are the most frequently used surfactants in laundry and dishwashing detergents. These molecules are frequently employed as wetting agents, emulsifiers, and foam stabilizers. Since nonionic surfactants do not have charge, they have less ability to form foam in waters with higher hardness and are less effective in cleaning than anionic. However, for sensitive individuals, they are preferred as they are less skin irritating. Most cleaning solutions combine different types of surfactants, such as anionic and nonionic surfactants, to improve product quality and to manage cleaning power with skin irritation risk (Gaubert et al. 2016). This combination also provides lower critical micelle concentration and reduces the required surfactant quantity to obtain the equal cleaning efficiency (Cheng et al. 2020). The surfactant additives in detergents must have certain properties. They should dissolve quickly in water, have high cleaning power, be biodegradable and be safe for the environment and should not degrade at washing temperature and pH (Cheng et al. 2020). In addition, the hardness of the water has a significant impact on the surfactant’s effectiveness.

3 Harms of Surfactants in Detergents Detergent use is generally associated with cleaning and preservation practices; however, some have risks to the environment and human health. Detergents can be defined as an aqueous solution of surfactant with cleaning properties. The task of surfactants, which have both hydrophilic and hydrophobic parts, in detergents is to facilitate the removal of stains from the surface by lowering the surface tension of the solutions. Soaps, subsets of surfactants, are metallic salts of fatty acids. Most detergents contain anionic or nonionic surfactants in addition to soaps. Anionic surfactants include alkyl sulfates; nonionic surfactants include long-chain alcohols. In addition to these, chelators, buffering agents, emulsifiers, and other additives are also used in detergents. Although soaps and surfactants differ in their composition, both can induce irritation to the skin, eyes, and respiratory systems by damaging the lipid cell membranes of living things. The harms of surfactants in detergents can be mild or severe through inhalation, direct contact, or ingestion. The majority of surfactants are skin irritants and the most common mode of exposure is direct contact, while prolonged exposure can cause severe irritation and dryness. Detergents of an alkaline nature can also cause severe chemical burns. Irritation of eyes is another common hazard that surfactants can cause. Again, almost all surfactants cause mild or severe eye irritation when they get into or come into contact with the eyes. With the annual use of surfactants in excess of 15 million tonnes worldwide and the release of the vast majority of these surfactants into the aquatic environment has detrimental effects on aquatic life (Ivanković and Hrenović 2010; Johnson et al. 2021). Chemically synthesized surfactants in detergents easily reach underground

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waters, fresh waters, and seas through wastewater, where they have harmful effects on living things and ecosystems. They greatly boost biological oxygen demand in water, damage fishes and crustaceans, and disrupt photochemical energy conversion in plants. With their antimicrobial properties, they also damage aquatic microorganisms putting a severe burden on sewage works, wastewater treatment, and the ecosystem, indirectly causing an increase in environmental pollution (Ivanković and Hrenović 2010). Discharge of surfactants into aquatic environments also causes foam production, decreasing the oxygen level and disrupting the ecosystem (Schilling and Zessner 2011). Due to their physical, chemical, and molecular structures, they are very difficult to remove after mixing with water. While their low degradability and persistence in aquatic environments are major problems, in some cases partial decomposition products can have a greater toxic effect (Siyal et al. 2020). Although the targets and effects of different surfactant types in living organisms vary, they all cause harm to life to different extents. For instance, while cationic ones bind to and disrupt bacterial cell membrane, anionic ones interact with DNA and enzymes (Cserháti et al. 2002; Olkowska et al. 2014). On the other hand, nonionic ones have antibacterial effect through attaching to different proteins in bacteria that induce cellular damage. Increasing public awareness and concern about the environmental risks related with synthetic surfactants has highlighted the need for environmentally friendly, natural substitutes for chemical surfactants in detergents (Johnson et al. 2021). As well as the environment, the widespread use of biosurfactants will undoubtedly be beneficial for human health. The use of biosurfactants in detergents will also eliminate skin irritations and allergic reactions caused by synthetic surfactants (Vecino et al. 2017).

4 Application of Biosurfactants as Detergent One of the recent application areas of biosurfactants is their use in the detergent industry. Biosurfactants function similarly to chemically produced synthetic surfactants. Biosurfactants attract the attention of the detergent industry with their wetting, detergency, dispersion, foaming, surface tension reducing, emulsifying properties, antimicrobial and antibiofilm potentials. With wetting properties, biosurfactants enable the penetration of detergent/washing solution into the pores to reach the soils. Detergent activity is one of the most important properties of biosurfactants and they have the potential to be incorporated into detergent formulations as an alternative to synthetic surfactants, as they are less toxic and more suitable for the skin (Banat et al. 2010). With their surface tension-reducing properties, biosurfactants disrupt the interface between washing solution/water and oils, thus facilitating the removal of soil. While emulsification activity is needed to keep the soil in the wash solution; dispersion activity is needed to prevent aggregation of soils. Biosurfactants disrupt the integrity of the cell membrane of microorganisms and increase its

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Table 1 Enzymes and biosurfactants used together as detergents Microorganism Bacillus licheniformis B. subtilis B. methylotrophicus Ochrobactrum intermedium Yarrowia lipolytica B. subtilis O9

Enzyme Protease Amylase Protease Alkaline amylase Lipase Lipase Protease

Biosurfactant Type Lipopeptide Lipopeptide

References Ramnani et al. (2005) Bhange et al. (2016)

Lipopeptide

Hmidet et al. (2019)

Glycolipopeptide

Ebrahimipour et al. (2017) Janek et al. (2020) Iglesias et al. (2019)

Lipopeptide Surfactin

permeability, and in this way, they show an antimicrobial effect (Bjerk et al. 2021). It is known that biosurfactants prevent biofilm formation due to their anti-adhesive and antimicrobial properties (Paraszkiewicz et al. 2021; Nalini et al. 2022). However, chemical surfactants that are used as a detergent ingredient are not effective in disrupting the biofilm structure at low temperatures (Marchant and Banat 2012). Therefore, the use of biosurfactants in detergent formulations is effective in preventing the formation of biofilm in washing machines and dishwashers at low temperatures. Biodegradable and eco-friendly biosurfactants must have certain properties in order to be included in the composition of detergents. Biosurfactants included in the detergents are influenced by a number of parameters such as temperature, pH, water hardness, enzymes, inhibitors, and other ingredients in detergents. Temperature and pH are the most important parameters in laundry or dishwashing (Grbavčić et al. 2011). Although the pH of washing conditions is almost always alkaline, the temperature may vary depending on the material to be washed. Therefore, the biosurfactant should be stable at alkaline pH values between pH 8–11. It should also be effective at lower temperatures for washing delicate wool and silk fabrics, and stable at high washing temperatures for cotton fabrics with stubborn stains such as oil stains. The choice of surfactant in detergent production also depends on the intended use. For example, while laundry detergents need a surfactant with high washing power that can create foam and can be easily removed, face washes need a surfactant with a high foaming feature that will not harm the skin (Furuta et al. 2004). Due to the increasing interest in the use of biosurfactants in the detergent industry globally, many studies have been carried out on the subject in recent years. Several studies have also focused on the use of biosurfactants with different enzyme additives to remove stains from fabrics. Protease, lipase, and amylase are the leading enzymes added to the detergents together with biosurfactants (Table 1). The detergents produced by combining cold-active enzymes (amylase, lipase, protease, and cellulase) and biosurfactants increase the cleaning ability (El-Khordagui et al. 2021). Bhange et al. (2016) reported that the combination of biosurfactant, keratinolytic protease, and amylase together with other detergent components can increase the stain removal power. Lipase and biosurfactants are intensively used together for the

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removal of stubborn oily stains from laundry and dishes. Zarinviarsagh et al. (2017) showed that alkaline thermostable lipase and biosurfactant from Ochrobactrum intermedium isolated from the hot springs in Ardebil, Iran were effective at oil removal from white cotton fabrics. This biosurfactant remained stable over a wide pH range (5.0–13.0), achieved best results at pH 10–13, and elevated temperatures of 60–90 °C, demonstrating potential for use as a washing component in laundry detergents. The crude extract of Bacillus methylotrophicus DCS1 containing both biosurfactants and amylase activity indicated that it could be used in detergent processing industries (Hmidet et al. 2019). The results indicated that the biosurfactant—enzyme mixtures performed higher than a single cleaning agent (Onaizi et al. 2009). In this way, they show a positive synergistic effect due to the attack of the surfactant and the enzyme on different bonds (physical and chemical). There are also many patents on the combined use of enzymes and biosurfactants for detergent purposes. A cleaner having enzymes (amylase, phosphatase, reductases, transferases, hydrolases, lyases, isomerases) from Laminaria digitata (brown algae or kelp) and sophorolipids produced by bacteria and yeasts have been patented (Furuta et al. 2004). This sophorolipid-containing detergent composition was especially designed for dishwashers, as it creates little and easily removable foam and has washing power over a wide temperature range. The detergent composition consisting of lipase in combination with rhamnolipid has been patented as a builder-free detergent formulation suitable for low washing temperatures and fast washing times, saving energy and time (Parry et al. 2013). In another patent study, powder and liquid detergents were developed by combining protease and biosurfactant, such as rhamnolipid or sophorolipid (Schilling et al. 2021). Biosurfactants that can remain stable in alkaline conditions are preferred in order to work in harmony with detergents during washing. The best source for these biosurfactants is the microorganisms adapted to alkaline environments. Jain et al. (2012, 2013) investigated a biosurfactant produced by the alkaliphilic bacterium Klebsiella sp. This thermostable biosurfactant has been reported to consist of protein, sugar, sulfate, and uronic acid and has been found to be compatible with detergents. It has improved washing performance and efficiency of oil removal from cotton fabrics, it has also been reported to have superior oil removal rate compared to synthetic surfactants like Tween 80 and SDS. A study was conducted on the compatibility and stability of cyclic lipopeptide biosurfactants produced from Bacillus subtilis in commercial detergents (Mukherjee 2007). It has been determined that these biosurfactants reduce the surface tension, form micelles and dissolve a wide variety of hydrophobic chemicals, while being non-toxic to aquatic organisms, especially fishes. It has also been reported that these biosurfactants remain stable in the pH range of 7–12 and at 80 °C for 60 minutes. When these stable cyclic lipopeptide biosurfactants are added to various commercial detergents as additives, they effectively remove oil and blood stains and improve washing performance. In a similar study, the addition of B. subtilis lipopeptide biosurfactant to a commercial detergent formulation and the compatibility of the biosurfactant with the other detergent ingredients was evaluated (Bouassida et al. 2018). Biosurfactant was tested in the formulation of a powder detergent containing

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sodium sulfate as the filler and sodium tripolyphosphate as the builder. The removal of a stain from cotton fabric was tested using a detergent composition produced with varied washing settings. It was reported that the lipopeptide biosurfactant improved washing performance by efficiently removing oil and tea stains (Bouassida et al. 2018). Glycolipid-type biosurfactants from Pseudozyma sp. NII 08165 were shown to be capable of removing blood, ketchup, and chocolate stains from the cotton clothes efficiently and could be used in laundry detergent formulations. It was also stable at high temperatures (60 °C and 80 °C for 2 h) and in alkaline pH (8.0–12.0) range, which favour their scope of application as laundry detergent additives (Sajna et al. 2013). In another study, it was determined that the biosurfactant produced by Serratiamarcescens UCP 1549 by solid-state fermentation has high stability at wide range of temperatures (0–100 °C), pH (2–12) and salinity (2–25%) (Dos Santos et al. 2021). Researchers suggested that this biosurfactant is suitable to be used in different industries such as detergent and bioremediation. Hydrocarbon pollution often results from oil spills from industrial storage facilities or tanks, spills during lubrication of equipment, transportation of petroleum products, and deliberate discharge of petroleum derivatives and by-products into ecosystem. Cleaning and removal of oil pollution are performed by directly using detergents or surfactants (Rocha e Silva et al. 2019). However, since their use also causes different pollutants to be released into the environment, the same process can also be accomplished by using biosurfactants (Farias et al. 2021). For instance, P. aeruginosa ATCC 10145 was used by Farias et al. (2021) to create a biodetergent aiming to eliminate heavy oil. The biosurfactant of P. aeruginosa was effective in reducing the surface tension, emulsification, and dispersion of motor oil and was used in detergent formulation. The toxicity of this detergent preparation was tested on Brassica oleracea seeds and microcrustacean Artemiasalina larvae, and it was reported to be low. Biosurfactant from Paenibacillus sp. D9 was found to be more efficient for removal of tomato sauce and coffee stains than chemical surfactants (SDS and Triton X-100) (Jimoh and Lin 2020). Biosurfactant synthesized using Cunninghamella echinulata UCP 1299 exhibited successful removal of 86% of burnt engine oil from cotton fabric (Andrade et al. 2018). Similarly, the anionic bioemulsifier synthesized by Stenotrophomonas maltophilia UCP 1601 perfectly removed the burnt engine oil from the sand (Nogueira et al. 2020). In addition to being effective in removing stains from laundry and dishes, biosurfactants have also a much lower toxic effect on the environment, human and animal health compared to synthetics. Fei et al. (2020) purified the lipopeptide surfactant from B. subtilis HSO121 for detergent formulations. They showed that surfactin is suitable for commercial detergent use because it is a biodegradable, low toxic, and non-skin irritant biosurfactant. Rhamnolipid from P. aeruginosa ATCC 10145 was able to completely remove heavy oil impregnated on metal, plastic, glass surfaces, floors, and heat exchanger plates (Farias et al. 2021, 2022). In addition, it has been also suggested that rhamnolipid produced by P. aeruginosa isolated from garage soil can be used as a detergent. Rhamnolipid was used to clean ketchup,

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blood, and chocolate stains on cotton fabric and showed the same activity as the commercial detergent. It was also determined that they are effective when rhamnolipid and commercial detergent are used together (Suryawanshi et al. 2021). Detergent which contains biosurfactant showed low toxicity in tests involving crustacean larvae of Artemia salina and seeds of cabbage and tomato. Khaje Bafghi and Fazaelipoor (2012) developed a formulation including rhamnolipids and tested the formulation on cotton fabrics to compare the results of removing cooking oil and stains. They compared these results with commercial detergents for the removal of cooking oil, chocolate, and albumin stains, and reported that rhamnolipid-containing detergent formulation is as effective as commercial ones, especially in removing hydrophilic stains. Sophorolipds are promising glycolipid biosurfactants produced by non-pathogenic yeast strains. Sophorolipids obtained from Candida bombicola are used in commercial household cleaning products due to their low toxicity and complete biodegradability. They are also used in hard surface cleaning, automatic dishwashing applications, and hand soaps (Develter and Lauryssen 2010). It was shown that biosurfactant obtained from C. sphaerica removed 41% of engine oil from contaminated cotton fabric (de Souza et al. 2013). In a similar experiment, biosurfactant produced by C. lipolytica UCP0988 removed 70% of the engine oil from cotton cloth in detergency tests (Santos et al. 2017). Cox et al. (2013) patented a foaming, skin-compatible detergent formulation containing sophorolipids. In recent years, washing applications at low temperatures have been on the agenda of companies due to economic and environmental concerns such as reducing carbon footprint. However, there may be some drawbacks of using low temperature for washing. Especially in washing with detergents that do not contain bleach and disinfectant, a temperature of 60 °C is typically required to inactivate most of the microorganisms such as bacteria, mould, and viruses. However, washing at 30 °C or lower temperature provides suitable conditions for microorganisms on clothes or glassware rising from human skin or from the environment. Especially for common biofilm-forming organisms such as Pseudomonas sp. and Bacillus sp., cold washing will not give the desired cleaning result. In such a case, detergent formulations, with biosurfactants having anti-adhesive, antimicrobial, and biofilm-degrading properties, can contribute to desired cleaning results. Washes performed with cold water require the use of cold-active biosurfactants in some cases. As the source of these cold-active biosurfactants, psychrophilic microorganisms are sought. Biosurfactants from psychrophilic microorganisms can improve washing performance at cold temperatures, which can reduce energy consumption by enabling laundry at lower temperatures (Perfumo et al. 2018). The use of very low wash water temperatures adversely affects detergent effectiveness and suppresses the cleaning impact (Laitala and Jensen 2010; Perfumo et al. 2018). Some of the surfactants become crystalline below the Krafft temperature, resulting in the failure of some surface activities including micelle-forming, emulsification, and dispersion (Tsujii and Mino 1978). It is known that the use of different surface-active agents together has a synergistic effect in detergents, especially in removing oil stains. This synergistic effect

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may be the use of different biosurfactants together, as well as the biosurfactants used together with synthetic surfactants. A plant-based biodetergent composed of saponin, cotton oil, and stabilizers has been reported to be non-toxic, stable, and highly efficient in removing heavy oil from metal and glass surfaces (Rocha e Silva et al. 2020). In the other case, biosurfactant and synthetic surfactant blends have additionally provided an alternative way to reduce cost and produce superior properties by maximizing their advantages with their synergistic use (Kong et al. 2018). In another example, mixture of synthetic surfactants and tea saponin demonstrated improved foaming and interface capabilities compared to when used alone (Jian et al. 2011). Biosurfactants show antiviral activity by disrupting the structures of the viral envelope and capsid and by affecting the receptor binding sites. Biosurfactants can be used in the formulations of hand washing and cleaning products to prevent the spread of viruses (herpes simplex virus, influenza virus, and COVID-19) since they have low toxicity (Smith et al. 2020; Ceresa et al. 2021; Daverey and Dutta 2021). Biosurfactants are also used in different applications such as personal care cleaning products. Surfactants are present in 10 to 40% by weight of the total shampoo formulations. In recent years, there has been an increase in the demand for sulfate-free or salt-free formulations of shampoos (Luengo et al. 2021). Due to the high cost of biosurfactants, they are mostly used in the production of high-value cosmetic products instead of being used in shampoo formulas. It is interesting to use natural cationic biosurfactants instead of cationic chemical polymers used in the shampoo composition. When the sophorolipid surfactant was mixed with chitosan, it increased the viscosity properties of the mixture, which is one of the required properties for shampoos (Pingali et al. 2020).

5 Approaches for Commercial Biosurfactant Production The versatile use of biosurfactants necessitates their large-scale production for industrial use. Besides the oil and detergent industry as the largest possible market, they are also useful in the pharmacological, medical, cosmetic, agricultural, and food industries. Although they have many commercially attractive superior properties, they are still expensive and limited to obtain commercially due to their low yields, high production costs, and tedious purification processes. The main factor hindering the commercialization of biosurfactants is associated with the high-cost production process. In order to make them competitive with chemical surfactants, they must be produced and purified on a large scale at comparable costs. The cost of a biosurfactant is more than ten times that of a synthetic surfactant on average (Drakontis and Amin 2020). Therefore, it is necessary to reduce the production costs of biosurfactants. Every year, an increasing amount of carboncontaining waste is generated in the world. In some cases, these wastes can be used as a carbon source for the fermentation processes to produce useful and valuable products. In order to obtain maximum yield of biosurfactants, it is important to determine the optimum fermentation conditions for the selected

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Selection of suitable reactor

Use of inexpensive raw materials/ substrates

Selection of appropriate microorganisms

Cost-effective biosurfactant production

Strains improvement

Effective purification

Optimization of the culture medium

Fig. 1 Strategies developed for effective biosurfactant production

microorganism species. Approximately 30–50% of the total cost of biosurfactant production is due to the cost of raw materials used in the production medium (Mohanty et al. 2021). Factors such as carbon and nitrogen sources, availability of micronutrients, inoculum concentration, temperature, pH, aeration, and agitation speed need to be considered (Jahan et al. 2020; Gurkok 2021; Gurkok and Ozdal 2021). The production of biosurfactants by microbial fermentation can be carried out using agro-industrial by-products or wastes (waste oils, molasses, corn maceration liquid, vegetable oils, and petroleum products). These substrates are used as inexpensive carbon sources (Ozdal et al. 2017a; Sarubbo et al. 2022). By employing this approach, while the production cost is reduced, environmental pollution is also minimized (Domínguez Rivera et al. 2019; Liepins et al. 2021). Nitrogen sources have an important effect on the growth of microorganisms and the synthesis of secondary metabolites such as biosurfactants (Ozdal et al. 2017b). The presence of other nutrients such as phosphorus, magnesium, calcium, manganese, sulphur, iron, and their ratios also affect the biosurfactants fermentation processes (Jimoh and Lin 2019; Gurkok and Ozdal 2021; Sarubbo et al. 2022). In order to incorporate biosurfactants into commercial-size detergent formulations, different strategies must be developed for their cost-effective manufacturing (Fig. 1). These include the selection of suitable biosurfactant-producing

374 Table 2 Biosurfactant-producing microorganisms

M. Ozdal et al. Bacteria • Pseudomonas • Bacillus • Acinetobacter • Rhodococcus • Serratia • Lactobacillus • Arthrobacter • Corynebacterium • Rhodococcus • Citrobacter • Paracoccus • Pediococcus • Stenotrophomonas • Franconibacter • Corynebacterium

Fungi and yeast • Candida • Saccharomyces • Ustilago • Curvularia • Phoma • Rhizopus • Mucor • Trichoderma • Penicillium • Fusarium • Aspergillus • Torulopsis • Kluyveromyces • Rhodotorula

microorganisms (Gurkok 2022). Genetically modified organisms can also be used to achieve higher yields. The choice of raw material is also crucial for commercial manufacturing. As mentioned earlier, the substrate cost accounts for a large part of the total cost, cheap raw materials or industrial wastes may be preferred. Optimization of culture conditions will undoubtedly increase production efficiency in biosurfactant production as in many processes. Efficiency can be increased considerably, especially with statistical optimization strategies that take into account the relationships between variable factors affecting the productivity (Ozdal et al. 2017a, 2017b; Bertrand et al. 2018; Ambaye et al. 2021; Onwosi et al. 2021; Gaur et al. 2022). Biosurfactants are produced extracellularly by eukaryotic (plant and fungi) and prokaryotic (bacteria) organisms. Species belonging to the genera Pseudomonas, Bacillus, and Candida are often used for commercial production of different types of biosurfactants (Patel et al. 2019; Singh et al. 2019). Other biosurfactant-producing microorganisms are shown in Table 2. The reasons for using microorganisms in the production of biosurfactants can be listed as follows; (i) is not seasonal, (ii) be able to use waste materials as substrate, (iii) be produced in small areas, and (iv) be produced in many different varieties.

6 Biosurfactant Market Size Biosurfactants are excellent compounds with a wide range of activities, and their manufacturing has progressed from the laboratory to the large scale. Although biosurfactants have been known for a very long time, their industrial production has increased in recent years (Gayathiri et al. 2022). Depending on the nature of biosurfactants, it offers opportunities for many applications such as pharmacology, agriculture, environment, food, cosmetics, and detergents (Gayathiri et al. 2022).

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Important studies have been carried out for the detergent industry, which is one of these areas. Although most of the biosurfactants and their production processes are patented, only some of them have been commercialized. Accordingly, there are only a very limited number of companies producing biosurfactants on a commercial scale. Among biosurfactants, the largest number of patents and scientific publications have been made on rhamnolipids (Randhawa and Rahman 2014). Today, biosurfactants are produced by certain companies in USA, UK, Germany, and China. The global market for biosurfactants was valued at $4.7 billion in 2021 and is estimated to reach $6.4 billion by 2026, growing at a CAGR of 6.4%. Increasing consumer preference for biologically based products, especially in Europe and North America, is a key factor in the growth of the market. Today, some biosurfactant-based detergents are produced by some multinational companies (Table 3). For example, SyntheZyme (USA) is developing bioscavengers of modified sophorolipids for cleaning purposes. Sophorolipids are also found in different cleaning products (soap, window freshener, and car wash cleaner) by Ecover. In addition, Henkel uses biosurfactants in its detergent products.

7 Conclusion Surfactants are used extensively in a variety of industries. Currently, chemically produced surfactants are the most common type of surface-active agents, but adverse effects on the environment and humans are widely known. Biosurfactants have several advantages, such as low toxicity, higher biodegradability, and biocompatibility, thereby reducing the problems caused by chemical surfactants. They can be produced by microorganisms using renewable and sustainable raw materials. However, the main obstacle of using biosurfactant as an alternative to chemically derived ones is the high production costs of biosurfactants. Although global industries serve the economy and the growing needs of a rapidly growing population, they also have responsibilities to the sustainability of the ecosystem, the environment, and human health. Therefore, it is important to produce biosurfactants in a cost-effective way to pave the way for their widespread use. Due to the green consumption trend, there is no doubt that studies, on the development of detergent formulations in which synthetic biosurfactants are replaced with microbial biosurfactants, will increase rapidly over the next years.

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Table 3 Biosurfactant manufacturing companies Company/ Country AGAE Technologies/USA Allied Carbon Solutions/ Japan BioFuture / Ireland Evonik/ Germany Ecover/ Belgium GlycoSurf

Henkel/ Germany Jeneil Biotech/USA KANEKA/ Japan Soliance/ France Logos Technologies / USA MG Intobio/ South Korea Saraya Co./ Japan TeeGene Biotech/UK TensioGreen

Product Name R90, R95 ACS-Sophor

BFL 5600SS, BFL Multi Clean Rewoferm® Sl One

GlycoSurf

Breff, Sidolin, Sonasol, Tenn Jeneil Kaneka Surfactin Sopholiance

Application Area Pharmaceuticals, cosmetics, personal care products, home cleaning products Detergent, agriculture

Biosurfactant Type Rhamnolipids Sophorolipids

Bioremediation, fruit and vegetable processing, household cleansers Shampoos, shower gels, and household cleansers Cleansing and detergent products

Sophorolipids

Anti-aging products, acne skin creams, hair care, medical device cleaning, surface cleaning Glass cleaning products, laundry products

Rhamnolipids

Soaps, shampoos, and contact lens cleaners Cleansing oil gel, organic cosmetic material

Sophorolipids

Sophorolipids Rhamnolipids Surfactin, Rhamnolipid Surfactin

Face cleanser, shower gel and make-up remover Personal care, industrial and household cleaning, medicine, agriculture

Sophorolipids

Sopholine

Cleaning products

Sophorolipids

Soforo

Cleaning products, hygiene products

Sophorolipids

https://www. teegene.co.uk

Detergent, cosmetics, antimicrobial, antiviral, bioremediation, waste management sectors Manufacturer

Rhamnolipid

NatSurFact

PAS, BBS

Rhamnolipids, Trehalolipids

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sustainable energy and bioproducts. Academic Press, Cambridge, pp 511–521. https://doi.org/ 10.1016/B978-0-12-818996-2.00023-5 Rocha e Silva NMP, Almeida FCG, Rocha e Silva FCP, Luna JM, Sarubbo L (2020) Formulation of a biodegradable detergent for cleaning oily residues generated during industrial processes. J Surfactant Deterg 23(6):1111–1123. https://doi.org/10.1002/jsde.12440 Rocha e Silva NMP, Meira HM, Almeida FCG, Soares da Silva RDCF, Almeida DG, Luna JM, Rufino RD, Santos VA, Sarubbo LA (2019) Natural surfactants and their applications for heavy oil removal in industry. Sep Purif Rev 48(4):267–281. https://doi.org/10.1080/15422119.2018. 1474477 Sajna KV, Sukumaran RK, Jayamurthy H, Reddy KK, Kanjilal S, Prasad RB, Pandey A (2013) Studies on biosurfactants from Pseudozyma sp. NII 08165 and their potential application as laundry detergent additives. Biochem Eng J 78:85–92. https://doi.org/10.1016/j.bej.2012. 12.014 Santos DK, Resende AH, de Almeida DG, Soares da Silva RDCF, Rufino RD, Luna JM, Banat IM, Sarubbo LA (2017) Candida lipolytica UCP0988 biosurfactant: potential as a bioremediation agent and in formulating a commercial related product. Front Microbiol 8:767. https://doi.org/ 10.3389/fmicb.2017.00767 Sarubbo LA, Silva MGC, Durval IB, Bezerra KGO, Ribeiro BG, Silva IA, Twigg MS, Banat IM (2022) Biosurfactants: production, properties, applications, trends, and general perspectives. Biochem Eng J 181:108377. https://doi.org/10.1016/j.bej.2022.108377 Schilling K, Zessner M (2011) Foam in the aquatic environment. Water Res 45(15):4355–4366. https://doi.org/10.1016/j.watres.2011.06.004 Schilling M, Davids I, Van Logchem MD, Wenk HH (2021) U.S. Patent No. 10, 988,713. Washington, DC, U.S. Patent and Trademark Office Schultz J, Rosado AS (2020) Extreme environments: a source of biosurfactants for biotechnological applications. Extremophiles 24(2):189–206 Randhawa SKK, Rahman PK (2014) Rhamnolipid biosurfactants—past, present, and future scenario of global market. Front Microbiol 5:454. https://doi.org/10.3389/fmicb.2014.00454 Singh P, Patil Y, Rale V (2019) Biosurfactant production: emerging trends and promising strategies. J Appl Microbiol 126(1):2–13. https://doi.org/10.1111/jam.14057 Siyal AA, Shamsuddin MR, Low A, Rabat NE (2020) A review on recent developments in the adsorption of surfactants from wastewater. J Environ Manag 254:109797. https://doi.org/10. 1016/j.jenvman.2019.109797 Smith ML, Gandolfi S, Coshall PM, Rahman PK (2020) Biosurfactants: a Covid-19 perspective. Front Microbiol 11:1341. https://doi.org/10.3389/fmicb.2020.01341 St. Laurent JB, de Buzzaccarini F, De Clerck K, Demeyere H, Labeque R, Lodewick R, van Langenhove L (2007) Laundry cleaning of textiles. In: Johansson I, Somasundaran P (eds) Handbook for cleaning/decontamination of surfaces. Elsevier, Amsterdam, pp 57–102 Suryawanshi Y, Kaur G, Mandavkar A, Jena B (2021) Biosurfactants from soil microorganisms as a possible detergent replacement. Int J Res Appl Sci Biotechnol 8(3):136–143 Tsujii K, Mino J (1978) Krafft point depression of some zwitterionic surfactants by inorganic salts. J Phys Chem 82:1610–1614 Vecino X, Cruz JM, Moldes AB, Rodrigues LR (2017) Biosurfactants in cosmetic formulations: trends and challenges. Crit Rev Biotechnol 37(7):911–923. https://doi.org/10.1080/07388551. 2016.1269053 Zarinviarsagh M, Ebrahimipour G, Sadeghi H (2017) Lipase and biosurfactant from Ochrobactrum intermedium strain MZV101 isolated by washing powder for detergent application. Lipids Health Dis 16(1):1–13. https://doi.org/10.1186/s12944-017-0565-8 Zhang W, Wu J, Xiao J, Zhu M, Yang H (2021) Compatibility and washing performance of compound protease detergent. Appl Sci 12(1):150. https://doi.org/10.3390/app12010150

Application of Biosurfactant in Petroleum Eduardo J. Gudiña, Jéssica Correia, and José A. Teixeira

Abbreviations AOR bpd BS CEOR CFS CMC E24 IEA IFT IFTo/w Nca OOIP PV ST

additional oil recovery barrels per day biosurfactant chemical enhanced oil recovery cell-free supernatant critical micelle concentration emulsifying activity after 24 h International Energy Agency interfacial tension interfacial tension between crude oil and the displacing fluid capillary number original oil in place pore volume surface tension

1 Introduction As demonstrated by the invasion of Ukraine by Russia in 2022, crude oil is crucial for our society, and geopolitical instabilities, which cause uncertainties about petroleum production and distribution, have a significant impact on the price of crude oil E. J. Gudiña · J. Correia · J. A. Teixeira (✉) CEB - Centre of Biological Engineering, University of Minho, Braga, Portugal LABBELS - Associate Laboratory, Braga/Guimarães, Portugal e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_18

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and, consequently on our daily life. According to the International Energy Agency (IEA), crude oil demand increased in 2021 (achieving 96 million barrels per day (bpd)), after the severe decline observed in 2020 due to the Covid-19 pandemic, and it is expected to continue increasing up to 105 million bpd by 2026 (IEA 2022). The main factors contributing to the expected increase in oil consumption are the continuous world population increase and sustained economic growth, which will result in an increase in energy demand in the upcoming years (Sakthipriya et al. 2021; Li et al. 2022). Furthermore, due to the delay in the development and implementation of renewable energies, nowadays they can only partially replace crude oil consumption (Datta et al. 2020; Mahmoud et al. 2021). As crude oil is a limited resource, it is necessary to develop technologies that allow its efficient exploitation. Known oil reserves are limited, and most of the oil produced worldwide is obtained from mature reservoirs that have been explored for a long time, which difficult the recovery of the remaining oil that they still contain (Couto et al. 2019; Nasiri and Biria 2020; Hoseini-Moghadam et al. 2021). Furthermore, in the last years, the discovery of significant new oil reserves has not been reported (Correia et al. 2021; Sakthipriya et al. 2021). Consequently, the exploitation of mature reservoirs and unconventional oil reserves, which is disadvantageous from an economical point of view, will be necessary in the near future in order to fulfil the growing energy demand (Hoseini-Moghadam et al. 2021; Xia et al. 2021). Oil recovery comprises several stages, as summarized in Table 1. In an early stage, petroleum is extracted using the natural pressure accumulated in the reservoir. When this is not enough to displace crude oil to the production wells, secondary recovery is applied. Although these figures change from reservoir to reservoir, as they are affected by parameters such as crude oil viscosity or oil reservoir permeability, primary and secondary recovery usually recover only between 30 and 40% of the original oil in place (OOIP), which means that between 60 and 70% stays in the reservoirs after these operations (Haloi et al. 2020; Sharma et al. 2020; Liu et al. 2021; Mahmoud et al. 2021; Onaizi et al. 2021; Sakthipriya et al. 2021; Al-Ghamdi et al. 2022; Wang et al. 2022). It is estimated that 7 × 1012 crude oil barrels remain entrapped in the oil reservoirs worldwide, which cannot be recovered through primary and secondary recovery (Haloi et al. 2020). In order to recover this oil, chemical-enhanced oil recovery (CEOR) strategies have been developed by the oil companies to modify the oil reservoir properties in order to improve oil recovery (Table 1).

2 Surfactant Flooding and Oil Recovery Chemical surfactants are widely used to mobilize the entrapped oil. Crude oil mobility inside oil reservoirs is regulated by the viscous and capillary forces. Their combined effect in the efficiency of oil mobilization is given by the capillary number (Nca):

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Table 1 Summary of the oil recovery phases and their main mechanisms Primary recovery Secondary recovery Tertiary recovery (CEOR)

Mechanism Natural reservoir pressure Pumps Immiscible gas injection Water flooding Miscible gas Natural gas injection CO2 CH4 N2 Chemical Polymers flooding Surfactants

Acids Solvents

Thermal

Gases Hot water injection Steam injection In situ combustion

Effect Mobilize crude oil to the extraction wells Repressurize the oil reservoir Push crude oil towards production wells Reduce oil density, oil viscosity, and IFT

Increase viscosity of injected water, improving mobility ratio and sweep efficiency Reduce the oil reservoir permeability Reduce IFT between crude oil/brine/reservoir rock Emulsify crude oil Alter reservoir rock wettability Modify reservoir porosity and permeability Reduce crude oil viscosity Modify reservoir porosity and permeability Reduce crude oil viscosity Reduce crude oil viscosity Increase reservoir temperature, reduce oil viscosity, improve oil mobility

CEOR chemical enhanced oil recovery, IFT interfacial tension Sources: Couto et al. (2019); Haloi et al. (2020); Sharma et al. (2020); Al-Ghailani et al. (2021); Al-Ghamdi et al. (2022)

Nca =

Viscousforces V ×μ = Capillaryforces σ × cos θ

ð1Þ

V: injected fluid speed (m/s); μ: injected fluid viscosity (mPa s); σ:interfacial tension (mN/m); θ: contact angle (Datta et al. 2020; Li et al. 2021). To achieve a substantial displacement of the immobilized oil, it is necessary to change Nca, from values around 10-6–10-7 (characteristic of mature oil reservoirs) to 10-3–10-1. According to Eq. (1), that can be achieved by increasing the speed and/or the viscosity of the injected fluid, and reducing the IFT and/or the affinity of the rocks for crude oil. Surfactants contribute to the release of entrapped oil due to a reduction of the capillary forces. The oil-water IFT (IFTo/w) is around 10–40 mN/m. However, in the presence of surfactants, that value can be reduced to 10-2–10-3mN/m. Furthermore, surfactants reduce the affinity of the reservoir rock surface for crude oil (measured as the contact angle), from values between 105–180° (oil-wet) to values below 75°

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(water-wet) (Hajibagheri et al. 2017; Zulkifli et al. 2019; Yun et al. 2020; Li et al. 2021). An oil-wet substrate means that oil has more affinity for the substrate than water, which is difficult for oil extraction. However, in a water-wet condition, the affinity of crude oil for the reservoir rock is reduced, and it is more easily recovered (Nasiri and Biria 2020; Hoseini-Moghadam et al. 2021; Dong et al. 2022). Finally, surfactants can emulsify the entrapped oil, allowing its mobilization by the aqueous phase (Hoseini-Moghadam et al. 2021; Onaizi et al. 2021; Dong et al. 2022). Chemical surfactants are classified according to the nature of their hydrophilic domain in ionic and nonionic. Ionic surfactants are categorized in anionic, cationic and zwitterionic (amphoteric), according to the dissociation of their polar head in aqueous solutions into anions, cations, or both of them, respectively (Negin et al. 2017; Hoseini-Moghadam et al. 2021). The main effect of anionic surfactants is IFT reduction. They are more suitable for application reservoirs formed by sandstone rocks, where they exhibit low adsorption, as rock surfaces have negative charge. Anionic surfactants commonly used in EOR include alkyl aryl sulfonates, alkyl benzene sulfonates, alkyl sulfates, n-ethoxy sulfonates, alcohol ether sulfates/sulfonates, alkyl ethoxy sulfates, alpha-olefin sulfates/sulfonates and petroleum sulfonates, among others. Those containing sulfate groups are usually more sensitive to high temperatures than those containing sulfonate groups. On the contrary, sulfonate surfactants are more sensitive to high salinities (Liu et al. 2006; Bai et al. 2014; Bera et al. 2014; Kumar and Mandal 2016; Negin et al. 2017; Hajibagheri et al. 2018; Zulkifli et al. 2019; Li et al. 2021). Cationic surfactants are more appropriate for application in carbonate reservoirs (positively charged rock surfaces), alone or in combination with nonionic surfactants. They are mainly used to alter the rock wettability towards water-wet. Some examples are cetyltrimethyl ammonium bromide, stearyltrimethyl ammonium chloride and ethoxylated alkyl amine (Bera et al. 2014; Kumar and Mandal 2016; Hajibagheri et al. 2017; Negin et al. 2017). Zwitterionic surfactants exhibit remarkable interfacial properties, are stable at high temperatures and salinities, and improve the properties of ionic and nonionic surfactants. However, their high manufacturing costs reduce their applicability in EOR. Some examples are sulfobetaines, carboxyl betaines and amino carboxylic acids (Zhao et al. 2015; Yun et al. 2020). Nonionic surfactants, due to their low interfacial activity, are commonly used as co-surfactants. Due to their tolerance to high salinities, they are more appropriate for application in oil reservoirs with high salinities. The combination of nonionic and ionic surface-active compounds usually reduces their critical micelle concentration (CMC) and their adsorption to the reservoir rock. Some examples of nonionic surfactants are alkyl ethoxycarboxylated, alkyl polyglycoside, poly (ethylene/propylene) glycol ether and triphenylmethane (Chen et al. 2013; Bera et al. 2014; Hajibagheri et al. 2017; Negin et al. 2017; Zulkifli et al. 2019). Some examples of chemical surfactants studied for application in CEOR are presented in Table 2. The selection of the most appropriate surfactant(s) for application in EOR requires previous studies taking into account the properties of the oil reservoir: rock composition; composition and pH of formation water (as they affect the charge of rock surfaces); temperature (the adsorption of nonionic surfactants onto rock

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Table 2 Properties of some chemical surfactants used or studied for application in CEOR

Type Anionic

Cationic

Zwitterionic

Nonionic

CMC (mg/L) 650

IFT (mN/m) * 0.5

Surfactant Alkyl (benzene/naphthalene/indane/phenanthrene) sulfonate Alkyl ether carboxylate (AEC)

36

Alkyl ether sulfate (AES)

18

Linear alkyl benzene sulfonate (LAS Na C16)

62

Sodium dodecyl benzene sulfonate (SDBS)

200

0.001 (4 g/L)a 3.0 (0.05 g/ L) 0.0003 (5 g/L)b 3.0

Sodium dodecyl sulfate (SDS)

3085

0.03

Sodium lauryl sulfate (SLS)

2364

2.0

9,10-dihydroxyl sodium stearate (SDHS)

3000

1.322c

9,10,12,13-tetrahydroxyl sodium stearate (STHS) Dodecyltrimethyl ammonium bromide (C12TAB) Hexadecyltrimethyl ammonium bromide (C16TAB)

2500

1.098c

150

–

426

0.01

Cocamidopropyl hydroxysultaine (CAHS)

–

Dimethylmyristylammonio hydroxyl propanesulfonate (C14HSB)

268

Dimethylmyristylammoniopropanesulfonate (C14SB)

84

Alkyl polyglycoside (APG1214)

61

Octoxynol (Triton X-100)

400

0.357 (2 g/L)d 0.0009 (0.3 g/ L)e 0.047 (0.3 g/ L)e 0.0013 (0.5 g/ L)f 2.3g

Polysorbate 80 (Tween 80)

18

0.142

Reference Bai et al. (2014) Zulkifli et al. (2019) Liu et al. (2006) Negin et al. (2017) Hajibagheri et al. (2018) Kumar and Mandal (2016) Bera et al. (2014) Li et al. (2021) Li et al. (2021) Hajibagheri et al. (2017) Kumar and Mandal (2016) Yun et al. (2020) Zhao et al. (2015) Zhao et al. (2015) Chen et al. (2013) Nezhad et al. (2021) Kumar and Mandal (2016) (continued)

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Table 2 (continued)

Type

Surfactant Tergitol 15-S-12 (secondary alcohol ethoxylate)

CMC (mg/L) 104

IFT (mN/m) * 1.0

Reference Bera et al. (2014)

CMC critical micelle concentration, IFT interfacial tension. Unless indicated otherwise, surfactant solutions were formulated in demineralized water. *When the concentration of surfactant used to measure the IFT was different from the CMC, that concentration is presented. –: Not reported a Surfactant dissolved in seawater (salinity 32 × 103 ppm) and measurements performed at 106 °C b Surfactant solution containing 15 g/L NaCl c Surfactant dissolved in formation water (salinity 10.5 × 103 ppm) and measurements performed at 60 °C d Surfactant dissolved in seawater (salinity 68 × 103 ppm) e Surfactant dissolved in reservoir water (salinity 11 × 104 ppm) and measurements performed at 90 °C f Surfactant solution containing 5 g/L Na2CO3 g Surfactant solution containing 4 g/L CaCl2.2H2O

surfaces increases as the temperature increases), etc. Surfactants can adsorb to the rock surface, which reduces their efficiency and causes economic losses (Li et al. 2022). In order to avoid that, sacrificial agents can be used to reduce the electrostatic interactions between them (Negin et al. 2017; Hoseini-Moghadam et al. 2021; Li et al. 2022). In some cases, a synergistic effect between surfactants and the salts present in the injected or formation water contributes to reduce the IFT up to one order of magnitude when compared with the values obtained with deionized water, achieving 10-3–10-4mN/m. This is applicable to ionic and nonionic surfactants (Chen et al. 2013; Bera et al. 2014; Zhao et al. 2015; Kumar and Mandal 2016; Yun et al. 2020). The presence of different salts can reduce the solubility of surfactants in the aqueous phase; as a result, surfactants arrange at the crude oil-aqueous phase interphase, improving IFT reduction. Furthermore, for ionic surfactants, the presence of ions can reduce the repulsive forces between surfactant molecules, resulting in their rearrangement at the interface, improving the IFT reduction (Bera et al. 2014; Zhao et al. 2015; Negin et al. 2017; Zulkifli et al. 2019; Yun et al. 2020). Most of these chemical surfactants are combined with different alkalis, which contribute to reduce the IFT values up to three orders of magnitude when compared with the values obtained with deionized water (Chen et al. 2013; Bai et al. 2014; Kumar and Mandal 2016). As it can be seen in Table 2, ultra-low IFT values were only obtained in the presence of salts or alkalis. Finally, as previously discussed, IFT reduction to ultra-low values is not the only mechanism responsible for mobilizing the entrapped oil. The incremental oil recoveries obtained with these surfactants can be due to changes in the rock wettability towards water-wet (Hajibagheri et al. 2017, 2018). Synergistic effects in IFT reduction can be obtained using cocktails of different surfactants. That can be due to the formation of mixed micelles, where the intercalation of different types of surfactants reduces the repulsive forces between the

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charged head groups of ionic surfactants, improving the packing of surfactant molecules and their orientation at the interface, consequently improving the IFT reduction. In the case of nonionic surfactants, this effect is attributed to a weakening of hydrogen bonds (Bera et al. 2014; Kumar and Mandal 2016; Hajibagheri et al. 2017, 2018; Hadia et al. 2019).

3 Biosurfactants as Green Alternatives to Chemical Surfactants Used in CEOR Although oil recovery cannot be considered an environmentally friendly activity, the replacement of chemical compounds commonly used in CEOR operations (which are environmentally hazardous and, in most cases, toxic to living organisms) by natural compounds can reduce its negative environmental impact (Akanji et al. 2021; Zhao et al. 2021; Li et al. 2022). In that sense, surfactants of microbial origin (biosurfactants) represent a promising alternative. Biosurfactants (BSs) are surface-active compounds of microbial origin. They are characterized by an amphipathic structure, comprising hydrophilic and hydrophobic domains in the same molecule, which provides them their characteristic surface activity. The most relevant comprise glycolipids (rhamnolipids, sophorolipids, threalose lipids) and lipopeptides (surfactin, lichenysin, fengycin, pumilacidin) (Ali et al. 2019; Akanji et al. 2021; Al-Ghailani et al. 2021; Haloi et al. 2021; Liu et al. 2021; Mahmoud et al. 2021). Due to their remarkable properties, BSs display a huge potential to be used in numerous fields, as substitutes of chemical surfactants (Gudiña and Teixeira 2022). The application of BSs in the petroleum industry has also been widely studied. Table 3 summarizes the main properties of the most relevant BSs studied for application in petroleum extraction. As it can be seen from Table 3, lipopeptide BSs (mainly surfactin) are the most widely studied BSs for application in oil recovery, followed by rhamnolipids. The producer microorganisms are mainly Bacillus spp., with special relevance for B. subtilis and Bacillus licheniformis (lipopeptide BS producers) (Table 3). In general, the BSs presented in Table 3 exhibit a remarkable surface activity (surface tension (ST) values lower than 30 mN/m), and show considerable emulsifying activity (between 45 and 94%) (Fig. 1). Although in some cases, the IFT values obtained are higher when compared with those reported for chemical surfactants (Table 2), it has to be taken into consideration that, as previously explained, ultralow IFT values are only achieved in the presence of salts or alkalis. As an example, B. subtilis W19 surfactin (100 mg/L) reduced the IFT up to 2 mN/m when dissolved in deionized water; however, in the presence of Na2CO3 (9 g/L), the IFT was reduced up to 0.025 mN/m (Al-Ghailani et al. 2021). One advantage of BSs when compared with chemical surfactants is their lower CMC values. Whereas for chemical surfactants the CMC values are usually between

Rhamnolipid

Lichenysin A Pumilacidin Lipopeptide

Fengycin

Biosurfactant Surfactin

Microorganism Bacillus amyloliquefaciens SAS-1 B. licheniformis AnBa7* B. subtilis AB2.0 B. subtilis AnPL-1* B. subtilis BR-15 B. subtilis BSFX026 B. subtilis SL B. subtilis W19 B. subtilis WD3* B. subtilis 22.2 B. tequilensis MK729017* B. velezensis H2O-1* Brevibacillus borstelensis YZ-2* B. licheniformisAli5 B. safensis CCMA-560 B. halotolerans XT-2* Luteimonas huabeiensis HB-2* B. licheniformis DS1* B. licheniformis L20 B. licheniformis WD2* Achromobacter sp. TMB1 Pseudomonas aeruginosa HAK01 26.2 56.0 25.5 26.7 – 42.0 27.1 27.0 28.1

29.9 24.7 28.5 20.2 28.0 25.6 28.0 25.7 – 30.0 24.8 30.1

8 16 30 – 36 154 100 40 250 90 39 60 21 96 46 32 157 – 60 90 120

ST (mN/m) 22.9

CMC (mg/L) –

66 – 85 90 94 62 50 – 67

65 45 71 74 60 67 – 72 – 66 – 70

– 3.8 2.29 – – 0.95 2.0 0.38 0.056 0.32 0.8 1.32 0.26 11.3 1.55 3.4 12.0 – 1.27 0.9 2.50

E24 (%) 78

IFT (mN/m) –

Table 3 Properties of the most relevant BSs studied for application in petroleum production

Mahmoud et al. (2021) Alvarez et al. (2020) Zhao et al. (2021) Sharma et al. (2018a) Hu et al. (2021) Wu et al. (2022) Al-Ghailani et al. (2021) Aboelkhair et al. (2022a) Hadia et al. (2019) Datta et al. (2020) Guimarães et al. (2021) Dong et al. (2022) Ali et al. (2019) de Araujo et al. (2019) Wang et al. (2022) Ke et al. (2018) Purwasena et al. (2019) Liu et al. (2021) Aboelkhair et al. (2022b) Haloi et al. (2021) Khademolhosseini et al. (2019)

50° → 17° – – – – 91° → 31° – 75° → 31° 106° → 7°

Reference Sharma et al. (2018a)

– – – – 60° → 18° – 83° → 60° – 71° → 35° 90° → 26° – 98° → 10°

Contact angle –

390 E. J. Gudiña et al.

54.0 31.0

125

23.7 28.0 14.0 40.8

350

– 120 120 13 76 68

–

60 78 – 55

0.18

– 0.8 0.5 –

Paul et al. (2022)

Sari et al. (2020)

– –

Sharma et al. (2018b) Haloi et al. (2020) Akanji et al. (2021) Mahmoud et al. (2021)

– 75° → 42° – –

*Indigenous microorganism. –: not reported. CMC critical micelle concentration, ST minimum surface tension value, IFT minimum interfacial tension value, E24 emulsifying activity after 24 h

Glycolipid + lipopeptide

Fatty acid

Sophorolipid Threalose lipid

P. aeruginosa PBS Pseudomonas sp. TMB2 Meyerozyma spp. MF138126 Rhodococcus erythropolis MN7* Halomonas meridiana BK-AB4 Pseudomonas mendocina IFE11

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Fig. 1 Mechanisms of action of BSs in petroleum extraction. (a) Surface/Interfacial tension reduction. (b) Crude oil emulsification. (c) Rock wettability change (left: oil-wet; right: water-wet)

100 and 3000 mg/L (Table 2), in the case of BSs they are usually between 10 and 200 mg/L (Table 3). Although this parameter has been less studied, surfactin, fengycin, lichenysin and rhamnolipids have the capacity of reducing the affinity of reservoir rocks for crude oil, making them water-wet (contact angle ≤75°) (Table 3 and Fig. 1), which contributes to improve oil recovery, as explained above. As it can be seen in

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Table 3, contact angles as low as 10° or 7° have been reported for fengycin and rhamnolipids (Khademolhosseini et al. 2019; Dong et al. 2022). In some cases, BSs displayed a better performance than chemical surfactants. For instance, Bacillus velezensis H2O-1 surfactin offered better results than the commercial chemical surfactant Ultrasperse II®in changing the wettability of calcite (Guimarães et al. 2021). As in the case of chemical surfactants, the combination of BSs with different properties can improve their surface activity. Al-Ghamdi et al. (2022) combined rhamnolipids (anionic) with two green co-surfactants: alkyl polyglucoside (APG) (nonionic) and lecithin (zwitterionic). Whereas rhamnolipids (200 mg/L) alone reduced the IFTo/w from 23 mN/m to 0.14 mN/m, the combination of rhamnolipids (400 mg/L) and lecithin (400 mg/L) reduced the IFTo/w to 0.019 mN/m. However, the combination of rhamnolipids and APG did not have a positive effect on the IFTo/ w values at any of the concentrations tested (Al-Ghamdi et al. 2022). The performance of BSs can be also improved by their combination with chemical surfactants. The combination of surfactin with Triton X-100 (ratio 70:30) resulted in a decrease of the CMC from 2.8 mM (surfactin alone) to 1.0 mM; at the same time, the minimum ST value was reduced from 28 mN/m to 16.5 mN/m, and the IFTo/ w achieved 0.19 mN/m (Mahmoud et al. 2021). The surfactin-Triton X-100 system also exhibited a good performance in wettability shift, reducing the affinity of rock surfaces for crude oil (contact angle 17.4°) (Mahmoud et al. 2021). Similar results were obtained when combining threalose lipids with Triton X-100 (Mahmoud et al. 2021). One important factor for the application of BSs in oil recovery is their performance and stability at the oil reservoir conditions, usually characterized by high temperatures, pressures and salinities. Several lipopeptide BSs maintained their surface-active properties at temperatures between 80 and 121 °C and salinities between 100 and 200 g/L (Datta et al. 2020; Guimarães et al. 2021; Liu et al. 2021; Mahmoud et al. 2021; Zhao et al. 2021; Aboelkhair et al. 2022a; Wang et al. 2022; Wu et al. 2022), and even at pressures as high as 100-270 bar (Guimarães et al. 2021; Sakthipriya et al. 2021). Also glycolipid BSs remained stable at temperatures as high as 121 °C, salinities between 40 and 200 g/L (Mahmoud et al. 2021; Sakthipriya et al. 2021), and pressures up to 80 bar (Khademolhosseini et al. 2019; Sakthipriya et al. 2021). In some cases, it has been reported a better performance for BSs at high temperatures and high pressures. The IFT of heavy crude oil/brine systems in the presence of sophorolipids produced by Meyerozyma spp. MF138126 decreased from 4.3 mN/m at 1 bar to 0.25 mN/m at 45 bar, and from 10 mN/m at 25 °C to 0.5 mN/m at 65 °C (Akanji et al. 2021). Another advantage of BSs is their lower toxicity when compared with synthetic surfactants. BSs produced by Pseudomonas mendocina IFE11 did not exhibit detrimental effects in phytotoxicity assays performed using Vigna radiata at concentrations up to 500 mg/L, and exhibited lower toxicity against animal cells when compared with the synthetic surfactant Tween 80 (Paul et al. 2022). Neither BSs produced by Bacillus safensis J2 displayed phytotoxicity against wheat seeds (Das and Kumar 2019). Aboelkhair et al. (2022b) performed a study of environmental and

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human health risk assessment regarding the use of B. subtilis WD3 and B. licheniformis WD2 to produce BSs for application in oil recovery, using data available in the literature. They concluded that, in both cases, a low risk to human health and the environment is expected (Aboelkhair et al. 2022b). BSs are usually produced as mixtures of compounds with different structures (e.g. with different compositions of the hydrophilic or the hydrophobic moieties, or both). Depending on their structure, different compounds display different surfaceactive properties (Hu et al. 2021). Accordingly, besides increasing the BS titres, the production of BSs with better properties for application in enhanced oil recovery can be achieved through genetic engineering (Hu et al. 2021). The overexpression of the gene encoding the thioesterase BTE in B. subtilis 168 improved surfactin production by 35%; at the same time, the relative proportion of nC14-surfactin was increased whereas C13- and C15-surfactin decreased. Although the surfactin extract obtained from B. subtilis BSFX026 (recombinant strain) displayed a similar CMC and minimum ST value when compared with the one obtained from the parental strain (B. subtilis 168), it exhibited better oil washing efficiency at low concentrations, higher emulsifying activity with long-chain n-alkanes and aromatics, and was more efficient in wettability alteration (Hu et al. 2021).

4 Application of Biosurfactants in Oil Recovery Tables 4 and 5 summarize the most relevant studies performed in BS flooding in the last five years (ex situ and in situ). In the ex situ approach (Table 4), the injection of BSs is performed following the same strategy used with the chemical surfactants. BSs are produced in flasks or bioreactors, using appropriate microorganisms, and after that, they are introduced into the models (sand-pack columns, core-flooding systems) to study their effect in oil recovery (Fig. 2) (Correia et al. 2021). The in situ approach is more complex. In this case, the models are “inoculated” with BS-producing microorganisms and nutrients, and subsequently they are maintained at the corresponding conditions for different time intervals, to allow microbial growth and BS production in situ (Table 5). Although the in situ strategy is considered less expensive than the ex situ, it has several limitations. First of all, the microorganisms used must be able to grow in the oil reservoir conditions, characterized by high temperatures and pressures, and by the lack of oxygen. Furthermore, uncontrolled microbial growth can occur inside the oil reservoir, which can result in harmful effects. Finally, it is not possible to associate the effect observed in oil recovery to a specific mechanism (Nasiri and Biria 2020; Correia et al. 2021). In ex situ assays, additional oil recoveries (AOR) between 5 and 66% were reported using BSs produced by different microorganisms (Table 4). As it can be seen from the studies presented in Table 4, surfactin and rhamnolipids were the most studied BSs for this application. However, due to the huge variability of the experimental conditions used (type of substrate (sand, rock cores), viscosity of

B. subtilis WD3

B. subtilis YB7

B. subtilis 22.2

Brevibacillus borstelensis YZ-2* B. licheniformis Ali5

Surfactin

Surfactin

Surfactin

Fengycin

Lipopeptide

Lipopeptide

Lipopeptide

Lichenysin A Pumilacidin

B. safensis CCMA560 B. halotolerans XT-2* B. licheniformis DS1* B. licheniformis WD2*

B. subtilis BR-15

Surfactin

Surfactin Surfactin

Microorganism B. amyloliquefaciens SAS-1 B. licheniformis L20 B. safensis J2

Biosurfactant Surfactin

Berea sandstone core Sandstone core (133 mD)

Berea sandstone (118 mD) Artificial core

Sand (100 mesh)

Sandstone core (187–205 mD) Silica sand (159 mD) Berea sandstone (153 mD) Core (42 mD)

Sand

Core Sand (45 mesh)

Substrate (permeability) Sand

69

–

60

50

– 1.5

42

130

1

10

1

1

–

– 11.3

69

45

1

30

25

80

1

7.7

1.2

22.8

1.5

64

1

–

5.1

200 1

80 –

– –

Pressure (bar) 1

Temperature (°C) –

Oil viscosity (cP) 5.1

Table 4 Results obtained in ex situ biosurfactant flooding assays

4

2.8

0.6

2.7

0.6

1.0

3.0

0.5

4.0

0.6

5.1 –

BS volume (PV) 0.6

CFS

BS (125 mg/ L) BS (200 mg/ L) BS-12 h

BS (120 mg/ L)-2 h CFS-1 day

BS (200 mg/ L) BS (1 g/L)

CFS

CFS-2 days

CFS BS

Treatment— shut-in time CFS-2 days

31

5

13

13

25

9

9

15

39

66

20 5

AOR (%) 57

(continued)

Purwasena et al. (2019) Aboelkhair et al. (2022b)

de Araujo et al. (2019) Wang et al. (2022)

Ali et al. (2019)

Dong et al. (2022)

Reference Sharma et al. (2018a) Liu et al. (2021) Das and Kumar (2019) Sharma et al. (2018a) Aboelkhair et al. (2022a) Sakthipriya et al. (2021) Hadia et al. (2019)

Application of Biosurfactant in Petroleum 395

Ochrobactrum anthropi HM-1 P. aeruginosa

P. aeruginosa CPCL

P. aeruginosa HAK01 P. aeruginosa PBS

Pseudomonas sp. TMB2 Pseudoxanthomonas sp. G3 –

Ochrobactrum pseudintermedium C1 + B. cereus K1 Clostridium sp. N-4 Halomonas meridiana BK-AB4 Pseudomonas mendocina IFE11

Rhamnolipid

Rhamnolipid

Rhamnolipid

Rhamnolipid

Glycolipid

*Indigenous microorganism

Lipopeptide + glycolipid

Glycoprotein Fatty acid

Rhamnolipid

Rhamnolipid

Rhamnolipid

Rhamnolipid

Microorganism B. subtilis WD3*

Biosurfactant Lipopeptide

Table 4 (continued)

Sand Berea sandstone core Sand

19.8

Berea sandstone core (176 mD) Sand (60–100 mesh)

96 65 –

–

70

52

50

1348 –

7.2

58.9

70

1

1.25

3.0 –

1.0

–

1 –

3

0.6

6.0

0.7

0.4

0.5

–

–

BS volume (PV) 4

138

1

41

1

–

– 17.7

1

25

1

242

80

30

– 22.8

1

–

– 1

Pressure (bar) 1

Temperature (°C) 60

Oil viscosity (cP) 1.5

Sand (50 mesh)

Core rock plug

Clay + sand (65– 100 mesh) Silica sand (154 mD) Glass micromodel Sand

Substrate (permeability) Sandstone core (206 mD) Sand

CFS

CFS BS-60 h

BS (120 mg/ L) Crude BS-1 day BS (5000 mg/L) Crude BS

BS (254 mg/ L) BS (200 mg/ L) BS (120 mg/ L) CFS-1 day

CFS

Treatment— shut-in time CFS

44

17 24

47

23

22

17

56

27

15

12

45

AOR (%) 39

Paul et al. (2022)

Arora et al. (2019) Sari et al. (2020)

Al-Ghamdi et al. (2022) Bhattacharya et al. (2019)

Astuti et al. (2019)

Câmara et al. (2019) Sakthipriya et al. (2021) Khademolhosseini et al. (2019) Sharma et al. (2018b) Haloi et al. (2020)

Reference Aboelkhair et al. (2022b) Ibrahim (2018)

396 E. J. Gudiña et al.

1348

–

358.9

10

3500

Surfactin

Surfactin

Glycoprotein

–

Lipopeptide

Rhamnolipid

Rhamnolipid

B. subtilis AnPL-1*

B. subtilis SL

Clostridium sp. N-4*

Clostridium spp.*

Luteimonas huabeiensis HB-2* Pseudomonas aeruginosa DQ3* Pseudomonas sp. WJ6

*Indigenous microorganism

45.4

Lipopeptide

17

8.6

Oil viscosity (cP) 130

Biosurfactant Lipopeptide

Microorganism Bacillus halotolerans XT-2* Bacillus sp. W5

Artificial core (331 mD) Berea sandstone (240 mD)

Artificial core

Core

Sand

Heterogeneous core (235 mDa) Sandstone core (390 mD) Core (14 mD)

Substrate (permeability) Artificial core

Table 5 Results obtained in in situ biosurfactant flooding assays

35

42

40

65

96

55

39

60

Temperature (°C) 42

1

1

80

89

1

30

100

1

Pressure (bar) 1

50

15

14

10

14

4

15

7

Shut-in time (days) 20

12

4

22

19

37

6

10

10

AOR (%) 25

Zhao et al. (2021) Wu et al. (2022) Arora et al. (2019) Sharma et al. (2020) Ke et al. (2018) Zhao et al. (2018) Xia et al. (2021)

Reference Wang et al. (2022) Qi et al. (2018)

Application of Biosurfactant in Petroleum 397

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E. J. Gudiña et al.

Fig. 2 Laboratory sand-pack column biosurfactant flooding assays

crude oil (between 1.2 and 1348 cP), BS concentration and purity, temperature (25–96 °C), pressure (1–200 bar) and shut-in time (0–2 days)), the results obtained in the different studies cannot be directly compared. Furthermore, few studies compared the performance of BSs and chemical surfactants. As an example, in sand-pack column assays, surfactin (AOR 15%) and rhamnolipids (AOR 15%) offered better results in oil recovery when compared with the chemical surfactants SDS (AOR 8.8%) and CTAB (AOR 7.0%) at the same concentration (200 mg/L) (Sakthipriya et al. 2021). Al-Ghailani et al. (2021) investigated the interaction of B. subtilis W19 surfactin with the biopolymer schizophyllan (produced by Schizophyllum commune ATCC38548) and the alkali Na2CO3 in oil recovery. Na2CO3 had a positive effect in the surface-active properties of surfactin, whereas surfactin (100 mg/L) alone reduced the IFTo/w to values around 1 mN/m, in combination with Na2CO3 (9 g/L), it was reduced up to 0.025 mN/m. Furthermore, schizophyllan maintained its viscoelastic properties in the presence of surfactin and Na2CO3, and the biopolymer did not affect the surface-active properties of the BS-alkali system. In core-flooding assays performed using Berea cores (oil viscosity 23 mPa s), the injection of 0.5 pore volumes (PV) of a formulation containing surfactin (400 mg/L), Na2CO3 (11 g/L) and schizophyllan (600 mg/L) resulted in an AOR of 32%, whereas in the absence of the biopolymer it was reduced to 17% (Al-Ghailani et al. 2021). As in the case of chemical surfactants, the adsorption of BSs to the rock surface is detrimental for oil recovery. The adsorption of rhamnolipids (anionic BSs) produced by Achromobacter sp. TMB1 to sand surface decreased as the temperature increased (from 30 to 60 °C), decreased as salinity decreased (from 15,000 to 1000 mg/L), and decreased as the pH increased (from 2 to 12) (Haloi et al. 2021). Accordingly, optimal conditions for rhamnolipid injection in oil recovery can be determined, taking into account these data, in order to minimize their adsorption to the reservoir

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Fig. 3 Biosurfactant-producing Bacillus spp. strains isolated from a Brazilian oil reservoir at 40 °C and 32 bars (Gudiña et al. 2012)

rock, which results in a decrease in rhamnolipid concentration and reduces the efficiency of the process. In order to reduce the adsorption of rhamnolipids to the rock surface, Li and co-workers (2022) synthesized derivatives of rhamnolipids through amidation to obtain nonionic rhamnolipid monoethanol amide (RL-MEA). This modification, which neutralizes the negative charge of the carboxylic groups of rhamnolipids, did not have negative effect on their surface activity. RL-MEA reduced the ST to similar values when compared with rhamnolipids (around 29 mN/m), exhibited lower CMC values than rhamnolipids, both in water (41 mg/L versus 75 mg/L) and 200 mM NaCl (30 mg/L versus 47 mg/L), and displayed better oil washing efficiency than rhamnolipids at low concentrations (100–500 mg/L). Furthermore, RL-MEA were more efficient in changing the wettability of crude oil-conditioned glass slides (reduced the contact angle to 46°) than rhamnolipids (contact angle 63°) at the same concentration (500 mg/L). Finally, the adsorption loss of RL-MEA on quartz sand was reduced by 20% when compared with rhamnolipids in assays performed at 60 °C and 200 mM NaCl (Li et al. 2022). Although the formation of stable emulsions between the displacing fluid and crude oil is advantageous to recover the entrapped oil, it represents a problem in downstream processing. Rhamnolipids (1 g/L) proved to form stable oil/water nanoemulsions both with light (13 mPa s) and heavy (394 mPa s) crude oil, with 0% phase separation after 30 days (Onaizi et al. 2021). Interestingly, these stable nanoemulsions are easily destabilized at low pH values, which can be easily performed in downstream processing by the addition of sulphuric acid, allowing the separation of crude oil and water (Onaizi et al. 2021). Some of the BS-producing microorganisms studied for application in oil recovery and presented in Tables 3, 4 and 5 were isolated from oil reservoirs with temperatures between 37 and 96 °C, and salinities up to 122,000 ppm. Those indigenous microorganisms belong to the genera Bacillus, Brevibacillus, Clostridium, Luteimonas, Pseudomonas and Rhodococcus. Most of them are facultatively anaerobic and thermophilic (growing at temperatures up to 49–65 °C) (Ke et al. 2018; Zhao et al. 2018; Purwasena et al. 2019; Datta et al. 2020; Sharma et al. 2020; Mahmoud et al. 2021; Zhao et al. 2021; Aboelkhair et al. 2022a, b). In some cases, they are also halophilic, growing at salinities up to 15–20% (Dong et al. 2022; Wang

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et al. 2022). Consequently, they are good candidates for application in in situ BS flooding (Fig. 3). Due to the presence of indigenous BS-producing microorganisms in oil reservoirs, BSs accumulate in oil reservoirs over the years, being mainly associated to the crude oil phase (Aboelkhair et al. 2022a). Nasiri and Biria (2020) demonstrated, in laboratory assays, that it is possible to extract those BSs from the oil phase to the aqueous phase using alkalis (e.g. ethylenediamine), which contributed to mobilize the entrapped oil, increasing oil recovery by 22% in glass micromodels (Nasiri and Biria 2020). Table 5 summarizes some of the studies performed for in situ BS flooding. AORs between 4 and 37% were reported, and the shut-in time ranged from 4 days to 50 days. In this case, the models were incubated at 35–96 °C and 1–100 bar (Table 5). In all the studies, bacterial growth and BS production were observed, meaning that these microorganisms could be used in in situ BS flooding assays in oil reservoirs with those conditions of pressure and temperature. A relationship between the shut-in time and the AOR values obtained was not observed, due to the different conditions used in the different assays. For instance, the highest and the lowest AOR values reported (37% and 4%) were obtained with similar shut-in times (14 and 15 days) (Table 5). Neither a relationship between the environmental conditions and AOR was observed (Table 5). In these experiments, BS production in situ by the injected microorganisms contributed to mobilize the entrapped oil. In core-flooding experiments performed with P. aeruginosa DQ3, 64 mg rhamnolipid/L were produced during the 15 days of incubation, which contributed to the AOR observed (Table 5) (Zhao et al. 2018). In assays performed with B. subtilis AnPL-1, 150 mg surfactin/L was produced in coreflood assays in situ, which decreased crude oil viscosity by 40%, improving oil recovery (Table 5) (Zhao et al. 2021). However, as previously discussed, in in situ assays, the increase observed in oil recovery can be the consequence of the action of more than one mechanism. Pseudomonas sp. WJ6, besides producing BSs (rhamnolipids and lipopeptides), degrades asphaltenes and heavy crude oil under anaerobic conditions. In coreflooding experiments performed with this strain, besides a slight reduction in the ST (from 41 to 34 mN/m), a crude oil viscosity reduction (about 50%) was observed, resulting from the degradation of heavy oil fractions (Xia et al. 2021). Luteimonas huabeiensis HB-2, which produces BSs under anaerobic conditions, reduced the ratio between long-chain (C20-C35) and short-chain n-alkanes (C10C19), and reduced the wax content of crude oil under anaerobic conditions; as a result, crude oil viscosity was reduced from 360 to 180 cP in core-flooding assays (Ke et al. 2018). In these cases, BS production and oil viscosity reduction contributed to the AOR observed (Table 5). Wang and co-workers (2022) compared the application of Bacillus halotolerans XT-2 in oil recovery in in situ and ex situ experiments using the same core system and operational conditions. AOR was higher in in situ assays (25%) than in ex situ assays (13%). Ex situ assays were performed through the injection of a solution containing 200 mg BS/L, whereas in in situ assays B. halotolerans XT-2 produced

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345 mg BS/L under oxygen-limited conditions during the 20 days of incubation. As a result, the IFT value achieved in in situ experiments (0.89 mN/m) was lower than in ex situ assays (1.17 mN/m). Furthermore, in in situ assays, crude oil viscosity was reduced from 112 to 66 cP, due to the consumption of n-alkanes with chains between C22 and C34, asphaltenes and paraffins by B. halotolerans XT-2, whereas in the ex situ experiments, only a slight decrease in crude oil viscosity (up to 102 mPa s) was observed (Wang et al. 2022). Among the results presented in Table 5, it is important to highlight the case of the hyper-thermophilic Clostridium sp. N-4, which optimum temperature for growth and BS production is 96 °C. In sand-pack column assays performed at 96 °C, Clostridium sp. N-4 allowed the recovery of 37% of the entrapped oil due to in situ BS (glycoprotein) production during the 14 days of incubation (Arora et al. 2019). Accordingly, this microorganism is a potential candidate for application in in situ BS flooding in oil reservoirs characterized by high temperatures. In some cases, the results obtained in laboratory in situ BS flooding assays were validated in field assays. Ke and co-workers (2018) performed a field assay using L. huabeiensis HB-2. A previously optimized combination of bacterial inoculum and nutrients was introduced in several oil wells. The average temperature and pressure of the oil reservoir were 40 °C and 80 bar, respectively. The field assay was extended for 16 months, and on average, oil production per day increased 3.6 times, confirming the results obtained in laboratory assays (Table 5) (Ke et al. 2018). Xia and collaborators (2021) performed field assays (reservoir temperature 35 °C) using Pseudomonas sp. WJ6. After the injection of the microorganism with the appropriate nutrients and a shut-in time of 21 days, oil production increased more than 11 times during the next 9 months. Furthermore, as in the laboratory assays, the viscosity of the crude oil produced was reduced between 50 and 65% (Xia et al. 2021).

5 Conclusions In laboratory assays, BSs demonstrated to be potential alternatives to synthetic surfactants to increment oil recovery. As discussed in this chapter, BSs display a similar or better performance than chemical surfactants, and they are more environmentally friendly. However, the main bottleneck for their widespread use is their high production costs. In the last years, due to the interest in BSs for application in several fields, considerable efforts have been performed in order to reduce their market price (Janek et al. 2021; Gudiña and Teixeira 2022). Furthermore, significant advances were performed in the optimization of the production process, which contributed to increase their productivity (Gudiña and Teixeira 2022). Finally, genetic engineering approaches allowed the modification of wild-type BS-producing strains, improving their production titres and directing the metabolic pathways to the production of BS congeners with better properties (Hu et al. 2021). The production of BSs in situ by indigenous or injected microorganisms is one of the most attractive approaches for their application in petroleum production, as it is

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expected to be a less expensive approach when compared with the ex situ strategy. Although BS production in situ exhibits some limitations, laboratory and field assays demonstrated its feasibility, as discussed in this chapter. Consequently, the screening of anaerobic, thermophile and barophile BS-producing microorganisms, together with a better understanding of the microbial processes that take place in the oil reservoirs, are crucial to develop this promising technology. Acknowledgements This study was funded by PARTEX Oil and Gas, the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechanical Systems, LA/P/0029/2020.

References Aboelkhair H, Diaz P, Attia A (2022a) Biosurfactant production using Egyptian oil fields indigenous bacteria for microbial enhanced oil recovery. J Pet Sci Eng 208:109601. https://doi.org/10. 1016/j.petrol.2021.109601 Aboelkhair H, Diaz P, Attia A (2022b) Environmental comparative study of biosurfactants production and optimization using bacterial strains isolated from Egyptian oil fields. J Pet Sci Eng 216:110796. https://doi.org/10.1016/j.petrol.2022.110796 Akanji LT, Rehman R, Onyemara CC, Ebel R, Jamal A (2021) A novel technique for interface analysis: behavior of sophorolipids biosurfactant obtained from Meyerozyma spp. MF138126 during low-salinity heavy-crude oil experiments. Fuel 297:120607. https://doi.org/10.1016/j. fuel.2021.120607 Al-Ghailani T, Al-Wahaibi YM, Joshi SJ, Al-Bahry SN, Elshafie AE, Al-Bemani AS (2021) Application of a new bio-ASP for enhancement of oil recovery: mechanism study and core displacement test. Fuel 287:119432. https://doi.org/10.1016/j.fuel.2020.119432 Al-Ghamdi A, Haq B, Al-Shehri D, Muhammed NS, Mahmoud M (2022) Surfactant formulation for green enhanced oil recovery. Energy Rep 8:7800–7813. https://doi.org/10.1016/j.egyr.2022. 05.293 Ali N, Wang F, Xu B, Safdar B, Ullah A, Naveed M, Wang C, Rashid MT (2019) Production and application of biosurfactant produced by Bacillus licheniformis Ali5 in enhanced oil recovery and motor oil removal from contaminated sand. Molecules 24(24):4448. https://doi.org/10. 3390/molecules24244448 Alvarez VM, Guimarães CR, Jurelevicius D, de Castillo LVA, de Sousa JS, da Mota FF, Freire DMG, Seldin L (2020) Microbial enhanced oil recovery potential of surfactin-producing Bacillus subtilis AB2.0. Fuel 272:117730. https://doi.org/10.1016/j.fuel.2020.117730 Arora P, Kshirsagar PR, Rana DP, Dhakephalkar PK (2019) Hyperthermophilic Clostridium sp. N-4 produced a glycoprotein biosurfactant that enhanced recovery of residual oil at 96°C in lab studies. Colloids Surf Biointerfaces 182:110372. https://doi.org/10.1016/j.colsurfb.2019. 110372 Astuti DI, Purwasena IA, Putri RE, Amaniyah M, Sugai Y (2019) Screening and characterization of biosurfactant produced by Pseudoxanthomonas sp. G3 and its applicability for enhanced oil recovery. J Pet Explor Prod Technol 9:2279–2289. https://doi.org/10.1007/s13202-019-0619-8 Bai Y, Xiong C, Shang X, Xin Y (2014) Experimental study of ethanolamine/surfactant flooding for enhanced oil recovery. Energy Fuel 28:1829–1837. https://doi.org/10.1021/ef402313n Bera A, Mandal A, Guha BB (2014) Synergistic effect of surfactants and salt mixture on interfacial tension reduction between crude oil and water in enhanced oil recovery. J Chem Eng Data 59: 89–96. https://doi.org/10.1021/je400850c

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Biosurfactants in Medical Industry Kongkona Saikia, Abiram Karanam Rathankumar, Suganyadevi Palanisamy, Rathi Muthaiyan Ahalliya, and Mariadhas Valan Arasu

Abbreviations ARDS cPLA2 DDS DPPC HSV MAS MDDS MELs PAP PC PG PI RDS SFTA SPs THL WHO

Acute respiratory distress syndrome Cytosolic Phospholipase-A2 Drug delivery systems Dipalmitoylphosphatidylcholine Herpes simplex virus Meconium aspiration syndrome Microemulsion drug delivery system Mannosylerythritol lipids Pulmonary alveolar proteinosis Phosphatidylcholine Phosphatidylglycerol Phosphatidylinositol Respiratory distress syndrome Surfactant-associated proteins Surfactant proteins Trehalose Lipid Biosurfactant World Health Organization

K. Saikia (*) · S. Palanisamy · R. M. Ahalliya Department of Biochemistry, FASCM, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India e-mail: [email protected] A. K. Rathankumar Department of Biotechnology, Faculty of Engineering, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu, India M. V. Arasu Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_19

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1 Introduction Biosurfactants or surface-active compounds produced by biological methods are amphiphilic compounds which have hydrophilic and hydrophobic entities (Rathankumar et al. 2019). Due to the advantages provided by biosurfactants, like limited use of chemicals, utilization of natural processes, environment friendliness, etc., the works on the production, purification and characterization of these compounds have been increasing substantially in the recent years (Das et al. 2014; Rathankumar et al. 2019, 2020). Moreover, owing to their simple structure and increased tolerance to salinity as compared to their chemical counterparts, along with their natural origin and the ability to form emulsions, biosurfactants are being utilized in the production of commercial products such as cosmetics, pharmaceuticals, antimicrobial agents, detergents and cleaning agents, food industry, agriculture, soil and water bioremediation and nanoscience, etc.(Gudiña et al. 2013; Sarubbo et al. 2022) (Fig. 1). The biosurfactants are generally diversified in their structural and chemical compositions, which include different types of glycolipids, phospholipids, lipoproteins, etc. (Rathankumar et al. 2021a). Due to their diversity and their sources of origin, the biosurfactants have varied physicochemical properties and thus, this opens the door towards various applications of these compounds (Gaur et al. 2022; Rathankumar et al. 2019). One of the commonly studied biosurfactants is the microbial biosurfactants. In order to improve the biosurfactants yield, various studies were performed in media optimization and development of engineered microorganisms. In spite of improved yield of biosurfactants, like rhamnolipids,

Fig. 1 Utilization of biosurfactants for the production of various commercial products (e.g., in medical industry, cosmetic industry, food processing industry, detergent industry, environmental applications, etc.)

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from these studies, most of the potential strains used were facultative pathogens which limit their utilization in high-end industries. Further, usage of expensive raw materials along with high operational and downstream processing costs makes the production process less economical (Blunt et al. 2022; Rathankumar et al. 2019). Although genetically modified microorganisms were introduced in order to address the issue of pathogenic microorganisms; however, genetically modified cells are not widely accepted by the society and the chemically synthesized media used for the cells may affect the quality of the products. Thus, the recent technique utilized for the production of biosurfactants is enzyme technology (Grüninger et al. 2019; Rathankumar et al. 2021b; Rathankumar et al. 2019). Various enzymatic processes have been developed for the production of surface-active compounds like rhamnolipids, sophorolipids, etc., (Grüninger et al. 2019; Rathankumar et al. 2019). The biocatalytic process of biosurfactant production not only improved the tensio-property, but also eased down the downstream processing and further aided in achieving purified biosurfactants (Torres-León et al. 2021). Thus, in the recent years, biosurfactants have become a noticeable trend in various industrial processes, including medical applications (Saimmai et al. 2020; Subramaniam et al. 2020; Ahamed and Prasad 2021; Joshi and Jadhao 2022). The properties and advantages over synthetic surfactants offer these bio-products, a wide range of applications in various fields (Rathankumar et al. 2019). With an aim to highlight the potential utilization of biosurfactants for medical applications, the present chapter discusses the role of different types of biosurfactants in various areas of medical industry, including drug delivery system (DDS), antimicrobial agents, replacement therapy, etc.

1.1

Classification of Biosurfactants

The biosurfactants can be majorly divided as cationic, anionic, or non-ionic surfactants. The hydrophilic entity of the surfactant can be amino acids, carbohydrates, peptide, or carboxyl acid; whereas, the hydrophobicity of the biosurfactant is contributed by long-chain fatty acids. Based on the chemical composition, biosurfactants can be classified as glycolipids, lipopeptides, fatty acids, neutral lipids, polymeric, and particulate surfactants (Shoeb et al. 2013; Ribeiro et al. 2020; Rathankumar et al. 2021a). The major classification of biosurfactants along with some of the examples is shown in Fig. 2.

2 Application of Biosurfactants in Medical Industry Biosurfactants are biodegradable, which contributes to circular economy, making these compounds a sustainable option towards a green bio-based economy. Biosurfactants are a diversified class of compounds with diversity in their structural

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Fig. 2 Classification of common biosurfactants (glucolipids, lipopeptides, fatty acids, neutral lipids, polymeric, particulate) and examples of various types of biosurfactants

properties due to which they can be used for environmental management, production of pharmaceuticals and therapeutics as DDS, and also to maintain hygiene and health (Shoeb et al. 2013; Rawat et al. 2020). Some of the potential applications of biosurfactants in the medical industry is discussed.

2.1 2.1.1

Biosurfactants for Replacement Therapy Role of Surfactants in Human Body

Pulmonary surfactant found in the human body lines the alveolar respiratory epithelium and comprises of a mixture of proteins and lipids. This surfactant is synthesized by alveolar pneumocytes and secreted by the lamellar bodies in the alveolar lumen (Rodrigues et al. 2006a; Santos et al. 2016). A detailed structure of the pulmonary surfactant in the alveolar lumen is shown in Fig. 3. The secreted pulmonary surfactant forms a surface-active film in the air-water surface which reduces this interface’s surface tension and thus, prevents the collapsing of the alveoli during expiration (Choi et al. 2020; Nkadi et al. 2009). The pulmonary surfactant comprises majorly of lipids, mainly dipalmitoylphosphatidylcholine (DPPC). Along with DPPC, pulmonary surfactant

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Fig. 3 Alveolar type II cell and lamellar body of the alveoli that releases the pulmonary surfactant layer (SP-A- surfactant protein A; SP-B- surfactant protein B; SP-C- surfactant protein C; SP-Dsurfactant protein D; DPPC- dipalmitoylphosphatidylcholine; PC- phosphatidylcholine; PGphosphatidylglycerol; PL- phospholipids)

also comprises of phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), and neutral lipids. Along with the lipid fraction, 8% of the total mass of the pulmonary surfactant consists of surfactant proteins (SPs)surface protein A (SP-A), surface protein B (SP-B), surface protein C (SP-C), and surface protein D (SP-D). SP-A and SP-D are mainly involved in innate immunity system (Nkadi et al. 2009). On the other hand, SP-B and SP-C are embedded in the phospholipid layers and are involved in the pulmonary surfactant interfacial adsorption and provide stability to the surfactant film during breathing (Nkadi et al. 2009; Lopez-Rodriguez et al. 2017; Lio et al. 2022).

2.1.2

Defects Due to Lack of Pulmonary Surfactant

The breakdown of the surfactant structure due to various reasons, like oxidation, proteolytic degradation, etc., may cause defects in surfactant metabolism and any defects in surfactant metabolism may lead to morbidity and mortality in neonates (Akella and Deshpande 2013). Some of the defects include respiratory distress syndrome (RDS), Meconium aspiration syndrome (MAS), pulmonary haemorrhage, acute respiratory distress syndrome (ARDS), pulmonary alveolar proteinosis (PAP), etc. Some of the defects caused due to the deficiency of surfactants are hereditary. Hereditary SP-B deficiency leads to surfactant dysfunction and lethal respiratory distress in infants which is caused due to autosomal recessive condition. Similarly, hereditary SP-C associated disorder is caused due to mutation in SP-C gene and causes acute and chronic lung disease. Further, mutation in the ABCA3 transporter gene leads to ARDS both in infants and adults (Nkadi et al. 2009; Willson et al. 2009).

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Use of Commercial Biosurfactants as Replacement for Pulmonary Surfactant

In order to address the problems faced due to the lack of pulmonary surfactants, surfactant-associated proteins (SFTA) are recently developed which have similar properties as pulmonary SPs (Mirastschijski et al. 2020). The SFTA are amphiphilic in nature, which influences the phagocytic activity. Pulmonary failure can also be caused due to cytokine storm and surfactant with anti-inflammatory properties can be applied to overcome this problem (Schicht et al. 2018; Mirastschijski et al. 2020; Das et al. 2022). Another important factor which leads to the pulmonary failure is the cytokine storm. In the recent years, various industries like LyomarkPharma GmbH, a German pharma house, are working on various synthetic lung biosurfactants (Das et al. 2022). These surfactants are deduced from either bovine or porcine lungs through tissue mincing which is followed by lipid fraction extraction process. These surfactants cover the surface of the alveoli and aids in the reduction of production of cytokine. Further, industrially produced pulmonary surfactants can be used in conjugation with supplemented oxygen as a respiratory distress therapy for neonatal RDS (Das et al. 2022).

2.2

Cytotoxic Effects and Antitumour Activity of Biosurfactants

Biosurfactants are produced via sustainable methods due to which they possess limited toxicity and are degradable in nature. These properties of biosurfactants make them potential candidates for various cellular functions (signal transduction, immune response, etc.) (Das et al. 2022). According to Duarte et al. (2014), some biosurfactants also have the ability to induce apoptosis in cancer cells. In this context, Cao et al. (2009b) studied the cytotoxic effects of lipopeptide biosurfactant from Bacillus natto TK-1 on the tumour cells and concluded that the produced biosurfactant suppressed the viability of the tumour cells and showed the common characteristics of apoptosis. In another study, trehaloselipid biosurfactant (THL) was produced from Nocardia farcinica by Christova et al.(2015) and was utilized to study its antitumour activity on cancer cells (JMSU-1, KE-37 (SKW-3), HL-60, BV-173, and HL-60/DOX). From this study, it was concluded that THL, which is a biologically active compound, have cytotoxic effects against malignant cells. Another biosurfactant, rhamnolipids, has also shown potential cytotoxic effects against cancer cells and can be used as antitumour drugs that can restrict cancer progression (Chen et al. 2017). In this contest, rhamnolipids showed promising antitumour activity against human breast cancer cell lines, human non-small lung cancer cells (H460 and MCF-7 cells), human hepatocellular carcinoma cells, etc., (Dey et al. 2015; Chen et al. 2017; Mishra et al. 2021). Mannosylerythritol lipids (MELs), produced mainly by Candida Antarctica and Pseudozyma aphidis, was also

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Fig. 4 Mechanism of antitumour activity of biosurfactants. Biosurfactants act on tumour cells by inhibiting cell proliferation, inducing apoptosis, activating natural killer T cells, etc.

studied recently for their antitumour activity against cancer cells. This biosurfactant showed antitumour activity against B16 melanoma cells by inducing apoptosis through endoplasmic reticulum stress (Coelho et al. 2020; Feuser et al. 2021). Further, most of these biosurfactants are produced from microbial source and the fatty acid chain present in the compound may act as a natural antioxidant, thus preventing free radical formation by scavenging them (Vecino et al. 2017). According to Christova et al. (2015), some of the biosurfactants possess high antioxidant capacity when compared to others, which make them potential antitumour agents. Some biosurfactants were also found to stimulate the formation of interleukin-1β and tumour necrosis factor-α. Some of the mechanisms by which biosurfactants induce antitumour activity are shown in Fig. 4.

2.3

Immunomodulatory Role of Biosurfactants

An organism’s immunity helps the organism to sustain any pathogenic attack as the host cells consist of membrane receptors for pathogen recognition which initiate a cascade of reactions for the activation of immune system (Sajid et al. 2020). In this context, biosurfactants have also shown efficiency in producing cationic proteins, lysozyme, and reactive oxygen species to induce inflammatory responses (Sajid et al. 2020). Glycolipids surfactants modulate the humoral and cellular immune response. Glycolipids like rhamnolipids induce various immunological responses like chemotactic recruitment of neutrophils along with increased oxidative responses, release of histamine, elevated release of serotonin and 12-hydroxyeicosatetraenoic acid, etc.

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(Kumar and Das 2018). Another glycolipid surfactant, sophorolipids, also possess anti-inflammatory properties and can be utilized as potential candidate for addressing diseases associated with IgE production (e.g., asthma, hay fever, atopic eczema, etc.) (Davereyet al. 2021). Various recent studies have also demonstrated that sophorolipids along with other antibiotics have the ability to enhance the killing of pathogens. Trehalose-containing glycolipids have also been shown to stimulate IL-1 and TNF-α production (Saavedra et al. 2006). In case of the lipopeptide surfactants, surfactin, produced by Bacillus sp., directly inhibits via cytosolic phospholipase-2 enzyme, thud inducing anti-inflammatory effects. Surfactin induces immunomodulatory properties by inhibiting CD80, CD40, CD54, and MHC class-II expression and suppresses the T cells activation and proliferation (Sajid et al. 2020). Surfactin also selectively inhibit cytosolic phospholipase-A2 (cPLA2), which is responsible for maintaining the arachidonic acid level in cells. Arachidonic acid is a precursor of various inflammatory mediators (eicosanoids) and thus, inhibition of cPLA2 affects the production of arachidonic acid, which reduces the inflammatory responses (Park and Kim 2009). Another surfactin, WH1fungin, has been found to induce humoral and cellular responses against Hepatitis-B surface antigens (Park and Kim 2009; Xu et al. 2016). The lipopeptide, TAN-1511-A, produced by Streptosporangium amethystogenes increases the secretion of granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Takizawa et al. 1995). Table 1 shows the immunomodulatory effects of various types of biosurfactants.

2.4

Antimicrobial Activity of Biosurfactants

Biosurfactants have also shown antibacterial, antifungal, and antiviral activities along with being potential antibiofilm agents (de Araujo et al. 2016; Rodrigues et al. 2006a; Kourmentza et al. 2021). According to World Health Organization (WHO, 2017), there is an alarming need to address the challenges posed by antibiotic resistance. In this context, biosurfactants are widely considered owing to their unique properties to inhibit microbial growth (Cao et al. 2009a). Recent studies show the potential of various microbial biosurfactants in inhibiting the growth of microbes like Klebsiella pneumonia, Escherichia coli, Bacillus subtilis, Vibrio cholera, etc. (Mani et al. 2016). The glycolipids, rhamnolipids, and sophorolipids have been studied extensively for their antimicrobial activities. Rhamnolipids can disrupt biofilm produced by Bacillus pumilus (Dusane et al. 2010) and can inhibit the growth of Listeria monocytogenes (de Araujo et al. 2016). Sophorolipids have also displayed antibacterial activity against gram-positive bacteria like Bacillus subtilis, Streptococcus faecium, etc., and gram-negative bacteria like Pseudomonas aeruginosa (Fontoura et al. 2020; Kumari et al. 2021). Although rhamnolipids and sophorolipids have antibacterial activities; however, the mechanism of action is different for both the biosurfactants. From previous studies, it was suggested that

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Table 1 Effects of various types of biosurfactants on the immune system S. No. 1

Type of surfactant Rhamnolipids

2

Sophoroplipids

3

Trehalose glycolipids

4

Surfactin

5

WH1 fungin

6

TAN-1511-A

Effects • Immunomodulation activity • Inhibits the phagocytic ability of macrophages • Induces lysis of macrophages • Induces lysis and necrosis of polymorphonuclear leukocytes • Anti-inflammatory properties • Enhances the killing of pathogens when conjugated with antibiotics • Stimulates IL-1 and TNF-α production • Induces expression of cytokines and chemokines • Enhances the production of antiinflammatory cytokines • Anti-inflammatory effects by inhibiting via cytosolic phospholipase-2 enzyme • Suppresses the T cells activation and proliferation • Induces humoral and cellular responses against Hepatitis-B surface antigens • Suppresses the production of pro-inflammatory cytokines • Increases the production of cytokines • Increases the secretion of G-CSF and GM-CSF

References Van Gennip et al. (2009)

Hardin et al. (2007)

Baeva et al. (2014); Chereshnev et al. (2010)

Hwang et al. (2008); Pirri et al. (2009)

Park and Kim (2009); Xu et al. (2016)

Takizawa et al. (1995)

rhamnolipids affect the exponential phase of cell growth by influencing the normal cell division process. Sophorolipids, on the contrary, influence the growth between the exponential and stationary phases (de Araujo et al. 2016; Fontoura et al. 2020). Lipopeptides also exhibit a wide range of antimicrobial activity. For instance, polymyxin B is active against Gram-negative pathogenic bacteria and it disrupts the cell membrane’s integrity through a micellar reaction (Roberts et al. 2015). Surfactin, produced by Bacillus sp., is a potential antibacterial agent. Surfactin changes the thickness of the bacterial membrane, thereby disturbing the integrity of the cell membrane, which may lead to cell death (de Araujo et al. 2016). Other lipopeptides like viscosin, are also studied extensively for their antimicrobial activities (Bonnichsen et al. 2015). Further, some probiotic strains, like Lactobacillus rhamnosus, which are active producers of biosurfactants, were found to restrict Escherichia coli adhesion to the intestinal cells by stimulating the expression of mucins (Hajfarajollah et al. 2018). Since these strains also produced biosurfactants, there might be a possibility that the biosurfactants produced also signal the host and bacterial cells interactions and prevent microbial adhesion, thus preventing any infection (Satpute et al. 2016; Hajfarajollah et al. 2018). Table 2 describes the antimicrobial activity of various microbial surfactants.

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Table 2 Sources and applications of different types of biosurfactants in providing antimicrobial activity against various pathogens S. No. 1

Biosurfactant Rhamnolipids

Source Pseudomonas aeruginosa

P. gessardii

2

Sophorolipids

Starmerella bombicola (Candida bombicola)

Metschnikowia churdharensis 3

Surfactin

Bacillus subtilis

B. methylotrophicus (B. velezensis) 4

Pumilacidin

B. pumilus

Application • Antibacterial activity against grampositive and gramnegative bacteria • Antifungal activity including plant pathogens • Anti-adhesive property against bacterial and yeast strains • Antiviral activity against Herpes Simplex Viruses and Coronaviruses • Antiviral activity against HIV virus, Influenza virus • Antibacterial activity against grampositive and gramnegative bacteria • Anti-adhesive property against bacterial strains • Antifungal activity against plant and human pathogens • Antibacterial activity against grampositive and gramnegative bacteria • Anti-adhesive property against bacterial strain • Mosquito larvicidal agents • Antiviral activity against pseudorabies virus, porcine parvovirus, new castle disease virus and bursal disease virus • Antifungal activity against plant pathogen • Antibacterial activity against food pathogens

Reference de Araujo et al. (2016)

Giugliano et al. (2021)

Fontoura et al. (2020); Valotteau et al. (2020)

Kumari et al. (2021)

Huang et al. (2006); Meena and Kanwar (2015); de Araujo et al. (2016); Kourmentza et al. (2021)

Toral et al. (2018)

Ceresa et al. (2016); Xiu et al. (2017); Saggese et al. (2022) (continued)

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Table 2 (continued) S. No.

Biosurfactant

Source

5

Iturin

B. subtilis

6

Fengycin

B. subtilis

B. amyloliquefaciens

7

2.4.1

Lichenysin

Lactobacillus sp.

Application • Inhibition of motility of Vibrio alginolyticus • Antiviral activity against Herpes simplex virus 1 • Antibacterial and antifungal activities against mycosis • Antiviral activity against cucumber mosaic virus, pseudorabies virus, porcine parvovirus, Newcastle disease virus, and bursal disease virus • Antibacterial activity against pathogenic bacteria • Anti-adhesive property against Candida sp.

Reference

Jasim et al. (2016); Kim et al. (2020) Huang et al. (2006); Kang et al. (2021)

Medeot et al. (2020)

Nelson et al. (2020)

Antifungal Activity of Biosurfactants

Due to the antifungal properties of biosurfactants, these compounds have found prominent utilization for the control of a wide range of yeasts and various fungi. Several lipopeptides, such as iturin, fengycin, kurstatin, etc., have shown promising antifungal activity (Table 2) (Jasim et al. 2016; Nelson et al. 2020). The interaction of iturin with the cell membrane of fungus increases the cell permeability which forms ion-conducting pores, thus, increasing the K+ ions permeability and leading to cell death (Kim et al. 2020). Fengycin is another class of lipopeptide biosurfactant which is active against filamentous fungi (Kang et al. 2021; Medeot et al. 2020). Viscosinamide, which is a cyclic depsipeptide produced by Pseudomonas sp., has also shown potential antifungal activity against Rhizoctonia solani by reducing the hyphal growth (Pandya and Saraf 2022). Further, most of the fungal-derived biosurfactants act as antifungal agents by inhibiting the fungal cell wall formation which is caused due to the inhibition of the fungal cell wall component, glucan (Meena and Kanwar 2015). Some fungal-derived biosurfactants were also studied for their activity against strains like Aspergillus sp., Candida sp., etc. The antifungal activity of biosurfactants allows the utilization of these components for agricultural purposes in order to act against phytopathogenic fungi invasion. Table 2 shows the antifungal activity of various biosurfactants.

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Antiviral Activity of Biosurfactants

Various glycolipids and lipopeptides biosurfactants have shown potential activities against various viruses like herpes simplex virus (HSV), HIV, Simian immunodeficiency virus, plant viruses, etc. In this context, Bacillus-derived surfactin has displayed antiviral activity against HSV, Vesicular stomatitis virus, Murine encephalomyocarditis virus, to name a few (Çelik et al. 2020; Giugliano et al. 2021). The inactivation of virus by surfactin depends on the length of the carbon chain (Isa et al. 2017). Moreover, it was observed that the activity of surfactin was more against enveloped viruses when compared to non-enveloped viruses (Huang et al. 2006). This difference in antiviral activity could be due to the physicochemical interactions between surfactin and the viral membrane, which leads to the permeation of surfactin into the viral envelop and disintegrating the membrane. Another lipopeptide, fengycin, has also been studied for its antiviral activity. This compound has shown noticeable activity against plant virus like cucumber mosaic virus. Fengycin inhibits viral development and replication, and cell-to-cell movement (Huang et al. 2006). The glycolipid, rhamnolipid, has been used as an antiviral agent against herpes simplex virus (HSV 1 and HSV 2), in which it inhibits the viral replication process. Recently, rhamnolipids was also found to exhibit antiviral activity against SARSCOV2 (Jin et al. 2021). These compounds exhibit antiviral activity by destabilizing the viral envelop which leads to its dissociation from the capsids. According to the study performed by Jin et al., rhamnolipids pose antiviral activity against both enveloped and non-enveloped viruses (Jin et al. 2021). Sophorolipids, have also shown promising results against SARS-COV2 virus. Due to the surface-active property, sophorolipids dissolve the viral envelop which leads to disintegration of the viral structure. Further, sophorolipids also act against HIV virus, and the degree of virulence is dependent on the carbon chain length (Daverey et al. 2021; Jin et al. 2021). Some examples of surfactants that exhibit antiviral properties are shown in Table 2.

2.4.3

Anti-adhesive Activity

Due to the amphipathic nature, biosurfactants can influence the adhesion of microorganisms to solid surfaces or any infection sites. Thus, utilization of biosurfactants for preventing the adherence of microorganisms to body tissues can provide a new platform to address various diseases (de Araujo et al. 2016). From previous works, it was observed that surfactin reduced the production of biofilm by various bacterial strains (e.g., Escherichia coli) (de Araujo et al. 2016; Isa et al. 2017; Nelson et al. 2020). Surfactin also has active anti-adhesive property against uro-pathogenic bacteria and yeast (Nahas et al. 2021). Rhamnolipids is another biosurfactant that had shown anti-adhesive activity against various bacterial and yeast strains (de Araujo et al. 2016; Dusane et al. 2010). A biosurfactant produced by Lactobacillus fermentum was found to inhibit the adherence of Streptococcus aureus to surgical

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implants (Gan et al. 2002; Rodrigues 2011). Thus, utilization of biosurfactants with anti-adhesive property along with antibiotics will provide a new strategy to increase the effectiveness of antibiotics as the biosurfactants can initiate the disruption of microbial membrane and facilitate antibiotics access. Some examples of biosurfactants that have potential anti-adhesive property are listed in Table 2.

2.5

Dermatological Activity of Biosurfactants

As an alternative to the chemical agents, biosurfactants can be used for dermatological purposes in order to reduce skin irritability and any allergic reactions (Madhu and Prapulla 2014; Bom et al. 2019). Due to their environmental friendliness and limited toxicity, along with anti-irritating properties, these compounds are being considered in high-end skin products, cosmetics and other tropical preparations (Totté et al. 2016). The applications of some of the important biosurfactants are shown in Table 3.

2.5.1

Biosurfactants for Wound Healing

From Sect. 2.5, it can be observed that biosurfactants are bioactive metabolites which have a wide range of dermatological applications, which also included wound healing. The biosurfactant produced by B. subtilis was evaluated for its wound-healing property by Zouari et al. (2016), and it was observed that there was an increased percentage of wound closure in case of the biosurfactant when compared with the control, CICAFLORA™ (Zouari et al. 2016). The biosurfactants tend to wound healing by epidermal regeneration. This property can be attributed to the free-radical scavenging ability of lipopeptides which aids in preventing inflammation and improves the formation of tissue along with epidermis differentiation (Jemil et al. 2017). According to Gupta et al. (2017) glycolipid obtained from Bacillus licheniformis enhanced the process of wound healing by re-epithelization. Moreover, it improves the proliferation of fibroblast cell and contributes to enhanced collagen deposition which aids in the process of wound healing (Gupta et al. 2017). Another biosurfactant, sophorolipids, has the ability to reduce the risk of infection during wound healing owing to their potential antimicrobial properties (Lydon et al. 2017).

2.5.2

Prebiotic Activity of Biosurfactants

Prebiotics are the compounds that can rebalance the microflora of the skin and biosurfactants mostly produced by lactic acid bacteria and Bacillus sp. are considered excellent prebiotics in the cosmetic industry (Vecino et al. 2017; Adu et al. 2020). This ability of biosurfactants to act as prebiotics can be attributed to their

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Table 3 Dermatological effects of various biosurfactants and their pharmacological activities S. No. 1

Type of biosurfactants Rhamnolipids

Pharmacological activity Antioxidant, Antimicrobial

2

Sophorolipids

Antibacterial

3

Surfactin

Antioxidant, Antibacterial

4

Saponins

Antioxidant, Antibacterial

5

Mannosyl erylthritol Lipids

Antioxidant, Antibacterial

Applications • Antiaging • Antidandruff and hair care products • Nail treatment • Moisturizers • Antacid formulations • Acne pads • Deodorants • Hair care products • Antiaging • Body odour treatment • Desquamating and depigmenting agents • Acne treatment • Moisturizers • Shower gels • Cellulite treatment agents • Collagen stimulants/Antiwrinkle agents • Antiaging • Moisturizers • Skin and hair care products • Antiaging • Moisturizers • Skin and hair care products • Antiaging • Moisturizers

References Hanno et al. (2015); Totté et al. (2016)

Aparajita and Ravikumar (2014); Archana et al. (2019)

Varvaresou and Iakovou (2015); Bhattacharya et al. (2017); Archana et al. (2019)

Vecino et al. (2017)

Naughton et al. (2019)

antimicrobial activity and anti-adhesive property (Rodrigues et al. 2006a). Thus, biosurfactants can aid in the inhibition of microbial growth in the skin and also promote restoration of healthy skin microbiome. The antimicrobial activity of biosurfactants is explained in detail in Sect. 2.4. Owing to the antimicrobial and

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anti-adhesive properties of biosurfactants, these compounds can be incorporated into dermatological formulations. Apart from being prebiotics, biosurfactants, like rhamnolipids and sophorolipids, also have the ability to act as anti-aging compounds (Aparajita and Ravikumar 2014; Hanno et al. 2015).

2.6

Biosurfactants for Oral Hygiene

Owing to the excellent antimicrobial property possessed by the biosurfactants, these compounds were also studied for their application in maintaining oral hygiene. The glycolipids, rhamnolipids and sophorolipids, have been studied extensively in this regard. According to Esteves et al., the incorporation of rhamnolipids in toothpastes inhibited the formation of biofilm by Streptococcus mutans (Esteves et al. 2022). Using similar formulations, Farias et al. studied the effect of biosurfactants in mouth washes and concluded that the mouth washes had the ability to inhibit the biofilm production (Farias et al. 2019). Although biosurfactants have shown potential in maintaining oral hygiene; however, there is limited work done in this area of research. Due to the multitude of properties of biosurfactants, these compounds can be exploited to substitute the chemical composition of toothpastes and mouth washes to make them bio-based and sustainable (Ali et al. 2022). Some examples of biosurfactants and their mechanism in maintaining oral hygiene are listed in Table 4.

2.7

Biosurfactants as Drug Delivery Systems

In order to improve the oral bioavailability of drugs which have low aqueous solubility, biosurfactants are being used as sustainable DDS. The potential of biosurfactants to constitute a robust DDS depends on its ability of micelle formation and emulsification (Ohadi et al. 2020; Bjerk et al. 2021). In the recent years, several studies have been done to improve the bioavailability of hydrophobic drugs and one of the most studied strategies is microemulsion DDS (MDDS), which has a concoction of surfactants, lipids, and cosurfactants (Gudiña et al. 2013; Ohadi et al. 2020). The most commonly used biosurfactants for preparation of MDDS are glycolipids and lipopeptides. MDDS can be used by various mode of administration- oral, nasal, topical, etc. A detailed list of MDDS using biosurfactants is given in Table 5. Another method of utilizing biosurfactants as delivery systems is via hydrogels. Hydrogels can be prepared by incorporating biosurfactants with antimicrobial activity and can be used for wound healing against skin drug-resistant infections. Hydrogels prepared from poly (vinyl alcohol), polyethylene oxide, and poly (acrylic acid) can be integrated for the delivery of topical drugs that contain biosurfactants (Bjerk et al. 2021).

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Table 4 Utilization of biosurfactants for maintaining oral hygiene and the action of biosurfactants during oral care Biosurfactant Glycoprotein-type biosurfactant

Microorganism Lactobacillus casei, L. acidophilus, L. reuteri, L. fermentum Streptococcus mitis

Biosurfactant action Downregulation of genes, which are important for biofilm formation, in S. mutans Increases the repulsive forces between the enamel surface and S. mutans

Uncharacterized biosurfactant

Candida lipolytica

Mixture of glycosidic residue and rhamnolipid-like biosurfactant Lipopeptide

–

Inhibition of biofilm formation by Lactobacillus sp. and S. mutans Growth inhibition against S. mutans

Bacillus tequilensis

Inhibition of biofilm formation by S. mutans

Emulsan

–

Control of dental plaque

Uncharacterized biosurfactant

2.8

Reference Savabi et al. (2014) Van Hoogmoed et al. (2006) Rufino et al. (2011) Rodrigues et al. (2006b) Pradhan et al. (2013) Eigen and Simone (1988)

Biosurfactants for Diagnostics

Nanobiosensors are biosensors incorporated with nanomaterials and can be used for advanced testing and diagnosis. In this context, in order to reduce the process toxicity and production cost of nanoparticles, microorganisms that can produce medically important molecules can be deposited on the sensors for the production of the required compounds either intracellularly or extracellularly (Kasture et al. 2007; Çelik et al. 2020). One of the biosurfactants that have been used for this process is sophorolipids. Cobalt nanoparticles capped with sophorolipids can be utilized for the generation of biocompatible surfaces by attaching bioactive components (e.g., lectins, glycosidases) and can be used for diagnostics and medical applications (Kasture et al. 2007; Darne and Prabhune 2021). Further, silver nanoparticles prepared with rhamnolipids have demonstrated effective antimicrobial activity against both gram-positive and gram-negative bacteria (Bharali et al. 2013). Utilization of nanotechnology for medical diagnostics has achieved prominence recently and utilization of biosurfactant-based nanoparticles will make the process sustainable and environmentally friendly.

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Table 5 Utilization of biosurfactants as a component of microemulsion drug delivery system (MDSS) and its function in MDDS S. No. 1

Type of surfactant Surfactin

Function Stabilizer

2

Surfactin

Stabilizer

3

Rhamnolipids/ Sophorolipids

4

Rhamnolipids

Stabilizer, Hydrophobic/ hydrophilic linker Stabilizer, Emulsifier

5

Mannosylerythritol lipids-B Rhamnolipids, Surfactin

7

Rhamnolipids

Vesicleformation Stabilizer, Hydrophobic/ hydrophilic linker Stabilizer

8

Rhamnolipids

Stabilizer

6

Microemulsion formulation Biosurfactant-based self-emulsifying system with vitamin E Biosurfactant-based self-emulsifying system with docosahexaenoic acid Glycolipids with oils

Rhamnolipids with alcohol, n-heptane, and water Functionalized glycolipids Poly (methylmethacrylate) core- biosurfactant (shell) nanoparticle Glycolipids-mediated silver nanoparticles Glycolipids-mediated nickel oxide nanoparticles

References Ohadi et al. (2020)

He et al. (2017)

Nguyen et al. (2010)

Xie et al. (2005)

Worakitkanchanakul et al. (2008) Hazra et al. (2014)

Xie et al. (2006); Kumar et al. (2010) Palanisamy and Raichur (2009)

3 Conclusion and Future Perspectives Biosurfactants have been utilized extensively in the recent years for various environmental applications due to their sustainability and minimal toxicity. These unique properties of biosurfactants have also opened new arenas towards the utilization of biosurfactants for various therapeutic applications. Various biosurfactants have immunosuppressive potential and this ability of the bio-based compounds can be exploited for addressing immune-related diseases like asthma, allergies, arthritis, etc. Moreover, due to their antimicrobial activities, biosurfactants can also be applied for the preparation of various antibiotic agents. With the outbreak of COVID-19, biosurfactants market has increased linearly due to increased utilization of hand wash and sanitizers, emulsifiers, disinfectants, etc., in which biosurfactants are an integral part. Moreover, they can be used for environmental applications as antifungal agents to prevent any possible phytopathogenic fungi invasion. In the recent years, biosurfactants have been widely explored for the formulation of microemulsion for safe drug delivery and to reduce cytotoxicity. Further, although these surface-active compounds can also be utilized for various cosmetics and

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dermatological applications; however, their large-scale production is limited owing to which the availability of biosurfactants for various industrial applications is constrained. In order to improve the obtainability, more research has to be focused towards reducing the production cost of surfactants and increasing the accessibility of raw materials. Further, to employ biosurfactants for various applications, new research areas have to be explored where biosurfactants can play an integral role. In order to increase the interaction of biosurfactants with pathogenic organisms, the biosurfactants can be engineered and tailor-made for specific reactions, which will provide an understanding of the interaction of biosurfactants with pathogens. Thus, with an ever-increasing biosurfactant market, these compounds can be utilized in various fields of research and industrial applicability. Apart from therapeutical applications, these compounds have a broad array of applications in environmental management, cosmetics, detergent and textile industries, etc., and have the potential to completely replace synthetic surfactants in the near future.

References Adu SA, Naughton PJ, Marchant R, Banat IM (2020) Microbial biosurfactants in cosmetic and personal skincare pharmaceutical formulations. Pharmaceutics 12:1099 Ahamed MI, Prasad R (2021) Microbial biosurfactants: preparation, properties and applications. Springer Nature, Singapore, pp 1–12 Akella A, Deshpande SB (2013) Pulmonary surfactants and their role in pathophysiology of lung disorders. Indian J Exp Biol 51:5–21 Ali N, Pang Z, Wang F, Xu B, El-Seedi HR (2022) Lipopeptide biosurfactants from Bacillus spp.: types, production, biological activities and applications in food. J Food Qual 2022:1 Aparajita V, Ravikumar P (2014) Liposomes as carriers in skin ageing. Int J Curr Pharm Res 6:1–7 Archana K, Sathi Reddy K, Parameshwar J, Bee H (2019) Isolation and characterization of sophorolipid producing yeast from fruit waste for application as antibacterial agent. Environ Sustain 2:107–115 Baeva T, Gein S, Kuyukina M, Ivshina I, Kochina O, Chereshnev V (2014) Effect of glycolipid Rhodococcus biosurfactant on secretory activity of neutrophils in vitro. Bull Exp Biol Med 157: 238–242 Bharali P, Saikia J, Paul S, Konwar B (2013) Colloidal silver nanoparticles/rhamnolipid (SNPRL) composite as novel chemotactic antibacterial agent. Int J Biolog Macromol 61:238–242 Bhattacharya B, Ghosh TK, Das N (2017) Application of bio-surfactants in cosmetics and pharmaceutical industry. Sch Acad J Pharm 6:320–329 Bjerk TR, Severino P, Jain S, Marques C, Silva AM, Pashirova T, Souto EB (2021) Biosurfactants: properties and applications in drug delivery, biotechnology and ecotoxicology. Bioengineering 8:115–119 Blunt W, Blanchard C, Morley K (2022) Effects of environmental parameters on microbial rhamnolipid biosynthesis and bioreactor strategies for enhanced productivity, vol 182. Biochem, Eng. J, p 108436 Bom S, Jorge J, Ribeiro H, Marto J (2019) A step forward on sustainability in the cosmetics industry: a review. J Clean Prod 225:270–290 Bonnichsen L, Svenningsen NB, Rybtke M, de Bruijn I, Raaijmakers JM, Tolker-Nielsen T, Nybroe O (2015) Lipopeptide biosurfactant viscosin enhances dispersal of Pseudomonas fluorescens SBW25 biofilms. Microbiology 161:2289

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Cao XH, Liao ZY, Wang CL, Yang WY, Lu MF (2009a) Evaluation of a lipopeptide biosurfactant from Bacillus natto TK-1 as a potential source of anti-adhesive, antimicrobial and antitumor activities. Brazilian J Microbiol 40:373–379 Cao XH, Liao ZY, Wang CL, Cai P, Yang WY, Lu MF, Huang GW (2009b) Purification and antitumour activity of a lipopeptide biosurfactant produced by Bacillus natto TK-1. Biotechnol Appl Biochem 52:97–106 Çelik PA, Manga EB, Çabuk A, Banat IM (2020) Biosurfactants’ potential role in combating COVID-19 and similar future microbial threats. Appl Sci 11(1):334–337 Ceresa C, Rinaldi M, Chiono V, Carmagnola I, Allegrone G, Fracchia L (2016) Lipopeptides from Bacillus subtilis AC7 inhibit adhesion and biofilm formation of Candida albicans on silicone. Antonie Van Leeuwenhoek 109:1375–1388 Chen J, Wu Q, Hua Y, Chen J, Zhang H, Wang H (2017) Potential applications of biosurfactant rhamnolipids in agriculture and biomedicine. Appl Microbiol Biotechnol 101:8309–8319 Chereshnev V, Gein S, Baeva T, Galkina T, Kuyukina M, Ivshina I (2010) Modulation of cytokine secretion and oxidative metabolism of innate immune effectors by Rhodococcus biosurfactant. Bull Exp Bio Med 149:734 Choi Y, Jang J, Park HS (2020) Pulmonary surfactants: a new therapeutic target in asthma. Curr Allerg Asthma Rep 20:1–8 Christova N, Lang S, Wray V, Kaloyanov K, Konstantinov S, Stoineva I (2015) Production, structural elucidation, and in vitro antitumor activity of trehalose lipid biosurfactant from Nocardia farcinica strain. J Microbiol Biotechnol 25(4):439–447 Coelho ALS, Feuser PE, Carciofi BAM, De Andrade CJ, De Oliveira D (2020) Mannosylerythritol lipids: antimicrobial and biomedical properties. Appl Microbiol Biotechnol 104(6):2297–2318 Darne P, Prabhune A (2021) Nanotechnology in sustainable agriculture. CRC Press, pp 191–210 Das P, Yang XP, Ma LZ (2014) Analysis of biosurfactants from industrially viable Pseudomonas strain isolated from crude oil suggests how rhamnolipids congeners affect emulsification property and antimicrobial activity. Front Microbiol 5:696 Das B, Kumar B, Begum W, Bhattarai A, Mondal MH, Saha B (2022) Comprehensive review on applications of surfactants in vaccine formulation, therapeutic and cosmetic pharmacy and prevention of pulmonary failure due to COVID-19. Chem Afr 5:1–22 Daverey A, Dutta K, Joshi S, Daverey A (2021) Sophorolipid: a glycolipid biosurfactant as a potential therapeutic agent against COVID-19. Bioengineering 12(2):9550–9560 De Araujo LV, Guimarães CR, Da Silva Marquita RL, Santiago VM, De Souza MP, Nitschke M, Freire DMG (2016) Rhamnolipid and surfactin: anti-adhesion/antibiofilm and antimicrobial effects. Food Control 63:171–178 Dey G, Bharti R, Sen R, Mandal M (2015) Microbial amphiphiles: a class of promising new-generation anticancer agents. Drug Discov Today 20(1):136–146 Duarte C, Gudiña EJ, Lima CF, Rodrigues LR (2014) Effects of biosurfactants on the viability and proliferation of human breast cancer cells. AMB Express 4(1):1–12 Dusane DH, Nancharaiah YV, Zinjarde SS, Venugopalan VP (2010) Rhamnolipid mediated disruption of marine Bacillus pumilus biofilms. Colloids Surf B Biointerfaces 81(1):242–248 Eigen E, Simone AJ (1988) Control of dental plaque and caries using emulsan, Google Patents Esteves GM, Esteves J, Resende M, Mendes L, Azevedo AS (2022) Antimicrobial and antibiofilm coating of dental implants—past and new perspectives. Antibiot 11(2):235 Farias JM, Stamford TCM, Resende AHM, Aguiar JS, Rufino RD, Luna JM, Sarubbo LA (2019) Mouthwash containing a biosurfactant and chitosan: an eco-sustainable option for the control of cariogenic microorganisms. Int J Bio Macromol 129:853–860 Feuser P, Coelho A, de Melo M, Scussel R, Carciofi B, Machado-de-Ávila R, de Oliveira D, de Andrade C (2021) Apoptosis induction in murine melanoma (B16F10) cells by Mannosylerythritol lipids-B; a glycolipid biosurfactant with antitumoral activities. Appl Biochem Biotechnol 193(11):3855–3866

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Biosurfactants: An Antiviral Perspective Sethuramalingam Balakrishnan, Marimuthu Ragavan Rameshkumar, Avoodaiappan Nivedha, Krishnan Sundar, Narasingam Arunagirinathan, and Mariadhas Valan Arasu

1 Introduction Biosurfactants are surface-active organic compounds mainly produced by environmental microorganisms (Santos et al. 2016). They are amphiphilic molecules in which hydrophilic moiety comprises polysaccharides and peptides, and the hydrophobic moiety comprises fatty acids (da Silva et al. 2021).Biosurfactants are biodegradable, non-toxic, and exhibit emulsification activity against hydrophobic molecules-(Nayarisseri et al. 2018). Some primary biosurfactant-producing bacterial genera are Bacillus, Streptomyces, Rhodococcus, Pseudomonas, Staphylococcus, Serratia, Paenibacillus and Stenotrophomonas (Yoshida et al. 2005; Cai et al. 2015; Rani et al. 2020). Biosurfactants possess important properties like tolerance

S. Balakrishnan · M. R. Rameshkumar Department of Microbiology and Biotechnology, Presidency College (Autonomous), Chennai, Tamil Nadu, India A. Nivedha Central Research Laboratory, Meenakshi Academy of Higher Education and Research (Deemed to be University), Chennai, Tamil Nadu, India K. Sundar Department of Biotechnology, Kalasalingam Academy of Research and Education, Srivilliputhur, Tamil Nadu, India N. Arunagirinathan (✉) Department of Microbiology and Biotechnology, Presidency College (Autonomous), Chennai, Tamil Nadu, India Central Research Laboratory, Meenakshi Academy of Higher Education and Research (Deemed to be University), Chennai, Tamil Nadu, India M. Valan Arasu Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 P. Kumar, R. C. Dubey (eds.), Multifunctional Microbial Biosurfactants, https://doi.org/10.1007/978-3-031-31230-4_20

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to temperature and pH and ready availability on a large scale, which make them highly suitable for application in various fields (Bhadoriya and Madoriya 2013). Biosurfactants are found to have antimicrobial, antioxidant, anti-aging, cytotoxic, and anti-inflammatory activities (Saravanakumari and Mani 2010; Sharma and Saharan 2016). Biosurfactant molecules can solubilize the viral membranes and act as antiviral agents against herpes simplex virus (HSV), retroviruses, and coronaviruses (Huang et al. 2006; Jin et al. 2021). Specifically, glycolipid and lipopeptide biosurfactants exhibit antiviral activity against HSV-1 &-2 and bovine coronavirus by interacting with their lipid membranes (Jin et al. 2021; Remichkova et al. 2008). Surfactin exhibits antiviral activity against HSV-1 and -2, simian immunodeficiency virus, vesicular stomatitis virus, murine encephalomyocarditis virus, semliki forest virus, and feline valicivirus. Surfactin inactivates enveloped viruses more efficiently than non-enveloped viruses. The interaction of surfactin with viral membrane and carbon chain length in surfactin are responsible for its antiviral activity (Vollenbroich et al. 1997; Seydlov et al. 2011; Meena and Kanwar 2015). In this chapter, microbial biosurfactants and their antiviral activities are discussed.

2 Types of Biosurfactants Biosurfactants are of two types, one is low-molecular weight and another one is high-molecular weight (Guerra-Santos Luis et al. 1986; Cooper and Goldenberg 1987) (Fig. 1).

Fig. 1 Types of biosurfactants

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Low-Molecular-Weight Biosurfactants

Biosurfactants like glycolipids (trehalolipids, sophorolipids, rhamnolipids) and lipopeptides (surfactin, gramicidin S and polymyxin) (Salihu et al. 2009) are lowmolecular-weight (LMW) biosurfactants.

2.1.1

Glycolipids

Glycolipids contain carbohydrates that combine with long-chain fatty acids. The glycolipid biosurfactants have hydrophilic moiety which is made up of carbohydrates (glucose, galactose, mannose, sophorose, trehalose, and rhamnose) and hydrophobic moiety which is made up of fatty acids (Müller and Hausmann 2011). Glycolipids decrease the surface and interfacial tension. Most important glycolipid biosurfactants are rhamnolipids, and trehalolipids. They possess antibacterial, antifungal, antiviral, antibiofilm, and anti-tumour properties (Desai and Banat 1997; Inès and Dhouha 2015).

Rhamnolipids Rhamnolipids are formed by combining rhamnose sugar attached to β-hydroxy decanoic acid. Rhamnolipids are used in food, healthcare, pharmaceutical, and petrochemical industries because of their low surface tension, non-toxicity, and biodegradability properties. Pseudomonas aeruginosa is the primary organism producing rhamnolipids (Abdel-Mawgoud et al. 2010). In their study, Thakur et al. (2021) reported that biosurfactants act as very good immunomodulators and they also exhibit anticancer activities, and prevent fungal growth, spore formation, and biofilm development. Rhamnolipid from P. aeruginosa BS01 was used as a stabilizing agent for synthesizing nanoparticles (Hazra et al. 2013).

Sophorolipids Sophorolipids are produced mainly by Candida sp. and Yarrowia lipolytica. They contain a disaccharide sophorose unit which is glycosidically attached to the surfaceactive glycolipids (Sen et al. 2017). Sophorolipids are mainly used as detergents in laundry and dishwasher cleaning agents. Sophorolipid-based detergents are biodegradable, ecologically safe, and contain no synthetic detergents. They exhibit antibacterial, antifungal, immunomodulatory, anticancer, and anti-HIV activities (Shah et al. 2005). A yeast strain, Rhodotorula babjevae YS3produced sophorolipid that exhibits antimicrobial activity against Colletotrichum gloeosporioides, Fusarium spp., Corynespora cassiicola, and Trichophyton rubrum (Sen et al. 2017).

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Trehalolipids Trehalolipids are the other glycolipids produced mainly by Rhodococcus sp. and Arthrobacter sp. (Franzetti et al. 2010). Other actinobacterial genera that produce trehalolipids are Corynebacterium, Mycobacterium, Tsukamurella, Nocardia, Skermania, Dietzia, Williamsia, and Gordonia (Lanéelle 1998; Kuyukina et al. 2015). Trehalolipids produced by Rhodococcus equi ATCC 33701, Rhodococcus ruber M1, Rhodococcus ruber IEGM 231, Rhodococcus opacus ATCC 21953 and Rhodococcus erythropolis SD-74 were found to have immunomodulatory activities (Isoda et al. 1996; Chereshnev et al. 2010; Harris et al. 2010; Kuyukina et al. 2015).

2.1.2

Lipopeptides

Lipopeptides are variants of heptapeptides and hydroxy fatty acids (surfactin, iturin, fengycin, viscosin, lichenysin, serrawettin, and phospholipids). Surfactin is one of the lipopeptides formed by Bacillus subtilis. It has the crucial property of lysing red blood cells (Arima et al. 1968), and high surface activity and is successfully used to enhance the oil recovery process (Mnif and Ghribi 2015). Lipopeptides produced by Bacillus sp. exhibit antifungal, anti-inflammatory, anti-tumoural, antiviral, and antiplatelet activities, making them potential therapeutic agents (Meena et al. 2020; Kourmentza et al. 2017; Meena and Kanwar 2015). The lipopeptides isolated from Bacillus sp., namely mycosubtilin and mycosubtilin, along with surfactin mixtures, showed antifungal activity against Byssochlamys fulva, Paecilomyces variotti, and Candida krusei (Kourmentza et al. 2021).

2.1.3

Phospholipids

Phospholipids are polar in nature and produced by many bacteria, fungi and yeasts. Thiobacillus thiooxidans and Rhodococcus erythropolis are major organisms producing phospholipids. The most abundant phospholipid is phosphatidylethanolamine which is highly present in the prokaryotic cells. The phospholipidbiosurfactant shows emulsification properties against various hydrocarbons (Gayathiri et al. 2022; McClements and Gumus 2016).

2.2

High-molecular-weight Biosurfactants

High-Molecular-Weight (HMW) biosurfactants are reported for their good bio-emulsifying activities. They are highly efficient in stabilizing emulsions and possess extensive substrate specificity. Lipopolysaccharides and amphipathic polysaccharides, mixtures of heteropolysaccharides, and proteins are examples of HMW biosurfactants. They are also known as extracellular polysaccharides (EPS)

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e.g. emulsan, alasan, liposan, sphingan, and xanthan gum (Salihu et al. 2009; Dastgheib et al. 2008). Microbial species producing HMW biosurfactants are Acinetobacter radioresistens, Acinetobacter calcoaceticus, Candidalipolytica IA 1055, Acinetobacter junii SC14, and Saccharomyces cerevisiae (Edosa et al. 2018).

2.2.1

Amphipathic Polysaccharides and Lipopolysaccharides

Amphipathic polysaccharides belong to the HMW category of biosurfactants, generally composed of emulsan, liposan, and mannoprotein. Acinetobacter calcoacetius RAG-1 produced a potent polyanionic amphipathic hetero-polysaccharide bioemulsifier. Emuslan is a very effective emulsifying agent for hydrocarbons in water. Liposan produced by C. lipolyticais made up of carbohydrates (83%) and protein (17%) (Edosa et al. 2018). Yarrowia lipolytica-produced liposan was found to contain carbohydrates and protein. Saccharomyces cerevisiae produced mannoprotein contains mannose sugar and protein molecules. Like-wise, Acinetobacter calcoaceticus BD4 secretes extracellular product capsule polysaccharide (Kaplan et al. 1985; Edosa et al. 2018).

2.2.2

Fatty Acids, Neutral Lipids and Lipoproteins

Yeasts and some bacterial genera produce fatty acid and phospholipid types of surfactants using n-alkanes. These biosurfactants are essential for biomedical applications (Vijayakumar and Saravanan 2015). Lipoproteins contain lipids and proteins, developing more complex forms with many lipids and proteins. The bacterial strain Streptomyces sp. DPUA1566 produces a lipoprotein called Bioelan. It effectively reduces the surface tension in different temperature, and pH ranges and salt concentrations (Santos et al. 2019). A yeast strain, C. lipolytica, produces anionic lipopeptide, which contains 50% protein, 20% lipid, and 8% carbohydrate (Rufino et al. 2014).

3 Microbial Biosurfactants Microbes are major producers of biosurfactants. Microbial surfactants are the replacement for chemical surfactants due to their high compatibility with the environment and numerous other advantages (Farn 2006; Santos et al. 2019). Biosurfactants are majorly used in the bioremediation of hydrocarbons since they increase the nutrient uptake of hydrocarbons by microbes which reduce the availability of hydrocarbon contaminants in the environment (Canet et al. 2001; Sotirova et al. 2009). Biosurfactants are found to be interrupting microbial adhesion and desorption. Hence, biosurfactants produced by probiotic bacteria are considered a defence

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against other colonizers in the gastrointestinal tract and medical devices (Rodrigues et al. 2004). Lactobacilli-produced biosurfactants were found to reduce the adhesion of disease-causing microorganisms on the glass (Rodrigues et al. 2006), silicone rubber (van Hoogmoed et al. 2004), surgical implants (Velraeds et al. 2000) and voice prostheses (Busscher et al. 1997; Gan et al. 2002). Furthermore, the biosurfactants delay the biofilm formation by pathogenic bacteria on catheters and also reduce the usage of synthetic drugs and chemicals (Falagas and Makris 2009; Rodrigues et al. 2004). Some important bacterial genera that produce biosurfactants are discussed below (Table 1).

3.1 3.1.1

Bacterial Biosurfactants Pseudomonas

Bacterial species of the genus Pseudomonas produce various biosurfactants like glycolipids and lipopeptides (Shahaliyan et al. 2015). Microbial rhamnolipids form emulsions with kerosene and crude oil. In our previous study, the biosurfactantproducing strain Pseudomonas mendocina ADY2b isolated from hydrocarboncontaminated coastal waters of Chennai harbour, showed 58.33% of emulsification index24 (EI24) towards diesel (Balakrishnan et al. 2022). Thavasi et al. (2011) characterized a biosurfactant produced by P. aeruginosa that could emulsify crude oil, kerosene, motor lubricant oil, anthracene, peanut oil, diesel, naphthalene, and xylene. It was identified as lipopeptide and its chemical compositions are protein (50.2%) and lipid (49.8%). P. aeruginosa-produced biosurfactant was reported for bioremediation of oil spills and could be an alternative to chemical surfactant (Prieto et al. 2008; Das et al. 2014).

3.1.2

Bacillus

Another most important biosurfactant-producing bacterial genus is Bacillus, and its biosurfactants are found to have applications in the microbial-enhanced oil recovery (MEOR) process. Biosurfactant-producing Bacillus spp. are found majorly in oil-contaminated soils. A Bacillus methylotrophicus USTBa strain isolated from a petroleum reservoir effectively removed crude oil (>90%) after 12 days of incubation. This bacterium produced a strong glycolipid biosurfactant which tolerates a wide range of pH and temperature (Chandankere et al. 2014). Parthipan et al. (2017) identified a B. subtilis strain A1 which produced a biosurfactant that completely degraded the LMW and 97% of HMW alkanes. It has shown excellent emulsification activity and is reported for application in oil spill remediation.

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Table 1 Biosurfactants produced by microorganisms with their biological properties Microorganism E. coli

Chemical composition Polysaccharides

Wickerhamiella domercqiae

Sophorolipid

B. subtilis natto T-2

Crude Cyclic Lipopeptides

Lactobacillus acidophilus

Biosurfactant

Exophiala dermatitidis

Biosurfactant

Rhodococcus aurantiacus B. subtilis Hs0121

Trehalose and lipids

C. lipolytica UCP 0988

Rufisan

Bacillus sp

Mycosubtilin (Lipopeptide)

Penicillium spiculisporum P. aeruginosa BN10

Spiculosporic acid

Lactobacillus paracasei subsp. paracasei A20

Glycoprotein

Lactobacillus jensenii and Lactobacillus rhamnosus

Biosurfactant

C15 Surfactin-Like Lipopeptide

Rhamnolipids

Properties Antibiofilm activity against E. coli, P. aeruginosa, K. pneumoniae, S. aureus, Staphylococcus epidermidis, and Enterococcus faecalis Anticancer activity against human liver cancer cells (H7402) Anticancer activity against leukaemia cancer cells (K562) Antibiofilm activity against S. aureus and S. epidermidis Anticancer activity against monocytic leukaemia cancer cells (U937) Lower the interfacial tension Anticancer activity against breast cancer cells (Bcap37) Antimicrobial activity against Streptococcus mutans, S. agalactiae, S. sanguis, S. oralis Antimicrobial activity against Paecilomyces variotti, Byssochlamys fulva, C. krusei Removal of heavy metal cations from water Anticancer activity against leukaemia cancer cells (BV-173) Cytotoxicity activity against breast cancer cells (T47D and MDA-MB231) Antimicrobial and antibiofilm activities against multidrug-resistant (MDR) strains of Acinetobacter baumannii, E. coli, and S. aureus

References Valle et al. (2006)

Chen et al. (2006)

Wang et al. (2007)

Walencka et al. (2008) Chiewpattanakul et al. (2010) Franzetti et al. (2010) Liu et al. (2010)

Rufino et al. (2011)

Kourmentza et al. (2021)

Wang et al. (2013) Christova et al. (2013) Duarte et al. (2014)

Sambanthamoorthy et al. (2014)

(continued)

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Table 1 (continued) Microorganism P. aeruginosa

Chemical composition Biosurfactant

C. bombicola ATCC 22214 B. subtilis fmbJ

Sophorolipid Fengycin

Properties Antimicrobial activity against Bacillus pumilus, Sarcina lutea, M. luteus, C. albicans Penicillium chrysogenum Bioaugmentation

Bacillus and Paenibacillus spp

Lipopeptides

Bacillus amyloliquefaciens and Bacillus thuringiensis Lactobacillus jensenii P6A and Lactobacillus gasseri P65

Lipopeptides

C. lipolytica Y-917

Sophorolipid

Anticancer activity against colon cancer cells(HT29) Antimicrobial activity against Klebsiella pneumonia, Enterobacter cloacae, Antimicrobial activity against Listeria monocytogenes, Bacillus cereus Antimicrobial and Antibiofilm activities against E. coli, Staphylococcus saprophyticus, K. pneumoniae, Enterobacter aerogenes, C. albicans Bioemulsifier

Arthrobacter calcoaceticus RAG-1 B. subtilis ATCC 21332 Bacillus atrophaeus AKLSR1 P. maritimus SAMP MCC 3013

Heteropolysaccharides

Bioemulsifier

Surfactin

Iron-remediation, antiinflammatory activity. Anticancer activity against lung cancer cells(A549)

Acinetobacter junii

Lipopeptide

P. maritimus SAMP MCC 3013

Glycolipid

P. maritimus SAMP MCC 3013

Glycolipid

Polysaccharide Biosurfactant

Cyclic Lipopeptides

Glycolipid

Anticancer activity against breast cancer cells (MCF7) Anticancer activity against epithelial cancer cells (KB) Anticancer activity against cervical cancer cells (HeLa) Anticancer activity against colon cancer cells (HCT)

References El-Sheshtawy and Doheim (2014)

Elshafie et al. (2015) Cheng et al. (2016) Cochrane and Vederas (2016)

Perez et al. (2017)

Morais et al. (2017)

Alizadeh-Sani et al. (2018) Abdelli et al. (2019)

Yea et al. (2019) Routhu et al. (2019)

Waghmode et al. (2020) Ohadi et al. (2020)

Waghmode et al. (2020) Waghmode et al. (2020) (continued)

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Table 1 (continued) Microorganism Rhodococcus sp. ST-5

Chemical composition Glycolipid

Candida tropicalis

Mannan-fatty acid

Rhodococcus erythropolis Bacillus velezensis strain T701

Trehalosedicarynomycolate Lipopeptide

Pseudomonas mendocina ADY2b

Heteropolysachharides

3.1.3

Properties Bioremediation and Biotransformation of chlorinated hydrocarbon Biostimulation Biorestoration for contaminated sediments. Anticancer activity against cervical cancer cells (HeLa) Biodegradation of petroleum products

References Cappelletti et al. (2020) Kuraoka et al. (2020) Cappelletti et al. (2020) Jiang et al. (2021)

Balakrishnan et al. (2022)

Rhodococcus

The Rhodococcus species can survive in different habitats (Finnerty 1992). Bicca et al. (1999) isolated two strains, Rhodococcus ruber and Rhodococcus erythropolis, from oil wells in Russia. They found that they utilize hydrocarbons as the carbon source for producing biosurfactants. Strain R. ruber emulsified diverse hydrocarbons with EI24 of 20–50% and was used for soil remediation. Zheng et al. (2009) identified a biosurfactant-producing strain, Rhodococcus ruber from Daqing Oilfield, China, which used alkanes as carbon source and its maximum biosurfactant yield was 13.34 g/L at 44 h. The biosurfactant is identified as glycolipid and it is involved in the MEOR process.

3.1.4

Halomonas

Several Halomonas species have the potential to produce exopolysaccharides (EPS) with anticancer activity (Calvo et al. 2002). EPS-producing thermophilic Halomonas nitroreducens isolated from a hot spring emulsifies aliphatic hydrocarbons (56–65%) and vegetable oils (68–85%). The EPS was predominantly made of glucose, galactose, and mannose (Chikkanna et al. 2018). Kokoulin et al. (2020) reported that Halomonas halocynthiae KMM1376 synthesized an EPS that showed cytotoxicity on human cancer cell line MDAMB- 231. Details of different bacterial genera and the biosurfactants produced by them are given in Fig. 2.

3.2

Fungal Biosurfactants

A Penicillium sp. obtained from Amazonian soil produced a biosurfactant which exhibits high emulsification index (EI24 > 50) (Sena et al. 2018). Kim et al. (2000)

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Fig. 2 Biosurfactants produced by different bacterial genera (Kumar et al. 2021; Santos et al. 2016; Shekhar et al. 2015)

isolated Nocardia sp. L-417, which produced two types of biosurfactants. Type I had a solid emulsifying property and type II reduced the surface tension of liquids. A Candida sp. SY16 produced a biosurfactant called mannosylerythritol lipid. It emulsified vegetable oil, crude oil and hydrocarbons at low concentrations (Kim et al. 2002). Loeto et al. (2021) isolated two halophilic yeasts, Debaryomyces hansenii and MK9 and Rhodotorula mucilaginosa SP6 from Makgadikgadi and Sua pans, Botswana, using Xanthocercis zambesiaca seeds as an inexpensive carbon substrate. Fourier Transform Infra-Red spectroscopy analysis indicated that biosurfactant produced by R. mucilaginosa SP6 was a rhamnolipid, whereas the surfactant produced by D. hansenii MK9 was a sophorolipid. These biosurfactants showed antimicrobial activity against Escherichia coli, Proteus vulgaris, Micrococcus luteus, Klebsiella pneumoniae, Staphylococcus aureus, Candida albicans, Cryptococcus neoformans, and Aspergillus niger. Polyol lipids are important type of fungal biosurfactants (Garay et al. 2017). The two important polyol lipids are liamocins produced by Aureobasidium pullulans (Price et al. 2013) and polyol fatty acid esters produced by Rhodotorula sp. (Cajka et al. 2016). Camargo et al. (2018) reported that the biosurfactant produced by the yeast Meyerozyma guilliermondii is of glycolipid type. It removes metals and solubilizes

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15.9% of cadmium in the sewage sludge. In another study by Ribeiro et al. (2020), it was reported that Saccharomyces cerevisiae strain produces a glycolipid biosurfactant. They suggested that it could be used to replace egg yolk for cookie formulation.

4 Antiviral Activity of Biosurfactants Since biosurfactants are amphiphilic substances, their hydrophobic domain interacts simultaneously with the viral lipid membrane and the hydrophilic substances. These unique properties disrupt the virus structure and deactivate it (Sandeep and Rajasree 2017). Additionally, biosurfactants can form micellar structures which directly target the virus and play a crucial application in drug delivery (Nakanishi et al. 2009). Studies reported that surfactin exhibits antiviral activity against HSV-1 and 2, simian immunodeficiency virus (SIV), newcastle disease virus (NDV), and vesicular stomatitis virus (VSV) (Vollenbroich et al. 1997; Yuan et al. 2019) (Table 2).

4.1

Herpes Simplex Virus (HSV)

HSV belongs to the Herpesviridae family and contains double-stranded DNA as genetic material. HSV-1 is transmitted by oral-to-oral contact, causes infection in or around the mouth, and sometimes causes genital herpes. However, HSV-2 is transmitted by sexual contact, and it causes genital herpes. WHO estimated that 3700 million people (aged above 50 years) have HSV-1, and 491 million people (aged between 15 and 49) have HSV-2 infections worldwide (WHO 2020). A crude mixture of sophorolipids produced by C. bombicola exhibited antiviral activity against Epstein - Barr virus (EBV) (Gross and Shah 2007). The Pseudomonas sp. S-17 produced rhamnolipid surfactant PS-17 and its complex with the polysaccharide alginate was tested against HSV-1 and -2. The IC50 of rhamnolipid PS-17 was 14.5 and 13 μg/mL against HSV-1 and -2, respectively. At the same time, IC50 of rhamnolipid PS-17 with alginate complex was 435 and 482 μg/mL against HSV-1 and -2, respectively. Hence, rhamnolipid PS-17 and its alginate complex would be useful in developing anti-HSV therapeutics (Remichkova et al. 2008). Phospholipids have been proven to prevent enveloped virus entry into the host cell by arresting the fusion of viral and cell membranes. In a study, a phospholipid, namely 5-(perylen-3-yl)ethynyl-2-deoxy-uridine arrested fusion of vesicular stomatitis virus (Colpitts et al. 2013). Rhamnolipids mixture secreted by Pseudomonas gessardii isolated from the Antarctic region was tested on herpes viruses. It was found that rhamnolipids completely inactivated HSV-1 and HSV-2 at 6 μg/mL (Giugliano et al. 2021). The new antimicrobial compound pumilacidin isolated from B. pumilus inhibited HSV-1 effectively (Naruse et al. 1990).

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Table 2 Antiviral activity of biosurfactants produced by microorganisms Microbes producing biosurfactants B. subtilis

Biosurfactants Surfactin

Type of study In vitro

B. subtilis OKB 105

Surfactin

In vitro

C. bombicola

Sophorolipid

C. bombicola

B. subtilis fmbj

Ethyl 17-L[(2′-O-β-Dglucopyranosyl-β-Dglucopyranosyl)oxy]-cis-9octadecenoate Lipopeptide

In vitro (Patent) In vitro

C. bombicola

Sophorolipid

In vitro

PRV, PPV, NDV and IBDV HIV

Pseudomonas sp.

Rhamnolipid and Rhamnolipid with Alginate Ethyl 17-L[(2′-O-β-Dglucopyranosylβ-Dglucopyranosyl)oxy]-cis-9octadecenoate New Peptide P18

In vitro

HSV-1 & 2

In vitro

EBV

Gross and Shah (2007)

Influenza virus

Starosila et al. (2017)

Influenza virus

Porcine Epidemic Diarrhea Virus (PEDV) and Transmissible gastroenteritis virus (TGV) PEDV

Hamamoto et al. (2013); Khan (2017) Yuan et al. (2018)

C. bombicola

In vitro

Tolipocladium inflatum

Cyclosporin A

In vitro and In vivo In vitro

B. subtilis

Surfactin

In vitro

Bacillus licheniformis Streptomyces sp.

Biosurfactants

In vitro

Enramycin A & B, Daptomycin

In silico

B. subtilis

Antiviral activity Semliki forest virus (SFV), HSV-1 &-2, SIV, Suid herpes virus (SHV-1), Feline calicivirus, murine encephalomyocarditis virus (EMCV), VSV VSV, SHV-1, and SFV Herpes virus HIV

References Vollenbroich et al. (1997)

Kracht et al. (1999) Gross and Shah (2007) Gross et al. (2004)

Huang et al. (2006) Shah et al. (2005) Remichkova et al. (2008)

Peng et al. (2019) Xia et al. (2021) (continued)

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Table 2 (continued) Microbes producing biosurfactants Paenibacillus sp. Bacillus sp. Pseudomonas sp.

Commercially purchased Microbes producing biosurfactants are not evaluated in this study. Retrieved biosurfactants from online sources and used for docking study

4.2

Biosurfactants Octapeptin C Polymixin B Iturin A, Surfactin A &C Xantholysin, Viscosin, Ferrocin A, Amphisin, Orfamide A and Fengycin Rhamnolipids Tsushimycin, Daptomycin Surfactin, Bacillomycin Iturin, LPD12

Type of study In silico In silico In silico

Antiviral activity SARS-CoV-2, RNA-dependent RNA polymerase

References

In vitro

HSV-1, SARS-CoV-2

In silico

SARS-CoV-2

Jin et al. (2021) Chowdhury et al. (2021)

Hepatitis Virus

Sakr et al. (2021) characterized a glycolipoprotein secreted by Lactobacillus plantarum which was isolated from cheese for potential antiviral, and anticancer activities. They identified that the glycolipoprotein showed weak activity against the Hepatitis A virus and exhibited cytotoxicity to colon carcinoma cells. The phospholipid 5-(perylen-3-yl) ethynyl-arabino-uridine was studied against hepatitis C virus (HCV). It exhibited significant antiviral activity against HCV (Colpitts et al. 2013).

4.3

Human Immunodeficiency Virus (HIV)

In a study, sophorolipid and its derivatives produced by C. bombicola were analysed for anti-HIV activity. It was found that sophorolipid derivatives expressed anti-HIV activity at the concentration of 3 mg/mL, which inactivated HIV in