Biobased surfactants : synthesis, properties, and applications [Second edition] 9780128127056, 0128127058

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BIOBASED SURFACTANTS

BIOBASED SURFACTANTS Synthesis, Properties, and Applications SECOND EDITION Edited by

Douglas G. Hayes Daniel K.Y. Solaiman Richard D. Ashby

Academic Press and AOCS Press Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2019 AOCS Press. Published by Elsevier Inc. All rights reserved. Published in cooperation with American Oil Chemists’ Society www.aocs.org Director, Membership and Publications, Janet Brown No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-12-812705-6 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Charlotte Cockle Acquisition Editor: Nancy Maragioglio Editorial Project Manager: Michelle Fisher Production Project Manager: Vignesh Tamil Cover Designer: Matthew Limbert Typeset by SPi Global, India

Contributors Edgar Acosta  Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada

Rudolf Hausmann  Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany

Sampson Anankanbil  Department of Engineering, Faculty of Science and Technology, Aarhus University, Aarhus, Denmark

Douglas G. Hayes  Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, TN, United States

Richard D. Ashby  Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, Wyndmoor, PA, United States

Yongjin He  Department of Engineering, Faculty of Science and Technology, Aarhus University, Aarhus, Denmark; College of Life Science, Fujian Normal University, Fuzhou, China

Long Bai  Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Aalto, Finland

Marius Henkel Department of Bioprocess Engineering, Institute of Food Science and ­ Biotechnology, University of Hohenheim, ­ Stuttgart, Germany

Alexander Beck  Institute of Interfacial Engineering and Plasma Technology IGVP, University of Stuttgart, Stuttgart, Germany

Krutika Invally  Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH, United States

Neil W. Boaz  Eastman Chemical Company, Kingsport, TN, United States

François Jérome  Ecole Nationale Superieure d’Ingenieurs de Poitiers, Institut de Chimie des Milieux et Materiaux de Poitiers (IC2MP, UMR7285), University of Poitiers, Poitiers Cedex, France

Jiazhi Chen  Guangdong Provincial Key Laboratory of Industrial Surfactant, Guangdong Research Institute of Petrochemical and Fine Chemical Engineering, Guangzhou, P. R. China

Lu-Kwang Ju  Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH, United States

Stephanie K. Clendennen  Eastman Chemical Company, Kingsport, TN, United States Mª del Carmen Morán  Biochemistry and Physiology Department, Physiology Section, Pharmacy and Food Sciences, Barcelona University, Barcelona, Spain

Toshio Kakui  LION Corporation, Sumida-ku, Tokyo, Japan Jingbo Li  Department of Engineering, Faculty of Science and Technology, Aarhus University, Aarhus, Denmark; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States

Boris Estrine  Agro Industrie Recherches et Développements, Pomacle, France Mareen Geissler  Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany

Sofie Lodens  Faculty of Bioscience Engineering, Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Ghent University, Ghent, Belgium

Zheng Guo  Department of Engineering, Faculty of Science and Technology, Aarhus University, Aarhus, Denmark



Sinisa Marinkovic  Agro Industrie Recherches et Développements, Pomacle, France

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CONTRIBUTORS

Kambiz Morabbi Heravi  Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany Lourdes Pérez  IInstitute for Advanced Chemistry of Catalonia, IQAC-CSIC, Barcelona, Spain Aurora Pinazo  Institute for Advanced Chemistry of Catalonia, IQAC-CSIC, Barcelona, Spain Ramon Pons  Institute for Advanced Chemistry of Catalonia, IQAC-CSIC, Barcelona, Spain Sang-Hyun Pyo  Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden Sophie Roelants  Faculty of Bioscience Engineering, Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Ghent University; Bio Base Europe Pilot Plant, Ghent, Belgium Orlando J. Rojas  Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Aalto, Finland George A. Smith  Sasol Performance Chemicals North America LLC, Westlake, LA, United States Wim Soetaert  Faculty of Bioscience Engineering, Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Ghent University; Bio Base Europe Pilot Plant, Ghent, Belgium Daniel K.Y. Solaiman Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, Wyndmoor, PA, United States

Cosima Stubenrauch Institute of Physical Chemistry, University of Stuttgart, Stuttgart, Germany Sang-Jin Suh  Department of Biological Sciences, Auburn University, Auburn, AL, United States Suryavarshini Sundar  Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada Blaise Tardy  Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Aalto, Finland Norio Tobori  LION Specialty Chemicals Co., Ltd., Sumida-ku, Tokyo, Japan Lisa Van Renterghem  Faculty of Bioscience Engineering, Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Ghent University, Ghent, Belgium Nicole Werner  Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart, Germany Wenchao Xiang  Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Aalto, Finland Ran Ye  Roha USA, St. Louis, MO, United States Susanne Zibek  Institute of Interfacial Engineering and Plasma Technology IGVP, University of Stuttgart; Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB, Stuttgart, Germany

Introduction As we wrote in the introduction of the First Edition of Biobased Surfactants in 2008, “for the majority of the 20th century, petroleum was utilized as the main feedstock for transportation and other fuels, chemical intermediates, and many co-products. The Surfactants and Detergents industrial sector, like many others, relied heavily upon petroleum as its main feedstock.” When the First Edition was being prepared, a major driving force for the interest in biobased surfactants was the concern of the increasing price and decreasing availability of petroleum. However, after the publication of the First Edition, the price of petroleum has decreased greatly due to its increased production as a result of improved “fracking,” tertiary oil recovery, and other technological advances, applied particularly to recover residual amounts of oil from previously retired oil wells in the Southwest United States and from the sand shales of North Dakota and Alberta. Yet, the manufacture and utilization of biobased surfactants has continued to increase over the past 10 years, now reaching 24% of the surfactant market, with continued growth expected. The increase is likely due to increased concern for climate change by consumers and retailers as extreme climatic changes and events have been observed to an increasing extent in recent years. The increased interest and utilization was observed in Europe first, followed by North America, and now is expected to follow suit in Asia. Since the publication of the First Edition, the production of biobased surfactants has



become more efficient and economically and environmentally friendly, partly as a result of technological developments in biotechnology, catalyst development, and green chemistry. Some examples include the increased performance of microorganisms to produce surfactants utilizing lower-cost carbon sources (“biosurfactants”) and production of ­medium-chain fatty acids by algae through metabolic engineering and improved catalysts (e.g., the Grubbs catalyst employed for metathesis and the Shvo catalyst for conducting ­oxidation-reduction) to enhance process selectivity. In addition, several new and emerging biobased surfactants have been developed, for example, new amino acid surfactants designed for several different nonfood applications. The developments described above motivated us to prepare the Second Edition. A few of the chapters are updated versions of chapters written for the First Edition, but even these chapters contain new information. We added several new chapters on important topics not directly addressed in the First Edition, including methyl ethyl sulfonates, fatty acid ethoxylates, betaines, phospholipids, and lipopeptide biosurfactants. We are thankful for the chapter authors for their diligence in preparing 15 outstanding chapters. We deeply appreciate the involvement and support of the American Oil Chemists’ Society. The original concept of this book originated after the first symposium on Biobased Surfactants at the AOCS Annual Meeting in Quebec City in 2007. This symposium has been given in every year

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xii INTRODUCTION since this date. We thank the editorial staff of Elsevier for their patience and kind assistance in preparing this book. We hope you find this book useful for your own research and development in biobased s­urfactants

and that it will inspire you to serve as an advocate for the preparation of chemicals, materials, and other products from renewable resources using green manufacturing principles. Douglas G. Hayes Daniel K.Y. Solaiman Richard D. Ashby

C H A P T E R

1 Biobased Surfactants: Overview and Industrial State of the Art Douglas G. Hayes*, George A. Smith† ⁎

Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, TN, United States †Sasol Performance Chemicals North America LLC, Westlake, LA, United States

1.1  WHAT ARE BIOBASED SURFACTANTS? Surfactants, molecules that adhere to interfaces (e.g., water-oil, liquid-gas, and solid-liquid or -gas) and lower their surface energy, have numerous applications in our everyday lives, including foods, medicines, toiletries, cleaners, automotive fluids, paints and coatings, and processing aids. Their surface activity is enabled by their molecular structure, consisting of separated hydrophilic and lipophilic domains. As one adds surfactant to a two-phase system (e.g., water and oil), the concentration of surfactant adsorbed at the fluid-fluid interface increases, and concurrently, the surface energy (i.e., interfacial tension) decreases until the interface becomes saturated in surfactant, known as the critical micelle concentration (CMC). As surfactant concentration exceeds the CMC, the surface tension remains relatively constant, and the excess surfactant frequently forms self-assembly systems such as micelles. The interfacial tension for a liquid-gas system is commonly referred to as the “surface tension.” Surfactants can be categorized by their chemistry, particularly that of their polar moiety, or “head group”: cationic, anionic, amphoteric, or nonionic. The relative strength of hydrophilic and lipophilic moieties in a surfactant (known as hydrophilic-lipophilic balance, or HLB) determines the nature of their surface activity, whether they are able to dissolve water into oil (more lipophilic), oil into water (more hydrophilic), or nearly balanced in hydrophilicity and lipophilicity, allowing them to form bicontinuous and lamellar structures. Surfactants’ HLB values can be tuned by environmental factors such as temperature (which increases the polarity of ionic surfactants and the lipophilicity of alkyl ethoxylate nonionic surfactants) and salinity (which decreases the hydrophilicity of ionic surfactants via Debye shielding). The ideal surfactant is described as inducing a low surface or interfacial tension and possessing low Krafft point temperature; high solubility in water or oil; insensitivity of its surface activity to temperature, salinity, or other environmental factors; fast kinetics for their self-assembly; high biodegradability and biocompatibility; an excellent environmental profile; and a low

Biobased Surfactants https://doi.org/10.1016/B978-0-12-812705-6.00002-2

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Copyright © 2019 AOCS Press. Published by Elsevier Inc. All rights reserved.

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1.  Biobased Surfactants: Overview and Industrial State of the Art

cost-to-performance ratio (Scheibel, 2007). The Krafft point is a critical temperature below which surfactants form crystalline structures and above which surfactants can form micelles and related self-assembly systems. Although HLB continues to be commonly employed for assessment of surfactants and detergents in preparing formulations, more sophisticated, versatile, and universal models such as the hydrophilic-lipophilic difference (HLD) are increasingly employed (Chapter 15). Biobased surfactants are derived “… in whole or significant part of biological products or renewable domestic agricultural materials (including plant, animal, and marine materials) or forestry materials” (US Senate Committee on Agriculture Nutrition and Forestry, 2006), are frequently fatty acid-based, and are valuable products attracting increased interest for the reasons described below. Recently, the European Commission of Standardization (CEN) derived a classification of biobased surfactants based on their biobased content (i.e., percentage of carbon atoms derived from renewable resources, as described in more detail in Section 1.2.1): wholly biobased (>95%), majority biobased (50%–94%), minority biobased (5%–49%), and nonbiobased (100°C), including the formation of polymers that can adversely affect human health, aldehydes, and ketones (Chang, 1988). Second, the high temperatures require excessive energy usage, leading to increased production of the greenhouse gas CO2. Third, the use of heterogeneous catalysts, acid/bases, and/or toxic solvents yield waste products that can lead to environmental harm and also present safety problems for workers. Fourth, often, these reactions produce broad product distributions of desired and undesired products, moreover, lower selectivity, which can impair product performance and impact the products’ biocompatibility and biodegradability. Enzymes can potentially play an important role in the manufacture of many biobased surfactants, although they are not currently employed on an industrial scale for this purpose (Karmee, 2008; Hayes, 2012) (Table 1.5). The use of enzymes provides many advantages compared with chemical processing, particularly for the upgrading of process sustainability: lower energy use (due to the use of lower temperatures), lower amounts of waste products and by-products, the absence of toxic metal catalysts or acids/bases, and safer operating conditions (Cowan et  al., 2008). The major disadvantages are the relatively high costs for enzymes compared with chemical catalysts (although this effect is reduced when enzymes are reused, which is enabled by immobilization) and the lower reaction rates that accompany many enzymatic reactions. In addition, inhibitory agents must be removed; thus, the starting materials must be purified. For instance, fatty acyl-containing material must not contain phospholipids, aldehydes/ketones, peroxides, and other contaminants. But due to an anticipated increase of energy costs, the importance of sustainability increases (to be driven by government regulation and/or consumer demand). Also, the capabilities of enzymes and their production systems will increase due to improved technology in screening, mutagenesis, protein engineering, recombinant DNA technology, immobilization, bioreactor design, etc. Therefore, enzymatic bioprocessing is anticipated to become more cost-competitive, which may lead to its employment on a larger, industrial scale in the future. For most of the applications listed in Table 1.5, the main role of the enzyme is the covalent attachment of hydrophile and lipophile. The ester bond is the most readily achieved linkage that is formed using enzymes (particularly hydrolases, such as lipases). Other bonds that can be formed include amide, carbonate, and ether (Table 1.5) (Stjerndahl et al., 2018). Oxidation of fatty alcohols to long-chain aldehydes and ketones via horse or yeast alcohol dehydrogenase may be useful for the chemoenzymatic synthesis of surfactants with acetal or ketal linkages (Orlich et al., 2000). Of the enzymes listed in Table 1.5, lipases are the “workhorse.” The employment of lipases in nonaqueous media is an established art, with over 30 years of research serving as a foundation. Lipases are abundant and relatively inexpensive enzymes that require no cofactors and are easily immobilized. Lipases from several thermophilic organisms have been isolated, cloned, and mass-produced via recombinant DNA technology in common vectors such as Escherichia coli.

I.  INTRODUCTION, IMPORTANCE, AND RELEVANCE OF BIOBASED SURFACTANTS



1.2  Comparison of Sustainability for Biobased Versus Fossil Fuel-Derived Feedstocks

17

TABLE 1.5  Enzymes Used for Bioprocessing of Biobased Surfactants and the Reactions and Products They Catalyze Enzyme

Surfactant Type

Biocatalytic Role

References

Alcohol dehydrogenase

Surfactants with acetal or ketal linkages

Oxidation of fatty alcohol to longchain aldehyde or ketone

Orlich et al. (2000)

α-Amylase

Alkyl polyglucosides

Simultaneous hydrolysis of starch and conjugation with fatty alcohol

Larsson et al. (2005)

Glucosidase

Alkyl glucosides

Formation of ether linkage between monosaccharide and fatty alcohol

Hansson and Adlercreutz (2002), van Rantwijk et al. (1999)

Glucosyl transferases

Alkyl polyglucosides

Addition of monosaccharide units to Svensson et al. (2009a,b) an alkyl glucoside surfactant

Lipase

Mono- and diacylglycerol (MAG and DAG, respectively)

Partial hydrolysis of triacylglycerol; esterification or transesterification between glycerol and FFA or FAME, respectively

Freitas et al. (2010), Watanabe and Shimada (2009), Zeng et al. (2010)

Polyol-fatty acid esters

Formation of ester bonds between fatty acyl group and hydroxyl(s) of polyol, such as MAGs and saccharide-fatty acid esters

Watanabe and Shimada (2009), Chapter 10

Lysophospholipid

Hydrolysis of phospholipid

Han and Rhee (1998), Xu et al. (2008), Guo et al. (2005)

Amino acid surfactants

Formation of amide bond between ε-amine group of lysine and fatty acyl group

Gardossi et al. (1991), Montet et al. (1990), Soo et al. (2003)

Amino acid surfactants

Conjugation of ε-OH group of homoserine and fatty acyl group

Nagao and Kito (1989)

Amino acid surfactants

Ester bond formation between free Chapter 13 fatty acids and hydroxyls of arginine esterified to glycerol

Polyglycerol polyricinoleate

Oligomerization of ricinoleic acid and esterification of oligo(ricinoleic acid) and polyglycerol

Ortega-Requena et al. (2014a,b)

Polyol-fatty acid carbonates

Alkyl exchange between biobased diethyl carbonate and fatty alcohol + polyol

Banno et al. (2007, 2010), Matsumura (2002), Lee et al. (2010)

Surfactants with amide linkages

N-acylation of alkanolamines and dialkanolamines

Otero (2009)

Amino acid surfactants

Ester or amide bond formation between arginine’s α-carboxylic acyl group and fatty alcohols, glycerol (polyols), or fatty amines, respectively

Chapter 13

Papain (and lipase)

(Continued) I.  INTRODUCTION, IMPORTANCE, AND RELEVANCE OF BIOBASED SURFACTANTS

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1.  Biobased Surfactants: Overview and Industrial State of the Art

TABLE 1.5  Enzymes Used for Bioprocessing of Biobased Surfactants and the Reactions and Products They Catalyze—cont’d Enzyme

Surfactant Type

Biocatalytic Role

References

Phospholipase A1 and A2

Lysophospholipid

Hydrolysis of phospholipid to create Daimer and Kulozik lysophospholipid (2009), Xu et al. (2008), Guo et al. (2005)

Phospholipases A1, A2, and D (and lipase)

Phospholipids

Tailor-making of phospholipids with Xu et al. (2008), Guo specific structure et al. (2005), Hossen and Hernandez (2005)

Reproduced from Hayes, D.G., 2012. Bioprocessing approaches to synthesize biobased surfactants and detergents. In: Dunford, N.T. (Ed.), Bioprocessing and Bio-Based Product Development. Wiley-Blackwell, Ames, IA, pp. 243–266, with permission from John Wiley and Sons, and updated herein.

1.3  COMPARISON OF CURRENT AND FUTURE PRICE AND AVAILABILITY FOR BIOBASED VERSUS FOSSIL FUEL-DERIVED FEEDSTOCKS The main barrier to biobased starting materials is their higher cost compared with fossil fuel-derived feedstocks. Moreover, there are typically no significant differences in manufacturing costs between the two (US Department of Energy, 1999), or biobased surfactants may be slightly less expensive (McCoy, 2007b). When the first edition of this book was published, in 2009, the price difference between biobased and fossil fuel-derived resources was lower and was continually decreasing. However, due to improvements in fracking and tertiary oil recovery, the supply of fossil fuels has increased (e.g., natural gas from the northeastern US states; tar shale from the northern Great Plains US states and Alberta, Canada; and residual oil released from previously abandoned wells in North America), leading to their lower costs (Institute for Energy Research, 2014; Erbach, 2014). But environmental concerns exist with the use of fracking technology, particularly relating to contamination of groundwater (Jackson et  al., 2014; Clough, 2018; Hirsch et  al., 2017). In addition, the long-term global supply is expected to decrease, and price of petroleum increase, as will market volatility in the short term (The Balance, 2018). And fossil fuel supply depends upon stability in the sociopolitical arena, which is never ensured. Additional government incentive programs such as the USDA BioPreferred program will enhance the increased utilization of biobased products. Therefore, interest in biobased surfactants will increase in the years to come.

1.4  FEEDSTOCKS FOR BIOBASED SURFACTANTS 1.4.1  Integration Within an Oleochemical Biorefinery Fig.  1.6 depicts the possible process streams that could be leveraged for the production of biobased surfactants in an oleochemical biorefinery. The biorefinery concept entails the utilization of biobased feedstocks (e.g., lignocellulosic biomass, oilseed crops, and aquatic organisms) for the production of fuels, chemical intermediates, fine chemicals, and materials resulting from the proper fractionation of the feedstock, analogous to the fractionation of

I.  INTRODUCTION, IMPORTANCE, AND RELEVANCE OF BIOBASED SURFACTANTS



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1.4  Feedstocks for Biobased Surfactants

Nutraceuticals & food products

Sterols/minor components Proteins Meal Polysaccharides (starch) Oilseed crops

Soapstock Refined oil

Glucose (sugars)

Amino acids Ethanol (alcohols)

Alcoholamines Biofuels

Sorbitol Phospholipids FFA

Biosurfactants Lysolecithin Fatty amines Fatty alcohols

FAME / FAEE MAG/DAG

Biodiesel

Carbonates

Propanediols Polyglycerol

Glycerine

Diethyl carbonate

Chemicals & materials

Glyceric acid

FIG.  1.6  Production of biobased surfactants according to an oleochemical biorefinery model. Underlined items refer to major biorefinery outputs. Italicized items are biorefinery chemicals that can be utilized to prepare surfactants. Modified from Hayes, D.G., 2017. Fatty acids-based surfactants and their uses. In: Ahmad, M.U. (Ed.), Fatty Acids. AOCS Press, Urbana, IL, pp. 355–384, with permission from AOCS Press.

petroleum at a petrochemical refinery (e.g., by distillation) into short- and medium-chain alkanes for fuels, long-chain alkanes for lubrication, and aromatics for preparation of chemicals and polymeric materials (Hatti-Kaul et al., 2007; Hill, 2007; Johansson and Svensson, 2001). As described earlier, fatty acyl groups serve as the principal biobased surfactant feedstock. The fatty acyl group is typically derived from oilseeds in triacylglycerol (TAG) form but also can be derived from oleochemical coproducts such as free fatty acid (FFA) or phospholipids (e.g., in soap stock, a coproduct formed during degumming) obtained during the refining process. Fatty acyl groups used as lipophilic building blocks for surfactants are typically used in the form of FFA or FA esters, obtained via hydrolysis or alcoholysis of TAG, respectively. Particularly attractive as acyl donors are FA methyl esters (FAME) due to their use as biodiesel. Many fatty acid-based surfactants contain an ester bond to conjugate the hydrophilic and lipophilic compounds. Ester bonds allow for biodegradability and biocompatibility, which are useful properties for surfactants used in foods, cosmetics, personal care products, and pharmaceuticals. However, ester bonds are quite labile, which prevents their utility for many product sectors, such as laundry detergents. More stable bonds are ethers, amides, and carbonates. To enable formation of the latter bonds, fatty acyl groups can be reduced to fatty alcohols or fatty amines (Biermann et al., 2011; Egan, 1968; Giraldo et al., 2010). Longchain carbonates are prepared through conjugation of fatty alcohol and diethyl carbonate (Banno et al., 2007), a chemical prepared from catalytic oxidation of ethanol (Rudnick, 2006).

I.  INTRODUCTION, IMPORTANCE, AND RELEVANCE OF BIOBASED SURFACTANTS

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1.  Biobased Surfactants: Overview and Industrial State of the Art

Conversion of fatty acids into acid chlorides (Bauer, 1946; Busch et al., 2004) allows for additional reactions to occur. In addition, the hydrophilic moiety of the surfactant can also be derived from oilseed components, such as polysaccharides and proteins. A desirable feedstock for the hydrophile is glycerol, an inexpensive coproduct derived from biodiesel production. Glycerol can be used directly (e.g., producing monoacylglycerols, or MAG, or their acylated or ethoxylated form); converted into other glycols such as glyceric acid (Habe et  al., 2009, Fukuoka et  al., 2012), 1,2- and 1,3-propanediol, and glycerol carbonate; or polymerized into polyglycerol (producing a heterogeneous mixture of linear, branched, and cyclic oligomers differing in degree of polymerization (Wenk and Meyer, 2009; Barrault et  al., 2004)), to enhance the diversity of biobased surfactant products that can be prepared. Sugars are a common biorefinery feedstock useful for preparing surfactant hydrophiles, including their derivatives, such as sugar alcohols (e.g., sorbitol and sorbitan, the latter produced from dehydration of the former) (Liu et  al., 2010; Tang et al., 2004; Wen et al., 2004), furfuryl (De Jong and Marcotullio, 2010; Park et al., 2016) and levoglucosanyl (Lakshmanan and Hoelscher, 1970) derivatives, and glucaric acid (Anonymous, 2014). Amino acids (discussed later) (or ethanolamine and isopropyl­­amine, derived from serine and threonine, respectively (Scott et al., 2007)) and DNA (Leal et al., 2006) are also useful biorefinery streams that can be used as feedstocks for the hydro­phile. Alternatively, oleochemical feedstocks can be utilized as carbon energy sources for mi­ croorganisms that ­produce biosurfactants such as sophorolipids and rhamnolipids (Chapters  2–6). Although ethoxylate groups are generally derived from petrochemicals, these important groups contained in nonionic surfactants can potentially be derived from biobased ethylene, produced from bioethanol derived from sugarcane (Gielen et al., 2008). Minor components in plants can also serve as the major feedstock of surfactants. One category of such components includes polyphenolics, phenolics, or their derivatives such as tannic acid (Negm et al., 2012, 2013), vanillin (Sayed et al., 2012; Negm et al., 2015) sterols (Johansson and Svensson, 2001), and lignin (Schmidt et al., 2017; Holladay et al., 2007; Gupta and Washburn, 2014; Zhang et  al., 2018). Rosin, a common waxy coproduct from conifers (pines), served as the source of dehydroabietylamine, which was quaternized to produce a cationic surfactant (Pei et al., 2013; Qi et al., 2015). Surfactants have also been prepared from lignocellulosic and food wastes (based on humic acid) (Quadri et  al., 2008) and municipal solid wastes (Baxter et al., 2014). Diacids are also useful building blocks for biobased surfactants. For instance, succinic acid, which can be derived from fossil fuels or via fermentation, is a component of Aerosol-OT (sodium bis(2-ethylhexyl) sulfosuccinate). Hydroxylated diacids, such as l-malic and l-tartaric acids, were employed to prepare ethoxylated nonionic surfactants through use of fatty acid chlorides (Altenbach et al., 2010).

1.4.2  Fatty Acid-Based Sources From Seed Oils For nonfood applications, the optimal fatty acyl feedstock will contain 10–14 carbons and no double bonds, the latter to enhance oxidative stability. The composition of common high-lauric acid oils employed for biobased surfactant synthesis is given in Table 1.6. The most frequently used sources are palm kernel (Chempro Gujarat India, 2016), palm stearin (a palmitic acid-rich coproduct produced from palm oil (Sellami et al., 2012)), and coconut (Pham, 2016) oils. There are no currently available high-lauric acid oils produced in North America or

I.  INTRODUCTION, IMPORTANCE, AND RELEVANCE OF BIOBASED SURFACTANTS



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1.4  Feedstocks for Biobased Surfactants

TABLE 1.6  Fatty Acid Composition of Selected Feedstocks Used to Prepare Biobased Surfactants Fatty Acid

Palm Kernela

Coconutb

Cupheac

10:0

3–7

4.4

77–84

12:0

40–52

44.5

2–3

14:0

14–18

18.6

2–4

16:0

7–9

12.0

32–45

18:0

1–3

4.8

18:1

11–19

18:2

0.5–2

Palma

Jatrophad

Beef Tallowe

Palm Stearinf

0.10 0.5–2

1.0–1.5

1.1

13.0–16.0

24.0–28.0

55.9

2–7

6.0–8.0

20.0–24.0

3.7

11.0

38–52

39.0–41.3

38.0–43.5

32.4

2.2

5–11

37.0–38.0

2.0–4.0

6.7

a

Chempro Gujarat India (2016). b Pham (2016). c McKeon (2016b) (PSR23, an interspecific hybrid of C. viscossima and C. lanceolate). d Barros et al. (2015) (J. curcas). e Alm (2017). f Sellami et al. (2012). Reproduced from Hayes, D.G., 2017. Fatty acids-based surfactants and their uses. In: Ahmad, M.U. (Ed.), Fatty Acids. AOCS Press, Urbana, IL, pp. 355–384, with permission from AOCS Press.

Europe, although cuphea, which contains 77%–84% capric acid in its oil (Table 1.6), has been investigated as a potentially valuable new oilseed crop (McKeon, 2016b). Another potential route to preparing medium-chain fatty acids (or their alkyl esters) is olefin metathesis, a genre of reactions involving the cleavage and reformation of carbon-­ carbon double bonds. The conversion was enabled by the development of homogeneous transition metal carbine catalysts in recent years, particularly Grubbs and Schrock catalysts, which led to Nobel prizes in chemistry for the inventors in 2005 (Montero de Espinosa and Meier, 2012). For example, cross metathesis of oleic acid and ethylene (the latter of which can be biobased, i.e., derived from sugar cane) will produce 9-dodecenoic acid (Fig. 1.7) (Rybak et al., 2008). 10-Undecenoic acid is readily prepared from ricinoleic acid (Van der Steen and Stevens Christian, 2009). Lard and tallow are inexpensive sources of C16- and C18-rich saturates (Table 1.6). Hydroxy acid-rich oils, particularly castor and lesquerella oils (which contain ricinoleic (R-18:1-9c, -OH-12) acid and its C20 homologue, lesquerolic acid, as their prominent fatty acyl group, respectively), have several specific applications as surfactants (described later) (Chen, 2016; McKeon, 2016a,c). Similarly, epoxy fatty acids may serve similar applications as hydroxy acids and can occur naturally (e.g., vernolic acid, 18:1-9, –epoxy-12S,13R) from Vernonia galamensis oil (McKeon, 2016c) or via chemical epoxidation of TAG containing unsaturated FA (Tan and Chow, 2010). For food-related applications, high-oleic acid oils such as corn, olive, cottonseed, palm, or soybean oils are commonly used as sources of lipophilic building blocks for biobased surfactants. However, jatropha and soapnut (Sapindus) oils, derived from a plants native to India that can be cultivated inexpensively on marginal agricultural land, may be a viable replacement for the common high-oleic acid oils described above (Table  1.6). Jatropha oil has attracted interest since 2000 as an economically viable feedstock for biodiesel (Barros et al., 2015).

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1.  Biobased Surfactants: Overview and Industrial State of the Art

FIG. 1.7  Production of medium-chain fatty acids via cross metathesis between oleic acid and ethylene. Modified from Hayes, D.G., 2017. Fatty acids-based surfactants and their uses. In: Ahmad, M.U. (Ed.), Fatty Acids. AOCS Press, Urbana, IL, pp. 355–384, with permission from AOCS Press.

If these oils are to be used in nonfood applications, they need to be hydrogenated (e.g., using Ni catalysts) to improve their oxidative stability (Veldsink et al., 1997).

1.4.3  Fatty Acid-Based Sources From Algae A potentially valuable source of fats and oils is algae, plant-like organisms that contain chlorophyll, which converts sunlight and CO2 into carbohydrates and oil through photosynthesis. There are over 100,000 different species of algae that live in salt water, fresh water, and on land. It is estimated that algae produce 70%–80% of the oxygen, and without algae, life on earth would not be possible. Interest in oil produced by algae increased greatly in the last decades of the 20th century. For almost two decades, the US Department of Energy funded the Aquatic Species Program (ASP) to study the feasibility of producing biodiesel from high-lipid-content algae grown in open ponds utilizing the waste CO2 from coal-fired power plants (Sheeham et al., 1998). The ASP collected over 3000 strains of algae and assessed their ability to produce oil as a function of temperature, pH, salinity, and environmental stress. Researchers at ASP were the first to isolate the acetyl-CoA carboxylase (ACCase) enzyme that catalyzes the production of oil and demonstrated that the gene that expresses the enzyme could be controlled through genetic engineering. ASP also demonstrated the feasibility of large-scale algae production in open ponds, but even with the most favorable assumptions concerning biological productivity, the cost of biodiesel from algae was about two times higher than petroleum diesel fuel costs. We have seen renewed interest in algae oil production in recent years. Oil companies such as Exxon Mobile and Chevron have spent millions of dollars researching biofuel ­production from algae. A number of different start-up firms are working on commercial production of biofuels from algae. Algenol Biofuels (Southwest Florida, United States) grows algae in closed photobioreactors and reports production yields two to three times higher than open ponds. Originally focused on ethanol and biofuel production, the company has branched out to produce natural colorants, protein for animal feed and food supplements, spirulina for

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1.5  Biobased Surfactants

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dietary supplements and nutraceuticals, ingredients for personal care, and biofertilizers and biostimulants for agricultural applications. Sapphire Energy (San Diego, CA, United States) is another company originally interested in producing biofuels from algae. The company developed a technology platform that uses nonpotable water and nonarable land that captures CO2 in open ponds. The company produced “green crude” that resembles many of the properties found in fossil crude oil. Early this year, Sapphire was sold and now focuses on oils enriched in ω-3 fatty acids for dietary supplements and animal feed. Solazyme (South San Francisco, CA, United States) is another company focused on the use of algae to make biofuels and dietary supplements. The technological approach employed by Solazyme is based on a strain of algae that converts plant sugars into carbohydrates, proteins, and oil. Grown in the dark in large industrial fermenters, the algae are reported to produce >30% biomass with 80% oil content. Solazyme have leveraged their intellectual property relating to recombinant DNA expression in algae and bioprocessing of algae to prepare oils tailored in their fatty acyl composition (Franklin et al., 2013). Solazyme produces high-lauric, high-capric, and high-myristic acid oils for biobased surfactants and other ingredients used in cosmetics (AlgaPur). Sugarcane, an inexpensive renewable resource, is commonly used as a carbon source for the fermentative AlgaPūr product. Microalgae oils are produced with low carbon, water, and land use impact. Early this year, Solazyme declared bankruptcy and was sold to Corbion, who is focused on algae for dietary supplements and cosmetic applications.

1.5  BIOBASED SURFACTANTS 1.5.1  Anionic Surfactants Anionic surfactants are the most frequently used category of surfactants due to their employment in laundry and personal care products. Some of the oldest and most common surfactants are fatty acid soaps (dating back to 2800 BCE in ancient Babylonia (Willcox, 2000)), formed via saponification of FFA. Recently, nanostructured soft matter has been prepared from fatty acid salts that employ organic cations (Fameau et al., 2011, 2013; Fameau and Zemb, 2014). Methyl ester sulfonates (MES, Fig.  1.1) are perhaps the most widely used biobased surfactant, with its major application being in powder and liquid laundry detergents as a replacement of the fossil fuel-derived surfactants, particularly LAS. The synthesis, properties, and applications of MES are thoroughly described in Chapter 9. Sodium coco sulfate is a biobased homologue of the commonly employed surfactant sodium lauryl (dodecyl) sulfate (n-C12H25OSO3Na; SLS or SDS), with the acyl groups derived from coconut oil, palm kernel oil, or another high-lauric acid oil. Recently, the replacement of the sodium counterion of SLS with biobased choline ((CH3)2N+(CH2)2OH) has been shown to improve water solubility and lower the surfactant’s Krafft point temperature (Klein et al., 2013). Sodium laureth sulfate (sodium lauryl ether sulfate, n-C12H25(OCH2CH2)nOSO3Na, SLES) is a common surfactant in many personal care products and is a more effective foaming agent than SDS. Amide ether carboxylates, prepared from ethoxylation of the free OH end of fatty acid-monoethanolamine, followed by attachment of a COO-end group via sodium monochloro acetate, have good properties for dermatologic formulation: compatible with

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1.  Biobased Surfactants: Overview and Industrial State of the Art

skin, biodegradable, low irritability with eyes and skin, good water solubility tolerance to water hardness, and good foam formation (Tsushima, 1997). Other biobased ionic surfactants include sodium methyl cocoyl taurate (a foam booster produced from medium-chain fatty acids and taurine, i.e., 2-aminoethanesulfonic acid, a common metabolite found in bile) and disodium coco sulfosuccinates (Pletnev, 2006). A deficiency of LAS and SDS is their performance in hard water. Typically, chelating agents or “builders” are required to neutralize divalent cations such as Mg2+ and Ca2+. The most common chelating agents are ethylenediaminetetraacetic acid (EDTA) and phosphates, which have negative environmental impacts (Doll and Erhan, 2009). Their bioaccumulation in waterways leads to a temporary surge of algal and bacterial growth, followed by oxygen depletion due to the thick “slime” layer that forms at the water-air interface that blocks oxygen and light transport from the air. This results in the loss of life for fish and aquatic wildlife and a source of drinking water. Biobased chelators with low ecotoxicity are currently under development (Doll and Erhan, 2009). Biobased sulfonate surfactants utilizing furans have recently been developed that perform better than LAS and SDS: lower Krafft point temperature and CMC, improved foaming behavior, faster wetting kinetics, and greater micelle stability in hard water (Park et al., 2016). The surfactant is synthesized by first forming the ketone 2-alkanoylfuran from furan, which can be derived from hemicellulose (xylose) (Perez and Fraga, 2014), and fatty acid anhydrides via Friedel-Crafts acylation, followed by sulfonation of the latter’s furan moiety (Fig.  1.8). Alternatively, the carbonyl group of 2-alkanoylfuran can be reduced using H2 and catalyst to produce 2-alkylfuran, followed by sulfonation (Fig. 1.8).

FIG. 1.8  Synthesis of furan-based surfactants.

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1.5  Biobased Surfactants

25

1.5.2  Cationic Surfactants Cationic surfactants, the majority of which are quaternary ammonium compounds, are most frequently employed as fabric softeners, antistatic agents, rinse aids (e.g., in haircare products), corrosion inhibitors, particle dispersants, and emulsifiers (e.g., for asphalt). In addition, cationics possess high biological activity against microorganisms (e.g., bacteria) due to their attraction to the negatively charged biomembranes of prokaryotes. Esterquats, quaternary ammonium compounds containing cleavable ester bonds to conjugate fatty acyl groups to a polar group containing the quaternary ammonium head group, are the most commonly used biobased cationic surfactant. The esterquat shown in Fig.  1.1 is produced by transesterification of FAME (e.g., from animal fats or vegetable oils) with triethanolamine at a controlled stoichiometric ratio (e.g., 2:1 mol ratio to prepare the diesterquat shown in Fig. 1.1, with smaller and larger ratios producing mono- and triesterquats, respectively) at 250°C for a few hours in vacuo (to remove the methanol coproduct) and is then quaternized with methylethylsulfate 98% SL purity) standards of SL mixtures, which are then used to quantify the same SL mixture in fermentation through HPLC-ELSD analysis (van Renterghem et  al., 2018; Roelants et al., 2016). For this methodology to prove adequate, care must be given to the ratios of the SL congeners that should remain (quasi) identical between the “standards” and the fermentation-derived unknowns. Validation of the quantification can then be performed by purifying the SL product from that specific fermentation to high purity and quantifying back the fermentation samples. This method gives rise to good quantification of SLs in fermentation samples. In addition, qualitative analysis (HPLC-ELSD, UV, etc.) should always be complemented with mass spectroscopy (MS) and/or NMR data. Because of the complex nature of SLs (see above), retention times are often shared between SL congeners, which can generate confusion about application data when products are assumed to be “identical.” A third issue, related with the second, is the importance of downstream processing (DSP) or purification of SLs, which is often underestimated. Considering that purification processes can make up to 60%–80% of the total production cost, it is crucial to investigate this, especially in light of commercialization (Fleurackers, 2013). Logically, the required purity and thus purification effort are determined by the anticipated application. However, in the research phase, where the basic characteristics of the compounds and their application potential are evaluated, it is of utmost importance that highly pure (see above for correct definition of “pure” in this field) products are evaluated. Minor contaminants (such as residual h ­ ydrophobic ­substrate)

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can completely change the behavior of the “product” and thus falsely display promising application potential. Another hurdle, already touched upon above, is that SL mixtures are produced with low uniformity. Acidic and lactonic SLs display completely different behavior (Roelants et al., 2016), thereby providing a p ­ ossible explanation for the varying reports on “SL properties” in the literature, which may be due to a disparity in the (unreported) ratios of the congeners. Therefore, attention must be given to the product composition when performing application research. The generation of more uniform (besides pure) samples, for example, 100% lactonic or 100% acidic, already simplifies this issue, which can be enabled through process development and/or genetic engineering (Lodens et al., 2018; van Renterghem et al., 2018; Roelants et al., 2016). A last issue is that an overwhelming number of original research papers and book chapters about SL production exist. Unfortunately, because of this, it is impractical to make general conclusions. This is primarily because of the highly variable parameters (basic medium composition, fed hydrophilic and/or hydrophobic substrate(s), incubation time, inoculation percentage, and fermentation technique ((fed) batch, cyclic, (semi-)continuous, and high cell density)) used to produce SLs. The variation of multiple influencing parameters makes it very difficult/impossible to make a valid comparison between production conditions. Moreover, doubts can arise with a number of research papers dealing with presumed SL production of certain microorganisms, but this will be elaborately discussed in Section 3.3. In conclusion, when consulting the literature on production and applications of SLs, one must thoroughly consult the methodology section to see how quantification and purification were performed and if a qualitative analysis of the SL product was completed (i.e., information about the specific SL congeners is supplied). Editors of journals should demand the presentation of such data to avoid further generation of confusing reports in the literature.

3.3  SOPHOROLIPID-PRODUCING STRAINS Sophorolipids were first discovered in 1961 as metabolites from the yeast Torulopsis magnoliae (Gorin et  al., 1961). Since SLs are produced in an “environmentally friendly” manner from renewable resources and consumers and governments are increasingly interested in shifting toward a more biobased economy, interest in SLs has risen over the last 10–15 years (Vanholme et al., 2013) giving rise to an increasing number of research papers. Within the last decade, new SL producers have been identified, and genetic engineering has enabled scientists to steer the production process and to modify the molecules that are produced. In the following paragraphs and in Table 3.1, an overview of reported native SL producers is given along with the major structural variants that are synthesized by those organisms. The same is done in Table 3.2 for genetically modified strains. As was recently mentioned in the review of Claus and van Bogaert (2017), one should read many research papers dealing with SL production with a certain amount of skepticism. The strains and the produced molecules must be correctly identified/characterized with appropriate techniques and with accurate interpretations in order to validate the results. Moreover, care should be taken when SL compositions are compared between species cultured under different media conditions and using different extraction/purification methods for analysis as this can also drastically alter SL

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TABLE 3.1  Overview of native SL producers and produced SL variants Structures of produces SL DiAc A MAc A C18:1 C18:1

NAc A DiAc L C18:1 C18:1

MAc L NAc L C18:1 C18:1

Candida riodocensis

✓

✓

✓

✓

Candida stellata

✓

✓

✓

Candida kuoi

✓

✓

✓

Starmerella bombicola ✓

Candida apicola

✓

Candida batistae

✓

Candida floricola

✓

Cryptococcus VITGBN2

✓

Kurtzman (2012), Kurtzman et al. (2010), and Price et al. (2012)

✓

Sometimes also C18:0 and C16:1 or C16:1 hydroxy fatty acid chain ✓

✓

Kurtzman et al. (2010) Hydroxy fatty acid is mostly ω-linked. Also small amounts of C18:0 and C18:2 hydroxy fatty acid chains

Konishi et al. (2008)

Imura et al. (2010) and Konishi et al. (2016, 2017)

Mono- and diacetylated C22:0 sophorolipids with hydroxylation on C13 ✓

Kurtzman et al. (2010), Tulloch et al. (1967), and Tulloch and Spencer (1968)

✓

✓

Pseudohyphozyma bogoriensis

References

Kurtzman et al. (2010)

✓

Candida rugosa

Rhodotorula mucilaginosa

✓

Remarks

Kurtzman et al. (2010)

✓

✓

Other structures

Doubtful identification of the yeast species

Chandran and Das (2011)

Doubtful identification of the yeast species

Chandran and Das (2011)

Ribeiro et al. (2012), Shin et al. (2010), Solaiman et al. (2015a), and Tulloch et al. (1968) Basak et al. (2014)

3.  PRODUCTION AND APPLICATIONS OF SOPHOROLIPIDS

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Strain



Nonacetylated lactonic C18:0 SL and diacetylated C16:0 lactonic SL

Lachancea thermotolerans

Production of acidic Further structural and lactonic SLs, no analysis necessary length of hydroxy fatty acid tail reported

Mousavi et al. (2015)

Rhodotorula babjevae

Nonacetylated acidic C11:0 and C13:1 SL and nonacetylated lactonic C13:1, C15:3, C16:0, and C18:2 SL and diacetylated lactonic C18:0 SL

Doubtful if SLs are produced, as Refs. Garay et al. (2017)) and Cajka et al. (2016)) claim production of extracellular polyol esters of fatty acids (PEFA)

Cajka et al. (2016), Garay et al. (2017), and Sen et al. (2017)

Wickerhamiella domercqiae = S. bombicola

Li et al. (2016)

Wickerhamiella domercqiae

Claimed produced molecules doubtful. Molar mass is not correct

Poomtien et al. (2013)

C20:4 monoacetylated lactonic SL (MW m/z 668) but aren’t these Na adducts of C18:1 monoacetylated lactonic SL?

Doubtful identification of the yeast species and doubtful structure analysis

Chandran and Das (2012)

Wickerhamiella anomalus

Thaniyavarn et al. (2008) claim several types of acidic SL

More recent publication, Souza et al. (2017) says the sugar moiety is definitely not sophorose

Souza et al. (2017) and Thaniyavarn et al. (2008)

Also the C18:2 and C18:0 variant detected

No strain Yang et al., 2012 identification was done and no other reports of C. albicans producing SL, so very doubtful

Candida albicans

A, acidic SL; L, lactonic SL.

✓

71

Candida tropicalis

3.3  Sophorolipid-Producing Strains

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Cyberlindnera samutprakamensis

Starmerella bombicola strain

Produced SL Fed substrates or glycolipid

72

TABLE 3.2  Overview of engineered S. bombicola strains and the produced glycolipid/SL products Fermentation technique (scale)

Titer (g/L)

Volumetric productivity (g/L h)

Carbon Purification yield (g/g) degree (%)

Purification yield (%)

Reference

Glucose, 1-dodecanol

C12:0-based SLs (medium chain)

B (SF)

29

0.12

0.21

n.a.

n.a.

van Bogaert et al. (2011) and Develter et al. (2013)

∆mfe-2

20-HETE, glucose

20-HETE based SLs

B (SF)

19

0.11

0.16

n.a.

n.a.

van Bogaert et al. (2013b)

∆at

Rapeseed oil, glucose

Nonacetylated B (SF) acidic and bolaform SLs

5

0.01

0.03

n.a.

n.a.

Saerens et al. (2011a, b, c)

∆cyp52m1

Oleic acid, glucose

No SL production

B (SF)

n.a.

n.a.

n.a.

n.a.

n.a.

van Bogaert et al. (2013a)

oe sble

Petroselinic acid, glucose

Petroselinicbased acidic SLs

FB (3 L BR)

40

0.18

0.19

98%

73%

Delbeke et al. (2016a)

oe sble

Oleic acid, glucose

Acetylated lactonic SLs

FB (150 L BR)

199

0.9

0.70

97%

n.a.

Delbeke et al. (2016b) and Roelants et al. (2016)

∆sble

Rapeseed oil, glucose

Acetylated acidic SLs

FB (150 L BR)

138

0.83

n.a.

n.a.

85%

Baccile et al. (2017) and Roelants et al. (2016)

∆ugtB1

Oleic acid, glucose

Acetylated glucolipid

FB (7 L BR)

258

0.7

0.60

n.a.

n.a.

∆at∆sble

HOSO, glucose Oleic acid, glucose

Bolaform SLs

FB (150 L BR) CF (10 L BR)

63 120a

0.22 0.63a

0.19 n.a.

95% n.a.

65% n.a.

Maes (2017) (unpublished results) van Renterghem et al. (2018) and Roelants et al. (2018)

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∆mfe-2



Oleyl alcohol, glucose

Bolaform SSs

FB (150 L BR)

62

0.28

0.41

93%

64%

van Renterghem et al. (2018) and Maes (2017) (unpublished results)

∆fao1

Tetradecanol, glucose

Bolaform SSs and alkyl SSs

B (SF)

27

0.23

n.a.

n.a.

n.a.

Takahashi et al. (2016)

Random mutagenesisb

Rapeseed oil, glucose

Lactonic and acidic SLs (up to 85% higher than WT)

FB (5 L BR)

135

0.79

0.72

n.a.

n.a.

Li et al. (2012)

∆pxa1, ∆pxa2, ∆fox2

Lauric acid ester (C12) to ethyl stearate ester (C18)

Lactonic and acidic SLs (up to 360% higher than WT)

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

Ichihara et al. (2015)

a

Continuous fermentation with cell retention was successfully performed, but the steady state for constant productivity could only be maintained for 10 days. Random mutagenesis by low-energy ion-beam implantation. This strain was later reclassified to S. bombicola based on sequence analysis (Li et al., 2016). WT, wild type; B, batch; FB, fed-batch; SF, shake flask; and BR, bioreactor. Volumetric productivity (g/L h) was calculated dividing end titer (g/L) by incubation time (h); yield (g/g, %) was defined as g of SL production per g of used substrate (hydrophobic and hydrophilic carbon source). b

3.3  Sophorolipid-Producing Strains

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

∆at∆sble ∆fao1

73

74

3.  PRODUCTION AND APPLICATIONS OF SOPHOROLIPIDS

uniformity (e.g., for S. bombicola, citrate has a very clear effect on the ratio of lactonic-to-acidic SLs, and extraction with ethyl acetate will give rise to predominant extraction of laconic SLs) (Roelants et al., 2016).

3.3.1  Native SL Producing Strains 3.3.1.1  Candida riodocensis, Candida stellata, and Candida kuoi In 2010, Kurtzman et al. tested several strains from the Starmerella clade for SL production, including Candida stellata, C. riodocensis, and Candida sp Y-27208 (Kurtzman et al., 2010), later identified as C. kuoi (Kurtzman, 2012). Candida stellata is commonly isolated from grape must and can be used in a cofermentation with Saccharomyces cerevisiae for wine production (Soden et al., 2000). Candida riodocensis was isolated from pollen-nectar provisions, larvae, and fecal pellet of Megachile sp. bees (Pimentel et al., 2005), whereas C. kuoi was isolated from concentrated grape juice in Cape Province, South Africa (Kurtzman, 2012). These three strains produce very little lactonized SL as compared with S. bombicola and C. apicola. The major SL produced from these three species is the diacetylated free acidic C18:1 congener along with smaller amounts of mono- and nonacetylated acidic C18:1 SL. Candida riodocensis and C. kuoi also produce monoacetylated lactonic ω-C18:1 SLs (Kurtzman et al., 2010; Kurtzman, 2012; Price et al., 2012). For C. stellata, it was not specified whether the hydroxy fatty acid was ω or ω-1 linked to the sophorose molecule. Candida kuoi also produces polymeric forms of SLs as identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectroscopy (MALDI-TOF-MS) (Fig. 3.2; Price et al., 2012). However, it is unlikely that this strain is the only one producing these polymeric SL forms, as most of the time MS scanning to high m/z ratios is not considered (molecular masses higher than 1000 g/mol) or extraction methods are not fitted for these hydrophilic compounds. Indeed, S. bombicola also produces so-called disophorolipids (van Bogaert et  al., 2016) and disophorosides (van Renterghem et al., 2018; Takahashi et al., 2016).

RO HO HO RO HO HO

RO O HO HO RO O O HO HO OH

O O

O

O

OH

RO O HO HO RO O O HO HO OH

O

HO HO RO O O O HO OH

O HO

OH

O OH

HOOC

O

RO O HO HO O RO O O HO OH

O

O

O=C

O=C

O

HO HO

RO

O

O

O=C

RO O HO HO RO O O O HO OH

O=C

RO

O

O=C

O=C

RO

RO O HO HO RO O O HO HO OH

O RO O HO HO RO O O O HO OH

OH

RO O HO HO RO O O O HO OH

O

HOOC

FIG. 3.2  Di- and trimeric SL forms as described by Price et al. (2012), which were found for C. kuoi by MALDITOF-MS. Seventeen polymeric sophorolipids were detected by MALDI-TOF-MS. From left to right, the structures shown are monoacyl disophorolipid, diacyl disophorolipid, diacyl trisophorolipid, and triacyl trisophorolipid. The R groups represent possible acetylation (R = H or COCH3).

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3.3.1.2  Starmerella bombicola Starmerella bombicola (initially referred as T. bombicola and C. bombicola) is the best-known SL producer and was first isolated from the honey of a bumblebee in 1970 (Spencer et al., 1970). At that time, it was only the third discovered SL-producing species, but it has become the yeast strain that is the most intensively studied nowadays, due to its high natural production titers (>200 g/L) (van Bogaert et al., 2015; Davila et al., 1997; Gao et al., 2013; Zhang et al., 2018) and productivities up to 3.7 g/L h (Gao et al., 2013). Many yeasts of the Starmerella clade are associated with bees or with substrates that are often visited by bees. It is thus believed that there is a mutually beneficial relationship between bumblebees and various species from the Starmerella clade (Rosa et al., 2003). Survival in this habitat with these high sugar concentrations classifies S. bombicola as an osmotolerant yeast. Standard fermentations and growth experiments are therefore often started with a glucose level of 100 g/L or more. The SL production pathway has been elucidated (Fig. 3.3), by systematically knocking out the genes responsible for SL production in the SL gene cluster (van Bogaert et  al., 2013a; Ciesielska et al., 2014; Saerens et al., 2010, 2011a, b, c). Typically, SLs produced by not only wild-type S. bombicola but also by other strains of the Starmerella clade contain a hydrophobic hydroxy fatty acid moiety that is ω-1 hydroxy-stearate (C18:0) or ω-1 hydroxyoleate (C18:1), but intermediates with 16 C atoms (C16:0 and C16:1) have also been detected (Tulloch et  al., 1962; Ashby et al., 2008). The specific chemical structures associated with SLs are driven by the specificity of the CYP52M1 cytochrome P450 monooxygenase enzyme, responsible for the first step of the production pathway (van Bogaert et  al., 2013a). The SLs may contain acetate groups at the 6′ and/or 6″ position (Tulloch et al., 1967), and a macrocyclic lactone structure may form between the 4″-hydroxyl group of the terminal glucose molecule and the carboxyl group of the hydroxy fatty acid moiety (Tulloch et  al., 1967). Although these SLs mainly consist of a C18 hydroxy fatty acid, (sub)terminally linked to a sophorose molecule, a clear structural diversity for the produced SLs was demonstrated by Kurtzman et al. for the Starmerella clade (Kurtzman et  al., 2010). In contrast to other species within the Starmerella clade, S. bombicola produces a major diacetylated lactone form plus a minor component of the free acid form, called lactonic and acidic SLs, respectively. Many different hydrophobic substrates have been reported for SL production using S. bombicola: alkanes, fatty acids, fatty esters, and so on (van Bogaert et al., 2015; van Bogaert and Soetaert, 2011; Lang et al., 2000; Paulino et al., 2016). The C16–C18 chain lengths have been primarily assessed, as this is the preference of the CYP52M1 enzyme (Ashby et al., 2008; Tulloch et al., 1962). Additionally, shorter or special unconventional substrates have been investigated. For example, petroselinic acid-based SLs (C18:1(n-12)) (Delbeke et al., 2016a), coconut (C12:0)-based or meadowfoam seed oil (mainly C20:1(n-5))-based SLs (van Bogaert et al., 2010), and eicosapentaenoic acid (EPA, C20:5(n-3))-based or docosahexaenoic acid (DHA, C22:6(n−3))-based SLs (Li et al., 2013) have all been reported in the literature. Medium-chain (C8–C14) SL production by a ∆mfe2 S. bombicola strain (suppressing β-oxidation and thereby improving SL production) was patented by Ecover, in cooperation with InBio.be (Develter et  al., 2013), and reported by van Bogaert et  al. (2011) when feeding with 1-­dodecanol. Later, arachidonic acid (ARA, C20:4 (n-6))-based SLs were successfully produced using the same modified S. bombicola strain (van Bogaert et  al., 2013b). Another strategy to bypass the CYP52M1 enzyme is to feed the wild-type S. bombicola with previously hydroxylated

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FIG. 3.3  (A) Illustration of chromosome II of S. bombicola containing the full SL biosynthetic gene cluster (±11 kb) and the gene responsible for lactonization (sble) at the other side of the same chromosome. (B) The full SL biosynthetic pathway consisting of (1) hydroxylation of a fatty acid by a CYP52M1 monooxygenase; (2) glucosylation of the hydroxy fatty acid by the first glucosyltransferase UGTA1; and (3) second glucosylation step of the formed glucolipid by a second glucosyltransferase UGTB1 giving rise to an acidic SL, which can be (4) acetylated by the action of an acetyltransferase AT. The different SLs are transported into the extracellular space by a multidrug transporter protein (MDR) (5). Lactonization (6) occurs extracellularly as the responsible enzyme sble is secreted. Adapted from Roelants, S.L.K.W., van Renterghem, L., Maes, K., Everaert, B., Redant, E., Vanlerberghe, B., Demaeseneire, S., Soetaert, W., 2018. Microbial biosurfactants: from lab to market. In: Banat, I.M., Thavasi, R. (Eds.), Microbial Biosurfactants and Their Environmental and Industrial Applications. CRC Press/Taylor & Francis, Boca Raton, Chapter 14. II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS



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substrates, like medium-chain alcohols or diols (1- or 2-dodecanol; 1,12-dodecanediol; and 1-tetradecanol) (van Bogaert et al., 2011; Brakemeier et al., 1995, 1998; Dengle Pulate et al., 2012). Even though the SL titers from feeding with these hydroxylated substrates are quite low, some novel glycolipid production was detected, and the SL products have been shown to possess very promising antimicrobial potential (Dengle Pulate et  al., 2012). Modified S. bombicola strains have been created to eliminate alcohol oxidation (van Renterghem et al., 2018; Takahashi et al., 2016), producing increasing amounts of these promising glycolipids (see Table 3.2 for more information). 3.3.1.3  Candida apicola The very first observation of SL production was accomplished by Gorin et al. (1961) in the ascomycetous yeast C. apicola (formerly known as T. magnoliae and T. apicola) (Tulloch and Spencer, 1968), which was originally isolated from sow thistle petals. This strain produces the most heterogeneous SL mixture, mainly consisting of lactonic SLs of which the mono- and nonacetylated forms are abundant, in addition to minor amounts of the free acidic forms (Kurtzman et al., 2010). Like in S. bombicola, the (C18:1) hydroxy fatty acid group is mainly ω-1 linked to the sophorose head group (Price et al., 2012). 3.3.1.4  Candida batistae Candida batistae was first described in 1999, when isolated from larval provisions, larvae, and pupae of the solitary bees Diadasina distincta and Ptilothrix plumata (Apidae) in Minas Gerais, Brazil (Rosa et al., 1999). The SLs produced by C. batistae consist of 75% ω-hydroxy fatty acids (mostly C18:1 and to a very small extent some C18:0 and C18:2), which is different from SLs produced by S. bombicola consisting of 65% (ω-1)-hydroxy fatty acids (Konishi et al., 2008). Also, C. batistae typically produces (more than 60%) acidic SLs, in contrast to S. bombicola that produces more than 65% lactonic SLs. It can thus be concluded that C. batistae primarily produces C18:1 (ω-linked) diacetylated acidic SLs (Konishi et al., 2008). 3.3.1.5  Candida floricola The first description of Candida floricola dates from 1987 (Tokuoka et al., 1987), when it was isolated from dandelion and azalea flowers. In 2010, Imura et al. described its ability to produce SLs (Imura et al., 2010). These SLs are preferentially diacetylated acidic SLs. This was confirmed in recent papers by Konishi et al. (2016, 2017), as new isolates from the C. floricola clade primarily produce ω-1 C18:1 diacetylated acidic SLs at a concentration of approximately 20 g/L when glucose and olive oil were simultaneously fed. Even though this organism could be interesting due to its specific production of acidic SLs, further strain and medium optimization are necessary to improve the end titers. Otherwise, the strains are not really relevant, considering a GMO SL-producing organism already exists that solely and very efficiently produces acidic SLs in high end titers (>100 g/L) (Roelants et al., 2016). However, non-GMObased production will be beneficial for a range of markets and applications. 3.3.1.6  Candida rugosa and Rhodotorula mucilaginosa Candida rugosa and Rhodotorula mucilaginosa, isolated from hydrocarbon contaminated sites, were shown to produce biosurfactants in the presence of 2% (v/v) diesel as sole source of carbon and energy (Chandran and Das, 2011). They claimed that R. mucilaginosa produces diacetylated acidic C18:1 SLs and that C. rugosa produces monoacetylated lactonic C18:1 SLs. II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

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It was not specified whether these hydroxy fatty acid chains are coupled via the ω or the ω-1 position. Unfortunately, due to some inconsistencies in the data, these proposed structures should be reconsidered, as the molar masses of m/z 728 and m/z 668 do not correspond to C18:1 SLs, but to the rather rare C20:4 acidic and lactonic SLs, respectively. It is possible that sodium adducts of the former compounds (C18:1 congeners) are detected, which would indeed give rise to these molar masses (Ribeiro et al., 2013), but this is nowhere stated. In addition, the identification of these two yeasts was done using a Vitek yeast card reader, which is not particularly accurate, so it could be that they were actually dealing with different yeasts than the ones mentioned in this particular report. 3.3.1.7  Pseudohyphozyma bogoriensis In 1968, Tulloch et al. first described the production of 13-[(2'-0-13-D-glucopyranosyl-8-nglucopyranosy1)-oxy] docosanoic acid 6′,6″-diacetate by C. bogoriensis (Tulloch et al., 1968). Later, its name was altered to R. bogoriensis (Nuñez et al., 2004), and recently, the name was again changed to Pseudohyphozyma bogoriensis (Wang et al., 2015). Candida bogoriensis was first isolated from the leaf surface of the Randia malleifera shrub in Indonesia. The SLs produced by P. bogoriensis contain 13-hydroxydocosanoic acid (13-OH-C22) as the lipid moiety (Solaiman et  al., 2015a) and are also called “branched C22 SLs” (Fig.  3.4). This is unique among the other SL-producing species, as they primarily produce SLs containing 16 or 18 carbon atoms (Shin et al., 2010). Ribeiro et al. (2012) discovered that the degree of acetylation depends on the length of the fermentation and the available carbon and nitrogen sources. Solaiman et al. (2015a) confirmed this and correlated it to glucose consumption; the less glucose left in the medium, the less acetylation and vice versa. This confirmed the observation of Esders and Light (1972) who observed more monoacetylated and less diacetylated C22 SLs in older cultures. Esders and Light also observed a decrease in acetyltransferase activity when the cells were in the stationary phase. Thus, it could be speculated that the decrease of diacetylated C22 SLs was probably due to the fact that the acetyltransferase was no longer active and/or catabolism of the SLs as also described for S. bombicola (Roelants, unpublished data). 3.3.1.8  Cryptococcus VITGBN2 In 2014, Basak et al. found that Cryptococcus sp. VITGBN2 is a potent producer of SLs in mineral salt media containing vegetable oil as additional carbon source (Basak et al., 2014). O O HO O HO

O

HO

HO

O O

O

O

OH

HO O

FIG. 3.4  The unique C22:0 diacetylated SLs produced by P. bogoriensis, also called branched C22 SLs, when it is fed with 13-hydroxydocosanoic acid. II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS



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The chemical structure of the purified biosurfactant was identified as diacetylated acidic C18:1 SL. It was not mentioned whether the C18:1 chain is ω or ω-1 linked to the sophorose head. 3.3.1.9  Cyberlindnera samutprakarnensis The production of SLs by Cyberlindnera samutprakarnensis was first described in 2013 (Poomtien et al., 2013). The strain was isolated from cosmetic industrial waste in Thailand. In the presence of 2% glucose and 2% palm oil, it produced 1.89 g/L SL. In that paper, the authors claimed to produce nonacetylated lactonic ω-1 C18:0 SL and diacetylated lactonic ω-1 C16:0 SL due to molecular weight peaks in the MALDI-TOF-MS analyses of m/z 574 and m/z 662. However, a molar mass peak of m/z 574 does not correspond to a nonacetylated lactonic C18:0 SL, so what is actually produced besides diacetylated C16:0 lactonic SL should be further investigated. A suggestion could be that these are C16:2 nonacetylated lactonic SLs. Furthermore, they claimed to produce a compound with a molar mass of m/z 662, but the chromatogram shows a peak at m/z 664 instead of m/z 662. The peak at m/z 664 might correspond to C18:1 monoacetylated acidic SL instead of diacetylated lactonic C16:0 SL. 3.3.1.10  Lachancea thermotolerans Recently, a new SL-producing microorganism was isolated from the gut of honeybee, Apis mellifera (Mousavi et al., 2015). In that paper, they reported the production of both acidic and lactonic SLs, but their conclusions were based solely on FTIR analysis. It was claimed that the production went up to 24 g/L when adding 100 g/L glucose and 100 g/L canola oil as substrates; however, more detailed analyses are vital in order to unequivocally confirm the identity of these SLs. 3.3.1.11  Rhodotorula babjevae Sen et  al. (2017) found proof of SL production in a novel yeast strain, R. babjevae YS3, which was isolated from an agricultural field in Assam, Northeast India. SL titers of 19.0 g/L were observed, which might be further increased after optimization of the growth parameters. The product was characterized as a heterogeneous SL product containing both lactonic and acidic congeners after analysis through TLC, Fourier-transform infrared spectroscopy (FTIR), and liquid chromatography-mass spectroscopy (LC-MS). The produced mixture was claimed to contain nonacetylated acidic C11:0 and C13:1 SL, as well as nonacetylated lactonic C13:1, C15:3, C16:0, and C18:2 SL and diacetylated lactonic C18:0 SL (Sen et al., 2017). However, these data should be treated critically, as another recent publication claimed production of extracellular polyol esters of fatty acids (PEFA) by R. babjevae (Garay et al., 2017), which was also claimed in an earlier publication by Cajka et al. (2016). In the latter publication, they claimed that the masses of the polar functions of the studied molecules gave indication of being sugar alcohols (polyols) as opposed to hexoses, which were observed in the SL control they used. It is thus doubtful that this species is an SL producer. 3.3.1.12  Wickerhamiella domercqiae In 2006, Chen et al. first reported a new SL-producing yeast with the name Wickerhamiella domercqiae, which was isolated from oil-containing waste water (Chen et  al., 2006a, b). II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

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Subsequently, Ma et al. (2011, 2012) described the influence of nitrogen (N) sources on the growth, production, and composition of SLs produced by W. domercqiae. More than 15 acidic SL molecules (nonacetylated C18:2, C18:1, and C18:0 acidic SL; monoacetylated C16:0, C16:1, C18:0, C18:1, and C18:2 acidic SL; and diacetylated C16:0, C16:1, C16:2, C18:1, and C18:2 acidic SL) and only 4 lactonic SL molecules (C18:1 and C18:2 mono- and diacetylated lactonic SL) were ­produced when ammonium sulfate was supplied as nitrogen source, whereas the use of organic N-sources promoted the formation of lactonic SLs. Later, the influence of metal ions on SL production was also studied: by adding Mg2+ ions, production of lactonic SLs was favored, whereas when Fe2+ was added, optimal acidic SL production was obtained (Chen et  al., 2014). This selective production was patented by Chen and Zhang (2014). In 2013, another patent was filed to produce SL using cottonseed molasses and cottonseed oil (Song, 2013). Several other papers described the effect of different factors on the SL production of this species (Li et  al., 2012, 2013; Liu et  al., 2016). However, in the beginning of 2016, the strain reported in the mentioned articles and patent (see below) was reclassified as S. bombicola based on sequence analysis, meaning all these results can be attributed to S. bombicola instead of W. domercqiae (Li et al., 2016). This again underlines the importance of thorough strain characterization. 3.3.1.13  Candida tropicalis Chandran and Das (2012) described SL production by C. tropicalis isolated from contaminated soil in India. It was claimed that this strain could produce SLs in the presence of diesel oil, making it an interesting and efficient degrader of diesel oil. However, the same remark should be made as for their study on C. rugosa and R. mucilaginosa (Section 3.3.1.6.) concerning the suspicious strain identification. Also, they claimed that the molar mass of m/z 668 that they discovered in their samples corresponded to C20:4 monoacetylated lactonic SL, which is doubtful, but similarly as our suggestion above for Section  3.3.1.6., this molar mass can correspond to the sodium adduct of lactonic C18:1 SL (Ribeiro et al., 2013). However, the SL titer and productivity only amount to 1 g/L and 0.003 g/L h, respectively, thus rendering this strain irrelevant for SL production at the industrial scale. 3.3.1.14  Wickerhamiella anomalus Wickerhamiella anomalus, formerly known as Pichia anomala PY1, was described to produce SLs in 2008 (Thaniyavarn et al., 2008). LC-MS analysis of the product revealed molar mass peaks that could be correlated to C20 and C18:1 SLs. However, in 2017, Souza et al. (2017) concluded that the molecules produced by this strain do not contain a sophorose moiety. The sugar moiety seemed to be coupled to one or three oleic acid molecules. It is thus advisable to reconsider the observations made by Thaniyavarn et al., as it is possible that these claimed molecules also do not contain a sophorose moiety and are similar to the ones described by Souza et al. 3.3.1.15  Candida albicans Candida albicans 0-13-1 was described for the first time as an SL-producing yeast in 2012 (Yang et al., 2012). Moreover, 108 g/L was produced. In this mixture, mostly, C18:1 diacetylated lactonic SLs were detected, although also the C18:2 and C18:0 variants were found. Later, the same research group published a new bioreactor design, enabling integrated SL production

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using C. albicans 0-13-1 (Zhang et al., 2018). However, as there are no additional reports of other research groups claiming SL production by C. albicans, it is likely that these authors are actually dealing with another strain as the identification of the producing strain was not mentioned in either of the two publications.

3.3.2  Modified SL Producing Strains Besides the abovementioned wild-type strains, genetic engineering of SL-producing strains has definitely made its entry. All currently available research papers on this subject are dealing with S. bombicola as the parental strain. This is mainly not only because it is one of the most efficient SL producers (see above) but also because it is quite challenging to enable efficient metabolic engineering of a nonconventional microorganism from scratch (Lodens et al., 2018). A lot of progress has been made in this field over the last ten years by the research group InBio.be in Ghent, Belgium, through the development of a molecular toolbox for this yeast and by Kao Corporation. An overview of modified strains and the produced SL and/or glycolipid variants is given in Table 3.2.

3.4  PRODUCTION OF SOPHOROLIPIDS SLs are secondary metabolites, so their production only initiates after a period of exponential growth of the yeast cells, when they attain their stationary phase and nitrogen and/or phosphorous sources are depleted (Albrecht et al., 1996; Alcon et al., 2004). For S. bombicola, almost all of the SL biosynthesis genes are present in a subtelomeric gene cluster (van Bogaert et al., 2013a), which is clearly upregulated in the stationary phase, indicating tight transcriptional regulation of gene expression (Ciesielska et al., 2013). Until today, the molecular basis of the glycolipid/SL biosynthesis has not yet been elucidated (Roelants et al., 2018), but similarity with other secondary metabolites is expected (Roelants et al., 2014). Most research papers on SL production deal with first-generation substrates such as glucose and vegetable oil, but an increasing number of research papers deal with second-generation or waste/ sidestream substrates. Both approaches will be described below together with the applied fermentation techniques.

3.4.1  First Generation Substrates There is an overwhelming number of papers and book chapters dealing with SL production using the wild-type S. bombicola, making it challenging to make general conclusions, especially since media and fermentation conditions vary between papers making a one-toone comparison impractical. A recent overview of the most efficient fermentation processing techniques can be consulted elsewhere (van Bogaert and Soetaert, 2011). For such “efficient” processes, typical yields between 30% and 70% and volumetric productivities between 1 and 2.5 g/L h are expected. One recent approach was based on high cell density that gave rise to a very promising volumetric productivity of 3.7 g/L h (Gao et al., 2013). Although such levels are relevant for industrial SL production, this also gave rise to increased foaming, which is a

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hurdle in industrial fermentation setups. Some important considerations for SL production with S. bombicola are summarized below: • Higher inoculation and/or higher cell density leads to improved titers, but only up to a certain maximum (Gao et al., 2013; Roelants et al., 2018). Gao et al. showed that by employing 15 units of YM broth (yeast extract, 3 g/L; malt extract, 3 g/L; peptone, 5 g/L; and dextrose, 10 g/L) and feeding continuously with glucose and rapeseed oil, a volumetric productivity of 3.8 g/L h could be obtained after 54 h of incubation in a 10 L bioreactor containing 80 g cell dry weight/L (Gao et al., 2013). • Sufficient aeration is a major factor for SL production (Guilmanov et al., 2002) and is a very important parameter to consider (Ribeiro et al., 2013). When changing the stirring and/or airflow, one can significantly influence SL composition. • If higher end titers are preferred, a combination of hydrophobic (such as oils, alkanes, fatty acids, and fatty esters) and hydrophilic substrates (such as glucose or sugar-rich molasses) should be used (van Bogaert et al., 2015). If no lipophilic substrate is added, S. bombicola must produce the necessary fatty acids for SL production de novo, which is a more time-consuming process in comparison with directly feeding with oils or fatty acids. • The hydrophobic substrate composition is crucial to tune the SL composition toward the desired applications (Paulino et al., 2016). However, one always must take into account that the specificity of the first step in SL production, the hydroxylation by the CYP52M1 monooxygenase, is decisive whether the substrate is implemented in SL production or into the β-oxidation pathway (thus metabolized to CO2). In general, for alkanes/fatty acid/fatty acid methyl esters, a preference for the oleic acid (C18:1) chain is observed. Therefore, high-oleic sunflower oil (HOSO) is a good option to feed (van Bogaert et al., 2015; van Renterghem et al., 2018), as it consists of 75% oleic acid. In comparison, rapeseed oil consists of only 65% oleic acid on average. The prices of the mentioned substrates vary considerably: HOSO €750/metric ton (MT), rapeseed oil €1600/MT, and pure oleic acid €2200/MT (Roelants et al., 2018). The “best” choice depends on the trade-off between the additional substrate cost and productivity increase. For bolaform SLs, it was shown that a more expensive substrate results in a decreased production cost by achieving higher SL productivities (Roelants et al., 2018). • Yeast extract (YE) is one of the most frequently used nitrogen sources, considering its richness in nitrogen, organics, metal ions, and vitamins. It has proved to be beneficial toward SL production, but care must be taken with the dosage. A compromise between biomass formation and SL production must be made. As not only mentioned by (Casas and García-Ochoa, 1999) but also noticed in-house (van Renterghem et al., 2019), lower concentrations of YE (1 g/L) seem to promote SL production, whereas too high YE (10 g/L) concentrations minimize SL production, and biomass is mainly produced. Additionally, the ratio of amino nitrogen over total nitrogen (AN/TN) could be an important parameter as well to optimize SL production (van Renterghem et al., 2019). • The importance of medium optimization cannot be understated. For example, bolaform SL productivity can be increased 10-fold by switching from corn steep liquor (CSL) and rapeseed oil to yeast extract (YE) and oleic acid (Roelants et al., 2018). To perform media optimization, an experimental design setup is key, in combination with high-throughput screening techniques allowing fast and parallel analysis using microtiter plates (MTPs)

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(Ribeiro et al., 2013). Papers employing a thorough factorial design (where multiple factors are simultaneously varied) and statistical analysis are increasingly reported (Daverey and Pakshirajan, 2010a; Jiménez-Peñalver et al., 2016; Minucelli et al., 2017). This can elucidate important factors in the production process, for example, that inoculum age (>2 days) can decrease SL production (Daverey and Pakshirajan, 2010a). A new type of mixture design (simple centroid design) has been suggested by Rispoli et al. (2010) to assess an impressive 16 parameters (various carbon, nitrogen, phosphorous, and lipid sources) in just 33 experiments. However, these authors defined the “standard SL production medium” as containing 10 g/L YE, therefore making YE the second-to-last important parameter in SL production. This is in contradiction with in-house experience (van Renterghem et al., 2019). It is also unclear what exactly the concentrations in the different performed experiments were, as they were not clearly stated. Thus, when employing factorial design, the baseline and level of each parameter of the experimental design must be chosen carefully through intensive literature review; otherwise, illogical results can be obtained. • Metal ions have proved to be important for SL production, more precisely for the SL production enzymes (Fig. 3.3 for S. bombicola). Unfortunately, when looking at these enzymes and their necessary cofactors, there is not much information available. However, one can identify some “suspects.” First, it is generally known that Fe2+ is necessary for P450 enzyme activity, so this metal ion can be crucial for the CYP52M1 enzyme to work in an efficient manner (Fig. 3.3). Mg2+ is presumably a cofactor for the glucosyltransferases UgtA1 and UgtB1, and it was also added in enzyme tests on S. bombicola cell lysates (added as MgCl2) (Saerens et al., 2011a, b, 2015). Similarly as reported for W. domercqiae (Chen et al., 2014), 20 mM Fe2+ or Zn2+ (both added as sulfate salts) optimized medium-chain SL production. However, an excess of metal ions decreased the SL production, as was previously demonstrated by Felse et al. (2007) when Ni2+ was used in excess, due to accumulation effects and negative impact on biomass formation. The influence of metal ions was also investigated for W. domercqiae (which is actually S. bombicola; see Section 3.3.1.12). When Cu2+ was added, a maximum titer of 149.9 g/L (0.89 g/L h) was obtained; by adding Mg2+ ions, production of lactonic SLs was favored, whereas Fe2+ enhanced acidic SL production (Chen et al., 2014). A patent application protecting this selective production was filed by Chen and Zhang (2014). Several fermentation techniques have been assessed, which will be further discussed below. 3.4.1.1  (Fed-)Batch Fermentation The most common reactor process for SL production is the two-stage fed-batch process where a cell growth stage is followed by a production stage whereby glucose and/or lipophilic substrates are fed in interrupted aliquots (Davila et al., 1994; van Renterghem et al., 2018; Roelants et  al., 2016; Solaiman et  al., 2007) or continuously (Davila et  al., 1997; Kim et al., 2009; Rau et al., 1996) to stimulate SL production. In contrast, in a batch fermentation, no additional substrate is provided during the stationary phase. In general, due to the high viscosity of the broth, the oxygen transfer rate becomes limiting, and the fermentation is stopped after 1 or 2 weeks. Numerous publications elaborate on not only increased SL production using wild-type S. bombicola (van Bogaert et  al., 2011; Davila et  al., 1992, 1997; Gao et  al., 2013; Rau et  al., 1996) (Table 3.3) but also engineered S. bombicola strains for the exclusive production of acidic,

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TABLE 3.3  Overview of important SL production processes over the last decade

Organism

Fed substrates

Fermentation scale

Titer (g/L)

Volumetric productivity (g/L h)

Carbon yield (g/g)

Reference

FED-BATCH FERMENTATION (FB) Starmerella bombicola WT

Various

SF—50 L BR

120–300

0.5–1.9

0.3–0.68

van Bogaert et al. (2015), Casas and García-Ochoa (1999), Kyung and Jung (1993), and Rau et al. (1996, 2001)

Starmerella bombicola WT

Rapeseed esters and glucose

4 L BR

317

2.1

0.65

Davila et al. (1997)

Starmerella bombicola WT

Glucose and 10 L BR rapeseed oil

>200

3.7

n.a.

Gao et al. (2013)a

Starmerella bombicola oe sble

Oleic acid, glucose

199

0.9

0.7

Roelants et al. (2016)

Starmerella bombicola WT

Single cell 3 L BR oil, rapeseed oil, glucose

422

0.80

0.84

Daniel et al. (1998a)

Pseudohyphozyma bogoriensis WT

Glucose

51

0.16

1.3

Solaiman et al. (2015a)

150 L BR

14 L BR

SEMICONTINUOUS FERMENTATION (SCF) Starmerella bombicola WT

Rapeseed ethyl ester and glucose

4 L BR

266 (cycle average)

1.38 (cycle average)

0.43 (cycle average)

Marchal et al. (1999)

Starmerella bombicola ∆ugtB1

Oleic acid and glucose

7 L BR

258

0.7

0.60

Maes (2017) (unpublished data)

90 (dilution rate 0.3 h−1)

n.a.

n.a.

Kim et al. (1997)

CONTINUOUS FERMENTATION (CF) Starmerella bombicola WT

Soybean oil and glucose

5 L BR

INTEGRATED FERMENTATION, IN SITU PRODUCT REMOVAL (IF, ISPR) Starmerella bombicola WT Candida albicans WTb

Sunflower oil and glucose Oleic acid and glucose

10 L BR

74

0.35

0.15

Kim et al. (1997)

5 L BR

477

1.59

0.60

Zhang et al. (2018)

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3.4  Production of Sophorolipids

TABLE 3.3  Overview of important SL production processes over the last decade—cont’d Fermentation scale

Titer (g/L)

Volumetric productivity (g/L h)

Carbon yield (g/g)

Organism

Fed substrates

Starmerella bombicola WT

Glucose, 3 L BR rapeseed oil

402

0.61

0.47

Dolman et al. (2017)

Starmerella bombicola ∆at∆sble

Oleic acid and glucose

120

0.63

n.a.

Maes (2017) (unpublished data)c

10 L BR

Reference

a

High cell density fermentation with up to 15 units of YM broth (yeast extract, 3.0 g/L; malt extract, 3.0 g/L; peptone, 5.0 g/L; and dextrose, 10.0 g/L). b It is highly questionable that the strain is indeed C. albicans and not mistaken for S. bombicola. c Steady state could only be maintained for 10 days. B, batch; FB, fed-batch; BR, bioreactor; SF, hake flask; WT, wild type. Volumetric productivity (g/L h) was calculated dividing end titer (g/L) by incubation time (h); yield (g/g, %) was defined as g of SL production per g of used substrate (hydrophobic and hydrophilic carbon source).

l­actonic, or bolaform SLs (Baccile et  al., 2017; Delbeke et  al., 2016c; van Renterghem et  al., 2018; Roelants et al., 2016) by applying fed-batch processes (Table 3.2). Feeding with fatty acid esters has proved to increase SL productivities compared with respective oils, as shown by (Davila et al., 1994, 1997). Indeed, when rapeseed fatty esters were used, SL titers amounted to 317 g/L, whereas when rapeseed oil was fed, 255 g/L was obtained in the same time frame. However, the fatty esters are generally more expensive compared with regular oils, as they are a derivatization product of the latter. More importantly, the release of ethanol or methanol when using these esterified substrates makes SL production more complicated and/or dangerous, so this is not preferred. As mentioned previously, Gao et al. (2013) developed a high cell density fermentation for increased SL production, whereby a 15-unit YM (yeast extract, 3.0 g/L; malt extract, 3.0 g/L; peptone, 5.0 g/L; and dextrose, 10.0 g/L) production medium was employed for optimal SL production with a cell density of 80 g/L (cell dry weight). Later, glucose and rapeseed oil were continuously added. A volumetric productivity of 3.7 g/L h was obtained, which is one of the highest ever reported for SL production. In the future, it would be worthwhile to assess whether this concentrated medium is an option to lower final production costs of SLs as a financial or cost evaluation had not been included. Additionally, the specific SL production (SL titer/total biomass) could also be compared with a regular (non-high cell density) fermentation to see if there is an effective improvement per amount of cell dry weight. A slightly different two-stage design for SL production was evaluated by Daniel et  al. (1998b) in a 100 L setup procedure: first, C. curvatus ATCC 20509 was grown on deproteinized whey concentrate to break down lactose; second, the single cell oil obtained from the first stage was autoclaved and used as growth and SL production medium for S. bombicola. SL production only amounted to 12 g/L (productivity of 0.06 g/L h), and the limiting factor was the amount of oil present as single cell oil in the second stage. By adding another extra 4 aliquots of 100 g/L of rapeseed oil in the second stage, the SL titer was increased to 422 g/L over 400 h (productivity of 0.8 g/L h) (Daniel et  al., 1999). However, the purification was crude and thus most likely an overestimation, as the SL amounts were obtained gravimetrically.

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3.  PRODUCTION AND APPLICATIONS OF SOPHOROLIPIDS

Fed-batch fermentations were also described for P. bogoriensis. Solaiman et al. (2015a) reported a successful scale-up to 14 L bioreactor scale (10 L working volume) where only glucose and no lipophilic carbon source was added (thus solely relying on de novo fatty acid synthesis of the organism for SL production). However, at bioreactor level, all glucose was depleted after 6 days. At that time, the main component was diacetylated C22 SLs (62%). Upon glucose starvation, incubation was prolonged to 13 days, and a complete metamorphosis of SL composition was observed as previously described. A final composition of monoacetylated C22 SLs (89.3%) and diacetylated C22 SLs (3.5%) was obtained. Thus, for more uniform production of C22 SLs, the glucose feeding strategy and the bioreactor termination times are crucial. This also required correct follow-up of SL composition by dedicated sampling and analysis. After crude purification, a titer of 51 g/L branched C22:0 SLs was obtained, corresponding to a volumetric productivity of 0.16 g/L h (Solaiman et al., 2015a). Several distinct disadvantages are associated with the above-described fed-batch process designs. In the first 24–48 h, limited SLs were produced, which negatively impacts the overall productivity of the process. Next to increased costs (cleaning and personnel), the downtime of the equipment is substantial as cleaning, and (lengthy) inoculum preparations are necessary for every batch (Dolman et al., 2017). 3.4.1.2  Semi-continuous Fermentation To solve the problem with the substantial downtime of the reactor equipment, cyclic fermentation was proposed by McCaffrey and Cooper (1995; no SL titers reported), and such a process was later patented by Marchal et al. (1999). This semicontinuous technique enables longer fermentation times, though several fed-batch cycles are executed without actually terminating the fermentation. Instead, once the optimal “cycle time” is reached, half of the broth is removed, fresh medium is resupplied into the bioreactor vessel, and the cycle is repeated. The repeated batch fermentation is only terminated once productivity starts to drop substantially. Another example where cyclic fermentation was applied for increasing glycolipid production was for the glucolipid-producing ∆ugtB1 S. bombicola strain (Baccile et al., 2016a, b; Saerens et al., 2011c) and recently also for a newly developed more efficient version of this strain (unpublished data). An important drawback of this method is that half of the produced biomass is lost in every cycle, which necessitates additional time and energy (from substrate) requirements to renew the biomass to the former level, before SL production can initiate again. In this method, the growth phase is relatively short as this technique actually enables the use of a very dense inoculum, but such a system is characterized by dynamic parameters and is thus difficult to (automatically) control/monitor. 3.4.1.3  Continuous and Integrated Fermentations (In Situ Product Removal) To eliminate the loss of cells throughout the process of cyclic fermentation and generate a steady-state system, continuous setups have been evaluated. As SLs are non-growth-­ associated secondary metabolites, this cannot occur through a classic chemostat setup. In a continuous setup for SL production, the yeast cells must be maintained in the bioreactor, while the product is constantly removed. Some older articles do report a full continuous setup in a chemostat fashion, where an optimized dilution rate of 0.03/h was employed to obtain the highest SL productivity of 1.75 g/L h (Fiehler et al., 1997; Kim et al., 1997). However, this low dilution rate mimics a batch fermentation. Moreover, no other reports have been II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS



3.4  Production of Sophorolipids

87

published since, and it is even mentioned that such setups for SL production do not work well (Marchal et al., 1999). As mentioned previously, full continuous systems require a simultaneous combination of cell recycling/immobilization and product removal/purification. This is called “integrated fermentation,” or “in situ product removal.” Fermentation and downstream processing do not necessarily need to occur in the same reactor nor at the same time. Next to the obvious downtime-reducing aspect of such setups, other important advantages are associated with such setups. Depending on the influence on productivity and the potential cost reduction of personnel, such processes can be associated with a decrease in the cost of goods. A positive influence on productivity can be expected due to the avoidance of product inhibition and/or the avoidance of the accumulation of toxic metabolites/medium components. Moreover, decomposition of the product is minimized, and DSP (mostly volume reduction) is simplified (Palme et al., 2010). Such effects lower capital expenses (CAPEX). However, operational expenses (OPEX) might be higher as a full continuous system typically requires more follow-up and is more sensitive to failure. Buffer tanks and parallel modules can solve this issue but represent higher CAPEX costs. Every situation has to be evaluated case by case for the selection of the best option. Palme et al. (2010) studied the effect of in situ cell separation by applying ultrasonic enhanced settling (UES) of S. bombicola. Ultrasonic radiation caused particle aggregation and improved cell settling. After two cycles (days), the separation procedure had virtually no effect on cell viability. For the optimal flow rate of 7.2 L/day, separation efficiency was 94.6%. Additionally, separation efficiency decreased with increasing hydrophobic substrate concentration, which should be dosed in small amounts. For the “optimized” setup, a settling vessel was coupled to the bioreactor, and UES was applied twice for 12 h. A volumetric productivity of 0.34 g/L h was obtained, but the yield of SL over substrate was only 0.15, which is relatively low. The technique seems promising, but UES is not applied continuously: easier DSP is only achieved for the SLs that were separated using the UES system, which was a small concentration (8 g/L) compared with the total SL concentration. Additionally, the effect of repeatedly sonicating the cells has not been investigated, but should be included in order to use it for SL production. In 2016, a novel bioreactor design based on gravimetric separation of SLs was developed and patented combining dual ventilation pipes and dual sieve plates for SL production using C. albicans (Jia et al., 2016; Zhang et al., 2018) (Fig. 3.5A). The sieve plates divide the tank into the typical cylindrical bioreactor area and a cone-shaped area at the bottom that functions as a separator. In both areas, a pipeline for oxygen supply is present. The separation process was activated after 70 h of incubation, when SLs began to accumulate at the bottom of the bioreactor. The purified SLs were collected in a two-stage separation process. More precisely, (genetically modified) soybean oil was added to the sediment layer to “extract” the SLs, and the remaining broth was recycled back to the reactor. However, three important issues remained in this system: first, it was unlikely that the producing strain was C. albicans but probably was, in fact, S. bombicola, as this is the only strain where such high titers have ever been reported. Second, the addition of soybean oil to the DSP procedure introduces another variable into the system and is expected to severely hinder the efficient purification of the SLs (although this was not reported). This hindrance is based on the similarity of the hydrophobic moieties between SLs and soybean oil, which complicates the separation of SLs (and other glycolipids) from their hydrophobic substrates (oils, free fatty acids (FFAs), alkanes, etc.). Combined with the fact that substrate fed in the fermentation should be used completely, especially to avoid II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

88

3.  PRODUCTION AND APPLICATIONS OF SOPHOROLIPIDS Sophorolipid depleted broth return Fermentation broth

(A)

Sophorolipid product

Sophorolipid depleted broth return Fermentation broth

Sophorolipid depleted broth return Sophorolipid product

Fermentation broth

Fi (0 ltra .2 tio µm n )

Sophorolipid enriched broth

(B)

(C) FIG. 3.5  Examples of integrated production and purification of SLs, employing S. bombicola. (A) Schematic diagram of the new bioreactor with dual ventilation pipe and double sieve plate (unit: mm) with detail of sieve (Jia et al., 2016). (B) Schematic setup of gravity separation setup in a separate gravity settling column. Solid black = SL phase. Broth is pumped from the bioreactor to the separator. The concentrated SL phase is removed on the bottom (B bottom) or top (B top) of the separator, depending on whether the SL-phase density is higher or lower than the fermentation broth. Next, cells and other media components are recirculated back to the bioreactor (Dolman et al., 2017). (C) Schematic representation of a full continuous setup for bolaform SL production and simultaneous purification (two-step filtration) (Roelants et al., 2018).

such problems with similar hydrophobicity, makes it an inadequate strategy, as it only makes the process costlier. Third, the SLs are quantified in a rough manner and are thus presumably overestimated; Zhang et al. report an end titer and productivity of 477 g/L and 1.59 g/L h, respectively (Zhang et al., 2018). Other examples of integrated fermentation techniques using gravimetric separation of SLs have been published (Guilmanov et al., 2002; Marchal et al., 1999). However, for industrial scale, gravity settling within the bioreactor vessel becomes impractical. Therefore, Dolman et al. (2017) proposed the use of a separate settling column, while the fermentation was running. Hereby, a reduction of 11% required fermenter volume was enabled, positively influencing CAPEX, and broth viscosity reduction enabled 34% less power for mixing. Finally, fermentation time was increased to 1023 h (±42 days), wherefrom 623 g of SLs could be extracted. Two different setups are possible depending on if the SL-phase density is lower or higher as compared with that of the fermentation broth (Fig. 3.5B). The authors state that the key to determining whether the SL layer ends up in the separator (top or bottom) is by controlling the glucose concentration above or below 50 g/L. To date, no integrated foam fractionation system for the purification of SLs has been reported, in contrast to rhamnolipids, surfactin, and hydrophobic proteins (Alonso and Martin, 2016; II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS



3.4  Production of Sophorolipids

89

Beuker et al., 2016; Winterburn et al., 2011). No foaming has been reported for exclusive lactonic SL production using an engineered S. bombicola Δsble overexpression strain (Roelants et  al., 2016), in contrast to strong foaming when only acetylated acidic SLs are produced using another engineered sble deletion strain S. bombicola ∆sble (Baccile et al., 2017; Ciesielska et al., 2016; Roelants et al., 2016). Considering the latter and that more novel glycolipids are produced with S. bombicola (and thus unknown foaming characteristics), this might be worthwhile to look at in the future. A last example is the full continuous setup of SL production with integrated fermentation (Fig. 3.5C) described by Roelants et al. (2018) for bolaform SL production, which had a positive influence on the productivity. In this setup, a cell retention system was applied to enable the separation by sedimentation of a crude (cell free) SL fraction from the culture broth, which was recycled back to the reactor to continue supporting the fermentative production of SL. “In-line” purification of the bolaform SLs can be accomplished by a two-step filtration process (van Renterghem et al., 2018) as shown in Fig. 3.5C. Within the first 10 days, productivity rose to 0.63 g/L h, which is 14 times higher compared with corn steep liquor (CSL) and rapeseed oil in a fed-batch process using the same S. bombicola ∆at ∆sble strain. However, after a successful period of 10 days, the steady state was lost, and productivity declined. 3.4.1.4  Solid-State Fermentation In the last decades, the concept of solid-state fermentation (SSF) has become increasingly popular (Thomas et al., 2013). In contrast to the “regular” submerged fermentation (SmF), the substrate is a solid. In SL fermentations, such solid substrates can be oil cakes derived from plant seeds after pressing. SL production by SSF holds one important advantage as compared with SmF: the problems associated with foaming can be avoided (Jiménez-Peñalver et al., 2016). However, bioreactor design for monitoring moisture and temperature and correct extraction protocols are more challenging. Similarly to SmF, care must be taken with the selective extraction of SLs. Starmerella bombicola naturally produces de novo fatty acids, so a hexane extraction should be performed after ethyl acetate to remove fatty acids and residual oil (when this was added as cosubstrate). Moreover, contaminants from the solid substrate can be extracted as well. More research (bioreactor design, medium optimization, and extraction protocols) is necessary in order to commercialize this fermentation technique for SL production in the future. This technique will be further discussed in the next section, as almost exclusively waste/sidestreams are employed for SSF processes.

3.4.2  Second Generation Substrates There are two main drivers for the employment of second-generation substrates and/or waste/sidestreams for SL production. First, the environmental impact associated with applying such streams is expected to be lower as compared with first-generation substrates. The European Union is set toward implementing legislation to push the model of circular bioeconomy instead of the traditional “cradle to grave” approach (Satpute et al., 2017). Another driving factor for this development is to achieve lower production costs of the biosurfactant, as raw materials account for 10%–30% of the overall cost (Mnif and Ghribi, 2016). Waste or sidestream substrates are indeed available in high quantities (Banat et  al., 2014a) and at a lower price. However, the associated costs with the use of these substrates, not associated with the use of first-generation substrates (e.g., lower efficiencies, Table 3.4), will generally result in higher as opposed to lower cost of goods for produced SLs. II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

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3.  PRODUCTION AND APPLICATIONS OF SOPHOROLIPIDS

TABLE 3.4  Overview of research reported over the last decade, using waste/sidestreams for SL production with S. bombicola in submerged (SmF) or solid-state fermentation (SSF)

Fed substrates

Fermentation technique (scale)

Incubation time (h)

Volumetric productivity (g/L h)

Carbon yield (g/g)

75

168

0.44

0.13

Solaiman et al. (2007)

Titer (g/L)

Reference

SUBMERGED FERMENTATION (SmF) Soy molasses, oleic acid

FB (12 L F)

Tallow fatty acid, glucose Coconut fatty acid, glucose

FB (SF)

120 40

240

0.50 0.17

0.42 0.15

Felse et al. (2007)

Deproteinized whey, glucose, oleic acid

B (3 L F)

34

192

0.18

0.17

Daverey and Pakshirajan (2010b)

Sugarcane molasses, soybean oil

B (5 L F)

60

192

0.31

0.6

Daverey and Pakshirajan (2010a)

Sugarcane molasses

B (5 L F)

23

120

0.19

0.10

Takahashi et al. (2011)

Jatropha oil, glycerol Karanja oil, glycerol Neem oila, glycerol

B (SF)

5 6 4

200

0.02 0.03 0.02

0.02 0.02 0.02

Bhangale et al. (2014)

Waste cooking oila, glucose

FB (2.5 L F)

56

240

0.23

0.40

Maddikeri et al. (2015)

Sweet sorghum bagassea, glucose, soybean oil Corn fiber hydrolysatea, glucose, soybean oil

B (SF)

85 16

240

0.35 0.07

n.a. n.a.

Samad et al. (2014)

Castor oil, glycerol

FB (5 L F)

40

192

0.21

0.11

Bajaj and Annapure (2015)

Corncob hydrolysatea, olive oil

B (1 L F)

49

96

0.51

0.51

Konishi et al. (2015)

Rice strawa, oleic acid

B (SF)

54

168

0.32

0.45

Liu et al. (2016)b

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3.4  Production of Sophorolipids

TABLE 3.4  Overview of research reported over the last decade, using waste/sidestreams for SL production with S. bombicola in submerged (SmF) or solid-state fermentation (SSF)—cont’d

Fed substrates

Fermentation technique (scale)

Titer (g/L)

Incubation time (h)

Volumetric productivity (g/L h)

Carbon yield (g/g)

Reference

Corn stover hydrolysatea, waste cooking oil

FB (3 L F)

52

168

0.31

0.05

Samad et al. (2017)

Rice bran, glucose, cottonseed oil

B (7 L F)

122

240

0.51

0.41

Haque et al. (2017)

Tapis oil, glucose Melita oil, glucose Ratawi oil, glucose

B (SF)

26 21 19

120

0.22 0.18 0.16

0.13 0.11 0.10

Shah et al. (2017)

Chicken fat, glucose

B (SF)

40

120

0.33

0.26

Minucelli et al. (2017)

Fermentation technique (scale)

Yield (g/g DM)

Incubation time (h)

Reference

Mango kernel fata, glucose, wheat bran powder (as support)

B (SF)

0.18

240

Parekh et al. (2012)

Sunflower oil cakea, soybean oil

B (n.a.)

0.40

384

Rashad et al. (2014b)

Sunflower oil cakea, motor oil

B (n.a.)

0.32

384

Rashad et al. (2014a)

Winterization oil cakea, sugar beet molasses

B (SF)

0.24

192

Jiménez-Peñalver et al. (2016)

Safflower oil cakea, soybean oil

B (n.a.)

0.48

336

Nooman et al. (2017)

SOLID-STATE FERMENTATION (SSF) Fed substrates

a

Pretreatment of the waste or sidestream was necessary before starting fermentation. This strain was later reclassified to S. bombicola ATCC 22214 (as mentioned in Section 3.3.1.12). B, batch; FB, fed-batch; F, fermenter; SF, shake flask. Volumetric productivity (g/L h) was calculated dividing end titer (g/L) by incubation time (h); yield (g/g, %) was defined as g of SL production per g of used substrate (hydrophobic and hydrophilic carbon source). b

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3.  PRODUCTION AND APPLICATIONS OF SOPHOROLIPIDS

Previous studies with S. bombicola include animal fat, waste (cooking/frying) oil, molasses, lignocellulosic biomass, and exotic oils (such as tapis, castor, or jatropha oil). Considering SSF processes for SL production using S. bombicola, several options were evaluated: mango kernel (Parekh et al., 2012), sunflower and safflower oil cakes (Nooman et al., 2017; Rashad et al., 2014a), and recently winterization oil cake (WOC) combined with sugar beet molasses (Jiménez-Peñalver et al., 2016). The latter discovered that intermittent (manual) mixing increased SL production by 31%, to a final yield of 0.235 g per g of dry matter after 10 days. Humidified air was continuously supplied to the reactor, in order to achieve a 0.30 L/kg/min aeration rate. In 2017, successful scale-up of SSF using S. bombicola at 40 L packed-bed bioreactor was reported. However, problems occurred with heat removal for scale-up to the 100 L intermittent mixed bioreactor level. Nevertheless, it was determined that oxygen consumption was an indirect way of monitoring the production of SLs during the scale-up to pilot plant, enabling online monitoring of SL production (Jiménez-Peñalver et al., 2016). Recently, Maddikeri et  al. (2015) proposed the use of ultrasound in a fed-batch fermentation using waste cooking oil in order to increase SL production using S. bombicola. Ultrasound pulses were given in the exponential growth phase, aiming to increase cell permeability, thereby enhancing substrate uptake. However, the difference in end titers with or without ultrasound was very small (50 vs 55 g/L after 240 h of incubation), suggesting that ultrasound does not improve SL production. Also, for some other SL-producing strains, the use of sidestreams was evaluated. Glycerol, an increasingly abundant waste material arising from biodiesel fuel production, was fed to C. floricola strains ZM1502, CBS7290, and NBRC10700. The strains were fed with 20% glycerol, and titers for these fermentations were below 4 g/L (only 20% of the production on glucose) after 1 week of cultivation (volumetric productivity of 20 based on Davis’ 1957 formula. Therefore, aside from HLB, the more commonly used parameters to gauge the suitability of a surfactant molecule for washing and cleaning applications are the critical micelle concentration (CMC) and the minimum surface tension (γmin) of water in the presence of the surfactant. Similar to the HLB values, the CMC and γmin of SL are also strongly governed by its structures and compositions, which in turn are influenced by its production and purification processes. In contrast to the HLB values, however, there are tensiometric procedures to conveniently determine the CMC and γmin values of an SL sample. Table 3.6 tabulates the CMC and γmin values of many different SL samples as determined under different solution conditions. It can be seen that the CMC and γmin values varied depending on the structural compositions of the biosurfactants. It is noteworthy, however, to point out that the γmin values of SLs listed in Table 3.6 only slightly varied. The vast majority of the γmin values clustered in a narrow range centered on mid-30’s mN/m despite the extensive structural change or modification of either the hydrophobic or the hydrophilic moiety of the SL molecules. It is interesting to note however that

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

Production Major components of sophorolipid

Fermentation

Isolation

Surfactant properties Minimum surface tension (γmin)

Reference

>200 mg/L

35 mN/m

Ashby et al. (2008)

35 mg/L

35 mN/m

• Fed-batch • Substrates: Glu and PA

SL-s (76% C18:0); 99% Lc

• Fed-batch • Substrates: Glu and SA

SL-l (29% C18:2; 35% C18:0); 98% Lc

• Fed-batch • Substrates: Glu an d LA

250 mg/L

36 mN/m

SL-o (94% C18:1); 100% Lc

• Fed-batch • Substrates: Glu and OA

140 mg/L

36 mN/m

Ashby et al. (2008) and Solaiman et al. (2017a)

SL-o (C18:1) + SL-s (C18:0); 99% Lc

• Fed-batch • Substrate: Glu + rapeseed oil;

45.1 ± 0.1 mg/L

33.9 ± 0.7 mN/m

Roelants et al. (2016)

112 ± 7 mg/L (mix acetylation)

38.2 ± 0.5 mN/m

245 ± 9 mg/L (nonacetylated)

40.9 ± 0.3 mN/m

Multistep ultrafiltration

n-Alkyl (C1–C6) esters of SL-o

Postharvest esterification of SL-o

3 (C6) to 40 μM (C1)a

34 (C6) to 39 mN/m (C1)a

SL-o, GL-o (C18:1); 100% Ac

• Fed-batch • Substrate: Glu + olive oil + oleic acid

160–170 μM (SLdiOAc, SL, GLOAc, and GL)

39.8–43.0 mN/m Imura et al. (2010) (SLdiOAc, SL, GLOAc, and GL)

• EtOAc extraction • Chromatography • Enzymatic reactions

Zhang et al. (2004)

3.  PRODUCTION AND APPLICATIONS OF SOPHOROLIPIDS

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

Critical micelle concentration (CMC)

SL-p (75% C16:0); 92% Lc

SL-o (C18:1) + SL-s (C18:0); 100% Ac

• Whole culture was lyophilized • EtOAc extraction • Purification by precipitation in hexane

98

TABLE 3.6  Selective surface tension activity measurements of sophorolipids

• Fed-batch • Substrate: Glu + SA

Various enzymatic reactions

• Whole culture was lyophilized • EtOAc extraction • Purification by precipitation in hexane

200 mg/L (SL-E2–12)b

31 mN/m (SL-E2–12) b

200 mg/L (GL-A2–12)

31 mN/m (GL-A2–12)

200 mg/L (SL-E2–12-di-3-OH-C10)b

27 mN/m (SL-E2–12-di-3-OH-C10)

150 mg/L (SLE2–12-mono/ di-17-OH-C18)b

50 mN/m (SLE2–12-mono/ di-17-OH-C18)

90 μM (SL-s OEt; ethyl ester of SL)

48 mN/m

Recke et al. (2013)

Zerkowski et al. (2006)

6 μM (pH 5.8) to 24 μM 37–42 mN/m (pH 9.1); OH-proline-SL (positive charge) 110 μM (pH 2.5) to 51 μM 43–49 mN/m (pH 5.8); N-propionylglutamic SL (negative charge) 14–17 μM (pH 2.5–9.1); 39–41 mN/m glutamic SL (zwitterion)

a

Calculated based on data in Zhang et al. (2004). Lipid moiety of all species was a 2-dodecanol (not a hydroxy fatty acid). SL-E2–12, 6′′-monoacetylated sophorose; GL-A2–12, glucose as the hydrophilic moiety; SL-E2–12-di-3-OH-C10, 6′,6′′-di(3-OH-C10)-esters of SL-E2–12; SL-E2–12-mono/di-17-OH-C18, 6′-mono (40%); and 6′,6′′-di(60%)-(17-OH-C18)-esters of SL-E2–12. (Note: More details in text.) Abbreviations: Ac, free acid form; EtOAc, ethyl acetate; GL, glucose lipid; Glu, glucose; LA, linoleic acid; Lc, lactone form; OA, oleic acid; OAc, acetyl group; PA, palmitic acid; SA, stearic acid; SL-o, SL containing C18:1; SL-p, SL containing C16; SL-s, SL containing C18. All measurements were conducted at room temperature, except for the ones by Zhang et al. (2004) (45°C).

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SL-s (C18:0); 100% ester/ Ac; variously charged amino acid attached to the sophorose head group

• Shake flask • Substrate: Glu + 2-C12H25OH



SL-alcohol (2 OH-C12)b

b

99

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the two ends of the γmin range were occupied by the esters of an unnatural SL-E2-12 in which a 2-hydroxy-dodecane (thus the E2-12 designation) instead of a hydroxy alkanoate (found in naturally occurring SLs) was the backbone of the hydrophobic moiety (Recke et al., 2013). At the low-value end of the γmin spectrum was SL-E2-12-di-3-OH-C10 (with γmin = 27 mN/m) in which the hydroxyl group at both C6′ and C6″ of the sophorose structure was esterified to the carboxylate of a 3-hydroxy-decanoate molecule. At the other end of the γmin spectrum was the mixture of SL-E2-12-mono (40% at C6′)/di (60% at C6′ and C6″)-17-OH-C18 (γmin = 50 mN/m) in which the esterified group was a 17-hydroxy-stearate. Rosen and Dahanayake (2000a) had tabulated an extensive list of γ values (note: not the γmin as reported in Table 3.6) for around 50 commercially available nonionic surfactants at 0.001, 0.01, and 0.1 wt%. The γ values on their list ranged from 20.8 mN/m for [(CH3)3SiO]2Si(CH3)(CH2)3(OC2H4)7.5OCH3 (at 0.1 wt%) to 58.2 mN/m for castor oil ethoxylate 40 (at 0.001 wt%) that contains a (OC2H4)40OH hydrophilic tail. In comparison with Rosen and Dahanayake’s list, the γmin values of the various SL compounds shown in Table 3.6 fall well within the range of the γ values of the commercial nonionic surfactants, suggesting that SLs are indeed functionally suitable for use as surfactants in commercial applications. Unlike the γmin values, the CMC values of SLs covered an extended range (Table 3.6). A conclusive summary and precise interpretation of a structural correlation with the listed CMC values (Table 3.6) are difficult due to the structural complexity of the SL preparations and the salient differences in the methods of CMC determination. Nevertheless, it could be seen that the CMC values of various SLs and their derivatives fall within the range of the CMCs of the commercial surfactants. In Rosen and Dahanayake’s report (Rosen and Dahanayake, 2000b), the lowest and highest CMCs were recorded at 3.1 × 10−6 M (at 0.00027 wt%) and 1.92 × 10−3 M (at 0.027 wt%) for C16H33(OC2H4)15OH and C9–11H19–23(OC2H4)8OH, respectively. It is however informative to study the variation of CMCs as a function of structural difference of SLs as reported by individual research groups. For example, based on the study of Ashby et al. (2008), on a group of lactonic SLs having different carbon chain lengths (C16 and C18) and degree of unsaturation (C18:0–C18:2) in their respective hydroxy fatty acid moiety, we could conclude that shorter chain length or higher degree of unsaturation would lead to a higher CMC value (Table 3.6). The data by Roelants et al. (2016) additionally showed that the lactonic form and the 6′- and/or 6″-acetylated derivatives of acidic SLs had lower CMC values than their respective counterparts (Table 3.6). The study by Zhang et al. (2004) also revealed to some degrees that the length of the alkyl chain in the acyl group of a series of SL esters significantly governed the CMC values; the longer the alkyl chain was, the lower the CMC value became (Table 3.6). Zerkowski et al. (2006) synthesized a series of SL stearyl ethyl esters in which variously charged amino acids were conjugated onto the sophorose head group of the SL. The CMC values of these amino acid-conjugated SLs showed that they were generally lower than the natural unmodified SLs (Table 3.6). These observations underlined the versatility of SL molecules to meet specific demands of a particular commercial application because of the vast possibility accorded to their structure and composition. Another aspect of the amphiphilic nature of SLs is the fact that they tend to form supramolecular structures, of which the types, sizes, and characteristics are highly dependent on not only small structural differences of the molecules but also the purity and the uniformity thereof. This aspect has been very thoroughly mapped by the research group of Baccile in France.

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3.5.2  Bioactive Agents (Antibacterial, Antifungal, Antiviral, and Anticancer) Many biosurfactants have been reported to possess bioactive properties. Among the myriads of biosurfactants, the lipopeptides, produced primarily by various members of the genus Bacillus, are the most reported in terms of antimicrobial activity. Bacillus subtilis is known to produce surfactin (Shaligram and Singhal, 2010), fengycin (Vanittanakom et  al., 1986) and iturin (Ahimou et al., 2000), while B. licheniformis and B. pumilus are known to produce lichenysin (Madslien et al., 2013) and pumilacidin (Naruse et al., 1990), respectively, each of which are known antimicrobial agents (Mnif and Ghribi, 2015a, b). Glycolipid biosurfactants also have antimicrobial properties. Both rhamnolipids (RL) and mannosylerythritol lipids (MEL) have been demonstrated effective against both gram-­ positive and gram-negative bacterial strains under appropriate conditions (Haba et al., 2003; Kitamoto et al., 1993). Sophorolipids are another member of the glycolipid family and are no exception. They have been demonstrated to be biologically active in the antibacterial, antiviral, antifungal, and anticancer realms. 3.5.2.1  Antibacterial Activity of SLs Sophorolipids have been studied extensively for their potential in antibacterial applications. From the late 1980s, researchers have used various methods including serial dilution, microtitration, and agar diffusion to determine the minimal inhibitory concentrations (MIC) or minimal lethal doses (MLD50; a 50% lethal dose based on a broth microdilution method) of SLs toward various bacterial strains. Lang et al. (1989) recognized the antibacterial activity of SLs against four gram-positive (B. subtilis, Staphylococcus epidermidis, Streptococcus faecium, and Propionibacterium acnes) and one gram-negative (Pseudomonas aeruginosa) bacterial strains. In that paper, the specific molecular type of SL being tested was defined as either a diacetylated, monoacetylated, or nonacetylated lactone from the SL derived from oleic acid (SL-o). Each structural variant was tested either individually or as mixtures. Results proved that B. subtilis and S. epidermidis were prone to SL-based inhibition with the lowest MIC values (6 μg/ml) derived in the presence of the diacetylated lactone. Pseudomonas aeruginosa was resistant to a mixture of SL-o acetylation derivatives showing no inhibition even at SL concentrations as high as 2 mg/mL. Finally, that paper established that growth inhibition against susceptible gram-positive bacteria was in the order of diacetylated>monoacetylated >nonacetylated SLs. Later, Kim et  al. (2002) compared the antibacterial activity of chromatographically pure lactonic and acidic SLs against four gram-positive bacterial strains. Contrary to the results of the Lang study, the acidic form of SL was more inhibitory than the lactonic form. Specifically, the inhibitory concentrations (IC) of the acidic versus lactonic SLs ranged from 0.5 to 4 ppm (against P. acnes) and 4 to 16 ppm (against B. subtilis), respectively. That same year, Gross and Shah summarized the inhibitory effects of lactonic, acidic, diacetylated, and monoacetylated SLs against S. agalactiae, B. subtilis, Micrococcus luteus (gram-positive) and Escherichia coli, Moraxella sp., Salmonella choleraesuis, Alcaligenes latus, and Ralstonia eutropha (gram-negative) bacteria (Gross and Shah, 2004). Their results revealed that the Moraxella sp. was more susceptible to the antibacterial action of SLs than the gram-positive strains and also that the acidic form of SLs displayed better antibacterial activity when compared with lactonic forms. These findings contradict the current consensus that the lactonic SLs generally performed better in antibacterial tests than the acidic forms (Delbeke et al., 2016c).

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In a subsequent study, Shah et  al. (2007) synthesized SLs containing altered sugar head groups using different C5 and C6 sugars (i.e., fructose, ribose, mannose, galactose, xylose, arabinose, and lactose; a disaccharide) as the hydrophilic substrate in the fermentation process. When these SLs were tested against a panel of gram-positive and gram-negative bacteria, the xylose-SL (identified by GC/MS) was most effective against Rhodococcus erythropolis (gram-positive) with an MLD50 of 6 μg/mL, followed by the lactose- and arabinose-SLs (MLD50 of 24 μg/mL) against B. subtilis (gram-positive) and Moraxella sp (gram-negative). In comparison, the MLD50s of the glucose-derived SLs in that study were 98 μg/mL for R. erythropolis, B. subtilis, S. agalactiae, and Moraxella sp and greater than 6250 μg/mL for S. epidermidis, P. putida, Enterobacter aerogenes, and E. coli. The antibacterial properties of SL have also been targeted toward specific clinically relevant bacteria. Sleiman et al. (2009) described the antibacterial activity of SLs against E. coli, S. aureus, Klebsiella pneumoniae, Proteus mirabilis, P. aeruginosa and S. pneumoniae and compared the results with standard antibiotics (i.e., ceftazidime and cefotaxime). It was determined that the ethyl ester diacetate derivatives of SLs resulted in MIC values greater than 128 μg/mL for all the bacterial strains tested. In contrast, the antibiotics alone resulted in MIC concentrations of, at most, 4 μg/mL against P. aeruginosa. The conclusion was that SLs did not demonstrate significant antibacterial activity in vitro at clinically relevant concentrations (Sleiman et al., 2009). However, a paper authored by Joshi-Navare and Prabhune (2013) demonstrated the improved synergistic antibacterial effects of a 75% lactone-25% free acid SL mixture when combined with tetracycline and cefaclor against S. aureus and E. coli, respectively. Elshikh et al. (2017) reported the natural antibacterial effects of lactonic SLs (92% C18:1, diacetylated) against the common oral bacteria S. mutans, S. oralis, S. sanguis, Neisseria mucosa, and Actinomyces naeslundii, common inhabitants of oral biofilms, and corroborated the conjugative antibacterial effects of lactonic SLs when used with popular antibiotics. The results showed that the lactonic SLs interfered with biofilm formation and when used in combination with tetracycline HCl, ciprofloxacin, and/or chlorhexidine improved the minimum inhibitory concentrations (MIC) of the antibiotics to as low as 0.03 μg/mL (Elshikh et al., 2017). That same year, Solaiman et al. (2017a) established the growth inhibition and the cell morphology of S. mutans, S. salivarius and S. sobrinus (oral streptococci), and Lactobacillus acidophilus and L. fermentum (oral lactobacilli) in the presence of lactonic (98%) SLs. It was hypothesized that during caries development, the streptococci (particularly S. mutans) produces a protective polysaccharide matrix in the form of a biofilm in which the acid-producing lactobacilli can flourish. This condition leads to a focused concentration of organic acids that can harm the integrity of the enamel and the bone that anchors the tooth in the mouth (Kleinberg, 2002; Tanzer et al., 2001). The results showed that lactonic SLs were more effective antibacterial agents against the oral streptococci than the lactobacilli, causing growth inhibition at a minimal concentration of 50 μg/mL for S. mutans versus 1 mg/mL for L. acidophilus. This finding was of particular significance because the growth of (and the consequent biofilm formation by) the streptococci could be repressed at low SL concentrations where lactobacilli (some species of which are beneficial probiotics) are unaffected. Solaiman et al. (2015a) first reported the antimicrobial activity of acidic form of the SL isolated from R. bogoriensis. Unlike the SLs isolated from S. bombicola, the SL from R. bogoriensis contains a 13-hydroxydodecanoic acid as the lipid moiety

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of the molecule (Nuñez et al., 2004; Tulloch et al., 1968). Solaiman et al. (2015a) showed that the R. bogoriensis SL immobilized on biodegradable membrane exhibited a stronger antimicrobial activity against P. acnes on a plate assay than the similarly immobilized S. bombicola SLs. Additional studies demonstrating the antibacterial nature of SLs include various reports in which select bacteria isolated from salted animal hides and skins (Birbir and Ilgaz, 1996; Aslan and Birbir, 2011, 2012) were subjected to the antibacterial properties of lactonic SLs. The results showed that SLs produced from glucose and stearic acid (SL-s) exhibited a slightly better antibacterial activity than SLs obtained using glucose and oleic acid (SL-o) or palmitic acid (SL-p) (Solaiman et al., 2016a). The target strains represented gram-­positive endospore-­ forming bacteria (B. licheniformis, B. pumilus, and B. mycoides), gram-positive nonspore-­forming bacteria (Enterococcus faecium, S. xylosus, Aerococcus viridans, S. cohnii, and S. equorum), and gram-negative bacteria (P. luteola, E. cloacae, Vibrio fluvialis, and E. sakazakii). Interestingly, results showed that the endospore-forming species were more sensitive to SLs than were the nonspore formers. MICs were generally measured at 19.5 μg/ml for all of the bacterial strains exposed to lactonic SL-p and SL-o. In contrast, MIC values as low as 5 μg/ml were seen with SL-s against B. licheniformis, B. pumilus, P. luteola, S xylosus, and B. mycoides. These low MIC values (even against gram-negative strains) could be the result of a cumulative effect of SL with the salt present in the media. Studies by Zhang et  al. (2016, 2017) and Olanya et  al. (2018) showed that SLs exhibited antibacterial activity toward food pathogens including E. coli O157-H7, S. enterica, and Listeria monocytogenes under appropriate conditions. Those studies once again confirmed that the lactonic form of SL was more effective against the bacterial strains tested than the free acid form, and while each test strain was susceptible to the antibacterial action of SLs, different SL ­concentrations were required to reduce bacterial counts. A significant reduction in bacterial counts was observed with all SLs tested (i.e., SL-o, SL-p, and SL-s) at a concentration of 0.1% w/v against the gram-positive L. monocytogenes. A higher concentration of SL-o (1% w/v) was required, however, to inhibit the growth of the gram-negative E. coli O157-H7 (Olanya et al., 2018; Zhang et al., 2017). Ashby et al. (2011) explored the use of biodegradable and biocompatible polymers to control the release of the antimicrobial SLs for potential slow-release applications. In that study, four kinds of biobased polymers were tested, that is, poly-3-hydroxybutyrate (PHB), PHBco-10%-3-hydroxyhexanoate (PHB/HHx), pectin/hyaluronate, and alginate/hyaluronate. Although all four biopolymers are suitable carriers to provide release of SLs against P. acnes on an agar-plate assay, the results showed that the pectin and alginate films were more transparent and therefore aesthetically more appealing than the PHB and PHB/HHx films for facial anti-acne applications to combat P. acnes. A subsequent study, however, indicated that in addition to the compatibility of blending SLs into the solution-cast PHB-based biofilms, the presence of SLs also conferred the beneficial effects of improving the thermomechanical properties of the short-chain-length PHA biofilms (Ashby and Solaiman, 2014). Solaiman et al. (2015b) compared the antimicrobial activity of SLs immobilized on solvent-cast biofilms of poly(L-lactic acid) (PLLA) and poly(ε-caprolactone) with that on PHB. The results showed that the width of the zone of inhibition in an agar-plate anti-P. acnes assay decreased in the order of SL-PCL>SL-PLLA>SL-PHB, indicating that PCL could be the best SL carrier matrix for the control release of SL.

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3.5.2.2  Antiviral Activity of SLs Sophorolipids appear to exhibit antiviral activity and acceptable cytotoxicity for use as therapeutic agents in preventing or treating viral infections. To date, they have been demonstrated as effective antiviral agents against both the human immunodeficiency virus (HIV) and the herpes virus. Shah et al. (2005) showed that SLs could be used as effective spermicidal and virucidal agents. In that study, the SLs were initially prepared by fermentation using C. bombicola ATCC 22214 with glucose and oleic acid as the carbon substrates. Then, the specific SL structural analogues were produced through chemical catalysis. The natural mixtures of lactonic and free acid variants resulted in spermicidal activity with a minimum effective concentration (MEC) of 0.8 mg/mL, while the lactonic and free acid forms alone showed MEC values of 1.0 and 3.8 mg/mL, respectively. In contrast, the best results were obtained using the diacetylated ethyl ester SL (DiOAc-EES) where the MEC value was 0.18 mg/mL that was analogous to the widely used commercial spermicide nonoxynol-9 (MEC = 0.25 mg/mL). Further tests showed that the DiOAc-EES completely immobilized the spermatozoa at a concentration of 0.08 mg/mL after a 30 second incubation, and this motility was unrecoverable in fresh medium without SL present. In addition to its spermicidal activity, SLs inactivated HIV infectivity. In fact, at 3 mg/mL, all of the SL derivatives displayed anti-HIV activity in less than 2 minutes. Again, the open-chain forms of the SLs were more potent inhibitors than the lactone forms, and rather fortunately, the DiOAc-EES was the most effective anti-HIV virucidal variant at a concentration of 0.09 mg/mL causing a reduction in infectivity greater than 5.2 log units. These results were further documented in a 2014 US patent (Gross et al., 2014). Further investigations into the antiviral character of SLs were performed against the herpes virus (Gross and Shah, 2007). The Epstein-Barr virus was used as a model to assess anti-herpes activity. Five different SL samples were tested including natural mixtures of lactonic and nonlactonic SLs along with the ethyl and methyl esters and the 6′,6″-diacetylated ethyl variants. All of the SLs tested showed some degree of anti-herpes activity, but the best results came from ethyl 17-L-[(2′-O-β-D-glucopyranosyl-β-D-glucopyranosyl)-oxy]-cis-9-octadecenoate. 3.5.2.3  Antifungal Activity of SLs Ito et al. (1980) demonstrated the effectiveness of lactonic SLs against five genera of yeast including various strains of Candida, Pichia, Debaryomyces, Saccharomycopsis, and Lodderomyces. By measuring growth inhibition, the results disclosed that C. lipolytica (1-day culture) and P. farinose (2-day culture) were the most sensitive to SLs and were completely inhibited at an SL concentration 0.4 mM. In contrast, C. parapsilosis, P. ohmeri, and P. scolyti were the most resistant to SLs even at twice the aforementioned SL concentration. The remaining 16 yeast strains experienced only partial growth inhibition. Krivobok et al. (1994) undertook a growth inhibitory study of SLs against five different classes of microorganisms, three of which were yeasts (seven strains), phytopathogenic fungi (five strains), and dermatophytic fungi (seven strains). The results of this study were in general agreement with the findings of Ito et  al. (1980) that the SLs were well tolerated by most yeasts, phytopathogens and dermatophytes (most resistant strain was the dermatophyte Trichophyton tonsurans with a 50% inhibition concentration (IC50) value of 3.52 g/L). In fact, among the yeasts and fungi tested, only C. parapsilosis was susceptible to the antifungal action of SLs exhibiting an IC50 value of 0.18 g/L.

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A 2005 report showed the response of specific plant pathogenic fungi to different concentrations of SLs. The authors focused on three strains of Phytophthora (P. nicotianae, P. capsici, and P. cactorum) and two strains of Pythium (P. ultimum and P. aphanidermatum) by looking at the mycelial growth and zoospore motility and lysis (Yoo et al., 2005). The results showed that 80% of mycelial growth was inhibited at 500 mg/L of SLs while 90% inhibition was observed with P. ultimum at 100 mg/L of SL. In addition, with the exception of P. aphanidermatum (200 mg/L), SLs inhibited the motility of the zoospores from each of the fungi at a concentration of 100 mg/L. Later, Kulakovskaya et  al. (2014) tested Sopholiance (a commercial preparation of SLs with unspecified structural composition) against Filobasidiella neoformans, C. tropicalis, and C. albicans. The MIC (defined as 50% inhibition of cell growth based on optical density measurement) values ranged between 1 mg/mL (F. neoformans) and 15 mg/mL (C. tropicalis and C. albicans). At the same time, Gross and Shah (2014) synthesized a series of n-alkyl esters (C1–C6) of SLs using both chemical and enzymatic means with various acetylation patterns at the 6′ and/or 6″ carbon of the sophorose head group. Using these derivatives, they demonstrated that the growth of several Candida species was completely inhibited by some of these SL-alkyl esters at a concentration of 5 mg/mL. Different Candida species appeared to respond differently to the SL-alkyl esters. For example, C. albicans was susceptible to the C1-ester, C2ester-diacetylated, and C2-ester-6′-monoacetylated derivatives, whereas C. tropicalis was best inhibited by the C1- and C6-esters and C2-ester-diacetylated derivatives. Haque et al. (2016, 2017) reported the inhibitory effects of SLs on biofilm formation and hyphal growth of select Candida strains. The MIC80 (defined as the lowest concentration of SL that inhibits 80% of the cell growth as compared with a control without SL) was determined for C. albicans (60 μg/mL), C. glabrata (120 μg/mL), C. tropicalis (60 μg/mL), and C. lusitaniae (30 μg/mL), which supported the notion that lactonic SLs may be effective biocontrol agents against biofilms containing potentially pathogenic yeasts. Lastly, Sen et al. (2017) reported the antifungal activity of SL produced by R. babjevae against certain plant and human pathogens including Colletotrichum gloeosporioides, Fusarium verticilliodes, F. oxysporum, Corynespora cassiicola, and T. rubrum. The MIC (described as the lowest concentration of SL at which no growth was observed) for each test strain was determined. MICs ranged from 62 μg/mL (C. gloeosporioides) to >2000 μg/mL (C. cassiicola). However, as mentioned earlier (see Table 3.1), some question have arisen as to whether R. babjevae actually produces SLs or polyol esters of fatty acids (PEFA) as described recently by Cajka et al. (2016) and Garay et al. (2017). The results of all these studies seem to indicate that SLs can be used in antifungal applications, but the effectiveness of the application is highly variable based on the type of SL used, the nature of the specific target yeast, and the duration of the application. 3.5.2.4  Anti-cancer Activity for SLs Sophorolipids have been established as molecular agents capable of combating different types of cancer. Cell death is generally accomplished through either necrosis or apoptosis. Necrotic cell death is a process by which cells are damaged resulting in lysis, while apoptosis involves the instigation of a cell suicide program through either intrinsic or extrinsic stimuli. Isoda et al. (1997) demonstrated that SLs could induce cell differentiation instead of cell proliferation in the human promyelocytic leukemia cell line HL60 at a concentration of 10 μg/mL, could increase nitroblue tetrazolium (NBT) reducing ability (which is a common

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­ ifferentiation-associated characteristic in monocytes and granulocytes), and also inhibit prod tein kinase C activity within the HL60 cells. Protein kinase C is an enzyme that is implicated in cell regulation, differentiation, and proliferation, so inhibitors of this enzyme are expected to be effective antitumor agents. Later, Chen et al. (2006a, b) demonstrated the cytotoxic effects of SL on cancer cells of H7402 (human hepatoma), A549 (human lung adenocarcinoma), HL60, and K562 (human chronic myeloid leukemia). In that study, SLs were tested, and an effective dose-dependent inhibition (based mainly on the development of apoptotic features) on cell viability was determined at SL concentrations greater than 62 μg/mL. Sophorolipids have also been demonstrated effective against human pancreatic cancer (HPAC) cells (Fu et al., 2008). Different structural analogues of SLs were tested for their cytotoxic response toward HPAC cells. All SL derivatives were tested between 50 and 200 μg with varying degrees of success. Cytotoxicity was dependent on SL dosing and the specific SL structural derivative. The cytotoxicity of the methyl ester derivative was consistently elevated (63% ± 5%) at all doses tested, while the ethyl ester diacetate form mediated the greatest toxicity at the lowest dose tested (50 μg). These results indicate a dose- and ­derivative-dependent response and likely kill cancer cells by necrosis. Further work focused on human esophageal cancer cells (KYSE 109 and KYSE 450) and demonstrated that the diacetylated lactone form containing oleic acid was the most cytotoxic (no viable cells at SL concentrations higher than 30 μg/mL) toward both KYSE 109 and KYSE 450 cells, while the monoacetylated lactonic derivative required an SL concentration of 60 μg/mL for complete inhibition. In addition, the acidic forms of the SLs, regardless of type of fatty acid chain and acetylation patterns, have little cytotoxicity toward KYSE 109 and KYSE 450 cells even at elevated concentrations (Shao et al., 2012). Further work by the same group demonstrated anticancer activity of SLs toward human cervical cancer by using HeLa and CaSki cells. The cytotoxicity measured as IC50 showed that the diacetylated lactonic SL containing oleic acid was the most cytotoxic by inducing apoptosis in both HeLa (IC50 = 12.2 μg/mL) and CaSki (IC50 = 25.5 μg/mL) cells (Li et  al., 2017). The apoptotic response was further clarified by Nawale et al. (2017) who showed that SL-induced apoptosis in HeLa cells was accomplished through depolymerization of the mitochondrial membrane and elevation in the intracellular calcium levels leading to the activation of caspase-3, caspase-8, and caspase-9, which play essential roles in programmed cell death.

3.5.3  Nonfood and Food Uses 3.5.3.1  SLs in Lubrication Applications Recently, a report of possible applications for microbial glycolipids in lubricant formulations was published (Solaiman et al., 2017b). From a lubricant perspective, glycolipid use can be classified into two areas: (1) uses where the glycolipids are used themselves as lubricant additives (i.e., biocides, emulsifiers, and friction modifiers) and (2) applications where chemically and/or enzymatically modified glycolipids (and/or the corresponding lipids and sugars) are developed into biobased base oils or lubricant additives. Recent literature has discussed SLs as a possible source for hydroxy fatty acids that can serve as precursors for estolides, well-known lubricant additives, and the use of intact SLs in lubricant formulations. Benefits include the relative ease of producing the ω-1 hydroxy

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fatty acids from the parental SL molecules and the ease of converting these molecules into derivatives suitable for lubrication applications. Estolides (intermolecular ester derivatives of hydroxy fatty acids that are generally composed of at least two fatty acid molecules) are typically considered as effective substitutes/additives in biobased functional fluids (Cermak and Isbell, 2004). In 2008, Zerkowski et al. described a chemical synthesis strategy to construct structured estolides by using as reactants the 17-hydroxy stearic acid and 17-hydroxy oleic acid that were released from S. bombicola SLs through acid-catalyzed hydrolysis (Zerkowski et al., 2008). Ester-forming reactions such as carbodiimide coupling and a modified Yamaguchi symmetrical anhydride method were used in specific orders, resulting in the ability to control the length and the sequence of the structured estolide products. In a 2016 report, Ashby et al. (2016) also demonstrated the synthesis of biobased estolides from the hydroxy fatty acids derived from SLs. Sophorolipids were produced via fermentation using glucose and either oleic acid or linoleic acid as the substrates to produce SL molecules containing high concentrations of unsaturated ω-1 hydroxy fatty acids (Ashby et  al., 2008). The fatty acids were liberated from the sophorose sugar through acid-catalyzed alcoholysis and used to synthesize both unsaturated and epoxy estolides, which can be used as additives in lubricant applications. In a more recent report, Sturms et al. (2017) examined the frictional properties of intact SL, two ω-1 hydroxy fatty acid methyl esters (FAMEs) produced from the parental SL, olive oil and olive oil FAMEs. Frictional coefficient, wear depth, and kinematic viscosity were compared with an API Group III base oil (NEXBASE 3043). Blends were prepared using 1% and 5% additive and were tested at room temperature using a reciprocating ball-on-flat tribometer with steel/steel and SiC/Al ball/flat friction surfaces. The coefficient of friction of the blends (1% and 5%) was slightly lower for the SiC-Al test compared with the pure base oil but showed no change for the steel/steel test. Wear depth results indicated lower wear values for the 5% blends relative to the pure base oil, with the wear depth increasing in the order olive oil ≈ SL FAMEs  800  mg/L (Dashtbozorg et  al., 2016). The congener adsorption at pH 6.5 showed the following order: R-C10-C12  > R-C10-C12:1 > RR-C10-C12:1 > RRC10-C12 > R-C10-C10 > RRC10-C10 > R-C8-C10 >  RR-C8-C10, indicating stronger effect of ­hydrocarbon-tail chain length than the number (1 or 2) of rhamnose groups. Similar to synthetic surfactants, rhamnolipids can reduce surface and interfacial tensions and offer detergent, emulsifying, foaming, and/or dispersing properties for different applications. The minimal surface tension in aqueous rhamnolipid solutions is generally reported at around 30 mN/m (although values as low as 23 mN/m were also reported) (Hamzah et al., 2013; Nitschke et al., 2005a; Renfro et al., 2014; Varjani and Upasani, 2016; Wei et al., 2005; Xia et al., 2011). Rhamnolipids have very low critical micelle concentrations (CMCs), which also depend strongly on molecular structures and environmental pH. For example, at pH 7, the CMC for dirhamnolipid RR-C10-C10 was reported as 5 mg/L; it increased to 40 mg/L for the monorhamnolipid R-C10-C10; and it increased further to about 200 mg/L for congeners with only one hydrocarbon tail such as R-C10 and RR-C10 (Nitschke et al., 2005a). Recently, Kłosowska-Chomiczewska et  al. (2017) compiled the CMC data for 97 rhamnolipids, predominantly mixtures, reported in articles appearing during 2000–16. The CMCs vary from 6.5 to 400 mg/L, but the larger values are outliers: only 1 > 250 mg/L and 3 others > 200 mg/L. They evaluated the effects of purity, pH, and fermentation-carbon-substrate hydrophobicity on the reported CMCs. Carbon-substrate hydrophobicity was found to be most influential: more hydrophobic substrates tended to give rhamnolipids with smaller CMCs (presumably due to the production of larger fractions of rhamnolipids with one rhamnose and/or longer lipid chains). Purity also has a significant effect: the less pure the rhamnolipids, the larger the CMCs. As expected, the CMC was affected by pH particularly in the range of pKa ± 2, with lower pH tending to give smaller CMCs, but the pH effect diminishes in less pure rhamnolipids (Kłosowska-Chomiczewska et al., 2017). The pKa of a monorhamnolipid mixture was reported to be 4.28 ± 0.16 and 5.50 ± 0.06 for concentrations below and above CMC, respectively (Lebrón-Paler et al., 2006). The pKa for dirhamnolipid RR-C10-C10 was reported as 4.20 (Aleksic et al., 2017).

5.2  BIOSYNTHESIS OF RHAMNOLIPIDS BY P. AERUGINOSA Rhamnolipids are produced by bacteria belonging to different phyla including Actinobacteria, Firmicutes, and Proteobacteria (Abdel-Mawgoud et al., 2010). However, because most of the biosynthetic pathway and the mechanisms of genetic regulation have been elucidated in P. aeruginosa, this section will focus on the biosynthesis of rhamnolipids in this bacterium. In addition, because several excellent review articles on rhamnolipid biosynthesis have been published in the past several years (Abdel-Mawgoud et al., 2010; Chong and Li, 2017; Dobler et al., 2016; Li, 2017; Müller et al., 2012; Reis et al., 2011), we will summarize here the recent findings and present a succinct description of rhamnolipid biosynthesis. Although P. aeruginosa produces more than 28 congeners of rhamnolipids, the most prevalent congeners are the mono- and dirhamnose R-C10-C10 and RR-C10-C10 (Deziel et  al., 1999) as shown in Fig. 5.1. The overall biosynthesis of rhamnolipids in P. aeruginosa is ­complex

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for two major reasons. Firstly, the precursors, dTDP-l-rhamnose and β-hydroxy fatty acids, are generated from the central metabolic carbohydrate pathways, de novo fatty acid biosynthetic pathway, and catabolism of lipids via β-oxidation (Abdel-Mawgoud et al., 2014, 2010; Blankenfeldt et al., 2000a; Giraud and Naismith, 2000; Rahim et al., 2000; Rehm et al., 1998; Zhu and Rock, 2008). Secondly, rhamnolipids are synthesized as the bacterial cells enter the stationary phase of growth. In P. aeruginosa, this results in the regulation of rhamnolipid production by highly complex and intricate global regulatory mechanisms including the Las, rhamnosyltransferase chain (Rhl), pseudomonas quinolone signal (PQS), and integrated quorum signal (IQS) quorum-sensing systems and alternative sigma factors RpoS and RpoN (Abdel-Mawgoud et  al., 2011, 2010; Aguirre-Ramirez et  al., 2012; Croda-García et  al., 2011; Dusane et al., 2010b; Müller and Hausmann, 2011; Soberon-Chavez et al., 2005). Rhamnolipid biosynthesis can be divided into three major steps: synthesis of the carbohydrate moiety, rhamnose; synthesis of the lipid tail, β-hydroxy fatty acids; and synthesis of rhamnolipids. The biosynthetic pathway of rhamnolipids in P. aeruginosa is shown in Fig. 5.2.

5.2.1  Biosynthesis of Precursors 5.2.1.1  Biosynthesis of dTDP-l-Rhamnose The carbohydrate moiety of rhamnolipids, rhamnose, can be synthesized from glucose that P. aeruginosa acquires from either the environment, degradation of intracellular glycogen, or gluconeogenesis (Olvera et al., 1999; Pham et al., 2004). For the utilization of exogenous glucose, P. aeruginosa typically converts glucose directly to 6-phosphogluconate during transport, which then enters the Entner-Doudoroff pathway. In this case, glucose-6-phosphate is subsequently synthesized from fructose-6-phosphate generated in the pentose phosphate pathway or from gluconeogenesis (Lessie et al., 1984). Alternatively, P. aeruginosa can phosphorylate intracellular glucose via glucokinase to generate glucose-6-phosphate (Lessie et al., 1984), which is then converted to glucose-1-phosphate by a phosphoglucomutase, AlgC (Olvera et  al., 1999). In the absence of exogenous glucose, P. aeruginosa has to generate this hexose via gluconeogenesis, a process that is critical for the biosynthesis of peptidoglycan in addition to other essential cellular compounds (Lessie and Neidhardt, 1967; Lessie et al., 1984). In addition to its role in rhamnolipid biosynthesis, AlgC plays a crucial role in alginate biosynthesis by catalyzing the conversion of mannose-6-phosphate to mannose-1-phosphate (Padgett and Phibbs, 1986). Glucose-1-phosphate generated by AlgC is then converted to dTDP-l-rhamnose in four sequential reactions catalyzed by the enzymes encoded by the rmlBDAC operon (Rahim et al., 2000). The first committed enzyme, RmlA, of the dTDP-l-­rhamnose biosynthetic pathway is a nucleotidyltransferase that converts glucose-1-phosphate to ­dTDP-d-glucose (Blankenfeldt et al., 2000b). The second enzyme, RmlB, is a d ­ TDP-d-glucose 4,6-dehydratase that catalyzes the oxidation and dehydration of dTDP-d-glucose to form d ­ TDP-4-keto-6-deoxy-d-glucose (Allard et al., 2000). The third enzyme, RmlC, is an epimerase that catalyzes epimerization at the C3 and C5 to generate dTDP-4-keto-6-deoxy-l-­mannose (Graninger et al., 1999). This substrate is then reduced to dTDP-l-rhamnose by RmlD, a d ­ TDP-4-keto-6-deoxy-l-mannose reductase (Graninger et  al., 1999). Pseudomonas aeruginosa uses dTDP-l-rhamnose as the precursor for the biosynthesis of rhamnolipids (Edwards and Hayashi, 1965; Hauser and Karnovsky, 1957; Jarvis and Johnson, 1949), lipopolysaccharide (LPS) (Rahim et al., 2000), Psl (Byrd et al., 2009), and b-type flagellin (Lindhout et al., 2009). II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS



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FIG. 5.2  Biosynthesis of rhamnolipids.

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5.2.1.2  Biosynthesis of β-Hydroxydecanoyl-β-Hydroxydecanoic Acid Although P. aeruginosa produces more than 28 congeners of rhamnolipids that differ in the chain length and the degree of saturation of the fatty acid moiety (Deziel et  al., 1999), the most prevalent rhamnolipids produced contain β-hydroxydecanoyl-β-hydroxydecanoic acid. Pseudomonas aeruginosa acquires the β-hydroxy fatty acid moiety via two mechanisms. The first mechanism is via the de novo fatty acid biosynthetic pathway (FAS II) in which a β-ketoacyl-ACP is converted to β-hydroxyacyl-ACP by a NADPH-dependent β-ketoacyl reductase (Campos-Garcia et al., 1998). Although the gene encoding this enzyme was initially identified as rhlG (Campos-Garcia et al., 1998), more recent data by Miller et al. (2006) suggest this step is likely catalyzed by FabG that has 2000-fold higher enzymatic activity than RhlG. In support of this finding, Zhu and Rock (2008) demonstrated through genetic and biochemical analyses that RhlG did not catalyze the reduction step and that it was not involved in rhamnolipid biosynthesis. Zhu and Rock’s assertion that RhlG is not involved in rhamnolipid biosynthesis was further supported by Bazire and Dufour (2014) when they determined that rhlAB and rhlG are inversely regulated and that RhlG does not play a substantial role in the synthesis of rhamnolipids. In order to account for these different results regarding the putative role of RhlG in rhamnolipid biosynthesis, Fig. 5.2 shows both FabG and RhlG for catalyzing the reduction of β-ketoacyl-ACP to β-hydroxyacyl-ACP. The second mechanism for generating the β-hydroxy fatty acid moiety for rhamnolipid biosynthesis is via β-oxidation of fatty acids (Hori et al., 2011; Kang et al., 2010; Zhang et al., 2012). This mechanism was first suggested when Kang et al. (2010) demonstrated that a fadD2 mutation resulted in an almost 40% decrease in rhamnolipid production. Subsequently, Hori et  al. (2011) demonstrated a relationship between the carbon number of rhamnolipids and β-hydroxy fatty acids. Given the evidence for the generation of β-hydroxy fatty acids via both de novo fatty acid synthesis and β-oxidation of fatty acids, both pathways were considered to contribute equally to rhamnolipid biosynthesis. However, in 2014, Abdel-Mawgoud et al. (2014) demonstrated that β-­oxidation of fatty acids is likely to be the major source of β-hydroxy fatty acids for rhamnolipid biosynthesis. In their study, they observed a sharp decrease in rhamnolipid production when P. aeruginosa was forced to use only the de novo fatty acid biosynthetic pathway to generate β-hydroxydecanoic acids. The study also demonstrated that trans-2-enoyl-CoA was the β-oxidation intermediate that was diverted to rhamnolipids by the enoyl-CoA hydratase/ isomerase activity of the RhlYZ enzyme complex (Abdel-Mawgoud et al., 2014).

5.2.2  Biosynthesis of Rhamnolipids Three enzymes that catalyze the production of rhamnolipids from the precursors are encoded by the rhlAB operon and the rhlC gene. Rhamnosyltransferase chain A (RhlA) catalyzes the formation of dimeric fatty acid moiety of β-hydroxydecanoyl-β-hydroxydecanoic acid by condensing two molecules of β-hydroxydecanoyl-ACP or by condensing β-hydroxydecanoyl-­ ACP and β-hydroxydecanoyl-CoA as shown in Fig. 5.2 (Abdel-Mawgoud et al., 2014; Deziel et al., 2003). Rhamnosyltransferase chain B (RhlB) is a membrane-bound enzyme that catalyzes the transfer of an activated l-rhamnose (dTDP-l-rhamnose) to a β-hydroxydecanoyl­β-hydroxydecanoic acid to form a monorhamnolipid (R-C10-C10) (Burger et  al., 1966; Ochsner et  al., 1994b). RhlC, located in a separate operon with a hypothetical transporter protein PA1131, catalyzes the transfer of a second l-rhamnose moiety to monorhamnolipids

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to generate dirhamnolipids (RR-C10-C10) (Rahim et al., 2001). Although PA1131 encodes for a hypothetical transporter protein belonging to the major facilitator superfamily (MFS), it is not yet clear whether this protein is involved in rhamnolipid secretion. It is interesting that rhlAB and rhlC are found on different loci in P. aeruginosa while Dubeau et al. (2009) found these three genes to be encoded in a single genetic cluster in Burkholderia thailandensis and B. pseudomallei. Although P. aeruginosa synthesizes both mono- and dirhamnolipids, the gene expression patterns in which rhlAB expression increased over 100-fold in the early stationary phase of growth while rhlC expression increased only 7.4-fold (Wagner et al., 2003) suggest that monorhamnolipids are made first early in the stationary phase followed by the conversion of some of these moieties into dirhamnolipids.

5.2.3  Regulation of Rhamnolipids Biosynthesis Biosynthesis of rhamnolipids in P. aeruginosa is tightly regulated by a complex network of cell-cell communication (quorum-sensing) systems and other physiological and environmental conditions, including carbon and nitrogen sources and iron availability (Fig. 5.3). Although rhamnolipids are synthesized from the precursors derived from major metabolic pathways, the majority of the regulatory studies have focused on the transcriptional regulation of the rhamnolipid biosynthetic genes, rhlAB and rhlC, by the hierarchical but intertwined cell-cell communication systems of P. aeruginosa. The regulatory mechanisms are further complicated by the fact that the biosynthesis of three of the signaling molecules for cell-cell communication systems shares the biosynthetic pathway as the rhamnolipids (Fig. 5.2). In the following section, some of the latest findings on genetic regulation of rhamnolipid production in P. aeruginosa are discussed. 5.2.3.1  Cell-Cell Communication Systems of P. aeruginosa To date, four cell-cell communication systems have been identified and characterized in P. aeruginosa: Las, Rhl, PQS, and IQS (Lee and Zhang, 2015). These cell-cell communication systems are more commonly known as quorum-sensing (QS) systems because they were initially characterized for coordinating gene expression as the function of the cell density in a population (Fuqua et  al., 1994). For each QS system, bacterial cells synthesize a diffusible signaling molecule termed an autoinducer (AI) that, when complexed with the cognate response regulator, induces the expression of the QS system. As the cell density in a population increases, the concentration of the extracellular AI increases until a “quorum” is reached and the molecules reenter the cell to complex with the cognate response regulator and initiate the QS regulatory cascade (Eberhard, 1972; Fuqua et al., 1994; Nealson, 1977; Nealson et al., 1970). In P. aeruginosa, the QS systems control diverse physiological mechanisms including virulence factor production, immune evasion, antibiotic resistance, motility, formation and architecture of biofilms, and response to environmental signals (please see Lee and Zhang (2015) for the latest comprehensive review on QS systems of P. aeruginosa). The four P. aeruginosa QS systems were initially identified for their roles in regulating the production of P. aeruginosa virulence factors. The first P. aeruginosa QS system discovered was the Las system composed of LasR and LasI, and it was implicated in the regulation of elastase, LasA protease, exotoxin A, and alkaline phosphatase productions (Gambello and Iglewski, 1991; Passador et al., 1993; Toder et al., 1991). LasR is a DNA-binding protein and a

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FIG. 5.3  Regulation of rhamnolipid biosynthesis.



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homologue of LuxR that regulates light production in Vibrio fischeri. LasI is a LuxI homologue with acyl-homoserine lactone synthase activity for the biosynthesis of the AI molecule N-3oxododecanoyl-homoserine lactone (OdDHL). The second QS system discovered in P. aeruginosa was the Rhl system composed of the response regulator, RhlR, and the acyl-homoserine lactone synthase, RhlI. RhlR was initially identified as a transcriptional regulator of the rhlAB operon for rhamnolipid biosynthesis (Ochsner et al., 1994b). Based on sequence analysis, it became clear that RhlR was another LuxR homologue. The rhlI gene located downstream of rhlR was then characterized to encode for the enzyme that synthesizes N-butyryl-homoserine lactone (BHL), the cognate AI for RhlR (Ochsner and Reiser, 1995; Pearson et al., 1994, 1995). Similar to the Las system, the Rhl system regulates the production of multiple virulence factors including several exoproducts such as elastase and LasA protease that are also regulated by the Las system (Brint and Ohman, 1995). Both Las and Rhl systems belong to the acyl-­ homoserine lactone-mediated cell-density-dependent cell-cell communication systems. The third P. aeruginosa QS system discovered was the Pseudomonas quinolone signal (PQS) system (Pesci et  al., 1999). PQS (2-heptyl-3-hydroxy-4-quinolone) was discovered when an increase in the expression of lasB, the gene encoding for elastase, was observed in a lasR null mutant upon addition of the spent culture medium of the wild-type culture. Addition of OdDHL or BHL to the lasR mutant failed to induce lasB expression indicating that induction was due to another potential cell-cell signaling system. PQS is synthesized by PqsABCD, PhnAB, and PqsH (Gallagher et al., 2002). PqsABCD and PhnAB are involved in the synthesis of 2-heptyl-4-quinolone (HHQ), an intercellular molecule that is converted to PQS by PqsH (Cao et al., 2001; Deziel et al., 2004; Dubern and Diggle, 2008; Gallagher et al., 2002). PQS acts by complexing with its cognate response regulator PqsR (multiple virulence factor regulator (MvfR)), an LysR-type transcriptional regulator (Gallagher et  al., 2002). Interestingly, both HHQ and PQS are able to complex with PqsR to activate the expression of the target genes (Diggle et al., 2007a; Xiao et al., 2006a,b). Although not all of the PQS regulon can be activated by the PqsR/HHQ complex, many of the genes can be regulated by both the PqsR/HHQ and the PqsR/PQS complexes. The PQS system also controls the production of multiple virulence factors including rhamnolipids. The PQS system was the first non-acyl-homoserine lactone-mediated cell-cell communication system characterized for P. aeruginosa. The latest QS system to be identified and characterized in P. aeruginosa was the integrated quorum signal (IQS) system that was determined to connect the Las system to phosphate limitation (Lee et al., 2013). Like the PQS system, the IQS system is a non-acyl-­homoserine lactone-based cellcell communication system in which the signaling molecule, IQS, is 2-(2-hydroxyphenyl)-­ thiazole-4-carbaldehyde. The IQS is synthesized by the gene products of a nonribosomal peptide synthesis operon ambBCDE (Lee et al., 2013). All four of the QS systems of P. aeruginosa are important for the virulence of the bacterium. Interestingly, the AIs for Las, Rhl, and PQS share biosynthetic pathway with the lipid portion of rhamnolipids (Müller and Hausmann, 2011). 5.2.3.2  Regulatory Network of P. aeruginosa QS Systems The regulatory circuit of QS systems in P. aeruginosa is complex and involves both hierarchical QS regulation and regulation by other global regulators (Lee and Zhang, 2015; Schuster and Greenberg, 2006; Venturi, 2006; Williams and Cámara, 2009). The QS regulatory hierarchy starts with the Las system. In addition to various genes in the Las regulon, the LasR/OdDHL

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complex activates transcription of lasI, rhlR, rhlI, pqsR, and rsaL. Transcriptional activation of lasI by the LasR/OdDHL complex leads to increased production of OdDHL to create a positive feedback loop of the Las system (Seed et al., 1995). lasI expression is also positively regulated by the RhlR/BHL complex to form a positive feedback loop between the Las and the Rhl QS systems (Dekimpe and Deziel, 2009). In contrast, rsaL encodes for a transcriptional repressor of lasI to create a negative feedback loop, which together with the induction of lasI by the LasR/OdDHL complex autoregulates the Las system (De Kievit et al., 1999; Rampioni et al., 2006). The expression of lasR is induced by two global regulators of gene expression, Vfr and GacA, in P. aeruginosa (Albus et al., 1997; Reimmann et al., 1997). Vfr is a homologue of the Escherichia coli’s cyclic AMP receptor protein (CRP) that was initially characterized for its role in regulating exotoxin A synthesis in P. aeruginosa (West et al., 1994a,b). GacA is the response regulator of the GacA/S two-component regulatory system that regulates multiple virulence factors in many γ-proteobacteria including Pseudomonas species (Lapouge et al., 2008; Laville et al., 1992; Reimmann et al., 1997). Interestingly, the activity of the Las system is also regulated by two orphan LuxR homologues, QscR and VqsR, which lack their own cognate AI synthases. QscR suppresses the transcriptional regulator activities of LasR and RhlR by forming heterodimers with these proteins (Ledgham et al., 2003). In addition to directly suppressing the Las and the Rhl QS systems, QscR also indirectly suppresses the Las QS system by complexing with OdDHL to regulate its own regulon (Lee et al., 2006; Lequette et al., 2006). VqsR, in contrast, indirectly activates the Las QS system through interfering with qscR expression (Liang et al., 2012). To complete the Las QS autoregulatory circuit, vqsR expression is a part of the Las QS regulon. The second QS system, the Rhl system, is activated by the LasR/OdDHL complex that induces the expression of both rhlR and rhlI (De Kievit et al., 2002; Latifi et al., 1996). The RhlR/BHL complex induces the expression of rhlI to form a positive feedback loop. In addition to LasR/ OdDHL, rhlR expression is activated by Vfr and GacA (Croda-García et  al., 2011; Reimmann et al., 1997). Thus, transcriptions of both lasR and rhlR are activated by Vfr and GacA, and the activities of LasR and RhlR are suppressed by QscR. In addition, transcriptions of rhlR and rhlI are indirectly activated by VqsR through its repression of qscR expression that allows the activity of the LasR/OdDHL complex (Liang et al., 2012). Furthermore, expression of both rhlI and rhlR is positively regulated by the PqsR/PQS complex (McKnight et al., 2000). Expression of the acyl-homoserine synthase-encoding genes lasI and rhlI is also regulated by two alternative sigma factors in P. aeruginosa, RpoN and RpoS. RpoN (σ54) is a global regulator involved in nitrogen limitation response and plays a role in various aspects of P. aeruginosa physiology including motility (Totten et al., 1990; Woods et al., 1980). In a rpoN mutant growing in complex growth media, elevated expression of both lasI and rhlI was observed in low-cell-density cultures indicating negative regulation by RpoN (Heurlier et al., 2003). This phenotype was believed to be due to the negative effect of RpoN on gacA expression. However, Medina et al. (2003a) and Thompson et al. (2003) characterized RpoN as an activator of rhlI expression when P. aeruginosa was grown in specific growth media. These data support the earlier observation that rhamnolipid production was increased under nitrogen-limiting conditions (Mulligan and Gibbs, 1989). RpoS is the general stress response regulator of P. aeruginosa that facilitates adaptation of the bacterium to a variety of environmental stress including nutrient starvation (Suh et al., 1999). The role of RpoS in rhamnolipid biosynthesis has also been less than clear with one group finding RpoS to repress rhlI expression (Whiteley et al., 2000) while another group finding that RpoS activates the expression of the rhlAB operon (Medina et al., 2003a). The rhlI gene is also negatively regulated at the II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS



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posttranscriptional level by a small RNA, DksA, which interferes with its translation (Branny et al., 2001; Jude et al., 2003). Finally, the Las and the Rhl systems are also positively regulated by the stringent response that is mounted by the cells in response to amino acid starvation and mediated by the intracellular level of (p)ppGpp (Van Delden et al., 2001). Regulatory mechanisms of the PQS system have been elucidated predominantly for the hierarchical regulation by the Las and the Rhl systems. The LasR/OdDHL complex induces the production of PQS by activating the expression of pqsR, the gene that encodes for the transcriptional activator of the pqsABCDE and the phnAB operons for the biosynthesis of HHQ, and pqsH, the gene that encodes for a flavin-dependent monooxygenase that converts HHQ into PQS in the last step of the PQS biosynthetic pathway (Cao et al., 2001; Deziel et al., 2004; Gallagher et al., 2002). In contrast, the PQS system is negatively regulated by the RhlR system due to repressed expression of pqsR by the RhlR/BHL complex (Wade et al., 2005; Xiao et al., 2006b). In turn, PqsR/PQS complex induces the expression of rhlI and rhlR to positively regulate the Rhl system (McKnight et al., 2000). Thus, the LasR/OdDHL activates both Rhl and PQS QS systems, but the level of PQS system response is autoregulated by a regulatory circuit between PQS and Rhl systems. In addition, the PQS system is activated by the recently characterized IQS system under phosphate-limiting conditions (Lee et al., 2013). The IQS system is positively regulated by the LasR/OdDHL complex to be a part of the P. aeruginosa QS hierarchy. Although the IQS system elevates the Rhl system under phosphate-limiting conditions, this action may be through the activation of the PQS system rather than by direct activity. 5.2.3.3  Regulation of Rhamnolipids Biosynthesis by P. aeruginosa QS Systems and Other Global Regulators Rhamnolipid biosynthesis is regulated by multiple regulators at the transcriptional and posttranscriptional levels. The role of QS systems in rhamnolipid biosynthesis in P. aeruginosa has been clearly established since RhlR was characterized to require the function of RhlI, an acyl-homoserine synthase, or exogenously added acyl-homoserine lactones, to activate the expression of the rhlAB operon involved in rhamnolipid synthesis (Ochsner and Reiser, 1995). Then in 2001, Rahim et al. (2001) demonstrated that rhlC, the gene that encodes for a rhamnosyl transferase for the synthesis of dirhamnolipids, was also regulated by the Rhl/BHL complex. Thus, three genes that encode for the last three enzymes for the synthesis of mono- and dirhamnolipids were determined to be regulated by the Rhl QS system. In 2012, Aguirre-Ramirez et al. (2012) demonstrated that the rmlBDAC operon involved in the biosynthesis of dTDP-l-­ rhamnose is also regulated by RhlR/BHL complex. This demonstrated that QS regulates the early steps of rhamnolipid biosynthesis in addition to the last three steps. Since the P. aeruginosa QS systems form a complex regulatory circuit as described in the previous section, all four of the QS systems are then involved in regulating the rhamnolipid biosynthesis in P. aeruginosa and not just the Rhl system. However, the regulation of rhamnolipid biosynthesis appears to be more complex than simply transposing the available information from QS regulatory circuit because there appears to be instances of apparent contradiction. For example, the rmlBDAC operon is regulated by both the Rhl system and the alternative sigma factor RpoS (AguirreRamirez et al., 2012; Medina et al., 2003b) from the major promoter (P2). In addition, RpoS was demonstrated to enhance the expression of rhlAB (Medina et al., 2003b). Given that RpoS has also been demonstrated to repress rhlI expression and, therefore, suppresses the Rhl QS system (Whiteley et al., 2000), exact mechanisms by which RpoS and Rhl QS act in concert to induce rhamnolipid biosynthesis remain to be elucidated. A similar apparent contradiction has been II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

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observed for RpoN between its role in repressing the expression of lasI and rhlI (Heurlier et al., 2003) while activating the expression of the rhlAB operon (Medina et al., 2003a). With the recent characterizations of the emergence and existence of “cheaters” in bacterial populations that take advantage of “public goods” produced by QS-mediated gene expression to gain advantage in fitness (Diggle et al., 2007b; Sandoz et al., 2007; Rumbaugh et al., 2009, 2012; Popat et al., 2012), it is clear that nuances of QS-mediated gene expression, including rhamnolipid biosynthesis, still remain to be elucidated (please see Whiteley et al. (2017) and Popat et al. (2012) for recent reviews on bacterial QS). In addition to the regulation of rhamnolipid biosynthetic genes by global regulators including the QS systems, the catalytic activity of RmlA that catalyzes the conversion of glucose-1-phosphate to dTDP-d-glucose is feedback inhibited by the end product of the pathway, dTDP-l-rhamnose (Blankenfeldt et al., 2000a). 5.2.3.4  Regulation of Rhamnolipids Biosynthesis by Physiological Conditions of P. aeruginosa As evidenced by the QS systems and their effect on rhamnolipid production, it is clear that rhamnolipid biosynthesis is intimately linked to physiological conditions of the bacterium. As secondary metabolites that are regulated by the QS systems, rhamnolipids are typically produced in the late logarithmic to the early stationary phase of growth when cells experience nutrient deprivations including nitrogen and phosphate limitations (Mulligan et  al., 1989; Mulligan and Gibbs, 1989). Nutrient limitation, specifically amino acid limitation, results in induction of the stringent response that indirectly affects rhamnolipid biosynthesis through its effect on the Las and the Rhl QS systems independent of the cell density (Van Delden et al., 2001). Iron limitation also affects the rhamnolipid biosynthesis indirectly through induction of the PQS and the Rhl systems (Glick et al., 2010; Jensen et al., 2006; Oglesby et al., 2008). In contrast, when iron is abundant, the Las QS system is suppressed (Bollinger et al., 2001). Thus, by extrapolation, abundance of iron would suppress the whole QS hierarchy and reduce the biosynthesis of rhamnolipids. In addition to the limitations of iron, nitrogen, and phosphate that enhance rhamnolipid biosynthesis, the nature of the carbon source provided to P. aeruginosa affects the production of this biosurfactant. Under most growth conditions, P. aeruginosa can utilize almost all available nutrients, including hydrophilic molecules such as carbohydrates and hydrophobic hydrocarbons as the carbon source to synthesize rhamnolipids (Varjani and Upasani, 2017). Recent evidence suggests that the carbon source may play a role in determining the relative ratio between mono- and dirhamnolipids (Nicolo et al., 2017). In this study, P. aeruginosa preferentially produced monorhamnolipids when grown with long-chain fatty acids and dirhamnolipids when grown with glucose or glycerol. This relative ratio of monoversus dirhamnolipids was due to the effect of carbon source on rhlC expression. These data suggest that P. aeruginosa produces mono- versus dirhamnolipids based on the relative availability of the β-hydroxy fatty acid moiety versus the rhamnose moiety.

5.3  RHAMNOLIPIDS PRODUCTION 5.3.1  Challenges in Rhamnolipids Production The major factors that affect or limit industrial production of rhamnolipids are discussed in the following sections.

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5.3.1.1  Substrate Cost For biosurfactants, raw materials are estimated to make up about 10–50% of the total manufacturing costs (Cameotra, 1998; Mulligan and Gibbs, 1993). To improve the economics of rhamnolipid production, researchers have studied inexpensive industrial by-products as substrates, which can be classified into four types: agro-industrial waste, dairy and distillery waste, food processing waste, and animal fat and oil industrial waste (Banat et  al., 2014). Among them, the by-products of oil industries such as soap stock, waste frying oil, oil mill effluent, and waste fatty acids are generally the most effective substrates for rhamnolipid production. As described above, the free fatty acids such as oleic acid, linoleic acid, and stearic acid present in these sources can be effectively converted via β-oxidation to the rhamnolipid precursors, β-hydroxy fatty acids (Abdel-Mawgoud et al., 2014). The reported rhamnolipid yield from carbon substrate varies in a wide range of 10%–62% (Henkel et  al., 2012). The yields from industrial by-products are summarized in Table  5.2. Rhamnolipid yield depends also on the bacterial strain and process conditions used for production. High yields of 45%–59% were found from P. aeruginosa LBI using soap stock as substrate (Benincasa et  al., 2002; Nitschke et  al., 2005a,b) The 45% yield was from a fermenter study with high aeration to ensure proper dissolved oxygen concentrations (DO); unexpectedly, the 59% yield was from a 144 h shake-flask cultivation. In these studies, rhamnolipid concentrations were estimated by multiplying the measured rhamnose concentrations by a factor of 3.4 (Benincasa et al., 2002) or 3.0 (Nitschke et al., 2005a,b). This might have introduced overestimations because, for example, the conversion factor for the rhamnolipid mixture shown in Table 5.1 is only about 2.3. Different methods used for rhamnolipid quantitation may contribute to the variation of yields reported in the literature. Nevertheless, this productive P. aeruginosa LBI strain gave clearly higher rhamnolipid yields from the soap stock rich in free fatty acids than from vegetable oils with predominantly triglycerides (Benincasa et al., 2002). TABLE 5.2  Rhamnolipids Yield from Agricultural and Industrial Byproducts as Substrates Using Different Bacterial Strains Bacterial Strain

Substrate

% Yielda

Ref.

Pseudomonas aeruginosa PAO1

Olive mill waste

0.2

(Moya et al., 2015) b

Pseudomonas aeruginosa GS3

Molasses

0.4

(Patel and Desai, 1997)

Pseudomonas sp. JAMM NCIB 40044

Olive oil mill effluents

1.4

(Mercad et al., 1993)

Pseudomonas aeruginosa MTCC 2297

Waste frying coconut oil

7.5

(George and Jayachandran, 2013)

Pseudomonas aeruginosa AT10

Waste free fatty acid

19

(Abalos et al., 2001)

Pseudomonas aeruginosa MTCC 2297

Orange peel

30.6

(George and Jayachandran, 2009)

Pseudomonas aeruginosa MA-1

Amurcawas (olive oil waste)

31

(Tazdait et al., 2018)

Pseudomonas aeruginosa LBI

Used soybean oil

38.2

(Nitschke et al., 2005a,b) c

Pseudomonas aeruginosa LBI

Soap stock

45.4

(Benincasa et al., 2002)

Pseudomonas aeruginosa LBI

Soybean oil soap stock

58.6

(Nitschke et al., 2005a,b)

a

Yield calculated from the reported data as a ratio of final rhamnolipid concentration to the substrate supplied. b Yield in the form of rhamnose equivalent. c 33% during 0–48 h and 70% during 48–54 h after addition of a fresh batch of substrate.

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Using industrial waste or by-products for rhamnolipid production thus offers potential advantages such as improved economics due to cheaper and abundant availability of these substrates and improved product yield particularly from those with high contents of free fatty acids. However, there are also limitations that must be carefully considered. These include requirement of pretreatments to make the substrate utilizable, need for additional separation and purification steps, and variability in composition of the agro-industrial by-­ products, which could cause varying yield and composition of the rhamnolipids produced (Banat et al., 2014). Technoeconomic analysis is necessary for specific target applications of the rhamnolipids. 5.3.1.2  Low Productivity and Yield Low productivity and yield are major setbacks for rhamnolipid production. As rhamnolipids are typically produced in the stationary phase of fermentation, it is desirable to maintain the culture in prolonged stationary phase. However, rhamnolipid productivity tends to decrease significantly beyond certain period. Ochsner et al. (1994a, 1995) demonstrated this productivity decline with wild-type and mutant P. aeruginosa strains and E. coli DH5α. A recent study also showed that the rhamnolipid production stopped in batch fermentations ­using P. aeruginosa E03-40 after about 190 h without any carbon-substrate limitation (Sodagari et al., 2018). This productivity decrease can be inferred from other publications, although not generally described/explained; some examples are given in Table 5.3. Recently, this rhamnolipid productivity decline over extended stationary phase was reported to be associated with long-term nitrogen source depletion (Sodagari, 2013). Soares dos Santos et al. (2016) postulated that the enzymes involved in rhamnolipid synthesis depend on nitrogen-nutrient assimilation and the formation of certain essential proteins. They found that fed-batch addition of both carbon and nitrogen sources resulted in higher cell yield and rhamnolipid volumetric productivity than the supplementation of only carbon source (Table 5.4). Sodagari (2013) also showed that the declining rhamnolipid production can be reinstated by partial broth replacement with fresh medium. Accordingly, steady and sustained rhamnolipid production may be obtained by supplementing suitable nitrogen source(s) periodically. Future studies are required to evaluate if nitrogen source is the only nutrient required for sustaining long-term production and, if so, the effects of different nitrogen supplementation strategies. It is important to determine the optimal rhamnolipid yield and productivity achievable by this approach and its potential impact to production economics.

TABLE 5.3  End-Time of Stationary-Phase Rhamnolipid Production without Carbon-Substrate Limitation in Fermentations Using Different P. aeruginosa Strains and Vegetable Oils as Substrate Strain

Carbon Substrate

Max. Rhamnolipid Conc. (g/L)

Total Process Time (h)

Production End-Time (h)

Ref.

PAO1

Sunflower oil

25.8

120

70

(Müller et al., 2011)

DSM 2874

Sunflower oil

30.8

120

90

(Müller et al., 2011)

44T1

Olive oil

9.1

140

80

(Manresa et al., 1991)

E03-40

Soybean oil

42.1

250

191

(Sodagari et al., 2018)

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TABLE 5.4  Fed-Batch Processes for Rhamnolipid Production Using P. aeruginosa PA1 Carbon Source Supplementation

Carbon + Nitrogen Source Supplementation

Rhamnolipid concentration (g/L)

7.76

10.93

Cell concentration (g/L)

3.34

4.78

Product yield (%)

35

33

Volumetric productivity (mg/L h)

33

47

The supplementation of both carbon and nitrogen sources resulted in higher volumetric productivity and yield than only carbon source addition (Soares dos Santos et al., 2016).

5.3.1.3  Excessive Foaming The inherent foaming issue in biosurfactant fermentations significantly increases the production costs (Winterburn and Martin, 2012). Foaming poses difficulties with the overall process control and may cause spilling of frothed broth (Etoc et al., 2006) and associated filter clogging and contamination. Managing the above problems generally includes the reduction of fermenter working volume to leave large headspace, which translates to higher capital costs. For example, even with heavy addition of a chemical antifoam and the use of a mechanical foam breaker in the fermenter headspace, Müller et al. (2010) used only about 36% of the total bioreactor volume as liquid culture for rhamnolipid fermentation. Use of chemical antifoam agents may also complicate the purification and other downstream processing steps. Foaming is therefore an important issue to address for industrial rhamnolipid production. To identify the contributions of broth components to foaming, Sodagari and Ju (2014) collected samples from fed-batch fermentations of P. aeruginosa E03-40, where pH was controlled at 6.7 ± 0.2 and soybean oil substrate was added periodically. The maximum cell and rhamnolipid concentrations reached were approximately 10 and 40 g/L, respectively. Foaming ability was evaluated for the whole broth samples, suspensions of washed cells in deionized water, and cell-free supernatants. The bacterial cells themselves were found to contribute the most to foaming throughout the fermentation, especially during the active growth phase with high oxygen consumption rates. For both the maximum foam volume and initial foaming rate, the contributions due to cells were much larger than those due to the rhamnolipid-containing cell-free supernatants, but the latter became increasingly more significant in the later stages. Foaming was strongly pH-dependent, decreasing significantly as pH was lowered from 6.5 to 5.0 (Sodagari and Ju, 2014). Long et al. (2016) later also did a mechanistic study on foaming in rhamnolipid fermentations. They, however, found that cells contributed less than 10% to overall foam height and foam stability and rhamnolipids played the dominant role in foaming. Their fermentations were done with P. aeruginosa ZJU211 (CCTCC M209237) using colza oil as carbon substrate and reached maximum cell and rhamnolipid concentrations of about 12 and 30 g/L, respectively. Their foaming study also showed that rhamnolipids had low foam stability in nonagitated solutions but imparted high foam stability under stirring; when compared at 20 g/L under stirring, rhamnolipids gave more stable foams than sodium dodecyl sulfate. They therefore attributed the severe foaming in their fermentations to the mechanical agitation of broths with high (up to 30 g/L) rhamnolipid concentrations.

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These studies indicated that cell surface properties can differ significantly depending on the strains. Future studies are warranted to compare surface properties of different rhamnolipid-­ productive species/strains and to evaluate whether these properties correlate with specific rhamnolipid productivities. The knowledge can be important for selecting or modifying rhamnolipid overproducers to allow better foaming control in fermentations. 5.3.1.4  Expensive Downstream Processing The downstream processing of biosurfactants can be expensive and often constitutes about 60%–80% of the manufacturing costs (Mukherjee et al., 2006). For rhamnolipids, the primary challenges can be attributed to the multicomponent complexity of fermentation broth, unknown thermodynamic data of different components, low concentration of target compound, and the need to process a large volume of broth (Weber and Zeiner, 2014). Different unit operations have been utilized in downstream processing of rhamnolipids, such as filtration, foam fractionation, precipitation, solvent extraction, adsorption, and chromatography (Hubert et al., 2012). Precipitation of rhamnolipids is one of the most commonly used methods for isolation of rhamnolipids from fermentation broth (Deziel et al., 1999). Acidification of fermentation broths to pH 2–3 eliminates the negative charge of rhamnolipids, thereby reducing their solubility in aqueous solutions (Weber and Zeiner, 2014). Phase formation can occur at low pH to complicate the desired rhamnolipid precipitation, when the residual oil present in the broth is in a large enough amount to cause emulsion/microemulsion formation (unpublished results). A recovery of 72% and a purity of 90% were obtained in downstream processing of a fermentation broth when the precipitation step was combined with ethyl acetate extraction (Wei et al., 2005). The rhamnolipid purity required depends on the end applications. For example, high purity is required when rhamnolipids are used in cosmetics or food applications, while for some agricultural applications and uses in enhanced oil recovery and environmental remediation, the purity may be less critical. Some used adsorption column for purification of rhamnolipids. For example, Haba et al. (2003) used an adsorption column made up of polystyrene resin for this purpose. The surface tension of column effluent was measured to detect occurrence of breakthrough, and methanol was used to elute out the adsorbed rhamnolipids. Others used chromatographic methods to get high-purity biosurfactants (Lin, 1996). Sim et al. (1997) used column chromatography, with 50 g activated silica gel (230–400 mesh), for separation of a larger volume (10 mL at 500 g/L) of crude rhamnolipid extract into mono- and dirhamnolipid groups. After sample loading, chloroform was first used to wash out all neutral lipids, and then, chloroform/methanol mobile phases were passed sequentially in ratios (v/v) of 50:3 (1000 mL), 50:5 (200 mL), and 50:50 (100 mL). The 50:3 mobile phase eluted all monorhamnolipids, while 50:5 and 50:50 mobile phases eluted dirhamnolipids. Purities of 100% and 95% were reported for mono- and dirhamnolipids, respectively. Miao et al. (2014) used a modified method, with a silica gel column (5 × 60 cm) and a single mobile-phase chloroform/methanol/water (100/23/3, v/v/v, 1.4 L), for purification of 10 mL concentrated crude extract. Two separate peaks appeared after about 0.4 L elution; the first contained monorhamnolipids and the second dirhamnolipids. While chromatographic methods can be used for rhamnolipid purification, they tend to be expensive. Systematic investigation and optimization of the various unit operations for downstream processing, particularly purification of rhamnolipids in large-scale industrial application, are still missing (Weber and Zeiner, 2014).

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5.3.2  Aerobic Fermentations Rhamnolipids are typically produced by aerobic fermentations. Rhamnolipids are secondary metabolites that are majorly produced during the stationary phase induced by exhaustion of at least one essential growth nutrient like N source (Guerra-Santos et al., 1984; Gunther IV et al., 2005; Mulligan et al., 1989), P source (Chayabutra et al., 2001; Mulligan et al., 1989), or trace elements (Manresa et  al., 1991). Water-soluble carbon substrates such as glycerol, glucose, mannitol, and ethanol can be used for production (Müller et al., 2011), but for most P. aeruginosa strains, nonpolar substrates like hydrocarbons and vegetable oils give higher rhamnolipid yields (Sodagari et al., 2018; Trummler et al., 2003). There are however exceptions reported that support strain-dependent preference of carbon substrate; for example, shake-flask cultures of P. aeruginosa EM1 were reported to produce more rhamnolipids with glucose and glycerol, particularly a mixture of both, as the carbon source, over olive oil, oleic acid, and soybean oil (Wu et al., 2008). Various bacterial strains have been employed for rhamnolipid production; most were isolated from oil-contaminated environments. A recent review by Chong and Li (2017) provides a comprehensive list of different rhamnolipid-producing strains used. Some of the more promising P. aeruginosa strains that have been reported to give higher concentrations and/or productivities are listed as examples in Table 5.5. Pseudomonas aeruginosa is however an opportunistic human pathogen with some health and safety concerns, although P. aeruginosa ATCC 9027 has recently been reported to be sensitive to antibiotics and completely avirulent in a mouse model (Grosso-Becerra et al., 2016). Rhamnolipid production by other nonpathogenic Pseudomonas species such as P. chlororaphis, P. putida, and P. fluorescens and bacteria from other genera like Burkholderia (e.g., B. thailandensis) has also been reported. The dominant rhamnolipid congener produced by B. thailandensis is RR-C14-C14 as compared with R-C10-C10 and RR-C10-C10 by P. aeruginosa (Costa et al., 2011; Hörmann et al., 2010). A comparison of percent identity and similarity of RhlA, RhlB, and RhlC proteins produced by B. thailandensis and P. chlororaphis versus P. aeruginosa is given in Table 5.6. This was done using the BlastP tool for comparing the amino acid sequence of RhlA, RhlB, and RhlC proteins produced by P. aeruginosa PAO1 with analogous proteins in the genome of B. thailandensis and P. chlororaphis. P. chlororaphis is deficient in rhlC gene that makes it incapable of producing dirhamnolipids. Most of the papers related to rhamnolipid production using bacteria other than P. aeruginosa focused on the synthesis genes and characterization of the congeners with little or no emphasis on process optimization or production kinetics. A summary of the volumetric TABLE 5.5  Some P. aeruginosa Strains Reported with High Rhamnolipid Concentrations and Productivities

Strain

Carbon Substrate

Maximum Rhamnolipid Concentration (g/L)

Specific Rhamnolipid Productivity (mg/g h)

Volumetric Rhamnolipid Productivity (mg/L h)

Ref.

PAO1

Sunflower oil

36.7

40

430

(Müller et al., 2011)

O-2-2

Soybean oil

70.6

14

730

(Zhu et al., 2012)

E03-40

Soybean oil

42.1

25

220

(Sodagari et al., 2018)

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TABLE 5.6  The Percent Identity and Similarity of RhlA, RhlB, and RhlC Proteins Produced by B. thailandensis and P. chlororaphis as Compared with P. aeruginosa PAO1 RhlA

RhlB

RhlC

% Identity

% Similarity

% Identity

% Similarity

% Identity

% Similarity

Burkholderia thailandensis

48

64

49

61

47

62

Pseudomonas chlororaphis

67

78

43

54

–

–

TABLE 5.7  Volumetric Productivity and Maximum Cell Concentration Reported for Bacteria Other Than P. aeruginosa for Rhamnolipid Production Rhamnolipid Producer

Carbon Substrate

Vol. Productivity (mg/L h)

Max. Cell Cone. (g/L)

Ref.

Burkholderia thailandensis E264

Canola oil

4.7

NA

(Dubeau et al., 2009)

Burkholderia plantarii DSM 9509

Glucose

1.8

5.78

(Hörmann et al., 2010)

Burkholderia glumae AU6208

Canola oil

6.9

NA

(Costa et al., 2011)

Pseudomonas chlororaphis NRRL B-30761

Glucose

8.3

NA

(Gunther IV et al., 2005)

Glucose

a

b

(Tuleva et al., 2002)

b

(Onwosi and Odibo, 2012)

Pseudomonas putida 21BN

25

4.5

Pseudomonas nitroreducens

Glucose

31.4

1.7

Acinetobacter calcoaceticus

Citrate

0.5

NA

(Hoskova et al., 2013)

Enterobacter asburiae

Citrate

0.6

NA

(Hoskova et al., 2013)

Note: The volumetric productivity was calculated as a ratio of final rhamnolipid concentration to the time required for the production. a In terms of rhamnose equivalent. b Cell concentration expressed as optical density at 600 nm.

rhamnolipid productivity and maximum cell concentration extracted from some of these reports is given in Table 5.7. Pseudomonas nitroreducens gave the highest volumetric productivity of 31.4 mg/L h, which was still far lower than the productivities of 220–730 mg/L h from the P. aeruginosa strains reported in Table 5.5. More work in process development is merited to improve the productivity of these bacteria.

5.3.3  Denitrifying Fermentations A different strategy of rhamnolipid production under anaerobic denitrifying conditions was investigated by Ju and coworkers (Chayabutra et al., 2001). This approach helps overcome the excessive foaming problem associated with aerobic fermentations. P. aeruginosa is a facultative aerobe that can utilize nitrate, nitrite, and arginine as alternative terminal electron acceptor (Mercenier et al., 1980; Payne, 1973; Vander Wauven et al., 1984). Nitrate is highly water-soluble and can potentially be delivered to support denitrifying respiration of cells with minimal or no bulk mixing or aeration. The denitrification of P. aeruginosa occurs in sequential steps as follows: NO3− ➔ NO2− ➔ NO (enzyme bound) ➔ N2O ➔ N2. Denitrification

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5.3  Rhamnolipids Production

187

enzymes are generally thought to be negatively affected by oxygen, but aerobic/microaerobic denitrification by P. aeruginosa has been shown to occur (Chen et al., 2003). Accordingly, denitrifying P. aeruginosa fermentation does not need a strictly anaerobic condition. For process monitoring/control, the culture’s switch of respiration modes, between aerobic and denitrifying respiration and when nitrate (and dissolved oxygen) becomes exhausted, can be detected online as step changes in the fluorescence (excitation, 340 nm, and emission, 460 nm) of NADH, that is, the reduced form of intracellular coenzyme nicotinamide adenine dinucleotide (Chen et al., 2003). The fluorometer for NADH fluorescence monitoring is however not commonly used in the fermentation industry. Further, denitrification is less energy-efficient compared with aerobic respiration: the energy yield per electron accepted via denitrification is estimated to be only about 69% of that via aerobic respiration (Chen et al., 2006). Chayabutra et  al. (2001) first reported the rhamnolipid production by denitrifying P.  ­aeruginosa cultures in phosphorous-limited media. The more common strategy of nitrogen limitation to induce stationary-phase production of rhamnolipids was not used because P. aeruginosa could use nitrate as the nitrogen source for cell growth. The specific productivity of rhamnolipids by resting cells suspended in a P-free medium under the denitrification conditions was reported to be about one-third of that in the same P-free medium under aerobic conditions but was similar to that in a N-free medium under aerobic conditions (Chayabutra et al., 2001). More recently, long-term rhamnolipid production under denitrifying conditions was demonstrated using immobilized P. aeruginosa E03-40 cells in a hollow-fiber bioreactor (Pinzon et al., 2013). The polysulfone hollow fibers had cutoff pore sizes of approximately 0.1 μm in the fiber wall, which prevented passage of bacterial cells but allowed permeation of nutrients and products. The setup used is illustrated in Fig. 5.4. The cells were lodged in the extracapillary space (ECS) outside the fibers, while the circulating culture medium was passed through the lumens of fibers. Glycerol was the carbon substrate, and the nitrate concentration in the circulating medium was maintained in 300–1800 mg/L NO3−-N by periodic addition of nitric acid or sodium nitrate. Both glycerol and nitrate concentrations in the ECS varied similarly to those in the circulating medium, confirming high enough nutrient permeation rates to support cell metabolism. On the other hand, owing to the limited permeation rate of rhamnolipid micelles, rhamnolipid concentrations could be significantly higher in the ECS than in the circulating medium. Long-term continuous rhamnolipid production was achieved for more than 3 months. This approach of production using immobilized cells under denitrifying conditions has a major advantage of minimizing oxygen transfer limitation that is commonly encountered in immobilized-cell systems. Hollow-fiber bioreactors are however

FIG.  5.4  Cell immobilization using hollow-fiber bioreactor for rhamnolipid production under denitrifying conditions.

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5.  Rhamnolipids: Pathways, Productivities, and Potential

complicated to prepare and operate and are unsuitable for large-scale production. Future work should focus on developing cell immobilization systems that can be more readily scaled up for long-term continuous rhamnolipid production under denitrifying conditions.

5.3.4  Rhamnolipids Production Using Heterologous Hosts Rhamnolipids can be produced by recombinant heterologous, nonpathogenic hosts such as P. fluorescens, P. putida, and E. coli. A comprehensive review by Dobler et al. (2016) includes a list of genetically engineered microorganisms for rhamnolipid production. Researchers expressed the rhlAB operon from P. aeruginosa in different hosts, using plasmids or transposon-­ mediated combination (Cabrera-Valladares et al., 2006; Wang et al., 2007). It is crucial that the host is effective in producing the precursors of rhamnolipids, namely, the activated rhamnose (dTDP-l-rhamnose) and β-hydroxy fatty acids. The nonpathogenic bacterial strain created by genetic engineering should also be capable of tolerating high concentrations of the overexpressed RhlA, RhlB, and RhlC proteins in order to be employed for rhamnolipid production (Toribio et al., 2010). The growth and rhamnolipid productivity characteristics of two recombinant strains that showed the highest productivity among reported/reviewed recombinant strains are given in Table 5.8. The genetic engineering significantly improved the volumetric productivity, from no larger than 31.4 mg/L h in Table 5.7 to 100–200 mg/L h here in Table 5.8, for bacteria other than P. aeruginosa. These productivities are still lower than but closer to the productivities of 220–730 mg/L h in Table 5.5, from the productive P. aeruginosa strains. While the main purpose of using genetically engineered strains for rhamnolipid production is to avoid pathogenicity and complex quorum-sensing control of P. aeruginosa, there are other secondary reasons. Zhao et al. (2015) cloned the rhlABRI genes into a facultative anaerobic bacteria Pseudomonas stutzeri DQ1 to produce rhamnolipids under denitrifying conditions. They reported the rhamnolipid productivity of the engineered strain under anaerobic denitrifying conditions (up to 1.6 g/L using glycerol as substrate) was comparable with that of the donor strain under aerobic conditions. Solaiman et al. (2015) inserted the rhlC gene into P. chlororaphis (NRRL B-30761) to enable it to produce dirhamnolipids, as only putative rhlA, rhlB, and rhlR genes, but not the rhlC gene, were detected in P. chlororaphis. Koch et al. (1988) inserted the lacZY gene from E. coli to P. aeruginosa, to enable it to utilize lactose and produce rhamnolipids from waste whey. Some researchers have also explored insertion of genes unrelated to rhamnolipid biosynthesis into host bacteria, to improve rhamnolipid production. For example, the Vitreoscilla hemoglobin gene vgb was transmitted using transposon integration into P. aeruginosa NRRL B-771, attempting to improve oxygen utilization under oxygen limitation (Kahraman and Erenler, 2012). TABLE 5.8  Growth and Rhamnolipid Productivity Characteristics of the Most Productive Recombinant Strains with Hosts Other Than P. aeruginosa Bacteria

Type of Gene Transfer

Carbon Substrate

Maximum Cell Volumetric Cone. (g/L) Productivity (mg/L h) Ref.

Pseudomonas putida KT2440

Plasmid with lov and rhlAB

Glucose

23

204

(Beuker et al., 2016)

Pseudomonas putida KCTC1067

Plasmid with rhlABRI

Soybean oil

NA

101

(Cha et al., 2008)

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189

The use of metabolic engineering to modify the central carbon metabolism and direct more carbon flux toward rhamnolipid production has also been investigated. Tiso et  al. (2016) ­reported that for a recombinant P. putida strain with inserted rhlAB operon, the deletion of polyhydroxyalkanoate (PHA) synthesis genes resulted in higher specific productivity of rhamnolipids. PHA and rhamnolipids are metabolically related as they compete for the β-­hydroxy fatty acid derivatives as precursors in both synthesis pathways (Chayabutra and Ju, 2001; Soberon-Chavez et al., 2005). It is therefore not surprising to find higher rhamnolipid production when PHA synthesis is restricted.

5.4  RHAMNOLIPIDS APPLICATIONS Various uses of rhamnolipids have been studied or proposed, including in industrial applications for emulsification, solubilization, detergency, wetting, foaming, dispersion, and antimicrobial and antiadhesive activities; in environmental applications for bioremediation, soil washing, and enhanced oil recovery (Benincasa, 2007; Nguyen et  al., 2008; Wang and Mulligan, 2004; Whang et al., 2008); in agriculture for controlling spread of infection by phytopathogenic fungi such as Phytophthora sp., Pythium sp., Plasmopara sp., and Colletotrichum sp. (Dashtbozorg et al., 2015; De Jonghe et al., 2005; Haba et al., 2003; Kim et al., 2000); and in wound healing (Piljac and Piljac, 2007; Stipcevic et al., 2006) and cosmetics to replace synthetic surfactants and for antimicrobial effects and better healing of skin lesions (Marchant and Banat, 2012). Some are described in more detail in the following sections.

5.4.1  Soil and Water Remediation of Heavy Metals and Pollutants Hydrocarbons and hydrophobic derivatives from the use of petroleum products are contaminants found in soil, groundwater, and surface waters. Hydrocarbons pose health hazards and are toxic to soil microorganisms, plants, and invertebrates (Labud et al., 2007; Mendoza, 1998). Rhamnolipids can facilitate removal and mobilization of hydrocarbon pollutants by solubilizing/emulsifying them in water (Benincasa, 2007; Wang et al., 2007). Rhamnolipidfacilitated removal of different hydrocarbon contaminants is summarized in Table  5.9. For biodegradation, rhamnolipids improve dispersion of hydrophobic compounds in aqueous medium, thereby increasing their availability to microorganisms (Zhang and Miller, 1992), and may increase microbial surface hydrophobicity, thus improving association of microorganisms with hydrophobic compounds (Zhang and Miller, 1994). A more recent study showed that surfactants like rhamnolipids could enhance enzyme activities of hydrogen peroxidases and invertases in the soil, resulting in improved degradation of polycyclic aromatic hydrocarbons and organochlorine pesticides (Wang et al., 2014). Zhong et al. (2016) studied the effects of low-concentration monorhamnolipids on the transport of P. aeruginosa cells, with low or high surface hydrophobicity due to cultivation in glucose- or hexadecane-based medium, in packed columns of glass beads with hydrophobic or hydrophilic surface. Effects were complicated, depending on monorhamnolipid concentration and both the surface hydrophobicity of cells and glass beads. Monorhamnolipids at 20 and, particularly, 40 mg/L enhanced the transport of hexadecane-grown cells through porous media of hydrophobic beads, which was originally very slow due to cell deposition on bead surface presumably driven

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TABLE 5.9  List of Hydrophobic Contaminants Reported With Rhamnolipid-Facilitated Removal Contaminant Class

Pollutant

Ref.

Saturated and unsaturated hydrocarbons

Hydrocarbon mixture

(Scheibenbogen et al., 1994)

Eicosane, pristine

(Sharma et al., 2015)

Polychlorinated biphenyls

(Fiebig et al., 1997)

Acenaphthene

(Sponza and Gök, 2010)

Pyrene

(Bordas et al., 2005; Sponza and Gök, 2010; Cao et al., 2012)

Phenanthrene

(Van Dyke et al., 1993; Maier and Soberon-Chavez, 2000; Zhao et al., 2011; Sharma et al., 2015)

Anthracene

(Van Dyke et al., 1993)

Atrazine

(Singh and Cameotra, 2014)

Aromatic hydrocarbons

Agrochemicals

by ­hydrophobic interaction, although at all tested concentrations (20, 40, and 80 mg/L) the monorhamnolipids reduced the transport rate of glucose-grown cells, which was originally much faster. The study results confirmed the positive effect (and one possible mechanism) of using rhamnolipids for bioremediation of soil contaminated by hydrophobic pollutants. Rhamnolipids can also remove heavy metal contamination by forming complexes with metals such as cadmium, lanthanum, lead, and zinc (Herman et  al., 1995; Tan et  al., 1994). Tan et al. (1994) reported a maximum complexation ratio of 45 mg Cd2+ per g rhamnolipids. Herman et al. (1995) demonstrated using an ion exchange column that rhamnolipids reduced the resin-bound cadmium from 71% to 3%–12% and correspondingly increased aqueous-phase cadmium concentrations. Besides the above counter-ion binding and ion exchange, other mechanisms proposed for heavy metal removal from soil by rhamnolipids include electrostatic interactions (Aşçi et al., 2012; Mulligan, 2005) and incorporation of heavy metals into surfactant micelles (Mulligan et al., 2001). A recent review by Liu et al. (2017) provides an exhaustive list of rhamnolipid applications in hydrocarbon and heavy metal remediation. Most studies of rhamnolipid-assisted soil washing for removal of hydrocarbons and heavy metals were done in laboratories with shake reactors or soil columns. However, there are some foreseeable challenges in field implementation. The strong sorption of rhamnolipids on soil can significantly reduce the aqueous-phase concentration available for solubilization or mobilization of contaminants and bacterial cells. The cost for ex situ rhamnolipid production (by microbial fermentation) remains high, making it difficult to compete with the use of less expensive synthetic surfactants. Studies to understand and resolve these issues and more field studies are warranted.

5.4.2  Antimicrobial and Biofilm Dispersing Activities of Rhamnolipids Surfactants have been proposed to disrupt cell membranes by modification of lipid organization, changing the integral protein arrangement, and disturbing the overall cell equilibrium (Manaargadoo-Catin et al., 2016). Surfactants may interact with cell membrane lipid bilayer

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191

in the following manners: The surfactant molecules may interact with and/or become incorporated in the outer monolayer; these incorporated surfactant molecules may translocate to the inner layer and become distributed in a transmembrane equilibrium between the inner and outer layers; as the membrane layer becomes saturated with surfactant molecules, micelles of mixed surfactant and membrane components may form and result in membrane solubilization (Kragh-Hansen et al., 1998; Lichtenberg et al., 2013). Surfactants that translocate rapidly to the inner monolayer are thought to cause faster solubilization of cell membrane. Surfactants that are ineffective in this translocation can become saturated on the outer monolayer causing a mass imbalance between the two layers and form mixed micelles with only the outer layer components resulting in partial membrane solubilization. Surfactants that do not translocate or translocate slowly to the inner layer are thought to have a slower solubilizing effect (Lichtenberg et al., 2013). In a recent study, rhamnolipids are compared with another common anionic synthetic surfactant, sodium dodecyl sulfate (SDS), for effects on a eukaryotic flagellated alga Ochromonas danica (Invally and Ju, 2017). SDS lysed O. danica cells at concentrations much lower than its CMC, while rhamnolipids showed biolytic effect at concentrations at least close to its CMC. While individual SDS molecules actively interact with cell membrane, rhamnolipids seem to do so only when the solubility limit as individual molecules is reached and micelles start to form. It is also possible that micelle formation is important for rhamnolipids to manifest its cell membrane solubilizing effect. The kinetics of cell membrane damage by the two surfactants, evaluated using a fluorescent dye propidium iodide, also showed rhamnolipids to be much slower in acting, while SDS effect was essentially instantaneous. The slower action of rhamnolipids may be due to slow translocation across lipid bilayer because of the large hydrophilic sugar moiety. Although rhamnolipids were less damaging than SDS to O. danica, the antimicrobial properties of rhamnolipids have been reported for a wide range of microorganisms including bacteria, yeast, fungi, and algae. They can cause cell lysis especially in organisms without protective cell walls (De Jonghe et al., 2005; Stanghellini and Miller, 1997; Varnier et al., 2009). Several studies have also reported the antifouling and antiadhesive properties of rhamnolipids against different microorganisms (Das et al., 2008; Mukherjee et al., 2009; Rodrigues et al., 2006). For examples, Dusane et al. (2010a) showed that rhamnolipids inhibited biofilm formation by Bacillus pumilus at 1.6 mM concentration, and Sodagari et al. (2013) showed that rhamnolipids can effectively reduce attachment of P. aeruginosa, E. coli, and B. subtilis on glass and octadecyltrichlorosilane-modified glass surface at both 10 mg/L and 200 mg/L.

5.4.3  Agricultural Biopesticide Rhamnolipids can potentially replace synthetic agrochemicals for some agricultural applications. Rhamnolipids are very effective in rupturing the wall-less zoospores of pathogenic fungi (oomycetes); for example, as low as 8–20 mg/L, rhamnolipids could almost instantaneously rupture zoospores of Phytophthora sojae (Dashtbozorg et al., 2015), a soilborne pathogen causing severe damages to plants especially soybean plants with annual loss of $1–2 billion (Canaday and Schmitthenner, 2010). Because of their motility in water, zoospores are commonly implicated in the spreading of these pathogens. The minimum inhibitory concentrations of rhamnolipids against zoospores of some phytopathogenic fungi are given in Table 5.10.

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TABLE 5.10  Minimum Rhamnolipid Concentrations Required for Inhibition of Zoospores of Phytopathogenic Fungi Pathogenic Fungi

Minimum Inhibitory Cone. (mg/L) Disease/Plants Affected

Ref.

Cercospora kikuchii

50

Leaf spot and blight seed stain in soybeans

(Kim et al., 2000)

Colletotrichum orbiculare

50

Infection in cucumber plants

(Kim et al., 2000)

Phytophthora capsica

10

Blight in peppers, through stem wound

(Kim et al., 2000)

Phytophthora cryptogea

25

Brown root rot in chicory

(De Jonghe et al., 2005)

Phytophthora sojae

20

Soybean root rot

(Dashtbozorg et al., 2015)

5.5 SUMMARY Rhamnolipid structures, mixture compositions, surfactant properties, and techniques for their characterization are becoming well established. The knowledge on rhamnolipid biosynthesis has also advanced significantly. In P. aeruginosa, it is complex and involves central metabolic pathways, de novo fatty acid biosynthesis, β-oxidation of fatty acids, four QS systems, alternative sigma factors, several two-component regulatory systems, and other global regulators of gene expression. A better understanding of the complex regulatory circuit that controls rhamnolipid synthesis should enable and facilitate future metabolic engineering efforts to optimize and maximize rhamnolipid production. Rhamnolipids have many important applications including some that utilize more unique/effective bioactivities such as wound healing, skin care, and antizoosporic effect. Commercial realization of these applications depends on the technology and cost of rhamnolipid production including purification. While not much technoeconomic analysis information is available in the literature, rhamnolipid cost is generally considered to be still high. It is important to continue addressing the major challenges described. P. aeruginosa remains as the most productive species. While P. aeruginosa is generally considered as an opportunistic pathogen, at least one strain (ATCC 9027) has recently tested nonvirulent in a rat model. Results from other nonpathogenic rhamnolipid-­ producing bacterial species showed low productivity. Genetic engineering significantly improved the productivity from nonpathogenic hosts, but the productivity is still lower than that of effective P. aeruginosa strains. Further developments are warranted.

References Abalos, A., Pinazo, A., Infante, M.R., Casals, M., García, F., Manresa, A., 2001. Physicochemical and antimicrobial properties of new rhamnolipids produced by Pseudomonas aeruginosa AT10 from soybean oil refinery wastes. Langmuir 17, 1367–1371. https://doi.org/10.1021/LA0011735. Abdel-Mawgoud, A.M., Lépine, F., Déziel, E., 2010. Rhamnolipids: diversity of structures, microbial origins and roles. Appl. Microbiol. Biotechnol. 86, 1323–1336. https://doi.org/10.1007/s00253-010-2498-2. Abdel-Mawgoud, A.M., Hausmann, R., Lépine, F., Müller, M.M., Deziel, E., 2011. Rhamnolipids: detection, analysis, biosynthesis, genetic regulation, and bioengineering of production. In: Biosurfactants. Heidelberg, Berlin, pp. 13–55. https://doi.org/10.1007/978-3-642-14490-5_2.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

REFERENCES 193

Abdel-Mawgoud, A.M., Lépine, F., Déziel, E., 2014. A stereospecific pathway diverts β-oxidation intermediates to the biosynthesis of rhamnolipid biosurfactants. Chem. Biol. 21, 156–164. https://doi.org/10.1016/j. chembiol.2013.11.010. Aguirre-Ramirez, M., Medina, G., Gonzalez-Valdez, A., Grosso-Becerra, V., Soberon-Chavez, G., 2012. The Pseudomonas aeruginosa rmlBDAC operon, encoding dTDP-L-rhamnose biosynthetic enzymes, is regulated by the quorum-sensing transcriptional regulator RhlR and the alternative sigma factor sigma S. Microbiology 158, 908–916. https://doi.org/10.1099/mic.0.054726-0. Albus, A.M., Pesci, E.C., Runyen-Janecky, L.J., West, S.E.H., Iglewski, B.H., 1997. Vfr controls quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179, 3928–3935. Aleksic, I., Petkovic, M., Jovanovic, M., Milivojevic, D., Vasiljevic, B., Nikodinovic-Runic, J., Senerovic, L., 2017. Antibiofilm properties of bacterial di-rhamnolipids and their semi-synthetic amide derivatives. Front. Microbiol. 8, https://doi.org/10.3389/fmicb.2017.02454. Allard, S.T., Giraud, M.F., Whitfield, C., Messner, P., Naismith, J.H., 2000. The purification, crystallization and structural elucidation of dTDP-d-glucose 4,6-dehydratase (RmlB), the second enzyme of the dTDP-l-rhamnose synthesis pathway from Salmonella enterica serovar Typhimurium. Acta Crystallogr. D Biol. Crystallogr. 56, 222–225. Aşçi, Y., Açikel, Ü., Açikel, Y.S., 2012. Equilibrium, hysteresis and kinetics of cadmium desorption from sodium-feldspar using rhamnolipid biosurfactant. Environ. Technol. 33, 1857–1868. https://doi.org/10.1080/09593330.2011.650219. Banat, I.M., Satpute, S.K., Cameotra, S.S., Patil, R., Nyayanit, N.V., 2014. Cost effective technologies and renewable substrates for biosurfactants’ production. Front. Microbiol. 5, 1–18. https://doi.org/10.3389/fmicb.2014.00697. Bazire, A., Dufour, A., 2014. The Pseudomonas aeruginosa rhlG and rhlAB genes are inversely regulated and RhlG is not required for rhamnolipid synthesis. BMC Microbiol. 14, 160–169. https://doi.org/10.1186/1471-2180-14-160. Benincasa, M., 2007. Rhamnolipid produced from agroindustrial wastes enhances hydrocarbon biodegradation in contaminated soil. Curr. Microbiol. 54, 445–449. https://doi.org/10.1007/s00284-006-0610-8. Benincasa, M., Contiero, J., Manresa, M.A., Moraes, I.O., 2002. Rhamnolipid production by Pseudomonas aeruginosa LBI growing on soapstock as the sole carbon source. J. Food Eng. 54, 283–288. https://doi.org/10.1016/ S0260-8774(01)00214-X. Beuker, J., Barth, T., Steier, A., Wittgens, A., Rosenau, F., Henkel, M., Hausmann, R., 2016. High titer heterologous rhamnolipid production. AMB Express 6, 4–10. https://doi.org/10.1186/s13568-016-0298-5. Blankenfeldt, W., Asuncion, M., Lam, J.S., Naismith, J.H., 2000a. The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA). EMBO J. 19, 6652–6663. https://doi. org/10.1093/emboj/19.24.6652. Blankenfeldt, W., Giraud, M.F., Leonard, G., Rahim, R., Creuzenet, C., Lam, J.S., Naismith, J.H., 2000b. The purification, crystallization and preliminary structural characterization of glucose-1-phosphate thymidylyltransferase (RmlA), the first enzyme of the dTDP-L-rhamnose synthesis pathway from Pseudomonas aeruginosa. Acta Crystallogr. D Biol. Crystallogr. 56, 1501–1504. Bollinger, N., Hassett, D.J., Iglewski, B.H., Costerton, J.W., McDermott, T.R., 2001. Gene expression in Pseudomonas aeruginosa: evidence of iron override effects on quorum sensing and biofilm-specific gene regulation. J. Bacteriol. 183, 1990–1996. https://doi.org/10.1128/JB.183.6.1990-1996.2001. Bordas, F., Lafrance, P., Villemur, R., 2005. Conditions for effective removal of pyrene from an artificially contaminated soil using Pseudomonas aeruginosa 57SJ rhamnolipids. Environ. Pollut. 138, 69–76. https://doi.org/10.1016/j. envpol.2005.02.017. Branny, P., Pearson, J.P., Pesci, E.C., Kohler, T., Iglewski, B.H., Van Delden, C., 2001. Inhibition of quorum sensing by a Pseudomonas aeruginosa dksA homologue. J. Bacteriol. 183, 1531–1539. https://doi.org/10.1128/ JB.183.5.1531-1539.2001. Brint, J.M., Ohman, D.E., 1995. Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family. J. Bacteriol. 177, 7155–7163. Burger, M.M., Glaser, L., Burton, R.M., 1966. Formation of rhamnolipids of Pseudomonas aeruginosa. Methods Enzymol. 8, 441–445. Byrd, M.S., Sadovskaya, I., Vinogradov, E., Lu, H., Sprinkle, A.B., Richardson, S.H., Ma, L., Ralston, B., Parsek, M.R., Anderson, E.M., Lam, J.S., Wozniak, D.J., 2009. Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production. Mol. Microbiol. 73, 622–638. https://doi.org/10.1111/j.1365-2958.2009.06795.x.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

194

5.  Rhamnolipids: Pathways, Productivities, and Potential

Cabrera-Valladares, N., Richardson, A.-P., Olvera, C., Treviño, L.G., Déziel, E., Lépine, F., Soberon-Chavez, G., 2006. Monorhamnolipids and 3(3-hydroxyalkanoyloxy) alkanoic acids (HAAs) production using Escherichia coli as a heterologous host. Appl. Microbiol. Biotechnol. 73, 187–194. https://doi.org/10.1007/s00253-006-0468-5. Cameotra, M.S.S., 1998. Synthesis of biosurfactants in extreme conditions. Appl. Microbiol. Biotechnol. 50, 520–529. Campos-Garcia, G., Caro, A.D., Najera, R., Miller-Maier, M., Al-Tahhan, A., Soberon-Chavez, G., 1998. The Pseudomonas aeruginosa rhlG gene encodes an NADPH-dependent beta-ketoacyl reductase which is specifically involved in rhamnolipid synthesis. J. Bacteriol. 180, 4442–4451. Canaday, C.H., Schmitthenner, A.F., 2010. Effects of chloride and ammonium salts on the incidence of Phytophthora root and stem rot of soybean. Plant Dis. 94, 758–765. https://doi.org/10.1094/PDIS-94-6-0758. Cao, H., Krishnan, G., Goumnerov, B., Tsongalis, J., Tompkins, R., Rahme, L.G., 2001. A quorum sensing-­associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-­regulatory mechanism. Proc. Natl. Acad. Sci. U. S. A. 98, 14613–14618. https://doi.org/10.1073/pnas.251465298. Cao, L., Wang, Q., Zhang, J., Li, C., Yan, X., Lou, X., Xia, Y., Hong, Q., Li, S., 2012. Construction of a stable genetically engineered rhamnolipid-producing microorganism for remediation of pyrene-contaminated soil. World J. Microbiol. Biotechnol. 28, 2783–2790. https://doi.org/10.1007/s11274-012-1088-0. Cha, M., Lee, N., Kim, M., Kim, M., Lee, S., 2008. Heterologous production of Pseudomonas aeruginosa EMS1 biosurfactant in Pseudomonas putida. Bioresour. Technol. 99, 2192–2199. https://doi.org/10.1016/j.biortech.2007.05.035. Chayabutra, C., Ju, L.-K., 2001. Polyhydroxyalkanoic acids and rhamnolipids are synthesized sequentially in hexadecane fermentation by Pseudomonas aeruginosa ATCC 10145. Biotechnol. Prog. 17, 419–423. https://doi. org/10.1021/bp010036a. Chayabutra, C., Wu, J., Ju, L.-K., 2001. Rhamnolipid production by Pseudomonas aeruginosa under denitrification: effects of limiting nutrients and carbon substrates. Biotechnol. Bioeng. 72, 25–33. https://doi. org/10.1002/1097-0290(20010105)72:13.0.CO;2-J. Chen, F., Xia, Q., Ju, L., 2003. Aerobic denitrification of Pseudomonas aeruginosa monitored by online NAD(P)H fluorescence. Appl. Environ. Microbiol. 69, 6715–6722. https://doi.org/10.1128/AEM.69.11.6715. Chen, F., Xia, Q., Ju, L.-K., 2006. Competition between oxygen and nitrate respirations in continuous culture of Pseudomonas aeruginosa performing aerobic denitrification. Biotechnol. Bioeng. 93, 1069–1078. https://doi. org/10.1002/bit. Chong, H., Li, Q., 2017. Microbial production of rhamnolipids: opportunities, challenges and strategies. Microb. Cell Factories 16, 137https://doi.org/10.1186/s12934-017-0753-2. Costa, S.G.V.A.O., Déziel, E., Lépine, F., 2011. Characterization of rhamnolipid production by Burkholderia glumae. Lett. Appl. Microbiol. 53, 620–627. https://doi.org/10.1111/j.1472-765X.2011.03154.x. Croda-García, G., Grosso-Becerra, V., Gonzalez-Valdez, A., Servín-González, L., Soberon-Chavez, G., 2011. Transcriptional regulation of Pseudomonas aeruginosa rhlR: role of the CRP orthologue Vfr (virulence factor regulator) and quorum-sensing regulators LasR and RhlR. Microbiology 157, 2545–2555. https://doi.org/10.1099/ mic.0.050161-0. Das, P., Mukherjee, S., Sen, R., 2008. Antimicrobial potential of a lipopeptide biosurfactant derived from a marine Bacillus circulans. J. Appl. Microbiol. 104, 1675–1684. https://doi.org/10.1111/j.1365-2672.2007.03701.x. Dashtbozorg, S.S., Miao, S., Ju, L., 2015. Rhamnolipids as environmentally friendly biopesticide against plant pathogen. Environ. Prog. 28, 404–409. https://doi.org/10.1002/ep. Dashtbozorg, S.S., Kohl, J., Ju, L.-K., 2016. Rhamnolipid adsorption in soil: factors, unique features, and considerations for use as green antizoosporic agents. J. Agric. Food Chem. 64, 3330–3337. https://doi.org/10.1021/acs. jafc.6b00215. De Jonghe, K., De Dobbelaere, I., Sarrazyn, R., Höfte, M., Hofte, M., 2005. Control of Phytophthora cryptogea in the hydroponic forcing of witloof chicory with the rhamnolipid-based biosurfactant formulation PRO1. Plant Pathol. 54, 219–226. https://doi.org/10.1111/j.1365-3059.2005.01140.x. De Kievit, T.R., Seed, P.C., Nezezon, J., Passador, L., Iglewski, B.H., 1999. RsaL, a novel repressor of virulence gene expression in Pseudomonas aeruginosa. J. Bacteriol. 181, 2175–2184. De Kievit, T.R., Kakai, Y., Register, J.K., Pesci, E.C., Iglewski, B.H., 2002. Role of the Pseudomonas aeruginosa las and rhl quorum-sensing systems in rhlI regulation. FEMS Microbiol. Lett. 212, 101–106. Dekimpe, V., Deziel, E., 2009. Revisiting the quorum-sensing hierarchy in Pseudomonas aeruginosa: the transcriptional regulator RhlR regulates LasR-specific factors. Microbiology 155, 712–723. Deziel, E., Lepine, F., Dennie, D., Boismenu, D., Mamer, O.A., Villemur, R., 1999. Liquid chromatography/mass spectrometry analysis of mixtures of rhamnolipids produced by Pseudomonas aeruginosa strain 57RP grown on

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

REFERENCES 195

mannitol or naphthalene. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1440, 244–252. https://doi.org/10.1016/ S1388-1981(99)00129-8. Deziel, E., Lepine, F., Milot, S., Villemur, R., 2003. rhlA is required for the production of a novel biosurfactant promoting swarming motility in Pseudomonas aeruginosa: 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors of rhamnolipids. Microbiology 149, 2005–2013. https://doi.org/10.1099/mic.0.26154-0. Deziel, E., Lepine, F., Milot, S., He, J., Mindrinos, M.N., Tompkins, R.G., Rahme, L.G., 2004. Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell communication. Proc. Natl. Acad. Sci. U. S. A. 101, 1339–1344. Diggle, S.P., Matthijs, S., Wright, V.J., Fletcher, M.P., Chhabra, S.R., Lamont, I.L., Kong, X., Hider, R.C., Cornelis, P., Camara, M., Williams, P., 2007a. The Pseudomonas aeruginosa 4-quinolone signal molecules HHQ and PQS play multifunctional roles in quorum sensing and iron entrapment. Chem. Biol. 14, 87–96. https://doi.org/10.1016/j. chembiol.2006.11.014. Diggle, S.P., Griffin, A.S., Campbell, G.S., West, S.A., 2007b. Cooperation and conflict in quorum-sensing bacterial populations. Nature 450, 411–414. https://doi.org/10.1038/nature06279. Dobler, L., Vilela, L.F., Almeida, R.V., Neves, B.C., 2016. Rhamnolipids in perspective: gene regulatory pathways, metabolic engineering, production and technological forecasting. New Biotechnol. 33, 123–135. https://doi. org/10.1016/j.nbt.2015.09.005. Dubeau, D., Déziel, E., Woods, D.E., Lépine, F., 2009. Burkholderia thailandensis harbors two identical rhl gene clusters responsible for the biosynthesis of rhamnolipids. BMC Microbiol. 9, 263. https://doi.org/10.1186/1471-2180-9-263. Dubern, J.F., Diggle, S.P., 2008. Quorum sensing by 2-alkyl-4-quinolones in Pseudomonas aeruginosa and other bacterial species. Mol. BioSyst. 4, 882–888. https://doi.org/10.1039/b803796p. Dusane, D.H., Nancharaiah, Y.V., Zinjarde, S.S., Venugopalan, V.P., 2010a. Rhamnolipid mediated disruption of marine Bacillus pumilus biofilms. Colloids Surf. B Biointerfaces 81, 242–248. https://doi.org/10.1016/j. colsurfb.2010.07.013. Dusane, D.H., Zinjarde, S.S., Venugopalan, V.P., Jc, R., Weber, M.M., Rahman, P.K.S.M., 2010b. Quorum sensing: implications on rhamnolipid biosurfactant production. Biotechnol. Genet. Eng. Rev. 27, 159–184. https://doi.org /10.1080/02648725.2010.10648149. Eberhard, A., 1972. Inhibition and activation of bacterial luciferase synthesis. J. Bacteriol. 109, 1101–1105. Edwards, J.R., Hayashi, J.A., 1965. Structure of a rhamnolipid from Pseudomonas aeruginosa. Arch. Biochem. Biophys. 111, 415–421. Etoc, A., Delvigne, F., Lecomte, J.P., Thonart, P., 2006. Foam control in fermentation bioprocess. Appl. Biochem. Biotechnol. 130, 392–404. https://doi.org/10.1385/ABAB:130:1:392. Fiebig, R., Schulze, D., Chung, J.-C., Lee, S.-T., 1997. Biodegradation of polychlorinated biphenyls (PCBs) in the presence of a bioemulsifier produced on sunflower oil. Biodegradation 8, 67–75. https://doi.org/10.102 3/A:1008256110136. Fuqua, W.C., Winans, S.C., Greenberg, E.P., 1994. Quorum sensing in bacteria: the LuxR-LuxI family of cell density-­ responsive transcriptional regulators. J. Bacteriol. 176, 269–275. Gallagher, L.A., McKnight, S.L., Kuznetsova, M.S., Pesci, E.C., Manoil, C., 2002. Functions required for extracellular quinolone signaling by Pseudomonas aeruginosa. J. Bacteriol. 184, 6472–6480. Gambello, M.J., Iglewski, B.H., 1991. Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression. J. Bacteriol. 173, 3000–3009. George, S., Jayachandran, K., 2009. Analysis of rhamnolipid biosurfactants produced through submerged fermentation using orange fruit peelings as sole carbon source. Appl. Biochem. Biotechnol. 158, 694–705. https://doi. org/10.1007/s12010-008-8337-6. George, S., Jayachandran, K., 2013. Production and characterization of rhamnolipid biosurfactant from waste frying coconut oil using a novel Pseudomonas aeruginosa D. J. Appl. Microbiol. 114, 373–383. https://doi.org/10.1111/ jam.12069. Giraud, M.F., Naismith, J.H., 2000. The rhamnose pathway. Curr. Opin. Struct. Biol. 10, 687–696. Glick, R., Gilmour, C., Tremblay, J., Satanower, S., Avidan, O., Deziel, E., Greenberg, E.P., Poole, K., Banin, E., 2010. Increase in rhamnolipid synthesis under iron-limiting conditions influences surface motility and biofilm formation in Pseudomonas aeruginosa. J. Bacteriol. 192, 2973–2980. https://doi.org/10.1128/JB.01601-09. Graninger, M., Nidetzky, B., Heinrichs, D.E., Whitfield, C., Messner, P., 1999. Characterization of dTDP-4-­ dehydrorhamnose 3,5-epimerase and dTDP-4-dehydrorhamnose reductase, required for dTDP-L-rhamnose biosynthesis in Salmonella enterica serovar Typhimurium LT2. J. Biol. Chem. 274, 25069–25077.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

196

5.  Rhamnolipids: Pathways, Productivities, and Potential

Grosso-Becerra, M.V., González-Valdez, A., Granados-Martínez, M.J., Morales, E., Servín-González, L., Méndez, J.L., Delgado, G., Morales-Espinosa, R., Ponce-Soto, G.-Y., Cocotl-Yanez, M., Soberon-Chavez, G., 2016. Pseudomonas aeruginosa ATCC 9027 is a non-virulent strain suitable for mono-rhamnolipids production. Appl. Microbiol. Biotechnol. 100, 9995–10004. https://doi.org/10.1007/s00253-016-7789-9. Guerra-Santos, L., Käppeli, O., Fiechter, A., 1984. Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source. Appl. Environ. Microbiol. 48, 301–305. Gunther IV, N.W., Nunez, A., Fett, W., Solaiman, D.K.Y., 2005. Production of rhamnolipids by Pseudomonas chlororaphis, a nonpathogenic bacterium. Appl. Environ. Microbiol. 71, 718–725. https://doi.org/10.1007/s00253-005-0150-3. Haba, E., Pinazo, A., Jauregui, O., Espuny, M.J., Infante, M.R., Manresa, A., 2003. Physicochemical characterization and antimicrobial properties of rhamnolipids produced by Pseudomonas aeruginosa 47T2 NCBIM 40044. Biotechnol. Bioeng. 81, 316–322. https://doi.org/10.1002/bit.10474. Hamzah, A., Sabturani, N., Radiman, S., 2013. Screening and optimization of biosurfactant production by hydrocarbon-­degrading bacteria. Sains Malaysiana 42, 615–623. https://doi.org/10.1016/j.fuel.2014.12.003. Hauser, G., Karnovsky, M.L., 1957. Rhamnose and rhamnolipids biosynthesis by Pseudomonas aeruginosa. J. Biol. Chem. 224, 91–105. Henkel, M., Müller, M.M., Kügler, J.H., Lovaglio, R.B., Contiero, J., Syldatk, C., Hausmann, R., 2012. Rhamnolipids as biosurfactants from renewable resources: concepts for next-generation rhamnolipid production. Process Biochem. 47, 1207–1219. https://doi.org/10.1016/j.procbio.2012.04.018. Herman, D.C., Artiola, J.F., Miller, R.M., 1995. Removal of cadmium, lead, and zinc from soil by a rhamnolipid biosurfactant. Environ. Sci. Technol. 29, 2280–2285. https://doi.org/10.1021/es00009a019. Heurlier, K., Denervaud, V., Pessi, G., Reimmann, C., Haas, D., 2003. Negative control of quorum sensing by RpoN (sigma54) in Pseudomonas aeruginosa PAO1. J. Bacteriol. 185, 2227–2235. Hori, K., Ichinohe, R., Unno, H., Marsudi, S., 2011. Simultaneous syntheses of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa IFO3924 at various temperatures and from various fatty acids. Biochem. Eng. J. 53, 196–202. https://doi.org/10.1016/j.bej.2010.10.011. Hörmann, B., Müller, M.M., Syldatk, C., Hausmann, R., 2010. Rhamnolipid production by Burkholderia plantarii DSM 9509T. Eur. J. Lipid Sci. Technol. 112, 674–680. https://doi.org/10.1002/ejlt.201000030. Hoskova, M., Schreiberova, O., Jezdík, R., Chudoba, J., Masak, J., Sigler, K., Rezanka, T., 2013. Characterization of rhamnolipids produced by non-pathogenic Acinetobacter and Enterobacter bacteria. Bioresour. Technol. 130, 510– 516. https://doi.org/10.1016/j.biortech.2012.12.085. Hubert, J., Ple, K., Hamzaoui, M., Nuissier, G., Hadef, I., Reynaud, R., Guilleret, A., Renault, J.H., 2012. New perspectives for microbial glycolipid fractionation and purification processes. C. R. Chim. 15, 18–28. https://doi. org/10.1016/j.crci.2011.11.002. Invally, K., Ju, L.-K., 2017. Biolytic effect of rhamnolipid biosurfactant and dodecyl sulfate against phagotrophic alga Ochromonas danica. J. Surfactant Deterg. 20, 1161–1171. https://doi.org/10.1007/s11743-017-2005-1. Jarvis, F.G., Johnson, M.J., 1949. A glycolipide produced by Pseudomonas aeruginosa. J. Am. Chem. Soc. 71, 4124–4126. https://doi.org/10.1021/ja01180a073. Jensen, V., Lons, D., Zaoui, C., Bredenbruch, F., Meissner, A., Dieterich, G., Munch, R., Haussler, S., 2006. RhlR ­expression in Pseudomonas aeruginosa is modulated by the Pseudomonas quinolone signal via PhoB-dependent and independent pathways. J. Bacteriol. 188, 8601–8606. https://doi.org/10.1128/JB.01378-06. Jude, F., Kohler, T., Branny, P., Perron, K., Mayer, M.P., Comte, R., Van Delden, C., 2003. Posttranscriptional control of quorum-sensing-dependent virulence genes by DksA in Pseudomonas aeruginosa. J. Bacteriol. 185, 3558–3566. Kahraman, H., Erenler, S.O., 2012. Rhamnolipid production by Pseudomonas aeruginosa engineered with the Vitreoscilla hemoglobin gene. Appl. Biochem. Biotechnol. 48, 212–217. https://doi.org/10.1134/S000368381202007X. Kang, Y., Zarzycki-Siek, J., Walton, C.B., Norris, M.H., Hoang, T.T., 2010. Multiple FadD acyl-CoA synthetases contribute to differential fatty acid degradation and virulence in Pseudomonas aeruginosa. PLoS One 5, e13557 https:// doi.org/10.1371/journal.pone.0013557. Kim, B.S., Lee, J.Y., Hwang, B.K., Andrieu, N., Genet, J.L., Jaworska, G., Bompeix, G., 2000. In vivo control and in vitro antifungal activity of rhamnolipid B, a glycolipid antibiotic, against Phytophthora capsici and Colletotrichum orbiculare. Pest Manag. Sci. 56, 1029–1035. https://doi.org/10.1002/1526-4998(200012)56:123.0.CO;2-Q. Kłosowska-Chomiczewska, I.E., Medryzycka, K., Hallmann, E., Karpenko, E., Pokynbroda, T., Macierzanka, A., Jungnickel, C., 2017. Rhamnolipid CMC prediction. J. Colloid Interface Sci. 488, 10–19. https://doi.org/10.1016/j. jcis.2016.10.055.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

REFERENCES 197

Koch, A.K., Reiser, J., Käppeli, O., Fiechter, A., 1988. Genetic construction of lactose utilizing strains of Pseudomonas aeruginosa and their application in biosurfactant production. Nat. Biotechnol. 6, 1335. https://doi.org/10.1038/ nbt1188-1335. Kragh-Hansen, U., Le Maire, M., Moller, J.V., 1998. The mechanism of detergent solubilization of liposomes and protein-­containing membranes. Biophys. J. 75, 2932–2946. https://doi.org/10.1016/S0006-3495(98)77735-5. Labud, V., Garcia, C., Hernandez, T., 2007. Effect of hydrocarbon pollution on the microbial properties of a sandy and a clay soil. Chemosphere 66, 1863–1871. https://doi.org/10.1016/j.chemosphere.2006.08.021. Lapouge, K., Schubert, M., Allain, F.H., Haas, D., 2008. Gac/Rsm signal transduction pathway of gamma-­ proteobacteria: from RNA recognition to regulation of social behaviour. Mol. Microbiol. 67, 241–253. https://doi. org/10.1111/j.1365-2958.2007.06042.x. Latifi, A., Foglino, M., Tanaka, K., Williams, P., Lazdunski, A., 1996. A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhlR (VsmR) to expression of the stationary-­ phase sigma factor RpoS. Mol. Microbiol. 21, 1137–1146. Laville, J., Voisard, C., Keel, C., Maurhofer, M., Defago, G., Haas, D., 1992. Global control in Pseudomonas fluorescens mediating antibiotic synthesis and suppression of black root rot of tobacco. Proc. Natl. Acad. Sci. U. S. A. 89, 1562–1566. Lebrón-Paler, A., Pemberton, J.E., Becker, B.A., Otto, W.H., Larive, C.K., Maier, R.M., Otto, W.H., Larive, C.K., Maier, R.M., 2006. Determination of the acid dissociation constant of the biosurfactant monorhamnolipid in aqueous solution by potentiometric and spectroscopic methods. Anal. Chem. 78, 7649–7658. https://doi.org/10.1021/ ac0608826. Ledgham, F., Ventre, I., Soscia, C., Foglino, M., Sturgis, J.N., Lazdunski, A., 2003. Interactions of the quorum sensing regulator QscR: interaction with itself and the other regulators of Pseudomonas aeruginosa LasR and RhlR. Mol. Microbiol. 48, 199–210. Lee, J., Zhang, L., 2015. The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell 6, 26–41. https://doi.org/10.1007/s13238-014-0100-x. Lee, J.H., Lequette, Y., Greenberg, E.P., 2006. Activity of purified QscR, a Pseudomonas aeruginosa orphan quorum-­ sensing transcription factor. Mol. Microbiol. 59, 602–609. https://doi.org/10.1111/j.1365-2958.2005.04960.x. Lee, J., Wu, J., Deng, Y., Wang, J., Wang, C., Wang, J., Chang, C., Dong, Y., Williams, P., Zhang, L.-H., 2013. A cellcell communication signal integrates quorum sensing and stress response. Nat. Chem. Biol. 9, 339. https://doi. org/10.1038/nchembio.1225. https://www.nature.com/articles/nchembio.1225#supplementary-information. Lépine, F., Déziel, E., Milot, S., Villemur, R., 2002. Liquid chromatographic/mass spectrometric detection of the 3-(3-hydroxyalkanoyloxy)alkanoic acid precursors of rhamnolipids in Pseudomonas aeruginosa cultures. J. Mass Spectrom. 37, 41–46. https://doi.org/10.1002/jms.244. Lequette, Y., Lee, J.H., Ledgham, F., Lazdunski, A., Greenberg, E.P., 2006. A distinct QscR regulon in the Pseudomonas aeruginosa quorum-sensing circuit. J. Bacteriol. 188, 3365–3370. https://doi.org/10.1128/JB.188.9.3365-3370.2006. Lessie, T., Neidhardt, F.C., 1967. Adenosine triphosphate-linked control of Pseudomonas aeruginosa glucose-6-­ phosphate dehydrogenase. J. Bacteriol. 93, 1337–1345. Lessie, T.G.G., Phibbs Jr., P.V., Phibbs, P.V., 1984. Alternative pathways of carbohydrate utilization in Pseudomonads. Annu. Rev. Microbiol. 38, 359–388. https://doi.org/10.1146/annurev.micro.38.1.359. Li, Q.X., 2017. Rhamnolipid synthesis and production with diverse resources. Front. Chem. Sci. Eng. 11, 27–36. https://doi.org/10.1007/s11705-016-1607-x. Liang, H., Deng, X., Ji, Q., Sun, F., Shen, T., He, C., 2012. The Pseudomonas aeruginosa global regulator VqsR directly inhibits QscR to control quorum-sensing and virulence gene expression. J. Bacteriol. 194, 3098–3108. https://doi. org/10.1128/JB.06679-11. Lichtenberg, D., Ahyayauch, H., Goñi, F.M., 2013. The mechanism of detergent solubilization of lipid bilayers. Biophys. J. 105, 289–299. https://doi.org/10.1016/j.bpj.2013.06.007. Lin, S., 1996. Review biosurfactants: recent advances. J. Chem. Technol. Biotechnol. 66, 109–120. Lindhout, T., Lau, P.C., Brewer, D., Lam, J.S., 2009. Truncation in the core oligosaccharide of lipopolysaccharide affects flagella-mediated motility in Pseudomonas aeruginosa PAO1 via modulation of cell surface attachment. Microbiology 155, 3449–3460. https://doi.org/10.1099/mic.0.030510-0. Liu, G., Zhong, H., Yang, X., Liu, Y., Shao, B., Liu, Z., 2017. Advances in applications of rhamnolipids biosurfactant in environmental remediation: a review. Biotechnol. Bioeng. 115, 1–19. https://doi.org/10.1002/bit.26517. Long, X., Sha, R., Meng, Q., Zhang, G., 2016. Mechanism study on the severe foaming of rhamnolipid in fermentation. J. Surfactant Deterg. 19, 833–840. https://doi.org/10.1007/s11743-016-1829-4.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

198

5.  Rhamnolipids: Pathways, Productivities, and Potential

Maier, R.M., Soberon-Chavez, G., 2000. Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications. Appl. Microbiol. Biotechnol. 54, 625–633. https://doi.org/10.1007/s002530000443. Manaargadoo-Catin, M., Ali-Cherif, A., Pougnas, J.L., Perrin, C., 2016. Hemolysis by surfactants—a review. Adv. Colloid Interf. Sci. 228, 1–16. https://doi.org/10.1016/j.cis.2015.10.011. Manresa, M.A., Bastida, J., Mercadé, M.E., Robert, M., De Andrés, C., Espuny, M.J., Guinea, J., 1991. Kinetic studies on surfactant production by Pseudomonas aeruginosa 44T1. J. Ind. Microbiol. 8, 133–136. https://doi.org/10.1007/ BF01578765. Marchant, R., Banat, I.M., 2012. Microbial biosurfactants: challenges and opportunities for future exploitation. Trends Biotechnol. 30, 558–565. https://doi.org/10.1016/j.tibtech.2012.07.003. McKnight, S.L., Iglewski, B.H., Pesci, E.C., 2000. The Pseudomonas quinolone signal regulates rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 182, 2702–2708. Medina, G., Juarez, K., Diaz, R., Soberon-Chavez, G., 2003a. Transcriptional regulation of Pseudomonas aeruginosa rhlR, encoding a quorum-sensing regulatory protein. Microbiology 149, 3073–3081. https://doi.org/10.1099/ mic.0.26282-0. Medina, G., Juarez, K., Soberon-Chavez, G., 2003b. The Pseudomonas aeruginosa rhlAB operon is not expressed during the logarithmic phase of growth even in the presence of its activator RhlR and the autoinducer N-butyrylhomoserine lactone. J. Bacteriol. 185, 377–380. https://doi.org/10.1128/JB.185.1.377-380.2003. Mendoza, R.E., 1998. Hydrocarbon leaching, microbial population, and plant growth in soil amended with petroleum. Biorem. J. 1, 223–231. https://doi.org/10.1080/10889869809351337. Mercad, M.E., Manresa, M.A.A., Robert, M., Espuny, M.J.J., de Andrés, C., Guinea, J., 1993. Olive oil mill effluent (OOME). New substrate for biosurfactant production. Bioresour. Technol. 43, 1–6. https://doi. org/10.1016/0960-8524(93)90074-L. Mercenier, A., Simon, J.P., Vander Wauven, C., Haas, D., Stalon, V., 1980. Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa. J. Bacteriol. 144, 159–163. Miao, S., Callow, N., Soltani Dashtbozorg, S., Salager, J.L., Ju, L.K., 2014. Ethylation of di-rhamnolipids: a green route to produce novel sugar fatty acid nonionic surfactants. J. Surfactant Deterg. 17, 1069–1080. https://doi. org/10.1007/s11743-014-1641-y. Miller, D.J., Zhang, Y.M., Rock, C.O., White, S.W., 2006. Structure of RhlG, an essential beta-ketoacyl reductase in the rhamnolipid biosynthetic pathway of Pseudomonas aeruginosa. J. Biol. Chem. 281, 18025–18032. https://doi. org/10.1074/jbc.M601687200. Moya, I., Tsaousi, K., Rudden, M., Marchant, R., Jurado, E., García, M., Banat, I.M., 2015. Rhamnolipid and surfactin production from olive oil mill waste as sole carbon source. Bioresour. Technol. 198, 231–236. https://doi. org/10.1016/j.biortech.2015.09.012. Mukherjee, S., Das, P., Sen, R., 2006. Towards commercial production of microbial surfactants. Trends Biotechnol. 24, 509–515. https://doi.org/10.1016/j.tibtech.2006.09.005. Mukherjee, S., Das, P., Sivapathasekaran, C., Sen, R., 2009. Antimicrobial biosurfactants from marine Bacillus circulans: extracellular synthesis and purification. Lett. Appl. Microbiol. 48, 281–288. https://doi. org/10.1111/j.1472-765X.2008.02485.x. Müller, M.M., Hausmann, R., 2011. Regulatory and metabolic network of rhamnolipid biosynthesis: traditional and advanced engineering towards biotechnological production. Appl. Microbiol. Biotechnol. 91, 251–264. https:// doi.org/10.1007/s00253-011-3368-2. Müller, M.M., Hörmann, B., Syldatk, C., Hausmann, R., 2010. Pseudomonas aeruginosa PAO1 as a model for rhamnolipid production in bioreactor systems. Appl. Microbiol. Biotechnol. 87, 167–174. https://doi.org/10.1007/ s00253-010-2513-7. Müller, M.M., Hörmann, B., Kugel, M., Syldatk, C., Hausmann, R., 2011. Evaluation of rhamnolipid production capacity of Pseudomonas aeruginosa PAO1 in comparison to the rhamnolipid over-producer strains DSM 7108 and DSM 2874. Appl. Microbiol. Biotechnol. 89, 585–592. https://doi.org/10.1007/s00253-010-2901-z. Müller, M.M., Kügler, J.H., Henkel, M., Gerlitzki, M., Hörmann, B., Pöhnlein, M., Syldatk, C., Hausmann, R., 2012. Rhamnolipids-Next generation surfactants? J. Biotechnol. 162, 366–380. https://doi.org/10.1016/j. jbiotec.2012.05.022. Mulligan, C.N., 2005. Environmental applications for biosurfactants. Environ. Pollut. 133, 183–198. https://doi. org/10.1016/j.envpol.2004.06.009. Mulligan, C.N., Gibbs, B.F., 1989. Correlation of nitrogen metabolism with biosurfactant production by Pseudomonas aeruginosa. Appl. Environ. Microbiol. 55, 3016–3019.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

REFERENCES 199

Mulligan, C.N., Gibbs, B.F., 1993. Factors influencing the economics of biosurfactants. In: Biosurfactants: Production, Properties and Applications. Mercel Dekker Inc., New York, pp. 329–371. Mulligan, C.N., Mahmourides, G., Gibbs, B.F., 1989. The influence of phosphate metabolism on biosurfactant production by Pseudomonas aeruginosa. J. Biotechnol. 12, 199–210. https://doi.org/10.1016/0168-1656(89)90041-2. Mulligan, C.N., Yong, R.N., Gibbs, B.F., 2001. Heavy metal removal from sediments by biosurfactants. J. Hazard. Mater. 85, 111–125. https://doi.org/10.1016/S0304-3894(01)00224-2. Nealson, K.H., 1977. Autoinduction of bacterial luciferase. Occurrence, mechanism and significance. Arch. Microbiol. 112, 73–79. Nealson, K.H., Platt, T., Hastings, J.W., 1970. Cellular control of the synthesis and activity of the bacterial luminescent system. J. Bacteriol. 104, 313–322. Nguyen, T.T., Youssef, N.H., McInerney, M.J., Sabatini, D.A., 2008. Rhamnolipid biosurfactant mixtures for environmental remediation. Water Res. 42, 1735–1743. https://doi.org/10.1016/j.watres.2007.10.038. Nicolo, M.S., Cambria, M.G., Impallomeni, G., Rizzo, M.G., Pellicorio, C., Ballistreri, A., Guglielmino, S.P.P., 2017. Carbon source effects on the mono/dirhamnolipid ratio produced by Pseudomonas aeruginosa L05, a new human respiratory isolate. New Biotechnol. 39, 36–41. https://doi.org/10.1016/j.nbt.2017.05.013. Nitschke, M., Costa, S., Contiero, J., 2005a. Rhamnolipid surfactants: an update on the general aspects of these remarkable biomolecules. Biotechnol. Prog. 21, 1593–1600. https://doi.org/10.1021/bp050239p. Nitschke, M., Costa, S., Haddad, R., Goncalves, L.A.G., Eberlin, M.N., Contiero, J., 2005b. Oil Wastes as unconventional substrates for rhamnolipid biosurfactant production by Pseudomonas aeruginosa LBI. Biotechnol. Prog. 21, 1562–1566. https://doi.org/10.1021/bp050198x. Ochsner, U.A., Reiser, J., 1995. Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 92, 6424–6428. https://doi.org/10.1073/pnas.92.14.6424. Ochsner, U.A., Fiechter, A., Reiser, J., 1994a. Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J. Biol. Chem. 269, 19787–19795. Ochsner, U.A., Koch, A.K., Fiechter, A., Reiser, J., 1994b. Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa. J. Bacteriol. 176, 2044–2054. https://doi. org/10.1128/jb.176.7.2044-2054.1994. Ochsner, U.A., Hembach, T., Fiechter, A., 1995. Production of rhamnolipid biosurfactants. In: Downstream Processing Biosurfactants Carotenoids. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 89–118. https://doi.org/10.1007/ BFb0102326. Oglesby, A.G., Farrow 3rd, J.M., Lee, J.H., Tomaras, A.P., Greenberg, E.P., Pesci, E.C., Vasil, M.L., 2008. The influence of iron on Pseudomonas aeruginosa physiology: a regulatory link between iron and quorum sensing. J. Biol. Chem. 283, 15558–15567. https://doi.org/10.1074/jbc.M707840200. Olvera, C., Goldberg, J.B., Sanchez, R., Soberon-Chavez, G., 1999. The Pseudomonas aeruginosa algC gene product participates in rhamnolipid biosynthesis. FEMS Microbiol. Lett. 179, 85–90. https://doi.org/10.1111/j.1574-6968.1999.tb08712.x. Onwosi, C.O., Odibo, F.J.C., 2012. Effects of carbon and nitrogen sources on rhamnolipid biosurfactant production by Pseudomonas nitroreducens isolated from soil. World J. Microbiol. Biotechnol. 28, 937–942. https://doi. org/10.1007/s11274-011-0891-3. Padgett, P.J., Phibbs, P.V., 1986. Phosphomannomutase activity in wild-type and alginate-producing strains of Pseudomonas aeruginosa. Curr. Microbiol. 14, 187–192. https://doi.org/10.1007/Bf01568516. Passador, L., Cook, J.M., Gambello, M.J., Rust, L., Iglewski, B.H., 1993. Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science (80-) 260, 1127–1130. Patel, R.M., Desai, A.J., 1997. Biosurfactant production by Pseudomonas aeruginosa GS3 from molasses. Appl. Microbiol. 25, 91–94. https://doi.org/10.1046/j.1472-765X.1997.00172.x. Payne, W.J., 1973. Reduction of nitrogenous oxides by microorganisms. Bacteriol. Rev. 37, 409–452. Pearson, J.P., Gray, K.M., Passador, L., Tucker, K.D., Eberhard, A., Iglewski, B.H., Greenberg, E.P., 1994. Structure of the autoinducer required for the expression of Pseudomonas aeruginosa virulence genes. Proc. Natl. Acad. Sci. U. S. A. 91, 197–201. Pearson, J.P., Passador, L., Iglewski, B.H., Greenberg, E.P., 1995. A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 92, 1490–1494. https://doi.org/10.1073/pnas.92.5.1490. Pesci, E.C., Milbank, J.B., Pearson, J.P., McKnight, S., Kende, A.S., Greenberg, E.P., Iglewski, B.H., 1999. Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 96, 11229–11234.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

200

5.  Rhamnolipids: Pathways, Productivities, and Potential

Pham, T.H., Webb, J.S., Rehm, B.H., 2004. The role of polyhydroxyalkanoate biosynthesis by Pseudomonas aeruginosa in rhamnolipid and alginate production as well as stress tolerance and biofilm formation. Microbiology 150, 3405–3413. Piljac, T., Piljac, G., inventors; Paradigm Biomedical Inc, assignee., 2007. Use of rhamnolipids in wound healing, treating burn shock, atherosclerosis, organ transplants, depression, schizophrenia and cosmetics. August 28. US patent US 7,262,171. Pinzon, N.M., Cook, A.G., Ju, L.-K., 2013. Continuous rhamnolipid production using denitrifying Pseudomonas aeruginosa cells in hollow-fiber bioreactor. Biotechnol. Prog. 29, 352–358. https://doi.org/10.1002/btpr.1701. Popat, R., Crusz, S.A., Messina, M., Williams, P., West, S.A., Diggle, S.P., 2012. Quorum-sensing and cheating in bacterial biofilms. Proc. Biol. Sci. 279, 4765–4771. https://doi.org/10.1098/rspb.2012.1976. Rahim, R., Burrows, L.L., Monteiro, M.A., Perry, M.B., Lam, J.S., 2000. Involvement of the rml locus in core oligosaccharide and O polysaccharide assembly in Pseudomonas aeruginosa. Microbiology 146, 2803–2814. https://doi. org/10.1099/00221287-146-11-2803. Rahim, R., Ochsner, U.A., Olvera, C., Graninger, M., Messner, P., Lam, J.S., Soberon-Chavez, G., 2001. Cloning and functional characterization of the Pseudomonas aeruginosa rhlC gene that encodes rhamnosyltransferase 2, an enzyme responsible for di-rhamnolipid biosynthesis. Mol. Microbiol. 40, 708–718. https://doi. org/10.1046/j.1365-2958.2001.02420.x. Rampioni, G., Bertani, I., Zennaro, E., Polticelli, F., Venturi, V., Leoni, L., 2006. The quorum-sensing negative regulator RsaL of Pseudomonas aeruginosa binds to the lasI promoter. J. Bacteriol. 188, 815–819. Rehm, B.H., Kruger, N., Steinbuchel, A., 1998. A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis. J. Biol. Chem. 273, 24044–24051. Reimmann, C., Beyeler, M., Latifi, A., Winteler, H., Foglino, M., Lazdunski, A., Haas, D., 1997. The global activator GacA of Pseudomonas aeruginosa PAO positively controls the production of the autoinducer N-butyrylhomoserine lactone and the formation of the virulence factors pyocyanin, cyanide, and lipase. Mol. Microbiol. 24, 309–319. Reis, R.S., Pereira, A.G., Neves, B.C., Freire, D.M.G., 2011. Gene regulation of rhamnolipid production in Pseudomonas aeruginosa—a review. Bioresour. Technol. 102, 6377–6384. https://doi.org/10.1016/j.biortech.2011.03.074. Renfro, T.D., Xie, W., Yang, G., Chen, G., 2014. Rhamnolipid surface thermodynamic properties and transport in agricultural soil. Colloids Surf. B Biointerfaces 115, 317–322. https://doi.org/10.1016/j.colsurfb.2013.12.021. Rodrigues, L., Banat, I.M., Teixeira, J., Oliveira, R., 2006. Biosurfactants: potential applications in medicine. J. Antimicrob. Chemother. 57, 609–618. https://doi.org/10.1093/jac/dkl024. Rumbaugh, K.P., Diggle, S.P., Watters, C.M., Ross-Gillespie, A., Griffin, A.S., West, S.A., 2009. Quorum sensing and the social evolution of bacterial virulence. Curr. Biol. 19, 341–345. https://doi.org/10.1016/j.cub.2009.01.050. Rumbaugh, K.P., Trivedi, U., Watters, C., Burton-Chellew, M.N., Diggle, S.P., West, S.A., 2012. Kin selection, quorum sensing and virulence in pathogenic bacteria. Proc. Biol. Sci. 279, 3584–3588. https://doi.org/10.1098/ rspb.2012.0843. Sandoz, K.M., Mitzimberg, S.M., Schuster, M., 2007. Social cheating in Pseudomonas aeruginosa quorum sensing. Proc. Natl. Acad. Sci. U. S. A. 104, 15876–15881. https://doi.org/10.1073/pnas.0705653104. Scheibenbogen, K., Zytner, R.G., Lee, H., Trevors, J.T., 1994. Enhanced removal of selected hydrocarbons from soil by Pseudomonas aeruginosa UG2 biosurfactants and some chemical surfactants. J. Chem. Technol. Biotechnol. 59, 53–59. https://doi.org/10.1002/jctb.280590109. Schuster, M., Greenberg, E.P., 2006. A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa. Int. J. Med. Microbiol. 296, 73–81. https://doi.org/10.1016/j.ijmm.2006.01.036. Seed, P.C., Passador, L., Iglewski, B.H., 1995. Activation of the Pseudomonas aeruginosa lasI gene by LasR and the Pseudomonas autoinducer PAI: an autoinduction regulatory hierarchy. J. Bacteriol. 177, 654–659. Sharma, D., Ansari, M.J., Al-Ghamdi, A., Adgaba, N., Khan, K.A., Pruthi, V., Al-Waili, N., 2015. Biosurfactant production by Pseudomonas aeruginosa DSVP20 isolated from petroleum hydrocarbon-contaminated soil and its physicochemical characterization. Environ. Sci. Pollut. Res. 22, 17636–17643. https://doi.org/10.1007/s11356-015-4937-1. Sim, L., Ward, O.P., Li, Z.Y., 1997. Production and characterisation of a biosurfactant isolated from Pseudomonas aeruginosa UW-1. J. Ind. Microbiol. Biotechnol. 19, 232–238. Singh, A.K., Cameotra, S.S., 2014. Influence of microbial and synthetic surfactant on the biodegradation of atrazine. Environ. Sci. Pollut. Res. 21, 2088–2097. https://doi.org/10.1007/s11356-013-2127-6. Soares dos Santos, A., Pereira Jr., N., Freire, D.M.G., 2016. Strategies for improved rhamnolipid production by Pseudomonas aeruginosa PA1. Peer J. 4, e2078https://doi.org/10.7717/peerj.2078.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

REFERENCES 201

Soberon-Chavez, G., Aguirre-Ramírez, M., Sánchez, R., 2005. The Pseudomonas aeruginosa RhlA enzyme is involved in rhamnolipid and polyhydroxyalkanoate production. J. Ind. Microbiol. Biotechnol. 32, 675–677. https://doi. org/10.1007/s10295-005-0243-0. Sodagari, M., 2013. Optimization of Production and Recovery of Rhamnolipid and Study of Their Effect on Bacterial Attachment. The University of Akron. Sodagari, M., Ju, L.-K., 2014. Cells were a more important foaming factor than free rhamnolipids in fermentation of Pseudomonas aeruginosa E03-40 for high rhamnolipid production. J. Surfactant Deterg. 17, 573–582. https://doi. org/10.1007/s11743-013-1535-4. Sodagari, M., Wang, H., Newby, B.M.Z., Ju, L.-K., 2013. Effect of rhamnolipids on initial attachment of bacteria on glass and octadecyltrichlorosilane-modified glass. Colloids Surf. B Biointerfaces 103, 121–128. https://doi. org/10.1016/j.colsurfb.2012.10.004. Sodagari, M., Invally, K., Ju, L.-K., 2018. Maximize rhamnolipid production with low foaming and high yield. Enzym. Microb. Technol. 110, 79–86. https://doi.org/10.1016/j.enzmictec.2017.10.004. Solaiman, D.K.Y., Ashby, R.D., Gunther IV, N.W., Zerkowski, J.A., 2015. Dirhamnose-lipid production by recombinant nonpathogenic bacterium Pseudomonas chlororaphis. Appl. Microbiol. Biotechnol. 99, 4333–4342. https://doi. org/10.1007/s00253-015-6433-4. Sponza, D.T., Gök, O., 2010. Effect of rhamnolipid on the aerobic removal of polyaromatic hydrocarbons (PAHs) and COD components from petrochemical wastewater. Bioresour. Technol. 101, 914–924. https://doi.org/10.1016/j. biortech.2009.09.022. Stanghellini, M.E., Miller, R.M., 1997. Bisosurfactants: their identity and potential efficacy in the biological control of zoosporic plant pathogens. Plant Dis. 81, 4–12. https://doi.org/10.1094/PDIS.1997.81.1.4. Stipcevic, T., Piljac, A., Piljac, G., 2006. Enhanced healing of full-thickness burn wounds using di-rhamnolipid. Burns 32, 24–34. https://doi.org/10.1016/j.burns.2005.07.004. Suh, S.J., Silo-Suh, L., Woods, D.E., Hassett, D.J., West, S.E., Ohman, D.E., 1999. Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. J. Bacteriol. 181, 3890–3897. Tan, H., Champion, J.T., Artiola, J.F., Brusseau, M.L., Miller, R.M., 1994. Complexation of cadmium by a rhamnolipid biosurfactant. Environ. Sci. Technol. 28, 2402–2406. https://doi.org/10.1021/es00062a027. Tazdait, D., Salah, R., Mouffok, S., Kabouche, F., Keddou, I., Abdi, N., Grib, H., Mameri, N., 2018. Preliminary evaluation of a new low-cost substrate (amurca) in production of biosurfactant by Pseudomonas aeruginosa isolated from fuel-contaminated soil. J. Mater. Environ. 9, 964–970. https://doi.org/10.26872/jmes.2018.9.3.107. Thompson, L.S., Webb, J.S., Rice, S.A., Kjelleberg, S., 2003. The alternative sigma factor RpoN regulates the quorum sensing gene rhlI in Pseudomonas aeruginosa. FEMS Microbiol. Lett. 220, 187–195. Tiso, T., Sabelhaus, P., Behrens, B., Wittgens, A., Rosenau, F., Hayen, H., Blank, L.M., 2016. Creating metabolic demand as an engineering strategy in Pseudomonas putida- Rhamnolipid synthesis as an example. Metab. Eng. Commun. 3, 234–244. https://doi.org/10.1016/j.meteno.2016.08.002. Toder, D.S., Gambello, M.J., Iglewski, B.H., 1991. Pseudomonas aeruginosa LasA: a second elastase under the transcriptional control of lasR. Mol. Microbiol. 5, 2003–2010. Toribio, J., Escalante, A.E., Soberon-Chavez, G., 2010. Rhamnolipids: production in bacteria other than Pseudomonas aeruginosa. Eur. J. Lipid Sci. Technol. 112, 1082–1087. https://doi.org/10.1002/ejlt.200900256. Totten, P.A., Lara, J.C., Lory, S., 1990. The rpoN gene product of Pseudomonas aeruginosa is required for expression of diverse genes, including the flagellin gene. J. Bacteriol. 172, 389–396. Trummler, K., Effenberger, F., Syldatk, C., 2003. An integrated microbial/enzymatic process for production of rhamnolipids and L-(+)-rhamnose from rapeseed oil with Pseudomonas sp. DSM 2874. Eur. J. Lipid Sci. Technol. 105, 563–571. https://doi.org/10.1002/ejlt.200300816. Tuleva, B.K., Ivanov, G.R., Christova, N.E., 2002. Biosurfactant production by a new Pseudomonas putida strain. Z. Naturforsch. 57, 356–360. https://doi.org/10.1515/znc-2002-3-426. Van Delden, C., Comte, R., Bally, M., 2001. Stringent response activates quorum sensing and modulates cell density-­ dependent gene expression in Pseudomonas aeruginosa. J. Bacteriol. 183, 5376–5384. https://doi.org/10.1128/ Jb.183.18.5376-5384.2001. Van Dyke, M.I., Couture, P., Brauer, M., Lee, H., Trevors, J.T., 1993. Pseudomonas aeruginosa UG2 rhamnolipid biosurfactants: structural characterization and their use in removing hydrophobic compounds from soil. Can. J. Microbiol. 39, 1071–1078. Vander Wauven, C., Pierard, A., Kley-Raymann, M., Haas, D., 1984. Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway. J. Bacteriol. 160, 928–934.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

202

5.  Rhamnolipids: Pathways, Productivities, and Potential

Varjani, S.J., Upasani, V.N., 2016. Carbon spectrum utilization by an indigenous strain of Pseudomonas aeruginosa NCIM 5514: production, characterization and surface active properties of biosurfactant. Bioresour. Technol. 221, 510–516. https://doi.org/10.1016/j.biortech.2016.09.080. Varjani, S.J., Upasani, V.N., 2017. Critical review on biosurfactant analysis, purification and characterization using rhamnolipid as a model biosurfactant. Bioresour. Technol. 232, 389–397. https://doi.org/10.1016/j.biortech.2017.02.047. Varnier, A.L., Sanchez, L., Vatsa, P., Boudesocque, L., Garcia-Brugger, A., Rabenoelina, F., Sorokin, A., Renault, J.H., Kauffmann, S., Pugin, A., Clement, C., Baillieul, F., Dorey, S., 2009. Bacterial rhamnolipids are novel MAMPs conferring resistance to Botrytis cinerea in grapevine. Plant Cell Environ. 32, 178–193. https://doi. org/10.1111/j.1365-3040.2008.01911.x. Venturi, V., 2006. Regulation of quorum sensing in Pseudomonas. FEMS Microbiol. Rev.. Wade, D.S., Calfee, M.W., Rocha, E.R., Ling, E.A., Engstrom, E., Coleman, J.P., Pesci, E.C., 2005. Regulation of Pseudomonas quinolone signal synthesis in Pseudomonas aeruginosa. J. Bacteriol. 187, 4372–4380. https://doi. org/10.1128/JB.187.13.4372-4380.2005. Wagner, V.E., Bushnell, D., Passador, L., Brooks, A.I., Iglewski, B.H., 2003. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J. Bacteriol. 185, 2080–2095. https:// doi.org/10.1128/JB.185.7.2080. Wang, S., Mulligan, C.N., 2004. An evaluation of surfactant foam technology in remediation of contaminated soil. Chemosphere 57, 1079–1089. https://doi.org/10.1016/j.chemosphere.2004.08.019. Wang, Q., Fang, X., Bai, B., Liang, X., Shuler, P.J., Goddard, W.A., Tang, Y., 2007. Engineering bacteria for production of rhamnolipid as an agent for enhanced oil recovery. J. Anat. 98, 842–853. https://doi.org/10.1002/bit. Wang, C.P., Li, J., Jiang, Y., Zhang, Z.Y., 2014. Enhanced bioremediation of field agricultural soils contaminated with PAHs and OCPs. Int. J. Environ. Res. 8, 1271–1278. Weber, A., Zeiner, T., 2014. Purification of biosurfactants. In: Biosurfactants: Production and Utilization—Processes, Technologies, and Economics. CRC Press, Boca Raton, FL, pp. 129–152. Wei, Y.H., Chou, C.L., Chang, J.S., 2005. Rhamnolipid production by indigenous Pseudomonas aeruginosa J4 originating from petrochemical wastewater. Biochem. Eng. J. 27, 146–154. https://doi.org/10.1016/j.bej.2005.08.028. West, S.E.H., Kaye, S.A., Hamood, A.N., Iglewski, B.H., 1994a. Characterization of Pseudomonas aeruginosa mutants that are deficient in exotoxin A synthesis and are altered in expression of regA, a positive regulator of exotoxin A. Infect. Immun. 62, 897–903. West, S.E.H., Sample, A.K., Runyen-Janecky, L.J., 1994b. The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family. J. Bacteriol. 176, 7532–7542. Whang, L.-M., Liu, P.-W.G., Ma, C.-C., Cheng, S.-S., 2008. Application of biosurfactants, rhamnolipid, and surfactin, for enhanced biodegradation of diesel-contaminated water and soil. J. Hazard. Mater. 151, 155–163. https://doi. org/10.1016/j.jhazmat.2007.05.063. Whiteley, M., Parsek, M.R., Greenberg, E.P., 2000. Regulation of quorum sensing by RpoS in Pseudomonas aeruginosa. J. Bacteriol. 182, 4356–4360. Whiteley, M., Diggle, S.P., Greenberg, E.P., 2017. Progress in and promise of bacterial quorum sensing research. Nature 551, 313. https://doi.org/10.1038/nature24624. Williams, P., Cámara, M., 2009. Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules. Curr. Opin. Microbiol. 12, 182–191. https://doi. org/10.1016/j.mib.2009.01.005. Winterburn, J.B., Martin, P.J., 2012. Foam mitigation and exploitation in biosurfactant production. Biotechnol. Lett. 34, 187–195. https://doi.org/10.1007/s10529-011-0782-6. Woods, D.E., Strauss, D.C., Johanson, W.G., Berry, V.K., Bass, J.A., 1980. Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells. Infect. Immun. 29, 1146–1151. Wu, J.Y., Yeh, K.-L., Lu, W.-B., Lin, C.-L., Chang, J.-S., 2008. Rhamnolipid production with indigenous Pseudomonas aeruginosa EM1 isolated from oil-contaminated site. Bioresour. Technol. 99, 1157–1164. https://doi.org/10.1016/j. biortech.2007.02.026. Xia, W.J., Dong, H.P., Yu, L., Yu, D.F., 2011. Comparative study of biosurfactant produced by microorganisms isolated from formation water of petroleum reservoir. Colloids Surfaces A Physicochem. Eng. Asp. 392, 124–130. https:// doi.org/10.1016/j.colsurfa.2011.09.044. Xiao, G., Deziel, E., He, J., Lepine, F., Lesic, B., Castonguay, M.H., Milot, S., Tampakaki, A.P., Stachel, S.E., Rahme, L.G., 2006a. MvfR, a key Pseudomonas aeruginosa pathogenicity LTTR-class regulatory protein, has dual ligands. Mol. Microbiol. 62, 1689–1699. https://doi.org/10.1111/j.1365-2958.2006.05462.x.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

REFERENCES 203

Xiao, G., He, J., Rahme, L.G., 2006b. Mutation analysis of the Pseudomonas aeruginosa mvfR and pqsABCDE gene promoters demonstrates complex quorum-sensing circuitry. Microbiology 152, 1679–1686. https://doi.org/10.1099/ mic.0.28605-0. Zhang, Y., Miller, R.M., 1992. Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant). Appl. Environ. Microbiol. 58, 3276–3282. Zhang, Y., Miller, R.M., 1994. Effect of a Pseudomonas rhamnolipid biosurfactant on cell hydrophobicity and biodegradation of octadecane. Appl. Environ. Microbiol. 60, 2101–2106. Zhang, L., Veres-Schalnat, T.A., Somogyi, A., Pemberton, J.E., Maier, R.M., 2012. Fatty acid cosubstrates provide beta-­ oxidation precursors for rhamnolipid biosynthesis in Pseudomonas aeruginosa, as evidenced by isotope tracing and gene expression assays. Appl. Environ. Microbiol. 78, 8611–8622. https://doi.org/10.1128/AEM.02111-12. Zhao, Z., Selvam, A., Wong, J.W.C., 2011. Effects of rhamnolipids on cell surface hydrophobicity of PAH degrading bacteria and the biodegradation of phenanthrene. Bioresour. Technol. 102, 3999–4007. https://doi.org/10.1016/j. biortech.2010.11.088. Zhao, F., Shi, R., Zhao, J., Li, G., Bai, X., Han, S., Zhang, Y., 2015. Heterologous production of Pseudomonas aeruginosa rhamnolipid under anaerobic conditions for microbial enhanced oil recovery. J. Appl. Microbiol. 118, 379–389. https://doi.org/10.1111/jam.12698. Zhong, H., Liu, G., Jiang, Y., Brusseau, M.L., Liu, Z., Liu, Y., Zeng, G., 2016. Effect of low-concentration rhamnolipid on transport of Pseudomonas aeruginosa ATCC 9027 in an ideal porous medium with hydrophilic or hydrophobic surfaces. Colloids Surf. B Biointerfaces 139, 244–248. https://doi.org/10.1016/j.colsurfb.2015.11.024. Zhu, K., Rock, C.O., 2008. RhlA converts beta-hydroxyacyl-acyl carrier protein intermediates in fatty acid synthesis to the beta-hydroxydecanoyl-beta-hydroxydecanoate component of rhamnolipids in Pseudomonas aeruginosa. J. Bacteriol. 190, 3147–3154. https://doi.org/10.1128/JB.00080-08. Zhu, L., Yang, X., Xue, C., Chen, Y., Qu, L., Lu, W., 2012. Enhanced rhamnolipids production by Pseudomonas aeruginosa based on a pH stage-controlled fed-batch fermentation process. Bioresour. Technol. 117, 208–213. https:// doi.org/10.1016/j.biortech.2012.04.091.

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6 Lipopeptide Biosurfactants From Bacillus Species Mareen Geissler, Kambiz Morabbi Heravi, Marius Henkel, Rudolf Hausmann Department of Bioprocess Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany

6.1 INTRODUCTION Bacillus species, especially Bacillus subtilis, are important microorganisms in industrial biotechnology due to their natural secretion of diverse proteins (van Dijl and Hecker, 2013). Among others, B. subtilis is used to produce enzymes such as proteases, cellulases, amylases, and laccases (Singh et  al., 2017). The natural secretion of these products into the medium offers advantages in their respective purification procedures (van Dijl and Hecker, 2013). In addition, as a well-studied or even the best-studied gram-positive bacterium, genetic manipulation of B. subtilis is well established, and therefore, it is a common host for large-scale production (Harwood, 1992). For instance, genetically engineered B. subtilis is used for the industrial production of riboflavin, an essential vitamin for growth and reproduction in both humans and animals (Schwechheimer et al., 2016). Furthermore, B. subtilis is a convenient industrial strain due to its excellent cultivation performances, high product titers, and the lack of toxic by-product formations (van Dijl and Hecker, 2013). Next to the already implemented industrial processes, great interest in Bacillus spp. has grown due to their ability to synthesize and secrete cyclic lipopeptides. Bacillus spp. produce five families of lipopeptides, with surfactin as the most relevant, followed by iturin and fengycin (Coutte et al., 2017). Each family of lipopeptides consists of different congeners, and although all lipopeptide families possess structural similarities, their chemical and physicochemical characteristics vary both inter- and intrafamily (Beltran-Gracia et al., 2017). The broad spectrum of these molecules and their characteristics ranging from surface-active properties to antimicrobial activities make them exceptional candidates for a variety of applications. However, in comparison with well-established industrial processes employing Bacillus strains to produce, for example, enzymes, the yields obtained for lipopeptides are currently

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comparably low. Hence, although often claimed as promising biobased surfactant, a stable and reliable process with high yields has not been realized so far. This chapter will therefore primarily give a firm background on the respective lipopeptide families, their synthesis by nonribosomal peptide synthetases (NRPS), and the underlying quorum-sensing mechanisms. Furthermore, the physicochemical properties and the concomitant potential applications of these promising biosurfactants will be given. In addition, this chapter evaluates current approaches toward large-scale production of lipopeptides that includes both up- and downstream processing. In the end, several challenges that must be considered in future research will be presented.

6.2  TYPES AND CLASSIFICATION OF LIPOPEPTIDES Cyclic lipopeptides are chemical compounds that generally possess an amphiphilic structure. The hydrophilic part, which is formed by an oligopeptide ring, is connected to a fatty acid chain. The huge structural variety of these compounds is based on the variation of the oligopeptide composition, the sequence of the polar head, or the diversity of the fatty acid tail (Mnif and Ghribi, 2015b). The fatty acid can be either linear or branched and varies in the length or degree of oxidation. Furthermore, lipopeptides are outstanding with respect to their structural diversity by often containing d-amino acids and iso- or anteiso-hydroxy fatty acids. Both Bacillus spp. and Pseudomonas spp. are known to produce the currently most interesting lipopeptides; however, also yeast and fungi can synthesize cyclic lipopeptides. Based on a survey in the database NORINE, Coutte et al. (2017) counted 263 different lipopeptides synthesized by 11 microbial genera. Within these genera, Pseudomonas spp., Bacillus spp., and Streptomyces spp. represent the most abundant lipopeptide producers with 78, 98, and 40 different lipopeptides, classified in 11, 5, and 6 lipopeptide families, respectively. An overview of these lipopeptide families and the subfamilies and the number of known lipopeptides within these subfamilies is given in Table 6.1. The data shown are based on a research in the database NORINE with the annotation search terms “lipopeptide” for “category” and the respective organism for “organism name” (Caboche et al., 2008; Flissi et al., 2016). The table furthermore illustrates the structure of the lipopeptides and is divided in cyclic, partial cyclic, and linear. In comparison with other lipopeptide producers, the amounts synthesized by natural Bacillus spp. are generally higher, and thus, these lipopeptides are more commonly in focus of research (Coutte et al., 2017). However, the non-Bacillus lipopeptides should also not be disregarded as they expand the structural diversity and potential application areas of Bacillus lipopeptides.

6.2.1  Excursion to Non-Bacillus Lipopeptides An overview of structures, characteristics, and biosynthesis of Pseudomonas lipopeptides is given by Raaijmakers et al. (2010). The Pseudomonas lipopeptides were first divided into four groups, namely, viscosin, amphisin, tolaasin, and syringomycin. As shown in Table 6.1, up to date, 11 families of Pseudomonas lipopeptides were described. A purified viscosin was reported to have excellent surface-active properties since it had a low critical micelle ­concentration (CMC) of 54 mg/L, which corresponds to 0.048 mmol/L, and reduced the surface tension

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TABLE 6.1  Overview of Lipopeptide Families, Subfamilies, and Number of Known Lipopeptides Within These Subfamilies Organism Name

Lipopeptide Families

Number of LP Within Family

Subfamilies

Peptide Structure

Pseudomonas

Orfamide

3

Orfamide

Cyclic

Amphisin

9

Arthrofactin

Cyclic

Tensin Corrugatin

1

Corrugatin

Linear

8

Plusbacin

Cyclic

3

Putisolvin

Partial cyclic

1

Pyoverdin

Linear

Syringafactin

6

Syringafactin

Linear

Syringomycin

11

Syringomycin

Partial cyclic

Plusbacin

a

Putisolvin Pyoverdin

b

Pseudomycin Syringopeptin

11

Syringopeptin

Partial cyclic

Tolaasin

11

Tolaasin

Partial cyclic

Corpeptin Viscosin

14

Viscosin

Partial cyclic

Massetolide c

Bacillus

Kurstakin

7

Kurstakin

Partial cyclic

Fengycin

3

Fengycin/plipastatin

Partial cyclic

Iturin

32

Iturin

Cyclic

Bacillomycin Mycosubtilin Polymyxin

19

Polymyxin

Partial cyclic

Surfactin

37

Surfactin

Cyclic

Lichenysin Streptomyces

A54145

8

A54145

Cyclic

Ca-dependent antibiotic

10

CDA

Cyclic

a

Arylomycin

12

Arylomycin

Partial cyclic

Daptomycin

4

Daptomycin

Partial cyclic

A21978 Enduracidin

2

Enduracidin

Partial cyclic

Friulimicin

4

Amphomycin

Partial cyclic

Data shown are based on a search in the database NORINE with the annotation search terms “lipopeptide” for “category” and respective organism for “organism name” (Caboche et al., 2008; Flissi et al., 2016). a Putative NRPS product. b Only peptide structure, no fatty acid moiety, but NRPS product. c Locillomycin as possible further family of LP.

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at the air/water interface to 28 mN/m (Saini et al., 2008). Ma et al. (2017) evaluated the effectiveness of a new class of Pseudomonas lipopeptides, namely, orfamide-like lipopeptides, synthesized by P. protegens, against the fungus Cochliobolus miyabeanus. This fungus causes brown spot disease on rice, and the lipopeptide was reported to trigger induced systemic resistance in rice plants and consequently was described as a promising biological agent in the agricultural area. The antibiotics daptomycin and colistin are lipopeptides already approved to be used in medicine. Daptomycin, for example, synthesized by S. roseosporus, showed potent antibacterial activities against many gram-positive organisms (Steenbergen et al., 2005). Although less studied, also several fungi can synthesize lipopeptides nonribosomally. Echinocandins are representative fungal lipopeptides produced by Aspergillus nidulans and were described to have a high potential of being implemented in antifungal drugs (Hüttel et al., 2016). In the recent past, interest in new natural isolates and their respective production of secondary metabolites has increased. Janek et  al. (2010), for example, extracted a cyclic lipopeptide from P. fluorescens BD5 and named it pseudofactin. Further techniques such as oil displacement test and drop collapse method were employed as analytic tools to screen for lipopeptides. With these techniques, new lipopeptides such as pontifactin from Pontibacter korlensis SBK-47 (Balan et al., 2016) or cystargamide from Kitasatospora cystarginea (Gill et al., 2014) were described. Kiran et al. (2017) identified a lipopeptide produced by Nesterenkonia sp. MSA31 and showed its potential application in the food industry. In their study, egg yolk and baking powder in a muffin recipe were replaced by different concentrations of this lipopeptide, and the texture was evaluated. Another lipopeptide interesting to the food industry was investigated by Li et al. (2018). The lipopeptide paenibacterin synthesized by Paenibacillus thiaminolyticus was evaluated to be able to control Listeria monocytogenes, a foodborne pathogen. To be more specific, paenibacterin downregulated biofilm formation in L. monocytogenes, combatting a major initiator of pathogenicity.

6.2.2  Bacillus Lipopeptides—Discovery and Structural Diversity Among all known microbial lipopeptides, lipopeptides produced by Bacillus species are the most studied. So far, Bacillus spp. are known to produce five different families of lipopeptides, among Fig. 6.1 displays the research articles published dealing with either surfactin, iturin, or fengycin alone or all three lipopeptides together, highlighting the increasing interest in the past decade. As illustrated in Fig. 6.1, research on the most studied Bacillus lipopeptide surfactin started in 1968, and in 2015, almost 100 articles were published dealing with this biosurfactant. For iturin and fengycin, research started 10 and 20 years later, respectively, and the number of articles counted was almost 40 for each of them in 2015. Mostly reported and studied producers of lipopeptides belong to the species B. subtilis. But also other strains such as B. circulans (Hsieh et al., 2008; Sivapathasekaran et al., 2010), B. licheniformis (Li et al., 2008), B. amyloliquefaciens (Borriss et al., 2011; Hsieh et al., 2008), and B. methylotrophicus (Jemil et al., 2017) are able to synthesize either one or more of the lipopeptides belonging to the surfactin, iturin, or fengycin family. As illustrated in Table  6.1, based on the data search, polymyxins were displayed as another family of Bacillus lipopeptides. Polymyxins were discovered in 1947

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100 Surfactin Iturin Fengycin Surfactin and iturin and fengycin

Amount of research articles

80

60

40

20

0 1970

1980

1990 Year

2000

2010

FIG.  6.1  Sum of articles per year from 1968 to 2016 taken from the peer-reviewed literature database Scopus (www.scopus.com) with respect to the keywords “surfactin,” “iturin,” “fengycin,” and “surfactin AND iturin AND fengycin.” Results limited to “article” and keywords mentioned in “article title, abstract, and keywords.”

and were used as antibiotics in hospitals between the 1950s and 1970s. Due to toxicological concerns, the application was reduced. Nowadays, polymyxins are again used in therapeutic applications for patients where other antibiotic treatments failed due to an increasing antibiotic resistance observed (Rabanal and Cajal, 2017). However, a study conducted in 1991 splits the order Bacillales into several families, creating among others the genus Paenibacillus. The strain B. polymyxia was afterward assigned as P. polymyxa (Grady et al., 2016). As this chapter focuses on lipopeptides synthesized by the genus Bacillus, polymyxins are not further described. Interestingly, in the recent years, two new families of Bacillus lipopeptides were discovered, namely, the kurstakin (Hathout et al., 2000), also displayed in Table 6.1, and the locillomycin (Luo et al., 2015) families. The next section will individually describe the respective lipopeptide families. Chemical structures of representatives of the three main families are given in Fig. 6.2. A summary of further different lipopeptide variants is given by Cochrane and Vederas (2016) and Mnif and Ghribi (2015b). 6.2.2.1 The Surfactin Family—Surfactin, Pumilacidin, and Lichenysin In 1968, Arima et  al. (1968) described surfactin, whose chemical structure was assigned as a lipopeptide consisting of l-aspartic acid, l-glutamic acid, l-valine, and two l-leucine and d-leucine residues in its peptide ring. Kakinuma et al. (1969a,b) published a more detailed structure of the first surfactin structure discovered 1 year later. This surfactin congener was determined to possess a 3-hydroxy-13-methyltetradecanoic (C15) fatty acid as lipid residue, which is connected to the first amino acid of the heptapeptide ring, namely, l-Glu.

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FIG. 6.2  Chemical structure of representative congeners of (A) surfactin, (B) iturin, and (C) fengycin lipopeptides.



6.2  Types and Classification of Lipopeptides

211

The ­peptide ring itself is composed of the amino acids in the sequence l-Glu, l-Leu, d-Leu, l-Val, l-Asp, d-Leu, and l-Leu. The first and last amino acids l-Glu and l-Leu form the lactone ring by an ester linkage. A main congener of surfactin is given in Fig. 6.2A. Up to now, more than 30 different surfactin congeners were reported that differ either in their amino acid composition or in their fatty acid residues. However, the chiral sequence for all surfactin molecules remains identical with lldlldl (Cochrane and Vederas, 2016). Pumilacidins were first discovered in 1990 in the strain B. pumilus with the variants A–F (Naruse et al., 1990). In these variants, the amino acid at position 7 is substituted by either l-Val or l-Ile. Peypoux et al. (1991) discovered a surfactin variant with l-Val at position 7 in the strain B. subtilis S499. Due to reported structural similarities, pumilacidin variants can be assigned to the surfactin family. Lichenysins were discovered in 1995 and named after their strain of origin, namely, B. licheniformis (Yakimov et al., 1995). Structurally, lichenysins were reported to be closely related to surfactin, with the main difference being the substitution of l-glutamic acid with l-glutamine at position 1. 6.2.2.2 The Iturin Family—Iturin, Bacillomycin, and Mycosubtilin The discovery of iturin dates back to 1952, and the structure of iturin A was elucidated in 1978 by Peypoux et  al. (1978). Similar to surfactin, iturins are cyclic heptapeptides. The amino acid sequence of the iturin A molecule is l-Asn, d-Tyr, d-Asn, l-Gln, l-Pro, d-Asn, and l-Ser. The lipid moiety of the first reported iturin A molecule was reported to be a mixture of 3-amino-12-methyltridecanoic acid and 3-amino-12-methyltetradecanoic acid (Peypoux et  al., 1978). Similar to surfactin, the fatty acid is attached to the first amino acid, and the ­cyclization occurs between the first and the last amino acid. However, in contrast to surfactin, the cyclization between the first and the last amino acid occurs by an amide bond as depicted in Fig. 6.2B. In addition to iturin, also mycosubtilin and bacillomycin were assigned to this family of lipopeptides due to structural similarities. 6.2.2.3 The Fengycin Family—Fengycin and Plipastatin While surfactin and iturin show structural similarities, fengycins, as illustrated in Fig. 6.2C, differ more. They are decapeptides, and the cyclization occurs between a phenol side chain of the third amino acid d-Tyr and the C-terminus of the amino acid at position 10 (Cochrane and Vederas, 2016). Hence, fengycin lipopeptides can be considered as partial cyclic. Fengycin A and B were first mentioned in 1986. Vanittanakom and Loeffler (1986) explored these molecules in the strain B. subtilis F-29-3 as antifungal lipopeptides. The amino acid sequence of fengycin A was determined as l-Glu, d-Orn, d-Tyr, d-Thr, l-Glu, d-Ala, l-Pro, l-Gln, l-Tyr, and l-Ile. For fengycin B, d-Ala at position 6 is replaced by d-Val (Schneider et al., 1999). Another lipopeptide, closely related to fengycin, was named plipastatin. While fengycin A is composed of d-Tyr at position 3 and l-Tyr at position 9, plipastatin A constitutes of l-Tyr and d-Tyr at positions 3 and 9, respectively (Cochrane and Vederas, 2016). However, up to now, fengycin and plipastatins are also often used as synonyms. 6.2.2.4 The Kurstakin and Locillomycin Families Lately, two novel lipopeptide families were discovered, namely, the kurstakin family (Hathout et al., 2000) and the locillomycin family (Luo et al., 2015). Hathout et al. (2000) investigated the structures of three representatives of the kurstakin family, isolated from a culture

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of the strain B. thuringiensis subs. Kustaki HD-1. Kurstakins are partial cyclic heptapeptides. The fatty acid with varying chain length is connected via the first amino acid. The residues of kurstakin are Thr, Gly, Ala, Ser, His, Gln, and Gln with the cyclization between the fourth and seventh amino acid (Cochrane and Vederas, 2016). The presence of histidine is rare in the structural diversity of lipopeptides explored so far (Hathout et al., 2000). Locillomycins were extracted from a culture of the strain B. subtilis 916. These molecules are nonapeptides with the amino acid sequence of Thr, Gln, Asp, Gly, Asn, Asp, Gly, Tyr, and Val (Luo et al., 2015). For both kurstakin and locillomycin family lipopeptides, the complete structural identification, for example, with respect to the presence of d- or l-amino acids, still needs to be further investigated.

6.3  LIPOPEPTIDE BIOSYNTHESIS 6.3.1  Nonribosomal Peptide Synthetases Lipopeptides from Bacillus species are encoded by nonribosomal peptide synthetases (NRPSs) or hybrid NRPSs. The synthesis of peptide structures by NRPSs has several unique features. For example, also noncanonical amino acids can be incorporated into peptides synthesized by NRPSs, which allows for a high structural diversity. However, NRPSs catalyze very specific reactions, and modifications with genetic engineering methods are currently not established compared with ribosomally synthesized peptides. The NRPSs encoding for the lipopeptides surfactin, iturin, and fengycin share some specific features. NRPSs are large multienzyme complexes organized in modules that possess iterative functions (Hamdache et  al., 2013). Each of these multidomain modules is responsible for a reaction cycle and is composed of different domains such as adenylation domain (A); peptidyl carrier domain (PCP), also often referred to as thiolation domain; and condensation domain (C). The operons encoding for surfactin, iturin A, and fengycin, with the respective arrangement of modules and domains, are illustrated in Fig. 6.3. The adenylation domain selects the amino acid and activates it to an aminoacyl adenylate by the consumption of adenosine triphosphate (ATP). The PCP domain is a transport unit where the amino acid adenylate is tethered by a thioester bond and binds to a conserved serine residue on the carrier protein domain. The condensation domain forms a new peptide bond between two adjacent modules and their respective aminoacyl substitutes (Finking and Marahiel, 2004; Hamdache et  al., 2013; Strieker et  al., 2010). While a linear arrangement of these domains is necessary to ensure for elongation of the oligopeptide structures, several modules hold further modifying domains, for example, for epimerization, hydroxylation, methylation, or cyclization (Finking and Marahiel, 2004). The last module for both surfactin, iturin, and fengycin synthetases comprises another domain, the thioesterase domain (TE). This domain initiates product release by cleaving the last thioester bond and cyclization of the oligopeptide chain (Finking and Marahiel, 2004; Wu et al., 2017). The following sections give a more detailed overview of the synthesis of the lipopeptides surfactin, fengycin, and iturin.

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6.3  Lipopeptide Biosynthesis

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213

FIG. 6.3  Schematic overview of the operons encoding for the nonribosomal peptide synthetases for the lipopeptides surfactin, iturin, and fengycin. The modules with the respective domains and the amino acids incorporated into the growing peptide chain by each module are illustrated.

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6.3.2  Surfactin Synthesis The NRPS responsible for surfactin synthesis is encoded by srfA, which consists of four open reading frames (ORF). The first three ORFs srfAA (402 kDa), srfAB (401 kDa), and srfAC (144 kDa) encode the NRPS subunits that elongate the oligopeptide chain (Kraas et al., 2010). As illustrated in Fig. 6.3, each of these subunits consists of modules with condensation, adenylation, and peptidyl carrier domains. The last modules of SrfAA and SrfAB additionally contain an epimerization domain that converts l-Leu to d-Leu. However, the subunits are only able to form a mature complex after posttranslational modification of the PCP domains. This is performed by a phosphopantetheinyl transferase encoded by the sfp gene, which is crucial for the surfactin biosynthesis. The sfp gene is located 4 kb downstream of the srfA operon and is responsible for the posttranslational phosphopantetheinylation of the PCP domains to convert them from the inactive apoform into the active holoform (Das et al., 2008; Reuter et al., 1999; Roongsawang et al., 2010; Wu et al., 2017). Sfp phosphopantetheinylates the PCP domains by transferring the phosphopantethein group from CoA and thereby introduces a reactive thiol terminus to each PCP domain. This enables both to load the amino acid on the domain and to form the peptide bond (Quadri et al., 1998). The forth ORF, srfAD (40 kDa), which is not directly involved in the elongation process of the peptide chain, is reported to synthesize an external type II thioesterase, with the function of recycling misprimed peptidyl carrier protein (PCP) domains (Koglin et al., 2008). Surfactin synthesis begins with a CoA-activated fatty acid. However, the NRPS cluster itself does not contain an acyl-CoA-ligase. Kraas et al. (2010) reported that two out of four identified CoA ligases, namely, LcfA and YhfL, play a major role in fatty acid activation. Interestingly, a mutant lacking all four identified ligases still produced surfactin, indicating the presence of further pathways that provide the activated fatty acid. The activated fatty acid is recognized by the C-domain of the first module of the srfAA subunit, which catalyzes the acylation with the amino group of the first amino acid to be incorporated, namely, l-Glu (Kraas et al., 2010; Steller et al., 2004; Wu et al., 2017). This amino acid itself was activated through adenylation by the adenylation domain within its respective module. Next, the peptide chain is elongated by the addition of two l-Leu within the SrfAA subunit. The PCP domains thereby allow the traveling of activated amino acids between the catalytic centers, and the condensation domain catalyzes the reaction of two neighboring amino acids and forms a peptide bond. The last module of SrfAA additionally contains an epimerization domain, which converts the second l-Leu to d-Leu. Afterward, three amino acids, namely, l-Val, l-Asp, and l-Leu, are incorporated in the nascent oligopeptide within the SrfAB subunit that is similar to SrfAA. Here, l-Leu is also converted to d-Leu by an epimerization domain located in the last module of SrfAB. The last subunit SrfAC is composed of the l-Leu peptide chain elongation domains and a thioesterase domain. This type I thioesterase catalyzes the reaction between the first and the last amino acid of the peptide chain by forming a macrolactone (Koglin et al., 2008). Release of the surfactin molecule from the enzyme complex then finalizes the biosynthesis of surfactin. Since no active transporter has been identified so far, Tsuge et al. (2001) postulated a passive diffusion of surfactin into the extracellular milieu.

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6.3  Lipopeptide Biosynthesis

215

6.3.3  Fengycin Synthesis As illustrated in Fig. 6.3, the biosynthesis of fengycin is encoded by five ORFs and is also initiated by the attachment of a β-hydroxy fatty acid to the first amino acid of the first module within FenC. The other ORFs encoding for fengycin are, in the following order, fenC (ppsA) (287 kDa), fenD (ppsB) (290 kDa), fenE (ppsC) (286 kDa), fenA (ppsD) (406 kDa), and fenB (ppsE) (146 kDa) (Tapi et al., 2010; Wu et al., 2007). The general organization of the functional domains of the respective modules is similar to the surfactin NRPS, with the linear arrangement of the peptidyl carrier protein domain, adenylation domain, condensation domain, and several epimerization domains. Each of FenC, FenD, and FenE adds two amino acids to the oligopeptide chain, whereas FenA and FenB add three and one amino acid, respectively (Ongena and Jacques, 2008; Wu et al., 2007). As reported for surfactin, the release of the molecule is initiated after macrocyclization, which is catalyzed by the TE-domain located in FenB. Fengycin lipopeptides are partial cyclic, and the lactone bond formation occurs between the third amino acid l-Tyr and the last amino acid l-Ile (Samel et al., 2006). Similar to surfactin, sfp is required for the biosynthesis of fengycin. As a result, a frameshift mutation in the sfp ORF hampered the biosynthesis of both surfactin and fengycin in B. subtilis 168 although it carries both the surfactin and fengycin biosynthesis operons (Coutte et al., 2010a).

6.3.4  Iturin Synthesis The biosynthesis of lipopeptides belonging to the iturin family differs from both surfactin and fengycin lipopeptides. Hitherto, Duitman et al. (1999) elucidated the structural arrangement of the mycosubtilin synthetase; the iturin A operon was investigated by Tsuge et al. (2001); and Moyne (2004) elucidated the structural arrangement of the operon encoding for bacillomycin D. All biosynthesis operons consist of four ORFs, namely, ituD, ituA, ituB, and ituC for the iturin A operon and fenF, mycA, mycB, and mycC for mycosubtilin. Similar to the surfactin operon, ituABC/mycABC encodes the NRPS. Unlike the synthesis of surfactin and fengycin, ituA/ mycA combines several functions. ituA/mycA are hybrids of a polyketide synthase and NRPS and have an additional PCP and C domain (Aron et al., 2005). The ituA/mycA subunits possess several functions. Duitman et al. (1999) proposed a model that illustrates the incorporation of the fatty acid into the assembly line of mycosubtilin. First, in a reaction dependent on ATP, CoA is coupled to a long-chain fatty acid by the acyl-CoA-ligase domain. This activated fatty acid is transferred to the 4-phosphopantetheine cofactor of the first PCP domain. A malonylCoA transacylase, which is encoded by fenF, catalyzes the reaction of a malonyl-CoA to the second PCP domain. In the next step, the malonyl and acyl thioester condensate. This reaction is catalyzed by the β-ketoacyl synthetase domain and results in a β-ketoacyl thioester. This thioester is then converted into a β-amino fatty acid. A condensation domain further catalyzes the transfer to a PCP domain prior to coupling to asparagine by another condensation domain. The subsequent elongation process is similar to the biosynthesis of surfactin and fengycin. However, there are differences in the arrangement of the subunit domains. For example, while both srfB/fenB and srfC/fenC start at the condensation domain, this domain is integrated in the respective afore subunit for ituB/mycB and ituC/mycC (Tsuge et al., 2001).

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6.4  REGULATION OF LIPOPEPTIDE BIOSYNTHESIS The surfactin synthetase is encoded by the tetracistronic srfA operon (srfAA, srfAB, srfAC, and srfAD) (Nakano et al., 1991). Expression of the srfA operon is directly linked to the cell density and the growth phase. During exponential growth, the srfA operon is repressed by AbrB, the transition state regulator, and its homolog Abh (Chumsakul et  al., 2011). Upon entrance of B. subtilis into the stationary phase, the cells begin to differentiate into different subpopulations including cannibals, biofilm or protease producers, sporulators, and competent cells. The competent cells, approximately 10%–20%, can take up extracellular DNA and produce surfactin (Hamoen et al., 2003). This connection is due to the presence of the comS ORF, which encodes an antiadaptor protein protecting ComK, the master regulator of competence, from posttranslational degradation, within the srfAB ORF (D’Souza et al., 1994; Hamoen et al., 2003). Induction of the srfA operon and comS only takes place in competent cells by a phosphorylated activator, ComA (Fig. 6.4). The ComA response regulator belongs to the ComPA two-component signal transduction system in which ComP is a sensor kinase that autophosphorylates and afterward phosphorylates ComA (Hamoen et  al., 2003). This phosphorylation happens when ComP senses the presence of a short extracellular quorumsensing peptide, ComX (Magnuson et  al., 1994; Pottathil et  al., 2008). The pre-ComX protein is intracellularly processed by ComQ and secreted into the medium. During the stationary phase, the cell density increases that results in a higher concentration of ComX and stimulation of ComPA, thereby activation of PsrfA by ComA~P (Hamoen et al., 2003). Hence, the surfactin production depends on quorum sensing (Hamoen et al., 1995). In addition to ComX-dependent quorum-sensing regulation, phosphatase regulator (Phr) peptides and their cognate response regulator aspartyl phosphatase (Rap) systems are involved in the regulation of PsrfA (Shank and Kolter, 2011). So far, 11 Rap enzymes have been identified in B. subtilis dephosphorylating their target proteins. Among them, RapC, RapD, RapF, RapH, RapK, and RapP are known to dephosphorylate ComA~P and thereby prevent its function (Omer Bendori et  al., 2015; Auchtung et  al., 2006). Here, the regulation of PhrC-RapC will be discussed in more detail as an example. PhrC, also known as CSF for competence and sporulation stimulating factor, is produced and processed intracellularly and secreted to the extracellular milieu. The mature form of PhrC is then taken up via an ATP-binding cassette (ABC) transport system, oligopeptide permease (Opp) (Perego et al., 1991). Transcription of phrC is activated by the sigma factor H (σH) (Hamoen et al., 2003). The sigH gene is regulated by the sporulation master regulator, Spo0A (Weir et al., 1991). Therefore, the physiological state of the cells is important for the expression of quorum-sensing factor, PhrC. The quantity and phosphorylation state of Spo0A determine its targets. Upon entrance to stationary phase, Spo0A activates the expression of sigH. Consequently, surfactin production depends on quorum sensing influenced by cell density of B. subtilis. In addition to the ComA(~P)-dependent pathway, other regulators are also involved in the regulation of PsrfA including DegU, CodY, Abh, PhoP, PerR, and Spx. Each of these regulators links the surfactin biosynthesis to one of the physiological states of B. subtilis. DegU is a global regulator that belongs to the DegS-DegU two-component signal transduction system. DegU is mainly known for its function as an activator for the genes encoding degradation enzymes, such as aprE. However, DegU has other roles, for instance, in the formation of biofilm or swarming. Depending on the physiological state of B. subtilis, the amount and

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6.4  Regulation of Lipopeptide Biosynthesis

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217

FIG. 6.4  Overview of molecular interactions in regulation of genes related to lipopeptide biosynthesis. Main regulatory proteins and transcription factors involved in regulation of the ComX-dependent quorum-sensing network are shown along with external and growth-dependent effects (top boxes). The connection to lipopeptide biosynthesis is given by expression of the srfA operon (surfactin biosynthesis), bmy genes (iturin biosynthesis), and pps operon (fengycin biosynthesis).

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­ hosphorylation state of DegU vary. DegU is phosphorylated by DegS(~P) in a reaction fap cilitated by DegQ (Marlow et al., 2014; Murray et al., 2009). In the undomesticated B. subtilis NCIB3610, it has been recently shown that deletion of degS has a positive effect on PsrfA. As a result, it is assumed that DegU(~P) represses the srfA promoter (Miras and Dubnau, 2016). On the other hand, PdegQ is activated by ComA(~P) (Msadek et  al., 1991) showing that increasing the concentration of DegU(~P) is one step after phosphorylation of ComA via quorum sensing. Therefore, in a model suggested by Miras and Dubnau (2016), activation of the ComA(~P) results in the expression of degQ that results in higher concentration of DegU(~P). As a result, DegU(~P) shuts down PsrfA and activates other differentiation pathways. Apart from the mentioned quorum-sensing pathways, expression of the srfA operon is also influenced by the availability of nutrients, such as phosphate and amino acids. PhoP belongs to the two-component signal transduction system PhoPR, which is activated during phosphate starvation conditions. In this signal transduction system, PhoR is the sensory histidine kinase, while PhoP is the response regulator. This system activates the production of enzymes and transporters dealing with the assimilation of extracellular phosphate (Pragai et al., 2004). Recently, genome-wide ChIP-on-chip analysis indicated that srfAA is activated by PhoP(~P). Surprisingly, comQ was also found as a new member of the PhoP(~P) regulon (Salzberg et al., 2015). This means that phosphate limitation activates surfactin biosynthesis directly via PhoP(~P) and indirectly via ComQ that finally activates a ComA(~P)-dependent pathway. Depletion of amino acids or GTP also upregulates the srfA operon because of CodY derepression (Serror and Sonenshein, 1996). CodY is a global regulator affecting different pathways in B. subtilis. CodY mainly represses genes that help the cell to grow during poor nutritional availability, and its affinity for DNA is enhanced in the presence of high intracellular GTP or branched-chain amino acids (isoleucine and valine) (Sonenshein, 2007). Therefore, during the exponential phase and in the presence of sufficient nutrients, PsrfA is repressed, whereas upon entrance to stationary phase, PsfrA is released from repression by CodY due to the low concentration of intracellular GTP and depletion of branched-chain amino acids. Finally, the presence of oxidative agents downregulates the expression of the srfA operon (Mostertz, 2004). This is due to the structural changes of peroxide response regulator (PerR), which positively regulates PsrfA (Hayashi et al., 2005). In the presence of H2O2, oxidation of the Fe atoms results in a structure of PerR that likely dissociates from the PsrfA DNA (Zuber, 2009). Moreover, the presence of diamide also induces the encoding gene of Spx, which inhibits the interaction between RNA polymerase and ComA(~P); thereby, it inhibits the activation of PsrfA (Nakano et al., 2003).

6.5  LIPOPEPTIDE PHYSICOCHEMICAL PROPERTIES AND CONCOMITANT COMMERCIAL ASPECTS Lipopeptides synthesized by Bacillus spp. are attractive to diverse industrial sectors. Several review articles (Banat et al., 2000; Kanlayavattanakul and Lourith, 2010; Geetha et al., 2018; Gudiña et al., 2013; Mnif and Ghribi, 2015b; Nitschke and Costa, 2007; Nitschke and Silva, 2018; Ongena and Jacques, 2008; Shafi et al., 2017; Shaligram and Singhal, 2014; Zhao et al., 2017) give an overview of chemical attributes and their possible applications such as in the petroleum industry; environmental applications; agricultural applications; laundry

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­ roducts; and the pharmaceutical, cosmetic, and food industry. The following chapter will p give a brief overview of several physicochemical properties of lipopeptides. In addition, selected application fields will be described in more detail.

6.5.1  Physicochemical Properties Surfactin is often claimed as a very efficient surface-active molecule. High-purity surfactin reduced the surface tension of water to 27.90 mN/m at a concentration of 0.005% (Arima et al., 1968). Other studies reported a reduction in surface tension of water from 70 to 36 mN/m at a CMC of 15.6 mg/L, when surfactin was used with a lower purity (AbdelMawgoud et al., 2008). In a study conducted by Deleu et al. (1999), the CMCs of surfactin, iturin A, and fengycin were reported to be 10, 20, and 11 mg/L, respectively. Other biosurfactants such as sophorolipids were described to have a CMC of 20 and 130 mg/L for pure and crude biosurfactant, respectively. At these concentrations, the surface tension of water was reduced to 36 and 39 mN/m (Otto et al., 1999). Further examples are summarized by Desai and Banat (1997). In general, biosurfactants have a lower CMC than chemical surfactants such as potassium oleate with 350 mg/L or fatty alcohol ether sulfate with 170 mg/L (Kosswig, 2012). The low CMCs of biosurfactants and especially for the lipopeptides surfactin, iturin A, and fengycin make them very interesting as smaller amounts of them are needed in comparison with petrochemically derived surfactants. Overall, even surfactin with a lower purity had a CMC lower than chemical counterparts, which is greatly beneficial for its application such as bioremediation or enhanced oil recovery. Here, the demand of high-purity products is less essential than, for example, in the pharmaceutical and food industry. Abdel-Mawgoud et al. (2008) characterized surfactin produced by a B. subtilis strain with respect to several chemical characteristics. The solubility in aqueous solution was given at pH >5, with the optimum at pH 8–8.5. Surfactin was stable in the pH range from 5 to 13. With respect to temperature and salinity, stability was not influenced after autoclaving and in salinities up to 6% NaCl (Abdel-Mawgoud et al., 2008). Next to the outstanding surface-active and chemical properties, lipopeptides hold bioactive properties. With this feature, they stand out from many other surfactants. The bioactive properties of biosurfactants and their concomitant potential in being used in food safety and therapeutic applications are summarized by Meena and Kanwar (2015). Surfactin, for example, showed both antiviral, antibacterial, and antitumor activities, while iturin displayed mostly antifungal activity with limited antibacterial properties. The manifold structures and concomitant attributes allow for several functions such as emulsifier, dispersant, foaming agent, thickening agent, detergent, viscosity-reducing agents, and antimicrobial ingredient. Therefore, several industrial sectors where an application is feasible will be presented in more detail.

6.5.2  Agricultural Applications Every year, plant diseases caused by environmental conditions or by pathogens account for a great loss of plants in the agriculture sector. To prevent loss of crops by diseases, research investigated the applicability of biosurfactants as an alternative to chemical fertilizers due to their eco-friendly, less invasive, and more sustainable character. The bacteria, to which

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both Bacillus spp. and Pseudomonas spp. belong, acting as an “active ingredient” are so-called plant growth-promoting rhizobacteria (PGPR). These PGBR can either protect plants against diseases or promote plant growth (Besset-Manzoni et al., 2018; Shameer and Prasad, 2018). Investigations have already demonstrated that these bacteria live in biofilms and their mode of action relies on a variety of physical triggers and microbial metabolites, such as the excretion of lipopeptides. In this field, Ongena and Jacques (2008) published a review highlighting the relevant facts regarding plant disease biocontrol employing Bacillus lipopeptides. Bacillus spp. is attributed to have antagonistic, inhibiting, spreading, and immuno-stimulating effects. Studies have already demonstrated that the individual lipopeptide families possess different characteristics and hence play different roles in the interaction with plants. Surfactin, for example, is attributed with many features such as being antibacterial and antifungal. Further influences are in the fields of biofilm formation, spreading and as signal for plant diseases. The actions of iturin are spreading and antifungal activities, and fengycin possesses antifungal activities and also acts as signal molecule for plant cells (Ongena and Jacques, 2008). In a study conducted by Malfanova et al. (2012) to evaluate the antifungal activity of lipopeptides synthesized by B. subtilis HC8, fengycin showed the highest antifungal activity. Cawoy et al. (2015) indicated that strains producing all three families of lipopeptides were overall the most effective producers in terms of fungi inhibition. Strains lacking the production of iturin but coproducing both surfactin and fengycin and strains incapable of producing any lipopeptides showed lower inhibition capacities. Results indicated that iturin is the most active ingredient followed by fengycin. Surfactin was reported to have synergistic effects, for example, supports root tissue colonization and promotes nutrient supply by surface wetting and detergent properties. This synergistic effect of surfactin is based on its strong surface-­ active properties that trigger biofilm formation. Biofilm formation is essential for swarming on plant tissues. Also Paraszkiewicz et al. (2017) confirmed that the presence of surfactin in sufficient amounts is necessary for biofilm formation. However, although iturin and fengycin showed higher antimicrobial activities in this report, also surfactin is attributed with this effect due to so-called detergent-like effects on membranes (Heerklotz and Seelig, 2007). Dimkić et al. (2017) used high-performance thin-layer chromatography (HPTLC) to separate individual lipopeptide mixtures and to test the antimicrobial activity against P. syringae pv. aptata, Xanthomonas arboricola pv. juglandis, and L. monocytogenes employing an agar overlay method. Iturin was determined to be the most effective antifungal family. Furthermore, the authors concluded that the antimicrobial activity of iturin was dependent on the homologues within the family. The inhibition zones for surfactin homologues were rather low, supporting the hypothesis that surfactin has synergistic effects and supports plant colonization. Due to the importance of synergistic effects causing plant benefits, lipopeptide family coproducers are very attractive in the agricultural sector to protect plants by diseases. In a recent study conducted by Le Mire et al. (2018), the effectiveness of surfactin to protect wheat by up to 70% against the fungi Zymoseptoria tritici was shown. Interestingly, surfactin itself did not show any antifungal activity, but was reported to stimulate salicylic acid- and jasmonic acid-depending signaling pathways. These acids are important regulators for plant defenses against biotic stresses. Also in a study conducted by Paraszkiewicz et  al. (2017), growth of fungi was reduced for all strains tested, which were surfactin single producers; surfactin and iturin producers; and surfactin, iturin, and fengycin coproducers. However, B. cinerea and A. flavus were only

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growth-inhibited by Bacillus coproducer strains. Likewise, Cawoy et al. (2015) explored that the production of individual congeners and the respective amounts within lipopeptide families vary depending on the target pathogen. Bais (2004) demonstrated that B. subtilis strain 6051, able to synthesize surfactin, forms a biofilm on Arabidopsis roots and shows antibacterial effect against P. syringae. While the wild-type strain B. subtilis 6051 was effective against the pathogens, the derivative containing a ΔsrfAA mutation was less effective.

6.5.3 Detergents Industrial processes are already implemented for sophorolipids (Soliance SA, Pomacle, France; Evonik Industries AG, Essen, Germany; and Saraya Co., Ltd., Osaka, Japan) and for rhamnolipids (AGAE Technologies, Corvallis, Oregon, the United States, and Biotensidon GmbH, Karlsruhe, Germany), and also Evonik Industries AG announced the commercialization of rhamnolipids in a press release from 2016 (Evonik Commercializes Biosurfactants, 2016). Lipopeptides produced by B. subtilis are also of great interest to this industrial sector, and currently, no products in the houseware sector are available containing these biosurfactants. Mukherjee (2007) investigated the compatibility and stability of cyclic lipopeptides in locally available laundry detergents. Results revealed that a mixture of detergent and crude biosurfactant led to an overall improved washing performance of up to 26% in removing sunflower oil or blood from cotton fabrics in comparison with the detergents itself. However, the crude biosurfactants alone had a lower efficiency than the detergents alone. Taira et al. (2017) further evaluated the effect of surfactin on subtilisin, a Bacillus protease often used in laundry formulations. While surfactants such as sodium dodecyl sulfate decreased the proteolytic activity, the presence of surfactin at low concentrations did not inhibit the subtilisin activity.

6.5.4  Nanoemulsions and Emulsions (Nano-)emulsions are important structures in different fields such as the pharmaceutical, cosmetic, and food industry. Such structures allow the incorporation of, for example, health beneficial molecules such as vitamins that are poorly soluble in water and instable at certain conditions. The surface-active molecules incorporated to stabilize these emulsions have to provide both physical and chemical stability. With respect to physical stability, fundamental research on surface-active, interfacial, and emulsifying properties of lipopeptides was performed by several research groups (Deleu et al., 1999; Iglesias-Fernández et al., 2015; Onaizi et al., 2016). Among the three main lipopeptide families, surfactin was reported to be the most effective regarding the time to reach the equilibrium at the oil/water interface and the final interfacial tension, whereas iturin A possessed the best resistance to creaming/flocculation, and fengycin was most stable against coalescence (Deleu et al., 1999). Hence, as each food emulsion features its own character and is accompanied by different destabilization mechanisms, a matrix-specific lipopeptide stabilizer has to be characterized. Onaizi et al. (2016) and Iglesias-Fernández et  al. (2015) also investigated the ability of surfactin to adsorb at solid-­ liquid and especially air-liquid interfaces. The latter investigations further increase the field of applications in, for example, aerated food products such as mousse.

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With respect to chemical stability, He et al. (2017) investigated the physical and oxidative stability of microemulsions with docosahexaenoic acid, one of the most important omega-3 polyunsaturated fatty acids. Results revealed that the microemulsion stabilized with surfactin showed an enhanced physical and antioxidative stability than a microemulsion with Tween 80. Furthermore, the experiments indicated that surfactin at a concentration of 0.2 mmol/L had a better emulsifying capability in o/w emulsions than Tween 80, lecithin, and sucrose fatty acid ester at equal concentrations. The incorporation of surfactin as stabilizer can have a further benefit over synthetic stabilizers depending on the application. As surfactin possesses antimicrobial properties, (nano-) emulsions stabilized by this biosurfactant have consequently antimicrobial effects. As a result, applications in the pharmaceutical industry, in cosmetics, or the food industry are conceivable. A nanoemulsion based on sunflower oil and stabilized by surfactin, for example, showed a high activity against Salmonella typhi and also against L. monocytogenes and Staphylococcus aureus (Joe et al., 2012). These findings give one example of application possibilities in the food safety area, as lipopeptides can be incorporated into the food matrix as protecting ingredient.

6.5.5  Food Industry With respect to applications as ingredient or additive, the presence of surfactin in various fermented food products such as in natto, a Japanese soybean dish, is highly beneficial for approval. Juola et al. (2014) examined different natto samples with respect to their surfactin content. Interestingly, the highest amounts detected were up to 2.2 mg/g, which correlates to an amount of 80–100 mg surfactin per 50 g of natto. Further studies need to be conducted in order to determine the acceptable daily intake (ADI) for surfactin to declare it as nontoxic. But, regarding the fact that surfactin is already consumed daily by numerous humans and as B. subtilis is generally considered being a generally accepted as safe (GRAS) organism, an implementation of surfactin in the food industry is very conceivable. One possible application of surfactin is in the reduction of biofilm formation on food processing equipment induced from food pathogens. Surfactin can adsorb on materials such as stainless steel, and the lipopeptide was able to reduce adhesion and biofilm formation of the pathogens L. monocytogenes and P. fluorescens. Further coeffects might be essential for the antimicrobial effects of surfactin (de Araujo et al., 2016). Few studies were performed so far to incorporate surfactin directly into a food matrix to replace other ingredients. One of these attempts was carried out by Zouari et al. (2016). Sesame peel flour was used for the preparation of cookies as partial replacement for conventional white wheat flour. Parameters such as hardness, moisture content, and spread factor became worse when more sesame peel flour was used. However, when 0.1% B. subtilis SPB1 biosurfactant was added, the texture profile was highly improved, even in comparison with the conventional used emulsifier glycerol monostearate (Zouari et al., 2016). Another possible application can be found during food processing, as investigated by Jiang et al. (2017). In this study, the potential of lipopeptides from Bacillus spp. to inhibit A. carbonarius and hence ochratoxin A contamination in the winemaking process was investigated. Ochratoxin A concentration should not exceed 2.0 μg/L in wine as it is a carcinogenic mycotoxin. Furthermore, this compound negatively affects the fermentation behavior of yeasts. The study revealed that the presence of lipopeptides during the winemaking process

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reduced A. carbonarius and hence ochratoxin A concentrations. Also in comparison with SO2, the lipopeptides showed better antifungal properties and also promoted the growth of yeasts and formation of esters and acids being involved in the aroma profile (Jiang et al., 2017).

6.5.6  Personal Care and Therapeutic Applications Biosurfactants can also be used in personal care and therapeutic applications. One attempt was made by Bouassida et al. (2017). Here, B. subtilis SPB1 lipopeptide was used as alternative to the conventional used surfactant sodium dodecyl sulfate in toothpaste formulation. Next to good foaming abilities, the toothpaste containing lipopeptides furthermore was reported to be advantageous due to the antimicrobial activities the lipopeptides come along with.

6.6  BIOTECHNOLOGICAL PRODUCTION OF SURFACTIN In the field of surfactin production, research often aims at increasing the yields to approach industrial implementation. The following chapter describes the recent progress regarding surfactin production and lipopeptide synthesis in general. Thereby, the chapter is divided into three categories. In the first part, strategies dealing with both medium and process parameter optimization will be presented. Secondly, techniques employing strain engineering are given, and the last part illustrates an overview of different cultivation strategies employed.

6.6.1  Optimization of Media Composition and Process Parameter The optimization of both the medium composition and process parameter is often addressed in studies dealing with the increase of lipopeptide yields. In this field, the review paper by Rangarajan and Clarke (2015) and Shaligram and Singhal (2010) give a fundamental summary. With respect to medium optimization, either defined or complex media are used as basis, and novel approaches incorporate the utilization of cheap substrates labeled as agro-­ industrial waste products or by-products.

6.6.2  Carbon, Nitrogen and Trace Elements With respect to media optimization, the well-established mineral salt medium used by Cooper et al. (1981) and the Landy medium (Landy et al., 1948) are often used as reference. By surveying literature, the complexity of surfactin and lipopeptide synthesis in general becomes obvious. In terms of growth, ammonium and glutamine are the preferred nitrogen sources for Bacillus spp. (Detsch, 2003; Fisher, 1999). Among different nitrogen sources tested for surfactin production, ammonium nitrate was shown to be a very good nitrogen source for the strain B. subtilis YRE207 (Fonseca et al., 2007), whereas this source was less suited for the strain B. subtilis BBG208 (Yaseen et al., 2017). The same study also indicated that ammonium nitrate is a promising alternative for glutamic acid for the synthesis of fengycin (Yaseen et al., 2017). In both studies, also urea was an interesting nitrogen source that also benefits from its low prize. Alanine had a positive effect on surfactin synthesis when glutamic acid in the original Landy medium was replaced, whereas the

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employment of other amino acids and complex media components significantly reduced the yield (Yaseen et al., 2017). Interestingly, while a replacement of glucose by different carbon sources did not enhance surfactin production, a significant increase in the concentration of fengycin was determined using mannitol (Yaseen et al., 2017). Depending on the strain, the C-N ratio is also critical. This ratio was addressed in a study conducted by Medeot et  al. (2017) with the strain B. subtilis MEP218. The optimal ratio was reported as 10:1, and best carbon and nitrogen sources tested were glucose and ammonium nitrate, respectively. The original Cooper and Landy media have a C-N ratio of 13:1 and 27:1, respectively. In general, medium optimization procedures come along with numerous experiments. Therefore, designs of experiment methods, such as response surface methodology or the Taguchi method, are useful tools to evaluate the effect of different components on the lipopeptide concentration. Sen (1997) improved the original Cooper medium with respect to glucose, NH4NO3, FeSO4, and MnSO4 concentrations. The optimal concentrations determined for maximal surfactin production with the strain B. subtilis DSM 3256 were 36.5, 4.5, 4 × 10−3, and 27.5 × 10−2 g/L, respectively. While this study confirmed a rather high glucose concentration being better suited, Willenbacher et al. (2015b) reported that a glucose concentration of 8 g/L gives better yields. Here, the surfactin concentration was increased from 0.7 to 1.1 g/L for the strain B. subtilis DSM10T. In addition, this study also addressed environmental concerns and the original chelating agent Na2EDTA of the Cooper medium was replaced by Na3-citrate. In contrast, a study addressing the production of lichenysin by the strain B. licheniformis WX-02 identified 30 g/L glucose as optimal concentration when testing different concentrations in the range of 10–50 g/L (Qiu et al., 2014). Employing design of experiment, the surfactin concentration obtained by B. subtilis ATCC 21332 was increased from 1.74 to 3.34 g/L with an optimized trace element composition (Wei et  al., 2007). Here, optimal concentrations of Mg2+, K+, Mn2+, Fe2+, and Ca2+ were 2.4, 10, 0.01, and 0.008 mM and 7 μM, respectively, instead of the original 0.8, 30, 0.2, 0.3, and 7 μM. Hence, concentrations of Mg2+, K+, and Mn2+ were increased, and especially, Mg2+ and K+ were found to have interactive effects. Mg2+ and K+ are directly involved in surfactin synthesis, with Mg2+ needed in the PCP domain (Reuter et al., 1999) and K+ stimulating surfactin secretion (Kinsinger et al., 2003). Other trace elements, such as Mn2+ and Fe2+, had a lower impact on surfactin production itself, but cell growth was drastically reduced in the absence (Wei et al., 2007). However, a study conducted by Huang et al. (2015) revealed a positive effect of Mn2+ when using NH4NO3 as nitrogen source. Bacillus spp. show diauxic consumption and use first NH 4 + followed by NO 3 − after depletion (Davis et al., 1999). An increase in Mn2+ to >0.05 mmol/L led to a shift toward nitrate utilization, and final concentrations of surfactin measured were 6.2-fold higher as well. Consequently, Mn2+ was shown to increase the activity of both nitrate reductase and glutamate synthetase. The latter one increases the ability of free amino acids (Huang et al., 2015). Design of experiments was also conducted by Motta Dos Santos et al. (2016) in microplate bioreactor. The statistics revealed that higher levels of glutamic acid, glucose, yeast extract and MgSO4, and l-tryptophan had a positive effect on surfactin production by the strain B. subtilis BBG131. Interestingly, higher amounts of trace elements were statistically not significant to enhance surfactin yields, when other chemicals with a higher effect were improved. After statistical analysis, the proposed medium for both optimal biomass production and optimal

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YP/X was compared with the original Landy medium in a bubbleless membrane bioreactor as shown in Coutte et al. (2013). Although no significant improvement in the surfactin concentration was obtained, the yield YP/X increased from 135.34 mg/g cell dry weight to 305 mg/g. Also Gudiña et al. (2015) illustrated the complexity of the effects of added trace elements. For example, the reference medium containing 10% corn steep liquor achieved a crude biosurfactant concentration, which contained surfactin congeners, of 1.3 ± 0.1 g/L. In a next setup, individual optimal concentrations were determined for FeSO4, MnSO4, and MgSO4. Here, for both 2.0 mM FeSO4 and 0.8 mM MgSO4, the optimal individual concentrations evaluated led to crude biosurfactant concentrations of 4.2 ± 0.1 and 3.5 ± 0.1 g/L. Interestingly, when combining these trace elements at their respective optimal concentrations, the concentration achieved was rather low with 2.7 ± 0.1 g/L. However, when these chemicals were added together with 0.2 mM MnSO4, the highest crude biosurfactant concentration within this study was reported with 4.8 ± 0.2 g/L.

6.6.3  Alternative Substrates for Lipopeptide Production Next to the optimization of single components in defined media, another approach to increase the yields is the utilization of waste streams or by-products such as cashew apple juice (Freitas de Oliveira et al., 2013), rapeseed meal (Jin et al., 2014), waste distiller’s grain (Zhi et  al., 2017), two-phase olive mill waste (alpeorujo) (Maass et  al., 2016), orange peel (Kumar et al., 2016), or cassava-flour-processing effluent (Nitschke and Pastore, 2004). The utilization of such media components is especially interesting for high-volume and low-value applications (Rangarajan and Clarke, 2015). However, while investigating the effect of such supplements on the final lipopeptide concentration, the authors often do not compare their results with a reference medium. Such approaches have both advantages and disadvantages. For example, the overall production costs may be reduced when using alternative substrates that do not compete with the food industry and open new opportunities regarding lucrative waste management. On the other hand, for several waste streams, it is difficult to maintain a constant composition, and the purity plays an important role. For example, de Andrade et al. (2016b) replaced analytic-grade glycerol by concentrated glycerol from the biodiesel industry and studied the effect on surfactin production. The utilization of this waste might also come along with other nutritional elements such as phosphorus or nitrogen, depending on the initial source of triglycerides used. In contrast to prior expectations, the utilization of concentrated glycerol resulted in an overall delay in cell growth compared with analytic-grade glycerol. This indicated that impurities such as remaining salts and methanol, still present in the by-product at low concentrations, might have a strong, and in this study negative, influence on cell growth. In terms of biosurfactant production, higher foaming and hence higher biosurfactant synthesis were recorded for the medium containing analytic-grade glycerol with 325 mg/L in comparison with 71 mg/L.

6.6.4  Influence of Process Parameter Experimental design methods were also employed to optimize process parameter. Jacques et al. (1999) investigated the effect of different temperatures (25–40°C), pH (5.5–8), shaking (100–200 rpm), and media components in shake flask cultures. The optimal process p ­ arameter

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to increase the synthesis of surfactin, iturin A, and fengycin by the strain B. subtilis S499 was 30°C, pH 7, and 200 rpm. Fahim et al. (2012) investigated different rpm and concomitant different kLa values in shake flask experiments. They revealed that surfactin production is superior for higher kLa values, while fengycin synthesis was better under moderate oxygen supply. In terms of oxygen supply, Rangarajan et al. (2015) also reported a higher selectivity toward the synthesis of fengycin by the strain B. megaterium MTCC 8280 under oxygen-­ limiting conditions. Yeh et al. (2006) investigated different kLa values in a 5 L jar fermenter with foam collector. The highest kLa, obtained by 300 rpm and 1.5 vvm, led to the highest surfactin concentration and overall production rate of 106 mg/(L h). Monteiro et al. (2016) investigated the influence of different culture media, temperatures (15, 25, and 30°C), and initial pH (5–9) on the production of the three lipopeptides surfactin, iturin, and fengycin using the strain B. amyloliquefaciens 629. During their investigations, only one of the four media, namely, PDB, a medium containing plant-derived nutrient, allowed for a simultaneous production of all three lipopeptides at 25°C and 30°C. Nevertheless, the lowest concentrations of surfactin and fengycin were detected in this medium in comparison with the other media tested (medium optimal for lipopeptide production, MB1, and Luria-Bertani (LB)). For surfactin, overall higher concentrations were monitored at 15°C and for fengycin at 25°C. With respect to pH, no lipopeptides were detected after cultivations with an initial pH of 5, 8, or 9. An initial pH of 6 was furthermore more suitable than pH 7. Slivinski et al. (2012) investigated the temperatures 25, 30, 37, and 45°C on the production of surfactin using a medium containing 50% sugarcane and 50% okara in a solid-state fermentation. Highest surfactin concentration was measured at 37°C after 60 h with 484 mg/L. At 45°C, the maximum concentration measured was 108 mg/L at 36 h. For both 25 and 30°C, the surfactin concentration continuously increased to a concentration of ~ 380 and 290 mg/L until 72 h.

6.6.5  Alternative Medium Optimization Approaches Both de Andrade et  al. (2016b) and Zhi et  al. (2017) provided interesting approaches to optimize surfactin that will be described briefly. To increase the yield of lipopeptides, de Andrade et  al. (2016a) proposed a process that aimed to cosynthesize two different target products, namely, surfactin and 2,3-butanediol. The latter one shows already actual and potential applications in printing inks; rubbers; and cosmetic, food, and pharmaceutical products. A cooptimization may show an impact on the economic viability, as the overall value increases, especially when the main product of interest such as surfactin is just synthesized in small amounts. In order to achieve higher concentrations, they investigated different concentrations of the three media components cassava wastewater, whey, and activated carbon. Interestingly, the employment of central composite rotational design revealed an almost identical composition of the three media components to achieve the maximum concentrations for both surfactin and 2,3-butanediol. Also Zhi et  al. (2017) examined an interesting approach to synthesize surfactin using a waste by-product. In their study, waste distiller’s grain was used as carbon source for culturing B. amyloliquefaciens. In the first step, strains were screened with respect to their ability to grow on distiller’s grain and their surfactin concentrations achieved. As a result, the highest

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concentration of surfactin was reached by the strain B. amyloliquefaciens MT45. However, this strain exhibited poor growth on the medium and a low carbon conversion. Therefore, the strains were also evaluated regarding their amylase, protease, and lipase activities. Strains exhibiting a high amylase activity in general reached higher cell counts when cultivating on waste distiller’s grain. Hence, the surfactin-producing strain MT45 was cocultured with strains exhibiting a high hydrolase activity. This coculturing enhanced the surfactin concentration from 1.04 to almost 1.6 g/L. Further optimization was performed by evaluating the inoculation ratio of the two strains MT45 and X82. The overall surfactin concentration was the highest when using a ratio of 1:0.5 with 2.54 g/L.

6.6.6  Strain Engineering As already illustrated in a previous chapter, the synthesis of surfactin is regulated by a complex quorum-sensing mechanism. Consequently, also strain engineering is often employed to enhance the surfactin yields by different approaches. A study by Coutte et al. (2010a) revealed that the disruption of the operon encoding for plipastatin in the strain B. subtilis 168, where a functional sfp gene was integrated, led to a fivefold increase in surfactin synthesis. The native promoter PsrfA was thereby replaced by a constitutive promoter, and activity measurements revealed an earlier expression. Interestingly, Willenbacher et al. (2016) demonstrated that an exchange of the native promoter PsrfA by a strong constitutive promoter was strain-dependent. An already relatively strong surfactin producer such as B. subtilis DSM10T was negatively affected by the exchange, while a minor surfactin producer benefited from the replacement. Also, Yaseen et  al. (2016) investigated the effect of different promoters on the synthesis of fengycin. The Pfen promoter of strain B. subtilis BBG21, a spontaneous mutant of the strain ATCC 21332, showed a very high efficiency compared with the promoter Ppps, which is located in the strains BBG111, a 168 derivative, and B. amyloliquefaciens FZB42. Interestingly, when the Ppps promoter in the strain BBG111 was replaced by the strong promoter Pfen of strain BBG21, a 10-fold increase in fengycin production was observed for BBG111, while the overall synthesis rate remained almost constant when integrating the Pfen promoter of strain ATCC 21332. Sequence analysis of Pfen in ATCC 21332 and its derivative BBG21 revealed that a point mutation in Pfen of BBG21 led to the overproduction of fengycin. Another study addressing promoter exchange was conducted by Qiu et al. (2014). The lichenysin synthesis of the strain B. licheniformis WX-02 was reported to be improved when the native promoter was replaced by the srf operon promoter Psrf. Consumption exhibited a similar pattern (Zhao et  al., 2012). Within the same group, a strain with a 12.77-fold increased fengycin expression was reported by genome shuffling (Zhao et al., 2016). In terms of metabolic engineering, Coutte et al. (2015) generated a model predicting the knockout of respective genes to overproduce leucine, an important precursor for surfactin synthesis. The engineered strain based on this model held a 20.9-fold increased surfactin synthesis. Using the same syntax developed in this study, Dhali et al. (2017) engineered strains lacking either the gene codY or lpdV, and surfactin synthesis was increased by 5.8- and 1.4-fold, respectively. Furthermore, the results revealed that the lpdV mutant overproduced mainly the C14 isoform. Such approaches are very interesting as the synthesis can be headed toward a specific isoform.

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6.6.7  Cultivation Strategies for Lipopeptide Production In Section 6.1, findings regarding media composition and fundamental parameter such as temperature and pH were presented that provide a basis for bioreactor cultivation. However, the production of lipopeptides at a larger scale goes along with a main challenge, namely, the severe foaming, that is often not addressed in shake flask experiments. The occurrence of foam can be considered as both advantage and drawback, as it not only provides the first purification step but also goes along with the loss of culture media and surfactin-producing cells (Coutte et al., 2017; Rangarajan and Clarke, 2015). In this sense, the following sections focus on process strategies that either integrate or avoid foaming. 6.6.7.1  Batch, Fed-Batch, and Continuous Strategies A batch process with the addition of antifoam was carried out by Davis et  al. (1999). Under glucose-limited conditions, the strain B. subtilis ATCC 21332 was synthesized up to 31.2 mg/L surfactin, and the YP/X obtained was 0.0068 g/g. Surfactin was thereby mainly produced when entering the stationary phase. Under nitrogen-limiting conditions, the maximum concentration of surfactin was 45.3 mg/L with an YP/X of 0.021 g/g. Here, surfactin was mainly synthesized when the strain used nitrate as nitrogen source instead of ammonium. In a subsequent study, Davis et  al. (2001) demonstrated that foaming is favorable. Highest surfactin enrichment was obtained when foaming was integrated in the cell culture stage instead of employing it as separate unit. In addition, the authors reported that lower stirrer speeds were more favorable than higher stirrer speeds. Employing 269 rpm, half of the media was lost due to foaming after 11 h, and hence, the time window for surfactin production was drastically reduced. Lower stirrer speeds consequently favored surfactin enrichment in the foam. This was confirmed by Yeh et al. (2006). In general, higher stirrer speed and aeration rates in a 5 L jar fermenter resulted in overall higher surfactin concentrations until 1.5 vvm and 300 rpm. A further increase in both parameters, on the contrary, decreased surfactin production rates. In another study employing the strain ATCC 21332, antifoam addition was avoided (Coutte et al., 2010b). Antifoams are often considered as undesired as they are costly and might have a negative effect on cell growth. Besides, they need to be addressed in product purification (Yeh et al., 2006). In a batch culture with the strain B. subtilis ATCC 21332 and Landy medium, foaming started after 6 h, and from the initial volume of 3 L, only 1.13 L was in the system at the end of cultivation (Coutte et  al., 2010b). Both surfactin and fengycin were continuously extracted in the foam with total amounts produced of 714 and 43 mg, respectively. For surfactin, 60 mg were extracted from the broth, while fengycin was not determined in broth samples. From the total biomass of 12.4 g, 1.4 g was extracted from the foam. Consequently, the foaming yielded higher concentrations than the process employing antifoam. Next to the rpm, also the position of the impeller affects the performance. Chenikher et al. (2010) proposed to have one Rushton turbine impeller in the medium to allow for a proper mixing, and a second impeller was positioned slightly above the liquid level. When the volume increased due to feeding, the impeller promoted mixing at the air-liquid interface that favored foaming. The loss of both medium and cells was addressed in a feeding profile established by Guez et al. (2007) for the synthesis of mycosubtilin with the strain B. subtilis BBG100. Here, the feeding rate was adapted to the foam overflow rate. Jin et al. (2015) also increased the iturin A p ­ roduction

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by employing a two-stage glucose feeding strategy. They addressed the cell-to-spore ratio and aimed at reducing the amount of spores, as spores are the final state in cell differentiation and consequently do not produce lipopeptides. A low feeding rate resulted in a relatively low iturin A concentration. Interestingly, by employing a rather high feeding rate, the ability of cells to consume glucose was found to change, and a glucose accumulation in the later stage of cultivation resulted in an increase of spores and thereby a reduction in the iturin A productivity. Hence, a combination of the feeding rates was tested. During the first stage, the high rate was employed, and in the late feeding stage, the feed rate was reduced. As a result, glucose accumulation did not occur, the cell-to-spore ratio was relatively constant, and the synthesis of iturin A was maintained during the whole time course of cultivation. Another approach was presented by Rangarajan et al. (2015) for the production of fengycin in the strain B. megaterium MTCC 8280. In their bioreactor batch cultivation, foaming was almost completely avoided due to a switch from submerged aeration to headspace aeration and applying a positive pressure. This enabled the reproduction of shake flask experiments where fengycin production was found to be the highest under oxygen-limiting conditions. For lipopeptide production, the transition from fed-batch to continuous processes is very narrow. Especially when foaming is integrated and a feed is applied, the process can also be considered as a continuous setup. Such a process was also presented by Chen et  al. (2006) in which foam fractionation was integrated and optimal conditions for surfactin production employing the strain B. subtilis BBK006 were obtained at a dilution rate of 0.2 L/h and a rather low glucose concentration in the feed with 0.25 g/L. Alonso and Martin (2016) also examined a continuous strategy in a small-scale experiment where the culture broth was actively pumped with a set flow rate into a foam trap. An air pump was mounted to the bottom of the foam column that allowed to produce foam. The overflow of the foam was set to a defined rate and hence allowed to maintain a constant volume in the foam column. 6.6.7.2  Alternative and Novel Process Set-Ups SOLID CARRIER ASSISTED SUBMERGED CULTIVATION

Yeh et  al. (2006) claimed that the addition of solid carriers to the cultivation process stimulates surfactin production using the strain B. subtilis ATCC 21332 as the additional surface might trigger cell growth and hence surfactin production as cells form biofilms at the surfaces. The optimized concentration of the activated carbon with 25 g/L as solid carrier led to a surfactin concentration of 3600 mg/L in comparison with the control without solid carriers of 100 mg/L. In a further study, Yeh et  al. (2008) investigated a bioreactor design with the previously obtained experiences of increasing surfactin production by adding solid carriers. This enabled them to handle the severe foaming, and an addition of antifoam was not necessary. Their bioreactor is composed of a jar connected to a foam collector, a cell recycler, and a surfactin precipitation unit. In the medium, they added activated carbon. After 60 h of cultivation, a maximum surfactin concentration was measured with 6.45 g/L, and the overall volumetric production rate was calculated to be 0.106 g/ (L h). The application of a cell-recycling device is furthermore advantageous, as a severe loss of volume and hence surfactin-producing cells is often an issue when processes involving foaming are created.

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ROTATING DISC BIOREACTOR

The rotating disk system described by Chtioui et al. (2010) is a bubble-free process and takes advantage of the ability of Bacillus strains to form biofilms. The setup was further investigated in 2012 for the simultaneous synthesis of surfactin and fengycin employing the strain B. subtilis ATCC 21332 (Chtioui et al., 2012). The disks rotated at a speed of 30 L/min, and the disks were mounted to be half immersed in the medium. Air was supplied by an inlet in the medium-free area. In their study, they investigated two different air flow rates and used either 7 or 14 disks. At a lower air flow rate of 100 L/h, more planktonic cells and more biofilm formation were obtained at the end of cultivation with 14 disks. In general, the total biomass obtained after 72 h of cultivation was rather low in all experimental setups with 2.2–3 g cell dry weight per liter, indicating a low growth rate. During the experiment, oxygen limitation occurred in the medium especially during the exponential growth phase. This resulted in the production of acetoin. Interestingly, fengycin concentrations were about 2.5- and 4.5-fold higher than surfactin concentrations. In this experimental setup, surfactin production started at the beginning of the stationary phase, and fengycin concentrations were relatively significant at that time. The high production of fengycin might be correlated to the low oxygenation levels or to the formation of biofilm. BUBBLELESS MEMBRANE BIOREACTOR

Coutte et al. (2010b) investigated the production of the lipopeptides surfactin and fengycin in a bubbleless membrane bioreactor using the strain B. subtilis ATCC 21332. In this way, foam formation was avoided by employing aeration by a hollow fiber membrane air-liquid contactor. Different aeration setups were tested with either external or submerged aeration modules. With respect to surfactin production, the concentrations in the different setups ranged between 188 and 242 mg/L. A higher difference was observed for fengycin production, where the concentrations ranged between 47 and 392 mg/L. Interestingly, for surfactin, the highest concentration was obtained in the setup using an external aeration with polyethersulfone, and for fengycin, this setup yielded the lowest concentration. The highest concentration was obtained with a submerged aeration. Interestingly, washing of the membranes in all three setups revealed that a high proportion of surfactin was adsorbed onto the membranes. On the contrary, only small amounts of fengycin adsorbed onto the membranes. However, the choice of a proper membrane is very important as results demonstrated the effect of adsorbed surfactin on the kLa. In addition, cells accumulated on the membranes. As similar overall amounts of both surfactin and fengycin were produced in comparison with a simple batch process with foam overflow, the usefulness of such a design was highlighted. In 2013, an improved and continuous process with cell recycling using the developed membrane bioreactor was presented by Coutte et al. (2013). In detail, the medium was fed into the bioreactor at a constant flow rate of 0.3 L/h, and the same amount was allowed to leave the system. This overflow passed a microfiltration unit, and cells were recycled into the system. The permeate containing, among others, surfactin was collected in a second tank. A further purification step was integrated by mounting an ultrafiltration membrane unit to tank 2. Thereby, surfactin was separated from other residual substrates and metabolites. In comparison with the batch process, an improved surfactin productivity is using the strain B. subtilis BBG131, a 168 derivative, of 54.7 mg/(L h) instead of 17.4 mg/(L h) in a fed-batch process. Under integration of cell recycling, the productivity was further increased to 110 mg/ (L h), when a dilution rate of 0.2 L/h was set.

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ANAEROBIC CULTIVATION

Bacillus subtilis was for long considered being a strict aerobic microorganism. However, B. subtilis is also able to grow under anaerobic conditions using nitrate or nitrite as final electron acceptor. In the absence of an alternative electron acceptor, B. subtilis undergoes fermentative growth with lactate, acetate, and ethanol as end products (Härtig and Jahn, 2012; Nakano et al., 1997). For surfactin synthesis, B. subtilis ATCC 21332 was shown to exhibit a better YP/X under nitrate-limited oxygen-depleted (0.075 g/g) conditions, and the maximal concentration attained was 439.0 mg/L in comparison with ammonium-limited oxygen-depleted (0.012 g/g and 53.2 mg/L) and carbon-limited oxygen-depleted (0.0069 g/g and 41.3 mg/L) conditions (Davis et al., 1999). The high YP/X also surpassed the values obtained during aerobic conditions. These findings were confirmed by Willenbacher et al. (2015a) employing a foam-free anaerobic cultivation with the strain B. subtilis DSM10T. In comparison with a batch process employing foam fractionation with an YP/X of 0.192 g/g, the anaerobic process gave a yield of 0.278 g/g.

6.6.8  Downstream Processing of Lipopeptides The choice of an appropriate downstream process is dependent on the target product and the concomitant purity needed. In general, the number of downstream processing steps increases with increasing purity, and consequently, also production costs rise. Mnif and Ghribi (2015a) and Rangarajan and Clarke (2016) give a broad overview of the downstream techniques applied and the respective advantages and disadvantages. Acid precipitation is the most frequently used procedure to obtain crude biosurfactant (Alvarez et al., 2012; Dunlap et al., 2011; Grover et al., 2010; Vater et al., 2002) and is greatly advantageous as it is both easy to conduct and a cheap technique. Direct solvent extraction is also often applied. Thereby, the proper selection of a solvent can target at a specific lipopeptide family (Cazorla et al., 2007; Dimkić et al., 2017; Geissler et al., 2017). As previously indicated, foam fractionation is often integrated into the process in order to handle the severe foaming (Davis et al., 2001; Willenbacher et al., 2014). When the foam fractionation is directly incorporated into the process, this process step is called in situ product removal (ISPR). ISPR was shown to be very promising as a first enrichment step can be obtained and the subsequent number of downstream processing steps can be reduced. Other techniques such as thin-layer chromatography (TLC) (Cazorla et al., 2007), high-performance liquid chromatography (HPLC) (Dunlap et al., 2011), adsorption on resins or charcoal, and membrane ultrafiltration are used among others. Ultrafiltration is a promising approach for the environmental friendly and less time-consuming recovery of biosurfactants with high yields, but equipment costs are high (Isa et al., 2007). Reversed phase HPLC, so far, is probably the most appropriate technique to obtain lipopeptides of high purity.

6.7  CHALLENGES, NEEDS, AND FUTURE TRENDS IN LIPOPEPTIDE PRODUCTION As highlighted in the previous sections, many studies were successfully employed to increase the yields of lipopeptides. Nevertheless, the complex regulatory system of Bacillus spp. still poses a main challenge that results in concentrations and yields to a level far below an industrial-scale application. II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

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Another obstacle often observed is the degradation of surfactin. While iturin and fengycin are mainly produced during the stationary phase, surfactin is synthesized during the late exponential phase (Ongena and Jacques, 2008). Consequently, when the surfactin concentration is monitored during the time course of cultivation, surfactin synthesis mostly strongly correlates with cell growth. As reported, in the transition to the stationary phase, a decrease in surfactin concentration was often reported (Dhanarajan et al., 2014; Maass et al., 2016; Yeh et al., 2006). Consequently, putative mechanisms must be present that degrade lipopeptides. Nitschke and Pastore (2004) proposed that proteolytic activities might lead to a degradation of surfactin. However, surfactin degradation in B. subtilis ATCC 21332 exhibiting a high proteolytic activity was not observed. Interestingly, when the crude biosurfactant obtained by this strain was inoculated in culture broth of a strain where a degradation was detected, the recent stable biosurfactant was degraded as well. Maass et  al. (2016) named three mechanisms that might be responsible for surfactin degradation. Additional supplements need to be supplied, proteases are present in the culture medium hydrolyzing the peptide moiety of the lipopeptide, or the bacteria itself consume the lipopeptide as these might possess an inhibitory effect at a certain concentration. As they did not observe a complete degradation, Maass et  al. (2016) concluded that the last option is the most reasonable. Yeh et  al. (2006) assumed that cells assimilate surfactin as carbon source after glucose depletion to maintain cell growth for a certain time. Nevertheless, these observations must be examined in further studies to figure out if the yields might be increased when surfactin is not degraded. Another aspect that may be addressed in future studies is the complex regulatory mechanism and the lifestyle of Bacillus spp. As reported in the study by Jin et al. (2015), a reduced number of spores led to an increase in iturin concentration. However, spore formation is only one out of several differentiation states that Bacillus spp. can undergo (Romero, 2013). As a matter of fact, once entered a differentiation state other than surfactin production, cells are no longer able to change their physiological state for surfactin production (Lopez et al., 2009). Further studies addressing this obstacle are necessary especially when aiming at high cell density cultivations. If cells differentiate, the ability to produce surfactin in sufficient amounts is highly limited. In addition to strain development and process improvement, more studies must be targeted at further real applications and at promoting toward an approval as ingredient in food, cosmetic, and other applications. It was shown that surfactin has a low toxicity (Juola et al., 2014), but although often claimed as being a highly promising biosurfactant, only a few concrete comparisons were made. In addition, more research must be performed exploring the properties of individual lipopeptide congeners, and hence, also appropriate purification techniques need to be established.

6.8  CONCLUSION AND OUTLOOK This chapter provided information regarding both the synthesis, chemical properties, possible application fields, and an overview of approaches to increase yields. In summary, it can be concluded that results obtained within a study addressing, for example, media optimization cannot be transferred directly to any other strain. In addition, an issue often facing is the presentation of results. Often, lipopeptide concentrations were d ­ etermined at a few or just one time point during cultivation. With respect to different growth behavior, it

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might be reasonable to assume that the point of maximum productivity was missed. This fact makes it complicated to compare research. Nevertheless, surfactin is a promising microbial-derived surfactant, and although a large number of potential applications in different industrial sectors have been published, the widespread use of surfactin and other lipopeptides is not conceivable in the foreseeable future. Much research still has to be performed coupling the presented methods and addressing the issues illustrated in this chapter.

References Abdel-Mawgoud, A.M., Aboulwafa, M.M., Hassouna, N.A.-H., 2008. Characterization of surfactin produced by Bacillus subtilis isolate BS5. Appl. Biochem. Biotechnol. 150 (3), 289–303. Alonso, S., Martin, P.J., 2016. Impact of foaming on surfactin production by Bacillus subtilis: implications on the development of integrated in situ foam fractionation removal systems. Biochem. Eng. J. 110, 125–133. Alvarez, F., Castro, M., Príncipe, A., Borioli, G., Fischer, S., Mori, G., Jofré, E., 2012. The plant-associated Bacillus amyloliquefaciens strains MEP218 and ARP23 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease. J. Appl. Microbiol. 112 (1), 159–174. Arima, K., Kakinuma, A., Tamura, G., 1968. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun. 31 (3), 488–494. Aron, Z.D., Dorrestein, P.C., Blackhall, J.R., Kelleher, N.L., Walsh, C.T., 2005. Characterization of a new tailoring domain in polyketide biogenesis: the amine transferase domain of MycA in the mycosubtilin gene cluster. J. Am. Chem. Soc. 127, 14986–14987. Auchtung, J.M., Lee, C.A., Grossman, A.D., 2006. Modulation of the ComA-dependent quorum response in Bacillus subtilis by multiple Rap proteins and Phr peptides. J. Bacteriol. 188 (14), 5273–5285. Bais, H.P., 2004. Biocontrol of Bacillus subtilis against infection of arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production. Plant Physiol. 134 (1), 307–319. Balan, S.S., Kumar, C.G., Jayalakshmi, S., 2016. Pontifactin, a new lipopeptide biosurfactant produced by a marine Pontibacter korlensis strain SBK-47: purification, characterization and its biological evaluation. Process Biochem. 51 (12), 2198–2207. Banat, I.M., Makkar, R.S., Cameotra, S.S., 2000. Potential commercial applications of microbial surfactants. Appl. Microbiol. Biotechnol. 53 (5), 495–508. Beltran-Gracia, E., Macedo-Raygoza, G., Villafaña-Rojas, J., Martinez-Rodriguez, A., Chavez-Castrillon, Y.Y., Espinosa-Escalante, F.M., Di Mascio, P., Ogura, T., Beltran-Garcia, M.J., 2017. Production of lipopeptides by fermentation processes: endophytic bacteria, fermentation strategies and easy methods for bacterial selection. In: Jozola, A.F. (Ed.), Fermentation Processes. InTech, Rijeka, pp. 199–222. (Chapter 11). Besset-Manzoni, Y., Rieusset, L., Joly, P., Comte, G., Prigent-Combaret, C., 2018. Exploiting rhizosphere microbial cooperation for developing sustainable agriculture strategies. Environ. Sci. Pollut. Res. 25 (30), 29953–29970. Borriss, R., Chen, X.-H., Rueckert, C., Blom, J., Becker, A., Baumgarth, B., Fan, B., Pukall, R., Schumann, P., Sproer, C., Junge, H., Vater, J., Puhler, A., Klenk, H.-P., 2011. Relationship of Bacillus amyloliquefaciens clades associated with strains DSM 7T and FZB42T: a proposal for Bacillus amyloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amyloliquefaciens subsp. plantarum subsp. nov. based on complete genome sequence comparisons. Int. J. Syst. Evolut. Microbiol. 61 (8), 1786–1801. Bouassida, M., Fourati, N., Krichen, F., Zouari, R., Ellouz-Chaabouni, S., Ghribi, D., 2017. Potential application of Bacillus subtilis SPB1 lipopeptides in toothpaste formulation. J. Adv. Res. 8 (4), 425–433. Caboche, S., Pupin, M., Leclère, V., Fontaine, A., Jacques, P., Kucherov, G., 2008. NORINE: a database of nonribosomal peptides. Nucleic Acids Res. 36 (1), D326–D331. Cawoy, H., Debois, D., Franzil, L., De Pauw, E., Thonart, P., Ongena, M., 2015. Lipopeptides as main ingredients for inhibition of fungal phytopathogens by Bacillus subtilis/amyloliquefaciens: lipopeptides as inhibitors of phytopathogens. Microb. Biotechnol. 8 (2), 281–295. Cazorla, F.M., Romero, D., Pérez-García, A., Lugtenberg, B.J.J., de Vicente, A., Bloemberg, G., 2007. Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity: characterization of antagonistic Bacillus. J. Appl. Microbiol. 103 (5), 1950–1959.

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234

6.  LIPOPEPTIDE BIOSURFACTANTS FROM BACILLUS SPECIES

Chen, C.-Y., Baker, S.C., Darton, R.C., 2006. Continuous production of biosurfactant with foam fractionation. J. Chem. Technol. Biotechnol. 81 (12), 1915–1922. Chenikher, S., Guez, J.S., Coutte, F., Pekpe, M., Jacques, P., Cassar, J.P., 2010. Control of the specific growth rate of Bacillus subtilis for the production of biosurfactant lipopeptides in bioreactors with foam overflow. Process Biochem. 45 (11), 1800–1807. Chtioui, O., Dimitrov, K., Gancel, F., Nikov, I., 2010. Biosurfactants production by immobilized cells of Bacillus subtilis ATCC 21332 and their recovery by pertraction. Process Biochem. 45 (11), 1795–1799. Chtioui, O., Dimitrov, K., Gancel, F., Dhulster, P., Nikov, I., 2012. Rotating discs bioreactor, a new tool for lipopeptides production. Process Biochem. 47 (12), 2020–2024. Chumsakul, O., Takahashi, H., Oshima, T., Hishimoto, T., Kanaya, S., Ogasawara, N., Ishikawa, S., 2011. Genomewide binding profiles of the Bacillus subtilis transition state regulator AbrB and its homolog Abh reveals their interactive role in transcriptional regulation. Nucleic Acids Res. 39 (2), 414–428. Cochrane, S.A., Vederas, J.C., 2016. Lipopeptides from Bacillus and Paenibacillus spp.: a gold mine of antibiotic candidates. Med. Res. Rev. 36 (1), 4–31. Cooper, D.G., Macdonald, C.R., Duff, S.J.B., Kosaric, N., 1981. Enhanced production of surfactin from Bacillus subtilis by continuous product removal and metal cation additions. Appl. Environ. Microbiol. 42 (3), 408–412. Coutte, F., Leclère, V., Béchet, M., Guez, J.-S., Lecouturier, D., Chollet-Imbert, M., Dhulster, P., Jacques, P., 2010a. Effect of pps disruption and constitutive expression of srfA on surfactin productivity, spreading and antagonistic properties of Bacillus subtilis 168 derivatives. J. Appl. Microbiol. 109, 480–491. Coutte, F., Lecouturier, D., Ait Yahia, S., Leclère, V., Béchet, M., Jacques, P., Dhulster, P., 2010b. Production of surfactin and fengycin by Bacillus subtilis in a bubbleless membrane bioreactor. Appl. Microbiol. Biotechnol. 87 (2), 499–507. Coutte, F., Lecouturier, D., Leclère, V., Béchet, M., Jacques, P., Dhulster, P., 2013. New integrated bioprocess for the continuous production, extraction and purification of lipopeptides produced by Bacillus subtilis in membrane bioreactor. Process Biochem. 48 (1), 25–32. Coutte, F., Niehren, J., Dhali, D., John, M., Versari, C., Jacques, P., 2015. Modeling leucine’s metabolic pathway and knockout prediction improving the production of surfactin, a biosurfactant from Bacillus subtilis. Biotechnol. J. 10 (8), 1216–1234. Coutte, F., Lecouturier, D., Dimitrov, K., Guez, J.-S., Delvigne, F., Dhulster, P., Jacques, P., 2017. Microbial lipopeptide production and purification bioprocesses, current progress and future challenges. Biotechnol. J. 12 (7), 1600566. D’Souza, C., Nakano, M.M., Zuber, P., 1994. Identification of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 91 (20), 9397–9401. Das, P., Mukherjee, S., Sen, R., 2008. Genetic regulations of the biosynthesis of microbial surfactants: an overview. Biotechnol. Genet. Eng. Rev. 25 (1), 165–186. Davis, D.A., Lynch, H.C., Varley, J., 1999. The production of surfactin in batch culture by Bacillus subtilis ATCC 21332 is strongly influenced by the conditions of nitrogen metabolism. Enzyme Microb. Technol. 25, 322–329. Davis, D.A., Lynch, H.C., Varley, J., 2001. The application of foaming for the recovery of surfactin from B. subtilis ATCC 21332 cultures. Enzyme Microb. Technol. 28 (4–5), 346–354. de Andrade, C.J., de Andrade, L.M., Bution, M.L., Dolder, M.A.H., Cavalcante Barros, F.F., Pastore, G.M., 2016a. Optimizing alternative substrate for simultaneous production of surfactin and 2,3-butanediol by Bacillus subtilis LB5a. Biocatal. Agric. Biotechnol. 6, 209–218. de Andrade, C.J., Resende Simiqueli, A.P., de Andrade, L.M., Mendes, A.M., Jauregi, P., Pastore, G.M., 2016b. Comparative study: bench-scale surfactin production from Bacillus subtilis using analytical grade and concentrated glycerol from the biodiesel industry. Int. J. Sci. World 5 (1), 28. de Araujo, L.V., Guimarães, C.R., da Silva Marquita, R.L., Santiago, V.M.J., de Souza, M.P., Nitschke, M., Freire, D.M.G., 2016. Rhamnolipid and surfactin: anti-adhesion/antibiofilm and antimicrobial effects. Food Control 63, 171–178. Deleu, M., Razafindralambo, H., Popineau, Y., Jacques, P., Thonart, P., Paquot, M., 1999. Interfacial and emulsifying properties of lipopeptides from Bacillus subtilis. Colloids Surf. A: Physicochem. Eng. Asp. 152 (1), 3–10. Desai, J.D., Banat, I.M., 1997. Microbial production of surfactants and their commercial potential. Microbiol. Mol. Biol. Rev. 61 (1), 47–64. Detsch, C., 2003. Ammonium utilization in Bacillus subtilis: transport and regulatory functions of NrgA and NrgB. Microbiology 149 (11), 3289–3297. Dhali, D., Coutte, F., Arias, A.A., Auger, S., Bidnenko, V., Chataigné, G., Lalk, M., Niehren, J., de Sousa, J., Versari, C., Jacques, P., 2017. Genetic engineering of the branched fatty acid metabolic pathway of Bacillus subtilis for the overproduction of surfactin C14 isoform. Biotechnol. J. 12 (7), 1600574.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

REFERENCES 235

Dhanarajan, G., Mandal, M., Sen, R., 2014. A combined artificial neural network modeling–particle swarm optimization strategy for improved production of marine bacterial lipopeptide from food waste. Biochem. Eng. J. 84, 59–65. Dimkić, I., Stanković, S., Nišavić, M., Petković, M., Ristivojević, P., Fira, D., Berić, T., 2017. The profile and antimicrobial activity of Bacillus lipopeptide extracts of five potential biocontrol strains. Front. Microbiol. 8 (925), 1–12. Duitman, E.H., Hamoen, L.W., Rembold, M., Venema, G., Seitz, H., Saenger, W., Bernhard, F., Reinhardt, R., Schmidt, M., Ullrich, C., 1999. The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc. Natl. Acad. Sci. U. S. A. 96 (23), 13294–13299. Dunlap, C.A., Schisler, D.A., Price, N.P., Vaughn, S.F., 2011. Cyclic lipopeptide profile of three Bacillus subtilis strains; antagonists of Fusarium head blight. J. Microbiol. 49 (4), 603–609. Evonik Industries AG Corporate Press, 2016. Evonik Commercializes Biosurfactants. Evonik. (Press Release). Available from: https://corporate.evonik.com/en/media/press_releases/pages/news-details.aspx?newsid=60276. [(Accessed October 9, 2018)]. Fahim, S., Dimitrov, K., Gancel, F., Vauchel, P., Jacques, P., Nikov, I., 2012. Impact of energy supply and oxygen transfer on selective lipopeptide production by Bacillus subtilis BBG21. Bioresour. Technol. 126, 1–6. Finking, R., Marahiel, M.A., 2004. Biosynthesis of nonribosomal peptides. Annu. Rev. Microbiol. 58 (1), 453–488. Fisher, S.H., 1999. Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence!. Mol. Microbiol. 32 (2), 223–232. Flissi, A., Dufresne, Y., Michalik, J., Tonon, L., Janot, S., Noé, L., Jacques, P., Leclère, V., Pupin, M., 2016. Norine, the knowledgebase dedicated to non-ribosomal peptides, is now open to crowdsourcing. Nucleic Acids Res. 44 (D1), D1113–D1118. Fonseca, R.R., Silva, A.J.R., De Franca, F.P., Cardoso, V.L., Sérvulo, E.F.C., 2007. Optimizing carbon/nitrogen ratio for biosurfactant production by a Bacillus subtilis strain. Appl. Biochem. Biotechnol. 136–140, 471–486. Freitas de Oliveira, D.W., Lima França, Í.W., Nogueira Félix, A.K., Lima Martins, J.J., Aparecida Giro, M.E., Melo, V.M.M., Gonçalves, L.R.B., 2013. Kinetic study of biosurfactant production by Bacillus subtilis LAMI005 grown in clarified cashew apple juice. Colloids Surf. B: Biointerfaces 101, 34–43. Geetha, S.J., Banat, I.M., Joshi, S.J., 2018. Biosurfactants: production and potential applications in microbial enhanced oil recovery (MEOR). Biocatal. Agric. Biotechnol. 14, 23–32. Geissler, M., Oellig, C., Moss, K., Schwack, W., Henkel, M., Hausmann, R., 2017. High-performance thin-layer chromatography (HPTLC) for the simultaneous quantification of the cyclic lipopeptides surfactin, iturin A and fengycin in culture samples of Bacillus species. J. Chromatogr. B 1044–1045, 214–224. Gill, K.A., Berrué, F., Arens, J.C., Kerr, R.G., 2014. Isolation and structure elucidation of cystargamide, a lipopeptide from Kitasatospora cystarginea. J. Nat. Prod. 77 (6), 1372–1376. Grady, E.N., MacDonald, J., Liu, L., Richman, A., Yuan, Z.C., 2016. Current knowledge and perspective of Paenibacillus: a review. Microb. Cell Fact. 15 (203). Grover, M., Nain, L., Singh, S.B., Saxena, A.K., 2010. Molecular and biochemical approaches for characterization of antifungal trait of a potent biocontrol agent Bacillus subtilis RP24. Curr. Microbiol. 60 (2), 99–106. Gudiña, E.J., Rangarajan, V., Sen, R., Rodrigues, L.R., 2013. Potential therapeutic applications of biosurfactants. Trends Pharmacol. Sci. 34 (12), 667–675. Gudiña, E.J., Fernandes, E.C., Rodrigues, A.I., Teixeira, J.A., Rodrigues, L.R., 2015. Biosurfactant production by Bacillus subtilis using corn steep liquor as culture medium. Front. Microbiol. 6 (59). Guez, J.S., Chenikher, S., Cassar, J.P., Jacques, P., 2007. Setting up and modelling of overflowing fed-batch cultures of Bacillus subtilis for the production and continuous removal of lipopeptides. J. Biotechnol. 131 (1), 67–75. Hamdache, A., Azarken, R., Lamarti, A., Aleu, J., Collado, I.G., 2013. Comparative genome analysis of Bacillus spp. and its relationship with bioactive nonribosomal peptide production. Phytochem. Rev. 12 (4), 685–716. Hamoen, L.W., Eshuis, H., Jongbloed, J., Venema, G., Sinderen, D., 1995. A small gene, designated comS, located within the coding region of the fourth amino acid-activation domain of srfA, is required for competence development in Bacillus subtilis. Mol. Microbiol. 15 (1), 55–63. Hamoen, L.W., Venema, G., Kuipers, O.P., 2003. Controlling competence in Bacillus subtilis: shared use of regulators. Microbiology 149 (1), 9–17. Härtig, E., Jahn, D., 2012. Regulation of the anaerobic metabolism in Bacillus subtilis. Adv. Microb. Physiol. 61, 195–216. Harwood, C.R., 1992. Bacillus subtilis and its relatives: molecular biological and industrial workhorses. Trends Biotechnol. 10, 247–256.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

236

6.  LIPOPEPTIDE BIOSURFACTANTS FROM BACILLUS SPECIES

Hathout, Y., Ho, Y.-P., Ryzhov, V., Demirev, P., Fenselau, C., 2000. Kurstakins: a new class of lipopeptides isolated from Bacillus thuringiensis. J. Nat. Prod. 63 (11), 1492–1496. Hayashi, K., Ohsawa, T., Kobayashi, K., Ogasawara, N., Ogura, M., 2005. The H2O2 stress-responsive regulator PerR positively regulates srfA expression in Bacillus subtilis. J. Bacteriol. 187 (19), 6659–6667. He, Z., Zeng, W., Zhu, X., Zhao, H., Lu, Y., Lu, Z., 2017. Influence of surfactin on physical and oxidative stability of microemulsions with docosahexaenoic acid. Colloids Surf. B: Biointerfaces 151, 232–239. Heerklotz, H., Seelig, J., 2007. Leakage and lysis of lipid membranes induced by the lipopeptide surfactin. Eur. Biophys. J. 36 (4–5), 305–314. Hsieh, F.-C., Lin, T.-C., Meng, M., Kao, S.-S., 2008. Comparing methods for identifying Bacillus strains capable of producing the antifungal lipopeptide iturin A. Curr. Microbiol. 56 (1), 1–5. Huang, X., Liu, J., Wang, Y., Liu, J., Lu, L., 2015. The positive effects of Mn2+ on nitrogen use and surfactin production by Bacillus subtilis ATCC 21332. Biotechnol. Biotechnol. Equip. 29 (2), 381–389. Hüttel, W., Youssar, L., Grüning, B.A., Günther, S., Hugentobler, K.G., 2016. Echinocandin B biosynthesis: a biosynthetic cluster from Aspergillus nidulans NRRL 8112 and reassembly of the subclusters Ecd and Hty from Aspergillus pachycristatus NRRL 11440 reveals a single coherent gene cluster. BMC Genomics 17 (570). Iglesias-Fernández, J., Darré, L., Kohlmeyer, A., Thomas, R.K., Shen, H.-H., Domene, C., 2015. Surfactin at the water/ air interface and in solution. Langmuir 31 (40), 11097–11104. Isa, M., Coraglia, D., Frazier, R., Jauregi, P., 2007. Recovery and purification of surfactin from fermentation broth by a two-step ultrafiltration process. J. Membr. Sci. 296 (1–2), 51–57. Jacques, P., Hbid, C., Destain, J., Razafindralambo, H., Paquot, M., De Pauw, E., Thonart, P., 1999. Optimization of biosurfactant lipopeptide production from Bacillus subtilis S499 by Plackett-Burman design. In: Twentieth Symposium on Biotechnology for Fuels and Chemicals. Springer, pp. 223–233. Janek, T., Łukaszewicz, M., Rezanka, T., Krasowska, A., 2010. Isolation and characterization of two new lipopeptide biosurfactants produced by Pseudomonas fluorescens BD5 isolated from water from the Arctic Archipelago of Svalbard. Bioresour. Technol. 101 (15), 6118–6123. Jemil, N., Manresa, A., Rabanal, F., Ben Ayed, H., Hmidet, N., Nasri, M., 2017. Structural characterization and identification of cyclic lipopeptides produced by Bacillus methylotrophicus DCS1 strain. J. Chromatogr. B 1060, 374–386. Jiang, C., Chen, X., Lei, S., Zhao, H., Liu, Y., Shi, J., 2017. Lipopeptides from Bacillus subtilis have potential application in the winemaking process: inhibiting fungal and ochratoxin A contamination and enhancing esters and acids biosynthesis. Aust. J. Grape Wine Res. 23 (3), 350–358. Jin, H., Zhang, X., Li, K., Niu, Y., Guo, M., Hu, C., Wan, X., Gong, Y., Huang, F., 2014. Direct bio-utilization of untreated rapeseed meal for effective iturin A production by Bacillus subtilis in submerged fermentation. PLoS One 9 (10), e111171. Jin, H., Li, K., Niu, Y., Guo, M., Hu, C., Chen, S., Huang, F., 2015. Continuous enhancement of iturin A production by Bacillus subtilis with a stepwise two-stage glucose feeding strategy. BMC Biotechnol. 15 (53). Joe, M.M., Bradeeba, K., Parthasarathi, R., Sivakumaar, P.K., Chauhan, P.S., Tipayno, S., Benson, A., Sa, T., 2012. Development of surfactin based nanoemulsion formulation from selected cooking oils: evaluation for antimicrobial activity against selected food associated microorganisms. J. Taiwan Inst. Chem. Eng. 43 (2), 172–180. Juola, M., Kinnunen, K., Nielsen, K.F., von Wright, A., 2014. Surfactins in Natto: the surfactin production capacity of the starter strains and the actual surfactin contents in the products. J. Food Prot. 77 (12), 2139–2143. Kakinuma, A., Sugino, H., Isono, M., Tamura, G., Arima, K., 1969a. Determination of fatty acid in surfactin and elucidation of the total structure of surfactin. Agric. Biol. Chem. 33 (6), 973–976. Kakinuma, A., Ouchida, A., Shima, T., Sugino, H., Isono, M., Tamura, G., Arima, K., 1969b. Confirmation of the structure of surfactin by mass spectrometry. Agric. Biol. Chem. 33 (11), 1669–1671. Kanlayavattanakul, M., Lourith, N., 2010. Lipopeptides in cosmetics. Int. J. Cosmet. Sci. 32 (1), 1–8. Kinsinger, R.F., Shirk, M.C., Fall, R., 2003. Rapid surface motility in Bacillus subtilis is dependent on extracellular surfactin and potassium ion. J. Bacteriol. 185 (18), 5627–5631. Kiran, G.S., Priyadharsini, S., Sajayan, A., Priyadharsini, G.B., Poulose, N., Selvin, J., 2017. Production of lipopeptide biosurfactant by a marine Nesterenkonia sp. and its application in food industry. Front. Microbiol. 8 (1138). Koglin, A., Löhr, F., Bernhard, F., Rogov, V.V., Frueh, D.P., Strieter, E.R., Mofid, M.R., Güntert, P., Wagner, G., Walsh, C.T., Marahiel, M.A., Dötsch, V., 2008. When the fold fits the function: the external thioesterase of the surfactinsynthetase. Nature 454, 907–911. Kosswig, K., 2012. Surfactants. In: Ullmann’s Encyclopedia of Industrial Chemistry. Wiley-VCH Verlag GmbH & Co. KGaA, pp. 431–505.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

REFERENCES 237

Kraas, F.I., Helmetag, V., Wittmann, M., Strieker, M., Marahiel, M.A., 2010. Functional dissection of surfactin synthetase initiation module reveals insights into the mechanism of lipoinitiation. Chem. Biol. 17 (8), 872–880. Kumar, A.P., Janardhan, A., Viswanath, B., Monika, K., Jung, J.-Y., Narasimha, G., 2016. Evaluation of orange peel for biosurfactant production by Bacillus licheniformis and their ability to degrade naphthalene and crude oil. 3 Biotech 6 (1), 1–10. Landy, M., Warren, H., Rosenman, S.B., Colio, L.G., 1948. Bacillomycin: an antibiotic from Bacillus subtilis active against pathogenic fungi. Proc. Soc. Exp. Biol. Med. 67 (4), 539–541. Le Mire, G., Siah, A., Brisset, M.-N., Gaucher, M., Deleu, M., Jijakli, M., 2018. Surfactin protects wheat against Zymoseptoria tritici and activates both salicylic acid- and jasmonic acid-dependent defense responses. Agriculture 8 (11), 1–14. Li, Y.-M., Haddad, N.I.A., Yang, S.-Z., Mu, B.-Z., 2008. Variants of lipopeptides produced by Bacillus licheniformis HSN221 in different medium components evaluated by a rapid method ESI-MS. Int. J. Pept. Res. Ther. 14 (3), 229–235. Li, R., Du, W., Yang, J., Liu, Z., Yousef, A.E., 2018. Control of Listeria monocytogenes biofilm by paenibacterin, a natural antimicrobial lipopeptide. Food Control 84, 529–535. Lopez, D., Vlamakis, H., Losick, R., Kolter, R., 2009. Paracrine signaling in a bacterium. Genes Dev. 23 (14), 1631–1638. Luo, C., Liu, X., Zhou, X., Guo, J., Truong, J., Wang, X., Zhou, H., Li, X., Chen, Z., 2015. Unusual biosynthesis and structure of locillomycins from Bacillus subtilis 916. Appl. Environ. Microbiol. 81 (19), 6601–6609. Ma, Z., Ongena, M., Höfte, M., 2017. The cyclic lipopeptide orfamide induces systemic resistance in rice to Cochliobolus miyabeanus but not to Magnaporthe oryzae. Plant Cell Rep. 36 (11), 1731–1746. Maass, D., Moya Ramírez, I., García Román, M., Jurado Alameda, E., Ulson de Souza, A.A., Borges Valle, J.A., Altmajer Vaz, D., 2016. Two-phase olive mill waste (alpeorujo) as carbon source for biosurfactant production. J. Chem. Technol. Biotechnol. 91 (7), 1990–1997. Magnuson, R., Solomon, J., Grossman, A.D., 1994. Biochemical and genetic characterization of a competence pheromone from B. subtilis. Cell 77 (2), 207–216. Malfanova, N., Franzil, L., Lugtenberg, B., Chebotar, V., Ongena, M., 2012. Cyclic lipopeptide profile of the plant-­ beneficial endophytic bacterium Bacillus subtilis HC8. Arch. Microbiol. 194 (11), 893–899. Marlow, V.L., Porter, M., Hobley, L., Kiley, T.B., Swedlow, J.R., Davidson, F.A., Stanley-Wall, N.R., 2014. Phosphorylated DegU manipulates cell fate differentiation in the Bacillus subtilis biofilm. J. Bacteriol. 196 (1), 16–27. Medeot, D.B., Bertorello-Cuenca, M., Liaudat, J.P., Alvarez, F., Flores-Cáceres, M.L., Jofré, E., 2017. Improvement of biomass and cyclic lipopeptides production in Bacillus amyloliquefaciens MEP218 by modifying carbon and nitrogen sources and ratios of the culture media. Biol. Control 115, 119–128. Meena, K.R., Kanwar, S.S., 2015. Lipopeptides as the antifungal and antibacterial agents: applications in food safety and therapeutics. BioMed Res. Int. 2015, 1–9. Miras, M., Dubnau, D., 2016. A DegU-P and DegQ-dependent regulatory pathway for the K-state in Bacillus subtilis. Front. Microbiol. 7 (1868), 1–14. Mnif, I., Ghribi, D., 2015a. Lipopeptide surfactants: production, recovery and pore forming capacity. Peptides 71, 100–112. Mnif, I., Ghribi, D., 2015b. Lipopeptides biosurfactants: mean classes and new insights for industrial, biomedical, and environmental applications. Biopolymers 104 (3), 129–147. Monteiro, F.P., de Medeiros, F.H.V., Ongena, M., Franzil, L., de Souza, P.E., de Souza, J.T., 2016. Effect of temperature, pH and substrate composition on production of lipopeptides by Bacillus amyloliquefaciens 629. Afr. J. Microbiol. Res. 10 (36), 1506–1512. Mostertz, J., 2004. Transcriptome and proteome analysis of Bacillus subtilis gene expression in response to superoxide and peroxide stress. Microbiology 150 (2), 497–512. Motta Dos Santos, L.F., Coutte, F., Ravallec, R., Dhulster, P., Tournier-Couturier, L., Jacques, P., 2016. An improvement of surfactin production by B. subtilis BBG131 using design of experiments in microbioreactors and continuous process in bubbleless membrane bioreactor. Bioresour. Technol. 218, 944–952. Moyne, A., 2004. Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D. FEMS Microbiol. Lett. 234 (1), 43–49. Msadek, T., Kunst, F., Klier, A., Rapoport, G., 1991. DegS-DegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ. J. Bacteriol. 173 (7), 2366–2377. Mukherjee, A.K., 2007. Potential application of cyclic lipopeptide biosurfactants produced by Bacillus subtilis strains in laundry detergent formulations. Lett. Appl. Microbiol. 45 (3), 330–335.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

238

6.  LIPOPEPTIDE BIOSURFACTANTS FROM BACILLUS SPECIES

Murray, E.J., Kiley, T.B., Stanley-Wall, N.R., 2009. A pivotal role for the response regulator DegU in controlling multicellular behaviour. Microbiology 155 (1), 1–8. Nakano, M.M., Magnuson, R., Myers, A., Curry, J., Grossman, A.D., Zuber, P., 1991. srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis. J. Bacteriol. 173 (5), 1770–1778. Nakano, M.M., Dailly, Y.P., Zuber, P., Clark, D.P., 1997. Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth. J. Bacteriol. 179 (21), 6749–6755. Nakano, S., Kuster-Schock, E., Grossman, A.D., Zuber, P., 2003. Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 100 (23), 13603–13608. Naruse, N., Tenmyo, O., Kobaru, S., Kamei, H., Miyaki, T., Konishi, M., Oki, T., 1990. Pumilacidin, a complex of new antiviral antibiotics. J. Antibiot. 43 (3), 267–280. Nitschke, M., Costa, S.G.V.A.O., 2007. Biosurfactants in food industry. Trends Food Sci. Technol. 18 (5), 252–259. Nitschke, M., Pastore, G.M., 2004. Biosurfactant production by Bacillus subtilis using cassava-processing effluent. Appl. Biochem. Biotechnol. 112 (3), 163–172. Nitschke, M., Silva, S.S., 2018. Recent food applications of microbial surfactants. Crit. Rev. Food Sci. Nutr. 58 (4), 631–638. Omer Bendori, S., Pollak, S., Hizi, D., Eldar, A., 2015. The RapP-PhrP quorum-sensing system of Bacillus subtilis strain NCIB3610 affects biofilm formation through multiple targets, due to an atypical signal-insensitive allele of RapP. In: O’Toole, G.A. (Ed.), J. Bacteriol. 197(3), 592–602. Onaizi, S.A., Nasser, M.S., Al-Lagtah, N.M.A., 2016. Self-assembly of a surfactin nanolayer at solid–liquid and air–­ liquid interfaces. Eur. Biophys. J. 45 (4), 331–339. Ongena, M., Jacques, P., 2008. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol. 16 (3), 115–125. Otto, R.T., Daniel, H.-J., Pekin, G., Müller-Decker, K., Fürstenberger, G., Reuss, M., Syldatk, C., 1999. Production of sophorolipids from whey. Appl. Microbiol. Biotechnol. 52 (4), 495–501. Paraszkiewicz, K., Bernat, P., Siewiera, P., Moryl, M., Paszt, L.S., Trzciński, P., Jałowiecki, Ł., Płaza, G., 2017. Agricultural potential of rhizospheric Bacillus subtilis strains exhibiting varied efficiency of surfactin production. Sci. Hortic. 225, 802–809. Perego, M., Higgins, C.F., Pearce, S.R., Gallagher, M.P., Hoch, J.A., 1991. The oligopeptide transport system of Bacillus subtilis plays a role in the initiation of sporulation. Mol. Microbiol. 5 (1), 173–185. Peypoux, F., Guinand, M., Michel, G., Delcambe, L., Das, B.C., Lederer, E., 1978. Structure of iturine A, a peptidolipid antibiotic from Bacillus subtilis. Biochemistry 17 (19), 3992–3996. Peypoux, F., Bonmatin, J.-M., Labbé, H., Das, B.C., Ptak, M., Michel, G., 1991. Isolation and characterization of a new variant of surfactin, the [Val7]surfactin. Eur. J. Biochem. 202, 101–106. Pottathil, M., Jung, A., Lazazzera, B.A., 2008. CSF, a species-specific extracellular signaling peptide for communication among strains of Bacillus subtilis and Bacillus mojavensis. J. Bacteriol. 190 (11), 4095–4099. Pragai, Z., Allenby, N.E.E., O’Connor, N., Dubrac, S., Rapoport, G., Msadek, T., Harwood, C.R., 2004. Transcriptional regulation of the phoPR operon in Bacillus subtilis. J. Bacteriol. 186 (4), 1182–1190. Qiu, Y., Xiao, F., Wei, X., Wen, Z., Chen, S., 2014. Improvement of lichenysin production in Bacillus licheniformis by replacement of native promoter of lichenysin biosynthesis operon and medium optimization. Appl. Microbiol. Biotechnol. 98 (21), 8895–8903. Quadri, L.E., Weinreb, P.H., Lei, M., Nakano, M.M., Zuber, P., Walsh, C.T., 1998. Characterization of Sfp, a Bacillus subtilis phosphopantetheinyl transferase for peptidyl carrier protein domains in peptide synthetases. Biochemistry 37, 1585–1595. Raaijmakers, J.M., De Bruijn, I., Nybroe, O., Ongena, M., 2010. Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol. Rev. 34 (6), 1037–1062. Rabanal, F., Cajal, Y., 2017. Recent advances and perspectives in the design and development of polymyxins. Nat. Prod. Rep. 34, 886–908. Rangarajan, V., Clarke, K.G., 2015. Process development and intensification for enhanced production of Bacillus lipopeptides. Biotechnol. Genet. Eng. Rev. 31 (1–2), 46–68. Rangarajan, V., Clarke, K.G., 2016. Towards bacterial lipopeptide products for specific applications—a review of appropriate downstream processing schemes. Process Biochem. 51 (12), 2176–2185. Rangarajan, V., Dhanarajan, G., Sen, R., 2015. Bioprocess design for selective enhancement of fengycin production by a marine isolate Bacillus megaterium. Biochem. Eng. J. 99, 147–155.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

REFERENCES 239

Reuter, K., Mofid, M.R., Marahier, M.A., Ficner, R., 1999. Crystal structure of the surfactin synthetase-activating enzyme Sfp: a prototype of the 4′-phosphopantetheinyl transferase superfamily. EMBO J. 18 (23), 6823–6831. Romero, D., 2013. Bacterial determinants of the social behavior of Bacillus subtilis. Res. Microbiol. 164 (7), 788–798. Roongsawang, N., Washio, K., Morikawa, M., 2010. Diversity of nonribosomal peptide synthetases involved in the biosynthesis of lipopeptide biosurfactants. Int. J. Mol. Sci. 12 (1), 141–172. Saini, H.S., Barragán-Huerta, B.E., Lebrón-Paler, A., Pemberton, J.E., Vázquez, R.R., Burns, A.M., Marron, M.T., Seliga, C.J., Gunatilaka, A.A.L., Maier, R.M., 2008. Efficient purification of the biosurfactant viscosin from Pseudomonas libanensis strain M9-3 and its physicochemical and biological properties. J. Nat. Prod. 71 (6), 1011–1015. Salzberg, L.I., Botella, E., Hokamp, K., Antelmann, H., Maaß, S., Becher, D., Noone, D., Devine, K.M., 2015. Genomewide analysis of phosphorylated PhoP binding to chromosomal DNA reveals several novel features of the PhoPRmediated phosphate limitation response in Bacillus subtilis. In: Silhavy, T.J. (Ed.), J. Bacteriol. 197(8), 1492–1506. Samel, S.A., Wagner, B., Marahiel, M.A., Essen, L.O., 2006. The thioesterase domain of the fengycin biosynthesis cluster: a structural base for the macrocyclization of a non-ribosomal lipopeptide. J. Mol. Biol. 359 (4), 876–889. Schneider, J., Taraz, K., Budzikiewicz, H., Deleu, M., Thonart, P., Jacques, P., 1999. The structure of two fengycins from Bacillus subtilis S499. Z. Naturforsch. C (54)859–865. Schwechheimer, S.K., Park, E.Y., Revuelta, J.L., Becker, J., Wittmann, C., 2016. Biotechnology of riboflavin. Appl. Microbiol. Biotechnol. 100 (5), 2107–2119. Sen, R., 1997. Response surface optimization of the critical media components for the production of surfactin. J. Chem. Technol. Biotechnol. 68, 263–270. Serror, P., Sonenshein, A.L., 1996. CodY is required for nutritional repression of Bacillus subtilis genetic competence. J. Bacteriol. 178 (20), 5910–5915. Shafi, J., Tian, H., Ji, M., 2017. Bacillus species as versatile weapons for plant pathogens: a review. Biotechnol. Biotechnol. Equip. 31 (3), 446–459. Shaligram, N.S., Singhal, R.S., 2010. Surfactin—a review on biosynthesis, fermentation, purification and applications. Food Technol. Biotechnol. 48 (2), 119–134. Shaligram, N.S., Singhal, R.S., 2014. Study of the surfactant properties of aqueous stream from the corn milling industry. J. Agric. Food Chem. 62 (24), 5451–5457. Shameer, S., Prasad, T.N.V.K.V., 2018. Plant growth promoting rhizobacteria for sustainable agricultural practices with special reference to biotic and abiotic stresses. Plant Growth Regul. 84 (3), 603–615. Shank, E.A., Kolter, R., 2011. Extracellular signaling and multicellularity in Bacillus subtilis. Curr. Opin. Microbiol. 14 (6), 741–747. Singh, R., Kumar, M., Mittal, A., Mehta, P.K., 2017. Microbial metabolites in nutrition, healthcare and agriculture. 3 Biotech 7 (15), 1–14. Sivapathasekaran, C., Das, P., Mukherjee, S., Saravanakumar, J., Mandal, M., Sen, R., 2010. Marine bacterium derived lipopeptides: characterization and cytotoxic activity against cancer cell lines. Int. J. Pept. Res. Ther. 16 (4), 215–222. Slivinski, C.T., Mallmann, E., de Araújo, J.M., Mitchell, D.A., Krieger, N., 2012. Production of surfactin by Bacillus pumilus UFPEDA 448 in solid-state fermentation using a medium based on okara with sugarcane bagasse as a bulking agent. Process Biochem. 47 (12), 1848–1855. Sonenshein, A.L., 2007. Control of key metabolic intersections in Bacillus subtilis. Nat. Rev. Microbiol. 5 (12), 917–927. Steenbergen, J.N., Alder, J., Thorne, G.M., Tally, F.P., 2005. Daptomycin: a lipopeptide antibiotic for the treatment of serious gram-positive infections. J. Antimicrob. Chemother. 55 (3), 283–288. Steller, S., Sokoll, A., Wilde, C., Bernhard, F., Franke, P., Vater, J., 2004. Initiation of surfactin biosynthesis and the role of the SrfD-thioesterase protein. Biochemistry 43 (35), 11331–11343. Strieker, M., Tanović, A., Marahiel, M.A., 2010. Nonribosomal peptide synthetases: structures and dynamics. Curr. Opin. Struct. Biol. 20 (2), 234–240. Taira, T., Yanagisawa, S., Nagano, T., Tsuji, T., Endo, A., Imura, T., 2017. pH-induced conformational change of natural cyclic lipopeptide surfactin and the effect on protease activity. Colloids Surf. B: Biointerfaces 156, 382–387. Tapi, A., Chollet-Imbert, M., Scherens, B., Jacques, P., 2010. New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction. Appl. Microbiol. Biotechnol. 85, 1521–1531. Tsuge, K., Akiyama, T., Shoda, M., 2001. Cloning, sequencing, and characterization of the iturin A operon. J. Bacteriol. 183 (21), 6265–6273. van Dijl, J., Hecker, M., 2013. Bacillus subtilis: from soil bacterium to super-secreting cell factory. Microb. Cell Fact. 12 (3). Vanittanakom, N., Loeffler, W., 1986. Fengycin—a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J. Antibiot. 39 (7), 888–901.

II.  BIOSURFACTANTS: BIOSYNTHESIS AND APPLICATIONS

240

6.  LIPOPEPTIDE BIOSURFACTANTS FROM BACILLUS SPECIES

Vater, J., Kablitz, B., Wilde, C., Franke, P., Mehta, N., Cameotra, S.S., 2002. Matrix-assisted laser desorption ­ionization-time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl. Environ. Microbiol. 68 (12), 6210–6219. Wei, Y.-H., Lai, C.-C., Chang, J.-S., 2007. Using Taguchi experimental design methods to optimize trace element composition for enhanced surfactin production by Bacillus subtilis ATCC 21332. Process Biochem. 42 (1), 40–45. Weir, J., Predich, M., Dubnau, E., Nair, G., Smith, I., 1991. Regulation of spo0H, a gene coding for the Bacillus subtilis sigma H factor. J. Bacteriol. 173 (2), 521–529. Willenbacher, J., Zwick, M., Mohr, T., Schmid, F., Syldatk, C., Hausmann, R., 2014. Evaluation of different Bacillus strains in respect of their ability to produce surfactin in a model fermentation process with integrated foam fractionation. Appl. Microbiol. Biotechnol. 98 (23), 9623–9632. Willenbacher, J., Rau, J.-T., Rogalla, J., Syldatk, C., Hausmann, R., 2015a. Foam-free production of Surfactin via anaerobic fermentation of Bacillus subtilis DSM 10T. AMB Express 5 (21), 1–9. Willenbacher, J., Yeremchuk, W., Mohr, T., Syldatk, C., Hausmann, R., 2015b. Enhancement of surfactin yield by improving the medium composition and fermentation process. AMB Express 5 (57), 1–9. Willenbacher, J., Mohr, T., Henkel, M., Gebhard, S., Mascher, T., Syldatk, C., Hausmann, R., 2016. Substitution of the native srfA promoter by constitutive Pveg in two B. subtilis strains and evaluation of the effect on surfactin production. J. Biotechnol. 224, 14–17. Wu, C.Y., Chen, C.L., Lee, Y.H., Cheng, Y.C., Wu, Y.C., Shu, H.Y., Götz, F., Liu, S.T., 2007. Nonribosomal synthesis of fengycin on an enzyme complex formed by fengycin synthetases. J. Biol. Chem. 282 (8), 5608–5616. Wu, Y.-S., Ngai, S.-C., Goh, B.-H., Chan, K.-G., Lee, L.-H., Chuah, L.-H., 2017. Anticancer activities of surfactin and potential application of nanotechnology assisted surfactin delivery. Front. Pharmacol. 8 (761), 1–22. Yakimov, M.M., Timmis, K.N., Wray, V., Fredrickson, H.L., 1995. Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50. Appl. Environ. Microbiol. 61 (5), 1706–1713. Yaseen, Y., Gancel, F., Drider, D., Béchet, M., Jacques, P., 2016. Influence of promoters on the production of fengycin in Bacillus spp. Res. Microbiol. 167 (4), 272–281. Yaseen, Y., Gancel, F., Béchet, M., Drider, D., Jacques, P., 2017. Study of the correlation between fengycin promoter expression and its production by Bacillus subtilis under different culture conditions and the impact on surfactin production. Arch. Microbiol. 199 (10), 1371–1382. Yeh, M.-S., Wei, Y.-H., Chang, J.-S., 2006. Bioreactor design for enhanced carrier-assisted surfactin production with Bacillus subtilis. Process Biochem. 41 (8), 1799–1805. Yeh, M.-S., Wei, Y.-H., Chang, J.-S., 2008. Enhanced production of surfactin from Bacillus subtilis by addition of solid carriers. Biotechnol. Prog. 21 (4), 1329–1334. Zhao, J., Li, Y., Zhang, C., Yao, Z., Zhang, L., Bie, X., Lu, F., Lu, Z., 2012. Genome shuffling of Bacillus amyloliquefaciens for improving antimicrobial lipopeptide production and an analysis of relative gene expression using FQ RTPCR. J. Ind. Microbiol. Biotechnol. 39 (6), 889–896. Zhao, J., Zhang, C., Lu, J., Lu, Z., 2016. Enhancement of fengycin production in Bacillus amyloliquefaciens by genome shuffling and relative gene expression analysis using RT-PCR. Can. J. Microbiol. 62 (5), 431–436. Zhao, H., Shao, D., Jiang, C., Shi, J., Li, Q., Huang, Q., Rajoka, M.S.R., Yang, H., Jin, M., 2017. Biological activity of lipopeptides from Bacillus. Appl. Microbiol. Biotechnol. 101 (15), 5951–5960. Zhi, Y., Wu, Q., Xu, Y., 2017. Production of surfactin from waste distillers’ grains by co-culture fermentation of two Bacillus amyloliquefaciens strains. Bioresour. Technol. 235, 96–103. Zouari, R., Besbes, S., Ellouze-Chaabouni, S., Ghribi-Aydi, D., 2016. Cookies from composite wheat–sesame peels flours: dough quality and effect of Bacillus subtilis SPB1 biosurfactant addition. Food Chem. 194, 758–769. Zuber, P., 2009. Management of oxidative stress in Bacillus. Ann. Rev. Microbiol. 63, 575–597.

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C H A P T E R

7 Phospholipid-Based Surfactants Jingbo Li⁎,†, Yongjin He⁎,‡, Sampson Anankanbil⁎, Zheng Guo⁎ ⁎

Department of Engineering, Faculty of Science and Technology, Aarhus University, Aarhus, Denmark †Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States ‡College of Life Science, Fujian Normal University, Fuzhou, China

7.1 INTRODUCTION Phospholipids (PLs, also refer to glycerophospholipids) are a subclass of lipids that generally consist of a glycerol backbone with I,2-positions connected to two fatty acyl groups via ester bonds. The third glycerol position is connected to a phosphate group. The phosphate group bears two ester bonds to glycerol backbone and a polar head group, such as choline, ethanolamine, inositol, serine, and glycerol. The polar head groups correspond to the subspecies of PLs: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylglycerol (PG), respectively. Structurally, all PLs are amphiphilic molecules, serving as the molecular basis for their biological functions and as emulsifiers in food, cosmetic, or drug products. The fundamental biological function of PLs is to form cellular membranes and act as a barrier for entry of compounds into cells. PLs also function as precursors of second messengers such as diacylglycerol (DG) and inositol-1,4,5-P3 (inositol trisphosphate). A third, and usually overlooked, function of PLs is the storage of energy in the form of fatty acyl components (Vance and Vance, 2008). Other specific functions are associated with specific organs and involve unique molecular species of PLs. Pulmonary surfactant PLs are essential for life. They are composed of a complex lipoprotein-like mixture that lines the inner surface of the lung to prevent alveolar collapse at the end of expiration (Agassandian and Mallampalli, 2013). PS is a component of the lipid‑calcium-phosphate complex for deposition during bone formation (Merolli and Santin, 2009), regulation of apoptosis (programmed cell death) (Devitt et  al., 2003), and blood coagulation (Lentz, 2003). A better understanding of the biological functions in the human body not only helps us to know the role and mechanism of PLs to maintain

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structure and function but also facilitates the development of ingredients or drug excipients of phospholipid analogues for better health or to address dysfunction or diseases occurring in tissues or organs. In 1956, using rat liver as an enzyme source, Kennedy and Weiss (1956) elucidated a pathway for the de novo biosynthesis of PE and PC. The PE and PC branches of this “Kennedy” pathway are based upon the formation of characteristic high-energy intermediates: cytidine diphosphate (CDP)-ethanolamine, for the synthesis of PE, and CDP-choline, for the s­ ynthesis of PC (Gibellini and Smith, 2010). After the Kennedy and Weiss study, a few researches shed some light on the importance and regulation of individual pathways, but progress in biochemical and molecular analysis was slow. Many enzymes in these pathways are membraneassociated and therefore difficult to purify, and kinetic analysis of reactions with insoluble substrates and products was not straightforward (Kent, 1995). However, with the advances in gene analysis, expression, and purification of protein and structural analysis of enzymes, the biosynthesis pathways of PLs are much more clear, and the branching pathways for biosynthesis of specific phospholipid species have been deciphered (Lykidis, 2007). With respect to PLs, the food engineers and application scientists have different contents from biologists or pharmaceutical researchers, which generally refer to “lecithins.” The word “lecithin” comes from the Greek “λέκιθος,” meaning “egg, the start of life.” Nowadays, lecithins often refer to “a complex mixture of acetone-insoluble phosphatides, which consist chiefly of PC, PE, PS, and PI, combined with various amounts of other substances such as triglycerides, fatty acids, and carbohydrates as isolated from a crude vegetable oil source. It contains > 50% of acetone-insoluble matter” (van Hoogevest and Wendel, 2014). Soybean, sunflower, and rapeseed are the major sources of vegetable lecithins. In general, PLs are excellent surface-active amphiphilic molecules, which are widely used as emulsifier, wetting agent, solubilizer, and as the major component of liposome. However, for some specific applications, the inherent property and characteristics of common PLs are not sufficient to meet performance requirements, or the availability of suitable natural source of a phospholipid species is insufficient. Structural modification of natural PLs or a de novo synthesis of PLs with defined structure can address the problem. There are many different ways to construct novel PLs. Based on the starting materials, the synthesis methods can be classified as the modification of natural PLs and de novo synthesis. The preparation methods can be categorized as enzymatic, chemical, or chemoenzymatic approaches (Guo et al., 2005; Servi, 1999). Enzymatic synthesis depends greatly on the activity and selectivity of phospholipases and lipases, which are widely used in the modification or semisynthesis of PLs. The synthesis of PLs that are pure with respect to the polar head group and fatty acid compositions and of the natural stereochemical configuration starts preferably from glycerylphosphorylcholine (GPC). On the other hand, the industrial application such as enzymatic degumming is also largely dependent on the price and availability of industrial enzymes, which undergo a hand-in-hand growth with our ability to clone and express the genes in microbial hosts with commercially attractive amounts (De Maria et al., 2007). In this chapter, we first summarize the latest progress and updated knowledge about the biosynthesis, biological functions, and physicochemical properties of phospholipids. Then, we present the production technology for industrial lecithins and related analysis and characterization techniques, especially 31P NMR technology for qualification and quantification of phospholipid molecular species. Discussion is given to the strategy, methodology, and

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­ rocessing technology for chemoenzymatic modification of natural PLs and synthesis of new p derivatives. The latest research progress in the design and synthesis of dual- or multifunctional PLs and synthesis of structured PLs is highlighted. Finally, the applications of PLs as natural surfactants in food, pharma, and cosmetic industries are briefly summarized.

7.2  BIOSYNTHESIS, BIOLOGICAL FUNCTIONS AND PHYSICOCHEMICAL PROPERTIES OF PLs 7.2.1  De Novo Biosynthesis of PLs PLs, as the major components of cell membrane, are de novo synthesized by cells catalyzed by different enzymes (Fig. 7.1). sn-Glycerol-3-phosphate acyltransferase (EC 2.3.1.15) catalyzes the formation of lysophosphatidic acid (LPA) from glycerol-3-phosphate. This step is the major step in the whole phospholipid synthesis and is important in regulating the phospholipid to protein ratio in the membrane. Lyso-PA acyltransferase further catalyzes the acylation of lyso-PA to form PA where the two hydroxyl groups on glycerol are all esterified by fatty acid moieties. PA can be hydrolyzed to form diglyceride and further form triglyceride catalyzed by another acyltransferase. PA is also converted to cytidine-5′-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol) and deoxycytidine-5′-diphospho-l,2-diacyl-sn-glycerol (dCDP-diacylglycerol) by CDPdiacylglycerol synthase. dCDP-diacylglycerol is the precursor of several other phospholipid species like PS, PI, and phosphatidylglycerophosphate (PGP). Syntheses of these PLs are catalyzed by individual specific enzymes, such as PS synthase, PI synthase, and PGP synthase. The CDP-ethanolamine (Kennedy or salvage) pathway that forms PE is commonly found in eukaryotic cells. However, it does not occur in E. coli. Instead, another pathway that involves the decarboxylation of PS becomes the leading pathway to form PE. It has been demonstrated that the decarboxylase has approximately two times higher enzymatic activity than of PS synthase. Thus, PS is rapidly converted to PE and never accumulates. This is probably the reason why the natural abundance of PS is lower than that of PE. PG is the product of hydrolyzed PGP catalyzed by PGP phosphatase. Similar to PS, PGP is converted rapidly to PG and never accumulates unless one of the phosphatases is mutationally defective or inhibited by Hg2+. The genes that encode the corresponding enzymes have been well summarized (Shibuya, 1992). The information can be used for bioengineering to produce more specific phospholipid species. In most eukaryotic cell membranes, the most abundant phospholipid is usually PC (Vance and Vance, 2004). In all nucleated mammalian cells, PC is synthesized by the CDP-choline pathway (Fig. 7.2). Choline cannot be made de novo in animal cells; therefore, diet supply of choline in the diet is required if this pathway is dominant. An alternative pathway contributes 30%–40% PC, which is synthesized from PE catalyzed by phosphatidylethanolamine N-methyltransferase (Fig.  7.2). Through the three-step reaction, three hydrogen atoms are replaced by three methyl groups on the nitrogen atom to form PC. The methyl groups are provided by S-adenosyl-l-methionine (Vance and Vance, 2004). The details for the biosynthetic pathways and the related enzymes, genes, regulations, etc. are not discussed in the current chapter.

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FIG. 7.1  The de novo biosynthesis pathways for the major phospholipids in E. coli, where the PI synthase was from yeast. R, R1, and R2 repre-

sent fatty acyl chains. G3P: glycerol-3-phosphate. PA: phosphatidic acid. CTP: cytidine triphosphate. CDP-DG synthase: cytidine-5′-diphospho-1,2-­ diacyl-sn-glycerol synthase. PS, phosphatidylserine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PGP, phosphatidylglycerophosphate; CL, cardiolipin.



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FIG. 7.2  The biosynthesis pathways of PC from choline in nucleated mammalian cells (1) and from PE catalyzed by PEMT in liver, yeast, bacterial, and Pseudomonas (2). R and R1 represent fatty acyl chains.

7.2.2  Biological Functions of PLs PLs, including glycerophospholipids (GPLs) and sphingolipids, constitute the bulk lipid components of all mammalian membranes (Fagone and Jackowski, 2009). Biological membranes form hydrophobic barriers that limit the distribution of aqueous macromolecules and metabolites. Membranes in cells have many different functions including segregation and protection of cells from the environment, compartmentalization of functions, energy production, storage, protein synthesis and secretion, phagocytosis, movement, and cell-cell interaction (Fagone and Jackowski, 2009). All of these functions are realized with the presence of PLs. Different types and compositions of lipids in membranes enable the versatility of biological membranes to control their structure and biophysical properties. Lipid composition differs not only between different organisms but also between organelles within the same cell and between the two leaflets of the same membrane (Fagone and Jackowski, 2009). The diversity of lipid composition may enable the well-organized communications between cells and organelles based on the signaling function of each individual phospholipid. PLs were found to be associated with the ignition of DNA replication (Shibuya, 1992). Cardiolipin specifically replaces the ADP tightly bound to DnaA protein with ATP to reactivate this component essential for initiation. PG with unsaturated fatty acids (UFAs) was also found to affect the exchange of ADP and ATP (Shibuya, 1992). Acidic PLs in membranes are vital for the replication initiation, for instance through the activation of DnaA protein. Membrane fluidity is important for the DNA replication as well (Shibuya, 1992). Although there are some contrasting results (Makise et al., 2002) regarding the exact function of PLs for DNA replication, PLs indeed impact the bioprocess. Acidic PLs increase the initial rate of spontaneous intermembrane transfer of sterol by 5–89%, depending on the specific phospholipid (Hapala et al., 1990). High ionic strength, CaCl2, and low pH suppressed the stimulation of spontaneous intermembrane sterol transfer by acidic PLs (Hapala et  al., 1990).

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PLs play an important role in intermembrane sterol transfer. Acidic PLs also govern the enhanced activation of IgG-B cell receptor (Chen et  al., 2015). There are much more specific biological functions of PLs. For example, decreased unsaturation in membrane PLs induces unfolding of proteins (Ariyama et al., 2010). Molecular genetic alterations in membrane lipid composition result in many phenotypes and uncovered direct lipid-protein interactions that govern dynamic structural and functional properties of membrane proteins (Dowhan, 2017).

7.2.3  Physicochemical Properties of PLs PLs are natural amphiphilic compounds that are composed of a glycerol backbone with two fatty acid chains and a phosphoric acid moiety (Fig. 7.1) (Li et al., 2017). The fatty acid chains function as the hydrophobic tail of the compounds, whereas the phosphoric acid moiety and any attached groups impart hydrophilicity (Pérez et al., 2017). Due to their amphiphilicity, PLs are used as emulsifiers to stabilize lipid droplets (Li et al., 2018). In an oil-inwater system, PLs adsorb to the interface of oil and water (Anankanbil et al., 2018) (Fig. 7.3). Fluorescent dyes dissolved in soybean PC and other phospholipid derivatives are directly observed on the interface of oil droplets. An illustration of the moiety location of a ­phospholipid Fluorophore Nile red

(A)

Soybean lecithin

C14

C18

(B)

(C)

(E)

(F)

NBD PE

(D)

FIG. 7.3  Wide-field fluorescence images showing interfacial location of soybean lecithin (A, D), 1-myristoyl-2-­lysosn-3-glycero-phosphatidylcholine (B, E), and 1-stearoyl-2-lyso-sn-3-glycero-phosphatidylcholine (C, F). Lipid droplets in emulsions were stained with Nile red (dark grey). Surface-active compounds were dyed with a fluorescence probe (light grey colored, NBD PE (ammonium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1, 3-benzoxadiazol-4-yl)), Thermo Fisher Scientific, Waltham, MA, the United States. Images were captured against a dark background. From Anankanbil, S., Pérez, B., Banerjee, C., Guo, Z., 2018. New phenophospholipids equipped with multi-functionalities: regiospecific synthesis and characterization. J. Colloid Interface Sci. 523, 169–178.

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FIG. 7.4  Illustration of oil-in-water emulsions stabilized by phospholipids.

compound is shown in Fig. 7.4. PLs may form either a monolayer or multilayers at the interface depending on the properties of the phospholipid and the emulsion system.

7.3  PRODUCTION, ANALYSIS, AND CHARACTERIZATION OF PLs 7.3.1  PLs Production Commercially available PLs, commonly referred to as lecithin, is produced from plant seed oils such as soybean, sunflower, and rapeseed oils and egg yolk in some cases. Lecithin, as a mixture of PLs and adherent glycolipids and oil, is the focus of this section. PLs are copressed out and coextracted with neutral lipids during food oil refining processes. Extracted soybean oil contains 800–1200 ppm phosphor, equivalent to 2%–3% commercial lecithin with an acetone insoluble content of 60% (Van Nieuwenhuyzen and Tomás, 2008). PLs are either present in a hydratable or a nonhydratable form. The former occurs when the PLs are associated with calcium, magnesium, or iron cations. A general flowchart for lecithin production and further refining is shown in Fig. 7.5. Acid treatment must be performed for conversion into hydratable forms. Then water is introduced to precipitate the gums (Li and Guo, 2016). This step is called degumming. Centrifugation is the most commonly used technique in industry for separating the hydrated and precipitated gums. The resultant wet gum contains around 40%–50% water. Wet gum needs to be dried to a preferred moisture content of  PS. Moreover, they further pointed out  that Tyr56 of PC-PLC structure was a key binding site determining its substrate specificity. Recently, many studies have focused on the biological functions of eukaryotic PIPLCs and PC-PLCs. As reported previously, PI-PLCs play pivotal roles in regulating ­membrane-associated second messenger (1,4,5-trisphosphate) to activate cell surface receptor, promoting macrophage differentiation and survival, and effectively inhibiting cancer cell growth (Cocco et al., 2015; Rebecchi and Pentyala, 2000). On the other hand, inhibiting PC-PLC activity by a pharmacological method can be an effective way to downregulate the human epidermal growth factor receptor-2 expression and reduce cancer differentiation (Podo et al., 2016). 7.4.2.6  Phospholipase D (PLD) PLD is a ubiquitous enzyme hydrolyzing the phosphodiester bond of PLs to yield PA and free head groups such as choline and ethanolamine. This enzyme can be found in bacteria, fungi, plants, and mammals (Guo et al., 2005; Li et al., 2009). More recently, physiological and pathophysiological roles of mammalian PLDs have received great interest from researchers because PLD undertakes key roles in autoimmune, infectious neurodegenerative, and cardiovascular disease, as well as in cancer (Jenkins and Frohman, 2005; Nelson and Frohman, 2015). The three-dimensional structures and catalytic mechanism of some microorganism-derived PLDs have been revealed (Leiros et al., 2000, 2004). It is notable that the typical feature of mammalian PLD has two HKD motifs, namely, HxxxxKxD sequence (H, histidine; x, any amino acid; K, lysine; and D, aspartic acid). In a review paper (Jenkins and Frohman, 2005), the catalytic mechanism of PLD using PC as substrate was summarized (Fig. 7.13): (1) The histidines of two HKD motifs are firstly protonated, (2) PC substrate enters the catalytic active site located around two HKD motifs of PLD, (3) choline is generated by liberating the amino-terminal proton, and a PLD-PA intermediate is formed, (4) water moleculars enter the carboxy-terminal HKD domain to provide a proton for releasing PA.

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7.5  SYNTHESIS OF LYSO-PLs AND STRUCTURED PLs

Choline

P C

H K D

P C

H K D

PLD H K D

H K D

H K D

H K D

P A

PLD

PLD

H2O

P A

H K D

P A

H K D

PLD FIG. 7.13  Scheme of PLD hydrolysis of PC to PA and choline (Jenkins and Frohman, 2005).

7.5  SYNTHESIS OF LYSO-PLs AND STRUCTURED PLs 7.5.1  Synthesis of Lyso-PLs Lyso-PLs contain one fatty acid distributed in the sn-1 position (2-lyso-PLs) or sn-2 position (1-lyso-PLs). Lyso-PL production can be carried out by lipase- or phospholipase-catalyzed hydrolysis, alcoholysis, and esterification (Schemes 7.1, 7.2, and 7.3, respectively). To develop desired lyso-PLs for nutritional and therapeutic applications, researchers are still attempting to exploit and/or modify the existing enzymatic biotechnologies for large-scale production of lyso-PL.

SCHEME 7.1  Hydrolysis of PLs to produce lyso-PL and free fatty acids. R1 and R2 represent fatty acyl groups.

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SCHEME 7.2  Alcoholysis of PLs to produce lyso-PL and fatty acid esters. R1 and R2 represent fatty acyl groups. R3 represents short-chain alcohols (e.g., method and ethanol).

SCHEME 7.3  Esterification of glycerophospholipids to produce lysophospholipids and water. R1 represents fatty acyl group.

7.5.1.1 Hydrolysis Enzymatic hydrolysis is one of the most common reactions to produce lyso-PLs. It has been shown that the yield and structures of the final products such as 1-lyso or 2-lyso-PL are largely dependent on the selected enzyme species, due to the substrate specificity as discussed above. Sonoda et al. (2002) successfully cloned and characterized a novel PA-selective phospholipase A1 from rat. Lyso-PA can be synthesized by the PLA1 using PA as substrate. LPC has been prepared by hydrolysis of PC with PLA1 from Trypanosoma brucei (Richmond and Smith, 2007). Moreover, the latter study further revealed that phenylmethanesulfonyl fluoride and diethyl-p-nitrophenyl phosphate had a negative impact on the Ser131 activity of PLA1, resulting in a low activity. Interestingly, Wang et al. (2010) reported that PLA1 (Lecitase Ultra, Novozymes A/S) possessed sn-1-regioselectivity similar to lipases in the hydrolysis of PLs. Mustranta et al. (1995) showed that for hydrolysis of soybean PC, lipases from

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Aspergillus niger and Penicillium cyclopium gave a higher hydrolysis rate than PLA1 from A. niger and PLA2 from A. niger and Lecitase. In addition, some lipases have fatty acid selectivity that discriminates against long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) such as docosahexaenoic acid (DHA). For example, Devos et al. (2006) screened 12 commercial lipases for concentrating DHA into lyso-PL by enzymatic hydrolysis of Isochrysis galbana-derived PLs and found that lipase from Rhizopus oryzae yielded the highest DHA recovery (85%) after 3 h of hydrolysis. 7.5.1.2 Alcoholysis Enzymatic alcoholysis is usually mediated by a short-chain alcohol such as methanol, ethanol, or propanol. Ethanol is a green solvent, which has been used to produce lyso-PL in the ethanolysis of PLs for food. Therefore, available studies have focused on the ethanolysis of PLs for lyso-PL production. In 1994, for the first time, lipase-catalyzed alcoholysis of PLs to produce lyso-PL was reported (Sarney et  al., 1994). They selected an immobilized lipase from Rhizomucor miehei (Lipozyme® IM-60, Novozymes A/S) as biocatalyst and soybean PC as substrate. Results showed that the types of alcohol and reaction temperature affected the performance of alcoholysis. Lipozyme® IM-60 yielded a higher yield of LPC using methanol as acyl acceptor compared with ethanol and 2-propanol. Moreover, in the ethanol and 2-propanol reaction systems, Lipozyme® IM-60 achieved a higher reaction rate at 22°C than those at 37 and 55°C. In the 1-butanol system, the greatest yield of LPC was obtained at 37°C. These findings can be explained by alcohol and temperature influencing the affinity between substrate and enzyme and interfacial activation. Lipozyme® IM 20 (from R. miehei, Novozymes A/S)-catalyzed alcoholysis of soybean PLs for lyso-PL production with various short- and long-chain alcohols (from C4 to C18) was conducted by Ghosh and Bhattacharyya (1997). The authors found that the yield of lyso-PL was in the range of 75.5–77.6 mol% after 24 h of alcoholysis for C4, C8, C10, and C12 alcohols. A low lyso-PL yield was achieved with C18 alcohol. In view of the fatty acid composition of lyso-PL product, the linoleic acid (C18:2) content of lyso-PLs (75.5%–76.7%) was significantly higher than that of original PLs (54.3%), indicating that Lipozyme® IM 20 may discriminate against LA during the alcoholysis. This study demonstrates that Lipozyme® IM 20 can be used to concentrate LA in modified PLs by the alcoholysis of natural PLs. More recently, Novozym® 435 (C. antarctica, Novozymes A/S, Bagsværd, Denmark), Lipozyme® TL IM (T. lanuginosus, Novozymes A/S, Bagsværd, Denmark), and Lipozyme® RM IM (R. miehei, Novozymes A/S, Bagsværd, Denmark) were found to be superior biocatalysts for producing LPC by ethanolysis of PC in neat ethanol or n-hexane-ethanol mixtures (Yang et al., 2015). Results showed that adding n-hexane reduced the reaction time for all three immobilized lipases. LPC yields for the three lipases were higher than 90% in the both solvent systems. 7.5.1.3 Esterification Lyso-PLs can be obtained by enzymatic esterification of l-α-glycerophospholipids (GPLs) and desired fatty acid(s). Virto and Adlercreutz (2000) synthesized 1-lauroyl-sn-­ glycerophosphocholine via Novozym® 435 esterification of GPC and lauric acid in solventfree and 50% (v/v) tert-butanol media. After 100 h of esterification, the yield of LPC + PC was 50 mol%, and the water content was 0.1 mmol for the solvent-free system. The value was very

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close to the result in 50% (v/v) tert-butanol system. It should be noted that an increase in water content (0.5 mmol) resulted in the significant decrease in the yield of LPC + PC (4 mol%). In previous studies, Novozym® 435 showed a higher activity at very low water content (Ghosh and Bhattacharyya, 1997; Yang et al., 2015). For lipase-catalyzed esterification, some water-mimic solvents such as formamide, dimethylformamide, and methanol can increase the LPC yield (Kim and Kim, 2000). Lipozyme® RM IM gave the highest yields of LPC for the esterification of GPC with oleic acid in the 10% (v/ wt) formamide, 4% (v/wt) dimethylformamide, and 2% (v/wt) methanol systems. Compared with formamide and methanol as a solvent, a higher LPC yield was achieved with dimethylformamide. The possible reasons can be that (1) dimethylformamide is capable of depriving water molecules surrounding the enzyme, improving the shift toward esterification to form LPC and (2) dimethylformamide promotes the solubility of substrate and increases the miscibility and viscosity during the esterification. In order to improve the nutritional value of LPC, lyso-PLs containing conjugated linoleic acid (CLA) were synthesized by esterification of GPC with CLA catalyzed by Novozym® 435, Lipozyme® TL IM, Lipozyme® RM IM, PLA1 from T. lanuginosus, and PLA2 from porcine pancreas (PLA1 and PLA2 were immobilized on Duolite A 567 resin) (Hong et al., 2011). Under identical reaction conditions, Novozym® 435 produced the highest yield of LPC, particularly under vacuum pressure (1–50 mmHg). The five enzymes exhibited fatty acid selectivity toward 9c,11t-CLA and 10t,12c-CLA. Novozym® 435 tended to incorporate 10t,12c-CLA more favorably into LPC (40.7%), while Lipozyme ®TL IM, Lipozyme® RM IM, and PLA1 preferentially incorporated 9c,11t-CLA (41.3%–43%). PLA2 possessed no selectivity toward 9c,11t-CLA (27.1%) or 10t,12c-CLA (27.7%). LPC rich in n-3 PUFAs from fish oil were obtained by enzymatic esterification of GPC using the five enzymes mentioned above (Liu et  al., 2017). The highest yield of LPC was obtained by Lipozyme® TL IM (67.31%) under identical reaction conditions. Based on the fatty acid composition of LPC product, Lipozyme® TL IM synthesized LPC with the highest docosapentaenoic acid (DPA, 7.31%) and DHA (66.12%) contents, while Lipozyme® RM IM produced LPC had the lowest contents of DPA (1.83%) and DHA (50.7%). Among these five enzymes, the LPC produced by Novozym® 435 possessed the highest EPA content (17.35%). These findings reveal that biocatalysts have different fatty acid selectivity toward n-3 fatty acids in the esterification of GPL with specific fatty acids.

7.5.2  Synthesis of Structured PLs 7.5.2.1  Structured PLs Containing Specific Fatty Acids Structured PLs (SPLs) are PLs that can be formed with specific fatty acyl groups in the sn-1 and/or 2 position(s) of PLs through enzymatic acidolysis catalyzed by PLA1, PLA2, and various lipases from bacteria and fungi. As can be seen in recent studies (Table 7.4), incorporation of specific fatty acids such as CLA, ALA, EPA, and DHA has attracted attention due to their benefits in health. In general, reaction parameters including enzyme loading, water content, substrate ratio, reaction temperature, enzyme source, organic medium, and reaction time influence the incorporation rate of specific fatty acids during enzymatic reaction (Table 7.4). The screening of five commercial immobilized lipases showed that Novozym® 435 was the most efficient for incorporating citronellic acid into egg-yolk PC (Rychlicka et al., 2018).

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TABLE 7.4  Recent Studies on Enzymatic Acidolysis of PLs to Prepare Structured PLs (SPLs)a PLs Source

Acyl Donor

Enzyme

Reaction Conditions

Yield (%)

Reference

Egg-yolk PC

Citronellic acid

Novozym® 435

Enzyme loading 30%, PC/ citronellic acid ratio 1:60 (mol/ mol), 30°C, 48 h, toluene

39%

(Rychlicka et al., 2018)

PC

DHA

PLA1

Water content 0.4%, enzyme loading 40%, pH 6.92, DHA/PC ratio 2.13 (g/g), 45°C, 24 h

20.9%

(Chen et al., 2017)

Soybean PC

Medium-chain fatty acids (caproic C6:0, caprylic C8:0, capric C10:0, and lauric C12:0)

PLA1

Enzyme loading 12%, PC/fatty acids 1:15 (mol/mol), 45°C, 24 h

42.52%

(OchoaFlores et al., 2017)

Soybean PC

EPA and DHA ethyl esters

PLA1

Water content 1.1%, enzyme 30.31% loading 15%, ethyl esters/PC ratio 6.8:1 (g/g), 56°C, 72 h, 0.2 kPa

(Li et al., 2016)

Egg-yolk PC

CLA

Lipozyme® RM IM

Enzyme loading 24%, CLA/PC ratio 8:1 (mol/mol), 45°C, 24 h, heptane

33.8%

(Niezgoda et al., 2016)

Egg-yolk PC

CLA

Novozym® 435

Enzyme loading 20%, PC/ pomegranate oil 1:3 (mol/mol), 50°C, 72 h

28.8%

(Chojnacka et al., 2016)

Soybean PC

n-3 PUFAs from fish oil

PLA1

Water content 1.0%, enzyme loading 20%, 55°C, 24 h

57.4%

(Zhao et al., 2014)

a

PC, phosphatidylcholine; CLA, conjugated linoleic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; n-3 PUFAs, n-3 polyunsaturated fatty acids; PLA1, phospholipase A1(Novozymes A/S); Novozym® 435, lipase B from Candida Antarctica (Novozymes A/S); Lipozyme® RM IM, lipase from Rhizomucor miehei (Novozymes A/S).

Niezgoda et al. (2016) developed an efficient process to incorporate 33.8% CLA into egg-yolk PC by Lipozyme® RM IM-catalyzed acidolysis. Since PLA1 (Lecitase Ultra, Novozymes A/S) has been commercialized and its price is around 17 €/kg in liquid form, utilizing this enzyme to modify PLs has been studied extensively. For instance, Li et al. (2016) have reported n-3 PUFA incorporation at 26.26% into soybean PC by PLA1-catalyzed acidolysis under the optimized conditions (Table 7.4). It is noted that the incorporation rate of n-3 PUFAs increased significantly to 30.31% when vacuum pressure at 0.2 kPa was applied. 7.5.2.2  Enzymatic Transphosphatidylation for Modifying Polar Head Groups in PLs PLD has been widely used to modify the polar head groups of PLs by enzymatic transphosphatidylation. Early research on PLD-catalyzed transphosphatidylation mainly replaced the choline head group of PC with serine, glycerol, and ethanolamine, producing PS, PG, and PE, respectively (Guo et al., 2005; Hama et al., 2015). Recently, to increase the potential application of PLs for food and drugs, numerous studies have focused on preparation of novel restructured PLs as summarized in Table 7.5.

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TABLE 7.5  Recent Studies of Phospholipase D (PLD)-Catalyzed Transphosphatidylation of PLs to Prepare Structured PLs (SPLs)a PL Source

PLD Source

Acceptor Alcohol

Yield

Reference

DOPC and PA

Streptomyces sp. and white cabbage

Diethanolamine, triethanolamine, tris(hydroxymethyl)-aminomethane, 2-[bis(2-hydroxyethyl)imino]-2(hydroxymethyl)-1,3-propanediol

3.1%–47.2% of modified PC

(Dippe et al., 2008)

Soybean PC

Streptomyces sp.

2-(4-Hydroxyphenyl)ethanol, 2-(3,4-dihydroxyphenyl)ethanol, 4-Hydroxybenzyl alcohol, hydroquinone, 2-(4-aminophenyl) ethanol, 2-phenylethanol, benzyl alcohol, 3-phenyl-1-propanol, 4-phenyl-1-butanol;, 5-phenyl-1pentanol, 2-(2-methylphenyl)ethanol, 2-(3-methylphenyl)ethanol, 2-phenyl1-propanol, 1-phenyl-2-propanol, 2-methyl-1-phenyl-2-propanol

13–90 mol% of modified PC

(Yamamoto et al., 2011)

PC

Streptomyces sp.

Glucose

95% of PC-Glu

(Song et al., 2012)

Hydrogenated Actinomadura PC (90%) sp.

2-(4-Hydroxyphenyl)ethanol (tyrosol)

81 (PC 83 mmol/L) and 157 (PC 167 mnol/L) mmol/L of phosphatidyl-tyrosol

(Casado et al., 2013)

Hydrogenated Actinomadura PC (90%) sp

Hydroxytyrosol

86% of modified PC-HT

(Casado et al., 2014)

DOPC

Streptomyces lividans TK24

Glucose

12.5% of 1-phosphatidyl- (Inoue et al., β-d-glucose 2016)

DOPC

Streptomyces lividans TK24

Threonine

32.5 mol% of phosphatidylthreonine

(Damnjanović et al., 2018)

a

PC, phosphatidylcholine; DOPC, Dioleoyl-sn-glycerophosphocholine; PA, phosphatidic acid; PC-Glu, phosphatidyl-glucose.

To confer an antioxidative antioxidant and anticancer properties of structured PLs, Yamamoto et al. (2011) synthesized novel phosphatidyl derivatives via PLD-catalyzed transphosphatidylation of PC with functional phenylalkanols. Another research team (Song et al., 2012) developed a green and novel reaction method to replace the choline of PC with glucose for phosphatidyl-glucose production using PLD (from Streptomyces sp.). Martin et al. (2014) evaluated the antioxidant activity of phosphatidyl-hydroxytyrosol (PHT) obtained by PLDcatalyzed transphosphatidylation of PC with hydroxytyrosol (HT). Results revealed that the protective effect of PHT for olive oil and diacylglycerol oil was superior to HT, while the value of lard oil by PHT was similar to HT. Moreover, compared with tocopherol and PC, PHT possessed a higher antioxidant activity index. Various novel phosphatidyl derivatives are being developed, and their biological functions are needed to be further investigated for foods, cosmetics, and health applications.

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269

7.5.2.3  Enzymatic Modification of PLs in Reactors To date, most of the reported studies have investigated the enzyme-catalyzed reactions of PLs to prepare lyso-PLs or SPLs in laboratory-scale vessels (Tables 7.4 and 7.5). To scale up the enzymatic modification of PLs, researchers have been long committed to find green and cost-effective routes using different bioreactors for desired PL production. So far, several bioreactor types for enzymatic modification of PLs have been investigated: stirred tank reactor, packed-bed reactor, and membrane reactor (Guo et al., 2005; Hama et al., 2015). For stirred tank reactor, the PLD-catalyzed transphosphatidylation of PC with tyrosol to prepare phosphatidyl-tyrosol in a biphasic medium was successfully conducted in a 1 L stainless steel reactor coupled to a semicontinuous separation system (Casado et al., 2013). The yield and purity of phosphatidyl-tyrosol attained by this process was 40 g and 94%, respectively. When the enzyme is in immobilized form, PL modification in a packed-bed reactor can be a promising approach. A jacketed stainless steel column (length, 200 mm; diameter, 21 mm; and length, 196 mm) loaded with 36 g of Lipozyme® TL IM was used to produce soybean PL containing caprylic acid by enzymatic acidolysis (Vikbjerg et al., 2005). A packed-bed reactor equipped with a glass tube (diameter, 25 mm) was established to synthesize PS via PLD-catalyzed transphosphatidylation of Triton™ X-100 (Dow Chemical Co., Midland, MI, the United States)-modified PC adsorbed to silica with L-threonine (Zhang et  al., 2017). A membrane reactor containing immobilized PLC from C. perfringens was constructed for ceramide production via enzymatic hydrolysis of sphingomyelin (Zhang et al., 2008). Besides, one of attractive advantages of this process was that sphingomyelin substrate in aqueous phase and ceramide product in organic phase were easily separated by the membrane. This enzyme membrane reactor can be an alternative way to produce modified PLs by different enzymatic reactions.

7.6  DESIGN AND SYNTHESIS OF FUNCTIONAL PLs GPLs such as PE, PC, PI, PS, and PG are essential components of biological membranes (Sano et al., 2015). In addition, other GPLs (e.g., PA) serve as precursors during biosynthesis of other GPLs (Dubots et  al., 2012). Chemical synthesis for natural and unnatural PLs has attracted attention due to various reasons. First, natural PLs are complex mixtures with different FA distributions and polar head groups. Second, there is often the need for pure PL products for specific applications in food, drugs, gene therapy, or cosmetic formulations. Third, the purity of PLs provided by chromatographic separations and/solvent extraction of natural PLs can be low. Therefore, research efforts are devoted toward the synthesis of highly pure PL products with defined structures and configurations (Eibl, 1980) to enhance potential biological and physiological activities. Consequently, PLs with functional moieties (e.g., vitamins, sugars, essential FA, antioxidants, and nucleotides) incorporated as either part of the head group or acyl units have been synthesized for applications in food, cosmetics, or drugs. Depending on applications, synthetic procedures can be derived to achieve desired PLs. Synthesis of PLs are categorized into total synthesis and semisynthesis. Total synthesis of PLs and their analogues requires the formation of ester or ether linkages between acyl groups and the glycerol backbone and attachment of the desired polar head groups. Sugar alcohols (e.g., d-mannitol) or isopropylidene glycerols are sources of glycerol

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7.  Phospholipid-Based Surfactants

(A)

(B) SCHEME 7.4  Multistep synthetic pathway of 1-lauroyl-2-oleoyl-sn-glycero-3-[phosphor-1-glycerol]. (A) Use of isopropylidene glycerol as starting material. (B) Use of d-mannitol as starting material. DMAP, 4-(dimethylamino) pyridine; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(Murakami et al., 1999).

backbone for PLs in total synthesis (Li et al., 2014). Typical total syntheses of PLs are complex multistep procedures involving several protection/deprotection sequences. In some cases, toxic solvents and reagents are employed (Scheme 7.4). The use of sugar alcohols (e.g., d-mannitol) as a three‑carbon chiral building block for total synthesis of PG was suggested to be advantageous compared with synthesis using III.  BIOBASED SURFACTANTS



7.6  DESIGN AND SYNTHESIS OF FUNCTIONAL PLs

271

isopropylidene glycerol (Murakami et  al., 1999). This was due to possible acyl migrations in synthesis using isopropylidene glycerol as starting material (Scheme 7.4A). In addition, sugar alcohols are readily available substrates compared with isopropylidene glycerol. Furthermore, fewer protection and deprotection steps are needed when sugar alcohols are employed as substrates. An example of this strategy was demonstrated in the synthesis of 1-lauroyl-2-oleoyl-sn-glycero-3-[phosphor-1-glycerol] in a 10-step synthetic procedure starting with d-mannitol (Scheme 7.4B). A yield of only 19% was obtained (Murakami et al., 1999). However, other studies used isopropylidene derivatized glycerol as starting building blocks for other PL products and realized higher yields. For instance, Wang et al. (2013) reported the synthesis of labeled PC fluorophores for fluorescence resonance energy transfer (FRET)dependent assay of enzyme kinetics. The synthetic strategy outlined in that study (Scheme 7.5)

SCHEME 7.5  Reagents and reaction conditions: (a) dicyclohexylcarbodiimide (DCC)-DMAP, 4-(dimethylamino) pyridine, CH2Cl2; (b) 0.4 M HCl, aq. dioxane, 2 h; (c) C6H5OCH2COCl, 2,4,6-trimethylpyridine, CHCl3, 0°C; (d) dihydropyran (DHP), pyridinium para-toluenesulfonate (PPTS), CH2Cl2, rt.; (e) tert-butylamine, CHCl3-MeOH (1:1), 0°C, 24 h; (f) (i) (ethylene chlorophosphate, (C2H5)3N, benzene, rt., 6 h, (ii) (CH3)3N, MeCN, 65°C, 24 h; (g) 0.15 M HCl, aq dioxane; (h) 12-(2_-naphthylacetyl)aminododecanoic acid-DCC-DMAP, CHCl3; and (j) (i) 12-N-fluorenylmethyloxycarbonyl chloride-aminododecanoic acid (12-N-FMOC-aminododecanoic acid)-DCC-DMAP, CHCl3, (ii) 5 eq. DBU, rt., 1 h and (iii) p-nitrophenyl-7-diethylaminocoumarin-3-carboxylate, DMAP,CHCl3. (Wang et al., 2013).

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consisted of (1) the use of 2, 3-O-isopropylidene-sn-glycerol (3) as a three‑carbon chiral backbone of the target PC products; (2) the acylation of the sn-1 hydroxyl followed by acid-­ catalyzed hydrolysis of the acetonide functionality to give the corresponding diol; (3) orthogonal protection of the hydroxyl groups of the diol using a base-labile phenoxyacetyl group at the primary hydroxyl position and an acid-labile tetrahydropyranyl moiety at the secondary hydroxyl position; and (4) phosphorylation using 2-chloro-2-oxo-1,3,2-dioxaphospholane followed by trimethylamine-mediated cleavage of the five-membered phosphodiester ring. The fluorescent LPC (11) was obtained in yields of 79%. Further derivatization of (11) with fluorescent labeled fatty acyl units yielded PC-1 and PC-2 products. Similarly, Duclos (2010) described the total synthesis of isotopically labeled 2-O-arachidonoyl1-O-stearoyl-sn-glycero-3-phosphocholine-1,3,1′-13C3 and − 2, 1′-13C2, an important biosynthetic precursor of arachidonic acid metabolites and endocannabinoid 2-­arachidonoylglycerol (2-AG) (Scheme 7.6). To optimize signal-to-noise intensities of 13C spectral bands from labeled glycerol carbons, it requires a synthetic strategy where no 13C13C couplings of adjacent labeled carbons would occur. As a result, a 50:50 mixture of ­glycerol-1,3-13C2 (1)

SCHEME 7.6  Synthetic pathway of 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine-1,3,1′-13C3 and -2, 1′-13C2 (11, SAPC-13C4). DCC, dicyclohexylcarbodiimide; DMAP, 4-(dimethylamino)pyridine; DSPC, 1,2-di-O-­ stearoyl-sn-glycero-3-phosphocholine; SAPC, 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine (Duclos, 2010).

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273

and glycerol-2-13C (2) were converted to the respective trityl derivative (3) in the first step of the synthesis. A diesterification of the diol (3) with stearic-1-13C acid yielded the racemic trityl-protected diacylglycerol (4). Subsequent deprotection of (4) gave (5) in 90%. Through a number of chemoenzymatic steps, (5) was converted into the required mixed chain PC product (11). Other examples of total syntheses of PL can be found in Table 7.6. The lower complexity of semisynthesis compared with total synthesis makes it more attractive. Semisynthesis of PLs involves alteration of polar head groups, hydrophobic tails, or both using natural PLs as starting materials. Compared with total synthesis, semisynthesis requires fewer steps. There are three types of semisynthesis of PL products. The first is hydrogenation of double bonds of natural PLs to obtain the more stable and high melting saturated PLs. The second is PLD-catalyzed transphosphatidylation to modify the head groups of PLs. The third is the acylation of GPC by desired acyl donors to give modified PLs (Li et al., 2014). The use of GPC as raw material for semisynthesis is attractive due to its ready availability and affordable price. Most crucially, GPC has the same chirality as the final PC products. However, its limited solubility has been traditionally addressed by employing it in its cadmium adduct form (Gupta et al., 1977; Singh, 1990). Toxicity associated with heavy metals made it important to seek alternatives (Ichihara et al., 2005). Recently, a few studies utilized GPC adsorbed to diatomaceous earth as dispersant to achieve catalyst-free semisynthesis of PLs (Anankanbil et al., 2018; Marrapu et al., 2015). Metal oxides in diatomaceous earth served as catalysts for de novo and solvent-free synthesis of lyso-PC, PC, and modified PC in the studies. An example of semisynthesis of modified PC products using GPC as starting material is illustrated in Scheme 7.7. In Scheme 7.7A, soy/egg PC was enzymatically hydrolyzed to yield the respective LPC that was further acylated (chemically or enzymatically) to generate the required PC products. Scheme 7.7B illustrates the conversion of GPC into LPC in the presence of molten FFA via metal oxide catalytic groups in diatomaceous earth (kieselguhr). The next step was the acylation of LPC with various phenolic acids (e.g., caffeic acid) to yield the required modified PC products. The desired properties of the final PC products were good surface activity and antioxidant activity. Therefore, synthetic procedures to achieve such multifunctionality were devised accordingly. By careful design of synthetic strategies, it is thus possible to obtain PL products for various applications.

7.6.1  Modifications of Acyl Units of PLs With Functional Components PUFA such as DHA and EPA are known bioactive compounds (Ruxton et  al., 2007). Enrichment of PLs with PUFA has therefore attracted attention. Due to the low oxidative stability of PUFA, enzymatic methods are preferred. Incorporation of PUFA into PLs improve bioavailability of PUFA due to the well-known emulsifying properties of PLs (Cansell et al., 2003). Besides PUFA, PLs have been modified with short- and medium-chain FA. Modification of PLs with medium-chain saturated fatty acids improved the oxidative and heat stability of PLs (Vikbjerg et al., 2007) . Furthermore, phenolic acids are effective antioxidants. However, their miscibility with apolar media is limited (Stamatis et  al., 1999). Particularly, phenolic acids tend to stay in aqueous phase of enriched food systems (fish oil-in-water emulsions), and hence, their potential ability to combat lipid oxidation at the interface is not realized (Stamatis et al., 1999).

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TABLE 7.6  Literature on Total and Semisynthesis of Functional PLsa Starting Material

Method b

Functional Unit

Type of Modification Reference

Applications

d-Mannitol

Chemical

Fatty acids

Acyl

(Murakami et al., 1999)

Medical/ biochemistry

Glycerol

Chemoenzymaticb Fatty acids

Acyl

(Duclos, 2010)

Biochemistry

Acyl/head groups

(Lindberg et al., 2002)

Various Biochemical

S-glycidol

b

Chemical

Various acyl units

Isopropylidene Chemicalb glycerol

Palmitic acid Acyl Aminododecanoic acid derivatives

(Rosseto et al., 2006)

Isopropylidene Chemicalb glycerol

Fluorescent labels

Acyl chain

(Wang et al., 2013) Medical/ biochemistry

Caffeic acid

Acyl chain

(Anankanbil et al., Antioxidant, surface2018) active agents

GPC

Chemicalc

Soy/egg PC

Chemoenzymaticc Various phenolic acids

Acyl chain

(Anankanbil et al., Antioxidant, surface2018) (Balakrishna active agents et al., 2017)

Soy PC

PLA1c,d

n-3 PUFAs

Acyl chain

(Zhao et al., 2014)

Nutritional

Soy PC

c,e

PLA1

CLA

Acyl chain

(Baeza-Jiménez et al., 2012)

Nutritional

Soy lecithin

Immobilized R. oryzae cellsc

Lauric acid

Acyl chain

(Hama et al., 2010, Nutritional 2011)

PC

Pancreatic PLA2c,f Caprylic acid

Acyl chain

(Vikbjerg et al., 2006, 2007)

Nutritional

Soy PC

PLD (Streptomyces Saccharides sp.)c

Head group

(Song et al., 2012)

Liposomes

Dioleoyl PC

PLD from Streptomyces sp. and cabbagec

Ethanolamine derivatives

Head group

(Dippe et al., 2008)

Potential use as emulsifiers/ excipients

Soy PC

Streptomyces sp. PLDc

Tyrosol

Head group

(Yamamoto et al., 2011)

Antioxidants, anticancer

Soy PC

Streptomyces sp. PLDc

Terpenes

Head group

(Yamamoto et al., 2008)

Potential anticancer agents

Soy PC

Streptomyces sp. PLDc

β-sitosterol

Head group

(Hossen and Hernandez, 2005)

Therapeutic agents

1,2-Dioeleoyl PC

Streptomyces sp. PLDc

Threonine

Head group

(Damnjanović et al., 2018)

Potential biological benefits

a

PL, phosphatidylcholine; PLD, phospholipase D; CLA, conjugated linoleic acid; PLA, phospholipase A; PUFA, polyunsaturated fatty acids. Total synthesis. c Semisynthesis. d Lecitase™ Ultra immobilized on Lewatit. e Lecitase™ Ultra immobilized on Duolite. f PLA2 immobilized on Amberlite. b

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275

7.6  DESIGN AND SYNTHESIS OF FUNCTIONAL PLs O

O R1

O

O

Lipozyme RM IM

R

90% EtOH, 7 h

O O P O O

O R1

O

OH O O P O O

O

N

N

GPC.kieselguhr complex

Egg/Soy PC

O

O R1

O

O

O O P O O

(a) chemical (b) enzymatic OH

80 °C, 50 mbar 10 h

OH

OH a OR

N HO

OH

HO

O O O b

O

1. EDC 2. DMAP

OH

O O

O

(A)

HO

O HO

R R = 11, 13, 15, 17 O O P O N O O

FFA

HO

(B)

OH

R

O O P O O

OH OH CH2Cl2 , rt, 12 h

N

SCHEME 7.7  Semisynthetic pathways to yield modified PC products with dual functionality (surface activity and antioxidant properties) (Anankanbil et al., 2018). (A) Egg/soy PC as starting material. (B) GPC as starting material. FFA, free fatty acids; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMAP, 4-(dimethylamino)pyridine.

Therefore, a few studies have incorporated phenolic acids into PLs to generate “phenolipids.” Such molecules have potential applications in food, drugs, or cosmetic formulations.

7.6.2  Alterations of Polar Head Groups With Functional Units The head group of PLs is considered biocompatible and hence can be used as a nontoxic carrier of functional units. PLD-catalyzed transphosphatidylation can therefore be used for modifying the head groups of PLs with functional units. Functionalization of PLs adds the biological and other physicochemical properties of natural PLs to acceptor molecules (Iwasaki and Yamane, 2004). For instance, phosphatidyl ascorbic acid (PAsA) was synthesized by transphosphatidylation. Ascorbic acid is a well-known antioxidant but is highly hydrophilic with no surface activity. As a result, lipophilization (attachment of hydrophobic side chains) is required to ensure maximal function in heterogeneous systems. Synthesized PAsA sufficiently localized ascorbate at water-lipid interfaces of liposomes, by taking advantage of the emulsifying properties of PLs. In that way, PAsA effectively inhibited oxidation (Nagao et al., 1991). Moreover, the antioxidant activity of α-tocopherol is attributed to its chromanol ring. PLDcatalyzed synthesis of phosphatidylchromanol (PCh) inhibited autoxidation of lard more efficiently than the parent compound. The formation of water-in-oil microemulsions and entrapment of trace metal ions in the aqueous nanodroplets was hypothesized to cause the antioxidation activity of PCh (Koga and Terao, 1994). Similarly, PL derivatized with arbutin and kojic acid showed inhibitory effects toward tyrosinase similar to the parent arbutin or kojic acid (Takami et  al., 1994). In addition, a phosphatidyl derivative, phosphatidylgenipin, was found to be 6–52 times more cytotoxic against human embryonic lung fibroblasts and MT-4 cells than genipin (Takami and Suzuki, 1994). Antiviral derivatives of PL were synthesized using both chemical and enzymatic means. N-acetylneuraminic acid (NeuAc) was introduced into PC, and the resultant liposome structures inhibited rotavirus infection at about 103–104-fold higher than underivatized NeuAc.

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276

7.  Phospholipid-Based Surfactants

The formation of bilayers by NeuAc on the surface on the new lipids was attributed to this effect (Koketsu, 1997). The transphospatidylation of PLs with terpenes including farnesol, geraniol, geranylgeraniol, and phytol in aqueous or biphasic systems was investigated (Yamamoto et  al., 2008). PLD from Streptomyces sp. was the only PLD among others screened that was capable of catalyzing the reactions. Under optimal reaction conditions, incorporation of terpenes into PL were 90%, 54%, 73% and 17% for geraniol, gerenylgeraniol, farnesol, and phytol, respectively. Similarly, functional phenylalkanols including tyrosol and hydroxytyrosol have also been introduced into PLs by PLD-catalyzed transphosphatidylation (Yamamoto et al., 2011). The introduction of functional units such as saccharides into head groups of PLs may be useful for the delivery of ingredients and maintaining the structural integrity of liposomes. Phosphatidyl-glucose was successfully synthesized using PLD from Streptomyces sp. Incorporation of 95% was attained after just 1.5 h, significantly higher than previous reports (Song et al., 2013). The examples described above indicate the possibility to use synthetic chemistry for the modification of natural PLs into functional molecules with applications in food, drugs, and cosmetics. Besides retaining the indigenous biological and physiological activity of PLs, modifications add a new dimension of useful attributes to PLs. The next section deals with applications of PLs as surfactants.

7.7  APPLICATIONS OF PLs AS SURFACTANTS Recently, lipid-based formulations have become important as delivery cargos for lipophilic phytochemicals, drugs, or nutraceuticals (Leong et al., 2015; Yucel et al., 2012). Encapsulation of ingredients in lipid formulations (e.g., oil-in-water [O/W] emulsions and nanoparticles) improves both bioavailability and stability (Berton-Carabin et  al., 2013; Yucel et  al., 2012). Typically, such systems consist of an oil phase dispersed in a continuous aqueous phase stabilized by surfactants. PLs are excellent emulsifying agents and therefore can stabilize O/W emulsions as delivery systems for food, cosmetic ingredients, and drugs (Yang et al., 2013). The presence of both hydrophilic head groups and hydrophobic tails in PLs confers amphiphilicity, thereby allowing for their emulsification, self-assembly, and wetting properties (Li et al., 2014). For instance, when placed in aqueous media, PLs have the tendency to form liposomes that serve as drug carriers (Cullis et al., 1986). Both natural and synthetic PLs have been used in lipid formulations for use in drugs, food, and cosmetics. Most studies have used PLs as mixtures (e.g., lecithin) in applications (Table 7.7). PL mixtures seem to have better emulsifying properties than single-component emulsifiers. However, the generation of lyso-PLs during emulsification and storage makes mixtures of PLs undesirable (Li et al., 2014). Increased proportions of lyso-PLs have a positive effect for food applications due to improved emulsification properties (Li et al., 2018). On the other hand, lyso-PLs in drug formulations have been associated with hemolysis after intravenous injections of lipid emulsions (Li et al., 2014). Therefore, the type of applications impacts the choice of PLs. Single-component and pure PLs are preferred in drug formulations, while mixtures of PLs are desirable in food applications.

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277

7.7  APPLICATIONS OF PLs AS SURFACTANTS

TABLE 7.7  Applications of PLs as Surfactantsa Encapsulated Ingredient

PL Surfactant

Mode of Field of Application Encapsulation

N/A

Sunflower lecithin

Food

O/W emulsions

(Mezdour et al., 2011)

Fish oil

Synthetic PC

Food

O/W emulsions

(Anankanbil et al., 2018)

N/A

Soy lecithin

Food

O/W emulsions

(Chung et al., 2017)

Fish oil

Rapeseed lecithin

Food

O/W emulsions

(Li et al., 2018)

N/A

Sunflower lecithin

Food

O/W emulsions

(Komaiko et al., 2015)

Omega-3 PUFA

Sunflower lecithin

Food

O/W emulsions

(Liang et al., 2017)

N/A

PE and PS

Food

O/W emulsions

(Samdani et al., 2018)

Cinnarizine

Egg lecithin

Drug

O/W emulsions

(Shi et al., 2010)

n-3 PUFA

Corn lecithin

Food

O/W emulsions

(Liu et al., 2018)

All-trans retinoic acid

Egg PC

Drug

O/W emulsions

(Chansri et al., 2006)

Chlorambucil

Egg PC

Drug

O/W emulsions

(Ganta et al., 2008)

Cetyl palmitate

Synthetic PL

Drug

SLN and NLC

(Pucek et al., 2017)

Ketoprofen

Egg lecithin

Drug

SLNs

(Kheradmandnia et al., 2010)

Ligustrazine

Lecithin

Drug

O/W emulsions

(Wei et al., 2012)

Penciclovir

Egg PC

Drug

SLN

(Lv et al., 2009)

α-Asarone

Soy lecithin

Medicinal/drug

O/W emulsions

(Ma et al., 2013)

Ref.

a

N/A, not available; O/W, oil-in-water emulsion; SLN, solid lipid nanoparticles; PC, phosphatidylcholine; NLC, nanostructured lipid carriers; PUFA, polyunsaturated fatty acids.

Processing is an important factor when considering the suitability of PLs for applications. Recently, the effects of different processing conditions of rapeseed lecithin on the emulsifying and oxidative stability of fish oil emulsions were investigated. Both chemical and water degumming improved the emulsifying properties of rapeseed lecithin compared with soy lecithin. Processing enriched lecithin with LPC, especially in the case of chemical degumming, due to alkali-mediated hydrolysis of PC. The higher content of LPC in processed rapeseed lecithin was responsible for the better emulsion stability compared with soy lecithin, which was solely composed of PC. In addition, rapeseed lecithin presented improved free radical scavenging activity and inhibition of lipid oxidation in emulsions. Perhaps, synergistic interactions between PLs and phenolic compounds in rapeseed lecithin were responsible for this effect (Li et al., 2018). The use of single-component synthetic PLs in conferring physical and oxidative stability to fish oil-enriched emulsions was investigated by Anankanbil et al. (2018). By taking advantage of the surface-active properties of PLs and the well-known antioxidants properties of PLs,

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278

7.  Phospholipid-Based Surfactants

new functional molecules, called “phenophospholipids,” were synthesized. The new phenophospholipids exhibited potent antioxidant properties in vitro and in emulsified systems. Introduction of caffeic acid into PC did not sacrifice the antioxidant potential of caffeic acid nor the emulsifying activities of PC. On the contrary, improved surface activity was observed for phenophospholipids compared with soy/egg PC (Anankanbil et al., 2018). The uses of PLs as emulsifiers in delivery systems for drugs and cosmetic formulations have been widely investigated (Li et al., 2014; Singh et al., 2017). Table 7.7 highlights some of those studies. In drug applications, the composition of PLs influences not only the surface properties of lipid emulsions but also the distribution and fate of emulsions in the body. The behavior of emulsions stabilized by egg PC (EPC), dimyristoyl PC (DMPC), dioleoyl PC (DOPC), dipalmitoyl PC (DPPC), and 1-palmitoyl-2-oleoyl PC (POPC) in rat was studied (Lenzo et al., 1988). The measure of the removal from plasma of injected emulsion lipids was slowest for emulsions stabilized by DPPC, while metabolism of emulsions stabilized by EPC and POPC was similar to that of natural chylomicrons. In typical metabolism of PL fat emulsions, triacylglycerol (TAG) are first hydrolyzed by lipoprotein lipase. The remnants are taken up by the liver, while the PLs are transferred to high-density lipoproteins (HDL). However, in the case of the DPPC emulsions, there was impaired hydrolysis of TAG, and PLs were not transferred to HDL. Therefore, TAG were eliminated slowly from the plasma. Different metabolic profiles were also observed for emulsions stabilized by the other PLs (Lenzo et al., 1988). Other studies observed similar effects. Therefore, careful consideration of both composition and properties of PLs is vital when deciding on an application (Redgrave et al., 1992).

7.8  CONCLUDING REMARKS AND PERSPECTIVES The research and utilization of PLs are progressing along two parallel paths. The first is a mechanism study concerning identification of biosynthetic and metabolic pathways and study of interactions between protein and PL bilayers. Pure PLs are required. Advances in gene and protein structure analysis and modern analytic tools such as lipidomics and imaging techniques have benefited this area of research. Moreover, the research progress in the biochemistry of PL area also witnessed significant progress, which strengthens the scientific basis for the development of PL-related drugs and excipients for health application. The second path is for applications of natural PL resources for better nutrition and health. This path involves development of advanced processing/production technology and new products with additional functionalities. It is clear that developments along with the first path have contributed greatly to this application-related research and vice versa. Lecithins are regarded as truly “natural” ingredients, “without tangible properties or detectable change of the organism, it quietly and gently, like nature herself, still brings forth the most desirable effects in the organism” (Eibl, 1984). Furthermore, Kunze et al. (Eibl, 1984) have termed lecithin a true natural physic that “like sun, light, air, water, or earth always acts beneficially and in very many acute afflictions is indicated as an aid to the physician’s treatment.” Natural PLs are described in pharmacopeias and relevant regulatory guidance documentation of the Food and Drug Administration (FDA) and European Medicines Agency (EMA). The World Health Organization (WHO) places no limit on the oral intake of l­ ecithin. Also, no

III.  BIOBASED SURFACTANTS

REFERENCES 279

limit for the ADI value (acceptable daily intake) for lecithin as a food additive (WHO, 1974) is given. After parenteral administration, soybean and egg lecithin (unsaturated and saturated) are well tolerated. The European Commission declares that lecithin is a food additive (E322) “generally permitted for use in foodstuffs” (Commission Regulation (EU), 2015). Also, no ADI value has been fixed for lecithin in Europe; the material may be used “quantum satis” (Scientific Committee on Food (EC), 2002). The US FDA assigned the generally recognized as safe (GRAS) affirmation for lecithin (FDA, 2018a) and enzyme-modified lecithin (FDA, 2018b). The tolerability of natural PLs in formulations is further underscored by the user in innumerous products on the market. The acceptance of lethins admitted by the rules of the United States and the Europe clear up the barriers in commercial markets of lecithins and derived products in food, cosmetic, and pharmaceutical fields, which is also increasing the price of lecithins Moreover, in Europe, the soybean lecithin market is facing another problem. Widespread planting of genetically modified (GM) soybeans in the United States and Latin American countries is a concern to consumers. As a result, lecithins from sunflower and rapeseeds have received increasing interests. In addition to the use of natural lecithins and their fractionated high-purity individual phosphatidyl species, an active research area is to develop multifunctional PLs by incorporating other natural segments, such as phenolics, polyphenols, and sugar alcohol, to integrate or add new functionalities (Anankanbil et al., 2018; Song et al., 2012, 2013). Consequently, synergetic or additive effects can be empowered to expand the multifaceted applications in complicated food emulsion system or as novel surface-active excipients to deliver drugs and nutrients.

Acknowledgments Jingbo Li would like to thank the International Postdoctoral Grant from Independent Research Fund Denmark (7026-00039B) for financial support.

References Agassandian, M., Mallampalli, R.K., 2013. Surfactant phospholipid metabolism. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1831 (3), 612–625. Anankanbil, S., Pérez, B., Banerjee, C., Guo, Z., 2018. New phenophospholipids equipped with multi-functionalities: regiospecific synthesis and characterization. J. Colloid Interface Sci. 523, 169–178. Ariyama, H., Kono, N., Matsuda, S., Inoue, T., Arai, H., 2010. Decrease in membrane phospholipid unsaturation induces unfolded protein response. J. Biol. Chem. 285 (29), 22027–22035. Baeza-Jiménez, R., González-Rodríguez, J., Kim, I.-H., García, H.S., Otero, C., 2012. Use of immobilized phospholipase A1-catalyzed acidolysis for the production of structured phosphatidylcholine with an elevated conjugated linoleic acid content. Grasas Aceites 63 (1), 44–52. Balakrishna, M., Kaki, S.S., Karuna, M.S.L., Sarada, S., Kumar, C.G., Prasad, R.B.N., 2017. Synthesis and in vitro antioxidant and antimicrobial studies of novel structured phosphatidylcholines with phenolic acids. Food Chem. 221, 664–672. Benfield, A.P., Goodey, N.M., Phillips, L.T., Martin, S.F., 2007. Structural studies examining the substrate specificity profiles of PC-PLCBc protein variants. Arch. Biochem. Biophys. 460 (1), 41–47. Berton-Carabin, C.C., Elias, R.J., Coupland, J.N., 2013. Reactivity of a model lipophilic ingredient in surfactant-­ stabilized emulsions: effect of droplet surface charge and ingredient location. Colloids Surf. A Physicochem. Eng. Asp. 418, 68–75. Caceres, C.C., Bansal, P.S., Navarro, S., Wilson, D., Don, L., Giacomin, P., Loukas, A., et al., 2017. An engineered cyclic peptide alleviates symptoms of inflammation in a murine model of inflammatory bowel disease. J. Biol. Chem. 292 (24), 10288–10294.

III.  BIOBASED SURFACTANTS

280

7.  Phospholipid-Based Surfactants

Cansell, M., Nacka, F., Combe, N., 2003. Marine lipid-based liposomes increase in vivo FA bioavailability. Lipids 38 (5), 551–559. Casado, V., Reglero, G., Torres, C.F., 2013. Production and scale-up of phosphatidyl-tyrosol catalyzed by a food grade phospholipase D. Food Bioprod. Process. 91 (4), 599–608. Casado, V., Reglero, G., Torres, C.F., 2014. Novel and efficient solid to solid transphosphatidylation of two phenylalkanols in a biphasic GRAS medium. J. Mol. Catal. B Enzym. 99, 14–19. Cesarini, S., Haller, R., Diaz, P., Nielsen, P., 2014. Combining phospholipases and a liquid lipase for one-step biodiesel production using crude oils. Biotechnol. Biofuels 7 (1), 29. Chansri, N., Kawakami, S., Yamashita, F., Hashida, M., 2006. Inhibition of liver metastasis by all-trans retinoic acid incorporated into O/W emulsions in mice. Int. J. Pharm. 321 (1–2), 42–49. Chen, X., Pan, W., Sui, Y., Li, H., Shi, X., Guo, X., Qi, H., et al., 2015. Acidic phospholipids govern the enhanced activation of IgG-B cell receptor. Nat. Commun. 6, 1–14. Chen, W., Guo, W., Gao, F., Chen, L., Chen, S., Li, D., 2017. Phospholipase A1-catalysed synthesis of docosahexaenoic acid-enriched phosphatidylcholine in reverse micelles system. Appl. Biochem. Biotechnol. 182 (3), 1037–1052. Chojnacka, A., Gładkowski, W., Gliszczyńska, A., Niezgoda, N., Kiełbowicz, G., Wawrzeńczyk, C., 2016. Synthesis of structured phosphatidylcholine containing punicic acid by the lipase-catalyzed transesterification with pomegranate seed oil. Catal. Commun. 75, 60–64. Chung, C., Sher, A., Rousset, P., Decker, E.A., McClements, D.J., 2017. Formulation of food emulsions using natural emulsifiers: utilization of quillaja saponin and soy lecithin to fabricate liquid coffee whiteners. J. Food Eng. 209, 1–11. Cocco, L., Follo, M.Y., Manzoli, L., Suh, P.-G., 2015. Phosphoinositide-specific phospholipase C in health and disease. J. Lipid Res. 56 (10), 1853–1860. Commission Regulation (EU), 2015. No 231/2012 of 9 March 2012 Laying Down Specifications for Food Additives Listed in Annexes II and III to Regulation (EC) No 1333/2008 of the European Parliament and of the Council (Text with EEA Relevance). Available at: https://publications.europa.eu/en/publication-detail/-/publication/ a42dd9b2-b63f-438b-a790-1fa5995b7d41/language-en. (Accessed October 25, 2018). Cullis, P.R., Hope, M.J., Tilcock, C.P.S., 1986. Lipid polymorphism and the roles of lipids in membranes. Chem. Phys. Lipids 40 (2–4), 127–144. Damnjanović, J., Matsunaga, N., Adachi, M., Nakano, H., Iwasaki, Y., 2018. Facile enzymatic synthesis of phosphatidylthreonine using an engineered phospholipase D. Eur. J. Lipid Sci. Technol. 120 (6), 1800089. Dawson, R.M., 1958. Studies on the hydrolysis of lecithin by a penicillium notatum phospholipase B preparation. Biochem. J. 70 (4), 559–570. De Maria, L., Vind, J., Oxenbøll, K.M., Svendsen, A., Patkar, S., 2007. Phospholipases and their industrial applications. Appl. Microbiol. Biotechnol. 74 (2), 290–300. De Schrijver, R., Vermeulen, D., 1991. Separation and quantification of phospholipids in animal tissues by Iatroscan TLC/FID. Lipids 26 (1), 74–77. Devitt, A., Pierce, S., Oldreive, C., Shingler, W.H., Gregory, C.D., 2003. CD14-dependent clearance of apoptotic cells by human macrophages: the role of phosphatidylserine. Cell Death Differ. 10, 371. Devos, M., Poisson, L., Ergan, F., Pencreac’h, G., 2006. Enzymatic hydrolysis of phospholipids from Isochrysis galbana for docosahexaenoic acid enrichment. Enzym. Microb. Technol. 39 (4), 548–554. Dippe, M., Mrestani-Klaus, C., Schierhorn, A., Ulbrich-Hofmann, R., 2008. Phospholipase D-catalyzed synthesis of new phospholipids with polar head groups. Chem. Phys. Lipids 152 (2), 71–77. Dowhan, W., 2017. Understanding phospholipid function: why are there so many lipids? J. Biol. Chem. 292 (26), 10755–10766. Dubots, E., Botté, C., Boudière, L., Yamaryo-Botté, Y., Jouhet, J., Maréchal, E., Block, M.A., 2012. Role of phosphatidic acid in plant galactolipid synthesis. Biochimie 94 (1), 86–93. Duclos, R.I., 2010. The total synthesis of 2-O-arachidonoyl-1-O-stearoyl-sn-glycero-3-phosphocholine-1,3,1′-13C3 and −2,1′-13C2 by a novel chemoenzymatic method. Chem. Phys. Lipids 163 (1), 102–109. Duncan, R.E., Sarkadi-Nagy, E., Jaworski, K., Ahmadian, M., Sul, H.S., 2008. Identification and functional characterization of adipose-specific phospholipase a 2 (AdPLA). J. Biol. Chem. 283 (37), 25428–25436. Eibl, H., 1980. Synthesis of glycerophospholipids. Chem. Phys. Lipids 26 (4), 405–429. Eibl, H., 1984. Phospholipids as functional constituents of biomembranes. Angew. Chem. Int. Ed. Engl. 23 (4), 257–271. Fagone, P., Jackowski, S., 2009. Membrane phospholipid synthesis and endoplasmic reticulum function. J. Lipid Res. 50 (Suppl), S311–S316.

III.  BIOBASED SURFACTANTS

REFERENCES 281

FDA, 2018a. 21CFR184.1400 Lecithin. In: Title 21—Food and Drugs Chapter I—Food and Drug Administration. Department of Health and Human Services Subchapter B—Food for Human Consumption. Available at: https:// www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=184.1400. (Accessed October 25, 2018). FDA, 2018b. 21CFR184.1063. In: Title 21—Food and Drugs Chapter I—Food and Drug Administration Department of Health and Human Services Subchapter B—Food For Human Consumption (Continued). Available at: https:// www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=184.1063. (Accessed October 25, 2018). Fine, J.B., Sprecher, H., 1982. Unidimensional thin layer chromatography of phospholipids on boric acid impregnated plates. J. Lipid Res. 23, 660–663. Ganta, S., Paxton, J.W., Baguley, B.C., Garg, S., 2008. Pharmacokinetics and pharmacodynamics of chlorambucil delivered in parenteral emulsion. Int. J. Pharm. 360 (1–2), 115–121. Ghosh, M., Bhattacharyya, D.K., 1997. Enzymatic alcoholysis reaction of soy phospholipids. J. Am. Oil Chem. Soc. 74 (5), 597–599. Gibellini, F., Smith, T.K., 2010. The Kennedy pathway—de novo synthesis of phosphatidylethanolamine and phosphatidylcholine. IUBMB Life 62 (6), 414–428. Golczak, M., Sears, A.E., Kiser, P.D., Palczewski, K., 2015. LRAT-specific domain facilitates vitamin a metabolism by domain swapping in HRASLS3. Nat. Chem. Biol. 11 (1), 26–32. Griffith, O.H., Ryan, M., 1999. Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1441 (2–3), 237–254. Guo, Z., Vikbjerg, A.F., Xu, X., 2005. Enzymatic modification of phospholipids for functional applications and human nutrition. Biotechnol. Adv. 23 (3), 203–259. Gupta, C.M., Radhakrishnan, R., Khorana, H.G., 1977. Glycerophospholipid synthesis: improved general method and new analogs containing photoactivable groups. Proc. Natl. Acad. Sci. U. S. A. 74 (10), 4315–4319. Hama, S., Yoshida, A., Nakashima, K., Noda, H., Fukuda, H., Kondo, A., 2010. Surfactant-modified yeast whole-cell biocatalyst displaying lipase on cell surface for enzymatic production of structured lipids in organic media. Appl. Microbiol. Biotechnol. 87 (2), 537–543. Hama, S., Miura, K., Yoshida, A., Noda, H., Fukuda, H., Kondo, A., 2011. Transesterification of phosphatidylcholine in sn-1 position through direct use of lipase-producing Rhizopus oryzae cells as whole-cell biocatalyst. Appl. Microbiol. Biotechnol. 90 (5), 1731–1738. Hama, S., Ogino, C., Kondo, A., 2015. Enzymatic synthesis and modification of structured phospholipids: recent advances in enzyme preparation and biocatalytic processes. Appl. Microbiol. Biotechnol. 99 (19), 7879–7891. Hapala, I., Butko, P., Schroeder, F., 1990. Role of acidic phospholipids in intermembrane sterol transfer. Chem. Phys. Lipids 56 (1), 37–47. Hong, S.I., Kim, Y., Kim, C.T., Kim, I.H., 2011. Enzymatic synthesis of lysophosphatidylcholine containing CLA from sn-glycero-3-phosphatidylcholine (GPC) under vacuum. Food Chem. 129 (1), 1–6. Hossen, M., Hernandez, E., 2005. Enzyme-catalyzed synthesis of structured phospholipids with conjugated linoleic acid. Eur. J. Lipid Sci. Technol. 107 (10), 730–736. Huang, S., Liang, M., Xu, Y., Rasool, A., Li, C., 2014. Characteristics and vegetable oils degumming of recombinant phospholipase B. Chem. Eng. J. 237, 23–28. Ichihara, K., Iwasaki, H., Ueda, K., Takizawa, R., Naito, H., Tomosugi, M., 2005. Synthesis of phosphatidylcholine: an improved method without using the cadmium chloride complex of sn-glycero-3-phosphocholine. Chem. Phys. Lipids 137 (1–2), 94–99. Inoue, A., Adachi, M., Damnjanović, J., Nakano, H., Iwasaki, Y., 2016. Direct enzymatic synthesis of 1-Phosphatidylβ-d-glucose by engineered phospholipase D. Chem. Select 1 (13), 4121–4125. Iwasaki, Y., Yamane, T., 2004. Enzymatic synthesis of structured lipids. Adv. Biochem. Eng. Biotechnol. 90, 151–171. Jenkins, G.M., Frohman, M.A., 2005. Phospholipase D: a lipid centric review. Cell. Mol. Life Sci. 62 (19–20), 2305–2316. Kahovcova, J., Odavic, R., 1969. A simple method for the quantitative analysis of phospholipids separated by thin layer chromatography. J. Chromatogr. Sci. 40, 90–96. Kennedy, E.P., Weiss, S.B., 1956. The function of cytidine coenzymes in the biosynthesis of phospholipids. J. Biol. Chem. 222, 193–214. Kent, C., 1995. Eukaryotic phospholipid biosynthesis. Annu. Rev. Biochem. 64 (1), 315–343. Khan, F.I., Lan, D.M., Durrani, R., Huan, W.Q., Zhao, Z.X., Wang, Y.H., 2017. The lid domain in lipases: structural and functional determinant of enzymatic properties. Front. Bioeng. Biotechnol. 5. Article 16.

III.  BIOBASED SURFACTANTS

282

7.  Phospholipid-Based Surfactants

Kheradmandnia, S., Vasheghani-Farahani, E., Nosrati, M., Atyabi, F., 2010. Preparation and characterization of ­ketoprofen-loaded solid lipid nanoparticles made from beeswax and carnauba wax. Nanomedicine 6 (6), 753–759. Kiełbowicz, G., Gładkowski, W., Chojnacka, A., Wawrzeńczyk, C., 2012. A simple method for positional analysis of phosphatidylcholine. Food Chem. 135 (4), 2542–2548. Kim, J., Kim, B.-G., 2000. Lipase-catalyzed synthesis of lysophosphatidylcholine using organic cosolvent for in situ water activity control. J. Am. Oil Chem. Soc. 77 (7), 791–797. King, J.W., Holliday, R.L., List, G.R., Snyder, J.M., 2001. Hydrogenation of vegetable oils using mixtures of supercritical carbon dioxide and hydrogen. J. Am. Oil Chem. Soc. 78 (2), 107–113. Koga, T., Terao, J., 1994. Antioxidant activity of a novel phosphatidyl derivative of vitamin-E in lard and its model system. J. Agric. Food Chem. 42 (6), 1291–1294. Koketsu, M., 1997. Synthesis of a novel sialic acid derivative (sialylphospholipid) as an antirotaviral agent. J. Med. Chem. 40 (21), 3332–3335. Komaiko, J., Sastrosubroto, A., McClements, D.J., 2015. Formation of oil-in-water emulsions from natural emulsifiers using spontaneous emulsification: sunflower phospholipids. J. Agric. Food Chem. 63 (45), 10078–10088. Leiros, I., Secundo, F., Zambonelli, C., Servi, S., Hough, E., 2000. The first crystal structure of a phospholipase D. Structure 8 (6), 655–667. Leiros, I., McSweeney, S., Hough, E., 2004. The reaction mechanism of phospholipase D from Streptomyces sp. strain PMF. Snapshots along the reaction pathway reveal a pentacoordinate reaction intermediate and an unexpected final product. J. Mol. Biol. 339 (4), 805–820. Lentz, B.R., 2003. Exposure of platelet membrane phosphatidylserine regulates blood coagulation. Prog. Lipid Res. 42 (5), 423–438. Lenzo, N.P., Martins, I., Mortimer, B.-C., Redgrave, T.G., 1988. Effects of phospholipid composition on the metabolism of triacylglycerol, cholesteryl ester and phosphatidylcholine from lipid emulsions injected intravenously in rats. Biochim. Biophys. Acta Lipids Lipid Metab. 960 (1), 111–118. Leong, W.F., Berton-Carabin, C.C., Elias, R.J., Lecomte, J., Villeneuve, P., Zhao, Y., Coupland, J.N., 2015. Effect of lipophilization on the distribution and reactivity of ingredients in emulsions. J. Colloid Interface Sci. 459, 36–43. Li, J., Guo, Z., 2016. Identification and quantification of phenolic compounds in rapeseed originated lecithin and antioxidant activity evaluation. LWT Food Sci. Technol. 73, 397–405. Li, M., Hong, Y., Wang, X., 2009. Phospholipase D- and phosphatidic acid-mediated signaling in plants. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1791 (9), 927–935. Li, J., Wang, X., Zhang, T., Wang, C., Huang, Z., Luo, X., Deng, Y., 2014. A review on phospholipids and their main applications in drug delivery systems. Asian J. Pharmaceut. Sci. 10 (2), 81–98. Li, D., Qin, X., Wang, W., Li, Z., Yang, B., Wang, Y., 2016. Synthesis of DHA/EPA-rich phosphatidylcholine by immobilized phospholipase A1: effect of water addition and vacuum condition. Bioprocess Biosyst. Eng. 39 (8), 1305–1314. Li, J., Vosegaard, T., Guo, Z., 2017. Applications of nuclear magnetic resonance in lipid analyses: an emerging powerful tool for lipidomics studies. Prog. Lipid Res. 68, 37–56. Li, J., Pedersen, J.N., Anankanbil, S., Guo, Z., 2018. Enhanced fish oil-in-water emulsions enabled by rapeseed lecithins obtained under different processing conditions. Food Chem. 264, 233–240. Liang, L., Chen, F., Wang, X., Jin, Q., Decker, E.A., McClements, D.J., 2017. Physical and oxidative stability of flaxseed oil-in-water emulsions fabricated from sunflower lecithins: impact of blending Lecithins with different phospholipid profiles. J. Agric. Food Chem. 65 (23), 4755–4765. Lindberg, J., Ekeroth, J., Konradsson, P., 2002. Efficient synthesis of phospholipids from glycidyl phosphates. J. Org. Chem. 67 (1), 194–199. Liu, J.J., Green, P., John Mann, J., Rapoport, S.I., Sublette, M.E., 2015. Pathways of polyunsaturated fatty acid utilization: implications for brain function in neuropsychiatric health and disease. Brain Res. 1597, 220–246. Liu, Y., Zhang, Q., Guo, Y., Liu, J., Xu, J., Li, Z., Wang, J., et al., 2017. Enzymatic synthesis of lysophosphatidylcholine with n−3 polyunsaturated fatty acid from sn-glycero-3-phosphatidylcholine in a solvent-free system. Food Chem. 226, 165–170. Liu, H., Liu, T., Fan, H., Gou, M., Li, G., Ren, H., Wang, D., et al., 2018. Corn lecithin for injection from deoiled corn germ: extraction, composition, and emulsifying properties. Eur. J. Lipid Sci. Technol. 120 (3), 1–11. Lv, Q., Yu, A., Xi, Y., Li, H., Song, Z., Cui, J., Cao, F., et al., 2009. Development and evaluation of penciclovir-loaded solid lipid nanoparticles for topical delivery. Int. J. Pharm. 372 (1–2), 191–198.

III.  BIOBASED SURFACTANTS

REFERENCES 283

Lykidis, A., 2007. Comparative genomics and evolution of eukaryotic phospholipid biosynthesis. Prog. Lipid Res. 46 (3–4), 171–199. Ma, W.C., Zhang, Q., Li, H., Larregieu, C.A., Zhang, N., Chu, T., Jin, H., et al., 2013. Development of intravenous lipid emulsion of α-asarone with significantly improved safety and enhanced efficacy. Int. J. Pharm. 450 (1–2), 21–30. Makise, M., Mima, S., Katsu, T., Tsuchiya, T., Mizushima, T., 2002. Acidic phospholipids inhibit the DNA-binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli. Mol. Microbiol. 46 (1), 245–256. Malz, F., Jancke, H., 2005. Validation of quantitative NMR. J. Pharm. Biomed. Anal. 38 (5), 813–823. Marrapu, B., Mallampalli, L.K., Kaki, S.S., Rachapudi, B.N.P., 2015. A novel method to synthesize 1-acyl-sn-­glycero-3phosphocholine and 1,2-diacyl-sn-glycero-3-phosphocholine. Eur. J. Lipid Sci. Technol. 117 (7), 1049–1055. Martin, D., Garcia-Serrano, A., Casado, V., Vázquez, L., Reglero, G., Torres, C.F., 2014. Antioxidant activity of phosphatidyl derivatives of hydroxytyrosol in edible oils. Eur. J. Lipid Sci. Technol. 116 (8), 1035–1043. Meng, X., Ye, Q., Pan, Q., Ding, Y., Wei, M., Liu, Y., van de Voort, F.R., 2014. Total phospholipids in edible oils by invial solvent extraction coupled with FTIR analysis. J. Agric. Food Chem. 62 (14), 3101–3107. Merolli, A., Santin, M., 2009. Role of Phosphatidyl-serine in bone repair and its technological exploitation. Molecules 14 (12), 5367–5381. Mezdour, S., Desplanques, S., Relkin, P., 2011. Effects of residual phospholipids on surface properties of a soft-refined sunflower oil: application to stabilization of sauce-types’ emulsions. Food Hydrocoll. 25 (4), 613–619. Murakami, K., Molitor, E.J., Liu, H.W., 1999. An efficient synthesis of unsymmetrical optically active phosphatidyl glycerol. J. Org. Chem. 64 (2), 648–651. Murayama, K., Kano, K., Matsumoto, Y., Sugimori, D., 2013. Crystal structure of phospholipase A1from Streptomyces albidoflavus NA297. J. Struct. Biol. 182 (2), 192–196. Mustranta, A., Forssell, P., Poutanen, K., 1995. Comparison of lipases and phospholipases in the hydrolysis of phospholipids. Process Biochem. 30 (5), 393–401. Nagao, A., Ishida, N., Terao, J., 1991. Synthesis of 6-phosphatidyi-l-ascorbic acid by phospholipase D. Lipids 26 (5), 390–394. Nelson, R.K., Frohman, M.A., 2015. Physiological and pathophysiological roles for phospholipase D. J. Lipid Res. 56 (12), 2229–2237. Niezgoda, N., Gliszczyńska, A., Gładkowski, W., Chojnacka, A., Kiełbowicz, G., Wawrzeńczyk, C., 2016. Lipasecatalyzed enrichment of egg yolk phosphatidylcholine with conjugated linoleic acid. J. Mol. Catal. B Enzym. 123, 14–22. Nzai, J., Proctor, A., 1998. Determination of phospholipids in vegetable oil by Fourier transform infrared spectroscopy. J. Am. Oil Chem. Soc. 75 (10), 1281–1289. Ochoa-Flores, A.A., Hernández-Becerra, J.A., Cavazos-Garduño, A., Vernon-Carter, E.J., García, H.S., 2017. Optimization of the synthesis of structured phosphatidylcholine with medium chain fatty acid. J. Oleo Sci. 66 (11), 1207–1215. Pang, X.-Y., Cao, J., Addington, L., Lovell, S., Battaile, K.P., Zhang, N., Rao, J.L.U.M., et al., 2012. Structure/function relationships of adipose phospholipase A2 containing a Cys-his-his catalytic triad. J. Biol. Chem. 287 (42), 35260–35274. Pardun, H., 1970. Phosphatides and their method for their preparation. US patent US3505074A. Pérez, B., Li, J., Guo, Z., 2017. Chemistry and properties of lipids and phospholipids. Food Lipids Chem. Nutr. Biotechnol., 37–72. Perumal, P., Raina, R., Arockiasamy, A., SundaraBaalaji, N., 2016. Crystal structure of Salmonella Typhi—OMPLA with Inherent Bound Cofactor. Available at: https://www.rcsb.org/structure/5DQX. (Accessed October 25, 2018). Podo, F., Paris, L., Cecchetti, S., Spadaro, F., Abalsamo, L., Ramoni, C., Ricci, A., et  al., 2016. Activation of ­phosphatidylcholine-specific phospholipase C in breast and ovarian cancer: impact on MRS-detected choline metabolic profile and perspectives for targeted therapy. Front. Oncol. 6. Available at: https://doi.org/10.3389/ fonc.2016.00171. (Accessed October 25, 2018). Pucek, A., Niezgoda, N., Kulbacka, J., Wawrzeńczyk, C., Wilk, K.A., 2017. Phosphatidylcholine with conjugated linoleic acid in fabrication of novel lipid nanocarriers. Colloids Surf. A Physicochem. Eng. Asp. 532, 377–388. Rebecchi, M.J., Pentyala, S.N., 2000. Structure, function, and control of phosphoinositide-specific phospholipase C. Physiol. Rev. 80 (4), 1291–1335. Redgrave, T.G., Rakic, V., Mortimer, B.C., Mamo, J.C.L., 1992. Effects of sphingomyelin and phosphatidylcholine acyl chains on the clearance of triacylglycerol-rich lipoproteins from plasma. Studies with lipid emulsions in rats. Biochim. Biophys. Acta Lipids Lipid Metab. 1126 (1), 65–72.

III.  BIOBASED SURFACTANTS

284

7.  Phospholipid-Based Surfactants

Restuccia, D., Spizzirri, U.G., Puoci, F., Cirillo, G., Vinci, G., Picci, N., 2012. Determination of phospholipids in food samples. Food Rev. Intl. 28 (1), 1–46. Richmond, G.S., Smith, T.K., 2007. The role and characterization of phospholipase A1 in mediating lysophosphatidylcholine synthesis in Trypanosoma brucei. Biochem. J. 405 (2), 319–329. Rios, L., Echeverri, D., Cardeño, F., 2013. Hydroxylation of vegetable oils using acidic resins as catalysts. Ind. Crop. Prod. 43 (1), 183–187. Rosseto, R., Bibak, N., Hajdu, J., 2006. A new approach to phospholipid synthesis using tetrahydropyranyl glycerol: rapid access to phosphatidic acid and phosphatidylcholine, including mixed-chain glycerophospholipid derivatives. Org. Biomol. Chem. 4 (12), 2358–2360. Roussel, A., Amara, S., Nyyssölä, A., Mateos-Diaz, E., Blangy, S., Kontkanen, H., Westerholm-Parvinen, A., et al., 2014. A cutinase from Trichoderma reesei with a lid-covered active site and kinetic properties of true lipases. J. Mol. Biol. 426 (22), 3757–3772. Ruxton, C.H.S., Reed, S.C., Simpson, M.J.A., Millington, K.J., 2007. The health benefits of omega-3 polyunsaturated fatty acids: a review of the evidence. Adv. Clin. Insights Bioceut. 20, 275–285. Rychlicka, M., Niezgoda, N., Gliszczyńska, A., 2018. Lipase-catalyzed acidolysis of egg-yolk phosphatidylcholine with citronellic acid. New insight into synthesis of isoprenoid-phospholipids. Molecules 23 (2), 314. Samdani, G.K., McClements, D.J., Decker, E.A., 2018. Impact of phospholipids and tocopherols on the oxidative stability of soybean oil-in-water emulsions. J. Agric. Food Chem. 66 (15), 3939–3948. Sano, S., Sumiyoshi, H., Handa, A., Tokizane, R., Nakao, M., 2015. A novel synthetic approach to glycerophospholipids via Horner-Wadsworth-Emmons reaction of mixed phosphonoacetate. Tetrahedron Lett. 56 (32), 4686–4688. Sarney, D.B., Fregapane, G., Vulfson, E.N., 1994. Lipase-catalyzed synthesis of lysophospholipids in a continuous bioreactor. J. Am. Oil Chem. Soc. 71 (1), 93–96. Scientific Committee on Food (EC), 2002. Opinion the Scientific Committee on Food on Impurities of 1,4-Dioxane, 2-Chloroethanol and Mono- and Diethylene Glycol in Currently Permitted Food Additives and in Proposed Use of Ethyl Hydroxyethyl Cellulose in Gluten-Free Bread. SCF/CS/ADD/EMU/198 Final. https://ec.europa.eu/ food/sites/food/files/safety/docs/sci-com_scf_out156_en.pdf. (Accessed October 25, 2018). Servi, S., 1999. Phospholipases as synthetic catalysts. Biocatal. Discov. Appl. Top. Curr. Chem., 127–158. Shi, S., Chen, H., Lin, X., Tang, X., 2010. Pharmacokinetics, tissue distribution and safety of cinnarizine delivered in lipid emulsion. Int. J. Pharm. 383 (1–2), 264–270. Shibuya, I., 1992. Metabolic regulations and biological functions of phospholipids in Escherichia coli. Prog. Lipid Res. 31 (3), 245–299. Singer, A.G., Ghomashchi, F., Le Calvez, C., Bollinger, J., Bezzine, S., Rouault, M., Sadilek, M., et al., 2002. Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2. J. Biol. Chem. 277 (50), 48535–48549. Singh, A., 1990. An efficient synthesis of phosphatidylcholines. J. Lipid Res. 31 (8), 1522–1525. Singh, R.P., Gangadharappa, H.V., Mruthunjaya, K., 2017. Phospholipids: unique carriers for drug delivery systems. J. Drug Deliv. Sci. Technol. 39, 166–179. Song, S., Cheong, L.Z., Guo, Z., Kristensen, K., Glasius, M., Jensen, H.M., Bertelsen, K., et al., 2012. Phospholipase D (PLD) catalyzed synthesis of phosphatidyl-glucose in biphasic reaction system. Food Chem. 135 (2), 373–379. Song, S., Cheong, L.Z., Falkeborg, M., Liu, L., Dong, M., Jensen, H.M., Bertelsen, K., et al., 2013. Facile synthesis of phosphatidyl saccharides for preparation of anionic nanoliposomes with enhanced stability. PLoS One 8 (9), 1–11. Sonoda, H., Aoki, J., Hiramatsu, T., Ishida, M., Bandoh, K., Nagai, Y., Taguchi, R., et al., 2002. A novel phosphatidic acid-selective phospholipase a 1 that produces Lysophosphatidic acid. J. Biol. Chem. 277 (37), 34254–34263. Stafforini, D.M., McIntyre, T.M., Zimmerman, G.A., Prescott, S.M., 1997. Platelet-activating factor acetylhydrolases. J. Biol. Chem. 272 (29), 17895–17898. Stamatis, H., Sereti, V., Kolisis, F.N., 1999. Studies on the enzymatic synthesis of lipophilic derivatives of natural antioxidants. J. Am. Oil Chem. Soc. 76 (12), 1505–1510. Szuhaj, B.F., 1983. Lecithin production and utilization. J. Am. Oil Chem. Soc. 60 (2), 306–309. Takami, M., Suzuki, Y., 1994. Enzymatic synthesis of novel phosphatidylgenipin, and its enhanced cytotoxity. Biosci. Biotechnol. Biochem. 58 (10), 1897–1898. Takami, M., Hidaka, N., Miki, S., Suzuki, Y., 1994. Enzymatic synthesis of novel phosphatidylkojic acid and phosphatidylarbutin, and their inhibitory effects on tyrosinase activity. Biosci. Biotechnol. Biochem. 58 (9), 1716–1717.

III.  BIOBASED SURFACTANTS

REFERENCES 285

Tan, C., Che Man, Y., 2002. Differential scanning calorimetric analysis of palm oil, palm oil based products and coconut oil: effects of scanning rate variation. Food Chem. 76 (1), 89–102. Tiesinga, J.J.W., van Pouderoyen, G., Nardini, M., Ransac, S., Dijkstra, B.W., 2007. Structural basis of phospholipase activity of staphylococcus hyicus lipase. J. Mol. Biol. 371 (2), 447–456. Touchstone, J.C., Chen, J.C., Beaver, K.M., 1980. Improved separation of phospholipids in thin layer chromatography. Lipids 15 (1), 61–62. van Hoogevest, P., Wendel, A., 2014. The use of natural and synthetic phospholipids as pharmaceutical excipients. Eur. J. Lipid Sci. Technol. 116 (9), 1088–1107. Van Nieuwenhuyzen, W., 1976. Lecithin production and properties. J. Am. Oil Chem. Soc. 53 (6), 425–427. Van Nieuwenhuyzen, W., Tomás, M.C., 2008. Update on vegetable lecithin and phospholipid technologies. Eur. J. Lipid Sci. Technol. 110 (5), 472–486. Vance, J.E., Vance, D.E., 2004. Phospholipid biosynthesis in mammalian cells. Biochem. Cell Biol. 82 (1), 113–128. Vance, J.E., Vance, D.E., 2008. In: Vance, J.E., Vance, D.E. (Eds.), Biochemistry of Lipids, Lipoproteins and Membranes. fifth ed. Elsevier Science, pp. 213–244. Vikbjerg, A.F., Mu, H., Xu, X., 2005. Parameters affecting incorporation and by-product formation during the production of structured phospholipids by lipase-catalyzed acidolysis in solvent-free system. J. Mol. Catal. B Enzym. 36 (1–6), 14–21. Vikbjerg, A.F., Rusig, J.Y., Jonsson, G., Mu, H., Xu, X., 2006. Strategies for lipase-catalyzed production and the purification of structured phospholipids. Eur. J. Lipid Sci. Technol. 108 (10), 802–811. Vikbjerg, A.F., Mu, H., Xu, X., 2007. Synthesis of structured phospholipids by immobilized phospholipase A2 catalyzed acidolysis. J. Biotechnol. 128 (3), 545–554. Virto, C., Adlercreutz, P., 2000. Two-enzyme system for the synthesis of 1-lauroyl-rac-glycerophosphate (lysophosphatidic acid) and 1-lauroyl-dihydroxyacetonephosphate. Chem. Phys. Lipids 104 (2), 175–184. Wang, Y., Zhao, M., Song, K., Wang, L., Tang, S., Riley, W.W., 2010. Partial hydrolysis of soybean oil by phospholipase A1 (lecitase ultra). Food Chem. 121 (4), 1066–1072. Wang, M., Pinnamaraju, S., Ranganathan, R., Hajdu, J., 2013. Synthesis of mixed-chain phosphatidylcholines including coumarin fluorophores for FRET-based kinetic studies of phospholipase A2 enzymes. Chem. Phys. Lipids 172–173, 78–85. Wei, L., Marasini, N., Li, G., Yong, C.S., Kim, J.O., Quan, Q., 2012. Development of ligustrazine-loaded lipid emulsion: formulation optimization, characterization and biodistribution. Int. J. Pharm. 437 (1–2), 203–212. Wei, W., Feng, F., Perez, B., Dong, M., Guo, Z., 2015. Biocatalytic synthesis of ultra-long-chain fatty acid sugar alcohol monoesters. Green Chem. 17 (6), 3475–3489. WHO, 1974. Toxicological Evaluation of Some Food Additives Including Anticaking Agents, Antimicrobials, Antioxidants, Emulsifiers and Thickening Agents. WHO Food Additives Series No. 5. Available at: http://www. inchem.org/documents/jecfa/jecmono/v05je42.htm. (Accessed October 25, 2018). Yamamoto, Y., Hosokawa, M., Kurihara, H., Miyashita, K., 2008. Preparation of phosphatidylated terpenes via phospholipase D-mediated transphosphatidylation. J. Am. Oil Chem. Soc. 85 (4), 313–320. Yamamoto, Y., Kurihara, H., Miyashita, K., Hosokawa, M., 2011. Synthesis of novel phospholipids that bind phenylalkanols and hydroquinone via phospholipase D-catalyzed transphosphatidylation. New Biotechnol. 28 (1), 1–6. Yang, R., Zhang, X., Li, F., Ding, L., Li, B., Sun, H., Gan, Y., 2013. Role of phospholipids and copolymers in enhancing stability and controlling degradation of intravenous lipid emulsions. Colloids Surf. A Physicochem. Eng. Asp. 436, 434–442. Yang, G., Yang, R., Hu, J., 2015. Lysophosphatidylcholine synthesis by lipase-catalyzed ethanolysis. J. Oleo Sci. 64 (4), 443–447. Yucel, U., Elias, R.J., Coupland, J.N., 2012. Solute distribution and stability in emulsion-based delivery systems: an EPR study. J. Colloid Interface Sci. 377 (1), 105–113. Zhang, L., Liang, S., Hellgren, L., Jonsson, G., Xu, X., 2008. Phospholipase C-catalyzed sphingomyelin hydrolysis in a membrane reactor for ceramide production. J. Membr. Sci. 325 (2), 895–902. Zhang, X., Li, B., Wang, J., Li, H., Zhao, B., 2017. High-yield and sustainable production of phosphatidylserine in purely aqueous solutions via adsorption of phosphatidylcholine on triton-X-100-modified silica. J. Agric. Food Chem. 65 (49), 10767–10774. Zhao, T., No, D.S., Kim, B.H., Garcia, H.S., Kim, Y., Kim, I.H., 2014. Immobilized phospholipase A1-catalyzed modification of phosphatidylcholine with n-3 polyunsaturated fatty acid. Food Chem. 157, 132–140.

III.  BIOBASED SURFACTANTS

286

7.  Phospholipid-Based Surfactants

Further Reading Avalli, A., Contarini, G., 2005. Determination of phospholipids in dairy products by SPE/HPLC/ELSD. J. Chromatogr. A 1071, 185–190. Helmerich, G., Koehler, P., 2003. Comparison of methods for the quantitative determination of phospholipids in lecithins and flour improvers. J. Agric. Food Chem. 51 (23), 6645–6651. Kaffarni, S., Ehlers, I., Gröbner, G., Schleucher, J., Vetter, W., 2013. Two-dimensional 31P, 1H NMR spectroscopic profiling of phospholipids in cheese and fish. J. Agric. Food Chem. 61, 7061–7069.

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C H A P T E R

8 Fatty Acid, Methyl Ester, and Vegetable Oil Ethoxylates George A. Smith Sasol Performance Chemicals North America LLC, Westlake, LA, United States

8.1 INTRODUCTION Surfactants based on fats and oils have been used since antiquity. The first and oldest biosurfactant is soap. Soap is the neutralized fatty acids obtained from saponification of fats and oils with caustic. One of the first written references to soap dates back to 2500 BC (Spitz, 2009). Soap was used to wash woolen garments in ancient Mesopotamia. Sumerian clay tablets from the time describe the preparation of soap from water, alkali, and palm oil. Soap was the main surfactant used for personal hygiene and washing of clothes up to the mid-20th century because of good detergency and emulsification properties. Soap is relatively easy to manufacture and uses low-cost biobased raw materials. With the advent of modern industrial chemistry, the reliance on soap has declined. Soap is most effective under alkaline conditions. In personal cleaning products, the high pH of soap can irritate the skin and eyes. Soap also suffers from sensitivity to salinity and hard water ions. In the presence of electrolytes, the detergency and foaming properties of soap are greatly diminished. In the presence of hard water ions, soap forms an insoluble precipitate that is difficult to remove after washing. A number of different surfactants based on fats and oils have been developed to replace soap. Foam stabilizers are prepared by reacting vegetable oil with an alkanolamine. Cocamide diethanolamine is produced by reacting coconut oil with diethanolamine (DEA). Cocamidopropyl betaine is produced by reacting coconut oil with dimethylaminopropylamine to obtain cocamidopropyl amide (CAPA) followed by quaternization with sodium chloroacetate. Cocamidopropyl amine oxide is made by oxidizing CAPA with hydrogen peroxide in aqueous solution. All of these are popular cosurfactants used to build viscosity and stabilize foam. Several different types of nonionic surfactants based on direct ethoxylation or esterification of fats and oils with ethylene oxide condensates are commercially available. While not

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Copyright © 2019 AOCS Press. Published by Elsevier Inc. All rights reserved.

288

8.  Fatty Acid, Methyl Ester, and Vegetable Oil Ethoxylates

being fully biobased, these materials contain a high renewable carbon content and have some useful and interesting properties in different applications.

8.2  RAW MATERIALS Fats and oils are a complex mixture of different lipids that come from plant and animal sources. Triglyceride is the main component in fats and oils, composed of three parts fatty acid and one part glycerin as shown in Fig.  8.1. Although often used interchangeably, the term “fat” refers to triglycerides that are solid at room temperature, whereas “oil” refers to materials that are liquid. Depending on the source of the triglyceride, the R group represents a long-chain hydrocarbon that can be saturated or unsaturated. Because of my vegetarian leanings, this review will deal primarily with fats and oil from plant sources. The alkyl chain distribution of a number of common vegetable oils is shown in Table 8.1 (Lusas, 2007). Coconut and palm kernel oil (PKO) are unique in that they contain a large proportion of C12 fatty acids and low levels of unsaturation. Coconut and PKO are the main feedstock used to make oleoalcohols for the detergent industry. They are grown primarily in Indonesia, Malaysia, and the Philippines. Other oils like soybean and canola oil are rich in C18 fatty acids with much higher levels of unsaturation. Both oils are grown in North America. The majority of the soybean oil comes from seeds that are genetically modified to be resistant to glyphosate herbicide. Castor oil is different than other oils. Castor oil is rich in ricinoleic acid, which has a hydroxyl group at the 12‑carbon position. Castor oil comes from India and China and is widely used to make emulsifiers for paints and coating, cosmetic, and agricultural applications. Regardless of the starting material, fats and oils need to be processed before use. For seed oils, the seeds are first cleaned and the hull removed before cooking at elevated temperature to rupture the cell walls. The crude oil in then extracted by passing through a screw press under high pressure or solvent extraction with hexane. The crude oil is further refined to remove undesirable impurities. Food grade oils are typically refined, bleached, and deodorized (RBD) before use (Gunstone, 2004). Refining consists of degumming to remove phospholipids and trace metals and neutralization to remove free fatty acids. Bleaching is used to reduce color by heating the oil under an inert atmosphere with a bleaching earth like bentonite to

FIG. 8.1  Structure of triglyceride. R refers to a fatty alkyl group.

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TABLE 8.1  Carbon Chain Distribution of Common Vegetable Oils Chain Length

Coconut

Palm Kernel Palm

Soybean

C6

1

1

C8

8

4

C10

7

4

C12

48

48

C14

18

16

1

1

C16

8

8

57

12

Corn

Peanut

Olive

Castor

4

11

7

12

2

2

1

1

C16 = 1 C18

2

2

5

4

2

2

2

2

C18 = 1

6

15

30

25

62

28

48

70

C18-OH C18 = 2

6 88

2

2

7

C18 = 3

51

22

58

32

13

7

10

1

11

1

3

100

100

100

100

100

100

100

100

100

Saponification number, mg KOH/g

250–264

245–255

195–205

189–195

196–204

187–193

188–195

188–196

176–187

Iodine value, g I2/100 g

7.5–10.5

14–23

44–54

120–141

105–125

103–128

84–100

80–88

81–91

Average carbon number

12.8

13.5

16.8

17.7

17.9

17.8

17.9

17.6

17.9

Average fatty acid MW, g/mol

211

221

267

277

280

278

279

277

324

Average triglyceride MW, g/mol

692

722

860

891

901

894

898

890

1032

289

Total

8.2  Raw Materials

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Canola

290

8.  Fatty Acid, Methyl Ester, and Vegetable Oil Ethoxylates

Screw press Oil seeds

Crude oil

Filtering degumming

Solvent extraction

Methyl esters

Bleaching deodorization Transesterification Glycerin

Fatty acids

RBD oil

Fat splitting

FIG. 8.2  Oleochemical process.

decompose hydroperoxides. Deodorization is used to improve odor and extend the shelf life by sparging with steam at elevated temperature and reduced pressure. A schematic of the process to make RBD vegetable oil is shown in Fig. 8.2. Fatty acids are produced by fat splitting at elevated temperature and pressure. Fat splitting can be done in batch mode or continuously. In the batch mode, fats and oils are hydrolyzed with water at elevated temperature (290–320°C). The reaction is believed to occur in the oil phase as the solubility of water in the triglyceride far exceeds the solubility of the oil in water (Mills and McCain, 1949). In the continuous mode, high temperature (250°C) and pressure (4.9–5.3 MPa) are used. Fat and water are mixed in a countercurrent fashion. The fatty acid and sweet water containing glycerin are removed at opposite ends of the reactor (Itiner, 1938). Ethylene oxide is made on an industrial scale by oxidizing ethylene with air or pure oxygen over a silver catalyst (Lefort, 1935). The process was first commercialized by Union Carbide who built the first production plant in 1937. The reaction is typically run at 230–280°C in a shell-and-tube reactor and is highly exothermic. The heat is removed by circulating water through the shell of the reactor to make steam. The typical conversion of ethylene to ethylene oxide is around 20%. The unreacted ethylene is recycled in a continuous fashion, and the ethylene oxide is purified by distilled to remove aldehydes (Satterfield, 1991). Ethylene oxide is used to make ethylene glycol (EG) for antifreeze and polyester fiber and used to manufacture water-soluble polymers and nonionic surfactants. Ethylene used to produce ethylene oxide is typically made by cracking ethane from shale gas or naphtha at high temperature in an ethylene cracker. In recent years, technology has been developed to produce biobased ethylene from sugar (Lundgren and Hjertberg, 2010). Molasses from sugar cane or corn is first converted to ethanol by fermentation using yeast. The ethanol is converted to ethylene by dehydration over an alumina bed. The ethylene can then be oxidized to ethylene oxide over a silver catalyst using conventional process technology. India Glycols Limited has used this technology to make biobased glycols and ethoxylates since 1989. More recently, Croda has commissioned a new bio-ethylene oxide plant at its Atlas Point facility. The plant uses ethanol as the starting raw material.

III.  BIOBASED SURFACTANTS



8.3  Fatty Acid Ethoxylates

291

8.3  FATTY ACID ETHOXYLATES Reaction of fatty acid with ethylene oxide converts the hydrophobic starting material to a surfactant. The fatty acid can be from any source, but C12 through C18 are the most common. The C18 fatty acids can be saturated or unsaturated. The reactions used to make fatty acid ethoxylates (FAEs) are shown in Eqs. (8.1) and (8.2) (Stockburger, 1979): (8.1)

(8.2)

Eq. (8.1) shows the direct ethoxylation of fatty acid with ethylene oxide. The reaction is run at 140–160°C under an inert atmosphere using a base catalyst. Potassium hydroxide is most commonly used, but sodium hydroxide and the corresponding methoxides can be used as well. At the beginning of the reaction, fatty acid and the catalyst are charged to the reactor and heated to reaction temperature. During the heating cycle, water from the fatty acid or the catalyst is removed by purging with nitrogen or pulling vacuum to minimize p ­ olyethylene glycol (PEG) formation. Once at the desired temperature, ethylene oxide is slowly added. The reaction is highly exothermic so the addition rate should not exceed the ability to remove heat to prevent a runaway reaction. At the end of the reaction, the product is cooled and the catalyst neutralized with acetic or lactic acid. The direct ethoxylation of fatty acids typically shows an induction period during which the reaction rate is relatively slow. The initial reaction produces primarily EG monoester. After about 1 mol of EO is added, the reaction rate increases, and the PEG and diester content in the product increases (Stockburger, 1979). The reaction of EO with fatty acid produces a broad oligomer distribution that follows a Poisson distribution (Shachat and Greenwald, 1966). Eq. (8.2) shows the esterification of fatty acid with PEG. PEG is made by reacting ethylene oxide with ethylene glycol (EG) or diethylene glycol (DEG) using a base catalyst. PEG is composed of a broad distribution of different oligomers. The esterification reaction is typically run using an acid catalyst. Sulfuric acid and p-toluene sulfonic acids (PTSA) are commonly used catalysts. The reaction is run at elevated temperature and the water of reaction removed to drive the reaction to completion. The catalyst is neutralized at the end of the reaction. Direct ethoxylation and esterification produce similar reaction products. At the temperatures used to make FAEs, ester interchange creates a complex reaction mixture consisting of monoester, diester, and PEG as shown in Eq. (8.3) (Wrigley et al., 1959):

(8.3)

III.  BIOBASED SURFACTANTS

292

8.  Fatty Acid, Methyl Ester, and Vegetable Oil Ethoxylates

The diester has hydrophobic groups at both ends of the molecule and has reduced s­olubility in aqueous solution. PEG is completely water-soluble but not surface-active. Formation of these species reduces the effective concentration of the monoester in the product and reduces the efficiency as a surfactant. On a molar basis, the ratio of monoester, diester, and PEG is typically around 2:1:1. Direct and indirect methods for determining the composition of FAEs have been described (Malkemus and Swan, 1957; Kudoh et al., 1984). Various approaches have been reported for increasing the content of monoester. The ethoxylation of hindered aliphatic fatty acids has been reported to produce higher levels of the monoester (Coopersmith and Maggart, 1969). PEG esters of various fatty acids with 80%–85% monoester content were prepared by reacting PEG with boric acid and transesterifying the borate ester with fatty acid. The boric acid protective group is removed by hydrolysis (Chandrasekhara Rao et al., 1977). Poloxamer fatty acid monoesters with greater than 95% monoester content have been reported using a homogeneous organotin catalyst (Nowicki et al., 2017). PEG esters of castor oil were prepared using lipase enzyme. The product had high monoester content, but the yields were low, especially for the higher-molecular-weight PEG adducts (Ghosh and Bhattacharya, 1998).

8.3.1 Properties FAEs range from oil-soluble to water-soluble depending on the length of the alkyl chain, the level of unsaturation, and the degree of ethoxylation. Lauric acid, oleic acid, steric acid, and tall oil fatty acids are common raw materials. Low-mole ethoxylates are soluble in nonpolar solvents. At higher degree of ethoxylation, FAEs become water-­ soluble (El-Shattory et al., 2011). FAEs show inverse water solubility with increasing temperature due to the removal of water molecules of hydration from the ethoxylate group and have cloud points similar to alcohol ethoxylates (AEs). Diesters are less water-soluble than monoesters. FAEs are relatively stable in pure water but hydrolyze under acid and base conditions. The surface properties of FAEs are similar to AE. The surface tension decreases with increasing concentration up to the critical micelle concentration (CMC) and then becomes relatively constant. Surface tension at the CMC increases with increasing alkyl chain length and degree of ethoxylation. FAEs are relatively low foaming, especially the diesters that have been used as defoamers in industrial applications. The wetting properties depend on alkyl chain length and degree of ethoxylation and are comparable with AE. FAEs are good detergents and show excellent emulsifying properties (Wrigley et al., 1957).

8.3.2 Uses FAEs are used as emulsifiers (Wadle et al., 2001) (Baseeth, 2018), softeners, wetting agents, lubricants (Cuff et al., 2018), cleaning agents, and dispersants (Stewart et al., 1998). They are used as textile and leather auxiliaries (Kimbrell, 2000), in metal working fluids (Muller et al., 2018), and crop protection (Chen, 2018). FAEs have also been used as corrosion inhibitors for steel in HCl and sulfuric acid (Hanna et al., 1989; El Sherbini, 1999).

III.  BIOBASED SURFACTANTS



293

8.4  Methyl Ester Ethoxylates

8.4  METHYL ESTER ETHOXYLATES Methyl ester ethoxylates (MEEs) are a relatively new class of nonionic surfactant. The development of MEE was driven by desire to use low-cost biobased raw materials and the need to reduce the undesirable by-products generated by the ester interchange reaction. Fatty acid methyl esters (FAMEs) are produced on an industrial scale by reacting fats and oils with methanol using a base catalyst as shown in Fig. 8.3. The reaction uses an excess of methanol to drive the reaction to completion and potassium hydroxide or methoxide as a catalyst. Glycerin and catalyst salts are removed by phase separation, and excess methanol is recovered from the organic phase for recycle. FAME is used as biofuel and as an intermediate for production of oleoalcohols. MEE is produced on an industrial scale using two main processes. Eq. (8.4) shows the direct ethoxylation of FAME. Special catalysts based on calcium or magnesium are used to insert ethylene oxide into the ester group (Behler and Folge, 1999; Leach et  al. 1993; Weerasooriya et al., 1995; Nakamura et al., 1994; Smith et al., 2009). Some of the different catalyst technologies have been recently reviewed (Anon, 2015). Products are produced in conventional pressure reactors. FAME and catalyst are heated to 160–180°C and dried before introducing EO. The reaction has an induction period of 30–60 min similar to fatty acids. Once the reaction occurs, the rate accelerates and approaches the kinetics of alcohol ethoxylation (Hreczuch, 1999). (8.4)

The catalysts used in the ester insertion reaction typically control the oligomer distribution of the resulting MEE (Hama et al., 1997a,b). This reduces the amount of unreacted ME and limits the amount of high-mole ethoxylates. The reaction appears to be a combination of

Vegetable oils

Methanol & KOH

Transesterification Crude biodiesel

Methanol recovery

Glycerin refining

ME refining

Biodiesel Crude glycerin

FIG. 8.3  Methyl ester process.

III.  BIOBASED SURFACTANTS

294

8.  Fatty Acid, Methyl Ester, and Vegetable Oil Ethoxylates

ethoxylation and transesterification. Low levels of polyethylene glycol methyl ether (MPEG) and diester are present in the reaction product. Like FAEs, MEE is relatively stable in pure water but will hydrolyze under acid or base conditions. The second way to prepare MEE is shown in Eq. (8.5). FAME is reacted with MPEG using a base catalyst (Gray et al., 2012). MPEG is produced by reacting methanol or diethylene glycol methyl ether (DEGME) with ethylene oxide using a conventional base catalyst. It is critical to remove any low-mole MPEG adducts in the final product to avoid toxicity concerns. The reaction is run at elevated temperature, and methanol is removed to drive the reaction to completion: (8.5)

MEE can also be prepared from fatty acids using an acid catalyst. Nonex 501 was one of the first commercial MEE prepared by reacting lauric acid with MPEG using PTSA as a catalyst. (Malik and Chand, 1969). The reaction is run at elevated temperature, and water is removed to drive the reaction to completion.

8.4.1 Properties The properties of MEE have been reported (Cox and Weerasooriya, 1997). Water solubility depends on the alkyl chain length, the amount of unsaturation and the degree of ethoxylation. MEE shows inverse solubility with increasing temperature and a well-­ defined cloud point. Compared with AE with the same alkyl chain length and degree of ethoxylation, MEE has a lower cloud point due to the lack of a terminal hydroxyl group capable of hydrogen bonding with water molecules. MEE also shows lower foam than AE for the same reason. MEE show surface properties similar to FAEs and AEs (Hama et al., 1997a,b). The ­surface tension decreases with increasing surfactant concentration up to the CMC and then becomes relatively constant. Compared with AE, the CMC of MEE is slightly higher, but the surface tension at the CMC is lower. Dynamic surface tension depends on the alkyl chain length and the degree of ethoxylates. Short-chain MEE shows lower dynamic surface ­tension that increases with increasing alkyl chain length (Smith and Weaver, 2011). MEE shows less tendency to form gel phases in water and improved liquidity compared with AE making them ideal for highly concentrated liquid detergent formulations. The ability to lower interfacial tension against oil depends strongly on the alkyl chain length and degree of ethoxylation (Liu et al., 2017). The performance properties of MEE have been compared with AE (Cox and Weerasoorriya, 1998). Short-chain MEE (C8–10) and low degree of ethoxylation show poor detergency in fabric cleaning. Mid-chain MEE (C12–18) with mid to high degrees of ethoxylation show cleaning performance to similar to AE. The opposite is observed for hard surface cleaning. Short-chain methyl esters with low degree of ethoxylation show better cleaning performance than alcohol or nonylphenol ethoxylates due to the solvency of the methyl ester.

III.  BIOBASED SURFACTANTS



8.5  Vegetable Oil Ethoxylates

295

Skin compatibility and ecotoxicity of MEE has been determined (Hama et al., 1998). Skin irritation testing indicates that MEE is less irritating than AE. Tests using human erythrocytes and keratinized epidermis cells show hemolysis and cytotoxicity of MEE to be lower than AE. Biodegradation, determined by measuring biological oxygen demand, increased with increasing alkyl chain length and decreased with increasing degree of ethoxylation. All of the MEE tested were found to be readily biodegradable. Acute toxicity to fish was about oneeighth of that for AE.

8.4.2  Applications of MEE MEE is used primarily in detergent products as a low-cost alternative to natural AE (Holzhauer and Johnson, 2018; Shearouse and Hibbard, 2017). Although commercially available for over 20 years, with a few exceptions, their use has failed to penetrate the market in a significant way. The hydrolytic stability of MEE in alkaline systems limits use in detergent powders containing soda ash and other alkaline builders. In addition, the lack of a gel phase also limits the ability of MEE to build viscosity in low active liquid formulations through the salt effect.

8.5  VEGETABLE OIL ETHOXYLATES The use of vegetable oil as a raw material for the production of surfactants has been practiced for many years. Partially hydrolyzed triglycerides are widely used as emulsifiers in foods and cosmetics. The products made by interesterification of fats or oils with glycerin are a complex mixture of mono-, di-, and triesters and have limited water solubility. Cosurfactants are usually added to help disperse these materials where they function by adsorbing at the oil-water interface to help stabilize the emulsion. The water solubility of partially hydrolyzed esters can be improved using polymerized glycerin. Polyglycerol esters (PGEs) are widely used as food emulsifiers (Lauridsen, 1976). The products are made up of glycerin oligomers esterified with fatty acid or interesterified with triglycerides (Benson, 1967). Polymerized glycerin is made by heating glycerin to over 230°C using a base catalyst. Water of reaction is removed to form glycerin oligomers with three or more repeating units (McIntyre, 1979). PGE form hexagonal liquid crystals and α crystalline gels in water that help to stabilize emulsions in food applications (Hemker, 1981). Purified diglycerol esters have been prepared and the surfactant properties determined (Kumar et al., 1989). Monoesters show better surface tension reduction, emulsification, and foaming properties than diesters. Short-chain esters show better properties than long-chain esters. Unsaturation in the acyl group reduced emulsion stability. A hydroxyl group on the acyl chain increased surface tension and reduced foaming. The water solubility and surfactant properties of triglycerides can be improved by adding ethylene oxide. Vegetable oil ethoxylates (VOEs) can be made in several different ways. At high temperature and alkaline conditions, triglycerides will hydrolyze to fatty acids and partially hydrolyzed glycerides. Ethylene oxide reacts with the fatty acids and

III.  BIOBASED SURFACTANTS

296

8.  Fatty Acid, Methyl Ester, and Vegetable Oil Ethoxylates

hydroxyl groups of the glyceride in a random fashion. The reaction product is a complex mixture of ethoxylated esters, glycerides, and PEG. This approach is used to make castor oil ethoxylates that are used as emulsifiers in cosmetics, textile processing, and crop protection (Porter, 1994). A second approach is to use ester insertion catalysts originally developed to ethoxylate methyl esters (Weerasooriya, 1999). The reaction shown in Eq. (8.6) requires high temperature (160–180°C) and exhibits an induction period. The reaction products are dark colored, hard waxy solids at room temperature. Soybean oil with 45 mol of EO (SOE-45) is completely water-soluble (Smith and Sneed, 2006). The CMC of these products is very low with surface tensions in the high 30 mN/m. Dynamic surface tension is high with relatively poor wetting kinetics. Interfacial tension against mineral oil is relatively low and decreases with increasing degree of ethoxylation. The foam height is about half that of AE and increases with increasing degree of ethoxylation. When tested in laundry, SOE-45 shows almost parity performance to mid-cut AE with 9 mol of EO:

(8.6) The water solubility of VOE can be improved by partially saponifying the ester with sodium hydroxide to obtain a roughly equal mixture of mono-, di-, and triglyceride ethoxylates; fatty acid soap; and glycerol ethoxylate (Cox and Weerasooriya, 2000). The reaction mixture shows good surface tension reduction with a low CMC. Microtox tests and human patch tests show the partially saponified VOEs to be exceptionally mild and nontoxic. Yet another approach is to react ethylene oxide with glycerin followed by esterification with fatty acid or methyl ester to produce polyoxyethylene glycerin esters. In the case of fatty acids, the reaction is run using a strong acid catalyst, and water of reaction is removed to drive the reaction to completion. This type of surfactant is widely used in cosmetic products as a foam boosters, emulsifier, and emollient to help reduce transdermal water loss. PEG-7 glyceryl cocoate is a well-known example of this type of surfactant. The wetting properties of polyoxyethylene glycerin esters have been determined by cotton disk wetting (Jurado et al., 2012). The wetting properties improve with decreasing moles of EO and show synergy with short-chain AE. A similar product is obtained by transesterifying triglycerides with ethoxylated glycerin using a conventional base catalyst. The reaction shown in Eq. (8.7) occurs at low temperature (100°C) and produces a light colored liquid product after bleaching. VOE prepared by this reaction are extremely water-soluble and exhibit almost no gel-phase in water.

III.  BIOBASED SURFACTANTS



8.5  Vegetable Oil Ethoxylates

297

(8.7) The reaction product is a complex mixture of mono-, di-, and triacyl ethoxylated glycerides; glycerin ethoxylate; and soap (Smith, 2013). The species distribution can be controlled by changing the ratio of triglyceride to ethoxylated glycerin used in the reaction. The main species formed is the monoacyl ethoxylated glyceride shown in Fig. 8.4. The reaction can be run with any purified triglyceride. The surface properties of coconut, olive, avocado, and soybean oil ethoxylates show low CMC values around 10 ppm and surface tension near 30 mN/m. CMC, surface tension, and foam increase with increasing degree of ethoxylation. Interfacial tension of soybean oil ethoxylates against mineral oil undergoes a minimum around 10 mol of EO. Detergency measurements under standard US wash conditions show optimum cleaning is inversely related to the interfacial tension.

FIG. 8.4  Structure of monoacyl ethoxylated glyceride.

III.  BIOBASED SURFACTANTS

298

8.  Fatty Acid, Methyl Ester, and Vegetable Oil Ethoxylates

VOE are extremely simple to make and consume 100% of the starting vegetable oil. The resulting product has very low levels of residual EO and 1,4-dioxane that are typically removed during the drying step. The US Environmental Protection Agency (EPA) classifies dioxane as a probable human carcinogen. Dioxane is irritating to the eyes and respiratory tract, and exposure may cause damage to the central nervous system, liver, and kidneys. Under California Proposition 65, dioxane is classified in the state of California to cause cancer. The properties of the product depend on the alkyl chain distribution and the degree of ethoxylation. Work was done to prepare a series of VOE surfactants using algal oil from microalgae (Smith, 2015). The algal oil was obtained from Solazyme (South San Francisco, CA, United States), which has developed a strain of microalgae that converts plant sugars into triglycerides in high yield. The carbon chain distribution of the oil can be controlled by genetically modifying the microalgae that are grown in large industrial fermenters. The alkyl chain distribution of high lauric and high oleic algal oil is shown in Table 8.2. Both strains of algal oil were converted into surfactants by reacting with ethoxylated glycerin with different degrees of ethoxylation using potassium hydroxide as a catalyst. Ethoxylated glycerin and catalyst were heated to 100°C and sparged with nitrogen to remove water from the catalyst. The algal oil was then slowly added over an hour and allowed to digest for 15 min. The reaction product was then cooled below 60°C and bleached with hydrogen peroxide to obtain light straw-colored, low-viscosity liquid products. The surface properties of algal oil ethoxylates (AOEs) were determined by measuring the surface tension as a function of surfactant concentration. The CMC was determined from the inflection in the surface tension isotherms. AOE prepared using high oleic algal oil shows normal behavior. CMC increases as the mole of EO in the molecule increases. AOE prepared using high lauric algal oil shows surprising behavior. The CMC decreases slightly as the moles of EO increase indicating that ethylene oxide is somehow acting as a hydrophobic material. The interfacial tension of AOE was determined using a drop volume tensiometer. The IFT shows a minimum around 10–15 mol of EO for both oils. At the minimum, the AOE made from high oleic oil is slightly lower than AOE made from high lauric oil. The IFT of AOE made from algal oil is comparable with that of AE. TABLE 8.2  Alkyl Chain Distribution of Algal Oil Fatty Acid

High Lauric Oil (wt%)

High Oleic Oil (wt%)

C10

15

0

C12

45

0

C14

14

0

C16

7

4

C18

1

5

C18–1

14

89

C18–2

4

2

C18–3

1

0

Total

100

100

III.  BIOBASED SURFACTANTS

REFERENCES 299

The cleaning performance of AOE was determined by measuring soil removal from standard soil swatches under standard US wash conditions. Dirty motor oil, dust sebum, olive oil, and clay on cotton and polyester cotton swatches were washed at 30°C in 150 ppm hard water in a six-pot terg-o-tometer at 200 ppm surfactant concentration in the wash water. The optical reflectance before and after washing was measured with a Hunter Lab spectrophotometer. All soils were run in triplicate and the results averaged. The results indicate that the AOE made using high oleic oil show slightly better detergency. For both algal oils, the detergency increased with increasing moles of EO up to about 15 EO, and further ethoxylation did not affect detergency. Compared with standard AE, AOE exhibits similar parity performance as AE.

8.6 CONCLUSIONS Nonionic surfactants made from fats and oils have a rich history. FAEs have been used for over 60 years in industrial applications and are relatively easy to make by direct ethoxylation or esterification and use inexpensive raw materials. The presence of PEG and diester in the product lowers the efficiency by diluting the concentration of active surfactant. Even so, FAEs are widely used in metal working and textile processing. MEEs are a relatively new class of surfactant made by direct ethoxylation of FAMEs. The ester insertion catalyst used to make MEE provides a narrow oligomer distribution and suppresses the ester interchange reaction that translates to improved detergent properties. The limited gel-phase region of MEE makes them ideal for highly concentrated liquid products. With a few exceptions, MEE has not gained widespread use in laundry liquids due to hydrolytic stability and the odor associated with low levels of residual methyl ester. VOEs consist of a complex mixture of different species. The relative proportions of the different species depend on how the product is prepared. Insertion of ethylene oxide into the ester gives hard waxy solid products that are somewhat difficult to handle on an industrial scale. Scrambling the triglyceride with ethoxylated glycerin gives liquid products with improved handling characteristics, but both versions of vegetable oil ethoxylate are prone to hydrolyze and have their own odor issues. VOE is currently used in personal care applications as mild surfactants based on natural oils.

References Anon, 2015. Catalyst for the ethoxylation of esters. J. Surfact. Deterg. 18, 913–918. Baseeth, S., 2018. Emulsifier for Solubilizing Polar Solvents in Oils and Polyols. USA, Patent No. 9,889,417. Behler, A., Folge, A., 1999. Process for Preparing Alkoxylated Fatty Acid Alkyl Esters. USA, Patent No. 6,008,392. Benson, F.R., 1967. In: Schick, M.J. (Ed.), Nonionic Surfactants. Marcel Dekker, New York, pp. 247–299. Chandrasekhara Rao, T., Sita Rama Sastry, Y., Subbarao, R., Lakshminarayana, G., 1977. Preparation and surface-­ active properties of polyoxyethyleneglycol. J. Am. Oil Chemists’ Soc. 54, 15–16. Chen, C.-Y.R., 2018. Fertilizer-Compatible Composition. USA, Patent No. 9,919,979. Coopersmith, M., Maggart, R.C., 1969. Monoethoxylation of hindered carboxylic acids. J. Am. Oil Chemists’ Soc. 46, 332–334. Cox, M.F., Weerasooriya, U., 1997. Methyl ester ethoxylates. J. Am. Oil Chemists’ Soc. 74 (7), 847–859. Cox, M.F., Weerasooriya, U., 2000. Partially saponified triglyceride ethoxylates. J. Surfact. Deterg. 3 (2), 213–220. Cox, M.F., Weerasoorriya, U., 1998. Impact of molecular structure on the performance of methyl ester ethoxylates. J. Surfact. Deterg. 1 (1), 11–22.

III.  BIOBASED SURFACTANTS

300

8.  Fatty Acid, Methyl Ester, and Vegetable Oil Ethoxylates

Cuff, T.J., Bowers, J.L., Olivier, E.J., Yao, K., 2018. Lubricating Compositions and Methods for the Use Thereof. USA, Patent No. 9,879,200. El Sherbini, E.F., 1999. Effect of some ethoxylated fatty acids on the corrosion behaviour of mild steel in sulphuric acid solution. Mater. Chem. Phys. 60 (3), 286–290. El-Shattory, Y.A., El-Wafa, G.A.A., Aly, S.M., 2011. Ethoxylation of fatty acids fractions of overused vegetable oils. J. Surfact. Deterg. 14, 151–160. Ghosh, M., Bhattacharya, D.K., 1998. Enzymatic preparation of polyethylene glycol esters of castor oil fatty acids and their surface-active properties. J. Surfact. Deterg. 1 (4), 503–505. Gray, L.M., Scheibel, J.J., Vinson, P.K., Binski, C.J., 2012. Alkyl-Capped Alkoxylated Esters and Compositions Comprising Same. USA, Patent No. 8,143,208. Gunstone, F.D., 2004. The Chemistry of Oils and Fats, first ed. Blackwell Publishing Ltd, Oxford. Hama, I., Sakaki, M., Sasamoto, H., 1997a. Nonionic surfactant properties of methoxypolyoxyethylene dodecanoate compared with polyoxyethylene dodecyl ether. J. Am. Oil Chemists’ Soc. 74 (7), 829–835. Hama, I., Sasamoto, H., Okamoto, T., 1997b. Influence of catalyst structure on direct ethoxylation of fatty methyl esters over Al–Mg composite oxide catalyst. J. Am. Oil Chemists’ Soc. 74 (7), 817–822. Hama, I., Sasamoto, H., Nakamura, T., Miura, K., 1998. Skin compatibility and ecotoxicity of ethoxylated fatty methyl ester nonionics. J. Surfact. Deterg. 1 (1), 93–97. Hanna, F., Sherbini, G.M., Barakay, Y., 1989. Commercial fatty acid ethoxylates as corrosion inhibitors for steel in pickling acid. Br. Corros. J. 24 (4), 269–272. Hemker, W., 1981. Associative structures of polyglycerol esters in food emulsions. J. Am. Oil Chemists’ Soc. 58, 114–119. Holzhauer, F.W., Johnson, K., 2018. Liquid Cleaning Compositions With Improved Enzyme Compatibility and/or Stability. USA, Patent No. 9,909,087. Hreczuch, W., 1999. Comparison of the kinetics and composition of ethoxylated methyl dodecanoate and ethoxylated dodecanol with narrow and broad distribution of homologs. J. Surfact. Deterg. 2 (3), 287–292. Itiner, M.H., 1938. Hydrolysis of Fats and Oils. USA, Patent No. 2,139,589. Jurado, E., Vicaria, J.M., Garcia-Martin, J.F., Garcia-Roman, M., 2012. Wettability of aqueous solutions of eco-friendly surfactants (ethoxylated alcohols and polyoxyethylene glycerin esters). J. Surfact. Deterg. 15, 251–258. Kimbrell, W.C., 2000. Method of Dyeing Low Pill Polyester. USA, Patent No. 6,113,656. Kudoh, M., Kotsuji, M., Fundano, S., Tsuji, K., 1984. Analysis of fatty acid exthoxylates by preparative high-­ performance liquid chromatography. J. Chromatogr. 295, 187–191. Kumar, T.N., Sastry, Y., Lakshminarayana, G., 1989. Preparation and surfactant properties of diglycerol esters of fatty acids. J. Am. Oil Chemists’ Soc. 66 (1), 153–157. Lauridsen, J.B., 1976. Food emulsifiers: surface activity, edibility, manufacture, composition, and application. J. Am. Oil Chemists’ Soc. 53, 400–407. Leach, B.E., et al., 1993. Process for the Alkoxylation of Esters and Products Produced Therefrom. USA, Patent No. 5,220,046. Lefort, T.E., 1935. Process for the Production of Ethylene Oxide. US, Patent No. 1,998,878. Liu, M., et al., 2017. Interfacial tensions of ethoxylated fatty acid methyl ester solutions against crude oil. J. Surfact. Deterg. 20, 961–967. Lundgren, A., Hjertberg, T., 2010. Ethylene from renewable resources. In: Kjellin, M., Johansson, I. (Eds.), Surfactants From Renewable Resources. John Wiley & Sons Ltd., Chichester, pp. 111–126. Lusas, E.W., 2007. Anegetable fats, oils, and waxesimal and V. In: Kent, J.A. (Ed.), Kent and Riefel’s Handbook of Industrial Chemistry and Biotechnology. Springer, New York, pp. 1549–1656. Malik, W.U., Chand, P., 1969. Critical middle concentration of non-ionic surfactants by polaro-graphic and spectrometric methods-a comparative study. J. Am. Oil Chemists’ Soc. 46, 285–288. Malkemus, J.D., Swan, J.D., 1957. Analysis of polyethylene glycol esters. J. Am. Oil Chemists’ Soc. 94, 342–344. McIntyre, R.T., 1979. Polyglycerol esters. J. Am. Oil Chemists’ Soc. 56, 835A–840A. Mills, V., McCain, H.K., 1949. Fat hydrolysis. Ind. Eng. Chem. 41 (9), 1982–1985. Muller, H., Maker, D., Hahnel, P., 2018. Esters for Drilling Emulsions and Metal Working Fluids. USA, Patent No. 9,896,613. Nakamura, H., Hama, I., Fujimora, Y., Nakamoto, Y., 1994. Method and Manufacturing of Fatty Acid Esters of Polyoxyalkylene Alkyl Ethers. USA, Patent No. 5,374,750.

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FURTHER READING

301

Nowicki, J., et  al., 2017. Selective synthesis of polyoxyethylene–polyoxypropylene block copolymer (poloxamer) fatty acid monoesters over homogeneous organotin catalyst. J. Surfact. Deterg. 20, 1475–1481. Porter, M.R., 1994. Handbook of Surfactants. second ed. Chapman& Hall, London. Satterfield, C.N., 1991. Catalytic oxidation. In: Heterogeneous Catalysis in Industrial Practice. McGraw-Hill Inc, New York, pp. 279–285. Shachat, N., Greenwald, H.L., 1966. Mechanism of ethylene oxide condensation. In: Schick, M.J. (Ed.), Nonionic Surfactants. Marcel Deffer, New York, pp. 8–43. Shearouse, W.C., Hibbard, D.L., 2017. Laundry Detergent Compositions Comprising Renewable Components. USA, Patent No. 9,796,948. Smith, G.A., 2013. Vegetable oil based surfactants: physical chemistry and performance properties. In: Montreal, 104th AOCS Annual Meeting & Expo. Smith, G.A., 2015. Surfactants based on algae oil. In: Istanbul, 10th World Surfactant Congress and Business Convention. Smith, G.A., Sneed, G., 2006. Natural based surfactants: physical chemistry and performance properties. In: St. Louis, 97th AOCS Annual Meeting & Expo. Smith, G.A., Weaver, P., 2011. Methyl Ester Ethoxylates: Physical Chemistry and Performance Properties. CESIO World Surfactant Congress, Vienna, Austria. Smith, G.A., O’Neill, J., Sneed, G., Whewell, C.J., 2009. Alkaline Earth-Based Alkoxylation Catalysts. USA, Patent No. 7,629,48. Spitz, L., 2009. The history of soaps and detergents. In: Spitz, L. (Ed.), Soap Manufacturing Technology. AOCS Press, Urbana, pp. 1–4. Stewart, R.E., Mark, H., Schumacher, O., Stewen, U., 1998. Stable Aqueous Dispersions of Dibutyltin Oxide. USA, Patent No. 5,807,802. Stockburger, G.J., 1979. Ethoxylation. J. Am. Oil Chemists’ Soc. 56, 774–777. Wadle, A., et  al., 2001. Fatty Acid Ethoxylates and Partial Glycerides for Preparing Phase Inversion Temperature Emulsions. USA, Patent No. 6,221,370. Weerasooriya, U., 1999. Ester alkoxylation technology. J. Surfact. Deterg. 2 (3), 373–381. Weerasooriya, U., et al., 1995. Process for Alkoxylation of Esters and Products Produced Therefrom. USA, Patent No. 5,386,045. Wrigley, A.N., Smith, F.D., Striton, A.J., 1957. Synthetic detergents from animal fats. VIII. The ethenoxylation of fatty acids and alcohols. J. Am. Oil Chemists’ Soc. 34 (1), 40–43. Wrigley, A.N., Smith, F., Stirton, A.J., 1959. Reaction of ethylene oxide or propylene oxide with long chain fatty acids. Mono- and diester formation. J. Am. Oil Chemists’ Soc. 36, 34–36.

Further Reading Buckingham, L.E., et al., 1995. Comparison of solutol HS 15, cremophor EL and novel ethoxylated fatty acid surfactants as multidrug resistance modification agents. Int. J. Cancer 62 (4), 436–442.

III.  BIOBASED SURFACTANTS

C H A P T E R

9 Methyl Ester Sulfonate Norio Tobori*, Toshio Kakui† *

LION Specialty Chemicals Co., Ltd., Sumida-ku, Tokyo, Japan †LION Corporation, Sumida-ku, Tokyo, Japan

9.1 INTRODUCTION Fatty acid methyl ester sulfonates (MESs) are oleochemical-based anionic surfactants derived from palm or coconut oil through transesterification and subsequent sulfonation. Linear alkylbenzene sulfonates (LASs), alkyl sulfonates (ASs), and alpha olefin sulfonates (AOSs) are generally used as detergent surfactants. However, because of the two oil crises in the 1970s, detergent manufactures have been interested in natural fat- and oil-based surfactants, rather than those that are petroleum-based, and have considered MESs as a possible detergent ingredient. MESs have good surface-active properties (Stirton et  al., 1962; Boucher et  al., 1968) and excellent detergency performance as a laundry detergent main ingredient (Okumura et al., 1976; Stirton et  al., 1954; Schambli and Schwuger, 1990), as well as good biodegradability (Maurer et al., 1977; Steber and Wierich, 1989). MESs were shown to be scum dispersant in the 1960s, and thus, their sulfonation mechanism (Stirton, 1968; Weil et al., 1953), characteristics, application, and manufacturing process (Stein and Baumann, 1975; Kapur et al., 1978) have been extensively studied. They have only recently been found to be applicable as a main component of laundry detergent products, and their manufacture on a commercial basis is now possible. There are problems in manufacturing MESs because of (a) the dark color of the sulfonated product, (b) the generation of disodium salts through sulfonation as a by-product, and (c) the low active ingredient content for a detergent raw material. It is thus difficult to produce high-quality MESs suitable for laundry detergents. Due to hydrolysis, MESs may possibly undergo degradation into the disodium the disodium salt on contact with alkaline components (Yamane and Miyawaki, 1989). The technology became available for producing high-quality MESs on a commercial basis in the early 1990s through improvements such as sulfonation, bleaching, and neutralization. This new technology was introduced into Japan, and a new compact powder detergent was produced in 1991. Further compact products were subsequently developed using the improved MESs. In the United States, Stepan Company developed coco-based MES and promoted their usage during the same period (Drozd and Desai, 1991; Smith, 1989). To date, there are many suppliers of MES in the world. Major global producers of MES are tabulated in Table 9.1. Biobased Surfactants https://doi.org/10.1016/B978-0-12-812705-6.00009-5

303

Copyright © 2019 AOCS Press. Published by Elsevier Inc. All rights reserved.

304

9.  Methyl Ester Sulfonate

TABLE 9.1  Global Major Producers of MES Companies

Location

Annual Capacity (MT)

Guangzhou Keylink Chemical Co.

China

40,000

Zhejiang Zanyu Technology Co., Ltd.

China

60,000

Stepan

United States

50,000

Dersa, Bogota

Colombia

15,000

KLK Oleo

Malaysia

100,000

Global Eco Chemicals Malaysia Sdn. Bhd

Malaysia

50,000

PT Global Eco Chemicals Indonesia

Indonesia

50,000

This chapter summarizes the application of MESs in laundry detergent formulations and their fundamental surfactant properties, solid structure, and biodegradability.

9.2  PHYSICAL AND CHEMICAL PROPERTIES OF METHYL ESTER SULFONATE MESs have the chemical structure shown in Fig. 9.1. The structure is that of a fatty acid methyl ester with a sulfonate group in the α-position. MESs are prepared by the direct sulfonation of fatty acid methyl esters from triglyceride through transesterification. The number of carbon atoms in MESs is generally 12–18 for detergent use.

9.2.1  Surfactant Properties 9.2.1.1  Basic Surfactant Properties (CMC, Krafft Point and Solubility) Fujiwara reported the basic properties of MESs and their water hardness tolerance (Fujiwara et  al., 1993). Solubility and the critical micellar concentration (CMC) curves as a function of temperature for C14-, C16-, and C18MES-Na are shown in Fig. 9.2 along with those of the corresponding calcium salts. The CMCs and Krafft points of MES-Na, MES-Ca, and AS-Na (sodium lauryl sulfate) are presented in Table 9.2. The solubility and the CMC were found to decrease with rising carbon number and a counterion change from Na to Ca. These features are characteristic of anionic surfactants (Hato and Shinoda, 1973). Table 9.2 shows that the increase in Krafft points of the MES compounds as a result of the counterion change is less than that for AS compounds (Hato and Shinoda, 1973; Shinoda et al., 1986).

FIG. 9.1  Chemical structures of MES and disodium salt. III.  BIOBASED SURFACTANTS



9.2  Physical and Chemical Properties of Methyl Ester Sulfonate

305

100 14 SFNa 16 SFNa 14 SFNa

10

Solubility (mM)

18 SFNa 1 16 SFCa

0.1

18 SFCa

0.01

0.001

0

10

20

30 40 Temp. (°C)

50

60

FIG. 9.2  Solubility and CMC curves of MES surfactants. Solubility for MES-Ca represents those of RCH(COOCH3)

SO3·1/2Ca. From Fujiwara, M., Miyake, M., Abe, Y., 1993. Colloidal properties of alpha-sulfonated fatty acid methyl esters and their applicability in hard water. Colloid Polym. Sci. 271, 782.

TABLE 9.2  Krafft Points and Critical Micellar Concentrations (CMCs) of Some Surfactants Surfactant

Krafft Point (°C)

CMC (mM)

Reference

C14MES-Na

6

2.8 (13°C)

Fujiwara et al. (1993)

C16MES-Na

17

0.73 (23°C)

Fujiwara et al. (1993)

C18MES-Na

30

0.18 (33°C)

Fujiwara et al. (1993)

28

0.66 (30°C)

Fujiwara et al. (1993)

C16MES-Ca

41

0.19 (45°C)

Fujiwara et al. (1993)

C18MES-Ca

49

0.042 (50°C)

Fujiwara et al. (1993)

C12AS-Na

9

8.1

Shinoda et al. (1986)

C14AS-Na

30

2.1

Shinoda et al. (1986)

C12AS-Ca

50

1.2

Hato and Shinoda (1973)

C14AS-Ca

71

0.68

Hato and Shinoda (1973)

From Fujiwara, M., Miyake, M., Abe, Y., 1993. Colloidal properties of alpha-sulfonated fatty acid methyl esters and their applicability in hard water. Colloid Polym. Sci. 271, 782.

III.  BIOBASED SURFACTANTS

306

9.  Methyl Ester Sulfonate

0 AS-Na –2 MES-Na

In CMC (mol/L)

–4

MES-Ca

–6

–8

–10

–12

6

8

10

12

14

16

18

No. of C-atoms

FIG. 9.3  Relationship between the logarithm of the CMC and the number of carbon atoms in the hydrophobic chain. The number of carbon atoms was plotted according to the following structures: AS = CnH2n + 1OSO3M and SF = Cn − 1H2(n − 1) + 1CH(COOCH3)-SO3M. Key: AS-Na◇, MES-Na●, and MES-Ca○. From Fujiwara, M., Miyake, M., Abe, Y., 1993. Colloidal properties of alpha-sulfonated fatty acid methyl esters and their applicability in hard water. Colloid Polym. Sci. 271, 782.

The CMC of MES-Ca is approximately 25% of that corresponding to MES-Na. Using the method of Shinoda, the logarithm of CMC was plotted against hydrophobic chain length (Fig. 9.3) and the logarithm of counterion concentration (Shinoda, 1963). Good linearity for MES-Na and MES-Ca was demonstrated, suggesting micelle formation by MES-Na and MES-Ca following the equation:

{

}

ln ( CMC ) = −mω / kT + K g / Zi ln ( 2000πσ 2 / DNkT ) − ln C g + constant where m is the number of calcium atoms, ω is the cohesive energy change for transferring one methylene group from a hydrophobic environment to aqueous medium, Kg is an experimental constant related to the degree of counterion binding by micelles, Kg/Zi is determined from the slope of the CMC as a function of Zi valent counterion concentration, D is the dielectric constant of the solution, N is Avogadro’s number, σ is the charge density on the micelle surface, and Cg is the total concentration of the counterion per liter. The energy calculated for MES-Na and MES-Ca was 1.1 and 0.93kT, respectively, and those values are almost equal to that of a typical ionic surfactant (e.g., AS-Na, 1.1kT). III.  BIOBASED SURFACTANTS



9.2  Physical and Chemical Properties of Methyl Ester Sulfonate

307

TABLE 9.3  Micellar Weights, Aggregation Numbers, and Second Virial Coefficients (B2) of Some Surfactants Surfactant

Medium

Molecular Weight of Micelles (g/mol)

Aggregation Number

B2 (mL/g)

C14MES-Na

0.01 N NaNO3

28,000

81

7.30 × 10−3

0.1 N NaNO3

32,800

95

8.73 × 10−3

0.4 N NaNO3

41,000

119

3.05 × 10−4

C16MES-Na

0.01 N NaNO3

31,600

85

5.20 × 10−3

C18MES-Na

0.01 N NaNO3

42,500

106

2.18 × 10−3

C14MES-Ca

H2O

32,600

96

1.87 × 10−3

0.003 N Ca(NO3)2

45,100

132

7.78 × 10−4

0.01 N Ca(NO3)2

42,200

124

−2.39 × 10−4

From Fujiwara, M., Miyake, M., Abe, Y., 1993. Colloidal properties of alpha-sulfonated fatty acid methyl esters and their applicability in hard water. Colloid Polym. Sci. 271, 783.

The dissociation of counterion for MES-Na and MES-Ca micelles was determined and was found to be essentially the same as that of typical anionic surfactants. The aggregation number of micelles is important for expressing the physicochemical properties of surfactant solutions. The micelle weight, aggregation number, and second virial coefficient for MES-Na and MES-Ca are listed in Table 9.3. The aggregation number of C14 MES increased from 81 to 119, and the second virial coefficient decreased from 7.30 × 10−3 to 3.05 × 10−4 with increasing electrolyte concentration. This behavior corresponds to that of C12AS-Na reported by Hayashi and Ikeda (1980). The aggregation number of C14MES is basically the same as that of C16MES, whereas C18MES number slightly exceeds that of C16 in the 0.01 N NaNO3 solution. The aggregation number of MES-Ca is larger than that of MES-Na even in distilled water and increases with electrolyte addition. 9.2.1.2  Water Hardness Tolerance Fujiwara proposed a mechanism for the good hardness tolerance of MESs based on the precipitation phase boundary for MES-Ca salts (Fujiwara et  al., 1993). The boundaries for C12AS-Na, C14MES-Na, and C16MES-Na are shown in Fig.  9.4. The boundary lines were obtained after 10 min, 1 h, 1 day, and in the equilibrium state, since precipitation was time-­ dependent. The equilibrium phase boundary for C12AS-Na, which has poor tolerance to hardness, agreed with that reported previously (Steliner and Scamehom, 1989). The phase boundary at equilibrium was quite close to that at 10 min, indicating that precipitation occurs very rapidly and equilibrium is easily attained. In activity, washing this surfactant would precipitate within 10 min, and surface activity would be lost. Precipitation region concentrations for C14MES-Na at equilibrium are low and that for C16MES-Na is spread over the range 3–10°DH (DH is German hardness, 1°DH = 10 ppm CaO). If washing is under the control of the precipitate boundary at equilibrium, all MES would precipitate with the loss of solution activity. The precipitation region of C14MES-Na and C16MES-Na in an actual washing was much smaller than that of AS-Na. The reason for the hardness tolerance of MES-Na may be due to the extremely slow precipitation rate of Ca salts at low temperature. III.  BIOBASED SURFACTANTS

308

9.  Methyl Ester Sulfonate

10–1

10–1

10 min

1 day

–2

10

Ca(NO3)2 (moles•L–1)

10–1

10 min

Precipi tation region –2

10–2

–3

10–3

10 10 min

1h

10 °DH –3

10

10

3 °DH

eq. 10–4 eq. Monophasic

Monophasic

region

–5

10

10

–5

10

(A)

eq.

region

–4

10

–3

10

–2

10

–1

10

C12AS-Na (moles･L–1)

10–5

(B)

Monophasic

10–5

–5

25°C 10–6

10–6

1 day

10–4

10–4

25°C 10–6 10–4

10–3

10–2

C14MES-Na (moles･L–1)

10–1

10–5

(C)

region

30°C 10–4

10–3

10–2

10–1

C16MES-Na (moles･L–1)

FIG. 9.4  Precipitation phase boundary diagrams of C12AS-Na, C14MES-Na, and C16MES-Na. From Fujiwara, M.,

Miyake, M., Abe, Y., 1993. Colloidal properties of alpha-sulfonated fatty acid methyl esters and their applicability in hard water. Colloid Polym. Sci. 271, 784.

9.2.2  Solid State Properties Few studies have been carried out on the solid or crystalline state of anionic surfactants apart from the monocrystalline X-ray diffraction analysis of the hydrated solid of sodium dodecyl sulfate (Kekicheff et al., 1989). To extend this to detergent powders, the crystalline state must be understood since features such as powder caking and the quick dissolving rate of a powder particle in water are closely related to the surfactant solid properties. Fujiwara investigated the crystalline state of C16MES-Na with regard to hydration, phase transition, and adsorption/desorption of moisture using the C16MES-Na as a model substance (Fujiwara et al., 1997). The phase behavior of MES-Na was studied by differential scanning colorimetry (DSC) for several C16MES-Na solid samples with different moisture contents. The results for samples with 0%–8.9% moisture content are shown in Fig. 9.5. For nonhydrated MES-Na, a single endothermic peak was noted. The phase transition temperature (Tc) and enthalpy change (ΔH) were 112°C and 22.1 kJ/mol, respectively. With increasing moisture, the endothermic peal gradually shifted to lower temperature with a concomitant decrease in size. At the same time, a new peak appeared at 68°C, and the size of this peak increased at constant temperature. The spectrum at 8.9% moisture content showed an endothermic peak with Tc and ΔH of 7°C and 67.5 kJ, respectively, which may possibly have been due to the phase transition of MES-Na·2H2O crystals from solid to liquid form since the molar ratio of [H2O]/[MES-Na] was 2.0. The X-ray diffraction patterns for these MES-Na samples are presented in Fig.  9.6. The MES-Na solid at 0% and 8.9% moisture content gave one peak corresponding to spacing of 26.9 and 30.1 Å, respectively. At 1.6% and 5.6% moisture content, however, two peaks of 26.9 and 30.1 Å appeared simultaneously. The two different crystal structures thus appear to coexist independently in solid MES-Na with moisture content from 0% to 8.9%.

III.  BIOBASED SURFACTANTS



309

9.2  Physical and Chemical Properties of Methyl Ester Sulfonate

Temperature (°C) 0

20

40

80

60

100

120

Moisture content (wt. %)

0 1.6 3.6 5.6

ENDO.

6.5 8.1 8.9

FIG. 9.5  DSC curves for C16MES-Na solids with moisture contents from 0 to 8.9 wt%. From Fujiwara, M., Okano, T., Amano, H., Asano, H., Oubu, K., 1997. Phase diagram of alpha-sulfonated palmitic acid methyl esters sodium salt/water system. Langmuir 13, 3346.

45K

Intensity (cps)

45K

22.5

22.5

5

6 2q (°)

0

7

(B)

5

6 2q (°)

45K

26.9 Å

30.1 Å

22.5 30.1 Å

30.1 Å

0

(A)

45K

26.9 Å

26.9 Å

7

0

(C)

5

6 2q (°)

22.5

7

0

(D)

5

6 2q (°)

7

FIG. 9.6  X-ray diffraction patterns of C16MES-Na solids with moisture contents from 0 to 8.9 wt%: (A) 0 wt%, (B)

1.6 wt%, (C) 5.6 wt%, and (D) 8.9 wt%. From Fujiwara, M., Okano, T., Amano, H., Asano, H., Oubu, K., 1997. Phase diagram of alpha-sulfonated palmitic acid methyl esters sodium salt/water system. Langmuir 13, 3346.

Fujiwara analyzed for the phase transition behavior quantitatively using the phase role and the Flory-Huggins-Scott equation (Wittman and Manley, 1977). C16MES-Na solids with 0%–8.9% moisture content were found to be a mixture of MES-Na crystals of anhydrate and 2 mol hydrate. C16MES-Na solids with greater moisture contents were also investigated. DSC of C16MES-Na solids at 8.9%–32.6% moisture content was carried out, and the results are shown in Fig. 9.7. Data similar to those in Fig. 9.6 were obtained, showing a shift of the endothermic peak corresponding to the phase transition of the MES-Na·2H2O to the lower-­ temperature side and the appearance of a new peak at 53°C. At 19.5% moisture content, a peak that apparently corresponds to the phase transition of the MES-Na·5H2O crystal

III.  BIOBASED SURFACTANTS

310

9.  Methyl Ester Sulfonate

0

20

Temperature (°C) 40 60 80

100

120

Moisture content (wt. %) 8.9 11.8 13.5 17.6

ENDO.

19.5 23.7 28.3 30.6 32.6

FIG. 9.7  DSC curves for C16MES-Na solids with moisture contents from 8.9 to 32.6 wt%. From Fujiwara, M., Okano, T., Amano, H., Asano, H., Oubu, K., 1997. Phase diagram of alpha-sulfonated palmitic acid methyl esters sodium salt/water system. Langmuir 13, 3347.

was observed. At 19.5%–32.6% moisture content, a shift of the endothermic peak corresponding to the phase transition of the MES-Na·5H2O crystals to the lower-­temperature side occurred, accompanied by a peak at 42°C, which was assigned to the phase transition of MES-Na·10H2O crystals. The transition temperature change in this moisture content range in all cases was attributed to the mixture of MES-Na·5H2O and MES-Na·10H2O crystals. Based on these findings, an overall phase diagram of the C16MES-Na/water system is proposed in Fig. 9.8, in which four different hydrated crystals (anhydrate, 2 mol hydrate, 5 mol hydrate, and 10 mol hydrate), two liquid crystals (lamellar and hexagonal), a micelle solution, and a monomer solution are presented. MES-Na crystals have several different hydrations, and this can be studied in relation to the atmosphere. The moisture content and X-ray diffraction spectra of MES-Na solid samples with 40% initial moisture content were determined at a constant relative humidity of 10% and ambient temperature (Fig. 9.9). The moisture content initially decreased rapidly for 5 h and then remained constant at 32 wt% up to 20 h. There was then a gradual decrease from 20 to 50 h, reaching 9 wt%, corresponding to the phase transition of MES-Na·2H2O. The moisture content then fell to 7 wt% and remained constant. The X-ray diffraction patterns indicated essentially the same results at from 40 to 10 wt% moisture content, where two peaks of 4.1 and 4.2 Å appeared. Finally, at 7 wt% moisture content, a single peak of 4.25 Å was observed.

III.  BIOBASED SURFACTANTS



311

9.2  Physical and Chemical Properties of Methyl Ester Sulfonate 120 M1

Temperature (°C)

100

L1

80

H1

+

L1

H1

La

+ La

H1

S2H2O

+La

60

S2H2O + La

S5H2O +H1?

40 20

S10H2O

S2H2O

S5H2O

+

S5H2O

+

Sum2o + M1

+ S2H2O

S10H2O

0 0

20

40

60

80

100

Surfactant concentration (wt. %)

FIG. 9.8  Phase diagram of the C16MES-Na-water system. H1, hexagonal liquid crystalline phase; L1, micellar solu-

tion phase; Lα, lamellar liquid phase; M1, monomer solution phase; S0H2, anhydrous MES; S2H2O, MES·2H2O; S5H2O, MES·5H2O; S10H2O, MES·10H2O. From Fujiwara, M., Okano, T., Amano, H., Asano, H., Oubu, K., 1997. Phase diagram of alpha-sulfonated palmitic acid methyl esters sodium salt/water system. Langmuir 13, 3345–3348.

Intensity (cps)

6K

3

Moisture content (wt. %)

40

20

6K

3

3 4.20 Å

0

30

6K

18

20

4.10 Å

22 2q (°)

4.10 Å 4.20 Å

4.10 Å 4.20 Å 24

26

0

18

20

22

2q (°)

24

26

0

18

20

6K

22 2q (°)

24

26

24

26

4.20 Å 4.25 Å

10H20

3 4.10 Å 0

5H20

18

20

6K

22

2q (°) 4.25 Å

10

0

3

2H20

50

100 Time (h)

150

0

60 days

18

20

22

24

26

2q (°)

FIG. 9.9  Time dependence of moisture content for the hydrated C16MES-Na solid. Conditions: initial moisture

content 40 wt%, 10% relative humidity, and room temperature. From Fujiwara, M., Okano, T., Amano, H., Asano, H., Oubu, K., 1997. Phase diagram of alpha-sulfonated palmitic acid methyl esters sodium salt/water system. Langmuir 13, 3348.

III.  BIOBASED SURFACTANTS

312

9.  Methyl Ester Sulfonate

Intensity (cps)

8.7K

8.7K 4.25 Å 4.25 Å 4.20 Å

4.35

4.10 Å 0 18

4.35

18

20

22 2q (°)

24

26

Intensity (cps)

8.7K

4.15 Å

4.35

22 2q (°)

24

0

26

18

20 22 2q (°)

24

26

8 4.20 Å 4.10 Å

0

20

Moisture content (wt. %)

Intensity (cps)

8.7K 4.25 Å

4.35

6

4

2

4.05 Å 0

18

20

22 2q (°)

24

26

0

10

20 Time (h)

30

40

120

FIG. 9.10  Time dependence of moisture content for the dry C16MES-Na solid. Conditions: initial moisture content

0.2 wt%, 80% relative humidity, and 50°C. From Fujiwara, M., Okano, T., Amano, H., Asano, H., Oubu, K., 1997. Phase diagram of alpha-sulfonated palmitic acid methyl esters sodium salt/water system. Langmuir 13, 3348.

MES-Na crystals in the dry state were also studied in a humid atmosphere. MES-Na solids with initial 0.2 wt% moisture content were maintained under 80% relative humidity at 50°C. Phase changes are shown in Fig. 9.10. The moisture content gradually increased up to 20 h and then leveled off near 25 h, where it became 8.6 wt%, corresponding to MES-Na·2H2O crystals. X-ray diffraction showed two peaks of 4.1 and 4.2 Å at 0.2–4 wt% moisture content and then a single peak of 4.25 Å at a moisture content less than 6 wt%. Recently, the crystalline structure of hydrated solids in mixed system composed of MES-Na and other surfactants (Watanabe et al., 2016) and the effect of the crystalline structure of MES induced by temperature and humidity history on the brittleness of grains (Watanabe et al., 2018) were also reported.

9.2.3 Biodegradability The biodegradability of MES-Na has been reported (Miura, 1991). Masuda et al. reported studies that used a different examination method and proposed a pathway of MES biodegradation by micrograms (Masuda et al., 1993a,b). These studies involved a shaking culture method, river die-away test, and biochemical oxygen demand measurement (Japanese Ministry of International Trade and Industry (MITI) test). Biodegradation was monitored by methylene blue active substances (MBAS), dissolved organic carbon (DOC), and biochemical

III.  BIOBASED SURFACTANTS



313

9.2  Physical and Chemical Properties of Methyl Ester Sulfonate

oxygen demand (BOD). Results from the shaking culture method are given in Fig. 9.11. It can be seen that MES and AOS-Na (alpha olefin sulfonate) lose more than 90% of methylene blue (MB) activity in a day and 100% within 2 days. LAS-Na lost MB activity in 5 days, and more than 40% of the dissolved organic carbon still remained after 15 days. MES-Na is thus shown to be readily biodegradable substances from the viewpoint of both primary and ultimate degradation under shaking culture conditions. The river die-away test, a biodegradation test using actual river water, was conducted on four different surfactants. Fig. 9.12 shows that MES-Na and AOS-Na surfactants quickly and 100

100

LAS DOC residue (%)

MBAS residue (%)

LAS

50 MES AOS

0

0

5

50 MES AOS

0

10

0

5

Time (day)

10

15

Time (day)

FIG. 9.11  Biodegradation of surfactants by shaking culture method. AOS, C11–C15AOS-Na; LAS, C10–C14LAS-Na;

and MES, C12–14MES-Na. From Masuda, M., Odake, H., Miuram K., Oba, K., 1993. Biodegradation of 2-sulfonatofatty acid methyl ester. I. J. Jpn. Oil Chem. Soc. 42, 645. 100 Surfactants 5 mg/L

MBAS residue (%)

80 AOS

60

MES LAS

Soap

40 20 0

0

1

2

3

4

5

Time (day)

FIG.  9.12  Biodegradation of surfactants in river water (river die-away test) detected by MBAS. Surfactants, 5 mg/L. AOS, C11–C15AOS-Na; LAS, C10–C14LAS-Na; MES, C12–14MES-Na; and Soap, C11–17COO-Na. From Masuda, M., Odake, H., Miuram K., Oba, K., 1993. Biodegradation of 2-sulfonatofatty acid methyl ester. I. J. Jpn. Oil Chem. Soc. 42, 646.

III.  BIOBASED SURFACTANTS

314

9.  Methyl Ester Sulfonate

100

100 mg/L

Surfactants Activated sludge

MES

30 mg/L

50

Biodegradability [(BOD/TOD)x100] (%)

LAS 0

0

10

20

30

100 MES 50

Surfactants Activated sludge

LAS 0

0

10

20

50 mg/L 30 mg/L

30

100 MES LAS

50

0

0

Surfactants Activated sludge

10

20

5 mg/L 10 mg/L

30

Time (day)

FIG. 9.13  Biodegradation of C12–14MES-Na and C10–C14LAS-Na detected by BOD in the MITI test. From Masuda, M., Odake, H., Miuram K., Oba, K., 1993. Biodegradation of 2-sulfonatofatty acid methyl ester. I. J. Jpn. Oil Chem. Soc. 42, 647.

easily undergo biodegradation, with total elimination of MB activity, within 3 days. For soap, 5 days were required for the loss of MB activity, suggesting that soap causes some kind of precipitation in river water whereby degradation is delayed. The results of the BOD/total oxygen demand (TOD) measurements in the MITI test are shown in Fig. 9.13. These results indicate that the biodegradation of MES-Na starts quickly and proceeds rapidly in the early stage at each surfactant concentration. For LAS-Na, degradation starts later and is in proportion to the increase in surfactant concentration. Results also indicate that both MES-Na and LAS-Na surfactants undergo ultimate biodegradation.

9.3  PRODUCTION OF METHYL ESTER SULFONATE The general MES-Na manufacturing process is shown in Fig. 9.14, and each stage is explained below (Itakura, 2004; Niikura et al., 2013):

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315

FIG. 9.14  MES-Na manufacturing process. ME refers to fatty acid methyl ester.

(1) A sulfonation stage, in which SO3 is chemisorbed by methyl ester (ME) to give an intermediate species. Generally, an SO3/ME molar ratio of 1.2 is used, and the reaction temperature is about 80°C. (2) An aging stage, in which the reaction mixture is kept at a high temperature in order to complete the reaction. Generally, it is performed at a minimum of 80°C. After aging, conversion of the initial ME is more than 98%. (3) An esterification stage, in which methanol is added to the reaction mixture, and the 1:2 adduct and disodium are converted into MES-H. The final yield of MES-H is generally over 90%. (4) A neutralization stage, in which aqueous sodium hydroxide is added to the esterified reaction mixture. (5) A bleaching stage, in which hydrogen peroxide is added to the neutralized reaction mixture. After bleaching, the color of the MES-Na product is generally less than 100. Acid bleaching prior neutralization is also established as an alternative to bleaching after neutralization.

9.4 APPLICATION 9.4.1  Powder Laundry Detergent The choice of surfactants for laundry detergents is based on factors such as performance, manufacturing technology, social considerations, and demands for feedstock. In Japan, in response to environmental concern in the 1970s, zeolite replaced polyphosphate as a detergent builder, and new surfactants from natural fats and oils attracted much attention. MESs, which appeared in 1991, were considered suitable for a compact detergent in Japan.

III.  BIOBASED SURFACTANTS

316

9.  Methyl Ester Sulfonate

FIG. 9.15  Relationship between detergency and concentration of surfactant. Conditions: Terg-O-Tometer, artificial soil (cotton), 25°C, water hardness 54 ppm (CaCO3), surfactant 270 ppm, sodium carbonate 135 ppm, and sodium silicate 135 ppm. Key: ◆C18MES-Na, ■C16MES-Na, *C14MES-Na, □C10–14LAS-Na, and ◇C12AS-Na.

The practical detergency and the surface activity of MESs are discussed in the following sections, along with formulations and the manufacturing process. 9.4.1.1 Detergency The detergency of MES-Na for different alkyl chain lengths has been evaluated for comparison with C10–14LAS-Na and C12AS-Na at low temperature and water hardness (Satsuki, 1992). Detergency was studied using the Terg-O-Tometer (US testing) and artificial soiled swatches as fabrics at 120 rpm, 25°C, and a 10 min wash plus two rinses of 3 min each. Sodium carbonate (135 ppm) and sodium silicate (35 ppm) were used in each washing test. Water hardness was controlled to 3°DH. Detergency was determined based on a swatch reflectance before and after washing and the Kubelka-Munk equation (Okumura et al., 1980). The swatches were prepared by soaking unsoiled swatches in an aqueous dispersion containing oil, protein, and mineral components, followed by drying in air. Detergency test results on three different surfactants as a function of concentration are shown in Fig.  9.15. C16MES-Na and C18MES-Na showed better detergency than LAS-Na or, in particular, AS-Na, and the best results were obtained at low surfactant concentrations. C16MES-Na gave the best detergency, followed by C18MES-Na and then C14MES-Na. Detergency followed the order C16MES-Na ≥ C18MES-Na > LAS-Na > AS-Na, C14MES-Na. The effects of temperature (Fig.  9.16) and cloth-to-liquor ratio (Fig.  9.17) on detergency have been evaluated (Satsuki, 1992). Detergency decreases with reduction in temperature, and results for MESs were best at low temperatures and low liquor ratio. The surface-active properties of the three surfactants were examined to determine in performance. Adsorption onto particle soil, dispersion of clay, emulsification of oily soil, and the zeta potential of oil droplets in the emulsion system were measured for C14–16MES-Na, LAS-Na, and AS-Na solution as a function of surfactant concentration. The MES-Na surfactants gave the best results in all cases as shown in Figs. 9.18 and 9.19 (adsorption and emulsification, respectively) (Satsuki, 1992). III.  BIOBASED SURFACTANTS



9.4 Application

317

FIG. 9.16  Effect of washing temperature on detergency. Conditions: Terg-O-Tometer, artificial soil, liquor ratio 30, water hardness 54 ppm (CaCO3), surfactant (AS, C12AS-Na; LAS, C10–14LAS-Na; and MES, C14–16MES-Na) 270 ppm, sodium carbonate 135 ppm, and sodium silicate 135 ppm.

FIG.  9.17  Effect of cloth-to-liquor ratio (w/w) on detergency. Conditions: Terg-O-Tometer, artificial soil, 25°C, water hardness 54 ppm (CaCO3), surfactant (AS, C12AS-Na; LAS, C10–14LAS-Na; and MES, C14–16MES-Na) 270 ppm, sodium carbonate 135 ppm, and sodium silicate 135 ppm. From Satsuki, T., Umehara, K., Yoneyama, Y., 1992. Performance and physicochemical properties of α-sulfo fatty acid methyl esters. J. Am. Oil Chem. Soc. 69, 675.

The adsorption of each surfactant on clay particles was determined for an aqueous dispersion prepared by agitating red-yellow diluvium for 3 h in the presence of each surfactant. In the emulsifying tests, emulsified oil was proportional to surfactant concentration. 9.4.1.2 Solubilization MES-Na has superior solubilization capacity, which is essential for characterizing surfactants. MES-Na was found to be highly capable of solubilizing oleic acid, which is usually present abundantly in natural soil. The solubilization capacity for LAS-Na, AS-Na, and C14–16MES-Na III.  BIOBASED SURFACTANTS

318

9.  Methyl Ester Sulfonate

FIG.  9.18  Adsorption of surfactants onto particle soil. Conditions: clay 0.4%, 25°C, and sodium carbonate 270 ppm. AS, C12AS-Na; LAS, C10–14LAS-Na; and MES, C14–16MES-Na. From Satsuki, T., Umehara, K., Yoneyama, Y., 1992. Performance and physicochemical properties of α-sulfo fatty acid methyl esters. J. Am. Oil Chem. Soc. 69, 676.

FIG. 9.19  Emulsification of oily soil by surfactants. Conditions: artificially oily soil 0.2%, 25°C, and sodium sulfate 270 ppm. AS, C12AS-Na; LAS, C10–14LAS-Na; and MES, C14–16MES-Na. From Satsuki, T., Umehara, K., Yoneyama, Y., 1992. Performance and physicochemical properties of α-sulfo fatty acid methyl esters. J. Am. Oil Chem. Soc. 69, 676.

was evaluated using oleic acid as a polar oil solubilizate and nonpolar n-­octane. Fujiwara investigated the solubilization of MES-Na and obtained the following results (Fujiwara et al., 1995). First, C14–16MES-Na had a larger solubilization capacity than LAS-Na. Second, capacity depended on the number of micelles and degree of solubilization per micelle. Third, micellar weight initially decreased for a small amount of solubilizate and then increased with increasing solubilizate amount. Fourth, the solubilization process was accompanied by the reconstitution of micelles.

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9.4 Application

319

9.4.1.3  Enzyme Stability Enzymes are essential components of detergents. Surfactants have significant influence on enzyme activity in detergents. A stable enzyme and surfactant system is desirable for optimal enzyme functioning in washing. Nonionic surfactants have little effect on enzyme activity, whereas cationic surfactants have considerable effect. Anionic surfactants exert an intermediate effect, the extent depending on the surfactant. The effects on three anionic surfactants on enzyme activity were examined using protease as the detergent enzyme (Satsuki, 1992; Satsuki et al., 1999). Activity stability in a washing liquor was assessed at low temperature, low water hardness, and weak alkaline pH. The results are shown in Fig. 9.20. Enzyme activity decreased with time for all the surfactants. In MES-Na solution, only a slight decrease in activity was noted, whereas that for LAS-Na and AS-Na solutions, it was significant; residual activity for either solution was below 50% after standing for 2 h at 25°C. The inhibition of enzyme activity due to surfactants is thought to be caused by adsorption of the surfactant onto the enzyme, leading to denaturation of the enzyme protein. 9.4.1.4 Formulations The best feature of MESs is that its surfactant content can be reduced without compromising performance. Formulations containing MES-Na are listed in Table 9.4 (Satsuki, 1986). Compared with the popular formulation of LAS-Na/AS-Na or LAS-Na/AOS-Na, those of MESs allow for sufficient detergency with low surfactant content, with no need for additional sequestering or alkaline builders. Detergent powders containing MES-Na as the main component have various technical problems, such as a low dissolving rate in low-temperature water, possibly due to the high Krafft point and high crystallinity of MES-Na. Ester bonds in the MES-Na molecule may easily undergo hydrolysis with consequent formation of the disodium salt, which has low surface activity. The hydrolysis of MES-Na in detergent powder may occur on contact with alkaline components.

FIG. 9.20  Effect of surfactants on protease activity. Conditions: surfactant 300 ppm, protease 0.008 AU/L, pH 10.5, and 40°C. AS, C12AS-Na; LAS, C10–14LAS-Na; and MES, C14–16MES-Na.

III.  BIOBASED SURFACTANTS

320

9.  Methyl Ester Sulfonate

TABLE 9.4  Formulations of MES-Based Detergents Component

LAS-Based

MES-Based

MES-/LAS-Based

Surfactant

30–40

30–40

30–40

(LAS/AOS and LAS/AS) Builder

15–25

15–25

15–25

Alkali

15–25

15–25

15–25

Enzyme

+

+

+

FWA

+

+

+

AS, C12AS-Na; LAS, C10–14LAS-Na; MES, C14–16MES-Na; and FWA, fluorescent whitening agent.

FIG. 9.21  Krafft point of MES/LAS mixture. LAS, C10–14LAS-Na; MES, C14–16MES-Na.

A new surfactant system, which uses LAS-Na with MES-Na, has been established, and good results obtained from it, as shown in Table 9.4. This system not only eliminates the disadvantages of MES-Na but also enhances washing detergency at low temperatures. The surfactant properties of the MES-Na/LAS-Na blend were investigated physicochemically by Krafft points determined from the cross temperature point of the solubility line and the CMC lines against temperature (Fig. 9.21). The value of the Krafft point was determined with increasing LAS-Na content. The blend was also studied by DSC using a surfactant powder with 5% moisture content. MES-Na powder is a 2 mol hydrate, judging from moisture content. Fig. 9.22 shows the DSC pattern for C14–16MES-Na. An endothermic peak can be seen at 39°C, lower than that of C16MES-Na (see Fig. 9.5). For LAS-Na powder, no endothermic peak was observed. MES/ LAS in a one-to-one ratio clearly showed a smaller DSC peak, indicating that the MES-Na powder no longer had high crystallinity. DSC and Krafft point data suggest that MES-Na

III.  BIOBASED SURFACTANTS



9.4 Application

321

FIG. 9.22  DSC profiles of C14–16MES-Na/C10–14LAS-Na mixture. Sample: surfactant powder, water content 5%, and DSC condition 1°C/min.

FIG. 9.23  Detergency of C14–16MES-Na/C10–14LAS-Na mixture. Conditions: Terg-O-Tometer, artificial soil (cotton), water hardness 54 ppm (CaCO3), surfactant (MES + LAS) 200 ppm, sodium carbonate 200 ppm, and zeolite 150 ppm.

should be mixed with LAS-Na at a molecular level to reduce the DSC endothermic peak and falloff of the Krafft points. This mixing would also enhance detergency, which was assessed under the usual conditions using artificial swatches (Fig. 9.23). Detergency was found to increase with the ratio of MES-Na to LAS-Na at 25°C; the detergency was a maximum at oneto-one blend ratio.

9.4.2  Liquid Laundry Detergent 9.4.2.1  Detergency at Neutral pH Liquid detergent has several advantages over powder detergents. Liquid detergents readily and completely dissolve in water, even in cold water; thus, liquid detergent is less messy than powder detergents. In the form of liquids, problem such as caking upon storage is eliminated as often seen with powders when exposed to moisture. However, the challenges arise

III.  BIOBASED SURFACTANTS

322

9.  Methyl Ester Sulfonate

FIG. 9.24  Solubilization temperatures for a combination of C16–18MES and other surfactants. The total concentration of the surfactants is 20 wt%, and the pH is set at 7.5.

in formulating a stable liquid detergent from C16–18MES-Na because of high Krafft point and low solubility at low temperature (Zulina et al., 2017). Tobori et  al. reported the formulation to decrease the solubilization temperature of the C16–18MES-Na solution, along with the combined use of other surfactants (Tobori, 2017; Kubozono et al., 2015). Fig. 9.24 shows the solubilization temperatures for a combination of C16–18MES-Na and other surfactants. When a combination of appropriate surfactants such as alcohol ethoxylate with 7 mol ethylene oxide (C12AE (7EO)), alcohol ether sulfate (C12AES (2EO)-Na), and C10–14LAS-Na was used, the solution containing 5 wt% MES-Na remained clear below 0°C (Fig. 9.24). Moreover, the detergency of MES-Na for sebum was higher than that of AES-Na or LAS-Na under liquid detergent conditions. We confirmed that the solubility of MES-Na could be improved by enhancing the detergency of sebum secreted by the sebaceous gland in humans in a liquid laundry detergent system. Based on these observations, we conclude that MES-Na has great potential to be used as an ingredient in liquid detergents, even in low-temperature conditions.

9.5 CONCLUSION Methyl ester sulfonates are derived from renewable sources. They have excellent surfactant properties. They are easily incorporated into formulations that meet many different requirements, such as cold-water washing, and they provide the advantages of low cost, excellent surfactant properties, low aquatic toxicity, and rapid biodegradability. In addition, they are mild and safe for the human skin. The employment of MES in detergents is increasing, and certainly, this trend will continue in the future.

III.  BIOBASED SURFACTANTS



9.5 Conclusion

323

References Boucher, E.A., Grinchuk, T.M., Zttlemoyer, A.C., 1968. Surface activity of sodium salts of alpha-sulfo fatty esters; the air-water interface. J. Am. Oil Chem. Soc. 45, 49–52. Drozd, J.C., Desai, D.D., 1991. Liquid laundry detergents based on soap and alpha-sulfo methyl esters. J. Am. Oil Chem. Soc. 68, 59–62. Fujiwara, M., Miyake, M., Abe, Y., 1993. Colloidal properties of alpha-sulfonated fatty acid methyl esters and their applicability in hard water. Colloid Polym. Sci. 271, 780–785. Fujiwara, M., Kaneko, Y., Oubu, K., 1995. Light scattering study on the micellar systems solubilizing a fatty acid. Colloid Polym. Sci. 273, 1055–1059. Fujiwara, M., Okano, T., Amano, H., Asano, H., Oubu, K., 1997. Phase diagram of alpha-sulfonated palmitic acid methyl esters sodium salt/water system. Langmuir 13, 3345–3348. Hato, M., Shinoda, K., 1973. The solubilities, critical micelle concentrations, and Krafft points of bivalent metal alkyl surfaces. Bull. Chem. Soc. Jpn. 46, 3889–3890. Hayashi, S., Ikeda, S., 1980. Micelle size and shape of sodium dodecyl sulfate in concentrated NaCl solution. J. Phys. Chem. 84, 744. Itakura, K., 2004. Powders, flakes, or pellets containing salts of alpha-sulfofatty acid alkyl esters in high concentrations, process for production thereof, granulated detergents, and process for production thereof, WO2004111166A1. Kapur, B.L., Solomon, J.M., Bluestein, B.R., 1978. Summary of the technology for the manufacture of higher a­ lpha-sulfo fatty acid esters. J. Am. Oil Chem. Soc. 55, 549–557. Kekicheff, P., Grabielle-Madelmont, C., Ollivon, M., 1989. Phase diagram of sodium dodecyl sulfate-water system: a calorimetric study. J. Colloid Interface Sci. 131 (1), 112–132. Kubozono, T., Morimoto, Y., Endo, C., Otsuka, S., Tobori, N., 2015. New features of methyl ester sulfonate (MES) for laundry detergent. In: Proceeding of the 10th World Surfactant Congress and Business Convention (CESIO), Istanbul, pp. 126–129. Masuda, M., Odake, H., Miuram, K., Oba, K., 1993a. Biodegradation of 2-sulfonatofatty acid methyl ester. I. J. Jpn. Oil Chem. Soc. 42, 643–647. Masuda, M., Odake, H., Miura, K., Ito, K., Yamada, K., Oba, K., 1993b. Biodegradation of 2-sulfonatofatty acid methyl ester. II. J. Jpn. Oil Chem. Soc. 42, 905–909. Maurer, E.W., Weil, J.K., Linfield, W.M., 1977. The biodegradation of esters of alpha-sulfo fatty acids. J. Am. Oil Chem. Soc. 54, 582–584. Miura, M., 1991. Performances of fatty acid alpha-sulfomethyl esters (3). Biodegradation. In: 3rd Annual Meeting of Home Economics. vol. 3, pp. 1099–1108. Niikura, F., Omine, M., Kimura, Y., Konta, H., Kageyama, M., Tobori, N., Araki, K., 2013. Coloration process in the sulfonation of fatty acid methyl ester with sulfur trioxide. J. Am. Oil Chem. Soc. 90 (6), 903–909. Okumura, O., Sakatani, T., Yamane, I., 1976. Mechanism of sulfonation of fatty acid esters with sulfur trioxide and properties of alpha-sulfo fatty acid esters. In: Proceedings of the 7th Committee International des Derives TensoActs. vol. 1. Mowcow, pp. 225–234. Okumura, O., Tokuyama, K., Sakatani, K., Tsuruta, T., 1980. J. Jpn. Oil Chem. Soc. 30, 432–441. Satsuki, T., 1986. Methyl ester sulfonates: a surfactant based on natural fats. In: Proceeding of the 3rd World Conference on Detergents, Monteux, pp. 135–140. Satsuki, T., 1992. Application of MES in detergents. Inform 3, 1099–1108. Satsuki, T., Tobe, S., Yoneyama, Y., Mukaiyama, K., 1999. Blending effect of different kind of surfactant on enzyme activity. Mater. Technol. 17, 119–125. Schambli, F., Schwuger, M.J., 1990. Physico-chemical properties of alpha-sulfo fatty acid methyl esters and alpha-sulfo fatty acid di-salts. Tenside Surfactant Deterg. 27 (6), 380–389. Shinoda, K., 1963. The formation of micelles. In: Colloidal Surfactants. Academic Press, New York, pp. 55–57. Shinoda, K., Maekawa, M., Shibata, M., 1986. Ionic surfactants soluble in hard water and hydrocarbons: behavior of organized surfactant solutions as a function of the hydrophilic-lipophilic balance. J. Phys. Chem. 90, 1228–1230. Smith, N.R., 1989. Alpha-sulfo methyl esters: a new alternative. Soap Cosmet. Chem. Spec 48–57. April. Steber, J., Wierich, D., 1989. The environmental fate of fatty acid alpha-sulfomethyl esters. Tenside Surfactant Deterg. 26, 406–411.

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324

9.  Methyl Ester Sulfonate

Stein, W., Baumann, H., 1975. Alpha-sulfonated fatty acids and esters; manufacturing process, properties and applications. J. Am. Oil Chem. Soc. 52, 323–329. Steliner, K.L., Scamehom, J.F., 1989. Hardness tolerance of anionic surfactant solutions I. Anionic surfactant with added monovalent electrolyte. Langmuir 5, 70–74. Stirton, A.J., 1968. Alpha-sulfo fatty acids and derivatives; synthesis, properties and use. J. Am. Oil Chem. Soc. 39, 490–496. Stirton, A.J., Weil, J.K., Bistline Jr., R.G., 1954. Surface-active properties of salts of alpha-sulfo fatty acid methyl esters and alpha-sulfo fatty acid and esters. J. Am. Oil Chem. Soc. 31, 13–16. Stirton, A.J., Bistline Jr., R.G., Weil, J.K., Maurer, J., 1962. Sodium salts of alkyl esters of alpha-sulfo fatty acids; wetting, lime soap dispersion and related properties. J. Am. Oil Chem. Soc. 39, 128–131. Tobori, N., 2017. The Use of Palm Oil-based Detergents toward Sustainable Society. In: Proceeding of the MPOB International Palm Oil Congress & Exhibition (PIPOC 2017), Kuala Lumpur, OS2, pp. 16–20. Watanabe, H., Morigaki, A., Kaneko, Y., Tobori, N., Aramaki, K., 2016. Effect of temperature and humidity history on brittleness of α-sulfonated fatty acid methyl ester salt crystals. J. Oleo Sci. 65 (2), 143–150. Watanabe, H., Morigaki, A., Yuba, M., Yamada, K., Miyake, M., Tobori, N., Aramaki, K., 2018. Structural analyses of hydrated crystals in mixer green surfactant systems: α-sulfonated fatty acid methyl ester salt and fatty acid soap mixture. J. Surfactant Deterg. 21, 221–229. Weil, J.K., Bistline Jr., R.G., Stirton, A.J., 1953. Sodium salts of alkyl alpha-sulfo-palmitate and stearates. J. Am. Oil Chem. Soc. 75, 4859–4860. Wittman, J.C., Manley, R.S.J., 1977. Polymer-monomer binary mixture. I. Euretic and epitaxial crystallization in poly(e-caprolactone)-trioxane mixtures. J. Polym. Sci. B Polym. Phys. 15, 1089–1100. Yamane, I., Miyawaki, Y., 1989. Manufacturing process of alpha-sulfomethyl esters and their application to detergents. In: Proceedings of 1989 International Palm Oil Development Conference: Chemistry, Technology and Marketing, Malaysia, pp. 132–141. Zulina, A.M., Zainab, I., Razmah, G., 2017. Performance of palm-based C16/18 methyl ester sulphonate (MES) in liquid detergent formulation. J. Oleo Sci. 66 (7), 677–687.

Further Reading Satsuki, T., Umehara, K., Yoneyama, Y., 1992. Performance and physicochemical properties of α-sulfo fatty acid methyl esters. J. Am. Oil Chem. Soc. 69, 672–677.

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C H A P T E R

10 Sugar Esters Sang-Hyun Pyo⁎, Jiazhi Chen†, Ran Ye‡, Douglas G. Hayes§ ⁎

Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden †Guangdong Provincial Key Laboratory of Industrial Surfactant, Guangdong Research Institute of Petrochemical and Fine Chemical Engineering, Guangzhou, P. R. China ‡Roha USA, St. Louis, MO, United States §Department of Biosystems Engineering and Soil Science, University of Tennessee, Knoxville, TN, United States

10.1  SUGAR ESTERS: PROPERTIES AND APPLICATIONS 10.1.1 Introduction Saccharide-fatty acid esters (sugar esters, SEs), value-added products derived from natural feedstocks such as corn or other plant oils and starch, cellulose, or other biobased polysaccharides, are nonionic surfactants commonly employed in foods, cosmetics, and pharmaceutical industries globally (Nakamura, 1997; Neta et  al., 2015; Szuts et  al., 2007) (Fig.  10.1). They possess good biocompatibility and environmental profiles and also biological activity. Their market size is projected to be $74.6 million by 2020, projected based on a 6% annual growth rate (Markets and Markets Inc., 2016). Europe possesses the largest market share, followed by North America and Asia-Pacific (Markets and Markets Inc., 2016). A market sector undergoing growth for SE consumption is the expanding middle-class population in the Asia-Pacific region, who are experiencing an increase of disposable income (Markets and Markets Inc., 2016). A list of manufacturers that produce SEs is given in Table 10.1. SEs exist as a liquid or a solid at room temperature, with monoesters existing as solids and polyesters and esters of unsaturated fatty acyl groups (e.g., oleyl, linoleyl, or linolenyl) existing as liquids. They are typically formed by creating ester bond(s) between sugars and fatty acids in vitro; however, they also naturally occur in some plants, such as the glandular trichomes found on the leaf surface of tobacco (Nicotiana tabacum) and other members of Solanaceae, where they occur as mixtures of glucose and sucrose esters containing short- and medium-chain acyl lengths that possess branching (Kroumova et  al., 2016). Common acyl acceptors include mono-, di-, and trisaccharides, such as glucose, fructose, xylose, ribose, sucrose, lactose, trehalose, and maltotriose, and sugar alcohols (e.g., sorbitol and xylitol, noting sorbitan esters are common commercial nonionic surfactants, Span). Recently, enzymes have

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Copyright © 2019 AOCS Press. Published by Elsevier Inc. All rights reserved.

326

10.  Sugar Esters

3

3

+ OHH

H

H

HO 6¢

O H

O OH

H

HO OH

OH

1¢

6

O H

H 2O

O

Oleic acid

O

Lipase

6

O H OH

O OH

H

H2O

H

HO OH

OH H

HO

OH

3

3

H

1¢

OH

6¢

O H

OH

OH H

HO

H

Sucrose-6-monooleate

H

Sucrose

O

H2O

Lipase

O OH

3

3

6

O H

O OH

H H2O

HO OH

H H

1¢

O 3

6¢

O H

OH

3

OH H

HO

H

Sucrose-6,1¢-dioleate

FIG. 10.1  Preparation of sugar esters (sucrose-oleic acid mono- and diesters). The enzyme lipase is employed in this example as biocatalyst.

TABLE 10.1  Sugar and Sugar Alcohol Ester Manufacturers Manufacturer

Location

Adana Foods

Liuzhou, China

BASF

Ludwigshafen, Germany

Emulgade

Compass Foods

Singapore

“Habo”

Croda

Cowick Hall, United Kingdom

Crodesta sugar esters, Span and Tween

Dai-Ichi Kogyo Seiyaku (DKS)

Kyoto, Japan

“DK Ester”

Evonik

Essen, Germany

Guangxi Gaotong Food Technology Company

Liuzhou, China

Hangzhou Jinhelai Food Additive

Hangzhou, China

Mitsubishi-Kagaku Foods

Tokyo, Japan

Sisterna B.V.

Roosendaal, The Netherlands

Stearinerie-Dubois

Boulogne, France

III.  BIOBASED SURFACTANTS

Product Information

“Ryoto”

Sucrose esters for cosmetics



10.1  Sugar Esters: Properties and Applications

327

been employed to acylate oligosaccharides, including dextrans and starches, (Alissandratos and Halling, 2012; van den Broek and Boeriu, 2013; Kaewprapan et al., 2012a,b; Zhao et al., 2008). Another recently used acyl acceptor is a saccharide conjugated enzymatically with an α,ω-diol at its reducing end, which contains a free primary OH group from the diol moiety that is readily acylated (Kurakake et al., 2011). Typical acyl donors consist of fatty acyl groups of 12–18 carbons. However, medium-chain acyl groups (e.g., butyrate and caprylate) have been employed to prepare diesters of sugar alcohols such as mannitol and sorbitol that have utility as gelators for the removal of petroleum spills in watersheds (Jadhav et al., 2010). The use of 4-hydroxyphenyl propionate acyl groups (i.e., derivatives of lignin) has also been investigated (Croitoru et al., 2011). Common forms of the acyl donor are free fatty acids (FFAs) or fatty acid methyl esters (FAME). This chapter provides an overview of SEs, describing chemical properties, applications, and their synthesis by chemical and enzymatic approaches.

10.1.2 Properties SEs are versatile surfactants that can emulsify water in oil or vice versa, by control of their hydrophilic-lipophilic balance (HLB) and also their melting point temperature, through the chemistry of their building blocks (e.g., chain length and degree of saturation for their fatty acyl moiety and degree of oligomerization or derivatization of hydroxyls for their saccharide component) and the number of fatty acyl groups per molecule (Sabeder et al., 2006; Csoka et al., 2007; Neta et al., 2015). HLB is calculated for nonionic surfactants by multiplying the ratio of the surfactant head group’s molecular weight to the molecular weight of the surfactant by 20 (Griffin’s method) (Rosen and Kunjappu, 2012). Surfactants with HLB ≥ 11 are more hydrophilic, hence water-soluble, and therefore commonly used to form oil-in-water (o/w) emulsions (e.g., SEs enriched in monoesters; employed in ice cream and cake batter) (Figs. 10.2 and 10.3). Alternatively, SEs possessing low HLB values (5–7) are oil-soluble and hence used to form w/o emulsions (e.g., SEs enriched in di- and triesters; employed in chocolates), while those possessing intermediate HLB values (8–10) are equally balanced in hydrophilicity and lipophilicity (used in chewing gum) (Figs. 10.2 and 10.3). Olestra, a fully acylated sugar, is not amphiphilic but previously served as a fat substitute (HLB ~ 3) (Lawson et al., 1997). SEs are well known to be effective surfactants, noted by the achievement of surface tension and critical micellar concentration (CMC) of 25–40 mN/m and 0.05–0.20 g/L, respectively (Nakamura, 1999; Garofalakis et al., 2000; Scheckermann et al., 1995; Husband et al., 1998; Soultani et al., 2003; Nelen and Cooper, 2004). CMC of SEs decreases with acyl chain length (Becerra et al., 2008) and the number of acyl groups per molecule (Husband et al., 1998), as would be expected from theory (Rosen and Kunjappu, 2012). They also are good foam stabilizers (Nelen and Cooper, 2004). SEs are stable in the 4–8 pH range and at temperatures below 140°C; at higher temperatures, discoloration can occur due to caramelization (Nelen and Cooper, 2004). SEs possessing acyl chain lengths of ≥14 carbons are poorly soluble in water (Polat and Linhardt, 2001). Their possession of ester bonds imparts high biodegradability and biocompatibility, which drives their utilization (EFSA Panel on Food Additives Nutrient Sources added to Food, 2010). In addition, SEs are odorless, flavorless, and nontoxic; do not irritate the skin; and can be easily digested by the stomach, readily undergoing hydrolysis into their fatty acid and saccharide constituents (EFSA Panel on Food Additives Nutrient Sources added to Food, 2010;

III.  BIOBASED SURFACTANTS

328 Fatty acyl group

10.  Sugar Esters

HLB 1

2

3

5

C12 C14

7

9

11

16

Detergent

Powdered milk

Fats & oils chocolate

15

Ice cream

C16

Curry roux

Wheat products dough

Confections

Tablet C18

Milk beverage Frozen dough

Chewing gum Cake batter

W/O emulsion O/W emulsion

Beverage C18:1

Sauce, dressing

Shortening chocolate

Ice cream

Seasoning

C22:1 C22

FIG. 10.2  Application map of sucrose fatty acid esters. Reprinted from Otomo, N., 2009. Basic properties of sucrose fatty acid esters and their application. In: Hayes, D.G., Kitamoto, D., Solaiman, D.K.Y., Ashby, R.D. (Eds.), Biobased Surfactants and Detergents: Synthesis, Properties, and Applications. AOCS Press, Champaign, IL, pp. 275–298, with permission from AOCS Press.

HLB 16

Mono Di Tri Tetra Penta Hexa Hepta Octa

11 7 3 1 0%

50%

100%

FIG. 10.3  Relationship between hydrophilic-lipophilic balance (HLB) and ester composition of sucrose stearates. Reprinted from Otomo, N., 2009. Basic properties of sucrose fatty acid esters and their application. In: Hayes, D.G., Kitamoto, D., Solaiman, D.K.Y., Ashby, R.D. (Eds.), Biobased Surfactants and Detergents: Synthesis, Properties, and Applications. AOCS Press, Champaign, IL, pp. 275–298, with permission from AOCS Press.

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329

10.1  Sugar Esters: Properties and Applications

Neta et al., 2015; Kobayashi, 2011). They have received “generally recognized as safe” (GRAS) status by US Food and Drug Administration and have an equivalent status in the European Union and other countries (EFSA Panel on Food Additives Nutrient Sources added to Food, 2010; US Food and Drug Administration, 2017). SEs possess a wide range of biological activities: anticancer, antitumor, antimicrobial, antibiotic, anti-insect, and plant growth inhibition (reviewed in Ye and Hayes, 2017). SEs’ antimicrobial activity may be due to its adsorption to cell membranes, where it may alter biomembrane permeability or induce cell lysis. Their activity against insects may be due to their ability to dewax, hence degrade, the protective coatings on insects.

10.1.3 Applications Applications of SEs are listed in Table 10.2. The industrial sector utilizing SEs to the greatest extent is food, followed by personal care products and detergents and cleaners (Markets and Markets Inc., 2016). The solid (powdered) form of SEs is the most abundantly used form, with the personal care sector using the powdered form almost solely (Markets and

TABLE 10.2  Applications of Sugar Esters and Their Derivatives Application

Sugar Esters Type

References

Food emulsifiersa

Various

Otomo (2009), Kralova and Sjöblom (2009), Nakamura (1999), Husband et al. (1998), Ferrer et al. (2002)

Dispersion of solids (e.g., calcium)

Sucrose monostearate

Otomo (2009)

Demulsification of heavy petroleum sludge

Ethoxylated glucose esters

Abdul-Raheim et al. (2013)

Skin and personal care products

Various

Polat and Linhardt (2001)

Gelators of organic solvents, oil spills

Diesters of trehalose, sugar alcohols

Jadhav et al. (2010), John et al. (2006)

Span surfactants

Sorbitan esters

Heuschkel et al. (2008)

Tween surfactants

Polyethoxylated sorbitan esters

Bhattacharya and Palepu (2004), Heuschkel et al. (2008)

Dental and oral care

Various

Reviewed in Szűts et al. (2010), Szűts and Szabó-Révész (2012)

FOODS AND PERSONAL CARE

Drug delivery systems (reviewed in Szűts et al., 2010; Szűts and Szabó-Révész, 2012) Transdermal

Sucrose esters

Csoka et al. (2007), Okamoto et al. (2005)

Tableting

Sucrose esters

Chansanroj and Betz (2010), Otomo (2009) Continued

III.  BIOBASED SURFACTANTS

330

10.  Sugar Esters

TABLE 10.2  Applications of Sugar Esters and Their Derivatives—cont’d Application

Sugar Esters Type

References

Nanoparticle encapsulation

Dextran decanoate esters

Kaewprapan et al. (2012b), AbdelMageed et al. (2012), Youan Bi-Botti et al. (2003)

Microemulsions

Sucrose esters

Fanun (2017)

Nanoemulsions

Klang et al. (2011)

ANTIMICROBIAL AGENTS Bacillus sp., Lactobacillus plantarum Lauric acid esters of sucrose and maltose

Ferrer et al. (2005a)

Spore-forming, heat-resistant bacteria (canned beverages)

Sucrose monopalmitate

Otomo (2009)

Sucrose monolaurate

Xiao et al. (2011)

Sucrose esters

Furukawa et al. (2010)

Escherichia coli (on spinach) b

Foodborne pathogenic bacteria

Insecticidal agents (reviewed in Ye and Hayes, 2017) Aphis glycines, Lymantria dispar

Diester of sucrose on octanoic acid

Song et al. (2006)

Sweet potato whiteflies, aphids

Diesters of sucrose, heptanoicnonanoic acid

Chortyk et al. (1996)

Whiteflies

Sucrose esters

Liu et al. (1996)

Tobacco aphid

Sucrose esters

Xia et al. (1998)

a

Several applications are provided in Fig. 10.2. Staphylococcus aureus, E. coli, Streptococcus mutans, and Listeria monocytogenes. Modified from Ye, R., Hayes, D.G., 2014. Recent progress for lipase-catalysed synthesis of sugar fatty acid esters. J. Oil Palm Res. 26(4), 355–365.

b

Markets Inc., 2016). Applications of SEs in foods are provided in Fig.  10.2. These include their role as oil-in-water emulsifiers (HLB > 9): beverages, batters, baked goods, coffee and whipping creams, yogurt, mayonnaise, sauces (e.g., of cheese and mushroom), cereal bars, chewy soft candies (e.g., fudge), baked foods (e.g., breads and sponge cake), confectionery foods, liqueurs, and fruit drinks (Nakamura, 1999; Nelen and Cooper, 2004). They also emulsify water into oil (HLB 3 are widely used in reactions such as the lipase-catalyzed interesterification of oils and fats to obtain new fats with improved physical and/or nutritional properties (Villeneuve, 2007). In addition, the stability of the enzyme is usually higher in the more hydrophobic solvents. However, sugar and polyol acyl acceptor substrates are poorly soluble in these solvents, which make them unsuitable. In contrast, solvents with lower log P values (e.g., pyridine, DMF, tert-butanol, and acetone) can partially cosolubilize acyl donors and many polar saccharides (Tables 10.7 and 10.8) and thus can be employed for polyol ester synthesis (Villeneuve, 2007). However, these solvents often inactivate the enzyme by their ability to remove tightly bound water molecules, which are essential for biocatalytic activity; promote accumulation of water in the reaction media, leading to hydrolysis and hence reduced product yield; and are often incompatible with food applications (Degn and Zimmermann, 2001; Ganske and Bornscheuer, 2005; Wang et al., 2016). The actual amount of bound water needed varies significantly depen­ ding on the origin and composition of the enzyme preparation (Yahya et al., 1998). The solvent’s polarity can also influence the rate and extent of acyl migration, in which acyl groups of polyol-fatty acid esters migrate between different hydroxyls of the acyl acceptor moiety (e.g., isomerization of 1- and 2-monoacylglycerols) (Compton et al., 2007). The effect of organic solvent type on the lipase-catalyzed esterification of saccharide and fatty acid will now be illustrated by discussing some recent examples in the literature. The monoesterification of glucose with stearic acid catalyzed by immobilized lipases from Candida sp. in acetone in the presence of molecular sieves was demonstrated, and the kinetics were described by a two-substrate Michaelis-Menten-based model, the Ping-Pong bi-bi mechanism, which predicts a time course consisting of a linear rapid conversion of substrate during the initial few hours, followed by a gradual reduction of the reaction rate as time is increased (Yu et al., 2008). When solvent is used, it is important to provide a means of removal for the released hydroxyl-containing products, water and n-alkanol (methanol) derived from FFA, and alkyl ester (FAME) acyl donors, respectively, to allow for a high polyol ester yield. Direct esterification of fructose with lauric acid in organic media catalyzed by Novozym 435 was investigated for degree of esterification (Li et al., 2015). Fructose laurate esters were synthesized for 12 h with 27.2% and 44.9% conversion of lauric acid in 2-methyl-2-butanol, 2M2B, (log P = 0.89), and methyl ethyl ketone, MEK, (log P = 0.29), respectively. Fructose possesses two primary hydroxyl groups, which serve as the major acyl acceptors for fatty acids in enzymatic esterification. A significant difference in the degree of esterification was observed between solvents: the diester/monoester molar ratio was 1.05:1 and 2.79:1 in 2M2B and MEK, respectively (Li et al., 2015). Moreover, selectivity toward monoesters is favored in the more polar solvent (2M2B) (Janssen et al., 1993). Mixtures of high and low log P solvents, which represent a compromise between enzyme activity and saccharide solubility, can be employed for lipase-catalyzed polyol ester synthesis (Degn and Zimmermann, 2001; Ferrer et  al., 2005b). The highest activity for the synthesis of saccharide-myristic acid esters catalyzed by Novozym 435 was observed for the apolar solvent mixture tert-butanol/pyridine 55:45 v/v (Degn and Zimmermann, 2001). For solvent systems that yielded low glucose solubility (80% of the initial activity was retained after 20 successive 6 h batch reactions. And Ferrer et al. (2005b) demonstrated with Lipozyme TL IM that the DMSO percentage in the solvent mixture substantially modified the final esterification degree. Thus, at DMSO concentrations ≤10%, the formation of diesters was favored, whereas at percentages higher than 15%, the formation of diesters was minimized (Ferrer et al., 1999), consistent with the favorable formation of monoesters in polar media as discussed above. Although there have been many successes on a laboratory scale, the use of organic solvents for the synthesis of sugar fatty acid esters has certain limitation for large-scale synthesis. And there are only a few solvents such as acetone, acetonitrile, tert-butanol, pyridine, 2M2B, DMF, or DMSO, which can solubilize saccharide to a significant extent, but the toxicity and environmental safety of these solvents is a concern. Also, many enzymes do not retain their catalytic activity in these solvents. Therefore, alternate methods that reduce the solvent amount or eliminate their need have been recently recommended. Reaction rate and conversion can be enhanced in nonaqueous media by the derivatization of fatty acyl groups and/or saccharides in organic solvents, the former through the “activation” provided by their covalent attachment to good leaving groups and the latter to enhanced solubilization (Coulon et al., 1999). The solubility of a sugar in an organic solvent can be raised using a hydrophobic sugar derivative instead of an unmodified sugar: (i) sugar acetals, (ii) phenylboronic acid esters, and (iii) n-alkyl glycosides (Kobayashi, 2011). Derivatization of sugar alcohols by isopropylidene, a common protective group for sugars, has led to enhanced solubilization of the polyol acyl acceptors, yielding higher rate and conversion, and to improved selectivity by the blockage of hydroxyl groups covalently bound to isopropylidine. 1-o-lauroyl-d-mannitol, a nonionic surfactant, was synthesized via a chemoenzymatic procedure that utilizes 1,2:4,5-di-o-isopropylidene-d-mannitol and vinyl laurate as substrates (Fig.  10.6) (Pinna et  al., 2004). The acyl acceptor was synthesized by reacting d-mannitol with 2 mol equivalents of 1,2-dimethoxypropane in 1,2-dimethoxyethane under neutral condition, yielding a product that had only two free hydroxyl groups (Chittenden, 1980). The reduced O

O H2C OH

H2C OC(CH2)10CH3

H2C OC(CH2)10CH3

H

O

CH3

H

O

CH3

H

OH

H

O

CH3

H

O

CH3

H

OH

HO

H

Vinyl laurate

H3C

O

H

Novozym 435

H3C

O CH2

HO

H

H3 C

O

H

H3C

O CH2

Acetic acid 90%

HO

H

HO

H

HO CH2

FIG. 10.6  Synthesis of 1-O-lauroyl-d-mannitol (Pinna et al., 2004). Reproduced with permission from Elsevier.

III.  BIOBASED SURFACTANTS



349

10.4  ENZYMATIC SYNTHESIS OF SEs

polarity of this compound compared with d-mannitol enabled it to solubilize in n-hexane and solvent-free reaction medium. 1-o-lauroyl-2,3:5,6-di-o-isopropylidene-d-mannitol was produced at a 92% yield in n-hexane by Novozym 435. The selective acid-catalyzed hydrolysis for the removal of the isopropylidene protective group gave the surfactant 1-o-lauroyl-d-mannitol with an overall yield of 80% and a high selectivity (Pinna et al., 2004). Another approach is to employ alkyl glycosides as acyl acceptors. Alkyl glycosides are readily formed through the formation of an acetal or ketal linkage with alcohols when employing an acidic catalyst. Alkyl glycoside esters were synthesized by immobilized C. antarctica A + B lipase (SP 382, Novozymes, Inc.)-catalyzed transesterification of methyl oleate and methyl glycoside, producing 58.6–100 mol% oleic acid incorporation (Mutua and Akoh, 1993). Employment of phenylboronic acid, a solubilizing agent for hydrophilic substrates in nonpolar solvents, resulted in enzymatic acylation of a single primary hydroxyl group of the saccharide (Schlotterbeck et  al., 1993; Oguntimein et  al., 1993; Ikeda and Klibanov, 1993; Scheckermann et  al., 1995). Organoboronic acids are known to solubilize sugar by forming a carbohydrate‑boronate complex through reversible condensation with carbohydrates (Oguntimein et al., 1993). Phenylboronic acid (2 mol equivalents) was found to effectively complex with α-d-glucose (1 mol equivalent) in benzene to give the corresponding 1,2:3,5-bis(phenylboronate) (Fig. 10.7). This complex, which, in contrast to free glucose, is soluble in a variety of organic solvents, was employed as a target for enzyme-catalyzed acylation in anhydrous tert-butyl alcohol by lyophilized Pseudomonas sp. lipase for 4 days (Ikeda and Klibanov, 1993). After removal of the lipase by filtration, distilled water (10 mL) was added to remove phenylboronic acid, and the overall yield was 77% (Ikeda and Klibanov, 1993). Although the derivatization of acyl donors and acceptors can enhance miscibility of substrates, thereby enhancing the reaction rate and final conversion, this approach has several disadvantages: an increase of costs and labor associated with protection and/or deprotection steps and the inability to readily remove the alcohol group used to form the ketal linkage of alkyl glycosides. CH2OH O

CH2OH O + PhB(OH)2

OH OH

OH

O

O

B

+ 4H2O O O B

Ph

OH

Ph

O H2C CHCOCH CH2 O

Lipase

O

CH2CH2CH O

CH2 H2O

OH OH

OH OH

CH2OCCH CH2 O B Ph

O

O O

O B

Ph

FIG. 10.7  Synthesis of 6-O-acryloylglucose via complex formation of d-glucose and 1,2:3,5-bi(pheny1boronate) at a 1:2 mole ratio in benzene (Ikeda and Klibanov, 1993). Reproduced with permission from John Wiley & Sons, Inc.

III.  BIOBASED SURFACTANTS

350

10.  Sugar Esters

10.4.2  Enzymatic Synthesis of SEs in Supercritical CO2 Supercritical carbon dioxide (SC-CO2) has several advantages over organic solvents for hosting enzymatic reactions since it is inert, nontoxic, nonflammable, and inexpensive. Since enzymes are insoluble in SC-CO2, the catalyst can be easily separated from the reaction mixture (Habulin et al., 2008). Novozym 435-catalyzed synthesis of fructose palmitate, fructose laurate, sucrose palmitate, and sucrose laurate resulted in 61% yield in 24 in 2M2B at atmospheric pressure and in SC-CO2 at 10 MPa (Habulin et al., 2008). Tsitsimpikou et al. (1998) investigated the acylation of glucose with lauric acid at a 1:5 mole ratio catalyzed by Novozym 435 (12.5 mg/cm3) in SC-CO2. Glucose conversions up to 60% at 60°C were observed. Since only lauric acid among the substrates and products is soluble in SC-CO2, the system offers the potential advantage of easy separation of the glucose laurate product from remaining substrate and enzyme upon completion of the reaction. Esterification of palmitic acid and glucose by Novozym 435 was performed in CO2-expanded acetone (3% (v/v)). Although SC-CO2 is a promising alternative to organic solvents, its employment has limitations. For instance, only nonpolar substrates are soluble at an acceptable level (Tsitsimpikou et al., 1998), and capital costs for system that include a high-pressure reactor and controller are high.

10.4.3  Enzymatic Synthesis of SEs in ILs Lipase-catalyzed production of SEs in ILs has also been of particular interest over the past 10 years (Mai et al., 2014). A variety of ILs have been reported to dissolve mono-, di-, oligo-, and polysaccharides to a greater concentration than in organic solvents (Mai et al., 2014). Most of the early work with enzymatic reactions have involved the use of 1-alkyl3-methyl imidazolium ILs, for example, [bmim][BF4] and [bmim][PF6], which are water-­ miscible and water-immiscible, respectively. The molecular structure and nomenclature for the imidazolium-based ILs are given in Fig. 10.8. Lee et al. (2008a,b) developed a process for preparing metastable suspensions of sugars in ILs that used water as a mediator. Supersaturated solutions prepared by this method contained glucose concentrations of 113 and 46.3 g/L at 25°C in [bmim][TfO] and [bmim] [Tf2N], respectively, 19 and 10 times higher than true solubility (6.1 and 4.8 g/L, respectively). A supersaturated [bmim][TfO]/[bmim][Tf2N] (1:1 v/v) mixture produced an even

FIG.  10.8  Structures of ionic liquids based on 1-alkyl–3-methylimidazolium salts. [emim] and [bmim] refer to R = C2H5 and n-C4H9, respectively.

III.  BIOBASED SURFACTANTS



351

10.4  ENZYMATIC SYNTHESIS OF SEs

100

Conversion (%)

80 60 40 20 0

0

2

4

6

8

10

12

Time (h)

FIG. 10.9  Time courses for lipase-catalyzed glucose-vinyl laurate transesterification of pure ILs and IL mixtures (Lee et al., 2008a). Reaction conditions: 222 mM glucose, 444 mM vinyl laurate, 0.5 mL IL, 50 mg Novozym 435, 40°C (♦, [bmim][TfO]; ●, 50% [bmim][TfO] with 50% [bmim][Tf2N]; ▲, [bmim][Tf2N]). Filled symbols represent the reaction with supersaturated glucose solution. Empty symbols represent the reaction with saturated solution with undissolved glucose crystals present. Reproduced with permission from Elsevier.

higher solubility at 25°C, 222 mM. Saturated and supersaturated solutions of glucose were compared for the Novozym 435-catalyzed transesterification of glucose and vinyl laurate in each of the two ILs in neat form and in a 1:1 v/v mixture. The conversion of glucose was increased four- to fivefold in the supersaturated IL solutions compared with saturated IL, with 85% and 65% conversion achieved in [bmim][TfO] and the 1:1 v/v mixture, respectively (Fig. 10.9) (Lee et al., 2008a,b). In contrast, the conversion achieved with the more apolar IL [bmim][Tf2N] was ~10% for both the saturated and supersaturated solutions. To measure enzyme stability, Novozym 435 was employed and then subsequently recovered and reused for five subsequent batch reactions. After the fifth batch reaction, Novozym 435 retained 86% of initial activity that remained when the [bmim][TfO]/[bmim][Tf2N] 1:1 v/v mixture was employed; in contrast, the residual activity using the pure polar IL [bmim][TfO] was only 36%. Reaction conditions were subsequently optimized for the transesterification reaction conducted in the IL mixture at 66.86°C, vinyl laurate/glucose molar ratio of 7.63, and enzyme load of 73.33 g/L, yielding 96.4% conversion, and the enzymes and ILs could be recycled and reused effectively for up to 10 cycles (Mai et al., 2014). The results indicate the feasibility of ILs as novel solvents for the biosynthesis of sugar fatty acid esters. ILs have both stabilizing and destabilizing effect on proteins, depending on its physicochemical properties (Patel et al., 2014; Zhao, 2015). Therefore, ILs (or their mixtures) can potentially be selected or derived to serve as solvent for enzymatic reactions while maintaining high enzyme activity retention. The employment of ILs for biocatalytic reactions on an industrial scale has been minimal due to the high price of ILs. However, material costs for ILs are predicted to decline as new applications for ILs continue to develop. The best opportunity for ILs in biocatalysis currently is for preparing value-added products (Villeneuve, 2007).

III.  BIOBASED SURFACTANTS

352

10.  Sugar Esters

10.4.4  Enzymatic Synthesis of SEs in Deep Eutectic Solvent Systems Deep eutectic solvents (DES), a new type of IL-inspired green solvent, have received attention in biocatalysis in recent years. DES is prepared by complexation of a quaternary ammonium salt (e.g., choline chloride) with a hydrogen-bond donor (e.g., amide, amine, alcohol, and carboxylic acid) (Zhao et al., 2016; Abbott et al., 2003). DES systems possess several attractive IL-like properties, such as low melting point and volatility, high thermal stability and solubility for various substances, and ability to serve as a “designer solvent.” But DES systems are less expensive and easier to prepare than ILs. Andler et al. (2017) employed CALB immobilized onto iron-based nanoparticles for the transesterification of glucose and vinyl laurate and esterification of lauric acid and glucose in choline chloride/urea 1:1 g/g. The immobilized lipase retained 70% of its activity in the DES mixture for 40 h at 50°C and exhibited >70-fold higher activity than Novozym 435, attributed by the authors to the higher internal surface area per lipase. Similarly, other recent reports cite low yields for SE synthesis in DES systems using Novozym 435 (Poehnlein et al., 2015; Siebenhaller et al., 2017; Zhao et al., 2016). Clearly, new lipases must be designed for DES systems that can withstand the mass transport limitations as a result of the high viscosity, and similarly to ILs, the economic and robust removal of accumulated water for esterification (or alcohol or acetaldehyde for fatty acid alkyl or vinyl ester acyl donors, respectively) needs to be addressed.

10.4.5  Enzymatic Synthesis of SEs in Solvent-Free System For reaction systems where the substrates are miscible, such as the lipase-catalyzed esterification of FFA and n-alkanols, the solvent-free approach can be quite successful (Foresti et al., 2007). However, for saccharide-fatty acid ester synthesis, the poor miscibility of the substrates makes the solventless approach challenging. SEs enhance the miscibility of sugars and FFA (Dang et al., 2005; Zhang and Hayes, 1999; Tsavas et al., 2002). A bioreactor system was developed to take advantage of the enhancement and was operated at 65°C. The solvent-free reaction medium (oleic acid plus SE, with the latter being present at 25% initially) was continuously recirculated through a packed-bed desorption column containing sugar and silica gel and a second column containing immobilized Rhizomucor miehei lipase (Lipozyme IM, Novozymes, Inc.) via a peristaltic pump (Pyo and Hayes, 2009). The closed loop initiated from and emptied into a reservoir opens to the atmosphere, which allowed for the evaporation of the coproduct water. An additional molecular sieve column was introduced into the closed loop after achieving ~60% conversion, to increase water removal at the approach of equilibrium. The desorption of saccharide from the packed column to solvent-free reaction medium at 65°C increased with an increase of either the liquid-phase SE concentration or the sugar mass fraction of the packed column (up to 70 wt%) (Pyo and Hayes, 2008). This approach led to 80%–85% conversion, with 85% of SEs being monoesters (Pyo and Hayes, 2009). However, the reaction occurred slowly (25 days) due to the small saccharide solubility in the reaction medium, ~0.1 wt% (Pyo and Hayes, 2009). To address the low solubility, metastable suspensions of sugar particles in solvent-free media were utilized, which enhanced the maximum concentration tenfold and stability of the suspended sugar particles. Solvent-free medium containing suspensions of fructose was operated in a closed-loop bioreactor system under continuous recirculation at 65°C (Ye and Hayes, 2012b).

III.  BIOBASED SURFACTANTS



353

10.4  ENZYMATIC SYNTHESIS OF SEs

CFA, 2

V0

CS, 1

V0 F

CFA, 1

G

H Vacuum

N2 E C B A

Vres

D

FIG. 10.10  Schematic diagram of bioreactor system employed for solvent-free synthesis of sugar esters. (A) Hot plate maintained at 85°C; (B) 25 mL Wheaton Celstir spinner double-side arm flask with stirring bar, serving as reservoir; (C) in-line filter; (D) peristaltic pump; (E) packed-bed bioreactor containing immobilized R. miehei lipase (Lipozyme IM, Novozymes, Inc.); (F) oven maintained at 78°C; (G) vacuum pressure gauge; (H) flowmeter. A vent is available for periodical addition of saccharide in the top of the reservoir. Reprinted from Ye, R., Hayes, D.G., 2012. Solvent-free lipase-catalysed synthesis of saccharide-fatty acid esters: closed-loop bioreactor system with in situ formation of metastable suspensions. Biocatal. Biotransform. 30(2), 209–216, with permission.

The system was similar to that described above, except that sugar was introduced into reservoir periodically under stirring through a vent and the reservoir was operated under vigor­ous stirring at 80°C to optimally form suspensions (Fig.  10.10). When reaching approximately 60 wt% ester, N2 bubbling and vacuum pressure was introduced into the reservoir, to reduce the liquid-phase water concentration. This approach led to ~85% conversion in 200 h (Fig. 10.11). In a related approach, the authors employed a similar bioreactor system but periodically formed suspensions (1–2 wt%, 10–100 μm) offline during the time course of reaction by isolating the reaction medium, adding saccharide, and stirring for several hours. The suspension-based ­medium was then returned to the bioreactor system and the reaction continued. This approach generated up to 92 wt% of esters within 132 h (Ye and Hayes, 2011, 2012a). In subsequent research, high-speed homogenization and high-intensity ultrasound were combined to decrease the particle size of sucrose crystals fivefold, enhancing the rate and extent of reaction (Ye, 2014). Lipases exhibited excellent activity retention in the solvent-free systems, showing no loss of activity during 22 successive days of operation (Ye and Hayes, 2012a). To further increase the conversion for the solvent-free approach, crude product (reaction medium) from the solvent-free system conducted in bioreactor systems was subjected to further enzymatic reaction to increase the ester content to >95%. (Saccharide and fatty acid were used at near-stoichiometric proportions.) Stirred batch reactions were conducted at low water activity, controlled through the use of CaSO 4 to control the reactor ’s air headspace and replacement of Lipozyme IM with III.  BIOBASED SURFACTANTS

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10.  Sugar Esters

100

Saccharide concentration (wt %)

1.8

Ester (wt %)

80

60

40

20

(A)

1.2 1 0.8 0.6 0.4 0.2 0

(B) Mass fraction of monoester among esters

Water concentration (wt %)

1.4

0 1

0.8

0.6

0.4

0.2

0

(C)

1.6

0

50

100 Time (h)

150

200

1

0.8

0.6

0.4

0.2

(D)

0

50

100

150

200

Time (h)

FIG. 10.11  Time course of reaction for lipase-catalyzed solvent-free synthesis of fructose oleate at 65°C using the bioreactor system of Fig. 10.10 operated under continuous recirculation (0.5 mL min−1). Concentrations depicted are those from the reservoir. (A) Ester content, (B) saccharide concentration, (C) water concentration, and (D) mass fraction of monoester among the esters. Error bars represent the standard deviations from two replicate runs. Reprinted from Ye, R., Hayes, D.G., 2012. Solvent-free lipase-catalysed synthesis of saccharide-fatty acid esters: closed-loop bioreactor system with in situ formation of metastable suspensions. Biocatal. Biotransform. 30(2), 209–216, with permission.

Novozym 435 to leverage the latter ’s more hydrophobic immobilization matrix (Ye et al., 2016). High-pressure homogenization was used to form metastable suspensions of 2.0–3.3 μm-sized saccharide particles. Under these conditions, the ester concentration was increased to 90% and 96% from 80% initially for oleic acid esters of sucrose and fructose, respectively (Ye et  al., 2016). The technical-grade sucrose and fructose oleic acid esters were recovered directly from the reaction medium and analyzed for properties. The technical-grade SEs possessed excellent emulsification capability and stability and antimicrobial activity (Ye et al., 2016).

10.4.6  Effects of Water Activity and Content on Enzymatic Synthesis of SEs As discussed above, water concentration must be carefully controlled during enzyme-­ catalyzed SE formation to balance the need for essential water molecules of hydration for

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10.4  ENZYMATIC SYNTHESIS OF SEs

the enzyme and the reduction of water concentration to maximize the conversion (Foresti et al., 2007). In situ water removal in organic solvent or solvent-free systems is often readily achieved through free evaporation from reaction vessels open to the atmosphere, but for more greater removal (e.g., at the approach of thermodynamic equilibrium), the following methods can be used: molecular sieves, vacuum pressure, azeotropic distillation, pervaporation, and dry gas bubbling (Fregapane et al., 1991; Napier et al., 1996; Kim et al., 1998; Sakaki et al., 2006). Yan et al. (1999) removed water during lipase-catalyzed SE synthesis by azeotropic distillation with the intention to develop a process that is practical on a large scale. Reaction medium, consisting of ethyl methyl ketone or acetone organic as solvent, glycerol as acyl acceptor, FFA or FAME as acyl donor, and Novozym 435 as biocatalyst, was placed in a 50 mL two-neck round-bottom flask equipped with a Soxhlet extractor/condenser apparatus, the latter of which was attached to a vacuum pump, and held at 60°C. Molecular sieves were placed in the Soxhlet extractor for the removal of coproducts (water, 3 Å; methanol, 5 Å). This system provided a 90% yield. However, one must be careful when employing molecular sieves in stirred batch mode, since the sieves are susceptible to abrasion by shear, leading to the formation of particulates that can adsorb to lipases and reduce their activity. Chamouleau et al. (2001) investigated the effect of water activity on fructose-palmitic acid esterification catalyzed in 2M2B at 60°C by Novozym 435. The initial water activity strongly affected the time course of reaction, with the lowest water activity (aw), 0.07, leading to the highest conversion yield (28.5%) and initial rate (4.9 g L−1 h−1). The water adsorption curve of Novozym 435 indicates that a small increase of water from 0 to 20 mg water/g dry catalyst would promote an increase of aw from 0.0 to 0.8, leading to hydrolysis rather than esterification (Fig. 10.12) (Chamouleau et al., 2001).

mg water/g dry catalyst

100 80 60 40 20 0 0

0.2 0.4 0.6 0.8 Measured water activity

1

FIG. 10.12  Water adsorption curve for Novozym 435 at 20°C (Chamouleau et al., 2001). Reproduced with permission from Elsevier.

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10.5 CONCLUSIONS The employment of SEs in the foods, cosmetics, personal care, and pharmaceutical industries is common and expanding, due to the biobased surfactants’ good surface activity, biodegradability, biocompatibility, and possession of biological activity. SEs’ versatility across the HLB scale, controlled by the selection of fatty acyl and saccharide components and the number of ester bonds per molecule, serves as an additional asset. The major challenge in forming SEs, the poor miscibility of acyl donor and acceptor substrates, has been traditionally addressed by the employment of organic solvent. However, green manufacturing approaches are leading to improved sustainable production of SEs, using new solvents such as ILs; solvent-free suspensions, melts, or microemulsions as reaction medium; and the use of enzymes, particularly lipases, as biocatalysts. Although challenges remain in the scaling up of the green manufacturing approaches, particularly the use of enzymes (e.g., lipases), additional research and development will yield improvements in these areas. We have already seen that increased interest in enzymatic saccharification of lignocellulosic biomass has led to reduced enzyme costs (National Renewable Energy Laboratory, 2018), giving rise to the hope that the newer “green” technological approaches will become more cost-effective in the future.

Acknowledgments Coauthors Pyo, Chen, and Hayes acknowledge financial support to write this chapter by the Swedish Research Council Formas for Environment, Agricultural Sciences and Spatial Planning (942-2016-63), the GDAS’ Special Project of Science and Technology Development (2017GDASCX-0855) and National Natural Science Foundation of China (21703042), and the University of Tennessee Institute of Agriculture, respectively.

References Abbott, A.P., Capper, G.D., DL, R., Tambyrajah, V., 2003. Novel solvent properties of choline chloride/urea mixtures. Chem. Commun. 70–71. Abdel-Mageed, H.M., El-Laithy, H.M., Mahran, L.G., Fahmy, A.S., Mader, K., Mohamed, S.A., 2012. Development of novel flexible sugar ester vesicles as carrier systems for the antioxidant enzyme catalase for wound healing applications. Process Biochem. 47 (7), 1155–1162. Abdulmalek, E., Hamidon, N.F., Abdul Rahman, M.B., 2016. Optimization and characterization of lipase catalysed synthesis of xylose caproate ester in organic solvents. J. Mol. Catal. B: Enzym. 132, 1–4. Abdul-Raheim, M.A., Abdel-Raouf, M.E., Maysour, N.E., El-Kafrawy, A.F., Mehany, A.Z., Abdel-Azim, A., 2013. Some sugar fatty ester ethoxylates as demulsifiers for petroleum sludge. J. Surfactant Deterg. 16 (3), 377–387. Akoh, C.C., Mutua, L.N., 1994. Synthesis of alkyl glycoside fatty acid esters: effect of reaction parameters and the incorporation of n-3 polyunsaturated fatty acids. Enzym. Microb. Technol. 16 (2), 115–119. Akoh, C.C., Swanson, B.G., 1987. One-stage synthesis of raffinose fatty acid polyesters. J. Food Sci. 52 (6), 1570–1576. Alissandratos, A., Halling, P.J., 2012. Enzymatic acylation of starch. Bioresour. Technol. 115 (1), 41–47. Allen, D.K., Tao, B.Y., 1999. Carbohydrate-alkyl ester derivatives as biosurfactants. J. Surfactant Deterg. 2 (3), 383–390. Andler, S.M., Wang, L.-S., Rotello, V.M., Goddard, J.M., 2017. Influence of hierarchical interfacial assembly on lipase stability and performance in deep eutectic solvent. J. Agric. Food Chem. 65 (9), 1907–1914. Ballesteros, A., Plou, F.J., Alcalde, M., Ferrer, M., Garcia-Arellano, H., Reyes-Duarte, D., Ghazi, I., 2007. Enzymatic synthesis of sugar esters and oligosaccharides from renewable resources. In: Patel, R.N. (Ed.), Biocatalysis in the Pharmaceutical and Biotechnology Industries. CRC Press, Boca Raton, FL, pp. 463–488. Barros, M.T., Petrova, K.T., Correia-da-Silva, P., Potewar, T.M., 2011. Library of mild and economic protocols for the selective derivatization of sucrose under microwave irradiation. Green Chem. 13 (7), 1897–1906.

III.  BIOBASED SURFACTANTS

REFERENCES 357

Becerra, N., Toro, C., Zanocco, A.L., Lemp, E., Günther, G., 2008. Characterization of micelles formed by sucrose 6-O-monoesters. Colloids Surf. A Physicochem. Eng. Asp. 327 (1), 134–139. Bhattacharya, S.C., Palepu, R.M., 2004. Micellization of nonionic surfactants. Tweens—a review. J. Surf. Sci. Technol. 20 (3–4), 159–177. Bouniot, A., Courand, R., Lartigau, G., 1981. Sucroglycerides. French Patent 2463152A1. Cerdeira, M., Martini, S., Hartel, R.W., Herrera, M.L., 2003. Effect of sucrose ester addition on nucleation and growth behavior of milk fat−sunflower oil blends. J. Agric. Food Chem. 51 (22), 6550–6557. Chamouleau, F., Coulon, D., Girardin, M., Ghoul, M., 2001. Influence of water activity and water content on sugar esters: lipase-catalyzed synthesis in organic media. J. Mol. Catal. B Enzym. 11 (4–6), 949–954. Chansanroj, K., Betz, G., 2010. Sucrose esters with various hydrophilic-lipophilic properties: novel controlled release agents for oral drug delivery matrix tablets prepared by direct compaction. Acta Biomater. 6 (8), 3101–3109. Chauvin, C., Plusquellec, D., 1991. A new chemoenzymatic synthesis of 6′-O-acylsucroses. Tetrahedron Lett. 32 (29), 3495–3498. Chauvin, C., Baczko, K., Plusquellec, D., 1993. New highly regioselective reactions of unprotected sucrose. Synthesis of 2-O-acylsucroses and 2-O-(N-alkylcarbamoyl)sucroses. J. Org. Chem. 58 (8), 2291–2295. Chen, I.H., Kou Kevin, G.M., Le Diane, N., Rathbun Colin, M., Dong Vy, M., 2014. Recognition and site-selective transformation of monosaccharides by using copper(II) catalysis. Chem. Eur. J. 20 (17), 5013–5018. Chittenden, G.J.F., 1980. Isopropylidenation of d-mannitol under neutral conditions. Carbohydr. Res. 87 (2), 219–226. Chortyk, O.T., Pomonis, J.G., Johnson, A.W., 1996. Syntheses and characterizations of insecticidal sucrose esters. J. Agric. Food Chem. 44 (6), 1551–1557. Chung, H.-Y., Park, J., Kim, J.-H., Kong, U.-Y., 1996. Preparation of sorbitol fatty acid polyesters, potential fat substitutes: optimization of reaction conditions by response surface methodology. J. Am. Oil Chem. Soc. 73 (5), 637–643. Claverie, V., Cecutti, C., Mouloungui, Z., Gaset, A., Le Hen Ferrenbach, C., 1998. Method for Preparation of Carbohydrate Partial Esters Via Alkali-Catalyzed Transesterification. German Patent 19717968A1. Cole, A.C., Jensen, J.L., Ntai, I., Tran, K.L.T., Weaver, K.J., Forbes, D.C., Davis, J.H., 2002. Novel Brønsted acidic ionic liquids and their use as dual solvent−catalysts. J. Am. Chem. Soc. 124 (21), 5962–5963. Compton, D.L., Vermillion, K.E., Laszlo, J.A., 2007. Acyl migration kinetics of 2-Monoacylglycerols from soybean oil via 1H NMR. J. Am. Oil Chem. Soc. 84 (4), 343–348. Corma, A., Hamid Sharifah, B.A., Iborra, S., Velty, A., 2008. Surfactants from biomass: a two-step cascade reaction for the synthesis of sorbitol fatty acid esters using solid acid catalysts. ChemSusChem 1 (1–2), 85–90. Coulon, D., Girardin, M., Ghoul, M., 1999. Enzymic synthesis of fructose monooleate in a reduced pressure pilot scale reactor using various acyl donors. Process Biochem. 34 (9), 913–918. Croitoru, R., van den Broek, L.A.M., Frissen, A.E., Davidescu, C.M., Peter, F., Boeriu, C.G., 2011. Lipase-Catalyzed Synthesis of Aromatic Esters of Sugar Alcohols. vol. 52. World Academy of Science, Engineering and Technology484–489. Cruces, M.A., Plou, F.J., Ferrer, M., Bernabe, M., Ballesteros, A., 2001. Improved synthesis of sucrose fatty acid monoesters. J. Am. Oil Chem. Soc. 78 (5), 541–546. Csoka, G., Marton, S., Zelko, R., Otomo, N., Antal, I., 2007. Application of sucrose fatty acid esters in transdermal therapeutic systems. Eur. J. Pharm. Biopharm. 65 (2), 233–237. Curtis, G.W., 1961. Sucrose Monoesters. US Patent 2999858. Dang, H.T., Obiri, O., Hayes, D.G., 2005. Feed batch addition of saccharide during saccharide-fatty acid esterification catalyzed by immobilized lipase: time course, water activity, and kinetic model. J. Am. Oil Chem. Soc. 82 (7), 487–493. Degn, P., Zimmermann, W., 2001. Optimization of carbohydrate fatty acid ester synthesis in organic media by a lipase from Candida antarctica. Biotechnol. Bioeng. 74 (6), 483–491. Dimakos, V., Garrett, G.E., Taylor, M.S., 2017. Site-selective, copper-mediated O-arylation of carbohydrate derivatives. J. Am. Chem. Soc. 139 (43), 15515–15521. EFSA Panel on Food Additives Nutrient Sources added to Food, 2010. Scientific opinion on the safety of sucrose esters of fatty acids prepared from vinyl esters of fatty acids and on the extension of use of sucrose esters of fatty acids in flavourings. EFSA J. 8 (3), 1512. Evtushenko, E.V., 2006. Regioselective monoacetylation of methyl pyranosides of pentoses and 6-deoxyhexoses by acetic anhydride in the presence of MoCl5. Synth. Commun. 36 (11), 1593–1599. Evtushenko, E.V., 2010. Regioselective benzoylation of glycopyranosides by benzoyl chloride in the presence of MoO2(acac)2. J. Carbohydr. Chem. 29 (8–9), 369–378.

III.  BIOBASED SURFACTANTS

358

10.  Sugar Esters

Fanun, M., 2017. Sugar Ester Microemulsions. Elsevier, Amsterdam. Ferrer, M., Cruces, M.A., Bernabe, M., Ballesteros, A., Plou, F.J., 1999. Lipase-catalyzed regioselective acylation of sucrose in two-solvent mixtures. Biotechnol. Bioeng. 65 (1), 10–16. Ferrer, M., Comelles, F., Plou, F.J., Cruces, M.A., Fuentes, G., Parra, J.L., Ballesteros, A., 2002. Comparative surface activities of di-and trisaccharide fatty acid esters. Langmuir 18 (3), 667–673. Ferrer, M., Perez, G., Plou, F.J., Castell, J.V., Ballesteros, A., 2005a. Antitumour activity of fatty acid maltotriose esters obtained by enzymatic synthesis. Biotechnol. Appl. Biochem. 42 (1), 35–39. Ferrer, M., Soliveri, J., Plou, F.J., Lopez-Cortes, N., Reyes-Duarte, D., Christensen, M., Copa-Patino, J.L., Ballesteros, A., 2005b. Synthesis of sugar esters in solvent mixtures by lipases from Thermomyces lanuginosus and Candida antarctica B, and their antimicrobial properties. Enzym. Microb. Technol. 36 (4), 391–398. Feuge, R.O., Zeringue, H.J., Weiss, T.J., Brown, M., 1970. Preparation of sucrose esters by interesterification. J. Am. Oil Chem. Soc. 47 (2), 56–60. Fitremann, J., Queneau, Y., Maître, J.-P., Bouchu, A., 2007. Co-melting of solid sucrose and multivalent cation soaps for solvent-free synthesis of sucrose esters. Tetrahedron Lett. 48 (23), 4111–4114. Food and Agriculture Organization (FAO) of the United Nations, 2007. Compendium of Food Additive Specifications (FAO JECFA Monographs 4). FAO, Rome. Foresti, M.L., Pedernera, M., Bucalá, V., Ferreira, M.L., 2007. Multiple effects of water on solvent-free enzymatic esterifications. Enzym. Microb. Technol. 41 (1), 62–70. Forsyth, S.A., MacFarlane, D.R., Thomson, R.J., von Itzstein, M., 2002. Rapid, clean, and mild O-acetylation of alcohols and carbohydrates in an ionic liquid. Chem. Commun. 7, 714–715. Fraile, J.M., Saavedra, C.J., 2017. Synthesis of isosorbide esters from sorbitol with heterogeneous catalysts. ChemistrySelect 2 (3), 1013–1018. Fregapane, G., Sarney, D.B., Vulfson, E.N., 1991. Enzymic solvent-free synthesis of sugar acetal fatty acid esters. Enzym. Microb. Technol. 13 (10), 796–800. Furukawa, S., Akiyoshi, Y., O’Toole, G.A., Ogihara, H., Morinaga, Y., 2010. Sugar fatty acid esters inhibit biofilm formation by food-borne pathogenic bacteria. Int. J. Food Microbiol. 138 (1–2), 176–180. Gangadharmath, U.B., Demchenko, A.V., 2004. Nickel(II) chloride-mediated regioselective benzylation and benzoylation of diequatorial vicinal diols. Synlett 2004 (12), 2191–2193. Ganske, F., Bornscheuer, U.T., 2005. Optimization of lipase-catalyzed glucose fatty acid ester synthesis in a two-phase system containing ionic liquids and t-BuOH. J. Mol. Catal. B Enzym. 36 (1–6), 40–42. Garofalakis, G., Murray, B.S., Sarney, D.B., 2000. Surface activity and critical aggregation concentrations of pure sugar esters with different sugar headgroups. J. Colloid Interf. Sci. 229, 391–398. Ghosh, B., Jones, A.D., 2017. Profiling, characterization, and analysis of natural and synthetic acylsugars (sugar ­esters). Anal. Methods 9 (6), 892–905. Habulin, M., Šabeder, S., Knez, Ž., 2008. Enzymatic synthesis of sugar fatty acid esters in organic solvent and in supercritical carbon dioxide and their antimicrobial activity. J. Supercrit. Fluids 45 (3), 338–345. Hang, F.-x., Shi, C.-r., Xu, Y.-s., Lu, H.-q., Xie, C.-f., Li, K., 2017. Green synthesis of sucrose laurate under different ultrasonic frequencies. Sugar Tech. 19 (3), 241–247. Hass, H.B., Snell, F.D., York, W.C., Osipow, L.I., 1959. Sugar Esters. US Patent 2893990. Hayes, R., Warr, G.G., Atkin, R., 2015. Structure and nanostructure in ionic liquids. Chem. Rev. 115 (13), 6357–6426. Heuschkel, S., Goebel, A., Neubert Reinhard, H.H., 2008. Microemulsions—modern colloidal carrier for dermal and transdermal drug delivery. J. Pharm. Sci. 97 (2), 603–631. Huang, D., Jiang, X., Zhu, H., Fu, X., Zhong, K., Gao, W., 2010. Improved synthesis of sucrose fatty acid monoesters under ultrasonic irradiation. Ultrason. Sonochem. 17 (2), 352–355. Husband, F., Sarney, D., Barnard, M., Wilde, P., 1998. Comparison of foaming and interfacial properties of pure sucrose monolaurates, dilaurate and commercial preparations. Food Hydrocoll. 12 (2), 237–244. Ikeda, I., Klibanov, A.M., 1993. Lipase-catalyzed acyiation of sugars solubilized in hydrophobic solvents by complexation. Biotechnol. Bioeng. 42 (6), 788–791. Jadhav, S.R., Vemula, P.K., Kumar, R., Raghavan, S.R., John, G., 2010. Sugar-derived phase-selective molecular gelators as model solidifiers for oil spills. Angewandte Chemie 49 (42), 7695–7698. S7695/1-S7695/15. Jager, M., Minnaard, A.J., 2016. Regioselective modification of unprotected glycosides. Chem. Commun. 52 (4), 656–664. Janssen, A.E.M., Klabbers, C., Franssen, M.C.R., van’t Riet, K., 1991. Enzymatic synthesis of carbohydrate esters in 2-pyrrolidone. Enzym. Microb. Technol. 13 (7), 565–572.

III.  BIOBASED SURFACTANTS

REFERENCES 359

Janssen, A.E.M., Van der Padt, A., van’t Riet, K., 1993. Solvent effects on lipase-catalyzed esterification of glycerol and fatty acids. Biotechnol. Bioeng. 42, 953–962. Janusz, N., Marcin, M., 2014. Ionic liquids as catalysts and reaction media in oleochemical raw materials processing: a review. Curr. Org. Chem. 18 (22), 2797–2807. Jiang, G., 2014. Method for Purification and Separation of Sucrose Fatty Acid Ester. Chinese Patent 104004033A. John, G., Zhu, G., Li, J., Dordick, J.S., 2006. Enzymatically derived sugar-containing self-assembled organogels with nano-structured morphologies. Angew. Chem. Int. Ed. 45 (29), 4772–4775. Kaewprapan, K., Baros, F., Marie, E., Inprakhon, P., Durand, A., 2012a. Macromolecular surfactants synthesized by lipase-catalyzed transesterification of dextran with vinyl decanoate. Carbohydr. Polym. 88 (1), 313–320. Kaewprapan, K., Inprakhon, P., Marie, E., Durand, A., 2012b. Enzymatically degradable nanoparticles of dextran esters as potential drug delivery systems. Carbohydr. Polym. 88 (3), 875–881. Kasori, Y., Yamazaki, T., 1992. Solventless Process for Preparing Sucrose Fatty Acid Esters. British Patent 2256869A. Kennedy, J.F., Kumar, H., Panesar, P.S., Marwaha, S.S., Goyal, R., Parmar, A., Kaur, S., 2006. Enzyme-catalyzed regioselective synthesis of sugar esters and related compounds. J. Chem. Technol. Biotechnol. 81 (6), 866–876. Khaled, N., Montet, D., Pina, M., Graille, J., 1991. Fructose oleate synthesis in a fixed catalyst bed reactor. Biotechnol. Lett. 13 (3), 167–172. Kidani, K., Akiyama, K., Kurihara, H., Tsukahara, Y., Okumura, H., 2016. Manufacturing Method of Sucrose Fatty Acid Ester With High Selectivity of Monoester. World Patent WO2016194887A1. Kim, J.E., Han, J.J., Yoon, J.H., Rhee, J.S., 1998. Effect of salt hydrate pair on lipase-catalyzed regioselective monoacylation of sucrose. Biotechnol. Bioeng. 57 (1), 121–125. Kim, J., Haam, S., Park, D.-W., Ahn, I.-S., Lee, T.G., Kim, H.-S., Kim, W.-S., 2004. Biocatalytic esterification of ­β-­methylglucoside for synthesis of biocompatible sugar-containing vinyl esters. Chem. Eng. J. 99 (1), 15–22. Klang, V., Matsko, N., Raupach, K., El-Hagin, N., Valenta, C., 2011. Development of sucrose stearate-based nanoemulsions and optimisation through γ-cyclodextrin. Eur. J. Pharm. Biopharm. 79 (1), 58–67. Kobayashi, T., 2011. Lipase-catalyzed syntheses of sugar esters in non-aqueous media. Biotechnol. Lett. 33 (10), 1911–1919. Kosaka, T., Yamada, T., 1977. New plant and new applications of sucrose esters. In: Hickson, J.L. (Ed.), Sucrochemistry (ACS Symp. Ser. Vol. 41). American Chemical Society, Washington, DC, pp. 84–96. Koyama, Y., Kawase, N., Yamamoto, H., Kawata, S., Kasori, Y., 1995. Process for Producing Sucrose Fatty Acid Esters. . European Patent 659760A2. Kralova, I., Sjöblom, J., 2009. Surfactants used in food industry: a review. J. Dispers. Sci. Technol. 30 (9), 1363–1383. Kroumova, A.B.M., Zaitlin, D., Wagner, G.J., 2016. Natural variability in acyl moieties of sugar esters produced by certain tobacco and other Solanaceae species. Phytochemistry 130, 218–227. Ku, M.A., Hang, Y.D., 1995. Enzymatic synthesis of esters in organic medium with lipase from Byssochlamys fulva. Biotechnol. Lett. 17 (10), 1081–1084. Kurakake, M., Ito, Y., Komaki, T., 2011. Synthesis of sugar fatty acid esters using a β-xylosidase from Aspergillus awamori for transxylosylation. Biotechnol. Lett. 33 (12), 2453–2456. Laane, C., Boeren, S., Vos, K., Veeger, C., 1987. Rules for optimization of biocatalysis in organic solvents. Biotechnol. Bioeng. 30, 81–87. Lawson, K.D., Middleton, S.J., Hassall, C.D., 1997. Olestra, a nonabsorbed, noncaloric replacement for dietary fat: a review. Drug Metab. Rev. 29 (3), 651–703. Lee, D., Taylor, M.S., 2011. Borinic acid-catalyzed regioselective acylation of carbohydrate derivatives. J. Am. Chem. Soc. 133 (11), 3724–3727. Lee, S.H., Ha, S.H., Hiep, N.M., Chang, W.-J., Koo, Y.-M., 2008a. Lipase-catalyzed synthesis of glucose fatty acid ester using ionic liquid mixtures. J. Biotechnol. 133 (4), 486–489. Lee, S.H., Nguyen, H.M., Koo, Y.-M., Ha, S.H., 2008b. Ultrasound-enhanced lipase activity in the synthesis of sugar ester using ionic liquids. Process Biochem. 43 (9), 1009–1012. Lee, D., Williamson, C.L., Chan, L., Taylor, M.S., 2012. Regioselective, borinic acid-catalyzed monoacylation, sulfonylation and alkylation of diols and carbohydrates: expansion of substrate scope and mechanistic studies. J. Am. Chem. Soc. 134 (19), 8260–8267. Li, Y., Li, J., Wei, S., Qin, Z., Ye, L., 2012. Method for Producing Sucrose Ester and Its Application in Confecting OilSpecific Antioxidant. Chinese Patent 102533454A.

III.  BIOBASED SURFACTANTS

360

10.  Sugar Esters

Li, L., Ji, F., Wang, J., Li, Y., Bao, Y., 2015. Esterification degree of fructose laurate exerted by Candida antarctica lipase B in organic solvents. Enzym. Microb. Technol. 69 (1), 46–53. Lin, X.-S., Zou, Y., Zhao, K.-H., Yang, T.-X., Halling, P., Yang, Z., 2016. Tetraalkylammonium ionic liquids as dual solvents–catalysts for direct synthesis of sugar fatty acid esters. J. Surfactant Deterg. 19 (3), 511–517. Liu, T.-X., Stansly, P.A., Chortyk, O.T., 1996. Insecticidal activity of natural and synthetic sugar esters against Bemisia argentifolii (Homoptera: Aleyrodidae). J. Econ. Entomol. 89 (5), 1233–1239. Lu, Y., Yan, R., Ma, X., Wang, Y., 2013. Synthesis and characterization of raffinose fatty acid monoesters under ultrasonic irradiation. Eur. Food Res. Technol. 237 (2), 237–244. Mai, N.L., Ahn, K., Bae, S.W., Shin, D.W., Morya, V.K., Koo, Y.-M., 2014. Ionic liquids as novel solvents for the synthesis of sugar fatty acid ester. Biotechnol. J. 9 (12), 1565–1572. Malik, S., Dixit, V.A., Bharatam, P.V., Kartha, K.P.R., 2010. A simple, mild, and regioselective method for the benzylation of carbohydrate derivatives promoted by silver carbonate. Carbohydr. Res. 345 (5), 559–564. Markets and Markets Inc., 2016. Sucrose Esters Market by Application (Food, Detergents and Cleaners, and Personal Care), Form (Powder, Liquid, and Pellet) and by Region (North America, Europe, Asia-Pacific, Latin America, and Rest of the World)-Global Trends and Forecast to 2020. Markets and Markets Inc, Pune, India. Martini, S., Puppo, M.C., Hartel, R.W., Herrera, M.L., 2002. Effect of sucrose esters and sunflower oil addition on crystalline microstructure of a high-melting milk fat fraction. J. Food Sci. 67 (9), 3412–3418. Matsumoto, S., Hatakawa, Y., Nakajima, A., 1989. Purification of Fatty Acid Esters Via Ultrafiltration. European Patent 338464A2. Molinier, V., Fitremann, J., Bouchu, A., Queneau, Y., 2004. Sucrose esterification under Mitsunobu conditions: evidence for the formation of 6-O-acyl-3′,6′-anhydrosucrose besides mono and diesters of fatty acids. Tetrahedron Asymmetry 15 (11), 1753–1762. Muramatsu, W., Takemoto, Y., 2013. Selectivity switch in the catalytic functionalization of nonprotected carbohydrates: selective synthesis in the presence of anomeric and structurally similar carbohydrates under mild conditions. J. Org. Chem. 78 (6), 2336–2345. Mutua, L.N., Akoh, C.C., 1993. Synthesis of alkyl glycoside fatty acid esters in non-aqueous media by Candida sp. lipase. J. Am. Oil Chem. Soc. 70 (1), 43–46. Nakamura, S., 1997. Using sucrose esters as food additives. Inform 8, 866–874. Nakamura, S., 1999. Application of sucrose fatty acid esters as food emulsifiers. Special Publication In: Industrial Applications of Surfactants IV. vol. 230. Royal Society of Chemistry, pp. 73–87. Nakamura, S., Nagahara, H., Kawaguchi, J., 1993. Production of High-Monoester Sucrose Higher Fatty Acid Esters by Solvent Precipitation. . European Patent 560081A1. Napier, P.E., Lacerda, H.M., Rosell, C.M., Valivety, R.H., Vaidya, A.M., Halling, P.J., 1996. Enhanced organic-phase enzymatic esterification with continuous water removal in a controlled air-bleed evacuated-headspace reactor. Biotechnol. Prog. 12 (1), 47–50. National Renewable Energy Laboratory, 2018. Reducing Enzyme Costs Increases Market Potential of Biofuels [Online]. Available from: https://www.nrel.gov/docs/fy10osti/47572.pdf. [(Accessed July 10, 2018)]. Nelen, B.A.P., Cooper, J.M., 2004. Sucrose esters’. In: Whitehurst, R.J. (Ed.), Emulsifiers in Food Technology. Blackwell, Oxford, UK, pp. 131–161. Neta, N.S., Teixeira, J.A., Rodrigues, L.R., 2015. Sugar Ester surfactants: enzymatic synthesis and applications in food industry. Crit. Rev. Food Sci. Nutr. 55 (5), 595–610. Nobile, L., 1963. Water Soluble and Insoluble Sucrose Esters. Belgian Patent 624765. Ntawukulilyayo, J.D., Demuynck, C., Remon, J.P., 1995. Microcrystalline cellulose-sucrose esters as tablet matrix forming agents. Int. J. Pharm. 121 (2), 205–210. Ntawukulilyayo, J.D., De Smedt, S.C., Demeester, J., Remon, J.P., 1996. Stabilisation of suspensions using sucrose esters and low substituted n-octenylsuccinate starch-xanthan gum associations. Int. J. Pharm. 128 (1), 73–79. Oguntimein, G.B., Erdmann, H., Schmid, R.D., 1993. Lipase catalysed synthesis of sugar ester in organic solvents. Biotechnol. Lett. 15 (2), 175–180. Okamoto, H., Sakai, T., Danjo, K., 2005. Effect of sucrose fatty acid esters on transdermal permeation of lidocaine and ketoprofen. Biol. Pharm. Bull. 28 (9), 1689–1694. Osipow, L.I., Rosenblatt, W., 1967. Micro-emulsion process for the preparation of sucrose esters. J. Am. Oil Chem. Soc. 44 (5), 307–309. Osipow, L., Snell, F.D., York, W.C., Finchler, A., 1956. Methods of preparation fatty acid esters of sucrose. Ind. Eng. Chem. 48 (9), 1459–1462.

III.  BIOBASED SURFACTANTS

REFERENCES 361

Otomo, N., 2009. Basic properties of sucrose fatty acid esters and their application. In: Hayes, D.G., Kitamoto, D.S. (Eds.), Biobased Surfactants and Detergents: Synthesis, Properties, and Applications. DKY & Ashby, RD, AOCS Press, Champaign, IL, pp. 275–298. Park, O.-J., Park, H.G., Yang, J.-W., 1996. Enzymatic transesterification of monosaccharides and amino acid esters in organic solvents. Biotechnol. Lett. 18 (4), 473–478. Parker, K.J., James, K., Hurford, J., 1977. Sucrose ester surfactants—a solventless process and the products thereof (ACS Symp. Ser. 41). In: Hickson, J.L. (Ed.), Sucrochemistry. American Chemical Society, Washington, DC, pp. 97–114. Patel, R., Kumari, M., Khan, A.B., 2014. Recent advances in the applications of ionic liquids in protein stability and activity: a review. Appl. Biochem. Biotechnol. 172 (8), 3701–3720. Pinna, M.C., Salis, A., Monduzzi, M., 2004. Novel mannitol based non-ionic surfactants from biocatalysis: Part two: Improved synthesis. J. Mol. Catal. B Enzym. 27 (4), 233–236. Poehnlein, M., Ulrich, J., Kirschhoefer, F., Nusser, M., Muhle-Goll, C., Kannengiesser, B., Brenner-Weiss, G., Luy, B., Liese, A., Syldatk, C., Hausmann, R., 2015. Lipase-catalyzed synthesis of glucose-6-O-hexanoate in deep eutectic solvents. Eur. J. Lipid Sci. Technol. 117 (2), 161–166. Polat, T., Linhardt, R.J., 2001. Syntheses and applications of sucrose-based esters. J. Surfactant Deterg. 4 (4), 415–421. Pyo, S.-H., Hayes, D.G., 2008. Desorption of fructose from a packed column to an oleic acid/fructose oleate mixture for employment in a bioreactor system. J. Am. Oil Chem. Soc. 85 (11), 1033–1040. Pyo, S.-H., Hayes, D.G., 2009. Designs of bioreactor systems for solvent-free lipase-catalyzed synthesis of fructose– oleic acid esters. J. Am. Oil Chem. Soc. 86 (6), 521–529. Queneau, Y., Chambert, S., Besset, C., Cheaib, R., 2008. Recent progress in the synthesis of carbohydrate-based amphiphilic materials: the examples of sucrose and isomaltulose. Carbohydr. Res. 343 (12), 1999–2009. Ren, K., Lamsal, B.P., 2017. Synthesis of some glucose-fatty acid esters by lipase from Candida antarctica and their emulsion functions. Food Chem. 214, 556–563. Rich, J.O., Bedell, B.A., Dordick, J.S., 1995. Controlling enzyme-catalyzed regioselectivity in sugar ester synthesis. Biotechnol. Bioeng. 45 (5), 426–434. Riva, S., Chopineau, J., Kieboom, A.P.G., Klibanov, A.M., 1988. Protease-catalyzed regioselective esterification of sugars and related compounds in anhydrous dimethylformamide. J. Am. Chem. Soc. 110 (2), 584–589. Rizzi, G.P., Taylor, H.M., 1976. Synthesis of Higher Polyol Fatty Acid Polyesters. US Patent 3963699A. Rizzi, G.P., Taylor, H.M., 1978. A solvent-free synthesis of sucrose polyesters. J. Am. Oil Chem. Soc. 55 (4), 398–401. Rosen, M.J., Kunjappu, J.T., 2012. Surfactants and Interfacial Phenomena, fourth ed. John Wiley and Sons, Hoboken, NJ. Sabeder, S., Habulin, M., Knez, Z., 2006. Lipase-catalyzed synthesis of fatty acid fructose esters. J. Food Eng. 77 (4), 880–886. Sakaki, K., Aoyama, A., Nakane, T., Ikegami, T., Negishi, H., Watanabe, K., Yanagishita, H., 2006. Enzymatic synthesis of sugar esters in organic solvent coupled with pervaporation. Desalination 193 (1), 260–266. Sarney, D.B., Vulfson, E.N., 2001. Enzymatic synthesis of sugar fatty acid esters in solvent-free media’ in enzymes in nonaqueous solvents: methods and protocols. In: Vulfson, E.N., Halling, P.J., Holland, H.L. (Eds.), Methods in Biotechnology. vol. 15. Humana Press, Totawa, NJ, pp. 531–543. Sasayama, T., Kamikanda, Y., Shibasaki-Kitakawa, N., 2018. Process design for green and selective production of biobased surfactant with heterogeneous resin catalyst. Chem. Eng. J. 334, 2231–2237. Scheckermann, C., Schlotterbeck, A., Schmidt, M., Wray, V., Lang, S., 1995. Enzymic monoacylation of fructose by two procedures. Enzym. Microb. Technol. 17 (2), 157–162. Schlotterbeck, A., Lang, S., Wray, V., Wagner, F., 1993. Lipase-catalyzed monoacylation of fructose. Biotechnol. Lett. 15 (1), 61–64. Shang, W., Mou, Z.-D., Tang, H., Zhang, X., Liu, J., Fu, Z., Niu, D., 2017. Site-selective O-arylation of glycosides. Angew. Chem. Int. Ed. 57 (1), 314–318. Sharma, A., Chattopadhyay, S., 1993. Lipase catalysed acetylation of carbohydrates. Biotechnol. Lett. 15 (11), 1145–1146. Shibatani, S., Kitagawa, M., Tokiwa, Y., 1997. Enzymic synthesis of vinyl sugar ester in dimethylformamide. Biotechnol. Lett. 19 (6), 511–514. Siebenhaller, S., Hajek, T., Syldatk, C., Muhle-Goll, C., Himmelsbach, M., Luy, B., Kirschhofer, F., Brenner-Weiss, G., Hahn, T., Zibek, S., 2017. Beechwood carbohydrates for enzymatic synthesis of sustainable glycolipids. Bioresour. Bioprocess. 4 (1), 25. Siebenhaller, S., Gentes, J., Infantes, A., Muhle-Goll, C., Kirschhoefer, F., Brenner-Weiß, G., Ochsenreithe, K., Syldatk, C., 2018. Lipase-catalyzed synthesis of sugar esters in honey and agave syrup. Front. Chem. https://doi. org/10.3389/fchem.2018.00024.

III.  BIOBASED SURFACTANTS

362

10.  Sugar Esters

Sin, Y.M., Cho, K.W., Lee, T.H., 1998. Synthesis of fructose esters by pseudomonas sp. lipase in anhydrous pyridine. Biotechnol. Lett. 20 (1), 91–94. Smidrkal, J., Cervenkova, R., Filip, V., 2004. Two-stage synthesis of sorbitan esters, and physical properties of the products. Eur. J. Lipid Sci. Technol. 106 (12), 851–855. Song, Z.-j., Li, S.-j., Xi, C., Liu, L.-m., Song, Z.-q., 2006. Synthesis of insecticidal sucrose esters. For. Stud. China 8 (3), 26–29. Soultani, S., Ognier, S., Engasser, J.-M., Ghoul, M., 2003. Comparative study of some surface active properties of fructose esters and commercial sucrose esters. Colloids Surf. A Physicochem. Eng. Asp. 227 (1), 35–44. Staroń, J., Dąbrowski, J.M., Cichoń, E., Guzik, M., 2018. Lactose esters: synthesis and biotechnological applications. Crit. Rev. Biotechnol. 38 (2), 245–258. Szűts, A., Szabó-Révész, P., 2012. Sucrose esters as natural surfactants in drug delivery systems—a mini-review. Int. J. Pharm. 433 (1), 1–9. Szuts, A., Pallagi, E., Regdon, G., Aigner, Z., Szabo-Revesz, P., 2007. Study of thermal behaviour of sugar esters. Int. J. Pharm. 336 (2), 199–207. Szűts, A., Budai-Szűcs, M., Erős, I., Otomo, N., Szabó-Révész, P., 2010. Study of gel-forming properties of sucrose esters for thermosensitive drug delivery systems. Int. J. Pharm. 383 (1), 132–137. Therisod, M., Klibanov, A.M., 1987. Regioselective acylation of secondary hydroxyl groups in sugars catalyzed by lipases in organic solvents. J. Am. Chem. Soc. 109 (13), 3977–3981. Thévenet, S., Descotes, G., Bouchu, A., Queneau, Y., 1997. Hydrophobic effect driven esterification of sucrose in aqueous medium. J. Carbohydr. Chem. 16 (4–5), 691–696. Thevenin, M.A., Grossiord, J.L., Poelman, M.C., 1996. Sucrose esters/cosurfactant microemulsion systems for transdermal delivery: assessment of bicontinuous structures. Int. J. Pharm. 137 (2), 177–186. Tsavas, P., Polydorou, S., Faflia, I., Voutsas, E.C., Tassios, D., Flores, M.V., Naraghi, K., Halling, P.J., Chamouleau, F., Ghoul, M., Engasser, J.-M., Ferrer, M., Plou, F.J., 2002. Solubility of glucose in mixtures containing 2-methyl-2-­ butanol, dimethyl sulfoxide, acids, esters, and water. J. Chem. Eng. Data 47 (4), 807–810. Tsitsimpikou, C., Stamatis, H., Sereti, V., Daflos, H., Kolisis, F.N., 1998. Acylation of glucose catalysed by lipases in supercritical carbon dioxide. J. Chem. Technol. Biotechnol. 71 (4), 309–314. US Food and Drug Administration, 2017. 21CFR172.859 [Title 21, Volume 3]. US Food and Drug Administration, Washington, DC. van den Broek, L.A.M., Boeriu, C.G., 2013. Enzymatic synthesis of oligo- and polysaccharide fatty acid esters. Carbohydr. Polym. 93 (1), 65–72. Vekariya, R.L., 2017. A review of ionic liquids: applications towards catalytic organic transformations. J. Mol. Liq. 227, 44–60. Vijai Kumar Reddy, T., Sandhya Rani, G., Prasad, R.B.N., Prabhavathi Devi, B.L.A., 2015. Green recyclable SO3Hcarbon catalyst for the selective synthesis of isomannide-based fatty acid monoesters as non-ionic bio-surfactants. RSC Adv. 5 (51), 40997–41005. Villeneuve, P., 2007. Lipases in lipophilization reactions. Biotechnol. Adv. 25 (6), 515–536. Vlahov, I.R., Vlahova, P.I., Linhardt, R.J., 1997. Regioselective synthesis of sucrose monoesters as surfactants. J. Carbohydr. Chem. 16 (1), 1–10. Vulfson, E., 2003. Enzymatic synthesis of surfactants. In: Holmberg, K. (Ed.), Novel Surfactants. Marcel-Dekker, New York, pp. 257–278. Wang, C.-C., Lee, J.-C., Luo, S.-Y., Kulkarni, S.S., Huang, Y.-W., Lee, C.-C., Chang, K.-L., Hung, S.-C., 2007a. Regioselective one-pot protection of carbohydrates. Nature 446, 896. Wang, Q., Zhang, S., Yang, J., 2007b. Regioselective formation of 6-O-acylsucroses and 6,3′-di-O-acylsucroses via the stannylene acetal method. Carbohydr. Res. 342 (17), 2657–2663. Wang, S., Meng, X., Zhou, H., Liu, Y., Secundo, F., Liu, Y., 2016. Enzyme stability and activity in non-aqueous reaction systems: a mini review. Catalysts 6 (2), 32. Ward, O.P., Fang, J., Li, Z., 1997. Lipase-catalyzed synthesis of a sugar ester containing arachidonic acid. Enzym. Microb. Technol. 20 (1), 52–56. Xia, Y., Johnson, A.W., Chortyk, O.T., 1998. Toxicity of synthetic sucrose esters against the tobacco aphid (Homoptera: Aphididae). J. Entomol. Sci. 33 (3), 292–299. Xiao, D., Ye, R., Davidson, P.M., Hayes, D.G., Golden, D.A., Zhong, Q., 2011. Sucrose monolaurate improves the efficacy of sodium hypochlorite against Escherichia coli O157:H7 on spinach. Int. J. Food Microbiol. 145 (1), 64–68.

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FURTHER READING

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Yahya, A.R.M., Anderson, W.A., Moo-Young, M., 1998. Ester synthesis in lipase-catalyzed reactions. Enzym. Microb. Technol. 23 (7), 438–450. Yamagishi, F., Endo, F., Ooi, H., Kozuka, Y., 1971. Sucrose Fatty Acid Esters. German Patent 2051766A. Yamamoto, T., Kinami, K., 1986. Sucrose Fatty Acid Polyester. British Patent 2161806A. Yan, Y., Bornscheuer, U.T., Cao, L., Schmid, R.D., 1999. Lipase-catalyzed solid-phase synthesis of sugar fatty acid esters: removal of byproducts by azeotropic distillation. Enzym. Microb. Technol. 25 (8), 725–728. Ye, R., Hayes, D.G., 2011. Optimization of the solvent-free lipase-catalyzed synthesis of fructose-oleic acid ester through programming of water removal. J. Am. Oil Chem. Soc. 88 (9), 1351–1359. Ye, R., Hayes, D.G., 2012a. Lipase-catalyzed synthesis of saccharide-fatty acid esters utilizing solvent-free suspensions: effect of acyl donors and acceptors, and enzyme activity retention. J. Am. Oil Chem. Soc. 89 (3), 455–463. Ye, R., Hayes, D.G., 2012b. Solvent-free lipase-catalysed synthesis of saccharide-fatty acid esters: closed-loop bioreactor system with in situ formation of metastable suspensions. Biocatal. Biotransform. 30 (2), 209–216. Ye, R., Hayes, D.G., 2017. Bioactive properties of sugar fatty acid esters. In: Aguilar, M.V., Otero, C. (Eds.), Frontiers in Bioactive Compounds, Volume 2: At the Crossroads Between Nutrition and Pharmacology. Bentham Science Publishers, Sharjah, UAE, pp. 124–145. Ye, R., Pyo, S.-H., Hayes, D.G., 2010. Lipase-catalyzed synthesis of saccharide-fatty acid esters using suspensions of saccharide crystals in solvent-free media. J. Am. Oil Chem. Soc. 87 (3), 281–293. Ye, R., Hayes, D.G., Burton, R., 2014. Effects of particle size of sucrose suspensions and pre-incubation of enzymes on lipase-catalyzed synthesis of sucrose oleic acid esters. J. Am. Oil Chem. Soc. 91 (11), 1891–1901. Ye, R., Hayes, G.D., Burton, R., Liu, A., Harte, M.F., Wang, Y., 2016. Solvent-free lipase-catalyzed synthesis of ­technical-grade sugar esters and evaluation of their physicochemical and bioactive properties. Catalysts 6 (6), 78. Youan Bi-Botti, C., Hussain, A., Nguyen Nga, T., 2003. Evaluation of sucrose esters as alternative surfactants in microencapsulation of proteins by the solvent evaporation method. AAPS PharmSci 5 (2), E22. Yu, J., Zhang, J., Zhao, A., Ma, X., 2008. Study of glucose ester synthesis by immobilized lipase from Candida sp. Catal. Commun. 9 (6), 1369–1374. Zhang, X., Hayes, D.G., 1999. Increased rate of lipase-catalyzed saccharide-fatty acid esterification by control of reaction medium. J. Am. Oil Chem. Soc. 76 (12), 1495–1500. Zhao, H., 2015. Protein stabilization and enzyme activation in ionic liquids: specific ion effects. J. Chem. Technol. Biotechnol. 91 (1), 25–50. Zhao, H., Baker, G.A., Song, Z., Olubajo, O., Crittle, T., Peters, D., 2008. Designing enzyme-compatible ionic liquids that can dissolve carbohydrates. Green Chem. 10 (6), 696–705. Zhao, K.-H., Cai, Y.-Z., Lin, X.-S., Xiong, J., Halling, J.P., Yang, Z., 2016. Enzymatic synthesis of glucose-based fatty acid esters in bisolvent systems containing ionic liquids or deep eutectic solvents. Molecules 21 (10), 1294. Zief, M., 1950. Unsaturated esters of sucrose. J. Am. Chem. Soc. 72 (3), 1137–1140.

Further Reading Ye, R., Hayes, D.G., 2014. Recent progress for lipase-catalysed synthesis of sugar fatty acid esters. J. Oil Palm Res. 26 (4), 355–365.

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C H A P T E R

11 Synthesis of Alkyl Polyglycosides From Glucose and Xylose for Biobased Surfactants: Synthesis, Properties, and Applications Boris Estrine⁎, Sinisa Marinkovic⁎, François Jérome† ⁎

Agro Industrie Recherches et Développements, Pomacle, France †Ecole Nationale Superieure d’Ingenieurs de Poitiers, Institut de Chimie des Milieux et Materiaux de Poitiers (IC2MP, UMR7285), University of Poitiers, Poitiers Cedex, France

11.1 INTRODUCTION In the chemical industry, surfactants represented about 14 million tons in 2011 for a turnover of around 23 billion US dollars per year (Transparency Market Research, 2012). In a context of uncertainty and variability of petroleum prices, the demand of consumers for environmentally friendly chemicals is rising, and thus, production and uses of surfactants from biomass receive greater attention. We should go on developing low-cost solutions that are multifunctional toward technical applications and low in hazards. Europe as an example represents a region where the annual growth rate of biobased surfactants was 3% between 2008 and 2013 with alkyl polyglycosides (APGs) registering the strongest growth (Bio-TicWorkshop, 2015; von Rybinski and Hill, 1998). Global demand for APGs has prompted major producers of APGs to increase their production capacities. APGs were first described in the late 1890s and then industrially developed by Henkel group one century later. Today, optimized process technologies enable large-scale production of APGs with consistent quality and a balanced price/performance ratio. APGs are produced from natural base stocks (from vegetable oils and starch or other carbohydrate-based materials) and are classified in the nonionic surfactant group where they address a very broad range of applications (food, detergent, cosmetic, agrochemicals, and pharmaceutical industries) (Steber et  al., 1995). It seems that APGs possess a range of advantages (dermatologic and ocular safety,

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­ iodegradability, wettability, foam production, and cleaning ability) when compared with b other classes of surfactants (Fukuda et al., 2001). This review provides some current research trends in production technologies that have allowed for increased employment of APGs on the market. Despite the previously mentioned benefits of the APG surfactants, it remains a challenge to produce APGs at costs comparable with petroleum-based surfactants. It is also difficult to produce APGs with an enlarged range of polarities. Indeed, conversion of biomass materials needs to be also sustainable, and research activities are still required to reach competitivity of this class of surfactants.

11.2  ALKYL POLYGLYCOSIDES BASED ON d-GLUCOSE AND/OR d-XYLOSE 11.2.1  Fischer Glycosylation APGs are produced by the so-called Fischer glycosylation. It is catalyzed either by enzymes or acid catalysts (Fischer, 1893). The first alkyl glycoside was synthesized from glucose and identified in the laboratory by Fischer (1893). APGs as Fischer glycosidation products are complex mixtures of α/β-anomers and pyranoside/furanoside isomers of glycosides and polyglycosides. The final composition depends on the saccharide type. The reducing sugar is reacted with an excess of an alcohol to mainly yield monoglycosides, along with smaller amounts of oligomeric glycosides. The final mixture composition results from the competition between many reactions (Fig. 11.1). The first step is sugar protonation, which leads to the formation of an oxonium cation after the loss of water. Its reaction with a fatty alcohol yields an alkyl glycoside. The activated sugar can also react with another glycoside molecule to form an oligoglycoside. Oligoglycosides can be activated at their reducing end to react either with the fatty alcohol or with other oligomers, the latter being possibly alkylated or nonalkylated. The composition of the final mixture is very complex, because each glycoside can exist in four cyclic forms (two furanosides and two pyranosides). Moreover, for APG oligoglycosides, two sugar units can be covalently linked in different ways. A diglycoside can thus adopt about 30 isomeric forms; this number rises to hundreds for triglucosides and to thousands for tetraglucosides. Deep analytic work has been performed to obtain precise range of compositions and to evaluate ranges of polymerization degrees (Fig. 11.2) (von Rybinski and Hill, 1998). The pyranoside forms are usually more thermodynamically stable than furanosides and are the main products obtained in the equilibrated mixture although some saccharides such as d-galactose or d-fructose tend to form higher amount of furanosides. In the case of d-glucose, for example, α-anomers are present in a higher concentration due to the so-called anomeric effect, the latter not completely suppressing the synthesis of β-glycosides that are sterically favored. Saccharide type and Fischer glycosylation parameters influence final chemical compositions of APGs and thus their physical and chemical behavior. Although Fischer glycosylation was known for a long time, its mechanism has been exhaustively studied, and clarifications came in the late 1960s (Capon, 1969). Formation of alkyl glycosides is considered to proceed through acetalization of the open form of the saccharide and the formation of kinetically favored furanosides that slowly rearrange to more stable pyranosides (Fig. 11.3).

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11.2  ALKYL POLYGLYCOSIDES BASED ON d-GLUCOSE AND/OR d-XYLOSE

HO HO HO

367

O

+

OH

HO

OH

Glucose

Fatty alcohol Å

H cat. H 2O

HO HO HO

O O OH

Alkyl monoglucoside + HO HO HO

O O OH O

HO HO

O OH

n

Alkyl polyglucoside

FIG. 11.1  Example of glucose glycosylation.

The common catalysts used for Fischer glycosylation are H2SO4, HCl, or H3PO4. At the end of the process, the acid catalyst is neutralized with a base such as sodium hydroxide (NaOH) (Borsotti, Glampiero and Tullio, 1996). Most of the catalysts induce saccharide polymer formation (polydextrose in the case of glucose) that confers high viscosity to the mixture of products and even at small concentration (Queneau et al., 2008). As a consequence, it results in product loss during separation and purification steps. The formation of polyglycose that is thermodynamically favored can be maintained as low as possible by the use of large amounts of alcohol during glycosylation, but this limits productivity and ends in higher plant operational and capital costs. In the case of d-glucose, the nature of catalyst can help lower the percentage of polyglycose. For example, glycosylation of d-glucose with H2SO4 yielded 20% polyglycosylation, and this level was lowered to 2% by the use of sterically hindered sulfonic acid (Borsotti, Glampiero and Tullio, 1996). The problem of polyglycose is obviously less consequent when saccharides offer less primary alcohol functional groups available such

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100 (wt%) HO O HO

H

O O OH DP-1

O

HO HO

O OH

50 DP =

¥ P1 P2 Pi ´1 + ´ 2 + ... = S ´i 100 100 i = 1 100

0 DP1

DP2

DP3

DP4

DP5

FIG. 11.2  Degree of polymerization (DP) for alkyl polyglycosides: weight percentage distribution of monoglycosides (DP1) and oligomers (DPn).

HO OH

HO O OH

OH

OH

RO

OH HO OH

OH

R—OH HÅ

OH

R—OH

-H2O CHO

HO HO

OH

HO

O

HO HO

HÅ

OH

RO

OH

R—OH HÅ

OH OH

HO

HO

HO

OH

OH

OH

OH

OH

OH

CH2OH

CH2OH

HO HO

-H2O

OH OH

OR OH

OR

OH

O

O

OR

O OH

HÅ

OH

Å O

OH

CH2OH R—OH HÅ

R—OH HO OH OH OH

FIG. 11.3  Fischer glycosylation mechanism.

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O

OR

OH



11.2  ALKYL POLYGLYCOSIDES BASED ON d-GLUCOSE AND/OR d-XYLOSE

369

2.5 DP min

2.2

DP max

2

1.5

1.5

1.4

1.4

1.1

1

1

0.5

0 Alkyl glucosides

Alkyl xylosides

Alkyl arabinosides

FIG. 11.4  Range of average degree of polymerization (DP) for various APG mixtures. Maximum and minimum DP occur for low and high fatty alcohol/sugar ratios, respectively (Martel et al., 2010).

as aldopentoses. For example, d-xylose is mainly available in solution in its pyranoside form, and consequently, degree of polymerization for alkylxylosides is lower than for their pendent hexosides (Fig. 11.4). When other saccharides are used, some particular by-products can be formed. For the case of fructose, it is particularly sensitive to acid and cyclodehydrates into 5-­hydroxymethylfurfural (Fig.  11.5). Unsaturated tar-like products are also produced under drastic conditions with glucose conferring dark color to APG mixtures that is incompletely removed by conventional filtration or bleaching techniques. Precedently, we highlighted that Fischer glycosylation needs to be thermodynamically oriented by the use of aglycone excess during reaction, while fine control of temperature is required to avoid the degradation of sugars. Pentoses are easier to handle in glycosylation as they are more reactive with less by-product formation (Fig. 11.6). Indeed, preparation of glucose-based APG usually requires operating over 120°C. In comparison, the grafting of “C5” sugars can be done under milder conditions (80°C), which lowers the degradation of the starting materials and end products. Self-etherification of fatty alcohol is also limited in this case. This results in a cost-effective process giving high-quality products.

11.2.2  Industrial Manufacture of Alkyl Polyglycosides APGs remained of mainly academic interest for a long time before their industrial development occurred. Rohm and Haas was the first company to market a d-glucose and octyl/ decyl alcohol-derived APG in commercial quantities. Due to their limited uses as hydrotropes in industrial and institutional cleaning, for example, glucose-based APG producers started research and development programs to develop C12/C14 alkyl polyglucosides in order to

III.  BIOBASED SURFACTANTS

370

11.  SYNTHESIS OF ALKYL POLYGLYCOSIDES FROM GLUCOSE AND XYLOSE

CH2OH

ion

sat

den

n l co

Soluble polymers

do Al

O

HO

OH

O

O HO

HO OH

HO OH

O

5-Hydroxymethylfurfural

OH Fructofuranose form

OH

O O Furfural

CH2OH Fructose

O OH

Insoluble humins

+

HO

O

Formic acid

O Levulinic acid

FIG. 11.5  Examples of by-product formation occurring during Fischer glycosylation of fructose. 100 D-Glucose

90 D-Xylose

80

L-Arabinose

Residual sugars (%)

70 60 50 40 30 20 10 0 0

50

100

150 200 Time (min)

250

300

350

FIG. 11.6  Comparative glycosylation of pentoses and d-glucose at 80°C.

a­ ddress applications in cosmetics and detergents. Significant pilot-scale developments were required before industrial-scale operations were feasible mainly due to polydextrose production mentioned before that complicated the product handling and lowered its quality (Fig. 11.7). In the late 1990s, Henkel company inaugurated two plants in the United States and Germany giving the start to a constant rise of production capacity of APG production till

III.  BIOBASED SURFACTANTS



11.2  ALKYL POLYGLYCOSIDES BASED ON d-GLUCOSE AND/OR d-XYLOSE

371

Starch or dextrose syrup

Anhydrous glucose or glucose monohydrate (dextrose)

Bu-OH Butanolysis H2O

Fatty alcohol

Fatty alcohol

Transacetalization Acetalization Bu-OH H2O

Neutralization

Distillation

Fatty alcohol in excess

H2O Dissolution

Bleaching

Alkyl polyglycosides

FIG.  11.7  Industrial production options for alkyl polyglycosides, direct glycosylation or glycosylationtransglycosylation.

now (global onstream capacity is estimated to be around 100,000 ton/year). Pioneer studies on alkyl polypentoside preparation by conventional Fisher-type glycosylation have been done by Agro-industrie Recherches et Développements (ARD) company in France following a pragmatic and cost-driven study (Fig. 11.8). This work allowed for the creation of the WHEATOLEO company in 2010 (wheatoleo website: http://www.wheatoleo.com/). Several sources of hemicelluloses have been studied, and noteworthy, the case of wheat by-product for the production of surfactants was the start of the development of alkyl polypentoside industrial products (Martel et al., 2010).

III.  BIOBASED SURFACTANTS

372 11.  SYNTHESIS OF ALKYL POLYGLYCOSIDES FROM GLUCOSE AND XYLOSE

III.  BIOBASED SURFACTANTS

FIG. 11.8  Industrial production of APGs from starch material or from lignocellulosic biomass residues.



11.2  ALKYL POLYGLYCOSIDES BASED ON d-GLUCOSE AND/OR d-XYLOSE

373

11.2.3  Latest Developments in the Synthesis of Alkyl Polyglycosides 11.2.3.1  Glycosylation Progress On the industrial scale, APGs are produced by Fischer glycosylation by following a process that requires precise parameters and elaborated techniques. However, this procedure suffers from some serious drawbacks such as the use of high amounts of corrosive or toxic acid catalysts and the careful management of the reaction pressure to limit the formation of polymerized sugars as mentioned above. The use of a large quantity of alcohol (a critical issue due to the high cost of alcohols) and high temperatures are required to reduce reaction times but finally lead to by-products (Rau and Speckman, 1984). Thus, neutralization and purification of the final product become an issue for these transformations. Furthermore, the reaction is more complicated when long-chain fatty alcohols are used due to low solubility of carbohydrates, requiring an intermediate glycosylation step with an alcohol such as methanol. Solvent or the use of ionic liquids has been considered; but they prevent access to an economical surfactant production by complicating the surfactant isolation. The immiscibility of glucose with fatty alcohols is one of the major obstacles hampering the production of APGs with high yield and productivity. Another alternative is to conduct the glycosylation of glucose with fatty alcohols under emulsion conditions. During the glycosylation reaction, carbohydrates can also partly polymerize. The performances of Aquivion perfluorosulfonic acid (PFSA), a solid superacid, in the catalytic glycosylation of glucose with fatty alcohols have been recently reported (Karam et al., 2017). Aquivion PFSA surpassed the catalytic performances of H2SO4 in terms of activity, selectivity, and reactor productivity. Under similar conditions to those industrially employed, Aquivion PFSA selectively converted glucose to alkyl polyglucosides (DP = 1.2) with 85% yield that corresponded to a productivity of 477 kg/m3/h (Fig. 11.9). Conversely to H2SO4, Aquivion PFSA was also capable of selectively producing alkyl polyglucosides directly from glucose syrup, a cheap source of glucose. This unprecedented result was ascribed to the amphiphilic nature of Aquivion PFSA that facilitated the Pickering-like emulsification of the biphasic reaction medium. Finally, owing to its high chemical and mechanical properties, Aquivion PFSA was highly stable under the working conditions and was recycled at least 10 times without significant loss of its catalytic performances. To the best of our knowledge, this work constitutes one of the rare examples where a solid acid catalyst surpasses the performances of H2SO4 in carbohydrate processing in terms of activity, selectivity, and productivity. Another work aiming at improving catalyst systems in APG manufacture was to use 2,5-furandicarboxylic acid or its n-decyl ester as cocatalyst in straightforward glycosylation of decanol with d-glucose under reduced quantities of sulfuric acid as catalyst (as low as 0.9 mol%) (van Es et al., 2013). Cocatalyst was employed to help reduce the amount of sulfuric acid and add phase transfer properties and thus help limit the formation of undesired products. Yield of decyl monoglucosides was highly improved by the use of the cocatalysts. Moreover, the presence of cocatalyst also limited the coloration of the reaction. Ultimate biodegradability of furan-2,5-dicarboxylic acid (FDCA) and its n-decyl ester formed or produced in the bulk-phase reaction medium was also studied so as to confirm its potential use in surfactant industry. Sulfoxides and sulfones have been used as solvents for the glycosidation of various carbohydrates with decanol in the absence of catalyst (Ludot et al., 2013). Organic acids produced

III.  BIOBASED SURFACTANTS

374

11.  SYNTHESIS OF ALKYL POLYGLYCOSIDES FROM GLUCOSE AND XYLOSE

AAG yield (%)

80

Yield 100%

Recyclability of Aquivion PSFA

60

H2SO4 SBA-15-S O3H

40

Amberly st 15

20 0

H-BEA Aquivion PFSA PW 98 1 2 3 4 5 6 7 8 9 10

Aquivion PFSA PW 87 Aquivion PFSA PW 66

100% Selectivity

600 kg/m3/h Productivity

FIG. 11.9  Performances of Aquivion PFSA catalyst in the production of APG.

by carbohydrate caramelization highly influenced the glycosidation kinetics. Other sulfur containing solvents were also effective. A decanol-sulfolane biphasic reaction medium has been designed for the production of decyl-d-xylosides in short reaction times and yields up to 83%. Sulfolane has been easily recovered by liquid-liquid separation and reused in the next three glycosidation reactions without any yield decrease (Fig. 11.10). 11.2.3.2 Transglycosylation of Polysaccharides in Fatty Alcohols The APG industry is looking for low-cost raw materials. Starch or glucose syrups have been extensively studied, but their use did not allow any production cost reduction and ­reduced the plant productivity (Martel et  al., 2010). The manufacture of pentoses from ­agricultural coproducts is well documented but remains problematic (Hausser et al., 2011). The processes usually require low loading of biomass and elevated temperature that raises the energy demand of the overall surfactant process. Alternatively, lower temperature and pressure lower equipment and energy costs but impose the need for high concentrations of acid (up to 100% based on carbohydrate material). This renders the overall cost of the process out of any market value. The transglycosylation of hemicelluloses in fatty alcohols was investigated by Estrine. This simple process allowed for the production of mixtures of alkyl polypentosides with good surface activities (Table 11.1). The content of arabinose in cereal products allows for the production of alkyl polypentosides in high yields, whereas wood-derived materials are less soluble in the fatty alcohols and thus are less reactive. The nature of xylan’s botanical origin in transglycosylation reaction strongly affected the reactivity (Ludot et  al., 2014) (Fig.  11.11). The use of a solvent such as dimethyl sulfoxide (DMSO) enhanced the solubility and raised the yield of alkylpentosides (Table 11.2). Huge efforts have been exerted also to produce APG from cellulose. Due to its crystalline structure, cellulose is a recalcitrant biopolymer, thus requiring elevated hydrolysis and glycosylation reaction temperatures that are not compatible with the stability of alkyl glycosides. Recently, the combination of catalytic ball milling with perfluorosulfonic acid polymer (Aquivion PFSA) allowed for the production of APGs in high yields (Jérôme et  al., 2017).

III.  BIOBASED SURFACTANTS



11.2  ALKYL POLYGLYCOSIDES BASED ON d-GLUCOSE AND/OR d-XYLOSE

375

FIG. 11.10  Uses of sulfoxides and sulfone in catalyst-free glycosylation reaction of d-xylose. (A) x-axis refers to the number of reactions performed with the same solvent. (B) Process flow chart.

III.  BIOBASED SURFACTANTS

376

11.  SYNTHESIS OF ALKYL POLYGLYCOSIDES FROM GLUCOSE AND XYLOSE

TABLE 11.1  One-Pot Hydrolysis-Glycosylation of Hemicelluloses With Various Alcoholsa Entry

Raw Material

Temperature (°C)

Time (min)

Alkyl Polypentosides Yield (%) References

1

Wheat bran

100

3

96

Marinkovic and Estrine (2010)

2

Wheat straw

110

5

95

Marinkovic et al. (2012)

3

Poplar

110

3

77

Ludot et al. (2014)

a

Maximal yields and the time to reach this yield are given in the table. Reaction conditions: biomass (3.5 g), alcohol (30 g), sulfuric acid, and water (respectively, 10 wt% and 6.4 wt% based on raw material weight).

O

O

Hydrolysis

OH

+

OH

O

OH

O HO

O

OH

O n

Transglycosylation OH

O OH

HO OH

H2SO4 + R-OH Heat

- H 2O

Glycosylation H2SO4 + R-OH Heat

OH HO HO

O O HO O

HO

H2SO4 + H2O Heat

O

OH

HO

HO HO

- H2O

H2SO4 + H2O Heat

OH

O

OH

+

O R

O HO

OH

O

R

Alkyl Monoglycosides Oligomérisation

Degradation reactions: Oligomerisation, furanics... HO

HO

O OH

O HO

O OH

O n

Alkyl polyglycosides

FIG. 11.11  Transglycosylation of hemicelluloses in fatty alcohol medium R: alkyl chain of fatty alcohol R-OH.

TABLE 11.2  One-Pot Hydrolysis-Glycosylation of Xylan From Beechwood: Effect of DMSOa Entry

DMSO

Temperature (°C)

Time (min)

Alkyl Polypentosides Yield (%)b

1

No

150

0.5

43.7

150

1

73.2

2

c

Yes

a

Maximal yields and the time to reach this yield are given in the table. Conversion based on weight of xylan recovered after filtration. Reaction conditions: beechwood xylan (10 g), decyl alcohol (30 g), sulfuric acid, and water (respectively, 10 wt% and 6.4 wt% based on raw material weight). b Based on the amount of pentose present in xylan. c 3.54 weight equivalent of DMSO was added related to the xylan weight for solubility enhancement.

III.  BIOBASED SURFACTANTS



11.3  Alkyl Polyglyosides: Properties, Applications, Toxicity and Environmental Profile

377

Cellulose was first depolymerized in a ball milling process with Aquivion PFSA for 3 h (mechanocatalytic depolymerization) and then reacted in n-dodecanol (glycosylation step). The overall yield of 70% for APGs was obtained productively (30 min of reaction). Interestingly, Aquivion PFSA was successfully recycled (mechanocatalytic depolymerization + glycosylation) at least five times without appreciable decrease of its performance.

11.3  ALKYL POLYGLYOSIDES: PROPERTIES, APPLICATIONS, TOXICITY AND ENVIRONMENTAL PROFILE 11.3.1  Physicochemical Properties and Their Relevance to Applications Physicochemical properties of APG products strongly depend on the molecular composition (von Rybinski and Hill, 1998). Commercially available APGs are complex mixtures of glycosides from various saccharide types, differing in their degree of polymerization (DP) and in alkyl chain length. DP is usually controlled by glycosylation parameters such as the molecular ratio of sugar to fatty alcohol and the acid catalyst type. Usually, the commercial APGs are constituted mainly of alkyl monoglycosides (50% or more), followed by the oligomers of up to heptaglycosides. The nature of the saccharide influences DP greatly. For example, glycosylation of pentoses produces lesser amounts of oligoglycosides due to the absence of a primary alcohol function on the pentopyranose ring. Lower DP confers lower hydrophilic/lipophilic balance and thus affects other properties and performances (Fig. 11.12). This property impacts water-air, water-oil, and water-solid interfacial behavior; solution behavior; and phase behavior and causes alkyl polyxylosides to act distinctly from the conventional nonionic surfactants. Furthermore, their solubility in water is reduced. Interfacial tension at the water/oil interface is of importance for emulsification processes. An emulsifier’s function is to stabilize unstable emulsions for a sufficient time, by adsorbing at the liquid/liquid interface. The emulsifying power generally increases with the length of the lipophilic chain of the APG and thus its oil solubility. APG’s water/oil behavior is recognized as being independent of temperature and of high effectiveness (low interfacial tension). Compared with ethoxylated products, APGs and more precisely alkyl polyxylosides are good emulsifiers (Table 11.3). C12/C14 and the C16/C18 APGs based on glucose are widely used as versatile self-­ emulsifying compositions in cosmetics, the latter displaying no toxicity. When microemulsions are developed, long-chain APGs show better results than the medium-chain C12/C14 APGs. APGs with pentoses as head group are interesting when high oil solubility is required, such as for the formulation of microemulsion or simplified process for vesicle production, as their solubility in oil is high also for medium-chain length (Smulek et al., 2017). Applications in oil fields, in cosmetics, or in agrochemicals are thus possible for such pentose-based APGs noteworthy when liquid emulsifiers are required (Ernenwein et  al., 2012; Ernenwein and Estrine, 2005). It is well known that critical micelle concentration (CMC) is the concentration above which surfactant molecules produce aggregates (micelles). CMC allows for assessing the efficiency of surfactants in cleaning applications such as detergents. As for interfacial tension, CMC of APGs is strongly influenced by the molecular structure and mixture compositions. It is considered that the CMC value of APGs decreases as the number of carbon atoms in the

III.  BIOBASED SURFACTANTS

378

11.  SYNTHESIS OF ALKYL POLYGLYCOSIDES FROM GLUCOSE AND XYLOSE

Alkyl polypentosides

Alkyl polyglucosides OH

R2 O

O

HO HO

R1

O OH

OH

n HLB

1,4 < Dp < 2,1

CMC

Glucosides (Wheat or corn starch origin)

O

HO

SOLUBILITY

Biodegradable

n

1,0 < Dp < 1,7 Pentosides (Wheat bran and straw origin)

Biodegradable g o/w

Mildness

DEGREASING

Mildness

Water solubility

WETTING

Oil solubility

FIG. 11.12  Main property differences between APG from glucose and xylose (Martel et al., 2010). TABLE 11.3  Emulsifying Properties of Hexadecyl Glycoside Compositions Obtained From Wheat Straw, Wheat Bran, and Pure d-Xylose Compared With Fatty Alcohol Polyethoxylates (Emulsifying Power Is the Quantity of Emulsifier Required to Obtain Stable Emulsion)a Fatty Alcohol Concentration (wt%)

Emulsifying Power (wt%)

Wheat straw

84

8

Wheat bran

83

4

Appyclean 6669

44

2

Galenol 16–18 AE

82

8

a

Results from Seguin et al. (2012).

­ ydrophobic group increases. For the same alkyl chain length, DP and saccharide type influh ence the HLB and thus the CMC. For APGs derived from raw lignocellulosic biomass, some polysaccharide residues can influence CMCs indirectly due to simplified process and so minerals produced by the use of higher amounts of catalysts and bases in the process (Table 11.4). As for other properties, foam production properties of APGs depend on the molecular structure. For example, APGs from xylose display maximum foam and stability for C10tailed xylosides, but the best foam properties occur for C12/C14 glucose-based APGs. APGs are considered moderate foam producers and are often associated with anionic surfactants and thus act as foam boosters. They are employed in cosmetics or in household detergents where rich and stable foam is required. As an example, in hand dishwashing detergents,

III.  BIOBASED SURFACTANTS



11.3  Alkyl Polyglyosides: Properties, Applications, Toxicity and Environmental Profile

379

TABLE 11.4  Surface and Foam Properties of Decyl Glycoside Compositions Obtained From Xylan, Poplar, Wheat Straw, Wheat Bran, and Pure d-Glucose and d-Xylose Origin

CMC (mg/L)

γCMC (mN/m)

References

Poplar

433

27

Ludot et al. (2014)

Wheat straw

483

28

Marinkovic et al. (2012)

Wheat bran

493

28

Marinkovic and Estrine (2010)

Xylan

230

27

Bouxin et al. (2010)

d-Glucose

994

26

Marinkovic et al. (2012)

d-Xylose

301

28

Marinkovic et al. (2012)

TABLE 11.5  Wetting Properties of Decyl Glycoside Compositions Obtained From d-Glucose and d-Xylose (Draves Test)a Origin

Wetting Power(s)

d-Glucose

194

d-Xylose

35

a

Results from Freville et al. (2013).

xylose-based surfactants displayed higher efficiency (10%–15% more plates washed following German Cosmetic, Toiletry, Perfumery and Detergent Association (IKW) testing) (SOFW Journal, 2002). The wetting power of a surfactant is important in various applications such as laundry and textile cleaning or in detergent fields (Freville et al., 2013). Also, in these applications, APG from d-xylose displayed improved efficiencies compared with glucose (Table 11.5 and Fig. 11.13). d-xylosides display high performance in wetting, and this property is also well exploited in crop protection where they allow higher performance of herbicides at lower dosage (Brancq, 2001). Their use has been assessed and revealed to lead to higher performance compared with glucose-based APGs (Fig. 11.14).

FIG.  11.13  Efficiency of oil droplet removal of C8/C10 APGs as a function of saccharide residue (d-xylose or d-glucose).

III.  BIOBASED SURFACTANTS

380

11.  SYNTHESIS OF ALKYL POLYGLYCOSIDES FROM GLUCOSE AND XYLOSE

10 9 8

Score

7 APP 8/10 = 45 g/L; gly = 90 g/L

6

APG 8/10 = 45 g/L; gly = 90 g/L

5

APP 8/10 = 90 g/L; gly = 180 g/L APG 8/10 = 90 g/L; gly = 180 g/L

4 3 2 1 0 2 Months

4 Months

FIG.  11.14  Glyphosate composition efficiencies: comparison of alkyl polyglycoside type (APG from glucose/ APP from pentoses; results from Brancq (2001)).

11.3.2  Toxicity and Environmental Data 11.3.2.1 Biodegradation APGs are widely used in household detergents and personal care products. They are also of interest in the food industry. For all of these applications, products are discharged into domestic wastewater after use and thus enter the aquatic environment. Usually, the environmental fate of surfactants is linked with their biodegradability. As for other surfactants, in the case of low degradation behavior while released in the environment, APGs have the potential risk to cause unacceptable adverse effects. Except for plant protection products where surfactants are directly released in the crop fields, the municipal wastewater treatment plant is the receiving system for surfactants. In all cases, biodegradation is the main mechanism for their ultimate removal. APG compounds differ in the alkyl chain length, in the nature of the saccharide residue, and in DP. Their biodegradation pathway has not been elucidated, but studies have hypothesized that degradation occurs releasing saccharides and fatty alcohols. Alkyl polyglycosides have shown biodegradation level of 60% at minimum in the Organization for Economic Cooperation and Development (OECD) biodegradation tests. As mentioned before, alkyl polypentosides are less hydrophilic and display higher effectiveness in most of the applications. Their biodegradation kinetics may differ from those of APGs due to differences in physicochemical properties compared with APG based on glucose. It was therefore essential to understand if these surfactants are compatible with wastewater treatment systems. The biodegradation tests with alkyl polypentosides were carried out along with OECD 301F guidelines. This test simulates the biodegradation process in a municipal sewage treatment plant. Pentose derivatives have good kinetics of biodegradation and were comparable with

III.  BIOBASED SURFACTANTS



11.3  Alkyl Polyglyosides: Properties, Applications, Toxicity and Environmental Profile

381

conventional surfactants (Martel et al., 2010). Their biodegradability occurred in a short time frame without any lag phase as it is the case for ethoxylated surfactants. 11.3.2.2 Toxicity and Irritability In the European Union, consumer products need to be classified and labeled according to the regulation on Classification, Labelling and Packaging (CLP). For cosmetics, this is not the case, even if they contain dangerous substances. Recently, studies have shown that a large proportion of cosmetics would lead to classification and labeling violations (Klaschka, 2012). Classification and labeling according to the CLP Regulation help product design by better selection of ingredients in formulation. The headgroup and hydrophobic tail of surfactants are both important structural determinants of surfactant-lipid interactions and, thus, their toxicity. Indeed, the HLB of surfactants influences their toxicological profile. Although APGs derived from all type of carbohydrates are not considered as toxic or harmful in acute toxicity tests, studies show differences in eye and skin irritation. In view of cosmetic application, this aspect tends to focus attention. Furthermore, correlation between a reduction of the CMC and skin irritation potential has been noticed for glucose-based APGs. In vitro testing such as cytotoxicity assessment can help in the ecoconception of surfactants and is preferred in classification of products although only in vitro testing is recognized in Registration, Evaluation and Authorization of Chemicals (REACH) regulation. Several studies have showed differences in APG irritability when glucose is substituted by xylose in the Fischer glycosylation reaction. In Table 11.6, xyloside displays maximum irritation around C8–C10 carbon tail length, while glucose derivative irritation is maximum for C14–C16. Red blood cell test is also considered to be a highly predictive tool for ocular irritation (H50/DI), dermal irritation, and changes in barrier function induced by surfactants (DI) (Mehling et al., 2007). In this test, xylose-based surfactant displays very low irritation potential. This low irritation is confirmed when in vivo testing is performed following OECD 405 test (Table 11.7). 11.3.2.3  Ecological Profile Although APGs possess good wettability, foam production, and cleaning ability as well as dermatologic and ocular safety for their use in cosmetics, they are not emission-free. Materials and energy inputs needed and process waste outputs released in the manufacture of APGs result in environmental impacts. In particular, agricultural activities carried out to produce necessary raw materials can be particularly impactful. Industrially, APGs

TABLE 11.6  Scores and Classifications Obtained by Ocular Irritation Methods for Glucose- and Xylose-Based APG (Martel et al., 2010) Surfactant

H50 ppma

% DIb

Classification

C8–C10 alkyl polyglycosides from d-xylose

317

0

Not irritating

C8–C18 alkyl polyglucosides

128

0.37

Not irritating

C12–C14 alkyl polyglucosides

317

0.66

Slightly irritating

a

H50: 50% of hemolysis. The lower is the value, the higher is the irritation score. DI%: denaturation index. The lower is the value, the milder is the chemical.

b

III.  BIOBASED SURFACTANTS

382

11.  SYNTHESIS OF ALKYL POLYGLYCOSIDES FROM GLUCOSE AND XYLOSE

TABLE 11.7  Eye Irritation Results Following OECD 405 for APG From Glucose or Xylose From Various Alkyl Chain Length Eye Irritation OECD 405 (From 0 (No Visible Response) to 4 (Severe Response))

APG or APP Cn

Active Substance (%)

APG C8-C10

Mean Values (24/48/72 h)

Conjunctive

Cornea

Iris

Erythema

Edema

70

1.80

0.60

2.40

–

APG C10-C12

50

1.78

1.00

3.00

1.11

APG C12-C14

50

0.78

0.22

2.11

0.67

APP C12

100

1.56

0.10

1.00

1.44

APP C8

70

2.00

0.33

1.30

1.57

APP C5

100

2.00

0.33

1.20

2.00

APP C16/C18

56

0.10

0.10

0.33

0.97

APG, alkyl polyglucosides; APP, alkyl polyxylosides.

are synthesized via the Fischer glycosidation, which comprises a reaction between plantbased fatty alcohols (typically sourced from palm or coconut oil) and carbohydrates in the presence of an acid catalyst. Carbohydrates used are generally refined syrup or powderlike glucose coming from cornstarch hydrolysis. Agriculture activities require inputs of water, fertilizers, pesticides, harvesting, transportation, and other energy and chemical intensive activities. Obviously, manufacturing APGs by transglycosylation of lignocellulosic biomass could substitute traditional cornstarch-based glucose glycosylation and limit impacts. As an example, the production of pentose-based surfactants (from lignocellulosic carbohydrates) requires 37%–41% less fertilizers and 36%–57% less nonrenewable energy compared with the production of glucose-based surfactants (Fig. 11.8) (Martel et al., 2010). On the one hand, biomass-based APGs would allow for the valorization of all polysaccharides available in lignocellulosic biomass like cellulose and hemicelluloses, lignin being burnt to provide heat and electricity. Few studies have considered environmental impacts of APGs. Recently, their use as an emulsifying surfactant in a cosmetic cream has been assessed (Guilbot et al., 2013). The results showed that APGs have relatively low impacts due to their relatively low concentration in products. Despite the limited contribution of APGs to the cream impacts, their environmental profile was examined and indicated a major environmental impact of the fatty alcohol part of the molecules. The carbon footprint of APGs can vary between 1.9 and 49.8 tons CO2 eq. per ton of APG depending on the cultivation mode of oil palms, the feedstock for fatty alcohols, and the land use. The glycosylation process accounts for only 2% of the carbon footprint of APGs, mainly attributable to the transport of raw materials. Future studies of APG using life cycle assessment will help evaluate how transglycosylation process, feedstock nature, and biorefinery environment induce less impacts than glucose-based APG conventional manufacture. Indeed, using an agricultural residue as a raw material, that is, wheat straw, would induce less impacts than glucose in conventional cornstarch-derived processes.

III.  BIOBASED SURFACTANTS

REFERENCES 383

11.4 CONCLUSION Production of biobased surfactant alkyl polyglycosides is of high interest and the use of lignocellulosic biomass and noteworthy of its two main components d-xylose and d-glucose as raw materials as been reviewed. APGs have been industrially developed since the 1980s, and recent developments have focused on d-xylose-based APGs. The recent progress on Fischer glycosylation and the conversion of lignocellulosic polysaccharides into various alkyl glycosides were addressed. The behavior of pentoses in the synthesis of glycosides allows for high kinetic rates, low temperature, and thus the reduction of processing costs. New processes have been designed with or without solvents so as to optimize lignocellulose reactivity, and new catalysts also displayed higher efficiency toward glycosylation or transglycosylation reactions. Indeed, direct conversion of lignocellulosic biomass or heteropolysaccharides through catalytic reactions is foreseen to become of great importance in industries for the production of specialty chemicals. Hydrolysis-glycosylation or transglycosylation reactions have been extensively studied recently, in particular to build up tailor-made surfactants of the APG type. In view of the potential economic impact of APGs, processes have been designed so as to remove cellulose recalcitrance, intensify hemicellulose conversion, and stabilize the so-formed APGs during the catalytic reaction. Alkyl polypentoside mixtures obtained from lignocellulosic biomass waste such as wheat straw, poplar, and beechwood display interesting physicochemical properties with promising performances in view of cleaner manufacture or environmentally safer products. Here again, the chemical composition and performances of alkyl polypentosides are depending on the botanical origin of biomass. Surfactant properties have been discussed displaying the wide range of APGs following the nature of the process, the sugar-based material (xylans, lignocellulosic biomass, and d-xylose and d-glucose monomers). APGs are nontoxic materials with compatible ecological profiles. Lignocellulosic biomass can be valorized through the development of new pentose-based surfactants, supporting the biorefinery model and green chemistry guidelines. The new class of alkyl pentosides is of good quality and shows promising tools in broad range of applications and fulfills the environmental requirements. Although enzymatic processes could improve the environmental sustainability, such processes need to improve in productivity rate to become industrially and economically viable. More generally, the transglycosylation reaction is a promising option in a biorefinery context since it can contribute to broadening the use of lignocellulosic sugars as surfactants in various applications in cosmetics and pharmaceuticals, by proposing lower production cost conditions while maintaining acceptable environmental and health profile of surfactants. New developments will be needed in the future to enlarge the HLB range of APG and thus their uses through new processes.

References Bio-Tic-Workshop, 2015. http://bio-tic-workshops.eu/biosurfactants/home. [(Accessed April 24, 2015)]. Borsotti, Glampiero, and Tullio, 1996. Process for preparing APGs. US Patent 5527892. Bouxin, F., Marinkovic, S., Bras, J.L., Estrine, B., 2010. Direct conversion of xylan into alkyl pentosides. Carbohydr. Res. 345, 2469–2473. Brancq, M., 2001. Composition herbicide comprenant du glyphosate et au moins un alkyl polyxyloside. EP1303190A1. Capon, B., 1969. Mechanism in carbohydrate chemistry. Chem. Rev. 69, 407–498.

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Ernenwein, C., Estrine, B., 2005. Novel emulsifiers and the use thereof for preparing vaporizable emulsions. WO2005110588. Ernenwein, C., Tranchant, J.F., Pouget, T., Estrine, B., Marinkovic, S., 2012. Preparations facilitated by vesicles using alkyl polypentosides and uses of said preparations. US2012027824. Fischer, E., 1893. Ueber die Glucoside der Alkohole. Ber. Dtsch. Chem. Ges. 26, 2400. Freville, V., van Hecke, E., Ernenwein, C., Salsac, A.V., Pezron, I., 2013. Effect of surfactants on the deformation and detachment of oil droplets in a model laminar flow cell. Oil Gas Sci. Technol. 69, 435–444. Fukuda, K., Olsson, U., Ueno, M., 2001. Microemulsion formed by alkyl polyglucoside and an alkyl glycerol ether with weakly charged films. Colloids Surf. B Biointerfaces 20, 129–135. Guilbot, J., Kerverdo, S., Milius, A., Escola, R., Pomrehn, F., 2013. Life cycle assessment of surfactants: the case of an alkyl polyglucoside used as a self emulsifier in cosmetics. Green Chem. 15, 3337–3354. Hausser, N., Marinkovic, S., Estrine, B., 2011. Improved sulfuric acid decrystallization of wheat straw to obtain high yield carbohydrates. Cellulose 18, 1521–1525. Jérôme, F., De Oliveira Vigier, K., Karam, A., Estrine, B., Marincovic, S., Oldani, C., 2017. Conversion of cellulose to amphiphilic alkyl glycosides catalyzed by Aquivion, a perfluorosulfonic acid polymer. ChemSusChem (18), 3604–3610. Karam, A., De Oliveira Vigier, K., Marinkovic, S., Estrine, B., Oldani, C., Jérôme, F., 2017. High catalytic performance of aquivion PFSA, a reusable solid perfluorosulfonic acid polymer, in the biphasic glycosylation of glucose with fatty alcohols. ACS Catal. 7, 2990–2997. Klaschka, U., 2012. Dangerous cosmetics—criteria for classification, labeling and packaging applied to personal care products. Environ. Sci. Eur. 24, 37. Ludot, C., Estrine, B., Le Bras, J., Hoffmann, N., Marinkovic, S., Muzart, J., 2013. Sulfoxides and sulfones as solvents for the manufacture of alkyl polyglycosides without added catalyst. Green Chem. 15, 3027–3030. Ludot, C., Estrine, B., Hoffmann, N., Bras, J.L., Marinkovic, S., Muzart, J., 2014. Manufacture of decyl pentosides surfactants by wood hemicelluloses transglycosidation: a potential pretreatment process for wood biomass valorization. Ind. Crop Prod. 58, 335–339. Marinkovic, S., Estrine, B., 2010. Direct conversion of wheat bran hemicelluloses into n-decyl-pentosides. Green Chem. 12, 1929–1932. Marinkovic, S., le Bras, J., Nardello-Rataj, V., Agach, M., Estrine, B., 2012. Acidic pretreatment of wheat straw in decanol for the production of surfactant, lignin and glucose. Int. J. Mol. Sci. 13, 348–357. Martel, F., Estrine, B., Plantier-Royon, R., Hoffmann, N., Portella, C., 2010. Development of agriculture left-overs: fine organic chemicals from wheat hemicellulose-derived pentoses. Top. Curr. Chem. 294, 79–115. Mehling, A., Kleber, M., Hensen, H., 2007. Comparative studies on the ocular and dermal irritation potential of surfactants. Food Chem. Toxicol. 45, 747–758. Queneau, Y., Chambert, S., Besset, C., Cheaib, R., 2008. Recent progress in the synthesis of carbohydrate-based amphiphilic materials: the examples of sucrose and isomaltulose. Carbohydr. Res. 343, 1999–2009. Rau, A.H., Speckman, D.T., 1984. Process of preparing alkylsaccharides. US Patent 4465828. Seguin, A., Marinkovic, S., Estrine, B., 2012. New pretreatment of wheat straw and bran in hexadecanol for the combined production of emulsifying base, glucose and lignin material. Carbohydr. Polym. 88, 657–662. Smulek, W., Kaczorek, E., Hricoviniova, S., 2017. Alkyl xylosides: physico-chemical properties and influence on environmental bacteria cells. J. Surfactant Deterg. 20, 1269–1279. SOFW Journal, 2002. Framework for testing performance for hand dishwashing detergents. 128, p. 15. Steber, J., Guhl, W., Stelter, N., Schröder, F.R., 1995. Alkyl polyglycosides-ecological evaluation of a new generation of nonionic surfactants. Tenside Surfactant Deterg. 32, 515–523. Transparency Market Research, 2012. Specialty Surfactants Market—global Scenario, Raw Material and Consumption Trends, Industry Analysis, Size, Share & Forecast 2011–2017. www.transparencymarketresearch.com. van Es, D.S., Marinkovic, S., Oduber, X., Estrine, B., 2013. Use of furandicarboxylic acid and its decyl ester as additives in the Fischer’s glycosylation of decanol by D-glucose: physicochemical properties of the surfactant compositions obtained. J. Surfactant Deterg. 16, 147. von Rybinski, W., Hill, K., 1998. Alkyl polyglycosides–properties and applications of a new class of surfactants. Angew. Chem. 110, 1394–1412.

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Further Reading Hausser, N., Marinkovic, S., Estrine, B., 2013. New method for lignocellulosic biomass polysaccharides conversion in butanol, an efficient route for the production of butyl glycosides from wheat straw or poplar wood. Cellulose 20, 2179–2184. Xu, W., Osei-Prempeh, G., Lema, C., Davis Oldham, E., Aguilera, R.J., Parkin, S., Rankin, S.E., Knutson, B.L., Lehmler, H.-J., 2012. Synthesis, thermal properties, and cytotoxicity evaluation of hydrocarbon and fluorocarbon alkyl β-D-xylopyranoside surfactants. Carbohydr. Res. 349, 12–23.

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C H A P T E R

12 Measuring the Interfacial Behavior of Sugar-Based Surfactants to Link Molecular Structure and Uses Wenchao Xiang⁎, Blaise Tardy⁎, Long Bai⁎, Cosima Stubenrauch†, Orlando J. Rojas⁎ ⁎

Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Aalto, Finland †Institute of Physical Chemistry, University of Stuttgart, Stuttgart, Germany

12.1 INTRODUCTION 12.1.1 Motivation There is growing interest in the development of material technologies that are based on renewable resources. The use of biomass as feedstock is of increasing industrial significance, particularly in the energy and commodity sectors. Mono- and polysaccharides, as widely available and tunable chemical starting materials, are estimated to make up three quarters of the world’s biomass. The efficient use of these resources is now recognized as a major future objective in a wide range of applications. It is necessary to identify secondary streams of saccharide by-products and to use these materials as the basis for biocompatible surfactants. Several research groups are advancing the knowledge on the structure-performance properties and facilitating the introduction of sugar-based surfactants in traditional markets dominated by nonrenewable nonionic surfactants. The general goal of the research work performed by these groups is to propose and test new structures for specific applications and to provide the basis for future strategies aimed at enhancing cost-effectiveness. More specific aims involve identifying molecular factors that determine the surface activity and facilitating the design and application of sugar-based surfactants as substitutes for conventional nonionic surfactants, such as those based on poly(ethylene oxide). Significant groundwork has already been done in this area (Kiraly and Findenegg, 2000; Kocherbitov et  al., 2002; Kumpulainen et  al., 2004a,b, 2005; Liljekvist et  al., 2001; Matsson

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et al., 2004; Muruganathan et al., 2003, 2004; Nickel et al., 1996; Persson et al., 2002, 2003a,b; Persson and Kumpulainen, 2004; Stubenrauch, 2001; Balzer and Luders, 2000; Ruiz, 2009). Three classes of surfactants with sugar or a polyol derived from sugar as polar head group have been widely investigated: alkyl polyglucosides (APGs, also known as alkylpolyglycosides, see Chapter 11), alkyl glucamides, and sugar esters (see Chapter 10) (Holmberg, 2001). These surfactants and biosurfactants produced by microorganisms, and other surfactants derived from renewable raw materials are coming progressively onto the market. Deleu and Paquot (2004) provided an interesting summary of trends, and for APGs, they reported a share of ca. 3% of the total production. The reader is referred to the book edited by Hill et al. (1997), for more detailed information about this subject. Overall, APGs have established themselves as natural surfactants of choice for diverse applications, ranging from emulsifiers for skin care to foaming agents. The possibility of future developments in the area of biofuels from cellulose may open up new opportunities, especially for secondary streams in related processes. We believe that four critical efforts need to be pursued in order to successfully expand the potential of sugar-based surfactants from such sources: • Understanding and rationalizing the state of the art in the area of saccharidebased surfactants. The existing, highly fragmented activities need to be systemized and analyzed in order to adequately respond to current changes in economic and environmental aspects related to surfactant production and use. • Identifying molecular and structural factors that govern the surface activity of these materials, so to facilitate their design and application in areas that currently utilize petroleum-based surfactants. This can be supported by studies with model systems, which will expand our understanding and allow targeting specific applications. • Designing and characterizing new sugar surfactants based on natural products and on new motifs (Blunk et al., 2006, 2009; Catanoiu et al., 2012) to demonstrate the economic and technical viability. • Exploring new synthetic approaches to utilize saccharides for the synthesis of new surfactants. Saccharides could be harvested from biomass processing (and related secondary streams) or from agricultural/food by-products. Better understanding of the current and projected economics of saccharide- and petroleum-based surfactant technologies will facilitate the identification of key future markets for the former. Recent research results show that glucose-based and sucrose-based surfactants possess a range of beneficial physical and performance properties, including high levels of surfactancy (surface and interfacial activity), very rapid biodegradability, low human and animal toxicity, effective emulsification properties, and surface interactions. Therefore, it is anticipated that one can rationally design saccharide-based surfactant structures, centered on the combination of potentially low-cost (based on natural starting materials), renewable saccharide components and appropriate renewable hydrophobic groups (natural fatty acids) that will enhance the understanding of the fundamental interactions governing their functionalities. Based on this understanding and on the synthetic approaches generating sugar-based surfactants, it is hoped that the interest in and the prospects for their utilization in a number of markets will be promoted, which would ultimately lead to a significant reduction in the use of petroleum-based materials in these sectors.

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12.1.2  Nonionic Surfactants Nonionic surfactants represent a major component material for applications ranging from personal care to a wide range of industrial uses. Structurally, nonionic surfactants combine uncharged hydrophilic and hydrophobic group that make them effective in wetting and spreading and as emulsifiers and foaming agents. Concurrently, they have minimal skin and eye irritation effects and exhibit a wide range of critical secondary performance properties. The hydrophilic component of nonionic surfactants is currently based largely on poly(ethylene oxide) (EO), which is petroleum-derived. In addition, a significant portion of the hydrophobic components of these materials is also petroleum-derived. The production costs of these commodity chemicals are closely linked to the highly volatile petroleum prices, which can be regarded as additional motivation to search for substitutes. In view of the enormous consumption of EO-based nonionic surfactants, it is expected that further replacement of the established market would be very attractive for a high-value utilization of sugars from biomass. A wide-ranging replacement of EO-based surfactants may be challenging, and there may be a market barrier that prevents a stronger penetration of a new generation of surfactants. Nevertheless, in our opinion, now is the time to further advance efforts by starting with highend applications such as drug emulsification and pharmaceutical formulations, which are industries that require these technologies and that can temporarily tolerate high prices. In summary, it is necessary to replace petroleum-based products with new classes of highly biodegradable, low-irritating, low-toxicity, and completely naturally derived nonionic surfactants. As superior performance properties of current and novel sugar-based surfactants are demonstrated, they will become further established in the future “green” markets.

12.1.3  Environmental and Product Performance Concerns There are environmental and toxicity issues prompting increased consideration of a wider use of sugar-based surfactants. For example, two of the most common types of surfactants used in large-scale applications are poly(ethylene oxide) alkyl ethers and fatty acid soaps. In particular, the use of nonylphenol ethoxylates is problematic. Nonylphenol (NP) is a mixture of isomeric monoalkyl phenols, predominantly para-substituted, and is found in the environment as a result of the biodegradation of nonylphenol ethoxylates. Nonylphenols that are used as nonionic surfactants are released to the environment through various waste streams in industrial processes. According to various reports, these surfactants can severely irritate the skin and eyes. Vapors cause a slight irritation of the eyes or respiratory system if present in high concentrations (ILO, 1998; NIOSH, 1983; Lewis, 1997). Furthermore, nonylphenols are believed to be endocrine disruptors, that is, they have adverse effects on the workings of the endocrine system in humans and animals (Ren et al., 1997). Many European countries have banned the use of NPs. Recent EU legislation on the use of nonylphenols (Directive 2003/53/EC) states that nonylphenol and nonylphenol ethoxylates “may not be placed on the market or used as a substance or constituent of preparations in concentrations equal to or higher than 0.1% by mass” (EPHA1). Therefore, it is imperative to find environmentally friendly replacements for NPs and NP ethoxylates as well as for other toxic surfactants. Our objective in this chapter is to introduce the 1

European Public Health Alliance, http://www.epha.org/a/926.

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interfacial characteristics of surfactants based on renewable materials (natural surfactants). In this way, the fundamental properties of sugar surfactants can be evaluated and fine-tuned for specific applications. Overall, it is expected that understanding the adsorption of biobased surfactants will not only solve important industrial and environmental problems but also open an avenue for a number of products, with unique properties that may be generated from biomass.

12.1.4  Significance of Bio-Based Surfactants There is a significant demand for new, less expensive, nontoxic, and biodegradable s­ urface-active materials. Their use, as conventional surfactants, is aimed at stabilizing liquid-­ liquid (emulsions), solid-liquid (dispersions) and gas-liquid (foams) interfaces. The economic impact that the introduction of sugar-based surfactants into the market can have is mirrored by the substantial consumption of emulsions worldwide in which the surfactant composition amounts to 0.1%–5% of the total mass of the emulsion. However, the use of surfactants is not limited to emulsions; there are a vast number of other applications that involve large quantities of (amphiphilic) stabilizers, including household, cleaning, food, cosmetic, pharmaceutical, and other specialty products. A number of studies were carried out only recently to examine the viscoelastic properties, surface tension, and surface properties of sugar surfactants. Most of those studies point to the overall feasibility of employing various sugar surfactants to replace their EO-based counterparts because of their superior surface physical responses and application performance, environmental compatibility, and chemical tunability (functionalization and modification). Thus, the sections that follow will provide a summary of results that can be used as basis for further developing of sugar-based surfactant systems. The final goal is to design biobased surfactants with properties superior to those of the traditionally used nonionic surfactants and thus to replace the latter whenever possible and feasible.

12.1.5  Interfacial Properties of Sugar-Based Surfactants A number of fundamental studies were conducted in the area of sugar surfactant properties. Four milestones that serve as recent evidence for the superior characteristics of ­sugar-based surfactants compared with EO-based counterparts are discussed below in the Sections 12.3.1–12.3.4. Before discussing these subjects, let us briefly outline the tools that were utilized in these experiments since most of these are not widely available in typical laboratories. Results from techniques used to measure surface tension, solution rheology, contact angle, detergency and emulsion properties are not covered in this chapter. Instead, we describe the interfacial properties of adsorbed layers of sugar surfactants by using the methods described below.

12.2 TECHNIQUES 12.2.1  Measurements and Analysis of Surface Interactions and Forces Force measurements were conducted using the noninterferometric surface force apparatus (Parker et al., 1989, Parker, 1994). This device, commonly known as measurement and analysis of

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surface interaction forces (MASIF), employs a bimorph force/deflection sensor that after calibration of the spring (deflection) constant yields the interaction force. One of the surfaces (bottom surface) is mounted on the edge of the bimorph and the other (the top one) at the end of a piezoelectric tube. The assembly is enclosed in a stainless steel cell of ca. 10 mL volume and mounted on a translation stage that is isolated from electric and sound noise. During a typical force measurement, the surfaces are driven closer until contact, and then, they are separated further apart. This is done by applying a triangular voltage wave to the piezo crystal. Simultaneously, the charge produced upon any deflection of the bimorph, due to repulsive or attractive forces, is recorded. Once the surfaces come into hard-wall contact, the linear movement of the piezo deflects the bimorph, thus enabling the force sensor to be calibrated against the known piezo crystal expansion and contraction as measured by a linear variable differential transformer (LVDT) sensor. Provided the deflection and the spring constant of the bimorph are known, the data are used to calculate the force-distance curves from Hooke’s law. The noninterferometric surface force apparatus does not allow absolute determination of the zero surface separation; however, the adsorbed surfactant layer thickness can be obtained from the magnitude of inward “jumps” that typically occur when a surfactant layer is pushed out from the contact area upon compression. It has been shown (Ederth et al., 1998) that flame-polished glass surfaces are smooth enough to enable accurate measurements of surface forces down to molecular separations. The surfaces used in each experiment were prepared by melting one end of a borosilicate glass rod (diameter 2 mm and length ca. 25 mm) in a butane‑oxygen burner until the tip formed into a sphere with a diameter of about 4 mm. The normal radii of curvature (r1 and r2) for each surface were determined more accurately at the end of the experiment by using a micrometer, and the local harmonic mean radius of the interaction, R, was then calculated as R = 2r1r2/(r1 + r2). The spring constant of the bimorph was measured at the end of each experiment by placing known weights on the bimorph spring and measuring the resulting deflection (usually about 100 N m−1). The force was then normalized by the local harmonic mean radius of interaction (F/R). The hydrophobic surfaces on which the adsorption of the surfactants was studied were obtained by silanization and thiolization of the hydrophilic glass surfaces. Despite being hydrophobic, the silanated glass carries a significant net negative charge. This charge results from the dissociation of unreacted silanol groups in the glass substrate that is not completely screened by the self-assembled silane layer. The thiolated surfaces, however, are completely uncharged. Details about the surface preparation can be found in previous reports (Ederth et al., 1998; Stubenrauch et al., 2004a). All procedures for assembling the measuring chamber and preparing the solutions were carried out in a laminar flow cabinet. At the beginning of each set of experiments, the interaction profiles were first determined in air to ensure that the system showed no signs of contamination. Then, background electrolyte solution was introduced into the measuring chamber, and the interaction profiles were again determined. Stock surfactant solution was then introduced through a 0.2 μm polytetrafluoroethylene (PTFE) filter until the desired concentration inside the chamber was attained. All measurements were carried out at 22 ± 1°C.

12.2.2  Thin Film Pressure Balance The most prominent method for investigating the interactions between two surfactant films at the air/water surface, that is, the interactions acting in foam films, is the thin-film

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pressure balance (TFPB) (Mysels and Jones, 1966; Exerowa and Scheludko, 1971; Claesson et al., 1996; Exerowa and Kruglyakov, 1998; Stubenrauch and von Klitzing, 2003) and its modified versions. In brief, a film is formed in a film holder consisting of a porous glass disk that is connected to a glass tube. A hole is drilled in the disk in which the film is formed. This film holder is fixed in a gas-tight cell, a pressure is applied to the cell via a syringe, and the film thickness h at this particular pressure is determined interferometrically. By calculating the disjoining pressure Π from the applied pressure, one obtains the characteristic Π-h curves.

12.2.3  Surface Light Scattering The surface light scattering (SLS) set up referred to in this study was reported by Rojas et al. (2005b), which is based on the original work of Hård and Neuman (1987). The respective surfactant solution was placed inside a closed 316 stainless steel double-walled thermostated cabinet sitting on an optical table. The temperature was monitored by temperature probes located inside the chamber and maintained by an external thermostated circulation bath (to ±0.1°C). The humidity was set close to saturation (ca. 90% RH) by placing filter papers moisted with water. In a typical experiment, the experimental autocorrelation function of the surface waves was recorded and fitted to an exponentially damped cosine function (Bouchiat and Meunier, 1971). The correlograms were Fourier-transformed and then fitted to a four-parameter Lorentzian function (Hård and Neuman, 1987; Bellman and Pennington, 1954). From these operations, we obtained the parameters of the power spectrum. The measured power spectrum was compared with the theoretical power spectrum (Kramer, 1971) for capillary waves in the presence of air. This theoretical power spectrum is described, among others, in terms of the surface tension γ, the transverse viscosity μ, the sum of interfacial shear elasticity and interfacial dilatational elasticity ε, and the sum of interfacial shear viscosity and interfacial dilatational viscosity κ. The experimental data, that is, the central frequency and the dampening coefficient (after correction for instrumental broadening), together with the bulk properties of the fluids were used to calculate the viscoelasticity coefficients from the power spectrum equation. Hence, the sum of real and imaginary part of the complex modulus (elasticity and viscosity) was obtained. We used polar diagrams for direct interpretation of the rheological parameters ε and κ. These plots were constructed from the dispersion equation for a given temperature, wave number, and surface tension (or surface pressure) using as parameters the normalized complex frequency (ωo/ωw and α/αw) where ωo is the experimental central frequency and α is the dampening coefficient. Here, the subscript “w” is used to denote the experimental values for a film-free surface (ε = 0 and κ = 0), that is, water in our case.

12.3  FUNDAMENTAL STUDIES ON INTERFACIAL PROPERTIES OF SUGAR-BASED SURFACTANTS A key aspect of our studies lies in a systematic comparison between petroleum-based surfactants and sugar-based surfactants. This is intended to highlight the benefits of sugar-based surfactant platforms. We compared an EO-based surfactant (hexaoxyethylene dodecyl ether, C12E6) III.  BIOBASED SURFACTANTS



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HO

n-Dodecyl-β-D-maltoside,β-C12 G2

O

HO OH

O

OH OH

O

O

OH

OH

Hexaoxyethylene dodecyl ether, C12 E6 O O

O

O

OH O

FIG. 12.1  n-Dodecyl-β-d-maltoside (β-C12G2, top) and hexaoxyethylene dodecyl ether (C12E6, bottom) are two nonionic surfactants with the same hydrophobic group that were compared regarding their interfacial properties.

with a sugar-based surfactant with the same hydrophobic group (n-dodecyl-β-d-maltoside, β-C12G2) (see Fig. 12.1) (Rojas et al., 2005a; Claesson et al., 2006). We consider here the interactions between nonpolar surfaces coated with either C12E6 or β-C12G2. As nonpolar surfaces, we chose the air/water surface, silanated glass, and thiolated gold surfaces. The most important results with respect to the comparison of different surfaces are summarized in this section.

12.3.1  Air/Water Interfaces The adsorption of nonionic surfactants at the air/water surface leads to a decrease of the surface charge of the interface (reviewed in Stubenrauch and von Klitzing, 2003). This decrease eventually results in a transition from an electrostatically stabilized common black film (CBF) to a Newton black film (NBF) that is stabilized by short-range repulsive forces. This phenomenon is illustrated in Fig. 12.2 with data obtained for the nonionic surfactant hexaoxyethylene dodecyl ether (C12E6). The formed NBF consists of two densely packed monolayers and creates a “force barrier” that prevents the film from rupturing and thus stabilizes a foam. Apart from densely packed monolayers, a sufficient monolayer cohesion is required for the formation of a stable NBF (Stubenrauch et al., 2004a,b). From the thickness of the NBF, one can estimate the thickness of one monolayer. Comparing Fig. 12.2 with the results obtained for n-dodecyl-β-d-maltoside (β-C12G2) shown in Fig. 12.3, one sees the same general trend. In both cases, film thicknesses were found to range from more than 80 nm to less than 5 nm, depending on the surfactant concentration and the applied pressure, which ranges from 200 to 9000 Pa. As was the case for C12E6 (Fig. 12.2), two different kinds of films were observed: thick common black films (CBF) stabilized by electrostatic repulsion and thin Newton black films (NBF) stabilized by short-range repulsion. The thicknesses of the CBFs decrease monotonically as Π increases. While the slope d(log Π)/dh is independent of the surfactant concentration, a significant shift of the curves toward lower disjoining pressures is observed when increasing the β-C12G2 concentration from 0.034 to 0.137 mM. Moreover, at the highest concentration, no CBF is formed at all, but the foam film drains directly down to the NBF. It has been demonstrated experimentally (reviewed in Stubenrauch and von Klitzing, 2003) and only recently also theoretically (Kudin and Car, 2008) that the air/water surface is

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Common black film, CBF

Newton black film, NBF

10 nm < h < 100 nm

h < 10 nm

10,000 0.1 mM

Π (Pa)

0.01 mM 1000

100

0.05 mM

0

20

40

60

80

100

h (nm)

FIG. 12.2  (Top) Pictures and schematic drawings of a common black film and a Newton black film, respectively. (Bottom) Disjoining pressure Π as a function of film thickness h measured for three concentrations of C12E6 in 10−4 M NaCl solution. The two lower concentrations are below; the highest is above the CMC (=0.073 mM). The solid lines are calculated according to the DLVO theory assuming interactions at constant charge (Stubenrauch et al., 2004a).

­ egatively charged. This charge is responsible for the long-range electrostatic repulsive forces n observed in foam films stabilized by nonionic surfactants. An increase in the nonionic surfactant concentration leads to a decrease of the surface charge density as more uncharged molecules (i.e., nonionic C12E6 and β-C12G2 surfactants) adsorb at an originally charged surface. The electrostatic forces acting in foam films stabilized by β-C12G2 were quantified by means of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory using constant charge boundary conditions and the theoretical Debye length of κ−1 = 30 nm. These calculations led to surface charge densities of q0 = 1.55 mC m−2 for the 0.035 mM solution and q0 = 0.95 mC m−2 for the 0.137 mM solution. The decrease in surface charge density destabilizes the CBF until no CBF is observed for c > CMC under the given experimental conditions. At these concentrations, it is the immediate formation of an NBF that is observed. The NBFs are very thin (ca. 5 nm) with an aqueous core of 1–2 nm assuming a length of ~2 nm for the surfactant. In other words, these films consist of two surfactant monolayers with only small amounts of water separating the head groups (mainly hydration water). As is the case for the force barrier between

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10,000

Π (Pa)

0.0340.034 mM mM 0.1370.137 mM mM 0.1700.170 mM mM

1000

100

(b) 0

10 20 30 40 50 60 70 80 90 100 h (nm)

FIG. 12.3  Disjoining pressure Π as a function of film thickness h measured for three concentrations of β-C12G2 in 10−4 M NaCl solution. The two lower concentrations are below; the highest is above the CMC (=0.14 mM). The solid lines are calculated according to the DLVO theory assuming interactions at constant charge (Stubenrauch et al., 2002).

nonpolar solid surfaces (to be presented later), densely packed monolayers are required to stabilize an NBF and thus to prevent contact between the two bare surfaces (i.e., film rupture). Hence, the presence of an NBF signifies the existence of a force barrier between two air/water surfaces in analogy to the force barrier between two solid surfaces (see Figs. 12.4 and 12.5). The remarkable similarities with regard to the Π-h curves of β-C12G2 and C12E6 are evident from the fact that the curves nearly lie on top of each other, which means that the surface charge densities q0 are nearly equal. Indeed, surface charge densities of q0 = 1.70 mC m−2 at c = 0.01 mM (0.12 CMC) for C12E6 and q0 = 1.55 mC m−2 at c = 0.035 mM (0.25 CMC) for β-C12G2 were calculated from the experimental data. As the surface charge density is a property of the bare air/water surface, these values mean that a surface concentration of 3.0 × 10−6 mol m−2 C12E6 (0.12 CMC) reduces the surface charge density to the same amount as a surface concentration of 4.0 × 10−6 mol m−2 β-C12G2 (0.25 CMC). Moreover, in both cases, the surface concentration is enough to stabilize a foam film up to 10,000 Pa. We suggest that it is the effective surface cross-sectional area covered per molecule that determines the reduction in surface charge density. Since the head group of C12E6 is larger than that of β-C12G2, a smaller number of C12E6 molecules would be needed to achieve a given reduction in the repulsive ­double-layer force. We conclude that for each nonionic surfactant, the surface charge density is reduced with increasing adsorption. More thorough discussion regarding the influence of the head groups and the chain length of the surfactant on film stability can be found in (Stubenrauch et al., 2002; Schlarmann and Stubenrauch, 2003).

12.3.2  Solid/Liquid Interfaces Silanated glass and thiolated gold surfaces were used as hydrophobic solid surfaces. Upon adsorption, at the lowest surfactant concentration, we noted that the interaction forces were

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2

F R−1 (mNm−1)

Liquid

Surfactant

1

Solid 0.010 mM C12 E6

0 Without C12 E6

0

10

20 30 D (nm)

40

50

FIG. 12.4  Force F normalized by the radius R as a function of relative surface separation D between two thiolated solid surfaces. The forces were measured across aqueous solutions containing 0.1 mM NaCl in the absence (filled circle) and in the presence of 0.01 mM nonionic surfactant C12E6 (open squares). Similar behavior was observed for βC12G2 (see Fig. 12.5). Modified from Stubenrauch, C., Rojas, O.J., Schlarmann, J., Claesson, P.M., 2004. Interactions between nonpolar surfaces coated with the nonionic surfactant hexaoxyethylene dodecyl ether C12E6 and the origin of surface charges at the air/water Interface. Langmuir 20(12), 4977–4988. See also Stubenrauch, C., Schlarmann, J., Rojas, O.J., Claesson, P.M., 2004. Comparison between interaction forces at air/liquid and solid/liquid interfaces in the presence of non-ionic surfactants. Tenside Surfactant Deterg. 41, 174–179; and Rojas, O.J., Stubenrauch, C., Schlarmann, J., Claesson, P.M., 2005. Interactions between non-polar surfaces coated with the non-ionic surfactant n-dodecyl-β-d-maltoside. Langmuir 21, 11836–11843.

F R−1 (mNm−1)

2

β-C12G2

1

C12E6 D (nm)

0 0

5

10

15

20

FIG. 12.5  Steric force barriers for 0.035 mM β-C12G2 and 0.01 mM C12E6, respectively, between solid thiolated surfaces. A stronger force barrier is observed for the sugar-based surfactant. This steric force is responsible for the stabilization of dispersions, where the solid particles are separated by liquid films. The stronger barrier seen in the case of the sugar surfactant is explained by a larger stiffness of its polar group as compared with the EO units (Rojas et al., 2005a).

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397

dominated by a steric barrier at short surface separation. By increasing the surfactant concentration, a more robust steric barrier was formed. Interestingly, the NBF formation observed for foam films (see Figs. 12.2 and 12.3) corresponded to the appearance of this force barrier between two solid surfaces (see Fig. 12.4 in the case of thiolated gold). The barrier shown in Fig. 12.4 is a measure of the force that is needed to remove surfactant from between the two surfaces. Because this barrier is located at a distance corresponding to the thickness of a surfactant bilayer, the analogy to the NBF formation is obvious. Note that a “removal” of this bilayer results in film rupturing in the case of foam films, whereas in the case of solid/liquid interfaces, it leads to a direct contact of the solid surfaces (Rojas et al., 2005a; Stubenrauch et al., 2004a). It is also concluded (data not shown) that the nature of the surface at which the surfactant adsorption takes place mainly influences the interaction forces at low surface coverage. Once a densely packed surfactant layer is formed, the surfactant itself determines the interaction forces.

12.3.3  Comparison Between EO- and Sugar-Based Surfactants: Structural Aspects, Interfacial Aspects, Packing at the Interface Since the surfactant can determine the interaction forces, depending on the surface coverage, it makes sense to inquire further on structural effects, for example, the influence of the polar head group on the interaction forces at the molecular level. Comparing data obtained for β-C12G2 and C12E6, we demonstrated that for similar bulk concentrations, the sugar surfactant forms a denser and more robust monolayer with higher cohesiveness (see Fig. 12.5). On the other hand, compared with the shorter tail homologue β-C10G2, β-C12G2 is anchored more strongly to hydrophobic surfaces. This is explained by a stronger interaction between the nonpolar tails within the monolayer. Our measurements indicated that the thickness of the β-C12G2 monolayer under compressive loads was marginally lower than 2 nm. This thickness value is in agreement with the surfactant molecular dimensions but smaller than the (compressed) monolayer thickness measured for C12E6. For both surfactants, the most distinctive feature in the force curve between surfactant-coated solid surfaces is the buildup of a steep and short-range repulsion as the surfactant adsorption approaches saturation. Boos et al. (2013a) measured the surface rheology of C12E6 and β-C12G2 under similar conditions and found that β-C12G2 has a higher surface elasticity, which, in turn, explains the observation that β-C12G2 forms much more stable foams than C12E6 (Stubenrauch et al., 2009, 2010; Boos et al., 2013b). In other words, the head group of the sugar-based surfactants is stiffer, less compressible, and more densely packed, which, in turn, seems to be favorable for the stabilization of foams and dispersions (Lu et al., 2007; Lu and Somasundaran, 2008). In summary, we have so far exclusively considered short-range repulsive interactions between surfactant layers. They are divided roughly into repulsive forces due to hydration and steric forces (Claesson et al., 2006). Of course, the net short-range interaction that is the only measurable quantity is due to a balance of attractive and repulsive force contributions. In the next sections, we will recapitulate some findings about hydration of polar groups and relate those findings to the short-range interactions between surfactant layers.

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12.3.4  Viscoelasticity Properties of Isomeric Sugar-Based Surfactants A wealth of information about surface tension of sugar-based surfactant is available in the literature, some of which are cited in this chapter. As expected, differences in surface activity, packing density, minimum surface tension, and critical micelle concentration have been observed when comparing sugar surfactants of different type or different structure. Less obvious is perhaps the fact that subtle changes in the stereochemistry of a given surfactant can lead to noticeable differences in the same properties mentioned above. Instead of discussing tensiometry data for sugar surfactants here, we only point out the case of an interrelated property, namely, the viscoelasticity of adsorbed monolayers. SLS by capillary waves was used to investigate the adsorption behavior of nonionic sugar surfactants at the air/water surface. The viscoelastic properties (surface elasticity and surface viscosity) of monolayers formed by the isomeric surfactants octyl β-glucoside, octyl α-glucoside, and 2-ethylhexyl α-glucoside (see Fig. 12.6) were quantified at submicellar concentrations (Rojas et al., 2005b). The same surfactant types were used in an earlier study by Waltermo who presented surface tension data and its relation with the foaming behaviors (Waltermo et al., 1996). Unfortunately, SLS data for C12E6 and β-C12G2 that were discussed in previous sections are not available; however, we used the surfactants illustrated in Fig. 12.6 to document the conclusion that differences in the configuration of the molecule affect the surface packing and thus the rheological behavior of the formed monolayers. Likewise, macroscopic properties such as foam ability and emulsion stability are also influenced.

OH

H HO

H HO

H

(A)

(B)

H HO

O

HO

H

H

O

HO O

H O

HO

H

H

O

OH

H

H

HO

H

O

HO H HO H H OH

(C)

FIG. 12.6  Isomeric sugar surfactants used to investigate the effects of the molecular structure on surface viscoelasticity: n-octyl-β-glucoside (A), n-octyl-α-glucoside (B), and 2-ethylhexyl-α-glucoside (C).

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The surfactants used (Fig.  12.6) were anomerically pure alkyl glucosides. The focus of this study was to uncover the difference between the β- and α-anomers of octyl glucoside (Fig. 12.6A and B, respectively). In addition, the effect of the structure of the surfactant’s hydrophobic part was addressed by comparing a linear (Fig. 12.6B) and a branched octyl chain (Fig. 12.6C). Readers interested in the processing of SLS data to calculate the surface rheological properties are referred to (Rojas et al., 2005b). In short, we used so-called helmet or polar plots in which radial and semicircular lines represent constant interfacial elasticity (ε) and constant interfacial viscosity (κ) or, in other words, isoelasticity and isoviscosity curves, respectively (see Fig. 12.7). The distance of an experimental data point from the origin (ε = 0 and κ = 0) in a polar plot is a measure of the surface elasticity and/or viscosity. Therefore, the surface elasticity and viscosity can simply be estimated by inspection of the location of the experimental data within the polar plot. Fig. 12.7 shows the experimental polar loci or the viscoelastic behavior of the three surfactants described earlier (at an SLS wave number of 78,508 m−1). The polar plots show first the limiting case of a surfactant-free liquid (no monolayer present), that is, pure water (ε = 0 and, κ = 0). As the surfactant concentration increases, the respective viscoelastic behavior can be identified by direct inspection of the curve starting from the value corresponding to zero surfactant concentration (ε = 0 and κ = 0). At low surfactant concentrations, the tendencies indicate a deviation from the perfectly elastic film behavior (κ = 0, contour line). In these cases, a maximum propagation velocity and dampening limit are attained. More importantly, in terms of the present discussion, is the fact that the polar plots indicate small yet distinctive differences in the viscoelasticity of the three isomeric (sugar) surfactants. Therefore, in agreement with other studies (Niraula et  al., 2004; Neimert-Andersson et  al., 2004), it can be concluded that the stereochemistry of the surfactant plays an important role in molecular packing, lateral interactions, and overall intermolecular forces, all of which affect the respective macroscopic surface behaviors. We note that the area per molecule for octyl α-glucoside and octyl β-glucoside surfactant was found to be 41.5 and 43.5 Å2, respectively (Rojas et  al., 2005b). Furthermore, the data presented in Fig.  12.7 demonstrate further important changes in molecular packing at the air/water surface for these isomeric surfactants. This is by virtue of very subtle configuration differences of the head group and the tail structure. One can therefore anticipate that by fine-tuning the molecular architecture of sugar surfactants, applications involving different performance requirements can be developed. For example, foamability and emulsion stabilization could be varied by changing the polar group (position and stereochemistry) and the structure of the hydrophobic tail, for example, via branching. Changes in the stereochemistry of the head group units are also expected to influence the interaction with the ions present in solution. Such effects were made clearer in the discussion presented in Section 3.1 on thin-film pressure balance experiments for other surfactants. It should be stressed, however, that dynamic effects (diffusion, relaxation, etc.) play important roles as well. At this point, it is meaningless to speculate further about the relationship between surfactant structure and properties until the nature of surface charge density, surfactant packing, and dynamic effects are better understood. An attempt to shed some light regarding the effects of surfactant structure will be covered in the next sections.

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e (mNm–1)

e (mNm–1)

Normalized damping aa 0–1

k (mgs–1)

Normalized central frequency ww 0–1

k (mgs–1)

Normalized central frequency ww 0–1

k (mgs–1)

Normalized central frequency ww 0–1

FIG. 12.7  Helmet or polar plots showing the experimental surface viscosity and elasticity obtained from the propagation (normalized central frequency and dampening) of capillary waves probed by surface light scattering for n-octyl-β-glucoside (bottom), n-octyl-α-glucoside (middle), and 2-ethylhexyl-α-glucoside (top) surfactants. The loci of the different curves show differences in the behavior of these isomeric surfactants (Rojas et al., 2005b).

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Normalized damping aa 0–1

e (mNm–1)



12.4  Molecular Prospects

401

12.4  MOLECULAR PROSPECTS It is very difficult to obtain experimental information on the structure of water adjacent to solutes. The main reason for this is that in most cases, the spectroscopic signal from the hydration shell is swamped by that of bulk water. However, recently, some progress has been made where an inherently surface-sensitive spectroscopic technique, vibrational sum frequency spectroscopy (VSFS), has been employed to investigate the hydration of sugar groups and oligo(ethylene oxide) chains anchored to the air/water surface. It was found that some very strong hydrogen bonds are formed between sugar and water, whereas the hydration of the oligo(ethylene oxide) chain is dominated by a clathrate-like structure (Tyrode et al., 2005; Claesson et al., 2006). Related arguments can be applied when considering short-range interactions between surfactant layers. The fact that hydrogen bonds can form between polar head groups of, for example, sugar-based surfactants does not directly lead to the conclusion that the short-range interaction across water should be attractive due to hydrogen bond formation. It is thus of some interest to compile available data on the depth of the interaction force minimum. In this case, the pull-off force needed to separate surfaces that are in contact (e.g., after bringing the surfaces together, as shown in Figs. 12.4 and 12.5) is measured. This force is related to the adhesion energy that in our case was measured between adsorbed nonionic surfactant layers. This will allow us to learn if the possibility of forming hydrogen bonds between two head groups has a bearing on the short-range attraction observed in these systems (see Table 12.1). We note that no direct hydrogen bonds can be formed between DDAO molecules in uncharged form, DDPO, and surfactants with ethylene oxide head groups (see Table  12.1). However, such interactions are possible for surfactants with polyhydroxyl head groups and also for amine head groups. By comparing the data compiled above, we note that the trend is that head groups that are able to form interlayer hydrogen bonds display a somewhat larger attractive short-range force component than layers that are unable to form such bonds. This could indicate that there is a contribution from interlayer hydrogen bond formation, but the extension of this effect is difficult to determine since the nature of the head group also influences other short-range interactions, for example, van der Waals forces. Due to the lack of systematic data, we cannot compare quantitatively the hydration numbers obtained for ethylene oxide and glucose head groups. A qualitative evaluation, however, might shed some light on the differences between these surfactants. From extensive SANS measurements in bicontinuous microemulsions, we know that the area per head group at the water/oil interface is 0.56 nm2 for a glucose unit (Kluge, 2000), which is comparable with that of a surfactant with four ethylene oxide units that is 0.54 nm2 (Sottmann et al., 1997). The hydration number of the glucose head group, as determined by NMR, is found to be 6 at a surfactant concentration of 3 wt%, whereas NMR estimates the hydration number to be 6 per ethylene oxide unit at a surfactant concentration of 10 wt% (i.e., 24 water molecules per tetraoxyethylene head group). Keeping in mind that the hydration number per ethylene oxide unit is expected to be even larger at 3 wt%, one can conclude that under similar conditions and for similar head group sizes, the hydration of ethylene oxide-based surfactants is significantly higher than that of sugar-based surfactants (Claesson et al., 2006). The short-range interaction between surfactant layers exposing the polar part toward solution is due to a complex interplay between van der Waals forces, hydration, and steric effects.

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TABLE 12.1  Depth of Force Minimum Between Various Nonionic Surfactant Layers (Claesson et al., 2006 and References Therein). Compound

Technique

Depth of Force Minimum F/R (mN m−1)

Substrate Surface and Remarks

n-Dodecyl amine oxide (DDAO) uncharged

SFA

0.25

Bilayer on mica

n-Dodecyl phosphine oxide (DDPO)

SFA

0.25

Hydrophobized mica. Force minimum increases with increasing temperature

Tetraoxyethylene dodecyl ether (C12E4)

MASIF

0

Silanated glass surfaces

Pentaoxyethylene dodecyl ether (C12E5)

SFA

0.1–0.2

Hydrophobized mica. At 20°C. Force minimum increases with increasing temperature

Hexaoxyethylene dodecyl ether (C12E6)

MASIF

1–1.4

Silanated glass surfaces

Hexaoxyethylene dodecyl ether (C12E6)

MASIF

0.2–0.4

Thiolated gold surfaces

Tetraoxyethylene dodecyl amide (C12ONHE4)

SFA

0.3–0.4

Hydrophobized mica and silanated glass (Herder, 1991)

Tetraoxyethylene dodecyl amine (C12NHE4)

SFA

0.1–0.2

Bilayer on mica, pH 9.4–9.9

n-Octyl-β-glucoside (β-C8G1)

SFA

1.2–1.5

Hydrophobized mica

n-Decyl-β-d-glucoside (β-C10G1)

MASIF

1

Thiolated gold surfaces. Estimated (Waltermo et al., 1993)

n-Decyl-β-d-maltoside (β-C10G2)

MASIF

0.9

Silanated glass surfaces

n-Dodecyl-β-d-maltoside (β-C12G2)

MASIF

1–2

Thiolated gold surfaces

Technical sugar surfactant mixture

SFA

2

Hydrophobized mica. Located on the repulsive side

n-Dodecyl lactobion amide (LABA), inner sugar ring is open

SFA

1.2

Hydrophobized mica

n-Dodecyl lactobion amide (LABA), inner sugar ring is open

MASIF

1.5

Silanated glass surfaces

Maltose 6′-O-dodecanoate (C12OG2)

MASIF

1.0

Silanated glass surfaces

0.5–1

Bilayer on mica, pH 9.5–10.1

n-Dodecyl amine (C12NH2) SFA Most molecules in the outer layer are uncharged

Data correspond to aqueous surfactant solutions at or above the respective CMC, that is, to a saturated monolayer. MASIF, measurement and analysis of surface interaction forces; SFA, surface force apparatus.

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12.5  Structure-Property Relationships

403

It is only the total force that can be measured, and this force displays both attractive and repulsive regimes. van der Waals force is the main attractive force, while hydrogen bonding within adsorbed layers, again directly or mediated by water molecules, increases the packing density within adsorbed surfactant layers, and this increases the van der Waals force. We note that both hydration and the mobility of surfactants are important for short-range interactions. The steric interaction between surfactants with oligomeric head groups also deserves further attention. It has been observed that as the head group size increases, the short-range repulsion goes from a hard-wall repulsion, via compressible excluded volume repulsion to a steric repulsion described by polymer brush theories (de Gennes, 1987). The experimental results discussed in this chapter help to demonstrate fundamental features of nonionic surfactants based on sugar polar groups (mainly glucoside and maltoside). These polar groups make them structurally more attractive in terms of their capacity to stabilize interfaces (liquid/liquid, solid/liquid, and gas/liquid) as compared with EO-based nonionic surfactants. Note that the OH groups in the sugar units can be used for chemical reactions, and thus, one has the possibility of engineering the surfactant structure and its stereochemistry (which, as has been demonstrated before, play a leading role for the properties). There are key molecular components (head group configuration, identity, number, tail length, and saturation) leading to subsequent packing aspects for “green” polysaccharide streams that can ultimately be used to manufacture nonionic surfactants. As mentioned in previous sections, nonionic, saccharide-based surfactants offer a veritable plethora of benefits for state-of-the-art surface, colloid, and condensed state research. Their solubility and phase behavior are typically insensitive to temperature changes in contrast to ethylene oxide-based surfactants. This allows much greater amplitude in their synthetic design without compromising desirable physical and chemical features such as small-­molecule (e.g., blood glucose) biosensor applications, biophysical recognition (such as antigen-­antibody mechanisms), host-guest chemistry, and a number of other smart functions. There is a need to use separation chemistries and chemically fine-tune the respective sugar surfactants with the appropriate physical characteristics (functional groups and/or ligands) to achieve the “smart” functionality we have described. The performance of sugar surfactants (vs EO surfactants) in dispersed systems (such as foams, emulsions, and dispersions) and their potential for the formation of new structures in these systems (such as unilamellar vesicles, ribbons, spirals, and tubes) depends on the detailed molecular structure. New surfactants can be synthesized that may offer unique templates for more sophisticated electronic, biomedical, and optical devices. Some strategies in the design of sugar surfactants to provide greater functionalities include the introduction of both mono- (glucoside) and di-(maltoside) saccharide moieties with various modes of attachments to the head group of synthesized surfactants and also the introduction of different degrees of hydrophobicity and crystallinity to the sugar surfactants by manipulating their aliphatic tails.

12.5  STRUCTURE-PROPERTY RELATIONSHIPS Efforts need to be devoted to the development of new surfactants based on structure-­ property relations. In the simplest form, a new sugar surfactant consists of three discrete parts, namely, (a) a polar head group (e.g., one or two glucosides or one maltoside), with or

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12.  MEASURING THE INTERFACIAL BEHAVIOR

without (b) a spacer (that could display the versatility of allowing the attachment of a second head group and two tails as well as two different tail placements for one group), and (c) an aliphatic tail (e.g., a fatty acid). How changes of these three parts affect the performance of the respective surfactants foaming, emulsifying, or dispersing agent is the key question. Some leads to answer such questions were presented in previous sections. What is exciting about all of the possible configurations is the inherent potential for providing a number of different surface interactions based on the hydrophilic and lipophilic balance (HLBG) parameter (Griffin, 1954), the variation in the packing based on steric interactions (Piispanen et al., 2004), and the packing variation based on other interactions (John and Vemula, 2006). The HLBG is useful to characterize the balance between the hydrophilic and hydrophobic parts of the surfactant molecule. The HLBG can be varied by changing the length of the tail of the surfactant. This can lead to different packing behaviors. The molecules will have a pronounced ability to engage in discrete regular packing motifs, as was shown in recent work (John et al., 2003). Typically, surfactant packing is dominated by the hydrophobic interactions between the hydrophobic chains, which, in turn, influences the flexibility, the orientation, and the surface energy of molecular assemblies. There is a serious need for a systematic study of structure-property relations that define sugar-based surfactants as the alternative of choice. Major issues in this area are the following: – Development of new products, production practices, and business in the biomass industry. This requires rapid changes in consumer demand for renewable products and alternative market solutions. – Replacement (even small fractions) of petroleum-based nonionic surfactants with sugarbased surfactants, which is important from an environmental and energetic point of view. – Synthetic strategies to reduce the costs of sugar-based surfactants as current barriers for acceptance of sugar-based surfactants are their costs. Linking fundamental studies with economics and market analysis may promote this goal. – Systematic studies of the properties of sugar-based surfactants as the lack of information about their properties and performance benefits may be an obstacle for their wide use. The use of nonbiodegradable, petroleum-based surfactants is a major drawback, and therefore, the development of biodegradable surfactants derived from agricultural resources is justified. Sugar-based surfactants represent a proved candidate, but any effort aimed at formulating dispersed systems using the saccharide platform entails that specific technical requirements in terms of their applications must be met. The performance of (new) surfactants in any give application is expected to be closely related to molecular factors, with the interfacial behaviors being some of the most important ones.

12.6  APPLICATIONS RELATED TO THE DEVELOPMENT OF BIO-BASED SYSTEMS Current applications of surfactants require more natural and sustainable surfactants to achieve efficient production, products with sufficiently long shelf lives despite environmental stresses, and also products that address growing environmental concerns. Sugar-based sur-

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12.6  Applications Related to the Development of Bio-Based Systems

405

factants address all of the above by being by-products of biomass processing, that is, they can result from biorefining streams. In today’s market, glycoside surfactants produced from fatty acid and mono- or multimeric saccharides are produced in the kiloton per annum with a large scope of applications in industries producing pharmaceutical products, food, personal care products, detergents, fiber products, agrochemicals and more recently for bioactive surfaces (Hill and Rhode, 1999; Kitamoto et al., 2002; von Rybinski and Hill, 1998; Mahon et al., 2010). Many of such applications involve the replacement of ethylene oxide surfactants that can result in hazardous pollutants as they are made from petrochemical derivatives. One of the most important market benefitting from sugar-based surfactants consists of oil-in-water edible emulsions, such as beverages and drinkable emulsion products. Among them, sophorolipids and rhamnolipids (covered in detail in Chapters 3 and 5, respectively) are a type of glycolipids that can be purified from certain microorganisms (e.g., Pseudomonas aeruginosa) using fermentation processes (de Oliveira et  al., 2015; Henkel et  al., 2012), leading to a fact that the composition and performance of such surfactants are largely dependent on microbial strain, fermentation conditions, and used substrates in culturing. The molecular structure of rhamnolipids includes one or two polar rhamnose units linked with a nonpolar fatty acid chain containing a β-hydroxyalkanoate and a carboxylic acid group as part of the hydrophilic regions, which enables the rhamnolipids an anionic character under appropriate pH conditions (Müller et al., 2012). Rhamnolipids have been successfully used to form oil-in-water emulsions containing antimicrobial essential oils (Haba et al., 2014). A nanoemulsion system has also been developed by using rhamnolipids and food-grade triglyceride oils via microfluidization (Bai and McClements, 2016). Particularly, the low concentration of rhamnolipids required to produce nanoemulsions makes them highly desirable in a variety of commercially food-grade emulsion-based products (Fig. 12.8A1, A2, and A3). Sophorolipids can be produced on an industrial scale using microbial fermentation processes (De et al., 2015) but at high production costs (approximately $2.54 kg−1 in 2014) (Ashby et  al., 2013), thereby making them more likely for niche applications (McClements et  al., 2017). Similar to rhamnolipids, sophorolipids have also been used to stabilize oil-in-water emulsions. The successful encapsulation of structured lipids (interesterified soybean and rice bran oils) into oil-in-water emulsions is facilitated by the efficient emulsifying ability of sophorolipids and yields food-grade emulsions with possible antioxidation properties (Xue et al., 2013). Sugar-based surfactants have also found applications through developments in solvent extraction in the petroleum and wastewater treatment industries where an important drawback in classic solvent extraction processes is the use of organic solvents, for example, principally high-boiling hydrocarbons. Such organic solvents can also bear toxic aromatic moieties (Adamczak et al., 1999). By utilizing surfactant micelles, small organic molecules and metal ions can be selectively trapped in the micellar pseudophase and separated with lower energy and pressure requirements in the so-called micellar-enhanced ultrafiltration (MEUF) (Purkait et al., 2006; Zaghbani et al., 2009). However, conventional monomeric and ionic surfactants are limited by retention through membrane pores and the presence of salts in large-scale, industrial settings. Therefore, biobased and biodegradable glycosidic surfactants, with less sensitivity to electrolytes, can be of special interest (Adamczak et al., 1999; Urbanski et al., 2000; Urbański et al., 2002; Samal et al., 2017).

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12.  MEASURING THE INTERFACIAL BEHAVIOR

FIG. 12.8  (a1) Visual appearance of rhamnolipid-stabilized nanoemulsions and effect of the ionic strength on their

stability. (a2) Molecular structures of the most common rhamnolipids available as biosurfactants, di-rhamnolipids. (a3) Influence of rhamnolipid concentration on the interfacial tension measured at the oil-water interface (Bai and McClements, 2016). Formation of volatile oxidation products in 5 wt% purified rapeseed oil emulsions stabilized with 1 wt% (b1) galactoglucomannans (GGM) and (b2) 5 wt% gum arabic (GA) after 13 days of storage at 40°C. The results are averages of n = 3 × 3 replicates with standard deviations, and the lines between the data points are drawn for guidance (Lehtonen et al., 2016). (c1) Schematic representation of the participation of the NSAID in the vesicle formation (Consola et al., 2007). (c2) In vitro percutaneous penetration of indomethacin after dermal application of aqueous solutions of the sodium salt and the vesicles of Indo12 (Consola et al., 2007).

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12.6  Applications Related to the Development of Bio-Based Systems

407

Sugar-based surfactants have also important implications in biology since several glycolipids have inherent functionalities due to the saccharide moieties (Mahon et al., 2010; Rosevear et al., 1980; Kiwada et al., 1985; Rico-Lattes and Lattes, 1997). Related properties have resulted in biological applications, such as the formation of bioreactors or carriers for drug delivery (Ollivon et al., 2000; Uchegbu and Vyas, 1998). The formation of surfactant (mixture) micelles helps with the solubilization and penetration of drugs to enhance drug effectiveness. By forming vesicles (Indo12) with amino sugar (Lhyd12) and nonsteroidal antiinflammatory drugs (NSAIDs) carboxylic acids, the antiinflammatory activity of indomethacin (an NSAID) on the skin could be effectively improved in vivo and for prolonged action due to a slow drug diffusion rate in the targeted sites (Fig. 12.8C1 and C2) (Consola et al., 2007). While the glucosidic surfactants applied in foams, detergents, and cleansers are mostly APGs (von Rybinski and Hill, 1998; Persson et  al., 2000), other species that are widely abundant and inexpensive need consideration. Foer instance, polysaccharides-based amphiphiles, such as gum arabic (GA), pectin, and modified starch, are of high commercial relevance. Gum arabic is a “golden standard” for the formulation of emulsions for the food industry. It is a branched polymer of anionic polysaccharide chains attached to a hydrophobic polypeptide backbone. It has low calorific values, resists intestinal enzymes, and is a nonstarch polysaccharide (Patel and Goyal, 2015). Apart from its outstanding emulsifying property (Klein et al., 2010), gum arabic has been used effectively and widely as a flavor encapsulating agent and stabilizer for oil emulsion concentrates (Yadav et al., 2007; Given, 2009) to produce soft drinks with the so-called flavor or cloud emulsions. Additionally, gum arabic provides beverages with controlled mouthfeel, flavor, color, and turbidity (Mirhosseini et al., 2008). These emulsions are highly stable under different pH, ionic strength, or temperatures (McClements et  al., 2017). Similar stabilization capabilities can be achieved with modified starch, which primarily consists of amylopectin (Piorkowski and McClements, 2014). Another widely applied sugar-based surfactant is pectin (e.g., beet pectin and citrus pectin). Due to the proteinaceous moiety attached to the carbohydrate portion of pectin, pectin emulsifying activities can vary (Nakauma et  al., 2008; Schmidt et  al., 2015a,b). The presence of carboxylate groups enables pectin to stabilize emulsions through both steric and electrostatic repulsion (Nakauma et  al., 2008). Therefore, with pectin, it is possible to produce emulsions that are more stable to external conditions than with gum arabic (Nakauma et  al., 2008). Other potential ­polysaccharide-based surfactants applied in emulsions include corn fiber gum (Yadav et  al., 2009; Cirre et  al., 2014), gum tragacanth (Farzi et  al., 2013), polysaccharide extracted from soy beans (Chivero et al., 2014), basil seeds (Osano et al., 2014), and wood (Lehtonen et  al., 2016; Mikkonen et  al., 2016a,b; Nypelö et  al., 2016). Further research and evaluation of the source-dependent reliability, economic viability, and environmental impact are needed for commercialization. For instance, galactoglucomannans (GGM), as an important by-product from forestry biorefinery industry, are attractive plant-based hydrocolloids that could fulfill the requirements necessary for efficient implementation. The lipid antioxidative capability of GGM (1 wt%) has been demonstrated to be better than the “standard” gum arabic (5 wt%) in rapeseed oil-in-water emulsion (Fig.  12.8B1 and B2) (Lehtonen et al., 2016).

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Acknowledgments O.J.R. acknowledges funding support from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 788489). C.S. is grateful for financial support of the European Commission under the 6th Framework Program, contract No. MRTN-CT-2004-512331-Project SOCON.

References Adamczak, H., Materna, K., Urbanski, R., Szymanowski, J., 1999. Ultrafiltration of micellar solutions containing phenols. J. Colloid Interface Sci. 218 (2), 359–368. Ashby, R.D., McAloon, A.J., Solaiman, D.K.Y., Yee, W.C., Reed, M., 2013. A process model for approximating the production costs of the fermentative synthesis of sophorolipids. J. Surfactant Deterg. 16, 683–691. Bai, L., McClements, D.J., 2016. Formation and stabilization of nanoemulsions using biosurfactants: rhamnolipids. J. Colloid Interface Sci. 479, 71–79. Balzer, D., Luders, H. (Eds.), 2000. Nonionic Surfactants: Alkyl Polyglucosides. Surfactant Science, vol. 91. Marcel Dekker, New York, ISBN: 0-8247-9390-0. Bellman, R., Pennington, R.H., 1954. Effects of surface tension and viscosity on Taylor instability. Q. Appl. Math. 12, 151–162. Blunk, D., Bierganns, P., Bongartz, N., Tessendorf, R., Stubenrauch, C., 2006. New speciality surfactants with natural structural motifs. New J. Chem. 30, 1705–1717. Blunk, D., Bongartz, N., Stubenrauch, C., Gärtner, V., 2009. Syntheses, amphitropic liquid crystallinity, and surface activity of new inositol-based amphiphiles. Langmuir 25, 7872–7878. Boos, J., Preisig, N., Stubenrauch, C., 2013a. Dilational surface rheology studies of n-dodecyl-β-D-maltoside, hexaethyleneglycol monododecyl ether, and their 1:1 mixture. Adv. Colloid Interf. Sci. 197–198, 108–117. Boos, J., Drenckhan, W., Stubenrauch, C., 2013b. Protocol for studying aqueous foams stabilized by surfactant mixtures. J. Surfactant Deterg. 16, 1–12. Bouchiat, M.A., Meunier, J., 1971. Spectre des fluctuations thermiques de la surface libre d’un liquide simple. J. Phys. 32, 561–571. Catanoiu, G., Blunk, D., Stubenrauch, C., 2012. Novel ethoxylated inositol derivatives—hybrid carbohydrate/oligoethylene oxide surfactants. J. Colloid Interface Sci. 371, 82–88. Chivero, P., Gohtani, S., Yoshii, H., Nakamura, A., 2014. Physical properties of oil-in-water emulsions as a function of oil and soy soluble polysaccharide types. Food Hydrocoll. 39, 34–40. Cirre, J., Al-Assaf, S., Phillips, G.O., Yadav, M.P., Hicks, K.B., 2014. Improved emulsification performance of corn fibre gum following maturation treatment. Food Hydrocoll. 35, 122–128. Claesson, P.M., Ederth, T., Bergeron, V., Rutland, M.W., 1996. Techniques for measuring surface forces. Adv. Colloid Interf. Sci. 67, 119–184. Claesson, P.M., Kjellin, M., Rojas, O.J., Stubenrauch, C., 2006. Short-range interactions between non-ionic surfactant layers. Phys. Chem. Chem. Phys. 8 (47), 5501–5514. Consola, S., Blanzat, M., Perez, E., Garrigues, J.C., Bordat, P., Rico-Lattes, I., 2007. Design of original bioactive formulations based on sugar–surfactant/non-steroidal anti-inflammatory catanionic self-assemblies: a new way of dermal drug delivery. Chem. Eur. J. 13 (11), 3039–3047. de Gennes, P.G., 1987. Polymers at an interface, a simplified view. Adv. Colloid Interf. Sci. 27, 189–209. de Oliveira, M.R., Magri, A., Baldo, C., Camilios-Neto, D., Minucelli, T., Pedrine Colabone Celligoi, M.A., 2015. Review: sophorolipids a promising biosurfactant and its applications. Int. J. Adv. Biotechnol. Res. 16, 161–174. De, S., Malik, S., Ghosh, A., Saha, R., Saha, B., 2015. A review on natural surfactants. RSC Adv. 5, 65757–65767. Deleu, M., Paquot, M., 2004. From renewable vegetables resources to microorganisms: new trends in surfactants. C.R. Chim. 7 (6–7), 641–646. Ederth, T., Claesson, P.M., Lindberg, B., 1998. Self-assembled monolayers of alkanethiolates on thin gold films as substrates for surface force measurements. Long-range hydrophobic interactions and electrostatic double-layer interactions. Langmuir 14, 4782–4789. Exerowa, D., Kruglyakov, P.M., 1998. In: Möbius, D., Miller, R. (Eds.), Foam and Foam Films—Theory, Experiment, Application. Studies in Interfacial Science, vol. 5. Elsevier, Amsterdam, ISBN: 9780444819222.

III.  BIOBASED SURFACTANTS

REFERENCES 409

Exerowa, D., Scheludko, A., 1971. Porous plate method for studying microscopic foam and emulsion films. C. R. Acad. Bulg. Sci. 24, 47–50. Farzi, M., Emam-Djomeh, Z., Mohammadifar, M.A., 2013. A comparative study on the emulsifying properties of various species of gum tragacanth. Int. J. Biol. Macromol. 57, 76–82. Given, P.S., 2009. Encapsulation of flavors in emulsions for beverages. Curr. Opin. Colloid Interface Sci. 14 (1), 43–47. Griffin, W.C., 1954. Calculation of HLB values of non-ionic surfactants. J. Soc. Cosmet. Chem. 5, 249–258. Haba, E., Bouhdid, S., Torrego-Solana, N., Marques, A.M., Espuny, M.J., García-Celma, M.J., Manresa, A., 2014. Rhamnolipids as emulsifying agents for essential oil formulations: antimicrobial effect against Candida albicans and methicillin-resistant Staphylococcus aureus. Int. J. Pharm. 476, 134–141. Hård, S., Neuman, R.D.J., 1987. Accurate laser light-scattering measurements of capillary waves. J. Colloid Interface Sci. 120 (1), 15–29. Henkel, M., Muller, M.M., Kugler, J.H., Lovaglio, R.B., Contiero, J., Syldatk, C., Hausmann, R., 2012. Rhamnolipids as biosurfactants from renewable resources: concepts for next-generation rhamnolipid production. Process Biochem. 47, 1207–1219. Herder, C.E., 1991. Interaction between phosphine oxide surfactant layers adsorbed on hydrophobed mica. J. Colloid Interface Sci. 143, 1–8. Hill, K., Rhode, O., 1999. Sugar-based surfactants for consumer products and technical applications. Eur. J. Lipid Sci. Technol. 101, 25–33. Hill, K., von Rybinski, W., Stoll, G. (Eds.), 1997. Alkyl Polyglycosides. Technology, Properties and Applications. VCH Verlagsgesellschaft, Weinheim. 242 pp. Holmberg, K., 2001. Natural surfactants. Curr. Opin. Colloid Interface Sci. 6, 148–159. ILO, International Labour Office, 1998. Encyclopaedia of Occupational Health and Safety, fourth ed. vols. 1–4 International Labour Office, Geneva, p. 104. John, G., Vemula, P.K., 2006. Design and development of soft nanomaterials from biobased amphiphiles. Soft Matter 2, 909–914. John, G., Minamikawa, H., Masuda, M., Shimizu, T., 2003. Liquid crystalline cardanyl β-D-glucopyranosides. Liq. Cryst. 30, 747–749. Kiraly, Z., Findenegg, G.H., 2000. Calorimetric study of the adsorption of short-chain nonionic surfactants on silica glass and graphite: dimethyldecylamine oxide and octyl monoglucoside. Langmuir 16, 8842–8849. Kitamoto, D., Isoda, H., Nakahara, T., 2002. Functions and potential applications of glycolipid biosurfactants-from energy-saving materials to gene delivery carriers. J. Biosci. Bioeng. 94, 187–201. Kiwada, H., Niimura, H., Fujisaki, Y., Yamada, S., Kato, Y., 1985. Application of synthetic alkyl glycoside vesicles as drug carriers. I. Preparation and physical properties. Chem. Pharm. Bull. 33, 753–759. Klein, M., Aserin, A., Svitov, I., Garti, N., 2010. Enhanced stabilization of cloudy emulsions with gum arabic and whey protein isolate. Colloids Surf. B Biointerfaces 77, 75–81. Kluge, K., 2000. Der Schlüssel zum Verständis von Mikroemulsionen aus Zuckertensiden: Die Interne Grenzfläche (Ph.D. thesis). University of Cologne, Cologne, ISBN: 3-89722-457-7. Kocherbitov, V., Söderman, O., Wadsö, L.J., 2002. Phase diagram and thermodynamics of the n-octyl beta-D-­ glucoside/water system. Phys. Chem. B 106, 2910–2917. Kramer, L., 1971. Theory of light scattering from fluctuations of membranes and monolayers. J. Chem. Phys. 55, 2097–2105. Kudin, K.N., Car, R., 2008. Why are water-hydrophobic interfaces charged? J. Am. Chem. Soc. 130, 3915–3919. Kumpulainen, A.J., Persson, C.M., Eriksson, J.C., 2004a. n-Decyl-glucopyranoside and n-decyl-maltopyranoside gibbs monolayers. Phase changes in the dilute liquid-expanded range. Langmuir 20, 10534–10541. Kumpulainen, A.J., Persson, C.M., Eriksson, J.C., 2004b. Headgroup and hydrocarbon tail effects on the surface tension of sugar-based surfactant solutions. Langmuir 20, 10935–10942. Kumpulainen, A.J., Persson, C.M., Eriksson, J.C., Tyrode, E.C., Johnson, C.M., 2005. Soluble monolayers of n-­decyl glucopyranoside and n-decyl maltopyranoside. Phase changes in the gaseous to the liquid-expanded range. Langmuir 21, 305–315. Lehtonen, M., Teräslahti, S., Xu, C., Yadav, M.P., Lampi, A.M., Mikkonen, K.S., 2016. Spruce galactoglucomannans inhibit lipid oxidation in rapeseed oil-in-water emulsions. Food Hydrocoll. 58, 255–266. Lewis, R.J. (Ed.), 1997. Hawley’s Condensed Chemical Dictionary, 13th ed. John Wiley and Sons, Inc, New York, NY, 805pp. ISBN: 978-0471292050.

III.  BIOBASED SURFACTANTS

410

12.  MEASURING THE INTERFACIAL BEHAVIOR

Liljekvist, P., Kjellin, M., Eriksson, J.C., 2001. The surface pressure effect of pentaoxyethylene and maltoside surfactant head groups. Adv. Colloid Interf. Sci. 89, 293–302. Lu, S., Somasundaran, P., 2008. Tunable synergism/antagonism in a mixed nonionic/anionic surfactant layer at the solid/liquid Interface. Langmuir 24 (8), 3874–3879. Lu, S., Bian, Y., Zhang, L., Somasundaran, P., 2007. pH dependence of adsorption of n-dodecyl-beta-D-maltoside on solids. J. Colloid Interface Sci. 316 (2), 310–316. Mahon, E., Aastrup, T., Barboiu, M., 2010. Dynamic glycovesicle systems for amplified QCM detection of ­carbohydrate-lectin multivalent biorecognition. Chem. Commun. 46, 2441–2443. Matsson, M.K., Kronberg, B., Claesson, P.M., 2004. Adsorption of alkyl polyglucosides on the solid/water interface: equilibrium effects of alkyl chain length and head group polymerization. Langmuir 20, 4051–4058. McClements, D.J., 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. Mikkonen, K.S., Merger, D., Kilpeläinen, P., Murtomäki, L., Schmidt, U.S., Wilhelm, M., 2016a. Determination of physical emulsion stabilization mechanisms of wood hemicelluloses via rheological and interfacial characterization. Soft Matter 12 (42), 8690–8700. Mikkonen, K.S., Xu, C., Berton-Carabin, C., Schroën, K., 2016b. Spruce galactoglucomannans in rapeseed oil-in-water emulsions: efficient stabilization performance and structural partitioning. Food Hydrocoll. 52, 615–624. Mirhosseini, H., Tan, C.P., Hamid, N.S.A., Yusof, S., 2008. Effect of arabic gum, xanthan gum, and orange oil contents on ζ -potential, conductivity, stability, size index, and pH of orange beverage emulsion. Colloids Surf. A Physicochem. Eng. Asp. 315, 47–56. Müller, M.M., Kügler, J.H., Henkel, M., Gerlitzki, M., Hörmann, B., Pöhnlein, M., Syldatk, C., Hausmann, R., 2012. Rhamnolipids-next generation surfactants? J. Biotechnol. 162, 366–380. Muruganathan, R.M., Krustev, R., Ikeda, N., Müller, H.-J., 2003. Temperature dependence of the gas permeability of foam films stabilized by dodecyl maltoside. Langmuir 19, 3062–3065. Muruganathan, R.M., Krustev, R., Müller, H.J., Möhwald, H., Kolaric, B., von Klitzing, R., 2004. Foam films stabilized by dodecyl maltoside. 1. Film thickness and free energy of film formation. Langmuir 20, 6352–6358. Mysels, K.J., Jones, M.N., 1966. Stability and some properties of the second black film. Discuss. Faraday Soc. 42, 42–50. Nakauma, M., Funami, T., Noda, S., Ishihara, S., Al-Assaf, S., Nishinari, K., Phillips, G.O., 2008. Comparison of sugar beet pectin, soybean soluble polysaccharide, and gum arabic as food emulsifiers. 1. Effect of concentration, pH, and salts on the emulsifying properties. Food Hydrocoll. 22 (7), 1254–1267. Neimert-Andersson, K., Blomberg, E., Somfai, P., 2004. Stereoselective synthesis of novel polyhydroxyl surfactants. stereochemical influence on langmuir monolayers. J. Organomet. Chem. 69, 3746–3752. Nickel, D., Forster, T., von Rybinski, W., 1996. Physicochemical properties of alkyl polyglycosides. In: Hill, K., von Rybinski, W., Stoll, G. (Eds.), Alkyl Polyglycoside, Technology, Properties and Applications. VCH, Weinheim, ISBN: 9783527294510. NIOSH, 1983. National Occupational Exposure Survey (NOES). http://www.cdc.gov/noes/. Niraula, B.B., Chun, T.K., Othman, H., Misran, M., 2004. Dynamic-interfacial properties of dodecyl-beta-D-­maltoside and dodecyl-beta-D-fructofuranosyl-alpha-D-glucopyranoside at dodecane/water interface. Colloids Surf. A Physicochem. Eng. Asp. 248, 157–166. Nypelö, T., Laine, C., Aoki, M., Tammelin, T., Henniges, U., 2016. Etherification of wood-based hemicelluloses for interfacial activity. Biomacromolecules 17 (5), 1894–1901. Ollivon, M., Lesieur, S., Grabielle-Madelmont, C., Paternostre, M., 2000. Vesicle reconstitution from lipid–detergent mixed micelles. Biochim. Biophys. Acta 1508 (1), 34–50. Osano, J.P., Hosseini-Parvar, S.H., Matia-Merino, L., Golding, M., 2014. Emulsifying properties of a novel polysaccharide extracted from basil seed (Ocimum bacilicum L.): effect of polysaccharide and protein content. Food Hydrocoll. 37, 40–48. Parker, J.L., 1994. Surface force measurements in surfactant systems. Prog. Surf. Sci. 47, 205–271. Parker, J.L., Christenson, H.K., Ninham, B.W., 1989. Device for measuring the force and separation between two surfaces down to molecular separations. Rev. Sci. Instrum. 60, 3135–3138. Patel, S., Goyal, A., 2015. Applications of natural polymer gum arabic: a review. Int. J. Food Prop. 18 (5), 986–998. Persson, C.M., Kumpulainen, A.J., 2004. Surface forces measured in sugar surfactant solutions between two hydrophobic thiolated silica spheres. Colloids Surf. A Physicochem. Eng. Asp. 233, 43–49. Persson, C.M., Claesson, P.M., Johansson, I., 2000. Interfacial behavior of n-octyl-β-D-glucopyranoside compared to that of a technical mixture consisting of octyl glucosides. Langmuir 16 (26), 10227–10235.

III.  BIOBASED SURFACTANTS

REFERENCES 411

Persson, C.M., Claesson, P.M., Lunkenheimer, K., 2002. Interfacial behavior of n-decyl-beta-D-maltopyranoside on hydrophobic interfaces and the effect of small amounts of surface-active impurities. J. Colloid Interface Sci. 251, 182–192. Persson, C.M., Kjellin, U.R.M., Eriksson, J.C., 2003a. Surface pressure effect of poly(ethylene oxide) and sugar headgroups in liquid-expanded monolayers. Langmuir 19, 8152–8160. Persson, C.M., Kumpulainen, A.J., Eriksson, J.C., 2003b. Adsorption of n-decyl-β-D-glucopyranoside and n-decyl-β-D-maltopyranoside mixtures at the liquid-vapor interface. Langmuir 19, 6110–6114. Piispanen, P.S., Persson, M., Claesson, P., Norin, T., 2004. Surface properties of surfactants derived from natural products. Part 1: syntheses and structure/property relationships-solubility and emulsification. J. Surfactant Deterg. 7, 147–159. Piorkowski, D.T., McClements, D.J., 2014. Beverage emulsions: recent developments in formulation, production, and applications. Food Hydrocoll 42 (1), 5–41. Purkait, M.K., DasGupta, S., De, S., 2006. Micellar enhanced ultrafiltration of eosin dye using hexadecyl pyridinium chloride. J. Hazard. Mater. 136 (3), 972–977. Ren, L., Marquardt, M.A., Lech, J., 1997. Estrogenic effects of nonylphenol on pS2, ER and MUC1 gene expression in human breast cancer cells-MCF-7. Chem. Biol. Interact. 104 (1), 55–64. Rico-Lattes, I., Lattes, A., 1997. Synthesis of new sugar-based surfactants having biological applications: key role of their self-association. Colloids Surf. A Physicochem. Eng. Asp. 123–124, 37–48. Rojas, O.J., Stubenrauch, C., Schlarmann, J., Claesson, P.M., 2005a. Interactions between non-polar surfaces coated with the non-ionic surfactant n-dodecyl-β-D-maltoside. Langmuir 21, 11836–11843. Rojas, O.J., Neuman, R.D., Claesson, P.M., 2005b. Viscoelastic properties of isomeric alkylglucoside surfactants studied by surface light scattering. J. Phys. Chem. B 109, 22440–22448. Rosevear, P., VanAken, T., Baxter, J., Ferguson-Miller, S., 1980. Alkyl glycoside detergents: a simpler synthesis and their effects on kinetic and physical properties of cytochrome c oxidase. Biochemistry 19, 4108–4115. Ruiz, C.C. (Ed.), 2009. Sugar-Based Surfactants: Fundamentals and Applications. Taylor & Francis, Boca Raton, ISBN: 978-1-4200-5166-7. Samal, K., Das, C., Mohanty, K., 2017. Application of saponin biosurfactant and its recovery in the MEUF process for removal of methyl violet from wastewater. J. Environ. Manag. 203, 8–16. Schlarmann, J., Stubenrauch, C., 2003. Stabilization of foam films with non-ionic surfactants: alkyl polyglycol ethers compared with alkyl polyglucosides. Tenside Surfactant Deterg. 40, 190–195. Schmidt, U.S., Koch, L., Rentschler, C., Kurz, T., Endress, H.U., Schuchmann, H.P., 2015a. Effect of molecular weight reduction, acetylation and esterification on the emulsification properties of citrus pectin. Food Biophys. 10, 217–227. Schmidt, U.S., Schmidt, K., Kurz, T., Endress, H.U., Schuchmann, H.P., 2015b. Pectins of different origin and their performance in forming and stabilizing oil-in-water-emulsions. Food Hydrocoll. 46, 59–66. Sottmann, T., Strey, R., Chen, H.-S., 1997. A small-angle neutron scattering study of nonionic surfactant molecules at the water-oil interface: area per molecule, microemulsion domain size, and rigidity. J. Chem. Phys. 106, 6483–6491. Stubenrauch, C., 2001. Sugar surfactants—aggregation, interfacial, and adsorption phenomena. Curr. Opin. Colloid Interface Sci. 6, 160–170. Stubenrauch, C., von Klitzing, R., 2003. Disjoining pressure in thin liquid foam and emulsion films. J. Phys. Condens. Matter 15, R1197–R1232. Stubenrauch, C., Schlarmann, J., Strey, R., 2002. A disjoining pressure study of n-dodecyl-β-D-maltoside films. Phys. Chem. Chem. Phys. 4, 4504–4513. Phys. Chem. Chem. Phys. 2003, 5, 2736–2737 (erratum). Stubenrauch, C., Rojas, O.J., Schlarmann, J., Claesson, P.M., 2004a. Interactions between nonpolar surfaces coated with the nonionic surfactant hexaoxyethylene dodecyl ether C12E6 and the origin of surface charges at the air/ water Interface. Langmuir 20 (12), 4977–4988. Stubenrauch, C., Schlarmann, J., Rojas, O.J., Claesson, P.M., 2004b. Comparison between interaction forces at air/ liquid and solid/liquid interfaces in the presence of non-ionic surfactants. Tenside Surfactant Deterg. 41, 174–179. Stubenrauch, C., Shrestha, L.K., Varade, D., Johansson, I., Olanya, G., Aramaki, K., Claesson, P.M., 2009. Aqueous foams stabilized by n-dodecyl-β-D-maltoside, hexaethyleneglycol monododecyl ether and their 1:1 mixture. Soft Matter 5, 3070–3080. Stubenrauch, C., Claesson, P.M., Rutland, M., Manev, E., Johansson, I., Pedersen, J.S., Langevin, D., Blunk, D., Bain, C.D., 2010. Mixtures of n-dodecyl-β-D-maltoside and hexaoxyethylene dodecyl ether: surface properties, bulk properties, foam films, and foams. Adv. Colloid Interf. Sci. 155, 5–18.

III.  BIOBASED SURFACTANTS

412

12.  MEASURING THE INTERFACIAL BEHAVIOR

Tyrode, E., Johnson, M.C., Kumpulainen, A., Rutland, M.W., Claesson, P.M., 2005. Hydration state of nonionic surfactant monolayers at the liquid/vapor Interface: structure determination by vibrational sum frequency spectroscopy. J. Am. Chem. Soc. 127, 16848–16859. Uchegbu, I.F., Vyas, S.P., 1998. Non-ionic surfactant based vesicles (niosomes) in drug delivery. Int. J. Pharm. 172 (1), 33–70. Urbanski, R., Draye, M., Cote, G., Szymanowski, J., 2000. Removal of bisphenol a from aqueous streams by micellar extraction and ultrafiltration. Solv. Extr. Ion Exch. 18 (3), 533–550. Urbański, R., Góralska, E., Bart, H.J., Szymanowski, J., 2002. Ultrafiltration of surfactant solutions. J. Colloid Interface Sci. 253 (2), 419–426. von Rybinski, W., Hill, K., 1998. Alkyl polyglycosides-properties and applications of a new class of surfactants. Angew. Chem. Int. Ed. 37 (10), 1328–1345. Waltermo, Å., Sjöberg, B., Anhede, B., Claesson, P.M., 1993. Adsorption of an ethoxylated amine surfactant on mica and its effect on the surface forces. J. Colloid Interface Sci. 156, 365–376. Waltermo, Å., Claesson, P.M., Simonsson, S., Manev, E., Johansson, I., Bergeron, V., 1996. Foam and thin-liquid-film studies of alkyl glucoside systems. Langmuir 12 (22), 5271–5278. Xue, C.L., Solaiman, D.K.Y., Ashby, R.D., Zerkowski, J., Lee, J.H., Hong, S.T., Yang, D., Shin, J.A., Ji, C.M., Lee, K.T., 2013. Study of structured lipid-based oil-in-water emulsion prepared with sophorolipid and its oxidative stability. J. Am. Oil Chem. Soc. 90, 123–132. Yadav, M.P., Igartuburu, J.M., Yan, Y., Nothnagel, E.A., 2007. Chemical investigation of the structural basis of the emulsifying activity of gum arabic. Food Hydrocoll. 21, 297–308. Yadav, M.P., Johnston, D.B., Hicks, K.B., 2009. Corn fiber gum: new structure/function relationships for this potential beverage flavor stabilizer. Food Hydrocoll. 23, 1488–1493. Zaghbani, N., Hafiane, A., Dhahbi, M., 2009. Removal of eriochrome blue black R from wastewater using ­micellar-enhanced ultrafiltration. J. Hazard. Mater. 168 (2), 1417–1421.

III.  BIOBASED SURFACTANTS

C H A P T E R

13 Arginine-Based Surfactants: Synthesis, Aggregation Properties, and Applications Aurora Pinazo⁎, Lourdes Pérez⁎, Mª del Carmen Morán†, Ramon Pons⁎ ⁎

Institute for Advanced Chemistry of Catalonia, IQAC-CSIC, Barcelona, Spain †Biochemistry and Physiology Department, Physiology Section, Pharmacy and Food Sciences, Barcelona University, Barcelona, Spain

13.1 INTRODUCTION Surfactants are amphiphilic molecules composed of a polar group (ionic or nonionic) and one or more hydrophobic chains (usually hydrocarbon). This duality confers a unique range of properties to surfactants, which are used in a variety of processes. These compounds have garnered ever-increasing interest owing to their interfacial activity, ability to self-­aggregate into myriad supramolecular motifs on the nanoscale, biological activities, and diverse applications. Nonetheless, research and development of new surfactants has shifted toward compounds with multifunctional benefits and must be carried out in accordance with acting regulations for human health and the environment (Holmberg, 2003). One of the key strategies to minimizing the toxicity and environmental impact of synthetic surfactants is to mimic natural surfactant structures such as lipoamino acids or their analogues, that is, surfactant-like peptides, all of which are found in cell membranes (Epand et  al., 1998; Yasuhara et al., 2005; Adams et al., 2007). Lipoamino acids constitute an important class of natural surface-active biomolecules of great interest to organic and physical chemists and to biologists with an unpredictable number of basic and industrial applications (Xia and Nnanna, 2001, Muriel-Galet et al., 2014, Burnett et al., 2017, Rostami et al., 2017, Bernal et al., 2018). Structurally, lipoamino acids are a very heterogeneous group of compounds but with a common advantage, in that they are relatively easy to design and synthesize. Often, these molecules combine charged or noncharged residues (i.e., glutamic acid (Glu), lysine (Lys),

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arginine (Arg), serine (Ser), leucine (Leu), phenylalanine (Phe), and alanine (Ala)) as the hydrophilic head group with a hydrophobic tail of different structure, length, and number (i.e., fatty acids, fatty alcohols, and fatty amines) as synthons for the amphiphilic structure (Takehara, 1989; Boyat et al., 2000; Zhang et al., 2005; Gerova et al., 2008; Vijay et al., 2008). This fact explains the diversity of amino acid-/peptide-based surfactants and the variety of their physicochemical and biological properties (Roy and Dey, 2006; Das et al., 2006; Varka et al., 2006; Ohta et al., 2008; Capone et al., 2008; Bordes and Holmberg, 2015; Pinheiro and Faustino, 2017). With the aim to develop biocompatible surfactants, our group, in collaboration with others, has synthesized and studied for more than three decades new monodisperse and chiral, lipoamino-acid-type surfactants of diverse structure and ionic character (Infante et al., 1985, 1997; Pinazo et al., 1993; Allouch et al., 1996; Infante and Moses, 1994; Castillo et al., 2004; Colomer et  al., 2011). Of particular interest are the arginine-based cationic surfactants that have been designed in accordance with four different amphiphilic structures: single-chain or monocatenary arginine surfactants, one arginine as a polar head or one arginine plus one lysine as a polar head, both of them with only one hydrophobic tail (Fig. 13.1, 1); Gemini arginine surfactants, two arginine polar heads and two hydrophobic tails per molecule linked by a spacer chain (Fig. 13.1, 2); glycerolipid arginine surfactants, one arginine polar head and one or two hydrophobic moieties linked together through a glycerol skeleton (Fig. 13.1, 3); and double-chain arginine surfactants, one arginine polar head bearing two hydrophobic moieties (Fig. 13.1, 4). All of these four structures are characterized by the presence of weak amide and/or ester bonds anywhere in the molecule (Infante et al., 1984; Pérez et al., 1996; Clapés et al., 1999; Morán et al., 2004a; Colomer et al., 2011; Pinazo et al., 2016). In terms of surfactant behavior, we have evaluated and compared their physicochemical properties of adsorption and self-aggregation in aqueous media at a range of concentrations and in the presence and absence of other components. The presence of the positively charged arginine in such amphiphilic structures gives an extensive series of compounds with a rich phase behavior properties and strong antimicrobial activity (Morán et al., 2004b). These compounds are water-soluble, nontoxic if orally administered, nonirritating, and biodegradable and have a minimal aquatic impact, thus guaranteeing their ultimate commercial development in the food and cosmetic sector and highlighting their potential for biochemical applications (Pérez et al., 2002a,b, 2005; Benavides et al., 2004; Martínez et al., 2006, Morán et al., 2009, 2010a,b, 2014). They can be regarded as an alternative to conventional synthetic surfactants in which fundamental requirements for industrial development are present: biodegradability, low toxicity, multifunctional performance, and renewable sources of raw materials. The authors’ work in this field has contained basic science and technology elements and has been of the most interdisciplinary nature possible, drawing on the expertise of researchers from chemical synthesis, biocatalysis, physical chemistry of colloids and surfaces, ecotoxicity, toxicology, and microbiology, and has relied on numerous industrial collaborators. In this chapter, the authors are going to present and analyze the data obtained from our group on the synthesis, physicochemical properties, and some potential applications of these four types of interesting biosurfactants derived from arginine.

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FIG. 13.1  Schematic structure of arginine-based surfactants.

13.2 SYNTHESIS 13.2.1  Monocatenary Arginine Surfactants Amino acids are linked to long aliphatic chains through the [α]-amino, [α]-carboxyl, or side chain groups. Thus, fatty acids or alkyl halides can react with amino groups yielding the corresponding N-acyl and N-alkyl derivatives, respectively. Alternatively, the carboxyl group of the amino acid can be condensed with alkylamines or aliphatic alcohols to give N-alkyl amides and O-alkyl esters. Among the different types of linkages between the long aliphatic chain and the amino acid, the Nα-acyl, Oα-alkyl esters, and Oα-alkyl amides of ­arginine

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have attracted much interest in our group to prepare low toxic, biodegradable antimicrobial monocationic surfactants. They selectively disrupt bacteria membranes at submicellar concentrations, but not erythrocytes or skin cell membranes. It has been demonstrated that the incorporation of ester functionality accelerates biodegradation (Infante et  al., 1984, 1992a; Morán et al., 2001b; Seguer et al., 1994). Thus Nα-acyl-arginine-methyl ester hydrochloride (Fig. 13.2, 1), arginine-Oα-alkyl ester dihydrochloride (Fig. 13.2, 2), and arginine-Oα-alkyl amide dihydrochloride (Fig. 13.2, 3) salts of different alkyl chain lengths have been synthesized by our group using chemical and biotechnological methodologies. Nα-acyl-arginine-methyl ester hydrochloride salts (n = 8, CAM; n = 10, LAM; n = 12, MAM; and n = 14, PAM) were prepared by Nα-acylation of the amino terminal arginine (Table 13.1). Fatty acids were condensed to arginine methyl ester hydrochloride using classical chemical methods (Infante et al., 1997). The application of biotechnological procedures was not efficient for these compounds (Clapés and Infante, 2002). However, papain from Carica papaya latex deposited onto solid support materials was found to be a suitable catalyst for the formation of amide and ester bonds between N-benzyloxycarbonyl-Arg-OMe (Cbz-Arg-OMe) and various long-chain fatty alcohols and amines to prepare in organic media arginine-O-­ alkyl ester dihydrochloride salts (n = 6, AOE; n = 8, ACE; and n = 10, ALE) and arginine-O-­alkyl amide dihydrochloride salts (n = 8, ACA; n = 10, ALA; and n = 12, AMA), respectively (Table 13.1) (Clapés et al., 1999). Changes in enzymatic activity and product yield were studied for the following variables: organic solvent, aqueous buffer content, support for the enzyme deposition, the presence of additives, enzyme loading, substrate concentration, and reaction temperature. The best yields (81%–89%) of arginine N-alkyl amide derivatives were obtained at 25°C in acetonitrile with an aqueous buffer content ranging from 0% to 1% (v/v) depending on the substrate concentration. The synthesis of arginine alkyl ester derivatives was carried out in solvent-free systems at 50°C or 65°C depending on the fatty alcohol chain length. In this case, product yields ranging from 86% to 89% were obtained with a molar ratio

FIG. 13.2  Structure of monocatenary arginine-based surfactants ((1), (2), and (3) refer to Nα-acyl, Oα-alkyl esters, and Oα-alkyl amides of arginine, respectively).

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TABLE 13.1  Acronyms and Chemical Names of the Four Types of Arginine-Based Surfactants Described in this Chapter Acronym

Chemical Name

Figure

α

CAM LAM MAM PAM

N -Caproyl-arginine-methyl ester HCl (n = 8) Nα-Lauroyl-arginine-methyl ester HCl (n = 10) Nα-Myristoyl-arginine-methyl ester HCl (n = 12) Nα-Palmitoyl-arginine-methyl ester HCl (n = 14)

13.2, 1

AOE ACE ALE

Arginine-Oα-octyl ester 2HCl (n = 6) Arginine-Oα-capryl ester 2HCl (n = 8) Arginine-Oα-lauroyl ester 2HCl (n = 10)

13.2, 2

ACA ALA AMA

Arginine-Oα-capryl amide 2HCl (n = 8) Arginine-Oα-lauroyl amide 2HCl (n = 10) Arginine-Oα-myristoyl amide 2HCl (n = 12)

13.2, 3

LAKM LALM

Nα-Lauroyl-arginine-Nε-lysine methyl ester Nα-Lauroyl-arginine-Nα-lysine methyl ester

13.3

C3(XA)2 C4(XA)2 C6(XA)2 C9(XA)2

Bis(Nα-acyl-arginine) α-ω propylene diamide 2HCl (s = 1) Bis(Nα-acyl-arginine) α-ω butylene diamide 2HCl (s = 2) Bis(Nα-acyl-arginine) α-ω hexylene diamide 2HCl (s = 4) Bis(Nα-acyl-arginine) α-ω nonylene diamide 2HCl (s = 7)

13.4

120RAc 140RAc

1-O-Lauroyl-glycero-3-O-(Nα-acetyl-arginine) HCl (n = 10) 1-O-Myristoyl-glycero-3-O-(Nα-acetyl-arginine) HCl (n = 12)

13.5A

1212RAc 1414RAc

1,2-Di-O-lauroyl-glycero-3-O-(Nα-acetyl-arginine) HCl (n = 10) 1,2-Di-O-myristoyl-glycero-3-O-(Nα-acetyl-arginine) HCl (n = 12)

13.5B

100R 120R 140R

1-O-Decyl-glycero-3-O-(Nα-arginine) 2HCl (n = 8) 1-O-Lauroyl-glycero-3-O-(Nα-arginine) 2HCl (n = 10) 1-O-Myristoyl-glycero-3-O-(Nα-arginine) 2HCl (n = 12)

13.6A

88R 1010R 1212R 1414R

1,2-Di-O-octyl-glycero-3-O-(Nα-arginine) 2HCl (n = 6) 1,2-Di-O-decyl-glycero-3-O-(Nα-arginine) 2HCl (n = 8) 1,2-Di-O-lauroyl-glycero-3-O-(Nα-arginine) 2HCl (n = 10) 1,2-Di-O-myristoyl-glycero-3-O-(Nα-arginine) 2HCl (n = 14)

13.6B

LANHC10 LANHC12 LANHC14 LANHC18

Nα-Lauroyl-arginine-decyl amide Nα-Lauroyl-arginine-lauroyl amide Nα-Lauroyl-arginine-myristoyl amide Nα-Lauroyl-arginine-octadodecanoyl amide

13.7

Cbz-Arg-OMe/fatty alcohol of 0.01. Papain deposited onto polyamide gave, in all cases, both the highest enzymatic activities and yields. Cationic monocatenary arginine surfactants combining two amino acids have also been prepared with two positive charges in the polar head. The two positive charges are provided by two basic amino acids bonded by peptide linkages: arginine-lysine, Nα-lauroyl-arginineNε-lysine methyl ester (LAKM, Fig. 13.3, 1), and arginine-lysine, Nα-lauroyl-arginine-Nα-lysine methyl ester (LALM, Fig. 13.3, 2) (Table 13.1). The general procedure for the preparation of these surfactants was described in Colomer et  al. (2011). The synthesis was carried out at room temperature, and typically, overall yields were 65%–75%.

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FIG. 13.3  Single-chain dicationic arginine-based surfactants. LAKM and LALM refer to Nα-lauroyl-arginine-Nεlysine methyl ester and Nα-lauroyl-arginine-Nα-lysine methyl ester, respectively.

13.2.2  Gemini Arginine Surfactants or bis(Args) With the aim of obtaining surfactants that are environmentally acceptable and with high performance, Pérez et al. (1996) described for the first time the synthesis of a novel class of Gemini cationic surfactants derived from the monocatenary long-chain Nα-acyl-arginine­ derivatives: the bis(Nα-acyl-l-arginine)α-ω-polymethylenediamide dihydrochloride salts named bis(Args) (Table 13.1) (Fig. 13.4) (Pérez et al., 1996). These compounds consist of two symmetrical monocatenary Nα-acyl-arginine structures of 8, 10, and 12 carbon atoms (Fig. 13.4, n = 6, 8, 10),

FIG.  13.4  Structure of bis(Args) Gemini cationic surfactant Cs (XA)2 where s (1–10) refers to the length of the spacer chain, 2 refers to the two symmetrical parts, and X (n = 6–10) refers to the length of the fatty acyl chains.

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connected through the α-carboxylic groups of the arginine residues by amide covalent bonds to an alpha-ω-diaminoalkane spacer chain of varying lengths (Fig. 13.4, s = 1–10). This particular diaminoalkane spacer chain was chosen to control the distance between the charged sites of the cation, which modifies the inter- and intrahydrophilic-hydrophobic interactions. The method used for this first approach involves chemical protecting groups, organic solvents, and chemical catalysts. Later, a strategy to reduce the environmental impact was developed. To this end, a novel chemoenzymatic synthesis of bis(Args) was described (Piera et al., 2000). Nα-acyl-l-arginine alkyl ester derivatives (Fig.  13.2) were the initial building blocks for the synthesis. The best strategy found consisted of two steps: first, the quantitative acylation of one amino group of the α,ω-diaminoalkane spacer by the carboxylic ester of the Nα-acyl-arginine took place spontaneously, at the melting point of the α,ω-diaminoalkane, in a solvent-free system. The second step was the papain-catalyzed reaction between another Nα-acyl-arginine alkyl ester and the free aliphatic amino group of the derivative formed in the first step. Reactions were carried out in solid-to-solid and solution systems using low toxic potential solvents. Changes in reaction performance and product yield were studied for the following variables: organic solvent, support for enzyme deposition, and substrate concentration. The best yields (70%) were achieved in solid-to-solid systems and in ethanol at a water content of 0.80% (w/w) equivalent to water activity of 0.07. Bis(Args) analogues of 8, 10, and 12 carbon atoms using 1,3-diaminopropane and 1,3-diamino-2-hydroxy-propane as hydrocarbon spacers were prepared at the 6–7 g level employing the methodology developed. The overall yields that include reaction and purification varied from 51% to 65% of pure (97%–98% by HPLC analysis) product.

13.2.3  Glycerolipid Arginine Surfactants Amino acid glyceride conjugates (i.e., glycero amino acids) constitute a novel class of lipoamino acids, which can be considered analogues of mono- and diacylglycerides and phospholipids. They consist of one or two aliphatic chains and one amino acid, as the polar head, linked together through ester bonds to the glycerol backbone. Our group has synthesized mono- and diacylglyceride derivatives from Nα-acetyl-arginine (Fig. 13.5A and B) and arginine (Fig. 13.6A and B) using chemical and chemoenzymatic methodologies (Morán et al., 2001a, 2002; Pérez et al., 2002a). The enzymatic preparation of mono- and diacylglyceride acetyl-arginine esters (Fig. 13.5) started with the preparation of the polar head 1-O-(N-α-acetyl-arginyl) glycerol and obtained by enzymatic methodology using hydrolases (Morán et al., 2001a). Proteases and lipases were found to be versatile catalysts for this reaction. A variety of protected amino acid glyceryl ester derivatives were obtained in 46%–98% yield under mild and selective conditions. In a second step, the free hydroxyl groups of the glyceryl moiety were acylated with fatty acids using lipases as catalyst. The authors have developed a novel methodology to obtain both 1-monoacyl- and 1,2-diacyl-3-aminoacyl glycerol (Morán et al., 2002, 2004a). Mono- and diacylation of amino acid glyceryl ester may be carried out using 1,3-selective lipases by taking advantage of the spontaneous intramolecular acyl-migration reaction that occurs in partial glycerides. Thus, the 1(3)-acylated product may undergo intramolecular 1(3) → 2 acyl migration, and the resulting 1,2(2,3)-isomer subsequently be acylated at the free primary hydroxyl group by the lipase. Accordingly, the yield of diacylated product will depend on both

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FIG.  13.5  N-acetyl-arginine mono- and diglyceride conjugates. (A) 1-O-Acyl-rac-glycero-3-O(Nα-acetyl-larginine) hydrochloride salts, n = 10, 120RAc, and n = 12, 140RAc. (B) 1,2-Di-O-acyl-rac-glycero-3-O(Nα-acetyl-larginine) hydrochloride salts, n = 10, 1212RAc, and n = 12, 1414RAc.

the rate of intramolecular acyl migration and the enzymatic esterification of the newly free primary hydroxyl of the monoacylated derivative. Both processes are influenced by the reaction conditions, such as solvent, support for enzyme immobilization, and buffer salts, and by the amino acid glyceryl ester derivative. All the enzymatic acylations were carried out in solvent-free media, at a temperature around the melting point of the corresponding fatty acid. We have found that the 1,2-diacyl-3-aminoacyl glycerol derivatives were in fact a mixture of two regioisomers: 1,2-diacyl-rac-glycero-3-(amino acid) derivative as the major one and 1,3-diacyl-glycero-2-(amino acid) derivative. With this methodology, a series of mono- and dilauroylated glycerol derivatives of acetyl arginine, aspartic acid, glutamic acid, asparagine, glutamine, and tyrosine were prepared. The monoacylglycerol and diacylglycerol-arginine compounds (Fig.  13.6A and B) were prepared using chemical methodologies. The synthesis of these surfactants consists of three steps: Step 1 corresponds to the preparation of 1-O-(N-Cbz-l-arginyl)rac-glycerol

FIG.  13.6  Arginine mono- and diglyceride conjugates, (A) 1-O-acyl-rac-glycero-3-O(Nα-l-arginine) hydrochloride salts, n = 10, 120R, and n = 12, 140R. (B) 1,2-Di-O-acyl-rac-glycero-3-O(Nα-l-arginine) hydrochloride salts, n = 10, 1212R, and n = 12, 1414R.

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­ onochloride (OORZ) by chemical esterification of the α‑carbonyl group of N-Cbz-l-arginine m HCl with the primary hydroxyl function of glycerol using boron trifluoroetherate as catalyst. The overall reaction yield was 95%. Step 2 consists of the synthesis of the 1-acyl-3-O-(NCbz-l-arginyl)rac-glycerol HCl (X0RZ) and 1,2-diacyl-3-O-(N-Cbz-l-arginyl)rac-glycerol HCl (XXRZ) by acylation of the hydroxyl groups of (00RZ) with the corresponding long-chain acid chloride. Finally, the Z-protecting group was removed by catalytic hydrogenation. The pro­ cedure for the preparation of these surfactants was described (Pérez et al., 2004a,b,c).

13.2.4  Double Chain Arginine Surfactants Four arginine-based surfactants with two alkyl chains of different lengths were synthesized (Pinazo et  al., 2016), Nα-lauroyl-arginine-O-decyl amide hydrochloride (LANHC10), Nα-dodecyl arginine-O-dodecyl amide hydrochloride (LANHC12), Nα-lauroyl-arginine-Omyristoyl amide hydrochloride LANHC14, and Nα-lauroyl-arginine-O-hexadecyl amide hydrochloride (LANHC16). The synthesis method was very simple. Nα-lauroyl-arginine-methyl ester hydrochloride (LAM) was dispersed in the desired fatty amine. The mixture was heated up to the melting point of the fatty amine and was stirred at this temperature for 3 h. After the completion of the reaction, the mixture was cooled to room temperature, and several crystallizations in methanol/acetonitrile were carried out to remove the excess of fatty amine. Under these conditions, the conversion is higher than 90% in the four assayed fatty amines, and the reaction does not require the use of any activating agent. This approach allowed us to perform the synthesis in a short time on a large scale and at a reasonable cost.

13.3  PHYSICOCHEMICAL PROPERTIES Molecular self-assembly of biomimetic molecules recently has attracted considerable attention for its use in the design and fabrication of advanced biocompatible materials with a wide range of applications in nanotechnology, medicine, and drug delivery systems (Gorbitz, 2007). The aggregation morphologies of the acylamino acids in water and especially the effect of the molecular structure of the amphiphiles on the formation and type of self-aggregate are a fascinating task. It has extensively been studied by different groups, for their potential uses in advanced industrial technologies (Imae et al., 2000; Yamashita et al., 2007; Roy and Dey, 2007; Oshimura et al., 2007; Gerova et al., 2008; Lee et al., 2008; Chandra and Tyagi, 2013; Joondan et al., 2017). In this section, the authors review the surface and aggregation properties of the four types of arginine-based surfactants whose acronyms are provided in Table 13.1.

13.3.1  Monocatenaries From Arginine The micellization properties in water solutions of the hydrochloride salts of Nα-acylarginine-methyl esters (Fig. 13.2, 1), arginine-Oα-alkyl esters (Fig. 13.2, 2), and arginine-Oα-­alkyl amides (Fig.  13.2, 3) have been studied by our group in collaboration with others. Notice that compounds in Fig. 13.2, 2, 3 have two positive charges in the hydrophilic moiety, one in the primary amino group and the other one in the guanidine group, whereas the hydrochloride salts of Nα-acyl-arginine-methyl ester in Fig. 13.2, 1 have only one positive charge in the

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guanidine group. The early studies of the micellization process of these cationic surfactants involved surface tension measurements with the dual purpose of investigating their behavior at the air-solution interface and determining the critical micelle concentration (CMC) values (Morán et al., 2001b; Pés, 1993). From the surface tension/concentration curves at 25°C, the CMC and surface tension at the CMC (γCMC) were determined. Using the Gibbs adsorption isotherm, the saturation adsorption (Γm) at the air-water interface and the area per molecule (Amin) were obtained (Rosen, 1988). As expected, the surfactant activity of all these compounds was similar to that of conventional quaternary cationic surfactants, CMC values in the range 1–30 mM and γCMC values being in the range 30–37 mN/m. The CMC values of the three families of monocatenary surfactants depend of the alkyl chain length; the more hydrophobic the molecule, the lower the CMC value. Contrarily, the nature of the hydrophilic group does not affect significatively the CMC and γCMC. The effect of the hydrophilic group on Am is most interesting. The authors found that the Am values for the arginine-Oα-alkyl ester dihydrochlorides (Fig. 13.2, 2) and arginine-Oα-alkyl amide dihydrochloride salts (Fig.  13.2, 3) (0.62–1.14 nm2 and 0.96–1.22 nm2, respectively) were higher than that for Nα-acyl-arginine-methyl ester compounds with the same alkyl chain length (Fig. 13.2, 1) (0.67–0.62 nm2) (Morán et al., 2001b). These results indicate that the arginine-Oα-alkyl ester and arginine-Oα-alkyl amide structures are packed less densely at the interface. The two charged groups in their molecular structure tend to spread them on the interface due to an increase in their electrostatic repulsion forces between the polar heads. A first study on self-aggregation of LAM, the hydrochloride salt of Nα-lauroyl-argininemethyl ester (Fig. 13.2, 1, for n = 10) at different concentrations, was carried out by Talmon’s group (Weihs et al., 2005). They examined the microstructures appearing in LAM solutions (typical of a single-tailed surfactant with a large head group) by cryogenic-temperature transmission electron microscopy (cryo-TEM) to image microstructures appearing as a function of the concentration. Data showed that LAM forms spheroidal micelles at low concentration; as the concentration increases and approaches the phase-transition concentration, elongated structures (cylindrical micelles) in an ordered array appear. A deeper insight into the micellization process of the LAM has been carried out using the pulsed-gradient spin echo (PGSE) NMR experiment and small-angle X-ray scattering (SAXS) (Pérez et al., 2007). The best form factor model fits for the SAXS scattering patterns were obtained for polydisperse spheres with a core-shell structure. The area per molecule value (Am), 0.65 ± 0.01 nm2, agrees roughly with those obtained from surface tension. The polar head and hydrophobic core electron densities are consistent with the molecular structure of the surfactant. Moreover, hydration per molecule obtained from the fits, 27 ± 5 mol of water per mole of surfactant, is also reasonable because in the molecular structure of the head group, several hydrogen bonding groups are present: the guanidinium cationic group and the chloride anion. The PGSE-NMR was used to determine the self-diffusion coefficients of LAM at 25°C using standard procedures (Stilbs, 1987). Fitting the two-site exchange model (Lindman et al., 1984) to the diffusion/concentration curve, we calculated the CMC; the intradiffusion coefficient of free monomers, Dfree; and that of the micellized molecules, Dmic. The CMC values obtained by NMR agree with those obtained previously by surface tension, and the structural micellar parameters determined by NMR are in accordance with those calculated from the SAXS data. Phase behavior including structural characterization of the monocatenaries, Nα-acylarginine-methyl ester hydrochloride salts CAM, LAM, MAM, and PAM (Fig. 13.2, 1) in binary

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423

water/surfactant and ternary water/surfactant/cosurfactant systems, has been systematically investigated as a function of the alkyl chain length of the surfactants and cosurfactants. These studies were carried out using phase diagrams, optical microscopy observations, light scattering, spectrofluorimetry, Fourier transform PGSE-NMR, and dielectric spectroscopy (Solans et al., 1989, 1990; Infante et al., 1992b; Fördedal et al., 1993). Solans showed by polarized light microscopy that lyotropic liquid crystals with hexagonal (CAM, LAM, MAM, and PAM), cubic (LAM, MAM, and PAM), and lamellar (MAM and PAM) structure appear as a function of the chain length. The phase behavior of monocatenary arginine surfactants in multicomponent systems was also investigated. To this end, ternary systems were studied using n-alcohols of different chain lengths (C5–C16) with the four N-acyl arginine homologues. In all systems, three monophasic regions were identified: micelles, reversed micelles, and lamellar liquid crystals. Microemulsion formation, in the presence of hydrocarbon components (hexadecane, squalane, and toluene), was also studied by Solans (Solans et  al., 1990; Pés, 1993). More interestingly, reversed vesicles in a system containing lecithin-LAM/squalene/water were also described by Kunieda as biocompatible systems to be applied in cosmetic and pharmaceutical formulations (Kunieda et al., 1992). Cationic liposomes are now recognized as potent vehicles for the delivery of DNA and other nucleic acids to cells. Cationic liposomes are formed either from a double-chain cationic surfactant in water or from a combination of a single cationic and single anionic amphiphile (catanionic systems), which, at a critical concentration and ratio, can form cationic vesicles. Combinations of different mixtures of ALA, the hydrochloride salt of arginine O-lauroyl amide (Fig. 13.2, 2, n = 10), and LAM with different anionic surfactants in water were studied. The ability of these systems to spontaneously form vesicles, cubosomes (dispersions of a cubic liquid crystalline phase), and hexosomes (dispersed particles with a hexagonal internal structure) has been described (Rosa et al., 2006). Lipoamino acid surfactants in which the polar head is constituted by two amino acids, arginine and lysine (LAKM and LALM, Fig. 13.3), have also been studied. Concerning the CMC values (26 and 25 mM, respectively), the details of the polar head group had only a minor effect, with CMC values differing by  1), low curvature and twisted ribbon structure occur. In all likelihood, ribbons form because of chirality of the amphiphiles and enhanced hydrogen bonding of the spacer with the surrounding water that leads to rigid filament-like structures.

13.3.3  Arginine Mono and Diacylglyceride Conjugates The micelle formation of mono- and diacylglyceride surfactants from arginine has been evaluated by conductivity, surface tension, and fluorescence measurements. The capability of the monoacyl arginine glycerides 100R, 120R, and 140R surfactants (Fig. 13.5A) to form micellar aggregates was studied by electric conductivity (Pérez et al., 2004a,b). The determined CMC values for the three homologues, 6 mM for 100R, 1.3 mM for 120R, and 0.2 mM for 140R, are approximately twice than those of the lysophosphatidylcholines with the same alkyl chain length (Yamanaka et al., 1997) and one order of magnitude lower than the CMC corresponding to the 12‑carbon straight-chain conventional cationic surfactants (Rosen, 1988). They also have slightly lower CMC than the Nα-acyl-arginine surfactants (Fig.  13.2, 1) (Morán et  al., 2001b). The difference between the monoglycerides from arginine and the Nα-acyl-arginine type is the glycerol group located between the arginine and the alkyl chain groups for the former, leading to an increased distance between the ionic group and the α‑carbon atom of the hydrophobic group, which often produces a lower CMC (Rosen, 1988). From the conductivity/concentration curves, the CMC of the diacyl arginine glyceride surfactants 88R, 1010R, 1212R, and 1414R was determined, and the following values were obtained: 5 mM for the 88R, 1.1 mM for 1010R, 0.3 mM for 1212R, and 0.25 mM for 1414R. The CMCs of the diacyl arginine glyceride surfactants are one order of magnitude higher than those published for the short-chain phospholipids (Tausk et al., 1974) with similar alkyl chain lengths. The second alkyl chain increases the hydrophobic content of the molecule, and consequently, the CMCs of these compounds are lower than those corresponding to the monoacyl arginine glycerides with the same alkyl chain. Curves of the log CMC versus chain length for mono- and diacylglycerides from arginine are nearly parallel. These results indicate that the value of the free energy of transfer from water to the micelle per methyl group (ΔG°(CH2)) is similar for the two families. This behavior has also been described for Gemini surfactants and their corresponding monomeric surfactants, such as the bis(Args) and LAM (Pérez et al., 1998), and for the bisQuats and monoQuats (Zana et al., 1991). The CMC was also measured by surface tension using Wilhelmy plate method and fluorescence for the 88R, 1010R, and the 1212R surfactants. Whereas the values obtained by fluorescence agree with those obtained by conductivity for all cases, the surface tension technique gave lower CMC values for the diacylglyceride arginine derivatives of Fig. 13.5B, 0.07 mM for the 88R, 0.006 for the 1010R, and 0.008 mM for the 1212R. A similar trend was obtained for the bis(Args) surfactants (Fig. 13.4). As discussed elsewhere (Pinazo et al., 1999), the bis(Args) compounds at very low concentrations form aggregates of substantial size to reduce the surface tension at the low concentrations. To further investigate the underlying cause of the difference of CMC values obtained by Wilhelmy plate and fluorescent methods, static light scattering was carried out to establish the aggregate type formed for these compounds at these low concentrations. The 1010R compound in particular underwent a vesicle-to-ribbon transition as the surfactant concentration increased. The scattered intensity of 1010R at concentrations as low as 0.001 mM showed that aggregates were already present in the solution.

III.  BIOBASED SURFACTANTS



13.3  Physicochemical Properties

427

Based on the form and size of the aggregates, polydisperse vesicles occur at low concentration and ribbons at millimolar concentration. The change of form was interpreted in terms of charging of the bilayer. At low concentration, surfactants dissociate into a chloride ion and the cationic species. The latter can further dissociate, with a proton being released. The highly charged surface that forms is unfavorable, and a pKa shift can be observed (Hagslatt et al., 1991), producing a more acidic behavior than that typically observed for the arginine. The pKa shift, up to 7 units, is larger compared with 1–2 units described in the literature. As the concentration increases, the proportion of charged species increases with a concomitant increase of Am, thus inducing a change in the curvature of the aggregates (Pinazo et al., 2004). The change induced by the intrinsic pH change was independently proved by acidification of a vesicle-forming concentration. The physicochemical properties of diacylglycerides from acetyl-arginine 1212RAc and 1414RAc, Fig. 13.5B, have been studied based on their ability to form monolayers and multilayers. Because they have two hydrocarbon chains, their aggregation in aqueous solution starts at very low concentrations. A change in the slope of conductivity versus concentration was observed for 1212RAc and 1414RAc near 0.1 mM. Because there was not significant difference in trend between the two compounds, it was hypothesized that this change did not correspond to a true CMC but to a change in the aggregate form (Pérez et al., 2004a), perhaps a transition from vesicles to ribbons (Pinazo et al., 2004). Dilauroyl glycerol acetyl-arginine conjugates can be considered as analogues of partial glycerides and phospholipids. During their preparation, spontaneous intramolecular acyl-migration reactions were observed, and both possible regioisomers were obtained: 1,2-dilauroyl-rac-glycero-3-(Nα-acetyl-l-arginine) (1212RAc) and 1,3-dilauroylglycero-2-(Nα-acetyl-l-arginine) (12RAc12). The presence of both regioisomers influences the phase behavior. The study of the thermotropic phase behavior in the dry state of pure 1,2-dilauroyl-rac-glycero-3-(Nα-acetyl-l-arginine) and two mixtures of both regioisomers showed that they arrange in multilamellar stacks. When observed by optical polarized microscopy, the typical texture for smectic systems was found and coincided with a characteristic peak ordering of the SAXS curves. At low temperature, the lamellar distance of the pure 1,2-compound was compatible with that of fully extended, all-trans, alkyl chains. For the mixtures, the difference in bilayer thickness was associated with a tilting of the hydrocarbon chains away from the bilayer normal caused by difficulties of packing. The higher the temperature, the shorter the lamellar distance. This change was associated with the introduction of a kink into the hydrocarbon chain that marks the onset of melting. Above the transition temperatures, all the samples show the same repeat distance that would correspond to the melted hydrocarbon chains in the lamellar liquid crystal phase (Morán et al., 2004b). When comparing this behavior with the monoacyl derivative, the immediate difference corresponds to the repeat distance of the corresponding smectic gel phases. While the diacyl derivatives (Fig. 13.5B) present repeat distances around 4.5 nm, the monoacyl derivatives’ (Fig. 13.5A) repeat distance is only 3.8 nm (Fig. 13.9). The coexistence of two lamellar orders is seen for the diacyl derivative, curve a. This was attributed to the coexistence of two lamellar phases caused by the crystallization from the solvent of the product composed of two enantiomers. The difference in repeat distances for the monoacyl and diacyl compounds is due to the preservation of a large area per molecule, while there is a strong increase in hydrophobic volume for the latter. Therefore, the monoacyl derivative self-assembles into a hydrocarbon

III.  BIOBASED SURFACTANTS

428

13.  Arginine-Based Surfactants: Synthesis, Aggregation Properties, and Applications

interdigitated lamellar phase, while the diacyl counterpart forms a normal bilayer arrangement. The self-assembly structures influence the head-group conformation; moreover, the diacyl compound expands to a shorter length (0.8 nm) than the monoacyl derivative (1.1 nm) (Morán et al., 2005). Monoacylated derivatives melt at 80°C, only slightly above the formation of the liquid crystalline phase (70°C), while the diacyl derivatives form a liquid crystal at a lower temperature (19°C) and melt at a higher temperature (93°C). This is probably due to the more compact packing of the chains in the monoacyl derivative compared with the diacyl derivative, for which pairs of hydrophobic chains are restricted by their polar head binding. Concerning the monolayer self-assembly behavior, it was shown that 1,2-diacylglycerol arginine-based surfactants mimic the self-assembly of phospholipids. The use of Brewster angle microscopy image analysis of the inner textures revealed that condensed phases of the dimyristoyl glycerol compounds exhibit hexatic order. Variations in the chain length introduce similar changes as those commonly found in lipid monolayers (Albalat et al., 2003). The compatibility studies of 1212RAc and 1414RAc with phospholipids assembling at the water-air interface show that the behavior of 1414RAc is similar to that of DPPC, that is, both products exhibit the gas, expanded liquid, compressed liquid, solid, and collapsed phases with increasing surface pressure. The behavior of 1212RAc is similar to that of 1,2-­dimyristoyl phosphatidylcholine (DMPC), which undergoes the same sequence of phase transitions with an increase of surface pressure as longer chain homologues but with the absence of the solid phase. The behavior of the long-chain homologues corresponds to that of an insoluble monolayer while that of the shorter chain corresponds to a fluid monolayer. The mixtures of the phospholipids with these lipoamino acids show miscibility over the full range of compositions except for the 1212RAc/DMPC mixtures, which were insoluble (Lozano et al., 2008). The surfactants 1212RAc and 1414RAc were able to stabilize both water-in-oil and oil-inwater droplets (Pérez et al., 2004a). This resulted in the stabilization of both types of droplets simultaneously when 50/50 v/v oil/surfactant mixtures were sheared in the presence of 0.2% surfactant. The emulsions were of the W/O/W type, consisting of small water droplets of around 5 μm in diameter that were dispersed in 50 μm-diameter oil droplets dispersed in a water medium. The ability to stabilize both types of droplets is not usual and may be related to the ability to form lamellar-type liquid crystalline phases.

13.3.4  Double-Chain Arginine Surfactants Double-chain arginine surfactants (Fig. 13.7) form vesicles in aqueous media (Pinazo et al., 2016). Their critical aggregation concentrations (CAC) are about one order of magnitude below the CMC of LAM, a single-chain analogue, with the carboxyl group esterified. Little

FIG. 13.7  Nα-Lauroyl-arginine-N-alkyl amide (LANHCx).

III.  BIOBASED SURFACTANTS



429

13.3  Physicochemical Properties

1010D (m2s−1)

3

2

1

0 0,1

1 10 C3(LA)2 Concentration (mM)

100

FIG. 13.8  Self-diffusion coefficients obtained using PGSE-NMR versus concentration for C3(LA)2. The line corresponds to the best fit of the two-site exchange model (equation 3 of Pérez et al., 2007). Nomenclature is referred to in Table 13.1. Reproduced from Pérez, L., Pinazo, A., Infante, M.R., Pons, R., 2007. Investigation of the micellization process of single and gemini surfactants from arginine by SAXS, NMR self-diffusion, and light scattering. J. Phys. Chem. B 111, 11379–11378 with permission of the American Chemical Society (ACS).

10,000

1000

I (A.U.)

(A) 100

10

(B)

1 0

1

2

3 q (nm−1)

4

5

6

FIG. 13.9  SAXS scattering intensity as a function of scattering vector modulus for (A) 1212RAc and (B) 120RAc dry products at 25°C. Arrows and double arrows show the position of the reflections attributed to the two coexisting lamellar phases of 1212RAc. Nomenclature is referred to in Table 13.1. From Morán, M.C., Pinazo, A., Clapés, P., Infante, M.R., Pons, R., 2004c. Investigation of the thermotropic behavior of isomer mixtures of diacyl arginine-based surfactants. Comparison of polarized light microscopy, DSC, and SAXS observations, J. Phys. Chem. B 108, 11080–11088. Reprinted with permission from the American Chemical Society (ACS).

430

13.  Arginine-Based Surfactants: Synthesis, Aggregation Properties, and Applications

influence of the length of the second alkyl chain on CAC values of decyl (LANHC10), myristoyl (LANHC14), and octadodecanoyl (LANHC18) compounds was observed. Vesicles of these compounds prepared by the method of film hydration and sonication were stable for weeks, being smaller in size and with smaller polydispersity if prepared at 50°C rather than at 25°C.

13.4 APPLICATIONS Due to their interesting properties, research on arginine-based surfactants has moved in the last years from fundamental research to the first generation of applications in life sciences. In this section, five different biological applications are discussed: antimicrobial activity, sequestration of membrane lipopolysaccharide, control of DNA compaction, drug delivery systems, and DNA gel particles.

13.4.1  Antimicrobial Properties After years of overuse of antibiotics and biocides, gram-positive and gram-negative organisms have developed a broad range of mechanisms to evade antimicrobial agents, resulting in a potential global health crisis (Heir et al., 1995). In order to overcome this rapid development of drug resistance, development of new classes of antimicrobial compounds has stimulated substantial research interest (Tan and Xiao, 2008; Ozdemir et al., 2007; Wright, 2016). Available microbicides and formulations differ considerably in their physical and chemical properties, effectiveness, and spectrum of activity although all agents must comply with the toxicological and environmental requirements. One important milestone in our research activity is the design and development of amino acid-based surfactants with a low toxicity profile and high antimicrobial activity (Infante et al., 1985, 1992a,b). Arginine derivatives present the best activity against bacteria due to the presence of the protonated guanidine group over a wide range of pH. The antimicrobial activity of all type of arginine derivatives was systematically determined “in vitro” on the basis of the minimum inhibitory concentration (MIC) values (Jones et al., 1980), defined as the lowest concentration of antimicrobial agent, which inhibits the visible growth of microorganisms after 24 h of incubation at 37°C. The results obtained for monocatenary arginine surfactants, monocatenary arginine surfactants with two cationic charges, double-chain arginine surfactants, Gemini arginine surfactants, and glycerolipid arginine surfactants are summarized in Tables 13.2–13.5, respectively. Data show that all the cationic surfactants from arginine possessed inhibition activities against a wide range of microorganisms. The surfactants exhibited moderate activity levels against bacteria, with MIC values of 4–256 mg/L. The low toxic and ecotoxic activity of these compounds is noteworthy. In general, gram-negative bacteria were more resistant to inhibition than the gram-positive bacteria, suggesting the former are suitable agents for biodegradation of these surfactants (Pérez et al., 2002b, 2005; Morán et al., 2001b). It is known that many gram-negative bacteria are relatively insensitive because their outer membranes are impermeable to many amphiphilic compounds (Rosen et al., 1999), thereby explaining their resistance to inhibition. Given that the MIC values occur at concentrations below the CMC of surfactants in water, it may be inferred that the surfactant monomers and not the aggregates

III.  BIOBASED SURFACTANTS



TABLE 13.2  Minimum Inhibitory Concentration (mg/L) of Monocatenary Arginine Surfactants

Grampositives

ALAa

AMAa

AOEa

ACEa

ALEa

CAMa

LAMa

MAMa

LAKMa

LALMa

Bacillus cereus var. mycoides

128

32

64

256

32

64

128

64

256

–

–

Bacillus pumilus

32

32

256

R

128

R

–

256

–

–

–

Bacillus subtilis

–

–

–

–

–

–

–

–

–

125

16

Staphylococcus aureus

32

16

64

256

16

32

256

32

128

250

31

Staphylococcus epidermidis

32

16

16

256

–

64

128

128

128

250

125

Micrococcus luteus

–

–

–

–

–

–

–

–

–

250

16

Candida albicans

64

16

32

256

128

64

128

64

128

250

125

Alcaligenes faecalis

32

16

32

256

64

32

128

64

128

–

–

Bordetella bronchiseptica

16

8

R

64

8

8

128

32

64

–

–

Citrobacter freundii

64

32

32

128

64

64

128

64

128

–

–

Serratia marcescens

64

32

64

R

128

64

–

128

–

–

–

Salmonella typhimurium

64

32

32

256

64

R

R

64

256

–

–

Streptococcus faecalis –

R

–

–

–

R

128

8

256

–

–

Escherichia coli

R

R

R

R

256

R

R

256

256

R

125

Klebsiella pneumoniae

32

16

R

256

256

R

256

128

256

R

125

Pseudomonas aeruginosa

128

64

64

R

256

128

R

128

256

R

125

Arthrobacter oxidans

64

4

4

128

32

64

128

64

256

–

–

Nomenclature of surfactants referred to in Table 13.1. MIC values from Morán et al., 2001b and Colomer et al., 2011.

431

a

ACAa

13.4 Applications

III.  BIOBASED SURFACTANTS

Gramnegatives

Microorganism

432

13.  Arginine-Based Surfactants: Synthesis, Aggregation Properties, and Applications

TABLE 13.3  Antimicrobial Activity Expressed as Minimum Inhibitory Concentration of the Double-Chain Arginine-Based Surfactants

Gram-positives

Gram-negatives

Microorganisms

LAMa (μM)

LANHC10 (μM)

LANHC12 (μM)

LANHC14 (μM)

LANHC18 (μM)

Micrococcus luteus

19

17

510

243

R

Bacillus subtilis

19

R

R

486

R

Staphylococcus aureus

–

17

R

486

R

Staphylococcus epidermidis

19

17

510

486

R

Pseudomonas aeruginosa

78

68

R

R

R

Escherichia coli

38

269

R

R

R

Klebsiella pneumoniae

78

R

R

R

R

Candida albicans

19

68

R

243

R

a

MIC values from Pinazo et al., 2016.

interact with the cells (Rosen et al., 1999). As a result of their antimicrobial activity, arginine surfactants are particularly useful as preservatives for food and active ingredients in pharmaceutical formulations in dermatology and personal care products. The antimicrobial action of cationic surfactants is based on their ability to disrupt the integral bacterial membrane by a combined hydrophobic and electrostatic adsorption phenomenon at the membrane/water interface followed by membrane disorganization. A great effort has been made to design and synthesize a large variety of arginine-based surfactants with different structures. The structural differences in the synthesized molecules consist of the number of cationic charges; the chemical structure of the head group; the number, length, and bonding positions of hydrophobic chains; and the symmetry of the molecule. In what follows, we report the most important contributions in the relationship between structure and antimicrobial activity. 13.4.1.1  Monocatenary Arginine Surfactants In all instances, both the alkyl chain length and the chemistry of the polar head group affect the bactericidal activity (Table 13.2). Data in Table 13.2 show that arginine O-alkyl amides (ACA, ALA, and AMA) and O-alkyl esters (AOE, ACE, and ALE) with two positive charges per head group possess the lowest MIC values (Morán et al., 2001b). The surfactants are strongly adsorbed to the bacterial cell walls due to the presence of two ionic charges in the molecule, triggering membrane disruption. On the other hand, for the three series, Nα-alkyl, Oα-alkyl amides, and Oα-alkyl esters, the best activity was obtained for the surfactants with alkyl chains of 12 carbons. The highest biological effect occurring for alkyl chains of 12 carbons for the monocatenary compounds has been reported frequently (Morán et al., 2001b; Pérez et al., 2005). This optimum acyl chain length (C12) can be attributed to the combination of several physicochemical properties: hydrophobicity, adsorption strength, CMC, and solubility in aqueous media. However, the optimum alkyl chain length for every homologous series depends on the surfactant structure (Thorsteinsson et al., 2003; Birnie et al., 2000; Tsatsaroni et al., 1987).

III.  BIOBASED SURFACTANTS



TABLE 13.4  Minimum Inhibitory Concentration (mg/L) of C3(OA)2, C3(CA)2, C6(CA)2, C9(CA)2, C3(LA)2, C6(LA)2, and C9(LA)2 Gram-positives

C3(CA)2

C6(CA)2

C9(CA)2

C3(LA)2

C6(LA)2

C9(LA)2

Bacillus cereus var. mycoides

128

16

64

>128

32

32

64

Bacillus subtilis

256

64

64

128

4

8

64

Staphylococcus aureus

256

8

64

64

64

8

>128

Staphylococcus epidermidis

32

8

64

128

>128

>128

>128

Micrococcus luteus

–

32

8

32

8

16

16

Candida albicans

128

16

16

32

16

16

128

64

16

8

128

>128

32

32

Bordetella bronchiseptica

32

128

16

128

>128

128

128

Citrobacter freundii

256

128

16

32

>128

>128

>128

Enterobacter aerogenes

256

64

64

128

>128

>128

>128

Salmonella typhimurium

128

64

16

32

64

128

>128

Streptococcus faecalis

–

32

8

16

8

128

128

Escherichia coli

>256

64

64

32

>128

>128

>128

Klebsiella pneumoniae

256

16

16

32

8

8

128

Pseudomonas aeruginosa

256

64

64

128

>128

>128

>128

Arthrobacter oxidans

8

32

64

64

8

8

128

Gram-negatives Alcaligenes faecalis

433

C3(OA)2

13.4 Applications

III.  BIOBASED SURFACTANTS

Microorganism

434

Gram-positives

III.  BIOBASED SURFACTANTS

Gram-negatives

a

Microorganisms

88Ra

1010Ra

1212Ra

1414Ra

100Ra

120Ra

140Ra

Bacillus cereus var. mycoides

64

16

256

>256

32

128

128

Bacillus subtilis

64

2

>256

>256

128

64

128

Staphylococcus aureus

4

16

>256

256

128

64

64

Staphylococcus epidermidis

8

>256

>256

16

128

128

64

Micrococcus luteus

16

1

4

1

128

64

64

Candida albicans

16

32

64

>256

32

64

128

Salmonella typhimurium

16

32

>256

>256

32

128

128

Pseudomonas aeruginosa

64

>256

>256

>256

256

256

64

Escherichia Coli

8

64

>256

>256

128

64

128

Arthrobacter oxidans

32

16

>256

>256

128

64

64

Streptococcus faecalis

8

4

2

0.5

128

128

128

Bordetella bronchiseptica

0.25

0.25

0.25

0.25

64

64

64

Citrobacter freundii

32

>256

>256

256

256

128

128

Alcaligenes faecalis

8

4

>256

256

256

256

128

Enterobacter aerogenes

32

>256

>256

16

256

128

128

Klebsiella pneumoniae v. preumonial

8

16

>256

4

128

64

128

Nomenclature referred to in Table 13.2. Data from Pérez, L., Clapés, P., Pinazo, A., Angelet, M., Vinardell, M.P., Infante, M.R., 2002. Synthesis and biological properties of dicationic arginine_diglicerides. New J. Chem. 26, 1221–1227; Pérez, L., Pinazo, A., García, M.T., Morán, C., Infante, M.R., 2004. Monoglyceride surfactants from arginine: synthesis and biological properties. New J. Chem. 28, 1326–1334.

13.  Arginine-Based Surfactants: Synthesis, Aggregation Properties, and Applications

TABLE 13.5  Minimum Inhibitory Concentration (mg/L) of the Mono- and Diglyceride Arginine Surfactants



13.4 Applications

435

Taking advantage of the antimicrobial properties of the monocatenary arginine-based surfactants, the compound Nα-lauroyl-arginine-ethyl ester hydrochloride (LAE) is produced at industrial scale for use as a preservative in food, food packaging, and cosmetic formulations (Contijoch et al., 1995; Urgell and Seguer, 2003; Higueras et al., 2013; Aznar et al., 2013; Becerril et al., 2013; Muriel-Galet et al., 2014). 13.4.1.2  Single Chain Surfactants With Arginine and Lysine in the Polar Head In general, the antimicrobial activity of the cationic surfactants is attributed to the hydrophiliclipophilic balance in which the net positive charge has a large influence. N-lauroyl surfactants with two positive charges in the polar head have been designed to improve antimicrobial activity. Results in Table 13.2 show that the position and the density of cationic charge play an important role in the antimicrobial activity (Colomer et al., 2011). The addition of lysine as a second amino acid reduced antimicrobial activity. The presence of two cationic charges on the polar head increases the hydrophilic character of the molecule and, consequently, reduced the surface activity and biocide activity. Studies that aim to elucidate the mechanisms involved in the antimicrobial activity of the monocatenary arginine-lysine surfactant, LALM, have been reported (Colomer et al., 2013). A simple membrane model compound (1,2-dipalmitoyl-sn-phosphatidylcholine, DPPC) was used to explore the monolayer properties at the air/liquid interface. Compression surface pressure-molecular area isotherms of the DPPC/LALM mixtures at different pH reflected an expansion of the DPPC monolayer, likely due to electrostatic interactions. Antimicrobial activity of LALM surfactant has also been assessed with transmission electron microscopy, observing the effects on Staphylococcus aureus and Escherichia coli. The surfactant caused damage to the bacteria tested, including alterations, leakage of internal material, and cell destruction. 13.4.1.3  Double-Chain Surfactants From Arginine Double-chain surfactants from arginine, LANHCx, have been synthesized using the monocatenary arginine surfactant LAM as starting material. The antimicrobial activity of the new compounds has been evaluated and compared with that of LAM (Pinazo et al., 2016). Results (Table 13.3) show that the activity of new double-chain surfactants is lower than that of LAM, probably due to their high hydrophobicity. The double-chain compounds form large and stable vesicles at room temperature. The low antimicrobial activity can also be related to their aggregation behavior. For LAM, MIC values are below the CMC. Therefore, the monomer form of surfactant interacts with bacterial cells. If the surfactants form vesicles (i.e., at concentrations above the CMC), the monomer concentration is very low, and the needed concentration to inhibit the growth of the bacteria is higher. 13.4.1.4  Gemini Surfactants Results in Table 13.4 demonstrate that the Gemini surfactants with spacer chain length of 3–9 and alkyl chain of 10 carbon atoms exhibit higher antibacterial activity than the corresponding single-chain homologue (CAM) whereas the bis(Args) with alkyl chains of 12 carbon atoms exert lower activity than their homologue LAM (Pérez et al., 2002b). When keeping the alkyl chain length constant, activity seems to decrease with values of s for s ≥ 5. When keeping the spacer chain length at s = 1, the relationship between the alkyl chain length and the activity is not linear, showing a maximum for the homologues C3(CA)2 (Pérez et al., 1996, 2002b; Piera et al., 2000). III.  BIOBASED SURFACTANTS

436

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Since the perturbation of the cell membrane by these compounds is directed primarily by physicochemical processes, model membrane systems can provide valuable information for understanding the mechanism of action of these molecules. The relationship between the antimicrobial activity of bis(Args), LAM, and other agents and the physicochemical process involved in the perturbation of the cell membrane has been studied (Castillo et al., 2004). To this end, the interaction of these surfactants with two biomembrane models, DPPC multilamellar lipid vesicles (MLV) and monolayers of DPPC, 1,2 dipalmitoyl phosphatidyl glycerol sodium salt (DPPG), and E. coli total lipid extract, was investigated using differential scanning calorimetry (DSC) and Langmuir monolayers. DSC results show that variations in both the transition temperature and the transition width at one-half of the height of the head absorption peak were consistent with the antimicrobial activity of the compounds. Penetration kinetics and compression isotherm studies indicated that both steric hindrance effects and electrostatic forces controlled the antimicrobial agent-lipid interactions. 13.4.1.5  Mono- and Diglycerides From Arginine All mono- and diglyceride arginine surfactant derivatives showed antimicrobial activities against a wide range of microorganisms, that is, they inhibited the growth of all the microorganisms tested (Table 13.5) (Pérez et al., 2004a,b, 2005). The power of monoglycerides from arginine does not change drastically with the alkyl chain length. For the diglyceride compounds, the antimicrobial activity decreased with an increase of alkyl chain length. Nevertheless, the activity of the shorter alkyl chain diglycerides, 88R and 1010R (average MIC value of 0.081 mM), is considerably higher that the activity of monoglyceride derivatives (average MIC value of 0.235 mM). The 88R and 1010R surfactants (Pérez et al., 2002a, 2005) were able to inhibit bacterial growth and even kill some bacteria at concentration as low as 4 mg/L, which is comparable with the activity showed by alkyldimethylbenzylammonium chloride or benzalkonium chloride, a well-known biocide compound, and that of new analogues of benzalkonium chlorides (Pernak et al., 1999). This demonstrates that the presence of two alkyl chain of 8–10 carbon atoms is effective in improving the intensity of the antimicrobial activity. In an attempt to explore the potential applications of 1212RAc and 1414Rac and the nonacetylated version of 1212R and 1414R surfactants in clinical settings, the antimicrobial and hemolytic activities were studied either as pure surfactants or after their formulation as pseudotetra-chain catanionic mixtures with phosphatidylglycerol and as cationic mixture with DPPC (Lozano et al., 2008, 2011). These systems form stable cationic vesicles by themselves, and the average diameter of the vesicles decreases as the alkyl chain length of the surfactant increases (Tavano et  al., 2014). The antimicrobial activity of the negatively charged vesicles against Acinetobacter baumannii was maintained with respect to the surfactant alone, while a significant improvement of the antimicrobial activity against S. aureus was observed, together with a strong decrease of hemolytic activity. These results constitute a proof of principle that tuning formulation can reduce the cytotoxicity of many surfactants, opening their possible biological applications.

13.4.2  Sequestration of Lipopolysaccharide Gram-negative sepsis is a common clinical problem (Gasche et  al., 1995), and the mortality due to septic shock reflects the absence of specific therapy aimed at the underlying

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pathogenetic mechanisms. Cationic hydrophobic compounds can interact with the toxic ­portion of the lipopolysaccharide (constituent of the outer membranes of the gram-negative bacteria) and reduce this serious problem. Bis(Args) bind to lipopolysaccharide and neutralize endotoxic activity in “in vitro” tumor necrosis factor-[α] and nitric oxide release assays (David et  al., 2001). The presence in the Gemini structure of two highly basic protonatable guanidinium functionalities separated by a spacer chain provides for excellent recognition of the bisphosphates on the lipopolysaccharide. In spite of these results, bis(Args) are themselves unlikely to be of therapeutic value due to their high cytotoxicity. However, this class of compounds offers an excellent point of departure for refining the design and development of less toxic analogues for the treatment of gram-negative sepsis.

13.4.3  Control of DNA Compaction DNA packaging in the living cellular environment is a very important phenomenon. DNA compactions by polyamines, like spermidine and spermine, are examples of events that occur in cells and are believed to be important in regulation of cell proliferation and differentiation. In the literature, one can find many studies of DNA compaction in aqueous solution. DNA molecules are known to undergo a discrete conformational transition from an extended to a collapsed state by interacting with single- or double-chain cationic amphiphiles. Cationic surfactants associate strongly with DNA and produce compaction but are often toxic. Since one of our main motivations consists in the development of new nontoxic biocompatible and biodegradable systems, we studied the interaction of the single-chain arginine-based surfactant ALA (Fig. 13.4, n = 10) with DNA (Rosa et al., 2007). The ability of this surfactant alone to compact DNA is compared with classical cationic surfactants by fluorescence microscopy. Furthermore, toxicity studies revealed that the incorporation of ALA in catanionic vesicle system transformed them into cell viable systems, extending therefore their use to drug and gene delivery systems. A precondensation step of DNA as a viable approach for liposome-based gene delivery has been also addressed (Rosa et al., 2008). To the best of our knowledge, ALA is the first cationic amphiphile based on an amino acid structure used in gene delivery. This approach consists in both the precondensation of plasmid DNA with an arginine-based cationic surfactant, ALA, and the incorporation of the blood protein transferrin into the formulations. Two cationic liposome formulations were used, one composed of a mixture of dioleoyl trimethylammoniopropane and cholesterol (DOTAP/Chol) and the other a pH-sensitive formulation constituted of DOTAP, Chol, dioleoyl phosphatidylethanolamine (DOPE), and cholesteryl hemisuccinate (CHEMS). The lipidic composition played an extremely important role in transfection efficiency. The pair of DOPE/CHEMS enhanced transfection in comparison with the complexes composed of DOTAP/Chol liposomes. Remarkable transfection results were obtained for ALA-CatpH complexes. A correlation between formulations that transfect poorly and large mean sizes was made. Overall, we demonstrated that the presence of ALA improved the transfection efficiency when conjugated with cationic liposome systems.

13.4.4  Drug Delivery Systems The method by which a drug is delivered can have a significant effect on its efficacy. Some drugs have an optimum concentration range within which maximum benefit is derived, and

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concentrations above or below this range can be toxic or produce no therapeutic benefit at all. On the other hand, the very slow progress in the efficacy of the treatment of severe diseases has suggested a growing need for an interdisciplinary approach (e.g., combining polymer science, pharmaceutics, bioconjugate chemistry, and molecular biology) to the delivery of therapeutics to targets in tissues, to generate new ideas on controlling the pharmacokinetics, pharmacodynamics, nonspecific toxicity, immunogenicity, biorecognition, and efficacy of drugs (Kaparissides et al., 2006). Colloidal drug carrier systems such as micellar solutions, vesicle and liquid crystal dispersions, and nanoparticle dispersions consisting of small particles of 10–400 nm diameter show great promise as drug delivery systems. When developing these formulations, the goal is to obtain systems with optimized drug loading and release properties, long shelf life, and low toxicity. Cationic vesicles based on positively charged surfactants are the most promising among other colloidal delivery systems. Among the classical cationic surfactants, quaternary ammonium compounds (QACs) and bis(QACs) are usually employed to form cationic liposomes (Aleandri et al., 2012) and cationic vesicles (Campanha et al., 2001; Sicchierolli et al., 1995). Unfortunately, a major problem related to the use of cationic vesicles based on QACs is their high degree of toxicity toward the host (Lv et al., 2006). In fact, QACs present acute toxicity, poor chemical and biological degradation, and hemolytic activity (Shirai et al., 2005). A strategy to increase the efficiency of surfactants and improve their environmental properties is to utilize amino acids as surfactant building blocks. Cationic colloidal formulations based on arginine-based surfactants (single or Gemini structures) and membrane additive compounds such as the phospholipid DLPC or cholesterol were prepared (Tavano et  al., 2013). The size of the aggregates depended on the molecular architecture of the surfactants and the concentration and type of components added to the formulation; aggregates of different dimensions and stability can be obtained starting from pure surfactant solutions or surfactant-additive mixtures. Single-chain surfactants and Gemini with short spacer chains (LAM and C6(LA)2, respectively) give rise to solutions with micellar aggregates, while Gemini with long spacers (C9(LA)2 and C12(LA)2) form large twisted ribbon aggregates. The addition of phospholipids or cholesterol drastically changed the aggregation behavior, leading to the formation of cationic vesicles that are smaller than the size of phospholipid- and cholesterol-free aggregates. All of the formulations had positive zeta-potential values and in general exhibited high colloidal stability. The antimicrobial and hemolytic activity of these formulations was strongly affected by the size of the aggregates; small aggregates penetrate more deeply into the cell surface and consequently have high antimicrobial activity and low toxicity. The length of the spacer chain for Gemini surfactants modulated the aggregation type and consequently the hemolytic and antimicrobial activities. Gemini with long spacer chains gave rise to large aggregates and had more difficulties interacting with biological membranes. The introduction of additives such as cholesterol or DLPC also affected the biological properties of these surfactants. The results obtained suggest new possible pharmaceutical delivery systems based on arginine surfactants as a viable alternative to the classical formulations, showing good stability, low hemolytic effects, and also a high antimicrobial activity, in contrast to conventional vesicles. Diacylglycerol-arginine surfactants (Fig. 13.5) have been also investigated for the preparation of cationic vesicular systems (Tavano et al., 2014). These surfactants formed stable cationic vesicles, and the average diameter of the vesicles increased as the alkyl chain length of

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the surfactant increased. The addition of DPPC also modified the physicochemical properties of these vesicles. The mean diameter of the vesicles diminished with the introduction of DPPC. The capability of the cationic vesicles on the encapsulation of therapeutic drug was considered. Ciprofloxacin and 5-fluorouracil (5-FU) were loaded onto these cationic systems, and their in vitro release from all formulations was effectively delayed with respect to the corresponding free drug solutions. The entrapment efficiency and the permeability of the vesicles appear to depend on the physicochemical properties of both vesicles and drug. Studies on the antimicrobial activity of empty and ciprofloxacin-loaded vesicles were determined against E. coli, S. aureus, and Klebsiella pneumoniae bacteria. The antimicrobial efficacy of the vesicles based only on cationic lipids is strongly affected by the hydrophobicity of lipids and the zeta-potential values of the dispersions. The introduction of DPPC strongly decreased the antimicrobial activity of these systems. On the other hand, the ciprofloxacin encapsulated in these vesicles preserves its antimicrobial activity.

13.4.5  DNA Gel Particles Hydrogels are typically composed of a hydrophilic organic polymer component that is cross-linked into a network by either covalent or noncovalent interactions (Gehrke, 1993; Hoffman, 2002). It is the cross-linking that provides for dimensional stability, while the high solvent content gives rise to the fluidlike transport properties. Hydrogels have been used to encapsulate proteins (Gombotz and Wee, 1998), cells (Goosen et al., 1985) and drugs (Lutolf et al., 2003) and then release them through the dissolution of the hydrogel structure. Crosslinks in this class of hydrogels often arise from noncovalent attractive forces between the polymer chains, such as hydrophobic interactions, hydrogen bonding, or ionic interactions. Polymer gels are important topics in colloids, polymer, and biological sciences and used in many technological applications. DNA gels have many applications, such as for the controlled delivery of DNA. We have prepared novel DNA gel particles based on associative phase separation and interfacial diffusion. By mixing solutions of DNA with solutions of different cationic agents, such as surfactants, proteins, and polysaccharides, the formation of DNA gel particles without adding any kind of cross-linker or organic solvent has been confirmed (Morán et al., 2009, 2010a, 2013, 2014). Surfactants with the cationic functionality based on the arginine structures were used to prepare biocompatible gel particles for the controlled encapsulation and release of DNA (Morán et al., 2010b). The DNA gel particles were prepared by mixing DNA either single- or double-stranded DNA (ss- and dsDNA, respectively), with two different single-chain amino acid-based surfactants: arginine-N-lauroyl amide dihydrochloride (ALA) and Nα-lauroylarginine-methyl ester hydrochloride (LAM). Studies on the degree of DNA entrapment, the swelling/deswelling behavior, and the DNA release kinetics have been studied as a function of both the number of charges in the polar head group of the amino acid-based surfactant and the secondary structure of the nucleic acid. It was shown that DNA was effectively entrapped by surfactant, protecting its secondary structure. Changes in the magnitude of the swelling behavior and the DNA release kinetics by differences in the structure of the surfactant were demonstrated. Analysis of the data indicates a stronger interaction of ALA with DNA compared with LAM, mainly attributable to the double charge carried by the former surfactant compared with the singly charged

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head group of the latter species. The stronger interaction with amphiphiles for ssDNA compared with dsDNA suggests the important role of hydrophobic interactions for complexation with DNA. Data on the microstructure of the complexes obtained from SAXS analysis of the particles strongly suggest a hexagonal packing. There exists a clear correlation between the DNA release rates and the lattice parameter obtained from SAXS analysis: the shorter the lattice parameter, the slower the release. These results can be explained by the complexation and neutralization of DNA in the DNA gel particles, confirmed by agarose gel electrophoresis measurements. This new generation of amino acid-based surfactant complexes with DNA contributes to the increasing demand for biocompatible vehicles for pharmaceutical applications.

13.5 CONCLUSIONS The proposed arginine-based surfactants will contribute to the field of biocompatible surfactant research and ultimately lead to the commercial advancement of biochemistry products for industrial use. Moreover, we believe that the results on the membrane interaction studies will elucidate new functions of the surfactants, thus expanding their arsenal of potential applications in biochemistry. Finally, the surfactants proposed in this review could be of great interest to the field of biology, specifically as substitutes for natural phospholipids.

References Adams, D.J., Holtzmann, K., Schneider, C., Michael, F., 2007. Self-assembly of surfactant-like peptides. Langmuir 23, 12729–12736. Albalat, R., Claret, J., Ignés-Mullol, J., Sagués, F., Morán, C., Pérez, L., Clapés, P., Pinazo, A., 2003. Langmuir monolayers of diacyl glycerol amino acid-based surfactants, effect of substitution pattern of the glycerol backbone. Langmuir 19, 10878–10884. Aleandri, S., Bonicelli, M.G., Bordi, F., Casciardi, S., Diociaiuti, M., Giansanti, L., Leonelli, F., Mancini, G., Perrone, G., Sennato, S., 2012. Structure and functionality of edible fats. Soft Matter 8, 5904–59015. Allouch, M., Seguer, J., Infante, M.R., Selve, C., 1996. Nonionic amphiphile compounds from aspartic and glutamic acids as molecular mimics of lecithins. J. Am. Oil Chem. Soc. 73, 87–96. Aznar, M., Gómez-Estaca, J., Vélez, D., Devesa, V., Nerín, C., 2013. Migrants determination and bioaccessibility study of ethyl lauroyl arginate (LAE) from a LAE based antimicrobial food packaging material. Food Chem. Toxicol. 56, 363–370. Becerril, R., Manso, S., Nerin, C., Gómez-Lus, R., 2013. Antimicrobial activity of Lauroyl Arginate Ethyl (LAE), against selected food-borne bacteria. Food Control 32, 404–408. Benavides, T., Martínez, V., Mitjans, M., Infante, M.R., Morán, M.C., Clapés, P., Clothier, R., Vinardell, M.P., 2004. Assessment of the potential irritation and photoirritation of novel amino acid-based surfactants by in vitro methods as alternative to the animal tests. Toxicology 201, 87–93. Bernal, C., Guzman, F., Illanes, A., Wilson, L., 2018. Selective and eco-friendly synthesis of lipoaminoacid-based surfactants for food, using immobilized lipase and protease biocatalysts. Food Chem. 239, 189–195. Birnie, C.R., Malamud, D., Schnaare, R.L., 2000. Antimicrobial evaluation of N-alkyl betaines and N-alkyl-N, N-dimethylamine oxides with variations in chain length. Antimicrob. Agents Chemother. 44, 2514–2517. Bordes, R., Holmberg, K., 2015. Amino acid-based surfactants—do they deserve more attention? Adv. Colloid Interf. Sci. 222, 79–91. Boyat, C., Rolland-Fulcrand, V., Roumestant, M.-L., Viallefont, P., Martinez, J., 2000. Chemo-enzymatic synthesis of new non ionic surfactants from unprotected carbohydrates. Prep. Biochem. Biotechnol. 30, 281–294.

III.  BIOBASED SURFACTANTS

REFERENCES 441

Burnett, C.L., et  al., 2017. Safety assessment of amino acid alkyl amides as used in cosmetics. Int. J. Toxicol. 36, 17S–56S. Campanha, M.T., Mamizuka, E.M., Carmona-Ribeiro, A.M., 2001. Interactions between cationic vesicles and Candida albicans. J. Phys. Chem. B 105, 8230–8236. Capone, S., Walde, P., Seebach, D., Ishikawa, T., Caputo, R., 2008. pH-sensitive vesicles containing a lipidic betaamino acid with two hydrophobic chains. Chem. Biodivers. 5, 16–30. Castillo, J.A., Pinazo, A., Carilla, J., Infante, M.R., Alsina, M.A., Haro, I., Clapes, P., 2004. Interaction of antimicrobial arginine-based cationic surfactants with liposomes and lipid monolayers. Langmuir 20, 3379–3387. Chandra, N., Tyagi, K., 2013. Synthesis, properties and applications of amino acids based surfactants: a review. J. Dispers. Sci. Technol. 34, 800–808. Clapés, P., Infante, M.R., 2002. Amino acid-based surfactants: enzymatic synthesis, properties and potential applications. Biocatal. Biotransformation 20, 215–233. Clapés, P., Morán, M.C., Infante, M.R., 1999. Enzymatic synthesis of arginine-based cationic surfactants. Biotechnol. Bioeng. 63, 333–343. Cocquyt, J., Olsson, U., Olofsson, G., Van der Meeren, P., 2004. Temperature quenched DODAB dispersions: fluid and solid state coexistence and complex formation with oppositely charged surfactant. Langmuir 20, 3906–3912. Colomer, A., Pinazo, A., Manresa, M.A., Vinardell, M.P., Mitjans, M., Infante, M.R., Pérez, L., 2011. Cationic surfactants derived from lysine: effects of their structure and charge type on antimicrobial and hemolytic activities. Med. Chem. 54, 989–1002. Colomer, A., Pinazo, A., García, M.T., Mitjans, M., Vinardell, M.P., Infante, M.R., Martínez, V., Pérez, L., 2012. pH-sensitive surfactants from lysine: assessment of their cytotoxicity and environmental behavior. Langmuir 28, 5900–5912. Colomer, A., Pérez, L., Pons, R., Infante, M.R., Perez-Clos, D., Manresa, A., Espuny, M.J., Pinazo, A., 2013. Mixed monolayer of DPPC and lysine-based cationic surfactants: an investigation into the antimicrobial activity. Langmuir 29, 7912–7921. Contijoch, A., Infante, M.R., Erra, P., 1995. Procedure for the synthesis of cationic surfactants derived from the condensation of fatty acids with esterified dibasic amino acids and their application as antimicrobial agents. ES2092958 (1995). USA 5 780 658 (1998), EPO 0 749 960 (2001),CA 2 185 165 (2001). Danino, D., Talmon, Y., Zana, R., 1995. Alkanediyl-alpha, omega-bis(dimethylalkylammonium bromide) surfactants (dimeric surfactants). 5. Aggregation and microstructure in aqueous-solutions. Langmuir 11, 1448–1456. Das, D., Dasgupta, A., Roy, S., Mitra, N.R., Debnath, S., Das, P.K., 2006. Water gelation of an amino acid-based amphiphile. Chem. Eur. J. 12, 5068–5074. David, S., Pérez, L., Infante, M.R., 2001. Sequestration of bacterial lipopolysaccharide by bis(Args) Gemini compounds. Bioorg. Med. Chem. Lett. 12, 357–360. Epand, R.F., Infante, M.R., Flanagan, T.D., Epand, R.M., 1998. Properties of lipoamino acids incorporated into membrane bilayers. Biochim. Biophys. Acta Biomembr. 1373, 67–75. Fördedal, H., Sjöblolm, S., Infante, M.R., 1993. Lipoamino acid association in the system na-lauroyl-l-arginine methyl ester/1-pentanol/water as studied by dielectric spectroscopy. Colloids Surf. A Physicochem. Eng. Asp. 79, 81–88. Gasche, Y., Pittet, D., Sutter, P., 1995. In: Sibbald, W.J., Vincent, J.L. (Eds.), Clinical Trials for Treatment of Sepsis. Sprinter, Berlin, pp. 35–36. Gehrke, S.H., 1993. Synthesis, equilibrium swelling, kinetics, permeability and applications of environmentally responsive gels. Adv. Polym. Sci. 110, 81–144. Gerova, M., Rodrigues, F., Lamere, J.F., Dobrev, A., Fery-Forgues, S., 2008. Self-assembly properties of some chiral N-palmitoyl amino acid surfactants in aqueous solution. J. Colloid Interface Sci. 319, 526–533. Gombotz, W.R., Wee, S., 1998. Protein release from alginate matrices. Adv. Drug Deliv. Rev. 31, 267–285. Goosen, M.F.A., OEShe, G.M., Gharapetian, H.M., Chou, S., Sun, A.M., 1985. Optimization of microencapsulation parameters: semipermeable microcapsules as a bioartificial pancreas. Biotechnol. Bioeng. 27, 146–150. Gorbitz, C.H., 2007. Microporous organic materials from hydrophobic dipeptides. Chem. Eur. J. 13, 1022–1031. Hagslatt, H., Soderman, O., Jonsson, B., 1991. Divalent surfactants. Experimental results and theoretical modeling of surfactant/water phase equilibria. Langmuir 10, 2177–2187. Heir, E., Sundheim, G., Holck, A.L., 1995. Resistance to quaternary ammonium compounds in staphylococcus spp. isolated from the food industry and nucleotide sequence of the resistance plasmid pst827. J. Appl. Bacteriol. 79, 149–156.

III.  BIOBASED SURFACTANTS

442

13.  Arginine-Based Surfactants: Synthesis, Aggregation Properties, and Applications

Higueras, L., López-Carballo, G., Hernandez-Muñoz, P., Gavara, R., Rollini, M., 2013. Development of a novel antimicrobial film based on chitosan with LAE (ethyl-Nα-dodecanoyl-L-arginate) and its application to fresh chicken. Int. J. Food Microbiol. 165, 339–345. Hoffman, A.S., 2002. Hydrogels for biomedical applications. Adv. Drug Deliv. Rev. 54, 3–12. Holmberg, K., 2003. In: Holmberg, K. (Ed.), Novel Surfactants: Preparation, Applications, and Biodegradability. second ed.. Marcel Dekker, New York, pp. 1–2. Imae, T., Hayashi, N., Matsumoto, T., Tada, T., Furusaka, M., 2000. Structures of fibrous supramolecular assemblies constructed by amino acid surfactants: investigation by AFM, SANS, and SAXS. J. Colloid Interface Sci. 225, 285–290. Infante, M.R., Moses, V., 1994. Synthesis and surface-activity properties of hydrophobic/hydrophilic peptides. Int. J. Pept. Protein Res. 43, 173–179. Infante, M.R., García Domínguez, J.J., Erra, P., Juliá, M.R., Prats, M., 1984. Surface active molecules: preparation and properties of long chain N[α]-acyl-l-[alpha]-amino-[omega]-guanidine alkylic acid derivatives. Int. J. Cosmet. Sci. 6, 275–282. Infante, M.R., Molinero, J., Erra, P., Juliá, M.R., García Domínguez, J.J., 1985. A comparative study on surface active and antimicrobial properties of some N[alpha]lauroyl-l-[alpha]-[omega]-dibasic amino acid derivatives. Eur. J. Lipid Sci. Technol. 8, 309–313. Infante, M.R., Molinero, J., Erra, P., 1992a. Lipopeptide surfactants. 2. Acidic and basic N-alpha-lauroyl-L-arginine dipeptides from pure amino-acids. J. Am. Oil Chem. Soc. 69, 647–652. Infante, M.R., Pés, M.A., Vilchez, A., Solans, C., 1992b. Síntesis y propiedades de tensioactivos catiónicos derivados de arginina. An. Quim. 88, 542–548. Infante, M.R., Pinazo, A., Seguer, J., 1997. Non-conventional surfactants from amino acids and glycolipids: structure, preparation and properties. Colloids Surf. A Physicochem. Eng. Asp. 123–124, 49–70. Jones, R.N., Barry, A.L., Gavan, T.L., Washington, J.A., 1980. In: Lennette, E.H., Ballows, A., Hauser, W.J., Shadomy, H.J. (Eds.), II Manual of Clinical Microbiology. American Society for Microbiology, Washington, DC. Joondan, N., Jhaumeer-Laulloo, S., Caumul, P., Akerman, M., 2017. Synthesis, physicochemical, and biological activities of novel N-acyl tyrosine monomeric and Gemini surfactants in single and SDS/CTAB-mixed micellar system. J. Phys. Org. Chem. 30, e3675. https://doi.org/10.1002/poc.3675. Kaparissides, C., Alexandridou, S., Kotti, K., Chaitidou, S., 2006. Recent advances in novel drug delivery systems. Azojomo J. Mater. Online https://doi.org/10.2240/azojono011. Kunieda, H., Nakamura, K., Infante, M.R., Solans, C., 1992. Reversed vesicles from biocompatible surfactants. Adv. Mater. 4, 291–293. Langevin, D., 1999. In: Sadoc, J.F., Revier, N. (Eds.), Foams and Emulsions. Kluwer, Boston. Lee, S., Kim, E., Seo, M.L., Do, Y., Lee, Y.-A., Lee, S.S., Jung, J.H., Kogiso, M., Shimizu, T., 2008. Self-assembled helical ribbon and tubes of alanine-based amphiphiles induced by two different formation mechanisms. Tetrahedron 64, 1301–1308. Lindman, B., Puyal, M.C., Kamenka, N., Rymden, R., Stilbs, P., 1984. Micelle formation of anionic and cationic surfactants from fourier transform proton and lithium-7 nuclear magnetic resonance and tracer self-diffusion studies. J. Phys. Chem. 88, 5048–5057. Lozano, N., Pérez, L., Pons, R., Luque-Ortega, J.R., Fernández-Reyes, M., Rivas, L., Pinazo, A., 2008. Interaction studies of diacyl glycerol arginine-based surfactants with DPPC and DMPC monolayers, relation with antimicrobial activity. Colloids Surf. A Physicochem. Eng. Asp. 319, 196–203. Lozano, N., Pérez, L., Pons, R., Pinazo, A., 2011. Diacyl glycerol arginine-based surfactants: biological and physicochemical properties of catanionic formulations. Amino Acids 40, 721–729. Lutolf, M.P., Raeber, G.P., Zisch, A.H., Tirelli, N., Hubbell, J.A., 2003. Cell-responsive synthetic hydrogels. Adv. Mater. 15, 888–892. Lv, H., Zhang, S., Wang, B., Cuis, S., Yan, J., 2006. J. Control Release 114 (1), 100–109. Martínez, V., Corsini, E., Mitjans, M., Pinazo, A., Vinardell, M.P., 2006. Evaluation of eye and skin irritation of arginine- derivative surfactants using different in vitro endpoints as alternatives to the in vivo assays. Toxicol. Lett. 164, 259–267. Morán, M.C., Infante, M.R., Clapés, P., 2001a. Synthesis of glycerol amino acid-based surfactants. Part 1. Enzymatic preparation of rac-1-O-(Nα-acetyl-L-aminoacyl) glycerol derivatives. J. Chem. Soc. Perkin Trans. 1, 2063–2070. Morán, M.C., Clapés, P., Comelles, F., García, M.T., Perez, L., Vinardell, M.P., Infante, M.R., 2001b. Chemical structure/properties relationship in single-chain arginine surfactants. Langmuir 17, 5071–5075.

III.  BIOBASED SURFACTANTS

REFERENCES 443

Morán, M.C., Infante, M.R., Clapés, P., 2002. Synthesis of glycerol amino acid-based surfactants. Part  2. Lipasecatalysed synthesis of 1-O-lauroyl-rac-glycero-3-(Nα-acetyl-L-amino acid) and 1,2-di-O-lauroyl-rac-glycero-3(Nα-acetyl-L-amino acid) derivatives. J. Chem. Soc. Perkin Trans. 1, 1124–1134. Morán, M.C., Pinazo, A., Pérez, L., Clapés, P., Pons, R., Infante, M.R., 2004a. Enzymatic synthesis and physicochemical characterization of glycerol arginine-based surfactants. C.R. Chim. 7, 169–176. Morán, M.C., Pinazo, A., Pérez, L., Clapés, P., Angelet, M., García, M.T., Vinardell, M.P., Infante, M.R., 2004b. “Green” amino acid-based surfactants. Green Chem. 6, 233–240. Morán, M.C., Pinazo, A., Clapés, P., Infante, M.R., Pons, R., 2005. The effect of molecular shape on the thermotropic liquid crystal behavior of monolauroylated amino acid glyceride conjugates. J. Phys. Chem. B 109, 22899–22908. Morán, M.C., Baptista, F.R., Ramalho, A., Miguel, M.G., Lindman, B., 2009. DNA gel nanoparticles: preparation and controlling the size. Soft Matter 5, 2538–2542. Morán, M.C., Miguel, M.G., Lindman, B., 2010a. DNA gel particles. Soft Matter 6, 3143–3156. Morán, M.C., Infante, M.R., Miguel, M.G., Lindman, B., Pons, R., 2010b. Novel biocompatible DNA gel particles. Langmuir 26, 10606–10613. Morán, M.C., Mitjans, M., Martínez, V., Nogueira, D.R., Vinardell, M.P., 2013. Current directions in DNA gel particles. In: Muñoz-Torrero, D., Cortés, A., Mariño, E.L. (Eds.), Recent Advances in Pharmaceutical Sciences III. Transworld Research Network, Kerala. Morán, M.C., Vinardell, M.P., Infante, M.R., Miguel, M.G., Lindman, B., 2014. DNA gel particles: an overview. Adv. Colloid Interf. Sci. 205, 240–256. Muriel-Galet, V., López-Carballo, G., Hernández-Muñoz, P., Gavara, R., 2014. Characterization of ethylene-vinyl alcohol copolymer containing lauril arginate (LAE) as material for active antimicrobial food packaging. Food Packag. Shelf Life 1, 10–18. Ohta, A., Toda, K., Morimoto, K.Y., Asakawa, T., Miyagishi, S., 2008. Effect of the side chain of N-acyl amino, acid surfactants on micelle formation: an isothermal titration calorimetry study. Colloids Surf. A Physicochem. Eng. Asp. 317, 316–322. Oshimura, E., Yuji, Y., Kazutami, S., 2007. Conformational behavior of N-acylamino acid oil and N-acylamino acid surfactant in aqueous solution. J. Oleo. Sci. 56, 115–121. Ozdemir, A., Turan-Zitouni, G., Kaplancikli, Z.A., Revial, G., Güven, K., 2007. Síntesis and antimicrobial activity of 1-(4-aryl-2-thiazoyl)-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives. Eur. J. Med. Chem. 42, 403–409. Pérez, L., Torres, J.L., Manresa, A., Solans, C., Infante, M.R., 1996. Synthesis, aggregation, and biological properties of a new class of gemini cationic amphiphilic compounds from arginine, bis(Args). Langmuir 12, 5296–5301. Pérez, L., Pinazo, A., Rosen, M., Infante, M.R., 1998. Surface activity properties at equilibrium of novel gemini cationic amphiphilic compounds from arginine, bis(Args). Langmuir 14, 2307–2315. Pérez, L., Clapés, P., Pinazo, A., Angelet, M., Vinardell, M.P., Infante, M.R., 2002a. Synthesis and biological properties of dicationic arginine_diglicerides. New J. Chem. 26, 1221–1227. Pérez, L., García, M.T., Ribosa, I., Vinardell, M.P., Manresa, A., Infante, M.R., 2002b. Biological properties of argininebased gemini cationic surfactants. Environ. Toxicol. Chem. 21, 1279–1285. Pérez, L., Infante, M.R., Pons, R., Morán, C., Vinardell, M.P., Mitjans, M., Pinazo, A., 2004a. A synthetic alternative to natural lecithins with antimicrobial properties. Colloids Surf. B: Biointerfaces 35, 235–242. Pérez, L., Pinazo, A., García, M.T., Morán, C., Infante, M.R., 2004b. Monoglyceride surfactants from arginine: synthesis and biological properties. New J. Chem. 28, 1326–1334. Pérez, L., Infante, M.R., Angelet, M., Clapes, P., Pinazo, A., 2004c. Glycerolipid arginine-based surfactants: synthesis and surface active properties. Progr. Colloid Polym. Sci. 123, 210–216. Pérez, N., Pérez, L., Infante, M.R., García, M.T., 2005. Biological properties of arginine-based glycerolipidic cationic surfactants. Green Chem. 7, 540–546. Pérez, L., Pinazo, A., Infante, M.R., Pons, R., 2007. Investigation of the micellization process of single and gemini surfactants from arginine by SAXS, NMR self-diffusion, and light scattering. J. Phys. Chem. B 111, 11379–11387. Pernak, J., Mirska, I., Kmiecik, R., 1999. Antimicrobial activities of new analogues of benzalkonium chloride. Eur. J. Med. Chem. 34, 765–771. Pés, A., 1993. Estudio del comportamiento fásico de sistemas con tensioactivos derivados de aminoácidos y caracterización structural de las diversas fases isótropas y anisótropas. (Ph.D thesis) Universidad de Barcelona. Piera, E., Infante, M.R., Clapés, P., 2000. Chemo-enzymatic synthesis of arg-based Gemini surfactants. Biotechnol. Bioeng. 70, 323–331.

III.  BIOBASED SURFACTANTS

444

13.  Arginine-Based Surfactants: Synthesis, Aggregation Properties, and Applications

Pinazo, A., Diz, M., Pés, A., Erra, P., Infante, M.R., 1993. Synthesis and properties of cationic surfactants containing a disulfide bond. J. Am. Oil Chem. Soc. 70, 37–42. Pinazo, A., Wen, X., Pérez, L., Infante, M.R., Franses, E.I., 1999. Aggregation behaviour in water of monomeric and Gemini cationic surfactants derived from arginine. Langmuir 15, 3134–3142. Pinazo, A., Pérez, L., Infante, M.R., Franses, E.I., 2001. Relation of foam stability to solution and surface properties of gemini cationic surfactants derived from arginine. Colloids Surf. A Physicochem. Eng. Asp. 189, 225–235. Pinazo, A., Pérez, L., Infante, M.R., Pons, R., 2004. Unconventional vesicle-to-ribbon transition behaviour of diacyl glycerol amino acid based surfactants in extremely diluted systems induced by pH-concentration effects. Phys. Chem. Chem. Phys. 6, 1475–1481. Pinazo, A., Petrizelli, V., Bustelo, M., Pons, R., Vinardell, M.P., Mitjans, M., Manresa, A., Pérez, L., 2016. New cationic vesicles prepared with double chain surfactants from arginine: role of the hydrophobic group on the antimicrobial activity and cytotoxicity. Colloids Surf. B: Biointerfaces 141, 19–27. Pinheiro, L., Faustino, C., 2017. Amino-Based Surfactants for Biomedical Applications. https://doi.org/10.5772/67977. Prud’homme, R.K., Khan, S.A., 1996. In: Prud’homme, R.K., Khan, S.A. (Eds.), Foams: Theory, Measurements, and Applications. Marcel Dekker, New York. Rosa, M., Infante, M.R., Miguel, M.G., Lindman, B., 2006. Spontaneous formation of vesicles and dispersed cubic and hexagonal particles in amino acid-based catanionic surfactant systems. Langmuir 22, 5588–5596. Rosa, M., Morán, M.C., Miguel, M.G., Lindman, B., 2007. The association of DNA and stable catanionic amino ­acid-based vesicles. Colloids Surf. A Physicochem. Eng. Asp. 301, 361–375. Rosa, M., Penacho, N., Simoes, S., Lima, M.C.P., Lindman, B., Miguel, M.G., 2008. DNA pre-condensation with an amino acid-based cationic amphiphile. A viable approach for liposome-based gene delivery. Mol. Membr. Biol. 25, 23–34. Rosen, M.J., 1988. Surfactants and Interfacial Phenomena, second ed. Wiley-Interscience Publication, New York65–67. Rosen, M.J., Fei, L., Zhu, Y.P., Morral, S.W., 1999. The relationship of the environmental effect of surfactants to their interfacial properties. J. Surfactant Deterg. 2, 343–347. Rostami, A., Hashemi, A., Takassi, M.A., Zadehnazari, A., 2017. Experimental assessment of lysine surfactant for enhanced oil recovery in carbonate rocks: mechanistic and core displacement analysis. J. Mol. Liq. 310–318. Roy, S., Dey, J., 2006. Self-organization properties and microstructures of sodium N-(11-acrylamidoundecanoyl)-Lvalinate and -L-threoninate in water. Bull. Chem. Soc. Jpn. 79, 59–66. Roy, S., Dey, J., 2007. Effect of hydrogen-bonding interactions on the self-assembly formation of sodium N-(11-acrylamidoundecanoyl)-L-serinate, L-asparaginate, and L-glutaminate in aqueous solution. J. Colloid Interface Sci. 307, 229–234. Seguer, J., Molinero, J., Manresa, A., Caelles, J., Infante, M.R., 1994. Physicochemical and antimicrobial properties of N-α-acyl-l-arginine dipeptides from acid-hydrolyzed collagen. J. Soc. Cosmet. Chem. 45, 53–63. Shirai, A., Maeda, T., Nagamune, H., Matsuki, H., Kaneshina, S., Kourai, H., 2005. Biological and physicochemical properties of Gemini quaternary ammonium compounds in which the position of a cross-linking sulfur in the spacer differ. Eur. J. Med. Chem. 40, 113–123. Sicchierolli, S.M., Mamizuka, E.M., Carmona-Ribeiro, A.M., 1995. Bacteria flocculation and death by cationic vesicles. In: Langmuir 11. pp. 2991–2995. Solans, C., Infante, M.R., Azemar, N., Warnheim, T., 1989. Phase behaviour of cationic lipoaminoacid surfactant systems. Progr. Colloid Polym. Sci. 79, 70–75. Solans, C., Pés, M.A., Azemar, N., Infante, M.R., 1990. Lipoaminoacid surfactants: phase behaviour of long Nα-acyl arginine methyl esters. Progr. Colloid Polym. Sci. 81, 144–150. Stilbs, P., 1987. Fourier transform pulsed-gradient spin-echo studies of molecular diffusion. Prog. Nucl. Magn. Reson. Spectrosc. 19, 1–45. Takehara, M., 1989. Properties and applications of amino-acid based surfactants. Colloids Surf. 38, 149–167. Tan, H., Xiao, H., 2008. Synthesis and antimicrobial properties of novel L-lysine Gemini surfactants pended with reactive groups. Tetrahedron Lett. 49, 1759–1761. Tausk, R.J.M., Karmiggelt, J., Oudshoorn, C., Overbeek, J.T.G., 1974. Physical chemical studies of short-chain lecithin homologues. I. Influence of the chain length of the fatty acid ester and of electrolytes on the critical micelle concentration. Biophys. Chem. 1, 175–183. Tavano, L., Infante, M.R., Abo-Riya, M., Pinazo, A., Vinardell, M.P., Mitjans, M., Manresa, M.A., Pérez, L., 2013. Role of aggregate size in the hemolytic and antimicrobial activity of colloidal solutions based on single and gemini surfactants from arginine. Soft Matter 9, 306–319.

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FURTHER READING

445

Tavano, L., Pinazo, A., Abo-Riya, M., Infante, M.R., Manresa, M.A., Muzzalupo, R., Pérez, L., 2014. Cationic vesicles based on biocompatible diacyl glycerol-arginine surfactants: physicochemical properties, antimicrobial activity, encapsulation efficiency and drug release. Colloids Surf. B: Biointerfaces 120, 160–167. Thorsteinsson, T., Masson, M., Kristinsson, K.G., Hjalmarsdottir, M.A., Hilmarsson, H., Loftsson, T., 2003. Soft antimicrobial agents: synthesis and activity of labile environmentally friendly long chain quaternary ammonium compounds. J. Med. Chem. 46, 4173–4181. Tsatsaroni, E., Pegiadou, S., Demertzis, G., 1987. Synthesis and properties of new cationic surfactants, II, odd homologous members. J. Am. Oil Chem. Soc. 64, 1444–1447. Urgell, J.B., Seguer, J., 2003. New preservative systems and their use in cosmetic preparations. International publication number: WO 03/013454 A1 Varka, E.M., Heli, M.G., Coutouli-Argyropoulou, E., Pegiadou, S.A., 2006. Synthesis and characterization of nonconventional surfactants of aromatic amino acid–glycerol ethers: effect of the amino acid moiety on the orientation and surface properties of these soap-type amphiphiles. Chem. Eur. J. 12, 8305–8311. Vijay, R., Angayarkanny, S., Bhasker, G., 2008. Amphiphilic dodecyl ester derivatives from aromatic amino acids: significance of chemical architecture in interfacial adsorption characteristics. Colloids Surf. A Physicochem. Eng. Asp. 317, 643–649. Weihs, D., Danino, D., Pinazo, A., Pérez, L., Franses, E.I., Talmon, Y., 2005. Self-aggregation in dimeric arginine-based cationic surfactants solutions. Colloids Surf. A Physicochem. Eng. Asp. 255, 73–78. Wright, G.D., 2016. Antibiotic adjuvants: rescuing antibiotics from resistance. Trends Microbiol. 24, 862–871. Xia, J., Nnanna, I.A., 2001. In: Nnanna, I.A., Xia, J. (Eds.), Protein-Based Surfactants. Synthesis, Physicochemical Properties, and Applications. Marcel Dekker, New York, pp. 1–14. Yamanaka, T., Ogihara, N., Ohhori, T., Hayashi, H., Muramatsu, T., 1997. Surface chemical properties of homologs and analogs of lysophosphatidylcholine and lysophosphatidylethanolamine in water. Chem. Phys. Lipids 90, 97–107. Yamashita, Y., Kunieda, H., Oshimura, E., et al., 2007. Formation of intermediate micellar phase between hexagonal and discontinuous cubic liquid crystals in brine/N-acylamino acid surfactant/N-acylamino acid oil system. J. Colloid Interface Sci. 312, 172–178. Yasuhara, K., Ohta, A., Asakura, Y., Kodama, T., Asakawa, T., Miyagishi, S., 2005. Unique incorporation behavior of amino acid-type surfactant into phospholipid vesicle membrane. J. Colloid Interface Sci. 283, 987–993. Zana, R., Xia, J. (Eds.), 2003. Gemini Surfactants: Synthesis, Interfacial and Solution-Phase Behavior, and applications. Surfactant Sciencevol. 117. Marcel Dekker, Inc., New York. Zana, R., Benrraou, M., Rueff, R., 1991. Alkanediyl-alpha, omega-bis(dimethylalkylammonium bromide) surfactants. 1. Effect of the spacer chain length on the critical micelle concentration and micelle ionization degree. Langmuir 7, 1072–1075. Zhang, X.M., Adachi, S., Watanabe, Y., Matsuno, R., 2005. Lipase-catalyzed synthesis of O-lauroyl L-serinamide and O-lauroyl L-threoninamide. Food Res. Int. 38, 297–300.

Further Reading Morán, M.C., Pinazo, A., Clapés, P., Infante, M.R., Pons, R., 2004c. Investigation of the thermotropic behavior of isomer mixtures of diacyl arginine-based surfactants. Comparison of polarized light microscopy, DSC, and SAXS observations. J. Phys. Chem. B 108, 11080–11088.

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C H A P T E R

14 Betaine Amphoteric Surfactants— Synthesis, Properties, and Applications Stephanie K. Clendennen, Neil W. Boaz Eastman Chemical Company, Kingsport, TN, United States

Abbreviations AEEA CAPB CAPHS CAS CBAHS CHOPSNA CIR CMC DCA DIMLA DMAE DMAP DMAPA EOR EPA EWG FDA GB GWP LB MCA SDS SLES SLS VCRP

aminoethylethanolamine cocamidopropyl betaine cocamidopropyl hydroxysultaine Chemical Abstract Service record number cocobutyramido hydroxysultaine 3-chloro-2-hydroxypropane sulfonate Cosmetic Ingredient Review critical micelle concentration dichloroacetate dimethyl laurylamine dimethylaminoethanol dimethylaminopropanol 3-dimethylaminopropylamine enhanced oil recovery US Environmental Protection Agency Environmental Working Group US Food and Drug Administration glycine betaine global warming potential lauryl betaine monochloroacetate sodium dodecyl sulfate sodium lauryl ether sulfate sodium lauryl sulfate Voluntary Cosmetic Registration Program

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14.1 INTRODUCTION There is an increasing need to incorporate renewable carbon responsibly in our everyday products. This is of urgent interest in consumer-facing industries such as personal and home care because consumers are becoming more educated about the impacts of carbon emissions and the persistence of chemicals in our environment. There is also significant regulatory and industrial interest in carbon footprinting, with sustainability goals being incorporated into many government and corporate missions. The tools to quantitatively assess environmental impacts of chemicals are becoming more widely applied to the ingredients that make up the products we use every day in our households. It’s generally held that bio-based content will reduce the environmental impact of a product, as biotic carbon uptake may be subtracted from the total carbon footprint. One obvious class of materials in consumer products that utilize a large fraction of biobased content is surfactants. The bio-based content in a surfactant is most typically the hydrophobic segment of the molecule, derived from tropical oils like coconut, palm, or palm-kernel oil, though bio-based hydrophiles based on sugars are growing. About 80% of all surfactants are based on an oleochemical hydrophobe, and the bio-based content does reduce the carbon footprint of the final surfactant. In 2017, a group composed of academic and industry partners published a comprehensive life cycle review of commercially important surfactants used in home care formulations (Schowanek et  al., 2017). The cradle-to-gate calculations focused on the comparative global warming potential (GWP) of a set of 15 commercially relevant surfactants and their precursors. When surfactants were compared that differed only in the source of the hydrophobe (synthetic vs bio-based), the use of a bio-based hydrophobe reduced the GWP of the final surfactant by 13%–50%. The source of the oleochemical hydrophobe made a difference in the GWP of the final surfactant; surfactants made using coconut oil products tended to have a negative total GWP, while palm and palm-kernel-derived products had a positive total GWP, reflecting a penalty related to land use change as animal habitat was converted to plantation acreage. Two amphoteric surfactants were included in the comparison, C8–C18 alkyl cocamidopropyl betaine (CAPB) and sodium cocoamphoacetate, not a true betaine but used in similar applications. The two amphoterics studied had similar GWP, between 2.4 and 2.5 metric tons of CO2 equivalents per metric ton of surfactant, comparable with other surfactant classes (Schowanek et al., 2017). While the bio-based component common to most betaine surfactants is an oleochemical hydrophobe precursor derived from tropical oils, the bio-based content for some surfactant types can be further increased by using additional bioderived intermediates. For example, alkyl hydroxysultaines are made using epichlorohydrin. When epichlorohydrin is made from glycerol, the overall renewable content of these amphoteric surfactants increases. Glycine betaine (GB) itself is typically derived from a natural source, sugar beets, and so, surfactants made from GB can take advantage of a higher bio-based content as well. Betaines are zwitterionic surfactants used in the personal and household care industries. The key functional groups in the chemical structure of betaines are the quaternized nitrogen and the carboxylic group. Betaines are characterized by a fully quaternized nitrogen atom and do not exhibit anionic properties in alkaline solutions, which means that betaines are present only as “zwitterions.” Betaines are uncharged at neutral pH but will exhibit a positive charge in extremely low pH conditions. This property makes betaines behave more like

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nonionic surfactants than ionic surfactants and allows them to be compatible in formulations with anionic surfactants in the pH range of most personal and home care formulations. Amphoteric (or zwitterionic) surfactants are used throughout the personal and household care industries. They are typically classified as specialty secondary or cosurfactants that complement the performance of the primary surfactants and are used in formulations that take advantage of their ability to stabilize foam and thicken the formulation using salt. Because so many surfactants are used in the home where they contact our skin, directly or indirectly, and contact surfaces used for food preparation, the safety of our personal and home care surfactants is also at the forefront of many consumers’ thoughts. Compared with most anionic surfactants and some nonionic surfactants, betaine surfactants are generally very mild to skin and mucous membranes. Amphoteric cosurfactants like betaines can also increase the mildness of the formulation by reducing irritation associated with purely ionic surfactants. This article will discuss betaine surfactants and several related amphoteric surfactants, both commercial and developmental, but is not meant to be comprehensive. Despite the large variety of amphoteric surfactants, in practice, only a small group of products is used because of factors such as cost, performance, availability of raw materials, and the complexity of the reactions involved. By volume, the most important groups of betaines today consist of alkyl amido betaines and to a lesser extent alkyl betaines, and these will be discussed in Section 14.2. A number of betaine or betaine-based surfactants have been proposed or characterized at the research level, but have not yet made a commercial impact, and a few of these will be discussed in Section 14.3. There are also betaine-like amphoteric surfactants that are often discussed alongside the betaines. Imidazoline derivatives and alkyl hydroxysultaines are two classes of betaine-like amphoterics that will be covered briefly in Section 14.4. Readers interested in more detail on the synthesis of commercial surfactants, including betaines, are referred to Zoller and Sosis (2008).

14.2  ALKYL BETAINES AND ALKYL AMIDO BETAINES Alkyl betaines and alkyl amido betaines will be discussed together in this section. The alkyl amido betaines are commercially the most important class of amphoteric surfactants in use today, specifically cocamidopropyl betaine, CAPB. CAPB is made at very large scale, and the economy of scale has lowered the cost of using CAPB to the extent that it has displaced other classes of amphoteric surfactants over time. CAPB growth has occurred over decades, and formulators like its performance as a foam booster and its favorable safety profile, especially in products for personal and home care. Alkyl betaines like lauryl betaine are still used in personal and home care for the same reasons but to a lesser extent. Based on the current routes and intermediates, there is no obvious opportunity to increase the bio-based content in alkyl betaines and alkyl amido betaines by substituting bio-based raw materials for either the linker or final reactant. The bio-based content is exclusive to the hydrophobic alkyl chain.

14.2.1  Synthesis of Alkyl Betaines and Alkyl Amido Betaines Alkyl and alkyl amido betaines differ primarily in the hydrophobe precursor and linker segments of the molecules. Both alkyl betaines and alkyl amido betaines are produced by

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multistep processes based on coconut or palm-kernel oil (Cowen and Bellis, 1965; Bade, 1985; Aigner, 1994; Domingo, 1996). Alkyl betaines rely on the use of a fatty alcohol, while alkyl amido betaines use a fatty acid or fatty acid ester. The surfactant precursor is a trialkyl amine or amido-trialkyl amine that is converted to the final betaine surfactant by reaction with sodium chloroacetate (SCA). The synthetic processes for alkyl betaines and alkyl amido betaines are summarized in Figs. 14.1 and 14.2, respectively. A bio-based hydrophobe is common to all commercially important amphoteric surfactants and is most often a bio-based alkyl chain derived from coconut, palm, or palm-kernel oil fatty acids. The bio-based hydrophobe used in most surfactants is an average of 12–14 carbons in length and can be based on a mixture of fatty acids (C8–C18) or may be fractionated to increase the proportion of C12 or C12 and C14 chain lengths. There is no branching in a biobased C12–C14 hydrophobe or in the trialkyl amine intermediate or in the surfactants made from it. Alkyl betaines are general purpose amphoteric surfactants used in a variety of applications, mostly as a secondary surfactant to boost foam. Lauryl betaine is shown in Fig. 14.1 where R is n-C12H25. For alkyl betaines, the oleochemical hydrophobe is a bio-based linear fatty alcohol produced via the hydrogenation of fatty acid methyl esters. In a typical process, tropical oils are subjected to (1) oil refining, bleaching and deodorizing, (2) methanolysis, (3) distillation of the fatty acid methyl esters, and (4) hydrogenation of fatty acid methyl ester to form fatty alcohols. As shown in Fig. 14.1, these bio-based fatty alcohols are then reacted with a secondary amine under reductive conditions to produce trialkylamines. A common trialkylamine hydrophobe is a dimethyl alkyl amine made from C12 and C14 fatty alcohols and dimethylamine, sometimes referred to as dimethyl laurylamine, or DIMLA. Lauryl betaine made from DIMLA is a mixture of lauryl betaine and myristyl betaine. Coco-betaine is made from a mixture of C8–C18 fatty alcohols reflective of the fatty acid composition of coconut oil.

FIG. 14.1  Synthesis of alkyl betaines (lauryl betaine, R = C12).

FIG. 14.2  Synthesis of amido betaines (cocamidopropyl betaine, CAPB, R = C7 to C17).

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For alkyl amido betaines, the hydrophobe is a blend of fatty acids from coconut oil, which are obtained from saponification (and sometimes hydrogenation) of the respective oil, and optionally distillation to fractionate the mixture. The most common alkyl amido betaine surfactant in use today is cocamidopropyl betaine (CAPB, Fig. 14.2), for which the hydrophobe is a natural mixture of fatty acids from coconut oil such that the alkyl chain length represented by the R group ranges from C7 to C17. For alkyl amido betaines, the reactive tertiary amine group is added to the hydrophobe precursor by way of an amidoamine linker molecule. The most common linker is 3-dimethylaminopropylamine, DMAPA, which is commercially produced via the reaction of dimethylamine with acrylonitrile to produce dimethylaminopropionitrile (DMAPN). Subsequent hydrogenation of DMAPN results in the desired DMAPA. To manufacture CAPB, the first step is the reaction of DMAPA with coconut fatty acids, with fatty acid methyl esters, or directly with coconut oil (fatty acid glycerol esters) (Fig. 14.2 depicting fatty acid reactants). The amidation reaction requires high temperatures (150– 175°C) for conversion and subsequent distillation to remove unreacted starting materials. These high reaction temperatures can generate by-products and impart color to the products, often requiring additional steps to remove the by-products and the color. Final step: The final alkyl and amidopropyl betaine surfactants are made by reacting the corresponding tertiary amine intermediate with monochloroacetic acid (MCA) to afford the zwitterionic surfactant. For the synthesis of betaines, MCA is used as the sodium salt, sodium chloroacetate (SCA), which is usually generated in situ by MCA neutralization with sodium hydroxide. Hydrolysis of SCA to sodium glycolate is a competing process, and the hydrochloric acid generated in the hydrolysis will protonate the amine reactant (rendering it unreactive) and thus limit the conversion to betaine. Thus, the reaction mixture must be kept basic (generally with sodium hydroxide) to afford high conversion. MCA is produced by the catalyzed chlorination of acetic acid with chlorine. Residual MCA can be removed from the final betaine surfactant by subsequent reaction with sodium metabisulfite (Seitz and Vybiral, 1994). MCA is always contaminated with a small amount of dichloroacetic acid (DCA), which is classified as a suspected carcinogen. DCA cannot be removed from the final aqueous surfactant solution, and it is very difficult to react away. Ultrahigh-purity MCA is required to produce betaines to minimize the amount of DCA in the final product. The reaction to form alkyl or alkyl amido betaine surfactants can be performed as a batch reaction or a continuous reaction. The presence of 1%–10% of the final surfactant in the reaction can help create a homogeneous reaction mixture between the hydrophobic surfactant intermediate and the aqueous base. The reaction temperature is moderate, 80–100°C for 2–8 h. The betaine solution will contain one molar equivalent of salt (sodium chloride, about 5% in the final product) and may contain residual MCA, residual amine, or glycolic acid (Lomel et al., 2014). The alkyl amido betaines like CAPB may also contain unreacted linker, DMAPA. When the triglyceride oil is used as the hydrophobe precursor for CAPB, glycerol and glycerol esters will be present in the final surfactant mixture. Salt content also varies and is one way that grades of betaine are distinguished—with low-salt preparations preferred due to their greater ease of formulation with less variability in formulation viscosity. The final reaction with SCA to form alkyl and alkyl amido betaines is performed in water, and the final surfactant is sold as a 30%–40% active solution in water, which can be stabilized

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14.  Betaine Amphoteric Surfactants—Synthesis, Properties, and Applications

or preserved as required. Alkyl betaines of different alkyl chain lengths are available. Lauryl betaine (CAS 683-10-3) and oleyl betaine (CAS 871-37-4) are commercially available alkyl betaines. Alkyl betaines made using natural or fractionated blend of fatty alcohols are also used. Coco-betaine, C10–C16 alkyl betaine (CAS 66455-29-6), is made from coconut fatty alcohols with a mix of alkyl chain length and is often confused with CAPB, cocamidopropyl betaine, but they are two distinct ingredients. Alkyl betaines are available from several suppliers under different trade names (e.g., Macat CB, Macat LB, and Macat OB (Pilot); Mackam CB-35 coco-betaine and Mackam LB-35 lauryl betaine (Solvay); Ampholak MSK-2 and Amphoteen 24 (AkzoNobel); Empicol XCT 10 and Empigen BB; and BB/HP (Huntsman)). CAPB and lauramidopropyl betaine are the most common commercially available alkyl amido betaines. CAPB (CAS 61789-40-0) is available from many suppliers under different trade names (e.g., Crodateric CAB 30, Dehyton K, Genagen CAB, TEGO Betain, Chembetaine, and OXITAINE CP 30). Also available, though not as widely used as CAPB, is lauramidopropyl betaine (CAS 4292-10-8, e.g., Amphitol 20AB, Cola Teric LMB, Anfodac LB, and Mackam LMB).

14.2.2  Properties of Alkyl Betaines and Alkyl Amido Betaines Amphoteric surfactants like betaines offer good detergency coupled with high foaming capacity and mildness to the skin. Most alkyl betaines and alkyl amido betaines have good solubility in water for formulating. Recall that the final step in their synthesis is performed in water and they are typically sold as 30%–40% solutions in water. Betaines may function as cationic surfactants at extremely low pH and may form precipitates with anionic surfactants but are unaffected by hard water. The hydrolytic stability of alkyl betaines tends to be higher than alkyl amido betaines, especially under extreme conditions of pH and temperature. The addition of alkyl amido betaines like CAPB to formulations containing anionic surfactants—especially sodium lauryl ether sulfate, SLES—increases the viscosity of the formulation, and the extent of viscosity increase depends greatly on the salt concentration. This phenomenon is called salt thickening and is used to create thick liquids that cling to surfaces like the hands, hair, sponges, and dishes. The salt-thickened formulations also tend to shear thin, and so, they are readily dispensed from a small-diameter opening of a squeeze bottle or pump. Historically, fatty acid alkanolamides were the principal secondary surfactants used to salt-thicken formulations containing SLES. Now, amphoterics including CAPB are used because of their excellent skin compatibility. No matter what the final applications may be, alkyl and alkyl amido betaines are often added to formulations principally to help increase or stabilize foam. Betaines are attractive as foaming agents because their foam is relatively resistant to the effects of hard water, unlike anionic surfactants. Betaines also foam even at extremes of pH, again, in contrast to anionics that tend to foam poorly at high pH. CAPB is one of the few amphoterics that show an increase in foam density and stability in hard water (Castan et al., 1997). Interestingly, foam stability of CAPB and lauryl betaine was greatly enhanced at low temperature (20°C) versus high temperature (60°C). This difference has implications in home care applications like dishwashing liquids and laundry liquids for which water temperature varies in use.

III.  BIOBASED SURFACTANTS



14.2  Alkyl Betaines and Alkyl Amido Betaines

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Alkyl and alkyl amido betaines show synergy with anionic surfactants as foam boosters by increasing the surfactant packing density and stabilizing the interface. Specifically studied for lauryl betaine (LB), it has been suggested that the zwitterionic surfactant helps screen the charge carried by the anionic surfactant at the air interface, resulting in closer packing of surfactants. The closer packing would increase the charge density and the resulting electrostatic repulsion, preventing film coalescence. Closer packing of surfactant molecules at the interface also increases the surface shear viscosity, leading to better foam stability. Molecular dynamics simulations support both the hypothesis of a specific synergistic interaction between the head groups of LB and a linear alpha olefin anionic surfactant (AOS-14) as well as tighter packing (Wang et al., 2017). Similarly, CAPB in combination with the anionic surfactant sodium dodecyl sulfate (SDS) showed excellent foam stability, even in the presence of oils (Osei-Bonsua et al., 2015). High viscosity of the film surface in the mixed surfactant system slowed liquid drainage and increased resistance to film thinning. Thicker films were less susceptible to interbubble diffusion or coalescence. As secondary surfactants in personal care formulations, amphoterics are often paired with an anionic primary surfactant, such as sodium lauryl sulfate or sodium lauryl ether sulfate. Betaine amphoterics can form complexes with and thus reduce the irritation potential of anionic surfactants. The mildness makes amphoterics particularly valuable in personal care products. Lauryl betaine was found to facilitate the penetration of model compounds in the skin of hairless mice, as well as penetrating the skin itself (Ridout et al., 1991). Lauryl betaine was recommended for further study as an adjuvant to increase dermal penetration of pharmaceuticals. Alkyl betaines have also been shown to have antimicrobial activity (Lindstedt et al., 1990; Birnie et al., 2000). Biodegradation and aquatic toxicity are important safety considerations for betaine surfactants, as in most applications, these surfactants will find their way into the environment. Aerobic biodegradation testing (ISO 14593, 1999) indicated that alkyl betaines are all readily biodegradable (at least 60% within 28 days) under aerobic conditions, with no obvious effect of alkyl chain length. Under the same conditions, CAPB showed both a high degree of biodegradation (100% after 28 days) and a high rate of mineralization, reaching the required 60% biodegradation in