Biosurfactants: Research Trends and Applications

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5 Biosurfactants and Bioemulsifiers from Marine Sources

Rengathavasi Thavasi and Ibrahim M. Banat

INTRODUCTION

Marine microbes are known for their many novel extra- and intracellular products such as antibiotics, enzymes, biopolymers, pigments, and toxins. Reports suggest that so far more than 10,000 metabolites with broad-spectrum biological activities and interest-ing medicinal properties have been isolated from marine microbes (Kelecom, 2002). However, due to the enormity of the marine biosphere, most of the marine microbial worlds remain unexplored. It has been estimated that <0.1% of marine microbial world has been explored or investigated (Ramaiah, 2005). Among various marine bioactive compounds, microbial biosurfactants (BSs) are of great importance due to their struc-tural and functional diversity and industrial applications (Banat et al., 1991; Banat, 1995a,b; Rodrigues et al., 2006a). Marine microbial BSs are such metabolites with many interesting properties. BSs are basically amphiphilic surface active agents pro-duced by bacteria, fungi, and actinomycetes. They belong to various classes including glycolipids, glycolipoproteins, glycopeptides, lipopeptides, lipoproteins, fatty acids, phospholipids, neutral lipids, lipopolysaccharides (Banat et al., 2010), and glycoglyc-erolipids (Wicke et al., 2000). The properties/applications of BSs include detergency, emulsification, foaming, dispersion, wetting, penetrating, thickening, microbial growth enhancement (e.g., oil-degrading bacteria), antimicrobial agents, metal sequestering, and resource recovering (oil recovery). These interesting properties allow BSs to have the ability to replace some of the most versatile chemical surfactants that are now in practice. In addition, BSs are promising natural surfactants that offer several advantages over chemically synthesized surfactants, such as in situ production using

CONTENTS

Introduction ............................................................................................................ 125Marine Biosurfactants ............................................................................................ 126Areas of Potential Applications of Marine Biosurfactants .................................... 134Conclusion ............................................................................................................. 139Acknowledgment ................................................................................................... 140References .............................................................................................................. 140

126 Biosurfactants: Research Trends and Applications


renewable substrates, lower toxicity, biodegradability, and ecological compatibility (Marchant and Banat, 2012a,b). Interest in BS research has been on the increase during the past two decades due to their interesting properties, yet the reason behind the pro-duction of BSs by many microorganisms remains mostly unknown. Several proposed physiological roles of BSs have been put forward including (1) increasing the surface area and bioavailability of hydrophobic water-insoluble substrates (e.g., oil-degrading microbes) (Ron and Rosenberg, 1999), (2) bacterial pathogenesis and quorum sensing and biofilm formation (e.g., Pseudomonas aeruginosa) (Davey et al., 2003), (3) antimi-crobial for self-defense (e.g., antimicrobial activity of rhamnolipids) (Stanghellini and Miller, 1997), and (4) cell proliferation in the producing bacteria (e.g., viscosinamide production by P. fluorescens) (Nielsen et al., 1999). To isolate BS-producing microbes, combination of various screening methodologies has been studied (Maneerat and Phetrong, 2007; Satpute et al., 2008; Thavasi et al., 2011c) and extensively reviewed (Nerurkar et al., 2009; Satpute et al., 2010). Microbial communities like Acinetobacter, Arthrobacter, Pseudomonas, Halomonas, Bacillus, Rhodococcus, Enterobacter, Azotobacter, Corynebacterium, Lactobacillus, and yeast have been reported to pro-duce BSs (Schulz et al., 1991; Passeri et al., 1992; Banat, 1993; Abraham et al., 1998; Maneerat et al., 2006; Thavasi et al., 2007, 2009, 2011a; Das et al., 2008a,b; Perfumo et al., 2010a). This chapter collates and highlights data search on isolation, culture methods, and potential applications for BSs from marine microbes.

MARINE BIOSURFACTANTS

Biosurfactants from marine microbes: A detailed list of BSs from marine microbes is described (Table 5.1) and a graphic representation of the number of publications and their percentages are illustrated in Figures 5.1 through 5.4. Earlier reports on BSs of marine origin mainly focused on environmental remediation applications of BSs such as emulsifiers and dispersants or BSs from oil-degrading microbes (Rosenberg et al., 1979; Schulz et al., 1991; Yakimov et al., 1998; Thavasi and Jayalakshmi, 2003, Thavasi et al., 2006, 2009, 2008, 2011a; Peng et al., 2007, 2008) and other potential applications such as medical and industrial sectors have not been studied extensively (Rodrigues et al., 2006b; Marchant and Banat, 2012a,b). Recently the trend has changed, and scientists started focusing on other potential application/properties of marine BSs such as antimicrobial (Mukherjee et al., 2009), biofilm disruption (Kiran et al., 2010a), and nanoparticle synthesis (Kiran et al., 2010b). A detailed description of marine microbial surfactants, their composition, and pro-ducing organism are described in the following sections.

Glycolipids and glycoglycerolipids: Glycolipids are the most studied surfactants among all other marine microbial BSs, that is, 48% of the publications on marine BSs are about glycolipid BSs (Figure 5.1). Glycolipid BSs include glycolipids, glucose lipid, trehalose lipids, trehalose tetraester, trehalose corynomycolates, rhamnolipids, mannosylerythritol lipids, sophorolipids, and extracellular polysaccharide-lipids (ESLs). Glycolipids are composed of a hydrophobic fatty acid moiety esterified to a hydrophilic carbohydrate moiety. The sugar components of the glycolipids may be one or two molecules of glucose, trehalose, mannose, sophorose, or rhamnose.

127Biosurfactants and Bioemulsifiers from Marine Sources


TABLE 5.1Types of Biosurfactants and Microbial Strains

Biosurfactant Type Microorganism Reference

Glycolipid Bacterial strain MM1 Passeri et al. (1992)

Nocardioides sp. Vasileva-Tonkawa and Gesheva (2005)

Aeromonas sp. Ilori et al. (2005)

Halomonas sp. ANT-3b Pepi et al. (2005)

Azotobacter chroococcum Thavasi et al. (2006)

Pseudomonas aeruginosa Thaniyavarn et al. (2006)

Pantoea sp. Vasileva-Tonkawa and Gesheva (2007)

Rhodococcus erythropolis Peng et al. (2007)

Bacillus megaterium Thavasi et al. (2008)

Brevibacterium casei Krian et al. (2010a)

Nocardiopsis lucentensis MSA04 Kiran et al. (2010d)

Bacillus pumilus

Serratia marcescens Dusane et al. (2011)

Lactobacillus delbrueckii Thavasi et al. (2011a)

Streptomyces sp. B3 Khopade et al. (2012)

Glycolipid and phospholipids Alcanivorax borkumensis Abraham et al. (1998)

Trehalose lipids Rhodococcus fascians DSM 20669 Yakimov et al. (1999)

Trehalose tetraester Arthrobacter sp. EK 1 Schulz et al. (1991)

Passeri et al. (1991)

Trehalose corynomycolates Arthrobacter sp. SI 1 Schulz et al. (1991)

Glucose lipid Alcaligenes sp. MM 1 Schulz et al. (1991)

Unidentified bacterial strain MM 1 Passeri et al. (1992)

Alcanivorax borkumensis gen. nov., sp. nov.

Yakimov et al. (1998)

Rhamnolipids Aspergillus sp. MSF1 Kiran et al. (2010c)

Mannosylerythritol lipids Pseudozyma hubeiensis Konishi (2010)

Glucoglycerolipids Microbacterium sp. DSM 12583 Wicke et al. (2000)

Bacillus pumilus strain AAS3 Ramm et al. (2004)

Micrococcus luteus Palme et al. (2010)

Emulsan Acinetobacter calcoaceticus RAG-1 Reisfeld et al. (1972)

Extracellular polysaccharide-lipid

Alcaligenes sp. PHY 9L-86 Goutx et al. (1987)

Sulfated heteropolysaccharide Halomonas eurithalina Calvo et al. (1998)

Extracellular polysaccharide Pseudomonas putida ML2 Bonilla et al. (2005)

Planococcus matriensis Anita I Kumar et al. (2007)

Glycolipopeptide Yarrowia lipolytica NCIM3589 Zinjarde et al. (1997)

Yarrowia lipolytica Amaral et al. (2006)

Corynebacterium kutscheri Thavasi et al. (2007)

Glycolipoprotein Pseudomonas nautica Husain et al. (1997)

Aspergillus ustus MSF3 Kiran et al. (2009)

(continued)



31

1

1

2

1

1

15

3

GlycoglycerolipidsMannosylerythritol lipids

GlycolipidGlycolipid andphospholipidsTrehalose lipidsTrehalose tetraesterTrehalosecorynomycolatesGlucose lipidRhamnolipids

Number of publications (28, 48%) on marineglycolipid biosurfactants

FIGURE 5.1 (See color insert.) Breakdown compilation of publication on marine glyco-lipid biosurfactants.

TABLE 5.1 (continued)Types of Biosurfactants and Microbial Strains

Biosurfactant Type Microorganism Reference

Trehaloselipid-o-dialkyl monoglycerides–protein

Pseudomonas fluorescence Desai et al. (1988)

Alasan Acinetobacter radioresistens Navon-Venezia et al. (1995)

Biodispersion Acinetobacter calcoaceticus Rosenberg and Ron (1998)

Carbohydrate–protein complex

Rhodotorula glutinis Oloke and Glick (2005)

Glycoprotein Antarctobacter sp. TG22 Gutiérrez et al. (2007a)

Halomonas sp. TG39 and 67 Gutiérrez et al. (2007b)

Lipopeptide Bacillus licheniformis BAS50 Yakimov et al. (1995)

Bacillus pumilus KMM150 Kalinovskaya et al. (1995)

Pseudomonas sp. MK90e85 and MK91CC8

Gerard et al. (1997)

Bacillus pumilus1364 Kalinovskaya et al. (2002)

Rhodococcus sp. TW53 Peng et al. (2008)

Nocardiopsis alba MSA10 Gandhimathi et al. (2009)

Azotobacter chroococcum Thavasi et al. (2009)

Brevibacterium aureum MSA13 Kiran et al. (2010b)

Pseudomonas aeruginosa Thavasi et al. (2011d)

Bacillus circulans DMS-2 Sivapathasekaran et al. (2011)

Ornithine lipids Myroides sp. SM1 Maneerat et al. (2006)

Surfactin Bacillus velezensis H3 Liu et al. (2010)

Proline lipid Alcanivorax dieselolei B-5 Qiao and Shao (2010)



Number of publications (11, 19%) on marine glycolipopeptideand glycolipoprotein biosurfactats

11

1

1

2 2

3Glycolipopeptide

Glycolipoprotein

Glycoprotein

Trehaloselipid-o-dialkylmonoglycerides–proteinAlasan

Biodispersion

Carbohydrate–proteincomplex

FIGURE 5.3 (See color insert.) Breakdown compilation of publication on marine glycoli-popeptide, glycolipoprotein, and glycoprotein biosurfactants.

Number of publications on (13, 22%) marine lipopeptide andlipoprotein biosurfactants

Lipopeptide

Surfactin

Proline lipid

Ornithine lipids

11

1

10

FIGURE 5.4 (See color insert.) Breakdown compilation of publication on marine lipopep-tide and lipoprotein biosurfactants.

Number of publications (6, 10%) on marine extracellularpolysaccharide and polysaccharide lipid biosurfactants

Extracellularpolysaccharide-lipid

Extracellularpolysaccharide

Emulsan

1

1 1

3

Sulfatedheteropolysaccharides

FIGURE 5.2 (See color insert.) Breakdown compilation of publication on marine extracel-lular polysaccharide and polysaccharide lipid biosurfactants.



The fatty acid moiety comprises one or two fatty acids with a chain length of C6–C20 with or without unsaturation depending on the source of the fatty acid and the microbial enzyme involved in processing the fatty acid part of the BS. Glycolipid-producing microbes were isolated from different marine sources such as Halomonas sp. ANT-3b isolated from Ross Sea, Antarctica (Pepi et al., 2005), which produced a glycolipid BS with a molecular weight of 18 kDa using n-hexadecane as the sole carbon source. P. aeruginosa A41 isolated from the gulf of Thailand was capable of utilizing wide range of carbon sources (oil, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid) and produced 6.58 g/L rhamnolip-ids with olive oil as the carbon substrate (Thaniyavarn et al., 2006). Azotobacter chroococcum, Bacillus megaterium, and Lactobacillus delbrueckii isolated from Tuticorin coastal waters (Tamil Nadu, India) using crude oil as the carbon source produced glycolipid BSs on crude oil, waste motor oil, and peanut oil cake carbon sources (Thavasi et al., 2006, 2008, 2011a). The highest BS production was observed with peanut oil cake as 4.6, 1.4, and 5.3 g/L, respectively, and was able to emulsify a wide range of hydrocarbons and vegetable oil in the order of waste motor lubricant oil > crude oil > peanut oil > kerosene > diesel > xylene > anthracene > naphthalene.

Among the glycolipid BSs, trehalose lipids, trehalose corynomycolates, and treha-lose tetraester are the second largest group reported (9%). Trehalose lipid production by an Antarctic Rhodococcus fascians DSM 20669 strain was reported by Yakimov et al. (1999), and the surfactant reduced the surface tension of water from 72 to 32 mN/m. Trehalose tetraester production was found with n-alkanes utilizing marine bacterium, Arthrobacter sp. EK1 (Passeri et al., 1991). The main fraction of the purified surfactant is an anionic 2,3,4,2′-trehalose tetraester. The chain lengths of fatty acids ranged from 8 to 14. Trehalose corynomycolates producing marine Arthrobacter sp. SI1 was iso-lated from North Sea, and it produced 2 g/L of BS. The isolated BSs showed significant interfacial and emulsifying properties (Schulz et al., 1991).

A marine sponge associated Aspergillus sp. MSF1 strain producing rhamnolip-ids was also isolated from the Bay of Bengal coast of India (Kiran et al., 2010c). Rhamnolipid BS isolated from Aspergillus sp. MSF1 showed potential activity against pathogenic yeast, Candida albicans and bacteria, Streptococcus sp., Micrococcus luteus, and Enterococcus faecalis. Konishi et al. (2010) recently isolated a mannosyl-erythritol lipid-producing Pseudozyma hubeiensis from a deep sea clam Calyptogena soyoae at 1156 m in Sagami Bay. The major component of the BS was MEL-C (4-o-[4′-o-acetyl-2′,3′-di-o-alka(e)noil-β-d-mannopyranosyl]-d-erythritol). Analysis of the lipid part of the BS revealed that major fatty acids of MEL-C were saturated fatty acids with chain lengths of C6, C10, and C12. The surface tension determination of the MEL-C showed a critical micelle concentration (CMC) of 1.1 × 10−5 M.

Another class of BSs that are not in the spotlight of general research is glycoglycerolipids. There are three reports available on glycoglycerolipids from marine bacterial strains. The marine bacterium Microbacterium sp. DSM 12583, isolated from the Mediterranean sponge Halichondria panicea, was able to form a glucosylmannosyl-glycerolipid (GGL 2), 1-o-acyl-3-[α-glucopyranosyl-(1–3)-(6-o-acyl-α-mannopyranosyl)]glycerol, when grown on a complex medium with glycerol (Wicke et al., 2000). Another marine strain, M. luteus was isolated from the North sea and reported to produce a dimannosyl-glycerolipid (GGL 5),



mannopyranosyl(1α-3)-6-acylman-nopyranosyl(1α-1)-3-acylglycerol on arti-ficial seawater supplemented with glucose, yeast extract, peptone, and nitrogen/phosphate sources (Palme et al., 2010). The third marine bacterium, B. pumilus strain AAS3 isolated from the Mediterranean sponge Acanthella acuta and synthesized a diglucosyl-glycerolipid (GGL 11), 1,2-o-diacyl-3-[β-glucopyranosyl-(1–6)-β-glucopyranosyl)]glycerol, when grown on artificial medium provided with glucose, yeast extract, peptone, and nitrogen/phosphate sources (Ramm et al., 2004).

Extracellular polysaccharide-lipids (ESL) and polysaccharides (PS): ESLs and PSs are another class of BSs produced by marine microbes, and reports on ESLs and PSs contribute 10% of total publications on marine BSs (Figure 5.2). Acinetobacter calcoaceticus RAG-1 strain (formally known as Arthrobacter RAG-1) was isolated from tar balls collected in beach soil (Rosenberg et al., 1979). RAG-1 strain produced an emulsifier called emulsan, an extracellular polyanionic amphipathic heteropolysac-charide. Its amphipathic and polymeric characteristics provide it with emulsifying as well as stabilizing activity of oil/water systems. The bioemulsifier exhibits specificity toward its hydrocarbon emulsifiable substrates. It tends to concentrate at the oil/water interface of the hydrocarbon droplets preventing their coalescence and allowing the formation of concentrated emulsanosols, which were found to be useful in enhancing oil degradation, binding heavy metal cations or constituting a low-viscosity stable oil-in-water emulsion suitable for oil pipeline transportation or direct combustion. Emulsan was also found to protect its producing cells against the toxic cationic deter-gent cetyltrimethylammonium bromide (Shabtai and Gutnick, 1986).

Another marine bacterium Alcaligenes sp. PHY 9 L-86 was isolated from hydro-carbon-polluted sea-surface waters that produced surface active exopolysaccharides (extracellular carbohydrates) and lipids on a medium containing 0.1% tetradecane. Chemical analysis revealed the composition of the extracellular lipids as phospholip-ids, free fatty acids, triglycerides, monoglycerides, esters, and free fatty acids (73%) as the major constituent (Goutx et al., 1987). A PS BS-producing P. putida ML2 strain was also isolated from hydrocarbon-polluted sediment collected at the Montevideo bay (Uruguay). Chemical composition of the PS BS revealed its sugar composition as rhamnose, glucose, and glucosamine in a 3:2:1 molar ratio with a molecular weight of 10–80 kDa. Another PS surfactant-producing marine Planococcus strain called P. maitriensis Anita I was isolated from coastal area of Gujarat, India (Kumar et al., 2007). The extracellular polymeric substance produced had a chemical composition of carbohydrate (12.06%), protein (24.44%), uronic acid (11%), and sulfate (3.03%) and effectively emulsified xylene and formed stable emulsions with jatropha, paraffin, and silicone oils. Its cell-free supernatant reduced the surface tension of water from 72 to 46.07 mN/m.

Glycolipopeptides, Glycolipoproteins, and Glycoproteins (GLPs): Glycolipopeptide and glycolipoprotein BSs are made of sugar–lipid–peptides or amino acids. GLPs are the third most studied surfactants among marine BSs, and reports on GLPs con-tribute 19% of total publications (Figure 5.3). Corynebacterium kutscheri, a marine bacterium isolated by Thavasi et al. (2007) from Tuticorin harbor, India, produced a glycolipopeptide BS. The BS was composed of carbohydrate (40%), lipid (27%), and protein (29%) and was able to emulsify waste motor lubricant oil, crude oil,



peanut oil, kerosene, diesel, xylene, naphthalene, and anthracene. Its emulsification activity was comparatively higher than the activity found with Triton x-100.

Amaral et al. (2006) isolated a yeast strain, Yarrowia lipolytica, from Guanabara Bay in Brazil, which produced a glycolipopeptide BS called Yansan in glucose-based medium. It was reported to contain 15% protein and 1% lipid. The fatty acid compo-sition of the lipid was palmitic acid (35.8%), stearic acid (21.4%), lauric acid (8.8%), and oleic acid (6.9%). However, there was no quantitative information available on its sugar content, but its monosaccharide composition was reported as arabinose, galactose, glucose, and mannose in a ratio of 1:6:17:31. The molecular weight of the BS was approximately 20 kDa with a CMC value of 0.5 g/L. Another marine yeast strains called Yarrowia lipolytica NCIM 3589 isolated by Zinjarde et al. (1997) pro-duced an emulsifier with a chemical composition of 75% lipid, 20% carbohydrate, and 5% protein. The lipid and carbohydrate part of the emulsifier comprised palmitic acid, mannose, and galactose, respectively, while the main amino acid components were aspartic acid, alanine, and threonine.

GLPs are another set of BSs produced by marine microbes composed of sugar and protein complexes. Gutiérrez et al. (2007a) reported the GLP bioemulsifier production by a marine bacterium, Antarctobacter sp. TG22, in a low-nutrient seawater medium supplemented with glucose. Chemical, chromatographic, and nuclear magnetic reso-nance spectroscopic analysis confirmed that the bioemulsifier has a high-molecular-weight (>2000 kDa) GLP with high uronic acid contents. The carbohydrate content of this emulsifier was reported as 15.4% ± 0.2%; further analysis of the carbohydrate com-ponent revealed the presence of hexoses (rhamnose, fructose, galactose, glucose, and mannose), amino sugars (galactosamine, glucosamine, and muramic acid), and uronic acids (galacturonic and glucuronic acids). The amino acid component was 5.0% ± 0.2% and mainly contained the three major amino acids, aspartic acid, glycine, and alanine.

Gutiérrez et al. (2007b) reported GLP BS production by two Halomonas species, TG39 and TG67. These strains produced two different GLP emulsifiers, called HE39 (strain TG39) and HE67 (strain TG67). The total carbohydrate content of HE39 and HE67 was 17.3% ± 1.0% and 22.7% ± 0.8%, respectively. The major monosaccharides identified in HE39 were rhamnose (31.7% ± 2.1%), glucuronic acid (27.9% ± 1.9%), and galactose (15.3% ± 0.5%). Carbohydrate content of HE67 showed that the main monosaccharides detected were glucuronic acid (58.8% ± 0.4%), glucosamine (10.9% ± 0.1%), and mannose (11.5% ± 0.5%), while rhamnose, galactose, galactos-amine, glucose, muramic acid, and galacturonic acid, each of them present at less than 10% of the total monosaccharide composition and together they contributed only 18% to the total carbohydrate content. The total amino acid content of HE39 and HE67 was 26.6% ± 1.0% and 40.5% ± 1.6%, respectively. Amino acid analysis of hydrolyzed surfactant identified the presence of four major amino acids in both emul-sifiers as aspartic acid, glutamic acid, glycine, and alanine, which in total contributed 45.1% and 50.7% to the total amino acid content of HE39 and HE67, respectively.

Lipopeptides: Lipopeptides are the second well-studied BS group from marine microorganisms that accounts for 22% of the total publications on marine BSs of which the genus Bacillus alone contributes 38% (Figure 5.4). Lipopeptide BSs are made of hydrophilic peptide head group and a hydrophobic lipid tail. A marine



bacterium, Azotobacter chroococcum (Thavasi et al., 2009) isolated from seawater, grew and produced a lipopeptide BS in a medium provided with crude oil, waste motor lubricant oil, and peanut oil cake. Peanut oil cake gave the highest BS pro-duction (4.6 mg/mL). The BS product emulsified waste motor lubricant oil, crude oil, diesel, kerosene, naphthalene, anthracene, and xylene. Preliminary character-ization using biochemical, Fourier transform infrared spectroscopy, and LC-MS analysis indicated that the BS was a lipopeptide with percentage lipid and protein proportion of 31.3:68.7. Sivapathasekaran et al. (2011) isolated a marine lipopeptide BS-producing B. circulans strain from seawater sample from Andaman and Nicobar Islands, India. Similar marine Bacillus strains, B. pumilus KMM150 isolated from an Australian marine sponge Ircinia sp. (Kalinovskaya et al., 1995) and B. pumilus KMM1364 isolated from the surface of the ascidian Halocynthia aurantium (Kalinovskaya et al., 2002), were also reported to produce lipopeptide BSs. Strain KMM150 produced a mixture of cyclic depsipeptides known as bacircines (1–5 frac-tions with different molecular weights). Bacircines 1, 2, and 3 had molecular masses of 1007, 1021, and 1021, respectively, and bacircines 4 and 5 had a molecular mass of 1035. The amino acid composition of the bacircines 1, 2, 3, 4, and 5 were simi-lar with a combination of Leu:Val:Asp:Glu with a ratio of 4:1:1:1. Bacircines 1, 2, and 3 has C13–C14–hydroxyacids in their lipid part whereas bacircines 4 and 5 had 3β-hydroxypentadecanoic acid (C15-β-hydroxyacid). B. pumilus KMM1364 also produced a mixture of lipopeptide analogs with major components with molecular masses of 1035, 1049, 1063, and 1077. The variation in molecular weight represents changes in the number of methylene groups in the lipid and/or peptide portions of the surfactant. Structurally, these lipopeptides differ from surfactin in the substitu-tion of the valine residue in position 4 by leucine and have been isolated as two carboxy-terminal variants, with valine or isoleucine in position 7. The lipid part of the surfactant is composed of β-hydroxy-C15-, β-hydroxy-C16-, and a high amount of β-hydroxy-C17 fatty acids. Liu et al. (2010) reported a marine B. velezensis strain producing nC14-surfactin and anteisoC15-surfactin. These compounds can reduce the surface tension of phosphate-buffered saline (PBS) from 71.8 to 24.8 mN/m. The CMCs of C14-surfactin and C15-surfactin in 0.1 M PBS (pH 8.0) were determined to be 3.06 × 10−5 and 2.03 × 10−5 M, respectively. The surface tension values at CMCs for C14-surfactin and C15-surfactin were 25.7 and 27.0 mM/m, respectively.

Kiran et al. (2010e) isolated a marine sponge (Dendrilla nigra)-associated Brevibacterium aureum MSA13 strain producing lipopeptide BS. The peptide part of the surfactant was made of short sequence of four amino acids including pro–leu–gly–gly coupled with a lipid moiety composed of octadecanoic acid methylester. Oil-degrading lipopeptide BS-producing Rhodococcus sp. was isolated from Pacific Ocean deep-sea sediments (Peng et al., 2008). The hydrophobic lipid part of the surfactant contained five types of fatty acids with chain lengths of C14–C19 and C16H32O2 as a major com-ponent making up 59.18% of the total. The hydrophilic fraction was composed of five kinds of amino acids with a sequence of Ala–Ile–Asp–Met–Pro.

Two strains of marine Pseudomonas, namely, MK90e85 and MK91CC8, pro-duced antimycobacterial cyclic depsipeptides and viscosin (Gerard et al., 1997). The MK90e85 strain isolated from a red alga produced massetolides A, B, C, and D and the other strain MK91CC8 isolated from a marine tubeworm produced massetolides



E, F, G, H, and viscosin. Massetolide A is an optically active molecule, with a mass of 1141 Da and a molecular formula of C55H97N9O16. Another marine Pseudomonas strain called P. aeruginosa producing lipopeptide BS was also isolated from coastal waters of Tamil Nadu, India (Thavasi et al., 2011d). The BS is composed of protein (50.2%) and lipid (49.8%), and at 1 mg/mL concentration, the BS was able to emulsify waste motor lubricant oil, crude oil, peanut oil, kerosene, diesel, xylene, naphthalene, and anthracene, and the emulsification activity was higher than that achieved by Triton x-100 at similar concentration. A marine bacterium, Myroides sp. SM1 capable of pro-ducing ornithine lipid bioemulsifier was isolated from seawater (Maneerat et al., 2006; Maneerat and Dikit, 2007). l-Ornithine lipids were composed of l-ornithine and two different iso-3-hydroxyfatty acid (C15–C17) and iso-fatty acid (C15 or C16) in a ratio of 1:1:1. Ornithine lipids exhibited emulsifying activity with crude oil in a broad range of pH, temperature, and salinity and showed high oil displacement activity.

AREAS OF POTENTIAL APPLICATIONS OF MARINE BIOSURFACTANTS

Biosurfactants and hydrocarbon degradation/remediation: Oil pollution in terrestrial and aquatic environments is a common phenomenon that causes significant ecologi-cal and social problems. The recent British Petroleum deepwater horizon oil spill at the Gulf of Mexico during the summer of 2010 is a poignant example. The traditional available treatment processes used to decontaminate polluted areas have on the main been limited in their application (Perfumo et al., 2010b). Physical collection methods such as booms, skimmers, and adsorbents typically recover no more than 10%–15% of the spilled oil, and the use of chemical surfactants as remediating agents is not favored due to their toxic effects on the existing biota in the polluted area. Therefore, despite decades of research, successful bioremediation of oil contaminated environ-ment remains a challenge (Perfumo et al., 2010a).

Oil pollution in marine environments stimulates the indigenous community of obligate hydrocarbonoclastic bacteria to flourish, becoming the majority of the total microbial population (Yakimov et al., 2007). Microorganisms involved in oil degrada-tion have adopted different strategies to enhance the bioavailability and gain access to hydrophobic compounds, such as hydrocarbons, including (1) BS-mediated solubili-zation, (2) direct access of oil drops, and (3) biofilm-mediated access (Hommel, 1990). The production of BSs and bioemulsifiers is generally involved in varying degrees, in all the strategies provided earlier. BS structural uniqueness resides in the coexistence of the hydrophilic (a sugar or peptide) and the hydrophobic (fatty acid chain) domains in the same molecule, allowing them to occupy the interfaces of mixed-phase systems (e.g., oil/water, air/water, and oil/solid/water) and consequently altering the forces governing the equilibrium conditions, which is a prerequisite for the occurrence of a broad range of surface activities including emulsification, dispersion, dissolution, solubilization, wetting, and foaming (Desai and Banat, 1997; Banat et al., 2000). Such advantage was reported by Thavasi et al. (2011a,b) investigating the effect of BS and fertilizer on biodegradation of crude oil by four marine oil-degrading bac-teria, B. megaterium, Corynebacterium kutscheri, P. aeruginosa, and Lactobacillus delbrueckii. It was reported that the addition of BS and fertilizer into the lab-scale



biodegradation system increased the degradation process from 19% to 37.7% as com-pared to the experiments where no BS and fertilizer was added. The BSs used in the earlier experiments were able to emulsify a range of different hydrocarbons.

Another example is the high-molecular exopolysaccharide-producing marine bacterium Alcaligenes sp. PHY 9L-86, which was able to use 0.1% tetradecane as the sole carbon and energy source and degrade 98% of the substrate within 48 h (Goutx et al., 1987). In the same way, emulsifiers from Halomonas sp. TG39 and TG67 showed significant emulsification activity with different edible oils as well as with hexadecane, and these emulsions remained stable for several months (Gutiérrez et al., 2007b). Low-molecular-weight BSs such as glycolipids produced by marine bacterium, Halomonas sp. ANT-3b, was able to degrade n-hexadecane and use it as the sole source of carbon to produce BSs (Pepi et al., 2005). In addition to the broad-spectrum emulsification ability to promote the biodegradation process, marine microbial BSs also exhibited higher stability at various conditions as reported by Thaniyavarn et al. (2006); the researchers isolated a P. aeruginosa A41 strain producing rhamnolipid BS that had good stability and activity at a wide range of temperatures (40°C–121°C), pH (2–12), and NaCl concentrations (0%–5%). Higher stability at various conditions and broad-spectrum emulsification activities found with marine BSs indicated their potential broad-spectrum application against vari-ous hydrocarbons and in different environments.

Besides the reports on the application of marine microbes and their BSs, there are few reports on the application of microbes, and their BSs isolated from nonmarine origin support the concept of the application of microbial BSs in oil bioremediation. Bioremediation of gasoline contaminated soil by bacterial consortium with poultry litter (PL), coir pith (CP), and rhamnolipid BS in an ex situ bioremediation system was reported by Rahman et al. (2002). In this study, the authors treated red soil (RS) with gasoline-spilled soil (GS) from a gasoline station, and different combinations of amendments were prepared using (1) mixed bacterial consortium (MC), (2) PL, (3) CP, and (4) rhamnolipid BS produced by Pseudomonas sp. DS10–129. The study was conducted for a period of 90 days during which bacterial growth, hydrocar-bon degradation, and growth parameters of Phaseolus aureus RoxB (mung bean planted on the oil-polluted soil) including seed germination, chlorophyll content, and shoot and root length were measured. Results from the biodegradation experiments revealed that 67% and 78% of the hydrocarbons were effectively degraded within 60 days in soil samples amended with RS + GS + MC + PL + CP + BS at 0.1% and 1%. Maximum seed germination, shoot length, root length, and chlorophyll content of the P. aureus were recorded after 60 days in the earlier amendments. This study suggests that using BSs or BS-producing microbes, oil-polluted agriculture soil can be restored to its original state, and the application of BS as a growth enhancer in agriculture lands polluted with hydrocarbons. Another examples is enhanced biore-mediation of n-alkane in petroleum sludge using bacterial consortium amended with rhamnolipid and micronutrients (Rahman et al., 2003). Results reported in this study showed that maximum hydrocarbon degradation was observed after the 56 days of treatment. Degradation of hydrocarbons with different carbon chain length showed that n-alkanes in the range of nC8–nC11 were degraded completely followed by nC12–nC21, nC22–nC31, and nC32–nC40 with percentage degradations of 100%,



83%–98%, 80%–85%, and 57%–73%, respectively. Addition of rhamnolipids into the biodegradation system significantly increased the microbial growth by promot-ing the efficient utilization of hydrocarbons.

Biosurfactants and microbially enhanced oil recovery (MEOR): BSs have extensive potential application in the petroleum industry such as emulsifiers, demulsifiers, and oil recovery agents. MEOR is a technique that either uses a crude preparation of BS or sterilized BS containing culture broth to liberate crude oil from a binding sub-strate (Marchant and Banat, 2012b). For example, Banat et al. (1991) carried out a crude oil sludge tank cleanup and oil recovery process in which BS-containing ster-ilized culture broth was used to clean up oil sludge from an oil storage tank. After 5 days of treatment involving energy addition and circulation to enhance the process of emulsification followed by a deemulsification, 91% of crude oil present in the oil storage tank was recovered. Hydrocarbon analysis for the recovered crude showed a 100% hydrocarbon content. This result indicated that MEOR process doesn’t require live microorganisms or pure BSs and that sterilized BS-containing broth is sufficient to mobilize and recover significant amount of oil from oil sludge deposits.

Biosurfactants and heavy metal remediation: Microbial BSs are known for their metal-complexing activities that have been reported to be effective in the remedia-tion of heavy metal-contaminated environments (Mulligan et al., 2001; Singh and Cameotra, 2004). The mechanisms behind metal binding are (1) anionic BSs create complexes with metals in a nonionic form by ionic bonds. These bonds are stronger than the metal’s bonds with the soil/sediment and metal–BS complexes are desorbed from the soil matrix to the solution due to the lowering of the interfacial tension and (2) the cationic BSs can replace the same charged metal ions by competition for some but not all negatively charged surfaces (ion exchange). Metal ions can be removed from soil surfaces by the BS micelles. The polar head groups of micelles bind metals that mobilize the metals in water (Mulligan and Gibbs, 2004).

Applications of marine BSs in heavy metal remediation have been reported by many researchers. Das et al. (2009b) studied the bacterial cells (B. circulans) and BS-mediated cadmium and lead metal binding and suggested that there was no cell-mediated metal binding, but that an increase in metal binding was observed with an increase in BS concentration from 0.5 × CMC to 5 × (CMC of the BS was 40 mg/L). The percentage removal at 0.5 × CMC was 76.6%, 53.18%, 56.63%, and 42.74%, 29.72%, 23.19% for lead and cadmium, respectively, while the percentage removal was increased to 100%, 95.75%, 87.69%, and 97.66%, 88.43%, 86.35% for lead and cadmium, respectively at 5.0 × CMC of BS. A complete removal of the metals was seen at 10 × CMC.

Gnanamani et al. (2010) reported the chromium reduction and trivalent chro-mium tolerance behavior of marine Bacillus sp. MTCC5514 through its extracellular enzyme reductase and BS production. The isolate reduces 10–2000 mg/L of hexava-lent chromium to trivalent chromium within 24–96 h, and the release of extracellular chromium reductase was responsible for the metal reduction. The role of the BS in this metal reduction process is to entrap the trivalent chromium in the micelle of BSs, which prevents microbial cells from exposure toward trivalent chromium. It was concluded that extracellular chromium reductase and BS mediate the remediation



process and keep the cells active and provide tolerance and resistance toward high concentration of hexavalent chromium and trivalent chromium. The earlier reports on the application of marine BSs in heavy metal binding and mobilization activities clearly indicated the potential application marine BSs in metal remediation.

Antimicrobial and antifouling agents: Marine microbial surfactants have been rec-ognized for their biological properties such as antimicrobial and antifouling/biofilm activities. Antimicrobial activity of lipopeptide BS produced by marine B. circu-lans was reported by Mukherjee et al. (2009). Significant inhibitory activity was seen against gram-positive bacteria like M. flavus, B. pumilus, and Mycobacterium smegmatis, and gram-negative bacteria like Escherichia coli, Serratia marcescens, Proteus vulgaris, Pseudomonas sp., and Klebsiella aerogenes. The BS also showed significant inhibitory action against fungal species such as Aspergillus niger, Aspergillus flavus, and C. albicans. The purified BS showed more antimicrobial activity, and its broad-spectrum activity against gram-positive and -negative and fun-gal cultures indicated its potential application as an antimicrobial agent in medical and household antimicrobial and disinfectant applications.

Gerard et al. (1997) reported the antimicrobial activity of cyclic depsipeptides (Massetolide A-H) and viscosin produced by Pseudomonas sp. MK90e85 and MK91CC8 strains. Massetolide A and viscosin showed antimicrobial action against Mycobacterium tuberculosis and Mycobacterium avium-intracellulare. Massetolide A showed a minimum inhibitory concentration (MIC) value of 5–10 μg/mL against M. tuberculosis and 2.5–5 μg/mL against M. avium-intracellulare. Viscosin had an MIC value of 10–20 μg/mL against M. tuberculosis and 10–20 μg/mL against M. avium-intracellulare. Even though these molecules are toxic to pathogenic micro-bial cells, massetolide A was nontoxic to mice at a dose of 10 mg/kg body weight. This nontoxic nature to mammalian cell property of the massetolide A indicates its potential application in treating infections caused by Mycobacterium sp. The pro-posed antimicrobial mechanism of BSs is membrane lipid order perturbation, which compromises the viability of microorganisms and their spores (Azim et al., 2006).

Similar to antimicrobial activity, BSs play an important role in the formation of biofilm especially attachment and detachment of cells. Das et al. (2009a) reported the antiadhesive and biofilm dislodging activities of a lipopeptide surfactant isolated from marine B. circulans strain. The concentration of BS used in both the experi-ments was 0.1–10 mg/mL, and the results suggested that maximum antiadhesive and biofilm dislodging activity was observed at 10 mg/mL concentration at which percentage antiadhesion activity was 89%, 88%, 84%, 87%, 86%, 88%, 87%, and 83% against E. coli, M. flavus, S. marcescens, Salmonella typhimurium, P. vulgaris, Citrobacter freundii, Alcaligenes faecalis, and K. aerogenes, respectively. The bio-film dislodging activity of the BS against bacterial strains was as follows: E. coli (59%), M. flavus (72%), S. marcescens (94%), S. typhimurium (89%), P. vulgaris (82%), Citrobacter freundii (77%), Alcaligenes faecalis (79%), and K. aerogenes (80%). Biofilm disruption potential of a glycolipid BS isolated from marine B. casei was reported by Kiran et al. (2010a). The biofilm-forming bacterial cultures used in the assay system were Vibrio parahaemolyticus MTCC 451, Vibrio harveyi MTCC 3438, Vibrio alginolyticus MTCC 4439, Vibrio alcaligenes MTCC 4442,



Vibrio vulnificus MTCC 1145, P. aeruginosa MTCC 2453, and E. coli MTCC 2339. Biofilm disruption results revealed that biofilm-forming capacity of both mixed cul-ture and individual strains were significantly inhibited at 30 mg/mL concentration. Dusane et al. (2011) reported the antibiofilm activity of a glycolipid BS produced by a tropical marine bacterium S. marcescens isolated from the hard coral, Symphyllia sp. The reported antibiofilm activity of the glycolipid BS was 89% and 90%, 75% and 88%, 76% and 82% at 50 and 100 μg/mL concentrations against B. pumilus, P. aeruginosa, and C. albicans, respectively.

Biosurfactants in medical/therapeutic applications: Antimicrobial activities of marine BSs reviewed in the previous section of this chapter make them relevant molecules for applications in combating many diseases and as therapeutic agents. In addition, their role as antiadhesive agents against several pathogens indicates their utility as suitable antiadhesive coating agents for medical insertional materials leading to a reduction in a large number of hospital infections without the use of synthetic drugs and chemicals. Recent reports suggest that microbial surfactants also have other important medicinal and therapeutic applications. The use and potential commercial application of BSs in the medical field have increased during the past decade. There are few reports avail-able on therapeutic applications of BSs of nonmarine origin. Example, Rodrigues et al. (2006a) reported the inhibition of microbial adhesion to silicone rubber treated with BS from Streptococcus thermophilus A. Pathogens used in this study were Rothia dentocariosa GBJ 52/2B and Staphylococcus aureus GB 2/1 (bacterial strains) and C. albicans GBJ 13/4A and C. tropicalis GB 9/9 (yeast strains). After 4 h of treatment of the silicon rubber with BS, a decrease in the initial cell deposition rate was observed for Rothia dentocariosa GBJ 52/2B and S. aureus GB 2/1 from 1937 ± 194 to 179 ± 21 microorganisms/cm2/s and from 1255 ± 54 to 233 ± 26 microorganisms/cm2/s, respec-tively, which was an 86% reduction of the initial deposition rate for both strains. The number of bacterial cells adhering to the silicone rubber with preadsorbed BS after 4 h was further reduced by 89% and 97% for the two strains, respectively. Antiadhesion activity observed against the two yeast strains showed 67% and 70% reduction, respec-tively, for C. albicans GBJ 13/4A and C. tropicalis GB 9/9.

Another example was reported by Rodrigues et al. (2006c) on antimicrobial cell adhesion activity of rhamnolipid BSs on voice prostheses to silicone rubber. After 4 h of treatment with (4 g/L concentration) rhamnolipids, an average of 66% antiadhesion activity occurred for S. salivarius GB 24/9 and C. tropicalis GB 9/9. Preformed microbial mat/biofilm had a 96% detachment of microor-ganisms adhered to the silicone rubber. They concluded that pretreatment with surface active compounds may be a promising strategy to reduce the microbial colonization rate of silicone rubber voice prostheses used after total laryngec-tomy surgery. Laryngectomy is a surgical treatment for the extensive cancer of larynx, which alters swallowing and respiration in patients, which is followed up with a surgical voice restoration procedure comprising tracheoesophageal punc-ture techniques with an insertion of a “voice prosthesis” to improve successful voice rehabilitation. After the surgery, microbial colonization on the silicon rub-ber voice prostheses is a major drawback of these devices. Antimicrobials are usually used to prevent the colonization of silicone rubber voice prostheses by



microorganisms, but long-term medication may induce the development of resis-tant strains (Rodrigues et al., 2007). However, antiadhesion results reported by Rodrigues et al. (2006a,b) suggest an alternate way of using microbial BSs as antiadhesive agent; in this process, the BSs used not necessarily have to be an antimicrobial agent and that approach may prevent the antimicrobial resistance development among the microbes involved in adhesion.

Another interesting example for the therapeutic application of BSs is sophoro-lipids, a family of natural and easily chemoenzymatically modified microbial gly-colipid, showed promising immune modulator activity in a rat model of sepsis. Sophorolipid administration after the induction of intra-abdominal sepsis signifi-cantly decreased the rat mortality in this model. This may be mediated in part by decreased macrophage nitric oxide production and modulation of inflammatory responses (Bluth et al., 2006).

Enhanced healing of full-thickness burn wounds using dirhamnolipid was reported by Stipcevic et al. (2006). In this study, treatment of full-thickness burn wounds with topical 0.1% dirhamnolipid accelerated the closure of wounds by 32% within 21 days of the treatment as compared to control where no rhamnolipids used. Dirhamnolipid was well tolerated by the animals (rat), which indicates the nontoxic-ity toward mammalian cells. This study indicated the possible potential application of dirhamnolipid in accelerating normal wound healing and perhaps in overcoming defects associated with healing failure in chronic wounds. BSs isolated from marine microbes may have similar properties, which requires further research. The earlier results clearly indicate that evaluation of marine BSs for therapeutic applications may bring more interesting properties into light and add more values to marine BSs.

Biosurfactants and nanotechnology: The biological synthesis of nanoparticles has gained considerable attention in view of their exceptional biocompatibility and low toxicity. The use of surfactants as nanoparticle stabilizing agents is an emerging field; however, synthetic surfactants are not environmentally friendly. The application of BSs as an alternate to synthetic surfactants for nanoparticle stabilization could be a green ecofriendly approach. BS-mediated nanoparticle syntheses have been reported for microbially produced rhamnolipids (Kumar et al., 2010) and sophorolipids (Kasture et al., 2008) isolated from nonmarine sources. Synthesis of silver nanoparticles by gly-colipid BS isolated from marine B. casei MSA19 was reported by Kiran et al. (2010b). They used an in situ water-in-oil microemulsion phase for the particle synthesis. The glycolipid BS was used as a particle-stabilizing agent by forming reverse micelles. The silver nanoparticles synthesized in this study were uniform and stable for 2 months. Therefore, the BS-mediated nanoparticle synthesis can be considered as “green” stabi-lizer of nanoparticles and could be extended to other marine microbial BSs.

CONCLUSION

Although BSs have been the subject of intense investigation during the past few decades, relatively small numbers of microorganisms and research output have focused on their production from marine microorganisms. Nevertheless, some marine microbial com-munities, including Acinetobacter, Arthrobacter, Pseudomonas, Halomonas, Bacillus,



Rhodococcus, Enterobacter, Azotobacter, Corynebacterium, and Lactobacillus, have been explored for the production of surface active molecules both BSs and bioemulsifi-ers. Such biological surfactants have important potential application in different indus-tries, and the marine ecosystems can provide an excellent opportunity to select unique and diverse producing microorganisms and chemical products. Effective screening and purification techniques are essential in order to explore and discover unique and effective BSs and bioemulsifiers able to be produced using cost-effective technology processes and at acceptable yields and quality. Promising recent biotechnological approaches coupled with highlighting of the importance of the marine resource for such novel compounds and their environmental credentials are expected to support both search and potential industrial application of BSs in many industrial applications.

ACKNOWLEDGMENT

The first author (Dr. R. Thavasi) is very much thankful to CSIR Government of India and the authorities of Annamalai University for their support to carry out the biosurfactant-related research.

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Biosurfactants: Research Trends and Applications