operties of surface active molecules nd numerous applications in various

. . .

2.1.1. Acinetobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439

Biotechnology Advances 28 (2010) 436450

Contents lists available at ScienceDirect

Biotechnology Advances

j ourna l homepage: www.e lsev ie r.com/ locate /b iotechadv2.1.2. Pseudomonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4402.1.3. Myroides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4412.1.4. Halomonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4412.1.5. Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4412.1.6. Streptomyces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4412.1.7. Antarctobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4412.1.8. Marinobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441

2.2. Glycolipid surface active molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4412.2.1. Alcaligenes sp. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4412.2.2. Arthrobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4412.2.3. Alcanivorax borkumensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4422.2.4. Rhodococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4422.2.5. Halomonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4422.2.6. Unidentied marine bacterium MM1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442 Corresponding author. Institute of Bioinformatics anfax: +91 2025690087.

E-mail address: directoribb@unipune.ernet.in (B.A. C

0734-9750/$ see front matter 2010 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2010.02.006. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437al resource of biosurfactant (BS)/bioemulsier (BE), exopolysaccharides (EPS) producers . . . . . . 438ers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4392. Marine microorganisms The rich natur2.1. Polymeric biosurfactant/bioemulsiKeywords:MarineBiosurfactantBioemulsierExopolysaccharidesGlycolipidsLipopeptidesApplications

Contents

1. Introduction . . . . . . . . . .(EPS). Due to the enormity of marine biosphere, most of the marine microbial world remains unexplored.The discovery of potent BS/BE producing marine microorganism would enhance the use of environmentalbiodegradable surface active molecule and hopefully reduce total dependence or number of new applicationoriented towards the chemical synthetic surfactant industry. Our present review gives comprehensiveinformation on BS/BE which has been reported to be produced by marine microorganisms and their possiblepotential future applications.

2010 Elsevier Inc. All rights reserved.d Biotechnology and Professor ofMicrobiology, University

hopade).

l rights reserved.n studied for production of BS/BE and exopolysaccharides

industries. Marine microorganisms such as Acinetobacter, Arthrobacter, Pseudomonas, Halomonas, Myroides,Corynebacteria, Bacillus, Alteromonas sp. have beeAccepted 3 February 2010Available online 19 February 2010

diversity. The versatile prReceived 1 June 2009Received in revised form 12 December 2009

functional commercial grabioemulsiers (BE) are attResearch review paper

Biosurfactants, bioemulsiers and exopolysaccharides from marine microorganisms

Surekha K. Satpute a, Ibrahim M. Banat c, Prashant K. Dhakephalkar d,Arun G. Banpurkar e, Balu A. Chopade a,b,a Department of Microbiology, University of Pune, Pune 411007, Maharashtra, Indiab Institute of Bioinformatics and Biotechnology, Pune 411007, Maharashtra, Indiac School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, N. Ireland, UKd Division of Microbial Sciences, Agharkar Research Institute, Pune 411004, Indiae Center for Advanced Studies in Materials Science and Condensed Matter Physics, Department of Physics, University of Pune, Pune 411007, Maharashtra, India

a b s t r a c ta r t i c l e i n f o

Article history: Marine biosphere offers wealthy ora and fauna, which represents a vast natural resource of imperativede products. Among the various bioactive compounds, biosurfactant (BS)/racting major interest and attention due to their structural and functionalof Pune, Pune 411007, Maharashtra, India. Tel.: +91 2025691333;

2.3. Lipopeptide surface active molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4422.4. Phospholipids and fatty acids surface active molecules. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4422.5. Glycolipopeptide surface active molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4422.6. Exopolysaccharides (EPS)/complex surface active polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

2.6.1. Bacillus producing exopolysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4432.6.2. Halomonas producing exopolysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4432.6.3. Planococcus producing exopolysaccharide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4432.6.4. Enterobacter producing exopolysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4432.6.5. Alteromonas producing exopolysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4432.6.6. Pseudoalteromonas producing exopolysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4442.6.7. Rhodococcus producing exopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4452.6.8. Zoogloea producing exopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4452.6.9. Cyanobacteria producing exopolysaccharide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4452.6.10. Vibrio producing exopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4452.6.11. Other exopolysaccharide producing bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445

3. Potential applications of marine microbial surface active agents and exopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . 446. .. .. .

allow solubilisation of hydrophobic substrates (Desai and Banat, overcome such spills, on the one hand, many petroleum based

437S.K. Satpute et al. / Biotechnology Advances 28 (2010) 4364501997). During the last decade they have been under investigation aspotential replacements for synthetic surfactants and are expected tohave many potential industrial and environmental applicationsrelated to emulsication, foaming, detergency, wetting, dispersionand solubilisation of hydrophobic compounds (Banat et al., 2000;

synthetic chemical surfactants often get used. Such syntheticcompounds on the other hand, however often have detrimentalecological effects (Smith, 1968; Smith et al., 1968). The use ofbiosurfactants and bioemulsiers therefore may represent a betteralternative to overcome the toxicity of synthetic compounds

Table 1Major features/aspects of bioactive compounds produced by marine microorganisms in comparison to those for other habitats.

Feature/aspect Marine Other resources

Source of isolation Sea, oceans, hypersaline, salty brine, and Antarctic areas Fresh water, industrial efuents, soil, air, wastewater, sewage, oil wells, and reneries

Probability to obtain the particular microbes Constant churning in the marine biosphere, difcultto obtain particular type of microbial population

More chances of obtaining particular type ofmicrobial biota from particular sampling site

Ability to cultivate Relatively difcult to culture in the laboratory Mostly easily cultured in the laboratoryMaintenance of environmental andphysiological requirement

Difcult to maintain humidity, salinity, and otherextreme conditions in the laboratory

Environmental condition for growth of biosurfactants/bioemulsier producers are mostly easy to maintain

Novelty in structures and functional properties Has diverse biological and functional properties Generally similar type of functional propertiesToxicity nature of biosurfactant/bioemulsier Relatively higher toxicity Less toxic and mostly biodegradablePossibility of culture contamination Lower chances due to extreme environments halophilic,

thermophilic, acidophilic or alkalophilicCan be easily contaminated4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

The hydrosphere marine environment represents the majorcomponent of the Earth's biosphere. It covers a majority (70%) partof earth's surface and makes up N90% of the volume of its crust. Themaximum depth of N11,000 m and 365 million km2 is attributed tothe hydrosphere oceanic system. Oceans represent a vast andexhaustive source of natural products in the globe, harbouring themost diverse groups of ora and fauna. Marine microorganisms havedeveloped uniquemetabolic and physiological capabilities to thrive inextreme habitats and produce novel metabolites which are not oftenpresent in microbes of terrestrial origin (Fenical, 1993). Therefore,this rich marine habitat provides a magnicent opportunity todiscover newer compounds such as antibiotics, enzymes, vitamins,drugs, biosurfactant (BS), bioemulsier (BE) and other valuablecompounds of commercial importance (Jensen and Fenical, 1994;Austin, 1989; Romanenko et al., 2001; Lang and Wagner, 1993).Among these various marine bioactive compounds, BS/BE are of greatimportance due to their structural and functional diversity andindustrial applications (Banat et al., 1991; Banat, 1995a,b; Rodrigueset al., 2006). Biosurfactants and bioemulsiers are amphiphiliccompounds containing both a hydrophilic and a hydrophobic moietyand therefore are able to display a variety of surface activities thatLiterature availability Limited (about 56 reports are avai. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447

Dastgheib et al., 2008). They generally can be grouped either as low orhigh molecular weight biosurfactants (Smyth et al., 2010a,b) theformer consisting of glycolipids and lipopeptides and the latter of highmolecular weight polymeric biosurfactants. To date, more than 225patents are available describing these microbial amphiphilic agents(Shete et al., 2006). More than 10,000 metabolites with broadspectrum biological activities and amazing medicinal propertieshave been isolated from marine microbes (Fusetani, 2000; Kelecom,2002). However, due to the enormity of the marine biosphere, most ofthe marine microbial world remained unexplored. It has beenestimated that b0.1% of marine microbial world has been exploredor investigated (Ramaiah, 2005). Such investigations are mainlyhindered, due to the non-culturable nature of most microorganismsunder laboratory conditions (Harayama et al., 2004). Some of themajor features of bioactive producing marine microorganisms incomparison to those for other habitats are presented in Table 1.

Despite the fact that marine environment represents a wealthybasin of diverse microorganisms, it is important to know that at thesame time they are suffering from anthropogenic pollution withdomestic and industrial wastes (Hassan et al., 1996). Large quantitiesof crude oil, hydrocarbons, petroleum oil products and halogenatedcompounds nds their way into the marine ecosystem throughaccidental spillage (Satpute et al., 2005). To treat, emulsify or simplylable) Quite large

(Edwards et al., 2003). It is important to note that, on thewhole, BS/BEproduced by soil or freshwater microorganisms are less toxic and aremostly biodegradable (Poremba et al., 1991a,b) which means they donot accumulate in the environment. Under such conditions, marinemicrobes offer a rich source of novel surface active compounds.Effective screening methods for biosurfactants and bioemulsiers aretherefore needed to tap into this vast resource. Combination ofvarious such screening methodologies to obtain potent BS producershas been recently reviewed by Satpute and co-workers (2008).Microbial communities like Acinetobacter, Arthrobacter, Pseudomonas,Halomonas, Bacillus, Rhodococcus, Enterobacter, and yeast have beenreported to produce BS/BE (Das et al., 2008a,b; Schulz et al., 1991; Passeriet al., 1992; Banat, 1993; Abraham et al., 1998; Maneerat et al., 2006;Perfumo et al., 2006). About 56 reports including 35 for bioemulsier, 12for glycolipid, and 9 for other types are available on different types ofBS/BE produced by marine microorganisms (Fig. 1). Few reviews areavailable on BS/BE and EPS production from marine microorganisms(Nerurkar et al., 2009; Zhenming and Yan, 2005; Maneerat, 2005;Weiner, 1997; Weiner et al., 1985; Bertrand et al., 1993). This reviewtherefore aims to report/collate up to date information available ondifferent types of BS/BE and EPS produced by different marinemicroorganisms and highlight their potential applications.

2. Marine microorganisms The rich natural resource ofbiosurfactant (BS)/bioemulsier (BE), exopolysaccharides(EPS) producers

Surface active molecules producing microorganisms are ubiqui-tous, inhabiting both water (sea, fresh water, and groundwater) and

land (soil, sediment, and sludge) as well as extreme environments(e.g. hypersaline sites, oil reservoirs), and thriving at a wide range oftemperatures, pH values and salinity. Microorganisms produce BS/BEto mediate solubilisation of hydrophobic compounds in theirenvironment to be able to utilize them as substrates (Margesin andSchinner, 2001; Olivera et al., 2003; Floodgate, 1978), however, thisfact may not be always true. Few microbes produce BS/BE on water-soluble substrates (Gunther et al., 2005; Turkovskaya et al., 2001). Ithas been suggested that the presence of surface active molecules onthe microbial cell surface increases the hydrophobicity of the cell andhelps it to survive in hydrophobic environment (Abraham et al., 1998;Perfumo et al., 2010). BS/BE produced by microbes may either beextracellularly released into the environment or may be localized onsurfaces i.e. become associated with the cell membrane. When BS/BEare associated with cell, the organisms itself behave as a BS/BE incontrolling the adherence property to water-insoluble substrates(Maneerat and Dikit, 2007). The synthesis of these surface activemolecules takes place by de novo pathway and/or assembly fromsubstrates (Syldatk and Wagner, 1987) which is diagrammaticallyrepresented by Satpute et al. (2010). Various BS/BE producingbacteria have been isolated and characterized from marine sitescontaminated with oil, petroleum or their by-products. The use ofdifferent separation techniques ensure the ability to efciently extracthigh and low-molecular weight BS/BE. Different screening methodsare available to identify BS/BE producing microorganisms. However,due to the diverse functional and chemical properties of each type ofmarine BS/BE, it is difcult to obtain BS/BE producers with singlescreening method. Several screening methodologies are thereforeessential to isolate and investigate potential marine BS producer

438 S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450Fig. 1. Summary of different types of marine biosurfactant/bioemulsier producing microorganisms.

(Satpute et al., in press). Isolation from petroleum contaminated sitesusing the screening protocol presented proved to be a rapid andeffective manner to identify bacterial isolates with potential industrialapplications (Batista et al., 2006). A wide variety of genera producingdiverse types of BS/BE (Tables 2 and 3) are associated with the marineenvironment and produce various types of molecular structures andcomposition described in detail as follows.

2.1. Polymeric biosurfactant/bioemulsiers

2.1.1. AcinetobacterSpecies of Acinetobacter are ubiquitous in nature and are one of the

commonly foundGram-negativebacteria inmarine environments. Theyare a predominant (followed by members of Pseudomonas) species inmarine ecosystem and contributes to the major micro ora of sea foods(Austin et al., 1979; Choi, 1995; Choi et al., 1996; Chen-hsing-Chen,1995; Thampuran and Gopakumar, 1993; Chang et al., 1996). Due totheir versatile nature, Acinetobacter sp. has attracted considerableattention by many researchers. Other than marine environment anumber of other sources has been studied in great detail (Patil andChopade, 2001a,b, 2003). Species ofAcinetobacterplay an important rolein hydrocarbon degradation (Juni, 1978) and have a key role in bio-remediation processes (MacCormack and Fraile, 1997). A. calcoaceticus

RAG-1 was isolated from the Mediterranean Sea (Reisfeld et al., 1972)and found to produce a surfactant (Rosenberg et al., 1979), which hasbeen exploited commercially as the product Emulsan. A number ofpotential applications of emulsan has been patented by Gutnick and co-workers (1981) and was marketed by Petrorm Inc. for cleaning of oilcontaminated vessels, oil spills and microbial enhanced oil recovery(MEOR) (Desai and Patel, 1994). Emulsan is a high molecular weightheteropolysaccharide protein containing repeating trisaccharide ofN-acetyl -D-galactosamine, N-acetylgalactosamine uronic acid and anunidentiedN-acetyl amino sugar. Fatty acids (FA) are covalently linkedto the polysaccharide through o-ester linkages (Belsky et al., 1979;Rosenberg et al., 1979; Zukerberg et al., 1979; Gorkovenko et al., 1997;Desai and Banat, 1997; Rosenberg and Kaplan, 1987). Even at very lowconcentration (b0.001 to 0.01%), emulsan can emulsify hydrocarbonsefciently and is the most powerful emulsion stabilizer having severalapplications in diverse elds (Belsky et al., 1979; Gutnick and Shabtai,1987; Zosim et al., 1982). Different Acinetobacter sp. produce proteinpolysaccharide complexes that possess surface active properties(Kaplan and Rosenberg, 1986; Shete, 2003). A great deal of studieshave been carried out regarding isolation, emulsifying, chemical, physicalproperties, and specicity to hydrocarbon substrate (Rosenberg et al.,1979). A. calcoaceticus represents an excellent candidate for theavailability of maximum number of reports in the application area.

Table 2Summary of marine polymeric biosurfactant/bioemulsier produced by different microorganisms.

Source organism Chemical composition Application property Reference

Acinetobacter calcoaceticus RAG1. Emulsan: polysaccharide and protein:backbone O-ester and N-acyl linkages

Powerful emulsion stabilizers: atlow concentration, effective emulsier(emulsionhydrocarbons 1:100 to 1:1000)

Belsky et al. (1979), Gutnickand Shabtai (1987),Zosim et al. (1982), Rosenberg et al.(1979, 1988a,b; 1989)

A. calcoaceticus A2 Biodispersion: four reducing sugars;glucosamine, 6-methylaminohexose,galactosamine uronic acid, unidentiedamino sugar (51.4 kDa)

Considerable substrate specicity Dispersion of limestone and titaniumdioxide; better dispersion of water

Rosenberg and Kaplan (1987),Rosenberg and Ron (1998)

A. radioresistens Alasan: protein polysaccharide (1 MDa)Contains covalent bound alanine

Lowers surface tension (6942 mN/m) Acts as surfactant Effective stabilizer oil-in-water emulsions

Navon-Venezia et al. (1995)

A. calcoaceticus BD4 BD4 emulsan : protein and polysaccharide Polysaccharide attach with protein(which is bound to hydrocarbon) andstabilization of oil-in-water emulsions

Kaplan et al. (1987)

Pseudomonas nautica Proteins, carbohydrates and lipids at Emulsifying activity Husain et al. (1997)

,

439S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450(35:63:2) ratioPseudomonas uorescence Trehaloselipid-o-dialkyl

monoglyceridesprotein

Myroids L-ornithine lipids and a different couple ofiso-3-hydroxyfatty acid (C15C17) andiso-fatty acid (C15 or C16) 1:1:1 ratio

Streptomyces Bioemulsier: protein (82%), reducing sugar (1%)and polysaccharide (17%)

Yarrowia lipolytica NCIM 3589 Bioemulsier: lipidcarbohydrateproteinYarrowia lipolytica IMUFRJ 50682 Yansan: glycoprotein complex

Protein (15%), fatty acids : palmitic acid (35.8%),stearic acid (21.4%), lauric acid (8.8%), oleic acid(6.9%). Monosaccharides arabinose, galactose,glucose, mannose (1:6:17:31).

Rhodotorula glutinis Carbohydrateprotein complex

Halomonas Emulsier HE39 and HE67High molecular weight glycoproteins with ahigh content of protein and uronic acids

Halomonas eurihalina Sulfated heteropolysaccharideAntarctobacter High-molecular-weight glycoprotein with high

uronic acids content, polyanionic structure High yield with gasoline Depending upon hydrocarbon source;different level of emulsication againstgasoline

Desai et al. (1988)

Superior surface activity as comparedwith other surfactants Higher stability; emulsication activity

Maneerat et al. (2006), Maneeratand Phetrong (2007), Maneeratand Dikit (2007)

Signicant emulsication activity Reduction of surface tension

Kokare et al. (2007)

Emulsication activity Zinjarde and Pant (2002) High emulsication activity Oil-in-water emulsions Yansan-based emulsions change theaging mechanisms of oil-in-wateremulsions from coalescence (at pH 3to molecular diffusion at pH 7) ascompared with gum Arabic(age by coalescence)

Trindade et al. (2008),Amaral et al. (2006)

Emulsies kerosene and crude oilefciently

Oloke and Glick (2005)

Highest emulsifying activities Highly stable at neutral, acidic pH andeven at high-temperature treatment

Gutirrez et al. (2007a)

Emulsication Calvo et al. (1998) Better stabilizing activity than emulsication Interesting functional properties ascompared with other biopolymers Emulsion-stabilizing agent: food oils

Gutirrez et al. (2007b)

pro

trehgth

ric

roxygeriecat wposi

oseturid (1id (

ither b

440 S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450Table 3Summary of marine glycolipid and other types of biosurfactant (BS)/bioemulsier (BE)

BS/BE type Source organism Chemical composition

Glycolipid Alcaligenes Non-toxic glucoselipid

Arthrobacter Trehalose tetraester glycolipid:an anionic trehalose lipids 2,3,4,2-tetraester with fatty acids chain lenform 8 up to 14

Alcanivorax borkumensis Anionic glucoselipid with tetrameoxyacyl side chain, N-terminallyesteried with glycine: four 3-hydacids linked with glucose containinglycine and -glucopyranoside est3-hydroxyhexanoic-octanoic and d

Rhodococcus Two different types of biosurfactanto the structure and chemical com

Halomonas Glycolipid: mannose (1.71), galactglucose (2.96); lipid: fatty acid mixa caprylic acid (18.85), myristic acpalmitic acid (9.68), palmitoleic acand oleic acid (1.26)

Unidentied marinebacterium MM1

Cell-bound, novel type glycolipid w3-OH-decanoic acids, linked by estA. calcoaceticusBD4 andBD413produce extracellular BD4 emulsan in theproduction medium supplemented with ethanol (2%). Amphipathicproperties of BD4 emulsan are due to the association of an anionichydrophilic polysaccharide with proteins. Selective digestion, deprotei-nization, reconstitution and chemical modication studies proved thatthe polysaccharide and protein fractions are essential for emulsicationactivity (Kaplan et al., 1987). An extracellular, anionic non-dialyzablepolysaccharide named biodispersion was reported from A. calcoaceticusA2. Biodispersion, the name itself suggests the dispersing property forwater-insoluble compounds (Rosenberg et al., 1988a,b, 1989; RosenbergandRon, 1998). Biodispersion has an averagemolecularweight of 51,400and along with the extracellular polysaccharide a rich protein is alsosecreted by A. calcoaceticus A2 species. Mutants' defective in proteinproduction gives enhanced yield of biodispersion as compared to theparent strain (Elkeles et al., 1994). There is another report suggesting thatAcinetobacter sp. A3 produces two proteins/polypeptides of molecularmasses of 26.5 kDa and 56 kDa when grown on crude oil as sole source.Acinetobacter sp. A3utilizes BombayHigh crudeoilwithout productionofany surface active agents. Hanson et al. (1994) proposed that the directcelloil interaction facilitates the crude oil utilization by the bacterium.Bioemulsier producing marine Acinetobacter calcoaceticus subsp. ani-tratus SM7 strain was isolated from oil contaminated seawater in

The lipophilic moiety is coupled glycoswith C-1 of glucose

Lipopeptide Bacillus circulans Novel type of lipopeptide biosurfactan

aBacillus licheniformis BAS50 Lipopeptide: lichenysin (1006 to 1034the lipid moiety: mixture of 14 linearbranched beta-hydroxy fatty acids ranin size from C12 to C17. Amino acids:acid as the N-terminal amino acid, aspvaline, leucine, and isoleucine as the Camino acid, at a ratio of 1.1:1.1:1.0:2.8respectively.

Azotobacter chroococcum Lipid and protein (31.3:68.7)

Phospholipidsand fatty acids

Myroides Bile acids with cholic acid, deoxycholicand their glycine conjugates

Glycolipopeptide Corynebacterium kutscheri Carbohydrate (40%), lipid (27%) andprotein (29%)

a Bacillus licheniformis BAS50 has been isolated from oil reservoir (at 1500 m) but it can grobacteria and can grow at a marine environment.duced by different microorganisms.

Application property Reference

Inhibition of the growth ofmicroagellates and microalgae

Poremba et al. (1991a)Poremba et al. (1991b)

alose Surfactant activities Effective interfacial andemulsifying properties

Schulz et al. (1991)Passeri et al. (1991)

decanoic

ed withnoic acids

Surface activity Abraham et al. (1998)

ith respecttion of BS

Enhanced the solubility of polycyclicaromatic hydrocarbons and thedegradation rate of hexadecane

Peng et al. (2007)

(1.00),e with.0),5.69)

Emulsifying properties Pepi et al. (2005)

fouronds.

Good surface active agent Passeri et al. (1992)Songkhla lagoon, Thailand. The emulsier emulsies n-hexadecaneeffectively at a wide pH (612) and temperatures ranges (30121 C)and in the presence of up to 12% NaCl. Phetrong et al. (2008) suggestedthat such emulsier with stable properties at broad range of pH,thermostability and salt tolerance may play a signicant role inenvironmental application, especially bioremediation of oil-pollutedseawater.

2.1.2. PseudomonasBS produced by marine microbes involved in the degradation of

hydrocarbons in surrounding environment. Coelho et al. (2003) re-ported production of BS by quinoline degrading marine Pseudomonassp. strain GU 104. Comparative studies of synthetic surfactant and BSproduced by Pseudomonas on the physiology of Perna viridis L, aneconomically important mussel used for food, a bivalve belonging tothe family Mytilidae indicate that BS does not affect the physiology ofP. viridis. There is an additional single report available on polymeric BSproducing Pseudomonas strain. P. nautica which was isolated from acoastal area of the Mediterranean Sea (Husain et al., 1997) andreported producing an extracellular BS exhibiting emulsifying activity.Depending upon the different type of carbon source/hydrocarbons,different types of emulsiers are produced by microorganism. This

idically

t Pronounced antimicrobial activity No haemolytic activity Antimicrobial activity

Das et al. (2008a)Mukherjee et al. (2009)

Da),andgingglutamicaragine,-terminal:1.0,

Surface tension of water from72 to 28 mN/m with CMC (criticalmicelle concentration): 12 mg/liter Powerful surface active agent comparedwith other surfactants Good antibacterial activity

Yakimov et al. (1995)

Emulsication of waste motor lubricant oil,crude oil, diesel, kerosene, naphthalene,anthracene and xylene

Thavasi et al. (2008)

acid Good surface active agent Maneerat et al. (2005)

Emulsication of different hydrocarbons Thavasi et al. (2007, 2008)

w at high salt up to 13% and optimum salt around seewater level so it resembles marine

441S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450fact is well proved by Desai et al. (1988) from the production oftrehalose lipido-dialkyl monoglyceridesprotein emulsier by hy-drocarbon degrading P. uorescens. Another similar contribution byChristensen et al. (1985) suggests the production of emulsier frommarine Pseudomonas sp. NCMB 2021 extracellularly at the end of theexponential growth phase and in the stationary phase of growth.Polysaccharide contains deoxy sugars and acetyl groups with out-standing solubility even at high concentrations of phenol, methanoland ethanol.

2.1.3. MyroidesIt is an aerobic, Gram-negative, non-motile, pigmented (yellow-

to-orange) rod-shaped bacterium usually found in marine ecosystem.The majority of work carried out on BE producing Myroides was onstrain sp. SM1 which was isolated from oil-spilled seawater inSongkhla Lake, Thailand (Maneerat et al., 2005). Myroides sp. SM1,grows well, emulsifying weathered crude oil and produce extracel-lular BE (complex of L-ornithine lipidsL-ornithine and a differentcouple of iso-3-hydroxy fatty acid and iso-fatty acid) that possesseshigh surface activity for oil displacement as compared with othersurfactants (Maneerat et al., 2006). BE from such an extremeenvironment shows higher stability at a broad temperature range.However, its emulsication activities diminish rapidly at extreme pHand high salt concentration (Maneerat and Phetrong, 2007). Cellassociated surface active molecules from Myroides sp. possess highemulsication activity by adhering to weathered crude oil (Maneeratand Dikit, 2007).

2.1.4. HalomonasGlycoprotein (protein and uronic acids) based bioemulsier are

also produced by Halomonas sp. and have been characterizedchemically and physically by Gutirrez et al. (2007a). BE from twonew Halomonas sp., TG39 and TG67 has been concentrated byultraltration, and precipitation. This is the rst report on the highestemulsifying activities of BE produced from Halomonas sp. Surfaceactive molecules from both strains are also highly stable at neutral,acidic pH and at high temperatures.

2.1.5. YeastYarrowia lipolytica is one of the well reported yeast for production

of lipidcarbohydrateprotein based bioemulsiers. This polysaccha-ride based bioemulsier can increase the hydrophobicity of the cellsduring the growth phase. Studies carried out by Zinjarde and Pant(2002) showed that extracellular production of BE does take placewhen cell enters into stationary phase. Yarrowia lipolytica NCIM 3589isolated from a marine sample has been shown to produce a cell wallassociated emulsier which is a complex of lipidcarbohydrateprotein in the presence of alkanes or crude oil. Yansan, is anothertype of bioemulsier produced from Brazilian wild strain of Yarrowialipolytica, IMUFRJ 50682 in the fermentation medium supplementedwith glucose. Trindade et al. (2008) carried out comparative studies ofaging mechanisms of oil-in-water emulsions with Yansan and gumArabic emulsions and observed dependence on the pH of theproduction medium. Studies proved that emulsions containing gumArabic age by coalescencewhile Yansan-based emulsions change theiraging mechanisms from coalescence at pH 3 to molecular diffusion atpH 7. Other than Yarrowia lipolytica, an unusual yeast isolate ofRhodotorula glutinis has also been reported to produce a bioemulsierwhich shows good emulsication index (80%) with kerosene andcrude oil. It has also been shown to remove crude oil (76%) frompollutants (Oloke and Glick, 2005).

2.1.6. StreptomycesMarine Streptomyces are known to produce protein polysaccha-

ride type bioemulsiers (Kokare et al., 2006). Out of 80 actinomy-

cetes strains isolated from Alibag, Janjira and Goa coastal regions ofIndia, six produced BE on oils and hydrocarbons as substrates.Streptomyces sp. S1 showed highest emulsication units (200 EU/ml)and reduced the surface tension of production medium up to42.6 mN/m with a CMC value of 0.3 mg/ml. The bioemulsiermolecules secreted by Streptomyces species mainly contain protein,polysaccharide and a small part of reducing sugar (Kokare et al.,2007).

2.1.7. AntarctobacterIt is amarine Gram-negative alpha proteobacterium generally seen

in marine ecosystem. Gutirrez et al. (2007b) isolated marineAntarctobacter sp. TG22, that produces extracellular (high-molecu-lar-weight fraction N2000 kDa) emulsifying agent named AE22. Thisbioemulsier is composed of uronic acids with high emulsicationand stabilizing activity. This glycoprotein AE22 represents a powerfulextracellular emulsion-stabilizing polymer that forms highly stableemulsions as compared with xanthan gum and gum Arabic. Due to theinteresting functional properties, AE22 can be preferred for industrialapplication as compared to xanthan gum and gum Arabic.

2.1.8. MarinobacterIt is a Gram-negative, rod-shaped (coccobacilli, straight/curved),

non-spore forming bacterium which shows motility by means ofsingle unsheathed polar agellum.Marinobacter (previously named asAlteromonas) produce large amounts of a non-dialyzable bioemulsi-er. This strain also degrades various liquid and solid hydrocarbons(Al-Mallah et al., 1990). Further taxonomic verication of Alteromonasstrain revealed that the organism represented a new sp. within a newgenus named, Marinobacter hydrocarbonoclasticus (Gauthier et al.,1992).

2.2. Glycolipid surface active molecules

This class of surface active molecules have been studied exten-sively due to their importance, quantities and extended applicationsas compared with other types of BS/BE.

Sugar based, cheaper, renewable substrates can be easilyemployed for the production of glycolipid BS. From a commercialpoint of view, glycolipids represents superior product for productionand utilization purposes (Kitamoto et al., 2002). Glycolipids BS arecarbohydrates in combination with long-chain aliphatic acids orhydroxyaliphatic acids. There are different types of glycolipid BSproduced by marine microorganisms as described below.

2.2.1. Alcaligenes sp.It is a Gram-negative, rods, cocci (usually single), motile,

obligatory aerobic, oxidase and catalase positive bacterium. Porembaet al. (1991a) was the rst to report the production of non-toxicglucoselipid BS from Alcaligenes sp. Biosurfactant produced byAlcaligenes sp. inhibits the growth of microagellates and microalgae(Poremba et al., 1991b). Schulz et al. (1991) isolated three marinebacterial strains and oil-degrading mixed cultures from marinesamples collected around the Isle of Helgoland (North Sea). Theyscreened and selected three marine bacteria from n-alkane degradingmicrobial population. One strain-identied as Alcaligenes strain MM1produced a novel glucoselipid while the other two strains belongedto Arthrobacter sp. as described below.

2.2.2. ArthrobacterTwo strains of Arthrobacter sp. viz., EK 1 and Arthrobacter SI 1

produce glycolipid type BS (Schulz et al., 1991). Arthrobacter EK 1produced trehalose tetraester glycolipid while culture, Arthrobacter SI1 produced an extracellular emulsifying agent. Both BS exhibit strongsurfactant activities due to their effective interfacial and emulsifying

properties. Passeri et al. (1991) also reported on the production and

hydrocarbons. Analysis of the molecules with GCMS, FAB-MS

degrades alkane, whereas Cycloclasticus degrades various aromatichydrocarbons. Such studies are important for developing in situ

degrading bacterium R. erythropolis strain 3C-9 from seaside soil of theIsland of Xiamen, located on the west bank of the Taiwan Strait. Two

together by ester bonds. The lipophilic moiety is coupled glycosidically

442 S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450with C-1 of glucose. This glycolipid act as a powerful biosurfactant byreductionof surface tensionofwater from72 to30 mN/mand interfacialdifferent types (structure and chemical composition) of biosurfactantsas a glycolipid and free fatty acids are produced by R. erythropolis.

2.2.5. HalomonasThese marine strains are mostly known for production of EPS with

emulsifying properties. There are few reports suggesting that theemulsifying surface active agents are also produced by Halomonasspecies. Detailed studies were carried out by Pepi and co-workers(2005). A bacterial strain, Halomonas ANT-3b was isolated from seaice seawater interface at Terra Nova Bay station, Ross Sea, Antarcticathat produces emulsifying glycolipids. This surface active compound isa mixture of fatty acids and sugars of a molecular weight of 18 kDawith mesophase (liquid crystal) organization. We isolated anotherstrain H. hydrothermalis IACS3 from marine water sample of CaspianSea (Iran) which produce glycolipid type biosurfactant that reducessurface tension of distilled water from 71 to 31 mN/m and emulsiesvarious plant oils efciently (Satpute, 2008).

2.2.6. Unidentied marine bacterium MM1Low and high-molecular surface active substances are produced by

n-alkane-utilizing bacteria. Passeri et al. (1992) screened marinebacteria for biosurfactant production and reported a Gram-negativemarine bacteriumMM1 synthesizing a novel low-molecular-mass cell-bound glycolipid with four 3-OH-decanoic acids, which are linkedbioremediation strategies for cleaning up marine oil spills (Harayamaet al., 2004).

2.2.4. RhodococcusDifferent species of Rhodococcus are known for production of

glycolipid surface active molecules. Peng et al. (2007) isolated an oil-indicated four 3-hydroxydecanoic acids linked together by esterbonds and coupled glycosidically with C-1 of glucose. It containsglycine and -glucopyranoside esteried with 3-hydroxyhexanoic,-octanoic and decanoic acids. Alcanivorax sp. exhibit active alkanedegradation properties. Yakimov et al. (1998) isolated six (AP1, MM1,SK, SK2, SK4A and SK7) n-alkane-degrading heterotrophic bacteriathat produces cell-bound and extracellular surface active glucoselipid. These strains were identied as A. borkumensis that wascharacterized as producers of novel class of glycolipid biosurfactants.Similar kind glycolipid BS production from A. borkumensis has beenreported by Passeri et al. (1992). BS produced by these marinebacteria reduced the surface tension of water from 72 to 29 mN/m.Alcanivorax and Cycloclasticus shows predominance in marine systemand are known as hydrocarbon degraders. Alcanivorax mainlycharacterization of marine biosurfactant (an anionic trehalosetetraester) from the marine bacterium Arthrobacter sp. EK1.

2.2.3. Alcanivorax borkumensisSpecies of Alcanivorax (gammaproteobacterium) are Gram-nega-

tive, non-motile aerobic bacteriawithmorphology of elongated rod. A.borkumensis produces a potent glucoselipid surfactant (Abrahamet al., 1998). A mixture of active biosurfactant, glucoselipids wereextracted from the growth medium supplemented with aliphatictension value with n-hexadecane to b5 mN/m.2.3. Lipopeptide surface active molecules

Several types of cyclic lipopeptide biosurfactants are producedmainly by members of the Bacillus sp., which have been isolated fromvarious resources (Arima et al., 1968; Yakimov et al., 1995). Recently,Das et al. (2008a) isolated B. circulans from the Andaman and NicobarIslands, India. Different functional groups and chemical bonds in theseBS have been determined by HPLC technique. Some of theselipopeptide possess some antimicrobial activity. Their studies alsoshowed that one of the HPLC fractions (nonhaemolytic) of the crudelipopeptide BS is responsible for its antimicrobial action. Therefore,lipopeptide biosurfactant have been implicated with some antimi-crobial chemotherapy applications. Biosurfactant produced by amarine Bacillus possesses good surface tension reduction with lowCMC and antimicrobial activity. Pure quality of product obviouslywidens the applications in diverse elds. It is also important to notethat the quality and quantity of biosurfactant production is dependenton the carbon substrate and fermentation medium. Studies by Das etal. (2009c) showed that biosurfactant possessing good antimicrobialactivity can be isolated in the productionmedium supplemented withglucose as compared with glycerol, starch and sucrose. RP-HPLCstudies were employed to resolve crude biosurfactants into severalfractions which indicated that one of the fractions bears signicantantimicrobial action.

Biosurfactant producing B. circulans has been shown to enhancethe bioavailability and degradation of a polyaromatic hydrocarbon(PAH) like anthracene. The presence of anthracene enhances thebacterial growth and biosurfactant production. Biosurfactant obtainedfrom such strain can emulsify various hydrocarbons efciently.Analytical studies are important to understand the role of biosurfac-tant in solubilisation of PAH (Das et al., 2008b). Newly introducedapproach PlackettBurman-based statistical screening procedure canbe employed to determine the nutritional requirement for the growthand biosurfactant production of bacterium. Statistical approaches arealso helpful in the formulation of production medium for microbialproduction of biosurfactant and may be crucial to enhance thequantity of the product obtained (Mukherjee et al., 2008).

A lipopeptide biosurfactant producing Azotobacter chroococcumisolated from marine environment of Tuticorin harbour (India) hasalso been reported to grow on crude oil, waste motor lubricant oil andpeanut oil cake and was capable of emulsifying waste motor lubricantoil, crude oil, diesel, kerosene, naphthalene, anthracene and xylene.Potential applications of such surface active molecule for bioremedia-tion were highlighted by Thavasi et al. (2006, 2009). Lipopeptide typebiosurfactant are also produced by marine actinomycetes. A biosurfac-tant from sponge-associated marine actinomycetes Nocardiopsis albaMSA10 has been characterized recently reported (Gandhimathi et al.,2009).

2.4. Phospholipids and fatty acids surface active molecules

Maneerat et al. (2005) reported the production of surface activebile acids byMyroides SM1 bacterial strains. These compoundswere ofsimilar structures to those produced by eukaryotic cells and arecomposed of cholic acid, deoxycholic acid and their glycine con-jugates. M. odoratus JCM7458 and M. odoramitimus JCM7460 alsoproduced such type of biosurfactants.

2.5. Glycolipopeptide surface active molecules

Renewable substrates like motor lubricant oil and peanut oil cakehas been used for BS production from microbes. Thavasi et al., 2007carried out biosurfactant production by using renewable, relativelyinexpensive and easily available resources. Two different species aCorynebacterium kutscheri and a Bacillus megaterium isolated from

Tuticorin harbour (India) produced complex glycolipopeptide capable

443S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450of emulsifying different hydrocarbons which is important to remediatehydrocarbon polluted sites (Thavasi et al., 2007, 2008). Similar kind ofsurface active compounds has been reported from in non marineCorynebacterium isolates (Zajic et al., 1977; Cooper et al., 1979).

2.6. Exopolysaccharides (EPS)/complex surface active polymer

Exopolysaccharides (EPS) are high molecular weight carbohydratepolymers. Many marine microorganisms produce extracellular poly-mers which form a layer surrounding the cells that helps them towithstand or resist adverse and extreme environmental conditions. Atthe same time it is also true that these extreme environments offernovel microbial biodiversity that produces varied and interestingtypes of EPS (Mancuso Nichols et al., 2005a,b; Margesin and Schinner,2001; Vincent et al., 1991). The release of intracellular andextracellular capsular material by marine bacteria in the oceanmight drive the dissolved organicmatter pool of themarine biosphere(Heissenberger et al., 1996). EPS may act as a cryoprotective agent inbrine channels of sea ice, where extremes of high salinity and lowtemperature impose pressures on microbial growth and survival(Mancuso Nichols et al., 2005b). EPS secreted by Vibrio parahaemo-lyticus shows good adhesive property, where cells are held together toform colonies, which helps the colonies to withstand physical forces(Jodi et al., 2000). Some of the key roles of microbial EPS arerepresented diagrammatically in Fig. 2. Such polysaccharide produ-cers have been isolated from hydrothermal deep-sea vents extremeenvironment. Mancuso Nichols et al. (2005b) analysed sea ice andwater samples obtained by a voyage of RSV Aurora Australis andreported near dominance of gamma proteobacteria and cytophagaexibacterbacteroides in these marine samples. These EPS areimportant in microbial interaction and emulsication of varioushydrophobic substrates (Yim et al., 2005; Maki et al., 2000; Perfumoet al., 2010). They are known to increase the viscosity of solutions at lowpH value and emulsify several hydrocarbon compounds (Guezennec etal., 1994;Calvo et al., 1998) andare intriguingmanyresearchers trying toharness their extraordinary properties and considerable potentialapplications in various elds (Weiner, 1997; Desai and Banat, 1997). Apolysaccharide namely PS 3a24 and PS 3a35 produced by marinebacteria has been shown tohavehigh specic viscosity, pseudoplasticity,and stability over a wide range of pH in the presence of a variety of salts(Boyle and Reade, 1983). Several Gram positive and Gram-negativemarine bacteria like Bacillus, Halomonas, Planococcus, Enterobacter,Alteromonas, Rhodococcus, Zoogloea, and Cyanobateria are known toproduce EPS (Zukerberg et al., 1979;Maugeri et al., 2002; Nicolaus et al.,2000) as listed below.

2.6.1. Bacillus producing exopolysaccharideVery few reports are available on EPS production by marine

Bacillus sp. Maugeri et al. (2002) isolated a Bacillus strain B3-15(halophilic, thermotolerant) from shallowwater, marine hot spring atVolcano Island (Eolian Islands, Italy). EPS production in Bacillus B3-15was carried out inmineral medium as supplementedwith glucose anddifferent type of EPS composed of tetrasaccharide repeating unit withsugars having a manno-pyranosidic conguration were produced.Another Bacillus strain B3-72, isolated from same shallow ventproduced structurally different EPS (Nicolaus et al., 2000). Thisindicates the diversity of EPS produced by same genus from thesimilar environment. Other few reports includes B. licheniformisisolated from Volcano Island produce EPS (Arena, 2004) andB. thermoantarcticus sea sand in Ischia Island (Nicolaus et al., 2000)for production of medical/pharmaceutically important polysacchar-ides. Arena et al. (2006) also reported novel type of EPS-1 poly-saccharide producing thermotolerant strain Bacillus licheniformis,which was isolated from a shallow marine hot spring of Volcano

Island (Italy). Antiviral and immunomodulatory effects of EPS-1studies showed that the EPS-1 treatment impaired HSV-2 replicationin human PBMC but not in WISH cells.

2.6.2. Halomonas producing exopolysaccharideSome microorganisms are capable of growing in extreme environ-

ments, where most other organisms are not able to survive. Amongthese extremophiles, halophiles are one of the major microbialcommunities that tolerates high salt concentrations and are highlysought after by many industries for their novel enzymes and productsthat has wider potential applications (Ventosa et al., 1998; Ventosaand Nieto, 1995).Halomonas is a Gram-negative bacterium, non-sporeforming, predominantly present in marine environments and areoften isolated from deep-sea sediments and hydrothermal vents.Halomonas sp. comprises a remarkably high percentage (up to N10%)of the total microbial community in their habitat (Kaye and Baross,2000). Most of the Halomonas spp. That has been reported for EPSproduction have been isolated from hypersaline environments withdifferent salt concentra (Calvo et al., 1995, 1998; Bjar et al., 1998;Martnez-Checa et al., 2002; Arias et al., 2003; Quesada et al., 1990,1993, 2004a,b; Bouchotroch et al., 2000, 2001). Gutierrez et al. (2009)isolated and studied physicochemical properties of EPS namely HMW-EPS from Halomonas sp. strain TG39 by growing on different types ofsubstrates. Culture medium supplemented with glucose and apeptone/yeast extract results EPS fraction (heterogeneous polymerHMW-glucose) that possess specic emulsifying activity(EI24=100%) as compared to culture medium supplemented withmannitol, sucrose, and malt extract for EPS production.

2.6.3. Planococcus producing exopolysaccharideA variety of marine environments harbour Planococcus abundant-

ly. These hydrocarbon-degrading microbes are signicant in control-ling the hydrocarbon contamination in marine environments(Engelhardt et al., 2001). Now it is known that Planococcus sp. arealso able to produce EPS which exhibit high emulsication property.Kumar et al. (2007) isolated P. maitriensis Anita I from the coastal seawater area of Bhavnagar district, India. The EPS has been precipitatedfrom cell-free supernatant using alcohol and was found to containcarbohydrate, protein, uronic acid and sulfate. The oil spreadingpotential of the puried EPS was comparable to Triton X100 andTween 80 and with good emulsifying and tensiometric propertieswhich make it a useful product in bioremediation, enhanced oilrecovery and cosmetics related applications.

2.6.4. Enterobacter producing exopolysaccharideThese are Gram-negative, motile, facultative anaerobic bacteria

which are widely distributed in nature. Enterobacter cloacae isolatedfrom marine sediment collected from Gujarat coast, India (Iyer et al.,2005) produce EPS 71a that emulsies various water-insolublesubstrates. As compared to commercial gums (Arabic, tragacanth,karaya and xanthan) EPS 71a form stable emulsion with xylene.Chemically EPS 71a is an acidic polysaccharide that contains highamount of uronic acid, fucose and sulfate. Such kind of EPS isuncommon in bacteria (Iyer et al., 2006).

2.6.5. Alteromonas producing exopolysaccharideThese are all Gram-negative (straight or curved rods), motile,

heterotrophic, mesophilic bacterium found in coastal water and theopen sea. Vincent et al. (1994) isolated Alteromonas sp. from Alvinellapompejana of deep-sea hydrothermal vent of East Pacic Rise.Alteromonas strain 1545 excretes EPS containing glucose, galactose,glucuronic acid, galacturonic acid and 4, 6-O-1-carboxyethilidene-galactose. From the same sampling site, an EPS producing Alteromonassp. strain 1644 was isolated by Bozzi et al., (1996a,b). Another EPS(p-11568) producing Alteromonas sp. was isolated from the South Seaof Korea and claimed to exhibit superior emulsifying activity as

compared with commercial available polysaccharides (Yim et al.,

444 S.K. Satpute et al. / Biotechnology Advances 28 (2010) 4364502005). Two types of structurally identical polysaccharides areproduced by marine microorganism Alteromonas haloplanktis KMM156. These polymers are acidic in nature, capsular and an O-speciccontains repeating tetrasaccharide units with L-rhamnose, 2-acet-amido-2-deoxy-D-glucose and a 3-O-[(R)-1-carboxyethyl]-D-glucose(Glc3Lac) residue (Gorshkova et al., 1993). Alteromonas macleodii2MM6 isolated from Intertidal zone of Halifax, Nova Scotia is reportedfor production of an acidic polysaccharide containing tetrasacchariderepeating units with D-galactose, 3-O-acetyl-2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-L-guluronic acid and 3,6-dideoxy-3-(4-hydroxybutyramido)-D-galactose residues showed the structure ofR:COCH2CH2CH2OH (Nazarenko et al., 1993).

Raguns et al. (1997) reported aerobic, mesophilic and hetero-trophic bacterium a new Alteromonas infernus sp. isolated from adeep-sea hydrothermal vent of Riftia pachyptila (Gulf of California).The strain secretes two types of unusual polysaccharides duringstationary phase in the culture medium supplemented with glucose.The EPS found is water-soluble containing monosaccharides likeglucose, galactose, galacturonic and glucuronic acids. Raguns et al.(1996) also isolated four bacteria from deep-sea hydrothermal ventsfound to produce four types of extracellular polymers having differentchemical composition and rheological properties.Alteromonasmacleodii

Fig. 2. Various roles played by exopolysaccharide (EPS) produsubsp. jiensis one of the new bacterium produce EPS similar to that ofxanthan containing glucose, galactose, mannose, glucuronic andgalacturonic acids.

2.6.6. Pseudoalteromonas producing exopolysaccharideChemically diverse types EPS are produced by Antarctic isolates

which mostly contains charged uronic acid residues and somecontains sulfate groups. Polymers produced by some of the isolatesfrom this microbial population are large polymers of approximately5.7 MDa in size (Mancuso Nichols et al., 2005c). EPS producingPseudoalteromonas sp. strain CAM025 and CAM036 were also isolatedfrom seawater and sea ice in southern ocean (Mancuso Nichols et al.,2004). Marine bacterium CAM025, isolated from sea ice sample shows30 times higher yield at 2 C and 10 C than at 20 C. Most of themarine bacterial isolates are psychrotolerant where their optimumgrowth occurs at about 20 C. However, these bacteria may produceoptimum EPS at lower temperature (Mancuso Nichols et al., 2005a).Recently, interesting work is reported by Gutirrez et al. (2008). Thisresearch group has described the production of polymeric emulsifyingagent from Pseudoalteromonas with the absorption property to metalions. The high-molecular-weight glycoprotein exopolymer PE12contains identical monosaccharide with exopolymers of bacteria.

ced by marine microorganisms in the marine ecosystem.

445S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450PE12 also contains uronic acid, protein similar to that of Pseudoalter-omonas/Alteromonas species. EPS producing Pseudoalteromonas strainHYD 721is isolated from deep-sea hydrothermal vent of East PacicRise which contains repeating unit of the polymer with a branchedoligosaccharide (Rougeaux et al., 1999a). Novel types of EPSproducing bacteria have been isolated from deep-sea hydrothermalvent (Rougeaux et al., 1996).

In the year of 1996, Raguns et al.'s group isolated twoPseudoalteromonas species from deep-sea hydrothermal vents pro-duce same types of polymer containing glucose, galactose, mannose,glucuronic and galacturonic acids. Studies with transmission electronmicroscopy (TEM) of immunogold-labeled indicated that the EPSproduction in Marine Pseudomonas sp. strain S9 is closely associatedwith the cell surface. EPS production takes place during the bacterialgrowth and energy-nutritional starvation condition (Wrangstadhet al., 1990). A previous study also reported secretion of EPS byPseudomonas sp. S9 during complete nutrient starvation and energy.When the cells were starved in agitated conditions, EPS secretionoccurs and different adhesion pattern to hydrophobic surfaces isobserved (Wrangstadh et al., 1986).

2.6.7. Rhodococcus producing exopolysaccharidesUrai et al. (2007) isolated marine R. erythropolis PR4 that produces

a large quantity of acidic EPS (FR1 and FR2). FR2 composed ofD-galactose, D-glucose, d-mannose, D-glucuronic acid, and pyruvicacid at a molar ratio of 1:1:1:1:1, and contained 2.9% (w/w) stearicacid and 4.3% (w/w) palmitic acid attached via ester bonds. It wasproposed that EPS plays a signicant role in the hydrocarbon toleranceof this bacterium.

2.6.8. Zoogloea producing exopolysaccharidesThese are Gram-negative, straight or slightly curved, plump rods,

non-spore or cyst forming free living bacteria in organically pollutedfresh and waste water bodies. Kwon et al. (1994) has reported theproduction of polysaccharide extracellularly from Zoogloea sp. Thismarine bacterium produces water-soluble polysaccharide: WSP (cell-free liquidmedium) and cell-bound polysaccharide: CBP (cell surface)containing glucose, galactose and mannose as main sugars and uronicacid. Zoogloea sp. KCCM10036 produces two types of EPS (CBP: cell-bound polysaccharide and WSP: water-soluble polysaccharide).Emulsions formed by cell-bound polysaccharide are highly stablewhen compared with other commercially available EPS such asxanthan gum, Tween series, Triton. Both types of EPS from Zoogloeasp. possess excellent occulating activity (Lim et al., 2007). Zoogloeasp. secretes water-soluble and the other cell-bound EPS possessingnon-Newtonian, pseudoplastic uid behaviour. Solutions of these EPSwere also reported to have low activation energies with goodrheological behaviour over broader pH range (212) and temperature(2080 C) in the presence of NaCl (Jang et al., 2002).

2.6.9. Cyanobacteria producing exopolysaccharideMucilaginous envelope coating is common around marine Cyano-

bacteria living in hypersaline environments and therefore, most ofthese cyanobacterial strains can produce EPS in the surroundingmedia (Campbell and Golubic, 1985). Fifteen EPS producing unicel-lular cyanobacterial strains have been isolated from saline environ-ments. Cyanothece EPS contains uronic acids, six to eightmonosaccharides, with one or two acidic sugars. Other chemicalgroups like acetyl, pyruvyl, and/or sulfate groups are also detected.EPS from such organisms are promising candidates for variousindustrial applications (De Philippis et al., 1998). Other EPS fromcyanobacterial strain Cyanothece 16Som2 which was isolated from aSomaliland saltpan contains various sugars like glucuronic acid,galacturonic acid, galactose, glucose, mannose, xylose and fucose.Nitrogen limitation condition favours the enhanced production of EPS

from Cyanothece sp. (De Philippis et al., 1993). EPS from Cyanothecesp. ATCC 51142 has high content of calcium and plays key role inremoving metals ions (Shah et al., 2000). Another report on EPSproducing Cyanothece sp. 113 isolated from salt lakes in China (Chiet al., 2007) reported novel type of -D-1,6-homoglucan. Othermarine cyanobacterial strains like Schizothrix sp and Oscillatoria sp.isolated from marine stromatolites, Bahamas have been reported forproduction of EPS (Kawaguchi and Decho, 2000).

2.6.10. Vibrio producing exopolysaccharidesVibrio is a Gram-negative marine bacterium and ubiquitous in

nature. Marine biofouling material were used to isolate EPS from V.alginolytics to carry out and physicochemical studies (Jayaraman andSeetharaman, 2003). Jodi et al. (2000) investigated the factor(s)responsible for the opaque and translucent phenotypes, the cellorganization within both colony types in V. parahaemolyticus. Theyshowed that both type cultures secrete EPS containing four sugars likeglucose, galactose, fucose and N-acetylglucosamine. They also notedthat the copious amount of EPS produced by the opaque strain lls theintercellular space within the colony, resulting in increased structuralintegrity and the opaque phenotype. The structure of the EPSproduced under laboratory conditions by Vibrio diabolicus (isolatedfrom a deep-sea hydrothermal vent of East Pacic Rise) showed thepresence of linear tetrasaccharide repeating unit (Rougeaux et al.,1999b). Raguns et al. (1996) reported production of EPS containingdifferent sugars like glucose, galactose, mannose, glucuronic andgalacturonic acids from the Vibrio species isolated from deep-seahydrothermal vents. An interesting patent is led by Guezennec et al.(2002) on genus Vibrio belonging to V. diabolicus for production anduses of water-soluble polysaccharides. Molecular weight of the EPSfrom this marine Vibrio is 800,000 Da and contains osamines (305%)with uronic acids (325%) and monosaccharide like glucuronic acid(11.2%), N-acetylglucosamine (18%), andN-acetylgalactosamine (7.9%).The EPS is similar to that of heparin and hence nds important applica-tions in pharmaceutics.

2.6.11. Other exopolysaccharide producing bacteriaOther than the above mentioned (2.6.1 to 2.6.10) marine bacterial

population, there are many other marine bacteria producing EPS.However, the number of reports available on each bacterial strain islimited. This shows the diversity in production of bioactive moleculesby microorganisms in the marine biosphere. For example, Hyphomo-nas strain MHS-3 isolated from shallow water sediments in PugetSound found to produce adherent form of exopolysaccharide (EPS)capsule associated with Glycine max lectin, Arachis hypogaea lectinand Bauhinia purpurea lectin (BPA). The capsule of Hyphomonas strainMHS-3 EPS has an effective adhesive property (Quintero and Weiner,1995). EPS namely, Marinactan from Flavobacterium uliginosum showsantitumor activity against sarcoma-180. Basically this bacteriumrequires typical marine sea water condition for growth. The noveltype of Marinactan is composed of glucose, mannose and fucose withthe proportion of 7:2:1 (Umezawa et al., 1983). It is possible todetermine the effect of surface on the yield and composition of EPSproduced by bacteria. This fact is well explained from thework on twomarine Desulfovibrio sp. Indl SRB-sulfate-reducing bacteria (SRB)isolated from severe corrosion failures. Bacterial isolates were grownin static batch cultures with and without the presence of carbon steelsurfaces. EPS of these Desulfovibrio sp. contained similar kind ofcarbohydrates, proteins and nucleic acids in with different quantities.Studies also showed that surface provided for the growth had effecton the type of EPS produced by Desulfovibrio sp. (Zinkevich et al.,1996). Highly mucoidal Mediterranean Sea isolate, Haloferax medi-terranei is an extreme halophilic archaebacterium which secretesheteropolysaccharide extracellularly. The EPS contains glucose,galactose, unidentied sugar, amino sugars and uronic acids. Therheological properties of EPS like pseudoplasticity and viscosity are

seen effectively at extreme ranges of pH, temperature and salinity.

446 S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450Therefore, such polymer has potential applications to enhance oilrecovery processes. The EPS can be used as potential thickening agentin various industries (Anton et al., 1988). Geobacillus thermodenitri-cans, a bacterial isolate recovered from vent of Vulcano Island (Italy)produce EPS-2 shows immunomodulatory and antiviral effect byhindering HSV-2 replication in human peripheral blood mononuclearcells (PBMC). The concentration or the dose of EPS-2 is important forits immunomodulatory and antiviral activity. Arena (2004) alsoclaimed that EPS-2 successful immunological disorders determinedby HSV-2 could be partially restored by its treatment. Geobacillus sp.4004, a thermophilic (60 C) strain isolated from sea sand at Maronti,near Sant' Angelo (Ischia Island), secretes high amount of EPSexocellularly in the medium supplemented with carbon sources likesucrose/trehalose. Purication of EPS resulted three fractions namelyEPS1, EPS2, and EPS3, where different sugars with different propor-tions were present. Among these three, detailed studies on EPS3shows the molecular weight of 1106 Da with a pentasacchariderepeating unit (Schiano Moriello et al., 2003). EPS producing Gram-negative bacterium, aerobic, Hahella chejuensis 96CJ10356(T), isisolated from marine sediment recovered from Marado, Cheju Island,Republic of Korea. To achieve optimum growth, presence of NaCl (2%)(w/v) is essential (Lee, et al., 2001). Rinker and Robertm (1996)studied a hyperthermophilic archaeon Thermococcus litoralis fordeveloping sulfur-free, dened growth medium, production of EPSand biolm formation. T. litoralis secretes soluble EPS (with thepresence of mannose) in an articial-seawater medium supplemen-ted with 16 different amino acids, vitamins and trace elements. EPSplays an important role in the biolm formation on polycarbonatelters and glass slides. The authors proposed that such kind of work isimportant in reveling information on the interactions among high-temperature organisms. Other moderately halophilic eubacteriumnamely Volcaniella eurihalina is reported for production of EPS. TheEPS produced by this bacterium is composed of glucose, rhamnoseand mannose and has interesting physical and chemical propertieswhich have enormous applications in industries (Calvo et al., 1995;Quesada et al., 1990, 1993, 1994).

3. Potential applications of marine microbial surface active agentsand exopolysaccharides

The promising progress in the eld of marine biotechnology hasattracted many researchers towards marine microbial surface activeagents and exopolysaccharides. To date, various marine microbes areknown for production of various bioactive compounds. Discoveries ofnew marine microbial BS/BE and EPS are helping to explore themarine ecosystem. Different industrial sectors like textile, pharma-ceutical, cosmetics, food, metal mining, oil recovery, and metalrecovery are continuously searching for novel bioactive products.Due to the structural and functional novelty of these marine bioactivecompounds numerous applications are developing in various elds.Tensiometric and emulsication properties of biosurfactant/bioemul-sier are important for application purposes (Baird et al., 1983;Sutherland and Ellwood, 1979; Sutherland, 1998). Some of the marinepolymeric biosurfactant/bioemulsier with their application proper-ties are summarised in Tables 2 and 3.

Initially, microbial surface active agents were explored asantibiotics (due to their antimicrobial activities). Today a number ofbiosurfactant/bioemulsier with antimicrobial activities are availableand exploited. Most of the common examples for antimicrobialactivity of biosurfactant are shared from Bacillus species. Das et al.(2008a) disclosed that the single nonhaemolytic fractions of lipopep-tide biosurfactant (from B. circulans) has a powerful antimicrobialactivity and hence these type of surface active agents have applica-tions in antimicrobial chemotherapy. Mukherjee et al. (2009) puried(by gel ltration) a biosurfactant from marine B. circulans in glucose

mineral salts (GMS) medium where the puried biosurfactantsexhibit enhanced surface and antimicrobial activities. Thus, authorsproposed that such kind of biosurfactant can serve as new potentialdrugs in antimicrobial chemotherapy.

The use of biodispersion in paper manufacturing industries is oneof interesting approach for routine applications. The addition ofbiodispersion as ller along with paper and limestone improves thequality of paper and signicantly reduces the time required for thegrinding process (Rosenberg et al., 1989). Marinemicrobial surfactantact as an effective antiadhesive. Recently, Das et al. (2009a)investigated the antiadhesive action of a lipopeptide biosurfactantfrom a marine bacterium and the effect of cultivation conditions onthe adhesion properties. Under oxygen limiting conditions staticcultures showed a good adhesion property which was conrmed withthe help of confocal laser scanning microscopy studies. Otherbiosurfactant producing marine bacterium has also been shown toremove metal from solutions with the efciency dependent on theconcentration of the metal as well as the biosurfactant. Das et al.(2009b) showed that concentrated biosurfactants with low CMC,removes 100 ppm of lead and cadmium. Various other techniques likeatomic absorption spectroscopy (AAS), Fourier transform infraredspectroscopy (FTIR) and transmission electron microscopy (TEM)equipped with energy dispersive X-ray spectroscopy (EDS) also play asignicant role in such investigations.

Polysaccharides of bacterial origin are very important in pharma-ceutical industries. EPS secreted by marine bacteria contains novelcombination which has potential applications in various industrialsectors. Various properties of EPS like thickening, coagulating,adhesion, stabilizing and gelling are utilized in various industries.EPS with good viscosity and pseudoplasticity properties are resistantto the extremities of temperature, pH and salinity. Examples ofsuch EPS an emulsier produced by HaheUa chejuensis gen. nov., sp.nov. (Lee et al., 2001), Haloferax mediterranei. Alteromonas sp. strain1545 producing acidic EPS with thickening property (Talmont et al.,1991) and EPS from Cyanothece sp. ATCC 51142 with gelling property(Shah et al., 2000). Other novel EPS produced by Antarctic bacteriacontains high uronic acid, and a sulfate content which is important inbiotechnological point of view. EPS namely PS3a24 and PS3a35 frombacteria show pseudoplastic owing properties at higher concentra-tions and stability at broader pH range. Such properties are seen inxanthan gum and therefore, these two EPS are very important in theapplication point of view (Boyle and Reade, 1983). EPS fromPseudomonas sp. strain NCMB 2021 can attach effectively to thesolid surfaces (Christensen et al., 1985) while those produced byHyphomonas adhaerens helps in biolm formation and give adhesiveproperty to cells and assist their growth in metal containingenvironment (Quintero and Weiner, 1995; Quintero et al., 2001).Such metal resistant properties are very much important frombiotechnological applications point of view.

Chemically modied (sulfation and acidic depolymerization) EPSsecreted by A. infernus has anticoagulant activity similar to heparinand therefore, is important for the treatment of lipemia andarteriosclerosis (Guezennec et al., 1998). Another heparin like EPS issecreted by Vibrio sp. and has been reported to have anticoagulant andantithrombotic properties and can be utilized as an antiviral,antitumour or antithrombotic agent (Guezennec et al., 2002). Thereis a similar report on anticoagulant properties of heparin by modiedEPS from Alteromonas infernos. Such pharmaceutically importantcompounds can be modied for suitable application purpose (Colliec-Jouault et al., 2004). EPS produced by Bacillus licheniformis andGeobacillus thermodenitricans can act as strong stimulators for Thlcell-mediated immunity. Such immunomodulatory agents can play amajor role in the treatment of immunocompromised patients (Arena,2004). Similarly it was recently reported that a Geobacillus thermo-denitricans obtained from a shallow marine vent of Volcano Island(Italy) produced an EPS-2 having immunomodulatory and antiviral

effects on immunocompetent cells (Arena et al., 2009). In addition

447S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450Vibrio diabolicus has been reported to secrete HE800 EPS whichpossess signicant bone healing property by formation of extracellu-lar matrix for the direct adhesion of osteoblasts, osteoprogenitor cellsand pericytes. This polysaccharide provides the ideal conditionsrequired for bones growth and healing process (Zanchetta et al.,2003). Furthermore Marinactan, an EPS produced by Flavobacteriumuliginosum has been shown to be an effective antitumour againstsarcoma-180 solid tumor inmice through inhibition of growth of solidsarcoma 180 in mice. Marinactan also inhibit the growth of solidsarcoma 180 even before and after tumor transplantation (Umezawaet al., 1983). Other marine Vibrio sp. producing EPS have beenreported to have an effective antitumor and immunostimulantactivities which are quite important from a medicinal point of view(Okutani, 1984, 1985).

EPS produced by marine microbes plays a crucial role in theremoval of pollutant metals and toxic elements from contaminatedsolutions. Their emulsication and surface active properties makesthem applicable for utilization in bioremediation and other oilrecovery and cosmetics related applications (Kumar et al., 2007).Strains such as Cyanothece sp. ATCC 51142 produce EPS which areessential for removal of metals (Shah et al., 2000). Other EPS rich inuronic acid content have a high afnity towards heavy-metals makingthem suitable for the removal and/or minimization of metal toxicity(Raguns et al., 1996). Similarly Alteromonas sp. strain 1644produces EPS with binding ability towards monovalent and divalentions (Bozzi et al., 1996b) while isolates from deep-sea hydrothermalvents produce EPS, which can bind and remove toxic metals like lead,cadmium, and zinc. They acts as a powerful chelating agent and hencethey are useful for wastewater treatment and other environmentalapplications (Loac et al., 1998).

4. Conclusions

In spite of a long history of BS/BE production frommicroorganisms,very few marine microbial communities viz., Acinetobacter, Arthro-bacter, Pseudomonas, Halomonas, Myroides, and Corynebacteria sp.have been explored for production of surface active molecules. BS/BEand EPS produced bymarinemicrobes have some novel structural andfunctional properties. Several high molecular weight polymer andglycolipid type BS/BE are produced by marine microbes and haveimportant potential application in different industries. Marineecosystems therefore provide an excellent opportunity to selectpotent microorganisms. Effective screening methodologies andimproved purication techniques are essential in order to achievedesired quantities and qualities of BS/BE. Promising recent biotech-nological approaches will support the search of potent BS producersfrom this important ecosystem.

Acknowledgements

Surekha K. Satpute, thanks the UGC, Govt. of India, for nancialsupport {(F.17-37/98(SA-I)}. This work is also supported by theresearch project sanctioned by DBT (BT/PR304/AAQ/03/155/2002),Govt. of India.

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