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Biotechnology Advances 28 (2010) 436450

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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v

Research 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, India


Institute of Bioinformatics and Biotechnology, Pune 411007, Maharashtra, India
School of Biomedical Sciences, University of Ulster, Coleraine, BT52 1SA, N. Ireland, UK
d
Division of Microbial Sciences, Agharkar Research Institute, Pune 411004, India
e
Center for Advanced Studies in Materials Science and Condensed Matter Physics, Department of Physics, University of Pune, Pune 411007, Maharashtra, India
b
c

a r t i c l e

i n f o

Article history:
Received 1 June 2009
Received in revised form 12 December 2009
Accepted 3 February 2010
Available online 19 February 2010
Keywords:
Marine
Biosurfactant
Bioemulsier
Exopolysaccharides
Glycolipids
Lipopeptides
Applications

a b s t r a c t
Marine biosphere offers wealthy ora and fauna, which represents a vast natural resource of imperative
functional commercial grade products. Among the various bioactive compounds, biosurfactant (BS)/
bioemulsiers (BE) are attracting major interest and attention due to their structural and functional
diversity. The versatile properties of surface active molecules nd numerous applications in various
industries. Marine microorganisms such as Acinetobacter, Arthrobacter, Pseudomonas, Halomonas, Myroides,
Corynebacteria, Bacillus, Alteromonas sp. have been studied for production of BS/BE and exopolysaccharides
(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 environmental
biodegradable surface active molecule and hopefully reduce total dependence or number of new application
oriented towards the chemical synthetic surfactant industry. Our present review gives comprehensive
information on BS/BE which has been reported to be produced by marine microorganisms and their possible
potential future applications.
2010 Elsevier Inc. All rights reserved.

Contents
1.
2.

Introduction . . . . . . . . . . . . . . . . . . . .
Marine microorganisms The rich natural resource of
2.1.
Polymeric biosurfactant/bioemulsiers . . . .
2.1.1.
Acinetobacter . . . . . . . . . . . .
2.1.2.
Pseudomonas . . . . . . . . . . . .
2.1.3.
Myroides . . . . . . . . . . . . . .
2.1.4.
Halomonas . . . . . . . . . . . . .
2.1.5.
Yeast . . . . . . . . . . . . . . . .
2.1.6.
Streptomyces . . . . . . . . . . . .
2.1.7.
Antarctobacter . . . . . . . . . . . .
2.1.8.
Marinobacter . . . . . . . . . . . .
2.2.
Glycolipid surface active molecules . . . . . .
2.2.1.
Alcaligenes sp. . . . . . . . . . . . .
2.2.2.
Arthrobacter . . . . . . . . . . . . .
2.2.3.
Alcanivorax borkumensis . . . . . . .
2.2.4.
Rhodococcus. . . . . . . . . . . . .
2.2.5.
Halomonas . . . . . . . . . . . . .
2.2.6.
Unidentied marine bacterium MM1 .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
biosurfactant (BS)/bioemulsier (BE), exopolysaccharides (EPS) producers
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Corresponding author. Institute of Bioinformatics and Biotechnology and Professor of Microbiology, University of Pune, Pune 411007, Maharashtra, India. Tel.: + 91 2025691333;
fax: + 91 2025690087.
E-mail address: directoribb@unipune.ernet.in (B.A. Chopade).
0734-9750/$ see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2010.02.006

S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

2.3.
2.4.
2.5.
2.6.

Lipopeptide surface active molecules . . . . . . . . . . . . . . . . . . . . .


Phospholipids and fatty acids surface active molecules. . . . . . . . . . . . .
Glycolipopeptide surface active molecules . . . . . . . . . . . . . . . . . .
Exopolysaccharides (EPS)/complex surface active polymer . . . . . . . . . . .
2.6.1.
Bacillus producing exopolysaccharide . . . . . . . . . . . . . . . .
2.6.2.
Halomonas producing exopolysaccharide . . . . . . . . . . . . . . .
2.6.3.
Planococcus producing exopolysaccharide. . . . . . . . . . . . . . .
2.6.4.
Enterobacter producing exopolysaccharide . . . . . . . . . . . . . .
2.6.5.
Alteromonas producing exopolysaccharide . . . . . . . . . . . . . .
2.6.6.
Pseudoalteromonas producing exopolysaccharide . . . . . . . . . . .
2.6.7.
Rhodococcus producing exopolysaccharides . . . . . . . . . . . . . .
2.6.8.
Zoogloea producing exopolysaccharides . . . . . . . . . . . . . . .
2.6.9.
Cyanobacteria producing exopolysaccharide . . . . . . . . . . . . .
2.6.10. Vibrio producing exopolysaccharides . . . . . . . . . . . . . . . . .
2.6.11. Other exopolysaccharide producing bacteria . . . . . . . . . . . . .
3.
Potential applications of marine microbial surface active agents and exopolysaccharides
4.
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
The hydrosphere marine environment represents the major
component of the Earth's biosphere. It covers a majority (70%) part
of earth's surface and makes up N90% of the volume of its crust. The
maximum depth of N11,000 m and 365 million km2 is attributed to
the hydrosphere oceanic system. Oceans represent a vast and
exhaustive source of natural products in the globe, harbouring the
most diverse groups of ora and fauna. Marine microorganisms have
developed unique metabolic and physiological capabilities to thrive in
extreme habitats and produce novel metabolites which are not often
present in microbes of terrestrial origin (Fenical, 1993). Therefore,
this rich marine habitat provides a magnicent opportunity to
discover newer compounds such as antibiotics, enzymes, vitamins,
drugs, biosurfactant (BS), bioemulsier (BE) and other valuable
compounds 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 great
importance due to their structural and functional diversity and
industrial applications (Banat et al., 1991; Banat, 1995a,b; Rodrigues
et al., 2006). Biosurfactants and bioemulsiers are amphiphilic
compounds containing both a hydrophilic and a hydrophobic moiety
and therefore are able to display a variety of surface activities that
allow solubilisation of hydrophobic substrates (Desai and Banat,
1997). During the last decade they have been under investigation as
potential replacements for synthetic surfactants and are expected to
have many potential industrial and environmental applications
related to emulsication, foaming, detergency, wetting, dispersion
and solubilisation of hydrophobic compounds (Banat et al., 2000;

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Dastgheib et al., 2008). They generally can be grouped either as low or


high molecular weight biosurfactants (Smyth et al., 2010a,b) the
former consisting of glycolipids and lipopeptides and the latter of high
molecular weight polymeric biosurfactants. To date, more than 225
patents are available describing these microbial amphiphilic agents
(Shete et al., 2006). More than 10,000 metabolites with broad
spectrum biological activities and amazing medicinal properties
have been isolated from marine microbes (Fusetani, 2000; Kelecom,
2002). However, due to the enormity of the marine biosphere, most of
the marine microbial world remained unexplored. It has been
estimated that b0.1% of marine microbial world has been explored
or investigated (Ramaiah, 2005). Such investigations are mainly
hindered, due to the non-culturable nature of most microorganisms
under laboratory conditions (Harayama et al., 2004). Some of the
major features of bioactive producing marine microorganisms in
comparison to those for other habitats are presented in Table 1.
Despite the fact that marine environment represents a wealthy
basin of diverse microorganisms, it is important to know that at the
same time they are suffering from anthropogenic pollution with
domestic and industrial wastes (Hassan et al., 1996). Large quantities
of crude oil, hydrocarbons, petroleum oil products and halogenated
compounds nds their way into the marine ecosystem through
accidental spillage (Satpute et al., 2005). To treat, emulsify or simply
overcome such spills, on the one hand, many petroleum based
synthetic chemical surfactants often get used. Such synthetic
compounds on the other hand, however often have detrimental
ecological effects (Smith, 1968; Smith et al., 1968). The use of
biosurfactants and bioemulsiers therefore may represent a better
alternative to overcome the toxicity of synthetic compounds

Table 1
Major 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

Probability to obtain the particular microbes

Constant churning in the marine biosphere, difcult


to obtain particular type of microbial population
Relatively difcult to culture in the laboratory
Difcult to maintain humidity, salinity, and other
extreme conditions in the laboratory
Has diverse biological and functional properties
Relatively higher toxicity
Lower chances due to extreme environments halophilic,
thermophilic, acidophilic or alkalophilic
Limited (about 56 reports are available)

Fresh water, industrial efuents, soil, air, waste


water, sewage, oil wells, and reneries
More chances of obtaining particular type of
microbial biota from particular sampling site
Mostly easily cultured in the laboratory
Environmental condition for growth of biosurfactants/
bioemulsier producers are mostly easy to maintain
Generally similar type of functional properties
Less toxic and mostly biodegradable
Can be easily contaminated

Ability to cultivate
Maintenance of environmental and
physiological requirement
Novelty in structures and functional properties
Toxicity nature of biosurfactant/bioemulsier
Possibility of culture contamination
Literature availability

Quite large

438

S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

(Edwards et al., 2003). It is important to note that, on the whole, BS/BE


produced by soil or freshwater microorganisms are less toxic and are
mostly biodegradable (Poremba et al., 1991a,b) which means they do
not accumulate in the environment. Under such conditions, marine
microbes offer a rich source of novel surface active compounds.
Effective screening methods for biosurfactants and bioemulsiers are
therefore needed to tap into this vast resource. Combination of
various such screening methodologies to obtain potent BS producers
has been recently reviewed by Satpute and co-workers (2008).
Microbial communities like Acinetobacter, Arthrobacter, Pseudomonas,
Halomonas, Bacillus, Rhodococcus, Enterobacter, and yeast have been
reported to produce BS/BE (Das et al., 2008a,b; Schulz et al., 1991; Passeri
et al., 1992; Banat, 1993; Abraham et al., 1998; Maneerat et al., 2006;
Perfumo et al., 2006). About 56 reports including 35 for bioemulsier, 12
for glycolipid, and 9 for other types are available on different types of
BS/BE produced by marine microorganisms (Fig. 1). Few reviews are
available 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 review
therefore aims to report/collate up to date information available on
different types of BS/BE and EPS produced by different marine
microorganisms and highlight their potential applications.
2. Marine microorganisms The rich natural resource of
biosurfactant (BS)/bioemulsier (BE), exopolysaccharides
(EPS) producers
Surface active molecules producing microorganisms are ubiquitous, 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 of
temperatures, pH values and salinity. Microorganisms produce BS/BE
to mediate solubilisation of hydrophobic compounds in their
environment to be able to utilize them as substrates (Margesin and
Schinner, 2001; Olivera et al., 2003; Floodgate, 1978), however, this
fact may not be always true. Few microbes produce BS/BE on watersoluble substrates (Gunther et al., 2005; Turkovskaya et al., 2001). It
has been suggested that the presence of surface active molecules on
the microbial cell surface increases the hydrophobicity of the cell and
helps it to survive in hydrophobic environment (Abraham et al., 1998;
Perfumo et al., 2010). BS/BE produced by microbes may either be
extracellularly released into the environment or may be localized on
surfaces i.e. become associated with the cell membrane. When BS/BE
are associated with cell, the organisms itself behave as a BS/BE in
controlling the adherence property to water-insoluble substrates
(Maneerat and Dikit, 2007). The synthesis of these surface active
molecules takes place by de novo pathway and/or assembly from
substrates (Syldatk and Wagner, 1987) which is diagrammatically
represented by Satpute et al. (2010). Various BS/BE producing
bacteria have been isolated and characterized from marine sites
contaminated with oil, petroleum or their by-products. The use of
different separation techniques ensure the ability to efciently extract
high and low-molecular weight BS/BE. Different screening methods
are available to identify BS/BE producing microorganisms. However,
due to the diverse functional and chemical properties of each type of
marine BS/BE, it is difcult to obtain BS/BE producers with single
screening method. Several screening methodologies are therefore
essential to isolate and investigate potential marine BS producer

Fig. 1. Summary of different types of marine biosurfactant/bioemulsier producing microorganisms.

S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

(Satpute et al., in press). Isolation from petroleum contaminated sites


using the screening protocol presented proved to be a rapid and
effective manner to identify bacterial isolates with potential industrial
applications (Batista et al., 2006). A wide variety of genera producing
diverse types of BS/BE (Tables 2 and 3) are associated with the marine
environment and produce various types of molecular structures and
composition described in detail as follows.
2.1. Polymeric biosurfactant/bioemulsiers
2.1.1. Acinetobacter
Species of Acinetobacter are ubiquitous in nature and are one of the
commonly found Gram-negative bacteria in marine environments. They
are a predominant (followed by members of Pseudomonas) species in
marine 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 to
their versatile nature, Acinetobacter sp. has attracted considerable
attention by many researchers. Other than marine environment a
number of other sources has been studied in great detail (Patil and
Chopade, 2001a,b, 2003). Species of Acinetobacter play an important role
in hydrocarbon degradation (Juni, 1978) and have a key role in bioremediation processes (MacCormack and Fraile, 1997). A. calcoaceticus

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RAG-1 was isolated from the Mediterranean Sea (Reisfeld et al., 1972)
and found to produce a surfactant (Rosenberg et al., 1979), which has
been exploited commercially as the product Emulsan. A number of
potential applications of emulsan has been patented by Gutnick and coworkers (1981) and was marketed by Petrorm Inc. for cleaning of oil
contaminated vessels, oil spills and microbial enhanced oil recovery
(MEOR) (Desai and Patel, 1994). Emulsan is a high molecular weight
heteropolysaccharide protein containing repeating trisaccharide of
N-acetyl -D-galactosamine, N-acetylgalactosamine uronic acid and an
unidentied N-acetyl amino sugar. Fatty acids (FA) are covalently linked
to 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 low
concentration (b0.001 to 0.01%), emulsan can emulsify hydrocarbons
efciently and is the most powerful emulsion stabilizer having several
applications in diverse elds (Belsky et al., 1979; Gutnick and Shabtai,
1987; Zosim et al., 1982). Different Acinetobacter sp. produce protein
polysaccharide complexes that possess surface active properties
(Kaplan and Rosenberg, 1986; Shete, 2003). A great deal of studies
have been carried out regarding isolation, emulsifying, chemical, physical
properties, and specicity to hydrocarbon substrate (Rosenberg et al.,
1979). A. calcoaceticus represents an excellent candidate for the
availability of maximum number of reports in the application area.

Table 2
Summary 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: at


low concentration, effective emulsier
(emulsionhydrocarbons 1:100 to 1:1000)

A. calcoaceticus A2

Biodispersion: four reducing sugars;


glucosamine, 6-methylaminohexose,
galactosamine uronic acid, unidentied
amino sugar (51.4 kDa)
Alasan: protein polysaccharide (1 MDa)
Contains covalent bound alanine

Considerable substrate specicity


Dispersion of limestone and titanium
dioxide; better dispersion of water

Belsky et al. (1979), Gutnick


and Shabtai (1987),
Zosim et al. (1982), Rosenberg et al.
(1979, 1988a,b; 1989)
Rosenberg and Kaplan (1987),
Rosenberg and Ron (1998)

A. radioresistens

A. calcoaceticus BD4

BD4 emulsan : protein and polysaccharide

Pseudomonas nautica

Proteins, carbohydrates and lipids at


(35:63:2) ratio
Trehaloselipid-o-dialkyl
monoglyceridesprotein

Pseudomonas uorescence

Myroids

Streptomyces
Yarrowia lipolytica NCIM 3589
Yarrowia lipolytica IMUFRJ 50682

L-ornithine lipids and a different couple of


iso-3-hydroxyfatty acid (C15C17) and
iso-fatty acid (C15 or C16) 1:1:1 ratio
Bioemulsier: protein (82%), reducing sugar (1%),
and polysaccharide (17%)
Bioemulsier: lipidcarbohydrateprotein
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 HE67


High molecular weight glycoproteins with a
high content of protein and uronic acids
Sulfated heteropolysaccharide
High-molecular-weight glycoprotein with high
uronic acids content, polyanionic structure

Halomonas eurihalina
Antarctobacter

Lowers surface tension (6942 mN/m)


Acts as surfactant
Effective stabilizer oil-in-water emulsions
Polysaccharide attach with protein
(which is bound to hydrocarbon) and
stabilization of oil-in-water emulsions
Emulsifying activity
High yield with gasoline
Depending upon hydrocarbon source;
different level of emulsication against
gasoline
Superior surface activity as compared
with other surfactants
Higher stability; emulsication activity
Signicant emulsication activity
Reduction of surface tension
Emulsication activity
High emulsication activity
Oil-in-water emulsions
Yansan-based emulsions change the
aging mechanisms of oil-in-water
emulsions from coalescence (at pH 3
to molecular diffusion at pH 7) as
compared with gum Arabic
(age by coalescence)
Emulsies kerosene and crude oil
efciently
Highest emulsifying activities
Highly stable at neutral, acidic pH and
even at high-temperature treatment
Emulsication
Better stabilizing activity than emulsication
Interesting functional properties as
compared with other biopolymers
Emulsion-stabilizing agent: food oils

Navon-Venezia et al. (1995)

Kaplan et al. (1987)

Husain et al. (1997)


Desai et al. (1988)

Maneerat et al. (2006), Maneerat


and Phetrong (2007), Maneerat
and Dikit (2007)
Kokare et al. (2007)
Zinjarde and Pant (2002)
Trindade et al. (2008),
Amaral et al. (2006)

Oloke and Glick (2005)


Gutirrez et al. (2007a)

Calvo et al. (1998)


Gutirrez et al. (2007b)

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S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

Table 3
Summary of marine glycolipid and other types of biosurfactant (BS)/bioemulsier (BE) produced by different microorganisms.
BS/BE type

Source organism

Chemical composition

Application property

Reference

Glycolipid

Alcaligenes

Non-toxic glucoselipid

Arthrobacter

Trehalose tetraester glycolipid:


an anionic trehalose lipids 2,3,4,2-trehalose
tetraester with fatty acids chain length
form 8 up to 14
Anionic glucoselipid with tetrameric
oxyacyl side chain, N-terminally
esteried with glycine: four 3-hydroxydecanoic
acids linked with glucose containing
glycine and -glucopyranoside esteried with
3-hydroxyhexanoic-octanoic and decanoic acids
Two different types of biosurfactant with respect
to the structure and chemical composition of BS

Inhibition of the growth of


microagellates and microalgae
Surfactant activities
Effective interfacial and
emulsifying properties

Poremba et al. (1991a)


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

Surface activity

Abraham et al. (1998)

Enhanced the solubility of polycyclic


aromatic hydrocarbons and the
degradation rate of hexadecane
Emulsifying properties

Peng et al. (2007)

Good surface active agent

Passeri et al. (1992)

Pronounced antimicrobial activity


No haemolytic activity
Antimicrobial activity
Surface tension of water from
72 to 28 mN/m with CMC (critical
micelle concentration): 12 mg/liter
Powerful surface active agent compared
with other surfactants
Good antibacterial activity

Das et al. (2008a)


Mukherjee et al. (2009)

Emulsication of waste motor lubricant oil,


crude oil, diesel, kerosene, naphthalene,
anthracene and xylene
Good surface active agent

Thavasi et al. (2008)

Emulsication of different hydrocarbons

Thavasi et al. (2007, 2008)

Alcanivorax borkumensis

Rhodococcus

Halomonas

Unidentied marine
bacterium MM1

Lipopeptide

Bacillus circulans

Bacillus licheniformis BAS50

Azotobacter chroococcum

Phospholipids
and fatty acids
Glycolipopeptide

Myroides
Corynebacterium kutscheri

Glycolipid: mannose (1.71), galactose (1.00),


glucose (2.96); lipid: fatty acid mixture with
a caprylic acid (18.85), myristic acid (1.0),
palmitic acid (9.68), palmitoleic acid (5.69)
and oleic acid (1.26)
Cell-bound, novel type glycolipid with four
3-OH-decanoic acids, linked by ester bonds.
The lipophilic moiety is coupled glycosidically
with C-1 of glucose
Novel type of lipopeptide biosurfactant

Lipopeptide: lichenysin (1006 to 1034 Da),


the lipid moiety: mixture of 14 linear and
branched beta-hydroxy fatty acids ranging
in size from C12 to C17. Amino acids: glutamic
acid as the N-terminal amino acid, asparagine,
valine, leucine, and isoleucine as the C-terminal
amino acid, at a ratio of 1.1:1.1:1.0:2.8:1.0,
respectively.
Lipid and protein (31.3:68.7)

Bile acids with cholic acid, deoxycholic acid


and their glycine conjugates
Carbohydrate (40%), lipid (27%) and
protein (29%)

Pepi et al. (2005)

Yakimov et al. (1995)

Maneerat et al. (2005)

a
Bacillus licheniformis BAS50 has been isolated from oil reservoir (at 1500 m) but it can grow at high salt up to 13% and optimum salt around see water level so it resembles marine
bacteria and can grow at a marine environment.

A. calcoaceticus BD4 and BD413 produce extracellular BD4 emulsan in the


production medium supplemented with ethanol (2%). Amphipathic
properties of BD4 emulsan are due to the association of an anionic
hydrophilic polysaccharide with proteins. Selective digestion, deproteinization, reconstitution and chemical modication studies proved that
the polysaccharide and protein fractions are essential for emulsication
activity (Kaplan et al., 1987). An extracellular, anionic non-dialyzable
polysaccharide named biodispersion was reported from A. calcoaceticus
A2. Biodispersion, the name itself suggests the dispersing property for
water-insoluble compounds (Rosenberg et al., 1988a,b, 1989; Rosenberg
and Ron, 1998). Biodispersion has an average molecular weight of 51,400
and along with the extracellular polysaccharide a rich protein is also
secreted by A. calcoaceticus A2 species. Mutants' defective in protein
production gives enhanced yield of biodispersion as compared to the
parent strain (Elkeles et al., 1994). There is another report suggesting that
Acinetobacter sp. A3 produces two proteins/polypeptides of molecular
masses of 26.5 kDa and 56 kDa when grown on crude oil as sole source.
Acinetobacter sp. A3 utilizes Bombay High crude oil without production of
any surface active agents. Hanson et al. (1994) proposed that the direct
celloil interaction facilitates the crude oil utilization by the bacterium.
Bioemulsier producing marine Acinetobacter calcoaceticus subsp. anitratus SM7 strain was isolated from oil contaminated seawater in

Songkhla lagoon, Thailand. The emulsier emulsies n-hexadecane


effectively at a wide pH (612) and temperatures ranges (30121 C)
and in the presence of up to 12% NaCl. Phetrong et al. (2008) suggested
that such emulsier with stable properties at broad range of pH,
thermostability and salt tolerance may play a signicant role in
environmental application, especially bioremediation of oil-polluted
seawater.
2.1.2. Pseudomonas
BS produced by marine microbes involved in the degradation of
hydrocarbons in surrounding environment. Coelho et al. (2003) reported production of BS by quinoline degrading marine Pseudomonas
sp. strain GU 104. Comparative studies of synthetic surfactant and BS
produced by Pseudomonas on the physiology of Perna viridis L, an
economically important mussel used for food, a bivalve belonging to
the family Mytilidae indicate that BS does not affect the physiology of
P. viridis. There is an additional single report available on polymeric BS
producing Pseudomonas strain. P. nautica which was isolated from a
coastal area of the Mediterranean Sea (Husain et al., 1997) and
reported 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

S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

fact is well proved by Desai et al. (1988) from the production of


trehalose lipido-dialkyl monoglyceridesprotein emulsier by hydrocarbon degrading P. uorescens. Another similar contribution by
Christensen et al. (1985) suggests the production of emulsier from
marine Pseudomonas sp. NCMB 2021 extracellularly at the end of the
exponential growth phase and in the stationary phase of growth.
Polysaccharide contains deoxy sugars and acetyl groups with outstanding solubility even at high concentrations of phenol, methanol
and ethanol.
2.1.3. Myroides
It is an aerobic, Gram-negative, non-motile, pigmented (yellowto-orange) rod-shaped bacterium usually found in marine ecosystem.
The majority of work carried out on BE producing Myroides was on
strain sp. SM1 which was isolated from oil-spilled seawater in
Songkhla Lake, Thailand (Maneerat et al., 2005). Myroides sp. SM1,
grows well, emulsifying weathered crude oil and produce extracellular BE (complex of L-ornithine lipidsL-ornithine and a different
couple of iso-3-hydroxy fatty acid and iso-fatty acid) that possesses
high surface activity for oil displacement as compared with other
surfactants (Maneerat et al., 2006). BE from such an extreme
environment shows higher stability at a broad temperature range.
However, its emulsication activities diminish rapidly at extreme pH
and high salt concentration (Maneerat and Phetrong, 2007). Cell
associated surface active molecules from Myroides sp. possess high
emulsication activity by adhering to weathered crude oil (Maneerat
and Dikit, 2007).
2.1.4. Halomonas
Glycoprotein (protein and uronic acids) based bioemulsier are
also produced by Halomonas sp. and have been characterized
chemically and physically by Gutirrez et al. (2007a). BE from two
new Halomonas sp., TG39 and TG67 has been concentrated by
ultraltration, and precipitation. This is the rst report on the highest
emulsifying activities of BE produced from Halomonas sp. Surface
active molecules from both strains are also highly stable at neutral,
acidic pH and at high temperatures.
2.1.5. Yeast
Yarrowia lipolytica is one of the well reported yeast for production
of lipidcarbohydrateprotein based bioemulsiers. This polysaccharide based bioemulsier can increase the hydrophobicity of the cells
during the growth phase. Studies carried out by Zinjarde and Pant
(2002) showed that extracellular production of BE does take place
when cell enters into stationary phase. Yarrowia lipolytica NCIM 3589
isolated from a marine sample has been shown to produce a cell wall
associated emulsier which is a complex of lipidcarbohydrate
protein in the presence of alkanes or crude oil. Yansan, is another
type of bioemulsier produced from Brazilian wild strain of Yarrowia
lipolytica, IMUFRJ 50682 in the fermentation medium supplemented
with glucose. Trindade et al. (2008) carried out comparative studies of
aging mechanisms of oil-in-water emulsions with Yansan and gum
Arabic emulsions and observed dependence on the pH of the
production medium. Studies proved that emulsions containing gum
Arabic age by coalescence while Yansan-based emulsions change their
aging mechanisms from coalescence at pH 3 to molecular diffusion at
pH 7. Other than Yarrowia lipolytica, an unusual yeast isolate of
Rhodotorula glutinis has also been reported to produce a bioemulsier
which shows good emulsication index (80%) with kerosene and
crude oil. It has also been shown to remove crude oil (76%) from
pollutants (Oloke and Glick, 2005).
2.1.6. Streptomyces
Marine Streptomyces are known to produce protein polysaccharide type bioemulsiers (Kokare et al., 2006). Out of 80 actinomycetes strains isolated from Alibag, Janjira and Goa coastal regions of

441

India, 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 to
42.6 mN/m with a CMC value of 0.3 mg/ml. The bioemulsier
molecules secreted by Streptomyces species mainly contain protein,
polysaccharide and a small part of reducing sugar (Kokare et al.,
2007).
2.1.7. Antarctobacter
It is a marine Gram-negative alpha proteobacterium generally seen
in marine ecosystem. Gutirrez et al. (2007b) isolated marine
Antarctobacter sp. TG22, that produces extracellular (high-molecular-weight fraction N2000 kDa) emulsifying agent named AE22. This
bioemulsier is composed of uronic acids with high emulsication
and stabilizing activity. This glycoprotein AE22 represents a powerful
extracellular emulsion-stabilizing polymer that forms highly stable
emulsions as compared with xanthan gum and gum Arabic. Due to the
interesting functional properties, AE22 can be preferred for industrial
application as compared to xanthan gum and gum Arabic.
2.1.8. Marinobacter
It is a Gram-negative, rod-shaped (coccobacilli, straight/curved),
non-spore forming bacterium which shows motility by means of
single unsheathed polar agellum. Marinobacter (previously named as
Alteromonas) produce large amounts of a non-dialyzable bioemulsier. This strain also degrades various liquid and solid hydrocarbons
(Al-Mallah et al., 1990). Further taxonomic verication of Alteromonas
strain revealed that the organism represented a new sp. within a new
genus named, Marinobacter hydrocarbonoclasticus (Gauthier et al.,
1992).
2.2. Glycolipid surface active molecules
This class of surface active molecules have been studied extensively due to their importance, quantities and extended applications
as compared with other types of BS/BE.
Sugar based, cheaper, renewable substrates can be easily
employed for the production of glycolipid BS. From a commercial
point of view, glycolipids represents superior product for production
and utilization purposes (Kitamoto et al., 2002). Glycolipids BS are
carbohydrates in combination with long-chain aliphatic acids or
hydroxyaliphatic acids. There are different types of glycolipid BS
produced 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. Poremba
et al. (1991a) was the rst to report the production of non-toxic
glucoselipid BS from Alcaligenes sp. Biosurfactant produced by
Alcaligenes sp. inhibits the growth of microagellates and microalgae
(Poremba et al., 1991b). Schulz et al. (1991) isolated three marine
bacterial strains and oil-degrading mixed cultures from marine
samples collected around the Isle of Helgoland (North Sea). They
screened and selected three marine bacteria from n-alkane degrading
microbial population. One strain-identied as Alcaligenes strain MM1
produced a novel glucoselipid while the other two strains belonged
to Arthrobacter sp. as described below.
2.2.2. Arthrobacter
Two strains of Arthrobacter sp. viz., EK 1 and Arthrobacter SI 1
produce glycolipid type BS (Schulz et al., 1991). Arthrobacter EK 1
produced trehalose tetraester glycolipid while culture, Arthrobacter SI
1 produced an extracellular emulsifying agent. Both BS exhibit strong
surfactant activities due to their effective interfacial and emulsifying
properties. Passeri et al. (1991) also reported on the production and

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S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

characterization of marine biosurfactant (an anionic trehalose


tetraester) from the marine bacterium Arthrobacter sp. EK1.

2.2.3. Alcanivorax borkumensis


Species of Alcanivorax (gammaproteobacterium) are Gram-negative, non-motile aerobic bacteria with morphology of elongated rod. A.
borkumensis produces a potent glucoselipid surfactant (Abraham
et al., 1998). A mixture of active biosurfactant, glucoselipids were
extracted from the growth medium supplemented with aliphatic
hydrocarbons. Analysis of the molecules with GCMS, FAB-MS
indicated four 3-hydroxydecanoic acids linked together by ester
bonds and coupled glycosidically with C-1 of glucose. It contains
glycine and -glucopyranoside esteried with 3-hydroxyhexanoic,
-octanoic and decanoic acids. Alcanivorax sp. exhibit active alkane
degradation properties. Yakimov et al. (1998) isolated six (AP1, MM1,
SK, SK2, SK4A and SK7) n-alkane-degrading heterotrophic bacteria
that produces cell-bound and extracellular surface active glucose
lipid. These strains were identied as A. borkumensis that was
characterized as producers of novel class of glycolipid biosurfactants.
Similar kind glycolipid BS production from A. borkumensis has been
reported by Passeri et al. (1992). BS produced by these marine
bacteria reduced the surface tension of water from 72 to 29 mN/m.
Alcanivorax and Cycloclasticus shows predominance in marine system
and are known as hydrocarbon degraders. Alcanivorax mainly
degrades alkane, whereas Cycloclasticus degrades various aromatic
hydrocarbons. Such studies are important for developing in situ
bioremediation strategies for cleaning up marine oil spills (Harayama
et al., 2004).

2.2.4. Rhodococcus
Different species of Rhodococcus are known for production of
glycolipid surface active molecules. Peng et al. (2007) isolated an oildegrading bacterium R. erythropolis strain 3C-9 from seaside soil of the
Island of Xiamen, located on the west bank of the Taiwan Strait. Two
different types (structure and chemical composition) of biosurfactants
as a glycolipid and free fatty acids are produced by R. erythropolis.

2.2.5. Halomonas
These marine strains are mostly known for production of EPS with
emulsifying properties. There are few reports suggesting that the
emulsifying surface active agents are also produced by Halomonas
species. Detailed studies were carried out by Pepi and co-workers
(2005). A bacterial strain, Halomonas ANT-3b was isolated from sea
ice seawater interface at Terra Nova Bay station, Ross Sea, Antarctica
that produces emulsifying glycolipids. This surface active compound is
a mixture of fatty acids and sugars of a molecular weight of 18 kDa
with mesophase (liquid crystal) organization. We isolated another
strain H. hydrothermalis IACS3 from marine water sample of Caspian
Sea (Iran) which produce glycolipid type biosurfactant that reduces
surface tension of distilled water from 71 to 31 mN/m and emulsies
various plant oils efciently (Satpute, 2008).

2.2.6. Unidentied marine bacterium MM1


Low and high-molecular surface active substances are produced by
n-alkane-utilizing bacteria. Passeri et al. (1992) screened marine
bacteria for biosurfactant production and reported a Gram-negative
marine bacterium MM1 synthesizing a novel low-molecular-mass cellbound glycolipid with four 3-OH-decanoic acids, which are linked
together by ester bonds. The lipophilic moiety is coupled glycosidically
with C-1 of glucose. This glycolipid act as a powerful biosurfactant by
reduction of surface tension of water from 72 to 30 mN/m and interfacial
tension value with n-hexadecane to b5 mN/m.

2.3. Lipopeptide surface active molecules


Several types of cyclic lipopeptide biosurfactants are produced
mainly by members of the Bacillus sp., which have been isolated from
various resources (Arima et al., 1968; Yakimov et al., 1995). Recently,
Das et al. (2008a) isolated B. circulans from the Andaman and Nicobar
Islands, India. Different functional groups and chemical bonds in these
BS have been determined by HPLC technique. Some of these
lipopeptide possess some antimicrobial activity. Their studies also
showed that one of the HPLC fractions (nonhaemolytic) of the crude
lipopeptide BS is responsible for its antimicrobial action. Therefore,
lipopeptide biosurfactant have been implicated with some antimicrobial chemotherapy applications. Biosurfactant produced by a
marine Bacillus possesses good surface tension reduction with low
CMC and antimicrobial activity. Pure quality of product obviously
widens the applications in diverse elds. It is also important to note
that the quality and quantity of biosurfactant production is dependent
on the carbon substrate and fermentation medium. Studies by Das et
al. (2009c) showed that biosurfactant possessing good antimicrobial
activity can be isolated in the production medium supplemented with
glucose as compared with glycerol, starch and sucrose. RP-HPLC
studies were employed to resolve crude biosurfactants into several
fractions which indicated that one of the fractions bears signicant
antimicrobial action.
Biosurfactant producing B. circulans has been shown to enhance
the bioavailability and degradation of a polyaromatic hydrocarbon
(PAH) like anthracene. The presence of anthracene enhances the
bacterial growth and biosurfactant production. Biosurfactant obtained
from such strain can emulsify various hydrocarbons efciently.
Analytical studies are important to understand the role of biosurfactant in solubilisation of PAH (Das et al., 2008b). Newly introduced
approach PlackettBurman-based statistical screening procedure can
be employed to determine the nutritional requirement for the growth
and biosurfactant production of bacterium. Statistical approaches are
also helpful in the formulation of production medium for microbial
production of biosurfactant and may be crucial to enhance the
quantity of the product obtained (Mukherjee et al., 2008).
A lipopeptide biosurfactant producing Azotobacter chroococcum
isolated from marine environment of Tuticorin harbour (India) has
also been reported to grow on crude oil, waste motor lubricant oil and
peanut oil cake and was capable of emulsifying waste motor lubricant
oil, crude oil, diesel, kerosene, naphthalene, anthracene and xylene.
Potential applications of such surface active molecule for bioremediation were highlighted by Thavasi et al. (2006, 2009). Lipopeptide type
biosurfactant are also produced by marine actinomycetes. A biosurfactant from sponge-associated marine actinomycetes Nocardiopsis alba
MSA10 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 active
bile acids by Myroides SM1 bacterial strains. These compounds were of
similar structures to those produced by eukaryotic cells and are
composed of cholic acid, deoxycholic acid and their glycine conjugates. M. odoratus JCM7458 and M. odoramitimus JCM7460 also
produced such type of biosurfactants.
2.5. Glycolipopeptide surface active molecules
Renewable substrates like motor lubricant oil and peanut oil cake
has been used for BS production from microbes. Thavasi et al., 2007
carried out biosurfactant production by using renewable, relatively
inexpensive and easily available resources. Two different species a
Corynebacterium kutscheri and a Bacillus megaterium isolated from
Tuticorin harbour (India) produced complex glycolipopeptide capable

S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

of emulsifying different hydrocarbons which is important to remediate


hydrocarbon polluted sites (Thavasi et al., 2007, 2008). Similar kind of
surface active compounds has been reported from in non marine
Corynebacterium isolates (Zajic et al., 1977; Cooper et al., 1979).
2.6. Exopolysaccharides (EPS)/complex surface active polymer
Exopolysaccharides (EPS) are high molecular weight carbohydrate
polymers. Many marine microorganisms produce extracellular polymers which form a layer surrounding the cells that helps them to
withstand or resist adverse and extreme environmental conditions. At
the same time it is also true that these extreme environments offer
novel microbial biodiversity that produces varied and interesting
types of EPS (Mancuso Nichols et al., 2005a,b; Margesin and Schinner,
2001; Vincent et al., 1991). The release of intracellular and
extracellular capsular material by marine bacteria in the ocean
might drive the dissolved organic matter pool of the marine biosphere
(Heissenberger et al., 1996). EPS may act as a cryoprotective agent in
brine channels of sea ice, where extremes of high salinity and low
temperature impose pressures on microbial growth and survival
(Mancuso Nichols et al., 2005b). EPS secreted by Vibrio parahaemolyticus shows good adhesive property, where cells are held together to
form colonies, which helps the colonies to withstand physical forces
(Jodi et al., 2000). Some of the key roles of microbial EPS are
represented diagrammatically in Fig. 2. Such polysaccharide producers have been isolated from hydrothermal deep-sea vents extreme
environment. Mancuso Nichols et al. (2005b) analysed sea ice and
water samples obtained by a voyage of RSV Aurora Australis and
reported near dominance of gamma proteobacteria and cytophaga
exibacterbacteroides in these marine samples. These EPS are
important in microbial interaction and emulsication of various
hydrophobic substrates (Yim et al., 2005; Maki et al., 2000; Perfumo
et al., 2010). They are known to increase the viscosity of solutions at low
pH value and emulsify several hydrocarbon compounds (Guezennec et
al., 1994; Calvo et al., 1998) and are intriguing many researchers trying to
harness their extraordinary properties and considerable potential
applications in various elds (Weiner, 1997; Desai and Banat, 1997). A
polysaccharide namely PS 3a24 and PS 3a35 produced by marine
bacteria has been shown to have high 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-negative
marine bacteria like Bacillus, Halomonas, Planococcus, Enterobacter,
Alteromonas, Rhodococcus, Zoogloea, and Cyanobateria are known to
produce EPS (Zukerberg et al., 1979; Maugeri et al., 2002; Nicolaus et al.,
2000) as listed below.
2.6.1. Bacillus producing exopolysaccharide
Very few reports are available on EPS production by marine
Bacillus sp. Maugeri et al. (2002) isolated a Bacillus strain B3-15
(halophilic, thermotolerant) from shallow water, marine hot spring at
Volcano Island (Eolian Islands, Italy). EPS production in Bacillus B3-15
was carried out in mineral medium as supplemented with glucose and
different type of EPS composed of tetrasaccharide repeating unit with
sugars having a manno-pyranosidic conguration were produced.
Another Bacillus strain B3-72, isolated from same shallow vent
produced structurally different EPS (Nicolaus et al., 2000). This
indicates the diversity of EPS produced by same genus from the
similar environment. Other few reports includes B. licheniformis
isolated from Volcano Island produce EPS (Arena, 2004) and
B. thermoantarcticus sea sand in Ischia Island (Nicolaus et al., 2000)
for production of medical/pharmaceutically important polysaccharides. Arena et al. (2006) also reported novel type of EPS-1 polysaccharide producing thermotolerant strain Bacillus licheniformis,
which was isolated from a shallow marine hot spring of Volcano
Island (Italy). Antiviral and immunomodulatory effects of EPS-1

443

studies showed that the EPS-1 treatment impaired HSV-2 replication


in human PBMC but not in WISH cells.
2.6.2. Halomonas producing exopolysaccharide
Some microorganisms are capable of growing in extreme environments, where most other organisms are not able to survive. Among
these extremophiles, halophiles are one of the major microbial
communities that tolerates high salt concentrations and are highly
sought after by many industries for their novel enzymes and products
that has wider potential applications (Ventosa et al., 1998; Ventosa
and Nieto, 1995). Halomonas is a Gram-negative bacterium, non-spore
forming, predominantly present in marine environments and are
often 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 EPS
production have been isolated from hypersaline environments with
different 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 HMWEPS from Halomonas sp. strain TG39 by growing on different types of
substrates. Culture medium supplemented with glucose and a
peptone/yeast extract results EPS fraction (heterogeneous polymer
HMW-glucose) that possess specic emulsifying activity
(EI24 = 100%) as compared to culture medium supplemented with
mannitol, sucrose, and malt extract for EPS production.
2.6.3. Planococcus producing exopolysaccharide
A variety of marine environments harbour Planococcus abundantly. These hydrocarbon-degrading microbes are signicant in controlling the hydrocarbon contamination in marine environments
(Engelhardt et al., 2001). Now it is known that Planococcus sp. are
also able to produce EPS which exhibit high emulsication property.
Kumar et al. (2007) isolated P. maitriensis Anita I from the coastal sea
water area of Bhavnagar district, India. The EPS has been precipitated
from cell-free supernatant using alcohol and was found to contain
carbohydrate, protein, uronic acid and sulfate. The oil spreading
potential of the puried EPS was comparable to Triton X100 and
Tween 80 and with good emulsifying and tensiometric properties
which make it a useful product in bioremediation, enhanced oil
recovery and cosmetics related applications.
2.6.4. Enterobacter producing exopolysaccharide
These are Gram-negative, motile, facultative anaerobic bacteria
which are widely distributed in nature. Enterobacter cloacae isolated
from marine sediment collected from Gujarat coast, India (Iyer et al.,
2005) produce EPS 71a that emulsies various water-insoluble
substrates. 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 high
amount of uronic acid, fucose and sulfate. Such kind of EPS is
uncommon in bacteria (Iyer et al., 2006).
2.6.5. Alteromonas producing exopolysaccharide
These are all Gram-negative (straight or curved rods), motile,
heterotrophic, mesophilic bacterium found in coastal water and the
open sea. Vincent et al. (1994) isolated Alteromonas sp. from Alvinella
pompejana 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-carboxyethilidenegalactose. From the same sampling site, an EPS producing Alteromonas
sp. strain 1644 was isolated by Bozzi et al., (1996a,b). Another EPS
(p-11568) producing Alteromonas sp. was isolated from the South Sea
of Korea and claimed to exhibit superior emulsifying activity as
compared with commercial available polysaccharides (Yim et al.,

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S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

Fig. 2. Various roles played by exopolysaccharide (EPS) produced by marine microorganisms in the marine ecosystem.

2005). Two types of structurally identical polysaccharides are


produced by marine microorganism Alteromonas haloplanktis KMM
156. These polymers are acidic in nature, capsular and an O-specic
contains repeating tetrasaccharide units with L-rhamnose, 2-acetamido-2-deoxy-D-glucose and a 3-O-[(R)-1-carboxyethyl]-D-glucose
(Glc3Lac) residue (Gorshkova et al., 1993). Alteromonas macleodii
2MM6 isolated from Intertidal zone of Halifax, Nova Scotia is reported
for production of an acidic polysaccharide containing tetrasaccharide
repeating units with D-galactose, 3-O-acetyl-2-acetamido-2-deoxy-Dglucose, 2-acetamido-2-deoxy-L-guluronic acid and 3,6-dideoxy-3(4-hydroxybutyramido)-D-galactose residues showed the structure of
R:COCH2CH2CH2OH (Nazarenko et al., 1993).
Raguns et al. (1997) reported aerobic, mesophilic and heterotrophic bacterium a new Alteromonas infernus sp. isolated from a
deep-sea hydrothermal vent of Riftia pachyptila (Gulf of California).
The strain secretes two types of unusual polysaccharides during
stationary phase in the culture medium supplemented with glucose.
The EPS found is water-soluble containing monosaccharides like
glucose, galactose, galacturonic and glucuronic acids. Raguns et al.
(1996) also isolated four bacteria from deep-sea hydrothermal vents
found to produce four types of extracellular polymers having different
chemical composition and rheological properties. Alteromonas macleodii

subsp. jiensis one of the new bacterium produce EPS similar to that of
xanthan containing glucose, galactose, mannose, glucuronic and
galacturonic acids.
2.6.6. Pseudoalteromonas producing exopolysaccharide
Chemically diverse types EPS are produced by Antarctic isolates
which mostly contains charged uronic acid residues and some
contains sulfate groups. Polymers produced by some of the isolates
from this microbial population are large polymers of approximately
5.7 MDa in size (Mancuso Nichols et al., 2005c). EPS producing
Pseudoalteromonas sp. strain CAM025 and CAM036 were also isolated
from seawater and sea ice in southern ocean (Mancuso Nichols et al.,
2004). Marine bacterium CAM025, isolated from sea ice sample shows
30 times higher yield at 2 C and 10 C than at 20 C. Most of the
marine bacterial isolates are psychrotolerant where their optimum
growth occurs at about 20 C. However, these bacteria may produce
optimum EPS at lower temperature (Mancuso Nichols et al., 2005a).
Recently, interesting work is reported by Gutirrez et al. (2008). This
research group has described the production of polymeric emulsifying
agent from Pseudoalteromonas with the absorption property to metal
ions. The high-molecular-weight glycoprotein exopolymer PE12
contains identical monosaccharide with exopolymers of bacteria.

S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

PE12 also contains uronic acid, protein similar to that of Pseudoalteromonas/Alteromonas species. EPS producing Pseudoalteromonas strain
HYD 721is isolated from deep-sea hydrothermal vent of East Pacic
Rise which contains repeating unit of the polymer with a branched
oligosaccharide (Rougeaux et al., 1999a). Novel types of EPS
producing bacteria have been isolated from deep-sea hydrothermal
vent (Rougeaux et al., 1996).
In the year of 1996, Raguns et al.'s group isolated two
Pseudoalteromonas species from deep-sea hydrothermal vents produce same types of polymer containing glucose, galactose, mannose,
glucuronic and galacturonic acids. Studies with transmission electron
microscopy (TEM) of immunogold-labeled indicated that the EPS
production in Marine Pseudomonas sp. strain S9 is closely associated
with the cell surface. EPS production takes place during the bacterial
growth and energy-nutritional starvation condition (Wrangstadh
et al., 1990). A previous study also reported secretion of EPS by
Pseudomonas sp. S9 during complete nutrient starvation and energy.
When the cells were starved in agitated conditions, EPS secretion
occurs and different adhesion pattern to hydrophobic surfaces is
observed (Wrangstadh et al., 1986).
2.6.7. Rhodococcus producing exopolysaccharides
Urai et al. (2007) isolated marine R. erythropolis PR4 that produces
a large quantity of acidic EPS (FR1 and FR2). FR2 composed of
D-galactose, D-glucose, d-mannose, D-glucuronic acid, and pyruvic
acid at a molar ratio of 1:1:1:1:1, and contained 2.9% (w/w) stearic
acid and 4.3% (w/w) palmitic acid attached via ester bonds. It was
proposed that EPS plays a signicant role in the hydrocarbon tolerance
of this bacterium.
2.6.8. Zoogloea producing exopolysaccharides
These are Gram-negative, straight or slightly curved, plump rods,
non-spore or cyst forming free living bacteria in organically polluted
fresh and waste water bodies. Kwon et al. (1994) has reported the
production of polysaccharide extracellularly from Zoogloea sp. This
marine bacterium produces water-soluble polysaccharide: WSP (cellfree liquid medium) and cell-bound polysaccharide: CBP (cell surface)
containing glucose, galactose and mannose as main sugars and uronic
acid. Zoogloea sp. KCCM10036 produces two types of EPS (CBP: cellbound polysaccharide and WSP: water-soluble polysaccharide).
Emulsions formed by cell-bound polysaccharide are highly stable
when compared with other commercially available EPS such as
xanthan gum, Tween series, Triton. Both types of EPS from Zoogloea
sp. possess excellent occulating activity (Lim et al., 2007). Zoogloea
sp. secretes water-soluble and the other cell-bound EPS possessing
non-Newtonian, pseudoplastic uid behaviour. Solutions of these EPS
were also reported to have low activation energies with good
rheological behaviour over broader pH range (212) and temperature
(2080 C) in the presence of NaCl (Jang et al., 2002).
2.6.9. Cyanobacteria producing exopolysaccharide
Mucilaginous envelope coating is common around marine Cyanobacteria living in hypersaline environments and therefore, most of
these cyanobacterial strains can produce EPS in the surrounding
media (Campbell and Golubic, 1985). Fifteen EPS producing unicellular cyanobacterial strains have been isolated from saline environments. Cyanothece EPS contains uronic acids, six to eight
monosaccharides, with one or two acidic sugars. Other chemical
groups like acetyl, pyruvyl, and/or sulfate groups are also detected.
EPS from such organisms are promising candidates for various
industrial applications (De Philippis et al., 1998). Other EPS from
cyanobacterial strain Cyanothece 16Som2 which was isolated from a
Somaliland 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 Cyanothece

445

sp. ATCC 51142 has high content of calcium and plays key role in
removing metals ions (Shah et al., 2000). Another report on EPS
producing Cyanothece sp. 113 isolated from salt lakes in China (Chi
et al., 2007) reported novel type of -D-1,6-homoglucan. Other
marine cyanobacterial strains like Schizothrix sp and Oscillatoria sp.
isolated from marine stromatolites, Bahamas have been reported for
production of EPS (Kawaguchi and Decho, 2000).
2.6.10. Vibrio producing exopolysaccharides
Vibrio 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 and
Seetharaman, 2003). Jodi et al. (2000) investigated the factor(s)
responsible for the opaque and translucent phenotypes, the cell
organization within both colony types in V. parahaemolyticus. They
showed that both type cultures secrete EPS containing four sugars like
glucose, galactose, fucose and N-acetylglucosamine. They also noted
that the copious amount of EPS produced by the opaque strain lls the
intercellular space within the colony, resulting in increased structural
integrity and the opaque phenotype. The structure of the EPS
produced under laboratory conditions by Vibrio diabolicus (isolated
from a deep-sea hydrothermal vent of East Pacic Rise) showed the
presence of linear tetrasaccharide repeating unit (Rougeaux et al.,
1999b). Raguns et al. (1996) reported production of EPS containing
different sugars like glucose, galactose, mannose, glucuronic and
galacturonic acids from the Vibrio species isolated from deep-sea
hydrothermal vents. An interesting patent is led by Guezennec et al.
(2002) on genus Vibrio belonging to V. diabolicus for production and
uses of water-soluble polysaccharides. Molecular weight of the EPS
from this marine Vibrio is 800,000 Da and contains osamines (30 5%)
with uronic acids (32 5%) and monosaccharide like glucuronic acid
(11.2%), N-acetylglucosamine (18%), and N-acetylgalactosamine (7.9%).
The EPS is similar to that of heparin and hence nds important applications in pharmaceutics.
2.6.11. Other exopolysaccharide producing bacteria
Other 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 is
limited. This shows the diversity in production of bioactive molecules
by microorganisms in the marine biosphere. For example, Hyphomonas strain MHS-3 isolated from shallow water sediments in Puget
Sound found to produce adherent form of exopolysaccharide (EPS)
capsule associated with Glycine max lectin, Arachis hypogaea lectin
and Bauhinia purpurea lectin (BPA). The capsule of Hyphomonas strain
MHS-3 EPS has an effective adhesive property (Quintero and Weiner,
1995). EPS namely, Marinactan from Flavobacterium uliginosum shows
antitumor activity against sarcoma-180. Basically this bacterium
requires typical marine sea water condition for growth. The novel
type of Marinactan is composed of glucose, mannose and fucose with
the proportion of 7:2:1 (Umezawa et al., 1983). It is possible to
determine the effect of surface on the yield and composition of EPS
produced by bacteria. This fact is well explained from the work on two
marine Desulfovibrio sp. Indl SRB-sulfate-reducing bacteria (SRB)
isolated from severe corrosion failures. Bacterial isolates were grown
in static batch cultures with and without the presence of carbon steel
surfaces. EPS of these Desulfovibrio sp. contained similar kind of
carbohydrates, proteins and nucleic acids in with different quantities.
Studies also showed that surface provided for the growth had effect
on the type of EPS produced by Desulfovibrio sp. (Zinkevich et al.,
1996). Highly mucoidal Mediterranean Sea isolate, Haloferax mediterranei is an extreme halophilic archaebacterium which secretes
heteropolysaccharide extracellularly. The EPS contains glucose,
galactose, unidentied sugar, amino sugars and uronic acids. The
rheological properties of EPS like pseudoplasticity and viscosity are
seen effectively at extreme ranges of pH, temperature and salinity.

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S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

Therefore, such polymer has potential applications to enhance oil


recovery processes. The EPS can be used as potential thickening agent
in various industries (Anton et al., 1988). Geobacillus thermodenitricans, a bacterial isolate recovered from vent of Vulcano Island (Italy)
produce EPS-2 shows immunomodulatory and antiviral effect by
hindering HSV-2 replication in human peripheral blood mononuclear
cells (PBMC). The concentration or the dose of EPS-2 is important for
its immunomodulatory and antiviral activity. Arena (2004) also
claimed that EPS-2 successful immunological disorders determined
by 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 EPS
exocellularly in the medium supplemented with carbon sources like
sucrose/trehalose. Purication of EPS resulted three fractions namely
EPS1, EPS2, and EPS3, where different sugars with different proportions were present. Among these three, detailed studies on EPS3
shows the molecular weight of 1106 Da with a pentasaccharide
repeating unit (Schiano Moriello et al., 2003). EPS producing Gramnegative bacterium, aerobic, Hahella chejuensis 96CJ10356(T), is
isolated 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 for
developing sulfur-free, dened growth medium, production of EPS
and biolm formation. T. litoralis secretes soluble EPS (with the
presence of mannose) in an articial-seawater medium supplemented with 16 different amino acids, vitamins and trace elements. EPS
plays an important role in the biolm formation on polycarbonate
lters and glass slides. The authors proposed that such kind of work is
important in reveling information on the interactions among hightemperature organisms. Other moderately halophilic eubacterium
namely Volcaniella eurihalina is reported for production of EPS. The
EPS produced by this bacterium is composed of glucose, rhamnose
and mannose and has interesting physical and chemical properties
which have enormous applications in industries (Calvo et al., 1995;
Quesada et al., 1990, 1993, 1994).
3. Potential applications of marine microbial surface active agents
and exopolysaccharides
The promising progress in the eld of marine biotechnology has
attracted many researchers towards marine microbial surface active
agents and exopolysaccharides. To date, various marine microbes are
known for production of various bioactive compounds. Discoveries of
new marine microbial BS/BE and EPS are helping to explore the
marine ecosystem. Different industrial sectors like textile, pharmaceutical, cosmetics, food, metal mining, oil recovery, and metal
recovery are continuously searching for novel bioactive products.
Due to the structural and functional novelty of these marine bioactive
compounds numerous applications are developing in various elds.
Tensiometric and emulsication properties of biosurfactant/bioemulsier are important for application purposes (Baird et al., 1983;
Sutherland and Ellwood, 1979; Sutherland, 1998). Some of the marine
polymeric biosurfactant/bioemulsier with their application properties are summarised in Tables 2 and 3.
Initially, microbial surface active agents were explored as
antibiotics (due to their antimicrobial activities). Today a number of
biosurfactant/bioemulsier with antimicrobial activities are available
and exploited. Most of the common examples for antimicrobial
activity of biosurfactant are shared from Bacillus species. Das et al.
(2008a) disclosed that the single nonhaemolytic fractions of lipopeptide biosurfactant (from B. circulans) has a powerful antimicrobial
activity and hence these type of surface active agents have applications 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 biosurfactants

exhibit enhanced surface and antimicrobial activities. Thus, authors


proposed that such kind of biosurfactant can serve as new potential
drugs in antimicrobial chemotherapy.
The use of biodispersion in paper manufacturing industries is one
of interesting approach for routine applications. The addition of
biodispersion as ller along with paper and limestone improves the
quality of paper and signicantly reduces the time required for the
grinding process (Rosenberg et al., 1989). Marine microbial surfactant
act as an effective antiadhesive. Recently, Das et al. (2009a)
investigated the antiadhesive action of a lipopeptide biosurfactant
from a marine bacterium and the effect of cultivation conditions on
the adhesion properties. Under oxygen limiting conditions static
cultures showed a good adhesion property which was conrmed with
the help of confocal laser scanning microscopy studies. Other
biosurfactant producing marine bacterium has also been shown to
remove metal from solutions with the efciency dependent on the
concentration 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 like
atomic absorption spectroscopy (AAS), Fourier transform infrared
spectroscopy (FTIR) and transmission electron microscopy (TEM)
equipped with energy dispersive X-ray spectroscopy (EDS) also play a
signicant role in such investigations.
Polysaccharides of bacterial origin are very important in pharmaceutical industries. EPS secreted by marine bacteria contains novel
combination which has potential applications in various industrial
sectors. Various properties of EPS like thickening, coagulating,
adhesion, stabilizing and gelling are utilized in various industries.
EPS with good viscosity and pseudoplasticity properties are resistant
to the extremities of temperature, pH and salinity. Examples of
such EPS an emulsier produced by HaheUa chejuensis gen. nov., sp.
nov. (Lee et al., 2001), Haloferax mediterranei. Alteromonas sp. strain
1545 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 bacteria
contains high uronic acid, and a sulfate content which is important in
biotechnological point of view. EPS namely PS3a24 and PS3a35 from
bacteria show pseudoplastic owing properties at higher concentrations and stability at broader pH range. Such properties are seen in
xanthan gum and therefore, these two EPS are very important in the
application point of view (Boyle and Reade, 1983). EPS from
Pseudomonas sp. strain NCMB 2021 can attach effectively to the
solid surfaces (Christensen et al., 1985) while those produced by
Hyphomonas adhaerens helps in biolm formation and give adhesive
property to cells and assist their growth in metal containing
environment (Quintero and Weiner, 1995; Quintero et al., 2001).
Such metal resistant properties are very much important from
biotechnological applications point of view.
Chemically modied (sulfation and acidic depolymerization) EPS
secreted by A. infernus has anticoagulant activity similar to heparin
and therefore, is important for the treatment of lipemia and
arteriosclerosis (Guezennec et al., 1998). Another heparin like EPS is
secreted by Vibrio sp. and has been reported to have anticoagulant and
antithrombotic properties and can be utilized as an antiviral,
antitumour or antithrombotic agent (Guezennec et al., 2002). There
is a similar report on anticoagulant properties of heparin by modied
EPS from Alteromonas infernos. Such pharmaceutically important
compounds can be modied for suitable application purpose (ColliecJouault et al., 2004). EPS produced by Bacillus licheniformis and
Geobacillus thermodenitricans can act as strong stimulators for Thl
cell-mediated immunity. Such immunomodulatory agents can play a
major role in the treatment of immunocompromised patients (Arena,
2004). Similarly it was recently reported that a Geobacillus thermodenitricans 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

S.K. Satpute et al. / Biotechnology Advances 28 (2010) 436450

Vibrio diabolicus has been reported to secrete HE800 EPS which


possess signicant bone healing property by formation of extracellular matrix for the direct adhesion of osteoblasts, osteoprogenitor cells
and pericytes. This polysaccharide provides the ideal conditions
required for bones growth and healing process (Zanchetta et al.,
2003). Furthermore Marinactan, an EPS produced by Flavobacterium
uliginosum has been shown to be an effective antitumour against
sarcoma-180 solid tumor in mice through inhibition of growth of solid
sarcoma 180 in mice. Marinactan also inhibit the growth of solid
sarcoma 180 even before and after tumor transplantation (Umezawa
et al., 1983). Other marine Vibrio sp. producing EPS have been
reported to have an effective antitumor and immunostimulant
activities which are quite important from a medicinal point of view
(Okutani, 1984, 1985).
EPS produced by marine microbes plays a crucial role in the
removal of pollutant metals and toxic elements from contaminated
solutions. Their emulsication and surface active properties makes
them applicable for utilization in bioremediation and other oil
recovery and cosmetics related applications (Kumar et al., 2007).
Strains such as Cyanothece sp. ATCC 51142 produce EPS which are
essential for removal of metals (Shah et al., 2000). Other EPS rich in
uronic acid content have a high afnity towards heavy-metals making
them suitable for the removal and/or minimization of metal toxicity
(Raguns et al., 1996). Similarly Alteromonas sp. strain 1644
produces EPS with binding ability towards monovalent and divalent
ions (Bozzi et al., 1996b) while isolates from deep-sea hydrothermal
vents produce EPS, which can bind and remove toxic metals like lead,
cadmium, and zinc. They acts as a powerful chelating agent and hence
they are useful for wastewater treatment and other environmental
applications (Loac et al., 1998).
4. Conclusions
In spite of a long history of BS/BE production from microorganisms,
very few marine microbial communities viz., Acinetobacter, Arthrobacter, Pseudomonas, Halomonas, Myroides, and Corynebacteria sp.
have been explored for production of surface active molecules. BS/BE
and EPS produced by marine microbes have some novel structural and
functional properties. Several high molecular weight polymer and
glycolipid type BS/BE are produced by marine microbes and have
important potential application in different industries. Marine
ecosystems therefore provide an excellent opportunity to select
potent microorganisms. Effective screening methodologies and
improved purication techniques are essential in order to achieve
desired quantities and qualities of BS/BE. Promising recent biotechnological approaches will support the search of potent BS producers
from this important ecosystem.
Acknowledgements
Surekha K. Satpute, thanks the UGC, Govt. of India, for nancial
support {(F.17-37/98(SA-I)}. This work is also supported by the
research project sanctioned by DBT (BT/PR304/AAQ/03/155/2002),
Govt. of India.
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