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Microbial Exopolysaccharides: Advances in Applications and Future Prospects

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 0311

Microbial Exopolysaccharides: Advances in

Applications and Future Prospects
PRASAD ANDHARE1, K ISHOR CHAUHAN2, M ARGI DAVE1 AND HILOR PATHAK1*

ABSTRACT

Microbial extracellular polymeric substances (EPS) are the renewable

and the most significant group of polymeric materials. The chemical
and structural diversity of EPS bestow on them the diversified roles in
various fields like microbial ecology, medicine, dairy industry, biofilms,
agricultural industry, cosmetics, neutraceuticals, etc. depicting their
biotechnological significance. The present chapter describes the advances
in the applications of microbial exopolysaccharides with reference to
the ir mar ket re levanc e. Consider ing the market potential of
exopolysaccharides, research interest towards EPS has been continuously
growing. The major research is focused on lowering the production cost
of EPS and tuning of the properties of EPS as per market requirements.
Owing to the versatility and scope of modifications in the properties of
exopolysaccharides, hopefully, the next coming years will witness a boom
in the usage of exopolysaccharides in nearly all the industries.
Key words: Microbial extracellular polymeric substance, EPS,
exopolysaccharide applications, hydrogels.

1
Department of Microbiology, P.D. Patel Institute of Applied Sciences, Charotar
University of science and Technology (CHARUSAT), Changa-388421,Gujarat, India
2
Department of Integrated Biotechnology, Ashok & Rita Patel Institute of Integrated
Studies and Research in Biotechnology and Allied Sciences, Sardar Patel University,
New Vallabh-Vidyanagar, 388 121 Gujarat, India
*Corresponding author: Email: hilorpathak.mi@ecchanga.ac.in
2 Biotechnology Vol. 3: Microbial Biotechnology

INTRODUCTION

The term exopolysaccharide (EPS) was coined by Sutherland (1972) to

described high molecular weight carbohydrate polymers produced by
many marine bacteria. Since then, EPS has also been used to designate
more broadly defined compounds called extracellular polymeric
substances (Nichols et al., 2005). The term exopolysaccharide or
extracellular polymeric substance is widely used for the polysaccharide
materials present external to the cell-wall and/or cell membrane of the
microbial cell and can be applied to polymers of very diverse composition
and of different physical types. The existence of EPS in case of microbial
cells can easily be spotted from the mucoid colony appearance as
depicted in Fig. 1 (Sutherland, 1990).

Fig. 1: Mucoid colony appearance on solid media by EPS producing

organisms
EPS is associated with both pr okaryotes (eubac teria and
archaebacteria) as well as eukaryotes (phytoplankton, algae and
seeweeds) and have various industrial and biotechnological applications
and thereby portray a great deal of research interest (Singha, 2012).
These biological polymers constitute a wide variety of high molecular
weight organic macromolecules like polysaccharides, proteins, nucleic
acids, phospholipids, uronic acids, humic substances, lipids, etc (Pal
and Paul, 2008). Owing to the diversified properties of EPS, inherent
biocompatibility and non-toxic nature (Nwodo et al., 2012); depict wide
spectrum of applications in several industries namely food industry,
agriculture industry, textile industry, bakery industry, pharmaceutical
industry, cosmetic industry, civil industry as well as oil industry (Van
Balken, 1997). Although, the polysaccharide production by plants and
seaweeds has been used since decades, which includes cellulose, starch
and pectin from plants and agar, alginate and carragenan from algae
and seeweeds (Singha, 2012); however, the exploration of novel
composition and promising properties of microbial exopolysaccharides
have captured the scientists’ attention.
Microbial Exopolysaccharides: Advances in Applications and Future Prospects 3

MICROBIAL POLYSACCHARIDE CATEGORIZATION

Based on their localization in the cell, the microbial polysaccharides

are divided into intracellular polysac charides, structural
polysaccharides and extracellular polysaccharides. The intracellular
polysaccharides are found more in mushrooms like Ganoderma spp.,
Inonotus spp., Phlebia spp., Pleurotus spp., Trametes versicolor,
Tricholoma mongolicum, etc. and in certain bacteria like Bacteroides
spp. (Fig. 2). Majority of the intracellular polysaccharides are reported
to have antioxidant and anti-cancer activities as well as
immunomodulatory effects and thereby impart medicinal significance
(Linder et al., 1979; Lee et al., 2007; Zhang et al., 2011 2013; Wu et al.,
2012; Wu et al., 2012; Silva et al.,). Structural polysaccharides include
the cellulose, chitin, peptidoglycan, etc. These structures provide
structural integrity i.e. these impart shape and firmness to the cell,
thereby aptly termed as structural polysaccharides.

Fig. 2: Electron micrograph of IPS from Bacteroides fragilis (Adapted from

Linner et al., 1979)

Extracellular polysacc harides produced by microbes are immense

and of diverse nature. These are further grouped into four major classes;
polysaccharides, slime and microcapsular polysaccharides, inorganic
 4

Table 1: Distribution of exopolysaccharides, their structure, properties and market potentials

Market Applications

Type of Source Monosaccharide composition and Properties Brands Products References

exopolysac- structure
charide

Mannuronic acid, guluronic acid and PROTANTAL: Anti-reflux (link-84; link-

acetate suspension, controlled 85; Rehm,
released tablets, wound 2009; Imeson,
Anionic charge, FMC Biopolymer dressing, dental impression 2011; Yang,
Hydrocolloid, Gelling material, denture fixatives, 2011; Rehm,
Alginate Pseudomonas spp capacity, Film viscosifiers, encapsulation, 2009 and Pena
and Azotobacter forming Non- films, foams. et al., 2008)
spp. Newtonian
M.W. (0.3–1.3) × 106
JOHNSON and SILVERCEL® Antimicrobial
JOHNSON Alginate Dressing

D – glucose SONY
CORPORATION Audio speakers diaphragms (link-85;
link-87;
Neutral charge, Non- JOHNSON and Brown, 1998;
Alphaproteobacteria, Newtonian Insoluble JOHNSON Pad for would care Rehm, 2009;
Betaproteobacteria, in most solvents, Yang, 2011;
AJINOMOTO CO Paper products Chawla et
Cellulose Gammaproteobacteria, High tensile strength,
Gram-positive bacteria Moldability and High CEKOL® Cellulose gum for al., 2009;
and Acetobacter spp. crystallinity food, cosmetics, personal Bae et al.,
M.W. ~ 106 care and pharmaceuticals, 2004 and
CPKelco CELLUFIX® Efficient Ivleva et al.,
binders developed for use in 2009)
building products, CELPOL®
PAC for oil and gas drilling.

CURDLAN 424: bulking (link-88;

Glucose NOSHLY agent, thickener, emulsifer, link-89;
Neutral charge, Gel- Stabilisers Rehm, 2009;
forming ability, Water Yang, 2009
Agrobacterium spp., insolubility, Edible FOREVER Vegan Seaweed Scallop and and de
Curdlan Rhizobium spp. and and Non-toxic has HEALTH FOOD in some Taiwan vegetarian Oliveira et
Cellulomonas spp biological activity CO. LTD foods al., 2007)
M.W. 1.0 × 106
Used in heavy metal removal and concrete additive
Biotechnology Vol. 3: Microbial Biotechnology

Table 1: Contd.
 Market Applications

Type of Source Monosaccharide composition and Properties Brands Products References

exopolysac- structure
charide

Glucose Dextran 40, 60 and 70 (link-90; link-

injections; Replacement of 91; line-92;
blood loss, Plasma Rehm, 2009;
PHARMASOSMOS substitution, Thrombosis Yang, 2011
Neutral charge, Non- prophylaxis, Volume and Rehm,
Dextran Leuconostoc spp. and ionic, good stability expansion, Rheological 2010)
Streptococcus spp. Newtonian fluid improvement
behavior
Iron Dextran Injection: For
M.W. 106 - 109 DEXTRAN Prevention and Treatment of
PRODUCTS LTD Iron Deficiency Anemia in
Baby Pigs
SIGMA-ALDRICH Used in chromatographic
(link-93;
media
Rehm, 2009;
Glucose, mannose, glucuronate, KELDENT® Toothpaste and Imeson,
pyruvate oral care application, 2011;
KELFLO® Food-grade for Ullrich, 2009
Anionic charge, Non- animal feed, KELTROL® and Yang,
Newtonian, Food and personal care 2011)
Hydrocolloid, High applications, KELZAN®
Xanthan Xanthomonas spp. viscosity, Stable over CP Kelco Industrial and oilfield
a wide temperature drilling applications,
pH and salt XANTURAL®
concentration ranges Pharmaceutical applications,
M.W. (2.0-5.0) × 106 XANVIS® Biopolymer for
Oilfield and horizontal
drilling fluids.

Glucose Rhamnose Glucuronic acid (link-94;

Acetate Glycerate GELZAN® specialty gellan Rehm, 2009;
Anionic charge, gum for growth matrix for Imeson;
Hydrocolloid - most clinical and non- 2011;
Stability over wide pH clinical organisms, Ullrich,
Gellan gum Sphingomonas spp. range, Gelling CP KELCO KELCOGEL® for use in 2009; Fialho
capacity, food, beverage, personal et al., 2008
care, oral care, household and Bajaj et
Microbial Exopolysaccharides: Advances in Applications and Future Prospects

cleaner formulations al., 2007

Table 1: Contd.
5
 6

Market Applications

Type of Source Monosaccharide composition and Properties Brands Products References

exopolysac- structure
charide

Glucose Galactose Acetate Pyruvate RHEOZAN®: thickening and

Succinate 3-hydroxybutyrate stabilizing agent with
SOLVAY pseudoplastic behavior used
NOVECARE in industrial and household (link-95; link-
cleaners 96; link-97;
Alcaligenes faecalis var Anionic charge, Viscous link-98; Rehm,
Succinoglycan myxogenes, shear thinning aqueous JOHNSON and Roc Retinol Correction Deep 2009 and
Sinorhizobium meliloti solutions, Acid stability JOHNSON Wrinkle Night Cream Simsek et al.,
M.W. 5 × 103 – 1 × 106
2009)
SHELL OIL Gravel packing process
COMPANY
RHEO ZAN®: Thickeners in
AMETECH food industry

Fructose
FRUCTANS: used in
(link-99;
by MONTANA cosmetics and film coating to
Neutral charge, Low POLYSACCHARI increase shelf life of food Rehm, 2009;
viscosity, High water DES CO increase shelf life of food de Oliveira et
Alcaligenes viscosus, solubility, Biological materials al., 2007 and
Levan Zymomonas activity: Anti-tumor Senthikumar,
mobilis, Bacillus subtilis activity and Anti- and
inflammatory, Adhesive Gunasekaran,
strength, Film-forming 2005)
capacity Also used as thickener and binder in pet feed.
M.W. 3.0 × 106

Glucuronic acid Acetylglucosmine NOVAENZYMES HYASIS®: Cosmetics,

Biomedicine
ELEVESS, HYDRELLE
American Society (Dermal Fillers): Reduce
of Plastic Surgeons Facial wrinkles (link-100;
Hyaluronan Anionic charge, Regenerist Micro-Sculpting link-101;
Streptococcus spp. and Biological activity, OLAY Cream: An anti-aging link-102;
(Hyaluronic Pasteurella multocida Highly hydrophilic, treatment for sagging skin link-103
acid) Biocompatible ANIGRAN: Deep wounds, and Rehm,
M.W. 2.0 × 106 skin defects, pressure sores, 2009)
burns, post-surgical wounds,
CONTIPRO difficult-toheal wounds,
PHARMA large abrasions, bites and
infected wounds in all animal
species
Biotechnology Vol. 3: Microbial Biotechnology
Microbial Exopolysaccharides: Advances in Applications and Future Prospects 7

polyanhydrides (such as polyphosphates), polyesters, and polyamides,

and have been collectively termed as EPS (Nwodo et al., 2012). The
microbial EPS are polymers that principally consist of carbohydrates
(with monosaccharides as the repeating units) with varying composition
and structure. Based on monomeric composition these EPS are either
homopolysaccharides (made up of single monomer linked by glycosidic
bond) or heteropolysaccharides (composed of more than two monomeric
units joined by glycosidic bond) they also contain a number of different
organic moieties like organic acids and amino acids and inorganic
constituents like phosphates and sulfates (Nanjani and Soni, 2012).

Majority of the microbial exopolysaccharides of industrial relevance

are observed to be homopolymers. These include the glucan polymer
group whose sole monosaccharide component is D-glucose. In spite of
the fact that the monomeric unit is same, these polymers exhibit
significantly diverse properties. The polymers that belong to the
homopolysaccharide group include cellulose, curdlan, dextran, pullulan
and scleroglucan. Curdlan and cellulose are linear homopolysaccharide
with ( 1  3 and  1  4) glycosidic linkage but several others are
branched homopolysaccharides, including scleroglucan ( 1  3 and
branch at  1  6 ) and pullulan ( 1  3 and branch at  1  6).
Dextran, another glucan, contains three different types of linkage (Table
1). Majority of the homopolysaccharides are composed of neutral sugars
however there are few exceptions like alginate that comprise of
polyanionic acid called the polysialic acid (Table 1) (Sutherland, 1988).

Most of the bacterial origin exopolysaccharides are

heteropolysaccharides. These comprise of three or four different
monosaccharide units with different degree of branching. Components
most usually found in EPS are pentoses (namely arabinose, ribose, and
xylose), hexoses (glucose, galactose, mannose, allose, rhamnose, fucose),
amino sugars (D-Glucosamine and D-Galactosamine) or uronic acids
(D-Glucuronic acids, D-Galacturonic acids). Organic or inorganic
alternative such as sulfate, phosphate, acetic acid, succinic acid and
pyruvic acid may also be present (Poli et al., 2010). Most microbial
exogenous layers contain neutral carbohydrates (mainly-hexose,
seldom-pentose) and uronic acids. The commonest extracellular
carbohydrates substituents are acetate esters, pyruvtes, formates and
succinates (Czaczyk and Myszka, 2007).

EXOPOLYSACCHARIDE PRODUCING MICROBES

EPS producing microorganisms are widespread in nature. Habitats

having a medium with prominent carbon/nitrogen ratio are known to
8 Biotechnology Vol. 3: Microbial Biotechnology

contain microorganisms producing polysaccharides, for example, sewer

water from the sugar, paper or food manufacture companies as well as
wastewater plants. Lactic acid bacteria (LAB) are the familiar
mesophilic group of EPS producer since decades. Among mesophilic
bacteria genera, Lactobacillus Bulgaricus, Lactobacillus helveticus,
Lactobacillus brevi, Lactococcus lactis, Leuconostoc mesenteroides and
Streptococcus spp. are the good EPS producer. The other potential
EPSproducers are Pseudomonas spp., Acetobacter spp., Aureobasidium
spp.,Sinorhizobium spp., Escherichia spp., Acetobacter spp., Bacillus
spp., etc.

Thermophilic microorganisms from marine hotsprings, both deep

and shallow, and terrestrial hot springs are Archaeoglobus fulgidu,
Thermococcus litoralis, Bacillus thermantarcticus, Geobacillus
thermodenitrificans, Bacillus licheniformis, Thermotoga maritima,
Thermotoga maritima, Methanococcus jannaschii, Geobacillus
tepidamans V264, etc.

Many halophilic Archaea were described as EPS producers such as

Haloferax, Haloarcula, Halococcus, Natronococcus and Halobacterium.
The most common halophilic EPS producing bacteria belong to genus
Halomonas, most importantly H. maura, H. eurihalina, H. ventosae
and H. anticariensis (Singha, 2012).

Characterization of Exopolysaccharides

The physical, chemic al and biological char acter istics of

exopolysaccharides cumulatively govern the properties of these
polymers. Thus prior to the evaluation of potential application of an
exopolysaccharide, it becomes inevitable to study its composition and
structure. The pre-requisite step in understanding the structure of
exopolysaccharide is its hydrolysis by acid or other agents. The
commonly used acids include sulfuric acid, hydrochloric acid,
trichloroacetic acid and trifluroacetic acid. The EPS hydrolyis leads to
cleavage of glycosidic linkages of the polymer and thus exposing the
monosaccharide constituents. These monomers are reduced to form
sugar alditols and further derivatized by acetylation with acetic
anhydride in the presence of pyridine. These volatile sugar derivatives
are subjected to gas chromatography coupled to mass spectrometry (GC-
MS) analysis and compared with sugar standards (Zhang et al., 2003;
Patel et al., 2010; Poli et al., 2011; Leivers, 2011). High Performance
Anion Exchange Chromatography with Pulsed Amperometric Detection
(HPAEC-PAD) provides an alternative to GC-MS to study monomeric
composition of EPS. The major advantage with this technique is, unlike
Microbial Exopolysaccharides: Advances in Applications and Future Prospects 9

GC-MS, it does not require any of the derivatisation steps. To study

the anomeric conformation of the monomers, the hydrolysate is
subjected to formation of acetylated glycosides of a chiral secondary
alcohol and investigated by GC-MS analysis. The linkage analysis of
the carbohydrate can be understood by methylation of the carbohydrate
components followed by separation with GC-MS. Another
instrumentation technique called nuclear magnetic resonance (NMR
spectroscopy) yields the information on glycosidic linkages, ring size
and anomeric configuration of monomeric units. It also provides
information about the relative orientations of the monomeric units
toeach other. 2D NMR like COSY, NOESY, TOCSY and HSQC are key
techniques for investigating the unequivocal chemical composition of a
said polysaccharide. For determination of molecular mass of the
polysaccharide, a more advanced and efficient method namely size
exclusion chromatography coupled with multi-laser light scattering
(SEC-MALLS) is employed (Patel et al., 2010). Apart from these,
thermoanalytical techniques like Thermal Gravimetric Analysis (TGA),
Differential Scanning Calorimetry (DSC), X-ray diffraction (XRD) and
deuterium induced differential isotope shift studies can aid significantly
in understanding the structure of polysaccharides (Patel et al., 2010;
Parikh and Madamwar, 2006). A comprehensive compilation of
instrumentation techniques for EPS characterization and the
subsequenct information elucidated is depicted in Table 2. The above
techniques can thus contribute significantly to studying the major
chemical characterization which includes estimation of total
carbohydrate content, identification of sugar constituents, identification
of non-sugar constituents, linkage studies, degree of branching and
molecular weight.
Table 2: Characterization techniques for exopolysaccharides
Sr. Type of technique Characterization References
No. details
1 Fourier Transform infrared Monosaccharide Parikh and
spectroscopy (FTIR) composition Madamwar, 2006
2 Viscometric analysis Viscosity
3 High pressure liquid Monosaccharide Sutherland 2003
chromatography (HPLC) composition (book)
4 Size exclusion chromatography Molecular weight Poli et al., 2011;
coupled with multi-laser light
scattering (SEC-MALLS)
5 Gas chromatography coupled to Monosaccharide Zhang et al., 2003;
mass spectrometry (GC-MS) composition, Patel et al., 2010;
monosaccharide Poli et al., 2011
configuration
Table 2: Contd...
10 Biotechnology Vol. 3: Microbial Biotechnology

Table 2: Contd...

Sr. Type of technique Characterization References

No. details
6 Capillary electrophoresis (CE) Monosaccharide Poli et al., 2011
composition (without
hydrolysis of
polysaccharide)
7 GC-MS and 2D NMR – COSY, Structural Hallack et al., 2010;
NOESY, TOCSY and HSQC composition, Linkage Shaun 2011;
analysis, molar ratio Poli et al., 2011
of monosaccharides
8 High Performance Anion Monosaccharide Shaun 2011-thesis
Exchange Chromatography with composition
Pulsed Amperometric Detection
(HPAEC-PAD)
9 X-ray diffraction (XRD) Molecular structure Sutherland 2003
and conformation, (book)
model building
10 Deuterium induced differential Linkage analysis Patel et al., 2010
isotope shift
11 Transmission electron microscopy Distribution of the Poli et al., 2011
(TEM), Atomic force microscopy polymer chain lengths,
(AFM) polymer chain
flexibility and mass
per unit length

Properties of Exopolysaccharides

Microbial EPS are chiefly composed of linear

molecules with side chains of variable length
and complexity (Sutherland, 1997). The
exopolysaccharides are high molecular
weight compounds usually synthesized
within the cell and exported outside with few
exceptions like levan and dextrans which are
synthesized outside the cell (Freitas et al.,
2011). The exopolysaccharides exhibit
viscous appearance (Fig. 3) especially after
extraction with appropriate solvents and
usually portray thinning performance with Fig. 3: Viscous
increasing shear rate (non-newtonion polysaccharide after
extraction with solvent
behavior). Majority of the exopolysaccharides
depict gel forming ability upon exposure to cations. The molecular mass
of EPS varies to some extent as it depends upon the strains and cultural
conditions employed, and on the methods of isolation and purification
of EPS.
Microbial Exopolysaccharides: Advances in Applications and Future Prospects 11

Physiological Role of EPS

The physiological role of EPS is influenced by the habitat of the

producing micro-organism and the ecological niches. In general the EPS
depicts a gluey behavior. Therefore, these polymers constitute the major
part of the biofilm formation process on solid substratum (Langilleet
al., 2000; Czaczyk and Myszka, 2007); minor portion being the proteins
and DNA. Furthermore, the exopolysaccharides impart structural
integrity to the biofilms. It has been well documented that the micro-
organisms forming biofilms are found to be 1000 times more resistant
to antibacterial compounds like antibiotics, surface active agents,
sanitizers than planktonic cells (Lindsay and Von Holy, 1999; Czaczyk
and Myszka, 2007). This response is attributed to the EPS matrix
surrounding the cells that proves to be an effective barrier that limits
the incursion of chemically reactive biocides (Nichols et al., 2005; Kruger
et al., 2008; Kumar et al., 2011). Apart from adhesion phenomenon, the
exopolysaccharides have also been reported to depict cell-cell cohesion
and cell - solid substratum cohesion interactions (Chen and Stewart,
2002). Moreover, with extremophiles, the EPS forms a protective layer
for the cells against the harsh external environmental condition like
high and low temperature, salinity, radiation, high and low pH, etc.
Thus, the EPS covering of extremophiles enables them to survive the
deleterious effect of the extreme environments (Poli et al., 2010). The
exopolysaccharide layer also enables the organisms’ protection from
environmental stresses like toxins and antibiotics. Thus, EPS formation
is fundamental to the existence of such micro-organisms (Kumar et al.,
2011). Difference of opinion exists on the role of exopolysaccharides as
mechanism of resistance to phages. Few studies have attributed the
phage resistance by lactic acid bacteria (LAB) to its exopolysaccharide
(Looijesteijn et al., 2001; Forde and Fitzgerald, 2003). The other reports
deny this hypothesis (Deveau et al., 2002; Rodríguez et al., 2008). This
inconsistency can be imputed to the heterogeneous structure of
exopolysaccharides as well as difference in the mechanism of cell surface
recognition by different phages (Broadbent et al., 2003). The
exopolysaccharides from the sea ice microbial communities depict its
role in strong association to particles and more importantly in
cryoprotection (Junge et al., 2004). Furthermore, most of the
exopolysaccharides from Antarctic bacterial isolates are rich in uronic
acids and sulfate groups and can thus act as ligands for cations like
trace metals such as iron (Fe +3 ) found in the Southern Ocean
environment, thereby providing nutrition to the residing microbial
communities which are usually limited by poor availability of trace
metals (Poli et al., 2010).
12 Biotechnology Vol. 3: Microbial Biotechnology

ADVANCES IN POTENTIAL APPLICATIONS OF

EXOPOLYSACCHARIDES

Food Industry

EPS producing cultures are widely used in dairy foods to provide

viscosity, stability and water-binding functions to dairy products which
is contributed positively to the mouth-feel, texture and taste perception
of fermented dairy products. One of the most important commercial
application of EPS is in yogurt manufacture in which Lactic Acid
Bacteria (e.g. S. thermophilus) is authoritatively used in dairy foods
(Broadbent et al., 2003). Lactobacillus helveticus has the characteristic
EPS which increases water absorption, dough development time and
dough stability in biscuit production process and thus the shelf life of
biscuit is lengthened (Hussein et al., 2010). Exopolysaccharides like
pullulan are used as a partial replacement for starch in pastas or baked
goods. Pullulan solutions are of relatively low viscosity, resembling gum
Arabic, thus used as low-viscosity filler in beverages and sauces.
Pullulan are used as a denture adhesive, a binder and stabilizer in
food pastes, and to adhere nuts to cookies. Pullulan films are formed
by drying a pullulan solution onto an appropriate smooth surface. This
property is used to protect fruits such as apples to improve their shelf
life Films have the thickness of about 5-60 um, these films are clear,
highly oxygen-impermeable and have excellent mechanical properties,
thus keeping the fruits fresh for long duration, prevent fruits from wear
and tear in transport period. As these films readily dissolve in water,
they melt in the mouth as edible food coatings and do not possess any
harmful effects as that of wax coatings normally used for fruits to keep
them fresh. Specialty films may include colors or flavors. Decorative
pullulan chips are produced for food uses. Alternatively, pullulan can
be applied directly to foods as a protective glaze (Leathers, 2003). A
novel high molecular weight heteropolysaccharide composed of fucose,
galactose and glucose and acyl groups (pyruvate, succinate and acetate)
impart a great potential for usage in food industry, namely as thickener,
stabilizer, emulsifier or binding agent like in icings, jams, jelly, pastries,
cheese, sauces, pet food, salad dressing, etc (Torres et al., 2010).
Microbial cellulose has also found significant space in the food industry
recently. Microbial cellulose extracted from Acetobacter xylinum, in the
food industries, was introduced as nata de coco which became a popular
snack in Philippines and other countries. It depicted cholesterol lowering
ability (Chawla et al., 2009). It was also introduced in the diet drinks of
Japan as Manchurian tea for health needs.
Microbial Exopolysaccharides: Advances in Applications and Future Prospects 13

Pharmaceutical Industry and Health Aspect

One of the most remarkable and useful features of a polysaccharide’s

swelling ability manifests itself when it is triggered by a change in the
environment surrounding the delivery system. Depending upon the
polymer, the environmental change can involve pH, temperature or
ionic strength and the system can either shrink or swell upon a change
in any of these environmental factors of these sensitive systems. Drug
release is accomplished only when the polymer swells. Many of the
potentially useful pH sensitive polymers swell at high pH values and
collapse at low pH values, trigger the drug delivery upon an increase
in the pH of the environment. Such materials are ideal for systems
such as oral delivery, in which the drug is not released at low pH values
in the stomach, but rather at high pH values in the upper small intestine
(Manjanna et al., 2010). Natural polysaccharides are extensively used
for the development of solid dosage forms. These polymers of
monosaccharides (sugars) are inexpensive and available in a variety of
structures with a variety of properties. They are highly stable, safe,
non-toxic and hydrophilic and gel forming in nature. Pectin, starch,
guar gum, amylose and karaya gum are a few polysaccharides commonly
used in dosage forms. Non-starch, linear polysaccharides remain intact
in the physiological environment of the stomach and the small intestine,
but are degraded by the bacterial inhabitants of the human colon which
make them potentially useful in targeted delivery systems to the colon
(Singh et al., 2011).

Recent estimates suggest that bacterial biofilms are accounting for

over 80% of microbial infections in the body. The traditional approach
to prevent biofilm formation in vivo is local administration of biocides.
Escherichia coli group II capsular polysaccharide was characterized to
exert broad-spectrum biofilm inhibition activity. A bacterial
exopolysaccharide (A101) not only inhibits biofilm formation of many
bacteria but also disrupts established biofilm of some strains. EPS holds
potential in the design of new therapeutics as it directly targets at the
component of biofilm strategies for bacterial biofilm-associated
infections and limiting biofilm formation on medical indwelling devices
(Jiang et al., 2011). Several exopolysaccharides such as pullulan and
its derivatives exhibit adhesive properties and can be used in wound-
healing compositions (Leathers, 2003). Chitosan refers to a series of
polymers that are deacetylated derivatives of the natural
polysaccharide, chitin, with different degrees of deacetylation and
molecular weights. Highly refined grades of chitosan have been used
in pharmaceutical formulations as a release-controlling agent in oral
preparations. Chitosan has been the subject of interest for its use as a
14 Biotechnology Vol. 3: Microbial Biotechnology

polymeric drug carrier material in dosage form design due to its

appealing properties such as biocompatibility, biodegradability, low
toxicity and relatively low production cost from abundant natural
sources. The scope of polymers used in dosage form design can be
increased by several approaches such as modification of their chemical
structure, by combining different polymers in physical mixtures or by
formation of polymer-polymer associations such as polyelectrolyte
complexes. Polyelectrolyte complexes combine unique physicochemical
properties of different polymers with the advantage of retaining high
biocompatibility. It is therefore not surprising that polyelectrolyte
complexes are gaining importance in modern pharmaceutical technology
(Hamman, 2010).

EPS plays an important role in the development of new

pharmaceuticals, not only because of their ability of forming polymeric
matrices, but also due to their inherent biological activity. For example,
xanthan, sulfated dextran and sulfated curdlan, are used as antiviral
and anticancer agents. As a result of high fucose content in some EPS,
Fucopol is seen as a product with potential to be used in antitumor,
anti-Inflammatory and immune-enhancer drugs (Freitas et al., 2011).
Mushroom polysaccharides have traditionally been used for the
prevention and treatment of a multitude of disorders like infectious
illnesses, cancers and various autoimmune diseases. A water-soluble
Morchella conica Polysaccharides (MCP) modulate nitric oxide
production in macrophages and promote splenocytes proliferation and
it act as a potent immunomodulatory agent (Su et al., 2013). Bacterial
polysaccharides are known to induce the immune response in
macrophages. The excessive use of antibiotics poses tremendous
selection pressure on microorganisms to develop drug resistance, which
eventually leads to incurable diseases. Recently it has been discovered
that immunomodulaters are potential substitute to antibiotics. Natural
products extracted from microorganisms, mushrooms, algae, lichens
and higher plants were known to induce positive immunological effects.
Polysaccharides in these extracts have been found to be the primary
factor for macrophage stimulation through induction of the immune
system of Toll-like receptors (Lin et al., 2011)

The interest in marine microalgae is growing increasingly, especially

because of the compounds they produce. An advantage of working with
microalgae is the fact that they are easy to grow and its culture and
harvest does not depend on the climate or season. Being easily
controlled, it enables the production of polysaccharides, or whichever
other compounds with similar properties, either chemical or physical,
all year. Polysaccharides in general and sulphated exopolysaccharides
Microbial Exopolysaccharides: Advances in Applications and Future Prospects 15

in particular, are released by many species of microalgae. They serve

as antiviral agents, health foods, nutraceuticals, antioxidants, anti-
lipidaemic agents, antiglycaemic agents, anti-inflammatory molecules,
immunomodulators and they may also be used as lubricants for bone
joints, used in eye drops as a lubricator and even antithrombotic agent
(Raposo et al., 2013).

Research

It is well known that agar is widely used as a gelling agent in various

media to provide solid surface for the better growth of microbes since
1892 in microbiology. It is a gelatinous compound in red algae but
research has shown that a group of microorganisms known as
uncultured microbes or yet-to-be cultured microbes fail to grow on agar
medium. These microorganisms can be grown on media which substitute
agar with EPS gellan gum. Studies have proved that some isolated
strains from freshwater don’t form any seeable colony on agar medium
but on gellan gum medium. Moreover, strains like Gemmatimonas
aurantiaca significantly grow in well colonies but not the same on agar
medium. Hence, EPS can substitute agar to provide some novel
applications in research (Tamaki et al., 2009). A new biopolymer-
exopolysaccharide acrylamide (EPAA) can be used as partial
substitution of agar-agar in solid nutrient media in microbiology. The
EPAA in concentration 6.0-12.0 g/l with agar in concentration 10.0 g/l
produces a gel which is as strong as agar in concentration of 15.0-20.0
g/l. The superlative feature of EPAA in nutrient media is that it does
not at all affect the growth, colony and cell morphology and biochemical
properties of microorganisms. The use of EPAA to prepare nutrient
media permits us to reduce expenditures for agar (Vashchenko et al.,
2004). Researchers have attempted to develop methods to permit 100%
preservation of diverse cellular specimens after freezing and thawing.
In an effort to improve cell preservation, cryoprotectants, including
glycerol or dimethyl sulfoxide, are utilized over a span of duration, Along
with these chemicals natural exopolysaccharides extracted from cold
adapted microorganisms from Arctic and Antarctic regions can be used
to improve cell preservation. The probable reason being minimization
of the cell water content thereby bolstering vitrification and protecting
the constitutive macromolecules of the cell. The primary ecologically
significant characteristic of exopolysaccharide of low temperature
habitat microorganisms is that it can form and maintain protective
microhabitats around microorganisms in aquatic and cold environments
(Kim and Yim, 2007). Other exopolysaccharides such as gellan gel is
used in nutrient media in the area of plant tissue culture (Doner and
16 Biotechnology Vol. 3: Microbial Biotechnology

Bécard, 1991). In chromatography, cross linked dextran Sephadex is

filled in columns and function as molecular sieve to separate various
compounds (Axen et al., 1970).

Cosmetics and Personal Care Products

In cosmetics, EPS are widely used in creams and lotions as moisturizer

as they have more water retention capacity and thus improve the skin
moisturisation. EPS SEAFILL which is a purified and completely
sequenced Marine Exopolysaccharide contains galactose, galacturonic
acid, glucose, glucuronic acid and mannose. Its melting texture comforts
the skin immediately after application. In the same time its novel
structure surface gives properties to fill in wrinkles and tightens the
cutaneous effectively after 15 minutes, it then works all day long [link-
21]. Exopolysaccharide from mushroom Grifola frondosa is used in sun
screen protection creams and lotions as it have photoprotective potential
and inhibitory action in photo aging skin (Bae et al., 2004).
Hyperthermophilic and mesophilic bacteria from deep-sea hydrothermal
vents found to be an extraordinary source of innovative molecules with
unusual biological properties. These properties have developed as
defense mechanisms enabling the marine organisms to adapt
themselves to extreme conditions of temperature, pressure and darkness
that prevail in the world’s seas. This property of marine microbes could
be of powerful value in the cosmetic industry by providing a new way
to protect the skin from environmental injuries (Thibodeau,. 2005).
Pullulan has applications in cosmetics. As the viscosity of pullulan
solutions is stable to heating, changes in pH and most metal ions,
pullulan has demonstrated uses in cosmetics creams, lotions, and
shampoos (Leathers, 2003). Xanthan have high cream stability, this
characteristic of xanthan is advisably used in cosmetic industry,
including toothpaste technology, facilitating the suspension of
ingredients (high viscosity) and the easy brushing onto and off the teeth
(high shear thinning). Uniform pigment dispersal along with other
ingredients and long-time stability make xanthan a good base for
shampoos (Morris and Harding, 2009). EPS used in hydrogel in skin
care fabrics, where they are implanted in fabrics. These hydrogels are
used to treate textiles to impart moisturizing, whitening, brightening,
and even anti-ageing effects on human skins (Hilor, 2012.)

Bioremediation and Bioleaching

In this urbanization era, environmental pollution has brought about

various social problems. Soil, air, rivers, seas, waste streams and various
sites have been polluted by the far-flung use of chemicals, such as
Microbial Exopolysaccharides: Advances in Applications and Future Prospects 17

solvents, herbicides, insecticides and many industrial chemicals. A large

number of organisms suffer from these environmental pollutants
causing their entry into consociated food chains. Bioremediation, is an
effective and practicable biological way of using microorganisms and
plants, has the potential to augment the removal of hazardous
compounds from foul sites. Animals used the EPS as a source of energy
and nutrition.

The labile nature of the bacterial EPS and its ability to bind heavy
metals route the bound metals through the marine food chain, thereby
transferring and aiding bioaccumulation of metal pollutants in the
higher trophic animals (Bhaskar and Bhosle, 2006). Polycyclic aromatic
hydrocarbons (PAHs) are persistent pollutants in the environment, and
are emitted during the combustion of fossil fuels and organic compounds.
Due to their potential toxicity, mutagenicity and carcinogenicity, PAHs
pose a serious risk to the environment and human health. EPS contain
large quantities of negatively charged functional groups and have strong
binding capabilities towards heavy metal ions and organic pollutants.
EPS isolated from Zoogloea spp. and Aspergillus niger helps in
degradation of pyrene in contaminated soils (Jia et al., 2011). Some
EPS producing bacteria like Bacillus cereus possess the ability of
biocorrosion of stainless steel thus are used in bioremediation to remove
excessive steel compounds in stainless steel industries (Bragadeeswaran
et al., 2011). Sphingomonads are peculiar due to the presence of many
large pleat-like structures on the cell surface the extraordinary
metabolic ability to degrade various refractory environmental
pollutants, notably xenobiotics such as dioxin, biphenyl and bisphenol;
and the production of useful biopolymers such as gellan and related
polysaccharides (Hashimoto et al., 2010).

Textile Industry

In textile industries EPS are used as a binding agent with color dyes or
hydrogel because of its property such as viscosity, stabilizer and its
cross linking ability with fabrics. Smart fabrics are one of the novel
applications of EPS. Hydrogel (Kulkarni et al., 2010) based on
biopolymers have distinctive physical characteristic of swelling and
shrinkage which is influenced with external component like pH,
temperature, solvent, electric field, light, stress, ionic strength, other
external chemical stimuli, etc. Biopolymers such as chitosan (Aranaz
et al., 2009) in combination with synthetic polymers are used to produce
hydrogel which are successfully embedded on fabric surfaces to have a
smart textile application. These fabrics are implemented as a deodorant
releasing agents at specific temperatures. Thus they are used in aroma
18 Biotechnology Vol. 3: Microbial Biotechnology

finishing of fabrics. The polymer -Cyclodextrin is capable of releasing

it with the change in external temperature or pH (Liu et al., 2009).
These textiles have another application i.e. display change in color with
the change in the external temperature (Fan et al., 2008) by applying
thermochromic materials like cholesterins. This textile can be used in
areas where there is a temperature fluctuations like ware house of
industries. Like this, photochromic textiles are also produced on which,
the irradiation with light causes breakage in the covalent bond between
the carbon and oxygen of photochromic compound to produce ionic pairs
which absorbs photons of visible light, and is colorful. Temperature
dependent delivery of drugs/nutrients like vitamins, Chinese herbs and
other therapeutic medicines can be carried out using these fabrics. The
property of swelling and shrinkage of polymer hydrogel enables a drug
or nutrient loaded in the hydrogel to be released in a controlled manner
(Pathak, 2012).

Petroleum industry and Oil recovery

The petroleum industry uses xanthan gum a bacterial EPS in oil drilling,
fracturing and pipeline cleaning (Chen and Stewart, 2002), and due to
its excellent compatibility with salt and resistance to thermal
degradation, it is also useful as an additive in drilling fluids (Sutherland,
1990). Microbial enhanced oil recovery (MEOR) is a petroleum
biotechnology for manipulating function and/or structure of microbial
environments existing in oil reservoirs for prolonged exploitation of
the largest source of energy. Microbial Enhanced Oil Recovery (MEOR)
is the use of microorganisms to retrieve additional oil from existing
wells, thereby enhancing the petroleum production of an oil reservoir.
In this technique, selected natural microorganisms are introduced into
oil wells to produce harmless by-products, such as slippery natural
substances or gases, all of which help propel oil out of the well. Because
these processes help to mobilize the oil and facilitate oil flow, they allow
a greater amount to be recovered from the well. A genetically engineered
Enterobacter cloacae is used in MEOR (Sun et al., 2011).

Agriculture

Fertility and productivity of soils can be improved by buildup of EPS-

producing bacteria in rhizosphere of the crop plants, since the EPS-
producing bacteria are involved in root adhering soil around- and, water
as well as nutrient flow across plant roots. Very high yields observed in
shoot and root growth of the crop plants grown in a salt affected soil
due to EPS-producing bacterial inoculation could be attributed to PGPR
effect since at later growth stages nutrient uptake and utilization would
Microbial Exopolysaccharides: Advances in Applications and Future Prospects 19

be the limiting factor for growth and yield of the crop plants (Ashraf et
al., 2004). Azotobacter EPS in the soil habitat play key roles in ecosystem
functioning through controlling nutrient cycling reactions essential for
maintaining soil fertility and also contributing to the genesis and
maintenance of soil structure under conventional. biotic, and abiotic
stressed soil environment (Gauri et al., 2012).

Some exopolysaccharides act as a biosurfactants. Biosurfactants are

low molecular weight surface-active compounds widely produced by
bacteria, yeast and fungi. Many rhizosphere and plant associated
microbes produce biosurfactant; these biomolecules play vital role in
motility, signaling and biofilm formation, indicating that EPS governs
plant–microbe interaction. In agriculture, EPS can be used for plant
pathogen elimination and for increasing the bioavailability of nutrient
for beneficial plant associated microbes. EPS can widely be applied for
improving the agricultural soil quality by soil remediation. These
biomolecules can replace the harsh surfactant presently being used in
million dollar pesticide industries. (Sachdev and Cameotra, 2013). The
proteobacteria Rhizobia comprise a very diverse group of nitrogen-fixing
symbiotic bacteria including Rhizobium, Sinorhizobium,
Mesorhizobium, Bradyrhizobium, Azorhizobium, Allorhizobium and
Methylobacterium, as well as Burkholderia and Cupriavidus. They
possess the ability to associate with leguminous plants and induce the
formation of special organs, termed nodules, on roots and stems, inside
of which atmospheric nitrogen is reduced to ammonia by the enzymatic
complex of nitrogenase. The outer surface of rhizobia consists of complex
polysaccharides, among these lipopolysaccharides (LPS), capsular (CPS)
and extracellular (EPS) polysaccharides. EPS are weakly associated
with the bacterial surface and are released in large amounts into the
environment. This surface polysaccharides play an essential role in
the establishment of an effective symbiosis, especially with host plants
that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia
and Medicago spp. (Janczarek, 2011). Exopolysaccharides gellan and
curdlan have potential agriculture importance as they act as
conditioners of soil which in turn results in improves water holding
capacity of cultivation land (Morris and Harding, 2009).

Concluding remarks

The EPS from plants, algae and animals have considerable share of
biopolymer for various application and industrial purposes in the
market. Nevertheless, an increasing awareness for environment and
new look for applications of greener technology will probably go for
microbes as a renewable resource of exopolysaccharides to replace
20 Biotechnology Vol. 3: Microbial Biotechnology

synthetic and other sources exopolysaccharide in the market. The

microbial exopolysaccharide have wide range of potential applications
with respect to human utilization mainly in pharmaceutical products,
cosmetics, health devices, food industries and so on, because of virtually
lack of toxicity problems. Comparably, bacterial genome is easy to
manipulate than higher organisms. Hence, to engineer their genetic
system and metabolic pathway to design microbial strains with
appreciable EPS yield as well as tailoring of EPS to impart specific
material properties of industrial interest becomes simple. Thus, in the
upcoming years, we can expect more research and technologies focusing
on high EPS production and its economical extraction with cheap
substrate usage that have added commercial significance.

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