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Introduction

Extracellular polymeric substances are metabolic products. Their production by selected microorganisms was first reported in the 1880s (Whitfield 1988). Extracellular polysaccharides (EPSs) accumulate on the microbial cell surface and provide protection to the cells by stabilizing membrane structure against the harsh external environment. EPSs also serve as carbon and energy reserves. EPSs are a heterogeneous matrix of polymers comprised of polysaccharides, proteins, nucleic acids, and (phospho) lipids (McSwain et al. 2005). Generally, EPSs have often been reported in bacteria and cyanobacteria (De Philippis et al. 2001; Parikh and Madamwar 2006; Chi et al. 2007); however, they have also been reported in the marine microalga Chroomonas sp. (Bermúdez et al. 2004), Dunaliella salina (Mishra and Jha 2009), the medicinal mushroom Phellinus linteus (Zou et al. 2006), yeast (Duan et al. 2008), basidiomycetes (Manzoni and Rollini 2001; Chi and Zhao 2003), and marine microorganisms (Satpute et al. 2010). EPS-producing microorganisms have been isolated from different natural sources of both aquatic and terrestrial environments, like freshwater, marine water, wastewater, soils, biofilms, and also extreme niches such as hot springs, cold environments, hypersaline and halophilic environments, salt lakes, and salterns (Maugeri et al. 2002; Nichols et al. 2005; Mata et al. 2006; Gerbersdorf et al. 2009; Satpute et al. 2010; Andersson et al. 2011; Kavita et al. 2011).

Microbial polysaccharides show biotechnological promise in pharmaceutical industries as immunomodulators and healing agents and in food-processing industries as gelling and thickening agents. Some EPSs are used as biosurfactants in detoxification of petrochemical oil-polluted areas. Some microbial exopolymers that are commonly used in industries are summarized in Table 5.1 . Exopolymers are used as thickeners and gelling agents to improve food quality and texture in food industries. They can be used as adjuvants to enhance nonspecific immunity, as hydrophilic matrix for development of bacterial vaccines and controlled release of drugs in pharmaceutical industry. Besides these, different environmental applications are also proposed as improvement of water-holding capacity of soil, detoxification of heavy metals and radionuclide-contaminated water, and removal of solid matter from water reservoirs for cyanobacterial EPSs (Bender and Phillips 2004). Some special applications like sludge settling and dewatering were demonstrated with EPSs (Subramanian et al. 2010). Bioremediation is one of most common application for EPSs as they have the capacity to adsorb oil and make it more easily biodegraded. Heavy metals and organic contaminants can be efficiently removed by EPSs as they have a large number of negatively charged functional groups (Liu et al. 2001; Sheng et al. 2005; Bhaskar and Bhosle 2006; Zhang et al. 2006) which form multiple complexes with heavy metal ions.

Table 5.1 Microbial exopolymers and their applications (Sutherland 1998; Kumar et al. 2007; Vu et al. 2009)
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Methods of EPSs extraction are very important as their physicochemical properties may get affected during isolation and purification. Several physical and chemical methods are available for the extraction of EPSs from different sources, such as cell suspension, biofilm, sludge, solid surfaces, and waters. Physical methods include centrifugation, sonication, heating, freeze thawing, while in chemical methods, different chemical agents, like ethylenediamine tetraacetic acid (EDTA), NaOH, and formaldehyde, are used for extractions. Structural diversity and physicochemical characteristics of biopolymers are exhibited by different EPSs. Several analytical methods have been adopted for the analysis of exopolysaccharides, including HPLC, FTIR, and NMR. Monosaccharide composition is generally analyzed by HPLC or advances methods of GC-MS, while functional groups are investigated by FTIR and NMR. Recently, MALDI TOF TOF (Mishra et al. 2011), AFM (Pletikapić et al. 2011), XRD (Mishra et al. 2011), etc., have been used for qualitative analysis of exopolymers. This chapter aims to present comprehensive information on microbial exopolymers, methods of analysis, and their potential future applications.

Sources of Exopolymers Producing Bacteria

Microbial polysaccharides are classified as (a) cell wall polysaccharides, (b) intracellular polysaccharides, and (c) extracellular polysaccharides. The first two are integral part of the cell, while exopolysaccharides are produced by numerous microorganisms, many of which are isolated from aquatic, especially marine ecosystems. About 134 strains have been isolated from 18 different saline habitats, including inland salterns, marine salterns, and soils, and a taxonomic study was reported by Martínez-Cánovas et al. (2004) in order to establish the relationship between exopolysaccharide-producing bacterial strains living in diverse hypersaline habitats. A halophilic, thermotolerant Bacillus strain B3-15 was isolated from water of a shallow, marine hot spring at Vulcano island (Eolian islands), Italy (Maugeri et al. 2002); similarly, Nicolaus et al. (2002) isolated a thermophilic bacteria from the genus Geobacillus from shallow, marine hydrothermal vents of flegrean areas (Italy) and an EPS-producing bacterium Alteromonas macleodii subsp. fijiensis; Pseudoalteromonas strain HYD 721 was isolated from a deep-sea hydrothermal vent (Raguénès et al. 1996; Rougeaux et al. 1999). Extreme marine habitats, deep-sea hydrothermal vents, volcanic and hydrothermal marine areas, and shallow submarine thermal springs were observed as a new source of EPS-producing bacteria (Poli et al. 2010). In contrast to thermal marine environments, little has been reported on EPS-producing bacteria from cold marine environments. Nichols et al. (2004) studied the EPS-producing bacterial strains CAM025 and CAM036, which were closely related to the genus Pseudoalteromonas and isolated from particulate material and melted sea ice, collected from the Southern Ocean. Similarly, a gamma-proteobacterium Pseudoalteromonas sp. SM9913 was isolated from sea sediment in the Bohai Gulf of the Yellow Sea of northeastern China (Qin et al. 2007). A flagella-containing EPS-producing, gamma-proteobacterium Colwellia psychrerythraea strain 34H was isolated from cold marine environments of Arctic and Antarctic sea ice (Marx et al. 2009).

Marine microorganisms are rich natural resource of exopolysaccharide producers, and several marine microorganisms were isolated from the sea for the EPS production (Satpute et al. 2010). A halophilic EPS-producing archaea Haloferax mediterranei was isolated from the Mediterranean Sea (Anton et al. 1988). A novel EPS-R-producing halophilic marine bacterium Hahella chejuensis was isolated from a marine sediment of Marado, Cheju Island, Republic of Korea (Lee et al. 2001), and a new halo-alkalophilic Halomonas alkaliantarctica strain CRSS was isolated from salt sediments near the salt lake in Cape Russell in Antarctica (Poli et al. 2007). Mata et al. (2008) studied three moderately halophilic, exopolysaccharide-producing bacteria belonging to the family Alteromonadaceae isolated from inland hypersaline habitats in Spain. Jiao et al. (2010) isolated an acidophile from natural microbial pellicle biofilms growing on acid mine drainage water and characterized their extracellular polymeric substances. Ortega-Morales et al. (2007) studied tropical intertidal biofilms that contained Microbacterium and Bacillus species as a source of novel exopolymers. Exopolysaccharides produced by Antarctic isolates, representing the genera Pseudoalteromonas, Shewanella, Polaribacter, and Flavobacterium were characterized by Nichols et al. (2005). They concluded that members of the Gammaproteobacteria and Cytophaga-Flexibacter-Bacteroides dominate polar sea ice and seawater microbial communities. Several marine thermophilic strains were isolated from shallow hydrothermal vents and marine hot springs, near Lucrino area (gulf of Pozzuoli, Naples, Italy), and around Ischia Island (Flegrean areas, Italy) and analyzed for exopolysaccharide production (Nicolaus et al. 2004). They also identified four new polysaccharides from thermophilic marine bacteria Bacillus thermantarcticus isolated from the crater of Mount Melbourne. Ganesh-Kumar et al. (2004) screened some heavily polluted soil samples from the mudflats surrounding the city of Inchon, Korea, and isolated a haloalkalophilic bacterium Bacillus sp. I-450 that was identified as an extracellular polysaccharide producer. Most of the marine producers of EPSs are Gram-negative bacteria such as Pseudomonas, Acinetobacter, Vibrio, and Alteromonas. EPSs from marine biofouling Vibrio species, namely, V. harveyi, V. alginolyticus, V. furnissii, and V. parahaemolyticus (Muralidharan and Jayachandran 2003; Bramhachari and Dubey 2006; Bramhachari et al. 2007; Kavita et al. 2011) were characterized in detail.

Biosynthesis and Genetic Regulation

EPSs are synthesized intracellularly either throughout growth or during late logarithmic or stationary phase. Rate of EPS production also depends on stresses, namely, nutrient imbalance, salt, temperature, pH, etc. Microbial exopolysaccharides are apparently synthesized in four steps (Stanford 1979) with the help of four groups of enzymes (Kumar et al. 2007) as shown in Fig. 5.1 . In the first step (uptake of substrate), a specific substrate, for example, glucose, is taken up by the bacterial cell. Entry of sugar moiety may be accomplished by active transport, diffusion, or group translocation (Roseman 1972). In the second step, metabolism of sugar proceeds, and the substrate is phosphorylated. The intracellular enzyme “hexokinase” phosphorylates glucose to glucose-6-phosphate which is subsequently converted to glucose-1-phosphate by “phosphoglucomutase.” The phosphorylated glucose may be utilized for energy (i.e., catabolism) or for the formation of polysaccharides (anabolism). Enzyme of group II “UDP-glucose pyrophosphorylase” convert glucose-1-phosphate to UDP-glucose (uridine diphosphate glucose). UDP-glucose is a key intermediate, which can be interconverted to other sugars and proceed toward catabolic pathways. It can also enter in third step of polymerization, where polysaccharides are synthesized by an anabolic pathway. Thus, fate of intermediate and formation of polysaccharides is controlled by the cell, which can be achieved by genetic regulation of synthesis or hydrolysis of precursors (Lieberman and Markovitz 1970). Bacterial polysaccharides are comprised of repeating units of sugars moieties, which are synthesized by third group of enzymes “glycosyltransferases.” They transfer a sugar moiety to a repeating unit, which is attached to a glycosyl carrier lipid, identified as isoprenoid alcohol. The terminal alcohol group of lipid carrier is linked through a pyrophosphate bridge to a monosaccharide unit. Thus, in this step, monomers are polymerized, and polysaccharides are synthesized. Individual repeating sugar units are linked onto a lipid carrier through glycosyltransferases, a key enzyme that catalyzes the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming a glycosidic bond (Campbell et al. 1997). Polysaccharides can be modified in fourth step by different enzymatic activities like acylation, acetylation, sulphation, and methylation. Modified polysaccharides are transported to the cell surface and finally exuded from the cell in the form of loose slime or a capsule (Margaritis and Pace 1985) with the help of group IV hydrophobic enzymes like flippase (Liu et al. 1996), permease (Daniels et al. 1998), or ABC transporters (Sutherland 2001).

Fig. 5.1
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Schematic representation of microbial exopolysaccharide biosynthesis

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Homology among the gene and gene products was noted for different polysaccharide-synthesizing systems, and a gene sequence of 12–17 kb (single or multiple copies) may be required (Sutherland 2001). Length and copy number of the responsible gene depends on the complexity of polysaccharide. Biosynthesis of alginate in Pseudomonas aeruginosa is under control of single operon includes algA, algC, algD, algE, and algK genes, while in Azotobacter vinelandii, it is regulated by three transcriptional units (Martinez-Salazar et al. 1996; Gacesa 1998). Regulatory systems comprise similar clusters of genes and gene products in both bacterial species.

Exopolysaccharide synthesis is generally controlled by gene(s) located on chromosomes, but in some bacteria, it is controlled through a two component system, that is, by megaplasmids and chromosome. In E. coli strains, generally one chromosomal segment controls synthesis of the sugar nucleotides required for polysaccharide formation along with enzymes for monosaccharide transfer and polymerase (Sutherland 2001). In contrast, biosynthesis of succinoglycan is regulated by a two component system (chromosomal and megaplasmid) comprised of about 30 genes (Cheng and Walker 1998) in Rhizobium meliloti, in which succinoglycan biosynthesis is fully characterized (Glucksman et al. 1993; Becker et al. 1995).

Biosynthesis of xanthan in Xanthomonas campestris is regulated by eight chromosomal gene loci, and gene loci eps7 contains the gum gene cluster, encoding functional enzymes required for assembly of the lipid-bound repeating unit (Tseng et al. 1999). Gum gene cluster is a linear sequence of 16 kb transcribed as a 12-cistron operon containing two promoters (Fig. 5.2a ), one upstream to gene gumB, and a second internal upstream to gene gumK (Katzen et al. 1998). It was observed that genes gumB, gumC, and gumJ showed homology with kpsD, exoP, and exoT, respectively, and are involved in translocation or export of the polymer. Gene gumD product shows similarity to bacterial glucosyl- and galactosyl-1-phosphate transferases and thus is probably involved in the first step of xanthan biosynthesis, while genes gumM, gumH, gumK, and gumI encode glycosyltransferases II, III, IV, and V, respectively (Katzen et al. 1998). These gene products are involved in the transfer of a glycosyl residue to growing lipid-linked oligosaccharide. Genes gumF and gumG are involved in acetylation of mannose and encode acetyltransferase, while gene gumL encodes ketal pyruvate transferase and thus is involved in further modification (Katzen et al. 1998).

Fig. 5.2
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Schematic representation of gene cluster involve in microbial exopolysaccharide biosynthesis. (a) gum gene cluster responsible for xanthan biosynthesis in Xanthomonas campestris and (b) eps gene cluster involve in EPS biosynthesis in Streptococcus thermophilus Sfi6

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Similar to the gum gene cluster, a 29-kb multicistronic locus was detected in Sphingomonas sp. and contains regulatory genes required for the biosynthesis of Sphingan S-88 (Yamazaki et al. 1996). Gene spsB of Sphingomonas showed homology with gumD and encodes a priming glucosyltransferase. Like xanthan biosynthesis, several genes are involved in acetan synthesis in Acetobacter xylinum. Nucleotide sugar synthesis is regulated by genes aceF and aceM, and gene cluster aceFA and aceBDCE is involved in the repeating unit synthesis, polymerization, and export. Enzymatic glycosyltransferases activity is controlled by genes aceA, aceB, and aceC (Griffin et al. 1996a, b, 1997a, b). It was observed that initial steps of formation of repeating oligosaccharide unit are controlled by the same set of genes encoding glycosyltransferases in both acetan and xanthan biosynthesis, as both have a similar structure (Griffin et al. 1996a; Katzen et al. 1998).

Biosynthesis of exopolysaccharides in Streptococcus thermophilus Sfi6 is regulated by eps gene cluster (Fig. 5.2b ) of 14.5-kb region comprised of 13 genes, namely, epsA to epsM (Stingele et al. 1996). Gene epsA located at the beginning of the cluster is involved in the regulation of EPS expression, and the central region (epsE, epsF, epsG, epsH, and epsI) of the gene cluster is involved in biosynthesis of the tetramer repeating unit. Gene epsE encodes the galactosyltransferase, catalyzing the first step of biosynthesis of the repeating unit. Genes upstream (epsC and epsD) and downstream (epsJ and epsK) of the central region regulate polymerization and export of the EPS.

EPS Composition and Analytical Techniques/Characterization

EPSs are comprised of repeating units of monosaccharides, which may link with proteins (glycoproteins), lipids (glycolipids), acids (e.g., glucuronic acid, galacturonic acid, or mannuronic acid), and/or extracellular DNA. EPSs mostly consist of a limited number of different monosaccharide types or their derivatives and show a high diversity through various combinations of monosaccharide units arranged in linear or branched configurations. The most common backbone linkages of monosaccharide sequences are 1,4-β- or 1,3-β-linkages, which exhibit characteristic structural rigidity, while other linkages like 1,2-α- or 1,6-α-linkages are considered to have flexible structures. Extracellular polysaccharides are associated with each other and can also interact with other components of the EPS matrix, like proteins, lipids, inorganic ions, and other macromolecules of the bacterial cell surface (Meisen et al. 2008). The common components of bacterial exopolysaccharides are shown in Table 5.2 .

Table 5.2 Common components of bacterial exopolysaccharides (Kenne and Lindberg 1983)
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Microbial extracellular polysaccharides can be divided into three groups on the basis of structural composition (Sutherland 1997). (1) Homopolysaccharides—These are comprised of a single structural unit that can be further categorized as (a) linear or (b) branched polymers. An example of a linear homopolysaccharide is bacterial cellulose (polyglucose). Branched homopolysaccharides are represented by levans (polyfructoses) and dextrans (polyglucose). (2) Heteropolysaccharides—These are composed of two or more structural repeating units with varying complexity. These have a regular structure and may also possess short side-chains. (3) Polysaccharides with irregular structure—A regular structure is not defined for this group of polysaccharides. Alginate is one of the best examples of this group. It has two structural units—d-mannuronic acid and l-guluronic acid and composed of (a) blocks of 1,4-linked β-d-mannuronic acid (M), (b) blocks of 1,4-linked α-l-guluronic (G), and (c) mixed blocks (-M-G-) of alternating mannuronic acid and guluronic acid residues. Basic structures of the common exopolysaccharides are listed in Table 5.3 .

Table 5.3 Common exopolysaccharides and structures
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A method was developed for determining the mass-molecular composition of microbial exopolysaccharides by Votselko et al. (1993). In this method, molecular mass composition of microbial exopolysaccharides (EPS) is determined by centrifuging EPS in a combined density gradient created by NaC1 and CsCl solutions, and molecular mass of dextran is used as standard. EPS is preliminary assayed for total carbohydrate content using the phenol sulfuric acid method with glucose as standard (Dubois et al. 1956), while uronic acids are assayed by a spectrophotometric micromethod of color reaction of methyl pentoses (Dische and Shettles 1948) with glucuronic acid as standard. Sulfated sugars are determined by measuring sulfates after hydrolysis of the polymer with K2SO4 as standard (Terho and Hartiala 1971). The protein content of the EPSs is determined by Lowry method where BSA is taken as standard (Lowry et al. 1951). Major constitutive unit of extracellular polymers are sugars which are generally analyzed after acid hydrolysis. High-performance liquid chromatography (HPLC) with refractive index (RI)/UV detection is used for the qualitative and quantitative determination of various monosaccharides, oligosaccharides, and uronic acids present in the microbial exopolysaccharide. Gas chromatography–mass spectroscopy (GC-MS) is another and sensitive method applied for the determination of monomer units, but it requires conversion of monosaccharides to the partially methylated alditol acetates (PMAA). Identification of the PMAA is made by comparison of the mass spectra obtained with those of known standards. The functional groups and bonds present in EPS are analyzed by Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). Infrared spectroscopy reveals the fact that molecules possess specific frequencies at which they can rotate or vibrate corresponding to discrete energy levels (vibrational modes). These resonant frequencies are determined by the shape of the molecular potential energy surface, by the mass of the atoms, and by the associated vibronic coupling. Variation in stretching and bending modes of vibration with single functional group is usually coupled with the vibration of adjacent group, as well as with the number of substitution(s) taking place on the molecule itself. This leads to the shifting or overlapping of the peaks of two or more functional groups in the same region of IR spectrum. The interpretation of infrared spectra involves the correlation of absorption bands in the spectrum of an unknown compound with the known absorption frequencies for different type of bonds. NMR spectroscopy is a powerful tool for studying the structure, function, and dynamics of biological macromolecules, widely used to determine chemical structure and conformational changes. The chemical structure of the EPS mucoidan was determined using 1H and 13C NMR spectroscopy and by conducting 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments (Urai et al. 2007). A FTIR and NMR spectrum of EPS is shown in Figs. 5.3 and 5.4 , respectively.

Fig. 5.3
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FT–IR spectrum of EPS extracted from marine bacterium Vibrio parahaemolyticus (Kavita et al. 2011)

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Fig. 5.4
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A typical NMR spectrum of EPSs isolated from Dunaliella salina showing range of chemical shift (1H, ppm) (Mishra et al. 2011)

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Energy dispersive X-ray spectroscopy (EDS or EDX) is one of the variants of X-ray fluorescence spectroscopy used for the elemental analysis of EPS. EDX relies on the investigation of a sample through interactions between electromagnetic radiation and matter, analyzing X-rays emitted by the matter in response to be hit with charged particles (Goldstein et al. 2003). A polymer can be considered crystalline or amorphous, and X-ray powder diffraction (XRD) is a rapid analytical technique most widely used for phase identification of EPS (Kavita et al. 2011; Mishra et al. 2011; Singh et al. 2011.

EPSs, obtained from marine bacterium Vibrio parahaemolyticus, seaweed associated-bacterium Bacillus licheniformis and microalgae Dunaliella salina are amorphous in nature with CI xrd 0.092, 0.397, and 0.12, respectively (Kavita et al. 2011; Mishra et al. 2011; Singh et al. 2011). Crystallinity index (CI xrd ) is calculated from the area under crystalline peaks normalized with corresponding total scattering area, that is, ratio of areas of peaks of crystalline phases to the sum of areas of crystalline peaks and amorphous profile (Eq. 5.1, Ricou et al. 2005). The crystalline domains act as a reinforcing grid and improve the performance of EPS over a wide range of temperatures. A typical XRD pattern of EPS extracted from B. licheniformis is shown in Fig. 5.5 :

$$ C{I_{{xrd}}} = \left( {\frac{{\sum {{A_{{crystal}}}} }}{{\sum {{A_{{crystal}}}} - \sum {{A_{{amorphous}}}} }}} \right) $$
(5.1)
Fig. 5.5
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XRD spectrum of EPS extracted from seaweed-associated bacterium B. licheniformis (Singh et al. 2011)

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The applicability of exopolysaccharides is largely dependent on their thermal and rheological behavior. Thermogravimetric analysis (TGA) is a simple analytical technique that measures the weight loss of a material as a function of temperature. As temperature increases, an amorphous solid will become less viscous, and at a particular temperature, the molecules obtain enough freedom of motion to spontaneously arrange themselves into a crystalline state, known as the crystallization temperature (Dean 1995). This transition from amorphous solid to crystalline solid is an exothermic process, and differential scanning calorimetric analysis showed a significant thermal transition of EPSs (Fig. 5.6 ).

Fig. 5.6
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A DSC thermogram of a polymer

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Matrix-assisted laser desorption—ionization mass spectroscopy is a convenient method for rapid and sensitive structural analysis of oligosaccharides (Harvey 1999). It was observed that MALDI TOF TOF mass spectrometric analysis reveal a series of masses (m/z) corresponding to pentose and hexose sugars (150, 180) individually or combined as disaccharides (2 pentose—approx. 300, 1 pentose +1 hexose—approx. 330, and 2 hexsose approx. 360) in midrange linear mode while positive ion reflector mode exhibited a higher range of masses m/z attributed to oligosaccharides comprised of hexose and pentose moiety linked in different combinations. Positive ion linear mode was found suitable for oligomers and reflector mode for polysaccharide analysis (Mishra et al. 2011). Recent progress in the surface and structure analysis of EPS has resulted from the development of advanced microscopy and spectroscopy techniques like atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM), infrared spectroscopy, nuclear magnetic resonance imaging (NMRI), Raman spectroscopy (RM), and scanning electron microscopy (SEM). The physicochemical properties and further applications of EPSs depend on its emulsifying activity in different solvents, rheological behavior under varying pH, temperature and stress, gel strength, binding ability toward metals, ions, biodegradability, immunogenicity, thermostability, nature, abundance (availability), and downstream processing (fermentation and commercialization).

Common Exopolysaccharides: Property and Applications

Microbial exopolysaccharides have a wide range of applications depending on their nature, composition, and structure. Generally a limited number of monosaccharides comprise EPS, and its structural diversity determines its possible applications. EPSs are used in the food, pharmaceutical, biomedical, bioremediation, and bioleaching fields because of their physical, rheological, some unique properties, and wide structural diversity. Medicinal applications include antitumor, antiviral, and immunostimulant activities of exopolysaccharides produced by marine Vibrio and Pseudomonas as well as several other bacterial genera (Okutani 1984, 1992). A low molecular weight heparin-like exopolysaccharide was extracted from Alteromonas infernus, isolated from deep-sea hydrothermal vents, which contains anticoagulant property (Colliec et al. 2001). EPSs are also reported for the prevention of tumor cell development, formation of white blood cells and in the treatment of the rheumatoid arthritis (Vanhooren and Vandamme 2000). Owing to their low immunogenic properties, some low molecular weight exopolysaccharides are used as integral components of vaccines, adjuvant, or as carriers of antigenic proteins. Most of the EPSs have applications in food industries as gelling agent, and it was reported that gelrite, an exopolysaccharide isolated from Pseudomonas sp., is superior to agar (Lin and Casida 1984). EPSs are also used as surfactants and emulsifiers, which attracted attention because of their biodegradability (Rosenberg and Ron 1997). The flocculation and metals binding ability of EPS in solutions make its wide applications in the removal of heavy metals from the environment (Pal and Paul 2008). Thus, bioremediation of targeted pollutants such as heavy metals, hydrocarbons, petroleum, polycyclic aromatic hydrocarbons, microaromatics, polychlorinated biphenyls, chlorinated phenols, and aliphatics is one of the most important applications of EPS (Lynch and Moffat 2005). Apart from these, it is also used to enhance oil recovery (Sun et al. 2011). Biofilm-mediated bioremediation is an effective and safe method for removing pollutants from water (Singh et al. 2006). Bacterial exopolysaccharides play a key role in biofilm formation, facilitating initial adhesion leading to development of complex architecture. In contrast, antibiofilm activity was observed with an exopolysaccharide obtained from a marine Vibrio sp. (Jiang et al. 2011).

Xanthan has widespread applications in diverse fields like crude-oil recovery, paints industry, pesticide and detergent formulations, cosmetics, pharmaceuticals, printing inks, and food sector (Vandamme and Soetaert 1995; Sutherland 1997; 1998; 1999; Kumar et al. 2007; Kumar and Mody 2009). Xanthan is an extracellular polysaccharide produced by the bacterium Xanthomonas campestris composed of homopolysaccharide d-glucose backbone with trisaccharide side chains, modified with differing proportions of O-acetyl and pyruvic acid acetal. Xanthan has remarkable emulsion stabilizing, particle suspension ability, and recoverable shear-thinning activity with high viscosities even at low concentration. Generally its viscosity does not change greatly on raising the temperature, and it is stable at both acid and alkaline pHs. It forms pseudoplastic dispersions in water and can also form gels in solution even with some nongelling polysaccharides. Xanthan gum has several applications and received GRAS (generally regarded as safe) listing for edible purposes. In the food sector, it is used in French dressing, for making cool packs and paper to wrap food stuffs, cottage cheese creaming emulsions, as a toothpaste stabilizer, cattle feed supplement, and calf milk replacer. Its shear-thinning characteristics is utilized in the paint industry as xanthan containing paints are highly viscous at low shear rates and thus will not drip from the brush. Its solution is also used as lubricant for drilling machines used in drilling muds at drilling oil wells.

Similar to xanthan, another and the only bacterial exopolysaccharide listed in GRAS is gellan, which is a product of the bacterium Sphingomonas paucimobilis (Sutherland 2002). The physicochemical property of gellan that is of considerable interest is that it is highly viscous and shows high thermal stability (Banik et al. 2000). It is an excellent gelling agent, making clear, brittle gels at a lower concentration than agar (Lin and Casida 1984). Because naturally gellan is nongelling, it must be modified for commercial purposes and is available under the trade name of gelrite or phytogel (Banik et al. 2000). It is widely used as replacement of agar in culture media. As an elastic gel, it holds particles in suspension without altering the viscosity of the solution (Kumar and Mody 2009). Furthermore, it shows thermal and acid stability, elasticity and rigidity, and high transparency (Sutherland 1998; Banik et al. 2000; Kumar and Mody 2009). Gellan is also used in pharmaceutical industries as a vehicle for ophthalmic drugs release (Carlfors et al. 1998). In the food sector, it is used in low calorie jams and jellies, as a fining agent for alcoholic beverages including beers, wines, and fortified wines (Sutherland 1998; Banik et al. 2000; Kumar and Mody 2009; Patel and Patel 2011).

Dextran is an important extracellular bacterial polysaccharide widely used as a molecular sieve for purification and separation of biomacromolecules, like proteins, nucleic acids, and polysaccharides, as matrices (e.g., SephadexTM) in size-exclusion chromatography (Naessens et al. 2005). Dextran is also used in clinical research as “clinical dextran,” a blood plasma substitute, in alleviate iron-deficiency anemia, and in confectionary to improve moisture retention, viscosity, and inhibit sugar crystallization (Sutherland 1997, 1998, 1999; Kumar et al. 2007; Kumar and Mody 2009).

Alginate or alginic acid is a well-known commercial product obtained from brown algae. It is also secreted by Pseudomonas aeruginosa and Azotobacter vinelandii as extracellular polysaccharides (Remminghorst and Rehm 2009). Applications of bacterial alginate include its uses as an emulsion stabilizer, gelling agent, thickener, foam stabilizer, suspending agent, viscosifier, film forming, or water-binding agent, and in the pharmaceutical industry for encapsulation of cells and enzymes for slow release and as wound dressings and dental materials. In the agriculture sector, it is used as a coating for roots of seedlings and plants to prevent desiccation, and as a microencapsulation matrix for fertilizers, pesticides, nutrients, etc. (Vandamme and Soetaert 1995; Sutherland 1998; Kumar and Mody 2009).

Hyaluronan or hyaluronic acid (HA) is a linear polymer and has significant structural, rheological, physiological, and biological functions. It is thought to possess properties that can affect angiogenesis, cancer, cell motility, wound healing, and cell adhesion and thus has a wide range of applications in the cosmetic and medicinal fields as skin moisturizers, in artificial tears, osteoarthritis treatment, as replacer of eye fluid in ophthalmic surgery, lubricant for the joints, for adhesion prevention in abdominal surgery, wound healing, and surface coating (Vandamme and Soetaert 1995; Yamada and Kawasaki 2005; Widner et al. 2005).

Exopolysaccharide curdlan has ability to form an elastic gel (gelatin) in aqueous suspension beyond approx. 55 °C (Dumitriu 2004) and is used in food and pharmaceutical industries to improve texture and stability of foods (heat processed food) and release of drugs, respectively (Gummadi and Kumar 2005; Kumar and Mody 2009). Succinoglucan is a glucose-galactose heteropolymer, which contains succinate and pyruvate moieties with similar applications as curdlan (Vandamme and Soetaert 1995). Since curdlan has no caloric value, it is useful in low-calorie foods (jams and jellies). During baking, it is used as a film to support immobilization of enzymes (Sutherland 1998). Bacterial cellulose is highly pure polymer and used in specific applications including food supplements such as a food matrix, dietary fiber, or thickening or suspending agents (Sutherland 1998). Besides this, it is used as a temporary artificial skin for healing burns and surgical wounds in human (Vandamme and Soetaert 1995). Future application of bacterial cellulose is in separation technology as membranes or hollow fibers and as a special paper source (Vandamme and Soetaert 1995). Another microbial EPS cyclosophorans has potential applications in encapsulation of drugs and food components (Vandamme and Soetaert 1995). EPS welan has stability and viscosity at elevated temperatures and thus is used at oil well-drilling sites. It is also use as a stabilizer and viscosifier in cement systems (Kumar and Mody 2009). Antibacterial, antifungal, and antitumor activity has been reported for the bacterial EPS, kefiran (Micheli et al. 1999; Kumar and Mody 2009). Kefiran is traditionally consumed and used in the formulation of self-carbonated, slightly alcoholic fermented milk (Duboc and Mollet 2001). In addition, it used to enhance viscosity of the dairy products. Recently, levans have drawn attention of researchers because of their prebiotic properties and wide scope in dairy industries. A cytotoxic effect against human cancer cell lines has been reported for a sulfated exopolysaccharide obtained from Pseudomonas sp. (Matsuda et al. 2003). Its application is now being extended toward development of new drugs to be used for pharmaceutical purposes (Laurienzo 2010).

Future Prospects

Microbial exopolysaccharides are preferred in industries owing to their novel functionality, reproducible physicochemical properties, stable cost, and supply. The increased demand of natural polymers for various industrial applications in recent past has led to an interest in EPS production by new sources. Most of the microbial world remains unexplored because of enormity of the biosphere and a large fraction of natural bacteria cannot be cultured by existing methods. Intelligent screening of microbes for novel exopolysaccharides is prerequisite for its further exploration toward commercialization. An advanced approach is needed for applications of exopolysaccharides in the medical or pharmaceutical field and food sectors. A multidisciplinary approach including microbiology, biochemistry, genetics, molecular biology, fermentation technology, dairy science, chemistry, etc., is imperative in order to develop novel industrially important biopolymers. Exopolysaccharides used in food sectors require further research and development to expand its use and to be listed in GRAS. In future, there will be further development of prebiotic and probiotic-based dairy products where bacterial exopolysaccharides will play a key role.

There are several reports regarding the novelty of exopolysaccharides extracted from marine bacteria and its pharmaceutical applications. Applicability and acceptability of exopolysaccharides in pharmaceutical industries has now opened a new avenue for the research to utilize novel bacteria that inhabit unexplored marine ecosystems. Microbial exopolysaccharides are constantly evolving, and advancement in biological techniques is required for its in vitro production. The main challenges for the commercialization of new microbial exopolysaccharides are the identification (of both strain and exopolysaccharides nature), improvement of original structures, cost of production, and development of downstream process. There is a need of a suitable downstream process for the production and commercialization of exopolysaccharides. There are several problems associated with fermentation processes for the production of EPSs because of the properties of exopolysaccharides, such as high gel strength, viscosity, and foaming. More research is needed on fermentation technology to overcome these limitations. Further developments are also required for genetic engineering of microbes so they can more efficiently convert inexpensive raw materials to exopolysaccharides. In the light of current knowledge, an emphasis should be given to discover new microbial exopolysaccharides with potential multifarious applicability. In future years, there will be a quest for renewable resources and to preserve the ecosystem.