Isolation and physico-chemical characterisation of extracellular polymeric substances produced by the marine bacterium Vibrio parahaemolyticus

A marine bacterial strain identified as Vibrio parahaemolyticus by 16S rRNA gene (HM355955) sequencing and gas chromatography (GC) coupled with MIDI was selected from a natural biofilm by its capability to produce extracellular polymeric substances (EPS). The EPS had an average molecule size of 15.278 μm and exhibited characteristic diffraction peaks at 5.985°, 9.150° and 22.823°, with d-spacings of 14.76661, 9.29989 and 3.89650 Å, respectively. The Fourier-transform infrared spectroscopy (FTIR) spectrum revealed aliphatic methyl, primary amine, halide groups, uronic acid and saccharides. Gas chromatography mass spectrometry (GCMS) confirmed the presence of arabinose, galactose, glucose and mannose. 1HNMR (nuclear magnetic resonance) revealed functional groups characteristic of polysaccharides. The EPS were amorphous in nature (CIxrd 0.092), with a 67.37% emulsifying activity, thermostable up to 250°C and displayed pseudoplastic rheology. MALDI-TOF–TOF analysis revealed a series of masses, exhibiting low-mass peaks (m/z) corresponding to oligosaccharides and higher-mass peaks for polysaccharides consisting of different ratios of pentose and hexose moieties. This is the first report of a detailed characterisation of the EPS produced by V. parahaemolyticus, which could be further explored for biotechnological and industrial use.

Introduction All surfaces exposed to the marine environment are subject to colonisation by marine organisms, a phenomenon known as biofouling, comprising microfouling and macrofouling (Aldred and Clare 2008;Dobretsov et al. 2009). In an aqueous environment, microbial cells grow in association with surfaces, leading to the formation of biofilms. Bacterial biofilms are sessile communities of microorganisms that coexist with highly differentiated extracellular polymeric substances (Dickschat 2010). The extracellular polymeric substances (EPS) are metabolic products that accumulate on the microbial cell surface and provide protection to the cells by stabilising their membrane structures against the harsh external environment and also serve as carbon and energy reserves during starvation. EPS, a heterogeneous matrix of polymers composed of polysaccharides, proteins, nucleic acids and (phospho)lipids (McSwain et al. 2005;Flemming et al. 2007), are renewable resources of biotechnological importance. The precise composition of a biofilm varies with the resident species and environmental conditions. The EPS of microorganisms possess a wide diversity of structural, physical, rheological and other unique properties (Vu et al. 2009). These exopolymers are used in food industries as thickeners and gelling agents to improve food quality and texture. In the pharmaceutical industry, exopolymers can be used as a hydrophilic matrix for the controlled release of drugs, the development of bacterial vaccines and to enhance nonspecific immunity (de Carvalho and Fernandes 2010). EPS are regarded as an abundant source of structurally diverse polysaccharides, some of which may possess unique properties for special applications such as sludge settling and dewatering (Subramanian et al. 2010).
Microbial EPS are preferred in industry owing to their novel functionalities, reproducible physicochemical properties, stable cost and abundant supply. The recent increase in demand for natural polymers in various industrial applications has motivated interest in developing new sources of EPS production. Marine microorganisms, which are currently used as a source of products of high aggregate value, such as pigments, metabolites, fatty acids and proteins, could also be exploited for their EPS as biosurfactants and/or bioemulsifiers (Satpute et al. 2010). Marine microorganisms such as Acinetobacter, Arthrobacter, Pseudomonas, Halomonas, Myroides, Corynebacterium, Bacillus, Alteromonas spp. have been studied for the production of biosurfactants and exopolysaccharides (Satpute et al. 2010).
Most of the marine microbial world remains unexplored due to the enormity of the marine biosphere (Satpute et al. 2010). The discovery of potent biofilm-producing marine microorganisms could enhance the use of environmentally biodegradable EPS molecules in industry and reduce dependence on biohazardous, nondegradable synthetic polymers. To date, the study of biofilm-forming bacterial species has been scanty, especially from the Indian west coast, and exopolymer-producing marine bacteria have rarely been cultured or identified. Vibrio species are ubiquitous in marine ecosystems and well known for biofilm formation (Yildiz and Visick 2008). Exopolymers produced by Vibrio spp. display huge diversity in composition and potential applications. Despite the immense potential of exopolymers produced by Vibrio spp., the characterisation of EPS by only a few species, viz., Vibrio harveyi, Vibrio alginolyticus and Vibrio furnissii (Muralidharan and Jayachandran 2003;Bramhachari and Dubey 2006;Bramhachari et al. 2007), has been reported so far. The present study is the first report of a detailed characterisation of planktonic EPS produced by the marine bacterium Vibrio parahaemolyticus. This work involved the isolation and physiochemical characterisation of planktonic EPS extracted from the biofilm-forming V. parahaemolyticus, which could be useful in biotechnological and industrial applications.

Materials and methods
Isolation and identification of a biofilm-forming bacterial strain Bacteria were isolated from a natural biofilm collected from the coastal region of Diu, India (latitude N 208 42 0 20.8 00 , longitude E 708 58 0 6.42 00 ) using Zobell 2216 agar medium (Kwon et al. 2002). The maximum EPSproducing bacterial strain was selected by quantifying the amounts of total EPS produced, as identified by fatty acid methyl ester profiling using MIDI (GC system-6850, Agilent technologies, USA) and 16S rRNA gene sequencing amplified by universal primers (fD1-5 0 -AGA GTT TGA TCC TGG CTC AG -3 0 and rP2-5 0 -ACG GCT ACC TTG TTA CGA CTT -3 0 ) (Weisburg et al. 1991). The sequences were evaluated and phylogenetic relationships were inferred by a neighbour joining (NJ) method in BLAST (Zhang et al. 2000).

Extraction and purification of EPS
V. parahaemolyticus was cultured in Zobell 2216 medium (500 ml) under controlled laboratory conditions at 30 + 28C (180 rpm). Bacterial cultures (3 days old) were centrifuged at 15,000 6 g for 20 min at 48C. The supernatant was filtered twice and concentrated to a volume of 100 ml using a rotary evaporator (Buchi, Switzerland) for 8-10 h. The exopolymer was precipitated with trichloroacetic acid (20%, 48C, 30 min) to remove proteins and nucleic acids (Andersson et al. 2009). The supernatant was gradually precipitated by adding two volumes of cold isopropanol and held at 48C for 12 h to obtain exopolysaccharides from crude EPS (Mishra and Jha 2009). The precipitate was washed with absolute alcohol and dissolved in 50 ml MilliQ water (Millipore, USA). Dissolved EPS were dialysed for 1 day against distilled water for purification, and purified EPS were lyophilised at -708C for 10-12 h (Chi et al. 2007).
Particle size measurement and emulsifying activity EPS (50% w/v) samples dissolved in water were sonicated for 5 min, and the particle size distributions were measured by laser diffraction (Malvern Mastersizer 2000, Malvern Ltd, Worcestershire, UK). Lyophilised EPS (1 mg) was dissolved in 0.5 ml deionised water, heated to 1008C for 15 min and allowed to cool to room temperature (25 + 28C). The volume was brought up to 2 ml using phosphate-buffered saline (PBS). The sample was vortexed for 1 min after the addition of 1 ml hexadecane. The absorbance at 540 nm was measured immediately before and after vortexing (A 0 ). The decline in absorbance was recorded after incubation at room temperature for 30 min (A t ). A control was run simultaneously with 2 ml PBS and 1 ml hexadecane. The emulsification activity was expressed as the percentage retention (t % ) of emulsion during incubation for time t, following Equation (1) (Mishra and Jha 2009).
Fourier-transform infrared spectroscopy The major structural groups of purified EPS were detected using FTIR spectroscopy. The pellet for infrared analysis was obtained by grinding a mixture of 2 mg EPS with 200 mg dry KBr followed by pressing the mixture into a 16 mm diameter mould. The FTIR spectra were recorded in the region of 4000-400 cm 71 on a GX FTIR system (PerkinElmer, USA).

Powder X-ray diffraction analysis
EPS (2 mg) were analysed by X-ray diffraction using an X-ray powder diffractometer (Philips X'pert MPD, The Netherlands) with 2y ranging from 2 to 808 at 258C. The irradiated length and specimen length were each 10 mm, with a receiving slit size of 0.2 mm at a 200 mm goniometer radius. The distance between the focus and divergence slit was 100 mm. Dried EPS samples were mounted on quartz substrata, and the intensity peaks of diffracted X-rays were continuously recorded with a scan step time of 1 s; d-spacings appropriate to the diffracted X-rays at each y value were calculated from Bragg's law ( Equation 2).
where y is half of the scattering angle measured from the incident beam. The crystallinity index (CI xrd ) was calculated as the ratio of the peak areas of the crystalline phases to the sum of the areas of crystalline peaks and the amorphous profile (Equation (3), Ricou et al. 2005).
Analytical gas chromatography mass spectrometry (GCMS) Exopolymers were assayed for total carbohydrate content using the phenol sulphuric acid assay with glucose as a standard (Dubois et al. 1956). The EPS were hydrolysed in 2.0 M H 2 SO 4 (1008C, 6-8 h), and sugar was converted to alditol acetates by reduction followed by methylation (Mehta et al. 2010). Alditol acetates were analysed and quantified on a GCMS-QP2010 (Shimadzu, Japan) using an SGE BP-225 capillary column (25 m length, 0.22 mm diameter and 0.25 mm thickness). The injector temperature was 2208C, and the carrier gas was helium at a flow rate of 1.0 ml min 71 ; the initial temperature of 1608C was held for 3.0 min, followed by a ramp from 160 to 2308C at 10 C min 71 and a final hold at 2308C for 10 min, all at a pressure of 131.8 kPa. The injection volume was 1 ml, the electron impact ionisation was 70 eV, the temperatures of the ion source and quadrupole were both 2408C; and the acquisition mode was scanning from m/z 60 to m/z 400 with a scan interval of 0.5 s and a scan speed of 714 per cycle.
MALDI TOF-TOF mass spectrometry EPS samples were prepared in acetonitrile (5% v/v; 1 mg ml 71 ) and mixed with an equal volume of the matrix (a-cyano-4-hydroxycinnamic acid; 10 mg ml 71 ). Matrix-assisted laser desorption/ionisationtandem time-of-flight (MALDI TOF-TOF) analysis was performed on an Applied Biosystems 4800 MALDI TOF-TOF analyser with an Nd-YAG (neodymium-doped yttrium aluminium garnet) laser (355 nm, 200 Hz) operated at an acceleration voltage of 20 kV. Each spectrum was collected in positive ion reflector as well as linear mode, with an average of 1400 laser shots per spectrum (Mishra et al. 2011). The reproducibility of the spectra was determined from six spot sets in each mode, and the spectra were analysed after centroid and deisotoping using the Data Explorer software (Applied Biosystems, USA).
Nuclear magnetic resonance (NMR) and scanning electron microscopy (SEM) The 1 HNMR spectra of the EPS were obtained in D 2 O with a Bruker Avance II 500 (Switzerland) operating at 500 MHz with net spinning at 5000 rpm. The sample was dissolved in water and dehydrated by sequential transfer to increasing concentrations of acetone up to anhydrous, and the morphology of the exopolymer was observed under a scanning electron microscope (SEM, LEO series VP1430, Germany) with an acceleration voltage of 20 kV.
Thermal gravimetric (TG) and differential scanning calorimetry (DSC) analysis TG and DSC analyses of EPS were carried out on a Mettler Toledo TGA/SDTA System (Greifensee, Switzerland). The EPS were enclosed in an aluminium vessel, and their energy level was scanned in the ranges of 30-4008C and 25-6008C for TGA and DSC, respectively, under a nitrogen atmosphere at a heating rate of 10 C min 71 . TG and DSC analyses were performed by gradually raising the temperature and plotting the weight (percentage) and heat flow, respectively, vs temperature. The activation energy (E a ) was determined from the Arrhenius equation (Equation (4)): where A is the frequency factor for the reaction, R is the universal gas constant, T is the temperature (in Kelvin) and k is the reaction rate coefficient.

Rheological studies
Lyophilised samples were dissolved in distilled water (0.4% w/v) (Khattar et al. 2010), and their viscosities were measured on a rheometer (RS1, Haake Instruments, Karlsruhe, Germany) at 258C at varying shear rates (50 to 950 s 71 ). The influence of pH on the viscosity was studied by comparing the viscosities at pH 3.0 and 7.0 (Be´jar et al. 1998). The influence of temperature (108C to 608C) was analysed at both pH values (Checa et al. 2007). All experiments were performed in triplicate, and slippage of the gel due to applied stress was carefully avoided by selecting appropriate operating parameters.

Molecular identification of bacterial strain and EPS extraction
A total of 11 different types of bacterial colonies were obtained from the natural biofilm on Zobell agar medium, among which one isolate was selected on the basis of EPS production (Gauri et al. 2009). Using conserved primers (Weisburg et al. 1991), a 1.42 kb 16S rRNA gene was amplified, sequenced and submitted to NCBI (GenBank Accession no. HM355955). Phylogenetic analysis of the 16S rRNA gene sequence revealed a close resemblance with V. parahaemolyticus (Supplementary Figure S1 [Supplementary material is available via a multimedia link on the online article webpage]). Fatty acid methyl ester profiling of the screened bacterial strain further confirmed that the isolated bacterium was V. parahaemolyticus (Supplementary Figure S2 [Supplementary material is available via a multimedia link on the online article webpage]). High EPS production was observed in the late log phase and stationary growth phase; 58.98 mg l 71 EPS was extracted from a V. parahaemolyticus culture grown in ZMB (Zobell marine broth) medium for 3 days. The yield of EPS was significantly greater compared with that produced by other Vibrio species; generally 27-30 mg l 71 EPS can be extracted from Vibrio species (Bramhachari and Dubey 2006;Bramhachari et al. 2007).

Particle size and emulsifying activity
The EPS consisted of particles with sizes ranging from 4.394 (d 0.1 ) to 40.248 (d 0.9 ) mm, with an average size of 15.278 mm (d 0.5 ) and a specific surface area of 0.728143 m 2 g 71 (Supplementary Figure S3 [Supplementary material is available via a multimedia link on the online article webpage]). The emulsifying activity of the exopolymer was determined by its strength in maintaining an emulsion. It was found to be 67.37% stable for up to 60 min, a value that decreased to 60.52% at 90 min. The exopolymer emulsion was more stable than that of the EPS produced by V. harveyi VB23, which had a stability of 34-40% (Bramhachari and Dubey 2006).

FTIR spectroscopy of the EPS
Fourier-transform infrared spectroscopy (FTIR) reveals the specific frequencies at which molecules can rotate or vibrate, and these resonant frequencies are determined by the nature of molecules with associated vibronic coupling. The interpretation of infrared spectra involves the correlation of absorption bands in the spectrum of an unknown compound with the known absorption frequencies of different types of bonds. The FTIR spectrum of the EPS obtained from the V. parahaemolyticus culture revealed characteristic functional groups (Figure 1). The broad stretch of frequency ranging from 3600-3200 cm 71 was assigned to the hydroxyl group. The weak absorption at 2928 cm 71 (2915-2935 cm 71 ) was attributed to the asymmetrical CÀ ÀH stretching vibration of an aliphatic CH 2 group, which revealed the presence of sugar content (Iyer et al. 2005). IR peaks observed in the range of 2350-2360 cm 71 may be due to CO 2 adsorption or the asymmetric stretching of the À ÀN¼C¼OÀ À group (Mishra and Jha 2009). An asymmetrical medium stretching peak was observed at 1642 cm 71 (1593-1662 cm 71 ), which corresponds to the ring stretching of galactose and mannose (Freitas et al. 2009); however, a peak at 1416 cm 71 represented the symmetric stretching of the À ÀCOOgroup (Pongjanyakul and Puttipipatkhachorn 2007;Freitas et al. 2009). The stretching of CÀ ÀOÀ ÀC and CÀ ÀO at 1000-1200 cm 71 corresponds to the presence of carbohydrates (Mishra and Jha 2009) correspond to the NÀ ÀH wag of primary amines and the CÀ ÀX stretch of alkyl halides, respectively (Mishra and Jha 2009). The FTIR spectrum of EPS confirmed the presence of aliphatic methyl groups, primary amines, halide groups, uronic acid and saccharides (viz., galactose and mannose).
Powder X-ray diffraction analysis X-ray powder diffraction (XRD) analysis is extensively used for the phase identification of polymers. The XRD profile of the EPS obtained from V. parahaemolyticus (Figure 2) exhibited characteristic diffraction peaks at 5. 9858, 9.1508 and 22.8238, with interplanar spacings (d-spacings) at 14.76661, 9.29989 and 3.89650 Å , respectively. The XRD pattern indicated that the EPS were amorphous in nature, with a CI of 0.092. The 9.2% content of crystalline domains acted as a reinforcing grid and improved the performance of the EPS over a wide temperature range, as observed in the calorimetric analysis. The XRD profile and interplanar spacing (d-spacing) are the basic characteristics of a polymer and are useful for comparing or studying the nature of EPS isolated from different sources.

Monosaccharide composition by GCMS and MALDI TOF-TOF
The content of total sugars was estimated by the procedure of Dubois et al. (1956) and was found to be 78.48 mg [mg EPS] 71 . The monosaccharide composition of the extracted EPS was analysed; GCMS analysis ( Figure 3 and Supplementary Figure S4 [Supplementary material is available via a multimedia link on the online article webpage]) revealed the presence of four monosaccharides: arabinose (7.9 mg [mg EPS] 71 ), galactose (19.2 mg [mg EPS] 71 ), glucose (19.9 mg [mg EPS] 71 ) and mannose (31.3 mg [mg EPS] 71 ). The sugar composition of the EPS consisted of hexoses (glucose, 25.4%, and galactose, 24.5%) and pentoses (mannose, 39.9%, and arabinose, 10.1%). The molar percentage of mannose was the highest, followed by glucose, galactose and arabinose. Glucose, galactose and fucose were previously detected as the major sugars in the EPS produced by V. parahaemolyticus (Enos-Berlage and McCarter 2000) grown in heart infusion medium, with negligible amounts ( 5 1%) of mannose and arabinose. In the present study, the sugars mannose and arabinose were obtained as the major sugars present in the EPS; this could possibly be due to the different culture medium used for the growth in present study; Zobell 2218 provides different carbon and nutrient sources than heart infusion medium, and this can determine the quality and quantity of polysaccharide formation (Cerning et al. 1994;Nourani et al. 1998). Heart infusion medium contains 1% beef heart, 1% tryptose and 0.5% sodium chloride (w/v), whereas, Zobell 2216 medium is composed of 0.5% peptone, 0.1% yeast extract and all essential minerals (ferric citrate 0.01%; sodium chloride, 1.945%; magnesium chloride, 0.88%; sodium sulphate, 0.324%; calcium chloride, 0.18%;  potassium chloride, 0.055%; sodium bicarbonate, 0.016%; potassium bromide, 0.008%; strontium chloride, 34 ppm; boric acid, 22 ppm; sodium silicate, 4 ppm; sodium fluoride, 2.4 ppm; ammonium nitrate 1.6, ppm and disodium phosphate, 8 ppm; (w/v)), closely duplicating the mineral composition of seawater. Peptone and yeast extract (in the Zobell medium) provide nitrogen, vitamins and minerals while the high salt content simulates seawater. Heart infusion broth is a nonselective, general-purpose medium used in the isolation and cultivation of a wide range of microorganisms, from a variety of clinical specimens and nutritionally fastidious microorganisms (Elliott et al. 1995;Atlas and Parks 1997). Zobell 2216 medium contains most of the nutrients necessary for the growth of marine bacteria. It is specifically used for cultivating heterotrophic marine bacteria (Kwon et al. 2002). The EPS produced by V. harveyi strainVB23 also contained the same monosaccharide constituents along with rhamnose, fucose, ribose, and xylose (Bramhachari and Dubey 2006).
MALDI TOF-TOF mass spectrometric analysis revealed a series of masses (m/z) corresponding to pentose and hexose sugars (150 and 180, respectively) individually or combined as disaccharides (ie 2 pentoses, *300; 1 pentose þ 1 hexose, *330 and 2 hexoses, *360) in midrange linear mode (Supplementary Figure S5a [Supplementary material is available via a multimedia link on the online article webpage]). The positive ion reflector mode exhibited a higher m/z range attributed to oligosaccharides comprised of hexose and pentose moieties linked in different combinations (Supplementary Figure S5b [Supplementary material is available via a multimedia link on the online article webpage]). Positive ion linear mode has been reported to be suitable for the analysis of oligomers, whereas reflector mode has been recommended for polysaccharide analysis (Mishra et al. 2011). This work contains the first report of a MALDI TOF-TOF analysis of bacterial EPS, whereas MALDI TOF mass spectroscopy for a bacterial EPS was recently reported (Gauri et al. 2009).

NMR and SEM
The 1 HNMR spectrum of the EPS revealed characteristic chemical shifts (ppm) and corresponding functional groups (Figure 4). The 4.8 ppm shift is attributed to the b-anomeric carbons of hexose or pentose (Mishra et al. 2011), and the chemical shift of the functional group R 2 CHOR was observed at 3.2-4.3 ppm. A stretching of the NÀ ÀH group was observed at 1.3 ppm and for alkanes at 0.8-1.2 ppm (CH 3 group)  and 1.1-1.5 ppm (CH 2 group). The spectrum at 2.0 ppm corresponded to the acetyl amines of hexose or pentose, and the peak at 2.6 ppm substantiates the presence of the functional group HÀ ÀCÀ ÀCOOH. The presence of acetyl groups rendered the EPS somewhat hydrophobic, which contributed to their emulsifying capacity (Mata et al. 2006), as observed in this study. It was clear from the SEM image (Supplementary Figure S6 [Supplementary material is available via a multimedia link on the online article webpage]) that the exopolymer was compact in nature with small pores. Compactness with porosity was also observed in the EPS extracted from an Azotobacter sp. (Gauri et al. 2009).

TG and DSC analysis
The thermal behaviour of EPS plays an important role in their commercial exploitation (Marinho-Soriano and Bourret 2005). The thermal degradation of the EPS occurred in two steps: 13% of the EPS was degraded on heating to 1608C, followed by a second phase of depolymerisation (33%) occurring at 3808C (Figure 5a). The transition from an amorphous solid to a crystalline solid was an exothermic process, and differential scanning calorimetric analysis showed a significant thermal transition in the EPS (Figure 5b). The DSC thermogram showed a transition characteristic of an exopolymer, with a crystallisation temperature (T c ) of 107.358C (onset temperature 102.498C) and a latent energy of crystallisation of 1542.00 mJ g 71 , followed by a melting transition. The melting temperature (T m ) of the EPS was determined to be 249.608C (onset temperature 274.518C), with a 51.94 mJ g 71 latent energy of melting. The activation energy (for the nth-order reaction) of the first transition (crystallisation) was 42.68 + 0.51 kJ mol 71 ; for the second transition (melting), it was 187.68 + 0.79 kJ mol 71 , which is lower than that of EPS isolated from cyanobacteria (Parikh and Madamwar 2006). The extracted EPS was found to be thermostable up to 2508C, thus making it a promising additive for industrial applications. A similar thermogram was observed with EPS extracted from Paenibacillus pabuli (Pooja and Chandra 2009).

Rheological studies
The viscosity (Z) of the EPS decreased concomitantly with shear rate (g), showing a pseudoplastic rheological behaviour (Figure 6a). The pseudoplasticity was more profound up to a shear rate of 250-300 s 71 ; thereafter, Newtonian behaviour was observed. The EPS showed shear thinning behaviour at both low and neutral pH, making them a promising additive for the food industry, as they might provide suspension and sensory qualities in food products (Enrı´quez et al. 1989). Shear thinning is mainly due to the breakdown of structural units in EPS by hydrodynamic forces, generated during shear. Similarly, viscosity decreased concurrently with temperature at both pH values (Figure 6b), following a pattern similar to that of the EPS extracted from V. alginolyticus (Muralidharan and Jayachandran 2003). The EPS extracted from V. parahaemolyticus showed a rheology independent of pH, whereas in the EPS produced by several bacterial species, high viscosity was observed at low pH (Be´jar et al. 1998;Gauri et al. 2009).

Conclusions
The biopolymer produced by the biofilm-forming marine bacterium V. parahaemolyticus was amorphous (CI xrd 0.092) and thermostable up to 2508C. The FTIR spectrum of the EPS revealed the presence of aliphatic Figure 6. Effect of (a) shear rate and (b) temperature on the viscosity of EPS isolated from V. parahaemolyticus. methyl groups, primary amines, halide groups, uronic acid and saccharides (galactose and mannose). Four monosaccharides (arabinose, galactose, glucose and mannose) were detected by GCMS. The EPS were also characterised by advanced analytical methods, including MALDI-TOF-TOF analysis, which constitutes the first report of this application for EPS analysis. The EPS showed a pH-independent pseudoplastic rheology and stable emulsifying activity, making the EPS a very promising candidate for commercial exploitation. However, further work is required to determine the suitability of this EPS for biotechnological applications.