Characterization of exopolysaccharide (EPS) produced by Weissella hellenica SKkimchi3 isolated from kimchi

Weissella hellenica SKkimchi3 produces the higher exopolysaccharide (EPS) on sucrose than lactose, glucose, and fructose at pH 5 and 20°C. Sucrose was exclusively used to cultivate SKkimchi3 in all experiments base on the EPS production tests. The molecular mass of EPS, as determined by gel permeation chroma-tography, was 203,000. ¹H and ¹³C NMR analysis indicated that the identity of EPS may be a glucan. When EPS, starch, and cellulose was treated with a-amylase, glucoamylase, glucosidase, and cellulase, glucose was produced from starch and cellulose but was not produced from EPS. Based on HPLC analysis, elemental analysis, ¹H and ¹³C NMR analysis, and enzymatic hydrolysis tests, EPS was estimated to be a glucan. EPS suspension was not precipitated even by centrifugation at 10,000×g for 60 min, and EPS made the fermented milk and bacterial culture viscous.     

Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants.

The composition of the Pseudomonas solanacearum lipolysaccharide (LPS) was found to be similar to that described for the LPS of enterobacteria. The lipid A contained fatty acids and glucosamine in a molar ratio of 5:2. The LPS fraction contained 2-keto-3-deoxyoctulosonic acid, L-glycero-D-mannoheptose, hexoses (glucose, rhamnose, and glucosamine), and a pentose (xylose). The LPSs from the wild-type strain (GMI1000), from the spontaneous rough mutant (GMI2000), and from their respective acridine orange-resistant (Acrr) mutants (GMI1178 and GMI2179) contained the same component sugars in their polysaccharide moieties, but the relative amounts of each sugar varied greatly. Spontaneous mutation to the rough type was characterized by a decrease in the ratio of rhamnose to glucose, whereas a reverse effect was seen for the acridine orange resistance mutation from the parent strains (GMI1000 and GMI2000) to the respective mutant strains (GMI1178 and GMI2179). The exopolysaccharide (EPS) from GMI1000 was found to be composed of two fractions: a heteropolysaccharide (galactosamine, glucose, and rhamnose) excluded from Sephadex G-50 and an additional glucan with a lower molecular weight. Strains GMI1000 and GMI1178 produced comparable amounts of EPS, GMI2179 synthesized less EPS, and GMI2000 produced no detectable EPS. High-pressure liquid chromatography and 13C nuclear magnetic resonance analyses revealed some differences between these EPSs. The glucan fraction seemed to be the major component of the EPS from GMI2179, whereas GMI1000 and GMI1178 EPSs contained both fractions and appeared to differ in the structures of their heteropolysaccharide fractions. Viscosity measurements confirmed differences between whole EPSs produced by the three strains.     

ENVIRONMENTAL EFFECTS ON EXOPOLYMER PRODUCTION BY MARINE BENTHIC DIATOMS: DYNAMICS,CHANGES IN COMPOSITION,AND PATHWAYS OF PRODUCTION1

Marine benthic diatoms excrete large quantities of extracellular polymeric substances (EPS), both as a function of their motility system and as a response to environmental conditions. Diatom EPS consists predominantly of carbohydrate‐rich polymers and is important in the ecology of cells living on marine sediments. Production rates, production pathways, and monosaccharide composition of water‐soluble (colloidal) carbohydrates, EPS, and intracellular storage carbohydrate (glucans) were investigated in the epipelic (mud‐inhabiting) diatoms Cylindrotheca closterium (Ehrenburg), Navicula perminta (Grün.) in Van Heurck, and Amphora exigua Greg. under a range of experimental conditions simulating aspects of the natural environment. Cellular rates of colloidal carbohydrate, EPS, and glucan production were significantly higher during nutrient‐replete compared with nutrient‐limited growth for all three species. The proportion of EPS in the extracellular carbohydrate pool increased significantly (to 44%–69%) as cells became nutrient limited. Cylindrotheca closterium produced two types of EPS differing in sugar composition and production patterns. Nutrient‐replete cells produced a complex EPS containing rhamnose, fucose, xylose, mannose, galactose, glucose, and uronic acids. Nutrient‐limited cells produced an additional EPS containing mannose, galactose, glucose, and uronic acids. Both EPS types were produced under illuminated and darkened conditions. ¹⁴C‐labeling revealed immediate production of ¹⁴C‐glucan and significant increases in ¹⁴C‐EPS between 3 and 4 h after addition of label. The glucan synthesis inhibitor 2,6‐dichlorobenzonitrile significantly reduced ¹⁴C‐colloidal carbohydrate and ¹⁴C‐EPS. The glucanase inhibitor P‐nitrophenyl β‐d ‐glucopyranoside resulted in accumulation of glucan within cells and lowered rates of ¹⁴C‐colloidal and ¹⁴C‐EPS production. Cycloheximide prevented glucan catabolism, but glucan production and EPS synthesis were unaffected.     

Partial characterization of extracellular polysaccharides produced by cyanobacterium Arthrospira platensis

In this study, the physico-chemical characteristics of extracellular polysaccharides (EPS) produced by Arthrospira platensis were evaluated. Elemental analysis and a bicinchoninic acid (BCA) reaction indicated that the EPS were heteropolysaccharides that contain carbohydrate (13%) and protein (55%) moieties. Analysis of the infrared spectrum and elemental analysis revealed the presence of a sulfate group (0.5%). The UV-visible spectrum showed high UV absorption at 190∼230 nm and a shoulder at 260∼280 nm. In addition, this spectrum indicated that EPS can form aggregates with mycosporine-like amino acids and/or scytonemin. Gas chromatography analysis of the carbohydrate portion of the EPS indicated that it was composed of seven neutral sugars: galactose (14.9%), xylose (14.3%), glucose (13.2%), frucose (13.2%), rhamnose (3.7%), arabinose (1%), and mannose (0.3%) and two uronic acids, galacturonic acid (13.5%) and glucuronic acid (0.9%).     

Optimization of extracellular glucan production from Pleurotus eryngii and its impact on angiogenesis

Pleurotus eryngii, an edible mushroom with therapeutic potential was optimized using response surface methodology of four-factor Box-Behnken design for maximum mycelial biomass and extracellular glucan (EPS) production. The model predicts to gain a maximal mycelial biomass and extracellular polysaccharide at 39.4 g/l; 36.04 g/l of glucose, 8.27 g/l; 7.51 g/l of yeast extract, pH 6.99; 7.07 and temperature 26.2°C; 25.84°C, respectively. The validation experiments showed that the model was significant and in close agreement with the model prediction. The evaluation of extracellular polysaccharide on angiogenesis by ex vivo CAM assay showed that there was significant inhibition in neo-vascularization.     

Production of extracellular polysaccharide by Bacillus megaterium RB-05 using jute as substrate

Bacillus megaterium RB-05 was grown on glucose and on “tossa-daisee” (Corchorus olitorius)-derived jute, and production and composition of extracellular polysaccharide (EPS) were monitored. An EPS yield of 0.065 ± 0.013 and of 0.297 g ± 0.054 g⁻¹ substrate after 72 h was obtained for glucose and jute, respectively. EPS production in the presence of jute paralleled bacterial cellulase activity. High performance liquid chromatography (HPLC), matrix assisted LASER desorption/ionization-time of flight (MALDI-ToF) mass spectroscopy, and fourier transform infrared (FT-IR) spectroscopy demonstrated that the EPS synthesized in jute culture (JC) differed from that synthesized in glucose mineral salts medium (GMSM). While fucose was only a minor constituent (4.9 wt.%) of EPS from GMSM, it a major component (41.9 wt.%) of EPS synthesized in JC. This study establishes jute as an effective fermentation substrate for EPS production by a cellulase-producing bacterium.     

The production of extracellular carbohydrates by estuarine benthic diatoms: the effects of growth phase and light and dark treatment

Epipelic diatoms are important constituents of estuarine microphytobenthic biofilms. Field‐based investigations have shown that the production of carbohydrates by such taxa is ecologically important. However, limited information exists on the dynamics of carbohydrate production by individual species of epipelic diatoms. The production of low and high molecular weight extracellular carbohydrates in axenic cultures of five species of benthic estuarine diatoms, Cylindrotheca closterium (Ehrenberg), Navicula perminuta (Grun.) in Van Heurck, Nitzschia frustulum (Kütz.) Grunow, Nitzschia sigma (Kütz.) Grunow, and Surirella ovata (Kütz.) Grunow, were investigated. All species produced colloidal (water‐soluble) carbohydrates during growth, with maximal production occurring during stationary phase. During logarithmic growth, approximately 20% of extracellular carbohydrates consisted of polymeric material (extracellular polymeric substances [EPS]), but during stationary phase, EPS content increased to 34%–50%. Pyrolysis–mass spectrophotometry analysis showed differences in the composition of EPS produced during logarithmic and stationary phase. All species synthesized glucan as a storage carbohydrate, with maximum glucan accumulation during the transition from log to stationary phase. Short‐term labeling with ¹⁴C‐bicarbonate found that between 30 and 60% of photoassimilates were released as colloidal carbohydrate, with EPS consisting of approximately 16% of this colloidal fraction. When cells were placed in darkness, EPS production increased, and between 85 and 99% of extracellular carbohydrate produced was polymeric. Glucan reserves were utilized in dark conditions, with significant negative correlations between EPS and glucan for N. perminuta and S. ovata. Under dark conditions, cells continued to produce EPS for up to 3 days, although release of low molecular weight carbohydrates rapidly ceased when cells were dark treated. Three aspects of EPS production have been identified during this investigation: (1) production during rapid growth, which differs in composition from (2) EPS directly produced as a result of photosynthetic overflow during growth limiting conditions and (3) EPS produced for up to 3 days in the dark using intracellular storage reserves (glucans). The ecological implications of these patterns of production and utilization are discussed.     

Fractionation of the beta-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were beta-linked as shown by H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.     

Structural studies of an exopolysaccharide produced by Streptococcus thermophilus THS

The structure of an extracellular polysaccharide (EPS) from Streptococcus thermophilus THS has been determined. A combination of component analysis, methylation analysis and NMR spectroscopy shows that the polysaccharide is composed of pentasaccharide repeating units. Sequential information was obtained by two-dimensional (1)H,(1)H-NOESY and (1)H,(13)C-HMBC NMR experiments. NMR data indicate different mobility within the EPS with a stiffer backbone and a more flexible side-chain.     

Chemical Composition of Two Exopolysaccharides from Bacillus thermoantarcticus

The thermophilic bacterium Bacillus thermoantarcticus produces two exocellular polysaccharides (EPS 1 and EPS 2), which can be obtained from the supernatant of liquid cultures by cold-ethanol precipitation, in yields as high as 400 mg liter(sup-1). The EPS fraction was produced with all substrates tested, although a higher yield was obtained with mannose as the carbon and energy source. The EPS content was proportional to the total biomass. On a weight basis, EPS 1 and EPS 2 represented about 27 and 71%, respectively, of the total carbohydrate fraction. EPS 1 is a sulfate heteropolysaccharide containing mannose and glucose in a relative molar proportion of 1.0 and 0.7, respectively. EPS 2 is a sulfate homopolysaccharide containing mannose as the major component. The absolute configurations of hexoses were shown to be d for both EPSs. Nuclear magnetic resonance spectra confirmed the presence of (alpha)-d-mannose and (beta)-d-glucose in EPS 1 and only (alpha)-d-mannose in EPS 2. In addition, (sup1)H nuclear magnetic resonance analysis and chemical analysis indicated the presence of pyruvic acid in EPS 2.     

Characterisation of Mesorhizobium huakuii cyclic beta-glucan

Periplasmic and extracellular glucans of Mesorhizobium huakuii were isolated and characterized by compositional and MALDI-TOF analyses, as well as 1H and 13C NMR spectroscopy. It was shown that M. huakuii produces a cyclic beta-glucan composed entirely of nonbranched glucose chains and unmodified by nonsugar substituents. The degree of polymerisation of the cyclic oligosaccharides was estimated to be in the range from 17 to 28. The most abundant glucan molecules contained 22 glucose residues. Glucose residues within the glucan were connected by beta-(1,2) glycosidic linkages. The cyclic glucan produced by M. huakuii is quite similar to the periplasmic beta-(1,2) glucans synthesized by Agrobacterium and Sinorhizobium genera. The synthesis of beta-glucan in M. huakuii is osmoregulated and this glucan could function as an osmoprotectant in free living cells.     

Increase in glucan formation by Botrytis cinerea and analysis of the adherent glucan

Summary Glucan production by Botrytis cinerea increased from 1 g/l to 3 g/l when KNO₃ or urea replaced asparagine as the nitrogen source. A further enhancement up to 5 g/l was obtained with nitrogen-limited medium or non-growing cells. Under these conditions an extracellular glucan layer was attached to the mycelium. The adherent glucan made up 60% of the total amount of glucan produced and thus increased the total glucan yield to 13 g/l. An enzymatic analysis of the adherent glucan indicated that only about every fifth molecule of the main chain was substituted by a glucose unit. In contrast, in the free glucan of culture filtrates glucose units were distributed at approximately every second to third residue of the main chain. Offprint requests to: P. Stahmann     

Fractionation of the β-Linked Glucans of Bradyrhizobium japonicum and Their Response to Osmotic Potential

Bradyrhizobium japonicum USDA 110 synthesized both extracellular and periplasmic polysaccharides when grown on mannitol minimal medium. The extracellular polysaccharides were separated into a high-molecular-weight acidic capsular extracellular polysaccharide fraction (90% of total hexose) and three lower-molecular-weight glucan fractions by liquid chromatography. Periplasmic glucans, extracted from washed cells with 1% trichloroacetic acid, gave a similar pattern on liquid chromatography. Linkage analysis of the major periplasmic glucan fractions demonstrated mainly 6-linked glucose (63 to 68%), along with some 3,6- (8 to 18%), 3- (9 to 11%), and terminal (7 to 8%) linkages. The glucose residues were β-linked as shown by ¹H-nuclear magnetic resonance analysis. Glucan synthesis by B. japonicum cells grown on mannitol medium with 0 to 350 mM fructose as osmolyte was measured. Fructose at 150 mM or higher inhibited synthesis of periplasmic and extracellular 3- and 6-linked glucans but had no effect on the synthesis of capsular acidic extracellular polysaccharides.     

Structural studies of the exopolysaccharide produced by Lactobacillus rhamnosus strain GG (ATCC 53103)

The structure of the extracellular polysaccharide (EPS) from Lactobacillus rhamnosus strain GG has been investigated. In combination with component analysis, NMR spectroscopy shows that the polysaccharide is composed of hexasaccharide repeating units. Sequential information was obtained by two-dimensional (1)H,(1)H-NOESY, and (1)H,(13)C-HMBC NMR techniques. The structure of the repeating unit of the EPS from Lactobacillus rhamnosus strain GG was determined as: [carbohydrate structure: see text]     

Partial characterization of an extracellular polysaccharide produced by the moderately halophilic bacterium Halomonas xianhensis SUR308

A moderately halophilic bacterium, Halomonas xianhensis SUR308 (Genbank Accession No. KJ933394) was isolated from a multi-pond solar saltern at Surala, Ganjam district, Odisha, India. The isolate produced a significant amount (7.87 g l^?1) of extracellular polysaccharides (EPS) when grown in malt extract–yeast extract medium supplemented with 2.5% NaCl, 0.5% casein hydrolysate and 3% glucose. The EPS was isolated and purified following the conventional method of precipitation and dialysis. Chromatographic analysis (paper, GC and GC-MS) of the hydrolyzed EPS confirmed its heteropolymeric nature and showed that it is composed mainly of glucose (45.74 mol%), galactose (33.67 mol %) and mannose (17.83 mol%). Fourier-transform infrared spectroscopy indicated the presence of methylene and carboxyl groups as characteristic functional groups. In addition, its proton nuclear magnetic resonance spectrum revealed functional groups specific for extracellular polysaccharides. X-ray diffraction analysis revealed the amorphous nature (CI_xrd, 0.56) of the EPS. It was thermostable up to 250°C and displayed pseudoplastic rheology and remarkable stability against pH and salts. These unique properties of the EPS produced by H. xianhensis indicate its potential to act as an agent for detoxification, emulsification and diverse biological activities.     

Evaluation of Arthrospira platensis extracellular polymeric substances production in photoautotrophic, heterotrophic and mixotrophic conditions

The kinetic study of Arthrospira platensis extracellular polymeric substances (EPS) production under different trophic modes??photoautotrophy (100???mol photons m^?2?s^?1), heterotrophy (1.5?g/L glucose), and mixotrophy (100???mol photons m^?2?s^?1 and 1.5?g/L glucose)??was investigated. Under photoautotrophic and heterotrophic conditions, the maximum EPS production 219.61?±?4.73 and 30.30?±?1.97?mg/L, respectively, occurred during the stationary phase. Under a mixotrophic condition, the maximum EPS production (290.50?±?2.21?mg/L) was observed during the early stationary phase. The highest specific EPS productivity (433.62?mg/g per day) was obtained under a photoautotrophic culture. The lowest specific EPS productivity (38.33?mg/g per day) was observed for the heterotrophic culture. The effects of glucose concentration, light intensity, and their interaction in mixotrophic culture on A. platensis EPS production were evaluated by means of 32 factorial design and response surface methodology. This design was carried out with a glucose concentration of 0.5, 1.5, and 2.5?g/L and at light levels of 50, 100, and 150???mol photons m^?2?s^?1. Statistical analysis of the model demonstrated that EPS concentration and EPS yield were mainly influenced by glucose concentration and that conditions optimizing EPS concentration were dissimilar from those optimizing EPS yield. The highest maximum predicted EPS concentration (369.3?mg/L) was found at 150???mol photons m^?2?s^?1 light intensity and 2.4?g/L glucose concentration, while the highest maximum predicted EPS yield (364.3?mg/g) was recorded at 115???mol photons m^?2?s^?1 light intensity and 1.8?g/L glucose concentration.     

Curdlan-like exopolysaccharide production by <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis> UNP3 during growth on hydrocarbon substrates

Cellulomonas flavigena UNP3, a natural isolate from vegetable oil contaminated soil sample has been studied for growth associated exopolysaccharide (EPS) production during growth on glucose, groundnut oil and naphthalene. The EPS showed matrix formation surrounding the cells during scanning electron microscopy. Cell surface hydrophobicity and emulsifying activity studies confirmed the role of EPS as bioemulsifier. Emulsifying activity was found to increase with time (0.2 U/mg for 10 min to 0.27 U/mg for 30 min). Emulsification index, E₂₄ value increased with the increase in EPS concentration. Degradation of polyaromatic hydrocarbons was confirmed using gas chromatography analysis. FTIR analysis showed presence of characteristic absorbance at 895.10 cm⁻¹ for β-configuration of glucan. NMR studies also revealed EPS produced by C. flavigena UNP3 as a linear β-1, 3-d-glucan, and a curdlan like polysaccharide.     

Structure of an acylated exopolysaccharide synthesized by Acinetobacter sp

The complex preparation ethapolan synthesized by Acinetobacter sp. consists of neutral (minor component) and two acidic exopolysaccharides (EPS) one of which is acylated. On the basis of chemical modification of EPS, solvolysis with anhydrous hydrogen fluoride resulting in a penta- and octasaccharide fragments, Smith degradation, 1H- and 13C NMR analysis the following structure of the acylated polysaccharide repeating unit has been established (scheme): It is suggested that in the acylated EPS at least one glucose residue and the galactose residue are O-acylated.     

Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans

Extracellular polymeric substances (EPS) play an important role in the formation and activity of biofilms in wastewater treatment (WWT). The EPS of the denitrifying biomarker Comamonas denitrificans strain 110, produced in different culture media and growth modes, were characterized. The EPS mainly contained protein (3–37%), nucleic acids (9–50%), and carbohydrates (3–21%). The extracellular DNA was found to be important for initial biofilm formation since biofilm, but not planktonic growth, was inhibited in the presence of DNase. The polysaccharide fraction appeared to consist of at least two distinct polymers, one branched fraction (A) made up of glucose and mannose with a molecular weight around 100 kDa. The other fraction (B) was larger and consisted of ribose, mannose, glucose, rhamnose, arabinose, galactose, and N-acetylglucosamine. Fraction B polysaccharides were mainly found in capsular EPS which was the dominant type in biofilms and agar-grown colonies. Fraction A was abundant in the released EPS, the dominant type in planktonic cultures. Biofilm and agar-grown EPS displayed similar overall properties while planktonic EPS showed clear compositional disparity. This study presents results on the physiology of a key WWT organism, which may be useful in the future development of improved biofilm techniques for WWT purposes.     

Glucan common to the microcyst walls of cyst-forming bacteria.

Chemical analysis indicated that D-glucose is tha major neutral monosaccharide present in the microcysts of a range of gram-negative bacteria. Varying amounts of other neutral sugars were found. The glucose was mainly present as a glucan that could be extracted from microcysts of representative strains with alkali or mild acid treatment. The glucan could be identified as an alpha-1,3-linked polymer on the basis of (i) periodate resistance of the extracted polymer and the material present in microcysts; (ii) lectin agglutination of the microcysts; (iii) lectin precipitation of the extracted glucans; and (iv) susceptibility of the glucan either in the walls or after extraction to a specific alpha-1,3-glucanase from Aspergillus nidulans, yielding glucose as the sole hydrolysis product. The galactosamine found in microcysts of Myxococcus xanthus by other workers is clearly a component of another polymer, distinct from the glucan. The presence of an alpha 1,3-linked glucan, common to microcyst walls of various bacterial genera, probably contributes to the rigidity of the walls of these forms and, inter alia, to their resistance to ultrasonic treatment. Preliminary experiments indicate that the gulcan is discarded on germination of the microcysts rather than being broken down by specific enzymes.