Characterization and fouling properties of exopolysaccharide produced by Klebsiella oxytoca

Klebsiella oxytoca produced a type of exopolysaccharide (EPS) with the average molecular weight (Mw) of 116,018 Da and the average size of 260 nm. The EPS monosaccharide components contained rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose and the molar ratio among them was 0.033:0.0411:0.0147:0.0051:0.2393:0.0986:0.1304. Typical EPS absorption peaks in FT-IR spectrum and pseudoplastic properties were also revealed. The polyvinylidenefluoride (PVDF) membrane showed a relatively larger flux decline resulted from the EPS fouling. The EPS filtration was dominated by more than one mechanism at the beginning phase and mainly by the cake formation at the later phase for both membranes. The pore blocking resistance had a predominant contribution to the filtration resistance and the cake resistance played a secondary role for both the membranes. The EPS adsorption resulted in a weak membrane fouling. The PVDF membrane exhibited a larger adsorption resistance than the polypropylene (PP) membrane.     

Culture Conditions Determine the Balance between Two Different Exopolysaccharides Produced by Lactobacillus pentosus LPS26

Lactobacillus pentosus LPS26, isolated from a natural fermentation of green olives, produces a capsular polymer constituted of two exopolysaccharides (EPS): EPS A, a high-molecular-weight (high-M_w) polysaccharide (1.9 × 10⁶ Da) composed of glucose and rhamnose (3:1), and EPS B, a low-M_w polysaccharide (3.3 × 10⁴ Da) composed of glucose and mannose (3:1). Fermentation experiments in a chemically semidefined medium with different carbon sources (glucose, fructose, mannitol, and lactose) showed that all of them except fructose supported EPS A production rather than EPS B production. The influence of temperature and pH was further analyzed. As the temperature dropped, increased synthesis of both EPS was detected. The control of pH especially enhanced EPS B production. With regard to this, the maximum total EPS production (514 mg liter⁻¹) was achieved at a suboptimal growth temperature (20°C) and pH 6.0. Continuous cultures showed that EPS A, synthesized mainly at low dilution rates, is clearly dependent on the growth rate, whereas EPS B synthesis was hardly affected. EPS production was also detected in supplemented skimmed milk, but no increase on the viscosity of the fermented milk was recorded. This could be linked to the high proportion of the low-M_w polysaccharide produced in these conditions in contrast to that observed in culture media. Overall, the present study shows that culture conditions have a clear impact on the type and concentration of EPS produced by strain LPS26, and consequently, these conditions should be carefully selected for optimization and application studies. Finally, it should be noted that this is, to our knowledge, the first report on EPS production by L. pentosus.     

Characterization of the LP28 strain-specific exopolysaccharide biosynthetic gene cluster found in the whole circular genome of Pediococcus pentosaceus

We have previously isolated a lactic acid bacterium (LAB), Pediococcus pentosaceus LP28, from the longan fruit Euphoria longana. Since the plant-derived LAB strain produces an extracellular polysaccharide (EPS), in this study, we analyzed the chemical structure and the biosynthesizing genes for the EPS.The EPS, which was purified from the LP28 culture broth, was classified into acidic and neutral EPSs with a molecular mass of about 50 kDa and 40 kDa, respectively. The acidic EPS consisted of glucose, galactose, mannose, and N-acetylglucosamine moieties. Interestingly, since pyruvate residue was detected in the hydrolyzed acidic EPS, one of the four sugars may be modified with pyruvate. On the other hand, the neutral EPS consisted of glucose, mannose, and N-acetylglucosamine; pyruvate was scarcely detected in the polysaccharide molecule.As a first step to deduce the probiotic function of the EPS together with the biosynthesis, we determined the whole genome sequence of the LP28 strain, demonstrating that the genome is a circular DNA, which is composed of 1,774,865 bp (1683 ORFs) with a GC content of 37.1%. We also found that the LP28 strain harbors a plasmid carrying 6 ORFs composed of 5366 bp with a GC content of 36.5%. By comparing all of the genome sequences among the LP28 strain and four strains of P. pentosaceus reported previously, we found that 53 proteins in the LP28 strain display a similarity of less than 50% with those in the four P. pentosaceus strains. Significantly, 4 of the 53 proteins, which may be enzymes necessary for the EPS production on the LP28 strain, were absent in the other four P. pentosaceus strains and displayed less than 50% similarity with other LAB species. The EPS-biosynthetic gene cluster detected only in the LP28 genome consisted of 12 ORFs containing a priming enzyme, five glycosyltransferases, and a putative polysaccharide pyruvyltransferase.     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

Exopolysaccharide production by a new Halomonas strain CRSS isolated from saline lake Cape Russell in Antarctica growing on complex and defined media

A haloalkalophilic Halomonas strain CRSS, isolated from salt sediments in Antarctica, produced exocellular polysaccharides (EPS) up to 2.9gg^-1 dry cells. Acetate was the most efficient carbon source for EPS production. The composition of media strongly affected the nature of the polymers; a mannan and a xylo-mannan, were obtained when cells were grown on complex media. Acetate was the most efficient carbon source for EPS production and in presence of this substrate, a new polysaccharide, a fructo-glucan, was produced. The EPS fraction was composed by glucose, fructose, glucosamine and galactosamine in relative proportions of 1:0.7:0.3:trace.Revisions requested; Revisions received 6 September 2004     

Microbial community developments and biomass characteristics in membrane bioreactors under different organic loadings

Microbial community developments and biomass characteristics (concentration, particle size, extracellular polymeric substances (EPS), and membrane fouling propensity) were compared when three MBRs were fed with the synthetic wastewater at different organic loadings. Results showed that the bacterial communities dynamically shifted in different ways and the EPS displayed dissimilar profiles under various organic loadings, which were associated with the ratios of food to microorganism and dissolved oxygen levels in the MBRs. The membrane fouling tendency of biomass in the low-loading MBR (0.57 g COD/L day) was insignificantly different from that in the medium-loading MBR (1.14 g COD/L day), which was apparently lower than that in the high-loading MBR (2.28 g COD/L day). The membrane fouling propensity of biomass was strongly correlated with their bound EPS contents, indicating cake layer fouling (i.e., deposition of microbial flocs) was predominant in membrane fouling at a high flux of 30 L/m² h.     

Microbial behaviors involved in cake fouling in membrane bioreactors under different solids retention times

Biomass characteristics and membrane performances in the MBRs operated at a high flux of 30 L/m² h under different SRTs (10, 30 days, and infinity) were monitored. Results showed that more serious cake-fouling happened in the SRT-infinity MBR, which correlated with the activated sludge characteristics such as smaller floc size and greater EPS amount. DGGE analysis indicated that the microbial community shifted in different ways under various SRTs, which also influenced EPS productions in the MBRs. Different microbial communities were developed on the membrane surfaces at various operating stages and SRTs. Possibly, the activated sludge characteristics (such as MLSS concentration, EPS properties) and hydrodynamic conditions influenced by the SRTs were associated with cake layer development and membrane fouling propensity. Insight into the EPS characteristics and deposition behaviors of bacterial flocs will be crucial to explore appropriate biofouling control strategies in MBRs.     

Spatial and Temporal Deposition of Hyphomonas Strain VP-6 Capsules Involved in Biofilm Formation

Hyphomonas strain VP-6 is a prosthecate bacterium isolated from the Guayamas vent region and is a member of a genus of primary and common colonizers of marine surfaces. It adheres to solid substrata as a first step in biofilm formation. Fine-structure microscopy and the use of specific stains and lectins reveal that it synthesizes two different extracellular polymeric substances (EPS). One is a temporally synthesized, polar holdfast EPS, and the other is a capsular EPS that is present during the complete life cycle and surrounds the entire cell, including the prosthecum. The timing and location of Hyphomonas strain VP-6 EPS elaboration correlate with adhesion to surfaces, suggesting that the EPS serves not only as the biofilm matrix but also as a primary adhesin. The temporality and polarity of VP-6 EPS expression substantially differ from those properties of Hyphomonas strain MHS-3 EPS expression.     

Membrane biofouling by chlorine resistant Bacillus spp.: effect of feedwater chlorination on bacteria and membrane biofouling

In this study, bacteria isolated from a lake were characterised for their chlorine resistivity and the effects of chlorination on growth, mortality, protein expression and attachment propensity towards membranes. Biofouling and membrane performance were analysed. All isolated chlorine resistant strains, characterised by 16s rRNA gene sequencing, belonged to the genus Bacillus. Chlorination caused limited effects on bacterial growth and mortality. B. safensis and B. lechinoformis suffered the maximum effects due to chlorination. Live-to-dead ratios immediately after chlorination were above 1.3, with some exceptions. The membrane pure water flux recovery was highly strain dependent. Irreversible membrane fouling was observed with B. aquimaris. Membrane flux decreased substantially during ultrafiltration of water containing chlorine resistant bacteria.     

PssP2 Is a Polysaccharide Co-Polymerase Involved in Exopolysaccharide Chain-Length Determination in Rhizobium leguminosarum

Production of extracellular polysaccharides is a complex process engaging proteins localized in different subcellular compartments, yet communicating with each other or even directly interacting in multicomponent complexes. Proteins involved in polymerization and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum are encoded within the chromosomal Pss-I cluster. However, genes implicated in polysaccharide synthesis are common in rhizobia, with several homologues of pss genes identified in other regions of the R. leguminosarum genome. One such region is chromosomally located Pss-II encoding proteins homologous to known components of the Wzx/Wzy-dependent polysaccharide synthesis and transport systems. The pssP2 gene encodes a protein similar to polysaccharide co-polymerases involved in determination of the length of polysaccharide chains in capsule and O-antigen biosynthesis. In this work, a mutant with a disrupted pssP2 gene was constructed and its capabilities to produce EPS and enter into a symbiotic relationship with clover were studied. The pssP2 mutant, while not altered in lipopolysaccharide (LPS), displayed changes in molecular mass distribution profile of EPS. Lack of the full-length PssP2 protein resulted in a reduction of high molecular weight EPS, yet polymerized to a longer length than in the RtTA1 wild type. The mutant strain was also more efficient in symbiotic performance. The functional interrelation between PssP2 and proteins encoded within the Pss-I region was further supported by data from bacterial two-hybrid assays providing evidence for PssP2 interactions with PssT polymerase, as well as glycosyltransferase PssC. A possible role for PssP2 in a complex involved in EPS chain-length determination is discussed.     

Improving polyhydroxyalkanoate production by knocking out the genes involved in exopolysaccharide biosynthesis in Haloferax mediterranei

Haloferax mediterranei is capable of producing large amounts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from many kinds of carbon sources, with exopolysaccharide (EPS) as a by-product. In this study, we identified a gene cluster involved in EPS biosynthesis in H. mediterranei. Knocking out the genes in this cluster encoding the putative UDP-N-acetylglucosamine 6-dehydrogenase (HFX_2145), glycosyltransferases (HFX_2146 and HFX_2147) and polysaccharide transporter (HFX_2148) eliminated EPS synthesis. The deficiency in EPS biosynthesis in the mutant strain remarkably decreased the viscosity of culture broth, and hence increased the dissolved oxygen content and decreased the foaming propensity. Compared with the wild-type (WT) strain, the PHBV production of the EPS-mutant strain was significantly enhanced (approximately 20 %), whereas the cell growth rate remained similar under the same culture conditions. These results indicated that the carbon sources used for synthesizing EPS were shifted to PHBV production. Thus, a novel engineered H. mediterranei strain was developed, which would be favorable for future industrial production of PHBV.     

Comparison between a moving bed membrane bioreactor and a conventional membrane bioreactor on membrane fouling

A membrane bioreactor filled with carriers instead of activated sludge named a moving bed membrane bioreactor (MBMBR) was investigated to minimize the effect of suspended solids on membrane fouling. The MBMBR and a conventional membrane bioreactor (CMBR) were operated in parallel for about two months. Unexpectedly, the rate of membrane fouling in MBMBR was about three times of that in CMBR. MBMBR showed a higher cake layer resistance than CMBR due to plenty of filamentous bacteria inhabited in suspended solids in MBMBR. Protein and polysaccharide contents of soluble EPS in MBMBR were obviously larger than those in CMBR. It could be speculated that the overgrowth of filamentous bacteria in MBMBR resulted in severe cake layer and induced a large quantity of EPS, which deteriorated the membrane fouling.     

Production of exopolysaccharides from a thermophilic microorganism isolated from a marine hot spring in flegrean areas

A thermophilic strain isolated from sea sand at Maronti, near Sant' Angelo (Ischia), is described. The organism grows well at an optimal temperature of 60 °C at pH 7.0. The thermophilic bacterium, named strain 4004, produces an exocellular polysaccharide (EPS) in yields of 90 mg/l. The EPS fraction was produced with all substrates tested, although a higher yield was obtained with sucrose or trehalose as sole carbon source. During growth, the EPS content was proportional to the biomass. Three fractions (EPS1, EPS2, EPS3) were obtained after purification. Quantitative monosaccharide analysis of the EPSs revealed the presence of mannose:glucose:galactose in a relative ratio of 0.5:1.0:0.3 in EPS1, mannose:glucose:galactose in a relative ratio of 1.0:0.3:trace in EPS2, and galactose:mannose:glucosamine:arabinose in a relative ratio of 1.0:0.8:0.4:0.2 in EPS3. The average molecular mass of EPS3 was determined to be 1×10⁶ Da. From comparison of the chemical shift values in ¹H and ¹³C spectra, we conclude that EPS3 presents a pentasaccharide repeating unit. Electronic Publication     

Relationships between Colony Morphotypes and Oil Tolerance in Rhodococcus rhodochrous

A mucoidal strain of Rhodococcus rhodochrous was resistant to 10% (vol/vol) n-hexadecane, while its rough derivatives were sensitive. When the extracellular polysaccharide (EPS) produced by the mucoidal strain was added to cultures of the rough strains, the rough strains gained resistance to n-hexadecane. Thus, EPS confer tolerance to n-hexadecane in members of the genus Rhodococcus.     

Factors affecting membrane fouling in filtration of Saccharomyces cerevisiae in an internal ceramic filter bioreactor

Filtration of ethanol fermentation medium and broth by using symmetric and asymmetric ceramic membranes has been studied in an internal filter bioreactor. Factors studied included membrane structure and pore size, medium sterilization, and concentrations of glucose, yeast extract in the medium, yeast cell and protein in broth. The aim was to determine the main factors responsible for the decline in filtration performance during ethanol fermentation by Saccharomyces cerevisiae. Flux index (Fi) of a new concept has been developed to evaluate the degree of flux decline during the membrane fouling process. Fi was defined as the ratio of the membrane flux at certain filtration time (t?=?t) to the initial (t?=??0) flux of pure water, not the initial (t?=?+0) flux of the test fluid. Flux with sterilized medium was approximately two-fold higher than that with unsterilized medium although the reason could not be explained clearly. Glucose, interaction between glucose and yeast extract, yeast cells, and proteins in fermentation broth were found to play an important part in membrane fouling. Fi of the symmetric membrane decreased to a less extent than that of the asymmetric membrane with increasing glucose concentration. But, the result with various yeast cell concentrations turned out to be contrary. Fouling was more serious for asymmetric membrane during the filtration of fermentation supernatant. This was thought to be due to different fouling mechanisms for the two types of membrane.     

Isolation of wildXanthomonas strains from agricultural produce, their characterization and potential related to polysaccharide production

One hundred fifty wild strains ofXanthomonas were isolated on five selective and nonselective media. The comparative effectivity of the five media has been discussed. Fifteen polysaccharide-producing strains shortlisted were identified up to species level. These were studied for polysaccharide production in shake flasks using YM media. The highest final yield of 21.3 g/L of the amount of carbon source supplied has been obtained in the optimum medium in shake-flask experiments fromXanthomonas campestris ICa-125 strain isolated from cabbage. Rheological properties of the exocellular polysaccharide (EPS) have been compared with standard xanthan. EPS fromX. campestris ICa-125 was found to be superior with respect to heat stability and effect of electrolytes as compared to standard xanthan.     

A Comparison of the Surface Polysaccharides from Rhizobium leguminosarum 128C53 smrif with the Surface Polysaccharides from Its Exo Mutant

The surface polysaccharides of Rhizobium leguminosarum 128C53 sm^rrif^r (parent) and its exo⁻¹ mutant were isolated and characterized. The parent carries out normal symbiosis with its host, pea, while the exo⁻¹ mutant does not nodulate the pea. The following observations were made. (a) The parent produces lipopolysaccharide (LPS), typical acidic extracellular polysaccharide (EPS), and three additional polysaccharides, PS1, PS2, and PS3. The PS1 and PS2 fractions are likely to be the capsular polysaccharide (CPS) and are identical in composition to the EPS. The PS3 fraction is a small-molecular-weight glucan. (b) The exo⁻¹ mutant produces LPS, EPS, and a PS3 fraction, but does not produce significant amounts of either PS1 or PS2. The LPS from the exo⁻¹ mutant appears to be identical to the parental LPS. Analysis of the EPS from exo⁻¹ shows that it consists of two polysaccharides. One polysaccharide is identical to the LPS and comprises 70% of the exo⁻¹ EPS. The second polysaccharide is identical to the exo⁻¹ PS3 and comprises 30% of the exo⁻¹ EPS. This result shows that the exo⁻¹ mutant does not produce any of the typical acidic parental EPS and that the major polysaccharide released into the media by the exo⁻¹ mutant is intact LPS. The exo⁻¹ mutant PS3 fraction was found to contain two polysaccharides, PS3-1 and PS3-2. The PS3-2 polysaccharide is identical to the parental PS3 described above. The PS3-1 polysaccharide has a composition similar to the polysaccharide portion of the LPS. This result suggests that the exo⁻¹ mutant produces LPS polysaccharide fragments. These LPS polysaccharide fragments are not produced by the parent strain.     

Linking microbial community structure to membrane biofouling associated with varying dissolved oxygen concentrations

In order to elucidate how dissolved oxygen (DO) concentration influenced the generation of extracellular polymeric substance (EPS) and soluble microbial products (SMP) in mixed liquor and biocake, 16S rDNA fingerprinting analyses were performed to investigate the variation of the microbial community in an aerobic membrane bioreactor (MBR). The function of microbial community structure was proved to be ultimately responsible for biofouling. Obvious microbial community succession from the subphylum of Betaproteobacteria to Deltaproteobacteria was observed in biocake. High concentration of EPS in biocake under the low DO concentration (0.5 mg L⁻¹) caused severe biofouling. The correlation coefficient of membrane fouling rate with EPS content in biocake (0.9941-0.9964) was much higher than that in mixed liquor (0.6689-0.8004).     

Characterization of Rhizobium leguminosarum Exopolysaccharide Glycanases That Are Secreted via a Type I Exporter and Have a Novel Heptapeptide Repeat Motif

The prsDE genes encode a type I protein secretion system required for the secretion of the nodulation protein NodO and at least three other proteins from Rhizobium leguminosarum bv. viciae. At least one of these proteins was predicted to be a glycanase involved in processing of bacterial exopolysaccharide (EPS). Two strongly homologous genes (plyA and plyB) were identified as encoding secreted proteins with polysaccharide degradation activity. Both PlyA and PlyB degrade EPS and carboxymethyl cellulose (CMC), and these extracellular activities are absent in a prsD (protein secretion) mutant. The plyA gene is upstream of prsD but appears to be expressed at a very low level (if at all) in cultured bacteria. A plyB::Tn5 mutant has a very large reduction in degradation of EPS and CMC. Cultures of plyB mutants contained an increased ratio of EPS repeat units to reducing ends, indicating that the EPS was present in a longer-chain form, and this correlated with a significant increase in culture viscosity. Thus, PlyB may play a role in processing of EPS. Analysis of the symbiotic properties of a plyA plyB double mutant revealed that these genes are not required for symbiotic nitrogen fixation and that nodulation was not significantly affected. PlyA and PlyB are similar to bacterial and fungal polysaccharide lyases; they contain 10 copies of what we propose as a novel heptapeptide repeat motif that may constitute a fold similar to that found in the family of extracellular pectate lyases. PlyA and PlyB lack the Ca²⁺-binding RTX nonapeptide repeat motifs usually found in proteins secreted via type I systems. We propose that PlyA and PlyB are members of a new family of proteins secreted via type I secretion systems and that they are involved in processing of EPS.     

Factors affecting the performance of membrane bioreactor for piggery wastewater treatment

This study was conducted to identify the factors affecting the performance of membrane bioreactor (MBR) for piggery wastewater treatment. The change of organic and nitrogen concentrations in piggery wastewater was studied to investigate the treatment efficiency. The increase of COD, BOD and NH₃–N from 1150 to 2050 mg/L, 683 to 1198 mg/L and 154 to 248 mg/L has led to the decrease of treatment efficiency. Removal efficiencies of COD, BOD and NH₃–N have decreased from 96.0% to 92.0%, 97.0% to 92.7% and 93.2% to 69.5%, respectively. The effects of biomass characteristics on membrane fouling were determined based on Pearson’s correlation coefficient (r_p). It was found that MLSS had a negative correlation with permeate flux (r_p = −0.745, at significant level of 0.05) while sludge floc size a positive correlation (r_p = 0.731, at significant level of 0.05). MLSS and sludge floc size were found to be the dominant factors that controlled the membrane filterability while sludge viscosity, EPS, SMP and SV₃₀ have taken as the sub-factors affecting membrane fouling.