Constituents Of Extracellular Polymeric Substances Research Articles

Using a stable isotope tracing technique to elucidate the effect of substrate C/N ratio on the formation of different constituents of extracellular polymeric substances in an aerobic-anoxic sequencing batch reactor

Carbon to nitrogen (C/N) ratios in wastewater vary with time and sources, which greatly affect the production and composition of extracellular polymeric substances (EPS) in biological treatment processes. In this study, the EPS yield and their compositional changes were examined using stable isotopes (13C-glucose and 15NH4Cl) during the operation of aerobic-anoxic sequencing batch reactors (SBR) with different C/N ratios (C/N = 5, 10, and 15). Spectroscopic characterization revealed that humic-like aromatic compounds tended to be more enriched in loosely bound EPS (LB-EPS) at a lower C/N ratio, whereas protein-like fluorophores prevailed in tightly bound EPS (TB-EPS) (>90 %) regardless of the feed C/N ratio. Variations in isotopic enrichment revealed that the rates of substrate assimilation into TB-EPS were different between organic carbon and nitrogen, with an earlier replenishment of nitrogen regardless of C/N ratios. Yield estimation based on individual carbon and nitrogen isotopes indicated that maximum 17.8 % of organic carbon was converted into TB-EPS at a lower C/N ratio, whereas nitrogenous EPS exhibited higher yield coefficients with a wider range from 26.0 % (C/N = 5) to 44.0 % (C/N = 15). The results suggest that varying C/N ratios might exert more pronounced effects on the production of nitrogenous EPS constituents than their carbon counterparts. This suggested isotope labeling approach can be further applied to determine the mass balances among the substrate, biomass, and bound/soluble EPS within activated sludge systems.

Variable pH and subsequent change in pCO2 modulates the biofilm formation, synthesis of extracellular polymeric substances, and survivability of a marine bacterium Bacillus stercoris GST-03

Biofilm-forming bacteria adhere to the substrates and engage in the nutrient cycling process. However, environmental conditions may interrupt the biofilm formation ability, which ultimately may affect various biogeochemical cycles. The present study reports the effect of varying pH and subsequent change in pCO2 on the survivability, biofilm formation, and synthesis of extracellular polymeric substances (EPS) of a biofilm-forming marine bacterium Bacillus stercoris GST-03 isolated from the Bhitarkanika mangrove ecosystem, Odisha, India. Understanding the pH-dependent alteration in EPS constituents, and associated functional groups of a marine bacterium will provide better insight into the adaptability of the bacteria in future ocean acidification scenarios. The strain was found to tolerate and form biofilm up to pH 4, with the maximum biofilm formation at pH 6. EPS yield and the synthesis of the key components of the EPS, including carbohydrate, protein, and lipid, were found maximum at pH 6. Changes in biofilm formation patterns and various topological parameters at varying pH/pCO2 conditions were observed. A cellular chaining pattern was observed at pH 4, and maximum biofilm formation was obtained at pH 6 with biomass of 5.28582 ± 0.5372 μm3/μm2 and thickness of 9.982 ± 1.5288 μm. Structural characterization of EPS showed changes in various functional groups of constituent macromolecules with varying pH. The amorphous nature of the EPS and the changes in linkages and associated functional groups (-R2CHOR, –CH3, and –CH2) with pH variation was confirmed. EPS showed a two-step degradation with a maximum weight loss of 59.147% and thermal stability up to 480 °C at pH 6. The present work efficiently demonstrates the role of EPS in providing structural and functional stability to the biofilm in varying pH conditions. The findings will provide a better understanding of the adaptability of marine bacteria in the future effect of ocean acidification.

Critical evaluation of heat extraction temperature on soluble microbial products (SMP) and extracellular polymeric substances (EPS) quantification in wastewater processes.

While soluble microbial products (SMP) and extracellular polymeric substances (EPS) in wastewater bioprocesses have been widely studied, a lack of standard quantification procedures make it difficult to compare results between studies. This study investigated the effect of temperature on SMP and EPS profiles for biological nutrient removal (BNR) sludges and aerobic membrane bioreactor sludge by adapting the commonly used heat extraction and centrifugation scheme, followed by colorimetric quantification of the carbohydrate and protein fractions using the phenol-sulfuric acid (PS) and the bicinchoninic acid (BCA) methods, respectively. To overcome known inconsistencies in colorimetry, total carbon (TC), total nitrogen (TN), and fluorometry analyses were performed in tandem. SMP samples marginally benefitted from heat extraction, owing to their mostly soluble nature, while EPS profiles were greatly influenced by temperature. 60 °C appears to be a suitable general-purpose extraction temperature near the lysis threshold for the sludges tested. The PS method's misestimation due to lack of specificity was observed and contrasted by TC analyses, while the TN analyses corroborated the BCA assays. Fluorometry proved to be a sensitive and rapid analytical method that provided semi-quantitative information on SMP and EPS constituents, particularly its proteinaceous components, with positive implications for robust wastewater process control.

Selective retention of extracellular polymeric substances induced by adsorption to and coprecipitation with ferrihydrite

Recent work shows that microbially-derived compounds constitute a significant fraction of the soil organic matter (OM) pool. These compounds include extracellular polymeric substances (EPS) whose mass can far exceed total microbial cell biomass. Sorption of EPS to soil minerals occurs via adsorption and coprecipitation and contributes to the preservation of OM in the soil environment. Little is known, however, about the sorption mechanisms of EPS and selective retention of different EPS constituents on iron (oxyhydr)oxides, especially during EPS adsorption versus coprecipitation with these reactive soil phases. This study examines how EPS interacts with the ubiquitous soil iron (oxyhydr)oxide ferrihydrite during EPS adsorption and coprecipitation and whether these different EPS-mineral association pathways affect EPS sorption and selective retention, and thus the mobility and fate of microbially-derived OM in the soil environment. We use several complimentary techniques to i) examine EPS-carbon, EPS-nitrogen and EPS-phosphorus sorption and fractionation, ii) visualize spatial relationships between EPS biomolecular classes and ferrihydrite using confocal laser scanning microscopy (CLSM), iii) determine EPS-C speciation and chemical fractionation with ferrihydrite using X-ray photoelectron spectroscopy (XPS) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and iv) determine functional group interactions with ferrihydrite using Fourier transform infrared spectroscopy (FTIR) combined with two-dimensional correlation spectroscopy (2D-COS) analysis. Results show that the coprecipitation of EPS does not change the ferrihydrite mineralogy, as the main mineral phase for EPS association, but it substantially increases the particle size of EPS-ferrihydrite. A substantial difference in the EPS mass fraction associated with the ferrihydrite is observed between the adsorption and coprecipitation experiments at an initial molar C/Fe ratio >1. The EPS-N is relatively enriched during the adsorption process, while more EPS-C and near-complete EPS-P are fixed in the coprecipitation process. XPS results show that the surface of the ferrihydrite formed through EPS adsorption is preferentially enriched with protein-like components, whereas, the surface of the ferrihydrite formed through EPS coprecipitation is enriched with polysaccharide-like components, which is visually confirmed with CLSM images. NEXAFS results reveal that the carboxylic/amide C-containing components are selectively retained during adsorption, with the aliphatic and O-alkyl C-containing components relatively enriched during coprecipitation. 2D-FTIR-COS results indicate that during EPS adsorption on ferrihydrite the PO functional groups are adsorbed faster than the amide and carboxylate functional groups, while during EPS coprecipitation with ferrihydrite the opposite trend is observed. The findings from this study indicate that the formation pathway of EPS-ferrihydrite associations substantially effects the sorption mechanisms and selective retention of EPS and may thus affect the mobility and fate of microbially-derived carbon (C), nitrogen (N) and phosphorus (P) in soils. These new insights on EPS behaviour at the mineral–water interface might be used to evaluate how microbially-derived compounds like EPS are stabilized by iron (oxyhydr)oxides and how EPS-iron (oxyhydr)oxide couplings might affect the reactivity and cycling of OM in natural environments.

Corrosion behavior of carbon steel in the presence of iron-oxidizing bacterium Pseudomonas sp. DASEWM1

The present study describes the microbiologically influenced corrosion (MIC) of carbon steel due to iron-oxidizing bacteria (IOB), isolated from the rust deposited on carbon steel’s surface exposed to river water. Bacterial isolates, meant for corrosion investigation, were identified as Pseudomonas sp. DASEWM1. In this work, corrosion tests were carried out to estimate the extent of corrosion. Corrosion products deposited on the steel’s surface were identified using X-ray diffraction and Fourier transformed infrared techniques. It is observed that the degree of corrosivity in inoculated nutrient broth (NB) depends upon bacterial concentration and the amount of the constituents of extracellular polymeric substances (EPS). The observance of a lesser amount of vivianite which is protective in nature, as corrosion product formed on corroded carbon steel exposed to inoculated NB, is suggested as an additional factor responsible for increased metal corrosion. Corrosion of steel in inoculated NB appears to be due to (1) oxidation of ferrous ions to ferric ions by iron-oxidizing bacteria (IOB), thereby decreasing the amount of ferrous ions available for vivianite formation, (2) some Fe3+ gets bound to EPS, thus offer electrons for the oxygen reduction reaction. Overall, this process leads to the increased alkalinity of NB. Lastly, a possible mechanism of MIC due to IOB has been proposed by considering the above mentioned reactions.

Study of Microbially Influenced Corrosion in the Presence of Iron-Oxidizing Bacteria (Strain DASEWM2)

The present study deals with the investigation of microbially influenced corrosion of mild steel by the iron-oxidizing bacterium (IOB), Pseudomonas sp. strain DASEWM2, using electrochemical tests, immersion tests, and surface analysis. The corrosion rate obtained by Tafel plots was higher after exposure of ~ 24 h to 72 h when the concentration of sessile cells and constituents of extracellular polymeric substances were high. In inoculated media, cyclic polarization curves show lower pitting potential, repassivation potential, and large area under the hysteresis loop, indicating higher susceptibility of steel to localized corrosion. Immersion tests show higher corrosion rate (0.252 ± 0.05 mpy) and large deep pits in the open area (maximum pit depth 140 ± 8 µm) and under crevice (maximum pit depth 95 ± 4 µm) indicating a higher degree of corrosion in inoculated media. Corrosion products formed on corroded steel samples were analyzed using EDAX, XRD, and FTIR techniques. Goethite and lepidocrocite are the main corrosion products in the case of inoculated media. In contrast, lepidocrocite and iron hydrogen phosphate are the main corrosion products in the case of control media. The absence of iron hydrogen phosphate, a protective type of corrosion product, on steel exposed to inoculated media may be responsible for observing a higher degree of corrosion due to bacteria.

Sulfur-based mixotrophic bio-reduction for efficient removal of chromium (VI) in groundwater

Organic matter and reduced sulfur compounds commonly coexist in groundwater aquifers and their respective roles in Cr(VI) bio-reduction have been well established, but Cr(VI) bio-reduction under mixotrophic condition, where organics and elemental sulfur simultaneously occur as co-donors of electrons, remains largely unknown. Herein a sulfur-based mixotrophic bio-reduction process is demonstrated to be effective to detoxify Cr(VI), with a removal efficiency of 95.5 ± 0.74% within 48 h at an initial concentration of 50 mg/L. In addition to direct reduction by heterotrophic Cr(VI) reducers such as Desulfovibrio and Desulfuromonas, volatile fatty acids (VFAs) produced from autotrophic sulfur oxidation served as electron donors for heterotrophic Cr(VI) reducers. Part of VFAs was also assimilated and accumulated as glycogen within cells, which enhanced their Cr(VI) removal capacity. Metabolic pathway analysis suggested that Cr(VI) was reduced to insoluble Cr(III) both extracellularly by cytochrome c and intracellularly by nicotinamide adenine dinucleotide in the presence of upregulated chrA gene. Constituents of extracellular polymeric substances (EPS) also contributed to Cr(VI) reduction enzymatically, through binding of toxic Cr(VI) by carboxyl and hydroxyl groups. Results from this study have important implications for understanding the biogeochemical behavior and environmental remediation of Cr(VI) in groundwater aquifers and sediments/soils.

Characterization of the extracellular polymeric substances (EPS) of Virgibacillus strains capable of mediating the formation of high Mg-calcite and protodolomite

The origin of dolomite –a common mineral in the geological record– is the subject of an ongoing debate. Among different hypotheses, it has been proposed that extracellular polymeric substances (EPS) excreted by microbes include organic molecules that catalyze the incorporation of Mg in the carbonate mineral. However, limited information exists on the composition of the EPS produced by CaMg carbonate-forming microbes, which in turn hampers a precise understanding of their role in the mineralization mechanism. Here, we present the results of laboratory experiments in which we cultured different strains of microbes, characterized their EPS, and identified components associated with carbonate minerals with high mol% Mg. Two Virgibacillus strains known to mediate the formation of Mg-rich carbonates, as well as a strain of Bacillus licheniformis–a negative control that does not mediate mineral formation but produces EPS, were grown under different salinities and temperatures, which caused them to produce EPS with different compositions. The EPS were subsequently characterized by measuring total carbohydrate (TCHO) and total protein (TP) contents, as well as by Fourier-transform infrared spectroscopy (FTIR). At the tested conditions, we found that Mg-carbonates with a mol% Mg higher than 40% (i.e., potential dolomite precursor phases) formed exclusively in association with EPS rich in carbohydrates (TCHO > than 75% of the total mass). FTIR spectra of CaMg carbonate-forming strains were distinct from those of the non-mineral-forming strain in areas associated with the protein structures responsible for the formation of hydrogels, which contribute to hydration or dehydration of ionic clusters; further differences have been observed in the regions of phosphoryl functional groups. These results provide insight on which fraction of organic molecules and specific functional groups are, among the many constituents of EPS, important for mineral nucleation and incorporation of Mg into carbonate minerals, a crucial step for the formation of dolomite in natural environments.

Influence of Pyrolytic Biochar on Settleability and Denitrification of Activated Sludge Process

Biochar is a massively produced by-product of biomass pyrolysis to obtain renewable energy and has not been fully used. Incomplete separation of sludge and effluent and insufficient denitrification of sewage are two of main factors that influence the efficiency of activated sludge process. In this work, we proposed a new utilization of biochar and investigated the effect of biochar addition on the performance of settleability and denitrification of activated sludge. Results show that the addition of biochar can improve the settleability of activated sludge by changing the physicochemical characteristics of sludge (e.g., flocculating ability, zeta-potential, hydrophobicity, and extracellular polymeric substances constituents). Moreover, the dissolved organic carbon released from biochar obtained at lower pyrolysis temperature can improve the nitrate removal efficiency to a certain extent.

The effects of substrate exposure history and carbon starvation-induced stress on the EPS synthesis of TCE degrading toluene oxidizing soil bacteria

A series of batch experiments was undertaken to explore the influence of growth conditions on the extracellular polymeric substances (EPS) production of five representative strains of trichloroethylene (TCE) degrading toluene oxidizing bacteria of importance to in situ bioremediation. EPS production for three distinct carbon sources (i.e., lactate, glucose, and toluene) was examined under starvation and non-starvation conditions. EPS extraction was accomplished using DOWEX™ (cation exchange resin; CER), and total carbohydrate, protein, and uronic acid were quantified as the main EPS constituents. Experimental results reveal that most bacteria grown on toluene synthesized more EPS per colony forming unit (CFU) than those grown on other substrates and that EPS production is enhanced with increasing length of starvation. These observations suggest that EPS may be produced under conditions of environmental stress and may be linked to enzyme deactivation. The results from this work additionally indicate that the mutant strain of Ralstonia pickettii PKO1 with the lack of gene encoding TbuX (an outer membrane protein) is more hydrophobic due to less EPS synthesis ability. The relatively lower amount of EPS synthesis in the mutant strain of R. pickettii PKO1 suggests that TbuX, an outer membrane protein encoded a part of the toluene catabolic regulation of R. pickettii PKO1, has a role in bacterial EPS production.

Effects of acid/alkaline pretreatment and gamma-ray irradiation on extracellular polymeric substances from sewage sludge

In order to investigate the mechanism of extracellular polymeric substances (EPS) influencing sludge characteristics, variations of extractable EPS from municipal sewage sludge by acid/alkaline pretreatment and gamma-ray irradiation were studied. The changes in constituents of EPS were analyzed by UV–vis spectra and SEM images. The effects of alkaline pretreatment and gamma-ray irradiation on the functional groups in EPS were investigated by Fourier transform infrared (FTIR) spectrometer. Results showed that the extractable EPS increased clearly with increasing irradiation dose from 0 to 15kGy. UV–vis spectra indicated that a new absorption band from 240nm to 300nm existed in all irradiated samples, apart from acid condition. The results of FTIR spectroscopic analysis indicated that, irradiation influenced major functional groups in EPS, such as protein and polysaccharide, and these effects were clearer under alkaline condition. SEM images provided that after alkaline hydrolysis, gamma-ray irradiation was more effective in resulting in the sludge flocs and cells broken, compared with acid pretreatment (pH 2.50).

Investigation of extracellular polymer substances (EPS) and physicochemical properties of activated sludge from different municipal and industrial wastewater treatment plants

This paper examines the chemical constituents of extracellular polymeric substances (EPS) and physicochemical properties of eight different sludge flocs from seven full-scale wastewater treatment plants. The physicochemical properties included floc properties (floc size, turbidity and effluent suspended solids (ESS) content of the supernatant), sludge volume index, capillary suction time and specific resistance to filtration. The relationships between the chemical constituents of EPS and the flocculation, settleability and dewaterability of sludge flocs were also assessed. The results showed that higher amounts of EPS were found in the municipal sludge flocs than in the industrial sludges. The content of tightly bound EPS (TB-EPS) was much greater than that of loosely bound EPS (LB-EPS). The amounts of total EPS, LB-EPS, TB-EPS and protein in LB-EPS were strongly related to ESS. The ratios of total protein to EPS and total carbohydrate to EPS showed positive correlation to the flocs size. It was surprising that there was no correlation between settleability or dewaterability and the chemical constituents of EPS.

Selection of commercial hydrolytic enzymes with potential antifouling activity in marine environments

In this work, the marine antifouling potential of some commercially available hydrolytic enzymes acting on the main constituents of extracellular polymeric substances (EPS) involved in bacterial biofilm formation was determined. The selected protease (i.e., alpha-chymotrypsin from bovine pancreas), carbohydrase (i.e., alpha-amylase from porcine pancreas) and lipase (from porcine pancreas) exhibited remarkable hydrolytic activities towards target macromolecules typically composing EPS under a wide range of pHs (6.5–9.0 for alpha-chymotrysin and alpha-amylase; 7.0–8.5 for the lipase) and temperatures (from 10 °C to 30 °C), as well as relevant half-lives (from about 2 weeks to about 2 months), in a marine synthetic water. The activity displayed by each enzyme was poorly affected by the co-presence of the other enzymes, thus indicating their suitability to be employed in combination. None of the enzymes was able to inhibit the formation of biofilm by an actual site marine microbial community when applied singly. However, a mixture of the same enzymes reduced biofilm formation by about 90% without affecting planktonic growth of the same microbial community. This indicates that multiple hydrolytic activities are required to efficiently prevent biofilm formation by complex microbial communities, and that the mixture of enzymes selected in this study has the potential to be employed as an environmental friendly antifouling agent in marine antifouling coatings.

Effect of algal extracellular polymer substances on UF membrane fouling

Low-pressure membrane processes, such as ultrafiltration (UF), are effective for treating algae-laden eutrophic source waters. However, fouling caused by algae excreted organics can be a major obstacle to the smooth operation of membrane processes. In this study, three algae species, with a similar cell size and shape, but a different amount of extracellular polymeric substances (EPS), were selected. The characteristics of the bound EPS, including its amount and constituents, mainly polysaccharide and protein, were analyzed. Cell suspensions were filtered through a dead-end stirred cell with a regenerated cellulose UF membrane. Based on the observations of permeate flux decline at higher transmembrane pressure (TMP) and critical flux values, it can be concluded that algae cell EPS do affect membrane fouling, and the constituents of EPS may also play a role.

Influence of Extracellular Polymeric Substances onPseudomonas aeruginosaTransport and Deposition Profiles in Porous Media

The impact of cell surface extracellular polymeric substances (EPS) on bacterial transport and retention profiles was investigated in saturated columns packed with glass beads. Three genetically well-defined isogenic Pseudomonas aeruginosa strains with different EPS secretion capability and EPS composition were used to systematically examine their deposition behavior over a range of solution chemistry. The presence of EPS on nonmucoid strain PA01 and mucoid strain PD0300 significantly increased bacterial adhesion over the EPS deficient PA01 psl pel mutant strain despite their similar surface charge as indicated by the zeta potential measurements. Retained bacterial profiles show the deposition rate coefficients with various shapes and degrees of deviation from those expected from the classic filtration theory. Non-monotonic deviations from the log-linear deposition pattern with the majority of the bacteria retained downgradient of the column inlet were observed when bacterial cells were encapsuled by EPS under both high and low ionic strength conditions. In contrast, the EPS-deficient strain exhibited monotonic deviation from theory only under low ionic strength conditions. The results demonstrate that the non-monotonic deviation from filtration theory observed in this study was driven by steric interactions between extracellular polymers and glass beads. Analysis of the retained polysaccharides (carbohydrates and uronic acids) and protein profiles suggests that bacterial re-entrainment and re-entrapment may have contributed to the downgradient movement of the maximum retained bacteria. The detachment of bacteria may leave behind various constituents of EPS as their "footprints," which can be a valuable tool for tracking the trajectory of bacterial transport.

The role and significance of extracellular polymers in activated sludge. Part I: Literature review

In biological wastewater treatment, bacteria tend to aggregate by forming flocs, biofilms and even granules. A large part of the floc structure in activated sludge is composed of extracellular polymers. The extracellular polymeric substances (EPS) are mainly responsible for the structural and functional integrity of biofilms/flocs and they are considered to be important for the physico-chemical properties of activated sludge. This review considers the composition of EPS and proportion between the constituents. Moreover it includes the relationship between the EPS constituents and sludge properties such as sludge retention time, hydrophobicity and surface charge.

Interactions of biopolymers with silica surfaces: Force measurements and electronic structure calculation studies

Pull-off forces were measured between a silica colloid attached to an atomic force microscope (AFM) cantilever and three homopolymer surfaces representing constituents of extracellular polymeric substances (EPS). The pull-off forces were −0.84 (±0.16), −0.68 (±0.15), and −2.37 (±0.31) nN as measured in water for dextran, phosphorylated dextran, and poly- l-lysine, respectively. Molecular orbital and density functional theory methods (DFT) were applied to analyze the measured pull-off forces using dimer clusters representing interactions between the three polymers and silica surfaces. Binding energies for each dimer were calculated with basis set superposition error (BSSE) and interpolated using corrections for silica surface hydroxyl density and silica charge density. The binding energies were compared with the normalized pull-off forces with the effective silica surface area contacting the polymer surfaces. The predicted binding energies at a −0.064 C/m 2 silica surface charge density corresponding to circum-neutral pH were −0.055, −0.029, and −0.338 × 10 −18 J/nm 2 for the dimers corresponding to the silica surface with dextran, phosphorylated dextran, and poly- l-lysine, respectively. Polarizable continuum model (PCM) calculations with different solvents, silanol vibrational frequency calculations, and orbital interaction analysis based on natural bonding orbital (NBO) showed that phosphate groups formed stronger H-bonds with neutral silanols than hydroxyl and amino functional groups of polymers, implying that phosphate containing polymers would play important roles in EPS binding to silica surfaces.