Characterization Of Extracellular Polymeric Substances Research Articles

Characterisation of extracellular polymeric substances from different cyanobacterial species and their influence on biocalcification processes

Environmental contextExtracellular polymeric substances provide a nucleation site for calcium carbonate and hence are important for bio-calcification processes, with implications for sediment formation and the global carbon cycle. We investigate the calcification potential of polymeric substances produced by five species of cyanobacteria. The results indicate that the protein content and alkaline functional groups of the extracellular polymeric substances may have a significant effect on cyanobacterial calcification. AbstractCyanobacterial calcification plays a crucial role in the formation of freshwater calcium carbonate precipitates, with cyanobacterial extracellular polymeric substances (EPSs) contributing significantly, partly by providing a nucleation site for calcium carbonate. Despite this, cyanobacterial EPS and their effect on calcification processes have not been completely characterised. In the present study, five cyanobacterial species were selected. First, EPS characteristics of these cyanobacterial species were examined, showing that proteins dominated both EPSs released in to solution (REPSs) and cell-surface bound (LEPSs). The major EPS functional groups included acidic groups, such as carboxyl groups, and highly alkaline groups, such as hydroxyl and amino groups. The calcification ability of different cyanobacterial species was found to vary dramatically. Solution pH increased during the calcification process, which was beneficial to the supersaturation of CaCO3, and could affect the calcification potential. Precipitation, however, was positively correlated with EPS protein content and the concentration of basic functional groups, such as amino or hydroxyl groups. These results suggest EPS protein content and alkaline functional groups may have a significant effect on cyanobacterial calcification. The results also provide a potential application in that EPS proteins of cyanobacteria may have beneficial positive applications in the removal of multivalent cations from wastewater.

Characterization of extracellular polymeric substances produced during nitrate removal by a thermophilic bacterium Chelatococcus daeguensis TAD1 in batch cultures

Positive correlation was observed between EPS production and nitrate removal efficiency during aerobic denitrification byChelatococcus daeguensisTAD1.

Characterization of extracellular polymeric substances of Bacillus amyloliquefaciens SQR9 induced by root exudates of cucumber.

Bacillus amyloliquefaciens SQR9 is a plant growth-promoting rhizobacterium (PGPRs) that forms biofilm on the roots of plants and protects them from a variety of pathogens. In this study, we reported the effect of root exudates produced by cucumber (Cucumis sativus L.) at different developmental stages on the biochemical composition of the biofilm matrix of SQR9. The results showed that the amino acids present in the root exudates of cucumber were responsible for triggering biofilm formation of SQR9. In addition, when root exudates harvested at different growth phases of cucumber were used as carbon sources for biofilm formation, the resulting biofilm matrixes differed both quantitatively and qualitatively. The biofilm matrix was mostly composed of amino groups observed by confocal laser scanning microscope (CLSM) hence the proteins formed the major component of the resulting extracellular polymeric substances (EPS). The potential use of amino acid-based dietary supplements to control biofilm formation in the plants may be a viable option to improve agricultural productivity by recruiting beneficial association with PGPRs in the manufacture of bio fertilizers or bio controls.

Characterization of extracellular polymeric substances and microbial diversity in anaerobic co-digestion reactor treated sewage sludge with fat, oil, grease

Performance of co-digesters, treated of sewage sludge (SS) with fat, oil and grease (FOG), were conducted semi-continuously in two mesophilic reactors over 180days. Compared with SS mono-digestion, biogas production and TS removal efficiency of co-digestion were significantly enhanced up to 35% and 26% by adding upper limit FOG (60% on VS). Enhancement in co-digestion performance was also stimulated by the release of extracellular polymeric substances (EPS), which was increased 40% in both loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) than that of mono-digester. Based on high-throughput sequencing (HTS), analysis of microbial 16S rRNA gene comprehensively revealed the dynamic change of microbial community. Results showed that both bacterial and archaeal undergone an apparent succession with FOG addition, and large amount of consortium like Methanosaeta and N09 were involved in the process. Redundancy analysis showed the acetoclastic genera Methanosaeta distinctly related with biogas production and EPS degradation.

Nitrogen and phosphorus removal from tertiary wastewater in an osmotic membrane photobioreactor

An osmotic membrane photobioreactor (OMPBR) was designed and operated for 162days for nitrogen and phosphorus removal from wastewater using Chlorella vulgaris. The removal efficiency for NH4+-N, NO3−-N and PO43−-P reached as high as 95%, 53% and 89%, whereas the maximum removal rates were 3.41mg/L-day, 0.20mg/L-day and 0.8mg/L-day, respectively. The microalgae exhibited high tendency to aggregate and attached to the bioreactor and membrane surfaces, and total biomass accumulation in the OMPBR was over 5g/L. Salt accumulation and biofouling had adverse effects on membrane filtration, but the performance could be recovered through periodic backwashing of the membranes. Extracellular polymeric substances characterization indicated higher fraction of polysaccharides as compared to proteins. The biomass in the OMPBR accumulated higher levels of carbohydrates and chlorophyll. These results indicate the suitability of OMPBR in wastewater treatment and in high-density microalgae cultivation.

Characterization of extracellular polymeric substances (EPS) extracted from Pseudomonas putida ATCC 11172 growths at different carbon concentration

The extracellular polymeric substances (EPS) extracted from Pseudomonas putida ATCC 11172 at different carbon concentration, P. putida was grown using Luria broth alone (LB) and supplemented with glucose at 0.5% w/v (LBG 0.5%) and 1.0% w/v (LBG 1%) in order to promote different EPS composition.. The Bound EPS were extracted by ethylene diamine tetra acetic acid (EDTA) methods and precipitated by cold ethanol 3:1; while free EPS extracted only by precipitate by cold ethanol 3:1. Study the effect of carbon concentration on the Protein and polysaccharides contains. Where found the extracted EPS had carbohydrate/ protein ratio between1.18-1.62; indicating the EPS extracted is pure. Low and stable values of 2-keto-3 deoxyoctonate (KDO) that meaning no significant cell rupture occurred during the extraction of EPS, the amount of Uronic acid was relatively high and similar in all EPS samples, this is indicative that the composition of EPS has only low contamination by intercellular material.

Characterization of Extracellular Polymeric Substances (Eps) Produced by Cloacibacterium normanense Isolated from Wastewater Sludge for Sludge Settling and Dewatering

In this study, extracellular polymeric substances (EPSs) producing strain was isolated from municipal wastewater sludge (MWWS). Growth profile and the EPS production by Cloacibacterium normanense using wastewater sludge as raw material in shake flask fermentation for 96 h were investigated. The highest concentration of S-EPS (13.0 ± 0.8 g/L) and C-EPS (0.3 ± 0.1 g/L) were attained at 48 h of fermentation. S-EPS revealed higher flocculation activity (94.2%) and dewaterability (59.9%) than other types of EPS in kaolin suspension. The dewaterability of MWWS with 2 g suspended solids (SS)/L was improved by 37.6% using 0.02 ± 0.01 g/L of S-EPS and 600 mg/L of Al2(SO4)3. The study showed a promising approach of new isolated strain to produce high concentration EPS in sludge with high flocculation activity as well as good settling.

Influence of solids retention time on membrane fouling: characterization of extracellular polymeric substances and soluble microbial products

The objective of this study was to investigate the influence of solids retention time (SRT) on membrane fouling and the characteristics of biomacromolecules. Four identical laboratory-scale membrane bioreactors (MBRs) were operated with SRTs for 10, 20, 40 and 80 days. The results indicated that membrane fouling occurred faster and more readily under short SRTs. Fouling resistance was the primary source of filtration resistance. The modified fouling index (MFI) results suggested that the more ready fouling at short SRTs could be attributed to higher concentrations of soluble microbial products (SMP). Fourier transform infrared (FTIR) spectra indicated that the SRT had a weak influence on the functional groups of the total extracellular polymeric substances (TEPS) and SMP. However, the MBR under a short SRT had more low-molecular-weight (MW) compounds (<1 kDa) and fewer high-MW compounds (>100 kDa). Aromatic protein and tryptophan protein-like substances were the dominant groups in the TEPS and SMP, respectively.

Spectroscopic characterization of extracellular polymeric substances from a mixed culture dominated by ammonia-oxidizing bacteria

Extracellular polymeric substances (EPS) of aerobic (AerAOB) and anaerobic ammonium-oxidizing bacteria (AnAOB) are expected to have a significant impact on the performance of autotrophic nitrogen removal in engineered systems. However, there are a few investigations of the EPS of AerAOB and AnAOB, and the results are contradictory. In this study, photometric measurements indicated that the EPS of AerAOB- (31.74 ± 1.48 mg/g-VSS, volatile suspended solids) and AnAOB-enriched cultures (30.12 ± 1.52 mg/g-VSS) contained more polysaccharides than did conventional activated sludge from a municipal wastewater treatment facility (10.76 ± 0.83 mg/g-VSS). In addition, the EPS of the AnAOB-enriched culture was dominated by proteins, leading to a considerably higher protein/polysaccharide ratio (2.64 ± 0.12) than those of the AerAOB-enriched culture (0.56 ± 0.03) and conventional activated sludge (1.96 ± 0.09). Characterization using Fourier transform infrared spectroscopy (FTIR) revealed the dominance of amide bands and/or polysaccharide-associated bands in the EPS of AnAOB and AerAOB. These results corroborate the data from the photometric measurements. In addition, the EPS of AnAOB (23.1% ± 1.2%) and AerAOB (21.9% ± 1.1%) had a higher portion of α-helices, which is the key protein secondary structure that determines flocculation or cell aggregation, in the amide I band than that of activated sludge (16.7% ± 0.8%). X-ray photoelectron spectroscopy (XPS) characterization also revealed significantly different functionalities among the EPS of the three mixed cultures; e.g., O-(C,H), which indicates the presence of polysaccharides, was richer in the EPS of AerAOB, whereas protonated amines, which are commonly found in amino acids and amino sugars, accounted for a large portion of the EPS of AnAOB. The results of this study can potentially expand our knowledge of the microbial aggregates responsible for autotrophic nitrogen removal.

Characterization of extracellular polymeric substances in the biofilms of typical bacteria by the sulfur K-edge XANES spectroscopy

A combined approach of physicochemical extraction and sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy was applied to characterize the extracellular polymeric substances (EPS) of typical bacterial biofilms in this study. Physicochemical analysis showed variation of the contents of DNA, polysaccharide and protein in different fractions of EPS in different mediums. The sulfur K-edge XANES analysis yielded a variety of spectra. Spectral fitting of the XANES spectra utilizing a large set of model compounds showed that there was more reduced sulfur in both LB-EPS (loosely bound EPS) and TB-EPS (tightly bound EPS) of all the biofilms in LB medium than in R2A medium. More oxidized sulfur was identified in LB-EPS than that in TB-EPS, suggesting different niches and physiological heterogeneity in the biofilms. Our results suggested that the sulfur K-edge XANES can be a useful tool to analyze the sulfur speciation in EPS of biofilms.

The effect of solids retention times on the characterization of extracellular polymeric substances and soluble microbial products in a submerged membrane bioreactor.

In this study, the effect of solids retention times (SRTs) on extracellular polymeric substances (EPS) and soluble microbial products (SMPs) were investigated in a membrane bioreactor (MBR) at SRTs of 10, 5 and 3 days. The results showed that more carbohydrates and proteins were accumulated at short SRT, which can due to the higher biomass activity in the reactor. The molecular weight (MW) distribution analysis suggested that macromolecules (MW>30 kDa) and small molecules (MW<1 kDa) were the dominant fraction of EPS and SMP, respectively. The reactor at shorter SRT had more small molecules and less macromolecules of carbohydrates. The MW distribution of total organic carbon (TOC) suggested that other organic moieties were exuded by microbes into the solution. The shorter SRT had more undefined microbial by-product-like substances and different O − H bonds in hydroxyl functional groups.

Environmental Applications of Excitation-Emission Spectrofluorimetry: An In-Depth Review II

: This part is devoted to the environmental applications of excitation-emission spectrofluorimetry (EES): Characterization of chromophoric dissolved organic matter (CDOM) in drinking waters, rivers, fog waters, lakes, oceans, leachates, wastewaters, sluge, bioreactor membrane foulants, and soils; characterization of extracellular polymeric substances in sludges; study of interactions between CDOM and organic pollutants in soils; and quantification of organic pollutants in waters and soils. This part includes the pretreatment of samples, and the environmental applications of the technique are discussed, including its application to bioremediation of wastewaters. More than 200 references (focusing mainly on the last 10 years) from all kinds of journals (environment, analytical chemistry, biotechnology) have been carefully revised and included in this part of the article.

Environmental Applications of Excitation-Emission Spectrofluorimetry: An In-Depth Review I

: This review attempts to cover the principles and environmental applications of excitation-emission spectrofluorimetry (EES). The article is divided into two parts and each part is divided into several sections. The first part includes the following: Introduction, advantages and drawbacks of EES, comparative EES versus other techniques, factors influencing EES signals, representation of EES spectra, relationships and changes in intensity of fluorescence, multivariate calibration combined with EES, quenching of fluorescence, and conclusions. Theoretical and practical considerations are included, and the possibilities and limitations are evaluated. The second part is devoted to the environmental applications of EES: Characterization of chromophoric dissolved organic matter (CDOM) in drinking waters, rivers, fog water, lakes, oceans, leachates, wastewaters, sludge, bioreactor membrane foulants and soils; characterization of extracellular polymeric substances in sluges; study of interactions between CDOM and organic pollutants in soils; and quantification of organic pollutants in waters and soils. This part includes the pretreatment of samples, and the environmental applications of the technique are discussed, including its application to biorremediation of wastewaters. More than 500 references (focusing mainly on the last 10 years) from all kinds of journals (environment, analytical chemistry, biotechnology) have been critically reviewed and included in this article.

Extraction and Characterization of Extracellular Polymeric Substances in Biofilm and Sludge via Completely Autotrophic Nitrogen Removal Over Nitrite System

Extracellular polymeric substances (EPS) were extracted from sludge and biofilm via the completely autotrophic nitrogen removal over nitrite (CANON) system. Tightly bound (TB)-EPS were extracted using four physical methods, namely, cationic exchange resin (CER), sonication, heating, and steaming. CER was the most effective and most suitable method for extraction among the four methods. Moreover, the ultraviolet-vis spectra of TB-EPS indicated that few cells were destroyed by the CER method. The major component contents of total EPS, proteins, carbohydrates, humic substances, and DNA in sludge were 60.77, 49.84, 21.63, and 9.01 mg/g volatile suspended solids (VSS) and 90.03, 29.01, 15.96, and 10.04 mg/g VSS in biofilm, respectively. The Fourier transform infrared (FT-IR) spectra results indicated differences in the EPS functional groups between biofilm and sludge. The results of the batch experiments showed that the biofilm activity was significantly higher than that of the sludge in the CANON system. Furthermore, biomass activity was probably influenced by the EPS composition and distribution in the sludge and biofilm.

Purification and Characterization of Extracellular Polymeric Substances (EPS) with Antimicrobial Properties from s Marine Epibiotic Pseudoalteromonas sp.

We present a study to purify and characterize the extracellular polymeric substances (EPS) produced by Pseudoalteromonas sp.isolated from fish epidermal surface. The purified EPS was recovered from the culture supernatant by using cold ethanol precipitation and dialysis method. Colorimetric analysis of EPS revealed that it was primarily composed of proteins, 0.0251g/L, carbohydrates, 0.0561g/L and uronic acid, 0.0217 g/L. The EPS also exhibited high emulsification activity when being tested with hydrophobic substances such as paraffin oil, (94.5 % and 79.4%), kerosene, (56.1% and 37.2%) and Tween 80 (46.6% and 32.9%). In addition, the EPS demonstrated discernible antimicrobial activity against Gram–negative bacteria like E.coli, P.aeruginosa and E.aerogenes. The SDS–PAGE profile revealed up to eight types of unknown proteins were detected within the EPS, with the molecular weight ranging from 15.486 kDa to 113.058 kDa. The results of the study would contribute to the elucidation of antimicrobial components and its mechanism inPseudoalteromonas sp.

Extraction and Characterization of Extracellular Polymeric Substances (EPS) from Waste Sludge of Pulp and Paper Mill

Secondary sludge (SS) and mixed sludge (MS) from a Canadian paper mill were characterized, and investigated for extracellular polymeric substances (EPS) to explore the potential opportunity for value added utilization of waste paper sludge as wood adhesive. After basic characterization which showed the availability of significant amount of organic matter, further study of wood adhesive extenders were carried out through extraction of EPS by using 4000g centrifugation, a physical control method, and a chemical extraction technique involving formaldehyde and sodium hydroxide reagents. Gravimetric analysis showed the chemical extraction technique as being efficient method by yielding up to 5 times more EPS compared to physical control method. SS sludge, as anticipated, contained significantly high amount of EPS compared to MS. Fourier transform infrared spectrometry (FTIR) studies on extracted EPS confirmed the presence of key functional groups related to potential adhesions like protein, glucose, lignin, and carbohydrates. The polymeric nature of EPS was further diagnosed through calorimetric studies

Step-by-step strategy for protein enrichment and proteome characterisation of extracellular polymeric substances in wastewater treatment systems

Extracellular polymeric substances (EPS) are keys in biomass aggregation and settleability in wastewater treatment systems. In membrane bioreactors (MBR), EPS are an important factor as they are considered to be largely responsible for membrane fouling. Proteins were shown to be the major component of EPS produced by activated sludge and to be correlated with the properties of the sludge, like settling, hydrophobicity and cell aggregation. Previous EPS proteomic studies of activated sludge revealed several problems, like the interference of other EPS molecules in protein analysis. In this study, a successful strategy was outlined to identify the proteins from soluble and bound EPS extracted from activated sludge of a lab-scale MBR. EPS samples were first subjected to pre-concentration through lyophilisation, centrifugal ultrafiltration or concentration with a dialysis membrane coated by a highly absorbent powder of polyacrylate-polyalcohol, preceded or not by a dialysis step. The highest protein concentration factors were achieved with the highly absorbent powder method without previous dialysis step. Four protein precipitation methods were then tested: acetone, trichloroacetic acid (TCA), perchloric acid and a commercial kit. Protein profiles were compared in 4-12% sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. Both acetone and TCA should be applied for the highest coverage for soluble EPS proteins, whereas TCA was the best method for bound EPS proteins. All visible bands of selected profiles were subjected to mass spectrometry analysis. A high number of proteins (25-32 for soluble EPS and 17 for bound EPS) were identified. As a conclusion of this study, a workflow is proposed for the successful proteome characterisation of soluble and bound EPS from activated sludge samples.

Characterization of extracellular polymeric substances (EPS) from phenol degrading aerobic granules

Extracellular polymeric substances (EPS) play essential role in determining efficiencies of sludge bio-granulation processes. Extraction methods and analysis technique on EPS from phenol degrading aerobic granules were examined. High pressure size exclusion chromatography (HPSEC) and excitation–emission matrix (EEM) spectroscopy were used to characterize the EPS extracted from phenol-fed aerobic granules using both physical and chemical extraction protocols. Ultrasound followed by chemical extraction using formaldehyde or formamide with NaOH effectively extracted EPS from aerobic granules of compact interior. The chemical composition, apparent molecular weight and EEM characteristic peaks revealed diverse nature of loosely bound, tightly bound and pellet fractions of the EPS. Chromatographic fingerprints of extracted EPS were compared.

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.

Characterization of extracellular polymeric substances from biofilm in the process of starting-up a partial nitrification process under salt stress

In this study, the characteristics of extracellular polymeric substance (EPS) fractions of biofilm during the process of establishing a partial nitrification under salt stress were analyzed in terms of concentrations, molecular weight distribution, and three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy. A partial nitrification was formed successfully with a salinity of 1%. Results indicated that the amount of total EPS increased from 54.2 mg g⁻¹ VSS⁻¹ on day 1 to 99.6 mg g⁻¹ VSS⁻¹ on day 55 due to the NaCl concentration changed from 0 to 10.0 g L⁻¹ in a biofilm reactor. The changes of loosely bound EPS (LB-EPS) compounds under different salt concentrations appeared to be more significant than those of the tightly bound EPS. A clear release of polysaccharides in the LB-EPS fraction was detected during the enhancement of salinity. This was considered as a protective response of bacteria to the salinity. Three fluorescence peaks were identified in the EEM fluorescence spectra of the EPS fraction samples. Two peaks were assigned to the protein-like fluorophores, and the third peak was located at the excitation/emission wavelengths of 275 nm/425-435 nm of the spectra of EPS fractions till the salinity maintained constant at 1%. This information is valuable for understanding the characteristics of EPS isolated from biomass in a saline nitrogen removal system.