Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges

The extracellular polymeric substances (EPS) extracted from three granular and one flocculant anaerobic sludges were characterised by size exclusion chromatography (SEC) using two serially linked chromatographic columns in order to obtain more detailed chromatograms. A Superdex peptide 10/300 GL (0.1–7 kDa) and Superdex 20010/300GL (10–600 kDa) from Amersham Biosciences were used in series with a mobile phase at pH 7 with an ionic strength of 0.223 M (phosphate buffer 50 mM and NaCl 150 mM). A part of the EPS molecules displays hydrophobic and/or ionic interactions with the column packing. Interactions could be modified by changing the mobile phase ionic strength or polarity (addition of acetonitrile). The detection wavelength (210 or 280 nm) affects strongly the EPS chromatogram. For a sludge originating from the same type of biofilms (i.e., anaerobic granules), the differences in EPS fingerprints are mainly due to differences in the absorbance of the chromatographic peaks, linked to EPS molecules content and composition. The EPS fingerprint changes significantly when the EPS originate from another type of anaerobic sludges. In addition, EPS fingerprints were affected by the extraction method used (centrifugation only; heat and centrifugation or cationic exchange resin and centrifugation). This phenomenon was observed mainly for the largest and smallest molecules and molecules which display interactions with column packing.     

Soluble microbial products (SMP) and soluble extracellular polymeric substances (EPS) from wastewater sludge

Laspidou and Rittmann (Water Research 36:2711–2720, 2002) proposed that the soluble extracellular polymeric substances (EPS) are identical to soluble microbial products (SMP) in sludge liquor. In this paper, we compared the physicochemical characteristics of the SMP and soluble EPS from original and aerobically or anaerobically digested wastewater sludge. The surface charges, particle sizes, residual turbidities of polyaluminum chloride (PACl) coagulated supernatant, and chemical compositions of the SMP and soluble EPS containing suspensions were used as comparison index. Experimental results revealed that the particles in SMP and soluble EPS fractions extracted from original wastewater sludge, before and after digestion, were not identical in all physicochemical characteristics herein measured. The current test cannot support the proposal by Laspidou and Rittmann (Water Research 36:2711–2720, 2002) that SMP is identical to the soluble EPS from a wastewater sludge.     

藻际环境中胞外聚合物的研究进展

微藻向细胞周围释放营养物质而形成了独特的藻际微环境,吸引了大量细菌的定殖。藻际环境中藻菌关系错综复杂,其间充斥着多样的物质交换与信息交流。以胞外聚合物(extracellularpolymeric substances,EPS)为代表的有机质在其中起着纽带作用。微藻和细菌都可以产生EPS,其过程受多种因素的调节。EPS在藻际环境中具有重要的生态功能,包括参与生物被膜(biofilm)的形成,影响藻菌共生关系的建立以及调节藻际微生物群落组成等。此外,EPS中的一大类别透明胞外聚合物颗粒(transparent exopolymer particles,TEP)还介导了海洋溶解有机碳向颗粒有机碳的转化,参与了海洋碳循环过程。本文以EPS的产生、组成以及对碳转化的影响为重点,综述了其在藻际生态位(Niche)中的生态功能,以期为深入理解藻际环境中的有机质特征和藻菌共生关系提供理论依据。     

A methodological review on the characterization of microalgal biofilm and its extracellular polymeric substances

Biofilm secreted by microalgae are extracellular polymeric substances (EPSs) composed mainly of polysaccharides, proteins, nucleic acids and lipids. These EPSs immobilize the cells and stabilize biofilm, mediating adhesion towards solid surfaces. The EPSs valorization through industrial exploitations and scientific works is becoming more popular, but the bottleneck of such studies is the lack of consensus among researchers on the selection of detection techniques to be used, especially for novice researchers. It is a daunting task for any inexperienced researcher when they fail to identify the right tools needed for microalgal biofilm studies. In this review, a well-refined analysis protocol about microalgal biofilm and EPSs were prepared including its extraction and characterization. Pros and cons of various detection techniques were addressed and cutting-edge methods to study biofilm EPSs were highlighted. Future perspectives were also presented at the end of this review to bridge research gaps in studying biofilm adhesion via EPSs production. Ultimately, this review aims to assist novice researchers in making the right choices in their research studies on microalgal biofilms in accordance to the available technologies and needs.     

Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review

A review concerning the definition, extraction, characterization, production and functions of extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment reactors is given in this paper. EPS are a complex high-molecular-weight mixture of polymers excreted by microorganisms, produced from cell lysis and adsorbed organic matter from wastewater. They are a major component in microbial aggregates for keeping them together in a three-dimensional matrix. Their characteristics (e.g., adsorption abilities, biodegradability and hydrophilicity/hydrophobicity) and the contents of the main components (e.g., carbohydrates, proteins, humic substances and nucleic acids) in EPS are found to crucially affect the properties of microbial aggregates, such as mass transfer, surface characteristics, adsorption ability, stability, the formation of microbial aggregates etc. However, as EPS are very complex, the knowledge regarding EPS is far from complete and much work is still required to fully understand their precise roles in the biological treatment process.     

Characterization of exopolymeric substances (EPS) produced by Aeromonas hydrophila under reducing conditions

The aim of this work was to investigate the production of extracellular polymeric substances (EPS) by Aeromonas hydrophila grown under anaerobic conditions. EPS composition was studied for planktonic cells, cells attached to carbon fibre supports using a soluble ferric iron source and cells grown with a solid ferric iron mineral (gossan). Conventional spectrophotometric methods, Fourier transform infrared (FTIR) and confocal laser scanning microscopy (CLSM) were used to determine the main components in the biofilm extracted from the cultures. The key EPS components were proteins, indicating their importance for electron transfer reactions. Carbohydrates were observed mostly on the mineral and contained terminal mannosyl and/or terminal glucose, fucose and N-acetylgalactosamine residues.     

Bismuth dimercaptopropanol (BisBAL) inhibits formation of multispecies wastewater flocs

Aims: To determine the ability of a bismuth thiol to control floc formation in a multispecies population of micro‐organisms obtained from the activated sludge unit of a wastewater treatment plant. The molecular level mechanisms by which bismuth‐2‐3‐dimercapto‐1‐propanol (BisBAL) inhibits bioaggregation are also elucidated. Methods and Results: Micro‐organisms were grown over a 3‐day period in a batch system by adding glucose as an electron donor to stimulate short‐term heterotrophic activity. Extracellular polymeric substances (EPS) produced by activated sludge micro‐organisms during exponential and stationary growth phases in the presence and absence of BisBAL were characterized using colorimetry, X‐ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FTIR) spectroscopy. BisBAL at its minimum inhibitory concentration (MIC, 10 μmol l^?1) was most effective in suppressing microbial floc formation. The principal effect of sub‐inhibitory concentrations of BisBAL was to decrease total EPS production while largely preserving homology. Conclusions: Antifouling and bactericidal properties of BisBAL arise from its ability to reduce EPS expression and preferentially suppressing acidic and O‐acetylated carbohydrates and certain protein secondary structures viz. β‐structures, random coils, and α‐and 3‐turn helices. As micro‐organisms exhibited a much weaker tendency to aggregate at lower concentrations of these specific EPS components, they also appear to be important for the formation of microbial flocs and bioaggregates. Significance and Impact of the Study: BisBAL was shown to be highly effective against multispecies microbial aggregation. Novel bismuth‐based biocides could also be potentially employed to control excess sludge production in wastewater treatment systems by inhibiting EPS expression.     

Mg2+ distribution in activated sludge and its effects on the nitrifying activity and the characteristics of extracellular polymeric substances and sludge flocs

Divalent cations act as bridges among extracellular polymeric substances (EPS) and form cross-linkage for the self-immobilization of microbial biomass. However, their effects on the nitrification performance during the biological nitrogen removal are still unclear. In the present study, the effects of Mg²⁺ on the nitrifying activity, EPS and floc characteristics were investigated using a lab-scale sequencing batch reactor. The distribution of Mg²⁺ was quantified at different level of sludge floc. The results indicated that the nitrification activity was significantly improved when influent Mg²⁺ was below 1.1 mmol/L, but suppressed at 3 mmol/L. The overall performance characterized by COD, NH₄⁺-N and TN, the particle size and sludge flocculation ability rapidly increased with the increase of Mg²⁺ concentration. Mg²⁺ was mainly distributed in the pellet and changed slightly in supernatant, LB-EPS and TB-EPS. The four fluorescence peaks detected by three-dimensional excitation-emission matrix spectra were attributed to PN-like substances and humic acid-like substances in the LB-EPS and TB-EPS. The results of XPS analysis demonstrated that LB-EPS and TB-EPS comprised similar elements. Therefore, the types of EPS functional groups was unchanged under varied Mg²⁺ concentrations, while their proportions changed and LB-EPS/EPS was key factor for the changes of bioflocculation.     

Enhanced aerobic granulation with extracellular polymeric substances (EPS)-free pellets

Extracellular polymeric substances (EPSs) were secreted by cells after they agglomerated into a compact aggregate. This study shows that the EPS initially embedded in seed sludge before granulation may sterically slow subsequent microbe–microbe contact, thereby delaying aerobic granulation. Three identical bioreactors were used in this study using glucose as the sole carbon and energy source. Reactor 1 (R1) was seeded with EPS-free pellets and operated in sequencing batch reactor (SBR) mode. Reactor 2 (R2) was seeded with the original sludge flocs and operated in SBR mode. Reactor 3 (R3) was seeded with EPS-free pellets and operated in continuously stirred tank reactor (CSTR) mode. Granulation occurred in R1 earlier than in R2; the granules that formed in R1 were larger and more compact than those in R2 at the same cultivation time. The few mature granules in R3 suggest that aerobic granulation can occur in a CSTR when a reactor is seeded with EPS-free pellets.     

Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms

In this study an enrichment culture developed from activated sludge was used to investigate the architecture of fully hydrated multispecies biofilms. The assessment of biofilm structure and volume was carried out using confocal laser scanning microscopy (CLSM). Bacterial cell distribution was determined with the nucleic acid-specific stain SYTO 60, whereas glycoconjugates of extracellular polymeric substances (EPS) were stained with the Alexa-488-labeled lectin of Aleuria aurantia. Digital image analysis was employed for visualization and quantification of three-dimensional CLSM data sets. The specific volumes of the polymeric and cellular biofilm constituents were quantified. In addition, gravimetric measurements were done to determine dry mass and thickness of the biofilms. The data recorded by the CLSM technique and the gravimetric data were then compared. It was shown that the biofilm thicknesses determined with both methods agree well for slow-growing heterotrophic and chemoautotrophic biofilms. In addition, for slow-growing biofilms, the volumes and masses calculated from CLSM and the biomass calculated from gravimetric measurements were also comparable. For fast-growing heterotrophic biofilms cultivated with high glucose concentrations the data sets fit to a lesser degree, but still showed the same common trend. Compared with traditional gravimetric measurements, CLSM allowed differential recording of multiple biofilm parameters with subsequent three-dimensional visualization and quantification. The quantitative three-dimensional results recorded by CLSM are an important basis for understanding, controlling, exploiting, and modeling of biofilms.     

Formation of extracellular polymeric substances from acidogenic sludge in H<Subscript>2</Subscript>-producing process

In this study, the formation of extracellular polymeric substances (EPS) and surface characteristics of an acidogenic sludge in anaerobic H₂-producing process was investigated. Results show that carbohydrates, proteins, and humic substances were the dominant components in bound EPS (BEPS), while in soluble EPS (SEPS), carbohydrates were the main component. The total content of BEPS initially increased but then kept almost unchanged during fermentation from 25 to 35 h; after that, it slightly decreased. The total content of SEPS increased to 172.5 ± 0.05 mg C g⁻¹ volatile suspended solid with the time that increased to 23.5 h, and then rapidly decreased until 43 h; thereafter, it kept almost unchanged. The SEPS had good correlations with the specific H₂ production rate, substrate degradation rate, and specific aqueous products formation rate, but the BEPS seemed to have no such correlations with these specific rates. Results also confirm that part of EPS could be utilized by the H₂-producing sludge. As the substrate was in short supply, the EPS would be hydrolyzed to sever as carbon and energy source.     

Distribution and composition of extracellular polymeric substances in membrane-aerated biofilm

Extracellular polymeric substances (EPS) are one of the main components of the biofilm and perform important functions in the biofilm system. In this study, two membrane-aerated biofilms (MABs) were developed for the thin and thick biofilms under different surface loading rates (SLRs). Supplies of oxygen and substrates in the MAB were from two opposite directions. This counter diffusion of nutrients resulted in a different growth environment, in contrast to conventional biofilms receiving both oxygen and substrates from the same side. The compositions, distributions and physicochemical properties (solubility and bindability) of EPS in the MABs of different thicknesses under different SLRs were studied. The effect of dissolved oxygen (DO) concentration within the MAB on EPS properties and distribution was investigated. Experimental results showed the different biofilm thicknesses produced substantially different profiles of EPS composition and distribution. Soluble proteins were more dominant than soluble polysaccharides in the inner aerobic layer of the biofilms; in contrast, bound proteins were greater than bound polysaccharides in the outer anoxic or anaerobic layer of the biofilms. The biofilm-EPS matrix consisted mainly of bound EPS. Bound EPS exhibited a hump-shaped profile with the highest content occurring in an intermediate region in the thin MAB and relatively more uniformly in the one half of the biofilm close to the membrane side and then declined towards the biofilm-liquid interface in the thick MAB. The profiles of soluble EPS presented a similar declining trend from the membrane towards the outer region in both thin and thick MABs. The study suggested that not only EPS composition but also EPS distribution and properties (solubility and bindability) played a crucial role in controlling the cohesiveness and maintaining the structural stability and stratification of the MABs.     

Characterisation of the mineral fraction in extracellular polymeric substances (EPS) from activated sludges extracted by eight different methods

This work characterises the mineral fraction of EPS extracts obtained using eight different methods from two activated sludges by total mineral content determination, Fourier Transformed Infrared spectrometry and with scanning electron microscopy coupled with an EDX probe. Despite EPS dialysis, the EPS extracts displayed a mineral fraction between 2% and 40% of the EPS dry weight depending on the extraction method used. The main mineral elements found in the EPS extract were Ca, Mg, Na, K, Al, Fe, Mn, P, Si and S, but their contents were strongly affected by the extraction method used. Some of the minerals are associated with the organic molecules within the EPS. The presence of mineral particles of various compositions and structures (clays, quartz or carbonate) in the EPS extract with a wide range in size was clearly demonstrated. Moreover, the association of metallic elements with the mineral particles in the EPS extract was highlighted.     

Characteristics of extracellular polymeric substances (EPS) fractions from excess sludges and their effects on bioflocculability

Extracellular polymeric substances (EPS) of biological origin are ubiquitous in excess sludges and can be applied as an underlying bioflocculant, owing to their high content of macromolecules and cations. However, low flocculating activity limits the feasibility of their practical applications. This study provides a novel EPS fractionation approach to improve their flocculability by extracting an active EPS fraction and removing the others with low flocculability. The results showed that for two excess sludges (called sludge A and sludge B), the tightly bound EPS (TB-EPS) fraction possessed a high flocculating rate to kaolin suspension compared with the other EPS fractions [i.e., supernatant, slime, and loosely bound EPS (LB-EPS) fraction] (>54.1 ± 1.4% vs <7.8 ± 1.6%). High bioflocculability of TB-EPS fraction could be attributable to high contents of macromolecules (330–1200 kDa) and trivalent cations (Fe³⁺ and Al³⁺). Further investigation reveals that the TB-EPS fraction caused aggregation of particles by bridging and sweep flocculation.     

Influences of extracellular polymeric substances (EPS) on the characteristics of activated sludge under non-steady-state conditions

Laboratory experiments were carried out on activated sludge (AS) to investigate the correlations between the content of extracellular polymeric substances (EPS) and the performance of biosolids–water separation, including sludge flocculation, sedimentation, compression, and dewatering, under non-steady-state conditions. On three stabilized AS reactors changes were made in sludge retention time (SRT), substrate composition, and loading rate, respectively, to bring about unstable operation to the reactors. A two-step heating method was used to extract from the sludge the easily extractable EPS, or loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS), respectively. The experimental results demonstrate dynamic changes in sludge characteristic and EPS production under the non-steady-state conditions. During the early phase of transition after a change was imposed, the sludge became generally worse in flocculation, compressibility, and dewaterability. With the acclimatization of the biomass to the new process conditions, biosolids–water separation showed a general trend of improvement. Changes in AS process condition also resulted in considerable variations in EPS production. The change of the LB-EPS content appeared to be more significant than that of the TB-EPS. Throughout the non-steady-state operation, the sludge flocculating behavior, settleability, compressibility, and dewaterability had a positive correlation with the LB-EPS content; however, no correlation could be found between these properties and the TB-EPS content. The results suggest that although EPS is essential to biofloc formation, excessive EPS in the form of LB-EPS would weaken cell attachment and deteriorate the AS floc structure, resulting in poor biosolids–water separation.     

Impact of sludge retention time on MBR fouling: role of extracellular polymeric substances determined through membrane autopsy

The impact of sludge retention time (SRT) on the biofouling of a membrane bioreactor (MBR) by extracellular polymeric substances (EPS) was investigated. The MBR was operated at 60 and 20 d SRT. The gel layer (recovered through optimized membrane autopsy methods) and the cake layer were analyzed for their content and profile of EPS proteins and polysaccharides. The change to a shorter SRT led to decreased membrane filterability, concomitant with a higher expression of EPS proteins in the cake layer, which were identified as being mainly related with biosynthesis and stress functions. The gel layer was more substantial in internal fibers, with polysaccharides being the major component in this layer. With the decrease in SRT (and filterability decrease), the overall polysaccharide content and sugar variety increased. In conclusion, SRT impacted not only on the quantity but also the composition of EPS molecules, and both were shown to be important in biofouling.     

Potential role of bacterial extracellular polymeric substances as biosorbent material for arsenic bioremediation

Carcinogenic effects of arsenic through consumption of contaminated water are an alarming threat and there is an emergent need to reduce extremely high levels of toxic arsenic from environment. Bacterial biofilms produce polyanionic extracellular polymeric substance (EPS) that is considered an excellent biosorbent material for the remediation of toxic metals and metalloids. This study was aimed to investigate the role of bacterial EPS in arsenic bioremediation. EPS was extracted from biofilm forming and arsenic reducer bacterial strains that were isolated from industrial waste water and characterized biochemically. Fourier transform infrared spectroscopy was also performed to study functional groups. Both Exiguobacterium profundum PT2 and Ochrobactrum ciceri SW1 exhibited enhanced EPS production in the presence of arsenic. Arsenic stress increased protein and carbohydrate contents in the EPS of both bacterial strains as indicated by the peaks of 1363 to 1613 and 1035 to 1218?cm^?1 wavenumbers, respectively to cope with arsenic present in the surroundings. Shifting of peaks in As⁵⁺ treated samples from 1363 to 1379, 847 to 800 and 1211 to 1134?cm^?1 demonstrated the involvement of proteins, carbohydrates and phosphates in the sequestration of arsenic. Scanning electron microscopic examination of EPS revealed structural alterations such as the presence of closely embedded large clumps with interstitial spaces between stacked layers of the EPS of E. profundum PT2 treated with As⁵⁺ displayed the enhanced polysaccharide content and arsenic sorption. Therefore, increased production of bacterial EPS with large number of polyanionic functional groups on its surface having tendency to sequester arsenic through electrostatic or covalent interactions presented EPS an excellent biosorbent material for arsenic bioremediation.     

EPS and SMP dynamics at different heights of a submerged anaerobic membrane bioreactor (SAMBR)

Membrane bioreactors (MBR) technology for wastewater offers many advantages over conventional technologies such as high effluent quality, less footprint and others. The main disadvantage of membrane bioreactors (MBR) is related to membrane fouling, which is mainly caused by extracellular polymeric substance (EPS) and soluble microbial products (SMP). This research studied EPS and SMP dynamics at different heights of a submerged anaerobic membrane bioreactor (SAMBR). The SAMBR was operated under two organic loading rates (OLR) (0.79 and 1.56 kg/m³ d) and was fed with synthetic wastewater with glucose as the carbon source. The results showed percentages of chemical oxygen demand (COD) removal above 95% and the highest COD removal rates were observed at the bottom of the reactor (>83%) for both OLR. The EPS showed a stratification with highest quantities in the supernatant. For the SMP the highest concentration was in the bottom of SAMBR where utilization predominated associated products whereas in the SAMBR supernatant predominated biomass associated products. The OLR change led to a significant increase in SMP accumulation but not in EPS. These facts showed that EPS and SMP dynamic in the SAMBR seemed to be mainly influenced by biological activity, total suspended solids concentration and substrate composition.     

Proteins dominate in the surface layers formed on materials exposed to extracellular polymeric substances from bacterial cultures

The chemical compositions of the surface conditioning layers formed by different types of solutions (from isolated EPS to whole culture media), involving different bacterial strains relevant for biocorrosion were compared, as they may influence the initial step in biofilm formation. Different substrata (polystyrene, glass, steel) were conditioned and analyzed by X-ray photoelectron spectroscopy. Peak decomposition and assignment were validated by correlations between independent spectral data and the ubiquitous presence of organic contaminants on inorganic substrata was taken into account. Proteins or peptides were found to be a major constituent of all conditioning layers and polysaccharides were not present in appreciable concentrations; the proportion of nitrogen which may be due to DNA was lower than 15%. There was no significant difference between the compositions of the adlayers formed from different conditioning solutions, except for the adlayers produced with tightly bound EPS extracted from D. alaskensis.