Electron microscopic examination of the extracellular polymeric substances in anaerobic granular biofilms

Scanning electron microscopy revealed that collapsed extracellular polymeric substances (EPS) surrounded bacteria present in granular sludge. Treatment of granular sludge with whole-cell antiserum and staining with polycationic ferritin demonstrated that bacteria were enveloped by extensive EPS. Antibody stabilization permitted a visualization of the EPS which more closely resembled its natural hydrated state. The EPS was seen to completely fill the intercellular spaces in the microcolonies. Both pure and mixed microcolonies were observed to be enclosed by EPS. The presence of these large amounts of EPS indicates that this extracellular layer is important in maintaining the structural integrity of granular sludge.     

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.     

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.     

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.     

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.     

Enhanced extraction of extracellular polymeric substances from biofilms by alternating current

The extraction of extracellular polymeric substances (EPS) including polysaccharides and proteins from aerobic biofilms using either EDTA or NaOH was enhanced by alternating current especially within the first 0.5 h of current application. At 60 mA and 50 Hz, the average extraction rate of polysaccharides within the first 0.5 h using EDTA was 8.3 mg h^–1 g TS^–1 (TS: total solid, dry wt), 1.6 times of that without current, and that of proteins was 2.5 mg h^–1 g TS^–1, 1.2 times higher. The extraction of polysaccharides was maximal at around 500 Hz while that of proteins was less affected by increasing the current frequency.     

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.     

Insights into the relationship between colony formation and extracellular polymeric substances (EPS) composition of the cyanobacterium Microcystis spp

Extracellular polymeric substances (EPS) were considered as fundamental substances in colony formation; however, the understanding of EPS composition remains limited. This study analyzed the content and composition of EPS fractions (soluble EPS, loosely bound EPS, and tightly bound EPS) of four Microcystis species from laboratory cultures in both unicellular and colonial morphologies, as well as colonies collected during Microcystis blooms, using fluorescence excitation - emission matrix spectroscopy combined with parallel factor analysis (EEM-PARAFAC). This method enables to make insight into protein-like and humic acid-like components but cannot detect polysaccharides. The EPS was successfully categorized into three humic acid-like components (C1 – C3) and a protein-like component (C4). Component C1 was discovered to be involved in colony formation and colony size growth of Microcystis. EPS content varied among Microcystis morphospecies, such as M. aeruginosa, M. wesenbergii and M. ichthyoblabe, and this was significantly affected by the environmental constraints rather than the morphospecies. The proportion of C1 relating to larger colony size was negatively correlated to temperature and concentrations of TN and TP. The tightly bound EPS directly promoted colony formation, but the soluble EPS or loosely bound EPS alone did not induce colony formation in Microcystis. These results advanced the current knowledge on the chemical materials involved in the colony formation of Microcystis and provided new clues in unicellular-multicellular transformation as well as colonial morphology changes in Microcystis.     

Contributions of functional groups and extracellular polymeric substances on the biosorption of dyes by aerobic granules

The contributions of loosely bound extracellular polymeric substances (LB-EPS), tightly bound EPS (TB-EPS), residual sludge (the sludge left after EPS extraction) and functional groups such as amine, carboxyl, phosphate and lipid on aerobic granules on biosorption of four different dyes (Reactive Brilliant Blue KN-R (KN-R), Congo Red (CR), Reactive Brilliant Red K-2G (RBR) and Malachite Green (MG)) were investigated. EPS may be responsible for biosorption of cationic dyes. However, residual sludge always made greater contribution than that of EPS. The biosorption mechanisms were dependent on the functional groups on aerobic granules and dyes’ chemical structures. The lipid and phosphate groups might be the main binding sites for KN-R biosorption. Amine, carboxyl, phosphate and lipid were all responsible for the binding of CR. The lipid fractions played an important role for RBR biosorption. For MG, the phosphate groups gave the largest contribution.     

Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans

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

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.     

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.     

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.     

Removal of microbial multi-species biofilms from the paper industry by enzymatic treatments

This study aimed to characterize biofilms from the paper industry and evaluate the effectiveness of enzymatic treatments in reducing them. The extracellular polymeric substances (EPS) extracted from six industrial biofilms were studied. EPS were mainly proteins, the protein to polysaccharide ratio ranging from 1.3 to 8.6 depending on where the sampling point was situated in the paper making process. Eight hydrolytic enzymes were screened on a 24-h multi-species biofilm. The enzymes were tested at various concentrations and contact durations. Glycosidases and lipases were inefficient or only slightly efficient for biofilm reduction, while proteases were more efficient: after treatment for 24 h with pepsin, Alcalase® or Savinase®, the removal exceeded 80%. Savinase® appeared to be the most adequate for industrial conditions and was tested on an industrial biofilm sample. This enzyme led to a significant release of proteins from the EPS matrix, indicating its potential efficiency on an industrial scale.     

Metal binding capabilities of Rhizobium etli and its extracellular polymeric substances

Extracellular polymeric substances (EPS) produced from a strain of Rhizobium etli demonstrated an ability to bind a variety of metals. Cells and capsular EPS rapidly bound Mn²⁺ ions preferentially to Pb²⁺ and Cu²⁺, but also showed an affinity for Pb²⁺. The binding capabilities of soluble EPS were affected by its extraction and processing. The results suggest potential applications in the field of bioremediation.     

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.     

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。     

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.     

Composition of extracellular polymeric substances influences the autoaggregation capability of hydrogen-producing bacterium Ethanoligenens harbinense

Non-spore-forming Ethanoligenens, a novel genus of hydrogen-producing bacteria, is endowed with great application potential in biohydrogen production due to acidophilic and autoaggregating growth. In order to elucidate the mechanism of autoaggregation of Ethanoligenens harbinense, extracellular polymeric substances (EPS) from YUAN-3 had been extracted and analyzed. The EPS was mainly produced during the exponential phase and with protein, carbohydrate and DNA as its main components, with yields of 21.0 ± 0.8 mg/g-cell dry weight (CDW), 16.9 ± 0.8 mg/g-CDW and 3.5 ± 0.5 mg/g-CDW, respectively. Compared with the EPS composition of semi-autoaggregating hydrogen-producing bacteria W1 and non-autoaggregating hydrogen-producing bacteria B49, carbohydrate and protein played an important part in the autoaggregation of YUAN-3.     

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.