Extraction of extracellular polymeric substances (EPS) of sludges

The efficacies of extracting extracellular polymeric substances (EPS) from aerobic, acidogenic and methanogenic sludges using EDTA, cation exchange resin and formaldehyde under various conditions were compared. Results show that formaldehye plus NaOH was most effective in extracting EPS for all sludges; only 1.1-1.2% of DNA in the sludge samples were detected, suggesting the EPS extracted were not contaminated by intracellular substances. For each gram of volatile solids, formaldehyde-NaOH extracted 165, 179 and 102 mg of EPS from aerobic, acidogenic and methanogenic sludges, respectively. All EPS were mainly composed of carbohydrate, protein and humic substance, plus small quantities of uronic acid and DNA. Carbohydrate was predominant in the acidogenic sludge (62% in the EPS extracted by formaldehyde-NaOH), whereas protein was predominant in the methanogenic sludge (41%). Humic substance, which has often been overlooked, accounted for 30.6, 8.4 and 22.8% of the extracted EPS from aerobic, acidogenic and methanogenic sludges, respectively. However, judging from EPS quantities estimated from confocal laser scanning microscopic observations, formaldehyde-NaOH extracted only a limited portion of EPS. Optimization of extraction procedures and/or development of a more effective extraction method are warranted.     

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.     

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.     

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.     

Effect of extracellular polymeric substances (EPS) on the attachment of activated sludge

Extracellular polymeric substances (EPS) were removed by mechanical (high-speed centrifugation) and chemical (EDTA treatment) methods. The number of attached microorganisms decreased from 33.0 2 10⁷ CFU/cm² to 17.5 2 10⁷ CFU/cm² and 12.5 2 10⁷ CFU/cm², respectively. When the activated sludge was treated with polysaccharide-oxidizing agent (sodium meta periodate) and protease (pronase E), the number of attached microorganisms decreased to 41% and 43.5%, respectively. Transmission electron microphotographs showed that polysaccharide-oxidizing agent and protease treatment caused the removal of filamentous extracellular structures.     

Stability of sludge flocs under shear conditions: roles of extracellular polymeric substances (EPS)

The roles of extracellular polymer substances (EPS) in the shear stability of aerobic and anaerobic flocs were investigated. Both pH and EDTA concentration had a significant effect on the floc stability. The sludge flocs became much weaker as the solution pH increase to above 10. Addition of 1 mM EDTA or more could cause considerable cell erosion and deflocculation of the anaerobic flocs, whereas more than 3 mM EDTA was needed to show its adverse effect on the stability of aerobic flocs. A fraction of the EPS, around 10 mg/g SS for the aerobic flocs and 15 mg/g SS for the anaerobic flocs, could be extracted by fluid shear when the dispersed mass concentration approached the equilibrium. This suggests that most of the dispersed particles were glued by a small amount of readily-extractable EPS fraction. In addition to the abundance of this EPS fraction, its proteins/carbohydrates ratio, about 0.22:1 for the aerobic flocs and 2.66:1 for the anaerobic flocs, also appeared to be an important factor governing the microbial floc stability. A lower content of the readily-extractable EPS fraction and a lower ratio of proteins/carbohydrates were responsible for the greater stability of microbial flocs. The total content of the EPS, however, did not show a direct correlation with the floc stability. A hypothesis about biological flocs with two distinct structural regions was proposed. The outer part contained dispersible cells loosely entangled by the readily-extractable EPS fraction. This part was layered and would become completely dispersed at an infinite shear intensity. On the other hand, the inner part contains biomass in a stable structure tightly glued by EPS, which could not be dispersed by shear except under unfavorable conditions.     

Contribution characteristics of the in situ extracellular polymeric substances (EPS) in Phanerochaete chrysosporium to Pb immobilization

Bioprocess and Biosystems Engineering - White rot fungi have been extensively reported to have strong adsorption capacity to heavy metal ions, whereas the knowledge of extracellular polymeric...     

Effect of extracellular polymeric substances (EPS) on Pb2+ accumulation by Aureobasidium pullulans

In Pb²⁺ accumulation by Aureobasidium pullulans, the time to reach an equilibrium state was not dependent on the initial cell dry weight. The Pb²⁺ accumulation capacity was increased from 56.9 to 215.6 mg Pb²⁺/g cell dry weight as the biomass was stored from 1 to 53 days, and correlated with the amount of excreted extracellular polymeric substances (EPS). It was observed that Pb²⁺ accumulated only on the surface of the intact cells of A. pullulans due to the existence of EPS, whereas Pb²⁺ penetrated into the inner cellular parts of the EPS-extracted cells.     

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.     

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.     

Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols

The characteristics of the extracellular polymeric substances (EPS) extracted with nine different extraction protocols from four different types of anaerobic granular sludge were studied. The efficiency of four physical (sonication, heating, cationic exchange resin (CER), and CER associated with sonication) and four chemical (ethylenediaminetetraacetic acid, ethanol, formaldehyde combined with heating, or NaOH) EPS extraction methods was compared to a control extraction protocols (i.e., centrifugation). The nucleic acid content and the protein/polysaccharide ratio of the EPS extracted show that the extraction does not induce abnormal cellular lysis. Chemical extraction protocols give the highest EPS extraction yields (calculated by the mass ratio between sludges and EPS dry weight (DW)). Infrared analyses as well as an extraction yield over 100% or organic carbon content over 1 g g⁻¹ of DW revealed, nevertheless, a carry-over of the chemical extractants into the EPS extracts. The EPS of the anaerobic granular sludges investigated are predominantly composed of humic-like substances, proteins, and polysaccharides. The EPS content in each biochemical compound varies depending on the sludge type and extraction technique used. Some extraction techniques lead to a slightly preferential extraction of some EPS compounds, e.g., CER gives a higher protein yield.     

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.     

Sorption of Cu(II) and Cd(II) by extracellular polymeric substances (EPS) from Aspergillus fumigatus

Sorption of Cu(II) and Cd(II) onto the extracellular polymeric substances (EPS) produced by Aspergillus fumigatus was investigated for the initial pH of the solution, EPS concentrations, contact time, NaCl concentration, initial metal ion concentration and the presence of other ions in the solution. The results showed that the adsorption of metal ions was significantly affected by pH, EPS concentrations, initial metal concentration, NaCl concentration and co-ions. The sorption of Cu(II) and Cd(II) increased with increasing pH and initial metal ion concentration but decreased with an increase in the NaCl concentration. The maximum sorption capacities of A. fumigatus EPS calculated from the Langmuir model were 40 mg g⁻¹ EPS and 85.5 mg g⁻¹ EPS for Cu(II) and Cd(II), respectively. The binary metal sorption experiments showed a selective metal binding affinity in the order of Cu(II) > Pb(II) > Cd(II). Both the Freundlich and Langmuir adsorption models described the sorption of Cu(II) and Cd(II) by the EPS of A. fumigatus adequately. Fourier transform infrared spectroscopy (FTIR) analysis revealed that carboxyl, amide and hydroxyl functional groups were mainly correlated with the sorption of Cu(II) and Cd(II). Energy dispersive X-ray (EDX) system analysis revealed that the ion-exchange was an important mechanism involved in the Cu(II) and Cd(II) sorption process taking place on EPS.     

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.     

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.     

Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods

The efficiency of eight extracellular polymeric substances (EPS) extraction methods was compared on two different activated sludges. Three chemical methods (EDTA, formaldehyde + NaOH, glutaraldehyde), four physical methods (sonication, cation exchange resin, sonication + cation exchange resin, heating) and a control method (centrifugation alone) were tested.EPS quantities extracted were more greater for chemical methods than those for physical methods. For the chemical methods used EPS contamination due to extracting reagents was pointed out by infra-red analysis. The EPS extracted by physical methods can show a different qualitative composition with protein and carbohydrate as predominant compounds. This study therefore underlines that the choice of EPS extraction method should not only be limited to extraction yield and nucleic acid content but should also consider that the EPS solution may be contaminated by extracting reagents and/or be greatly modified by the extraction protocol.     

Influence of Zn(II) stress-induction on component variation and sorption performance of extracellular polymeric substances (EPS) from Bacillus vallismortis

Bioprocess and Biosystems Engineering - Bacillus vallismortis (B. vallismortis), an aerobic heterotrophic bacteria, was screened in a laboratory pilot study, to assess the interaction between the...     

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.     

Role of extracellular polymeric substance (EPS) in toxicity response of soil bacteria Bacillus sp. S3 to multiple heavy metals

Heavy metal resistant bacteria are of great interest because of their potential use in bioremediation. Understanding the survival and adaptive strategies of these bacteria under heavy metal stress is important for better utilization of these bacteria in remediation. The objective of this study was to investigate the role of bacterial extracellular polymeric substance (EPS) in detoxifying against different heavy metals in Bacillus sp. S3, a new hyper antimony-oxidizing bacterium previously isolated from contaminated mine soils. The results showed that Bacillus sp. S3 is a multi-metal resistant bacterial strain, especially to Sb(III), Cu(II) and Cr(VI). Toxic Cd(II), Cr(VI) and Cu(II) could stimulate the secretion of EPS in Bacillus sp. S3, significantly enhancing the adsorption and detoxification capacity of heavy metals. Both Fourier transform infrared spectroscopy (FTIR) and three-dimensional excitation–emission matrix (3D-EEM) analysis further confirmed that proteins were the main compounds of EPS for metal binding. In contrast, the EPS production was not induced under Sb(III) stress. Furthermore, the TEM–EDX micrograph showed that Bacillus sp. S3 strain preferentially transported the Sb(III) to the inside of the cell rather than adsorbed it on the extracellular surface, indicating intracellular detoxification rather than extracellular EPS precipitation played an important role in microbial resistance towards Sb(III). Together, our study suggests that the toxicity response of EPS to heavy metals is associated with difference in EPS properties, metal types and corresponding environmental conditions, which is likely to contribute to microbial-mediated remediation.