Extracellular Polymeric Substances Extraction Research Articles

Composition of EPS fractions from suspended sludge and biofilm and their roles in microbial cell aggregation

The adhesion and aggregation properties of microbial cell are closely related to extracellular polymeric substances (EPS). In this work, the composition and physicochemical characteristics of EPS in biofilm and suspended sludge (S-sludge) were determined to evaluate their roles in microbial cell aggregation. Raman spectroscopy and three-dimensional fluorescence spectra have been employed to reveal each EPS fraction in different composition. The flocculating capacity of each EPS fraction in the S-sludge shows extraordinary activity, comparing its counterpart in biofilm. Microbial cell surfaces present high hydrophobicity and increased zeta potentials upon EPS extraction. In addition, the respective contribution of EPS to cell aggregating was elucidated. The contribution of combined SEPS and LB-EPS was 23% for S-sludge sample, whereas that was negligible for biofilm sample. The contribution of LB-EPS and TB-EPS were 16% and 30% for S-sludge sample, and −6% and negligible for biofilm sample, respectively. Therefore, EPS promoted the S-sludge cells to aggregate, while in contrast, they showed a negligible or negative effect on the biofilm cells aggregating.

Cross-Flow Microfiltration of Bacillus Subtilis Broths under Various Culture Times

Bacillus subtilis broths under different culture times are filtered in a cross-flow microfilter. The operating condition effects, such as cross-flow velocity, transmembrane pressure, and broth culture time, on the filtration flux, cake properties, and extracellular polymeric substances (EPS) transmissions are discussed thoroughly. The culture broths contain B. subtilis cells and EPS which is characterized as polysaccharides (hydrocarbons) and proteins. An increase in broth culture time leads to higher concentrations of cells, soluble and extractable EPS. The total protein to polysaccharide concentration ratio in the broths is ca 0.2. However, the soluble polysaccharide concentration is 10-fold higher than that of soluble proteins. The filtration flux increases with increasing cross-flow velocity or transmembrane pressure. However, the impact of cross-flow velocity is more significant. The filter cake resistance formed by B. subtilis cells and EPS flocs plays the most important role in determining the overall filtration resistance. The mass and average specific filtration resistance of cake can be estimated using a force balance model and empirical equations. The cake structure and thickness are analyzed using SEM. A thicker and more compact cake may be formed under longer broth culture time. Most soluble polysaccharide and protein molecules have the opportunity to penetrate through the cake and membrane into the filtrate because the solute transmissions are measured as high as 0.75–1.0. The influences of operating conditions on the polysaccharide and protein transmissions are negligible. Therefore, to enhance filtration flux by increasing transmembrane pressure or cross-flow velocity is beneficial to improve separation efficiency, especially by increasing cross-flow velocity.

Measuring the soil-microbial interface: Extraction of extracellular polymeric substances (EPS) from soil biofilms

Many soil microbes exist in biofilms. These biofilms are typified by variable quantities of extracellular polymeric substances (EPS: predominantly polysaccharides, glycoconjugates, and proteins) and the embedded microbial cells. A method to measure soil-EPS (the biofilm exclusive of microbial cells) has not yet been described. The present work investigates the potential of five extraction methods to estimate changes in soil-EPS content. A rationale for selection of appropriate EPS extraction and methodology is discussed, including the crucial consideration of both intracellular and extracellular contamination.EPS was developed in situ by provision of labile C (glycerol) to the microbial biomass of a moist soil and then applying desiccation stress. Only two out of the five extraction methods showed statistically significant increases in polysaccharide production responding to substrate addition. Humified organic matter, estimated by its humic acid equivalent (HAE) was used to indicate the degree of extracellular contamination, and/or creation of humic artefacts – both of which affect detection of changes in EPS. The HAE concentration was very high when applying original and modified methods designed to extract glomalin related soil protein (GRSP). Extraction methods involving heating with dilute sulphuric acid appeared to overestimate EPS-polysaccharide. Using microbial ATP as an indicator of cell-lysis, confidence could only be ascribed to EPS extraction with cation exchange resin. Using this method, the expected increases in EPS-polysaccharide were clearly apparent. The HAE/protein ratios of EPS extracts were also lowest with cation exchange – indicating this method did not cause excessive contamination from humified soil organic matter or create related artefacts.

Fate of extracellular polymeric substances during flocculation of anaerobically digested sludge using biogenic flocculant

This study investigated the fate of extracellular polymeric substances (EPS) of anaerobically digested saline sewage sludge during its conditioning with a biogenic flocculant produced by Acidithiobacillus ferrooxidans culture before dewatering. Sludge was conditioned for 24h with the biogenic flocculant in presence and absence of Fe2+ as energy substrate. During the sludge conditioning process, pH dropped from 7.24 to 3.12. The capillary suction time of sludge was <10s and specific resistance to filtration (SRF) was reduced by more than 94% of its original SRF in the biogenic flocculent treatments within 4h of incubation with or without FeSO4 indicating a significant (P<0.001) improvement in sludge dewaterability. A significant decrease in extractable EPS was observed in treatments with biogenic flocculant and the EPS contents showed a significant negative correlation with dewaterability of sludge (P<0.05). The results suggest that the biogenic flocculant effectively improved sludge dewaterability through modification of sludge EPS.

Critical assessment of extracellular polymeric substances extraction methods from mixed culture biomass

Extracellular polymeric substances (EPS) have a presumed determinant role in the structure, architecture, strength, filterability, and settling behaviour of microbial solids in biological wastewater treatment processes. Consequently, numerous EPS extraction protocols have recently been published that aim to optimize the trade off between high EPS recovery and low cell lysis. Despite extensive efforts, the obtained results are often contradictory, even when analysing similar biomass samples and using similar experimental conditions, which greatly complicates the selection of an extraction protocol. This study presents a rigorous and critical assessment of existing physical and chemical EPS extraction methods applied to mixed-culture biomass samples (nitrifying, nitritation-anammox, and activated sludge biomass). A novel fluorescence-based method was developed and calibrated to quantify the lysis potential of different EPS extraction protocols. We concluded that commonly used methods to assess cell lysis (DNA concentrations or G6PDH activities in EPS extracts) do not correlate with cell viability. Furthermore, we discovered that the presence of certain chemicals in EPS extracts results in severe underestimation of protein and carbohydrate concentrations by using standard analytical methods. Keeping both maximum EPS extraction yields and minimal biomass lysis as criteria, it was identified a sonication-based extraction method as the best to determine and compare tightly-bound EPS fractions in different biomass samples. Protein was consistently the main EPS component in all analysed samples. However, EPS from nitrifying enrichments was richer in DNA, the activated sludge EPS had a higher content in humic acids and carbohydrates, and the nitritation-anammox EPS, while similar in composition to the nitrifier EPS, had a lower fraction of hydrophobic biopolymers. In general, the easily-extractable EPS fraction was more abundant in carbohydrates and humic substances, while DNA could only be found in tightly bound EPS fractions. In conclusion, the methodology presented herein supports the rational selection of analytical tools and EPS extraction protocols in further EPS characterization studies.

Influence of extracellular polymeric substances on rheological properties of activated sludge

Influence of extracellular polymeric substances (EPS) on rheological properties of activated sludge (AS) was conducted by comparing properties of AS before and after EPS extraction at a TSS of 54g/L. Slime, loosely and tightly bound EPS (LB- and TB-EPS) were stratified by centrifugation and ultrasound method. The results showed that sludge after LB-EPS extraction produced a higher hysteresis loop area (Hla), limiting viscosity (η∞), yield stress (τy), energy of cohesion of network sludge (Ec), and storage modulus (G′0), than three other sludge samples, indicating that sludge with TB-EPS exhibited stronger network structure. Strain amplitude sweep (SAS) and frequency sweep (FS) tests revealed that AS before and after EPS extraction produced a gel-like structure in the linear viscoelastic (LVE) domain. TB-EPS appeared to have a positive effect on the gel-like structure of AS.

Effect of short-time aerobic digestion on bioflocculation of extracellular polymeric substances from waste activated sludge

The effect of short-time aerobic digestion on bioflocculation of extracellular polymeric substances (EPSs) from waste activated sludge (WAS) was investigated. Bioflocculation of the EPS was found to be enhanced by 2∼6 h of WAS aerobic digestion under the conditions of natural sludge pH (about 7), high sludge concentration by gravity thickening, and dissolved oxygen of about 2 mg/L. With the same EPS extraction method, the total suspended solid content reduction of 0.20 and 0.36 g/L and the volatile suspended solid content reduction of 0.19 and 0.26 g/L were found for the WAS samples before and after aerobic digestion of 4 h. It indicates that more EPS is produced by short-time aerobic digestion of WAS. The scanning electron microscopy images of the WAS samples before and after aerobic digestion of 4 h showed that more EPS appeared on the surface of zoogloea by aerobic digestion, which reconfirmed that WAS aerobic digestion induced abundant formation of EPS. By WAS aerobic digestion, the flocculating rate of the EPS showed about 31 % growth, almost consistent with the growth of its yield (about 34 %). The EPSs obtained before and after the aerobic digestion presented nearly the same components, structures, and Fourier transform infrared spectra. These results revealed that short-time aerobic digestion of WAS enhanced the flocculation of the EPS by promoting its production.

Effects of Microwave Irradiation on Dewaterability and Extracellular Polymeric Substances of Waste Activated Sludge

The effects of microwave irradiation on filterability and dewaterability of waste activated sludge measured by capillary suction time (CST) and dry solids in sludge cake were investigated. The results showed that the optimum irradiation time improved filterability, but that further increase of the time was detrimental. Dewaterability was enhanced significantly and increased with microwave time. Filterability and dewaterability were improved 25 to 28% and 1.3 times at the optimum times of 30 and 90 seconds for the sludge of 5 g total suspended solids (TSS)/L and 7 g TSS/L, respectively. The floc size decreased slightly. Loosely bound extracellular polymeric substances (LB-EPS) decreased under optimum time, but tightly bound extracellular polymeric substances did not change significantly after short irradiation time. The results implied that LB-EPS played a more important role in the observed changes of filterability and dewaterability and that the double-layered extracellular polymeric substances extraction method showed marked implications to dewaterability.

Effect of sludge properties on the filtration characteristics of self-forming dynamic membranes (SFDMs) in aerobic bioreactors: Formation time, filtration resistance, and fouling propensity

This study attempts to elucidate the effect of sludge properties on the formation time, filtration resistance, and fouling propensity of self-forming dynamic membranes (SFDMs), developed on coarse-pore filters in aerobic wastewater treatment bioreactors. Short-term filtration experiments were performed with twenty aerobic sludge samples having twelve sludge properties characterized. Statistical analysis showed that mean particle diameter (Dvm) associated with particle size distribution strongly affected both formation time (rp=−0.803, p=0.000) and filtration resistance (rp=−0.733, p=0.000) of SFDMs, whereas relative hydrophobicity (RH) exhibited a moderate correlation with SFDM formation time (rp=−0.487, p=0.029). In contrast, four other different sludge properties, i.e. viscosity (rp=0.811, p=0.000), extractable extracellular polymeric substances (eEPS) (rp=0.723, p=0.000), the carbohydrate content of eEPS (rp=0.766, p=0.000), and specific oxygen uptake rate (SOUR) (rp=−0.610, p=0.004), were identified as having significant impact on the fouling propensity of fresh SFDMs. Besides statistical analysis, more detailed experimental evidences were obtained by directly examining the identified important properties of fresh and initially fouled SFDMs. Sludge deposition during SFDM formation appears to be dominated by permeate drag force and hydrophobic interaction, whereas the subsequent fouling rate turns out to be controlled by the content of eEPS, particularly carbohydrates, which significantly correlated with viscosity and SOUR.

Influence of extraction method on size exclusion chromatography fingerprints of EPS from wastewater sludges

Extracellular polymeric substances (EPS) were separated using two serial-linked size exclusion chromatography (SEC) columns to obtain detailed fingerprints. The chromatographic profile results were influenced by the nature of biological sludge (activated sludges, anaerobic granules, anaerobic flocculated sludges). Furthermore, our results highlight that EPS fingerprints are also highly dependent on the extraction method. If physical extractions modify only the relative absorbance of the chromatographic peaks, heating during extraction induces significant modifications of the fingerprints, probably owing to better organic matter extraction efficiency as well as an increase in hydrolysis for some compounds but not for EPS extracted from anaerobic granular sludges. This confirms that thermal treatment is a proper method to extract EPS from anaerobic granular sludges. The use of chemical extraction results in major changes on the EPS fingerprints. This work demonstrates that some chromatographic peaks are due to residues from the chemical reagent (such as EDTA, glutaraldehyde) which can modify or form complexes with some EPS macromolecules. As a result, due to its sensitivity to sludge origin and/or extraction procedure, SEC appears to be a suitable tool for an accurate qualitative EPS characterization.

Effects of solution conditions on the physicochemical properties of stratification components of extracellular polymeric substances in anaerobic digested sludge

The composition and effects of solution conditions on the physicochemical properties of the stratification components of extracellular polymeric substances (EPS) in anaerobic digested sludge were determined. The total EPS in anaerobic digested sludge were extracted by the cation exchange resin method. Another EPS extraction method, the centrifugation and sonication technique was employed to stratify the EPS into three fractions: slime, loosely bound (LB)-EPS, and tightly bound (TB)-EPS from the outside to the inside of the anaerobic digested sludge. Proteins and polysaccharides were dispersed uniformly across the different EPS fractions, and humic-like substances were mainly partitioned in the slime, with TB-EPS second. Protein was the major constituent of the LB-EPS and TB-EPS, and the corresponding ratios ranged from 54.0% to 65.6%. The hydrophobic part in the EPS chemical components was primarily comprised of protein and DNA, while the hydrophilic part was mainly composed of polysaccharide. In the slime, the hydrophobic values of several EPS chemical components (protein, polysaccharide, humic-like substances and DNA) were all below 50%. The protein/polysaccharide ratio had a significant influence on the Zeta potentials and isoelectric point values of the EPS: the greater the protein/polysaccharide ratio of the EPS was, the greater the Zeta potential and the higher the isoelectric point value were. All Zeta potentials of the EPS showed a decreasing trend with increasing pH. The corresponding isoelectric point values (pH) were 2.8 for total EPS, 2.2 for slime, 2.7 for LB-EPS, and 2.6 for TB-EPS. As the ionic strength increased, the Zeta potentials sharply increased and then gradually became constant without charge reversal. In addition, as the temperature increased (< 40°C), the apparent viscosity of the EPS decreased monotonically and then gradually became stable between 40 and 60°C.

Metal binding properties of extracellular polymeric substances extracted from anaerobic granular sludges

Extracellular polymeric substances (EPS) were extracted from four anaerobic granular sludges with different procedures to study their involvement in biosorption of metallic elements. EPS extracts are composed of closely associated organic and mineral fractions. The EPS macromolecules (proteins, polysaccharides, humic-like substances, nucleic, and uronic acids) have functional groups potentially available for the binding of metallic elements. The acidic constants of these ionizable groups are: pKa1 (4-5) corresponding to the carboxyl groups; pKa2 (6-7) corresponding to the phosphoric groups; pKa3 (8-10) and pKa4 (≈10) corresponding to the phenolic, hydroxyl, and amino groups. The polarographic study confirms the higher affinity of the EPS to bind to lead than to cadmium. Moreover, the binding of these metallic compounds with the EPS is a mix of several sorption mechanisms including surface complexation, ion exchange, and flocculation. Inorganic elements were found as ions linked to organic molecules or as solid particles. The mineral fraction affects the binding properties of the EPS, as the presence of salts decreases the EPS binding ability. Calcite and apatite particles observed on SEM images of EPS extracts can also sorb metallic elements through ion exchange or surface complexation.

Controlled activity of mouse astrocytes on electrospun PCL nanofiber containing polysaccharides from brown seaweed

The central nervous system (CNS), once injured, rarely recovers original function mainly due to its limited regeneration ability. Astrocytes are cells that play critical roles in neural regeneration. Several biomaterials have been studied to replace and regenerate lost tissues within injured CNS. Seaweeds have extracellular polymeric substances (EPS) with bioactive properties such as antiviral and antioxidant properties. In this study, astrocyte activity was assessed, after being cultured on an electrospun polycaprolactone (PCL) nanofibrous mat containing a brown seaweed EPS. Laminarin and fucoidan, two main components of EPS extract from the brown seaweed, were concluded to increase or decrease astrocyte activity with respect to their concentration. When the concentration was under 10 μg/ml, the astrocytes tended to increase their viability. In contrast, over 10 μg/ml EPS in media suppressed the viability of astrocytes. In addition, when contained in PCL nanofiber, the EPS extract was also proven to influence astrocyte activity in the same way as the case when astrocytes were exposed to EPS in solution. This implies that the brown seaweed EPS-PCL nanofiber mat can be used for temporal control of astrocyte activity by EPS concentration. Through this research, we propose that the electrospun EPS-PCL nanofiber could be used as a nanomedicine or scaffold to treat CNS injuries.

A novel integrated approach to quantitatively evaluate the efficiency of extracellular polymeric substances (EPS) extraction process

A novel integrated approach is developed to quantitatively evaluate the extracellular polymeric substances (EPS) extraction efficiency after taking into account EPS yield, EPS damage, and cell lysis. This approach incorporates grey relational analysis and fuzzy logic analysis, in which the evaluation procedure is established on the basis of grey relational coefficients generation, membership functions construction, and fuzzy rules description. The flocculation activity and DNA content of EPS are chosen as the two evaluation responses. To verify the feasibility and effectiveness of this integrated approach, EPS from Bacillus megaterium TF10 are extracted using five different extraction methods, and their extraction efficiencies are evaluated as one real case study. Based on the evaluation results, the maximal extraction grades and corresponding optimal extraction times of the five extraction methods are ordered as EDTA, 10 h > formaldehyde + NaOH, 60 min > heating, 120 min > ultrasonication, 30 min > H₂SO₄, 30 min > control. The proposed approach here offers an effective tool to select appropriate EPS extraction methods and determine the optimal extraction conditions.

Characterization of extracellular polymeric substances (EPS) from periphyton using liquid chromatography-organic carbon detection–organic nitrogen detection (LC-OCD-OND)

A protocol was developed to extract, fractionate, and quantitatively analyze periphyton extracellular polymeric substances (EPS), which obtains both information on the molecular weight (M r) distribution and protein and polysaccharide content. The EPS were extracted from freshwater periphyton between July and December 2011. Organic carbon (OC) compounds from different EPS extracts were analyzed using liquid chromatography-organic carbon detection–organic nitrogen detection (LC-OCD-OND), and total protein and polysaccharide content were quantified. Four distinct OC fractions, on the basis of M r, were identified in all extracts, corresponding to high M r biopolymers (≥80–4 kDa), degradation products of humic substances (M r not available), low M r acids (10–0.7 kDa), and small amphiphilic/neutral compounds (3–0.5 kDa). Low C/N ratios (4.3 ± 0.8) were calculated for the biopolymer fractions, which represented 16–38 % of the measured dissolved organic carbon (DOC), indicating a significant presence of high M r proteins in the EPS. Protein and polysaccharide represented the two major components of EPS and, when combined, accounted for the measured DOC in extracts. Differences in specific OC fractions of EPS extracts over the course of the study could be quantified using this method. This study suggests that LC-OCD-OND is a new valuable tool in EPS characterization of periphyton.Electronic supplementary materialThe online version of this article (doi:10.1007/s11356-012-1228-y) contains supplementary material, which is available to authorized users.

Comparison of extracellular polymeric substances (EPS) extraction from two different activated sludges

The characteristics of extracellular polymeric substances (EPS) extracted with five different extraction protocols from two different activated sludges were studied. The results showed that the major EPS constituent extracted by centrifugation was protein for the sludge in sequencing batch reactor treating chemical wastewater, and nucleic acid for the sludge in moving bed biofilm reactor treating synthetic urban wastewater. The order of EPS extraction amounting from the two sludges was formaldehyde + NaOH > formaldehyde + heating > EDTA > heating > centrifugation. The different extraction methods, the wastewater type, and activated sludge source greatly affected the amount and composition of EPS. The chemical extracted methods were more effective than the physical methods in extracting EPS for the two sludges. Moreover, formaldehyde combined NaOH was most effective in extracting EPS for the two sludges. However, chemical extraction could contaminate the EPS solution, which was pointed out by infra-red analysis and was also proved by cell lyses during EPS extraction and carrying over of the chemical extractant. Therefore, this study highlights that the choice of EPS extraction method should consider both the extraction yield and content and the contamination of extracting reagents to the EPS solution. The extraction procedures should be optimized and most effective.

Effects of extracellular polymeric substances on granulation of anoxic sludge in sequencing batch reactor

Variations of extracellular polymeric substances (EPS) and its components with sludge granulation were examined in a lab-scale sequencing batch reactor (SBR) which was fed with sodium nitrate and sodium acetate. Ultrasonication plus cation exchange resin (CER) were used as the EPS extraction method. Results showed that after approximately 90 d cultivation, the sludge in the reactor was almost granulated. The content of extracellular polysaccharides increased from 10.36 mg/g-VSS (volatile suspended solids) at start-up with flocculent sludge to 23.18 mg/g-VSS at 91 d with matured granular sludge, while the content of extracellular proteins were almost unchanged. Polysaccharides were the major components of EPS in anoxic granular sludge, accounting for about 70.6-79.0%, while proteins and DNA accounted for about 16.5-18.9% and 4.6-9.9%, respectively. It is proposed that EPS play a positive role in anoxic sludge granulation and polysaccharides might be strongly involved in aggregation of flocs into granules.

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

Selection of effective methods for extracting extracellular polymeric substances (EPSs) from Bacillus megaterium TF10

Bacterial extracellular polymeric substances (EPSs) play an important role in the formation and stabilization of bioaggregates, such as biofilm, microbial flocs and granules. Thus, the selection of appropriate extraction method is of critical importance, which may affect the yield, compositions and properties of EPS. In this work, six different methods used for EPS extraction, including ultrasonication, heating, formaldehyde+NaOH, H2SO4, glutaraldehyde, and EDTA methods, from Bacillus megaterium TF10, a bacterium with a high EPS-producing capacity isolated from a soil sample, are investigated. These EPS extraction methods are compared in terms of EPS yields and compositions, cell lysis, and flocculation activities and spectrum characteristics of extracted EPS. The results show that both EPS yield and cell lysis generally increase with the extraction time. The heating, formaldehyde+NaOH and H2SO4 methods lead to a high EPS yield compared to the ultrasonication or EDTA methods, while the ultrasonication and H2SO4 methods cause much more cell lysis than the formaldehyde+NaOH treatment. The flocculation activities of EPS, which can quantitatively reflect the EPS disruption during extraction, are also evaluated. The flocculation efficiencies of the extracted EPS are found in the order of: formaldehyde+NaOH (30-min formaldehyde treatment and then 60-min NaOH treatment), 92.4%>EDTA (10h), 92.2%>heating (120min), 91.6%>glutaraldehyde (15min), 90.9%>ultrasonication (30min), 81.0%>H2SO4 (45min), 73.2%>control, 59.9%. The IR and EEM spectra suggest that the EPS compositions and structures also vary significantly with the extraction method. Among the six methods, the EDTA method with an extraction time of 10h was identified to be most effective method to extract EPS from B. megaterium TF10.

Extracellular Polymeric Substances (EPS) from Aerobic Granular Sludges: Extraction, Fractionation, and Anionic Properties

A multi-method protocol previously proposed for the extraction of extracellular polymeric substances (EPS) from flocculated sludges was investigated on dense aerobic granules. The protocol combines mechanical disruption by sonication and chemical extraction using the Tween detergent and the cation chelator, EDTA. Polysaccharides were mainly recovered during the first sonication step while proteins were recovered all along the extractive procedure with a high prevalence in the EDTA step. These data confirmed the interest of the multi-method protocol for harvesting a diversified pool of EPS from dense granules and for fractionation of the polymers according to their physicochemical properties. In addition, the high extractability of proteins with EDTA confers a specific behavior of the aerobic granules towards the multi-method extraction protocol, supporting the idea that proteins are associated in the granule matrix through ionic interactions involving divalent cations. Analysis of the extracted EPS by anionic exchange chromatography confirmed the presence of highly anionic proteins that were specifically detected in the extracts obtained from granules. One important question is now to investigate whether these highly anionic proteins are involved in the aggregation and densification process and if their presence is related to the cohesive properties of these particles.