Extracellular Polymeric Substances Of Activated Sludge Research Articles

Genetic potential for exopolysaccharide synthesis in activated sludge bacteria uncovered by genome-resolved metagenomics

A good floc formation of activated sludge (AS) is crucial for solid-liquid separation and production of clean effluent during wastewater treatment. Floc formation is partly controlled by self-produced extracellular polymeric substances (EPS) such as exopolysaccharides, proteins, and nucleic acids. Little is known about the composition, structure, and function of EPS in AS and which bacteria produce them. To address this knowledge gap for the exopolysaccharides, we took advantage of 1083 high-quality metagenome-assembled genomes (MAGs) obtained from 23 Danish wastewater treatment plants. We investigated the genomic potential for exopolysaccharide biosynthesis in bacterial species typical in AS systems based on genome mining and gene synteny analyses. Putative gene clusters associated with the biosynthesis of alginate, cellulose, curdlan, diutan, hyaluronic acids, Pel, poly-β-1,6-N-acetyl-d-glucosamine (PNAG), Psl, S88 capsular polysaccharide, salecan, succinoglycan, and xanthan were identified and linked to individual MAGs, providing a comprehensive overview of the genome-resolved potential for these exopolysaccharides in AS bacteria. The approach and results provide a starting point for a more comprehensive understanding of EPS composition in wastewater treatment systems, which may facilitate a more refined regulation of the activated sludge process for improved stability.

Effects of Waste Activated Sludge Extracellular Polymeric Substances on Biosorption

Extracellular polymeric substances (EPS) reportedly make up approximately half of the organic matter in activated sludge (AS), and therefore strongly influence AS properties. This study evaluated the component fractions of EPS normalized to volatile suspended solids (VSS) in waste activated sludge (WAS) from a trickling-filter-solids contact process (TF/SC) and its ability to biosorb organic matter from raw wastewater with 30 min of contact time. Biosorption is the process in which organic matter (carbohydrates, proteins, humic acids, DNA, uronic acids, and lipids) in a sorbate, such as raw wastewater, sorbs onto a sorbent such as WAS. A statistically significant correlation was found between both the total concentration of EPS and the proteins component of the EPS and the biosorption removal of soluble chemical oxygen demand (sCOD) and truly soluble COD (ffCOD). Thus, the biosorption of soluble forms of COD can accurately be predicted by quantifying just the amount of proteins in WAS-associated EPS. No significant correlations were found for the biosorption of colloidal COD (cCOD). WAS biosorbed 45–75 mg L−1 of COD in 30 min. WAS absorbed or stored the proteins fraction of the soluble microbial products (SMP) during the biosorption process. Higher concentrations of humic acids were found in the biosorption process effluent than in the untreated wastewater, which warrants further study. Longer cation exchange resin (CER) extraction times yielded more total EPS from the sludge: 90 ± 9, 158 ± 3, and 316 ± 44 mg g−1 VSS, for 45-min, 4-h, and 24-h extraction times, respectively. Thus, EPS extracted represented only 9%, 15.8%, and 31.6% of the VSS, respectively, raising questions about whether the accurate characterization of EPS can be performed using the typical extraction time of 45 min due to different extraction rates for different components. It was found that the humic acids fraction was extracted much more slowly than the other fractions.

Chemical extraction methods for activated sludge extracellular polymeric substances (EPS): A review

Waste activated sludge (WAS) is formed by a diverse microorganisms, organic and inorganic compounds merged and mixed together in an extracellular polymeric substances (EPS) network. EPS is a complex high-molecular weight macromolecules in WAS that happens to be one of the common analysis to determine the efficiency of treatment. Therefore, different extraction methods has been applied in order to achieve better EPS extraction yield. This paper serves as a base to review the commonly used chemical extraction methods to extract EPS components. The mechanisms, conditions and efficiencies of each of the chemical extraction methods were discussed and compared accordingly. The possible use of different chemical extraction methods for different type of activated sludge were summarized.

Interactions between activated sludge extracellular polymeric substances and model carrier surfaces in WWTPs: A combination of QCM-D, AFM and XDLVO prediction

To understand the biofilm formation of biofilm-based processes in wastewater treatment plants (WWTPs), the interaction mechanisms between extracted extracellular polymeric substances (EPS) and three model carrier surfaces (i.e., negatively charged hydrophilic silica, positively charged hydrophilic alumina, and neutral charged hydrophobic polystyrene) were investigated employing a laboratory quartz crystal microbalance with dissipation monitoring equipment (QCM-D) and an atomic force microscope (AFM). The data suggested that surface charge and hydrophobicity of both EPS and carriers played significant roles in the interaction behaviors. Moreover, increases in ionic strength could lead to the increasing zeta potential and hydrophobicity of EPS. It is worth noting that long-range DLVO forces dominated the EPS deposition on carriers in lower ionic strength while short-range Lewis acid-base (AB) interaction controlled the adhesion behaviors in higher ionic strength. Besides, the presence of calcium ions contributed to the adhesion behaviors because of strong charge neutralization and hydrophobic effect. Bound EPS (BEPS) showed higher affinity to model carriers than dissolved EPS (DEPS), which conformed to XDLVO prediction rather than classical DLVO model. Overall, these results provide insights into the influence mechanisms of carrier characteristics, ionic strength, calcium ion and EPS components on the interaction between EPS and representative carriers, contributing to predict and regulate biofilm formation in biofilm-based processes.

Stress-responses of activated sludge and anaerobic sulfate-reducing bacteria sludge under long-term ciprofloxacin exposure

The activated sludge (AS) and sulfate-reducing bacteria (SRB) sludge systems were continuously operated for 200 days in laboratory to investigate the stress-responses of these two sludge systems under ciprofloxacin (CIP) exposure. It was found that CIP was effectively removed by SRB sludge via adsorption and biodegradation, but little biodegradation in AS system. The CIP biodegradation by SRB sludge made the SRB sludge system more sustainable and tolerant to long-term CIP exposure than AS system with significant (p < 0.05) CIP desorption and decrease of CIP removal. CIP shaped the microbial communities in AS and SRB sludge, and significantly (p < 0.05) inhibited the family Nitrosomonadaceae (ammonia-oxidizing bacteria (AOB)) and genus Nitrospira (nitrite-oxidizing bacteria (NOB)/complete ammonia oxidizer(comammox)) and the nitrogen removal in AS system. Moreover, CIP posed the increase of genus Zoogloea-like organisms and the non-filamentous bulking of AS, e.g. 313 ± 12 mL/g of sludge volume index (SVI) at phase V (influent CIP = 5000 μg/L). The genus Desulfobacter was enriched in SRB sludge system under long-term CIP exposure, and stimulated chemical oxygen demand (COD) removal and sulfate reduction. The increase of genera Zoogloea, Acinetobacter and Flavobacterium in AS, and Zoogloea and Acinetobacter in SRB sludge systems under CIP exposure promoted extracellular polymeric substances (EPS) production and CIP adsorption for self-protection of microbes against CIP toxicity. The functional groups of N–H, O–H, C–O–C and C=O in EPS of AS and SRB sludge provided adsorption sites for CIP and impeded CIP impact on microbial cells. The findings of this study provide an insight into the stress-responses of AS and SRB sludge under long-term CIP exposure, and exhibit the great potential of treating CIP-laden wastewater by SRB sludge system.

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.

Monthly variation of activated sludge extracellular polymeric substances and their correlation with the removal of anthropogenic micropollutants

Monthly variation of activated sludge extracellular polymeric substances and their correlation with the removal of anthropogenic micropollutants

Application of metaproteomic analysis for studying extracellular polymeric substances (EPS) in activated sludge flocs and their fate in sludge digestion

Metaproteomic analysis, comprising protein separation and identification, was applied to study extracellular proteins in activated sludges and to track their fate in sludge digestion under both anaerobic and aerobic conditions. The complex sludge proteins were first separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and further analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to search their identification. Base extraction and cation exchange resin (CER) method were used to extract EPS from sludges at 0, 12 and 30 days of batch digestion. Several important observations were made during this study. Firstly, protein bands were well separated by extraction/SDS-PAGE protocol used in this study. Secondly, numerous protein bands remained after digestion, indicating that these proteins are not easily degradable in sludge digestion. Thirdly, protein bands detected following anaerobic and aerobic digestion differed, suggesting that proteins degraded in two different digestion environments are not the same. Finally, protein bands that emerged distinctively following anaerobic digestion was found to be subunits of methyl-coenzyme M reductase, the enzyme involved in methane generation, in Methanosarcina barkeri. These results demonstrated that metaproteomic investigation on activated sludge EPS is useful for studying floc formation in activated sludges and their degradation in various digestion environments.

Amyloid-Like Adhesins Produced by Floc-Forming and Filamentous Bacteria in Activated Sludge

Amyloid proteins (fimbriae or other microbial surface-associated structures) are expressed by many types of bacteria, not yet identified, in biofilms from various habitats, where they likely are of key importance to biofilm formation and biofilm properties. As these amyloids are potentially of great importance to the floc properties in activated sludge wastewater treatment plants (WWTP), the abundance of amyloid adhesins in activated sludge flocs from different WWTP and the identity of bacteria producing these were investigated. Amyloid adhesins were quantified using a combination of conformationally specific antibodies targeting amyloid fibrils, propidium iodide to target all fixed bacterial cells, confocal laser scanning microscopy, and digital image analysis. The biovolume fraction containing amyloid adhesins ranged from 10 to 40% in activated sludge from 10 different WWTP. The identity of bacteria producing amyloid adhesins was determined using fluorescence in situ hybridization with oligonucleotide probes in combination with antibodies or thioflavin T staining. Among the microcolony-forming bacteria, amyloids were primarily detected among Alpha- and Betaproteobacteria and Actinobacteria. A more detailed analysis revealed that many denitrifiers (from Thauera, Azoarcus, Zoogloea, and Aquaspirillum-related organisms) and Actinobacteria-related polyphosphate-accumulating organisms most likely produced amyloid adhesins, whereas nitrifiers did not. Many filamentous bacteria also expressed amyloid adhesins, including several Alphaproteobacteria (e.g., Meganema perideroedes), some Betaproteobacteria (e.g., Aquaspirillum-related filaments), Gammaproteobacteria (Thiothrix), Bacteroidetes, Chloroflexi (e.g., Eikelboom type 1851), and some foam-forming Actinobacteria (e.g., Gordonia amarae). The results show that amyloid adhesins were an abundant component of activated sludge extracellular polymeric substances and seem to have unexpected, divers functions.

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.

Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and complexation properties of Pb and Cd with EPS: Part II. Consequences of EPS extraction methods on Pb 2+ and Cd 2+ complexation

To study the effect of extraction protocols on extracellular polymeric substances (EPS) metal binding ability, EPS from two activated sludges were extracted by eight extraction protocols: three chemical treatments, four physical treatments and a control. Two p K a, for each EPS, were determined: p K a1 may be specific for carboxyl and phosphoric and p K a2 may be attributed to phenolic and amino functional groups according to EPS composition and IR spectra. EPS p K a values could be affected by the presence of extraction reagents and/or the modifications of EPS by extraction reagents. Complexation study performed at pH 7 by a polarographic method has always showed a greater affinity of EPS for Pb 2+ than for Cd 2+. The complexation properties of EPS extracted by chemical methods were greatly modified. Concerning EPS extracted by physical methods, their complexation properties were close except for EPS obtained by heating. Standardized extraction methods must be established as a function of the aims of the EPS study.

A new method for extraction of extracellular polymeric substances from biofilms and activated sludge suitable for direct quantification of sorbed metals

A method for extraction of extracellular polymeric substances (EPSs) with a dicyclohexyl-18-crown-6 ether was developed to determine levels of organic and inorganic contaminants sorbed to EPS. The crown ether selectively binds alkaline and alkaline earth metals but not heavy metals. The effectiveness of the extraction procedure was higher than that of 2 other methods tested and comparable with that of a method based on a cation exchange resin. On average it was possible to extract 20% of the TOC, 12% of the total protein content, and 4% of the total carbohydrate content of sludge or biofilm biomass. Metal sorption studies in activated sludge showed no influence of exposure time on the fractionation of metals within the biomass. Metals sorbed mostly to cellular material. In biofilms 12.2% of the cadmium and 9.1% of the zinc added was found in the EPS. In activated sludge EPS contained only 2.9% zinc. The distribution of metals within the biomass was dose dependent. The percentage of metals found in EPS decreased with increasing metal concentration. This indicates a higher affinity of metals for cellular binding sites. Time course experiments in a rotating biofilm annular reactor, which consisted of an external cylinder with removable slides and an internal solid drum, revealed a gradual change in zinc concentration associated with EPS, although the total zinc concentration in the biomass remained constant. Concurrently, the amount of extractable EPS decreased. This was a consequence of a microbial population shift, with bacterial counts decreasing and algal and fungal biomass increasing. Using confocal laser scanning microscopy and the fluorescent metal complexing agent Newport Green for in situ detection of zinc it was shown that metals were bound to algae and fungi in the latter part of the experiment. The biofilm became more and more heterogeneous coinciding with a decrease in EPS. To summarize, the observed sorption behavior of metals cannot be explained with the conventional paradigm of EPS as hydrophilic gel. Obviously, different binding mechanisms must be invoked to explain the role of EPS in the sorption and removal of toxic substances in activated sludge and biofilm systems. It is important to consider the microbial population to understand differences in sorption in different matrices.

Changes in the composition of extracellular polymeric substances in activated sludge during anaerobic storage.

Changes in the chemical composition of organic compounds in total activated sludge, activated sludge extracellular polymeric substances (EPS), and sludge bulk water during anaerobic storage (12 days) were studied. The background for the study was that anaerobic storage of activated sludge, which often takes place at wastewater treatment plants before dewatering, causes a deterioration of the dewaterability. The reasons are not known at present, but may be related to changes in exopolymer composition of the flocs. The results showed that a fast decrease in total sludge protein and carbohydrate took place within 3 days of anaerobic storage as a result of degradation processes, which accounted for approximately 20% of the organic fraction. The amount of uronic acids and humic compounds remained almost constant in the sludge. The EPS were extracted from the floc matrix using a cation-exchange resin. In the EPS matrix a similar initial (2-3 days) degradation of proteins and carbohydrate took place, whereas the content of DNA and uronic acids showed minor changes. The extractability of humic compounds increased during the first 3 days of storage. No changes in extractability of the carbohydrate were observed. A fraction of the EPS protein was found to be difficult to extract but was observed to be degraded during the anaerobic storage. The EPS composition was further characterized by high-performance size-exclusion chromatography analysis obtained by on-line UV detection and post-column detection of proteins, carbohydrates, humic compounds and DNA. Four fractions of polysaccharides were found, of which only one was responsible for the decrease in the carbohydrate content observed with storage time. The fraction was presumably of low molecular mass. Humic compounds and volatile fatty acids (acetate and propionate) were released to the bulk water from the flocs during the storage. A possible mechanism to explain the reduced dewaterability developed during anaerobic storage, partly because of the observed changes in EPS, is discussed.