Application of enzymes, sodium tripolyphosphate and cation exchange resin for the release of extracellular polymeric substances from sewage sludge. Characterization of the extracted polysaccharides/glycoconjugates by a panel of lectins

The study describes extraction of extracellular polymeric substances (EPS) from sewage sludge by applying enzymes and enzymes combined with sodium tripolyphosphate (STPP). Additionally, a systematic study of two non-enzymatic extraction agents is described. The assessment of the released products is made by colorimetrical methods and polysaccharides/glycoconjugates identification by the interaction with four immobilized lectins. Bio-sludge from Helsingborg (Sweden) and Damhus?en (Denmark) were used as two case studies for testing enzymatic extractability and thereby to make useful prediction of sludge bio-digestibility. From Helsingborg sludge the enzymes extracted about 40% more of EPS than from Damhus?en. The polysaccharides/glycoconjugates in both sludges maintained the same level, and showed substantial different interaction motifs with lectins panel. Damhus?en enzymatic extracted EPS had an enhanced amount of suspended material that was post-hydrolysed by the use of polygalacturonase and lysozyme resulting in pectin like polymers and petiptidoglycans. Petiptidoglycan is a marker from bacterial cell debris. STPP and cation exchange resin (CER) released different quantities of EPS. The CER released polysaccharides/glycoconjugates had higher molecular weight and stronger affinity towards Concanavalin A than the one released by the action of STPP. Independent of the extraction conditions, STPP released elevated amounts of polyvalent cations and humic substances in contrast to the very low amounts of released by CER.     

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.     

The use of a new mobile phase,with no multivalent cation binding properties,to differentiate extracellular polymeric substances (EPS), by size exclusion chromatography (SEC), from biomass used for wastewater treatment

The fingerprints of extracellular polymeric substances (EPS) extracted from different types of biomass used for wastewater treatment (i.e., activated sludge, filamentous activated sludge, anaerobic granular sludge, anaerobic flocculated sludge) were studied by size exclusion chromatography (SEC) with Amersham Biosciences Superdex 200 10/300 GL column with a theoretical resolving range of 10–600 kDa. A new mobile phase, which does not display binding properties for multivalent cations, was previously optimized. This mobile phase contained 75 mM Hepes buffer at pH 7 with 15% acetonitrile (v/v) and was selected to minimize ionic and hydrophobic interactions between the molecules that make up the EPS and the column packing.When EPS extracted from similar sludges is analyzed using different mobile phases, the number of chromatographic peaks obtained is quite similar, and differences are mainly observed in the relative absorbance of the chromatographic peaks. However, very different chromatograms (number and relative absorbance of chromatographic peaks) are obtained for EPS extracted from different types of sludges. Furthermore, when dysfunctions, such as filamentous bulking in the activated sludge, occur in a bioreactor, they also induce strong variations in chromatographic profiles.     

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.     

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.     

Selectively inducing the synthesis of a key structural exopolysaccharide in aerobic granules by enriching for <Emphasis Type="BoldItalic">Candidatus</Emphasis> “<Emphasis Type="BoldItalic">Competibacter phosphatis</Emphasis>”

A gel-forming exopolysaccharide was previously shown to play an important structural role in aerobic granules treating nutrient-rich industrial wastewater. To identify whether this exopolysaccharide performs a similar role in other granular biomass and if conditions favouring its production can be more precisely elucidated, extracellular polymeric substances (EPS) were extracted from granules grown under four different operating conditions. ¹H nuclear magnetic resonance (NMR) spectroscopy of their EPS indicated that the gel-forming exopolysaccharide was expressed in two granular sludges both enriched in Candidatus “Competibacter phosphatis”. In contrast, it was not expressed in granules performing denitrification with methanol as a carbon source and nitrate as the electron acceptor or granules enriched in Candidatus “Accumulibacter phosphatis” performing enhanced biological phosphorus removal from synthetic wastewater. In one of the first two sludges, the exopolysaccharide contained in the seeding granular sludge continued to be a major component of the granule EPS while Competibacter was being enriched. In the second sludge, a floccular sludge not containing the gel-forming exopolysaccharide initially was also enriched for Competibacter. In this sludge, an increase in particle size was detected coinciding with a yield increase of EPS. NMR spectroscopy confirmed its yield increase to be attributable to the production of this structural gel-forming exopolysaccharide. The results show that (1) the particular gel-forming exopolysaccharide previously identified is not necessarily a key structural exopolysaccharide for all granule types, and (2) synthesis of this exopolysaccharide is induced under conditions favouring the selective enrichment of Competibacter. This indicates that Competibacter may be involved in its production.     

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 Pb2+ and Cd2+ 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 pK_a, for each EPS, were determined: pK_a1 may be specific for carboxyl and phosphoric and pK_a2 may be attributed to phenolic and amino functional groups according to EPS composition and IR spectra. EPS pK_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 Pb²⁺ than for Cd²⁺. 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.     

Membrane biofouling by extracellular polymeric substances or soluble microbial products from membrane bioreactor sludge

This study extracted the soluble microbial products and loosely bound and tightly bound extracellular polymeric substances (EPS) from suspended sludge from a membrane bioreactor, original and aerobically/anaerobically digested, and compared their fouling potentials on a microfiltration membrane. The resistance of cake layer accounts for 95–98% of the total filtration resistances when filtering the whole sludges, with anaerobically digested sludge presenting the highest resistance among the three tested sludges. The tightly bound EPS has the highest potential to foul the membrane; however, the loosely bound EPS contribute most of the filtration resistances of the whole sludges. The foulants corresponding to the irreversible fouling have chemical fingerprints similar to those from loosely bound EPS, which have a greater predilection to proteins and humic substances than to polysaccharides.     

Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-mum cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 +/- 12 ml g(-1), and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 +/- 2 ml g(-1). EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.     

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na₂CO₃) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca²⁺). From the six extraction methods used, only the Na₂CO₃ extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca²⁺. The Ca²⁺-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.     

Hydrophobic features of EPS extracted from anaerobic granular sludge: an investigation based on DAX-8 resin fractionation and size exclusion chromatography

The hydrophobic fractionation of extracellular polymeric substances (EPS) extracted from anaerobic granular sludge was performed on the DAX-8 resin (two elution pH conditions, i.e., pH 2 and pH 5 were tested). The impact of seven different EPS extraction methods on EPS hydrophobicity features was assessed. The results showed that the extraction methods and bulk solution pH influenced dramatically the biochemical composition of the EPS, and in turn, the hydrophobicity determined. Besides, EPS extracting reagents i.e., formaldehyde, ethanol, sodium dodecyl sulfate (SDS), and Tween 20 not only introduced extra carbon content in the total organic carbon (TOC) measurement but also interacted with the DAX-8 resin. By comparing the apparent molecular weight (aMW) distribution of untreated and pH-adjusted EPS samples, more complete EPS aMW information was preserved at pH 5. Thus, elution at pH 5 was preferred in this study for the qualitative analysis of EPS hydrophobic features. The hydrophobic fraction of EPS retained by the resin at pH 5 was ascribed to a wide aMW range, ranging from >440 to 0.3 kDa. Within this range, EPS molecules ranging from 175 to 31 kDa were mostly retained by the DAX-8 resin, which indicates that these EPS molecules are highly hydrophobic.

     

Evidence of compositional differences between the extracellular and intracellular DNA of a granular sludge biofilm

Aims: Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. Methods and Results: Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR‐based denaturing gradient‐gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. Conclusions: Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. Significance and Impact of the Study: The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms.     

Dynamic Membrane Formation in Anaerobic Dynamic Membrane Bioreactors: Role of Extracellular Polymeric Substances

Dynamic membrane (DM) formation in dynamic membrane bioreactors plays an important role in achieving efficient solid-liquid separation. In order to study the contribution of extracellular polymeric substances (EPS) to DM formation in anaerobic dynamic membrane bioreactor (AnDMBR) processes, EPS extraction from and re-addition to bulk sludge were carried out in short-term filtration tests. DM formation behaviors could be well simulated by cake filtration model, and sludge with EPS re-addition showed the highest resistance coefficient, followed by sludge after EPS extraction. The DM layers exhibited a higher resistance and a lower porosity for the sludge sample after EPS extraction and for the sludge with EPS re-addition. Particle size of sludge flocs decreased after EPS extraction, and changed little with EPS re-addition, which was confirmed by interaction energy analysis. Further investigations by confocal laser scanning microscopy (CLSM) analysis and batch tests suggested that the removal of in-situ EPS stimulated release of soluble EPS, and re-added EPS were present as soluble EPS rather than bound EPS, which thus improved the formation of DM. The present work revealed the role of EPS in anaerobic DM formation, and could facilitate the operation of AnDMBR processes.     

Chemical characterization of humic substances extracted from organic-waste-amended soils

Humic substances were extracted from a soil treated, in a 4-year experiment, at different rates with a sludge from anaerobic treatment of combined civil and industrial wastes, and with agricultural manure. Elemental and chemical analyses, molecular weight (MW) distribution and infrared (IR) spectroscopy were performed on the purified humic acids (HA). Organic wastes significantly increased the HA content of the treated soils and improved CEC. The C/N, C/H and C/O ratios of HA extracted from the original wastes showed a higher degree of humification for sludge than for manure. This difference was also noticed for the C/N ratio of soil humic extracts, indicating a faster humification process for the sludges in soil. The content of oxygen-containing functional groups was lower than the ‘model’ HA reported in the literature, and even more so for HA from sludges, reflecting their anaerobic formation. MW distribution and E₄/E₆ ratios showed that the sludge material had a higher molecular complexity than manure, supporting the high degree of humification attributed to the former. HA extracted from sludge-treated soils revealed a molecular dimension increasing with the application doses of waste material. Infrared spectra showed that HA formed in soils after waste additions reflected the chemical composition of the original organic material, which was rich in aliphatic groups and peptides for sludge and in carbohydrates for manure. On the basis of this study, it is concluded that not only are organic waste additions able to build up the HA content in soils but the HA formed assume the chemical characteristics and the degree of humification of the original material.     

Composition and Distribution of Extracellular Polymeric Substances in Aerobic Flocs and Granular Sludge

Extracellular polymeric substances (EPS) were quantified in flocculent and aerobic granular sludge developed in two sequencing batch reactors with the same shear force but different settling times. Several EPS extraction methods were compared to investigate how different methods affect EPS chemical characterization, and fluorescent stains were used to visualize EPS in intact samples and 20-μm cryosections. Reactor 1 (operated with a 10-min settle) enriched predominantly flocculent sludge with a sludge volume index (SVI) of 120 ± 12 ml g⁻¹, and reactor 2 (2-min settle time) formed compact aerobic granules with an SVI of 50 ± 2 ml g⁻¹. EPS extraction by using a cation-exchange resin showed that proteins were more dominant than polysaccharides in all samples, and the protein content was 50% more in granular EPS than flocculent EPS. NaOH and heat extraction produced a higher protein and polysaccharide content from cell lysis. In situ EPS staining of granules showed that cells and polysaccharides were localized to the outer edge of granules, whereas the center was comprised mostly of proteins. These observations confirm the chemical extraction data and indicate that granule formation and stability are dependent on a noncellular, protein core. The comparison of EPS methods explains how significant cell lysis and contamination by dead biomass leads to different and opposing conclusions.     

Effects of physico-chemical factors on the viscosity evolution of anaerobic granular sludge

The rheological properties of anaerobic granular sludge samples from four full-scale and one lab-scale anaerobic bioreactors were characterized by determining their “limit viscosity” values. These values were deducted from the evolution of the apparent viscosity of granular sludge samples (20 mL) at steady shear rate (200 s⁻¹) recorded using rotation tests with a wings type measurement cell stirrer Anton-Parr reference: ST24-1D/2V-Q0. The limit viscosity values depended on the applied shear rate, indicating a non-Newtonian behavior of the anaerobic granular sludge types investigated. The effect of variations of physico-chemical parameters such as pH (involving surface charge change), size, surface roughness and TSS content on the evolution of the limit viscosity of an anaerobic granular sludge suspension was investigated. This showed the importance of both quantitative (number of particles in a given volume) as well as qualitative (surface charge or shape) granule-granule interactions on this rheological parameter. Moreover, the origin of the granular sludge strongly influenced the limit viscosity value according with different granules characteristics. This work confirms the ability of the rheological parameter “limit viscosity” as an overall parameter to describe the physico-chemical characteristics (TSS, granulometry, origin, and charge) of anaerobic granular sludge and showed this holds for both sieved (500 μm) and unsieved sludges.     

Bonding Form Analysis of Metals and Sulfur Fractionation in Methanol‐Grown Anaerobic Granular Sludge

This study investigates the metal and sulfur bonding form distribution in mesophilic (30 °C, pH 7) methanol‐grown anaerobic granular sludge from upflow anaerobic sludge bed reactors operating at an organic loading rate of 3.8 g CH₃OH‐COD/L d. This was achieved by applying a modified Tessier sequential extraction scheme to investigate the metal bonding forms and a sequential extraction scheme for sulfur and simultaneously extracted metals to granular sludge samples of the reactors after 0, 22, 35 and 43 days of operation. Metals were also determined in the sulfur extracts. Co and Ni predominated in their oxidizable bonding forms, which increased together with the pseudo‐total content during reactor operation. An omission of Co and Ni from the influent led to only a minor decline of the pseudo‐total content in the sludge, mainly from the acid‐soluble fraction. The ratio of simultaneously extracted metals (Co, Fe, Mn, Ni) to acid‐volatile sulfides was lower than 1, indicating that the sludge contained sufficient sulfide to bind the metals as metal monosulfides. The bioavailability of metals in the methanol‐grown anaerobic granular sludge investigated is therefore mainly controlled by sulfide formation/dissolution.     

Enzyme activities in activated sludge flocs

This study quantified the activities of enzymes in extracellular polymeric substances (EPS) and in pellets. Seven commonly adopted extraction schemes were utilized to extract from aerobic flocs the contained EPS, which were further categorized into loosely bound (LB) and tightly bound (TB) fractions. Ultrasonication effectively extracted the EPS from sludge flocs. Enzyme assay tests showed that the protease activity was localized mainly on the pellets, α-amylase and α-glucosidase activities were largely bound with LB-EPS, and few protease, α-amylase, or α-glucosidase activities were associated with the TB-EPS fraction. There exists no correlation between the biochemical compositions of EPS and the distribution of enzyme activities in the sludge matrix. The 44–65% of α-amylase and 59–100% of α-glucosidase activities noted with the LB-EPS indicate heterogeneous hydrolysis patterns in the sludge flocs with proteins and carbohydrates.     

Relationship between Methanogenic Cofactor Content and Maximum Specific Methanogenic Activity of Anaerobic Granular Sludges

In this study we investigated whether a relationship exists between the methanogenic activity and the content of specific methanogenic cofactors of granular sludges cultured on different combinations of volatile fatty acids in upflow anaerobic sludge blanket or fluidized-bed reactors. Significant correlations were measured in both cases between the contents of coenzyme F₄₂₀−2 or methanopterin and the maximum specific methanogenic activities on propionate, butyrate, and hydrogen, but not acetate. For both sludges the content of sarcinapterin appeared to be correlated with methanogenic activities on propionate, butyrate, and acetate, but not hydrogen. Similar correlations were measured with regard to the total content of coenzyme F₄₂₀−4 and F₄₂₀−5 in sludges from fluidized-bed reactors. The results indicate that the contents of specific methanogenic cofactors measured in anaerobic granular sludges can be used to estimate the hydrogenotrophic or acetotrophic methanogenic potential of these sludges.     

Scanning electron microscopy of biofilm development in anaerobic fixed-bed reactors: Influence of the inoculum

Summary Scanning electron microscopy was applied to evaluate the influence of inoculum on efficiency of initial biofilm formation and reactor performance. Five anaerobic fixed-bed reactors were inoculated with anaerobic sludges from different sources and operated in parallel under identical conditions with defined wastewater and acetate, propionate and butyrate as constituents In all sludges Methanothrix sp. was the predominant acetotroph. The reactors inoculated with anaerobic sludge adapted to the wastewater achieved the highest space loading with 21.0 g COD/l·d after 58 days. The inoculation with granular sludge from an upflow anaerobic sludge blanket (UASB) reactor resulted in significantly less reactor efficiency. Time course of biofilm formation and biofilm thickness (ranging from 20–200 m) depended on the type of inoculum.