Characteristics of extracellular polymeric substances (EPS) fractions from excess sludges and their effects on bioflocculability

Extracellular polymeric substances (EPS) of biological origin are ubiquitous in excess sludges and can be applied as an underlying bioflocculant, owing to their high content of macromolecules and cations. However, low flocculating activity limits the feasibility of their practical applications. This study provides a novel EPS fractionation approach to improve their flocculability by extracting an active EPS fraction and removing the others with low flocculability. The results showed that for two excess sludges (called sludge A and sludge B), the tightly bound EPS (TB-EPS) fraction possessed a high flocculating rate to kaolin suspension compared with the other EPS fractions [i.e., supernatant, slime, and loosely bound EPS (LB-EPS) fraction] (>54.1 ± 1.4% vs <7.8 ± 1.6%). High bioflocculability of TB-EPS fraction could be attributable to high contents of macromolecules (330–1200 kDa) and trivalent cations (Fe³⁺ and Al³⁺). Further investigation reveals that the TB-EPS fraction caused aggregation of particles by bridging and sweep flocculation.     

Enhanced dewatering of waste-activated sludge by composite hydrolysis enzymes

The feasibility of composite hydrolysis enzymes in enhanced dewatering of waste-activated sludge (WAS) was verified in this study. A Pearson correlation analysis was conducted to explore the roles of different extracellular polymeric substance (EPS) fractions on WAS dewaterability. The results indicated that tightly bound EPS (TB-EPS) was released into the liquid phase consistently during enzymatic hydrolysis to form soluble EPS (S-EPS) and loosely bound EPS and that the TB-EPS content was positively correlated with the capillary suction time of WAS. A kinetic analysis was carried out to gain further insights into the kinetic variation in TB-EPS removal. It was found that TB-EPS reduction fit a first-order kinetic model and that mild temperature (25–30 °C) and a slightly acidic condition were favorable for the improvement of enzyme activity. Solid phase extraction combined with UV–Vis spectroscopy analysis was used to characterize the processes of migration and transformation of the hydrophobic (HPO), transphilic and hydrophilic (HPI) fractions in EPS during the enzymatic process. The results revealed that HPO and HPI were mainly composed of PN and PS, respectively, and that the enzymatic hydrolysis could enhance the transformation of HPI from TB-EPS to S-EPS, which was the dominant mechanism of improving WAS dewaterability.     

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.     

New sludge pretreatment method to improve dewaterability of waste activated sludge

The enhancements of electrolysis-pretreated conditioning were investigated in this study. Normalized capillary suction time (CST) was used to evaluate sludge dewaterability. Extracellular polymeric substance (EPS) concentration, viscosity and scanning electron microscopy (SEM) were determined to explain the observed changes in conditioning process. It indicated that pretreatment at 50 v and 5 min with Ti/RuO₂ anode was determined to be the optimal condition, which generated the lowest normalized CST and optimal soluble EPS concentration, leading to the decreasing of viscosity. EPS had positive correlation with the normalized CST. Subjecting to a combination of electrolysis pretreatment and flocculants conditioning, 50% dosage of cationic polyacrylamide (PAM) could be reduced. When co-conditioned with electrolysis and polymerization ferric sulfate (PFS), it did not present any clear advantages over PFS conditioning alone. Furthermore, SEM investigation indicated that electrolysis pretreatment could rupture sludge, release the interstitial water and extracellular substances, especially protein and polysaccharide, and consequently enhance its dewaterability.     

Dewaterability of waste activated sludge with ultrasound conditioning

This study investigated the potential benefits of ultrasound-conditioned sludge dewatering treatments with specific energy dosages from 0 to 35,000 kJ/kg total solids (TS). Capillary suction time (CST) and specific resistance of filtration (SRF) were used to evaluate sludge dewaterability. Sludge water distribution was measured by the drying test and mechanical separation methods. Both extracellular polymeric substance (EPS) content and sludge particle size were determined in an attempt to explain the observed changes in sludge dewaterability. The results indicated that application of low specific energy dosages (<4400 kJ/kg TS) slightly enhanced sludge dewaterability, but larger specific energy dosages (>4400 kJ/kg TS) significantly deteriorated sludge dewaterability. The optimal specific energy to give maximal dewaterability characteristics was found to be 800 kJ/kg TS, which generated sludge with optimal EPS concentration (400-500 mg/l) and particle size distribution (80-90 microm diameter). Subjecting sludge to a combination of cationic polymer and ultrasound pretreatments did not present any clear advantages over polymeric conditioning alone for improving sludge dewaterability. The effects of cationic polymer treatment predominated over those of ultrasound pretreatment when both were used together.     

Mg2+ distribution in activated sludge and its effects on the nitrifying activity and the characteristics of extracellular polymeric substances and sludge flocs

Divalent cations act as bridges among extracellular polymeric substances (EPS) and form cross-linkage for the self-immobilization of microbial biomass. However, their effects on the nitrification performance during the biological nitrogen removal are still unclear. In the present study, the effects of Mg²⁺ on the nitrifying activity, EPS and floc characteristics were investigated using a lab-scale sequencing batch reactor. The distribution of Mg²⁺ was quantified at different level of sludge floc. The results indicated that the nitrification activity was significantly improved when influent Mg²⁺ was below 1.1 mmol/L, but suppressed at 3 mmol/L. The overall performance characterized by COD, NH₄⁺-N and TN, the particle size and sludge flocculation ability rapidly increased with the increase of Mg²⁺ concentration. Mg²⁺ was mainly distributed in the pellet and changed slightly in supernatant, LB-EPS and TB-EPS. The four fluorescence peaks detected by three-dimensional excitation-emission matrix spectra were attributed to PN-like substances and humic acid-like substances in the LB-EPS and TB-EPS. The results of XPS analysis demonstrated that LB-EPS and TB-EPS comprised similar elements. Therefore, the types of EPS functional groups was unchanged under varied Mg²⁺ concentrations, while their proportions changed and LB-EPS/EPS was key factor for the changes of bioflocculation.     

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.     

Extracellular polymeric substances enhanced mass transfer of polycyclic aromatic hydrocarbons in the two-liquid-phase system for biodegradation

The objective was to elucidate the role of extracellular polymeric substances (EPS) in biodegradation of polycyclic aromatic hydrocarbons in two-liquid-phase system (TLPs). Therefore, biodegradation of phenanthrene (PHE) was conducted in a typical TLPs—silicone oil–water—with PHE-degrading bacteria capable of producing EPS, Sphingobium sp. PHE3 and Micrococcus sp. PHE9. The results showed that the presence of both strains enhanced mass transfer of PHE from silicone oil to water, and that biodegradation of PHE mainly occurred at the interfaces. The ratios of tightly bound (TB) proteins to TB polysaccharides kept almost constant, whereas the ratios of loosely bound (LB) proteins to LB polysaccharides increased during the biodegradation. Furthermore, polysaccharides led to increased PHE solubility in the bulk water, which resulted in an increased PHE mass transfer. Both LB-EPS and TB-EPS (proteins and polysaccharides) correlated with PHE mass transfer in silicone oil, indicating that both proteins and polysaccharides favored bacterial uptake of PHE at the interfaces. It could be concluded that EPS could facilitate microbial degradation of PHE in the TLPs.     

Dewaterability characteristics of sludge conditioned with surfactants pretreatment by electrolysis

The potential benefits of electrolysis-conditioned sludge dewaterability treatments with surfactants were investigated in this study. Capillary suction time (CST) and specific resistance of filtration (SRF) were used to evaluate the sludge dewaterability. Extracellular polymeric substance (EPS) content, viscosity and zeta potential were determined in an attempt to explain the observed changes in the conditioning process. The results indicated that SDS (Sodium Dodecyl Sulphate) and Triton X-100 have negative effect on the dewaterability of sludge pretreated both with and without electrolysis. However, with a combination of CTAB (Cetyl Trimethyl Ammonium Bromide) and electrolysis pretreatment presented clear advantages over surfactant conditioning alone for improving sludge dewaterability. The optimal dosage of CTAB to give maximal dewaterability was found to be 2000 mg/L, which generated sludge with optimal EPS concentration (150-300 mg/L), viscosity (55-62 mpa s) and zeta potential (−2.12 to −1.19 mV).     

微生物接种密度和矿物收集时间对生物沥浸中次生铁矿物形成的影响

在嗜酸性氧化亚铁硫杆菌Acidithiobacillus ferrooxidans作用下, 污泥生物沥浸体系中常会有次生铁矿物形成, 这些矿物对污泥脱水和重金属溶出有重要影响。在FeSO4-K2SO4-H2O生物成矾临界点模拟生物沥浸过程, 考察了Acidithiobacillus ferrooxidans菌接种密度和矿物收集时间对次生铁矿物的影响。结果表明, 微生物接种密度和矿物收集时间对生物沥浸过程中次生铁矿物的重量及其类型均有一定影响, 随矿物收集时间的推迟, 溶液中含有的一价阳离子(如K+等)可导致施氏矿物向黄铁矾发生转变, 并成为影响铁矿物类型的主导因素。     

Correlation of extracellular polymeric substances and microbial community structure in denitrification biofilm exposed to adverse conditions

Microbial community may respond to different adverse conditions and result in the variation of extracellular polymeric substances (EPS) in denitrification biofilm; this study discovered the role of EPS in accordance with the analysis of cyclic diguanylate (c-di-GMP) and electron equilibrium (EE) under low organic loading rate, shock organic loading rate and low temperature conditions. Good nitrate removal performance could be achieved under shock organic loading rate and low temperature conditions; however, owing to the low organic loading rate, the carbon source was preferentially utilized for biomass growth. Tightly bound EPS (TB-EPS) contents progressively increased and facilitated cell adhesion and biofilm formation. The stable TB protein (TB-PN) content in TB-EPS built a cross-linked network to maintain internal biofilm structure and led to the rapid biosynthesis of polysaccharides, which could further enhance microbial adhesion and improve nitrate removal. C-di-GMP played an important role in biomass retention and biofilm formation, based on the correlation analysis of c-di-GMP and EPS. TB polysaccharide (TB-PS) contents presented a significant positive correlation with c-di-GMP content, microbial adhesion and biofilm stabilization was further enhanced through c-di-GMP regulation. In addition, a remarkable negative correlation between electron deletion rate (EDR) and TB-PN and TB-PS was discovered, and TB-PS was required to serve as energy source to enhance denitrification according to EE analysis. Surprisingly, dynamic microbial community was observed due to the drastic community succession under low temperature conditions, and the discrepancy between the dominant species for denitrification was found under shock organic loading rate and low temperature conditions. The notable increase in bacterial strains Simlicispira, Pseudomonas and Chryseobacterium was conducive to biofilm formation and denitrification under shock organic loading rate, while Dechloromonas and Zoogloea dramatically enriched for nitrate removal under low temperature conditions. The high abundance of Dechloromonas improved the secretion of EPS through the downstream signal transduction, and the c-di-GMP conserved in Pseudomonas concurrently facilitated to enhance exopolysaccharide production to shock organic loading rate and low temperature conditions.     

Investigation of energy gene expressions and community structures of free and attached acidophilic bacteria in chalcopyrite bioleaching

In order to better understand the bioleaching mechanism, expression of genes involved in energy conservation and community structure of free and attached acidophilic bacteria in chalcopyrite bioleaching were investigated. Using quantitative real-time PCR, we studied the expression of genes involved in energy conservation in free and attached Acidithiobacillus ferrooxidans during bioleaching of chalcopyrite. Sulfur oxidation genes of attached A. ferrooxidans were up-regulated while ferrous iron oxidation genes were down-regulated compared with free A. ferrooxidans in the solution. The up-regulation may be induced by elemental sulfur on the mineral surface. This conclusion was supported by the results of HPLC analysis. Sulfur-oxidizing Acidithiobacillus thiooxidans and ferrous-oxidizing Leptospirillum ferrooxidans were the members of the mixed culture in chalcopyrite bioleaching. Study of the community structure of free and attached bacteria showed that A. thiooxidans dominated the attached bacteria while L. ferrooxidans dominated the free bacteria. With respect to available energy sources during bioleaching of chalcopyrite, sulfur-oxidizers tend to be on the mineral surfaces whereas ferrous iron-oxidizers tend to be suspended in the aqueous phase. Taken together, these results indicate that the main role of attached acidophilic bacteria was to oxidize elemental sulfur and dissolution of chalcopyrite involved chiefly an indirect bioleaching mechanism.     

Influence analysis for the behavior of dewaterability of excess sludge in a two-stage vermifilter

To improve excess sludge dewaterability, a two-stage vermifilter was developed to qualitatively and quantitatively analyze sludge physico-chemical properties (fractal dimension, zeta potential, extracellular polymeric substances (EPS), particle size distribution, etc.) and to correlate them with sludge dewatering characteristics (specific resistance to filtration (SRF) and capillary suction time (CST)). Results demonstrated that sludge dewatering performance was significantly improved after the primary vermifilter VF₁ and the second-stage vermifilter VF₂. In addition, the further VF₂ treatment exhibited higher effects on sludge dewatering performance. The particle boundary of sludge after VF₂ treatment was clearer and smoother than VF₁ sludge (VF₁S), apart from the fact that sludge morphological structure got denser and more compact. Comparing with VF₁S, the fractal dimension D₁ calculated within 1D topological space was closer to 1 after VF₂ treatment, and the fractal dimension D₂ within 2D topological space closer to 2, indicating a better dewatering performance after VF₂ treatment. Additionally, the changes of sludge floc surface properties (such as zeta potential and EPS) resulted in small particles agglomerating into larger ones and then the increase of particle diameter. In summary, the two-stage vermifilter got a better sludge dewatering performance, and thus beneficial for subsequent processing of sludge.

     

Ultrasonic and Thermal Pretreatments on Anaerobic Digestion of Petrochemical Sludge: Dewaterability and Degradation of PAHs

Effects of different pretreatment methods on sludge dewaterability and polycyclic aromatic hydrocarbons (PAHs) degradation during petrochemical sludge anaerobic digestion were studied. Results showed that the total biogas production volume in the thermal pretreatment system was 4 and 5 times higher than that in the ultrasound pretreatment and in the control system, and the corresponding volatile solid removal efficiencies reached 28%, 15%, and 8%. Phenanthrene, paranaphthalene, fluoranthene, benzofluoranthene, and benzopyrene removal rates reached 43.3%, 55.5%, 30.6%, 42.9%, and 41.7%, respectively, in the thermal pretreatment system, which were much higher than those in the ultrasound pretreatment and in the control system. Moreover, capillary suction time (CST) of sludge increased after pretreatment, and then reduced after 20 days of anaerobic digestion, indicating that sludge dewaterability was greatly improved after anaerobic digestion. The decrease of protein and polysaccharide in the sludge could improve sludge dewaterability during petrochemical sludge anaerobic digestion. This study suggested that thermal pretreatment might be a promising enhancement method for petrochemical sludge solubilization, thus contributing to degradation of the PAHs, biogas production, and improvement of dewaterability during petrochemical sludge anaerobic digestion.     

Acidophilic microbial communities catalyzing sludge bioleaching monitored by fluorescent in situ hybridization

Biological autotrophic sulfur oxidation processes have been proposed to remove heavy metals from wastewater treatment sludge by bioleaching. We made a characterization of the microbial population in batch and continuous sludge bioleaching reactors using fluorescent in situ hybridization of fluorescently-labeled oligonucleotidic probes targeting rRNA in a ‚top to bottom approach’. Batch incubations of sludge with 0.2% (w/v) elemental sulfur resulted in a pH value of 5. Alpha-Proteobacteria hybridizing with probe ALF1b were dominant in this incubation. Members of the Acidophilium-group (hybridizing with probe Acdp821) of Nitrospira/Leptospirillum phylum (Ntspa712 probe) and from the archaeal domain (ARCH915) were also detected. When sludge was incubated with 1% elemental sulfur in batch or continuous reactor experiments, final pH values were always below 2. Active microbial communities consisted almost exclusively of gamma-Proteobacteria (hybridizing with probe GAM42a). However, further hybridization experiments with probe Thio820 targeting Acidithiobacillus ferroxidans and Acidithiobacillus thioxidans gave negative results. A new probe, named THIO181, encompassing all known members of the genus was designed. Hybridization perfomed with THIO181 and GAM42a showed a perfect co-localization of the hybridization signals. Further hybridization experiments with probe THIO181 and THC642, specific for the species Acidithiobacillus caldus, confirmed that this bacteria was largely responsible for the sulfur oxidation reaction in our acidophilic sludge bioleaching reactors.     

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 cationexchange 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.     

剩余活性污泥完全资源化利用微生物集成技术

剩余活性污泥完全资源化利用微生物集成技术包括: 使用土著PHA合成菌回注法驯化并发酵活性污泥, 生产生物降解材料聚羟基脂肪酸酯(PHA); 采用土著嗜酸性氧化亚铁硫杆菌和氧化硫硫杆菌进行生物淋滤, 去除重金属; 以解磷菌和解钾菌为菌种, 进行固态发酵, 生产生物菌肥。结果表明, 500 L中试PHA占挥发性悬浮固体的20%以上; 重金属含量达到国家排放要求; 生物菌肥中活菌数大于1×108 个/g以上。实现了剩余活性污泥的近零排放。     

The Effects of Bacterial Leaching on Metal Partitioning in Sewage Sludge

The partitioning of Mn, Al, Zn, Cu and Ti ions in municipal sewage sludge was investigated before and after bioleaching processes effectuated by Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. Oxidation–reduction potential increase and pH decrease were obtained as a result of bacterial activity. A less pronounced and constant decrease was obtained with A. ferrooxidans, whereas A. thiooxidans presented a lag phase before a steep pH decrease. Metal solubilization was accomplished in experimental systems supplemented with energy source, Fe²⁺ for A. ferrooxidans and S⁰ for A. thiooxidans. Solubilization efficiency differed for each metal except for Al, and was relatively similar for either organism. Metal partitioning was conducted using a five-step sequential extraction procedure before and after the bioleaching. The results indicated that Zn and Mn ions were mostly associated with the organic fraction, whereas Cu, Al and Ti ions with the sulphide/residue fraction. The bioleaching process caused prompt solubilization of metals mostly associated with the more labile fractions (exchangeable, adsorbed and organically bound metals), whereas those associated to the less labile ones (EDTA and sulphide/residue fractions) were exchanged towards more labile fractions.     

Tolerance to copper and zinc of Acidithiobacillus thiooxidans isolated from sewage sludge

The effect of copper and zinc ions on sulphur oxidation by Acidithiobacillus thiooxidans, strain SFR01, isolated from anaerobic sewage sludge was assessed, resulting in tolerance levels up to 20 and 200 mmol l^–1 for copper and zinc, respectively. The tolerance levels obtained were higher than the concentration of copper and zinc usually found in the collected sewage sludge. The tolerance levels obtained indicate no constraints for sludge bioleaching of those metals due to their toxicities to the indigenous A. thiooxidans.     

Characteristics of extracellular polymeric substances from sludge and biofilm in a simultaneous nitrification and denitrification system under high salinity stress

The composition and distribution of extracellular polymeric substance (EPS) both from suspended sludge and attached biofilm were investigated in a simultaneous nitrification and denitrification (SND) system with the increase of the salinity from 1.0 to 3.0 %. Fourier-transform infrared (FTIR) spectroscopy and three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectroscopy were used to examine proteins (PN), polysaccharides (PS) and humic substances (HS) present in EPS. High total nitrogen removal (above 83.9 %) via SND was obtained in the salinity range of 1.0–2.5 %. Total EPS in the sludge increased from 150.2 to 200.6 mg/gVSS with the increase of salinity from 1.0 to 3.0 %, whereas the corresponding values in the biofilm achieved the maximum of 288.6 mg/g VSS at 2.0 % salinity. Dominant composition of EPS was detected as HS in both sludge and biofilm, having the percentages of 50.6–68.6 and 41.1–69.9 % in total EPS, respectively. Both PN and PS contents in soluble EPS (S-EPS), loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) of sludge and biofilm increased with the increased salinity. The FTIR spectrum and 3D-EEM fluorescence spectroscopy of S-EPS, LB-EPS and TB-EPS in the sludge and biofilm showed the changes of functional groups and conformations of the compositions in EPS with the increase of salinity. The results demonstrated that the characteristics of EPS varied from sludge to biofilm. The obtained results could provide a better understanding of the salinity effect on the EPS characteristics in a SND system.