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.     

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.     

荧光定量PCR监测五氯酚对好氧颗粒污泥和活性污泥中氨氧化细菌数量的影响

通过特异引物扩增环境中氨氧化细菌16S rDNAV2保守区域,将该片段克隆到T-easy载体上,PCR产物经测序和定量PCR扩增体系鉴定,证实PCR扩增产物为氨氧化细菌16S rDNA保守序列,以含该序列的重组质粒作为定量PCR监测氨氧化细菌数量的DNA标准品。用荧光定量PCR技术比较了五氯酚(PCP)对好氧颗粒污泥和活性污泥中氨氧化细菌数量的影响。结果表明,不加PCP的反应器中,好氧颗粒污泥和活性污泥中氨氧化细菌的数量分别为4.28×107±5.44×106cells/(g干污泥)和2.51×109±8.61×108cells/(g干污泥)。随着PCP浓度的增加(0~50mg/L),PCP对氨氧化细菌数量的影响不大(P>0.05),而且,污泥中氨氧化细菌的数量与氨氮的去除率无直接的正相关关系(P>0.05),PCP主要是抑制氨氧化细菌的代谢活性导致污泥氨氮去除效率降低。     

Comparison of biological removal via nitrite with real-time control using aerobic granular sludge and flocculent activated sludge

The process of nitrification–denitrification via nitrite for nitrogen removal under real-time control mode was tested in two laboratory-scale sequencing batch reactors (SBRs) with flocculent activated sludge (R1) and aerobic granular sludge (R2) to compare operational performance and real-time control strategies. The results showed that the average ammonia nitrogen, total inorganic nitrogen (TIN), and chemical oxygen demand (COD) removal during aeration phase were 97.6%, 57.0%, and 90.1% in R2 compared with 98.6%, 48.7%, and 88.1% in R1. The TIN removed in both SBRs was partially due to the presence of simultaneous nitrification–denitrification via nitrite, especially in R2. The specific nitrification and denitrification rates in R2 were 0.0416 mgNH₄⁺–N/gSS-min and 0.1889 mgNO_X⁻–N/gSS-min, which were 1.48 times and 1.35 times that of R1. The higher rates for COD removal, nitrification, and denitrification were achieved in R2 than R1 with similar influent quality. Dissolved oxygen (DO), pH, and oxidization reduction potential, corresponding to nutrient variations, were used as diagnostic parameters to control the organic carbon degradation and nitrification–denitrification via nitrite processes in both SBRs. The online control strategy of granular SBR was similar to that of the SBR with flocculent activated sludge. However, a unique U-type pattern on the DO curve in granular SBR was different from SBR with flocculent activated sludge in aerobic phase.     

Heterogeneous diffusion in aerobic granular sludge

Aerobic granular sludge (AGS) technology allows simultaneous nitrogen, phosphorus, and carbon removal in compact wastewater treatment processes. To operate, design, and model AGS reactors, it is essential to properly understand the diffusive transport within the granules. In this study, diffusive mass transfer within full-scale and lab-scale AGS was characterized with nuclear magnetic resonance (NMR) methods. Self-diffusion coefficients of water inside the granules were determined with pulsed-field gradient NMR, while the granule structure was visualized with NMR imaging. A reaction-diffusion granule-scale model was set up to evaluate the impact of heterogeneous diffusion on granule performance. The self-diffusion coefficient of water in AGS was ∼70% of the self-diffusion coefficient of free water. There was no significant difference between self-diffusion in AGS from full-scale treatment plants and from lab-scale reactors. The results of the model showed that diffusional heterogeneity did not lead to a major change of flux into the granule (<1%). This study shows that differences between granular sludges and heterogeneity within granules have little impact on the kinetic properties of AGS. Thus, a relatively simple approach is sufficient to describe mass transport by diffusion into the granules.     

Successful bacterial incorporation into activated sludge flocs using alginate

Bioaugmentation experiments with the aerobic denitrifier Microvirgula aerodenitrificans were performed in an aerobic continuous stirred tank reactor (CSTR) treating urban wastewater. The fate of the added bacteria was monitored by a specific fluorescent oligonucleotide probe targeting 16S rRNA. The first addition of the strain led to its rapid disappearance because of grazing. Bacteria were then embedded within an alginate matrix before inoculation. Alginate fragments adhered to the existing flocs and were progressively colonized by the indigenous flora. Thereafter, microcolonies of the exogenous bacterium were found to be incorporated into existing flocs.     

Degrading high-strength phenol using aerobic granular sludge

Aerobic granules were adopted to degrade high-strength phenol wastewater in batch experiments. The acclimated granules effectively degraded phenol at a concentration of up to 5,000 mg l⁻¹ without severe inhibitory effects. The biodegradation of phenol by activated sludge was inhibited at phenol concentrations >3,000 mg l⁻¹. The granules were composed of cells embedded in a compact extracellular matrix. After acid or alkaline pretreatment, the granules continued to degrade phenol at an acceptable rate. The polymerase chain reaction-denaturing gradient gel electrophoresis technique was employed to monitor the microbial communities of the activated sludge and the aerobic granules following their being used to treat high concentrations of phenol in batch tests.     

PPCPs removal by aerobic granular sludge membrane bioreactor

An aerobic granular sludge membrane bioreactor (GMBR) was applied to the treatment of pharmaceutical and personal care products (PPCPs) wastewater. The influence of granular sludge on five antibiotic and antiphlogistic PPCPs wastewater and the removal effect of methyl alcohol and conventional organic matter were investigated while constantly reducing the density of inflow organic matter. The results showed that the sludge granulation process in the system was rapid but unstable, and that the system exhibits a dissolution–reunion dynamic equilibrium. The reactor demonstrated varying removal effects of PPCPs on different objects. The use of a GMBR was more effective for the removal of prednisolone, naproxen, and ibuprofen; the first two drugs were lower the average removal rate of which reached 98.46 and 84.02 %, respectively; whereas the average removal rate of ibuprofen was 63.32 %. By contrast, the GMBR has an insignificant degradation effect on antibiotics such as amoxicillin, indicating that such antibiotic medicine is not easily degraded by microorganisms, which plays different roles in system operation. Because of the different chemical structures and characteristics of drugs that result in various degradation behavior. During the GMBR granulation process, the value of mixed liquor volatility suspended solids (MLVSS) gradually increases from 1.5 to 4.1 g/L during the GMBR granulation process, and the removal rate of COD_Cr reaches up to 87.98 %. After reducing the density of organic matter is reduced, the removal rates of NH₃-N and TP both reach more than 90 %, respectively. Moreover, the proposed technique is considerably effective in the removal of methanol.     

Factors influencing the density of aerobic granular sludge

In the present study, the factors influencing density of granular sludge particles were evaluated. Granules consist of microbes, precipitates and of extracellular polymeric substance. The volume fractions of the bacterial layers were experimentally estimated by fluorescent in situ hybridisation staining. The volume fraction occupied by precipitates was determined by computed tomography scanning. PHREEQC was used to estimate potential formation of precipitates to determine a density of the inorganic fraction. Densities of bacteria were investigated by Percoll density centrifugation. The volume fractions were then coupled with the corresponding densities and the total density of a granule was calculated. The sensitivity of the density of the entire granule on the corresponding settling velocity was evaluated by changing the volume fractions of precipitates or bacteria in a settling model. Results from granules originating from a Nereda reactor for simultaneous phosphate COD and nitrogen removal revealed that phosphate-accumulating organisms (PAOs) had a higher density than glycogen-accumulating organisms leading to significantly higher settling velocities for PAO-dominated granules explaining earlier observations of the segregation of the granular sludge bed inside reactors. The model showed that a small increase in the volume fraction of precipitates (1–5 %) strongly increased the granular density and thereby the settling velocity. For nitritation–anammox granular sludge, mainly granular diameter and not density differences are causing a segregation of the biomass in the bed.     

Microbial populations and sludge characteristics in thermophilic aerobic sewage sludge digestion

Summary Estimates of bacterial numbers from raw sewage sludge and sludge treated by thermophilic aerobic digestion were compared with simple indicators of sludge quality and concentrations of potential substrates. Significant differences were found between sludge types for all but one of the variables examined (frequency of dividing cells). During a stable period of digestor operation, the average number of viable obligate thermophiles present in digested sludge (1.63 × 10⁶ ml^–1) was approximately 10²-fold greater than in feed sludge (1.10 × 10⁴ ml^–1). Total numbers of bacteria were slightly greater in digested sludge (3.24 × 10¹⁰ ml^–1) than in feed sludge (2.39 × 10 ml^–10), as were viable counts of bacteria at incubation temperatures of 37°C and 55°C. Significant correlation was found between viable counts of bacteria at 37°C and 55°C for digested sludge, and 65°C and 55°C for feed sludge. The numbers of obligate thermophiles present and the total of bacteria present were related to the temperature and pH of the digested sludge and inversely related to the numbers ofEscherichia coli and coliforms present, which were not detected at temperatures greater than 50°C.     

Organics and nitrogen removal and sludge stability in aerobic granular sludge membrane bioreactor

Novel aerobic granular sludge membrane bioreactor (GMBR) was established by combining aerobic granular sludge technology with membrane bioreactor (MBR). GMBR showed good organics removal and simultaneous nitrification and denitrification (SND) performances for synthesized wastewater. When influent total organic carbon (TOC) was 56.8-132.6 mg/L, the TOC removal of GMBR was 84.7-91.9%. When influent ammonia nitrogen was 28.1-38.4 mg/L, the ammonia nitrogen removal was 85.4-99.7%, and the total nitrogen removal was 41.7-78.4%. Moreover, batch experiments of sludge with different particle size demonstrated that: (1) flocculent sludge under aerobic condition almost have no denitrification capacity, (2) SND capacity was caused by the granular sludge, and (3) the denitrification rate and total nitrogen removal efficiency were enhanced with the increased particle size. In addition, study on the sludge morphology stability in GMBR showed that, although some granular sludge larger than 0.9 mm disaggregated at the beginning of operation, the granular sludge was able to maintain the stability of its granular morphology, and at the end of operation, the amount of granular sludge (larger than 0.18 mm) stabilized in GMBR was more than 56-62% of the total sludge concentration. The partial disaggregation of large granules is closely associated with the change of operating mode from sequencing batch reactor (SBR) system to MBR system.     

Comparison of soluble microbial products released from activated sludge and aerobic granular sludge systems in the presence of toxic 2,4-dichlorophenol

Bioprocess and Biosystems Engineering - The objective of this study was to compare the release of soluble microbial products (SMP) from activated sludge (AS) and aerobic granular sludge (AGS) in...     

Microbial composition and structure of aerobic granular sewage biofilms

Aerobic activated sludge granules are dense, spherical biofilms which can strongly improve purification efficiency and sludge settling in wastewater treatment processes. In this study, the structure and development of different granule types were analyzed. Biofilm samples originated from lab-scale sequencing batch reactors which were operated with malthouse, brewery, and artificial wastewater. Scanning electron microscopy, light microscopy, and confocal laser scanning microscopy together with fluorescence in situ hybridization (FISH) allowed insights into the structure of these biofilms. Microscopic observation revealed that granules consist of bacteria, extracellular polymeric substances (EPS), protozoa and, in some cases, fungi. The biofilm development, starting from an activated sludge floc up to a mature granule, follows three phases. During phase 1, stalked ciliated protozoa of the subclass Peritrichia, e.g., Epistylis spp., settle on activated sludge flocs and build tree-like colonies. The stalks are subsequently colonized by bacteria. During phase 2, the ciliates become completely overgrown by bacteria and die. Thereby, the cellular remnants of ciliates act like a backbone for granule formation. During phase 3, smooth, compact granules are formed which serve as a new substratum for unstalked ciliate swarmers settling on granule surfaces. These mature granules comprise a dense core zone containing bacterial cells and EPS and a loosely structured fringe zone consisting of either ciliates and bacteria or fungi and bacteria. Since granules can grow to a size of up to several millimeters in diameter, we developed and applied a modified FISH protocol for the study of cryosectioned biofilms. This protocol allows the simultaneous detection of bacteria, ciliates, and fungi in and on granules.     

Neural network analysis of the diffusional limitations in activated sludge flocs

This work deals with the development of a comprehensive but simplified version of floc model for the activated sludge process. The rate expression of the type used by the IAWPRC Task Group was employed in this study to represent the reaction kinetics inside the floc matrix. The intraparticle mass transport was then incorporated to account for the effects of the diffusional limitations. Solution to the floc model was obtained by orthogonal collocation technique. Since the actual reactions are not only related to the sludge floc size and the bulk characteristics but are also associated with the floc size distribution, the concept of overall effectiveness factor was therefore used. Evaluation of overall effectiveness factors using neural network analysis was carried out and potential control strategies were discussed.     

Bacterial diversity in thermophilic aerobic sewage sludge

Summary Using -amylase as an example, extremely thermophilic Bacilli isolated from heat-treated sewage sludge are shown to be a source for enzymes stable and active at high temperatures. The isolates which are classified as subspecies of Bacillus stearothermophilus differ from each other in protein composition indicating the heterogeneiety of that subspecies. Media are evaluated for good growth and high enzyme productivity. Best media are those composed of three or four different complex components like combinations of peptone, soy grist, and malt extract, -amylase production on simple carbon sources is negligible. From the cultivation supernatants crude -amylase extracts are prepared and their behaviour at high temperatures is described. The optimal temperature of all tested enzymes is 80°C. They are stable at suboptimal temperatures for over 20 h and at 95° C 50% of their activity is lost within 2 h. The activity at 95° C is however preserved for over 3 h in presence of starch. The products of the starch digestion are maltotriose, maltose, and some glucose. The amylases can therefore compete in activity and stability with commercially available -amylases from Bacillus licheniformis.     

Proteolytic activity in stored aerobic granular sludge and structural integrity

Aerobic granules lose stability during storage. The goal of this work was to highlight the main cause of stability loss for stored granules as intracellular protein hydrolysis. The quantity of extracellular proteins was noted to be significantly lower during granule storage, and protease enzyme activities were correspondingly higher in the cores of stored granules. The proteolytic bacteria, which secrete highly active protease enzymes, were for the first time isolated and characterized by analyzing 16S rDNA sequences. The proteolytic bacteria belonged to the genera Pseudomonas, Raoultella, Acinetobacter, Pandoraea, Klebsiella, Bacillus and uncultured bacterium, and were grouped into Proteobacteria, Enterobacteria and Firmicutes. The PB1 (Pseudomonas aeruginosa) strain, which exhibited very high proteolytic activity during the skim milk agar test, was located at the core regime with active protease enzymes, and was close to the obligate anaerobic strain Bacteroides sp. Hence, the extracellular proteins in stored granules were proposed to be hydrolyzed by enzymes secreted by proteolytic bacteria with the hydrolyzed products ultimately being used by nearby anaerobic strains. This process gradually digests the protein core, and eventually consumes the entire granule.     

Improved phosphate removal by selective sludge discharge in aerobic granular sludge reactors

Two lab-scale aerobic granular sludge sequencing batch reactors were operated at 20 and 30°C and compared for phosphorus (P) removal efficiency and microbial community composition. P-removal efficiency was higher at 20°C (>90%) than at 30°C (60%) when the sludge retention time (SRT) was controlled at 30 days by removing excess sludge equally throughout the sludge bed. Samples analyzed by fluorescent in situ hybridization (FISH) indicated a segregation of biomass over the sludge bed: in the upper part, Candidatus Competibacter phosphatis (glycogen-accumulating organisms--GAOs) were dominant while in the bottom, Candidatus Accumulibacter phosphatis (polyphosphate-accumulating organisms--PAOs) dominated. In order to favour PAOs over GAOs and hence improve P-removal at 30°C, the SRT was controlled by discharging biomass mainly from the top of the sludge bed (80% of the excess sludge), while bottom granules were removed in minor proportions (20% of the excess sludge). With the selective sludge removal proposed, 100% P-removal efficiency was obtained in the reactor operated at 30°C. In the meantime, the biomass in the 30°C reactor changed in color from brownish-black to white. Big white granules appeared in this system and were completely dominated by PAOs (more than 90% of the microbial population), showing relatively high ash content compared to other granules. In the reactor operated at 20°C, P-removal efficiency remained stable above 90% regardless of the sludge removal procedure for SRT control. The results obtained in this study stress the importance of sludge discharge mainly from the top as well as in minor proportions from the bottom of the sludge bed to control the SRT in order to prevent significant growth of GAOs and remove enough accumulated P from the system, particularly at high temperatures (e.g., 30°C).