Microbial communities in activated sludge performing enhanced biological phosphorus removal in a sequencing batch reactor

Microbial communities of activated sludge in an anaerobic/aerobic sequencing batch reactor (SBR) supplied with acetate as sole carbon source were analyzed to identify the microorganisms responsible for enhanced biological phosphorus removal. Various analytical methods were used such as electron microscopy, quinone, slot hybridization, and 16S rRNA gene sequencing analyses. Electron photomicrographs showed that coccus-shaped microorganisms of about 1 microm diameter dominated the microbial communities of the activated sludge in the SBR, which had been operated for more than 18 months. These microorganisms contained polyphosphate granules and glycogen inclusions, which suggests that they are a type of phosphorus-accumulating organism. Quinones, slot hybridization, and 16S rRNA sequencing analyses showed that the members of the Proteobacteria beta subclass were the most abundant species and were affiliated with the Rhodocyclus-like group. Phylogenetic analysis revealed that the two dominating clones of the beta subclass were closely related to the Rhodocyclus-like group. It was concluded that the coccus-shaped organisms related to the Rhodocyclus-like group within the Proteobacteria beta subclass were the most dominant species believed responsible for biological phosphorus removal in SBR operation with acetate.     

A conceptual ecosystem model of microbial communities in enhanced biological phosphorus removal plants

The microbial populations in 25 full-scale activated sludge wastewater treatment plants with enhanced biological phosphorus removal (EBPR plants) have been intensively studied over several years. Most of the important bacterial groups involved in nitrification, denitrification, biological P removal, fermentation, and hydrolysis have been identified and quantified using quantitative culture-independent molecular methods. Surprisingly, a limited number of core species was present in all plants, constituting on average approx. 80% of the entire communities in the plants, showing that the microbial populations in EBPR plants are rather similar and not very diverse, as sometimes suggested. By focusing on these organisms it is possible to make a comprehensive ecosystem model, where many important aspects in relation to microbial ecosystems and wastewater treatment can be investigated. We have reviewed the current knowledge about these microorganisms with focus on key ecophysiological factors and combined this into a conceptual ecosystem model for EBPR plants. It includes the major pathways of carbon flow with specific organic substances, the dominant populations involved in the transformations, interspecies interactions, and the key factors controlling their presence and activity. We believe that the EBPR process is a perfect model system for studies of microbial ecology in water engineering systems and that this conceptual model can be used for proposing and testing theories based on microbial ecosystem theories, for the development of new and improved quantitative ecosystem models and is beneficial for future design and management of wastewater treatment systems.     

Glycerol as a sole carbon source for enhanced biological phosphorus removal

Wastewaters with low organic matter content are one of the major causes of EBPR failures in full-scale WWTP. This carbon source deficit can be solved by external carbon addition and glycerol is a perfect candidate since it is nowadays obtained in excess from biodiesel production. This work shows for the first time that glycerol-driven EBPR with a single-sludge SBR configuration is feasible (i.e. anaerobic glycerol degradation linked to P release and aerobic P uptake). Two different strategies were studied: direct replacement of the usual carbon source for glycerol and a two-step consortium development with glycerol anaerobic degraders and PAO. The first strategy provided the best results. The implementation of glycerol as external carbon source in full-scale WWTP would require a suitable anaerobic hydraulic retention time. An example using dairy wastewater with a low COD/P ratio confirms the feasibility of using glycerol as an external carbon source to increase P removal activity. The approach used in this work opens a new range of possibilities and, similarly, other fermentable substrates can be used as electron donors for EBPR.     

Modelling biological and chemically induced precipitation of calcium phosphate in enhanced biological phosphorus removal systems

The biologically induced precipitation processes can be important in wastewater treatment, in particular treating raw wastewater with high calcium concentration combined with Enhanced Biological Phosphorus Removal. Currently, there is little information and experience in modelling jointly biological and chemical processes. This paper presents a calcium phosphate precipitation model and its inclusion in the Activated Sludge Model No 2d (ASM2d). The proposed precipitation model considers that aqueous phase reactions quickly achieve the chemical equilibrium and that aqueous-solid change is kinetically governed. The model was calibrated using data from four experiments in a Sequencing Batch Reactor (SBR) operated for EBPR and finally validated with two experiments. The precipitation model proposed was able to reproduce the dynamics of amorphous calcium phosphate (ACP) formation and later crystallization to hydroxyapatite (HAP) under different scenarios. The model successfully characterised the EBPR performance of the SBR, including the biological, physical and chemical processes.     

Seasonal bacterial community dynamics in a full-scale enhanced biological phosphorus removal plant

Activated sludge is one of the most abundant and effective wastewater treatment process used to treat wastewater, and has been used in developed countries for nearly a century. In all that time, several hundreds of studies have explored the bacterial communities responsible for treatment, but most studies were based on a handful of samples and did not consider temporal dynamics. In this study, we used the DNA fingerprinting technique called automated ribosomal intergenic spacer region analysis (ARISA) to study bacterial community dynamics over a two-year period in two different treatment trains. We also used quantitative PCR to measure the variation of five phylogenetically-defined clades within the Accumulibacter lineage, which is a model polyphosphate accumulating organism. The total bacterial community exhibited seasonal patterns of change reminiscent of those observed in lakes and oceans. Surprisingly, all five Accumulibacter clades were present throughout the study, and the total Accumulibacter community was relatively stable. However, the abundance of each clade did fluctuate through time. Clade IIA dynamics correlated positively with temperature (ρ = 0.65, p < 0.05) while Clade IA dynamics correlated negatively with temperature (ρ = −0.35, p < 0.05). This relationship with temperature hints at the mechanisms that may be driving the seasonal patterns in overall bacterial community dynamics and provides further evidence for ecological differentiation among clades within the Accumulibacter lineage. This work provides a valuable baseline for activated sludge bacterial community variation.     

Enhanced biological phosphorus removal by granular sludge: From macro- to micro-scale

In this study, phosphorus accumulating microbial granules were successfully cultivated in a sequencing batch reactor (SBR) using synthetic wastewater. The average diameter of the granules was 0.74 mm and the diameter distribution fitted well with normal distribution with a correlation coefficient of 0.989. Good performance of biological phosphorus removal (BPR) was obtained in the granular system. The average phosphorus removal efficiency was over 94.3% and the level of phosphorus in the effluent was below 0.50 mg/L during 300 days of operation. Particle analysis showed that positive charged particles were formed with the release of phosphorus in the anaerobic stage. These particles served as the cores of granules and stimulate the granulation. The maturated granules had a well-formed micro-pore structure with an average pore width between 291.5 nm and 446.5 nm. The spatial distribution of phosphorus decreased gradually from the surface to the center of the granules. Smaller granules had a higher specific area, pore width and phosphorus removal activity than bigger granules.     

生物除磷机理与新工艺

综述了生物除磷的PAO和DPB原理,介绍了PASF、Dephanox和A3N-SBR三种新的脱氮除磷工艺,从而达到提高脱氮除磷效果,消除水环境污染的目的。     

In situ identification and characterization of the microbial community structure of full-scale enhanced biological phosphorous removal plants in Japan

Fluorescent in situ hybridization (FISH) and polyphosphate (polyP) staining methods were used to characterize the microbial community structure of 13 activated sludge samples taken from nine different Japanese wastewater treatment plants with and without enhanced biological phosphorous removal (EBPR) activities. FISH with published rRNA-targeted oligonucleotide probes for important bacterial groups involving in the EBPR process revealed that Rhodocyclus-related polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms from a gammaproteobacterial lineage GB were the predominant populations detected, representing 4-18% and 10-31% of EUBmix-stained cells, respectively, in those samples. However, a considerable proportion of Rhodocyclus-related PAO cells were observed with no polyP granules accumulated based on polyP staining. This was further supported by a poor correlation between Rhodocyclus-related PAO population and sludge total phosphorous (TP) contents. In contrast, high correlations between polyP-stained cells and sludge TP contents were observed. In particular, among those polyP-stained cells in samples Ariake_A2O and Nakano_AO, more than 85% of them could not be targeted by probe PAOmix. These non-Rhodocyclus-related PAOs included populations from other bacterial divisions and members of the Betaproteobacteria other than those in Rhodocyclus-related group.     

Biochemical model for enhanced biological phosphorus removal

Enhanced biological phosphorus (bio-P) removal from wastewater is a promising technology for which the fundamental mechanisms are still unclear. The purpose of this paper is to present a biochemical model that explains bio-P removal mechanisms occurring under anaerobic, aerobic and anoxic conditions of the process. A bio-P bacterium is referred to as one that can store both polyphosphate and carbon (as poly-β-hydroxybutyrate for example). In this communication, observations from the literature are first reviewed and mechanisms of bacterial bioenergetics and membrane transport are summarized. The model for bio-P metabolism under anaerobic, aerobic and anoxic conditions is then presented. The role of polyphosphate under anaerobic conditions is suggested to be as a source of energy both for the reestablishment of the proton motive force, which would be consumed by substrate transport and for substrate storage. The role of the anaerobic zone is to maximize the storage of organic substrates in bio-P bacteria. For this purpose the supply of readily available substrates should be maximized and the presence of electron acceptors (molecular oxygen or oxidized nitrogen) minimized. Under subsequent aerobic or anoxic conditions, bio-P bacteria will accumulate polyphosphates in response to the availability of electron acceptors (oxygen or oxidized nitrogen) for energy production. Carbon reserves in bio-P bacteria should provide energy for growth and for soluble phosphate accumulation as polyphosphate reserves.     

Long-term impact of anaerobic reaction time on the performance and granular characteristics of granular denitrifying biological phosphorus removal systems

Removal of nitrogen and phosphorus (P) from wastewater is successfully and widely practiced in systems employing both granular sludge technology and enhanced biological P removal (EBPR) processes; however, the key parameter, anaerobic reaction time (AnRT), has not been thoroughly investigated. Successful EBPR is highly dependent on an appropriate AnRT, which induces carbon and polyphosphate metabolism by phosphorus accumulating organisms (PAOs). Therefore, the long-term impact of AnRT on denitrifying P removal performance and granular characteristics was investigated in three identical granular sludge sequencing batch reactors with AnRTs of 90 (R1), 120 (R2) and 150 min (R3). The microbial community structures and anaerobic stoichiometric parameters related to various AnRTs were monitored over time. Free nitrite acid (FNA) accumulation (e.g., 0.0008–0.0016 mg HNO₂–N/L) occurred frequently owing to incomplete denitrification in the adaptation period, especially in R3, which influenced the anaerobic/anoxic intracellular intermediate metabolites and activities of intracellular enzymes negatively, resulting in lower levels of poly-P and reduced activity of polyphosphate kinase. As a result, the Accumulibacter-PAOs population decreased from 51 ± 2.5% to 43 ± 2.1% when AnRT was extended from 90 to 150 min, leading to decreased denitrifying P removal performance. Additionally, frequent exposure of microorganisms to the FNA accumulation and anaerobic endogenous conditions in excess AnRT cases (e.g., 150 min) stimulated increased extracellular polymeric substances (EPS) production by microorganisms, resulting in enhanced granular formation and larger granules (size of 0.6–1.2 mm), but decreasing anaerobic PHA synthesis and glycogen hydrolysis. Phosphorus removal capacity was mediated to some extent by EPS adsorption in granular sludge systems that possessed more EPS, longer AnRT and relatively higher GAOs.     

A comparative study of fouling-related properties of sludge from conventional and membrane enhanced biological phosphorus removal processes

The physical and biochemical properties of activated sludge mixed liquor, including floc size distribution, zeta potential, relative hydrophobicity, and bound and unbound (soluble) extracellular polymeric substances (EPS), were examined in this study to evaluate their relationship to membrane fouling. Mixed liquors from a membrane enhanced biological phosphorus removal (MEBPR) process and a conventional enhanced biological phosphorus removal (CEBPR) process were compared. It was found that the floc size distribution and the amount of soluble EPS in the mixed liquor were the most important properties that significantly influenced the fouling propensity of sludge. Contrary to the literature, the content of EPS bound in activated sludge flocs was not found to be directly associated with membrane fouling, and sludge surface properties such as zeta potential and relative hydrophobicity were not closely related to the observed differences in the fouling tendencies of the two types of sludge.     

Improved biological phosphorus removal performance driven by the aerobic/extended-idle regime with propionate as the sole carbon source

Our previous studies proved that biological phosphorus removal (BPR) could be achieved in an aerobic/extended-idle (AEI) process employing two typical substrates of glucose and acetate as the carbon sources. This paper further evaluated the feasibility of another important substrate, propionate, serving as the carbon source for BPR in the AEI process, and compared the BPR performance between the AEI and anaerobic/oxic (A/O) processes. Two sequencing batch reactors (SBRs) were operated, respectively, as the AEI and A/O regimes for BPR using propionate as the sole substrate. The results showed that the AEI-reactor removed 2.98 ± 0.04-4.06 ± 0.06 mg of phosphorus per g of total suspended solids during the course of the steady operational trial, and the phosphorus content of the dried sludge was reached 8.0 ± 0.4% after 56-day operation, demonstrating the good performance of phosphorus removal. Then, the efficiencies of BPR and the transformations of the intracellular storages were compared between two SBRs. It was observed that the phosphorus removal efficiency was maintained around 95% in the AEI-reactor, and about 83% in the A/O-reactor, although the latter showed much greater transformations of both polyhydroxyalkanoates and glycogen. The facts clearly showed that BPR could be enhanced by the AEI regime using propionate as the carbon source. Finally, the mechanisms for the propionate fed AEI-reactor improving BPR were investigated. It was found that the sludge cultured by the AEI regime had more polyphosphate containing cells than that by the A/O regime. Further investigation revealed that the residual nitrate generated in the last aerobic period was readily deteriorated BPR in the A/O-SBR, but a slight deterioration was observed in the AEI-SBR. Moreover, the lower glycogen transformation measured in the AEI-SBR indicated that the biomass cultured by the AEI regime contained less glycogen accumulating organisms activities than that by the A/O regime.     

The efficiency of enhanced biological phosphorus removal from real wastewater affected by different ratios of acetic to propionic acid

The effect of different ratios of propionic to acetic acid on the efficiency of enhanced biological phosphorus removal (EBPR) from real wastewater supplemented with volatile fatty acids (VFAs) was investigated. Two sequencing batch reactors (SBRs) were used to acclimate two types (SBR1 and SBR2) of biomass. They were cultured and studied using real wastewater with an average propionic to acetic acid carbon molar ratio of 0.16 and 2.06, respectively. The laboratory results showed that for a given long-term cultured biomass the more the soluble ortho-phosphate (SOP) was released in the anaerobic stage, the higher the SOP was taken up in the aerobic phase. However, the SBR2 biomass had a much greater SOP uptake to release ratio than SBR1, which resulted in a higher SOP removal efficiency than SBR1 (average 87.3% versus 76.9% in SBRs experiments, and 93.5% against 68.1% in batch tests). The SBR2 biomass therefore had a higher SOP uptake ability than the SBR1 for a given amount of SOP release. In addition, the SBR1 had a higher secondary SOP release following VFAs uptake. It was found that the SBR2 biomass synthesized and utilized less observable polyhydroxyalkanoates (PHAs) during the anaerobic and aerobic stage respectively than SBR1. The apparent PHAs utilization efficiency for SOP uptake with the SBR2 biomass was much greater than with the SBR1, and the SBR2 biomass synthesized less glycogen during aerobiosis than SBR1, which might mean a higher PHAs fraction was used for SOP removal, resulting in the increased efficiency with the long-term cultured SBR2 biomass. Higher propionic acid content led to superior EBPR in long-term cultivation, but was transiently detrimental in the short term.     

Advancing post-anoxic denitrification for biological nutrient removal

The objective of this research was to advance a fundamental understanding of a unique post-anoxic denitrification process for achieving biological nutrient removal (BNR), with an emphasis on elucidating the impacts of surface oxygen transfer (SOT), variable process loadings, and bioreactor operational conditions on nitrogen and phosphorus removal. Two sequencing batch reactors (SBRs) were operated in an anaerobic/aerobic/anoxic mode for over 250 days and fed real municipal wastewater. One SBR was operated with a headspace open to the atmosphere, while the other had a covered liquid surface to prevent surface oxygen transfer. Process performance was assessed for mixed volatile fatty acid (VFA) and acetate-dominated substrate, as well as daily/seasonal variance in influent phosphorus and ammonia loadings. Results demonstrated that post-anoxic BNR can achieve near-complete (>99%) inorganic nitrogen and phosphorus removal, with soluble effluent concentrations less than 1.0 mgN L⁻¹ and 0.14 mgP L⁻¹. Observed specific denitrification rates were in excess of typical endogenous values and exhibited a linear dependence on the glycogen concentration in the biomass. Preventing SOT improved nitrogen removal but had little impact on phosphorus removal under normal loading conditions. However, during periods of low influent ammonia, the covered reactor maintained phosphorus removal performance and showed a greater relative abundance of polyphosphate accumulating organisms (PAOs) as evidenced by quantitative real-time PCR (qPCR). While GAOs were detected in both reactors under all operational conditions, BNR performance was not adversely impacted. Finally, secondary phosphorus release during the post-anoxic period was minimal and only occurred if nitrate/nitrite were depleted post-anoxically.     

生物法除磷的研究进展

叙述了生物法利用聚磷菌和反硝化聚磷菌除磷原理,研究了不同运行方式、有机物浓度及其种类、厌氧段NO3-浓度和污泥龄等因素对生物除磷产生的影响,并指出现阶段生物除磷存在的问题,以期对生物除磷的试验和实际生产提供帮助。     

The effect of pH on the competition between polyphosphate-accumulating organisms and glycogen-accumulating organisms

In enhanced biological phosphorus removal (EBPR) processes, glycogen-accumulating organisms (GAOs) may compete with polyphosphate-accumulating organisms (PAOs) for the often-limited carbon substrates, potentially resulting in disturbances to phosphorus removal. A detailed investigation of the effect of pH on the competition between PAOs and GAOs is reported in this study. The results show that a high external pH ( approximately 8) provided PAOs with an advantage over GAOs in EBPR systems. The phosphorus removal performance improved due to a population shift favouring PAOs over GAOs, which was shown through both chemical and microbiological methods. Two lab-scale reactors fed with propionate as the carbon source were subjected to an increase in pH from 7 to 8. The phosphorus removal and PAO population (as measured by quantitative fluorescence in situ hybridisation analysis of "Candidatus Accumulibacter phosphatis") increased in each system, where the PAOs appeared to out-compete a group of Alphaproteobacteria GAOs. A considerable improvement in the P removal was also observed in an acetate fed reactor, where the GAO population (primarily "Candidatus Competibacter phosphatis") decreased substantially after a similar increase in the pH. The results from this study suggest that pH could be used as a control parameter to reduce the undesirable proliferation of GAOs and improve phosphorus removal in EBPR systems.     

Effect of soluble microbial products on microbial metabolisms related to nutrient removal

In this study, the effect of soluble microbial products (SMP) on the metabolisms related to phosphate or nitrogen removal of activated sludge was investigated. Two anaerobic-aerobic activated sludge processes were operated, one with a hydraulic retention time (HRT) of 48 h (RunL) and the other 6.4 h (RunS). The longer HRT of RunL was intended to promote the accumulation of SMPs in the supernatant. With the sludge from RunS and the supernatant from both of the runs, supernatant exchange batch experiments (SEBEs) were conducted, in which the acetate uptake rate and phosphate release rates under anaerobic conditions and the phosphate uptake rate under aerobic conditions were measured as these metabolisms are related to enhanced biological phosphorus removal. The nitrification rate was also measured. The statistical analyses of the results from the SEBEs showed that the supernatant from RunL had an inhibitory effect on the anaerobic acetate uptake and nitrification of the sludge from RunS. The cause of which was attributed to SMPs in the supernatant from RunL. As a result, the inhibitory effect of SMPs on nitrification and anaerobic acetate uptake was confirmed.     

污水除磷及回收技术

介绍了当前污水中除磷的主要方法,阐述了各种除磷方法的原理、优缺点以及常用的处理工艺,对磷回收的原理、工艺做了简单的论述,以加强对除磷技术的研究,实现磷的可持续发展。     

Phosphorus release-storage reaction and organic substrate behavior in biological phosphorus removal

This paper represents the results of an experimental investigation conducted on the mechanism of biological phosphorus removal. The relationship between phosphorus release-storage reaction, and behavior of extracellular and intracellular organic substrates under anaerobic-aerobic conditions is studied in detail.The results obtained are as follows: (1) the amount of intracellular carbohydrate increases under the presence of extracellular glucose, but decreases when extracellular glucose is depleted in the anaerobic condition. (2) The amount of intracellular poly-β-hydroxybutylate (PHB) gradually increases under the anaerobic condition. The increase in intracellular PHB content appears to be related to the decrease in intracellular carbohydrate content when extracellular glucose is depleted. (3) Rate of phosphorus release under the anaerobic condition is related to the amount of releasable phosphorus in the cells. The observed ratios of postulated “maximum phosphorus storage capacity” to total amount of intracellular phosphorus are similar to those of the low molecular weight polyphosphate fraction in the cells. (4) Release of phosphorus under the anaerobic condition appears to be related to both ingestion of extracellular organic substrates and formation of intracellular PHB. (5) Release of phosphorus under the anaerobic condition appears to be limited once a fixed portion of intracellular phosphorus is released, even if substantial amount of extracellular organic substrate still remains available. (6) The amount of intracellular PHB increases in the subsequent aerobic condition under presence of a sufficient amount of extracellular organic substrate, but the amount decreases when the extracellular organic substrate is depleted. Similarly, the amount of intracellular carbohydrate initially increases, then gradually decreases following the decrease in PHB content. (7) Ingestion rate for phosphorus in the aerobic condition appears to be dependent on unsaturated storage capacity of intracellular phosphorus as well as on the concentration of extracellular phosphorus.     

Kinetics of anaerobic orthophosphate release and substrate uptake in enhanced biological phosphorus removal from synthetic wastewater

This paper presents some new results about the kinetics of orthophosphate release and substrate uptake occurring under anaerobic conditions in a lab-scale activated sludge plant consisting of an anaerobic first and an aerobic second step A/O-process. The synthetic wastewater contained acetate, peptone and yeast extract. The mixed culture was enriched from activated sludge samples from a large-scale plant with enhanced biological phosphorus removal. Anaerobic batch experiments were carried out with this enriched mixed culture using acetate as the only source for carbon and energy. In the first experiments, high acetate concentration was only partly consumed by the bacterial culture, resulting in a nearly total emptying of the poly-P-store (poly-P-limitation). In the next experiments, only a relatively low acetate concentration was added, resulting in a total acetate uptake and in partial emptying of the poly-P-store (acetate-limitation). This experimental strategy was successful in studying the complex kinetics of the anaerobic process in enhanced phosphorus removal. Most of the calculated kinetic coefficients depend on temperature; a dependence on pH cannot be postulated with certainty. A comparison with known results shows conformity but also differences.