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Ca. Accumulibacter was the predominant microorganism (relative FISH bio-abundance of 67 ± 5%) in a lab-scale sequential batch reactor that accomplished enhanced biological phosphorus removal (EBPR) while using glucose and acetate as the carbon sources (1:1 COD-based ratio). Both organic compounds were completely anaerobically consumed. The reactor's performance in terms of P/C ratio, phosphorous release and uptake, and overall kinetic and stoichiometric parameters were on the high end of the reported spectrum for EBPR systems (100:9.3 net mg phosphate removal per mg COD consumed when using glucose and acetate in a 1:1 ratio). The batch tests showed that, to the best of our knowledge, this is the first time a reactor enriched with Ca. Accumulibacter can putatively utilize glucose as the sole carbon source to biologically remove phosphate (COD:P (mg/mg) removal ratio of 100:6.3 when using only glucose). Thus, this research proposes that Ca. Accumulibacter directly anaerobically stored the fed glucose primarily as glycogen by utilizing the ATP provided via the hydrolysis of poly-P and secondarily as PHA by balancing its ATP utilization (glycogen generation) and formation (PHA storage). Alternative hypotheses are also discussed. The reported findings could challenge the conventional theories of glucose assimilation by Ca. Accumulibacter, and can be of significance for the biological removal of phosphorus from wastewaters with high contents of fermentable compounds or low VFAs.

Although simultaneous P-removal and nitrate reduction has been observed in laboratory studies as well as full-scale plants, there are contradictory reports on the ability of PAO I to efficiently use nitrate as electron acceptor. Such discrepancy could be due to other microbial groups performing partial denitrification from nitrate to nitrite. The denitrification capacities of two different cultures, a highly enriched PAO I and a PAO I-GAO cultures were assessed through batch activity tests conducted before and after acclimatization to nitrate. Negligible anoxic phosphate uptake coupled with a reduction of nitrate was observed in the highly enriched PAO I culture. On the opposite, the PAO I-GAO culture showed a higher anoxic phosphate uptake activity. Both cultures exhibited good anoxic phosphate uptake activity with nitrite (8.7 ± 0.3 and 9.6 ± 1.8 mgPO4-P/gVSS.h in the PAO I and PAO I-GAO cultures, respectively). These findings suggest that other microbial populations, such as GAOs, were responsible to reduce nitrate to nitrite in this EBPR system, and that PAO I used the nitrite generated for anoxic phosphate uptake. Moreover, the simultaneous denitrification and phosphate removal process using nitrite as electron acceptor may be a more sustainable process as can: i) reduce the carbon consumption, ii) reduce oxygen demand of WWTP, and iii) due to a lower growth yield contribute to a lower sludge production.

Phosphate accumulating organisms (PAO) are assumed to use nitrate as external electron acceptor, allowing an efficient integration of simultaneous nitrogen and phosphate removal with minimal organic carbon (COD) requirements. However, contradicting findings appear in literature regarding the denitrification capacities of PAO due to the lack of clade specific highly enriched PAO cultures. Whereas some studies suggest that only PAO clade I may be capable of using nitrate as external electron acceptor for anoxic P-uptake, other studies indicate that PAO clade II may be responsible for anoxic P-removal. In the present study, a highly enriched PAO clade IC culture (>99% according to FISH) was cultivated in an SBR operated under Anaerobic/Oxic conditions and subsequently exposed to Anaerobic/Anoxic/Oxic conditions using nitrate as electron acceptor. Before and after acclimatization to the presence of nitrate, the aerobic and anoxic (nitrate and nitrite) activities of the PAO I culture were assessed through the execution of batch tests using either acetate or propionate as electron donor. In the presence of nitrate, significant P-uptake by PAO I was not observed before or after acclimatization. Using nitrite as electron acceptor, limited nitrite removal rates were observed before acclimatization with lower rates in the acetate fed reactor without P-uptake and slightly higher in the propionate fed reactor with a marginal anoxic P-uptake. Only after acclimatization to nitrate, simultaneous P and nitrite removal was observed. This study suggests that PAO clade IC is not capable of using nitrate as external electron acceptor for anoxic P-removal. The elucidation of the metabolic capacities for individual PAO clades helps in better understanding and optimization of the relation between microbial ecology and process performance in enhanced biological phosphate removal processes.

The influence of different carbon sources (acetate to propionate ratios), temperature and pH levels on the competition between polyphosphate- and glycogen-accumulating organisms (PAO and GAO, respectively) was evaluated using a metabolic model that incorporated the carbon source, temperature and pH dependences of these microorganisms. The model satisfactorily described the bacterial activity of PAO (Accumulibacter) and GAO (Competibacter and Alphaproteobacteria-GAO) laboratory-enriched cultures cultivated on propionate (HPr) and acetate (HAc) at standard conditions (20 degrees C and pH 7.0). Using the calibrated model, the effects of different influent HAc to HPr ratios (100-0, 75-25, 50-50 and 0-100%), temperatures (10, 20 and 30 degrees C) and pH levels (6.0, 7.0 and 7.5) on the competition among Accumulibacter, Competibacter and Alphaproteobacteria-GAO were evaluated. The main aim was to assess which conditions were favorable for the existence of PAO and, therefore, beneficial for the biological phosphorus removal process in sewage treatment plants. At low temperature (10 degrees C), PAO were the dominant microorganisms regardless of the used influent carbon source or pH. At moderate temperature (20 degrees C), PAO dominated the competition when HAc and HPr were simultaneously supplied (75-25 and 50-50% HAc to HPr ratios). However, the use of either HAc or HPr as sole carbon source at 20 degrees C was not favorable for PAO unless a high pH was used (7.5). Meanwhile, at higher temperature (30 degrees C), GAO tended to be the dominant microorganisms. Nevertheless, the combined presence of acetate and propionate in the influent (75-25 and 50-50% HAc to HPr ratios) as well as a high pH (7.5) appear to be potential factors to favor the metabolism of PAO over GAO at higher sewage temperature (30 degrees C).

The anaerobic acetate (HAc) uptake stoichiometry of phosphorus-accumulating organisms (PAO) in enhanced biological phosphorus removal (EBPR) systems has been an extensive subject of study due to the highly variable reported stoichiometric values (e.g. anaerobic P-release/HAc-uptake ratios ranging from 0.01 up to 0.93 P-mol/C-mol). Often, such differences have been explained by the different applied operating conditions (e.g. pH) or occurrence of glycogen-accumulating organisms (GAO). The present study investigated the ability of biomass highly enriched with specific PAO clades (‘Candidatus Accumulibacter phosphatis’ Clade I and II, hereafter PAO I and PAO II) to adopt a GAO metabolism. Based on long-term experiments, when Poly-P is not stoichiometrically limiting for the anaerobic VFA uptake, PAO I performed the typical PAO metabolism (with a P/HAc ratio of 0.64 P-mol/C-mol); whereas PAO II performed a mixed PAO-GAO metabolism (showing a P/HAc ratio of 0.22 P-mol/C-mol). In short-term batch tests, both PAO I and II gradually shifted their metabolism to a GAO metabolism when the Poly-P content decreased, but the HAc-uptake rate of PAO I was 4 times lower than that of PAO II, indicating that PAO II has a strong competitive advantage over PAO I when Poly-P is stoichiometrically limiting the VFA uptake. Thus, metabolic flexibility of PAO clades as well as their intrinsic differences are additional factors leading to the controversial anaerobic stoichiometry and kinetic rates observed in previous studies. From a practical perspective, the dominant type of PAO prevailing in full-scale EBPR systems may affect the P-release processes for biological or combined biological and chemical P-removal and recovery and consequently the process performance.

In this study the following was evaluated: (a) application of a complex activated sludge model on a full-scale plant (Phostrip ® -like process), (b) influent and sludge characterization procedures for bio-P modeling, (c) the use of batch tests for model evaluation and (d) different alternative BPR process configurations (A/O, UCT and BCFS ® ). An integrated model for aerobic and denitrifying biological phosphorus removal (Delft bio-P model) replaced the module for P removal of ASM No. 2 and was combined with retained equations for COD and N conversion of the ASM No 2. This combined model proved well capable of describing the wastewater treatment plant (wwtp) Haarlem Waarderpolder with adjustment of only three (out of the sixty) default parameters. Some of batch tests were satisfactorily described by the model too. Batch tests proved as a useful tool for sludge characterization and model validation. The standard Dutch procedure for influent and sludge characterization proved satisfactory for the model construction. Modeling of alternative BPR process configuration showed that good P removal was achieved by all three alternative process configurations, by the latest one with comparatively lowest construction and operational costs (lower building costs and no acetate addition). Valuable experience from practical application of the model was obtained; study indicated where the model should be improved; the plant operation and treatment processes were better understood and plant performance was further optimized.

This paper describes the impact of long-term (weeks) temperature changes on stoichiometry and kinetics of the anaerobic and aerobic phases of the biological phosphorus removal process. Steady state conversion of relevant compounds for biological phosphorus removal was studied at 20, 30, 20, 10 and 5°C, following chronological order. Integrated in the process study, two methods (electron-microscopy and dry denaturing gradient gel electrophoresis) were applied to investigate the complexity of the bacterial community of biological phosphorus removing sludge cultivated at different temperatures. The coefficient for metabolic conversions obtained from long-term temperature tests was similar to the temperature coefficient observed in short-term (hours) tests ( θ =1.085 versus θ =1.078, respectively).Temperature had a moderate impact on the aerobic P-uptake process rate ( θ =1.031) during long-term tests. However, a strong temperature effect on other metabolic processes of the aerobic phase, such as polyhydroxyalkanoate consumption ( θ =1.163), oxygen uptake ( θ =1.090) and growth ( θ >1.110), was observed. Different temperature coefficients were obtained for the aerobic phase from long-term and short-term tests, probably due to a change in population structure. This change was also visible from molecular ecological studies. The different temperature coefficient found for P-uptake compared to the other metabolic processes of the aerobic phase underlines that, in complex processes such as BPR, it is dangerous to draw conclusions from easily observable parameters (like phosphate) only. Such consideration can easily lead to underestimation of the temperature dependency of other metabolic processes of the aerobic phase of BPR.

It has been reported that deterioration of biological phosphorus removal (BPR) efficiency at some wastewater treatment plants (WWTPs) regularly occurred after heavy rainfall or weekends. The deterioration has been attributed to low plant loading that took place during such events. However, it is hypothesized in this study that the cause of such deterioration may have been the excessive aeration that took place at some of those plants due to inadequate control of aeration system during weekends and rainfall periods. In order to prove this hypothesis, the influence of excessive aeration (aeration during starvation conditions) on BPR processes was studied using a laboratory anaerobic-aerobic-settling sequencing batch reactor (SBR). It was clearly demonstrated that the phosphorus uptake stops due to a gradual depletion of poly-hydroxy-butyrate (PHB) in an over-aerated process. If organic substrate is introduced to the system, phosphorus release is immediately at its maximal rate. However, the released phosphorus cannot be taken-up fully again because the PHB content limits the uptake rate. Consequently, incomplete phosphorus uptake leads to temporary reduction of BPR efficiency. This causal effect can explain the deterioration of BPR efficiency after heavy rainfall or weekends. Since excessive aeration clearly negatively affects the BPR processes, the aeration should be properly controlled at sewage treatment plants. Some other findings of this study deserve to be mentioned. • • It was confirmed that the presence of acetate under aerobic conditions provokes phosphorus release. This may also contribute to deterioration of the BPR efficiency. • • The aerobic phosphate uptake was found to depend not only on the PHB but also on polyphosphate (poly-P) content of the cells. • • A maximal poly-P (0.18 g-P/g-VSS) and minimal PHB content of the cells (2.11 mg-COD/g-VSS) were observed in the enriched sludge during excessive aeration experiments. • • It was shown that, under aerobic starvation conditions, glycogen can not replace PHB for phosphate uptake and is only used for maintenance. During this period, no oxygen consumption due to decay processes has been observed. © 1998 Elsevier Science Ltd. All rights reserved

Hong Kong has practiced seawater toilet flushing since 1958, saving 750,000 m3 of freshwater every day. A high sulfate-to-COD ratio (>1.25 mg SO 4 2 − /mg COD) in the saline sewage resulting from this practice has enabled us to develop the S ulfate reduction, A utotrophic denitrification and N itrification I ntegrated (SANI®) process with minimal sludge production and oxygen demand. Recently, the SANI® process has been expanded to include Enhanced Biological Phosphorus Removal (EBPR) in an alternating anaerobic/limited-oxygen (LOS-EBPR) aerobic sequencing batch reactor (SBR). This paper presents further development – an anaerobic/anoxic denitrifying sulfur cycle-associated EBPR, named as DS-EBPR, bioprocess in an alternating anaerobic/anoxic SBR for simultaneous removal of organics, nitrogen and phosphorus. The 211 day SBR operation confirmed the sulfur cycle-associated biological phosphorus uptake utilizing nitrate as electron acceptor. This new bioprocess cannot only reduce operation time but also enhance volumetric loading of SBR compared with the LOS-EBPR. The DS-EBPR process performed well at high temperatures of 30 °C and a high salinity of 20% seawater. A synergistic relationship may exist between sulfur cycle and biological phosphorus removal as the optimal ratio of P-release to SO 4 2 − -reduction is close to 1.0 mg P/mg S. There were no conventional PAOs in the sludge.

A full-scale sewage treatment plant designed for biological phosphorus removal may experience short- or long-term shortage in potassium of the influent. In this study, using an anaerobic-aerobic sequenced batch reactor system, inoculation sludge from laboratory-, pilot- and full-scale phosphorus removal plants was exposed to different potassium-phosphorus ratios in the influent. By simulating the conditions which may occur in practice, it was shown that potassium is an essential factor in biological phosphorus removal processes. When the system was exposed to severe shortage of potassium in the influent: (a) phosphorus removal was absent, (b) polyphosphate concentration in the biomass decreased exponentially due to sludge wasting and (c) the anaerobic phosphorus release and the related acetate uptake was only affected after several days of potassium absence, likely due to insufficient content of polyphosphate in the biomass to allow full acetate uptake under anaerobic conditions. In contrast, the system achieved complete phosphorus removal when potassium was present in excess amounts.

Hong Kong has practiced seawater toilet flushing since 1958, saving 750,000 m 3 freshwater every day. A high sulfate-to-COD ratio (>1.25 mg SO 4 /mg COD) in the saline sewage resulting from this practice has enabled us to develop the S ulfate reduction A utotrophic denitrification and Nitrification I ntegrated (SANI ® ) process with minimal sludge production. This study seeks to expand the SANI process into an enhanced biological phosphorus removal (EBPR) process. A sulfur cycle associated EBPR was explored in an alternating anaerobic/oxygen-limited aerobic sequencing batch reactor with acetate fed as sole electron donor and sulfate as sulfur source at a total organic carbon to sulfur ratio of 1.1–3.1 (mg C/mg S). Phosphate uptake and polyphosphate formation was observed in this reactor that sustained high phosphate removal (20 mg P/L removed with 320 mg COD/L). This new EBPR process was supported by six observations: 1) anaerobic phosphate release associated with acetate uptake, poly-phosphate hydrolysis, poly-hydroxyalkanoate (PHA) (and poly-S 2− /S 0 ) formation and an “aerobic” phosphate uptake associated with PHA (and poly-S 2− /S 0 ) degradation, and polyphosphate formation; 2) a high P/VSS ratio (>0.16 mg P/mg VSS) and an associated low VSS/TSS ratio (0.75) characteristic of conventional PAOs; 3) a lack of P-release and P-uptake with formaldehyde inactivation and autoclaved sterilized biomass; 4) an absence of chemical precipitated P crystals as determined by XRD analysis; 5) a sludge P of more than 90% polyphosphate as determined by sequential P extraction; and 6) microscopically, observed PHA, poly-P and S globules in the biomass.

The influence of operating and environmental conditions on the microbial populations of the enhanced biological phosphorus removal (EBPR) process at seven full-scale municipal activated sludge wastewater treatment plants (WWTPs) in The Netherlands was studied. Data from the selected WWTPs concerning process configuration, operating and environmental conditions were compiled. The EBPR activity from each plant was determined by execution of anaerobic-anoxic-aerobic batch tests using fresh activated sludge. Fractions of Accumulibacter as potential phosphorus accumulating organisms (PAO), and Competibacter, Defluviicoccus-related microorganisms and Sphingomonas as potential glycogen accumulating organisms (GAO) were quantified using fluorescence in situ hybridization (FISH). The relationships among plant process configurations, operating parameters, environmental conditions, EBPR activity and microbial populations fractions were evaluated using a statistical approach. A well-defined and operated denitrification stage and a higher mixed liquor pH value in the anaerobic stage were positively correlated with the occurrence of Accumulibacter. A well-defined denitrification stage also stimulated the development of denitrifying PAO (DPAO). A positive correlation was observed between Competibacter fractions and organic matter concentrations in the influent. Nevertheless, Competibacter did not cause a major effect on the EBPR performance. The observed Competibacter fractions were not in the range that would have led to EBPR deterioration. Likely, the low average sewerage temperature (12+/-2 degrees C) limited their proliferation. Defluviicoccus-related microorganisms were seen only in negligible fractions in a few plants (

In biological nitrogen removal processes, nitrite can be formed and accumulated through both nitrification and denitrification. Despite the fact that, in practice, biological phosphate removal (BPR) is often combined with biological nitrogen removal, there are only a few publications reporting the effect of nitrite on BPR. In this study, phosphate-accumulating organisms (PAO) were cultivated in an anaerobic–anoxic–aerobic sequencing batch reactor (SBR). The effect of nitrite on the enrichment of the sludge with PAO, the phosphate uptake rates and the sludge respiration was investigated. The results indicate that (1) presence of nitrite inhibits both aerobic and anoxic (denitrifying) phosphate uptake, (2) aerobic phosphate uptake was more affected than anoxic phosphate uptake, (3) presence of nitrite could be one of the factors enhancing the presence of glycogen accumulating organisms (GAO)—competitors to PAO for substrate in the anaerobic phase, and (4) it is required to monitor and control nitrite accumulation in a full-scale wastewater treatment plants.