Enhanced Biological Phosphate Removal Research Articles

Denitrifying phosphorus removal (DPR) performance and metabolic mechanisms of Tetrasphaera at long-term nitrite exposure

Tetrasphaera has been identified as the main polyphosphate-accumulating organism (PAOs) in enhanced biological phosphate removal (EBPR), but the ability of Tetrasphaera to use nitrite as electron acceptor for denitrifying phosphorus removal (DPR) is still unclear. This study investigated the DPR performance and mechanism, microbial structures, and FNA inhibition of Tetrasphaera-dominated culture in the long-term acclimatization at different nitrite concentrations (10, 20, 40 and 50 mgN/L). The results showed that the most optimal influent nitrite concentration was 20 mgN/L (C/N = 6), and the removal efficiency of nitrite and phosphorus reached above 90 % and 95 %, respectively. Fluorescence In Situ Hybridization-Flow Cytometry (FISH-FCM) showed that the population abundance of Tetrasphaera was 80.95 %, in which Tet2-892 clade was the most abundant and mainly responsible for DPR with nitrite as electron acceptor. When the nitrite concentration reached 50 mgN/L (FNA = 12.80 µgHNO2-N/L, pH = 7.0 ± 0.1), electron supply deficiency and FNA inhibition resulted in only 43.0 % and 68.4 % of nitrite and phosphorus removal efficiency, respectively. The intracellular amino acid and glycogen metabolism were inhibited by FNA under this condition. A significant accumulation of N2O was observed, resulting from a combination of the insufficient electron supply, absence of nos-Z gene, and long-term FNA inhibition. DPR dominated by Tetrasphaera with appropriate nitrite as electron acceptor provides a new way to achieve nitrogen and phosphorus simultaneous removal. In addition, this study found that Tet2-892 clade of Tetrasphaera was more tolerant to FNA compared to Accumulibacter and GAOs, which provides a strategy for inhibiting GAOs activity to maintain the stability of the EBPR system.

Turnover of the extracellular polymeric matrix of granules performing biological phosphate removal

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. 13C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d−1 respectively) was higher than the expected value based on the solid removal rate (0.132 d−1), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d−1.Key Points• Proteins showed a higher turnover rate than carbohydrates.• Turnover of EPS was similar to the turnover of intracellular proteins.• EPS is not preferentially consumed by flanking populations.

Design of a Soft Sensor for Monitoring Phosphorous Uptake in an EBPR Process

Phosphorus (P) is a key nutrient targeted for removal by wastewater treatment, increasingly being achieved using biological processes such as enhanced biological phosphate removal (EBPR). However, commercial instrumentation for automated measurement of P is costly and provides only limited temporal resolution, constraining implementation of real-time controls in EBPR processes. This study designs a soft sensor for real-time controls using a suite of relatively low-cost sensors (ion-selective electrodes) to monitor P removal by measuring (1) secondary cations tightly coupled to bioP metabolism and (2) process bulk chemistry. Data collected from a highly instrumented lab scale reactor are used to evaluate which and how many sensors are required to achieve this goal. Machine learning (ML) approaches (support vector machines, nonlinear logistic regression, random forest, and Bayesian classification) are evaluated for sensor data fusion and coupled with a decision metric to operationalize the algorithm for reactor controls. Two key results emerge: (1) use of the slope of sensor data (mV/min) rather than raw data (mV) as the predictor signals significantly improves accuracy and resilience of the soft sensor-based system and (2) the K+ ion-selective electrode, in combination with any of the four ML algorithms, is sufficient to detect completion of P removal (within study sampling granularity of 4 min) with 100% accuracy in real time. High accuracy is maintained even as process chemistry is varied to increase the interference experienced by the sensors, indicating that this soft sensor is viable for use in real wastewater applications.

Quantification of Biologically and Chemically Bound Phosphorus in Activated Sludge from Full-Scale Plants with Biological P-Removal.

Phosphorus (P) is present in activated sludge from wastewater treatment plants in the form of metal salt precipitates, extracellular polymeric substances, or bound into the biomass, for example, as intracellular polyphosphate (poly-P). Several methods for a reliable quantification of the different P-fractions have recently been developed, and this study combines them to obtain a comprehensive P mass-balance of activated sludge from four enhanced biological phosphate removal (EBPR) plants. Chemical characterization by ICP-OES and sequential P fractionation showed that chemically bound P constituted 38–69% of total P, most likely in the form of Fe, Mg, or Al minerals. Raman microspectroscopy, solution state 31P NMR, and 31P MAS NMR spectroscopy applied before and after anaerobic P-release experiments, were used to quantify poly-P, which constituted 22–54% of total P and was found in approximately 25% of all bacterial cells. Raman microspectroscopy in combination with fluorescence in situ hybridization was used to quantify poly-P in known polyphosphate-accumulating organisms (PAO) (Tetrasphaera, Candidatus Accumulibacter, and Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. Interestingly, only 1–13% of total P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are known.

Characterization of domestic wastewater released from ‘green’ households and field study of the performance of onsite septic tanks retrofitted into aerobic bioreactors in cold climate

This study aimed to investigate the efficacy of private septic systems retrofitted into aerobic bioreactors with ‘SludgeHammer’ technology. In addition, the study attempted to characterize the strength of domestic wastewater released from ‘green’ households practicing water conservation strategies. Ten retrofitted onsite septic systems were studied in the Edmonton area, Alberta (AB) Canada during winter. These systems could remove BOD5 and TSS by 92 ± 5 and 92 ± 6% respectively which, according to Albertan regulatory standards, were characteristic removal efficiencies of the secondary treatment in the subsequent drain field. These removal efficiencies were remarkable given the strength of the influent wastewater. The raw wastewater carried significantly high pollutant concentrations (1160 ± 350 mg BOD5/L, 1653 ± 1174 mg TSS/L, 99 ± 19 mg NH4+-N/L, 100 ± 56 mg TN/L, and 39 ± 28 mg PO43−-P/L), characterizing it as high-strength domestic wastewater. Mixing provided by the aerator could only suspend 1/34th (3% m/m) of the solids in the bioreactor and consequently released significantly low solid concentrations (195 ± 206 mg TSS/L) into the final treatment component. As such, this technology did not impair the natural function of septic tanks or did not create any unintended excessive solid loading on drain field as a consequence of the added mixing energies provided by the active aeration. Nitrogen balance suggested the possibility of simultaneous nitrification and denitrification (SND) in the aerobic bioreactors. In some cases, PO43−-P removal efficiency was as high as that in enhanced biological phosphate removal (EBPR) process (81–97%). Phosphorus balance estimated that non-assimilative pathways (i.e., EBPR + biologically induced phosphate precipitation (BIPP)) contributed 50–99% to overall phosphorus removal in the system. Long HRTs, high influent BOD5 and anaerobic/aerobic zoning in the bioreactor most likely provided favorable conditions for SND and high phosphorus removal efficiencies in the retrofitted onsite wastewater treatment systems (OWTS).

Biological phosphorus removal in seawater-adapted aerobic granular sludge

Seawater can be introduced or intrude in sewer systems and can thereby negatively influence biological wastewater treatment processes. Here we studied the impact of artificial seawater on the enhanced biological phosphate removal (EBPR) process performance by aerobic granular sludge (AGS) with synthetic wastewater. Process performance, granule stability and characteristics as well as microbial community of a seawater-adapted AGS system were observed. In seawater conditions strong and stable granules formed with an SVI5 of 20 mL/g and a lower abrasion coefficient than freshwater-adapted granules. Complete anaerobic uptake of acetate, anaerobic phosphate release of 59.5 ± 4.0 mg/L PO43--P (0.35 mg P/mg HAc), and an aerobic P-uptake rate of 3.1 ± 0.2 mg P/g VSS/h were achieved. The dominant phosphate accumulating organisms (PAO) were the same as for freshwater-based aerobic granular sludge systems with a very high enrichment of Ca. Accumulibacter phosphatis clade I, and complete absence of glycogen accumulating organisms. The effect of osmotic downshocks was tested by replacing influent seawater-based medium by demineralized water-based medium. A temporary decrease of the salinity in the reactor led to a decreased phosphate removal activity, while it also induced a rapid release of COD by the sludge, up to 45.5 ± 1.7 mg COD/g VSS. This is most likely attributed to the release of osmolytes by the cells. Recovery of activity was immediately after restoring the seawater feeding. This work shows that functioning of aerobic granular sludge in seawater conditions is as stable as in freshwater conditions, while past research has shown a negative effect on operation of AGS processes with NaCl-based wastewater at the same salinity as seawater.

Development of aerobic granular sludge under tropical climate conditions: The key role of inoculum adaptation under reduced sludge washout for stable granulation

Aerobic granular sludge (AGS) is a promising technology for wastewater treatment. However, the success of the process depends on the formation of stable granular biomass, which is associated with the microbiological aspects of the sludge and reactor operating conditions. In this study, the development of AGS from a poor nitrifying flocculent sludge obtained in a sewage treatment plant designed only for organic matter removal was assessed in a sequencing batch reactor (SBR) under tropical climate conditions (temperatures of 28 ± 4 °C). The results showed that, despite the alternating anaerobic-aerobic conditions during the granules selection phase under high sludge washout rates (low settling time), readily biodegradable organic matter was mainly removed aerobically. The formed granules were unstable, exhibiting a substantial amount of filaments and pasty consistency. The biomass characteristics (e.g., sludge volume index, density, diameter and settling velocity) were negatively impacted as complete granulation was reached, while biomass loss and degranulation became inevitable. Poor nitrification and no enhanced biological phosphate removal (EBPR) were observed. Implementation of a new operational strategy incorporating an adaptation of the seed sludge under reduced washout conditions (high settling time) prior to the granules selection stage enabled most of the influent organics to be removed anaerobically. Besides allowing a feast-famine regime to be established in the reactor, the sludge acclimation phase favoured the development of slow-growing organisms and suppressed the appearance of filamentous-like structures. Fast-settling granules with regular shape remained stable in the long-term, while high ammonium (>95%) and total nitrogen removal (>90%) was obtained. However, EBPR activity was very unstable, most likely due to the high temperatures. The findings of this study are important for the spreading of the AGS technology worldwide, especially in developing countries where the conditions are different in all aspects.

Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased In Vivo Polyphosphate Levels.

Bacteria synthesize inorganic polyphosphate (polyP) in response to a wide variety of stresses, and production of polyP is essential for stress response and survival in many important pathogens and bacteria used in biotechnological processes. However, surprisingly little is known about the molecular mechanisms that control polyP synthesis. We have therefore developed a novel genetic screen that specifically links growth of Escherichia coli to polyP synthesis, allowing us to isolate mutations leading to enhanced polyP production. Using this system, we have identified mutations in the polyP-synthesizing enzyme polyP kinase (PPK) that lead to dramatic increases in in vivo polyP synthesis but do not substantially affect the rate of polyP synthesis by PPK in vitro These mutations are distant from the PPK active site and found in interfaces between monomers of the PPK tetramer. We have also shown that high levels of polyP lead to intracellular magnesium starvation. Our results provide new insights into the control of bacterial polyP accumulation and suggest a simple, novel strategy for engineering bacteria with increased polyP contents.IMPORTANCE PolyP is an ancient, universally conserved biomolecule and is important for stress response, energy metabolism, and virulence in a remarkably broad range of microorganisms. PolyP accumulation by bacteria is also important in biotechnology applications. For example, it is critical to enhanced biological phosphate removal (EBPR) from wastewater. Understanding how bacteria control polyP synthesis is therefore of broad importance in both the fields of bacterial pathogenesis and biological engineering. Using Escherichia coli as a model organism, we have identified the first known mutations in polyP kinase that lead to increases in cellular polyP content.

Enhanced biological phosphate removal from wastewater and clade-level population dynamics of “Candidatus Accumulibacter phosphatis” under free nitrous acid inhibition: Linked with detoxication

“Candidatus Accumulibacter” are the dominant polyphosphate-accumulating organism (PAOs) to perform enhanced biological phosphate removal (EBPR) from wastewater. Free nitrous acid (FNA) as inhibitor is an intermediate of nitrification and denitrification commonly present in wastewater treatment plants (WWTPs) and lab-scale reactors. Impact of FNA on EBPR and community structures of Accumulibacter in domestic wastewater treatment was investigated. FNA complete inhibit concentration on aerobic P uptake at pH 6.5 was 6.85×10−3mg HNO2-N/L, higher than that at pH 7.0. Under complete inhibition, poly-β-hydroxyalkanoates degradation was used for nitrite reduction and detoxication rather than P-uptake. The P fraction existed in the forms of complex-P, interstitial-P, chemical participated-P and ortho-P at the end of aerobic phase, and complex-P proportion declined in comparison to non-inhibition. Dominant Accumulibacter clade was IIC, accounting for 70% of total Accumulibacter. Decline of IIF proportion indicated its sensitivity to FNA. Increase of IID percentage implied its strong tolerance to FNA. Under inhibition, Shannon index decreased from 3.45 of seed sludge to 1.92 at pH 7.0, indicating decline of biodiversity. Therefore, FNA inhibition weakened EBPR performance, and remarkably changed clade-level community structures of Accumulibacter.

A Comparative Study of the Bacterial Community in Denitrifying and Traditional Enhanced Biological Phosphorus Removal Processes

Denitrifying phosphorus removal is an attractive wastewater treatment process due to its reduced carbon source demand and sludge minimization potential. Two lab-scale sequencing batch reactors (SBRs) were operated in alternating anaerobic-anoxic (A-A) or anaerobic-oxic (A-O) conditions to achieve denitrifying enhanced biological phosphate removal (EBPR) and traditional EBPR. No significant differences were observed in phosphorus removal efficiencies between A-A SBR and A-O SBR, with phosphorus removal rates being 87.9% and 89.0% respectively. The community structures in denitrifying and traditional EBPR processes were evaluated by high-throughput sequencing of the PCR-amplified partial 16S rRNA genes from each sludge. The results obtained showed that the bacterial community was more diverse in A-O sludge than in A-A sludge. Taxonomy and β-diversity analyses indicated that a significant shift occurred in the dominant microbial community in A-A sludge compared with the seed sludge during the whole acclimation phase, while a slight fluctuation was observed in the abundance of the major taxonomies in A-O sludge. One Dechloromonas-related OTU outside the 4 known Candidatus "Accumulibacter" clades was detected as the main OTU in A-A sludge at the stationary operation, while Candidatus "Accumulibacter" dominated in A-O sludge.

Effects of side-stream, low temperature phosphorus recovery on the performance of anaerobic/anoxic/oxic systems integrated with sludge pretreatment

Introduction of sludge reduction practices in enhanced biological phosphate removal (EBPR) often leads to a decrease in phosphorous (P) removal efficiency. In this study, an attempt has been made to develop sustainable P removal in an anaerobic/anoxic/oxic (AAO) system integrated with sludge reduction by incorporating side-stream P recovery. Two AAO reactors, one acting as a control (CAAO) and the other as an experimental system (EAAO), were used in our experiment. The average P solubilisation efficiency and its recovery from thermally pre-treated sludge were found to be 28% and 99%, respectively. The P recovery process prevented the biomass in the EAAO system from reaching its threshold level and resulted in sustainable P removal throughout the study period. Thermochemical pre-treatment, at a rate of 1.5% Q in the EAAO reactor, was responsible for a 28% reduction in the amount of sludge produced. The yield observed (Y obs) values for the system with and without pre-treatment were found to be 0.28 and 0.22 kg MLSS/kg COD, respectively. An initial 50 days of reactor operation was utilised to stabilise the systems (Phase 1). The total nitrogen removal during Phase 2 (51-225 days) was slightly higher than that in Phase 1 (76-80% and 68-75%, respectively). The MLSS/MLVSS ratios of both systems were identical and were between 78% and 83% for both the CAAO and EAAO. The effluent COD concentration was not significantly affected by the proposed method of treatment. From the results of the present study, it is concluded that the proposed mode of treatment was capable of both sustainable removal of P and control of excess sludge production.

To Remove Phosphate from Municipal Wastewater with Insufficient Volatile Fatty Acids

Tow plug flow reactors were operated with municipal wastewater according to reversed A2/O process. The only difference was in sludge return ratio. Despite that more VFAs were consumed by denitrification, phosphate removal was enhanced rather than impaired by a higher sludge return ratio of 300%. It seems that higher phosphate removal can be achieved even more nitrate was reduced which means more carbon source consumption. This is contradictory with the well accepted enhanced biological phosphate removal (EBPR) theory, which regards sufficient volatile fatty acids in the wastewater as the indispensable premise. With less phosphate was released in the anaerobic zones, the final phosphate concentration in the effluent was lower when higher sludge return ratio was applied. The results indicated that neither carbon source nor phosphate release can directly determine phosphate uptake ability.

生物学的リン除去活性の変動過程におけるグリコーゲン蓄積細菌群の酢酸摂取速度の急激な上昇

An A/O SBR fed with acetate was operated at pH 7.0 after the inoculation of activated sludge capable of enhanced biological phosphate removal (EBPR). Glycogen-accumulating organisms (GAOs) were predominant in acetate uptake on the 19th day of operation. The operational pH was then increased to 8.2. Accordingly, the acetate uptake activity of GAOs gradually decreased as intended, whereas phosphate release activity improved. To estimate acetate uptake rate with addition of acetate more than that in the A/O SBR operation, an anaerobic batch test was conducted. Higher amounts of acetate added led to significantly high acetate uptake rates that were not consistent with phosphate release rates only when the acetate uptake activity of GAOs was relatively low in the A/O SBR operation. In other words, an acetate concentration higher than the usual influent concentration probably stimulates the acetate uptake activity of GAOs. These results suggest that a rapid increase in the acetate uptake rate of GAOs affects the sudden deterioration of EBPR.

Ecology of the Microbial Community Removing Phosphate from Wastewater under Continuously Aerobic Conditions in a Sequencing Batch Reactor

All activated sludge systems for removing phosphate microbiologically are configured so the biomass is cycled continuously through alternating anaerobic and aerobic zones. This paper describes a novel aerobic process capable of decreasing the amount of phosphate from 10 to 12 mg P liter(-1) to less than 0.1 mg P liter(-1) (when expressed as phosphorus) over an extended period from two wastewaters with low chemical oxygen demand. One wastewater was synthetic, and the other was a clarified effluent from a conventional activated sludge system. Unlike anaerobic/aerobic enhanced biological phosphate removal (EBPR) processes where the organic substrates and the phosphate are supplied simultaneously to the biomass under anaerobic conditions, in this aerobic process, the addition of acetate, which begins the feed stage, is temporally separated from the addition of phosphate, which begins the famine stage. Conditions for establishing this process in a sequencing batch reactor are detailed, together with a description of the changes in poly-beta-hydroxyalkanoate (PHA) and poly(P) levels in the biomass occurring under the feed and famine regimes, which closely resemble those reported in anaerobic/aerobic EBPR processes. Profiles obtained with denaturing gradient gel electrophoresis were very similar for communities fed both wastewaters, and once established, these communities remained stable over prolonged periods of time. 16S rRNA-based clone libraries generated from the two communities were also very similar. Fluorescence in situ hybridization (FISH)/microautoradiography and histochemical staining revealed that "Candidatus Accumulibacter phosphatis" bacteria were the dominant poly(P)-accumulating organisms (PAO) in both communities, with the phenotype expected for PAO. FISH also identified large numbers of betaproteobacterial Dechloromonas and alphaproteobacterial tetrad-forming organisms related to Defluviicoccus in both communities, but while these organisms assimilated acetate and contained intracellular PHA during the feed stages, they never accumulated poly(P) during the cycles, consistent with the phenotype of glycogen-accumulating organisms.

Biological phosphate removal using a degradable carbon source produced by hydrothermal treatment of excess sludge

Abstract - The possibility of reusing excess sludge treated by hydrothermal reaction for the purpose of improving the efficiency of the enhanced biological phosphate removal (EBPR) process was investigated. Excess sludge from a fish-processing industry located in Japan was treated in high-temperature and high-pressure water, at a reaction temperature ranging from 200 to 400 o C, a pressure of 1.8 to 30MPa and a constant reaction time of 7 min. For the conditions tested, the results showed that when the reaction temperature was increased the content of readily biodegradable substrate in the total COD Cr increased. In addition, the amount of some volatile fatty acids (VFAs) produced by the hydrothermal reaction increased as reaction temperature increased. From the phosphate release tests under anaerobic conditions, it was possible to demonstrate that not only the VFAs, but also the readily and slowly biodegradable substrates are used as potential carbon source by the phosphate-accumulating organisms (PAOs).

Which are the polyphosphate accumulating organisms in full-scale activated sludge enhanced biological phosphate removal systems in Australia?

To see if the compositions of the microbial communities in full scale enhanced biological phosphorus removal activated sludge systems were the same as those from laboratory scale sequencing batch reactors fed a synthetic sewage. Biomass samples taken from nine full scale enhanced biological phosphate removal (EBPR) activated sludge plants in the eastern states of Australia were analysed for their populations of polyphosphate (polyP)-accumulating organisms (PAO) using semi-quantitative fluorescence in situ hybridization (FISH) in combination with DAPI (4'-6-diamidino-2-phenylindole) staining for polyP. Very few betaproteobacterial Rhodocyclus related organisms could be detected by FISH in most of the plants examined, and even where present, not all these cells even within a single cluster, stained positively for polyP with DAPI. In some plants in samples from aerobic reactors the Actinobacteria dominated populations containing polyP. The PAO populations in full-scale EBPR systems often differ to those seen in laboratory scale reactors fed artificial sewage, and Rhodocyclus related organisms, dominating these latter communities may not be as important in full-scale systems. Instead Actinobacteria may be the major PAO. These findings illustrate how little is still known about the microbial ecology of EBPR processes and that more emphasis should now be placed on analysis of full-scale plants if microbiological methods are to be applied to monitoring their performances.

Effect of pH change on the performance and microbial community of enhanced biological phosphate removal process

An acetate-rich wastewater, containing 170 mg/L of total organic carbon (TOC), 13 mg/L of N, and 15 mg/L of P, was treated using the enhanced biological phosphate removal (EBPR) process operated in a sequencing batch reactor. A slight change of pH of the mixed liquor from 7.0 to 6.5 led to a complete loss of phosphate-removing capability and a drastic change of microbial populations. The process steadily removed 94% of TOC and 99.9% of P from the wastewater at pH 7.0, but only 93% TOC and 17% of P 14 days after the pH was lowered to pH 6.5. The sludge contained 8.8% P at pH 7.0, but only 1.9% at pH 6.5. Based on 16S rDNA analysis, 64.8% of the clones obtained from the sludge at pH 7.0 were absent in the pH 6.5 sludge. The missing microbes, some of which were likely responsible for the phosphate removal at pH 7.0, included beta-Proteobacteria, Actinobacteria, Bacteriodetes/Chlorobi group, plus photosynthetic bacteria and Defluvicoccus of the alpha-Proteobacteria. Among them, the last two groups, which represented 9.3% and 10.1% of the EBPR sludge at pH 7.0, have rarely been reported in an EBPR system.

The large PAO cells in full-scale EBPR biomass samples are not yeast spores but possibly novel members of the β-Proteobacteria

Large, homogenous clusters of coccobacilli were found to be abundant in the biomasses from a conventional plant at Rosebud, Victoria, Australia. The identity and the in situ physiology of these dominant microorganisms were investigated in this study. These large clustered cells were revealed to be neither Gram positive nor Gram negative bacteria and contain polyP granules. Cells with similar features were also observed in some enhanced biological phosphate removal (EBPR) systems and reported as yeast spores and Rhodocyclus-related polyphosphate accumulating organisms (PAOs). In this study, fluorescent in situ hybridization (FISH) probing showed these cells were prokaryotic and members of the beta-Proteobacteria. However, these large clustered cells did not respond to the PAO mix FISH probes. The in situ physiology of these large cells was studied with FISH in combination with microautoradiography (MAR) in order to understand their substrate assimilation abilities under different conditions as well as their phosphate uptake ability. These cells were able to take up acetate, glutamate and aspartate, but not glucose under both aerobic and anaerobic conditions. Nile Blue A staining in combination with MAR showed that cells incubated under anaerobic conditions contained polyhydroxyalkanoates (PHA) granules. In addition, MAR showed aerobic 33Pi assimilation with all these substrates, consistent with them supporting an EBPR capacity in these large cells. As well as raising doubts about a role for yeasts in EBPR, this study suggests that much still needs to be learned about the identity and level of biodiversity of the PAO in EBPR systems, and emphasizes the benefits of using techniques like FISH/MAR and PHA staining/MAR to resolve the in situ physiology of the populations of interest there.

Verification of enhanced phosphate removal capability in pure cultures ofAcinetobacter calcoaceticus under anaerobic/aerobic conditions in an SBR

Laboratory experiments were conducted using pure cultures ofAcinetobacter under anaerobic/aerobic cyclic conditions to explain the release and uptake of soluble phosphate in an activated sludge process showing enhanced biological phosphate removal (EBPR). Under anaerobic/aerobic cyclic conditions in a Sequencing Batch Reactor (SBR), COD uptake concurrent with soluble phosphate release byAcinetobacter was not significant during the anaerobic periods, indicating that EBPR would not be established in pure cultures. However,Acinetobacter cells accumulated higher phosphate content (5.2%) in SBR than that obtained (4.3%) from batch experiments. These results suggest thatAcinetobacter sp. may not follow the proposed pattern of behavior of poly-P bacteria in EBPR activated sludge plants.

Characterization of an acetate-degrading sludge without intracellular accumulation of polyphosphate and glycogen

A sequencing batch reactor (SBR) was operated in the conventional anaerobic–aerobic mode for enhanced biological phosphate removal (EBPR) using acetate as the sole substrate. Results showed that, however, the reactor was unable to remove phosphate from wastewater. The sludge containing 1.65% of phosphate did not exhibit the typical characteristics of polyphosphate-accumulating organisms (PAO) or glycogen-accumulating organisms (GAO). Phylogenetic analysis, based on 16S rDNA sequences of individual microorganisms, showed that the microbial community of this acetate-degrading sludge was closely related to Comamonas testosteroni (43.8% of total population) of β-1-proteobacteria, Zoogloea resiniphila (25.0%) of β-2-proteobacteria, and Xanthomonas maltophilia (19.8%) of γ-proteobacteria. Results of this study imply that GAO might not be the sole group of bacteria responsible to the deterioration of phosphate removal efficiency in an EBPR reactor.