Identification and comparison of aerobic and denitrifying polyphosphate-accumulating organisms

Two laboratory-scale sequencing batch reactors (SBRs) were operated for enhanced biological phosphorus removal (EBPR) in alternating anaerobic-aerobic or alternating anaerobic-anoxic modes, respectively. Polyphosphate-accumulating organisms (PAOs) were enriched in the anaerobic-aerobic SBR and denitrifying PAOs (DPAOs) were enriched in the anaerobic-aerobic SBR. Fluorescence in situ hybridization (FISH) demonstrated that the well-known PAO, "Candidatus Accumulibacter phosphatis" was abundant in both SBRs, and post-FISH chemical staining with 4,6-diamidino-2-phenylindol (DAPI) confirmed that they accumulated polyphosphate. When the anaerobic-anoxic SBR enriched for DPAOs was converted to anaerobic-aerobic operation, aerobic uptake of phosphorus by the resident microbial community occurred immediately. However, when the anaerobic-aerobic SBR enriched for PAOs was exposed to one cycle with anoxic rather than aerobic conditions, a 5-h lag period elapsed before phosphorus uptake proceeded. This anoxic phosphorus-uptake lag phase was not observed in the subsequent anaerobic-aerobic cycle. These results demonstrate that the PAOs that dominated the anaerobic-aerobic SBR biomass were the same organisms as the DPAOs enriched under anaerobic-anoxic conditions.     

Model-based analysis of anaerobic acetate uptake by a mixed culture of polyphosphate-accumulating and glycogen-accumulating organisms

An increasing number of studies shows that the glycogen-accumulating organisms (GAOs) can survive and may indeed proliferate under the alternating anaerobic/aerobic conditions found in EBPR systems, thus forming a strong competitor of the polyphosphate-accumulating organisms (PAOs). Understanding their behaviors in a mixed PAO and GAO culture under various operational conditions is essential for developing operating strategies that disadvantage the growth of this group of unwanted organisms. A model-based data analysis method is developed in this paper for the study of the anaerobic PAO and GAO activities in a mixed PAO and GAO culture. The method primarily makes use of the hydrogen ion production rate and the carbon dioxide transfer rate resulting from the acetate uptake processes by PAOs and GAOs, measured with a recently developed titration and off-gas analysis (TOGA) sensor. The method is demonstrated using the data from a laboratory-scale sequencing batch reactor (SBR) operated under alternating anaerobic and aerobic conditions. The data analysis using the proposed method strongly indicates a coexistence of PAOs and GAOs in the system, which was independently confirmed by fluorescent in situ hybridization (FISH) measurement. The model-based analysis also allowed the identification of the respective acetate uptake rates by PAOs and GAOs, along with a number of kinetic and stoichiometric parameters involved in the PAO and GAO models. The excellent fit between the model predictions and the experimental data not involved in parameter identification shows that the parameter values found are reliable and accurate. It also demonstrates that the current anaerobic PAO and GAO models are able to accurately characterize the PAO/GAO mixed culture obtained in this study. This is of major importance as no pure culture of either PAOs or GAOs has been reported to date, and hence the current PAO and GAO models were developed for the interpretation of experimental results of mixed cultures. The proposed method is readily applicable for detailed investigations of the competition between PAOs and GAOs in enriched cultures. However, the fermentation of organic substrates carried out by ordinary heterotrophs needs to be accounted for when the method is applied to the study of PAO and GAO competition in full-scale sludges.     

Identifying causes for N2O accumulation in a lab-scale sequencing batch reactor performing simultaneous nitrification, denitrification and phosphorus removal

The recently described process of simultaneous nitrification, denitrification and phosphorus removal (SNDPR) has a great potential to save capital and operating costs for wastewater treatment plants. However, the presence of glycogen-accumulating organisms (GAOs) and the accumulation of nitrous oxide (N(2)O) can severely compromise the advantages of this process. In this study, these two issues were investigated using a lab-scale sequencing batch reactor performing SNDPR over a 5-month period. The reactor was highly enriched in polyphosphate-accumulating organisms (PAOs) and GAOs representing around 70% of the total microbial community. PAOs were the dominant population at all times and their abundance increased, while GAOs population decreased over the study period. Anoxic batch tests demonstrated that GAOs rather than denitrifying PAOs were responsible for denitrification. N(2)O accumulated from denitrification and more than half of the nitrogen supplied in a reactor cycle was released into the atmosphere as N(2)O. After mixing SNDPR sludge with other denitrifying sludge, N(2)O present in the bulk liquid was reduced immediately if external carbon was added. We therefore suggest that the N(2)O accumulation observed in the SNDPR reactor is an artefact of the low microbial diversity facilitated by the use of synthetic wastewater with only a single carbon source.     

Metabolic influence of lead on polyhydroxyalkanoates (PHA) production and phosphate uptake in activated sludge fed with glucose or acetic acid as carbon source

Sludge in a sequential batch reactor (SBR) system was used to investigate the effect of lead toxicity on metabolisms of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) communities fed with acetic acid or glucose as their sole carbon source, respectively. Results showed that the effect of lead on substrate utilization of both PAOs and GAOs was insignificant. However, lead substantially inhibited both of phosphate release and uptake of PAOs. In high concentration of acetic acid trials, an abnormal aerobic phosphate release was observed instead of phosphate uptake and the release rate increased with increasing lead concentration. Results also showed that PAOs could normally synthesize polyhydroxybutyrate (PHB) in the anaerobic phase even though lead concentration was 40 mg L⁻¹. However, they could not aerobically utilize PHB normally in the presence of lead. On the other hand, GAOs could not normally metabolize polyhydroxyvalerate (PHV) in both the anaerobic and aerobic phases.     

Metabolic Characteristics of a Glycogen-Accumulating Organism in Defluviicoccus Cluster II Revealed by Comparative Genomics

Glycogen-accumulating organisms (GAOs) may compete with phosphate-accumulating organisms (PAOs) for short-chain fatty acids (VFAs) in anaerobic polyhydroxyalkanoates (PHA) synthesis, but no consequently aerobic polyphosphate accumulation in enhanced biological phosphorus removal (EBPR) process, thus deteriorating the EBPR process. They are detected frequently in the deteriorated EBPR process, but their metabolisms are still far from our comprehensions for there is seldom pure culture. In this study, a nearly complete draft genome of a GAOs in Defluviicoccus cluster II, GAO-HK, is recruited from the metagenome of activated sludge in a full-scale industrial anoxic/aerobic wastewater plant. Comparative genomics reveal similar metabolisms of PHA and glycogen in GAOs of GAO-HK, Defluviicoccus tetraformis TFO71 (TFO71) and Competibacter phosphatis clade IIA (CPIIA), and PAOs of Accumulibacter clade IIA UW-1 (UW-1) and Tetrasphaera elongata Lp2 (Lp2). Although there are similar gene cassettes related with polyphosphate metabolism in these GAOs and PAOs, especially for Defluviicoccus-relative bacteria and UW-1, ppk1 in GAOs are diverse from those in the identified PAOs, implying the difference of polyphosphate metabolism in GAOs and PAOs. Additionally, genes related to the dissimilatory denitrification are absent in TFO71 and GAO-HK, implying that additional nitrate or nitrite may favor PAOs over Defluviicoccus-relative GAOs. Therefore, PAOs suffering from competition of Defluviicoccus-relative GAOs might be rescued with the additional nitrate/nitrite, which is important to improve the stability of EBPR processes.     

Decolorization of azo-reactive dye by polyphosphate- and glycogen-accumulating organisms in an anaerobic-aerobic sequencing batch reactor

An anaerobic-aerobic sequencing batch reactor with a sludge age of 8 days and anaerobic + aerobic + settling times of 18 + 5 + 1 h, was used to decolorize an azo-reactive dye wastewater. The nutrient broth (NB) and sodium acetate (SA) solution at 500 + 0, 350 + 150, 250 + 250 and 0 + 500 mg/l as COD was fed to the system to promote the polyphosphate-accumulating organisms (PAOs), while only glucose (500 mg/l COD) was used as a glycogen-accumulating organisms (GAOs) promoting substrate. The decolorization capability of the process was about 73-77 and 59-64% in terms of ADMI for the systems which the PAOs and GAOs proliferated, respectively. The color reduction was mainly achieved within the first 2 h of the anaerobic stage.     

生物除磷系统中聚糖菌代谢机理的研究进展

强化生物除磷（EBPR）被认为是一种最经济、可持续的污水除磷工艺。近年来大量研究报道,系统中聚糖菌的大量繁殖会使除磷工艺性能变差或完全失败。介绍了聚糖菌的代谢机理和影响聚糖菌与聚磷菌之间竞争的因素（如进水基质、P／C、pH值、温度和泥龄等）,便于更好地理解聚糖菌的特性,从而实现提高生物除磷系统运行的性能与稳定性。     

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called ‘Candidatus Accumulibacter phosphatis’ was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge ‘biography’ be well understood – i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked. This revised version was published online in August 2006 with corrections to the Cover Date.     

A new interpretation of ASM2d for modeling of SBR performance for enhanced biological phosphorus removal under different P/HAc ratios

This study evaluated the prediction capability of Activated Sludge Model No. 2d (ASM2d), for the enhanced biological phosphorus removal (EBPR) performance of a sequencing batch reactor (SBR) receiving variable influent phosphate load. For this purpose, a laboratory-scale SBR was operated with a synthetic feed containing acetate as the sole carbon source. The experiments were conducted in four different Runs to ensure a range of different phosphate/acetate ratios in the influent. Model evaluations were carried out using concentration profiles measured throughout a representative cycle at steady state. An iterative calibration methodology was developed based on sensitivity analysis and applied to four different sets of experimental data on relevant model parameters reflecting SBR performance. ASM2d was able to predict the steady state behavior of the SBR system receiving variable influent phosphate loads only with the recalibrated parameter set. The regular changing pattern of the coefficients could be interpreted with the ability of the SBR system to sustain glycogen accumulating microorganisms, GAOs, which can store substrate under anaerobic conditions without polyphosphate energy, but deriving energy from the degradation of glycogen. Thus they are capable of prevailing at lower P/Ac ratios. The results indicate the need to include glycogen and GAOs as model components for processes involving both phosphate accumulating organisms, (PAOs) and GAOs, in order to obtain a better prediction of X(PHA) and oxygen uptake rate (OUR) profiles in the system.     

Comparison of acetate and propionate uptake by polyphosphate accumulating organisms and glycogen accumulating organisms

Enhanced biological phosphorus removal (EBPR) performance is directly affected by the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigates the effects of carbon source on PAO and GAO metabolism. Enriched PAO and GAO cultures were tested with the two most commonly found volatile fatty acids (VFAs) in wastewater systems, acetate and propionate. Four sequencing batch reactors (SBRs) were operated under similar conditions and influent compositions with either acetate or propionate as the sole carbon source. The stimulus for selection of the PAO and GAO phenotypes was provided only through variation of the phosphorus concentration in the feed. The abundance of PAOs and GAOs was quantified using fluorescence in situ hybridisation (FISH). In the acetate fed PAO and GAO reactors, "Candidatus Accumulibacter phosphatis" (a known PAO) and "Candidatus Competibacter phosphatis" (a known GAO) were present in abundance. A novel GAO, likely belonging to the group of Alphaproteobacteria, was found to dominate the propionate fed GAO reactor. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to take up acetate and propionate. PAOs enriched with acetate as the sole carbon source were immediately able to take up propionate, likely at a similar rate as acetate. However, an enrichment of GAOs with acetate as the sole carbon source took up propionate at a much slower rate (only about 5% of the rate of acetate uptake on a COD basis) during a short-term switch in carbon source. A GAO enrichment with propionate as the sole carbon source took up acetate at a rate that was less than half of the propionate uptake rate on a COD basis. These results, along with literature reports showing that PAOs fed with propionate (also dominated by Accumulibacter) can immediately switch to acetate, suggesting that PAOs are more adaptable to changes in carbon source as compared to GAOs. This study suggests that the PAO and GAO competition could be influenced in favour of PAOs through the provision of propionate in the feed or even by regularly switching the dominant VFA species in the wastewater. Further study is necessary in order to provide greater support for these hypotheses.     

Metabolic model for acetate uptake by a mixed culture of phosphate- and glycogen-accumulating organisms under anaerobic conditions

This paper proposes a new metabolic model for acetate uptake by a mixed culture of phosphate- and glycogen-accumulating organisms (PAOs and GAOs) under anaerobic conditions. The model uses variable overall stoichiometry based on the assumption that PAOs may have the ability of using the glyoxylate pathway to produce the required reducing power for polyhydroxyalkonate (PHA) synthesis. The proposed model was tested and verified by experimental results. A sequencing batch reactor system was operated for enhanced biological phosphorus removal (EBPR) with acetate as the sole carbon source at different influent acetate/phosphate ratios. The resulting experimental data supported the validity of the proposed model, indicating the presence of GAOs for all tested HAc/P ratios, especially under P-limiting conditions. Strong agreement is observed between experimental values and model predictions for all model components, namely, PHB production, PHA composition, glycogen utilization, and P release.     

Fully coupled activated sludge model (FCASM): Model development

A sub-microscopic mechanism model named Fully Coupled Activated Sludge Model (FCASM) about biological nutrient removal in the wastewater treatment process was developed in the present study. The functional organisms existing simultaneously in the activated sludge system were separated into eight groups, including aerobic heterotrophic organisms, nitrite reducing organisms, nitrate reducing organisms, ammonium oxidizing autotrophs, nitrite oxidizing autotrophs, non-denitrifying phosphorus-accumulating organisms (PAOs), denitrifying phosphorus-accumulating bacteria (DPB), and glycogen-accumulating organisms (GAOs). In FCASM, the interaction relationships of the eight functional microorganisms were taken fully into account. FCASM could model biological nitrogen removal via nitrite by splitting nitrification process and denitrification process into two-step reactions, and the autotrophs and denitrifying organisms were divided into two groups, respectively. What’s important, FCASM included the anaerobic maintenance processes of sequential utilization of polyphosphate followed by glycogen for PAOs and DPB and glycolysis of the intracellular stored glycogen for GAOs.     

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).     

Modeling and experimental study on the anaerobic/aerobic/anoxic process for simultaneous nitrogen and phosphorus removal: The effect of acetate addition

A mathematical model based on the simulation software AQUASIM was developed to validate an anaerobic/aerobic/anoxic (AOA) process that enables simultaneous nitrogen and phosphorus removal in a single reactor by adding external organic carbon to preclude excess aerobic phosphate uptake by polyphosphate-accumulating organisms (PAOs) and provide phosphate for denitrifying PAOs (DNPAOs). Aerobic batch tests after anaerobic phosphate release with different chemical oxygen demand (COD) concentrations indicated that the effect of COD concentration on the phosphate uptake preclusion could be expressed by a simple formula. The reduction factor reflecting the formula, which retards the aerobic phosphate uptake in the presence of COD, was added to the process rates of aerobic polyphosphate storage and PAOs growth in the model. The improved model, which included the reduction factor, reasonably matched the experimental result regarding aerobic phosphate uptake behavior whereas the model without it did not; thus, the former precisely predicts the AOA process behavior.     

Experimental evaluation of decrease in the activities of polyphosphate/glycogen‐accumulating organisms due to cell death and activity decay in activated sludge

Decrease in bacterial activity (biomass decay) in activated sludge can result from cell death (reduction in the amount of active bacteria) and activity decay (reduction in the specific activity of active bacteria). The goal of this study was to experimentally differentiate between cell death and activity decay as the cause of decrease in bacterial activity. By means of measuring maximal anaerobic phosphate release rates, verifying membrane integrity by live/dead staining and verifying presence of 16S rRNA with fluorescence in situ hybridization (FISH), the decay rates and death rates of polyphosphate‐accumulating organisms (PAOs) in a biological nutrient removal (BNR) system and a laboratory phosphate removing sequencing batch reactor (SBR) system were determined, respectively, under famine conditions. In addition, the decay rate and death rate of glycogen‐accumulating organisms (GAOs) in a SBR system with an enrichment culture of GAOs were also measured under famine conditions. Hereto the maximal anaerobic volatile fatty acid uptake rates, live/dead staining, and FISH were used. The experiments revealed that in the BNR and enriched PAO‐SBR systems, activity decay contributed 58% and 80% to the decreased activities of PAOs, and that cell death was responsible for 42% and 20% of decreases in their respective activities. In the enriched GAOs system, activity decay constituted a proportion of 74% of the decreased activity of GAOs, and cell death only accounted for 26% of the decrease of their activity. Biotechnol. Bioeng. 2010; 106: 399–407. © 2010 Wiley Periodicals, Inc.     

聚磷菌和聚糖菌的竞争影响因素研究进展

目前, 强化生物除磷工艺(EBPR)以其经济有效而得到广泛的应用, 该工艺关键在于聚磷菌的富集。然而已经发现, 有一类细菌—聚糖菌(GAOs)能够和聚磷菌(PAOs)竞争, 导致除磷效果恶化。关于PAOs-GAOs的竞争, 研究已经很多, 但是其结论有趋同也有矛盾, 有必要对此进行分析讨论。根据近年来国内外的相关报道, 阐述了聚磷菌与聚糖菌的竞争影响因素, 其中碳磷比、碳源种类、温度、pH值是关键因素, 而污泥龄、溶解氧以及水力停留时间等因素对于PAOs和GAOs的竞争也起一定的作用。此外, 在EBPR系统中, 缺氧条件下, 存在反硝化聚磷菌(DPB)和反硝化聚糖菌(DGAO)也会对聚磷菌富集和系统除磷产生影响。最后对EBPR系统未来的发展方向进行了展望。     

Microbial selection on enhanced biological phosphorus removal systems fed exclusively with glucose

The microbial selection on an enhanced biological phosphorus removal (EBPR) system was investigated in a laboratory-scale sequencing batch reactor fed exclusively with glucose as the carbon source. Fluorescence In Situ Hybridization analysis was performed to target two polyphosphate accumulating organisms (PAOs) (i.e., Candidatus Accumulibacter phosphatis and Microlunatus phosphovorus) and two glycogen accumulating organisms (GAOs) (i.e., Candidatus Competibacter phosphatis and Micropruina glycogenica). The results show that glucose might not select for Candidatus Accumulibacter phosphatis. However, Microlunatus phosphovorus, Candidatus Competibacter phosphatis, and Micropruina glycogenica might be selected. The highest percent relative abundance (% RA) of Candidatus Accumulibacter phosphatis was about 42%; this occurred at the beginning of the experimental period when phosphorus removal was efficient. However, the % RA of these bacteria decreased, reaching below 4% at the end of the run. The maximum % RA of Microlunatus phosphovorus, Candidatus Competibacter phosphatis, and Micropruina glycogenica was about 21, 37, 17%, respectively. It appears that a higher glucose concentration might be detrimental for Microlunatus phosphovorus and Micropruina glycogenica. Results also indicate a dominance of GAOs over PAOs when EBPR systems are fed with glucose. It is possible that the GAOs outcompete the PAOs at low pH values; it has been reported that at low pH, GAOs use glycogen as the energy source to uptake glucose. As a result, P-removal deteriorated. Therefore, glucose is not a strong candidate as a carbon source to supplement EBPR systems that do not contain sufficient volatile fatty acids.     

Competition between polyphosphate and glycogen accumulating organisms in enhanced biological phosphorus removal systems with acetate and propionate as carbon sources

Enhanced biological phosphorus removal (EBPR) is a widely used process for achieving phosphorus removal from wastewater. A potential reason for EBPR failure is the undesirable growth of glycogen accumulating organisms (GAOs), which can compete for carbon sources with the bacterial group responsible for phosphorus removal from wastewater: the polyphosphate accumulating organisms (PAOs). This study investigates the impact of carbon source on EBPR performance and the competition between PAOs and GAOs. Two sequencing batch reactors (SBRs) were operated during a 4-6 month period and fed with a media containing acetate or propionate, respectively, as the sole carbon source. It was found that the acetate fed SBR rarely achieved a high level of phosphorus removal, and that a large portion of the microbial community was comprised of "Candidatus Competibacter phosphatis", a known GAO. The propionate fed SBR, however, achieved stable phosphorus removal throughout the study, apart from one brief disturbance. The bacterial community of the propionate fed SBR was dominated by "Candidatus Accumulibacter phosphatis", a known PAO, and did not contain Competibacter. In a separate experiment, another SBR was seeded with a mixture of PAOs and a group of alphaproteobacterial GAOs, both enriched with propionate as the sole carbon source. Stable EBPR was achieved and the PAO population increased while the GAOs appeared to be out-competed. The results of this paper suggest that propionate may provide PAOs with a selective advantage over GAOs in the PAO-GAO competition, particularly through the minimisation of Competibacter. Propionate may be a more suitable substrate than acetate for enhancing phosphorus removal in EBPR systems.     

Ecophysiology of a group of uncultured Gammaproteobacterial glycogen-accumulating organisms in full-scale enhanced biological phosphorus removal wastewater treatment plants

The presence of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) plants can seriously deteriorate the biological P-removal by out-competing the polyphosphate-accumulating organisms (PAOs). In this study, uncultured putative GAOs (the GB group, belonging to the Gammaproteobacteria) were investigated in detail in 12 full-scale EBPR plants. Fluorescence in situ hybridization (FISH) revealed that the biovolume of the GB bacteria constituted 2-6% of total bacterial biovolume. At least six different subgroups of the GB bacteria were found, and the number of dominant subgroups present in each plant varied between one and five. Ecophysiological investigations using microautoradiography in combination with FISH showed that, under aerobic or anaerobic conditions, all subgroups of the GB bacteria could take up acetate, pyruvate, propionate and some amino acids, while some subgroups in addition could take up formate and thymidine. Glucose, ethanol, butyrate and several other organic substrates were not taken up. Glycolysis was essential for the anaerobic uptake of organic substrates. Polyhydroxyalkanoates (PHA) but not polyphosphate (polyP) granules were detected in all GB bacterial cells. Polyhydroxyalkanoate formation after anaerobic uptake of acetate was confirmed by measuring the increase in fluorescence intensity of PHA granules inside GB bacterial cells after Nile blue staining. One GB subgroup was possibly able to denitrify, and several others were able to reduce nitrate to nitrite. PAOs were also enumerated by FISH in the same treatment plants. Rhodocyclus-related PAOs and Actinobacteria-related PAOs constituted up to 7% and 29% of total bacterial biovolume respectively. Rhodocyclus-related PAOs always coexisted with the GB bacteria and showed many physiological similarities. Factors of importance for the competition between the three groups of important bacteria in EBPR plants are discussed.