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.     

Improvement strategy on enhanced biological phosphorus removal for municipal wastewater treatment plants: full-scale operating parameters, sludge activities, and microbial features

The poor quality of effluent discharged by municipal wastewater treatment plants (WWTPs) is threatening the safety of water ecology. This study, which integrated a field survey, batch tests, and microbial community identification, was designed to improve the effectiveness of the enhanced biological phosphorus removal (EBPR) process for WWTPs. Over two-thirds of the investigated WWTPs could not achieve total P in effluent lower than 0.5 mg/L, mainly due to the high ratio of chemical oxygen demand to P (28.6-196.2) in the influent. The rates of anaerobic P release and aerobic P uptake for the activated sludge varied from 0.22 to 7.9 mg/g VSS/h and 0.43 to 8.11 mg/g VSS/h, respectively. The fraction of Accumulibacter (PAOs: polyphosphate accumulating organisms) was 4.8 ± 2.0% of the total biomass, while Competibacter (GAOs: glycogen-accumulating organisms) accounted for 4.8 ± 6.4%. The anaerobic P-release rate was found to be an effective indicator of EBPR. Four classifications of the principal components were identified to improve the EBPR effluent quality and sludge activity.     

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.     

Characterizing the biochemical activity of full-scale enhanced biological phosphorus removal systems: A comparison with metabolic models

The metabolism of polyphosphate accumulating organisms (PAOs) has been widely studied through the use of lab-scale enrichments. Various metabolic models have been formulated, based on the results from lab-scale experiments using enriched PAO cultures. A comparison between the anaerobic stoichiometry predicted by metabolic models with that exhibited by full-scale sludge in enhanced biological phosphorus removal (EBPR) wastewater treatment plants (WWTPs) was performed in this study. Batch experiments were carried out with either acetate or propionate as the sole carbon source, using sludges from two different EBPR-WWTPs in Australia that achieved different phosphorus removal performances. The results support the hypothesis that the anaerobic degradation of glycogen is the primary source of reducing equivalents generated by PAOs, however, they also suggested a partial contribution of the tricarboxylic acid (TCA) cycle in some cases. The experimental results obtained when acetate was the carbon source suggest the involvement of the modified succinate-propionate pathway for the generation of poly-beta-hydroxyvalerate (PHV). Overall, the batch test results obtained from full-scale EBPR sludge with both substrates were generally well described by metabolic model predictions for PAOs.     

Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. We investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigations have shown that anaerobic phosphate release depends on the extracellular pH. We observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty acid (VFA) uptake. Acidification of the sludge inhibited anaerobic phosphate release even in the presence of VFA. From these observations, we postulate that an additional possible role of anaerobic polyphosphate degradation in EBPR is for intracellular pH control. Intracellular pH control may be a metabolic feature of EBPR, not previously considered, that could have some use in the control and optimisation of EBPR. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 507–515, 1999.     

Methanol‐driven enhanced biological phosphorus removal with a syntrophic consortium

The presence of suitable carbon sources for enhanced biological phosphorus removal (EBPR) plays a key role in phosphorus removal from wastewater in urban WWTP. For wastewaters with low volatile fatty acids (VFAs) content, an external carbon addition is necessary. As methanol is the most commonly external carbon source used for denitrification it could be a priori a promising alternative, but previous attempts to use it for EBPR have failed. This study is the first successful report of methanol utilization as external carbon source for EBPR. Since a direct replacement strategy (i.e., supply of methanol as a sole carbon source to a propionic‐fed PAO‐enriched sludge) failed, a novel process was designed and implemented successfully: development of a consortium with anaerobic biomass and polyphosphate accumulating organisms (PAOs). Methanol‐degrading acetogens were (i) selected against other anaerobic methanol degraders from an anaerobic sludge; (ii) subjected to conventional EBPR conditions (anaerobic + aerobic); and (iii) bioaugmented with PAOs. EBPR with methanol as a sole carbon source was sustained in a mid‐term basis with this procedure. Biotechnol. Bioeng. 2013; 110: 391–400. © 2012 Wiley Periodicals, Inc.     

Ecophysiology of polyphosphate-accumulating organisms and glycogen-accumulating organisms in a continuously aerated enhanced biological phosphorus removal process

Aims: To investigate the ecophysiology of populations of polyphosphate-accumulating organisms (PAO) and glycogen-accumulating organisms (GAO) in communities of a novel acetate fed process removing phosphate from wastewater. Attempts were made to see if acetate could be replaced by an alternative carbon source which did not support the growth of the GAO. Methods and Results: A continuously aerated sequencing batch reactor was operated with different acetate feed levels. Fluorescence in situ hybridization (FISH) showed that Defluviicoccus GAO numbers increased at lower acetate feed levels. With FISH/microautoradiography (MAR) both detected morphotypes of Defluviicoccus assimilated a wider range of substrates aerobically than Accumulibacter PAO. Their uptake profile differed from that reported for the same phylotype in full scale anaerobic : aerobic EBPR plants. Conclusions: This suggests that replacing acetate with another substrate is unlikely to provide Accumulibacter with a selective advantage in this process. Why Defluviicoccus appeared to out-compete Accumulibacter at lower acetate concentrations was not clear. Data suggest physiological and morphological diversity may exist within a single Defluviicoccus phylotype. Significance and Impact of the Study: This study implies that the current FISH probes for Defluviicoccus GAO may not reveal the full extent of their biodiversity, and that more information is required before strategies for their control can be devised.     

Analysis of the microbial community structure and function of a laboratory scale enhanced biological phosphorus removal reactor

A laboratory scale sequencing batch reactor (SBR) operating for enhanced biological phosphorus removal (EBPR) and fed with a mixture of volatile fatty acids (VFAs) showed stable and efficient EBPR capacity over a four-year-period. Phosphorus (P), poly-beta-hydroxyalkanoate (PHA) and glycogen cycling consistent with classical anaerobic/aerobic EBPR were demonstrated with the order of anaerobic VFA uptake being propionate, acetate then butyrate. The SBR was operated without pH control and 63.67 +/- 13.86 mg P l-1 was released anaerobically. The P% of the sludge fluctuated between 6% and 10% over the operating period (average of 8.04 +/- 1.31%). Four main morphological types of floc-forming bacteria were observed in the sludge during one year of in-tensive microscopic observation. Two of them were mainly responsible for anaerobic/aerobic P and PHA transformations. Fluorescence in situ hybridization (FISH) and post-FISH chemical staining for intracellular polyphosphate and PHA were used to determine that 'Candidatus Accumulibacter phosphatis' was the most abundant polyphosphate accumulating organism (PAO), forming large clusters of coccobacilli (1.0-1.5 micro m) and comprising 53% of the sludge bacteria. Also by these methods, large coccobacillus-shaped gammaproteobacteria (2.5-3.5 micro m) from a recently described novel cluster were glycogen-accumulating organisms (GAOs) comprising 13% of the bacteria. Tetrad-forming organisms (TFOs) consistent with the 'G bacterium' morphotype were alphaproteobacteria, but not Amaricoccus spp., and comprised 25% of all bacteria. According to chemical staining, TFOs were occasionally able to store PHA anaerobically and utilize it aerobically.     

Microautoradiographic study of Rhodocyclus-related polyphosphate-accumulating bacteria in full-scale enhanced biological phosphorus removal plants

The ecophysiology of uncultured Rhodocyclus-related polyphosphate-accumulating organisms (PAO) present in three full-scale enhanced biological phosphorus removal (EBPR) activated sludge plants was studied by using microautoradiography combined with fluorescence in situ hybridization. The investigations showed that these organisms were present in all plants examined and constituted 5 to 10, 10 to 15, and 17 to 22% of the community biomass. The behavior of these bacteria generally was consistent with the biochemical models proposed for PAO, based on studies of lab-scale investigations of enriched and often unknown PAO cultures. Rhodocyclus-related PAO were able to accumulate short-chain substrates, including acetate, propionate, and pyruvate, under anaerobic conditions, but they could not assimilate many other low-molecular-weight compounds, such as ethanol and butyrate. They were able to assimilate two substrates (e.g., acetate and propionate) simultaneously. Leucine and thymidine could not be assimilated as sole substrates and could only be assimilated as cosubstrates with acetate, perhaps serving as N sources. Glucose could not be assimilated by the Rhodocyclus-related PAO, but it was easily fermented in the sludge to products that were subsequently consumed. Glycolysis, and not the tricarboxylic acid cycle, was the source that provided the reducing power needed by the Rhodocyclus-related PAO to form the intracellular polyhydroxyalkanoate storage compounds during anaerobic substrate assimilation. The Rhodocyclus-related PAO were able to take up orthophosphate and accumulate polyphosphate when oxygen, nitrate, or nitrite was present as an electron acceptor. Furthermore, in the presence of acetate growth was sustained by using oxygen, as well as nitrate or nitrite, as an electron acceptor. This strongly indicates that Rhodocyclus-related PAO were able to denitrify and thus played a role in the denitrification occurring in full-scale EBPR plants.     

Ozonolysate of excess sludge as a carbon source in an enhanced biological phosphorus removal for low strength wastewater

Potential use of the municipal sludge ozonolysate as a carbon source was examined for phosphorus removal from low strength wastewater in a modified intermittently decanted extended aeration (IDEA) process. At ozone dosage of 0.2 g O₃/g solids, readily biodegradable COD accounted for about 36% of COD from sludge ozonolysate. The denitrification potential of ozonolysate as a carbon source was comparable to that of acetate. Although, the first order constant for phosphorus release with the ozonolysate was half that of acetate, it was much higher than that of wastewater. Continuous operation of the modified IDEA process showed that the removals of nitrogen and phosphorus were simultaneously enhanced by addition of the ozonolysate. Phosphorus release was significantly induced after complete denitrification indicating that phosphorus release was strongly depended on nitrate concentration. Effectiveness of the ozonolysate as a carbon source for EBPR was also confirmed in a track study of the modified IDEA.     

Production of polyhydroxybutyrate by activated sludge performing enhanced biological phosphorus removal

In this study, polyhydroxybutyrate (PHB) – a biodegradable plastics material – was produced by activated sludge performing enhanced biological phosphorus removal (EBPR) in batch experiments under anaerobic, aerobic and anaerobic/aerobic conditions. Under anaerobic conditions, the maximum PHB content of the dry biomass was 28.8% by weight, while under aerobic or anaerobic/aerobic conditions, the maximum PHB content was about 50%. The PHB production rate with respect to the volatile suspended solids (VSS) was: (i) 70 mg/(g VSS) h under aerobic conditions that followed anaerobic conditions, (ii) 156 mg/(g VSS) h under anaerobic condition, and (iii) 200 mg/(g VSS) h under aerobic conditions with energy also supplied from polyphosphate. A side stream, with initially anaerobic conditions for PHB accumulation and phosphorus release, and then aerobic conditions for PHB accumulation, was proposed. In this side stream, biomass with a high PHB content and a high PHB production rate could be both achieved.     

传统厌氧/好氧生物除磷与厌氧/缺氧反硝化除磷效能的比较

采用序批式反应器(SBR),对比厌氧/好氧(A/O)和厌氧/缺氧(A/A)2种运行模式对模拟生活和工业混合污水同时脱氮除磷的效能。结果表明:反硝化聚磷菌完全可以在厌氧/缺氧交替运行条件下得到富集,稳定运行的2种模式对有机物和P的去除率分别保持在90%和85%以上,且A/A SBR具有更强的释磷能力,其释磷量比A/O SBR高出1.2倍。进一步试验表明:磷的释放在有无硝酸盐的情况下效果是不同的。2个系统内污泥均有反硝化除磷能力,A/A SBR中所含反硝化聚磷菌(DPAO)的比例是A/O SBR的4.56倍。2种模式出水水质都能取得较好的效果,且能实现同步除磷脱氮,而反硝化除磷在生物除磷方面更具优势。     

强化生物除磷系统主要微生物及其代谢机理研究进展

强化生物除磷(enhanced biological phosphorus removal,EBPR)工艺在废水除磷处理中应用广泛.主要功能微生物及其代谢机理的研究是有效调控EBPR工艺稳定运行与效能提升的基础.本文选取EBPR系统中最主要的两类微生物(聚磷菌和聚糖菌),从底物吸收机制、糖酵解途径、TCA途径的贡献以及聚磷菌和聚糖菌的代谢相似性等方面对这些微生物的代谢机理进行综述,评价了分子生物学技术在研究EBPR系统微生物学及其代谢机理方面的应用现状,在此基础上对EBPR系统今后的研究方向进行了展望.
     

Transfer of energy pathway genes in microbial enhanced biological phosphorus removal communities

Background

Lateral gene transfer (LGT) is an important evolutionary process in microbial evolution. In sewage treatment plants, LGT of antibiotic resistance and xenobiotic degradation-related proteins has been suggested, but the role of LGT outside these processes is unknown. Microbial communities involved in Enhanced Biological Phosphorus Removal (EBPR) have been used to treat wastewater in the last 50 years and may provide insights into adaptation to an engineered environment. We introduce two different types of analysis to identify LGT in EBPR sewage communities, based on identifying assembled sequences with more than one strong taxonomic match, and on unusual phylogenetic patterns. We applied these methods to investigate the role of LGT in six energy-related metabolic pathways.

Results

The analyses identified overlapping but non-identical sets of transferred enzymes. All of these were homologous with sequences from known mobile genetic elements, and many were also in close proximity to transposases and integrases in the EBPR data set. The taxonomic method had higher sensitivity than the phylogenetic method, identifying more potential LGTs. Both analyses identified the putative transfer of five enzymes within an Australian community, two in a Danish community, and none in a US-derived culture.

Conclusions

Our methods were able to identify sequences with unusual phylogenetic or compositional properties as candidate LGT events. The association of these candidates with known mobile elements supports the hypothesis of transfer. The results of our analysis strongly suggest that LGT has influenced the development of functionally important energy-related pathways in EBPR systems, but transfers may be unique to each community due to different operating conditions or taxonomic composition.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1752-5) contains supplementary material, which is available to authorized users.     

Anaerobic metabolism of propionate by polyphosphate-accumulating organisms in enhanced biological phosphorus removal systems

Propionate, a carbon substrate abundant in many prefermenters, has been shown in several previous studies to be a more favorable substrate than acetate for enhanced biological phosphorus removal (EBPR). The anaerobic metabolism of propionate by polyphosphate accumulating organisms (PAOs) is studied in this paper. A metabolic model is proposed to characterize the anaerobic biochemical transformations of propionate uptake by PAOs. The model is demonstrated to predict very well the experimental data from a PAO culture enriched in a laboratory-scale reactor with propionate as the sole carbon source. Quantitative fluorescence in-situ hybridization (FISH) analysis shows that Candidatus Accumulibacter phosphatis, the only identified PAO to date, constitute 63% of the bacterial population in this culture. Unlike the anaerobic metabolism of acetate by PAOs, which induces mainly poly-beta-hydroxybutyrate (PHB) production, the major fractions of poly-beta-hydroxyalkanoate (PHA) produced with propionate as the carbon source are poly-beta-hydroxyvalerate (PHV) and poly-beta-hydroxy-2-methylvalerate (PH2MV). PHA formation correlates very well with a selective (or nonrandom) condensation of acetyl-CoA and propionyl-CoA molecules. The maximum specific propionate uptake rate by PAOs found in this study is 0.18 C-mol/C-mol-biomass . h, which is very similar to the maximum specific acetate uptake rate reported in literature. The energy required for transporting 1 carbon-mole of propionate across the PAO cell membrane is also determined to be similar to the transportation of 1 carbon-mole of acetate. Furthermore, the experimental results suggest that PAOs possess a similar preference toward acetate and propionate uptake on a carbon-mole basis.     

Identity and ecophysiology of uncultured actinobacterial polyphosphate-accumulating organisms in full-scale enhanced biological phosphorus removal plants

Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAO) in a full-scale enhanced biological phosphorus removal (EBPR) plant. The results showed that, in addition to uncultured Rhodocyclus-related PAO, two morphotypes hybridizing with gene probes for the gram-positive Actinobacteria were also actively involved in uptake of orthophosphate (Pi). Clone library analysis and further investigations by MAR-FISH using two new oligonucleotide probes revealed that both morphotypes, cocci in clusters of tetrads and short rods in clumps, were relatively closely related to the genus Tetrasphaera within the family Intrasporangiaceae of the Actinobacteria (93 to 98% similarity in their 16S rRNA genes). FISH analysis of the community biomass in the treatment plant investigated showed that the short rods (targeted by probe Actino-658) were the most abundant (12% of all Bacteria hybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up P(i) aerobically only if, in a previous anaerobic phase, they had taken up organic matter from wastewater or a mixture of amino acids. They could not take up short-chain fatty acids (e.g., acetate), glucose, or ethanol under anaerobic or aerobic conditions. The storage compound produced during the anaerobic period was not polyhydroxyalkanoates, as for Rhodocyclus-related PAO, and its identity is still unknown. Growth and uptake of Pi took place in the presence of oxygen and nitrate but not nitrite, indicating a lack of denitrifying ability. A survey of the occurrence of these actinobacterial PAO in 10 full-scale EBPR plants revealed that both morphotypes were widely present, and in several plants more abundant than the Rhodocyclus-related PAO, thus playing a very important role in the EBPR process.     

Long-term population dynamics and in situ physiology in activated sludge systems with enhanced biological phosphorus removal operated with and without nitrogen removal

Quantitative fluorescence in situ hybridization (FISH) and the combination of FISH with microautoradiography (MAR) were used in order to study the long-term population dynamics (2.5 years) and the in situ physiology in two parallel activated sludge pilot systems with enhanced biological phosphorus removal (EBPR). The two systems received the same influent wastewater, but were differently operated (with and without nitrogen removal, respectively). Both systems showed a significant P removal that increased when different substrates (phosphorus (P), acetate and glucose, respectively) were added to the influent wastewater. Rhodocyclus-related bacteria were present in both systems in significant numbers (ranging from 4 to 28%) throughout the whole period. This supports the hypothesis that these bacteria occur in significant numbers in different types of well-operating EBPR activated sludge processes. However, we observed a lower correlation (< 0.5) for the amount of Rhodocyclus-related bacteria to the P content in activated sludge than previous studies (> 0.9). The Actinobacteria were the only additional group of bacteria which showed a similar degree of correlation to the P content in activated sludge as the Rhodocyclus-related bacteria--but only for the system without nitrogen removal. Significant amounts (< or = 12%) of glycogen-accumulating bacteria (GAOs) were detected in the system with nitrogen removal (but not in the other system), but had no, in contrast to previous observations, apparent negative effect on the overall EBPR performance. FISH-MAR indicated that a significant part of the Betaproteobacteria (part of them identified as Rhodocyclus-related bacteria) as well as the Actinobacteria were able to take up 33Pi, [3H]-acetate and [3H]-glucose under anaerobic-aerobic conditions. The contribution of anoxic 33Pi uptake under alternating anaerobic-anoxic conditions was significantly lower. Interestingly, not all Rhodocyclus-related bacteria showed uptake of these three radioactive substrates. This may be due to differences in metabolic state, physiological potential or genotype, not detectable by the present probe set for Rhodocyclus-related bacteria. Comparison of the 33Pi, [3H]-acetate and [3H]-glucose uptake by activated sludge after different fixation and incubation procedures showed that a part of the observed 33Pi uptake may have been caused by a combination of a biological and chemical or biologically induced chemical P adsorption.     

Effect of aspartate and glutamate on the fate of enhanced biological phosphorus removal process and microbial community structure

This study investigated the fate of enhanced biological phosphorus removal (EBPR) and changes in microbial speciation in a sequencing batch reactor (SBR) fed with aspartate and glutamate. It involved SBR operation for 288 days, batch tests for observation of metabolic functions together with microscopic and phylogenetic analyses. Polyphosphate accumulating organisms (PAOs) were observed in abundance with complete removal of phosphorus. Fluorescence in situ hybridization (FISH) combined with 4′,6-dia-midino-2-phenylindole (DAPI) staining confirmed the accumulation of polyphosphate by Rhodocyclus-related and Actinobacterial PAOs. Aspartate seemed to favor the competitive growth of Rhodocyclus-related PAOs since EBPR population used the common biochemical pathways followed by Rhodocyclus-related PAOs in the aspartate fed batch tests. In the glutamate fed batch reactors, however, Actinobacterial PAOs appeared to be competitively selected which explains the lower levels of PHA generation. Even though operational conditions did not change, effective EBPR could not be maintained during the latter part of the study.     

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.     

Dynamics of microbial community structure of and enhanced biological phosphorus removal by aerobic granules cultivated on propionate or acetate

Aerobic granules are dense microbial aggregates with the potential to replace floccular sludge for the treatment of wastewaters. In bubble-column sequencing batch reactors, distinct microbial populations dominated propionate- and acetate-cultivated aerobic granules after 50 days of reactor operation when only carbon removal was detected. Propionate granules were dominated by Zoogloea (40%), Acidovorax, and Thiothrix, whereas acetate granules were mainly dominated by Thiothrix (60%). Thereafter, an exponential increase in enhanced biological phosphorus removal (EBPR) activity was observed in the propionate granules, but a linear and erratic increase was detected in the acetate ones. Besides Accumulibacter and Competibacter, other bacterial populations found in both granules were associated with Chloroflexus and Acidovorax. The EBPR activity in the propionate granules was high and stable, whereas EBPR in the acetate granules was erratic throughout the study and suffered from a deterioration period that could be readily reversed by inducing hydrolysis of polyphosphate in presumably saturated Accumulibacter cells. Using a new ppk1 gene-based dual terminal-restriction fragment length polymorphism (T-RFLP) approach revealed that Accumulibacter diversity was highest in the floccular sludge inoculum but that when granules were formed, propionate readily favored the dominance of Accumulibacter type IIA. In contrast, acetate granules exhibited transient shifts between type I and type II before the granules were dominated by Accumulibacter type IIA. However, ppk1 gene sequences from acetate granules clustered separately from those of propionate granules. Our data indicate that the mere presence of Accumulibacter is not enough to have consistently high EBPR but that the type of Accumulibacter determines the robustness of the phosphate removal process.