Involvement of Rhodocyclus-related organisms in phosphorus removal in full-scale wastewater treatment plants

The participation of organisms related to Rhodocyclus in full-scale enhanced biological phosphorus removal (EBPR) was investigated. By using fluorescent in situ hybridization techniques, the communities of Rhodocyclus-related organisms in two full-scale wastewater treatment plants were estimated to represent between 13 and 18% of the total bacterial population. However, the fractions of these communities that participated in polyphosphate accumulation depended on the type of treatment process evaluated. In a University of Cape Town EBPR process, the percentage of Rhodocyclus-related cells that contained polyphosphate was about 20% of the total bacterial population, but these cells represented as much as 73% of the polyphosphate-accumulating organisms (PAOs). In an aerated-anoxic EBPR process, Rhodocyclus-related PAOs were less numerous, accounting for 6% of the total bacterial population and 26% of the total PAO population. In addition, 16S ribosomal DNA sequences 99.9% similar to the sequences of Rhodocyclus-related organisms enriched in acetate-fed bench-scale EBPR reactors were recovered from both full-scale plants. These results confirmed the involvement of Rhodocyclus-related organisms in EBPR and demonstrated their importance in full-scale processes. In addition, the results revealed a significant correlation between the type of EBPR process and the PAO community.     

Progress Toward Understanding the Distribution of Accumulibacter Among Full-Scale Enhanced Biological Phosphorus Removal Systems

This study investigated the role of Accumulibacter-related bacterial populations and factors influencing their distribution in enhanced biological phosphorus removal (EBPR) systems in the USA. For this purpose, five full-scale wastewater treatment facilities performing EBPR were surveyed. The facilities had different configurations but were all treating primarily domestic wastewater. Two facilities had history of poor EBPR performance. Batch-scale acetate uptake and inorganic phosphate (P_i) release and uptake experiments were conducted to evaluate the EBPR activity of each sludge. Typical P_i and acetate profiles were observed, and EBPR activity was found to be positively correlated to polyphosphate (polyP)-accumulating organism (PAO) abundance, as determined by staining intracellular polyP. The abundance of Accumulibacter-related organisms was investigated using fluorescent in situ hybridization. Accumulibacter-related organisms were present in all full-scale EBPR facilities, at levels ranging from 9 to 24% of total cells. More than 80% of Accumulibacter-related organisms were estimated to have high polyP content, confirming their involvement in EBPR in these five facilities. However, Accumulibacter-related PAOs were only a fraction (40–69%) of the total PAO population. The variation of Accumulibacter-related PAO abundance among these EBPR systems suggests that multiple interacting factors such as wastewater characteristics and operational conditions are structuring PAO communities.     

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.     

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.     

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 (P_i). 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 P_i 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.     

Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation

Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were β-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the β-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing ≥98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the β-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.     

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.     

Microbial population analysis of nutrient removal-related organisms in membrane bioreactors

Membrane bioreactors (MBR) are an important and increasingly implemented wastewater treatment technology, which are operated at low food to microorganism ratios (F/M) and retain slow-growing organisms. Enhanced biological phosphorus removal (EBPR)-related organisms grow slower than ordinary heterotrophs, but have never been studied in detail in MBRs. This study presents a comprehensive analysis of the microorganisms involved in EBPR in pilot- and full-scale MBRs, using fluorescence in situ hybridization (FISH), as well as an overall assessment of other relevant microbial groups. The results showed that polyphosphate accumulating organisms (PAOs) were present at similar levels in all studied MBRs (10% ± 6%), even those without a defined anaerobic zone. Glycogen accumulating organisms were also detected, although rarely. The FISH results correlated well with the observed P removal performance of each plant. The results from this study suggest that a defined anaerobic zone is not necessarily required for putative PAO growth in MBRs, since polyphosphate storage may provide a selective advantage in fulfilling cell maintenance requirements in substrate-limited conditions (low F/M).     

Widespread detection of Candidatus Accumulibacter phosphatis,a polyphosphate-accumulating organism,in sediments of the Columbia River estuary

Enhanced biological phosphorus removal (EBPR) exploits the metabolism of polyphosphate-accumulating organisms (PAOs) to remove excess phosphorus (P) from wastewater treatment. Candidatus Accumulibacter phosphatis (Accumulibacter) is the most abundant and well-studied PAO in EBPR systems. In a previous study, we detected polyphosphates throughout peripheral bay sediments, and hypothesized that an estuary is an ideal setting to evaluate PAOs in a natural system, given that estuaries are characterized by dynamic dissolved oxygen fluctuations that potentially favour PAO metabolism. We detected nucleotide sequences attributable to Accumulibacter (16S rRNA, ppk1) in sediments within three peripheral bays of the Columbia River estuary at abundances rivalling those observed in conventional wastewater treatment plants (0.01%–2.6%). Most of the sequences attributable to Accumulibacter were Type I rather than Type II, despite the fact that the estuary does not have particularly high nutrient concentrations. The highest diversity of Accumulibacter was observed in oligohaline peripheral bays, while the greatest abundances were observed at the mouth of the estuary in mesohaline sediments in the spring and summer. In addition, an approximately 70% increase in polyphosphate concentrations observed at one of the sites between dawn and dusk suggests that PAOs may play an important role in P cycling in estuary sediments.     

From macro to lab-scale: Changes in bacterial community led to deterioration of EBPR in lab reactor

A laboratory scale sequencing batch reactor (SBR), fed with synthetic wastewater containing a mixture of organic compounds, was operated for nearly six months. Despite maintaining the same operational conditions, a deterioration of enhanced biological phosphorus removal (EBPR) occurred after 40 days of SBR operation. The P_rel/C_upt ratio decreased from 0.28 to 0.06 P-mol C-mol^?1, and C requirements increased from 11 to 32 mg C h^?1 g^?1 of mixed liquor suspended solids. A FISH analysis showed that the percentage of Accumulibacter in an overall community of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) dropped from 93% to 13%. An increase in abundance of Gammaproteobacteria (from 2.6% to 22%) and Alphaproteobacteria (from 1.8% to 10%) was observed. The number of Competibacter increased from 0.5% to nearly 9%. Clusters 1 and 2 of Defluviicoccus-related GAOs, not detected before deterioration, constituted 35% and 27% of Alphaproteobacteria, respectively. We concluded that lab-scale experiments should not be extended implicitly to full-scale EBPR systems because some bacterial groups are detected mainlyin lab-scale reactors. Well-defined, lab-scale operational conditions reduce the number of ecological niches available to bacteria.     

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.     

Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation

Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were beta-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the beta-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing >/=98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the beta-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.     

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.     

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.     

“Candidatus Dechloromonas phosphoritropha” and “Ca. D. phosphorivorans”, novel polyphosphate accumulating organisms abundant in wastewater treatment systems

Members of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish and global wastewater treatment plants. The two most abundant species worldwide revealed in situ dynamics of important intracellular storage polymers, measured by FISH-Raman in activated sludge from four full-scale EBPR plants and from a lab-scale reactor fed with different substrates. Moreover, seven distinct Dechloromonas species were determined from a set of ten high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for polyphosphate (poly-P), glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic–aerobic cycling, legitimately placing these microorganisms among the important PAOs. They are potentially involved in denitrification showing niche partitioning within the genus and with other important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphoritropha and Candidatus Dechloromonas phosphorivorans.Subject terms: Water microbiology, Microbial ecology     

增强型生物除磷过程中聚磷酸盐积累微生物的研究进展

从磷污染控制、污水脱磷和磷资源角度论述了生物除磷的作用,并着重论述了增强型生物除磷过程中聚磷酸盐微生物(PAO)的研究历史、代谢特征及研究方法．聚磷酸盐广泛存在于自然界,但只有少数PAO微生物被分离、培养、鉴定出来．培养基能否分离出PAO和PAO能否在实验室条件下表现出polyP积累特征,均至关重要．糖原积累微生物(GAO)与PAO对碳源存在竞争关系,影响EBPR的效率．原位荧光分子杂交、激光共聚焦扫描电镜、微量放射自显影术、活体核磁共振光谱等现代科学技术的发展。使我们能够观察原位微生物群落组成、空间结构和功能变化．对PAO的深入研究,可改进污水脱磷的效率,提高对磷在环境中迁移转化的认识     

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.     

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

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.     

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.     

Changes in respiratory quinone profiles of enhanced biological phosphorus removal activated sludge under different influent phosphorus/carbon ratio conditions

Changes in the microbial community of an enhanced biological phosphorus removal (EBPR) activated sludge system under different influent phosphorus/carbon (P/C) ratio conditions were investigated through evaluation of population respiratory quinone profiles. A total of 13 types of respiratory quinone homologs consisting of 3 types of ubiquinones (UQ) and 10 types of menaquinones (MK) were identified in this study. The dominant quinones were UQ-8 and MK-7 throughout the operational period. A higher P/C ratio (0.1) in the influent stimulated an increase in the mole fractions of UQ-8, MK-7, MK-8(H₄), MK-9(H₄) and MK-8(H₈), suggesting that actinobacterial polyphosphate-accumulating organisms (PAO) containing partially hydrogenated MK, mainly MK-8(H₄), were contributing to EBPR. However, when the P/C ratio gradually decreased from 0.1 to 0.01, the mole fractions of UQ-8 increased from 0.46 to 0.58, while MK-7, MK-8(H₂), MK-8(H₄), MK-9(H₄), MK-8(H₈) and MK-9(H₆) markedly decreased. These changes in the respiratory quinone profiles suggest that glycogen-accumulating organisms corresponding to some Gammaproteobacteria had become dominant populations with a decrease in actinobacterial PAO. On the other hand, increasing abruptly the P/C ratio to 0.1 further caused an increase in the mole fraction of UQ-8, indicating that Rhodocyclus-related organisms were important PAO.