Expanding our view of genomic diversity in Candidatus Accumulibacter clades

Enhanced biological phosphorus removal (EBPR) is an important industrial wastewater treatment process mediated by polyphosphate‐accumulating organisms (PAOs). Members of the genus Candidatus Accumulibacter are one of the most extensively studied PAO as they are commonly enriched in lab‐scale EBPR reactors. Members of different Accumulibacter clades are often enriched through changes in reactor process conditions; however, the two currently sequenced Accumulibacter genomes show extensive metabolic similarity. Here, we expand our understanding of Accumulibacter genomic diversity through recovery of eight population genomes using deep metagenomics, including seven from phylogenetic clades with no previously sequenced representative. Comparative genomic analysis revealed a core of shared genes involved primarily in carbon and phosphorus metabolism; however, each Accumulibacter genome also encoded a substantial number of unique genes (> 700 genes). A major difference between the Accumulibacter clades was the type of nitrate reductase encoded and the capacity to perform subsequent steps in denitrification. The Accumulibacter clade IIF genomes also contained acetaldehyde dehydrogenase that may allow ethanol to be used as carbon source. These differences in metabolism between Accumulibacter genomes provide a molecular basis for niche differentiation observed in lab‐scale reactors and may offer new opportunities for process optimization.     

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.     

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.     

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.     

Fine-scale population structure of Accumulibacter phosphatis in enhanced biological phosphorus removal sludge

To investigate the diversities of Accumulibacter phosphatis and its polyhydroxyalkanoate (PHA) synthase gene (phaC) in enhanced biological phosphorus removal (EBPR) sludge, an acetate-fed sequencing batch reactor was operated. Analysis of microbial communities using fluorescence in situ hybridization and 16S rRNA gene clone libraries showed that the population of Accumulibacter phosphatis in the EBPR sludge comprised more than 50% of total bacteria, and was clearly divided into two subgroups with about 97.5% sequence identity of the 16S rRNA genes. PAO phaC primers targeting the phaC genes of Accumulibacter phosphatis were designed and applied to retrieve fragments of putative phaC homologs of Accumulibacter phosphatis from EBPR sludge. PAO phaC primers targeting G1PAO, G2PAO, and G3PAO groups produced PCR amplicons successfully; the resulting sequences of the phaC gene homologs were diverse, and were distantly related to metagenomic phaC sequences of Accumulibacter phosphatis with 75-98% DNA sequence identities. Degenerate NPAO (non-PAO) phaC primers targeting phaC genes of non- Accumulibacter phosphatis bacteria were also designed and applied to the EBPR sludge. Twenty-four phaC homologs retrieved from NPAO phaC primers were different from the phaC gene homologs derived from Accumulibacter phosphatis, which suggests that the PAO phaC primers were specific for the amplification of phaC gene homologs of Accumulibacter phosphatis, and the putative phaC gene homologs by PAO phaC primers were derived from Accumulibacter phosphatis in the EBPR sludge. Among 24 phaC homologs, a phaC homolog (G1NPAO-2), which was dominant in the NPAO phaC clone library, showed the strongest signal in slot hybridization and shared approximately 60% nucleotide identity with the G4PAO group of Accumulibacter phosphatis, which suggests that G1NPAO-2 might be derived from Accumulibacter phosphatis. In conclusion, analyses of the 16S rRNA and phaC genes showed that Accumulibacter phosphatis might be phylogenetically and metabolically diverse.     

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.     

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.     

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.     

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.     

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.     

Monitoring the impact of bioaugmentation on the start up of biological phosphorus removal in a laboratory scale activated sludge ecosystem

The acclimatisation of activated sludge to enhanced biological phosphorus removal (EBPR) conditions requires a period of about 40–100 days but its output remains hazardous. The impact of bioaugmentation on the start-up of a laboratory scale EBPR sequencing batch reactor was evaluated by process parameters measurement and microbial community dynamics monitoring using 16S rDNA targeted polymerase chain reaction-single strand conformation polymorphism electrophoresis (PCR-SSCP). Bioaugmentation: (1) speeded up the installation of good and stable EBPR in the bioaugmented reactor by about 15 days; (2) correlated with the transient enrichment of the sludge in the added microbial populations; and (3) favoured the long-term enrichment of the sludge in the phosphorus-accumulating organism (PAO) Candidatus Accumulibacter phosphatis. However, despite a lag time period, the control non-bioaugmented reactor ended up with comparable reactor parameters and microbial community evolution, suggesting that the same PAO populations were already present from the beginning in the original non-P-accumulating seed sludge. The potential of a true installation of the added microbial populations within the bioaugmented reactor compared to their substitution by indigenous similar populations is discussed. Competition between PAOs and the antagonistic glycogen accumulating organism Candidatus Competibacter phosphatis is also highlighted during EBPR start-up.     

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.     

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.     

Net P-removal deterioration in enriched PAO sludge subjected to permanent aerobic conditions

Recently, some research in the field of enhanced biological phosphorus removal (EBPR) has been focused on studying systems where the electron donor (substrate) and the electron acceptor (nitrate or oxygen) are present simultaneously. This can occur, for example, in a full scale wastewater treatment plant during heavy rainfall periods when the anaerobic hydraulic retention time is temporarily shortened. To study this situation that could induce EBPR failure, the operation of a sequencing batch reactor (SBR) working under alternating anaerobic-aerobic conditions with an enriched EBPR population (50% Candidatus Accumulibacter phosphatis and less than 1% Candidatus Competibacter phosphatis) was shifted to strict aerobic operation. Seven cycle studies were performed during the 11 days of aerobic operation. Net P-removal was observed in this aerobic SBR during the first 4 days of operation but the system could not achieve net-P removal after this period, although the microbial composition, in terms of percentage of Accumulibacter and Competibacter, did not change significantly. The observed changes in the different compounds analysed (phosphorus, acetate, glycogen and PHB) as well as in the OUR profile indicate that metabolic changes are produced for the adaptation of PAO to aerobic conditions.     

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.     

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.     

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     

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.     

‘Candidatus Competibacter'-lineage genomes retrieved from metagenomes reveal functional metabolic diversity

The glycogen-accumulating organism (GAO) ‘Candidatus Competibacter'' (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-‘feast'': aerobic-‘famine'' regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, ‘Candidatus Competibacter denitrificans'' and ‘Candidatus Contendobacter odensis'', as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden–Meyerhof–Parnas and Entner–Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes—identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems.     

A metagenome of a full-scale microbial community carrying out enhanced biological phosphorus removal

Enhanced biological phosphorus removal (EBPR) is widely used for removal of phosphorus from wastewater. In this study, a metagenome (18.2 Gb) was generated using Illumina sequencing from a full-scale EBPR plant to study the community structure and genetic potential. Quantitative fluorescence in situ hybridization (qFISH) was applied as an independent method to evaluate the community structure. The results were in qualitative agreement, but a DNA extraction bias against gram positive bacteria using standard extraction protocols was identified, which would not have been identified without the use of qFISH. The genetic potential for community function showed enrichment of genes involved in phosphate metabolism and biofilm formation, reflecting the selective pressure of the EBPR process. Most contigs in the assembled metagenome had low similarity to genes from currently sequenced genomes, underlining the need for more reference genomes of key EBPR species. Only the genome of ‘Candidatus Accumulibacter'', a genus of phosphorus-removing organisms, was closely enough related to the species present in the metagenome to allow for detailed investigations. Accumulibacter accounted for only 4.8% of all bacteria by qFISH, but the depth of sequencing enabled detailed insight into their microdiversity in the full-scale plant. Only 15% of the reads matching Accumulibacter had a high similarity (>95%) to the sequenced Accumulibacter clade IIA strain UW-1 genome, indicating the presence of some microdiversity. The differences in gene complement between the Accumulibacter clades were limited to genes for extracellular polymeric substances and phage-related genes, suggesting a selective pressure from phages on the Accumulibacter diversity.