Accumulibacter Phosphatis Research Articles

Effect of Free Nitrous Acid on Nitrous Oxide Production and Denitrifying Phosphorus Removal by Polyphosphorus-Accumulating Organisms in Wastewater Treatment.

The inhibition of free nitrous acid (FNA) on denitrifying phosphorus removal has been widely reported for enhanced biological phosphorus removal; however, few studies focus on the nitrous oxide (N2O) production involved in this process. In this study, the effects of FNA on N2O production and anoxic phosphorus metabolism were investigated using phosphorus-accumulating organisms (PAOs) culture highly enriched (91 ± 4%) in Candidatus Accumulibacter phosphatis. Results show that the FNA concentration notably inhibited anoxic phosphorus metabolism and phosphorus uptake. Poly-β-hydroxyalkanoate (PHA) degradation was completely inhibited when the FNA concentration was approximately 0.0923 mgHNO2-N/L. Higher initial FNA concentrations (0.00035 to 0.0103 mgHNO2-N/L) led to more PHA consumption/TN (0.444 to 0.916 mmol-C/(mmol-N·gVSS)). Moreover, it was found that FNA, rather than nitrite and pH, was likely the true inhibitor of N2O production. The highest proportion of N2O to TN was 78.42% at 0.0031 mgHNO2-N/L (equivalent to 42.44 mgNO2-N/L at pH 7.5), due to the simultaneous effects of FNA on the subsequent conversion of NO2 into N2O and then into N2. The traditional nitrite knee point can only indicate the exhaustion of nitrite, instead of the complete removal of TN.

Phage-host associations in a full-scale activated sludge plant during sludge bulking.

Sludge bulking, a notorious microbial issue in activated sludge plants, is always accompanied by dramatic changes in the bacterial community. Despite large numbers of phages in sludge systems, their responses to sludge bulking and phage-host associations during bulking are unknown. In this study, high-throughput sequencing of viral metagenomes and bacterial 16S rRNA genes were employed to characterize viral and bacterial communities in a sludge plant under different sludge conditions (sludge volume index (SVI) of 180, 132, and 73ml/g). Bulking sludges (SVI>125ml/g) taken about 10months apart exhibited similar bacterial and viral composition. This reflects ecological resilience of the sludge microbial community and indicates that changes in viral and bacterial populations correlate closely with each other. Overgrowth of "Candidatus Microthrix parvicella" led to filamentous bulking, but few corresponding viral genotypes were identified. In contrast, sludge viromes were characterized by numerous contigs associated with "Candidatus Accumulibacter phosphatis," suggesting an abundance of corresponding phages in the sludge viral community. Notably, while nitrifiers (mainly Nitrosomonadaceae and Nitrospiraceae) declined significantly along with sludge bulking, their corresponding viral contigs were identified more frequently and with greater abundance in the bulking viromes, implying that phage-mediated lysis might contribute to the loss of autotrophic nitrifiers under bulking conditions.

Population dynamics of “Candidatus Accumulibacter phosphatis” under the modes of complete nitrification and partial nitrification (nitritation) in domestic wastewater treatment system

In order to understand the enhanced biological phosphorus removal in a continuous-flow domestic wastewater treatment system, population dynamics of “Candidatus Accumulibacter phosphatis” using polyphosphate kinase (ppk1) gene as genetic marker was investigated under the modes of complete nitrification and partial nitrification (nitritation). Nitritation was achieved at hydraulic retention time (HRT) of 6.49h and dissolved oxygen (DO) of 0.5mg O2/L. Denitrifying phosphate removal via nitrite pathway caused low PHAs consumption. Regardless of change of nitrifying modes, phosphorus removal always maintained at a high level of about 100%. Abundance of total Accumulibacter increased along with rising of carbon to nitrogen ratios (C/N). Under good performance of complete nitrification and nitritation, IIC and IID clade affiliated with type II (including clades II A-I) of Accumulibacter lineage was dominant (70%–80%), respectively. Clade IID exhibited high tolerance to starving, oxygen-limiting and high nitrite accumulation, and thus became dominant clade under insufficient carbon sources, low DO and nitritation mode. Clade IIC had a positive correlation with C/N ratios, and IIF had a positive correlation with temperature (p<0.05). Shannon index under nitrification and nitritation mode was 3.54 and 2.38, respectively. That indicated the change of Accumulibacter diversities under different operational conditions.

Dynamics of microbial community structure and nutrient removal from an innovative side-stream enhanced biological phosphorus removal process

Biological phosphorous (P) and nitrogen (N) removal from municipal wastewater was studied using an innovative anoxic-aerobic-anaerobic side-stream treatment system. The impact of influent water quality including chemical oxygen demand (COD), ammonium and orthophosphate concentrations on the reactor performance was evaluated. The results showed the system was very effective at removing both COD (>88%) and NH4+-N (>96%) despite varying influent concentrations of COD, NH4+-N, and total PO43--P. In contrast, it was found that the removal of P was sensitive to influent NH4+-N and PO43--P concentrations. The maximum PO43--P removal of 79% was achieved with the lowest influent NH4+-N and PO43--P concentration. Quantitative PCR (qPCR) assays showed a high abundance and diversity of phosphate accumulating organisms (PAO), nitrifiers and denitrifiers. The MiSeq microbial community structure analysis showed that the Proteobacteria (especially β-Proteobacteria, and γ-Proteobacteria) were the dominant in all reactors. Further analysis of the bacteria indicated the presence of diverse PAO genera including Candidatus Accumulibacter phosphatis, Tetrasphaera, and Rhodocyclus, and the denitrifying PAO (DPAO) genus Dechloromonas. Interestingly, no glycogen accumulating organisms (GAOs) were detected in any of the reactors, suggesting the advantage of proposed process in term of PAO selection for enhanced P removal compared with conventional main-stream processes.

Enrichment and characterization of a psychrophilic ‘Candidatus Accumulibacter phosphatis’ culture

Enhanced Biological phosphorous removal (EBPR) processes, often operated at low temperatures, are utilised world-wide, but currently little is known regarding enrichment cultures and the characteristics of active organisms (“Candidatus Accumulibacter phosphatis” (Accumulibacter)) under psychrophilic conditions. This study assesses the long-term performance, metabolic activity, microbial community characteristics and sludge morphology in an EBPR community enriched from activated sludge at 10 °C. Long solid retention times (SRT) and low temperatures resulted in the dominance of Accumulibacter type II over type I. Despite changes in the microbial community, P-removal efficiencies did not show obvious differences and although no specific measures were implemented, the enriched Accumulibacter-PAO culture formed stable dense granules. A high level of Alginate-like exopolysaccharides (ALE) were observed, with a large number of Guluronic acid-Guluronic acid (GG) blocks derived from the biomass at 10 °C. This characteristic favors sludge granulation, increasing the mechanical strength of granules formed, which encourages solid-liquid separation and consequently, contributes to the stable operation of EBPR systems.

Complete Nutrient Removal Coupled to Nitrous Oxide Production as a Bioenergy Source by Denitrifying Polyphosphate-Accumulating Organisms.

Coupled aerobic-anoxic nitrous decomposition operation (CANDO) is a promising emerging bioprocess for wastewater treatment that enables direct energy recovery from nitrogen (N) in three steps: (1) ammonium oxidation to nitrite; (2) denitrification of nitrite to nitrous oxide (N2O); and (3) N2O conversion to N2 with energy generation. However, CANDO does not currently target phosphorus (P) removal. Here, we demonstrate that denitrifying polyphosphate-accumulating organism (PAO) enrichment cultures are capable of catalyzing simultaneous biological N and P removal coupled to N2O generation in a second generation CANDO process, CANDO+P. Over 7 months (>300 cycles) of operation of a prototype lab-scale CANDO+P sequencing batch reactor treating synthetic municipal wastewater, we observed stable and near-complete N removal accompanied by sustained high-rate, high-yield N2O production with partial P removal. A substantial increase in abundance of the PAO Candidatus Accumulibacter phosphatis was observed, increasing from 5% of the total bacterial community in the inoculum to over 50% after 4 months. PAO enrichment was accompanied by a strong shift in the dominant Accumulibacter population from clade IIC to clade IA, based on qPCR monitoring of polyphosphate kinase 1 (ppk1) gene variants. Our work demonstrates the feasibility of combining high-rate, high-yield N2O production for bioenergy production with combined N and P removal from wastewater, and it further suggests a putative denitrifying PAO niche for Accumulibacter clade IA.

Community Assembly and Ecology of Activated Sludge under Photosynthetic Feast-Famine Conditions.

Here, we demonstrate that photosynthetic oxygen production under light-dark and feast-famine cycles with no mechanical aeration and negligible oxygen diffusion is able to maintain phosphorus cycling activity associated with the enrichment of polyphosphate accumulating organisms (PAOs). We investigate the ecology of this novel system by conducting a time series analysis of prokaryotic and eukaryotic biodiversity using the V3-V4 and V4 regions of the 16S and 18S rRNA gene sequences, respectively. In the Eukaryotic community, the initial dominant alga observed was Desmodesmus. During operation, the algal community became a more diverse consortium of Desmodesmus, Parachlorella, Characiopodium, and Bacillariophytina. In the Prokaryotic community, there was an initial enrichment of the PAO Candidatus Accumulibacter phosphatis (Accumulibacter) Acc-SG2, and the dominant ammonia-oxidizing organism was Nitrosomonas oligotropha; however, these populations decreased in relative abundance, becoming dominated by Accumulibacter Acc-SG3 and Nitrosomonas ureae. Furthermore, functional guilds that were not abundant initially became enriched including the putative Cyanobacterial PAOs Obscuribacterales and Leptolyngbya and the H2-oxidizing denitrifying autotroph Sulfuritalea. After a month of operation, the most-abundant prokaryote belonged to an uncharacterized clade of Chlorobi classified as Chlorobiales;SJA-28 Clade III, the first reported enrichment of this lineage. This experiment represents the first investigation into the ecological interactions and community assembly during photosynthetic feast-famine conditions. Our findings suggest that photosynthesis may provide sufficient oxygen to drive polyphosphate cycling.

Long-term effects of sulphide on the enhanced biological removal of phosphorus: The symbiotic role of Thiothrix caldifontis

Thiothrix caldifontis was the dominant microorganism (with an estimated bio-volume of 65 ± 3%) in a lab-scale enhanced biological phosphorus removal (EBPR) system containing 100 mg of sulphide per litre in the influent. After a gradual exposure to the presence of sulphide, the EBPR system initially dominated by Candidatus Accumulibacter phosphatis Clade I (98 ± 3% bio-volume) (a known polyphosphate accumulating organism, PAO) became enriched with T. caldifontis. Throughout the different operating conditions studied, practically 100% phosphate removal was always achieved. The gradual increase of the sulphide content in the medium (added to the anaerobic stage of the alternating anaerobic-aerobic sequencing batch reactor) and the adjustment of the aerobic hydraulic retention time played a major role in the enrichment of T. caldifontis. T. caldifontis exhibited a mixotrophic metabolism by storing carbon anaerobically as poly-β-hydroxy-alkanoates (PHA) and generating the required energy through the hydrolysis of polyphosphate. PHA was used in the aerobic period as carbon and energy source for growth, polyphosphate, and glycogen formation. Apparently, extra energy was obtained by the initial accumulation of sulphide as an intracellular sulphur, followed by its gradual oxidation to sulphate. The culture enriched with T. caldifontis was able to store approximately 100 mg P/g VSS. This research suggests that T. caldifontis could behave like PAO with a mixotrophic metabolism for phosphorus removal using an intracellular sulphur pool as energy source. These findings can be of major interest for the biological removal of phosphorus from wastewaters with low organic carbon concentrations containing reduced S-compounds like those (pre-)treated in anaerobic systems or from anaerobic sewers.

Phosphorus (P) recovery coupled with increasing influent ammonium facilitated intracellular carbon source storage and simultaneous aerobic phosphorus & nitrogen removal

Under decreasing C/N (from 8.8 to 3.5) conditions, an alternating anaerobic/aerobic biofilter (AABF) was used to remove nitrogen and accumulate/recover phosphorus (P) from synthetic wastewater. The AABF was periodically (every 10 days) fed with an additional carbon source (10 L, chemical oxygen demand (COD) = 900 mg L−1 sodium acetate (NaAC) solution) in the anaerobic phase to induce the release of P sequestered in the biofilm. An increase in PHA storage in the biofilm was observed and characterized with TEM and a GC-MS method. The accumulation of P and removal of total nitrogen occurred primarily in the aerobic phase. As the NH4+-N loading rate increased from 0.095 to 0.238 kg m−3 d−1 at a total empty bed retention time (EBRT) of 4.6 h, the TN removal in AABF was reduced from 91.2% to 43.4%, while the P removal or recovery rate remained unaffected. The high-throughput community sequencing analysis indicated that the relative abundance of Candidatus Competibacter, Nitrospira and Arcobacter increased while the Accumulibacter phosphatis decreased with an increase of ammonium loading rate within a short operational period (30 days). A putative N and P removal pattern via simultaneous nitrification and PHA-based denitrification, as well as P accumulation in the biofilm was proposed. The research demonstrated that an efficient N removal and P recovery process, i.e., simultaneous nitrification and denitrification, P accumulation and carbon source-regulated P recovery can be achieved by the symbiotic functional groups in a single biofilm reactor.

Metabolic Response of "Candidatus Accumulibacter Phosphatis" Clade II C to Changes in Influent P/C Ratio.

The objective of this study was to investigate the ability of a culture highly enriched with the polyphosphate-accumulating organism, “Candidatus Accumulibacter phosphatis” clade IIC, to adjust their metabolism to different phosphate availabilities. For this purpose the biomass was cultivated in a sequencing batch reactor with acetate and exposed to different phosphate/carbon influent ratios during six experimental phases. Activity tests were conducted to determine the anaerobic kinetic and stoichiometric parameters as well as the composition of the microbial community. Increasing influent phosphate concentrations led to increased poly-phosphate content and decreased glycogen content of the biomass. In response to higher biomass poly-phosphate content, the biomass showed higher specific phosphate release rates. Together with the phosphate release rates, acetate uptake rates also increased up to an optimal poly-phosphate/glycogen ratio of 0.3 P-mol/C-mol. At higher poly-phosphate/glycogen ratios (obtained at influent P/C ratios above 0.051 P-mol/C-mol), the acetate uptake rates started to decrease. The stoichiometry of the anaerobic conversions clearly demonstrated a metabolic shift from a glycogen dominated to a poly-phosphate dominated metabolism as the biomass poly-phosphate content increased. FISH and DGGE analyses confirmed that no significant changes occurred in the microbial community, suggesting that the changes in the biomass activity were due to different metabolic behavior, allowing the organisms to proliferate under conditions with fluctuating phosphate levels.

Dominant Candidatus Accumulibacter phosphatis Enriched in Response to Phosphate Concentrations in EBPR Process.

Candidatus Accumulibacter phosphatis (Accumulibacter), which plays an important role in enhanced biological phosphorus removal in wastewater treatment plants, is phylogenetically classified into two major types (Types I and II). Phosphate concentrations affect the Accumulibacter community of the biomass enriched in treatment plants. Therefore, in the present study, Accumulibacter enrichments were conducted using a down-flow hanging sponge reactor under five conditions and a wide range of controlled phosphate concentrations in order to investigate how phosphate governs the community. We found that excessive phosphate levels inhibited Accumulibacter activity, that this inhibitory effect was greater for Type II. In addition, the affinity of Type II for phosphate was higher than that of Type I. Type IIA-B dominated at a phosphate concentration less than 5 mg P L−1, while Type IA was dominant at 50 and 500 mg P L−1. These patterns of enrichment may be explained by an inhibition kinetics model.

Sulphide effects on the physiology of Candidatus Accumulibacter phosphatis type I.

Sulphate-rich wastewaters can be generated due to (i) use of saline water as secondary-quality water for sanitation in urban environments (e.g. toilet flushing), (ii) discharge of industrial effluents, (iii) sea and brackish water infiltration into the sewage and (iv) use of chemicals, which contain sulphate, in drinking water production. In the presence of an electron donor and absence of oxygen or nitrate, sulphate can be reduced to sulphide. Sulphide can inhibit microbial processes in biological wastewater treatment systems. The objective of the present study was to assess the effects of sulphide concentration on the anaerobic and aerobic physiology of polyphosphate-accumulating organisms (PAOs). For this purpose, a PAO culture, dominated by Candidatus Accumulibacter phosphatis clade I (PAO I), was enriched in a sequencing batch reactor (SBR) fed with acetate and propionate. To assess the direct inhibition effects and their reversibility, a series of batch activity tests were conducted during and after the exposure of a PAO I culture to different sulphide concentrations. Sulphide affected each physiological process of PAO I in a different manner. At 189mg TS-S/L, volatile fatty acid uptake was 55% slower and the phosphate release due to anaerobic maintenance increased from 8 to 18mg PO4-P/g VSS/h. Up to 8mg H2S-S/L, the decrease in aerobic phosphorus uptake rate was reversible (Ic60). At higher concentrations of sulphide, potassium (>16mg H2S-S/L) and phosphate (>36mg H2S-S/L) were released under aerobic conditions. Ammonia uptake, an indicator of microbial growth, was not observed at any sulphide concentration. This study provides new insights into the potential failure of enhanced biological phosphorus removal sewage plants receiving sulphate- or sulphide-rich wastewaters when sulphide concentrations exceed 8mg H2S-S/L, as PAO I could be potentially inhibited.

Denitrification of nitrate and nitrite by ‘Candidatus Accumulibacter phosphatis’ clade IC

Phosphate accumulating organisms (PAO) are assumed to use nitrate as external electron acceptor, allowing an efficient integration of simultaneous nitrogen and phosphate removal with minimal organic carbon (COD) requirements. However, contradicting findings appear in literature regarding the denitrification capacities of PAO due to the lack of clade specific highly enriched PAO cultures. Whereas some studies suggest that only PAO clade I may be capable of using nitrate as external electron acceptor for anoxic P-uptake, other studies indicate that PAO clade II may be responsible for anoxic P-removal. In the present study, a highly enriched PAO clade IC culture (>99% according to FISH) was cultivated in an SBR operated under Anaerobic/Oxic conditions and subsequently exposed to Anaerobic/Anoxic/Oxic conditions using nitrate as electron acceptor. Before and after acclimatization to the presence of nitrate, the aerobic and anoxic (nitrate and nitrite) activities of the PAO I culture were assessed through the execution of batch tests using either acetate or propionate as electron donor. In the presence of nitrate, significant P-uptake by PAO I was not observed before or after acclimatization. Using nitrite as electron acceptor, limited nitrite removal rates were observed before acclimatization with lower rates in the acetate fed reactor without P-uptake and slightly higher in the propionate fed reactor with a marginal anoxic P-uptake. Only after acclimatization to nitrate, simultaneous P and nitrite removal was observed. This study suggests that PAO clade IC is not capable of using nitrate as external electron acceptor for anoxic P-removal. The elucidation of the metabolic capacities for individual PAO clades helps in better understanding and optimization of the relation between microbial ecology and process performance in enhanced biological phosphate removal processes.

Prevalence of 'Candidatus Accumulibacter phosphatis' type II under phosphate limiting conditions.

P-limitation in enhanced biological phosphorus removal (EBPR) systems fed with acetate, has generally been considered as a condition leading to enrichment of organisms of the genotype’ Candidatus Competibacter phosphatis’ expressing the glycogen-accumulating organisms (GAO) phenotype. Recent studies have demonstrated in short-term experiments that organisms of the genotype ‘Candidatus Accumulibacter phosphatis’ clade I and II, known to express the polyphosphate-accumulating organisms (PAO) phenotype can switch to the GAO phenotype when poly-P is absent, but are performing the HAc-uptake at lower kinetic rates, where clade I showed the lowest rates. The objective of this study was to verify whether organisms of the genotype ‘Candidatus Accumulibacter phosphatis’ can also be enriched under P-limiting conditions while expressing a GAO phenotype and more specifically to see which specific clade prevails. A sequencing batch reactor was inoculated with activated sludge to enrich an EBPR culture for a cultivation period of 128 days (16 times the solids retention time) under P-limiting conditions. A mixed culture was obtained comprising of 49 % ‘Candidatus Accumulibacter phosphatis’ clade II and 46 % ‘Candidatus Competibacter phosphatis’. The culture performed a full GAO metabolism for anaerobic HAc-uptake, but was still able to switch to a PAO metabolism, taking up excessive amounts of phosphate during the aerobic phase when it became available in the influent. These findings show that P-limitation, often used as strategy for enrichment of ‘Candidatus Competibacter phosphatis’, does not always lead to enrichment of only ‘Candidatus Competibacter phosphatis’. Furthermore, it demonstrates that ‘Candidatus Accumulibacter phosphatis’ are able to proliferate in activated sludge systems for periods of up to 128 days or longer when the influent phosphate concentrations are just enough for assimilation purposes and no poly-P is formed. The ‘Candidatus Accumulibacter phosphatis’ retain the ability to switch to the PAO phenotype, taking up phosphate from the influent as soon as it becomes available.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0214-z) contains supplementary material, which is available to authorized users.

"Candidatus Propionivibrio aalborgensis": A Novel Glycogen Accumulating Organism Abundant in Full-Scale Enhanced Biological Phosphorus Removal Plants.

Enhanced biological phosphorus removal (EBPR) is widely used to remove phosphorus from wastewater. The process relies on polyphosphate accumulating organisms (PAOs) that are able to take up phosphorus in excess of what is needed for growth, whereby phosphorus can be removed from the wastewater by wasting the biomass. However, glycogen accumulating organisms (GAOs) may reduce the EBPR efficiency as they compete for substrates with PAOs, but do not store excessive amounts of polyphosphate. PAOs and GAOs are thought to be phylogenetically unrelated, with the model PAO being the betaproteobacterial “Candidatus Accumulibacter phosphatis” (Accumulibacter) and the model GAO being the gammaproteobacterial “Candidatus Competibacter phosphatis”. Here, we report the discovery of a GAO from the genus Propionivibrio, which is closely related to Accumulibacter. Propionivibrio sp. are targeted by the canonical fluorescence in situ hybridization probes used to target Accumulibacter (PAOmix), but do not store excessive amounts of polyphosphate in situ. A laboratory scale reactor, operated to enrich for PAOs, surprisingly contained co-dominant populations of Propionivibrio and Accumulibacter. Metagenomic sequencing of multiple time-points enabled recovery of near complete population genomes from both genera. Annotation of the Propionivibrio genome confirmed their potential for the GAO phenotype and a basic metabolic model is proposed for their metabolism in the EBPR environment. Using newly designed fluorescence in situ hybridization (FISH) probes, analyses of full-scale EBPR plants revealed that Propionivibrio is a common member of the community, constituting up to 3% of the biovolume. To avoid overestimation of Accumulibacter abundance in situ, we recommend the use of the FISH probe PAO651 instead of the commonly applied PAOmix probe set.

Comparative genomics analyses on EPS biosynthesis genes required for floc formation of Zoogloea resiniphila and other activated sludge bacteria

Activated sludge (AS) process has been widely utilized for municipal sewage and industrial wastewater treatment. Zoolgoea and its related floc-forming bacteria are required for formation of AS flocs which is the key to gravitational effluent-and-sludge separation and AS recycling. However, little is known about the genetics, biochemistry and physiology of Zoogloea and its related bacteria. This report deals with the comparative genomic analyses on two Zoogloea resiniphila draft genomes and the closely related proteobacterial species commonly found in AS. In particular, the metabolic processes involved in removal of organic matters, nitrogen and phosphorus were analyzed. Furthermore, it is revealed that a large gene cluster, encoding eight glycosyltransferases and other proteins involved in biosynthesis and export of extracellular polysaccharides (EPS), was required for floc formation. One of the two asparagine synthase paralogues, associated with this EPS biosynthesis gene cluster, was required for floc formation in Zoogloea. Similar EPS biosynthesis gene cluster(s) were identified in the genome of other AS proteobacteria including polyphosphate-accumulating Candidatus Accumulibacter phosphatis (CAP) and nitrifying Nitrosopira and Nitrosomonas bacteria, but the gene composition varies interspecifically and intraspecifically. Our results indicate that floc formation of desired AS bacteria, including CAP strains, facilitate their recruitment into AS and gradual enrichment via repeated AS settling and recycling processes.

Candidatus Accumulibacter phosphatis clades enriched under cyclic anaerobic and microaerobic conditions simultaneously use different electron acceptors

Lab- and pilot-scale simultaneous nitrification, denitrification and phosphorus removal-sequencing batch reactors were operated under cyclic anaerobic and micro-aerobic conditions. The use of oxygen, nitrite, and nitrate as electron acceptors by Candidatus Accumulibacter phosphatis during the micro-aerobic stage was investigated. A complete clade-level characterization of Accumulibacter in both reactors was performed using newly designed qPCR primers targeting the polyphosphate kinase gene (ppk1). In the lab-scale reactor, limited-oxygen conditions led to an alternated dominance of Clade IID and IC over the other clades. Results from batch tests when Clade IC was dominant (i.e., >92% of Accumulibacter) showed that this clade was capable of using oxygen, nitrite and nitrate as electron acceptors for P uptake. A more heterogeneous distribution of clades was found in the pilot-scale system (Clades IIA, IIB, IIC, IID, IA, and IC), and in this reactor, oxygen, nitrite and nitrate were also used as electron acceptors coupled to phosphorus uptake. However, nitrite was not an efficient electron acceptor in either reactor, and nitrate allowed only partial P removal. The results from the Clade IC dominated reactor indicated that either organisms in this clade can simultaneously use multiple electron acceptors under micro-aerobic conditions, or that the use of multiple electron acceptors by Clade IC is due to significant microdiversity within the Accumulibacter clades defined using the ppk1 gene.

Ancestral genome reconstruction identifies the evolutionary basis for trait acquisition in polyphosphate accumulating bacteria.

The evolution of complex traits is hypothesized to occur incrementally. Identifying the transitions that lead to extant complex traits may provide a better understanding of the genetic nature of the observed phenotype. A keystone functional group in wastewater treatment processes are polyphosphate accumulating organisms (PAOs), however the evolution of the PAO phenotype has yet to be explicitly investigated and the specific metabolic traits that discriminate non-PAO from PAO are currently unknown. Here we perform the first comprehensive investigation on the evolution of the PAO phenotype using the model uncultured organism Candidatus Accumulibacter phosphatis (Accumulibacter) through ancestral genome reconstruction, identification of horizontal gene transfer, and a kinetic/stoichiometric characterization of Accumulibacter Clade IIA. The analysis of Accumulibacter's last common ancestor identified 135 laterally derived genes, including genes involved in glycogen, polyhydroxyalkanoate, pyruvate and NADH/NADPH metabolisms, as well as inorganic ion transport and regulatory mechanisms. In contrast, pathways such as the TCA cycle and polyphosphate metabolism displayed minimal horizontal gene transfer. We show that the transition from non-PAO to PAO coincided with horizontal gene transfer within Accumulibacter's core metabolism; likely alleviating key kinetic and stoichiometric bottlenecks, such as anaerobically linking glycogen degradation to polyhydroxyalkanoate synthesis. These results demonstrate the utility of investigating the derived genome of a lineage to identify key transitions leading to an extant complex phenotype.

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

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

The microbial community in a high-temperature enhanced biological phosphorus removal (EBPR) process

Abstract An enhanced biological phosphorus removal (EBPR) process operated at a relatively high temperature, 28 °C, removed 85% carbon and 99% phosphorus from wastewater over a period of two years. This study investigated its microbial community through fluorescent in situ hybridization (FISH) and clone library generation. Through FISH, considerably more Candidatus “Accumulibacter phosphatis” (Accumulibacter)-polyphosphate accumulating organisms (PAOs) than Candidatus ‘Competibacter phosphatis’ (Competibacter)-glycogen accumulating organisms were detected in the reactor, at 36 and 7% of total bacterial population, respectively. A low ratio of Glycogen/Volatile Fatty Acid of 0.69 further indicated the dominance of PAOs in the reactor. From clone library generated, 26 operational taxonomy units were retrieved from the sludge and a diverse population was shown, comprising Proteobacteria (69.6%), Actinobacteria (13.7%), Bacteroidetes (9.8%), Firmicutes (2.94%), Planctomycetes (1.96%), and Acidobacteria (1.47%). Accumulibacter are the only recognized PAOs revealed by the clone library. Both the clone library and FISH results strongly suggest that Accumulibacter are the major PAOs responsible for the phosphorus removal in this long-term EBPR at relatively high temperature.