Expanding the Diversity of Accumulibacter with a Novel Type and Deciphering the Transcriptional and Morphological Features among Co-Occurring Strains.

"Candidatus Accumulibacter" is the major polyphosphate-accumulating organism (PAO) in global wastewater treatment systems, and its phylogenetic and functional diversity have expanded in recent years. In addition to the widely recognized type I and II sublineages, we discovered a novel type enriched in laboratory bioreactors. Core gene and machine learning-based gene feature profiling supported the assertion that type III "Ca. Accumulibacter" is a potential PAO with the unique function of using dimethyl sulfoxide as an electron acceptor. Based on the correlation between ppk1 and genome similarity, the species-level richness of Accumulibacter was estimated to be over 100, suggesting that the currently recognized species are only the tip of the iceberg. Meanwhile, the interstrain transcriptional and morphological features of multiple "Ca. Accumulibacter" strains co-occurring in a bioreactor were investigated. Metatranscriptomics of seven co-occurring strains indicated that the expression level and interphasic dynamics of PAO phenotype-related genes had minimal correlation with their phylogeny. In particular, the expression of denitrifying and polyphosphate (poly-P) metabolism genes exhibited higher interstrain and interphasic divergence than expression of glycogen and polyhydroxyalkanoate metabolic genes. A strategy of cloning rRNA genes from different strains based on similar genomic synteny was successfully applied to differentiate their morphology via fluorescence in situ hybridization. Our study further expands the phylogenetic and functional diversity of "Ca. Accumulibacter" and proposes that deciphering the function and capability of certain "Ca. Accumulibacter" should be tailored to the environment and population in question. IMPORTANCE In the last 2 decades, "Ca. Accumulibacter" has garnered significant attention as the core functional but uncultured taxon for enhanced biological phosphorus removal due to its phylogenetic and functional diversity and intragenus niche differentiation. Since 2002, it has been widely known that this genus has two sublineages (type I and II). However, in this study, a metagenomic approach led to the discovery of a novel type (type III) with proposed novel functional features. By comparing the average nucleotide identity of "Ca. Accumulibacter" genomes and the similarity of ppk1, a phylogenetic biomarker largely deposited in databases, the global species-level richness of "Ca. Accumulibacter" was estimated for the first time to be over 100. Furthermore, we observed the co-occurrence of multiple "Ca. Accumulibacter" strains in a single bioreactor and found the simultaneous transcriptional divergence of these strains intriguing with regard to their niche differentiation within a single community. Our results indicated a decoupling feature between transcriptional pattern and phylogeny for co-occurring strains.

Turnover of the extracellular polymeric matrix of granules performing biological phosphate removal.

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. 13C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d-1 respectively) was higher than the expected value based on the solid removal rate (0.132 d-1), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d-1. KEY POINTS: • Proteins showed a higher turnover rate than carbohydrates. • Turnover of EPS was similar to the turnover of intracellular proteins. • EPS is not preferentially consumed by flanking populations.

Long term operation of a phototrophic biological nutrient removal system: Impact of CO2 concentration and light exposure on process performance.

The utilization of non-aerated microalgae-bacterial consortia for phototrophic biological nutrient removal (photo-BNR) has emerged as an alternative to conventional wastewater treatment. Photo-BNR systems are operated under transient illumination, with alternating dark-anaerobic, light-aerobic and dark-anoxic conditions. A deep understanding of the impact of operational parameters on the microbial consortium and respective nutrient removal efficiency in photo-BNR systems is required. The present study evaluates, for the first time, the long-term operation (260 days) of a photo-BNR system, fed with a COD:N:P mass ratio of 7.5:1:1, to understand its operational limitations. In particular, different CO2 concentrations in the feed (between 22 and 60 mg C/L of Na2CO3) and variations of light exposure (from 2.75 h to 5.25 h per 8 h cycle) were studied to determine their impact on key parameters, like oxygen production and availability of polyhydroxyalkanoates (PHA), on the performance of anoxic denitrification by polyphosphate accumulating organisms. Results indicate that oxygen production was more dependent on the light availability than on the CO2 concentration. Also, under operational conditions with a COD:Na2CO3 ratio of 8.3 mg COD/mg C and an average light availability of 5.4 ± 1.3 W h/g TSS, no internal PHA limitation was observed, and 95 ± 7%, 92 ± 5% and 86 ± 5% of removal efficiency could be achieved for phosphorus, ammonia and total nitrogen, respectively. 81 ± 1.7% of the ammonia was assimilated into the microbial biomass and 19 ± 1.7% was nitrified, showing that biomass assimilation was the main N removal mechanism taking place in the bioreactor. Overall, the photo-BNR system presented a good settling capacity (SVI ∼60 mL/g TSS) and was able to remove 38 ± 3.3 mg P/L and 33 ± 1.7 mg N/L, highlighting its potential for achieving wastewater treatment without the need of aeration.

Quantification of Biologically and Chemically Bound Phosphorus in Activated Sludge from Full-Scale Plants with Biological P-Removal.

Phosphorus (P) is present in activated sludge from wastewater treatment plants in the form of metal salt precipitates, extracellular polymeric substances, or bound into the biomass, for example, as intracellular polyphosphate (poly-P). Several methods for a reliable quantification of the different P-fractions have recently been developed, and this study combines them to obtain a comprehensive P mass-balance of activated sludge from four enhanced biological phosphate removal (EBPR) plants. Chemical characterization by ICP-OES and sequential P fractionation showed that chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals. Raman microspectroscopy, solution state 31P NMR, and 31P MAS NMR spectroscopy applied before and after anaerobic P-release experiments, were used to quantify poly-P, which constituted 22-54% of total P and was found in approximately 25% of all bacterial cells. Raman microspectroscopy in combination with fluorescence in situ hybridization was used to quantify poly-P in known polyphosphate-accumulating organisms (PAO) (Tetrasphaera, Candidatus Accumulibacter, and Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. Interestingly, only 1-13% of total P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are known.

Enrichment of phosphate-accumulating organisms (PAOs) in a microfluidic model biofilm system by mimicking a typical aerobic granular sludge feast/famine regime.

Wastewater treatment using aerobic granular sludge has gained increasing interest due to its advantages compared to conventional activated sludge. The technology allows simultaneous removal of organic carbon, nitrogen, and phosphorus in a single reactor system and is independent of space-intensive settling tanks. However, due to the microscale, an analysis of processes and microbial population along the radius of granules is challenging. Here, we introduce a model system for aerobic granular sludge on a small scale by using a machine-assisted microfluidic cultivation platform. With an implemented logic module that controls solenoid valves, we realized alternating oxic hunger and anoxic feeding phases for the biofilms growing within. Sampling during ongoing anoxic cultivation directly from the cultivation channel was achieved with a robotic sampling device. Analysis of the biofilms was conducted using optical coherence tomography, fluorescence in situ hybridization, and amplicon sequencing. Using this setup, it was possible to significantly enrich the percentage of polyphosphate-accumulating organisms (PAO) belonging to the family Rhodocyclaceae in the community compared to the starting inoculum. With the aid of this miniature model system, it is now possible to investigate the influence of a multitude of process parameters in a highly parallel way to understand and efficiently optimize aerobic granular sludge-based wastewater treatment systems.Key points• Development of a microfluidic model to study EBPR.• Feast-famine regime enriches polyphosphate-accumulating organisms (PAOs).• Microfluidics replace sequencing batch reactors for aerobic granular sludge research.

Enhancing biological nitrogen and phosphorus removal performance in aerobic granular sludge sequencing batch reactors by activated carbon particles.

Long start-up periods for aerobic granular sludge (AGS) formation and establishment of P removal pathways are challenges for widespread implementation of AGS process. External additives such as activated carbon (AC) attracted interest for accelerating AGS formation. However, the roles of AC in granulation and biological nutrient removal (BNR) are not understood. Here, the role of AC was investigated in decreasing start-up periods in AGS formation and BNR under different carbon substrate conditions (i.e., acetate (HAc), propionate (HPr) and HAc-HPr) in sequencing batch reactors (SBRs). AC addition increased aggregation index and settleability of activated sludge (AS) inoculum which minimized AS washout from SBRs. AC addition hastened AGS formation and establishment of BNR pathways by facilitating AS retention and biofilm formation. Feeding HAc or HAc-HPr supported better granulation (MLSS: 6-7 g l-1, SVI: 30-40 ml g-1) than HPr (MLSS: 4 g l-1, SVI: 70). The start-up periods for efficient total nitrogen (TN) removals were decreased to 22 and 16 d from 38 to 25 d, respectively, in AC augmented SBRs fed with either HAc or HAc-HPr. TN removals were higher at ≥95% in HAc or HAc-HPr fed SBRs. Total phosphorus (TP) removals were also higher in AC-augmented SBRs at 80% and ≥90% in HAc and HAc-HPr fed SBRs, respectively. In contrast, TN and TP removals were lower at 70% and 35%, respectively, in HPr fed SBR. Ammonium was primarily removed via nitritation-denitritation pathway. Phosphorus removal was at 1.7 to 2-fold higher in AC augmented SBRs and driven by enhanced biological phosphorus removal (EBPR) pathway. MiSeq sequencing and qPCR revealed higher enrichment of polyphosphate accumulating organisms (PAOs), denitrifying PAOs, and ammonia oxidizers in AC-augmented SBRs fed with HAc or HAc-HPr. This study demonstrates that AC addition can be considered for enrichment of PAOs and establishment of EBPR in aerobic granular SBRs.

Inhibition of free nitrous acid and free ammonia on polyphosphate accumulating organisms: Evidence of insufficient phosphorus removal through nitritation-denitritation.

The purpose of this study is to investigate the effect of Free Nitrous Acid (FNA) and Free Ammonia (FA) on enhanced biological phosphorus removal (EBPR) and in particular on the aerobic phosphorus uptake rate (PUR). To this end, a PAO-enriched biomass was developed at a lab-scale reactor in order to fuel a series of ex-situ batch experiments to test the effect of various nitrite or ammonium concentrations on the phosphorus uptake rate at different pH values. FNA was found to be a strong inhibitor of EBPR, in agreement with other studies with PUR being inhibited by 50 % under 1.5 µg HNO2-N L-1 and 100 % at 13 µg HNO2-N L-1. FA was also found to inhibit EBPR with PUR being inhibited by 50 % under 6.4 mg NH3-N L-1. The results of this study suggest that EBPR under high nitrogen loading alongside nitritation-denitritation may not be a viable option.

Revealing the Metabolic Flexibility of "Candidatus Accumulibacter phosphatis" through Redox Cofactor Analysis and Metabolic Network Modeling.

Environmental fluctuations in the availability of nutrients lead to intricate metabolic strategies. "Candidatus Accumulibacter phosphatis," a polyphosphate-accumulating organism (PAO) responsible for enhanced biological phosphorus removal (EBPR) from wastewater treatment systems, is prevalent in aerobic/anaerobic environments. While the overall metabolic traits of these bacteria are well described, the nonavailability of isolates has led to controversial conclusions on the metabolic pathways used. In this study, we experimentally determined the redox cofactor preferences of different oxidoreductases in the central carbon metabolism of a highly enriched "Ca Accumulibacter phosphatis" culture. Remarkably, we observed that the acetoacetyl coenzyme A reductase engaged in polyhydroxyalkanoate (PHA) synthesis is NADH preferring instead of showing the generally assumed NADPH dependency. This allows rethinking of the ecological role of PHA accumulation as a fermentation product under anaerobic conditions and not just a stress response. Based on previously published metaomics data and the results of enzymatic assays, a reduced central carbon metabolic network was constructed and used for simulating different metabolic operating modes. In particular, scenarios with different acetate-to-glycogen consumption ratios were simulated, which demonstrated optima using different combinations of glycolysis, glyoxylate shunt, or branches of the tricarboxylic acid (TCA) cycle. Thus, optimal metabolic flux strategies will depend on the environment (acetate uptake) and on intracellular storage compound availability (polyphosphate/glycogen). This NADH-related metabolic flexibility is enabled by the NADH-driven PHA synthesis. It allows for maintaining metabolic activity under various environmental substrate conditions, with high carbon conservation and lower energetic costs than for NADPH-dependent PHA synthesis. Such (flexible) metabolic redox coupling can explain the competitiveness of PAOs under oxygen-fluctuating environments.IMPORTANCE Here, we demonstrate how microbial storage metabolism can adjust to a wide range of environmental conditions. Such flexibility generates a selective advantage under fluctuating environmental conditions. It can also explain the different observations reported in PAO literature, including the capacity of "Ca Accumulibacter phosphatis" to act like glycogen-accumulating organisms (GAOs). These observations stem from slightly different experimental conditions, and controversy arises only when one assumes that metabolism can operate only in a single mode. Furthermore, we also show how the study of metabolic strategies is possible when combining omics data with functional cofactor assays and modeling. Genomic information can only provide the potential of a microorganism. The environmental context and other complementary approaches are still needed to study and predict the functional expression of such metabolic potential.

Effect of anaerobic phases length on denitrifying dephosphatation biocenosis - a case study of IFAS-MBSBBR.

BACKGROUND: The study aimed to evaluate the influence of the duration times of anaerobic phases on the bacterial biocenosis characterisation while denitrifying dephosphatation in the Integrated Fixed-Film Activated Sludge - Moving-Bed Sequencing Batch Biofilm Reactor (IFAS-MBSBBR). The experiment was conducted in a laboratory model. The study consisted of four series, which differed in terms of the ratio of the anaerobic phases. duration concerning the overall reaction time in the cycle. The anaerobic phases covered from 18 to 30% of the whole cycle duration. During the reactor performance that took 9 months, the influent and effluent were monitored by analysis of COD, TKN, NH4-N, NO2-N, NO3-N, TP, PO4-P, pH, alkalinity and the phosphorus uptake batch tests. Characterisation of the activated sludge and the biofilm biocenosis was based on fluorescent in situ hybridisation (identification of PAO and GAO) and the denaturing gradient gel electrophoresis patterns. RESULTS: The organic compounds removal was high (more than 95.7%) independently of cycle configuration. The best efficiency for nitrogen (91.1%) and phosphorus (98.8%) removal was achieved for the 30% share of the anaerobic phases in the reaction time. Denitrifying PAO (DPAO) covered more than 90% of PAO in the biofilm and usually around 70% of PAO in the activated sludge. A substantial part of the polyphosphate accumulating organisms (PAO) community were Actinobacteria. The denitrifying dephosphatation activity was performed mainly by Accumulibacter phosphatis. CONCLUSIONS: High nutrient removal efficiencies may be obtained in IFAS-MBSBBR using the denitrifying dephosphatation process. It was found that the length of anaerobic phases influenced denitrification and the biological phosphorus removal. The extension of the anaerobic phases duration time in the reaction time caused an increase in the percentage share of denitrifying PAO (DPAO) in PAO. The biocenosis of the biofilm and the activated sludge reveal different species patterns and domination of the EBPR community.

Population Structure and Morphotype Analysis of "Candidatus Accumulibacter" Using Fluorescence In Situ Hybridization-Staining-Flow Cytometry.

"Candidatus Accumulibacter" is the dominant polyphosphate-accumulating organism (PAO) in denitrifying phosphorus removal (DPR) systems. In order to investigate the community structure and clade morphotypes of "Candidatus Accumulibacter" in DPR systems through flow cytometry (FCM), denitrifying phosphorus removal of almost 100% using nitrite and nitrate as the electron acceptor was achieved in sequencing batch reactors (SBRs). An optimal method of flow cytometry combined with fluorescence in situ hybridization and SYBR green I staining (FISH-staining-flow cytometry) was developed to quantify PAOs in DPR systems. By setting the width value of FCM, bacterial cells in a sludge sample were divided into three groups in different morphotypes, namely, coccus, coccobacillus, and bacillus. Average percentages that the three different PAO populations accounted for among total bacteria from SBR1 (SBR2) were 42% (45%), 14% (13%), and 4% (2%). FCM showed that the ratios of PAOs to total bacteria in the two reactors were 61% and 59%, and the quantitative PCR (qPCR) results indicated that IIC was the dominant "Candidatus Accumulibacter" clade in both denitrifying phosphorus removal systems, reaching 50% of the total "Candidatus Accumulibacter" bacteria. The subdominant clade in the reactor with nitrite as the electron acceptor was IID, accounting for 31% of the total "Candidatus Accumulibacter" bacteria. The FCM and qPCR results suggested that clades IIC and IID were both coccus, clade IIF was coccobacillus, and clade IA was bacillus. FISH analysis also indicated that PAOs were major cocci in the systems. An equivalence test of FCM-based quantification confirmed the accuracy of FISH-staining-flow cytometry, which can meet the quantitative requirements for PAOs in complex activated sludge samples.IMPORTANCE As one group of the most important functional phosphorus removal organisms, "Candidatus Accumulibacter," affiliated with the Rhodocyclus group of the Betaproteobacteria, is a widely recognized and studied PAO in the field of biological wastewater treatment. The morphotypes and population structure of clade-level "Candidatus Accumulibacter" were studied through novel FISH-staining-flow cytometry, which involved denitrifying phosphorus removal (DPR) achieving carbon and energy savings and simultaneous removal of N and P, thus inferring the different denitrifying phosphorus removal abilities of these clades. Additionally, based on this method, in situ quantification for specific polyphosphate-accumulating organisms (PAOs) enables a more efficient process and more accurate result. The establishment of FISH-staining-flow cytometry makes cell sorting of clade-level noncultivated organisms available.

Simultaneous nitrification, denitrification and phosphorus recovery (SNDPr) - An opportunity to facilitate full-scale recovery of phosphorus from municipal wastewater.

Sewage treatment plants are a potential point source for recycling of phosphorus (P). Several technologies have been proposed to biologically recover P from wastewater. The majority of these technologies are side-stream processes and rely on an external source of soluble organic carbon to facilitate P recovery. To date, no studies have demonstrated the potential to facilitate main-stream recovery of P, using carbon that is naturally present in wastewater. Simultaneous nitrification, denitrification and phosphorus removal (SNDPR) is an elegant process that can uptake influent carbon and effectively remove both nitrogen (N) and P from wastewater. SNDPR studies to date, however, have failed to facilitate an end-of-anaerobic-phase P rich liquor, that enables economies of scale to recover influent P. Therefore, this study examined the feasibility of achieving a P rich liquor (e.g. > 70 mg-P/L) in a granular SNDPR process. A synthetic influent that replicated the nutrient and carbon concentrations of municipal wastewater was used to investigate whether carbon in the influent wastewater could enable both nutrient removal and P recovery from wastewater. Our granular SNDPR process was able to facilitate an end-of-anaerobic-phase liquor with P enriched to approximately 100 mg-P/L. A dissolved oxygen (DO) concentration of 0.5 mg/L in a sequencing batch reactor (SBR) was found to be essential to achieve complete nutrient removal and a high P concentration at the end of the anaerobic phase. At this steady state of reactor operation, the abundance of polyphosphate accumulating organisms (PAOs) was 2.6 times the abundance of glycogen accumulating organisms (GAOs). The study also demonstrated the importance of denitrifying polyphosphate accumulating organisms (DPAOs) and glycogen accumulating organisms (DGAOs) to achieve complete removal of N from the effluent. Compared to nitrifying bacteria, the polyphosphate accumulating organisms (PAOs) had a higher affinity towards DO. This study, for the first time, showed that the mainstream recovery of P is feasible using a SNDPR process.

Improvement of carbon usage for phosphorus recovery in EBPR-r and the shift in microbial community.

Enhanced biological phosphorus removal and recovery (EBPR-r) is a biofilm process that makes use of polyphosphate accumulating organisms (PAOs) to remove and recover phosphorus (P) from wastewater. The original process was inefficient, as indicated by the low P-release to carbon (C)-uptake (Prel/Cupt) molar ratio of the biofilm. This study successfully validated a strategy to improve the Prel/Cupt ratio by at least 3-fold. With an unchanged supply of carbon in the recovery stream, an increase in the hydraulic loading in stages I, II and III (7.2, 14.4 and 21.6 L, respectively) resulted in a 43% increase in the Prel/Cupt ratio (0.069, 0.076 and 0.103, respectively). The ratio further increased by 150% (from 0.103 to 0.255) when the duration of the P uptake period was increased from 4 h (stage III) to 10 h (stage IV). Canonical correspondence analysis showed that, correlated to the 3-fold increase in the Prel/Cupt ratio, there was an increase in the abundance of PAOs ("Candidatus Accumulibacter" Clade IIA) and a decrease in the occurrence of glycogen accumulating organisms (GAOs) (family Sinobacteraceae). However, the four stage operation impaired denitrification, resulting in a 5-fold reduction in the Nden/Pupt ratio. The decline in denitrification was consistent with a decrease in the abundance of denitrifiers including denitrifying PAOs (family Comamonadaceae and "Candidatus Accumulibacter" Clade IA). Overall, a strategy to facilitate more efficient use of carbon was validated, enabling a 3-fold carbon saving for P recovery. The new process enabled up to 80% of the wastewater P to be captured in a P-enriched stream (>90 mg/L) with a single uptake/release cycle of recovery.

Enhanced biological phosphorus removal with different carbon sources.

Enhanced biological phosphorus removal (EBPR) process is one of the most economical and sustainable methods for phosphorus removal from wastewater. However, the performance of EBPR can be affected by available carbon sources types in the wastewater that may induce different functional microbial communities in the process. Glycogen accumulating organisms (GAOs) and polyphosphate accumulating organisms (PAOs) are commonly found by coexisting in the EBPR process. Predominance of GAO population may lead to EBPR failure due to the competition on carbon source with PAO without contributing phosphorus removal. Carbon sources indeed play an important role in alteration of PAOs and GAOs in EBPR processes. Various types of carbon sources have been investigated for EBPR performance. Certain carbon sources tend to enrich specific groups of GAOs and/or PAOs. This review summarizes the types of carbon sources applied in EBPR systems and highlights the roles of these carbon sources in PAO and GAO competition. Both single (e.g., acetate, propionate, glucose, ethanol, and amino acid) and complex carbon sources (e.g., yeast extract, peptone, and mixed carbon sources) are discussed in this review. Meanwhile, the environmental friendly and economical carbon sources that are derived from waste materials, such as crude glycerol and wasted sludge, are also discussed and compared.

Multilevel correlations in the biological phosphorus removal process: From bacterial enrichment to conductivity-based metabolic batch tests and polyphosphatase assays.

Enhanced biological phosphorus removal (EBPR) from wastewater relies on the preferential selection of active polyphosphate-accumulating organisms (PAO) in the underlying bacterial community continuum. Efficient management of the bacterial resource requires understanding of population dynamics as well as availability of bioanalytical methods for rapid and regular assessment of relative abundances of active PAOs and their glycogen-accumulating competitors (GAO). A systems approach was adopted here toward the investigation of multilevel correlations from the EBPR bioprocess to the bacterial community, metabolic, and enzymatic levels. Two anaerobic-aerobic sequencing-batch reactors were operated to enrich activated sludge in PAOs and GAOs affiliating with "Candidati Accumulibacter and Competibacter phosphates", respectively. Bacterial selection was optimized by dynamic control of the organic loading rate and the anaerobic contact time. The distinct core bacteriomes mainly comprised populations related to the classes Betaproteobacteria, Cytophagia, and Chloroflexi in the PAO enrichment and of Gammaproteobacteria, Alphaproteobacteria, Acidobacteria, and Sphingobacteria in the GAO enrichment. An anaerobic metabolic batch test based on electrical conductivity evolution and a polyphosphatase enzymatic assay were developed for rapid and low-cost assessment of the active PAO fraction and dephosphatation potential of activated sludge. Linear correlations were obtained between the PAO fraction, biomass specific rate of conductivity increase under anaerobic conditions, and polyphosphate-hydrolyzing activity of PAO/GAO mixtures. The correlations between PAO/GAO ratios, metabolic activities, and conductivity profiles were confirmed by simulations with a mathematical model developed in the aqueous geochemistry software PHREEQC.

Effect of different carbon sources on the biological phosphorus removal by a sequencing batch reactor using pressurized pure oxygen.

The effect of different carbon source on the efficiency of enhanced biological phosphorus removal (EBPR) from synthetic wastewater with acetate and two ratios of acetate/starch as a carbon source was investigated. Three pressurized pure oxygen sequencing batch reactor (POSBR) experiments were operated. The reactors (POSBR1, POSBR2 and POSBR3) were developed and studied at different carbon source ratios of 100% acetate, 75% acetate plus 25% starch and 50% acetate plus 50% starch, respectively. The results showed that POSBR1 had a higher phosphate release-to-uptake ratio and, respectively, in a much higher phosphorus removal efficiency (93.8%) than POSBR2 (84.7%) and POSBR3 (77.3%) within 30 days of operation. This indicated that the phosphorus removal efficiency decreased the higher the starch concentration was. It was also found that POSBR1 produced more polyhydroxyalkanoates (PHAs) than the other reactors. Based on the effect of the carbon source on the PHA concentration and consumption, the conditions of POSBR1 were favourable for the growth of polyphosphate-accumulating organisms and therefore, beneficial for the biological phosphorus removal process.

A review of the phosphorus removal of polyphosphate-accumulating organisms in natural and engineered systems.

Increasing eutrophication has led to a continuous deterioration of many aquatic ecosystems. Polyphosphate-accumulating organisms (PAOs) can provide insight into the human response to this challenge, as they initiate enhanced biological phosphorus removal (EBPR) through cyclical anaerobic phosphorus release and aerobic phosphorus uptake. Although the limiting environmental factors for PAO growth and phosphorus removal have been widely discussed, there remains a gap in the knowledge surrounding the differences in the type and phosphorus removal efficiencies of natural and engineered PAO systems. Furthermore, due to the limitations of PAOs in conventional wastewater treatment environments, there is an urgent need to find functional PAOs in extreme environments for better wastewater treatment. Therefore, it is necessary to explore the effects of extreme conditions on the phosphorus removal efficiency of PAOs as well as the types, sources, and characteristics of PAOs. In this paper, we summarize the response mechanisms of PAOs, denitrifying polyphosphate-accumulating organisms (D-PAOs), aerobic denitrifying polyphosphate-accumulating organisms (AD-PAOs), and sulfur-related PAOs (S-PAOs). The mechanism of nitrogen and phosphorus removal in PAOs is related to the coupling cycles of carbon, nitrogen, phosphorus, and sulfur. The genera of PAOs differ in natural and engineered systems, but PAOs have more diversity in aquatic environments and soils. Recent studies on the impact of several parameters (e.g., temperature, carbon source, pH, and dissolved oxygen) and extracellular polymer substances on the phosphorus removal efficiency of PAOs in natural and engineered systems are further discussed. Most of the PAOs screened under extreme conditions still had high phosphorus removal efficiencies (>80.0 %). These results provide a reference for searching for PAOs with different adaptations to achieve better wastewater treatment.

Simultaneous decarbonization and phosphorus removal by Tetrasphaera elongata with glucose as carbon source under aerobic conditions.

Previous researches have recognized the vital role of Tetrasphaera elongata in enhanced biological phosphorus removal systems, but the underlying mechanisms remain under-investigated. To address this issue, this study investigated the metabolic characteristics of Tetrasphaera elongata when utilizing glucose as the sole carbon source. Results showed under aerobic conditions, Tetrasphaera elongata exhibited a glucose uptake rate of 136.6 mg/(L·h) and a corresponding phosphorus removal rate of 8.6 mg P/(L·h). Upregulations of genes associated with the glycolytic pathway and oxidative phosphorylation were observed. Noteworthily, the genes encoding the two-component sensor histidine kinase and response regulator transcription factor exhibited a remarkable 28.3 and 27.4-fold increase compared with the group without glucose. Since these genes play a pivotal role in phosphate-specific transport systems, collectively, these findings shed light on a potential mechanism for simultaneous decarbonization and phosphorus removal by Tetrasphaera elongata under aerobic conditions, providing fresh insights into phosphorus removal from wastewaters.

Reevaluating the accuracy and specificity of EDTA-based polyphosphate quantification method.

Feng et al. (2020) developed a simple, nondestructive, and cost-effective method to quantify polyphosphate (poly-P) in poly-P-accumulating organism (PAO)-enriched sludge samples through 30-h anaerobic exposure to 1 % (w/v) ethylenediaminetetraacetic acid (EDTA). This study optimized the N/P ratio (∼2) of the PAO culture medium in order to provide excess P for poly-P formation in PAO cells. Subsequently, the fluorescence microscopic observation of stained cells confirmed that Corynebacterium glutamicum was a PAO species capable of heterotrophic nitrification. Finally, this study reevaluated the accuracy and specificity of the EDTA-based quantification method, using two confirmed PAO biomass, three confirmed non-PAO biomass, and two sludge samples. The 1 % (w/v) EDTA treatment appears destructive to non-PAO cells, causes the release of other P forms, and is not effective for all PAO species. Under the conditions, the actual P release amount should be calculated by subtracting approximately 8 mg P g-1 total suspended solids from the determination. The amounts of P released from sludge samples was determined not only by the PAO fractions described by Feng et al. but also by PAO community structure and sludge P content.

Demystifying polyphosphate-accumulating organisms relevant to wastewater treatment: A review of their phylogeny, metabolism, and detection.

Currently, the most cost-effective and efficient method for phosphorus (P) removal from wastewater is enhanced biological P removal (EPBR) via polyphosphate-accumulating organisms (PAOs). This study integrates a literature review with genomic analysis to uncover the phylogenetic and metabolic diversity of the relevant PAOs for wastewater treatment. The findings highlight significant differences in the metabolic capabilities of PAOs relevant to wastewater treatment. Notably, Candidatus Dechloromonas and Candidatus Accumulibacter can synthesize polyhydroxyalkanoates, possess specific enzymes for ATP production from polyphosphate, and have electrochemical transporters for acetate and C4-dicarboxylates. In contrast, Tetrasphaera, Candidatus Phosphoribacter, Knoellia, and Phycicoccus possess PolyP-glucokinase and electrochemical transporters for sugars/amino acids. Additionally, this review explores various detection methods for polyphosphate and PAOs in activated sludge wastewater treatment plants. Notably, FISH-Raman spectroscopy emerges as one of the most advanced detection techniques. Overall, this review provides critical insights into PAO research, underscoring the need for enhanced strategies in biological phosphorus removal.

Nitrate and nitrite utilization during denitrifying phosphorus removal: Electron acceptor preference and feasible process combinations.

Denitrifying phosphorus removal (DPR), which is dominated by denitrifying polyphosphate-accumulating organisms (DPAOs), is a promising process for nitrogen and phosphorus removal. Denitrifying glycogen-accumulating organisms (DGAOs) and DPAOs typically coexist in the DPR sludge, complicating the study of DPAOs' denitrification capacity. In this study, two reactors were fed with nitrate and nitrite during the anoxic phase to cultivate nitrate-DPR and nitrite-DPR sludge. Both reactors yielded high and low DGAO abundance sludges, enabling the evaluation of the denitrification capacity of DPAOs. For the nitrate-DPR sludge, the nitrite reduction rate was 1.63 times higher than the nitrate reduction rate when DPAOs were the primary denitrifiers. For the nitrite-DPR sludge, the reduction rate of nitrite was more than three times that of nitrate, irrespective of DGAO abundance. These findings indicated that DPAOs preferred nitrite to nitrate and were well suited to reduce nitrite rather than reduce nitrate to supply nitrite.