Biological Phosphorus Removal Sludge Research Articles

A potential phosphorus fertilizer to alleviate the coming “phosphorus crisis”-biochar derived from enhanced biological phosphorus removal sludge

The coming crisis of phosphate rock depletion initiates the development of various solid waste derived P fertilizer. Enhanced biological phosphorus removal (EBPR) sludge is ideal waste biomass to produce biochar-P-fertilizer. Here, the form and transformation pattern of released phosphorus (P) of EBPR sludge biochar pyrolyzed at different temperatures were comprehensively investigated. As pyrolysis temperature increased, the proportion of released polyphosphates (Poly-P) increased. The main Poly-P released from low-temperature biochar was tripolyphosphates (Tri-P), while those released from high-temperature were Tri-P and cyclic Poly-P. The presence of Ca2+ could strongly inhibit P-release of low-temperature biochar (e.g., pyrolyzed at 400 °C, E400) but had little effect on that of high-temperature biochar (e.g., 700 °C, E700). All the P species released from E400 and E700 could be efficiently utilized by Pseudomonas putida. Except for the cyclic Poly-P released from E700, the other P species could also be efficiently utilized by Escherichia coli. In short, Poly-P in biochar could hardly precipitate with Ca2+ and can be utilized by certain soil microorganisms. Therefore, high-temperature EBPR sludge biochar (>600 °C) containing a high proportion of Poly-P could be ideal P fertilizer. This study provides a new insight on pyrolysis way to recover P from the sludge.

Interaction of perfluorooctanoic acid with extracellular polymeric substances - Role of protein

Perfluorooctanoic acid (PFOA) is nonbiodegradable, and adsorption is the main pathway for its removal in wastewater treatment plants (WWTPs). This study compared the capability of three types of sludge on adsorbing PFOA and investigated the role of extracellular polymeric substances (EPS) in the adsorption process. Results show that enhanced biological phosphorus removal (EBPR) sludge had the highest adsorption capacity for PFOA. Studies on the interaction between EPS and PFOA reveal that proteins play a crucial role in binding PFOA to EPS/sludge. Specifically, the aromatic and amide groups on the structure of protein can attract the C–F chains and carboxylic head of PFOA via hydrophobic interaction and electrostatic attraction, respectively. EPS of EBPR sludge has the highest amount of protein and binding sites, thus exhibits the highest adsorption capability for PFOA. This study reveals the interaction mechanism between PFOA and sludge EPS and provides new insight into the function of EPS in perfluoroalkyl substances removal in WWTPs.

The effect of divalent cation complexation on anaerobically digested enhanced biological phosphorus removal sludge dewatering performance.

The effect of mono- and divalent cation concentrations on digested sludge dewatering characteristics was investigated in a full-scale study at the Harrisonburg-Rockingham Regional Sewer Authority, VA (HRRSA) and at an additional POTW (POTW B). At HRRSA, anaerobic digesters were fed with primary sludge, enhanced biological phosphorus removal (EBPR) WAS, and trucked-in DAF solids from a poultry processing facility, and digested sludge was dewatered by belt filter press. The POTW B digesters received EBPR WAS and primary sludge, and dewatering was by centrifuge. Visual MINTEQ 3.1 (Gustafsson, 2014, Visual MINTEQ 3.1. User's manual) was used to determine cation speciation and struvite, tricalcium phosphate, vivianite, and ferrous sulfide formation at a range of digestion conditions. Significant Mg and Ca complexation was predicted at all conditions evaluated, and the majority of the complexes were monovalent cations. Complexation reduced the divalent cation concentration on the order of 40%. The HRRSA and POTW B data both showed that dewatered cake total solids (TS) increased linearly with uncomplexed divalent cation concentration and that there was an increase of 1.0%-1.1% TS for every additional 1.0meq/L of uncomplexed divalent cations. Because POTW B used centrifuge dewatering, this relationship could be independent of dewatering technique. Further, the relationship between uncomplexed divalent cation concentration and dewaterability suggests that the selectivity of exocellular polymeric substances cation exchange sites is much greater for divalent cations so that monovalent cations only bind to them when their concentrations are much higher than those of the uncomplexed divalent cations. Thus, monovalent cation concentration may not be important in anaerobic digestion or other high alkalinity environments. PRACTITIONER POINTS: It is important to account for complexation in high alkalinity environments such as anaerobic digesters; The findings of this study suggest that dewatering characteristics of anaerobically digested enhanced biological phosphorus removal (EBPR) waste sludge can be significantly improved by preserving cation-mediated exocellular polymeric substances bonding in the solids stream by reducing struvite formation; Data from two full-scale POTW's indicated that dewatered cake TS was increased by 1.0%-1.1% for every 1.0meq/L of dissolved uncomplexed divalent cations in the anaerobic digestate bulk solution; This relationship existed for dewatering both by a belt filter press and by a centrifuge suggesting that this relationship is independent of dewatering technology; Dissolved monovalent cation concentrations may not be important in anaerobic digestion or other high alkalinity environments unless their concentrations are much greater than those of the dissolved divalent cations.

Biotransformation of phosphorus in enhanced biological phosphorus removal sludge biochar

Biochar derived from enhanced biological phosphorus removal (EBPR) sludge could be a potential phosphorus (P) fertilizer. Soil microorganisms play a regulating role on the turnover of P in soil. When the EBPR sludge biochar is added to soil, it would inevitably interact with soil microorganisms. Thus, for the wise use of the EBPR sludge biochar, it is imperative to understand the interaction between the biochar and soil microorganisms. In this study, Pseudomonas putida (P. putida), a common soil microorganism, was applied to investigate the biotransformation of P in two EBPR sludge biochars. The results reveal that P released from biochar produced at 700 °C (E700) was more easily absorbed by P. putida than that released from biochar produced at 400 °C (E400). This is attributed to the higher polyphosphates (poly-P) content in E700 and poly-P has higher affinity to P. putida surface compared to orthophosphates. Furthermore, E400 has a negative effect on intracellular poly-P formation in P. putida, which is probably caused by the oxidative stress induced by the free radicals from E400. As intracellular poly-P plays a critical role on bacteria survival and their interaction with surrounding environment, high-temperature biochar (E700) in this case would be more suitable for soil remediation.

Comparison and optimization of extraction protocol for intracellular phosphorus and its polyphosphate in enhanced biological phosphorus removal (EBPR) sludge

Intracellular polyphosphate (poly-P) plays important roles in Enhanced biological phosphorus removal (EBPR) process, but an effective and reliable protocol for extracting intracellular P and its poly-P in EBPR sludge without hydrolysis of poly-P has not been setup yet. In the study, it was revealed that the severe hydrolysis of intracellular poly-P occurred during the different extraction processes, such as acid (i.e., HClO4, H2SO4 and HCl), basic (i.e., NaOH and KOH) and freezing-grind (under different solid-liquid ratios), but it did not occur during ultrasonic extraction process. The optimal extraction process of the ultrasonic protocol was 10 w/mL of ultrasonic power density and 15 min of ultrasonic time, when the extraction efficiency of intracellular P was 88.24 ± 1.56%. In addition, the extraction efficiency of intracellular P could be furtherly improved by that the 0.75 mol/L LiCl solution was used to resuspend the bacterial cell before ultrasonic extraction (i.e., LiCl-ultrasonic protocol). The ultrasonic protocol was more suitable to extract the intracellular P and its poly-P of EBPR sludge than the other 4 protocols (i.e., PCA-NaOH, EDTA-NaOH, freezing-grind and LiCl-ultrasonic), which had the technical characteristics of (i) with relatively high extraction efficiency of intracellular P, (ii) without hydrolysis of intracellular poly-P, (iii) with weak noise signal in 31P NMR spectrum and (iv) with simple extraction process and short extraction time. It was founded by the ultrasonic protocol that there was the high content (82.88%–89.79% of intracellular P content) of intracellular poly-P with long average chain length (376.4–383.2) in the EBPR sludges. Importantly, it was confirmed that the EBPR process was related to the combined action of extracellular and intracellular poly-P using a new fractionation method of P in EBPR sludge, which included the ultrasonic protocol at high power density for extracting the intracellular P and its poly-P.

Low-temperature-steam activation of phosphorus in biochar derived from enhanced biological phosphorus removal (EBPR) sludge

The property and release behavior of phosphorus in biochar derived from enhanced biological phosphorus removal sludge (EBPR sludge) were investigated. A low-temperature-steam (LTS) activation method was developed to increase the P availability in the biochar. The results demonstrate that the P content in the biochar is comparable to that in typical P fertilizers. The biochar contained a considerable portion of fast-release P. Polyphosphates (poly-P) were the predominant P species in the biochar. LTS greatly improved P availability in biochar produced at 700 °C by hydrolyzing insoluble poly-P to soluble pyro-P and ortho-P. In addition, the presence of Ca2+ could greatly reduce the P-release from biochar produced at lower temperature, e.g. 400 °C, due to that Ca2+ could facilitate the precipitation/adsorption of orthophosohates/pyrophosphates released from the low temperature biochars. Such phenomena was not observed with high temperature biochar, as the soluble poly-P released from 700 °C biochar could complex with Ca2+ rather than precipitated with it. Other environmental conditions, i.e., environmental pH, ionic strength of the soil porewater, and presence of low-molecular-weight acid, only had minor or negligible effects on the P-release of most studied biochars. This study concludes that LTS-activated-700 °C biochar could be a promising P fertilizer given its high, rapid, and unaffected P-release under various environmentally relevant conditions.

Electricity generation and in situ phosphate recovery from enhanced biological phosphorus removal sludge by electrodialysis membrane bioreactor

In this study, a novel electrodialysis membrane bioreactor was used for EBPR sludge treatment for energy and phosphorus resource recovery simultaneously. After 30days stable voltage outputting, the maximum power density reached 0.32W/m3. Over 90% of phosphorus in EBPR sludge was released while about 50% of phosphorus was concentrated to 4mmol/L as relatively pure phosphate solution. Nitrogen could be removed from EBPR sludge by desalination and denitrification processes. This study provides an optimized way treating sludge for energy production and in situ phosphorus recovery.

Phosphate release involving PAOs activity during anaerobic fermentation of EBPR sludge and the extension of ADM1

Anaerobic fermentation of the enhanced biological phosphorus removal (EBPR) sludge was investigated in terms of phosphate release and volatile fatty acids (VFAs) production regarding polyphosphate accumulating organisms (PAOs) activity. PAOs decay rate during fermentation was determined as 0.35±0.03d−1. Sludge lysis was enhanced with an increase in polyhydroxyalkanoate (PHA) content. Moreover, the phosphate release profiles and the VFAs production as well as the individual VFA fractions varied with different acetate concentrations added initially. Based on these observations, anaerobic digestion model No. 1 was extended and modified by introducing: (1) processes that PAOs store 4 VFA species as PHA, which can be degraded into varied fractions of individual VFA in dependence on PHA composition, (2) the effect of PHA content on disintegration rate, and (3) phosphorus precipitation. The proposed model adequately fitted a multi-experiment, multi-variable data set, indicating that it plays an important role in predicting phosphate and VFAs variations during EBPR sludge fermentation.

Study on the Characteristics of Biological Phosphorus Removal Sludge in Different Operation Mode of Reactor

The characteristics of the P removal sludge were studied in 3 different operation modes (AO, AOA and A2O mode) of the sequencing batch membrane reactor (SBMBRs). The results showed that the sludge P content was positively correlated with the P removal ability. The relative P content of the saturated P uptake sludge was 30.6%, 36.7% and 42.9%, respectively in the 3 modes. PHB was synthesized in anaerobic activated sludge and the polyp-P granules were decreased. The opposite trend appeared in aerobic condition. The amount of PHB change in anaerobic stage was proportional to the P release capacity. The FISH detection showed that the more proportion of PAOs to the whole cell the more P removal ability in the system.

Metabolic modelling of full-scale enhanced biological phosphorus removal sludge

This study investigates, for the first time, the application of metabolic models incorporating polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) towards describing the biochemical transformations of full-scale enhanced biological phosphorus removal (EBPR) activated sludge from wastewater treatment plants (WWTPs). For this purpose, it was required to modify previous metabolic models applied to lab-scale systems by incorporating the anaerobic utilisation of the TCA cycle and the aerobic maintenance processes based on sequential utilisation of polyhydroxyalkanoates, followed by glycogen and polyphosphate. The abundance of the PAO and GAO populations quantified by fluorescence in situ hybridisation served as the initial conditions of each biomass fraction, whereby the models were able to describe accurately the experimental data. The kinetic rates were found to change among the four different WWTPs studied or even in the same plant during different seasons, either suggesting the presence of additional PAO or GAO organisms, or varying microbial activities for the same organisms. Nevertheless, these variations in kinetic rates were largely found to be proportional to the difference in acetate uptake rate, suggesting a viable means of calibrating the metabolic model. The application of the metabolic model to full-scale sludge also revealed that different Accumulibacter clades likely possess different acetate uptake mechanisms, as a correlation was observed between the energetic requirement for acetate transport across the cell membrane with the diversity of Accumulibacter present. Using the model as a predictive tool, it was shown that lower acetate concentrations in the feed as well as longer aerobic retention times favour the dominance of the TCA metabolism over glycolysis, which could explain why the anaerobic TCA pathway seems to be more relevant in full-scale WWTPs than in lab-scale systems.

Genome reconstruction and gene expression of "Candidatus Accumulibacter phosphatis" Clade IB performing biological phosphorus removal.

We report the first integrated metatranscriptomic and metagenomic analysis of enhanced biological phosphorus removal (EBPR) sludge. A draft genome of Candidatus Accumulibacter spp. strain HKU-1, a member of Clade IB, was retrieved. It was estimated to be ∼90% complete and shared average nucleotide identities of 83% and 88% with the finished genome CAP IIA UW-1 and the draft genome CAP IA UW-2, respectively. Different from CAP IIA UW-1, the phosphotransferase (pap) in polyphosphate metabolism and V-ATPase in orthophosphate transport were absent from CAP IB HKU-1. Additionally, unlike CAP IA UW-2, CAP IB HKU-1 carried the genes for carbon fixation and nitrogen fixation. Despite these differences, the key genes required for acetate uptake, glycolysis and polyhydroxyalkanoate (PHA) synthesis were conserved in all these Accumulibacter genomes. The preliminary metatranscriptomic results revealed that the most significantly up-regulated genes of CAP IB HKU-1 from the anaerobic to the aerobic phase were responsible for assimilatory sulfate reduction, genetic information processing and phosphorus absorption, while the down-regulated genes were related to N2O reduction, PHA synthesis and acetyl-CoA formation. This study yielded another important Accumulibacter genome, revealed the functional difference within the Accumulibacter Type I, and uncovered the genetic responses to EBPR stimuli at a higher resolution.

Impact of nitrite on aerobic phosphorus uptake by poly-phosphate accumulating organisms in enhanced biological phosphorus removal sludges

Impact of nitrite on aerobic phosphorus (P) uptake of poly-phosphate accumulating organisms (PAOs) in three different enhanced biological phosphorus removal (EBPR) systems was investigated, i.e., the enriched PAOs culture fed with synthetic wastewater, the two lab-scale sequencing batch reactors (SBRs) treating domestic wastewater for nutrient removal through nitrite-pathway nitritation and nitrate-pathway nitrification, respectively. Fluorescence in situ hybridization results showed that PAOs in the three sludges accounted for 72, 7.6 and 6.5% of bacteria, respectively. In the enriched PAOs culture, at free nitrous acid (FNA) concentration of 0.47 × 10(-3) mg HNO₂-N/L, aerobic P-uptake and oxidation of intercellular poly-β-hydroxyalkanoates were both inhibited. Denitrifying phosphorus removal under the aerobic conditions was observed, indicating the existence of PAOs using nitrite as electron acceptor in this culture. When the FNA concentration reached 2.25 × 10(-3) mg HNO2-N/L, denitrifying phosphorus removal was also inhibited. And the inhibition ceased once nitrite was exhausted. Corresponding to both SBRs treating domestic wastewater with nitritation and nitrification pathway, nitrite inhibition on aerobic P-uptake by PAOs did not occur even though FNA concentration reached 3 × 10(-3) and 2.13 × 10(-3) mg HNO₂-N/L, respectively. Therefore, PAOs taken from different EBPR activated sludges had different tolerance to nitrite.

Species of phosphorus in the extracellular polymeric substances of EBPR sludge

In this study, the species of extracellular phosphorus and their transformation during extracellular polymeric substances (EPS) extraction were explored by using (31)P nuclear magnetic resonance spectroscopy. Results show that the extraction methods had a substantial influence on the phosphorus species in the extracted EPS. Cation exchange resin method was more appropriate for extracting EPS from the enhanced biological phosphorus removal (EBPR) sludge. Orthophosphate, pyrophosphate and polyphosphate were the main species of phosphorus found to be present in the EPS, which together accounted for about 6.6-10.5% of the total phosphorus in the EBPR sludge. The high percentage of extracellular phosphorus and their diverse species might reveal a new insight into the characteristics of the phosphorus in EPS in EBPR system.

Phosphorus release mechanisms during digestion of EBPR sludge under anaerobic, anoxic and aerobic conditions

Three laboratory-scale digesters were operated in parallel under anaerobic, anoxic and aerobic conditions to reveal the release mechanisms of phosphorus when digesting enhanced biological phosphorus removal (EBPR) sludge. The variation rates of the parameters associated with phosphorus release were calculated and compared with that of a typical EBPR anaerobic process. The results show that both phosphorus-accumulating organisms (PAOs) and denitrifying phosphorus-accumulating organisms (DPAOs) played important roles in the phosphorus release during the digestion processes. Under anaerobic conditions, the PAOs hydrolyzed internal polyphosphorus (poly-P) into PO4(3-)-P concurrent with synthesis of polyhydroxyalkanoates (PHA). Under anoxic or aerobic conditions, PAOs and/or DPAOs assimilated part of the PO4(3-)-P from the digestive liquid using nitrate or oxygen as terminal electron acceptors. Nevertheless, the biological activities of PAOs under anaerobic conditions and DPAOs under anoxic conditions were limited. Moreover, it was the biomass hydrolysis degree that determined the phosphorus release capacity of the sludge, regardless of whether anaerobic, anoxic or aerobic conditions were adopted. Assuming that nitrate was the sole electron acceptor during anoxic digestion of EBPR biomass, the relationship between the consumption of nitrate and uptake of PO4(3-)-P associated with the denitrifying phosphorus removal (DPR) can be expressed as ΔP = 0.11 × ΔN.

Short-Chain Fatty Acid Production from Different Biological Phosphorus Removal Sludges: The Influences of PHA and Gram-Staining Bacteria

Recently, the reuse of waste activated sludge to produce short-chain fatty acids (SCFA) has attracted much attention. However, the influences of sludge characteristics, especially polyhydroxyalkanoates (PHA) and Gram-staining bacteria, on SCFA production have seldom been investigated. It was found in this study that during sludge anaerobic fermentation not only the fermentation time but also the SCFA production were different between two sludges, which had different PHA contents and Gram-negative bacteria to Gram-positive bacteria (GNB/GPB) ratios and were generated respectively from the anaerobic/oxic (AO) and aerobic/extended-idle (AEI) biological phosphorus removal processes. The optimal fermentation time for the AEI and AO sludges was respectively 4 and 8 d, and the corresponding SCFA production was 304.6 and 231.0 mg COD/g VSS (volatile suspended solids) in the batch test and 143.4 and 103.9 mg COD/g VSS in the semicontinuous experiment. The mechanism investigation showed that the AEI sludge had greater PHA content and GNB/GPB ratio, and the increased PHA content accelerated cell lysis and soluble substrate hydrolysis while the increased GNB/GPB ratio benefited cell lysis. Denaturing gradient gel electrophoresis profiles revealed that the microbial community in the AEI sludge fermentation reactor was dominated by Clostridium sp., which was reported to be SCFA-producing microbes. Further enzyme analyses indicated that the activities of key hydrolytic and acids-forming enzymes in the AEI sludge fermentation reactor were higher than those in the AO one. Thus, less fermentation time was required, but higher SCFA was produced in the AEI sludge fermentation system.

Identification of a novel subgroup of uncultured gammaproteobacterial glycogen-accumulating organisms in enhanced biological phosphorus removal sludge

The presence of glycogen-accumulating organisms (GAO) has been hypothesized to be a cause of deterioration in enhanced biological phosphorus removal (EBPR) processes due to their abilities to out-compete polyphosphate-accumulating organisms (PAO). Based on 16S rRNA gene sequences, new members of uncultured gammaproteobacterial GAO (GB) were identified from sludge samples of a lab-scale sequencing batch reactor used for EBPR. The new GB formed a phylogenetic lineage (GB8) clearly distinct from the previously reported seven GB subgroups. Because the new GB8 members were not targeted by the known fluorescence in situ hybridization (FISH) oligonucleotide probes, a GB8-specific FISH probe (GB429) and a new FISH probe (GB742) targeting all eight GB subgroups were designed, and the phenotypic properties of the new GB8 members were investigated. FISH and microautoradiography approaches showed that GB429-targeted cells (GB8) were large coccobacilli (2-4 µm in size) with the ability to take up acetate under anaerobic conditions, but unable to accumulate polyphosphate under the subsequent aerobic conditions, consistent with in situ phenotypes of GB. FISH analyses on several sludge samples showed that members of GB8 were commonly detected as the majority of GB in lab- and full-scale EBPR processes. In conclusion, this study showed that members of GB8 could be a subgroup of GB with an important role in EBPR deterioration. Designs of FISH probes which hybridize with broader GB subgroups at different hierarchical levels will contribute to studies of the distributions and ecophysiologies of GB in lab- or full-scale EBPR plants.

Microbial Community Analysis on Oxic-Settling-Anaerobic Process by Using PCR-DGGE Assay

In situ sludge reduction of the oxic-settling-anaerobic (OSA) process was investigated in this study and microbial community diversity in the system was analyzed by using a PCR-denaturing gradient gel electrophoresis (DGGE) approach. Comparing to the conventional activated sludge method, the production of excessive sludge in the OSA process was shown to be efficiently reduced by 44-50 % and the organic loading rate was observed to have a slight impact on sludge yield. As demonstrated by the slight variation of Shannon diversity indices (SDI), the dominant microbial composition remained stable in the OSA sludge with the increase ofNs. About 63% of clones represented by predominant bands in the DGGE pattern were affiliated with the subclass ofb-proteobacteria. A number of bacteria in the OSA process were phylogenetically related to uncultured bacteria isolated from enhanced biological phosphorus removal (EBPR) sludge.

Denitrification capabilities of two biological phosphorus removal sludges dominated by different ‘Candidatus Accumulibacter’ clades

The capability of "Candidatus Accumulibacter" to use nitrate as an electron acceptor for phosphorus uptake was investigated using two activated sludge communities. The two communities were enriched in Accumulibacter clade IA and clade IIA, respectively. By performing a series of batch experiments, we found that clade IA was able to couple nitrate reduction with phosphorus uptake, but clade IIA could not. These results agree with a previously proposed hypothesis that different populations of Accumulibacter have different nitrate reduction capabilities, and they will help to understand the ecological roles that these two clades provide.

Free Nitrous Acid Inhibition on Nitrous Oxide Reduction by a Denitrifying-Enhanced Biological Phosphorus Removal Sludge

Nitrite has generally been recognized as an inhibitor of N2O reduction during denitrification. This inhibitory effect is investigated under various pH conditions using a denitrifying-enhanced biological phosphorus removal (EBPR) sludge. The degree of inhibition was observed to correlate much more strongly with the free nitrous acid (FNA) concentration than with the nitrite concentration, suggesting that FNA, rather than nitrite, is likely the true inhibitor on N2O reduction. Fifty percent inhibition was observed at an FNA concentration of 0.0007-0.001 mg HNO2-N/L (equivalent to approximately 3-4 mg NO2(-) -N/L at pH 7), while complete inhibition occurred when the FNA concentration was greater than 0.004 mg HNO2-N/L. The results also suggest that the inhibition on N2O reduction was not due to the electron competition between N2O and NO2- reductases. The inhibition was found to be reversible, with the rate of recovery independent of the duration of the inhibition, but dependent on the concentration of FNAthe biomass was exposed to during the inhibition period. A higher FNA concentration caused slower recovery.

“ Candidatus Accumulibacter” Population Structure in Enhanced Biological Phosphorus Removal Sludges as Revealed by Polyphosphate Kinase Genes

We investigated the fine-scale population structure of the "Candidatus Accumulibacter" lineage in enhanced biological phosphorus removal (EBPR) systems using the polyphosphate kinase 1 gene (ppk1) as a genetic marker. We retrieved fragments of "Candidatus Accumulibacter" 16S rRNA and ppk1 genes from one laboratory-scale and several full-scale EBPR systems. Phylogenies reconstructed using 16S rRNA genes and ppk1 were largely congruent, with ppk1 granting higher phylogenetic resolution and clearer tree topology and thus serving as a better genetic marker than 16S rRNA for revealing population structure within the "Candidatus Accumulibacter" lineage. Sequences from at least five clades of "Candidatus Accumulibacter" were recovered by ppk1-targeted PCR, and subsequently, specific primer sets were designed to target the ppk1 gene for each clade. Quantitative real-time PCR (qPCR) assays using "Candidatus Accumulibacter"-specific 16S rRNA and "Candidatus Accumulibacter" clade-specific ppk1 primers were developed and conducted on three laboratory-scale and nine full-scale EBPR samples and two full-scale non-EBPR samples to determine the abundance of the total "Candidatus Accumulibacter" lineage and the relative distributions and abundances of the five "Candidatus Accumulibacter" clades. The qPCR-based estimation of the total "Candidatus Accumulibacter" fraction as a proportion of the bacterial community as measured using 16S rRNA genes was not significantly different from the estimation measured using ppk1, demonstrating the power of ppk1 as a genetic marker for detection of all currently defined "Candidatus Accumulibacter" clades. The relative distributions of "Candidatus Accumulibacter" clades varied among different EBPR systems and also temporally within a system. Our results suggest that the "Candidatus Accumulibacter" lineage is more diverse than previously realized and that different clades within the lineage are ecologically distinct.