Full-scale Enhanced Biological Phosphorus Removal Plants Research Articles

\u201cCandidatus Dechloromonas phosphoritropha\u201d and \u201cCa. D. phosphorivorans\u201d, novel polyphosphate accumulating organisms abundant in wastewater treatment systems

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

Resolving the individual contribution of key microbial populations to enhanced biological phosphorus removal with Raman\u2013FISH

Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms. Candidatus Accumulibacter conform to the classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers under in situ conditions, and applied it to eight full-scale EBPR plants. Besides Ca. Accumulibacter, members of the genus Tetrasphaera were found to be important microbes for P accumulation, and in six plants they were the most important. As these Tetrasphaera cells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populations in situ in other ecosystems and might become a valuable tool for many environmental microbiologists.

"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.

A metabolic model for members of the genus Tetrasphaera involved in enhanced biological phosphorus removal

Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to 'Candidatus Accumulibacter phosphatis' is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by 'Candidatus Accumulibacter phosphatis', and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation.

‘Candidatus Halomonas phosphatis’, a novel polyphosphate‐accumulating organism in full‐scale enhanced biological phosphorus removal plants

Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAOs) in full-scale enhanced biological phosphorus removal (EBPR) plants. Clone library analyses and application of MAR-FISH using newly designed probes revealed that small rods related to uncultured Halomonas within the gammaproteobacterial family Halomonadaceae were actively involved in uptake of orthophosphate. Although deeply branched in the Gammaproteobacteria, they were not targeted by the gammaproteobacterial probe (GAM42a). A part of them were also not targeted with the general bacterial probes (EUBmix). They could take up short-chain fatty acids (e.g. acetate and propionate) and ethanol under both anaerobic and aerobic conditions. Polyhydroxyalkanoate storage was observed under anaerobic conditions. There was no indication of a denitrifying capability. A survey of the occurrence of these Halomonas-PAOs in 23 full-scale EBPR plants revealed that they made up 0.5-5.7% of all bacteria in the plants, and were often in higher abundance than the well-described PAOs 'Candidatus Accumulibacter phosphatis'. This indicates a potentially important role for these uncultured Halomonas bacteria in the EBPR process in full-scale plants and we propose to name them 'Candidatus Halomonas phosphatis'.

Identification of glucose-fermenting bacteria in a full-scale enhanced biological phosphorus removal plant by stable isotope probing

Microbiology in wastewater treatment has mainly been focused on problem-causing filamentous bacteria or bacteria directly involved in nitrogen and phosphorus removal, and to a lesser degree on flanking groups, such as hydrolysing and fermenting bacteria. However, these groups constitute important suppliers of readily degradable substrates for the overall processes in the plant. This study aimed to identify glucose-fermenting bacteria in a full-scale enhanced biological phosphorus removal (EBPR) wastewater treatment plant (WWTP), and to determine their abundance in similar WWTPs. Glucose-fermenting micro-organisms were identified by an in situ approach using RNA-based stable isotope probing. Activated sludge was incubated anaerobically with (13)C(6)-labelled glucose, and (13)C-enriched rRNA was subsequently reverse-transcribed and used to construct a 16S rRNA gene clone library. Phylogenetic analysis of the library revealed the presence of two major phylogenetic groups of gram-positive bacteria affiliating with the genera Tetrasphaera, Propionicimonas (Actinobacteria), and Lactococcus and Streptococcus (Firmicutes). Specific oligonucleotide probes were designed for fluorescence in situ hybridization (FISH) to specifically target the glucose-fermenting bacteria identified in this study. The combination of FISH with microautoradiography confirmed that Tetrasphaera, Propionicimonas and Streptococcus were the dominant glucose fermenters. The probe-defined fermenters were quantified in 10 full-scale EBPR plants and averaged 39 % of the total biovolume. Tetrasphaera and Propionicimonas were the most abundant glucose fermenters (average 33 and 4 %, respectively), while Streptococcus and Lactococcus were present only in some WWTPs (average 1 and 0.4 %, respectively). Thus the population of actively metabolizing glucose fermenters seems to occupy a relatively large component of the total biovolume.

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.

High diversity and abundance of putative polyphosphate-accumulating Tetrasphaera-related bacteria in activated sludge systems

The diversity of the putative polyphosphate-accumulating genus Tetrasphaera in wastewater treatment systems with enhanced biological phosphorus removal (EBPR) was investigated using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. 16S rRNA actinobacterial gene sequences were retrieved from different full-scale EBPR plants, and the sequences belonging to the genus Tetrasphaera (family Intrasporangiaceae) were found to form three clades. Quantitative FISH analyses of the communities in five full-scale EBPR plants using 10 new oligonucleotide probes were carried out. The results showed that the probe-defined Tetrasphaera displayed different morphologies and constituted up to 30% of the total biomass. It was shown that active uptake of orthophosphate and formation of polyphosphate took place in most of the probe-defined Tetrasphaera populations. However, aerobic uptake of orthophosphate only took place after uptake of certain carbon sources under anaerobic conditions and these were more diverse than hitherto assumed: amino acids, glucose, and for some also acetate. Tetrasphaera seemed to occupy a slightly different ecological niche compared with 'Candidatus Accumulibacter' contributing to a functional redundancy and stability of the EBPR process.

Modelling the population dynamics and metabolic diversity of organisms relevant in anaerobic/anoxic/aerobic enhanced biological phosphorus removal processes

In this study, enhanced biological phosphorus removal (EBPR) metabolic models are expanded in order to incorporate the competition between polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) under sequential anaerobic/anoxic/aerobic conditions, which are representative of most full-scale EBPR plants. Since PAOs and GAOs display different denitrification tendencies, which is dependent on the phylogenetic identity of the organism, the model was separated into six distinct biomass groups, constituting Accumulibacter Types I and II, as well as denitrifying and non-denitrifying Competibacter and Defluviicoccus GAOs. Denitrification was modelled as a multi-step process, with nitrate (NO 3), nitrite (NO 2), nitrous oxide (N 2O) and di-nitrogen gas (N 2) being the primary components. The model was calibrated and validated using literature data from enriched cultures of PAOs and GAOs, obtaining a good description of the observed biochemical transformations. A strong correlation was observed between Accumulibacter Types I and II, and nitrate-reducing and non-nitrate-reducing PAOs, respectively, where the abundance of each PAO subgroup was well predicted by the model during an acclimatisation period from anaerobic–aerobic to anaerobic–anoxic conditions. Interestingly, a strong interdependency was observed between the anaerobic, anoxic and aerobic kinetic parameters of PAOs and GAOs. This could be exploited when metabolic models are calibrated, since all of these parameters should be changed by an identical factor from their default value. Factors that influence these kinetic parameters include the fraction of active biomass, relative aerobic/anoxic fraction and the ratio of acetyl-CoA to propionyl-CoA. Employing a metabolic approach was found to be advantageous in describing the performance and population dynamics in such complex microbial ecosystems.

Abundance and ecophysiology of Defluviicoccus spp., glycogen-accumulating organisms in full-scale wastewater treatment processes

The activity of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) wastewater treatment plants has been proposed as one cause of deterioration of EBPR. Putative GAOs from the Alphaproteobacteria, Defluviicoccus spp. (including D. vanus), were studied in full-scale EBPR plants to determine their distribution, abundance and ecophysiology. Fluorescence in situ hybridization (FISH) demonstrated that Defluviicoccus spp. were generally low in abundance; however, in one plant surveyed, Cluster 2 Defluviicoccus constituted 9 % of all Bacteria. FISH combined with microautoradiography revealed that both Cluster 1 and Cluster 2 Defluviicoccus were capable of taking up a narrow range of substrates including acetate, propionate, pyruvate and glucose under anaerobic and aerobic conditions. Formate, butyrate, ethanol and several other substrates were not taken up. Cluster 2 Defluviicoccus demonstrated a phenotype consistent with the current metabolic model for GAOs--anaerobic assimilation of acetate and reduction to polyhydroxyalkanoates (PHA) using the glycolytic pathway, and aerobic consumption of PHA. Polyphosphate-accumulating organisms (PAOs, 'Candidatus Accumulibacter phosphatis') and other putative GAOs ('Candidatus Competibacter phosphatis') co-existed in two plants with Cluster 2 Defluviicoccus, but in both plants, the latter organisms were more abundant. Thus Cluster 2 Defluviicoccus can be relatively abundant and could be carbon competitors of PAOs and other GAOs in EBPR plants.

Examining substrate uptake patterns of Rhodocyclus-related PAO in full-scale EBPR plants by using the MAR-FISH technique

While recognised as the important population responsible for enhanced biological phosphorus removal (EBPR), detailed knowledge on the physiology of Rhodocyclus-related polyphosphate accumulating organisms (PAO) has yet to be grasped. The objective of this study was to examine the in situ substrate uptake patterns of Rhodocyclus-related PAO present in full-scale EBPR plants by the combined technique of microautoradiography-fluorescent in situ hybridization (MAR-FISH). The presence of these PAO in the four investigated plants was confirmed by FISH and they constituted 17%, 9%, 8%, and 7% of the sludge community. By using MAR-FISH technique, Rhodocyclus-related PAO in all the plants demonstrated similar anaerobic substrate uptake patterns. They were capable of assimilating acetate, aspartate and glutamate under anaerobic condition but they showed negative uptake with palmitate. A significant fraction of the MAR-positive cells assimilated acetate, aspartate or glutamate was found to be Rhodocyclus-related PAO. Dual staining with DAPI and FISH showed that these PAO also accumulated polyphosphate aerobically with aspartate and glutamate as carbon source. The ability of assimilating amino acids besides acetate strongly indicates the versatile physiology of Rhodocyclus-related PAO, which could benefit them to achieve predominance in EBPR activated sludge.

Fine-scale differences between Accumulibacter-like bacteria in enhanced biological phosphorus removal activated sludge

A lab-scale sequencing batch reactor (SBR) and six full-scale wastewater treatment plants (WWTPs) performing enhanced biological phosphorus removal (EBPR) were surveyed. The abundance of Accumulibacter-related organisms in the full-scale plants was investigated using fluorescent in situ hybridization. Accumulibacter-related organisms were present in all of the full-scale EBPR plants, at levels ranging from 9% to 24% of total cells. The high percentage of Accumulibacter-related organisms seemed to be associated with configurations which minimize the nitrate recycling to the anaerobic zone and low influent BOD:TP ratios. PCR-based clone libraries were constructed from the community 16S rRNA gene plus the internally transcribed spacer region amplified from the SBR and five of the full-scale WWTPs. Comparative sequence analysis was carried out using Accumulibacter-related clones, providing higher phylogenetic resolution and revealing finer-scale clustering of the sequences retrieved from the SBR and full-scale EBPR

Ecophysiology of a group of uncultured Gammaproteobacterial glycogen‐accumulating organisms in full‐scale enhanced biological phosphorus removal wastewater treatment plants

The presence of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) plants can seriously deteriorate the biological P-removal by out-competing the polyphosphate-accumulating organisms (PAOs). In this study, uncultured putative GAOs (the GB group, belonging to the Gammaproteobacteria) were investigated in detail in 12 full-scale EBPR plants. Fluorescence in situ hybridization (FISH) revealed that the biovolume of the GB bacteria constituted 2-6% of total bacterial biovolume. At least six different subgroups of the GB bacteria were found, and the number of dominant subgroups present in each plant varied between one and five. Ecophysiological investigations using microautoradiography in combination with FISH showed that, under aerobic or anaerobic conditions, all subgroups of the GB bacteria could take up acetate, pyruvate, propionate and some amino acids, while some subgroups in addition could take up formate and thymidine. Glucose, ethanol, butyrate and several other organic substrates were not taken up. Glycolysis was essential for the anaerobic uptake of organic substrates. Polyhydroxyalkanoates (PHA) but not polyphosphate (polyP) granules were detected in all GB bacterial cells. Polyhydroxyalkanoate formation after anaerobic uptake of acetate was confirmed by measuring the increase in fluorescence intensity of PHA granules inside GB bacterial cells after Nile blue staining. One GB subgroup was possibly able to denitrify, and several others were able to reduce nitrate to nitrite. PAOs were also enumerated by FISH in the same treatment plants. Rhodocyclus-related PAOs and Actinobacteria-related PAOs constituted up to 7% and 29% of total bacterial biovolume respectively. Rhodocyclus-related PAOs always coexisted with the GB bacteria and showed many physiological similarities. Factors of importance for the competition between the three groups of important bacteria in EBPR plants are discussed.

Identity and Ecophysiology of Uncultured Actinobacterial Polyphosphate-Accumulating Organisms in Full-Scale Enhanced Biological Phosphorus Removal Plants

Microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) was used to screen for potential polyphosphate-accumulating organisms (PAO) in a full-scale enhanced biological phosphorus removal (EBPR) plant. The results showed that, in addition to uncultured Rhodocyclus-related PAO, two morphotypes hybridizing with gene probes for the gram-positive Actinobacteria were also actively involved in uptake of orthophosphate (Pi). Clone library analysis and further investigations by MAR-FISH using two new oligonucleotide probes revealed that both morphotypes, cocci in clusters of tetrads and short rods in clumps, were relatively closely related to the genus Tetrasphaera within the family Intrasporangiaceae of the Actinobacteria (93 to 98% similarity in their 16S rRNA genes). FISH analysis of the community biomass in the treatment plant investigated showed that the short rods (targeted by probe Actino-658) were the most abundant (12% of all Bacteria hybridizing with general bacterial probes), while the cocci in tetrads (targeted by probe Actino-221) made up 7%. Both morphotypes took up P(i) aerobically only if, in a previous anaerobic phase, they had taken up organic matter from wastewater or a mixture of amino acids. They could not take up short-chain fatty acids (e.g., acetate), glucose, or ethanol under anaerobic or aerobic conditions. The storage compound produced during the anaerobic period was not polyhydroxyalkanoates, as for Rhodocyclus-related PAO, and its identity is still unknown. Growth and uptake of Pi took place in the presence of oxygen and nitrate but not nitrite, indicating a lack of denitrifying ability. A survey of the occurrence of these actinobacterial PAO in 10 full-scale EBPR plants revealed that both morphotypes were widely present, and in several plants more abundant than the Rhodocyclus-related PAO, thus playing a very important role in the EBPR process.

Microautoradiographic study of Rhodocyclus-related polyphosphate-accumulating bacteria in full-scale enhanced biological phosphorus removal plants.

The ecophysiology of uncultured Rhodocyclus-related polyphosphate-accumulating organisms (PAO) present in three full-scale enhanced biological phosphorus removal (EBPR) activated sludge plants was studied by using microautoradiography combined with fluorescence in situ hybridization. The investigations showed that these organisms were present in all plants examined and constituted 5 to 10, 10 to 15, and 17 to 22% of the community biomass. The behavior of these bacteria generally was consistent with the biochemical models proposed for PAO, based on studies of lab-scale investigations of enriched and often unknown PAO cultures. Rhodocyclus-related PAO were able to accumulate short-chain substrates, including acetate, propionate, and pyruvate, under anaerobic conditions, but they could not assimilate many other low-molecular-weight compounds, such as ethanol and butyrate. They were able to assimilate two substrates (e.g., acetate and propionate) simultaneously. Leucine and thymidine could not be assimilated as sole substrates and could only be assimilated as cosubstrates with acetate, perhaps serving as N sources. Glucose could not be assimilated by the Rhodocyclus-related PAO, but it was easily fermented in the sludge to products that were subsequently consumed. Glycolysis, and not the tricarboxylic acid cycle, was the source that provided the reducing power needed by the Rhodocyclus-related PAO to form the intracellular polyhydroxyalkanoate storage compounds during anaerobic substrate assimilation. The Rhodocyclus-related PAO were able to take up orthophosphate and accumulate polyphosphate when oxygen, nitrate, or nitrite was present as an electron acceptor. Furthermore, in the presence of acetate growth was sustained by using oxygen, as well as nitrate or nitrite, as an electron acceptor. This strongly indicates that Rhodocyclus-related PAO were able to denitrify and thus played a role in the denitrification occurring in full-scale EBPR plants.

The effect of GAOs (glycogen accumulating organisms) on anaerobic carbon requirements in full-scale Australian EBPR (enhanced biological phosphorus removal) plants

Glycogen-accumulating organisms (GAOs) were present in six full-scale plants investigated and in all but one made a significant contribution to the amount of volatile fatty acid (VFA) taken up anaerobically. While most plants surveyed contain GAOs, it was demonstrated that it is possible for a full-scale plant to operate with an insignificant GAO population. "Candidatus Accumulibacter phosphatis" were the significant polyphosphate-accumulating organisms (PAOs) in all plants surveyed. "Candidatus Competibacter phosphatis" were found in all plants along with other possible GAOs that were observed but not identified. A significant GAO population will increase the carbon requirements by removing VFA that could otherwise have been used by PAOs. Process optimization minimizing GAOs in full-scale plants would lead to a more efficient use of VFA.

Environmental factors contributing to the “G bacteria” population in full-scale EBPR plants

A survey of several enhanced biological phosphorus removal (EBPR) plants within Australia has demonstrated that a group of bacteria known as the "G" bacteria are able to proliferate under a broad range of plant configurations. The diverse designs and operational parameters of these plants did not permit definitive determination of the factor(s) contributing to the proliferation of G bacteria. Two plants were monitored over time to assess the G bacteria and phosphorus accumulating organisms (PAO) populations in relation to key operational parameters. The mixed liquor biomass and operational parameters were compared to other plants successfully and unsuccessfully reducing phosphorus from the wastewater. Two critical factors recognised in this study were the dissolved oxygen concentration in the aerobic zone and the type and amount of carbon source in the anaerobic zone.

Sleep and Alertness

<h3>Abstract</h3> Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms. <i>Candidatus</i> Accumulibacter conform to classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers under <i>in situ</i> conditions, and applied it to eight full-scale EBPR plants. Besides <i>Ca</i>. Accumulibacter, members of the genus <i>Tetrasphaera</i> were found to be important microbes for P accumulation, and in six plants they were the most important. As these <i>Tetrasphaera</i> cells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populations <i>in situ</i> in other ecosystems and might become a valuable tool for many environmental microbiologists.