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.

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Presence of Rhodocyclus in a full-scale wastewater treatment plant and their participation in enhanced biological phosphorus removal

Water Science & Technology ◽

10.2166/wst.2002.0467 ◽

2002 ◽

Vol 46 (1-2) ◽

pp. 123-128 ◽

Cited By ~ 26

Author(s):

J.L. Zilles ◽

C.-H. Hung ◽

D.R. Noguera

Keyword(s):

Wastewater Treatment ◽

Phosphorus Removal ◽

Fluorescent In Situ Hybridization ◽

Wastewater Treatment Plants ◽

Full Scale ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Polyphosphate Accumulating Organisms ◽

Biological Phosphorus

The objective of this research was to assess the relevance of organisms related to Rhodocyclus in enhanced biological phosphorus removal in full-scale wastewater treatment plants. The presence of these organisms in full-scale plants was first confirmed by fluorescent in situ hybridization. To address which organisms were involved in phosphorus removal, a method was developed which selected polyphosphate-accumulating organisms from activated sludge samples by DAPI staining and flow cytometry. Sorted samples were characterized using fluorescent in situ hybridization. The results of these analyses confirmed the presence of organisms related to Rhodocyclus in full-scale wastewater treatment plants and supported the involvement of these organisms in enhanced biological phosphorus removal. However, a significant fraction of the polyphosphate-accumulating organisms were not related to Rhodocyclus.

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

Water Science & Technology ◽

10.2166/wst.2003.0584 ◽

2003 ◽

Vol 47 (11) ◽

pp. 37-43 ◽

Cited By ~ 91

Author(s):

A.M. Saunders ◽

A. Oehmen ◽

L.L. Blackall ◽

Z. Yuan ◽

J. Keller

Keyword(s):

Phosphorus Removal ◽

In Situ Hybridisation ◽

Full Scale ◽

Fluorescence In Situ Hybridisation ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Accumulibacter Phosphatis ◽

Polyphosphate Accumulating Organisms ◽

Biological Phosphorus

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. Enhanced biological phosphorus removal (EBPR), fluorescence in situ hybridisation (FISH), glycogen accumulating organism (GAO); polyphosphate accumulating organism (PAO);

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Resolving the individual contribution of key microbial populations to enhanced biological phosphorus removal with Raman-FISH

10.1101/387795 ◽

2018 ◽

Cited By ~ 3

Author(s):

Eustace Y. Fernando ◽

Simon Jon Mcllroy ◽

Marta Nierychlo ◽

Florian-Alexander Herbst ◽

Markus C. Schmid ◽

...

Keyword(s):

Phosphorus Removal ◽

Absolute Quantification ◽

Full Scale ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Important Player ◽

The Individual ◽

Polyphosphate Accumulating Organisms ◽

Biological Phosphorus

AbstractEnhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms.CandidatusAccumulibacter 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 underin situconditions, and applied it to eight full-scale EBPR plants. BesidesCa. Accumulibacter, members of the genusTetrasphaerawere found to be important microbes for P accumulation, and in six plants they were the most important. As theseTetrasphaeracells 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 populationsin situin other ecosystems and might become a valuable tool for many environmental microbiologists.

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Microautoradiographic Study of Rhodocyclus-Related Polyphosphate-Accumulating Bacteria in Full-Scale Enhanced Biological Phosphorus Removal Plants

Applied and Environmental Microbiology ◽

10.1128/aem.70.9.5383-5390.2004 ◽

2004 ◽

Vol 70 (9) ◽

pp. 5383-5390 ◽

Cited By ~ 125

Author(s):

Yunhong Kong ◽

Jeppe Lund Nielsen ◽

Per Halkjær Nielsen

Keyword(s):

Electron Acceptor ◽

Phosphorus Removal ◽

Tricarboxylic Acid Cycle ◽

Reducing Power ◽

Full Scale ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Polyphosphate Accumulating Organisms ◽

Biological Phosphorus

ABSTRACT 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.

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

Water Science & Technology ◽

10.2166/wst.2006.378 ◽

2006 ◽

Vol 54 (1) ◽

pp. 111-117 ◽

Cited By ~ 29

Author(s):

S. He ◽

A.Z. Gu ◽

K.D. McMahon

Keyword(s):

Phosphorus Removal ◽

Wastewater Treatment Plants ◽

Batch Reactor ◽

Comparative Sequence Analysis ◽

Full Scale ◽

Rrna Gene ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Phylogenetic Resolution ◽

Biological Phosphorus

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 plants.

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

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.4076-4085.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 4076-4085 ◽

Cited By ~ 176

Author(s):

Yunhong Kong ◽

Jeppe Lund Nielsen ◽

Per Halkjær Nielsen

Keyword(s):

Phosphorus Removal ◽

Treatment Plant ◽

Full Scale ◽

16S Rrna Genes ◽

Rrna Genes ◽

Fish Analysis ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Polyphosphate Accumulating Organisms ◽

Biological Phosphorus

ABSTRACT 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 Pi 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.

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Global warming readiness: Feasibility of enhanced biological phosphorus removal from wastewater at 35°C

10.1101/2021.02.08.429585 ◽

2021 ◽

Author(s):

Guanglei Qiu ◽

Yingyu Law ◽

Rogelio Zuniga-Montanez ◽

Yang Lu ◽

Samarpita Roy ◽

...

Keyword(s):

Phosphorus Removal ◽

Municipal Wastewater ◽

Feeding Strategy ◽

Full Scale ◽

Carbon Uptake ◽

Rrna Gene ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Biological Phosphorus ◽

P Removal

AbstractRecent research has shown enhanced biological phosphorus removal (EBPR) from municipal wastewater at warmer temperatures around 30°C to be stable in both laboratory-scale reactors and full-scale treatment plants. In the context of a changing climate, the feasibility of EBPR at even higher temperatures is of interest. We operated two lab-scale EBPR sequencing batch reactors with alternating anaerobic and aerobic phases for over 300 days at 30°C and 35°C, respectively, and followed the dynamics of the communities of phosphorus accumulating organisms (PAOs) and competing glycogen accumulating organisms (GAOs) using a combination of 16S rRNA gene metabarcoding, quantitative PCR and fluorescent in-situ hybridization analyses. Stable and nearly complete P removal was achieved at 30°C; similarly, long term P removal was stable at 35°C with effluent PO43−-P concentrations < 0.5 mg/L on half of all monitored days. Diverse and abundant Ca. Accumulibacter amplicon sequence variants were closely related to those found in temperate environments, suggesting that EBPR at this temperature does not require a highly specialized PAO community. The slow-feeding strategy used effectively limited the carbon uptake rates of GAOs, allowing PAOs to outcompete GAOs at both temperatures. Candidatus Competibacter was the main GAO, along with cluster III Defluviicoccus members. These organisms withstood the slow-feeding regime, suggesting that their bioenergetic characteristics of carbon uptake differ from those of their tetrad-forming relatives. This specific lineage of GAOs warrants further study to establish how complete P removal can be maintained. Comparative cycle studies at two temperatures for each reactor revealed higher activity of Ca. Accumulibacter when the temperature was increased from 30°C to 35°C, suggesting that the stress was a result of the higher carbon (and/or P) metabolic rates of PAOs and GAOs, the resultant carbon deficiency, and additional community competition. An increase in the TOC to PO43--P ratio (from 25:1 to 40:1) effectively eased the carbon deficiency and benefited the proliferation of PAOs. In general, the slow-feeding strategy and sufficiently high carbon input benefited a high and stable EBPR at elevated temperature and represent basic conditions for full-scale applications.

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Characterisation of enhanced biological phosphorus removal activated sludges with dissimilar phosphorus removal performances

Water Science & Technology ◽

10.2166/wst.1998.0719 ◽

1998 ◽

Vol 37 (4-5) ◽

pp. 567-571 ◽

Cited By ~ 14

Author(s):

Philip L. Bond ◽

Jürg Keller ◽

Linda L. Blackall

Keyword(s):

Phosphorus Removal ◽

Batch Reactor ◽

In Situ Hybridisation ◽

Chemical Transformations ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Reactor Performance ◽

Biological Phosphorus ◽

P Removal

A sequencing batch reactor (SBR) was operated for enhanced biological phosphorus removal (EBPR) and dramatic differences in the P removing capabilities were obtained in different stages of the operation. At one stage extremely poor P removal occurred and it appeared that bacteria inhibiting P removal overwhelmed the reactor performance. Changes were made to the reactor operation and these led to the development of a sludge with high P removing capability. This latter sludge was analysed by fluorescent in situ hybridisation (FISH) using a probe specific for Acinetobacter. Very few cells were detected with this probe indicating that Acinetobacter played an insignificant role in the P removal occurring here. Analysis of the chemical transformations of three sludges supported the biochemical pathways proposed for EBPR and non-EBPR systems in biological models. A change in operation that led to the improved P removal performance included permitting the pH to rise in the anaerobic periods of the SBR cycle.

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Oligotyping and Genome-Resolved Metagenomics Reveal DistinctCandidatusAccumulibacter Communities in Full-Scale Side-Stream versus Conventional Enhanced Biological Phosphorus Removal (EBPR) Configurations

10.1101/596692 ◽

2019 ◽

Cited By ~ 2

Author(s):

Varun N. Srinivasan ◽

Guangyu Li ◽

Dongqi Wang ◽

Nicholas B. Tooker ◽

Zihan Dai ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Phosphorus Removal ◽

Full Scale ◽

Rrna Gene ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Side Stream ◽

First Time ◽

Biological Phosphorus

AbstractCandidatusAccumulibacter phosphatis (CAP) and its sub-clades-level diversity has been associated and implicated in successful phosphorus removal performance in enhanced biological phosphorus removal (EBPR). Development of high-throughput untargeted methods to characterize clades of CAP in EBPR communities can enable a better understanding of Accumulibacter ecology at a higher-resolution beyond OTU-level in wastewater resource recovery facilities (WRRFs). In this study, for the first time, using integrated 16S rRNA gene sequencing, oligotyping and genome-resolved metagenomics, we were able to reveal clade-level differences in Accumulibacter communities and associate the differences with two different full-scale EBPR configurations. The results led to the identification and characterization of a distinct and dominant Accumulibacter oligotype - Oligotype 2 (belonging to Clade IIC) and its matching MAG (RC14) associated with side-stream EBPR configuration. We are also able to extract MAGs belonging to CAP clades IIB (RCAB4-2) and II (RC18) which did not have representative genomes before. This study demonstrates and validates the use of a high-throughput approach of oligotyping analysis of 16S rRNA gene sequences to elucidate CAP clade-level diversity. We also show the existence of a previously uncharacterized diversity of CAP clades in full-scale EBPR communities through extraction of MAGs, for the first time from full-scale facilities.

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A Full-Scale Comparative Study of Conventional and Side-Stream Enhanced Biological Phosphorus Removal Processes

10.20944/preprints201808.0250.v1 ◽

2018 ◽

Cited By ~ 1

Author(s):

Dongqi Wang ◽

Nicholas B. Tooker ◽

Varun Srinivasan ◽

Guangyu Li ◽

Peter Schauer ◽

...

Keyword(s):

Phosphorus Removal ◽

Tca Cycle ◽

Full Scale ◽

Enhanced Biological Phosphorus Removal ◽

Biological Phosphorus Removal ◽

Solid Retention Time ◽

Side Stream ◽

Significant Difference ◽

Polyphosphate Accumulating Organisms ◽

Biological Phosphorus

In this study, a full-scale pilot testing was performed with side-by-side operation of a conventional enhanced biological phosphorus removal (EBPR) process and a side-stream EBPR (S2EBPR) process. A comparison of the performance, activities and population dynamics of key functionally relevant populations between the two configurations were carried out. The results demonstrated that, with the same influent wastewater characteristics, S2EBPR configuration showed more effective and stable orthophosphate (PO4-P) removal performance (up to 94% with average effluent concentration down to 0.1 mg P/L) than conventional EBPR, especially when the mixers in side-stream reactor were operated intermittently. Mass balance analysis illustrated that both denitrification and EBPR performance have been enhanced in S2EBPR configuration through diverting primary effluent to anoxic zone and producing additional carbon (~40%) via fermentation in side-stream reactor. Microbial characterization showed that there was no significant difference in the relative abundances of Ca. Accumulibacter (~5.9%) and Tetrasphaera (~16%) putative polyphosphate-accumulating organisms (PAOs) between the two configurations. However, lower relative abundance of known GAOs was observed in S2EBPR configuration (1.1%) than the conventional one (2.7%). A relatively higher PAO activity and increased degree of dependence on glycolysis pathway than TCA cycle was observed in S2EBPR configuration using P release and uptake batch test. Adequate anaerobic solid retention time (SRT) and conditions that generate continuous and slow feeding/production of volatile fatty acid (VFA) with higher composition percentage of propionate in the side-stream reactor of S2EBPR process likely provide a competitive advantage for PAOs over GAOs.

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