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

2005 ◽  
Vol 71 (7) ◽  
pp. 4076-4085 ◽  
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.

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

2007 ◽  
Vol 73 (18) ◽  
pp. 5865-5874 ◽  
Author(s):  
Shaomei He ◽  
Daniel L. Gall ◽  
Katherine D. McMahon
Keyword(s):  
Population Structure ◽  
16S Rrna ◽  
Genetic Marker ◽  
Phosphorus Removal ◽  
Full Scale ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Biological Phosphorus

ABSTRACT 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.

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

2002 ◽  
Vol 46 (1-2) ◽  
pp. 123-128 ◽  
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.

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

2003 ◽  
Vol 47 (11) ◽  
pp. 37-43 ◽  
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);

Construction and analysis of a metagenomic library from an enhanced biological phosphorus removal biomass

2006 ◽  
Vol 54 (1) ◽  
pp. 277-284
Author(s):  
C. Yeates ◽  
L.L. Blackall
Keyword(s):  
Phosphorus Removal ◽  
In Situ Hybridisation ◽  
Metagenomic Library ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Metagenomic Data ◽  
Fluorescence In Situ Hybridisation ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Biological Phosphorus

The enhanced biological phosphorus removal (EBPR) process is regularly used for the treatment of wastewater, but suffers from erratic performance. Successful EBPR relies on the growth of bacteria called polyphosphate-accumulating organisms (PAOs), which store phosphorus intracellularly as polyphosphate, thus removing it from wastewater. Metabolic models have been proposed which describe the measured chemical transformations, however genetic evidence is lacking to confirm these hypotheses. The aim of this research was to generate a metagenomic library from biomass enriched in PAOs as determined by phenotypic data and fluorescence in situ hybridisation (FISH) using probes specific for the only described PAO to date, “Candidatus Accumulibacter phosphatis”. DNA extraction methods were optimised and two fosmid libraries were constructed which contained 93 million base pairs of metagenomic data. Initial screening of the library for 16S rRNA genes revealed fosmids originating from a range of non-pure-cultured wastewater bacteria. The metagenomic libraries constructed will provide the ability to link phylogenetic and metabolic data for bacteria involved in nutrient removal from wastewater.

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

Microbiology ◽  
2011 ◽  
Vol 157 (8) ◽  
pp. 2287-2296 ◽  
Author(s):  
Jeong Myeong Kim ◽  
Hyo Jung Lee ◽  
Dae Sung Lee ◽  
Kangseok Lee ◽  
Che Ok Jeon
Keyword(s):  
Phosphorus Removal ◽  
Batch Reactor ◽  
Full Scale ◽  
Rrna Gene ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Phenotypic Properties ◽  
Polyphosphate Accumulating Organisms ◽  
Biological Phosphorus

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.

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

2018 ◽  
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.

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

2004 ◽  
Vol 70 (9) ◽  
pp. 5383-5390 ◽  
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.

scholarly journals Analysis of the Fine-Scale Population Structure of “Candidatus Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge, Using Fluorescence In Situ Hybridization and Flow Cytometric Sorting

2010 ◽  
Vol 76 (12) ◽  
pp. 3825-3835 ◽  
Author(s):  
Jeong Myeong Kim ◽  
Hyo Jung Lee ◽  
Sun Young Kim ◽  
Jae Jun Song ◽  
Woojun Park ◽  
...  
Keyword(s):  
16S Rrna ◽  
Phosphorus Removal ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Fine Scale ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Accumulibacter Phosphatis ◽  
Biological Phosphorus

ABSTRACT To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “Ca. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for “Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that “Ca. Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.

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

2018 ◽  
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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