Optimization and Application of Fluorescence in Situ Hybridization Assay for Detecting Polyphosphate - Accumulating Microorganisms

2011 ◽  
Vol 183-185 ◽  
pp. 1369-1373 ◽  
Author(s):  
Yan Hui Ge ◽  
Lin Zhao ◽  
Ruo Chun Zhang ◽  
Yun Jie Liu
Keyword(s):  
Phosphorus Removal ◽  
Orthogonal Design ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Hybridization Temperature ◽  
Hybridization Time ◽  
Formamide Concentration ◽  
Activated Sludge Processes ◽  
Biological Phosphorus

Laboratory-scale sequencing batch reactors (SBRs) were operated on activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Polyphosphate-accumulating microorganisms (PAOs) play an important role during the enhanced biological phosphorus removal (EBPR) process. Fluorescence in situ hybridization (FISH) was applied to assess the proportions of microorganisms in the sludge. The aim of this study was to optimize hybridization of PAOMIX and RHC439 probes by orthogonal design. Orthogonal optimization test of the four factors were conducted under the individual three levels. The optimal hybridizition conditions were as follow: hybridization temperature 46°C, hybridization time 2.5h, washing time 15min, formamide concentration 35%(PAOMIX probe); hybridization temperature 50°C, hybridization time 2.5h, washing time 20min, formamide concentration 20% (RHC439 probe).

Morphology, In-Situ characterization with rRNA targetted probes and respiratory quinone profiles of enhanced biological phosphorus removal sludge

1998 ◽  
Vol 38 (8-9) ◽  
pp. 69-76 ◽  
Author(s):  
I. M. Sudiana ◽  
T. Mino ◽  
H. Satoh ◽  
T. Matsuo
Keyword(s):  
Carbon Source ◽  
Activated Sludge ◽  
Microbial Communities ◽  
Phosphorus Removal ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
In Situ Characterization ◽  
Respiratory Quinone ◽  
Biological Phosphorus

The microbial communities in activated sludge acclimated with either acetate or glucose as the major carbon source under phosphorus limited or rich conditions were investigated morphologically, phylogenetically and chemotaxonomically. The sludge with a minimized polyphosphate content was dominated by tetrad shaped bacteria, which were suspected to be ‘glycogen accumulating bacteria (GAOs) or G bacteria’ The sludge containing high polyphosphate was dominated by cluster forming coccus bacteria. Quinone analyses suggested that all the sludge tested contained various ubiquinones and menaquinones, of which the ubiquinones Q-8 and Q-10 were dominant. Analyses with rRNA targeted probes showed that beta sub class of Proteobacteria was most predominant in all sludges tested. Morphological, phylogenetic and chemotaxonomic investigation all indicated that both high and low P sludges are microbiologically diverse.

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);

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.

Population dynamics in wastewater treatment plants with enhanced biological phosphorus removal operated with and without nitrogen removal

2002 ◽  
Vol 46 (1-2) ◽  
pp. 163-170 ◽  
Author(s):  
N. Lee ◽  
J. la Cour Jansen ◽  
H. Aspegren ◽  
M. Henze ◽  
P.H. Nielsen ◽  
...  
Keyword(s):  
Population Dynamics ◽  
Activated Sludge ◽  
Nitrogen Removal ◽  
Phosphorus Removal ◽  
The Other ◽  
Enhanced Biological Phosphorus Removal ◽  
Biological Phosphorus Removal ◽  
Biological Phosphorus ◽  
P Removal

The population dynamics of activated sludge in a pilot plant with two activated sludge systems, both designed for enhanced biological phosphorus removal (EBPR), but one of them with (BNP) and the other without (BP) nitrogen removal, was monitored during a period of 2.5 years. The influent water to the pilot plant was periodically manipulated by external addition of phosphorus (P), acetate and glucose, respectively. The population dynamics and the in situ physiology were monitored by quantitative fluorescence in situ hybridization (FISH) and microautoradiography. Significant P removal was observed in both systems throughout the whole period, with significant increases of the P removal when substrates were dosed. The activated sludge in both systems contained large amounts of dense clusters of gram-negative, methylene-blue staining coccoid rods during the whole period. A large part of the clusters belonged to the β Proteobacteria, whereas the rest of the clusters belonged either to the Actinobacteria or to the α Proteobacteria. The relative abundance of Rhodocyclus-related bacteria in the activated sludge varied significantly in both systems during the whole period (from 6 to 18% in BNP, and from 4 to 28% in BP). However, no statistically significant correlation of the Rhodocyclus-related nor any of the other investigated bacterial groups to the P content of the activated sludge (correlation for all groups investigated was always < 0.5) was observed. A significant 33Pi uptake was observed by the β Proteobacteria (part of them Rhodocyclus-related, the identity of the rest unknown) and the Actinobacteria. However, not all of the Rhodocyclus-related bacteria showed 33Pi uptake. The P removal in the investigated plants is thus believed to be mediated by a mixed population consisting of a part of the Rhodocyclus-related bacteria, the Actinobacteria and other, yet unidentified bacteria.

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

1998 ◽  
Vol 37 (4-5) ◽  
pp. 567-571 ◽  
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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