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Title 

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

Authors 

J M KimH J LeeS Y KimJae Jun SongW ParkC O Jeon

Publisher 

American Society for Microbiology

Issue Date 

2010

Citation 

Applied and Environmental Microbiology, vol. 76, no. 12, pp. 3825-3835

Keywords 

16S rRNA geneAccumulibacterActivated sludgeCombined techniquesEnhanced biological phosphorus removalFish analysisFlow-cytometerFluorescence in situ hybridizationGenBank databaseLaboratory scaleMicroautoradiographyMicrobial characterizationMorphotypesPCR primersPhylogenetic analysisPhylogenetic treesPolyphosphate-accumulating organismsPolyphosphatesPopulation structuresSequencing batch reactorsSodium acetateSole carbon source

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 (ppkI) gene homologs were amplified by using a ppkl-specific PCR primer set for "Ca. Accumulibacter." The phylogenetic tree based on sequences of the ppkl gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppkl 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.

ISSN 

0099-2240

Link 

http://dx.doi.org/10.1128/AEM.00260-10

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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