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
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.