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

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 mu m, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 mu m 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.