Polyphasic approaches to the identification of predominant polyphosphate-accumulating organisms in a laboratory-scale anaerobic/aerobic activated sludge system

By combination of denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA (PCR-DGGE), quinone profiling, and 16S rRNA-targeted fluorescence in situ hybridization (FISH), a polyphosphate-accumulating organism (PAO) responsible for phosphate (P)-removal was identified in activated sludge with high P-removal ability from a laboratory-scale anaerobic/aerobic continuous flow reactor. The DNA fragment from the most dense band on the DGGE gel was closely related to that of 'Candidatus Accumulibacter phosphatis' (beta-Proteobacteria). Quinone profiling also suggested the predominance of beta-Proteobacteria. FISH with a specific oligonucleotide probe designed for the sequence showed that the targeted bacterium was dominant in the activated sludge, and the accumulation and consumption of polyphosphate were observed by dual staining with 4',6-diamidino-2-phenylindole. The bacterium was concluded to be the responsible PAO in the reactor. However, when the P-removal ability per cell slightly decreased, the dominance of the PAO greatly diminished in the activated sludge. Such sludge might be dominated by other types of PAOs.