A study was conducted to clarify phylogenetic affiliations of polyhydroxyalkanoate (PHA)-accumulating microorganisms in full-scale activated sludge processes. Activated sludge samples obtained from three full-scale activated sludge processes were aerobically incubated with excess acetate to increase their PHA content. The buoyant density separation method was applied to selectively collect PHA-accumulating cells, which were then analysed by the group-level FISH and the PCR-DGGE-sequencing methods, and possible PHA-accumulating microbial groups were screened. A set of oligonucleotide probes targeting the microbial groups suspected to accumulate PHA was introduced, and seven oligonucleotide probes were newly designed for this purpose. PHA accumulation of probe-positive cells was confirmed by the post-FISH PHA staining method, wherein PHA staining with Nile Blue A (NBA) was applied after FISH. As a result, the following seven bacterial groups were found to have PHA: Dechloromonas, Accumulibacter, Thauera, Zoogloea, Comamonas, Competibacter and a novel cluster in Beta-proteobacteria. Based on the results of the post-FISH PHA staining method, these seven bacterial groups were estimated to account for around four-tenths to two-thirds of total PHA-accumulating microorganisms.

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