Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor

The microbial diversity of a deteriorated biological phosphorus removal rea ctor was investigated by methods not requiring direct cultivation. The reac tor was fed with media containing acetate and high levels of phosphate (P/C weight ratio, 8:100) but failed to completely remove phosphate in the effl uent and showed very limited biological phosphorus removal activity. Denatu ring gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA was used to investigate the bacterial diversity. Up to 11 DGGE bands repre senting at least 11 different sequence types were observed; DNA from the 6 most dominant of these bands was further isolated and sequenced. Comparativ e phylogenetic analysis of the partial 16S rRNA sequences suggested that on e sequence type was affiliated with the alpha subclass of the Proteobacteri a, one was associated with the Legionella group of the gamma subclass of th e Proteobacteria, and the remaining four formed a novel group of the gamma subclass of the Proteobacteria with no close relationship to any previously described species. The novel group represented approximately 75% of the PC R-amplified DNA, based on the DGGE band intensities. Two oligonucleotide rR NA probes for this novel group were designed and used in a whole-cell hybri dization analysis to investigate the abundance of this novel group in situ, The bacteria were coccoid and 3 to 4 mu m in diameter and represented appro ximately 35% of the total population, suggesting a relatively close agreeme nt with the results obtained by the PCR-based DGGE method. Further, based o n electron microscopy and standard staining microscopic analysis, this nove l group was able to accumulate granule inclusions, possibly consisting of p olyhydroxyalkanoate, inside the cells.