Flow cytometric sorting and RFLP analysis of phosphate accumulating bacteria in an enhanced biological phosphorus removal system

Phosphate accumulating organisms (PAOs) stained with 4′,6-diamidino-2-phenylindol dihydrochloride (DAPI) at polyphosphate probing concentration were sorted from enhanced biological phosphorus removal (EBPR) sludge by flow cytometric sorting. All the genome DNA was extracted from the sorted bacteria and the 16S rDNA genes were cloned. Cloned 16S rDNA was PCR-amplified and analyzed by restriction fragment length polymorphism (RFLP) analysis. Eighty eight clones were analyzed and the RFLP patterns which appeared more than twice were classified into seven groups. The most dominant group (Group 1) contained four clones and accounted for 4.5% of the total clones. Four groups (from Group 2 to Group 5) contained three clones. Group 6 and 7 consisted of two clones. Sixty-eight clones gave unique RFLP patterns. By sequencing 16S rDNA in seven groups, Group 1, 2 and 5 were Rhodocyclus relatives (11%, 10/88). Rhodocyclus relatives were suggested to be one of the bacteria responsible for EBPR in this sludge. Groups 6 and 7 were related to b- or g-Proteobacteria. Group 4 belonged to e-Proteobacteria. Group 3 was related to green nonsulfur bacteria. Considering the complex RFLP pattern and the existence of the groups not related to Rhodocyclus by sequence analysis, in this EBPR system, together with Rhodocyclus relatives, some other bacteria might also play a role as PAOs.