Analysis of the Microbial Community in an Activated Sludge Enriched with an Inorganic Nitrite Medium

Abstract

To enrich nitrite-oxidizing bacteria, an activated sludge sample was transferred serially into an inorganic nitrite medium.Following the transfer, the culture maintained nitrite-oxidizing activity for over three months.Nitrite-oxidizing bacteria slightly decreased, but maintained their number at 10⁴MPN/ml, and 10⁶-10⁷cfu/ml of heterotrophic bacteria were also detected.Random cloning and analysis of amplified 16S rDNA using a universal primer set for bacteria showed that a culturable Pseudomonas putida-related strain was dominant in the culture, though the bacterium did not oxidize nitrite.The most dominant bacterial group estimated from the proportion of clones that showed identical pattern of restriction fragment length polymorphism belonged to the γ-subdivision of Proteobacteria.This was partly consistent with the results from whole-cell hybridization using group-specific fluorescent probes.Further limiting dilutions of the enriched culture produced a nitrite-oxidizing system with low numbers of heterotrophs.Molecular analysis suggested that the members were different from those in the enriched culture, and several kinds of Proteobacteria belonging to the β-, γ-, and α-subdivision, as well as bacteria in the high G+C Gram-positive phylum, existed.But in 16S rDNA sequence, none showed close similarity to any known autotrophic nitrite oxidizers.These results indicated that the population in serially transferred culture and limiting dilution culture is rather diverse, with some heterotrophic bacteria, and suggested the occurrence of an unidentified species of nitrite-oxidizing bacteria.