Microbiology of a nitrite-oxidizing bioreactor

Abstract

The microbiology of the biomass from a nitrite-oxidizing sequencing batch reactor (NOSBR) fed with an inorganic salts solution and nitrite as the sole energy source that had been operating for 6 months was investigated by microscopy, by culture-dependent methods, and by molecular biological methods, and the seed sludge that was used to inoculate the NOSBR was investigated by molecular biological methods. The NOSBR sludge comprised a complex and diverse microbial community containing gram-negative and gram-positive rods, cocci, and filaments. By culture-dependent methods (i.e., micromanipulation and sample dilution and spread plate inoculation), 16 heterotrophs (6 gram positive and 10 gram negative) were identified in the NOSBR sludge (RC), but no autotrophs were isolated. 16S ribosomal DNA clone libraries of the two microbial communities revealed that the seed sludge (GC) comprised a complex microbial community dominated by Proteobacteria (29% beta subclass; 18% gamma subclass) and high G + C gram-positive bacteria (10%). Three clones (4%) were closely related to the autotrophic nitrite-oxidizer Nitrospira moscoviensis. The NOSBR sludge was overwhelmingly dominated by bacteria closely related to N. moscoviensis (89%). Two clone sequences were similar to those of the genus Nitrobacter. Near-complete insert sequences of eight RC and one GC N. moscoviensis clone were determined and phylogenetically analyzed. This is the first report of the presence of bacteria from the Nitrospira phylum in wastewater treatment systems, and it is hypothesized that these bacteria are the unknown nitrite oxidizers in these processes.

FIG. 1

Diagrammatic representation of the banding profiles of the 13 OTUs in the RC clone library and their closest matches by BLAST comparisons with partial 16S rDNA sequencing of inserts.

FIG. 2

Evolutionary distance tree of the Nitrospira phylum and other known nitrite oxidizers in the domain Bacteria based on a comparative analysis of 1,030 nucleotides. Most bootstrap values greater than 92% from 100 resamplings for distance (numbers above branches) and parsimonious (numbers below branches) analyses are presented at the nodes. The outgroup, Bacteroides fragilis, is not shown in the tree. The bar represents 0.1 estimated change per nucleotide.