Historically, Nitrosomonas europaea has generally been believed to be the bacterium responsible for ammonia oxidation, as it was commonly isolated from nitrifying systems by traditional culturing techniques ( 3). AOB are responsible for the first step in nitrification, the oxidation of ammonia to nitrite, and are generally members of the β subdivision of the class Proteobacteria except for the marine genus Nitrosococcus, which belongs to the γ subdivision ( 37). Recent studies of many environments have demonstrated a large amount of diversity among ammonia-oxidizing bacteria (AOB) ( 4, 14, 21, 31, 39). Nitrosomonas marina-like AOB were 2% or less of the cells detected by fluorescence in situ hybridization analysis in aquaria in which nitrification was well established. Furthermore, if the Nitrosomonas marina-like AOB strain was present in the original enrichment, even one with other AOB, only the Nitrosomonas marina-like AOB strain was present in aquaria after nitrification was established.
Enrichments containing the Nitrosomonas marina-like AOB strain were most efficient at accelerating ammonia oxidation in newly established aquaria. Enrichments of the AOB strains were added to newly established aquaria to determine their ability to accelerate the establishment of ammonia oxidation. Oligonucleotide probes and PCR primer sets specific for the four AOB strains were developed and used to confirm the presence of the AOB strains in the enrichments. Ammonia concentration in the reactors determined which AOB strain dominated in an enrichment. Surveys of cloned rRNA genes from the enrichments revealed four major strains of AOB which were phylogenetically related to the Nitrosomonas marina cluster, the Nitrosospira cluster, or the Nitrosomonas europaea-Nitrosococcus mobilis cluster of the β subdivision of the class Proteobacteria. Reactors were seeded with biomass from freshwater nitrifying systems and enriched for AOB under various conditions of ammonia concentration. Culture enrichments and culture-independent molecular methods were employed to identify and confirm the presence of novel ammonia-oxidizing bacteria (AOB) in nitrifying freshwater aquaria.