Detection of the Presence of γ-Subclass Proteobacteria in a Sequencing Batch Reactor and the Determination of Probe Dissociation Temperatures for Nsc128 and Nso190

Abstract

We tested the presence of γ and β subclass of the Proteobacteria in samples collected on different dates from a Sequencing Batch Reactor fed with ammonium as the primary energy source. The bacteria were identified by the 16S rRNA-targeted oligonucleotide DNA probes Nsc128 and Nso190 respectively. Nitrosococcus halophilus and N.oceani, which belong to the γ subclass of the Proteobacteria, were absent from the samples as no signal was detected using the Nsc128 probe. Therefore, Nitrosococcus of the γ subclass of the Proteobacteria did not contribute to the continuous production of nitrite in the bioreactor. Isolated RNA samples that hybridized with Nso190 were used to determine the probe dissociation o temperature. The experimental probe dissociation temperature ranged from 44.1 C to o o 51.9 C, which deviated from the predicted melting temperature (60 C). The lower experimental dissociation temperatures might be due to the incomplete matching of Nso190 and the 16 rRNA sequence of the targeted bacteria.