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Abstract
Nitrobacter species are responsible for the oxidation of nitrite to nitrate in the environment. Studies indicated that there is an inconsistent correlation between the amount of nitrifying activity in the bioreactor and the amount of nitrifying bacteria detected by the NIT3 and Nb1000 16S rRNA probes. This study investigates the explanation for the inconsistency, focusing on whether there are any sequence mismatches between the probes and the Nitrobacter 16S ribosomal RNA present in the environmental sample that preclude the detection of Nitrobacter. The samples used in this study were collected from a lab scale completely stirred tank reactor system seeded from the Kent wastewater treatment plant. The techniques employed to analyze samples included RNA isolation, radioactive labelling of 16S rRNA oligonucleotide probes, slot blot hybridization, and the determination of the dissociation temperature for both probes. Nucleic acid hybridization results suggested that Nitrobacter was present at a very low concentration in the stirred tank reactor, which was below the limit of detection using the radioactively labelled NIT3 and Nb1000 probes. Using RNA isolated from pure Nitrobacter strain, the experimentally measured dissociation temperature for the Nb1000 probe was 43ºC and the minimum dissociation temperature for the NIT3 probe was 54ºC. It was concluded that the amount of RNA immobilized onto the membrane should be increased by approximately 400-fold in order to obtain a detectable signal for Nitrobacter.