Comparisons and Analyses of Probe Temperature Elution Profiles of Nitrospira

It was recently discovered in sequencing batch reactors (SBR) that Nitrospira, which is the dominant nitrifying bacteria in bioreactors, cannot be detected in cultures where the activity of nitrite oxidation is high. In order to investigate this problem, RNA was isolated from the SBR samples collected on different dates. The isolated RNA was immobilized onto a nylon membrane by slot blotting, and then radioactively labelled with Ntspa 454 and Ntspa 685 probes. The probe elution profiles of each of the samples were characterized by washing the individual bands at a series of increasing wash temperatures. It was found that the dissociation temperature ( T ) for the SBR samples were lower than the pure culture d Nitrospira moscoviensis RNA. This difference in T explains why Nitrospira could not be d detected despite the high nitrifying activities in the SBR. The probes were washed at the theoretical T, a higher temperature than the actual T of the bioreactor samples, which d d causes most of the probes to elute off, leaving only trace amounts of probe bound on the membrane. The minimal amounts of bound probe left on the membrane create the misconception that Nitrospira is not present. Comparing the autoradiogram and the elution profiles of the two probes, it is suspected that the dominant organism present in the bioreactor is Nitrospira marina. However, it is also possible that the probes are binding to novel, modified strains of Nitrospira. Further research must be performed to determine the dominant bacteria present in the bioreactors.