Complete Detection of the β-subclass of the Ammonia-Oxidizing Proteobacteria in a Sequence Batch Reactor Requires a Multi-Probe Approach

Abstract

During the operation of a sequencing batch reactor to study the nitrification process, poor correlation in data generated from the chemical analyses and molecular probes initiated an investigation into the potential mismatches between the probes and a 16S rDNA library. Analysis of 98 clones generated four (8%) positive clones that hybridized with the Nso190 and Nso1225 probes. These probes are used to identify the ammonia oxidizing β – proteobacteria thought to be responsible for the conversion of ammonia to nitrite in the sequencing batch reactor. Only one of these positive clones was observed to be positive for both probes. Sequencing of the positive clones showed mismatches that were found to be consolidated in the near center of a slightly destabilizing nature and the terminal ends of the target sites in 16S rDNA sequences. As the Nso190 and Nso1225 probes were found to target multiple genera within the β -proteobacteria division, a multi-probe approach is required to identify all potential ammonia oxidizers to better reduce the discrepancy in the activity seen in the SBR. On a technical note, the observation of the singular orientation insertion of 16S rDNA sequences into the selected vector by Topoisomerase I in transformed Escherichia coli cells has not been seen in 16S rDNA analysis using similar cloning systems used previously in similar studies.