Diversity and quantification analysis of functional genes in a lab scale denitrifying quinoline-degrading bioreactor

Abstract:

Abstract:[Objective] In order to study the diversity and distribution of Benzoyl coenzyme A reductase (bcrA) and oxygenase components of 1H-2-oxoquinoline 8-monooxygenase(oxoO) gene in a lab scale denitrifying bioreactors for treating quinoline-containing wastewater. [Methods] Genomic microbial DNA was extracted from the biofilm samples of bioreactor. Based on the known oxoO genes sequences in GenBank, we designed primers for oxoO gene amplification. Using our designed oxoO gene primers and a pair of degenerate primers of bcrA gene obtained from literature, we amplified the DNA samples. And the amplicons were used for constructing clone libraries of oxoO and bcrA gene. We also performed quantification analysis of these two genes in bioreactor by using Real-time qPCR method. [Results] The quantification analysis showed gradual increase of the bcrA gene abundance and reduction of the oxoO gene abundance along with time. The clone library analysis of these two genes indicated that most clones in bcrA gene clone library having more than 97% sequence similarities to the known bcrA gene of Thauera bacteria and others only having 74%~86% sequence similarities to bcrA gene sequences. Whereas, only a few clones in the oxoO gene clone library have 99% sequence similarities to that gene of Pseudomonas putida. But the sequences of most clones only distantly related with known oxoO genes. [Conclusions] This study showed a high bcrA and oxoO gene diversity, with some new gene sequences, in the lab scale denitrifying bioreactors. The abundance of bcrA and oxoO gene changed remarkably during the running of bioreactor, and closely related with the performance of bioreactor. Therefore, bcrA and oxoO genes have potential to be used as molecule markers to monitor the process of treating quinoline containing wastewater.

Key words:Keywords: quinoline; bcrA gene; oxoO gene; quantification; diversity analysis