Estimation of the Diversity of Denitrifying Bacteria in a Membrane Bioreactor by PCR Amplification with nir Gene Probes

Abstract

Reduction of nitrite by nitrite reductase, encoded by either nirS or nirK genes, is the key step in the denitrification pathway. In this study, nirS and nirK are used as the target genes to detect the nirS- and nirK-containing denitrification bacteria of the activated sludge and membrane biofilm of an intermittent aerated sequencing batch membrane bioreactor (SBMBR) using both the cultured and noncultured methods. Pure cultured method results showed that 44.4% of the isolates contained six different nirS genetic polymorphisms. These nirS-contained isolates were identified as six strains, in which three strains, that is, Paracossus, Thauera, and Acidovorax, are considered as denitrifying bacteria. Only 9.4% of the isolates contained five different nirK genetic polymorphisms, which were further classified into five strains, including the denitrifying genera Mesorphizobium, Paracossus, and Thauera. This implies that 17.5% of the denitrifying bacteria contained the nirK gene. With the cloning method, six and two genetic polymorphisms in the activated sludge were identified as nirS and nirK genes. For the membrane biofilm, on the other hand, nine and four genetic polymorphisms were found to be nirS and nirK genes, respectively. Irregardless of whether the pure cultured method or the gene cloning method was used, the diversity of the nirS genetic polymorphisms was higher than the nirK genetic polymorphisms. Finally, from the phylogenetic analysis, it can be observed that although the genes were obtained from different samples (activated sludge or membrane biofilm) and using different isolation methods (cultured method or gene cloning), their phylogenetic relationship was very similar.