Real-time PCR quantification of nitrite reductase (nirS) genes in a nitrogen removing fluidized bed reactor

Molecular approaches were applied to identify and enumerate denitrifying bacteria subsisting in a fluidized bed reactor (FBR). The FBR was continuously operated as a unit for the removal of nitrogen from the effluents of domestic sewage treatment plant, with an additional supply of methanol as a carbon source. By denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S ribosomal RNA genes, Thauera group was found to be dominant among the denitrifying bacteria in the FBR sludge. Oligonucleotide probe THA155 for fluorescence in situ hybridization (FISH) was newly designed for specifically targeting the Thauera group. However, the THA155 signal obtained from the sludge was only 0.9-5.7% of the DAPI-stained total cells. The real-time polymerase chain reaction (PCR) targeting the sequences of nitrite reductase (NIR) gene, a key enzyme of denitrification processes, was performed to quantify the cells of denitrifying bacteria cells including the Thauera group in FBR sludge. An excellent correlation was obtained between the numbers of nirS genes and the activity of denitrifiers in the FBR sludge.