Molecular approaches were applied to study the identification and enumeration of denitrifying bacteria in a fluidized bed reactor (FBR). The FBR was continuously operated as a unit for the removal of nitrogen from domestic sewage treatment plant effluents, with a supplementary supply of methanol as a carbon source. By denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S ribosomal DNA, it was revealed that the Thauera group was dominant among the denitrifying bacteria in the biofilms obtained from the FBR. The oligonucleotide probe THA155 for fluorescence in situ hybridization (FISH) was newly designed for specifically targeting the Thauera group. THA155 was able to detect Thauera cells from the biofilm sludge, but only 0.9-5.7 percent of DAPI-stained total cells was hybridized. The real-time polymerase chain reaction (PCR) targeting of the gene sequences of nitrite reductase gene, a key enzyme of denitrification processes, was performed to quantify denitrifying bacteria cells including the Thauera group. An excellent correlation was found between the number of nirS genes in the biofilm sludge and the activity of denitrifiers in the FBR. Therefore, the quantification of the nirS gene by real-time PCR can be an appropriate tool for monitoring denitrifying bacteria in nitrogen removal reactors.
