Functional diversity in the denitrifying biofilm of the methanol-fed marine denitrification system at the Montreal Biodome.

Nitrate is a serious problem in closed-circuit public aquariums because its accumulation rapidly becomes toxic to many lifeforms. A moving bed biofilm denitrification reactor was installed at the Montreal Biodome to treat its 3,250-m(3) seawater system. Naturally occurring microorganisms from the seawater affluent colonized the reactor carriers to form a denitrifying biofilm. Here, we investigated the functional diversity of this biofilm by retrieving gene sequences related to narG, napA, nirK, nirS, cnorB, and nosZ. A total of 25 sequences related to these genes were retrieved from the biofilm. Among them, the corresponding napA1, nirK1, cnorB9, and nosZ3 sequences were identical to the corresponding genes found in Hyphomicrobium sp. NL23 while the narG1 and narG2 sequences were identical to the two corresponding narG genes found in Methylophaga sp. JAM1. These two bacterial strains were previously isolated from the denitrifying biofilm. To assess the abundance of denitrifiers and nitrate respirers in the biofilm, the gene copy number of all the narG, napA, nirS, and nirK sequences found in biofilm was determined by quantitative PCR. napA1, nirK1, narG1, and narG2, which were all associated with either Methylophaga sp. JAM1 or Hyphomicrobium sp. NL23, were the most abundant genes. The other genes were 10 to 10,000 times less abundant. nirK, cnorB, and nosZ but not napA transcripts from Hyphomicrobium sp. NL23 were detected in the biofilm, and only the narG1 transcripts from Methylophaga sp. JAM1 were detected in the biofilm. Among the 19 other genes, the transcripts of only two genes were detected in the biofilm. Our results show the predominance of Methylophaga sp. JAM1 and Hyphomicrobium sp. NL23 among the denitrifiers detected in the biofilm. The results suggest that Hyphomicrobium sp. NL23 could use the nitrite present in the biofilm generated by nitrate respirers such as Methylophaga sp. JAM1.