Microbiological community structure of the biofilm of a methanol-fed, marine denitrification system, and identification of the methanol-utilizing microorganisms

We demonstrated in a previous study that the biofilm of the methanol-fed fluidized marine denitrification reactor at the Montreal Biodome was composed of at least 15 bacterial phylotypes. Among those were 16S ribosomal RNA (rDNA) gene sequences affiliated to Hyphomicrobium spp., and Methylophaga spp.; the latter made up 70% of a clone library. By using fluorescent in situ hybridization (FISH), we investigated the structure of the biofilm during the colonization process in the denitrification reactor by targeting most of the bacterial families that the 16S rDNA gene library suggested would occur in the biofilm. Our results revealed that gamma-Proteobacteria (mostly Methylophaga spp.) accounted for up to 79% of the bacterial population, confirming the abundance of Methylophaga spp. within the biofilm. alpha-Proteobacteria represented 27-57% of the population, which included Hyphomicrobium spp. that appeared after 20 days of colonization and represented 7-8% of the population. We noticed a great abundance and diversity of eukaryotic cells, which made up 20% of the biomass at the beginning of the colonization but decreased to 3-5% in the mature biofilm. We then used FISH combined with microautoradiography (MAR-FISH) to identify the methylotrophs in the biofilm. The results showed that alpha-Proteobacteria used C-14 methanol in the presence of nitrate, suggesting their involvement in denitrification. Despite their abundance, Methylophaga spp. did not assimilate methanol under those conditions.