Methanotrophic bacteria in oilsands tailings ponds of northern Alberta. uncultured bacterium

We have investigated methanotrophic bacteria in alkaline surface water (pH 7.5-8.8) of oilsands tailings ponds in Fort McMurray, Canada. These large lakes (up to 10 km2) contain silt, clay, processing chemicals, and residual hydrocarbons, which were not recovered in oilsands mining. They are strongly methanogenic, but have an aerobic surface layer. Aerobic methane oxidation was measured in the surface water at rates up to 200 nmol CH4 ml-1 water d-1. Microbial diversity was investigated via pyrotag sequencing of amplified 16S rRNA genes as well as by analysis of methanotroph-specific pmoA genes using both pyrosequencing and microarray analysis. The predominantly detected methanotroph in all tailings ponds at all sampling times was an uncultured species related to the gammaproteobacterium Methylocaldum, although a few other methanotrophs were also detected, including Methylomonas. Active species were identified via 13CH4 stable isotope probing (SIP) of DNA, combined with pyrotag sequencing and complete shotgun metagenomic sequencing of heavy 13C-DNA. The SIP results demonstrated that the Methylocaldum and Methylomonas strains actively consumed methane in fresh tailings. Metagenomic analysis of DNA from the heavy SIP fraction verified the PCR-based results and identified additional pmoA genes not detected via PCR. The metagenome indicated that the overall methylotrophic community possessed known pathways for formaldehyde oxidation, carbon fixation, and detoxification of nitrogenous compounds, but appeared to possess only particulate methane monooxygenase, not soluble methane monooxygenase.