Identification of methanotrophic bacteria community in the Jastrzebie-Moszczenica coal mine by fluorescence in situ hybridization and PCR techniques

The aim of our study was to identify the methanotrophic group of bacteria inhabiting coal mine rocks at the Jastrzębie-Moszczenica (Jas-Mos) coal mine, by application of molecular techniques: polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH). In the first stage of the experiment, methanotrophic activity (AM) of the rock material at different temperatures (20 and 30ºC) and methane (CH₄) concentrations (1, 5, 10, 20%) was determined. AM noted both in 20 and 30ºC reached a similar level equaling c.a. 0.2 µM CH₄ g⁻¹·day⁻¹. The PCR reactions were performed using primers specific for pmoA gene (encoding a key subunit of particulate methane monooxygenase) – A189f and mb661, whereas FISH was realized with Mg705, Mg84 (Type I of methanotrophs), and Ma450 (Type II of methanotrophs) probes. Sequence analysis of pmoA gene demonstrated that microorganisms, being present in the investigated rocks material were similar with 95-100% identity to the following methanotrophs genera: Methylosinus, Methylobacter, Methylocystis. Concentrations of CH₄ between 1-10% did not affect on bacterial variability, but in combination with 20% of CH₄, significant dominance of methanotrophic bacteria Type I was stated.