A novel methanogenic archaeon, strain MC-15(T), was isolated from a floating biofilm on a sulphurous subsurface lake in Movile Cave (Mangalia, Romania). Cells were non-motile sarcina-like cocci with a diameter of 2-4 µm, occurring in aggregates. The strain was able to grow autotrophically on H2/CO2. Additionally, acetate, methanol, monomethylamine, dimethylamine and trimethylamine were utilized, but not formate or dimethyl sulfide. Trypticase peptone and yeast extract were not required for growth. Optimal growth was observed at 33 °C, pH 6.5 and a salt concentration of 0.05 M NaCl. The predominant membrane lipids of MC-15(T) were archaeol and hydroxyarchaeol phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol as well as hydroxyarchaeol phosphatidylserine and archaeol glycosaminyl phosphatidylinositol. The closely related species, Methanosarcina vacuolata and Methanosarcina horonobensis, had a similar composition of major membrane lipids to strain MC-15(T). The 16S rRNA gene sequence of strain MC-15(T) was similar to those of Methanosarcina vacuolata DSM 1232(T) (sequence similarity 99.3%), Methanosarcina horonobensis HB-1(T) (98.8%), Methanosarcina barkeri DSM 800(T) (98.7%) and Methanosarcina siciliae T4/M(T) (98.4%). DNA-DNA hybridization revealed 43.3% relatedness between strain MC-15(T) and Methanosarcina vacuolata DSM 1232(T). The G+C content of the genomic DNA was 39.0 mol%. Based on physiological, phenotypic and genotypic differences, strain MC-15(T) represents a novel species of the genus Methanosarcina, for which the name Methanosarcina spelaei sp. nov. is proposed. The type strain is MC-15(T) ( = DSM 26047(T) = JCM 18469(T)).
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