Phylogenetically and physiologically diverse methanogenic archaea inhabit the Indian hot spring environments

Mesophilic and thermophilic methanogens belonging to the hydrogenotrophic, methylotrophic, and acetotrophic groups were isolated from Indian hot spring environments using BY and BCYT growth media. Following initial Hinf I-based PCR-RFLP screening, 70 methanogens were sequenced to ascertain their identity. These methanogens were phylogenetically and physiologically diverse and represented different taxa distributed across three physiological groups, i.e., hydrogenotrophs (53), methylotrophs (14) and acetotrophs (3). Overall, methanogens representing three families, five genera, and ten species, including two putative novel species, were recognized. The highest number and diversity of methanogens was observed at 40 ℃, dominated by Methanobacterium (10; 3 species), Methanosarcina (9; 3 species), Methanothermobacter (7; 2 species), Methanomethylovorans (5; 1 species) and Methanoculleus (3; 1 species). Both putative novel methanogen species were isolated at 40 ℃ and belonged to the genera Methanosarcina and Methanobacterium. At 55 ℃, limited diversity was observed, and resulted in the isolation of only two genera of methanogens, i.e., Methanothermobacter (28; 2 species) and Methanosarcina (4; 1 species). At 70 ℃, only members of the genus Methanothermobacter (5; 2 species) were isolated, whereas no methanogen could be cultured at 85 ℃. Ours is the first study that documents the extensive range of cultivable methanogenic archaea inhabiting hot springs across various geothermal provinces of India.