Diverse methanotrophs from alpha and gammaproteobacteria dwell in a stone quarry: a study from Western India wetland

Freshwater wetlands are a rich habitat for aerobic or micro-aerophilic methanotrophs. In the present study, we sampled a wetland ecosystem from Western India, a stone quarry with water situated amidst a hill. The wetland is surrounded by the macrophyte Typha sp. (cat-tail). To study the methanotroph diversity in this ecosystem, we collected mud samples from the mini-lakes and rhizospheric soil of Typha by random sampling. A serial dilution enrichment in liquid followed by isolation on agarose plates in a methane-air atmosphere was used. The high abundance of methanotrophs was indicated by the fact that growth was obtained till dilutions: 10-9 or 10-10. In Typha root-associated soil, Type I methanotrophs: Methylomonas koyamae and uncultured Methylococcus were isolated from the lower dilutions: 10-3-10-6. Type II methanotrophs Methylocystis sp. and Methylosinus spp were isolated from the higher dilutions. The Methylococcus strain isolated was about 97.4% (16S rRNA gene similarity) related to Methylococcus capsulatus and Methylococcus geothermalis and was predicted to represent a putative novel species. Three more methanotrophs, non-axenic, related to Methylomagnum-Methylocaldum spp, Methylocucumis oryzae, and Methylocystis spp, were cultured. Their affiliation was predicted based on the pmoA gene sequence. The mud samples showed relatively less diversity and mainly yielded two Methylomonas strains related to M. koyamae.