Cultivation of Diverse Type I and Type II Methanotrophs from Tropical Wetlands in India, Including Rare Taxa (Methylocucumis and Methylolobus)

Wetlands are the most important natural sources of methane. Studies on the distribution and diversity of methanotrophs, especially in tropical wetlands, are limited. The studies on wetland methanotrophs help bridge the gap in the literature for understanding the community structure of methanotrophs in tropical wetlands. Our present study documents the methanotroph diversity from various wetland habitats across Western India. Samples from various sites, such as freshwater ponds, lake sediments, mangroves, etc., located in Western India, were collected and enriched for methanotroph isolation. An established protocol for the isolation of methanotrophs from Indian rice fields, involving serial dilution and long-term incubations, was slightly modified and used. Obtaining entirely pure cultures of methanotrophs is a labor-intensive and technically challenging process. Hence, for primary level characterization, ‘methanotroph monocultures’, which have a single methanotroph culture with minimal contamination, were established. Twenty monocultures and eight pure cultures of methanotrophs were obtained in this study. The pmoA gene has been used for the phylogenetic characterization of methanotrophs for the last 25 years. Monocultures were from seven genera: the Methylomonas, Methylocystis, Methylosinus, Methylocaldum, Methylocucumis, Methylomagnum, and Methylolobus genera. Eight pure cultures were obtained, which were strains of Methylomonas koyamae, Methylosinus sporium, and Methylolobus aquaticus. A maximum number of cultures belonged to the Type I genus Methylomonas and to the Type II genus Methylocystis. Thus, the cultivation-based community studies of methanotrophs from wetland habitats in India expanded the current knowledge about the methanotroph diversity in such regions. Additionally, the cultivation approach helped us obtain new methanotrophs from this previously unexplored habitat, which can be used for further biotechnological and environmental applications. The isolated monocultures can either be used as MMCs (mixed methanotroph consortia) for environmental applications or further purified and used as pure cultures.