Methylotrophic bacteria (non-methanotrophic methanol oxidizers) consuming reduced carbon compounds containing no carbon–carbon bonds as their sole carbon and energy source have been found in a great variety of environments. Here, we report a unique moderately thermophilic methanol-oxidising bacterium (strain LS7-MT) that grows optimally at 55 °C (with a growth range spanning 30 to 60 °C). The pure isolate was recovered from a methane-utilizing mixed culture enrichment from an alkaline thermal spring in the Ethiopia Rift Valley, and utilized methanol, methylamine, glucose and a variety of multi-carbon compounds. Phylogenetic analysis of the 16S rRNA gene sequences demonstrated that strain LS7-MT represented a new facultatively methylotrophic bacterium within the order Hyphomicrobiales of the class Alphaproteobacteria. This new strain showed 94 to 96% 16S rRNA gene identity to the two methylotroph genera, Methyloceanibacter and Methyloligella. Analysis of the draft genome of strain LS7-MT revealed genes for methanol dehydrogenase, essential for methanol oxidation. Functional and comparative genomics of this new isolate revealed genomic and physiological divergence from extant methylotrophs. Strain LS7-MT contained a complete mxaF gene cluster and xoxF1 encoding the lanthanide-dependent methanol dehydrogenase (XoxF). This is the first report of methanol oxidation at 55 °C by a moderately thermophilic bacterium within the class Alphaproteobacteria. These findings expand our knowledge of methylotrophy by the phylum Proteobacteria in thermal ecosystems and their contribution to global carbon and nitrogen cycles.

Aerobic moderately thermophilic and thermophilic methane-oxidizing bacteria make a substantial contribution in the control of global warming through biological reduction of methane emissions and have a unique capability of utilizing methane as their sole carbon and energy source. Here, we report a novel moderately thermophilic Methylococcus-like Type Ib methanotroph recovered from an alkaline thermal spring (55.4 °C and pH 8.82) in the Ethiopian Rift Valley. The isolate, designated LS7-MC, most probably represents a novel species of a new genus in the family Methylococcaceae of the class Gammaproteobacteria. The 16S rRNA gene phylogeny indicated that strain LS7-MC is distantly related to the closest described relative, Methylococcus capsulatus (92.7% sequence identity). Growth was observed at temperatures of 30–60 °C (optimal, 51–55 °C), and the cells possessed Type I intracellular membrane (ICM). The comparison of the pmoA gene sequences showed that the strain was most closely related to M. capsulatus (87.8%). Soluble methane monooxygenase (sMMO) was not detected, signifying the biological oxidation process from methane to methanol by the particulate methane monooxygenase (pMMO). The other functional genes mxaF, cbbL and nifH were detected by PCR. To our knowledge, the new strain is the first isolated moderately thermophilic methanotroph from an alkaline thermal spring of the family Methylococcaceae. Furthermore, LS7-MC represents a previously unrecognized biological methane sink in thermal habitats, expanding our knowledge of its ecological role in methane cycling and aerobic methanotrophy.

Aerobic thermotolerant methane oxidizers utilize methane as a sole carbon and energy source, and predominantly they are associated with the phylum Proteobacteria. Here we present two further new strains (HGS-45: coccusshaped and HGF-47: rod-shaped and vibrioid) of thermotolerant obligate proteobacterial methanotrophic bacteria, which were isolated from an abandoned tropical natural gas field wet soil sample in the northeast of Bangladesh. Strains are Gram-negative, nonmotile, and capable of growth on methane and methanol as their energy sources. Isolates are thermotolerant and could grow up to 52oC, optimally at 42oC, but show no growth at 55 or 15oC. Based on 16S rRNA gene sequence analyses and phylogenetically, HGS-45 is most closely related to the obligate Type Ib methanotroph Methylococcus capsulatus of the family Methylococcaceae, whereas HGF-47 is affiliated to Type IIa methanotroph Methylocystis sp. of the family Methylocystaceae and possesses Type II intracytoplasmic membrane (ICM) systems. Genes of particulate methane monooxygenase (pMMO) and the methanol dehydrogenase (MDH) were detected by PCR. Southern-blot analyses of genomic DNA from both strains were positive, implying the aerobic biological oxidation process from methane to methanol by the pMMO. Each strain presumably represents a novel species. Furthermore, both strains will increase our knowledge of thermotolerant methanotrophic proteobacterial diversity, cohabitation, and their participation to global carbon cycles as well as signifying biological methane sinks in the terrestrial natural gas field ecosystems. Bangladesh J Microbiol, Volume 38, Number 2, December 2021, pp 63-71