Coupling methane oxidation and N2 fixation under methanogenic conditions in contrasting environments

Microbial methane oxidation under widespread suboxic environment is crucial for understanding methane emission. However, the role of aerobic methanotrophs in mediating methane oxidation and nitrogen fixation is less understood in oxygen-limiting environments. In this study, we identified diazotrophic methanotrophs under oxygen-limited conditions (initial O₂ of 6–8 μM) in two contrasting habitats (paddy soil and marine sediment) using DNA-based stable isotope probing combined with amplicon sequencing. Consistently, we documented significant ¹³CH₄ oxidation and ¹⁵N₂ fixation after 740 days of suboxic isotope labeling. Sequencing analysis revealed the predominance of Methylobacter–affiliated aerobic methanotrophs in the ¹³C-labeled DNA fractions. These Methylobacter-like OTUs accounted for 97.86 % in paddy soil and 99.49 % in marine sediment of the total pmoA gene sequences; while relative abundances for the nifH gene sequences were 91.59 % in paddy soil and 99.49 % in marine sediment. Taken together, our analyses revealed that Methylobacter is responsible for methane oxidation and nitrogen fixation under oxygen limitation in both habitats, demonstrating convergent emergence of this aerobic methanotroph under oxygen deficiency.