The clumped isotope signatures of multiple methanogenesis metabolisms

Methane is a potent greenhouse gas, an important energy source, and a potential biosignature on extraterrestrial planetary bodies. The relative abundances of doubly substituted (“clumped”) methane isotopologues ( ¹³ CH ₃ D and ¹² CH ₂ D ₂ ) offer important information on the sources and sinks of methane. However, the clumped isotope signatures of microbially produced methane from different methanogenic pathways lack a systematic investigation. In this study, we provide a dataset encompassing the relative isotopologue abundances produced by hydrogenotrophic, methylotrophic, acetoclastic, and methoxydotrophic methanogenesis. We find that a statistical “combinatorial effect” generates significant differences in ¹² CH ₂ D ₂ compositions between hydrogenotrophic methanogenesis and other pathways. The thermodynamic drive of methanogenic reactions and phylogenetic affiliation may also influence the isotope compositions of methane. Our study provides new experimental constraints on the isotope signatures of different microbial methanogenic pathways, and evidence of the mechanisms responsible for the observed differences.