Methane is a potent greenhouse gas, an important energy source, and an important part of the global carbon cycle. The relative abundances of doubly substituted ("clumped") methane isotopologues (13CH3D and 12CH2D2) 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 data set encompassing isotopic signatures of hydrogenotrophic, methylotrophic, acetoclastic, and methoxydotrophic methanogenesis. We find that a statistical "combinatorial effect" generates significant differences in 12CH2D2 compositions between hydrogenotrophic methanogenesis and the other pathways, while variations in the fractionation factors of clumped isotopologues result in differences in 13CH3D compositions between the methylotrophic, acetoclastic, and methoxydotrophic pathways. The energy yield of methanogenesis and the energy conservation approaches implemented by different microbial strains may also influence the isotope values of methane. Further analysis suggests that previously observed isotopic signatures of methane in freshwater environments are potentially due to mixing between hydrogenotrophic and other methanogenesis pathways. This study provides new experimental constraints on the isotope signatures of different microbial methanogenic pathways and evidence of the mechanisms responsible for the observed differences. This enables a better understanding of the sources and sinks of methane in the environment.
Keywords: clumped isotopologues; combinatorial effect; methane; methanogenesis pathways.