Extreme methane clumped isotopologue bio-signatures of aerobic and anaerobic methanotrophy: Insights from the Lake Pavin and the Black Sea sediments

Microbial methane oxidation - or methanotrophy - is a key control of the global methane budget on Earth, and perhaps in other planetary systems. Here, we explore the potential role of mass-18 isotopologues of methane, expressed as Δ13CH3D and Δ12CH2D2 values, in tracking both aerobic and anaerobic methanotrophy in nature. We examine two well documented methanotrophic environments: the Lake Pavin (France) water column, where methane degradation is dominated by aerobic methanotrophy (AeOM), and the Black Sea sediments (offshore Romania), dominated by anaerobic methanotrophy (AOM) coupled to sulfate-reduction. In both settings, lighter isotopologues are preferentially consumed, generating elevated 13CH4/12CH4, 12CH3D/12CH4, 13CH3D/12CH4 and 12CH2D2/12CH4 ratios. This results in increasing of δ13C and δD values in the residual methane for both settings, as observed commonly in systems dominated by methanotrophy. As a result, AeOM and AOM cannot be easily distinguished by the development of δ13C and δD. In contrast, the Δ13CH3D and Δ12CH2D2 (departure from stochastic) values have opposite trajectories, with minimal decreases in the case of the AeOM-dominated system, but dramatic increases in the case of AOM, with Δ13CH3D and Δ12CH2D2 reaching values as high as 15.7 ‰ and 76.6 ‰, respectively. This contrasting behavior of clumped isotopologues signatures illustrates fundamental distinction between the two processes and the way they segregate methane isotopologues. These data demonstrate that both AeOM and AOM have distinctive kinetic isotope effects in natural settings, consistent with preliminary laboratory work. In particular, we find that γ-values (which measure the deviation to the product of ‘normal’ bulk isotope fractionation factors) are close to unity in the case of AeOM (i.e. a negligible clumped isotope effect), but significantly below unity in the case of AOM (i.e. strong clumped isotope effect). In addition, our data also illustrate how AOM under low-sulfate conditions may promote methane isotopologue equilibration. Taken together, we suggest these data and apparent isotopologue fractionation factors extrapolated from these two environments may help refine the potential bio-signatures of methane affected by methanotrophy.