Δ13CH3D and Δ12CH2D2 signatures of methane aerobically oxidized by Methylosinus trichosporium with implications for deciphering the provenance of methane gases

Aerobic oxidation of methane (MOx) is an important biologically mediated process that consumes methane in a wide range of environments. Here we report results of culture experiments with the aerobic methane-oxidizing bacterium Methylosinus trichosporium (OB3b) that are used to characterize the mass-18 isotopologue (Δ13CH3D and Δ12CH2D2) signatures of MOx in residual methane gases. MOx activity was confirmed by simultaneous decrease of methane and oxygen in the bulk gas headspace. Bulk carbon (13C/12C) and hydrogen (D/H) isotope ratios of the methane gas increased while both Δ13CH3D and Δ12CH2D2 decreased as the oxidation proceeded. The corresponding fractionation factors (α) calculated from our experimental results are 0.98485 ± 0.00006 for 13C/12C, 0.7265 ± 0.0010 for D/H, 0.7141 ± 0.0011 for 13CH3D/12CH4, and 0.4757 ± 0.0023 for 12CH2D2/12CH4. Deviations of the mass-18 fractionation factors from the Rule of the Geometric Mean (RGM) expressed as γ values are 0.9981 ± 0.0017 for 13CH3D/12CH4 and 0.9013 ± 0.0045 for 12CH2D2/12CH4. Our α and γ values suggest that while MOx fractionates 13CH3D within error of the RGM, the Δ13CH3D and Δ12CH2D2 trajectories are very sensitive to even small deviations in 13CH3D/12CH4 from the RGM. Fractionation of 12CH2D2 deviates considerably from RGM, and this causes dramatic and robust effects on the trajectories of residual methane in Δ13CH3D vs. Δ12CH2D2 space. Our models suggest that Δ13CH3D and Δ12CH2D2 could potentially mimic microbial methanogenesis signatures in an environment that exhibits a strong Rayleigh Distillation process with little to no replenishment of methane during oxidation. However, in closed or open systems where oxidation is attended by simultaneous methane production, we find that modest increases in Δ13CH3D and dramatic increases in Δ12CH2D2 are to be expected, thus resulting in isotopologue signatures distinct from microbial methanogenesis. The overall trend in these conditions suggest that methane altered by MOx is distinguishable from other methane sources in Δ13CH3D and Δ12CH2D2 space.