Gas hydrates stored in the continental margins of the world’s oceans represent the largest global reservoirs of methane. Determining the source and history of methane from gas hydrate deposits informs the viability of sites as energy resources, and potential hazards from hydrate dissociation or intense methane degassing from ocean warming. Stable isotope ratios of methane (13C/12C, D/H) and the molecular ratio of methane over ethane plus propane (C1/C2+3) have traditionally been applied to infer methane sources, but often yield ambiguous results when two or more sources are mixed, or when compositions were altered by physical (e.g., diffusion) or microbial (e.g., methanotrophy) processes. We measured the abundance of clumped methane isotopologue (13CH3D) alongside 13C/12C and D/H of methane, and C1/C2+3 for 46 submarine gas hydrate specimens and associated vent gases from 11 regions of the world’s oceans. These samples are associated with different seafloor seepage features (oil seeps, pockmarks, mud volcanoes, and other cold seeps). The average apparent equilibration temperatures of methane from the Δ13CH3D (the excess abundance of 13CH3D relative to the stochastic distribution) geothermometer increase from cold seeps (15 to 65 ℃) and pockmarks (36 to 54 ℃), to oil-associated gas hydrates (48 to 120 ℃). These apparent temperatures are consistent with, or a few tens of degrees higher than, the temperature expected for putative microbial methane sources. Apparent methane generation depths were derived for cold seep, pockmark, and oil seep methane from isotopologue-based temperatures and the local geothermal gradients. Estimated methane generation depths ranged from 0.2 to 5.3 kmbsf, and are largely consistent with source rock information, and other chemical geothermometers based on clay mineralogy and fluid chemistry (e.g., Cl, B, and Li). Methane associated with mud volcanoes yielded a wide range of apparent temperatures (15 to 313℃). Gas hydrates from mud volcanoes the Kumano Basin and Mediterranean Sea yielded δ13C-CH4 values from -36.9 to -51.0‰, typical for thermogenic sources. Δ13CH3D values (3.8 to 6.0‰) from these sites, however, are consistent with prevailing microbial sources. These mud volcanoes are located at active convergent plate margins, where hydrogen may be supplied from basement rocks, and fuel methanogenesis to the point of substrate depletion. In contrast, gas hydrate from mud volcanoes located on km-thick sediments in tectonically less active or passive settings (Black Sea, North Atlantic) yielded microbial-like δ13C-CH4 and C1/C2+3 values, and low Δ13CH3D values (1.6 to 3.3‰), which may be due to kinetic isotope effects. This study is the first to document the link between methane isotopologue-based temperature estimates and key submarine gas hydrate seepage features, and validate previous models about their geologic driving forces.

Stable isotope analysis has been widely used to aid the source identification of methane. However, the isotopic (13C/12C and D/H) and isotopologue (13CH3D and 12CH2D2) signatures of microbial methane in natural environments are often different from those in laboratory cultures in which methanogens are typically grown under optimal conditions. Growth phase and hydrogen (H2) concentration have been proposed as factors controlling the isotopic compositions of methane, but their effects on the relationship among carbon, hydrogen and doubly-substituted “clumped” isotopologue systems have not been assessed in a quantitative framework. Here we experimentally investigate the bulk (δ13C and δD) and clumped (∆13CH3D) isotopologue compositions of methane produced by hyperthermophilic hydrogenotrophic (CO2-reducing) methanogens using batch and fed-batch systems at different growth phases and H2 mixing ratios (Methanocaldococcus bathoardescens at 82 or 60 °C and on 80 or 25% H2; Methanothermobacter thermautotrophicus ∆H at 65 °C and on 20, 5 or 1.6% H2). We observed a large range (18 to 63‰) of carbon isotope fractionations, with larger values observed during later growth phase, consistent with previous observations. In contrast, hydrogen isotope fractionations remained relatively constant at –317 ± 25‰. Linear growth was observed for experiments with M. bathoardescens, suggesting that dissolution of gaseous H2 into liquid media became the rate limit as cell density increased. Accordingly, the low (and undersaturated) dissolved H2 concentrations can explain the increased carbon isotope fractionations during the later growth phase. The δD and Δ13CH3D values indicated departure from equilibrium throughout experiments. As the cell density increased and dissolved H2 decreased, Δ13CH3D decreased (further departure from equilibrium), contrary to expectations from previous models. Our isotopologue flow network model reproduced the observed trends when the last H-addition step is less reversible relative to the first three H-addition steps (up to CH3-CoM). In this differential reversibility model, carbon, hydrogen and clumped isotopologue fractionations are largely controlled by the reversibility of the first three H-addition steps under high H2 concentrations; the last H-addition step becomes important under low H2. The magnitude of depletion and decreasing trend in Δ13CH3D values were reproduced when a large (≥6‰) secondary clumped kinetic isotope effect was considered in the model. This study highlights the advantage of combined bulk and clumped isotope analyses and the importance of physiological factors (growth phase) and energy availability (dissolved H2 concentration) when using isotope analyses to aid the source identification of methane.

In high-pH ($\text{pH}>10$) fluids that have participated in low-temperature ($<150\,^{\circ}\text{C}$) serpentinization, the dominant form of C is often methane (CH$_{4}$), but the origin of this CH$_{4}$ is uncertain. To assess CH$_{4}$ origin during low-temperature serpentinization, we pumped fluids from aquifers within the Samail Ophiolite, Oman. We determined fluid chemical compositions, analyzed taxonomic profiles of fluid-hosted microbial communities, and measured isotopic compositions of hydrocarbon gases. We found that 16S rRNA gene sequences affiliated with methanogens were widespread in the aquifer. We measured clumped isotopologue ($^{13}$CH$_{3}$D and $^{12}$CH$_{2}$D$_{2}$) relative abundances less than equilibrium, consistent with substantial microbial CH$_{4}$ production. Further, we observed an inverse relationship between dissolved inorganic C concentrations and $\delta^{13}\text{C}_{\text{CH}_{4}}$ across fluids bearing microbiological evidence of methanogenic activity, suggesting that the apparent C isotope effect of microbial methanogenesis is modulated by C availability. A second source of CH$_{4}$ is evidenced by the presence of CH$_{4}$-bearing fluid inclusions in the Samail Ophiolite and our measurement of high $\delta^{13}\text{C}$ values of ethane and propane, which are similar to those reported in studies of CH$_{4}$-rich inclusions in rocks from the oceanic lithosphere. In addition, we observed 16S rRNA gene sequences affiliated with aerobic methanotrophs and, in lower abundance, anaerobic methanotrophs, indicating that microbial consumption of CH$_{4}$ in the ophiolite may further enrich CH$_{4}$ in $^{13}$C. We conclude that substantial microbial CH$_{4}$ is produced under varying degrees of C limitation and mixes with abiotic CH$_{4}$ released from fluid inclusions. This study lends insight into the functioning of microbial ecosystems supported by water/rock reactions.