In the context of the shift toward clean, carbon-free energy, hydrogen (H2) has received growing attention as an energy carrier. We monitored a simulated leakage of gaseous molecular H2, e.g., from a pipeline. H2 was injected into a shallow aquifer, and the resulting biogeochemical processes were monitored. For the first time, stable isotopes of hydrogen were used to track in situ H2 transport and consumption. Isotopic composition of injected H2 was δ2H = −161.1 ± 0.4‰. During the injection, initial shifts in the isotope signature of about Δ2H = +8‰ in well D06 (1 m from injection) and Δ2H = −120‰ in well D04 (2 m from injection) were observed, probably caused by a mass-dependent isotope effect associated with the pressure of the injection and the migration of the gas phase through pores and channels in the aquifer. After the injection, H2 concentrations decreased and an equilibrium isotope exchange with water led to an isotopic depletion of H2 (δ2H = −710.7 ± 2.7‰) within 28 days, presumably catalyzed by hydrogenase enzymes of microbes. The theoretical equilibrium between H2 and water was however not reached. We hypothesize that a continuous isotopic shift in available H2 as a result of physical transport processes resulted in a new isotope equilibrium with water, catalyzed by hydrogenases. Acetate detected in groundwater samples indicates in situ H2 oxidation by microbial homoacetogenesis. In laboratory experiments using H2-amended sediments sampled from the same site, microbial H2 oxidation was accompanied by equilibrium isotope exchange with water and homoacetogenesis and ferric iron reduction were the main microbial H2-consuming processes. Overall, the H2 isotope ratio was considerably impacted by physical and microbial processes occurring in the shallow aquifer. Monitoring of the equilibrium isotope exchange between H2 and water could be used as a proxy for ongoing microbial H2 oxidation.

We determined 2 H stable isotope fractionation at natural abundances associated with hydrogenase activity by whole cells of Desulfovibrio vulgaris strain Miyazaki F expressing a NiFe(Se) hydrogenase. Inhibition of sulfate reduction by molybdate inhibited the overall oxidation of hydrogen but still facilitated an equilibrium isotope exchange reaction with water. The theoretical equilibrium isotope exchange δ 2 H-values of the chemical exchange reaction were identical to the hydrogenase reaction, as confirmed using three isotopically different waters with δ 2 H-values of – 62, +461, and + 1533‰. Expected kinetic isotope fractionation of hydrogen oxidation by non-inhibited cells was also superimposed by an equilibrium isotope exchange. The isotope effects were solely catalyzed biotically as hydrogen isotope signatures did not change in control experiments without cells of D. vulgaris Miyazaki.