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Hydrogen and carbon isotope systematics of H2O–H2–CO2–CH4 in hydrogenotrophic methanogenesis and their relation to H2 availability were investigated. Two H2-syntrophic cocultures of fermentatively hydrogenogenic bacteria and hydrogenotrophic methanogens under conditions of <102 Pa-H2 and two pure cultures of hydrogenotrophic methanogens under conditions of ~105 Pa-H2 were tested. Carbon isotope fractionation between CH4 and CO2 during hydrogenotrophic methanogenesis was correlated with pH2, as indicated in previous studies. The hydrogen isotope ratio of CH4 produced during rapid growth of the thermophilic methanogen Methanothermococcus okinawensis under high pH2 conditions (~105 Pa) was affected by the isotopic composition of H2, as concluded in a previous study of Methanothermobacter thermautotrophicus. This “δDH2$$ {\updelta \mathrm{D}}_{{\mathrm{H}}_2} $$ effect” is a possible cause of the diversity of previously reported values for hydrogen isotope fractionation between CH4 and H2O examined in H2-enriched culture experiments. Hydrogen isotope fractionation between CH4 and H2O, defined by (1000 + δDCH4$$ {\updelta \mathrm{D}}_{{\mathrm{CH}}_4} $$)/(1000 + δDH2O$$ {\updelta \mathrm{D}}_{{\mathrm{H}}_2\mathrm{O}} $$), during hydrogenotrophic methanogenesis of the H2-syntrophic cocultures was in the range 0.67–0.69. The hydrogen isotope fractionation of our H2-syntrophic dataset overlaps with those obtained not only from low-pH2 experiments reported so far but also from natural samples of “young” methane reservoirs (0.66–0.74). Conversely, such hydrogen isotope fractionation is not consistent with that of “aged” methane in geological samples (≥0.79), which has been regarded as methane produced via hydrogenotrophic methanogenesis from the carbon isotope fractionation. As a possible process inducing the inconsistency in hydrogen isotope signatures between experiments and geological samples, we hypothesize that the hydrogen isotope signature of CH4 imprinted at the time of methanogenesis, as in the experiments and natural young methane, may be altered by diagenetic hydrogen isotope exchange between extracellular CH4 and H2O through reversible reactions of the microbial methanogenic pathway in methanogenic region and/or geological methane reservoirs.

progress in earth and planetary science

Hydrogen and carbon isotope systematics in hydrogenotrophic methanogenesis under H2-limited and H2-enriched conditions: implications for the origin of methane and its isotopic diagnosis