Hydrogen isotope fractionation by Methanothermobacter thermoautotrophicus in coculture and pure culture conditions

We grew a hydrogen-utilizing methanogen, Methanothermobacter thermoautotrophicus strain ΔH, in coculture and pure culture conditions to evaluate the hydrogen isotope fractionation associated with carbonate reduction under low (< several tens of μM; coculture) and high (>6 mM; pure culture) concentrations of H2 in the headspace. In the cocultures, which were grown at 55 °C with a thermophilic butyrate-oxidizing syntroph, the hydrogen isotopic relationship between methane and water was well represented by the following equation:δDCH4=0.725(±0.003)·δDH2O-275(±3),δDCH4=0.725(±0.003)·δDH2O-275(±3),in which the hydrogen isotope fractionation factor (αH) was 0.725 ± 0.003. The relationship was consistent with the isotopic data on methane and water from terrestrial fields (a peat bog in Washington State, USA, and a sandy aquifer in Denmark), where carbonate reduction was reported to be the dominant pathway of methanogenesis. In the pure cultures, grown at 55 and 65 °C, the αH values were 0.755 ± 0.014 and 0.749 ± 0.014, respectively. Dependence of αH on growth temperature was not observed. The αH value at 55 °C in the pure culture was slightly higher than that in the coculture, a finding that disagrees with a hypothesis proposed by Burke [Burke, Jr. R. A. (1993) Possible influence of hydrogen concentration on microbial methane stable hydrogen isotopic composition. Chemosphere26, 55–67] that hydrogen isotope fractionation between methane and water increases (and αH decreases) with increasing H2 concentration.