Hyperthermophilic hydrogenotrophic methanogens are considered to be one of the most predominant primary producers in hydrogen (H-2 )-abundant hydrothermal environments in the present-day ocean and throughout the history of the Earth. However, the nitrogen sources supporting the development of microbial communities in hydrothermal environments remain poorly understood. We have investigated, for the first time, methanogenic archaea commonly found in deep-sea hydrothermal environments to understand their physiological properties (growth kinetics, energetics, and metal requirements) and isotopic characteristics during the fixation of dinitrogen (N-2), which is an abundant but less-bioavailable compound in hydrothermal fluids. Culture experiments showed that Methanocaldococcus strain (Mc 1-85N) (Topt = 85 degrees C) and Methanothermococcus strain (Mt 5-55N) (Topt = 55 degrees C) assimilated N-2 and ammonium, but not nitrate. Previous phylogenetic studies have predicted that the Methanocaldococcus and Methanothermococcus lineages have nitrogenases, key enzymes for N-2 fixation, with biochemically uncharacterised active site metal cofactors. We showed that Mt 5-55N required molybdenum for the nitrogenase to function, implying a molybdenum-bearing cofactor in the strain. Molybdenum also stimulated diazotrophic (i.e., N-2-fixing) growth of Mc 1-85N, though further experiments are required to test whether the strain contains a molybdenum-dependent nitrogenase. Importantly, Mc 1-85N exhibited an apparently lower requirement of and higher tolerance to molybdenum and iron than Mt 5-55N. Furthermore, both strains produced more 15 N-depleted biomass (-4 parts per thousand relative to N-2) than that previously reported for diazotrophic photosynthetic prokaryotes. These results demonstrate that diazotrophic hyperthermophilic methanogens can be broadly distributed in seafloor and subseafloor hydrothermal environments, where the availability of transition metals is variable and where organic carbon, organic nitrogen, and ammonium are generally scarce. The emergence and function of diazotrophy, coupled with methanogenesis, in the early Earth is also consistent with the nitrogen isotopic records of 3.5 billion-year-old hydrothermal deposits. (C) 2014 Elsevier Ltd. All rights reserved.