Growth Kinetics, Carbon Isotope Fractionation, and Gene Expression in the Hyperthermophile Methanocaldococcus jannaschii during Hydrogen-Limited Growth and Interspecies Hydrogen Transfer

Hyperthermophilic methanogens are often H2 limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H2 conditions and interspecies H2 transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80 to 83 μM) and low (15 to 27 μM) aqueous H2 concentrations and in coculture with the hyperthermophilic H2 producer Thermococcus paralvinellae. The purpose was to measure changes in growth and CH4 production kinetics, CH4 fractionation, and gene expression in M. jannaschii with changes in H2 flux. Growth and cell-specific CH4 production rates of M. jannaschii decreased with decreasing H2 availability and decreased further in coculture. However, cell yield (cells produced per mole of CH4 produced) increased 6-fold when M. jannaschii was grown in coculture rather than monoculture. Relative to high H2 concentrations, isotopic fractionation of CO2 to CH4 (eCO2-CH4) was 16‰ larger for cultures grown at low H2 concentrations and 45‰ and 56‰ larger for M. jannaschii growth in coculture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2 availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2 availability significantly affects the CH4 and biomass production and CH4 fractionation by hyperthermophilic methanogens in their native habitats. IMPORTANCE Hyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae. With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments.