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 H(2) limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H(2) conditions and interspecies H(2) transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80 to 83 μM) and low (15 to 27 μM) aqueous H(2) concentrations and in coculture with the hyperthermophilic H(2) producer Thermococcus paralvinellae. The purpose was to measure changes in growth and CH(4) production kinetics, CH(4) fractionation, and gene expression in M. jannaschii with changes in H(2) flux. Growth and cell-specific CH(4) production rates of M. jannaschii decreased with decreasing H(2) availability and decreased further in coculture. However, cell yield (cells produced per mole of CH(4) produced) increased 6-fold when M. jannaschii was grown in coculture rather than monoculture. Relative to high H(2) concentrations, isotopic fractionation of CO(2) to CH(4) (ε(CO2-CH4)) was 16‰ larger for cultures grown at low H(2) concentrations and 45‰ and 56‰ larger for M. jannaschii growth in coculture on maltose and formate, respectively. Gene expression analyses showed H(2)-dependent methylene-tetrahydromethanopterin (H(4)MPT) dehydrogenase expression decreased and coenzyme F(420)-dependent methylene-H(4)MPT dehydrogenase expression increased with decreasing H(2) availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H(2) availability significantly affects the CH(4) and biomass production and CH(4) fractionation by hyperthermophilic methanogens in their native habitats. IMPORTANCE Hyperthermophilic methanogens and H(2)-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H(2) can be very low in these environments, and there is a gap in our knowledge about the origin of CH(4) in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H(2) flux, growth rates, and CH(4) production rates decreased. The same trend was observed increasingly with interspecies H(2) transfer between M. jannaschii and the hyperthermophilic H(2) producer Thermococcus paralvinellae. With decreasing H(2) availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH(4) with a concomitant decrease in H(2)-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F(420)-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H(2) transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH(4) in natural environments.