Changes in growth rate, methanogenesis, growth yield (Y CH4 ), and methane gene transcription have been correlated with changes in the supply of H 2 to Methanobacterium thermoautotrophicum â¬H cells growing on H 2 plus CO 2 in fed-batch cultures. Under conditions of excess H 2 , biomass and methanogenesis increased exponentially and in parallel, resulting in cultures with a constant Y CH4 and transcription of the mth and mrt genes that encode the H 2 -dependent N 5 ,N 10 -methenyltetrahydromethanopterin (methenyl-H 4 MPT) reductase (MTH) and methyl coenzyme M reductase II (MRII), respectively. Reducing the H 2 supply, by decreasing the percentage of H 2 in the input gas mixture or by reducing the mixing speed of the fermentor impeller, decreased the growth rate and resulted in lower and constant rates of methanogenesis. Under such H 2 -limited growth conditions, cultures grew with a continuously increasing Y CH4 and the mtd and mcr genes that encode the reduced coenzyme F 420 -dependent N 5 ,N 10 -methenyl-H 4 MPT reductase (MTD) and methyl coenzyme M reductase I (MRI), respectively, were transcribed. Changes in the kinetics of growth, methanogenesis, and methane gene transcription directed by reducing the H 2 supply could be reversed by restoring a high H 2 supply. Methane production continued, but at a low and constant rate, and only mcr transcripts could be detected when the H 2 supply was reduced to a level insufficient for growth. ftsA transcripts, which encode coenzyme F 390 synthetase, were most abundant in cells growing with high H 2 availability, consistent with coenzyme F 390 synthesis signaling a high exogenous supply of reductant.Most methanogens can grow and generate energy in a mineral salts medium with CO 2 and H 2 as the sole carbon and energy sources (14,41), and this is the only known lifestyle for Methanobacterium thermoautotrophicum â¬H. There are seven steps in the energy-generating H 2 -dependent pathway of CO 2 reduction to CH 4 (7, 33) and the enzymes that catalyze these reactions have been characterized, in detail, from strains of M. thermoautotrophicum (2,8,10,13,27,42). The methane genes that encode these enzymes have been cloned and sequenced (3,8,10,13,18,21,22,24,32,35,36), leading to the discovery of two [Ni,Fe]-hydrogenases, designated the methyl viologenreducing hydrogenase (MVH) and the coenzyme F 420 -reducing hydrogenase (FRH) (1, 25); two formylmethanofuran dehydrogenases, one containing tungsten and one containing molybdenum (2, 13); two N 5 ,N 10 -methenyltetrahydromethanopterin (methenyl-H 4 MPT) reductases, designated MTH and MTD (18,22,42); and two methyl coenzyme M reductases, designated MRI and MRII (4,24,27). These enzymes facilitate the uptake of the reductant H 2 and catalyze steps 1, 4, and 7, respectively, in the reduction of CO 2 to CH 4 (7, 33). This complexity presumably provides M. thermoautotrophicum cells with the ability to adjust to changes in the availability of the substrates and of the metals employed as enzyme cofactors in methanogenesis.In 1980, Schönh...