Hydrogen regulation of growth, growth yields, and methane gene transcription in Methanobacterium thermoautotrophicum deltaH

Abstract

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 ⌬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 ⌬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...

Functionally distinct genes regulated by hydrogen limitation and growth rate in methanogenic Archaea

“…Comparison with our results suggests that both effects may have occurred, and that similar patterns of gene expression may occur in diverse species of methanogens. Among those genes of methanogenesis that we found to be markedly up-regulated by hydrogen limitation in M. maripaludis, studies in Methanothermobacter thermautotrophicus (4,22) led to a similar conclusion for homologs of Mtd, methylenetetrahydromethanopterin reductase, (24). In batch culture low hydrogen coincides with low growth rate, and the effects may have been a result of growth rate differences, consistent with our observation in M. maripaludis of decreased hmd mRNA levels with lower growth rate.…”

Coupling ofMethanothermobacter thermautotrophicusMethane Formation and Growth in Fed-Batch and Continuous Cultures under Different H2Gassing Regimens

“…Thus, methane formation and growth become more tightly coupled under conditions of reduced hydrogen availability. This finding is in agreement with observations by others (7,8,14,17,18,27,33), although the hydrogen concentrations had not been explicitly measured by those authors. A novel analytical approach applied to fed-batch growth suggested that Y CH4 max can take two distinct values, approximately 3 and 7 g mol Ϫ1 of methane, respectively.…”

“…However, growth experiments with media containing all biological relevant trace elements in excess seem to rule out this possibility. Quite remarkably, a shift toward linear growth was always also preceded by discrete decreases of the specific methane-forming activity at the end of the exponential phase in cultures supplemented with the complete trace set of elements (Table 1 (14) demonstrated that mrt and hmd were expressed during exponential growth, whereas mcr and mdh transcripts were observed when the growth rate declined and the rate of methanogenesis became constant, which is typical for linear growth. It remains to be verified, however, if the differential expression of the above and other methane genes (13,15,16,18,20,34) is connected to hydrogen deprivation, as has often been assumed, or to the shift in growth phases.…”

“…In fed-batch systems, dissolved hydrogen partial pressures (p H2 ) continuously change over time as the result of increasing consumption rates by a growing biomass. Many authors observed that specific growth yields were relatively low when growth proceeded under hydrogen excess and that yields were highest under conditions of hydrogen limitation (7,8,12,14,18,24,27,33,34). Apparently, the degree of coupling between methanogenesis and growth depends on the in situ hydrogen concentration.…”

Association between Archaeal Prolyl- and Leucyl-tRNA Synthetases Enhances tRNAPro Aminoacylation

“…Preparation of M. thermautotrophicus mRNA-Six cultures of M. thermautotrophicus were grown in 1.5-liter bioreactors at 55°C in a minimal salts medium (24). A gas mixture of 89% H 2 , 11% CO 2 was supplied to the cultures at a flow rate of 200 ml/min.…”