Effects of CO2 and H2 limitations on Methanococcus maripaludis.

Hydrogenotrophic methanogens play a key role in anaerobic ecosystems by catalyzing the bioconversion of hydrogen (H2) and carbon dioxide (CO2) into methane (CH4) and water. This metabolic process is the basis of biological power-to-methane (PtM) technology, a promising solution for the long-term storage of surplus renewable energy as CH4. Its successful application can be improved through a deeper understanding of methanogen physiology, particularly the metabolic response to intermittent substrate supply of H2 or CO2. In batch cultures of PtM-relevant strains, mesophilic Methanococcus maripaludis and thermophilic Methanothermobacter marburgensis, we determined the apparent Km for dissolved inorganic carbon (DIC) to be 0.60 and 1.65 mmol L-1, respectively, which correspond to near-maximum CH4 production rates Vmax when CO2 partial pressures exceed 350 Pa at 30°C or 1.95 kPa at 60°C, at pH 7. Focusing on M. maripaludis, cultivation in chemostats under either H2 or CO2 limitation did not affect the yield or activity of methanogenesis. However, differences emerged when M. maripaludis was completely starved of either CO2 or H2. M. maripaludis demonstrated a higher tolerance to H2 starvation compared to CO2 starvation, resulting in shorter lag times and increased methanogenesis activity upon revival with replenished substrates. CO2-starved cells displayed higher intracellular F420H2/F420 and NADH/NAD+ ratios and were more susceptible to O2 inactivation, indicating that the accumulation of reducing equivalents may be linked to cell damage. These insights can help stabilize the operation of PtM processes by adjusting the ratio of H2 to CO2 feed during intermittent operations or shutdowns.IMPORTANCEMicrobial physiology has increasingly been studied under dynamic substrate conditions, expanding beyond the classical focus on balanced growth. While starvation is typically associated with a halt in cellular growth and activity, how cells enter starvation influences their recovery dynamics. We demonstrate that starvation caused by a lack of electron donor vs electron acceptor results in distinctly different revival behaviors in the methanogenic Archaea, Methanococcus maripaludis. Methanogens play a crucial role in the global carbon cycle, participating in the anaerobic breakdown of organic matter to methane and carbon dioxide. They are also of biotechnological significance, being central to anaerobic digestion processes and power-to-gas technology. Thus, our results showing improved recovery from electron donor over electron acceptor starvation may prove essential for optimizing methanogenesis processes across various applications.