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 K_m for dissolved inorganic carbon (DIC) to be 0.60 and 1.65 mmol L^-1, respectively, which correspond to near-maximum CH₄ production rates V_max 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.