Developing a robust biomethanation process by improving oxygen tolerance of Methanothermobacter wolfeii strain BSEL

Application of biomethanation for conversion of CO₂ and H₂ to renewable natural gas (RNG) is constrained by the strong O₂ sensitivity of methanogenic archaea. In biogas facilities, O₂ enters through leaks, sampling ports, and intentional micro-aeration for hydrogen sulfide control, creating intermittent oxidative conditions that can destabilize a downstream biomethanation process for biogas upgrading to RNG. An O₂-tolerant biocatalyst reduces reliance on strict anaerobic techniques for handling the biocatalyst as well as purification of the gases used for operating biomethanation units. For reliable large-scale operation, biocatalysts must tolerate O₂ contamination in the gas streams while maintaining high CH₄ productivity. Adaptive laboratory evolution (ALE) is a method for improving microbial abilities such as O₂ tolerance. In this study, we applied ALE in serial batch and continuous cultures to increase the O₂ tolerance of Methanothermobacter wolfeii BSEL, a hydrogenotrophic methanogen. In batch experiments, a robust culture capable of growing with 2000 parts per million by volume (ppmv) O₂ was obtained. In a periodic gas-vented continuous (PGVC) bioreactor, the evolved culture maintained methanogenesis at up to 3600 ppmv O₂ and an oxidation-reduction potential (ORP) between -212 and -174 mV, and showed CH₄ productivity comparable to controls without O₂ added. Increasing O₂ concentrations affected the cells by decreasing their size while forming structured biofilms without affecting CH₄ productivity. These results demonstrate that M. wolfeii BSEL can adapt to elevated O₂ concentrations in continuous cultivation while maintaining high CH₄ production, expanding the redox operating range for biomethanation, directly impacting the future of biomethanation processes.