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

Abstract

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.