Hydrogen Oxidation Benefits Alphaproteobacterial Methanotrophs Under Severe Methane Limitation

Hydrogen (H₂) and methane (CH₄) are produced in the anoxic layers of wetlands and sediments. In the overlaying oxygenated surface layers, these gases become available for oxidation by aerobic hydrogenotrophic and methanotrophic microorganisms. While H₂ oxidation by verrucomicrobial methane-oxidising bacteria (MOB) is extensively studied, less is known about this metabolism in MOB from the class Alphaproteobacteria, which frequently inhabit wetlands. We show that Methylocystis bryophila H2s^T, Methylocapsa aurea KYG^T, and "Methylosinus acidophilus" 29 encode diverse hydrogenases, instantly oxidise H₂ when cultivated under CH₄-limited and low-oxygen conditions, under which hydrogenase transcription is upregulated compared to CH₄-replete conditions. H₂ exposure accelerated the maximum H₂ oxidation rates but caused no upregulation of hydrogenases. Furthermore, while CH₄ oxidation activity was affected by substrate-limited growth conditions, H₂ oxidation rates remained unaffected, and H₂ supply to CH₄-limited chemostats caused increased biomass yield. Moreover, CH₄ oxidation was severely inhibited by sulfide (H₂S), while H₂ and methanol oxidation rates were only moderately affected. In summary, the ability to conserve energy from H₂ oxidation increases resilience and enhances growth of alphaproteobacterial methanotrophs in CH₄-limited environments, which revises the ecological role of these MOB in ecosystems with naturally fluctuating CH₄ and H₂ concentrations.