Nitrous oxide respiration in acidophilic methanotrophs

Abstract Methanotrophic bacteria mitigate methane (CH 4 ) emissions from natural environments. Although aerobic methanotrophs are considered strict aerobes, they are often highly abundant in extremely hypoxic and even anoxic environments. Despite the presence of denitrification genes, it remains to be verified whether denitrification contributes to their growth. Here, we revealed that two acidophilic methanotrophs encoding N 2 O reductase (clade I and type II nosZ, respectively): Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6, respired N 2 O and grew anaerobically on diverse non-methane substrates, including methanol, C-C substrates, and hydrogen. However, NO 3 − and NO 2 − could be reduced during methanol oxidation in Methylocella tundrae T4 and Methylocella silvestris BL2 without significantly increasing cell biomass. The lack of growth on methanol + NO 3 − or NO 2 − was likely due to the production of toxic reactive nitrogen species and C1 metabolites. However, the oxidation of pyruvate, a C3 electron donor, combined with NO 3 − or NO 2 − reduction resulted in anaerobic growth of Methylocella tundrae T4 and Methylocella silvestris BL2. In the extreme acidophile, Methylacidiphilum caldifontis IT6, N 2 O respiration supported cell growth at an extremely acidic pH of 2.0. In Methylocella tundrae T4, simultaneous consumption of N 2 O and CH 4 was observed in suboxic conditions, both in microrespirometry and growth experiments, indicating the robustness of its N 2 O reductase activity in the presence of O 2 . Furthermore, CH 4 oxidation per O 2 reduced in O 2 -limiting conditions increased when N 2 O was added, indicating that cells of T4 can direct more O 2 towards methane monooxygenase when respiring N 2 O as a terminal electron acceptor. Upregulation of nosZ and distinct repertories of methanol dehydrogenase-encoding genes (XoxF- and MxaFI-type) in Methylocella tundrae T4 cells grown anaerobically on methanol with N 2 O as the sole electron acceptor indicated adaptation mechanisms to anoxia. Our findings demonstrate that some methanotrophs can respire N 2 O independently or in tandem with O 2 , significantly expanding their potential ecological niche and paving the way for enhanced growth and survival in dynamic environments. This metabolic capability has application potential for simultaneously mitigating the emissions of the key greenhouse gases, CO 2 , CH 4, and N 2 O, from natural and engineered environments.