Carbon Reallocation Driven by Aerobic–Anoxic Alternation during Methanotrophic Metabolism

Methanotrophs are of fundamental importance in the global methane cycle and hold promise for the bioconversion of methane into valuable products. This study elucidates the impact of alternating aerobic-anoxic conditions on metabolism of the two phylogenetically distinct groups of methanotrophs: <i>Methylomonas koyamae</i> (type I, γ-proteobacteria) and <i>Methylocystis bryophila</i> (type II, α-proteobacteria). Using batch cultivations with single and repeated oxygen pulses, we demonstrated that cyclic aerobic-anoxic alternation significantly increased the secretion of organic compounds. Notably, acetate secretion by the type II methanotroph <i>M. bryophila</i> was observed for the first time. Genome-scale metabolic modeling and flux balance analysis revealed that acetate secretion serves as an auxiliary ATP-generating pathway for <i>M. bryophila</i> under oxygen limitation. We further uncovered a dynamic mechanism: aerobic phases trigger methane uptake and build up intracellular organic carbon pools, while anoxic phases redirect these pools toward acetate and/or hydrogen release to sustain survival under oxygen limitation. This cyclic regimen effectively channels more methane-derived carbon into extracellular acetate. Our findings underscore that redox conditions critically shape methanotrophic metabolism, influencing both the biological methane cycle and the global climate.