A dual methane production mechanism mediated by enzymatic catalysis and the Fenton reaction in the wood-rotting fungus Schizophyllum commune

ABSTRACT Understanding biological sources of methane is critical for elucidating the global methane cycle and addressing climate change. While traditional research has predominantly focused on prokaryotic methanogens, emerging evidence suggests that eukaryotes, particularly fungi, can produce methane through both enzymatic and non-enzymatic pathways. However, no eukaryotic organism has been documented to possess both pathways simultaneously. Here, we provide preliminary evidence suggesting that the wood-decaying fungus Schizophyllum commune strain 20R-7-F01, isolated from sediment approximately 2 km below the seafloor, employs dual mechanisms for methane production. By comparing wild-type strains with mct1 (methyl chloride transferase 1) and dh3 (dehalogenase 3) deficient strains under iron-replete (50 μM Fe³ + , to facilitate Fenton reactions) and iron-free conditions, we estimated that enzymatic pathways account for roughly 82%–85% of total methane production. The remaining ~15%–18% of methane output is consistent with a contribution from Fenton-type chemistry, and this quantitative partitioning of pathway contributions was derived from cross-condition yield comparisons. Environmental factors such as oxygen, temperature, and substrate type significantly influence enzymatic efficiency, whereas the efficacy of the non-enzymatic pathway depends on the concentration of methyl donors, particularly dimethyl sulfoxide. Additionally, both pathways are activated by hydrogen peroxide, indicating that reactive oxygen species positively regulate methane synthesis. This study reveals a novel co-existing “enzymatic-Fenton” mechanism for methane production in fungi, highlighting their crucial role in the global methane budget and carbon cycling. IMPORTANCE Methane is a key driver of climate change, yet our understanding of its biological sources has long been limited to bacteria and archaea. Here, we reveal that a fungus, Schizophyllum commune 20R-7-F01, isolated from deep below the seafloor, produces methane through two distinct mechanisms operating simultaneously: an enzymatic pathway and a Fenton reaction-driven non-enzymatic pathway. This dual system, previously unreported in any eukaryote, redefines the potential contribution of fungi to the global methane budget. Our findings challenge the traditional prokaryote-centric view of methanogenesis and open new avenues for understanding methane cycling in oxygen-rich and iron-rich environments, with implications for climate modeling and carbon cycle research.