Multiple modes of methanogenesis in deep hydrothermally-influenced subsurface sediments

Deep marine sediments are the largest reservoir of methane on Earth. Yet, the pathways and activity of methanogenesis in deep, hot (>50℃) subsurface sediments remain poorly understood. We quantified methanogenic activity using five 14C-labeled substrates and integrated geochemical data to identify dominant pathways and their environmental controls in the subsurface sediments of the Guaymas Basin, Gulf of California, where temperatures are exceptionally high due to magmatic sill intrusions. Thermodynamic calculations and C1/C2+ ratios indicate that methane in the relatively cooler (<60℃) and shallower layers (<250 meters below seafloor; mbsf), is predominantly of biogenic origin. Radiotracer experiments revealed multiple pathways of hydrogenotrophic, acetoclastic, and methylotrophic methanogenesis from 3 to 80°C, highlighting an unexpectedly high metabolic versatility of methanogens in thermally-altered sediments. Methanogenesis peaked in near-surface sediments, declined at 40–60°C, and re-emerged at 80°C, reflecting a shift from mesophilic to thermophilic communities. Methylotrophic methanogenesis, fueled by abundant methylated compounds, persisted to 320 mbsf and dominated up to 60°C, while deeper sill-influenced sediments were dominated by hydrogenotrophic and acetoclastic pathways driven by active microbes and reactive organic matter. These results reveal simultaneous activity of multiple pathways, advancing understanding of methane production in hydrothermally heated sediments.