The deep continental biosphere represents one of Earth’s least explored ecosystems and a major frontier in biology and paleobiology, despite hosting a large proportion of the Earth’s biomass. Microorganisms inhabit fractures within the bedrock, where they drive subsurface biogeochemical cycles. Methane is a key product in this environment, and seeps of methane from the subsurface can contribute to atmospheric greenhouse gases on Earth. Although methanogenesis is an ancient metabolic pathway, its metabolism and long-term evolution in the deep biosphere remains poorly constrained. 

The aim of this thesis was to characterize methane-forming processes, associated biogeochemical cycles and microbial community structure in the terrestrial deep subsurface. A multi-disciplinary dataset integrating dissolved gases, groundwater, microbial biomass and fracture-filling minerals was utilized. Stable isotope geochemistry, clumped isotopes, U-Pb dating, and microbial analysis were combined to resolve the origin, timing and pathways of microbial methane formation at Siljan impact structure, COSC-2 borehole and Forsmark. 

The Siljan impact structure, methane accumulations are dominated by a microbial component, fueled by freshwater infiltration and hydrocarbon biodegradation, facilitated by fracture networks that enhance porosity and substrate supply. Anaerobic enrichment cultures from the Siljan impact structure provide direct evidence of active methylotrophic methanogenesis, mediated by syntrophic consortia. At Forsmark, groundwater at 480 m depth in the crystalline basement shows signatures for active acetoclastic methanogenesis from a methanogen of the family Methanobacteriaceae, possibly fueled by acetogens utilizing dissolved organic carbon. 

At the COSC-2 borehole, fracture-filling calcite δ¹³C values mark ancient microbial methanogenesis at up to 1907 m depth. U-Pb dating reveals that this process was episodic, spanning several hundreds of million years, linked to the reactivation of fractures during the exhumation of the Caledonian mountain range. Modern groundwaters at COSC-2 are highly isolated, contain only minor methanogens (Methanobactericeae) and are instead dominated by the sulfate reducer Candidatus Desulforudis audaxviator.

Together, these systems demonstrate the heterogeneity of the terrestrial deep biosphere, the resilience and adaptability of deep microbial life. The findings expand the known depth and temporal range of subsurface microbial methane formation and show the importance of methanogenic biogeochemical cycles in the crystalline bedrock.