Microbial methane generation (methanogenesis) is an important metabolic process in the terrestrial deep biosphere and is an analog to early Earth as it is proposed to be one of the most ancient metabolisms on Earth. Signs of methanogenesis in meteorite impact craters are of particular interest in this respect as these settings are proposed hot spots for deep microbial colonization of the upper crust. Yet, reports of active deep rock-hosted methanogenesis are scarce, particularly for methylotrophic methanogenesis, while reports from terrestrial meteorite impact craters are completely lacking. Here, we used indigenous communities in cultures enriched from 400-m deep fluids to confirm and characterize active methane production from several carbon donors, including indigenous oil, in a terrestrial impact crater at Siljan, Sweden. Metagenomic and metatranscriptomic data of the methane-producing cultures revealed a consortium dominated by Acetobacterium sp. KB-1 and Candidatus Methanogranum gryphiswaldense, mediating methanogenesis solely via the methyl-reduction pathway, and resulting in a δ¹³Cmethanol-methane isotope enrichment of up to 98.6‰. These results provide insights into methylotrophic methanogenesis in deep subsurface environments in general, and in particular in fractured meteorite impact structures.

IMPORTANCE: This study revealed that microbes enriched from groundwater in a 380-m deep borehole within the Siljan meteorite impact crater in Sweden were capable of producing methane, a key greenhouse gas. This is especially significant because it is the first proof of active methanogens in an impact crater and showing a specific pathway of methane production—methylotrophic methanogenesis—is present in the deep terrestrial subsurface, an environment that is typically hard to study. These findings shed light on life in extreme conditions on Earth and show that meteorite craters can be biological hotspots, rich with ancient life processes.