Active anaerobic methane oxidation and sulfur disproportionation in the deep terrestrial subsurface

Microbial life is widespread in the terrestrial subsurface and present down to several kilometers depth, but the energy sources that fuel metabolism in deep oligotrophic and anoxic environments remain unclear. In the deep crystalline bedrock of the Fennoscandian Shield at Olkiluoto, Finland, opposing gradients of abiotic methane and ancient seawater-derived sulfate create a terrestrial sulfate-methane transition zone (SMTZ). We used chemical and isotopic data coupled to genome-resolved metaproteogenomics to demonstrate active life and, for the first time, provide direct evidence of active anaerobic oxidation of methane (AOM) in a deep terrestrial bedrock. Proteins from Methanoperedens (formerly ANME-2d) are readily identifiable despite the low abundance ([≤]1%) of this genus and confirm the occurrence of AOM. This finding is supported by 13C-depleted dissolved inorganic carbon. Proteins from Desulfocapsaceae and Desulfurivibrionaceae, in addition to 34S-enriched sulfate, suggest that these organisms use inorganic sulfur compounds as both electron donor and acceptor. Zerovalent sulfur in the groundwater may derive from abiotic rock interactions, or from a non-obligate syntrophy with Methanoperedens, potentially linking methane and sulfur cycles in Olkiluoto groundwater. Finally, putative episymbionts from the candidate phyla radiation (CPR) and DPANN archaea represented a significant diversity in the groundwater (26/84 genomes) with roles in sulfur and carbon cycling. Our results highlight AOM and sulfur disproportionation as active metabolisms and show that methane and sulfur fuel microbial activity in the deep terrestrial subsurface. Significance StatementThe deep terrestrial subsurface remains an environment in which there is limited understanding of the extant microbial metabolisms, despite its reported large contribution to the overall biomass on Earth. It is much less well studied than deep marine sediments. We show that microorganisms in the subsurface are active, and that methane and sulfur provide fuel in the oligotrophic and anoxic subsurface. We also uncover taxonomically and metabolically diverse ultra-small organisms that interact with larger host cells through surface attachment (episymbiosis). Methane and sulfur are commonly reported in terrestrial crystalline bedrock environments worldwide and the latter cover a significant proportion of the Earths surface. Thus, methane- and sulfur-dependent microbial metabolisms have the potential to be widespread in the terrestrial deep biosphere.