Exploring the bottom of the deep biosphere nearly 2.5 km below the seafloor. subsurface metagenome

Extensive microbial populations inhabit deeply buried marine sediments along ocean margins, but the lower boundaries of this spacious ecosystem and the factors limiting its extent are poorly constrained. Here we provide evidence for the existence of microbial communities in sediment associated with lignite coalbeds buried nearly ~2.5 km below the seafloor off the Shimokita Peninsula, Japan, where ongoing microbial formation of methane is indicated by isotopic data of methane and carbon dioxide, methanogenic biomarkers, cultivation and gas composition results. Rigorous protocols aimed at minimizing and correcting for sample contamination resulted in concentration estimates as low as 1 cell cm 3 in numerous samples below 1.5 km subseafloor depth where temperatures range from ~40 to 60°C; only in coalbed-bearing sediments were cellular concentrations substantially elevated. The microbial ecosystem at this site appears situated close to the lower boundary of the subseafloor biosphere and is taxonomically distinct from shallower ecosystems at this location.