The oceanic basaltic crust is the largest aquifer on Earth and has the potential to harbor substantial subsurface microbial ecosystems, which hitherto remains largely uncharacterized and is analogous to extraterrestrial subsurface habitats. Within the sediment-buried 3.5 Myr old basaltic crust of the eastern Juan de Fuca Ridge flank, the circulating basement fluids have moderate temperature (∼65 °C) and low to undetectable dissolved oxygen and nitrate concentrations. Sulfate, present in high concentrations, is therefore expected to serve as the major electron acceptor in this subsurface environment. This study focused on the availability and potential sources of two important electron donors, methane (CH4) and hydrogen (H2), for the subseafloor biosphere. High integrity basement fluids were collected via fluid delivery lines associated with Integrated Ocean Drilling Program (IODP) Circulation Obviation Retrofit Kits (CORKs) that extend from basement depths to outlet ports at the seafloor. Two new CORKs installed during IODP 327 in 2010, 1362A and 1362B, were sampled in 2011 and 2013. The two CORKs are superior than earlier style CORKs in that they are equipped with coated casing and polytetrafluoroethylene fluid delivery lines, reducing the interaction between casing materials with the environment. Additional samples were collected from an earlier style CORK at Borehole 1301A.

The basement fluids are enriched in H2 (0.05–1.8 μmol/kg), suggesting that the ocean basaltic aquifer can support H2-driven metabolism. The basement fluids also contain significant amount of CH4 (5–32 μmol/kg), revealing CH4 as an available substrate for subseafloor basaltic habitats. The δ13C values of CH4 from the three boreholes ranged from −22.5 to −58‰, while the δ2H values ranged from −316 to 57‰. The isotopic compositions of CH4 and the molecular compositions of hydrocarbons suggest that CH4 in the basement fluids is of both biogenic and abiotic origins, varying among sites and sampling times. The δ2H values of CH4 in CORK 1301A fluid samples are much more positive than found in all other marine environments investigated to date and are best explained by the partial microbial oxidation of biogenic CH4. In conclusion, our study shows that CH4 and H2 are persistently available to fuel the deep biosphere and that CH4 is both produced and potentially consumed by microorganisms in the oceanic basement.