In Situ Growth of Halophilic Bacteria in Saline Fracture Fluids from 2.4 km below Surface in the Deep Canadian Shield.

Energy derived from water-rock interactions such as serpentinization and radiolysis, among others, can sustain microbial ecosystems deep within the continental crust, expanding the habitable biosphere kilometers below the earth's surface. Here, we describe a viable microbial community including sulfate-reducing microorganisms from one such subsurface lithoautotrophic ecosystem hosted in fracture waters in the Canadian Shield, 2.4 km below the surface in the Kidd Creek Observatory in Timmins, Ontario. The ancient groundwater housed in fractures in this system was previously shown to be rich in abiotically produced hydrogen, sulfate, methane, and short-chain hydrocarbons. We have further investigated this system by collecting filtered water samples and deploying sterile in situ biosampler units into boreholes to provide an attachment surface for the actively growing fraction of the microbial community. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, and DNA sequencing analyses were undertaken to classify the recovered microorganisms. Moderately halophilic taxa (e.g., ) were recovered from all sampled boreholes, and those boreholes that had previously been sealed to equilibrate with the fracture water contained taxa consistent with sulfate reduction (e.g., and hydrogen-driven homoacetogenesis (e.g., ). In contrast to this "corked" borehole that has been isolated from the mine environment for approximately 7 years at the time of sampling, we sampled additional open boreholes. The waters flowing freely from these open boreholes differ from those of the long-sealed borehole. This work complements ongoing efforts to describe the microbial diversity in fracture waters at Kidd Creek in order to better understand the processes shaping life in the deep terrestrial subsurface. In particular, this work demonstrates that anaerobic bacteria and known halophilic taxa are present and viable in the fracture waters presently outflowing from existing boreholes. Major cations and anions found in the fracture waters at the 2.4 km level of the mine are also reported.