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

Abstract

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., Marinobacter, Idiomarina, Chromohalobacter, Thiobacillus, Hyphomonas, Seohaeicola) 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., Desulfotomaculum) and hydrogen-driven homoacetogenesis (e.g., Fuchsiella). 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.

Keywords: deep life observatory; groundwater; microbial diversity; subsurface biosphere.

Figure 1

Study location: Kidd Creek Observatory in the Superior Province of the Canadian Shield north of Timmins, Ontario, Canada. Thin blue vertical lines represent the shaft and cage system for accessing the subsurface, while spiraling blue lines represent the ramp road descending from the surface to the Kidd Creek Observatory located at 2.4 km (L. 7850) below surface.

Figure 2

(A) SEM image and (B) detailed view of a biofilm feature in FW12299, which has been sealed from the mine environment via a packer or “CORK” (circulation obviation retrofit kit). (C) Energy-dispersive X-ray spectroscopy chemical analysis (EDS) spectrum of the biofilm. (D) Biofilm from hydrogen- and methane-rich waters from Copper Cliff South mine for comparison [25]; shown with permission from Taylor & Francis Ltd., www.tandfonline.com. (E–H) EDS imaging of elements identified within the biofilm as collected by secondary electron detector.

Figure 3

(A) SEM image and (B) detailed view of a second biofilm morphology from FW12299 with (C–E) EDS elemental imaging biofilm as collected by secondary electron detector.

Figure 4

16S classification by phylum and class for abundant operational taxonomic units (OTUs) (those representing >0.5% total sequences) on the basis of the V4 hypervariable region of the 16S gene. Bacteroidetes were enriched in the borehole that was recently exposed to the mine environment (FW12322). Clostridia were enriched in boreholes sealed from the mine environment (FW12887A and FW12299).

Figure 5

Agglomerative clustering of Kidd Creek filter and slide sample DNA results on the basis of community similarity using Ward’s method. Agglomerative coefficient = 0.73. Samples from packered boreholes isolated from the mine environment (FW12287A, FW12299) clustered together while those from a borehole freely discharging into the mine atmosphere (FW12322) clustered separately. Filtered water samples from FW12299 clustered only with negative controls, suggesting too little biomass was present to overcome background signal.

Figure 6

Genera within Gammaproteobacteria showing predominance of the moderately halophilic chemoorganotroph Chromohalobacter in FW12322, which has been continuously open to exchange with the mine environment as the fluids discharge.