Collaborative Research: ETBC: Deep Crustal Biosphere: Microbial Cycling of Carbon
Recent studies of the continental subsurface microbial ecosystem present in the Witwatersrand Basin, South Africa have shown that with increasing depth and fracture water age and salinity, biogenic methane diminishes and abiogenic hydrocarbons and H2 increase (7, 10) and the concentration of planktonic cells slowly declines (6) . Similarly studies of the 16S rRNA gene of the planktonic community revealed a decrease in the relative abundance of methanogens and an increase in the relative abundance of low G+C Firmicutes with increasing depth (4) . Metagenome analyses of one member of this Firmicutes community, D. Audaxviator, indicated that it was capable of both heterotrophic and chemoautotrophic activity (1) . An in situ incubation experiment suggests that some of the Firmicutes are acetogens that may support the aceticlastic methanogens (8) . Geochemical and isotopic analyses appear to indicate that the abiogenic hydrocarbons are not being utilized by the microbial community, but that a chemoautotrophic ecosystem is being sustained by radiolytic generation of H2 and oxidants (5) . Abiogenic hydrocarbon gases and elevated H2 have also been reported from the Canadian and Fennoscandian Precambrian shields (7) and from ocean floor vents. The subsurface ecosystem present in the Witwatersrand Basin, therefore, is potentially wide spread in both the continental and marine crust, yet quite distinct from continental sedimentary basins or coastal margin sea floor sediments where degradation of photosynthetically produced organic matter deposited within the strata is believed to limit the extent of the subsurface microbial ecosystem (2, 3) . Alternatively, organic acids, thermogenically produced ~2 by ago from the thin shale layers present in the Witwatersrand Basin, could be sustaining the microbial community observed today.
The proposed project will determine whether the subsurface microbial community in the Witwatersrand Basin (Fig. 1) and in the Bushveld Igneous Complex (BIC) (Fig. 2) is deriving its organic substrates from chemolithoautrophy, i.e the SLiMeS hypothesis of Stevens and McKinley (9), or from the abiogenic hydrocarbons either by syntrophic consortia or SRB capable of hydrocarbon degradation, or from thermogenic degradation of the organic matter deposited in the 2.9 Ga shale or of the abiogenic hydrocarbons. In the case of the BIC, thermogenic degradation of organic matter is not an option since by its igneous nature photosentate is not present and abiogenic hydrocarbons have yet to be detected. To distinguish between these alternative we will utilize a combination of C and H stable isotope and 14C analyses of dissolved hydrocarbons, inorganic carbon and organic acids, C and H stable isotope and 14C analyses of lipids, stable isotope probes, SIP, of the 16S rDNA and 16S rRNA genes, functional gene analyses and GCMS characterization of the TOC/DOC of fracture water. These analyses will be performed on fracture water and planktonic cells collected from boreholes in South African mines. Sites will be selected to represent a range of ages from millions of years to modern and differing biogeochemical processes (sulfate reduction and methanogenesis). Groundwater age will be quantified by noble gas analysis. Cells will be quantified by total cell counts, LIVE/DEAD BacLight (dead cells) and CARD-FISH (metabolically active cells). Microorganisms involved in the production of organic acids and in the degradation of hydrocarbons will be identified by SIP analyses.
Broader Impacts. A broad scientific impact of this study will to develop techniques that can be applied to other environments, e.g., deep-sea sediment and ocean crust. The project will engage undergraduate and graduate students in hands-on interdisciplinary research, with the aim of recruiting underrepresented minority students. Undergrads will contribute through molecular biology approaches and thus will have ownership of a significant project component. The grad student ‘s project will integrate geochemistry and modeling. Students will participate in fieldwork. The project will build upon ongoing collaborations with South African scientists and students. A publicly accessible web site will archive SEM photographs of subsurface microorganisms, photos and videos of the underground safaris.