Introduction: The potential for liquid water in the subsurface of Mars supports the possibility of an active deep subsurface biosphere. Given that the majority of prokaryotic life on Earth may exist in deep subsurface biofilms (Flemming and Wuertz, 2019), it is critical to understand the ecology of these communities to inform the search for life on Mars.
Minerals are likely an important energy source for the deep continental biosphere in which oxygen and organic carbon are limited. However, microbe-mineral interactions in these environments are not well understood in part due to sampling challenges. The Deep Mine Microbial Observatory (DeMMO) in Lead, South Dakota, offers a unique portal to the deep biosphere, providing access to fracture fluids that host microbial life to a depth of 4,850 ft (1478 m) (Osburn et al. 2017). This iron-rich Mars analog system consists of a diversity of minerals that may support microbial metabolisms. Biofilm formation on fracture surfaces may facilitate these metabolisms by allowing close contact between cells and minerals that is often required by microbes for extracellular electron transport (EET) (Shi et al. 2016). Here, we describe in situ cultivation experiments with minerals targeting mineral-hosted deep subsurface biofilm communities.
Methods: An array of flow through colonization reactors were filled with minerals representative of DeMMO lithology (pyrite, hematite, magnetite, siderite, pyrolusite, muscovite, gypsum, and calcite) or inert control substrates (glass beads, glass wool, and sand). Reactors were connected to borehole outflows at three sites located at depths of 800, 2,000, and 4,850 ft for 2-8 months to allow for colonization by biofilm communities prior to harvesting.
We identified biofilm community members using 16s rRNA gene amplicon sequencing. Sequences were binned into operational taxonomic units and assigned taxonomy with QIIME (Caporaso et al. 2010) and the SILVA132 taxonomy reference database (Quast et al. 2012). We used scanning electron microscopy to document biofilm cell morphologies and estimate cell densities on polished mineral chips and glass slide controls. We interpret biofilm community composition and colonization patterns on each substrate in the context of thermodynamic models of metabolic reactions. Models were generated from DeMMO geochemical data using SPECE8 (Bethke et al. 2009) and CHNOSZ (Dick 2008). Results: Mineral experiments enriched for dramatically different communities than fluids and from inert control substrates, suggesting that members of the biofilms may be using minerals as both a surface for attachment and a metabolic energy source. Specifically, experiments with pyrolusite (MnO2) yielded the highest cell densities of all minerals types and enriched for members of the Desulfobulbaceae and Thermodesulfovibrionia. Further, sheath-like structures were observed on the pyrolusite which may play a role in EET. These results align with thermodynamic models of DeMMO metabolisms, which suggest that pyrolusite is a highly favorable electron acceptor, especially when coupled to the oxidation of elemental sulfur or hydrogen. Further, our community data suggest the attached vs. suspended lifestyles of several candidate divisions, including Latescibacteria (WS3) and Omnitrophica (OP3) which appear to be important members of the biofilm and fluid communities, respectively.
Conclusion: Our in situ cultivation approach successfully targeted the growth of deep subsurface biofilm communities, offering insight into the metabolic and biomass potential of the terrestrial deep biosphere across a broad range of depths and geochemical conditions. Additionally, our results shed light on the lifestyles of candidate phyla that comprise microbial dark matter in the deep biosphere. We are working to further characterize DeMMO biofilm metabolisms and the potential for EET through laboratory-based cultivation experiments, CARD-FISH probing, and atomic force microscopy. Our findings underscore the potential for a mineral-hosted deep subsurface biosphere on Mars.
References: Bethke, C. et al. (2009) Geochemist’s Workbench: Release 8.0 GWB Essentials Guide. Caporaso, J. G. et al. (2010) Nat. Methods, 7. Dick, J. M. (2008), Geochemical Transactions, 9. Flemming, H. C. and Wuertz, S. (2019) Nat. Rev. Microbio., 1. Osburn, M. R. et al. (2017) AbSciCon, Abstract#3205.