In Situ Electrochemical Studies of the Terrestrial Deep Subsurface Biosphere at the Sanford Underground Research Facility, South Dakota, USA

ABSTRACTThe terrestrial deep subsurface is host to significant and diverse microbial populations. However, these microbial populations remain poorly characterized, partially due to the inherent difficulty of sampling,in situstudies, and isolating of thein situmicrobes. Motivated by the ability of microbes to gain energy from redox reactions at mineral interfaces, we here presentin situelectrochemical colonization (ISEC) as a method to directly study microbial electron transfer activity and to enable the capture and isolation of electrochemically active microbes. We installed a potentiostatically controlled ISEC reactor containing four working electrodes 1500 m below the surface at the Sanford Underground Research Facility. The working electrodes were poised at different redox potentials, spanning anodic to cathodic, to mimic energy-yielding mineral reducing and oxidizing reactions predicted to occur at this site. We present a 16S rRNA analysis of thein situelectrode-associated microbial communities, revealing the dominance of novel bacterial lineages under cathodic conditions. We also demonstrate that thein situelectrodes can be further used for downstream electrochemical laboratory enrichment and isolation of novel strains. Using this workflow, we isolatedBacillus,Anaerospora,Comamonas,Cupriavidus, andAzonexusstrains from the electrode-attached biomass. Finally, the extracellular electron transfer activity of the electrode-oxidizingComamonasstrain (isolated at −0.19 V vs. SHE and designated WE1-1D1) and the electrode-reducingBacillusstrain (isolated at +0.53 V vs. SHE and designated WE4-1A1-BC) were confirmed in electrochemical reactors. Our study highlights the utility ofin situelectrodes and electrochemical enrichment workflows to shed light on microbial activity in the deep terrestrial subsurface.SIGNIFICANCEA large section of microbial life resides in the deep subsurface, but an organized effort to explore this deep biosphere has only recently begun. A detailed characterization of the resident microbes remains scientifically and technologically challenging due to difficulty in access, sampling, and emulating the complex interactions and energetic landscapes of subsurface communities with standard laboratory techniques. Here we describe an in situ approach that exploits the ability of many microbes to perform extracellular electron transfer to/from solid surfaces such as mineral interfaces in the terrestrial subsurface. By deploying and controlling the potential of in situ electrodes 4850 ft below the surface at the Sanford Underground Research Facility (South Dakota, USA), we highlight the promise of electrochemical techniques for studying active terrestrial subsurface microbial communities and enabling the isolation of electrochemically active microbes.