ABSTRACT
The 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 situ studies, and isolating of the in situ microbes. Motivated by the ability of microbes to gain energy from redox reactions at mineral interfaces, we here present in situ electrochemical 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 the in situ electrode-associated microbial communities, revealing the dominance of novel bacterial lineages under cathodic conditions. We also demonstrate that the in situ electrodes can be further used for downstream electrochemical laboratory enrichment and isolation of novel strains. Using this workflow, we isolated Bacillus, Anaerospora, Comamonas, Cupriavidus, and Azonexus strains from the electrode-attached biomass. Finally, the extracellular electron transfer activity of the electrode-oxidizing Comamonas strain (isolated at −0.19 V vs. SHE and designated WE1-1D1) and the electrode-reducing Bacillus strain (isolated at +0.53 V vs. SHE and designated WE4-1A1-BC) were confirmed in electrochemical reactors. Our study highlights the utility of in situ electrodes and electrochemical enrichment workflows to shed light on microbial activity in the deep terrestrial subsurface.
SIGNIFICANCE A 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.
