Continental subsurface environments can present significant energetic challenges to the resident microorganisms. While these environments are geologically diverse, potentially allowing energy harvesting by microorganisms that catalyze redox reactions, many of the abundant electron donors and acceptors are insoluble and therefore not directly bioavailable. Extracellular electron transfer (EET) is a metabolic strategy that microorganisms can deploy to meet the challenges of interacting with redox-active surfaces. Though mechanistically characterized in a few metal-reducing bacteria, the role, extent, and diversity of EET in subsurface ecosystems remains unclear. Since this process can be mimicked on electrode surfaces, it opens the door to electrochemical techniques to enrich for and quantify the activities of environmental microorganisms in situ. Here, we report the electrochemical enrichment of microorganisms from a deep fractured-rock aquifer in Death Valley, CA, USA. In experiments performed in mesocosms containing a synthetic medium based on aquifer chemistry, four working electrodes (WEs) were poised at different redox potentials (272, 373, 472, 572 mV vs. SHE) to serve as electron acceptors, resulting in anodic currents coupled to the oxidation of acetate during enrichment. The anodes were dominated by Betaproteobacteria from the families Comamonadaceae and Rhodocyclaceae. A representative of each dominant family was subsequently isolated from electrode-associated biomass. The EET abilities of the isolated Delftia strain (designated WE1-13) and Azonexus strain (designated WE2-4) were confirmed in electrochemical reactors using WEs poised at 522 mV vs. SHE. The rise in anodic current upon inoculation was correlated with a modest increase in total protein content. Both genera have been previously observed in mixed communities of microbial fuel cell enrichments, but this is the first direct measurement of their electrochemical activity. While alternate metabolisms (e.g., nitrate reduction) by these organisms were previously known, our observations suggest that additional ‘hidden’ interactions with external electron acceptors are also possible. Electrochemical approaches are well positioned to dissect such extracellular interactions that may be prevalent in the subsurface.

1Continental subsurface environments can present significant energetic challenges to the resident 2 microorganisms. While these environments are geologically diverse, potentially allowing energy 3 harvesting by microorganisms that catalyze redox reactions, many of the abundant electron 4 donors and acceptors are insoluble and therefore not directly bioavailable. Extracellular electron 5 transfer (EET) is a metabolic strategy that microorganisms can deploy to meet the challenges of 6 interacting with redox-active surfaces. Though mechanistically characterized in a few metal-7 reducing bacteria, the role, extent, and diversity of EET in subsurface ecosystems remains 8 unclear. Since this process can be mimicked on electrode surfaces, it opens the door to 9 electrochemical techniques to enrich for and quantify the activities of environmental 10 microorganisms in situ.

Low oxygen availability in soil can impair root function, thereby decreasing agronomic productivity. Without oxygen to support mitochondrial respiration, energy levels in roots may limit mineral nutrient (N, P, K) transport. Traditional methods for measuring soil oxygenation include the use of redox electrodes and polarographic oxygen sensors. These approaches are limited to measuring oxygen concentrations at specific locations and cannot determine the bio-availability of that oxygen to a growing root. An innovative approach has been developed for direct measurement of oxygen bioavailability. Instead of building the sensor to measure oxygen concentrations, it is possible to construct an electrochemical system in a manner that makes it sensitive to changes in oxygen transport and availability in the soil. Furthermore, it is even possible to construct the sensor to match the oxygen consumption characteristics of a specific root tip, which is important because most metabolism and nutrient uptake occurs here. This is accomplished by using a conductive-gel membrane system that allows sensor profiles to be engineered to match the metabolic profiles of specific species. If constructed to these standards, the sensor will biomimetically replicate biophysical oxygen depletion profiles that are analogous to those found in the rhizosphere of a growing root tip. The biomimetic root oxygen bio-availability (ROB) sensor integrates all biotic and abiotic factors in the soil that limit oxygen transport to the root, while providing real-time sensing. This new sensor technology could be used as a research tool or as a closed-loop control system for field and greenhouse irrigation.