OCE-PRF Uncovering the Genetic Basis of Novel Oxidative Extracellular Electron Transfer Mechanisms in an Electrochemically Active Marine Bacterial Consortium

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).

Recently, it was discovered that some microbes are capable of respiring (‘breathing’) or deriving energy (‘eating’) from solid-phase minerals located outside the cell. This is facilitated by extracellular electron transfer (EET): the process by which some microbes shuttle electrons into and out of their cells from/to solid materials. EET can be evaluated in a laboratory setting, in which microbes are grown on electrodes and the generation of biological current – the flow of electrons to/from the electrode – can be measured.

In these experiments, the electrodes are poised at specific voltages (redox potentials) that function as proxies for mineral surfaces. Much of our knowledge of EET has been obtained from just a few model organisms capable of respiratory mineral reduction, in which electrons are transferred out of the cell onto oxidized solid-phase materials, such as iron and manganese oxides, which are prevalent in freshwater and marine sediments.

However, little is known about the mechanism or ecological implications of oxidative EET, the process by which electrons are transferred from reduced minerals into the cell as a source of energy (‘eating’). Evidence from microbial community surveys suggests that organisms inhabiting a wide variety of habitats, including both freshwater and marine environments, may be capable of EET.

This project will expand upon our understanding of the genetic basis of oxidative EET in microbes isolated from marine sediments grown individually and in a co-culture consortium. Further, this information will provide insight into the role of EET in global elemental cycling and may inform alternative clean energy generation, bioremediation, and wastewater treatment efforts using electrochemically active organisms.

This project will provide educational and training opportunities to undergraduate students, who will be recruited to aid with research tasks. Community outreach activities, particularly in underserved areas of Cincinnati, will be organized to provide middle-school aged children with hands-on educational opportunities focused around electromicrobiology and bioenergy production.

The discovery of extracellular electron transfer (EET) has important implications for a wide range of biogeochemically important processes. In marine subsurface sediments, where microbial biomass is estimated to account for 0.6% of the total biomass on Earth, lithotrophic metabolisms are difficult to detect since the genetic basis of oxidative EET remains largely unknown and uncharacterized, and ‘omics’ studies fail to identify genes or proteins involved in this process.

This significant knowledge gap, coupled with direct measurements of microbial metabolism in the ‘deep’ and ‘dark’ ocean far exceeding the expected models based on the influx of organic carbon to the system, strongly suggest that the importance of lithotrophic metabolisms, and the mechanisms by which these metabolisms operate, has been overlooked and understanding and detection of these processes in nature is crucial. Furthermore, the ecology of EET in polymicrobial communities is poorly understood, but a variety of data imply that diverse microbial communities are important for stable and processive environmental function.

Thus, the overarching goal of this project is to gain insight into the genetic basis of EET in oxidative processes, and into the ecological implications of these processes, in two genetically tractable, electrode-oxidizing lithotrophic marine sediment bacteria in pure culture and in co-culture. Cutting-edge, targeted high-throughput sequencing techniques will be used to identify genes putatively involved in oxidative EET and genes that facilitate interspecies electron transfer in co-cultures.

Gene deletion mutants will be constructed, and mutant strains will be assayed for their capacity for oxidative EET and interspecies electron transfer. Not only will this allow for a better understanding of microbial community ecology in marine systems, but this study will also provide insight into oxidative EET in a wide array of habitats, including marine and terrestrial subsurface environments and engineered environments where reduced electron sources are prevalent.

From an applied and biotechnological standpoint, results from this work will enhance the capacity for evaluating microbe-electrode interactions in genetically tractable organisms, and may inform industrial applications of electrochemically active organisms, such as clean energy generation, wastewater treatment, and electrosynthesis.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.