(Invited) Using the Model Bacterium Geobacter Sulfurreducens to Understand Microbial Electrochemical Conversion of Nitrogen Gas into Ammonium
Authors: Medina Juan Fausto Ortiz , Poole Mark , Grunden Amy M , Call Doug F 
Published: September 2023

There is a growing need to decrease reliance on the Haber-Bosch process. To convert nitrogen gas (N2) into ammonia (NH3) for fertilizers, this temperature- and pressure-intensive process consumes roughly 2–3% of global energy and releases over 740 million tons of CO2 each year. Many microorganisms naturally convert or “fix” N2 into ammonium (NH4
+) at room temperature and pressure using an enzyme called the nitrogenase. Attempts to increase NH4
+ yields from microorganisms through genetic engineering are seeing signs of success, but we lack methods to increase NH4
+ generation rates and overcome one of the largest challenges: O2 gas. Increasing O2 gas concentrations typically increases metabolic rates of aerobic microorganisms; however, in the case of microorganisms that fix N2 (called diazotrophs), O2 can shut down the nitrogenase and stop cell growth. To overcome these challenges, we are using electricity to drive N2 fixation in exoelectrogenic bacteria. These bacteria have the unique ability to naturally transfer electrons to anode electrodes in microbial electrochemical technologies (METs). Increasing the voltage applied to METs increases the metabolic rates of exoelectrogens. The anode chamber is kept anaerobic, which eliminates O2-driven inhibition of the nitrogenase. We previously showed that the N2 fixation rates of a mixed microbial community in a MET increased more than three times when the applied whole-cell potential increased from 0.7 V to 1.0 V. By adding a chemical inhibitor that prevented the incorporation of NH4
+ into larger biomolecules, NH4
+ was excreted by the cells and into the medium. Based on the acetylene reduction assay (a proxy for N2 fixation), we estimated that we recovered about 10% of the theoretical NH4
+generated by the cells. If close to 100% of the theoretical NH4
+ could be excreted from the cells and recovered, we predicted an energy demand of around 3 MJ/mol-NH4
+, which is close to the range of value reported for the Haber-Bosch process. To develop METs that generate NH4
+ at competitive production rates and energy demands, we are now focusing on the model exoelectrogenic diazotroph Geobacter sulfurreducens. Using a single organism with a fully sequenced genome allows us to understand how the N2 fixation process responds to electrochemical variables. Towards these efforts, we conducted a transcriptomic analysis of G. sulfurreducens at two fixed anode potentials: +0.15 V and −0.15 V. This approach revealed which genes are turned on and off in response to these potentials. The presence of NH4
+ decreased the expression of genes associated with N2 fixation which was expected due to the known sensitivity of nitrogenases to NH4
+. On the other hand, the two anode potentials had a dramatic and unexpected impact on the expression levels of N2 fixation genes. At −0.15 V, nitrogenase genes were significantly up-regulated, as were genes associated with NH4
+ uptake and transport, such as glutamine and glutamate synthetases. Considering that −0.15 V provides less energy to the cells relative to +0.15 V (based on thermodynamic predictions), the results suggest that the cells responded to this highly energy-constrained environment by increasing expression of N2 fixation pathways. Based on our new knowledge of how G. sulfurredcuens regulates N2 fixation and NH4
+ production, we have developed new strains of G. sulfurreducens that can excrete NH4
+ during N2 fixation. The new engineering toolkit that we have created in this bacterium can now be leveraged to maximize NH4
+ production in METs.