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(Invited, Digital Presentation) Using Electricity to Enhance Microbial Fixation of Nitrogen Gas and Generation of Ammonium

There is a growing need to replace the Haber-Bosch process of converting nitrogen gas (N2) into ammonia (NH3) for fertilizers and fuels. To generate the 190 million tons of NH3 each year through this process, roughly 2–3% of global energy is consumed and over 740 million tons of CO2 released. Microbial processes may provide a low-energy and sustainable alternative. The nitrogenase enzymes in many free-living microorganisms convert N2 into ammonium (NH4
 +), generating fixed nitrogen for cellular growth. Attempts to increase NH4
 + yields from microorganisms through genetic engineering are seeing signs of success, but there is a lack of methods to increase NH4
 + generation rates. For aerobic microorganisms, providing sufficient oxygen gas as a terminal electron acceptor can increase these rates; however, oxygen gas can also shut down the nitrogenase enzyme. To overcome this limitation, we are exploring the ability of electricity to drive N2 fixation in anaerobic, exoelectrogenic bacteria. These bacteria have the unique ability to respire on electrodes in microbial electrochemical technologies (METs). Applying a voltage to METs increases the respiration rates of exoelectrogens. Since the most abundant exoelectrogenic bacteria in METs are affiliated with the Geobacter genus and many known Geobacter species are N2 fixers in soils and sediments, we hypothesized that 1) anode biofilms would exhibit N2 fixation activity, 2) N2 fixation rates would respond to an applied voltage, and 3) NH3 could be generated by inhibiting NH4
 + uptake pathways. To test our hypothesis, we set up lab-scale microbial electrolysis cells (MECs) and promoted the growth of a Geobacter-rich anode biofilm using defined medium and operating conditions. We found that MET anode biofilms are capable of fixing N2 while maintaining high current densities over several months. N2 fixation rates increased more than three times to a max of 26 [nmol-ethylene (a proxy for N2 fixation) / min] when the applied voltage increased from 0.7 V to 1.0 V. By adding an NH4
 + uptake inhibitor, NH3 was generated and recovered from the MET. Using a pure culture of G. sulfurreducens, we also conducted transcriptomics under N2 fixation conditions in METs operated at two different fixed anode potentials. This approach allowed us to identify what genes were up- and down-regulated under the two conditions. 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 the low anode potential [−0.15 V vs. standard hydrogen electrode (SHE)], 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. sulfurreducens regulates N2 fixation and NH4
 + production, we are currently developing new strains of G. sulfurreducens using the CRISPR platform that can excrete NH4
 + during N2 fixation.