Nitrogen Fixation and Ammonium Assimilation Pathway Expression of Geobacter sulfurreducens Changes in Response to the Anode Potential in Microbial Electrochemical Cells

Nitrogen gas (N₂) fixation in the anode-respiring bacterium Geobacter sulfurreducens occurs through complex, multistep processes. Optimizing ammonium (NH₄⁺) production from this bacterium in microbial electrochemical technologies (METs) requires an understanding of how those processes are regulated in response to electrical driving forces. In this study, we quantified gene expression levels (via RNA sequencing) of G. sulfurreducens growing on anodes fixed at two different potentials (-0.15 V and +0.15 V versus standard hydrogen electrode). The anode potential had a significant impact on the expression levels of N₂ fixation genes. At -0.15 V, the expression of nitrogenase genes, such as nifH, nifD, and nifK, significantly increased relative to that at +0.15 V, as well as genes associated with NH₄⁺ uptake and transformation, such as glutamine and glutamate synthetases. Metabolite analysis confirmed that both of these organic compounds were present in significantly higher intracellular concentrations at -0.15 V. N₂ fixation rates (estimated using the acetylene reduction assay and normalized to total protein) were significantly larger at -0.15 V. Genes expressing flavin-based electron bifurcation complexes, such as electron-transferring flavoproteins (EtfAB) and the NADH-dependent ferredoxin:NADP reductase (NfnAB), were also significantly upregulated at -0.15 V, suggesting that these mechanisms may be involved in N₂ fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N₂ fixation rates. We hypothesize that at -0.15 V, they increase N₂ fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use. IMPORTANCE Biological nitrogen fixation coupled with ammonium recovery provides a sustainable alternative to the carbon-, water-, and energy-intensive Haber-Bosch process. Aerobic biological nitrogen fixation technologies are hindered by oxygen gas inhibition of the nitrogenase enzyme. Electrically driving biological nitrogen fixation in anaerobic microbial electrochemical technologies overcomes this challenge. Using Geobacter sulfurreducens as a model exoelectrogenic diazotroph, we show that the anode potential in microbial electrochemical technologies has a significant impact on nitrogen gas fixation rates, ammonium assimilation pathways, and expression of genes associated with nitrogen gas fixation. These findings have important implications for understanding regulatory pathways of nitrogen gas fixation and will help identify target genes and operational strategies to enhance ammonium production in microbial electrochemical technologies.