Bioelectrochemical nitrogen fixation to extracellular ammonium by Pseudomonas stutzeri

Diazotrophs can produce bioavailable nitrogen from inert N2 gas by bioelectrochemical nitrogen fixation (e-BNF), which is emerging as an energy-saving and highly selective strategy for agriculture and industry. However, current e-BNF technology is impeded by requirements for NH4 +-assimilation inhibitors to facilitate intracellular ammonia secretion and precious metal catalysts to generate H2 as the energy-carrying intermediate. Herein, we initially demonstrate inhibitor- and catalyst-less extracellular NH4 + production by the diazotroph Pseudomonas stutzeri A1501 using an electrode as the sole electron donor. Multiple lines of evidence revealed that P. stutzeri produced 2.32±0.25 mg/L of extracellular NH4 + at a poised potential of -0.3 V (vs. standard hydrogen electrode (SHE)) without the addition of inhibitors or expensive catalysts. The electron uptake mechanism was attributed to the endogenous electron shuttle phenazine-1-carboxylic acid, which was excreted by P. stutzeri and mediated electron transfer from electrodes into cells to directly drive N2 fixation. The faradaic efficiency was 20%±3% which was 2-4 times that of previous e-BNF using the H2-mediated pathway. This study reports a diazotroph capable of producing secretable NH4 + via extracellular electron uptake, which has important implications for optimizing the performance of e-BNF systems and exploring the novel nitrogen-fixing mode of syntrophic microbial communities in the natural environment.IMPORTANCE Ammonia greatly affects the global ecology, agriculture and the food industry. Diazotrophs with an enhanced capacity of extracellular NH4 + excretion have been proven to be more beneficial to the growth of microalgae and plants, whereas most previously reported diazotrophs produce intracellular organic nitrogen in the absence of chemical suppression and genetic manipulation. Here, we demonstrate that Pseudomonas stutzeri A1501 is capable of extracellular NH4 + production without chemical suppression or genetic manipulation when the extracellular electrode is used as the sole electron donor. We also reveal the electron uptake pathway from the extracellular electron-donating partner to P. stutzeri A1501 via redox electron shuttle phenazines. Since both P. stutzeri A1501 and potential electron-donating partners (such as electroactive microbes and natural semiconductor minerals) are abundant in diverse soils and sediments, P. stutzeri A1501 has broader implications on the improvement of nitrogen fertilization in the natural environment.