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. Here, we initially demonstrate inhibitor- and catalystless 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/liter extracellular NH4+ at a poised potential of −0.3 V (versus 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 to 4 times that of previous e-BNF attempts 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 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.
Free-living nitrogen fixation (FLNF) is a vital source of nitrogen for the initiation and development of ecosystems on bare lands. This process is catalyzed by the enzyme nitrogenase and is energy intensive. Of the three nitrogenase isoforms, molybdenum nitrogenase (Mo-Nase) is more efficient than its vanadium (V) and iron (Fe) counterparts at room temperature. However, the acquisition of Mo, one of the scarcest biometals in soils, represents a major resource investment for soil diazotrophs. In a vegetation restoration chronosequence developed on barren (low carbon and nitrogen) soils of a high-V region, we tested the following two hypotheses. First, the FLNF rate would be limited by the supply of energy. Second, high V availability would cut the cost of V acquisition, and it would be beneficial for diazotrophs to use V-Nase as a complement of Mo-Nase. For the soils collected along the chronosequence, the addition of a carbon cocktail remarkably stimulated the FLNF rate by relieving the energy constraint and by enriching the diazotrophic genus Paenibacillus . Despite the high V content in the soils, we failed to detect the vnf genes of V-Nase by metagenomic sequencing. Meanwhile, we observed a limited contribution of V-Nase activity to FLNF by checking the R ratio (ratio of FLNF rates measured by acetylene reduction and 15 N 2 incorporation) and the production of ethane during acetylene reduction. The reason might be that the Mo availability and temperature in our study area did not reach the thresholds for the onset of V-Nase activity. Overall, our results highlight the importance of carbon supply for FLNF in barren soils, indicating that plants may effectively regulate rhizosphere diazotrophs by secreting root exudates. This may be an important mechanism by which nonnitrogen-fixing plants survive on nitrogen-poor soils at the beginning of primary succession.
Summary Bacteria of the genus Pseudomonas consume preferred carbon substrates in nearly reverse order to that of enterobacteria, and this process is controlled by RNA-binding translational repressors and regulatory ncRNA antagonists. However, their roles in microbe-plant interactions and the underlying mechanisms remain uncertain. Here we show that root-associated diazotrophic Pseudomonas stutzeri A1501 preferentially catabolizes succinate, followed by the less favorable substrate citrate, and ultimately glucose. Furthermore, the Hfq/Crc/CrcZY regulatory system orchestrates this preference and contributes to optimal nitrogenase activity and efficient root colonization. Hfq has a central role in this regulatory network through different mechanisms of action, including repressing the translation of substrate-specific catabolic genes, activating the nitrogenase gene nifH posttranscriptionally, and exerting a positive effect on the transcription of an exopolysaccharide gene cluster. Our results illustrate an Hfq-mediated mechanism linking carbon metabolism to nitrogen fixation and root colonization, which may confer rhizobacteria competitive advantages in rhizosphere environments.
危险品化学品经营许可证（带存储）