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Abstract：
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.
摘要：
阳极呼吸细菌中的氮气（N2）固定是通过复杂的，多步骤过程。在微生物电化学技术（MET）中优化该细菌的铵（NH4）生产需要了解如何响应电驱动力来调节这些过程。在这项研究中,我们定量了在固定在两个不同电位（-0.15V和+0.15V相对于标准氢电极）的阳极上生长的硫还原菌的基因表达水平（通过RNA测序）。阳极电位对N2固定基因的表达水平有显著影响。在-0.15V时，固氮酶基因的表达，比如nifH，nifD,还有nifK,相对于+0.15V时显著增加，以及与NH4+吸收和转化相关的基因，如谷氨酰胺和谷氨酸合成酶。代谢物分析证实，这两种有机化合物在-0.15V时均以显着更高的细胞内浓度存在。N2固定率（使用乙炔还原测定法估算并标准化为总蛋白）在-0.15V时显着更大。表达基于黄素的电子分叉复合物的基因，例如电子转移黄素蛋白（EtfAB）和NADH依赖性铁氧还蛋白：NADP还原酶（NfnAB），在-0.15V时也显著上调，这表明这些机制可能与该电位的N2固定有关。我们的结果表明，在能量受限的情况下(即，低阳极电位)，细胞增加了每个细胞的呼吸和N2固定率。我们假设在-0.15V时，它们增加N2固定活性以帮助维持氧化还原稳态，他们利用电子分叉作为优化能源产生和使用的策略。重要性生物固氮与铵回收相结合，为碳提供了可持续的替代方案-，水-,和能源密集型Haber-Bosch工艺。氧气对固氮酶的抑制作用阻碍了好氧生物固氮技术。厌氧微生物电化学技术中的电驱动生物固氮克服了这一挑战。使用硫化还原Geobacter作为模型外电生重氮菌，我们表明，微生物电化学技术中的阳极电位对氮气固定速率有重大影响，铵同化途径，以及与固氮相关的基因的表达。这些发现对于理解氮气固定的调节途径具有重要意义，并将有助于确定目标基因和操作策略，以提高微生物电化学技术中铵的产量。
