Nitrogen Fixation and Ammonium Assimilation Pathway Expression of Geobacter sulfurreducens Changes in Response to the Anode Potential in Microbial Electrochemical Cells
Nitrogen Fixation and Ammonium Assimilation Pathway Expression of Geobacter sulfurreducens Changes in Response to the Anode Potential in Microbial Electrochemical Cells
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DOI：
10.1128/aem.02073-22
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发表时间：
2023-03-28
影响因子：
4.4
通讯作者：
Call，Douglas F.
中科院分区：
文献类型：
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作者：
Ortiz-Medina，Juan F.;Poole，Mark R.;Call，Douglas F.
氮气（N2）固定在阳极呼吸细菌硫还原地杆菌发生复杂的，多步骤的过程。在微生物电化学技术（METs）中优化这种细菌的铵（NH4+）生产需要了解这些过程是如何响应电驱动力进行调节的。在这项研究中，我们通过RNA测序，定量了生长在两种不同电位（与标准氢电极相比为- 0.15 V和+0.15 V）的阳极上的G.硫还原菌的基因表达水平。阳极电位对氮固定基因的表达水平有显著影响。与+0.15 V相比，−0.15 V时氮酶基因nifh、nifD、nifk以及与NH4+摄取和转化相关的基因谷氨酰胺、谷氨酸合成酶的表达量显著增加。代谢物分析证实，在−0.15 V时，这两种有机化合物在细胞内的浓度显著提高。n2固定率（使用乙炔还原法估计并归一化到总蛋白）在−0.15 V时显著较大。表达基于黄素的电子分叉复合物的基因，如电子转移黄蛋白（EtfAB）和nadh依赖的铁氧化还蛋白：NADP还原酶（NfnAB），在−0.15 V下也显著上调，表明这些机制可能参与了该电位下的n2固定。我们的研究结果表明，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氮固定率。我们假设在- 0.15 V时，它们增加n2固定活性以帮助维持氧化还原稳态，并且它们利用电子分岔作为优化能量产生和使用的策略。生物固氮与铵回收相结合，为碳、水和能源密集型的Haber-Bosch工艺提供了一种可持续的替代方案。好氧生物固氮技术受到氧气对固氮酶的抑制作用的阻碍。电驱动厌氧微生物的生物固氮电化学技术克服了这一挑战。以硫还原地杆菌（Geobacter sulfate reducens）为模型外电重氮养菌，我们发现微生物电化学技术中的阳极电位对固氮速率、铵同化途径和固氮相关基因的表达有显著影响。这些发现对于理解固氮调控途径具有重要意义，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的氨产量。
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 N2fixation genes. At −0.15 V, the expression of nitrogenase genes, such asnifH,nifD, andnifK, 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. N2fixation 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 N2fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N2fixation rates. We hypothesize that at −0.15 V, they increase N2fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use.IMPORTANCEBiological 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.