Collaborative Research: Bioavailability of mineral-associated molybdenum as a cofactor of Nif nitrogenase for N2 fixation

氮(N)是所有生命所必需的。然而，地球上大部分的氮以大气中氮的形式存在。为了在生物上被吸收，N2需要转化为氨（NH3）。N2到NH3的生物转化被称为N2固定，是由特定的微生物进行的。这些微生物产生一定的蛋白质，称为氮酶，催化N2固定反应。然而，功能性氮酶在其结构中需要钼元素。在海洋缺氧的远古早期地球上，微生物不可能获得钼来制造钼基氮酶，因为钼只存在于固体矿物中。然而，地质和生物证据表明，钼基氮酶是存在的。这个项目的目标就是解决这个矛盾。研究人员假设，当溶解的Mo有限时，能够固定N2的细菌发展出生化策略，直接从固体矿物质中释放和提取Mo。实验将在模拟早期地球条件下使用微生物培养物和含钼矿物的组合进行。微生物策略获取固相Mo将确定使用先进的分析技术。该项目将揭示微生物如何与矿物质相互作用，对养分循环、能量流动、土壤肥力和水质具有重要意义。该项目的结果还可能揭示：1)金属和稀土元素（ree）在环境中的迁移性；2)金属矿床和稀土矿床的形成；3)从尾矿中回收金属和稀土元素。在大氧化事件之前，当含钼矿物和岩石高度不溶时，钼基氮酶的出现引发了一个明显的悖论。该项目的目标是通过测试以下假设来解决这一悖论：在溶解Mo浓度有限的情况下，固氮细菌已经开发出直接从矿物和岩石中提取Mo的策略，用于Mo基氮酶。将设计三组实验来验证这一假设。在第一个实验中，将使用两种固氮细菌，一种是好氧细菌，一种是厌氧细菌，来评估通过分泌Mo结合代谢物从含Mo矿物中提取Mo的效果。将测量n2固定率以确定Mo的生物利用度。在第二个实验中，将两个厌氧培养物（一个Fe（II）氧化剂和一个甲烷菌）与矿物质孵育，研究矿物溶解对Mo释放的影响。在第三个实验中，我们将构建一个简单的微生物群落，以确定微生物相互作用对Mo生物利用度的重要性。互补分析技术将用于测量微生物代谢物，包括ICP-MS， LC-MS和LC-ICP-MS。N2固定率将通过ARA测定、15N标记实验和纳米sims成像来确定。将采用RT-qPCR方法将特定功能基因的表达水平与N2固定率联系起来。XRD， SEM和TEM将用于表征矿物变化。含钼岩石微生物风化的生物特征将由TOF-SIMS和XPS测定。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Nitrogen (N) is essential for all life. However, most N on Earth is in the form of atmospheric N2. To be biologically available for uptake, N2 needs to be converted to ammonia (NH3). Biological conversion of N2 to NH3 is known as N2 fixation and is carried out by specific microbes. These microbes produce certain proteins, called nitrogenase enzymes, to catalyze the N2 fixation reaction. Functional nitrogenases however require the element molybdenum (Mo) in their structures. On the ancient early Earth when the oceans lacked oxygen, Mo should not have been available to microbes to make Mo-based nitrogenase, because Mo was found only in solid minerals. However, geological and biological evidence suggests that Mo-based nitrogenase was present. The goal of this project is to resolve this paradox. The researchers hypothesize that when dissolved Mo is limited, bacteria able to fix N2 developed biochemical strategies to release and extract Mo directly from solid minerals. Experiments will be performed using combinations of microbial cultures and Mo-bearing minerals under simulated conditions of early Earth. Microbial strategies for acquiring solid-phase Mo will be determined using advanced analytical techniques. Insights from this project will reveal how microorganisms interact with minerals, with important implications for nutrient cycling, energy flow, soil fertility, and water quality. The outcome of this project may also shed light on: 1) mobility of metals and rare earth elements (REEs) in the environment; 2) the formation of metal and REE deposits; and 3) recovery of metals and REEs from mine tailings.The emergence of the Mo-based nitrogenase before the Great Oxidation Event when Mo-bearing minerals and rocks were highly insoluble, raises an apparent paradox. The objective of this project is to resolve this paradox by testing the following hypothesis: under limiting concentrations of dissolved Mo, N2-fixing bacteria have developed strategies to extract Mo directly from minerals and rocks to use in Mo-based nitrogenase. Three sets of experiments will be designed to test this hypothesis. In the first experiment, two N2-fixing bacteria, one aerobic, and one anaerobic, will be used to assess extraction of Mo from Mo-bearing minerals via secretion of Mo binding metabolites. N2-fixation rate will be measured to determine Mo bioavailability. In the second experiment, two anaerobic cultures, one Fe(II) oxidizer and one methanogen, will be incubated with the minerals to study the effects of mineral dissolution on Mo release. In the third experiment, a simple microbial community will be constructed to determine the importance of microbial interaction on Mo bioavailability. Complementary analytical techniques will be used to measure microbial metabolites, including ICP-MS, LC-MS, and LC-ICP-MS. N2 fixation rates will be determined by the ARA assay, 15N labelling experiments, and nano-SIMS imaging. RT-qPCR will be performed to correlate expression levels of specific functional genes with N2 fixation rate. XRD, SEM, and TEM will be used to characterize mineralogical changes. Biosignatures from microbial weathering of Mo-bearing rocks will be determined by TOF-SIMS and XPS.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.