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

合作研究：矿物相关钼作为 Nif 固氮酶辅助因子固定 N2 的生物利用度

• 批准号: 1937423
• 负责人: Hailiang Dong
• 金额: $ 31.19万
• 依托单位: Miami University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1937423&HistoricalAwards=false
• 关键词: Collaborative; Research; Bioavailability; mineral; associated

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.

氮（N）是所有生命所必需的。然而，地球上的大部分N是以大气N2的形式存在的。为了在生物学上可用于吸收，N2需要转化为氨（NH3）。N2到NH3的生物转化被称为N2固定，并且由特定的微生物进行。这些微生物产生某些蛋白质，称为固氮酶，以催化N2固定反应。然而，功能性固氮酶在其结构中需要元素钼（Mo）。在远古地球早期，当海洋缺氧时，钼不应该被微生物利用来制造钼基固氮酶，因为钼只存在于固体矿物中。然而，地质和生物学证据表明，钼基固氮酶存在。这个项目的目标就是解决这个悖论。研究人员假设，当溶解的Mo有限时，能够固定N2的细菌开发了直接从固体矿物中释放和提取Mo的生化策略。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。将使用先进的分析技术来确定微生物获取固相钼的策略。该项目的见解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。该项目的研究成果还可以揭示：（1）金属和稀土元素在环境中的活动性;（2）金属和稀土矿床的形成;（3）从尾矿中回收金属和稀土元素。钼基固氮酶在大氧化事件之前的出现，当含钼矿物和岩石高度不溶时，提出了一个明显的悖论。本项目的目的是通过测试以下假设来解决这个悖论：在溶解的Mo的限制浓度下，N2固定细菌已经开发出直接从矿物和岩石中提取Mo以用于Mo基固氮酶的策略。将设计三组实验来检验这一假设。在第一个实验中，两个N2固定细菌，一个好氧，一个厌氧，将被用来评估提取钼含钼矿物通过分泌钼结合代谢产物。将测量N2固定率以确定Mo生物利用度。在第二个实验中，两个厌氧培养物，一个Fe（II）氧化剂和一个产甲烷菌，将与矿物一起孵育，以研究矿物溶解对Mo释放的影响。在第三个实验中，将构建一个简单的微生物群落，以确定微生物相互作用对钼生物利用度的重要性。将使用补充分析技术测量微生物代谢物，包括ICP-MS、LC-MS和LC-ICP-MS。将通过ARA测定、15 N标记实验和纳米SIMS成像确定N2固定率。将进行RT-qPCR以将特定功能基因的表达水平与N2固定率相关联。XRD、SEM和TEM将用于表征矿物学变化。含钼岩石微生物风化的生物特征将通过TOF-SIMS和XPS确定。该奖项反映了NSF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。

项目成果

Bioavailability of mineral‐associated trace metals as cofactors for nitrogen fixation by Azotobacter vinelandii

• DOI: 10.1111/gbi.12552
• 发表时间: 2023-02
• 期刊: Geobiology
• 影响因子: 3.7
• 作者: S. Srivastava;Hailiang Dong;O. Baars;Yizhi Sheng