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.

National Science Foundation (NSF)
Division of Earth Sciences (EAR)
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Philip Bennett
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Miami University Oxford
United States
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