Date of Graduation

7-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Lessner, Daniel

Committee Member

Pinto, Ines

Second Committee Member

Sakon, Josh

Third Committee Member

Ivey, Mack

Fourth Committee Member

Kral, Timothy

Keywords

Metal, Nitrogen Fixation, Cellular Physiology, Methanosarcina acetivorans

Abstract

Nitrogen is an essential element required by all living organisms and found in cellular compounds such as protein and DNA. While N2 gas comprises a majority of the atmosphere, the gas itself is highly unreactive and cannot be used directly by humans, animals or plants for biosynthesis. Biological nitrogen fixation is the process by which N2 is reduced to the biologically relevant NH3, catalyzed by the energetically demanding nitrogenase enzymes. Nitrogenases are only encoded by a subset of bacteria and archaea and because nitrogenases are responsible for nearly 50% of bioavailable (fixed) nitrogen, the study of nitrogenases is of great importance. This study used model methanogen Methanosarcina acetivorans to study the effects of nitrogen fixation on the host, and how the availability of metals affects nitrogen fixation. To characterize the nitrogen sources that can support growth, it was observed that M. acetivorans has a limited range of fixed nitrogen sources. When provided limiting concentration of an external fixed nitrogen source, it was observed that M. acetivorans will readily fix nitrogen to increase cell yield. A decrease of the genomic copies per cell (ploidy) was observed in nitrogen fixing cells as compared to cells provided ammonium chloride. We propose a decrease in ploidy as an adaptation to the energetically costly process of nitrogen fixation, minimizing the energetic and nitrogen demands associated with DNA synthesis. M. acetivorans encodes for three functionally similar but genetically distinct nitrogenases, being the molybdenum nitrogenase (Mo-nitrogenase), vanadium nitrogenase (V-nitrogenase) and the iron nitrogenase (Fe nitrogenase), each named for the metal in the active site. H2 evolution is an obligate process during nitrogen fixation, and in M. acetivorans the Mo-nitrogenase was observed to produce the least H2 per N2 fixed, followed by the V- and Fe- nitrogenases. Furthermore, the availability of N2 varied the relative production of H2 and NH3 during nitrogen fixation. High concentrations of tungstate to molybdate in the growth medium are well described to be inhibitory to nitrogen fixation. This study shows the first evidence that M. acetivorans can fix nitrogen in the presence of 1000x tungstate/ molybdate with no defect in growth rate as compared to molybdate alone. Overall, this dissertation highlights adaptions of M. acetivorans to nitrogen fixing conditions and provides insight into the reactions of methanogen nitrogenases.

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