Date of Graduation

5-2025

Document Type

Thesis

Degree Name

Bachelor of Science in Biology

Degree Level

Undergraduate

Department

Biological Sciences

Advisor/Mentor

Dr. Daniel Lessner

Committee Member

Dr. Christian Tipsmark

Second Committee Member

Dr. Byron Zamboanga

Third Committee Member

Dr. Lucy Brown

Abstract

Nitrogen is a crucial element in biomolecules such as proteins and DNA, essential for all cellular life. Biological nitrogen fixation, a process utilized by certain bacteria and archaea, reduces dinitrogen (N₂) to ammonia (NH₃), enabling growth in the absence of fixed nitrogen sources like NH₃. This process is driven by the metalloenzyme nitrogenase, which catalyzes the reaction: N₂ + 16ATP + 8e⁻ + 8H⁺ → 2NH₃ + H₂ + 16ADP + 16Pi. Due to its high energy demand, nitrogenase production and activity are tightly regulated. Among archaea, nitrogenase is found exclusively in methanogens, where its regulation remains poorly understood. We employed Methanosarcina acetivorans as a model organism to investigate fixed nitrogen source utilization and nitrogenase regulation in methanogens. Using a mutant strain of M. acetivorans incapable of fixing N₂, we assessed the utilization of various nitrogen sources, including all 20 common amino acids and biologically relevant inorganic nitrogen compounds (e.g., nitrate). Surprisingly, only glutamine, trimethylamine (also a carbon source), and NH₃ supported the growth of the mutant strain, indicating a narrow range of fixed nitrogen source utilization in M. acetivorans. Notably, growth with glutamine as the sole nitrogen source was poor compared to NH₃. Further investigation using another mutant strain deficient in hydrogenase activity revealed H₂ production during growth with glutamine, suggesting functional nitrogenase activity despite the presence of glutamine as a fixed nitrogen source. Thus, in the absence of NH₃ or trimethylamine, M. acetivorans resorts to energy-intensive N₂ fixation, even when provided with a significant concentration of glutamine, a usable fixed nitrogen source and key intermediate in nitrogen assimilation. However, upon reexamining growth with glutamine as the sole nitrogen source, the results differed from initial observations. The previously noted poor growth was attributed to an inadequate glutamine concentration. Future studies will investigate how increased glutamine levels affect nitrogenase activity.

Keywords

Methanogen; Nitrogen Fixation; Nitrogenase; CRISPR

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