Year: 2023
Type: dissertation
Abstract: The global nitrogen cycle is largely powered by microorganisms. While mediating these processes, microorganisms leave behind stable isotopic signatures that can be used to identify and trace processes in the nitrogen cycle. However, significant variability has been observed for the stable isotope effects (15ε) associated with denitrification, the process that returns bioavailable nitrogen (nitrate) from ecosystems to the atmosphere. The first step of denitrification, dissimilatory nitrate reduction to nitrite, is catalyzed by the NapA and/ or NarG enzyme. As microorganisms use nitrate respiration as a source of energy for growth, the key to understanding the variability of isotope effects associated with denitrification lies within the interaction between microbial physiology and the NapA and NarG enzymes. In my dissertation I used a combination of stable isotope geochemistry, microbiology, and molecular genetics to measure the specific isotope effects of both NapA and NarG. Specifically, in the second chapter of my dissertation, I showed that 18ε/15ε proportionalities are largely controlled by nitrate reduction with either NarG (18ε/15ε value of ~1) or NapA (18ε/15ε value of ~0.5) and that the regulation of these enzymes is an important component to ecosystem level trends of 18ε/15ε values. In the third chapter, I used chemostat experiments to investigate how microbial growth rates impact the magnitude of 15ε in NapA based nitrate reduction and observed a range of 19.1 – 25.1‰ in 15ε across growth rates. Using proteomic analyses I also found that the enzyme specific rates of nitrate reduction rather than cell specific rates are key for predicting 15ε overall. The proteomic data from these chemostat experiments also indicated the simultaneous presence of dissimilatory nitrate reduction to ammonium (DNRA) and denitrifying proteins. Thus, in the fourth chapter of my dissertation I examined the proteomic data closer to determine the possibility of these pathways coexisting. In the final chapter of my dissertation, I assessed the robustness of enzymatically produced stable isotopic signatures by developing a strain of Pseudomonas aeruginosa with a mutated NarG enzyme and by culturing bacteria outside of the Proteobacteria phylum. Though no change to 18ε/15ε values occurred in these strains suggesting that the 18ε/15ε signature itself is robust, a 6‰ increase in 15ε occurred in the mutant strain suggesting that stable isotopic signatures may be sensitive to small mutations through time. By characterizing the isotopic variability associated with nitrate reduction, the findings from this research stand to improve our ability to trace denitrification in natural ecosystems, and therefore, enhance our overall understanding of global nitrogen cycling.
Author: Asamoto, Ciara Kathryn
Language: English
Subfield: Pollution
Field: Environmental Science
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W6981574568