The Impact of Metals and Other Stress Factors on Microbial Ammonia Oxidation Physiology and Isotope Effects

Donald Edward Martocello, III, Ph.D., 2019
Scott D. Wankel, Advisor

Microbially-mediated cycling processes play central roles in regulating the speciation and availability of nitrogen, a vital nutrient with wide implications for agriculture, water quality, ecosystem health, and climate change. Ammonia (NH3) oxidation, the first and rate-limiting step of nitrification, is carried out by bacteria (AOB) and archaea (AOA). Despite more than a century of research into the physiology of AOB, and only more recently AOA, fundamental questions remain about the ammonia oxidation reaction mechanism and relevant stress factors that regulate environmental rates. Ammonia oxidizing organisms (AOO) require the trace metal micronutrients copper (Cu) and iron (Fe) for growth and metabolic catalysis. Ammonia oxidation is directly affected by pH in regulating the relative availability of ammonium (NH4+) and NH3. Also, photoinhibition of AOO is widely reported. The mechanism is unknown, although links to reactive oxygen species (ROS) cycling seem likely. We present detailed investigations of three environmentally relevant AOO stress factors: metal micronutrient limitation, pH changes, and ROS. Central to these studies were analyses of stable isotope fractionation and how changes in AOO physiology impact expression of these isotope effects. In turn, these tools facilitate probing of the ammonia oxidation reaction mechanism and we propose an initial obligatory coordinated NH4+-NH3 uptake step based on isotope mass balance. In addition, we studied nitrification and related environmental chemistry in the Northern Guaymas hydrothermal vent basin in the Gulf of California. This region hosts a unique juxtaposition of hydrothermal vent emissions enriched in NH4+ underlying an extensive regional oxygen deficient zone (ODZ). Most environmental studies of nitrification have focused on the mesopelagic (high oxygen, comparably warmer temperatures, low NH4+, and low metal micronutrient availability). The Northern Guaymas Basin is functionally the opposite. We suggest ultimate limitation by temperature and, surprisingly, potential proximal limitation by NH+4, Fe, and Cu. In total, this work emphasizes the recently recognized role of Fe and Cu in environmental limitation of nitrification, challenges key axioms of AOO cell culturing and physiology, and proposes revisions to canonical ammonia oxidation reaction mechanisms.