Isotopic effects of microbially mediated processes are often considered immutable parameters of biogeochemical cycles, yet few microbial fractionation factors are truly constant. Decades of work on key isotope effects in the sulfur and carbon cycle have revealed that a number of physiological constraints, such as substrate flux, electron acceptor availability, temperature variations and nutrient limitations, can all lead to significant changes in the net isotope fractionation observed at the organism level. In the case of the nitrogen cycle, culture-based evidence from the past decade has likewise revealed the potential for significant isotopic variation in both of the two main isotopic levers of the global nitrogen cycle: nitrogen fixation converting atmospheric N2 into bioavailable nitrogen, and denitrification/anammox returning nitrogen back to the N2 pool. These microbially-mediated geochemical reactions and their relative fluxes are jointly responsible for setting the N isotopic signature of marine and terrestrial ecosystems. Here we present data from batch and continuous culture experiments with genetically altered diazotrophs and nitrate reducers to shed light onto the kinetic isotopic effects that underly enzymatic fractionation in these systems. These experiments bring us closer to understanding how enzymatic differences and the interactions of individual reaction steps modulate the observed isotope fractionation, and may explain the isotopic variations observed in Earth’s nitrogen cycle today and in the geologic past. Specifically we applied continuous culture (chemostat) techniques to precisely control the biophysical environment, nutrient availability and growth rate of the several model nitrate-reducers. Under the tested experimental conditions, we observed nitrogen isotope effects ranging from 5‰ to 25‰ that are strongly correlated with cell specific nitrate reduction rate regardless of other physiological constraints. Experiments with deletion mutants of different nitrate reductases coupled to dual N and O isotope measurements further revealed purely enzyme-based isotopic differences. Likewise, we examined nitrogenase mutants of the model diazotroph Azotobacter vinelandii to isolate the specific mechanisms that explain the small &lt; 3‰ nitrogen isotope effects typically observed for diazotrophy based on Mo nitrogenase and the known isotopic differences between diazotrophs using Mo vs. alternative V and Fe-only nitrogenase isoforms.

Closer look at the isotopic levers of the nitrogen cycle: kinetic isotope effects in denitrification and nitrogen fixation

ACS Spring 2022

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