Physiological and environmental controls on the nitrogen and oxygen isotope fractionation of nitrate during its assimilation by marine phytoplankton

To strengthen the foundation for isotopic studies of the nitrogen cycle in the modern and past ocean, the controls on nitrogen (N) and oxygen (O) isotopic fractionation associated with the growth of marine phytoplankton on nitrate were investigated. N and O isotopic fractionation associated with each step of nitrate assimilation (nitrate reduction to nitrite, nitrate uptake into the cell, and nitrate efflux out of the cell) was measured, and that context used to interpret the connection between net N and O isotopic fractionation and cell physiology in whole cell culture. The enzyme responsible for nitrate reduction to nitrite, eukaryotic assimilatory nitrate reductase eukNR), was found to reduce nitrate with an N isotope effect of 26.6 +/- 0.2permil and with near equivalent fractionation of N and O isotopes.;The N isotope effects for nitrate uptake and efflux, measured in the marine diatom Thalassiosira weissflogii, were low relative to that associated with reduction (2.0 +/- 0.3 / and 1.2 +/- 0.4permil, respectively). The ratio of O-to-N isotopic fractionation was greater than 1 for both processes (1.4 +/- 0.4 for uptake and 2.3 +/- 0.9 for efflux). Finally, whole cell N and O isotope effects were measured in continuous culture of T. weissflogii. The N isotope effect showed little variation (5.1 - 5.8permil) across a 4-fold change in growth rate under phosphate limitation. In contrast, under light limitation, the N isotope effect increased by a factor of 3 (to 17.5permil). Equivalent N and O isotope fractionation was observed under all conditions. In sum, these results validate the existing physiological model for isotopic fractionation during nitrate assimilation where intracellular reduction is the dominant fractionating step. They provide the first step in understanding the cellular basis for the equivalent fractionation of N and O associated with nitrate assimilation in the ocean. Results from this and previous studies suggest that (i) little variation in N isotope effect may be expected under most environmental conditions due to cellular control over the relative rates of steps in nitrate assimilation and (ii) irradiance may be the dominant driver of variability in the N isotope effect in the ocean.