The contributions of nitrate uptake and efflux to isotope fractionation during algal nitrate assimilation

Abstract

In order to strengthen environmental application of nitrate N and O isotopes, we measured the N and O isotopic fractionation associated with cellular nitrate uptake and efflux in the nitrate-assimilating marine diatom Thalassiosira weissflogii. We isolated nitrate uptake and efflux from nitrate reduction by growing the cells in the presence of tungsten, which substitutes for molybdenum in assimilatory nitrate reductase, yielding an inactive enzyme. After growth on ammonium and then N starvation, cells were exposed to nitrate. Numerical models fit to the evolution of intracellular nitrate concentration and N and O isotopic composition yielded distinct N isotope effects (15ε) for nitrate uptake and nitrate efflux (2.0±0.3‰ and 1.2±0.4‰, respectively). The O isotope effects (18ε) for nitrate uptake and nitrate efflux were indistinguishable (2.8±0.6‰), yielding a ratio of O to N isotopic fractionation for uptake of 1.4±0.4 and for efflux of 2.3±0.9. The 15ε for nitrate uptake can account for at most 40% of the organism-level N isotope effect (15εorg) measured in laboratory studies of T. weissflogii and in the open ocean (typically 5‰ or greater). This observation supports previous evidence that most isotope fractionation during nitrate assimilation is due to intracellular nitrate reduction, with nitrate efflux allowing the signal to be communicated to the environment. An O to N fractionation ratio (18εorg:15εorg) of ∼1 has been measured for nitrate assimilation in algal cultures and linked to the N and O isotope effects of nitrate reductase. Our results suggest that the ratios of O to N fractionation for both nitrate uptake and efflux may be distinct from a ratio of 1, to a degree that could cause the net 18εorg:15εorg to rise appreciably above 1 when 15εorg is low (e.g., yielding a ratio of 1.1 when 15εorg is 5‰). However, field and culture studies have consistently measured nearly equivalent fractionation of N and O isotopes in association with low isotope effects, calling for isotopic studies of nitrate transport by other phytoplankton strains.