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

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 ((15)epsilon) for nitrate uptake and nitrate efflux (2.0 +/- 0.3 parts per thousand and 1.2 +/- 0.4 parts per thousand, respectively). The O isotope effects ((18)epsilon) for nitrate uptake and nitrate efflux were indistinguishable (2.8 +/- 0.6 parts per thousand), 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 (15)epsilon for nitrate uptake can account for at most 40% of the organism-level N isotope effect ((15)epsilon(org)) measured in laboratory studies of T. weissflogii and in the open ocean (typically 5 parts per thousand 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 ((18)epsilon(org): (15)epsilon(org)) of similar to 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 (18)epsilon(org): (15)epsilon(org) to rise appreciably above 1 when (15)epsilon(org) is low (e.g., yielding a ratio of 1.1 when (15)epsilon(org) is 5 parts per thousand). 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. (C) 2013 Elsevier Ltd. All rights reserved.