Measurement of the Oxygen Isotopic Composition of Nitrate in Seawater and Freshwater Using the Denitrifier Method

TLDR

The denitrifier method, originally developed for 15N/14N analysis of nitrate, has been adapted for 18O/16O studies but requires addressing fractionation and oxygen exchange during conversion to N2O. The study reports a novel method for measuring the oxygen isotopic composition (18O/16O) of nitrate in both seawater and freshwater. The method uses cultured denitrifying bacteria to convert nitrate to nitrous oxide, whose isotope ratio is analyzed while accounting for oxygen fractionation and exchange with water. Experiments show that water exchange contributes less than 10% (often <3%) of the oxygen atoms in the N2O product, that fractionation and exchange are consistent within batches, and that reference materials can correct the measured 18O/16O ratios; this first seawater method offers higher sensitivity (down to 10 nmol and 1 µM NO3−), no interference from other solutes, and easier sample preparation.

Abstract

We report a novel method for measurement of the oxygen isotopic composition (18O/16O) of nitrate (NO3-) from both seawater and freshwater. The denitrifier method, based on the isotope ratio analysis of nitrous oxide generated from sample nitrate by cultured denitrifying bacteria, has been described elsewhere for its use in nitrogen isotope ratio (15N/14N) analysis of nitrate.1 Here, we address the additional issues associated with 18O/16O analysis of nitrate by this approach, which include (1) the oxygen isotopic difference between the nitrate sample and the N2O analyte due to isotopic fractionation associated with the loss of oxygen atoms from nitrate and (2) the exchange of oxygen atoms with water during the conversion of nitrate to N2O. Experiments with 18O-labeled water indicate that water exchange contributes less than 10%, and frequently less than 3%, of the oxygen atoms in the N2O product for Pseudomonas aureofaciens. In addition, both oxygen isotope fractionation and oxygen atom exchange are consistent within a given batch of analyses. The analysis of appropriate isotopic reference materials can thus be used to correct the measured 18O/16O ratios of samples for both effects. This is the first method tested for 18O/16O analysis of nitrate in seawater. Benefits of this method, relative to published freshwater methods, include higher sensitivity (tested down to 10 nmol and 1 μM NO3-), lack of interference by other solutes, and ease of sample preparation.