Using dual-bacterial denitrification to improve δ15N determinations of nitrates containing mass-independent 17O

The bacterial denitrification method for isotopic analysis of nitrate using N₂O generated from Pseudomonas aureofaciens may overestimate δ¹⁵N values by as much as 1–2‰ for samples containing atmospheric nitrate because of mass-independent ¹⁷O variations in such samples. By analyzing such samples for δ¹⁵N and δ¹⁸O using the denitrifier Pseudomonas chlororaphis, one obtains nearly correct δ¹⁵N values because oxygen in N₂O generated by P. chlororaphis is primarily derived from H₂O. The difference between the apparent δ¹⁵N value determined with P. aureofaciens and that determined with P. chlororaphis, assuming mass-dependent oxygen isotopic fractionation, reflects the amount of mass-independent ¹⁷O in a nitrate sample. By interspersing nitrate isotopic reference materials having substantially different δ¹⁸O values with samples, one can normalize oxygen isotope ratios and determine the fractions of oxygen in N₂O derived from the nitrate and from water with each denitrifier. This information can be used to improve δ¹⁵N values of nitrates having excess ¹⁷O. The same analyses also yield estimates of the magnitude of ¹⁷O excess in the nitrate (expressed as Δ¹⁷O) that may be useful in some environmental studies. The 1-σ uncertainties of δ¹⁵N, δ¹⁸O and Δ¹⁷O measurements are ±0.2, ±0.3 and ±5‰, respectively.