Correcting for background nitrate contamination in KCl-extracted samples during isotopic analysis of oxygen and nitrogen by the denitrifier method

Abstract

Previous research has shown that the denitrifying bacteria Pseudomonas chlororaphis ssp. aureofaciens (P. aureofaciens) can be used to measure the δ(15)N and δ(18)O values of extracted soil nitrate (NO3(-)) by isotope ratio mass spectrometry. We discovered that N2O production from reference blanks made in 1 M KCl increased relative to blanks made of deionized water (DIW). Further investigation showed that isotopic standards made in KCl yielded δ(15)N and δ(18)O values different from the standards prepared in DIW. Three grades of crystalline KCl were dissolved in DIW to create solutions of increasing molarity (0.1 M to 2 M), which were added to P. aureofaciens broth and measured as blanks. Reference standards USGS-32, USGS-34, and USGS-35 were then dissolved in a range of KCl concentrations to measure isotopic responses to changing KCl molarity. Reference blanks and standards created in DIW were analyzed as controls to measure the impact of KCl on the δ(15)N and δ(18)O values. The amount of N2O in the KCl blanks increased linearly with increasing molarity, but at different rates for each KCl grade. The isotopic values of the reference standards measured in KCl were systematically different from those measured in DIW, suggesting contamination by background NO3(-) in the KCl reagents. However, we also noted reduced conversion of NO3(-) into N2O as the KCl molarity increased, suggesting there is a physiological response of P. aureofaciens to KCl. There is a small amount of NO3(-) present in crystalline KCl, which can bias isotopic measurement of NO3(-) at low sample concentrations. This can be minimized by making standards and blanks in the same KCl as is used in samples, diluting all samples and standards to the appropriate NO3(-) concentration using matched KCl solutions, and adding samples and standards to the broth at a constant volume to standardize the KCl molarity in the reaction vial.