Nitrogen and oxygen isotope-ratio analysis of nitrate by the denitrifier method using continuous flow isotope-ratio mass spectrometry

The total isotopic composition of nitrate is used for identifying the origin and fate of nitrate in atmospheric, terrestrial and aquatic systems. The analysis of δ18O and δ15N values each give important and unique information about the sources and sinks of nitrate in these systems. This denitrifier method is based on the isotope ratio analysis of nitrous oxide generated through reduction of nitrate by cultured denitrifying bacteria. Here, preliminary data is presented on δ 15N and δ18O generated by the bacterial production of N2O from NO3-. During the process of denitrification, nitrates are converted to nitrogen gas via a series of intermediate gaseous nitrogen oxide compounds. Denitrification occurs due to the presence of heterotrophic bacteria or autotrophic denitrifier pathways in select bacteria. Thus, we have chosen a distinct bacterium for the investigation of nitrate reduction for this study. Pseudomonas aureofaciens contains the copper-containing nitrite reductase necessary for the reduction of nitrate. Our efforts focus on the conversion of NO 3 to N2O using techniques adapted from Cascotti22 . In this study, a customized headspace extraction system was built in order to analyze δ18O and δ15N of N2O from NO3-. Successful purification of Pseudomonas aureofaciens, conversion of NO3 - to N2O, and the standardization of nitrate was the result of many experiments. Tests of the impact on isotopic composition by pre-concentration methods have been performed including freeze-drying/evaporation and ion chromatography. The denitrifier method has been implemented in the Michalski Stable Isotope Lab in order to simultaneously detect the stable isotope composition of oxygen and nitrogen in nitrate samples