Isotopic analysis of N and o in nitrite and nitrate by sequential selective bacterial reduction to N2O

Nitrite is an important intermediate species in the biogeochemical cycling of nitrogen, but its role in natural aquatic systems is poorly understood. Isotopic data can be used to study the sources and transformations of NO2- in the environment, but methods for independent isotopic analyses of NO2- in the presence of other N species are still new and evolving. This study demonstrates that isotopic analyses of N and O in NO2- can be done by treating whole freshwater or saltwater samples with the denitrifying bacterium Stenotrophomonas nitritireducens, which selectively reduces NO2- to N2O for isotope ratio mass spectrometry. When calibrated with solutions containing NO2- with known isotopic compositions determined independently, reproducible delta15N and delta18O values were obtained at both natural-abundance levels (+/-0.2-0.5 per thousand for delta15N and +/-0.4-1.0 per thousand for delta18O) and moderately enriched 15N tracer levels (+/-20-50 per thousand for delta15N near 5000 per thousand) for 5-20 nmol of NO2- (1-20 micromol/L in 1-5 mL aliquots). This method is highly selective for NO2- and was used for mixed samples containing both NO2- and NO3- with little or no measurable cross-contamination. In addition, mixed samples that were analyzed with S. nitritireducens were treated subsequently with Pseudomonas aureofaciens to reduce the NO3- in the absence of NO2-, providing isotopic analyses of NO2- and NO3- separately in the same aliquot. Sequential bacterial reduction methods like this one should be useful for a variety of isotopic studies aimed at understanding nitrogen cycling in aquatic environments. A test of these methods in an agricultural watershed in Indiana provides isotopic evidence for both nitrification and denitrification as sources of NO2- in a small stream.