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 bio-geochemical 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 nitritire-ducens, which selectively reduces NO2 - to N2O for isotope ratio mass spectrometry. When calibrated with solutions containing NO2- with known isotopic compositions determined independently, reproducible delta N-15 and delta O-18 values were obtained at both natural-abundance levels (+/- 0.2-0.5 parts per thousand for delta N-15 and +/- 0.4-1.0 parts per thousand for delta O-18) and moderately enriched N-15 tracer levels ( +/- 20-50 parts per thousand for delta N-15 near 5000 parts per thousand) for 5-20 nmol of NO2- (1-20 mu mol/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.