A new 15N tracer method to determine N turnover and denitrification of Pseudomonas stutzeri

Although denitrification is one of the key processes of ecosystem N turnover, the understanding of the regulation of the denitrification pathway is still limited due to the lack of feasible methods for the quantification of N2 formation. Based on the previously developed isotope pairing method, we present a new in vitro 15N tracer method for the quantification of N2 released from denitrification by bacterial cultures. The application of the new method was enabled by replacing the background air in the sample flasks with a gas mixture of He and O2 with an approximately 50-fold reduced N2 background (1.7% v/v), allowing for a direct and sensitive quantification of N2 formation with isotope-ratio mass spectrometry after 15N-labelling on the one hand, but leaving the method relatively insensitive to intrusion of ambient N2 on the other hand. The method was tested on bacterial cultures of Pseudomonas stutzeri grown at different oxygen levels. Additionally, NO and N2O formation were determined with a chemoluminescence analyser and a gas chromatograph, respectively. Following labelling with 15N-ammonium and 15N-nitrate, it could be shown that P. stutzeri used ammonium preferably for biomass build-up, and nitrate preferably as electron acceptor. Between 84-107% of the total available N could be recovered. Due to the high sensitivity of the new method only low levels of 15N tracer were necessary, minimising substrate-induced effects and making this method also an appropriate tool for the use on soil cores. By that it offers a new method for studying denitrification in terrestrial ecosystems.