Continuous measurements of nitrous oxide isotopomers during incubation experiments

Nitrous oxide (N2O) is an important and strong greenhouse gas in the atmosphere. It is produced by microbes during nitrification and denitrification in terrestrial and aquatic ecosystems. The main sinks for N2O are turnover by denitrification and photolysis and photo-oxidation in the stratosphere. In the linear N=N=O molecule N-15 substitution is possible in two distinct positions: central and terminal. The respective molecules, (NNO)-N-14-N-15-O-16 and (NNO)-N-15-N-14-O-16, are called isotopomers. It has been demonstrated that N2O produced by nitrifying or denitrifying microbes exhibits a different relative abundance of the isotopomers. Therefore, measurements of the site preference (difference in the abundance of the two isotopomers) in N2O can be used to determine the source of N2O, i.e., nitrification or denitrification. Recent instrument development allows for continuous position-dependent delta N-15 measurements at N2O concentrations relevant for studies of atmospheric chemistry. We present results from continuous incubation experiments with denitrifying bacteria, Pseudomonas fluorescens (producing and reducing N2O) and Pseudomonas chlororaphis (only producing N2O). The continuous measurements of N2O isotopomers reveals the transient isotope exchange among KNO3, N2O, and N-2. We find bulk isotopic fractionation of -5.01% +/- 1.20 for P. chlororaphis, in line with previous results for production from denitrification. For P. fluorescens, the bulk isotopic fractionation during production of N2O is -52.21% +/- 9.28 and 8.77% +/- 4.49 during N2O reduction. The site preference (SP) isotopic fractionation for P. chlororaphis is -3.42% +/- 1.69. For P. fluorescens, the calculations result in SP isotopic fractionation values of 5.73% +/- 5.26 during production of N2O and 2.41% +/- 3.04 during reduction of N2O. In summary, we implemented continuous measurements of N2O isotopomers during incubation of denitrifying bacteria and believe that similar experiments will lead to a better understanding of denitrifying bacteria and N2O turnover in soils and sediments and ultimately hands-on knowledge on the biotic mechanisms behind greenhouse gas exchange of the globe.