Further optimization of the denitrifier method for the rapid 15 N and 18 O analysis of nitrate in natural water samples.

RATIONALE This study aims to develop a simplified denitrifier method for the δ15 N and δ18 O analysis of nitrate (NO3- ) in natural water samples combining the method of Zhu et al [14] and the original denitrifier method of Sigman et al [12] . Unlike in the abovementioned methods, the aerobic cultivation was performed without the addition or removal of nitrate in the liquid medium. We remove the nitrate contained in the nutrient medium as N2 O in the gas phase by an additional purging step after incubation overnight before the water sample is injected. This eliminates the need for another preparation step, thus saving working time. METHODS The δ15 N and δ18 O values of dissolved NO3- were determined using a Delta V Plus isotope ratio mass spectrometer coupled to a GasBench II sample preparation device that included a denitrification kit. RESULTS After optimising the influencing factors (i.e., purging gas, purging time, and type of crimp seals), the method yielded high accuracy and precision (standard deviations were generally ≤ 0.7‰ for δ18 O values and ≤ 0.3‰ for δ15 N values), confirming the suitability of this procedure. Finally, the potential applicability of the method was demonstrated by measuring the isotopic composition of NO3- in natural water samples. CONCLUSIONS The denitrifier method for converting NO3- to N2 O for isotope analysis was optimized. This allowed the sample preparation time to be further reduced. The complete working time for sample preparation, including all steps, takes 10 minutes per vial if 60 vials are prepared in one run. The water samples are ready for isotope analysis on the fourth day after preparation has started. Isotope measurements can be performed up to 14 days after preparation.