Isotopic Confirmation of Occurrence of Microbial Denitrification Based on N2 and N2O Production Monitored by Gas Chromatography/Isotope Ratio Mass Spectrometry and Gas Chromatography/Mass Spectrometry

A new N-15-labled procedure based on isotopic ratio monitoring of N-2 by GC-isolink-IRMS (isotope artio mass spectrometer) with high precision and of N2O by GC-MS in SIM mode with high sensitivity was developed for the identification and confirmation of in vitro microbial denitrification. Gas metabolites, produced by Alcaligenes faecalis, including atmospheric gases in the confined cultivation tube, were analyzed using GS-CarbonPlot column, and a satisfactory baseline separation of N-2/O-2, CO2, N2O and water vapour was obtained in a single run, which eliminated CO2 and H2O interference contributed to isotopic determination of N-2 and N2O. In delta N-15 analysis of N-2 combustion oven in GC isolink can remove all of the O-2 in the sample gases, and precise and accurate delta N-15 measurement can be conducted. The delta(15) N value of N-2 of N-15-labled sample, N-15-natural abundance control, and N-15-KNO3 blank control were (2.394 +/- 0.261)parts per thousand, (0.022 +/- 0.044)parts per thousand and (0.315 +/- 0.045)parts per thousand, respectively. In addition, significant increase in relative isotopic abundance of (N2O)-N-14.15 and (N2O)-N-15.15 (RT=2.99 min) to (N2O)-N-14.14 was observed, indicating N-2 and N2O production from denitrification by A. faecalis. This proposed identification procedure, providing isotopic evidence of N-2 and/or N2O production based on the marked enrichment in the N isotope ratio, is rapid, sensitive, accurate and precise to indentify and confirm the occurrence of microbial denitrificaiton, especially for new isolated and screened ones. We have confirmed the denitrifying activity by several strains of microorganism screened from the environment using this procedure. This procedure has also been applied in confirming the N-2 production by nitrifier denitrification under defined conditions.