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

Abstract

Abstract In this study, a new 15N-labeled procedure based on isotopic ratio monitoring of N2 by gas chromatography/isolink/isotope ratio mass spectrometry with great precision and of N2O by gas chromatography/mass spectrometry in SIM mode with high sensitivity was developed and proposed for the identification and confirmation of in vitro microbial denitrification. The mixture of gaseous metabolites produced by Alcaligenes faecalis and atmospheric gases in the confined cultivation tube was analyzed on a GS-CarbonPlot column. A baseline separation of N2/O2, CO2, N2O and water vapour was obtained in a single run, which eliminated CO2 and H2O interference with isotopic analysis of N2 and N2O. In δ15N analysis of N2, combustion oven/interface in GC isolink can remove all of the O2 in the sample gases, thereby providing accurate δ15N measurement. The δ15N value of N2 in 15N-labled sample, 15N-natural abundance control and 15N-KNO3 blank control were 2.394% ± 0.261%, 0.022% ± 0.044% and 0.315% ± 0.045%, respectively. Besides, significant increases in isotopic abundance of 14,15N2O and 15,15N2O (RT = 2.99 min) relative to 14,14N2O were observed, indicating N2 and N2O production from denitrificaiton by A. faecalis. This procedure provides isotopic evidence of N2 and/or N2O production based on the marked increase in the 15N isotope abundance, and is rapid, sensitive and accurate to indentify and confirm the occurrence of microbial denitrification. We have confirmed the denitrifying activity of several strains of microorganisms screened from the environment using this procedure. This procedure has also been applied to confirm the N2 formation by nitrifier denitrification under defined conditions.