Simultaneous characterization of methane and carbon dioxide produced by cultured methanogens using gas chromatography/isotope ratio mass spectrometry and gas chromatography/mass spectrometry

Abstract

The stable carbon isotope ratios of methanogen-produced CH4 and CO2 are useful information for identifying and quantifying methanogenic pathways. Isotope ratio mass spectrometry combined with gas chromatography (GC/IRMS) is a very attractive tool for performing high-precision compound-specific isotope analysis. However, no GC/IRMS techniques have yet been available or been validated that give baseline separation of methanogen-produced CH4 and CO2 from N2/N-oxides and H2O vapor at ambient temperature and compatibility with analysis by mass spectrometry. Microbe-produced CH4 and CO2 in headspace gases were separated from N2/N-oxides and H2O vapor in a single run on a GS-CarbonPLOT column at 35°C and with a maximum operating temperature of 120-140°C. The simultaneous characterization of CH4 and CO2 was then performed by GC/IRMS using an optimized backflush time to eliminate the interference from N2/N-oxides and H2O vapor, and by GC/MS due to there being no interference from O2 gas in the culture. GC/MS and GC/IRMS were used to calculate the ionization efficiency of CO2 as 8.22-8.84 times that of CH4 in GC/MS analysis, and it was deduced that the N-oxides, which can potentially interfere with δ(13)C analysis, were probably produced mainly in the source of the isotope ratio mass spectrometer. We also determined the aceticlastic methanogenic pathway. The established GC/MS and GC/IRMS techniques are suitable for characterizing the gaseous carbon-containing compounds produced by microbial cultures. Through high-precision carbon isotope analysis by GC/IRMS combined with low concentrations of (13)C-labelled substrates, the technique has great potential for identifying and quantifying methanogen-mediated carbon metabolic processes and pathways.