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

RATIONALE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.