A simple and rapid GC/MS method for the simultaneous determination of gaseous metabolites
Introduction
Gaseous compounds such as H2, N2, O2, CO, NO, CH4, CO2, and N2O are commonly produced and/or consumed by microorganisms during normal metabolic processes (Hughes, 1985, Conrad, 1996). Since the microbial production and consumption of these gaseous compounds via a variety of reductive and oxidative processes are interconnected, the development of a method for the simultaneous quantification of multiple gas species is highly relevant to the studies of microbial physiology and ecology.
To date, these common gas species have been quantified mainly by gas chromatography (GC) in combination with a number of detection techniques (reviewed by Crill et al., 1995), with individual or related groups of gas species requiring separate detection with specific detectors. For example, H2, N2, O2, and CO2 have been typically detected using a thermal conductivity detector, N2O by an electron capture detector, and CO and CH4 using a flame ionization detector. To overcome this inconvenience and facilitate more rapid sample processing, techniques for the simultaneous detection of multiple biogenic gas species using a single apparatus have been developed. For example, a GC system equipped with multiple columns and detectors with flow switching valves has been constructed (Hedley et al., 2006, Sitaula et al., 1992, Wang and Wang, 2003, Yoh et al., 1998); however, such GC systems consist of a number of components and require high technical skill to operate the complex assembly for redirecting the gas flow into the associated chromatographic systems.
Presently, GC-mass spectrometry (GC/MS) is used largely for the identification of microbial metabolites such as amino acids, sugars, and organic acids (Koek et al., 2006, Strelkov et al., 2004, Tian et al., 2009), but has also been applied for the detection some gaseous metabolites produced by microbes (Amano et al., 2007, Amano et al., 2011, Bazylinski et al., 1986, DeRito et al., 2005, Garber and Hollocher, 1982, Goretski and Hollocher, 1990, Liou et al., 2008, Liu et al., 2006, Shoun and Tanimoto, 1991, Waki et al., 2010). With the advent of GC/MS technology, particularly its enhanced sensitivity, the applicability of GC/MS has expanded to include the quantification of environmental ambient gasses existing in extremely low concentrations, such as CH4, CO2, and N2O (Ekeberg et al., 2004). One of the advantages of GC/MS over GC is that it can be applied in stable isotope tracer experiments to evaluate metabolic fluxes in cellular and ecosystem processes.
Despite the high potential utility of GC/MS in gas analyses, the configuration of GC/MS systems has not yet been optimized for the rapid and broad-dynamic-range quantification of gaseous microbial metabolites. In addition, it has not been optimized for determination of wide variety of gas species. Considering that multiple gas species at various concentration levels are produced and consumed by microbes and microbial communities, the importance of the modification and optimization for these purposes are highly obvious. It would be noteworthy that although such gas species as N2 and O2 are major substrates/metabolites for microbes, it is difficult to obtain accurate and reproducible results when attempting to quantify N2 and O2 that are present in samples at as low level as 100 ppm. This would be largely because contamination of such gas species from ambient air invariably occurs when gaseous samples are injected through an injection port. Thus, preventing air contamination during sample injection is the key technique to achieve the purposes.
Here, we first made an attempt to prevent air contamination by modifying the injection port of a commercially available GC-quadrupole MS instrument. Second, we demonstrated the sensitivity and dynamic range of several gas species commonly found in microbial gas metabolism with an optimized analytical conditions which would enable the rapid and simultaneous quantification of a variety of gas species ranging from the level of ppm to percent (in volume) using a single GC/MS apparatus. We then examined the usefulness of the new method and modified instrument during a denitrification experiment with Pseudomonas aureofaciens ATCC 13985T.
Section snippets
Instrumentation and operating conditions
Fig. 1 shows a scheme of the quadrupole GC/MS system (GCMS-QP2010 Plus, Shimadzu, Kyoto, Japan) equipped with a CP-PoraPLOT Q-HT column (25 m × 0.32 mm; Varian, Inc., CA, USA) used in this study. Several modifications were made to the commercial GC/MS model to minimize the contamination of ambient air into the analytical samples. The injection port (EN2SI, ZF2SI, and SI4G, Valco Instruments Co. Inc., Houston, TX) and the 8-port valve (2C8WE-PH, Valco Instruments Co. Inc.) were covered by a jacket
Simultaneous detection of nine gas species
A mixture of H2, N2, O2, Ar, 13CO, 15NO, CH4, CO2, and N2O was prepared in a He matrix, and each of the nine gas species was identified using the modified GC/MS instrument by the combination of retention time and mass-to-charge ratios (m/z) (Fig. 2). The mass chromatogram of the mixture of the nine gas species in the He matrix is presented in Fig. 2a–i in the order: H2, N2, O2, Ar, 13CO, 15NO, CH4, CO2, and N2O. The stable isotope labeled compounds, 13CO (m/z 29) and 15NO (m/z 31), which can be
Conclusions
Here, we established a novel GC/MS-based analytical method for the rapid, simultaneous quantification of most of the gaseous compounds found in microbial metabolism in a mixed gas sample. We showed that several gasses could be accurately quantified in a single analysis within 2.5 min (Fig. 2), with high sensitivity and a wide dynamic range (Table 2 and Fig. 3). It would be noteworthy that most abundant atmospheric gas species, N2 and O2, can be successfully determined as well (Fig. 2, Fig. 3),
Acknowledgements
This work was supported by the Grant-in-Aid, P07024, of the New Energy and Industrial Technology Development Organization (NEDO) of Japan.
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