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[BGM02-P02] Quantification method for position specific 13C-label distribution of formate and acetate by GC-MS analysis
Keywords:GC-MS, 13C-label, Formate, Acetate, Acetogen, Least-square method
Introduction: Low molecular organic acids, especially acetic acid, are key intermediates in anaerobic microbial decomposition of organic matters in the subsurface environments. Almost all methanogenic conversion processes of organic matter involve acetate and molecular hydrogen [1]. Therefore, understanding of metabolic pathways including acetogenesis can shed light on carbon cycle in the deep biosphere. Stable isotope tracers are powerful tool for visualizing a specific reaction pathway. The determination of isotope enrichment of target compounds is required in the isotopic labeling experiments. Gas chromatography-Mass spectrometry (GC-MS) technique with direct injection of aqueous sample has been a simple and rapid method for determining the isotope enrichment of underivatized short-chain fatty acids [2]. However, the applicability of 13C-tracer analysis by GC-MS to formate has still not been investigated adequately. Furthermore, the previous calculation method which estimates the 13C-labeled mass isotopomer ratio of acetate by using ion intensities of m/z 60, 61 and 62 cannot distinguish between two kinds of singly 13C-labeled isotopomers, i.e., CH313COOH and 13CH3COOH [2]. In this study, we developed quantification method for position specific 13C enrichment of formate and acetate in aqueous solutions by using GC-MS data and applied our method for determining the 13C enrichment of acetate in culture samples.
Methods: Standard solutions were prepared from sodium formate and sodium acetate reagents. Pure standard solutions (50 to 1000 μmol/L) were individually prepared; unlabeled formic acid (HCOOH), labeled formic acid (H13COOH), unlabeled acetic acid (CH3COOH), singly labeled acetic acids (CH313COOH and 13CH3COOH) and doubly labeled acetic acid (13CH313COOH). Then, mixture solutions in which two or more isotopomers presented were prepared by mixing of these pure standards.
Incubation experiments were performed with a pure culture of Acetobacterium woodii DSMZ1030 which was well-studied acetogen [3]. Methanol (CH3OH) and sodium bicarbonate (NaHCO3) were used as substrates and cultures were incubated at 30 °C under an anaerobic condition. Four treatments were prepared; (i) CH3OH + NaHCO3, (ii) 13CH3OH + NaHCO3, (iii) CH3OH + NaH13CO3 and (iv) 13CH3OH + NaH13CO3. Culture solutions were sampled at incubation times of 0, 2 and 7 days for GC-MS analysis.
The position specific 13C enrichment of acetate and formate was calculated from GC-MS measurements of ion peak intensities. In this study, the 13C enrichment of target compounds was expressed in terms of the mixing ratio of isotopomers. For example, acetate has four isotopomers, i.e., an unlabeled isotopomer and three 13C-labeled isotopomers. We determined relative mixing ratio of these four isotopomers by using the least-square approach.
Results and Discussion: The linearity of 13C enrichment analysis was tested by measuring series of standard solutions containing one or more acetate (or formate) isotopomers with various mixing ratios. The calculated mixing ratio of each isotopomer well corresponded with the theoretical values, indicating that our new calculation method is useful for determining the position specific 13C enrichment of acetate and formate dissolved in aqueous samples. The results of 13C enrichment analysis of acetate from acetogen incubations were as follows. For Exp. (ii), 13CH3COOH > 13CH313COOH > CH313COOH. For Exp. (iii), CH313COOH > 13CH313COOH > 13CH3COOH. For Exp. (iv) 13CH313COOH > CH313COOH ~ 13CH313COOH. These results were corresponding with the result of previous 13C-labeling study and agreed with the expected metabolic pathway [4]. We demonstrated that our GC-MS method is applicable and useful for study of metabolic pathway.
References: [1] Kamagata & Sekiguchi, 2002, Nippon Nogeikagaku Kaishi; [2] Mulat & Feilberg, 2015, Talanta; [3] Kremp et al., 2018, Environ microbiol; [4] Paulo et al., 2003, Appl Microbial Biotechnol.
Methods: Standard solutions were prepared from sodium formate and sodium acetate reagents. Pure standard solutions (50 to 1000 μmol/L) were individually prepared; unlabeled formic acid (HCOOH), labeled formic acid (H13COOH), unlabeled acetic acid (CH3COOH), singly labeled acetic acids (CH313COOH and 13CH3COOH) and doubly labeled acetic acid (13CH313COOH). Then, mixture solutions in which two or more isotopomers presented were prepared by mixing of these pure standards.
Incubation experiments were performed with a pure culture of Acetobacterium woodii DSMZ1030 which was well-studied acetogen [3]. Methanol (CH3OH) and sodium bicarbonate (NaHCO3) were used as substrates and cultures were incubated at 30 °C under an anaerobic condition. Four treatments were prepared; (i) CH3OH + NaHCO3, (ii) 13CH3OH + NaHCO3, (iii) CH3OH + NaH13CO3 and (iv) 13CH3OH + NaH13CO3. Culture solutions were sampled at incubation times of 0, 2 and 7 days for GC-MS analysis.
The position specific 13C enrichment of acetate and formate was calculated from GC-MS measurements of ion peak intensities. In this study, the 13C enrichment of target compounds was expressed in terms of the mixing ratio of isotopomers. For example, acetate has four isotopomers, i.e., an unlabeled isotopomer and three 13C-labeled isotopomers. We determined relative mixing ratio of these four isotopomers by using the least-square approach.
Results and Discussion: The linearity of 13C enrichment analysis was tested by measuring series of standard solutions containing one or more acetate (or formate) isotopomers with various mixing ratios. The calculated mixing ratio of each isotopomer well corresponded with the theoretical values, indicating that our new calculation method is useful for determining the position specific 13C enrichment of acetate and formate dissolved in aqueous samples. The results of 13C enrichment analysis of acetate from acetogen incubations were as follows. For Exp. (ii), 13CH3COOH > 13CH313COOH > CH313COOH. For Exp. (iii), CH313COOH > 13CH313COOH > 13CH3COOH. For Exp. (iv) 13CH313COOH > CH313COOH ~ 13CH313COOH. These results were corresponding with the result of previous 13C-labeling study and agreed with the expected metabolic pathway [4]. We demonstrated that our GC-MS method is applicable and useful for study of metabolic pathway.
References: [1] Kamagata & Sekiguchi, 2002, Nippon Nogeikagaku Kaishi; [2] Mulat & Feilberg, 2015, Talanta; [3] Kremp et al., 2018, Environ microbiol; [4] Paulo et al., 2003, Appl Microbial Biotechnol.