10:45 AM - 12:15 PM
[BCG06-P08] Novel quantitative method for determining the individual isotopomer of organic acids in 13C tracer experiments
Keywords:GC-MS, 13C tracer, Anaerobic metabolism, Acetogen, Least-squares method
Acetate is an important intermediate metabolite of anaerobic microbial methanogenesis. Therefore, understanding the metabolic mechanisms of acetate-producing bacteria (acetogen), which play a key role in the dynamics of acetate, will contribute to elucidate the global carbon cycle including the deep subsurface biosphere. Indeed, the carbon fixation mechanisms of acetogens have attracted great interest in various research fields, including the investigation of bioengineering solutions to global warming (e.g., Katsyv & Müller, 2020), primitive metabolic pathways, and origin of life (Martin, 2011). Recent studies have proposed and demonstrated novel carbon fixation pathways involving acetate-mediated reaction (e.g., Sánchez-Andrea et al., 2020), suggesting that more diverse mechanisms of acetate metabolism and carbon fixation may present in the natural environment than previously thought. Therefore, there is a need to establish an efficient and rapid method for evaluating acetogenesis. Isotope tracer methods using radiocarbon (14C) or stable carbon isotope (13C) are essential for elucidating carbon fixation mechanisms because they provide direct evidence for the presence of specific pathways. In 13C tracer experiments, there are four major acetate isotopomers; CH3COOH, CH313COOH, 13CH3COOH, and 13CH313COOH. The previous analytical method requires at least two independent measurements to distinguish and quantify the four acetate isotopomers, which is often time- and effort-consuming (Wood & Harris, 1952). Therefore, our goal was to develop a simple and rapid analytical method for determining the relative abundance of four acetate isotopomers by a gas chromatography-mass spectrometry (GC-MS) technique. In addition, we illustrate the application of the developed method and provide a framework for the quantitative investigation of carbon flow in the metabolic reactions of acetogens.
We modified the previously established GC-MS method that allows the direct injection of an aqueous sample into the GC-MS system without derivatization of organic acids (Mulat & Feilberg, 2015). In this study, in order to analyze formate as well as acetate, phosphoric acid was used for the acidification of aqueous samples because oxalic acid used for acidification in the previous method produce formic acid in the injector set at high temperature (Mulat & Feilberg, 2015). The novelty of our method is that the relative abundance of individual isotopomer is determined by a least-squares approach using the intensity data of molecular ion and fragment ions obtained from GC-MS measurements. To verify the validity of our calculation method for quantifying the relative abundance of individual isotopomers, we analyzed standard solution with known mixtures of unlabeled and 13C-labeled analytes. The developed method was applied to study the carbon fixation mechanism of the well-known acetogen Acetobacterium woodii grown in methanol and bicarbonate. We performed a series of experiments using a combination of 13C-labeled and unlabeled substrates; (i) 13CH3OH + HCO3−, (ii) CH3OH + H13CO3−, (iii) 13CH3OH + H13CO3− and (iv) CH3OH + HCO3−.
The validity of the method was demonstrated by determining known mixtures of unlabeled and 13C-labeled analytes. In the analysis of acetate and formate in aqueous solutions, the calculated mixing ratios of analyte isotopomers agreed well with the theoretical mixing ratios. The developed method was applied to the study of metabolic pathway of A. woodii. We provided a quantitative reaction model for methanol metabolism of A. woodii, which indicated that methanol was not the sole carbon precursor of the acetate methyl group and that 20% of the methyl group was formed from CO2. In contrast, the carboxyl group of acetate appeared to form exclusively by CO2 fixation. Our method can be a useful tool not only to investigate the detailed metabolism of well-known acetogens but also to reveal unprecedented carbon fixation pathways possibly present in nature.
We modified the previously established GC-MS method that allows the direct injection of an aqueous sample into the GC-MS system without derivatization of organic acids (Mulat & Feilberg, 2015). In this study, in order to analyze formate as well as acetate, phosphoric acid was used for the acidification of aqueous samples because oxalic acid used for acidification in the previous method produce formic acid in the injector set at high temperature (Mulat & Feilberg, 2015). The novelty of our method is that the relative abundance of individual isotopomer is determined by a least-squares approach using the intensity data of molecular ion and fragment ions obtained from GC-MS measurements. To verify the validity of our calculation method for quantifying the relative abundance of individual isotopomers, we analyzed standard solution with known mixtures of unlabeled and 13C-labeled analytes. The developed method was applied to study the carbon fixation mechanism of the well-known acetogen Acetobacterium woodii grown in methanol and bicarbonate. We performed a series of experiments using a combination of 13C-labeled and unlabeled substrates; (i) 13CH3OH + HCO3−, (ii) CH3OH + H13CO3−, (iii) 13CH3OH + H13CO3− and (iv) CH3OH + HCO3−.
The validity of the method was demonstrated by determining known mixtures of unlabeled and 13C-labeled analytes. In the analysis of acetate and formate in aqueous solutions, the calculated mixing ratios of analyte isotopomers agreed well with the theoretical mixing ratios. The developed method was applied to the study of metabolic pathway of A. woodii. We provided a quantitative reaction model for methanol metabolism of A. woodii, which indicated that methanol was not the sole carbon precursor of the acetate methyl group and that 20% of the methyl group was formed from CO2. In contrast, the carboxyl group of acetate appeared to form exclusively by CO2 fixation. Our method can be a useful tool not only to investigate the detailed metabolism of well-known acetogens but also to reveal unprecedented carbon fixation pathways possibly present in nature.