Novel quantitative method for individual isotopomer of organic acids from 13C tracer experiments determines carbon flow in acetogenesis.

Anaerobic microbial acetogenesis is ubiquitous on Earth, and thus plays an important role in the global carbon cycle. The mechanism of carbon fixation in acetogens has attracted great interest from various studies for combatting climate change, and even for studying ancient metabolic pathways. Here, we developed a new, simple method for investigating carbon flows in the metabolic reaction of acetogen by conveniently and accurately determining the relative abundance of individual acetate- and/or formate-isotopomers formed in 13C labeling experiments. We measured the underivatized analyte by gas chromatography-mass spectrometry (GC-MS) coupled with a direct aqueous sample injection technique. The individual abundance of analyte isotopomers was calculated by the mass spectrum analysis using the least-squares approach. The validity of the method was demonstrated by determining 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 on methanol and bicarbonate. 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 20-22% of the methyl group was formed from CO2. In contrast, the carboxyl group of acetate appeared to form exclusively by CO2 fixation. Thus, our simple method, without laborious analytical procedures, has broad utility for the study of biochemical and chemical processes related to acetogenesis on Earth.

The Relationship Between Microbial Community Structures and Environmental Parameters Revealed by Metagenomic Analysis of Hot Spring Water in the Kirishima Area, Japan.

Diverse microorganisms specifically inhabit extreme environments, such as hot springs and deep-sea hydrothermal vents. To test the hypothesis that the microbial community structure is predictable based on environmental factors characteristic of such extreme environments, we conducted correlation analyses of microbial taxa/functions and environmental factors using metagenomic and 61 types of physicochemical data of water samples from nine hot springs in the Kirishima area (Kyusyu, Japan), where hot springs with diverse chemical properties are distributed in a relatively narrow area. Our metagenomic analysis revealed that the samples can be classified into two major types dominated by either phylum Crenarchaeota or phylum Aquificae. The correlation analysis showed that Crenarchaeota dominated in nutrient-rich environments with high concentrations of ions and total carbons, whereas Aquificae dominated in nutrient-poor environments with low ion concentrations. These environmental factors were also important explanatory variables in the generalized linear models constructed to predict the abundances of Crenarchaeota or Aquificae. Functional enrichment analysis of genes also revealed that the separation of the two major types is primarily attributable to genes involved in autotrophic carbon fixation, sulfate metabolism and nitrate reduction. Our results suggested that Aquificae and Crenarchaeota play a vital role in the Kirishima hot spring water ecosystem through their metabolic pathways adapted to each environment. Our findings provide a basis to predict microbial community structures in hot springs from environmental parameters, and also provide clues for the exploration of biological resources in extreme environments.