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Background  Environmental processes in ecosystems are dynamically altered by several metabolic responses in microorganisms, including intracellular sensing and pumping, battle for survival, and supply of or competition for nutrients. Notably, intestinal bacteria maintain homeostatic balance in mammals via multiple dynamic biochemical reactions to produce several metabolites from undigested food, and those metabolites exert various effects on mammalian cells in a time-dependent manner. We have established a method for the analysis of bacterial metabolic dynamics in real time and used it in combination with statistical NMR procedures.    Methodology/Principal Findings  We developed a novel method called real-time metabolotyping (RT-MT), which performs sequential  1 H-NMR profiling and two-dimensional (2D)  1 H,  13 C-HSQC (heteronuclear single quantum coherence) profiling during bacterial growth in an NMR tube. The profiles were evaluated with such statistical methods as Z-score analysis, principal components analysis, and time series of statistical TOtal Correlation SpectroScopY (TOCSY). In addition, using 2D  1 H,  13 C-HSQC with the stable isotope labeling technique, we observed the metabolic kinetics of specific biochemical reactions based on time-dependent 2D kinetic profiles. Using these methods, we clarified the pathway for linolenic acid hydrogenation by a gastrointestinal bacterium,  Butyrivibrio fibrisolvens . We identified  trans 11,  cis 13 conjugated linoleic acid as the intermediate of linolenic acid hydrogenation by  B. fibrisolvens , based on the results of  13 C-labeling RT-MT experiments. In addition, we showed that the biohydrogenation of polyunsaturated fatty acids serves as a defense mechanism against their toxic effects.    Conclusions  RT-MT is useful for the characterization of beneficial bacterium that shows potential for use as probiotic by producing bioactive compounds.

Evaluation and Characterization of Bacterial Metabolic Dynamics with a Novel Profiling Technique, Real-Time Metabolotyping