Characterization of Metabolites in IntactStreptomyces citricolorCulture Supernatants Using High-Resolution Nuclear Magnetic Resonance and Directly Coupled High-Pressure Liquid Chromatography–Nuclear Magnetic Resonance Spectroscopy

A novel NMR spectroscopic approach to the direct biochemical characterization of bacterial culture broths is presented. A variety of one- and two-dimensional1H NMR spectroscopic methods were used to characterize low-molecular-weight organic components of broth supernatants from cultures ofStreptomyces citricolor.By applying1H NMR spectroscopy to analyze whole, untreated culture supernatants, it was possible to identify and monitor simultaneously a range of media substrates and excreted metabolites. Identified metabolites include 2-phenylethylamine, trehalose, succinate, acetate, uridine, and aristeromycin, a secondary metabolite with antibiotic properties. Directly coupled HPLC–NMR spectroscopy was also applied to the analysis of broth supernatants for the first time, to aid spectral assignments, especially where signals were extensively overlapped in the1H NMR spectra of the whole broth mixtures. Two-dimensional NMR methods such as1H–1H correlation spectroscopy,1H–13C heteronuclear single quantum correlation, and1H–13C heteronuclear multiple bond correlation aided the structure elucidation and peak assignments of individual components in the mixtures by providing information on1H–1H coupling networks and13C chemical shifts. This work shows that high-resolution NMR spectroscopic methods provide a rapid and efficient means of investigating microbial metabolism directly without invasive or destructive sample pretreatment.