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In situ proton-NMR analyses of Escherichia coli HB101 fermentations in ¹H₂O and in D₂O

Institute of Organic Chemistry, Technical University Graz, Stremayrgasse 16, A-8010 Graz, Austria¹
Institute of Biotechnology, Technical University Graz, Petersgasse 12, A-8010 Graz, Austria²

Author for correspondence: Douglas W. Ribbons. Tel: +43 316 873 8241. Fax: +43 316 873 8740. e-mail: sekretariat{at}orgc.tu- graz.ac.at

Experiments using one-dimensional Fourier-transform proton-NMR spectrometry for non-invasive analyses of microbial fermentations in situ, in vivo and in normal aqueous buffer are described. Analyses of the ‘mixed acid’ fermentation during growth of Escherichia coli on glucose and citrate were performed to identify and quantitatively estimate the concentrations of the two substrates provided and of the six products formed without sampling from the NMR tube. Identification of fermentation substrates and products was achieved by coincidence of selected diagnostic proton signals of individual compounds in the same solvent. The complete time course of growth of E. coli in the NMR tube correlated well with that of the same culture grown outside the magnet, with samples taken for proton-NMR analyses. The entire course of these in situ proton measurements during growth over 16–24 h was obtained automatically, usually unattended overnight. Thus, the utilization and formation of eight substances in the fermentation were monitored simultaneously, in normal ¹H₂O, without sampling and individual analysis. Several metabolic changes could be readily detected during the fermentations. Additionally, the pH changes were estimated from the chemical shifts of the acetate signal as growth progressed. The effect of varying D₂O concentrations in the solvent on growth rates and product yields was examined, and the increase in the complexity of signals given by these fermentations is described. This versatile and rapid method for the simultaneous, direct and automatic analysis of mixtures of many compounds has the potential to be extended to routine on-line analyses of industrial fermentations.

Keywords: proton-NMR, in situ analysis, deuterium-labelled products, Escherichia coli fermentations

Abbreviations: FT, Fourier-transform

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