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

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' fermen tation during growth of Escherichia coli on glucose and citrate were perfor med to identify and quantitatively estimate the concentrations of the two s ubstrates provided and of the six products formed without sampling from the NMR tube. Identification of fermentation substrates and products was achie ved by coincidence of selected diagnostic proton signals of individual comp ounds 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 t he 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 form ation of eight substances in the fermentation were monitored simultaneously , in normal (H2O)-H-1, without sampling and individual analysis. Several me tabolic changes could be readily detected during the fermentations. Additio nally, the ph changes were estimated from the chemical shifts of the acetat e signal as growth progressed. The effect of varying D2O concentrations in the solvent on growth rates and product yields was examined, and the increa se 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.