Chemical-defined medium

NMR Experiments - cells were harvested in mid-logarithmic growth phase (A600 = 2.2), centrifuged, washed twice, and suspended to a protein concentration of 16.5 mg/ml in 50 mM KPi or Mes/KOH buffer, pH 6.5, for 13C or 31P NMR experiments, respectively.
Quantification o ... f Products - Lactate, acetoin, acetate, 2,3-butanediol, ethanol, and formate were quantified in NMR sample extracts by 1H NMR [1]. The concentration of minor products (e.g. pyruvate, ethanol, diacetyl) and metabolic intermediates that remained inside the cells (PEP, 3-PGA) was determined from the analysis of 13C spectra of NMR sample extracts as previously described [2]. Intracellular metabolite concentrations were calculated using a value of 2.9 l/mg protein for the intracellular volume of L. lactis [3]. All NMR spectra of living cells were run at 30 °C with a quadruple-nucleus probe head on a Bruker DRX500 spectrometer. Acquisition of 31P NMR and 13C NMR spectra was performed as described by Neves et al. [2]; however, acquisition parameters for 13C NMR were modified as follows: data size, 16,000; recycle delay, 0.3 s; number of transients, 480. Carbon and phosphorus chemical shifts are referenced to the resonances of external methanol or H3PO4 (85%) designated at 49.3 ppm and 0.0 ppm, respectively.

---------References---------
[1] de Vos, W.M (1999). Curr. Opin, Microbiol. 2, 289-295. http://doi.org/dztzb8
[2] Neves, A. R., Ramos, A., Nunes, M. C., Kleerebezem, M., Hugenholtz, J., de Vos, W. M., Almeida, J., and Santos, H. (1999). Biotechnol. Bioeng. 64, 200–212. http://doi.org/fqrv8j
[3] Poolman, B., Smid, E. J., Veldkamp, H., and Konings, W. N. (1987). J. Bacteriol. 169, 1460–1468.