Minimal medium: 2 g of sodium acetate or 4 g of glucose per liter, 48 mM Na2HPO4, 22 mM KH2PO4, 10 mM NaCl, and 30 mM (NH4)2SO4. The following components were sterilized separately and then added (per liter of final medium): 1 ml of 1 M MgSO4, 1 ml of 0.1 mM CaCl2, 1 ml of 1 mg of vitamin B1 per liter (filter sterilized), and 10 ml of trace element solution containing (per liter) 0.55 g of CaCl2, 1 g of FeCl3, 0.1 g of MnCl2·4H2O, 0.17 g of ZnCl2, 0.043 g of CuCl2·2H2O, 0.06 g of CoCl2·6H2O, and 0.06 g of Na2MoO4·2H2O.

Labeling experiments were initiated after the culture reached a steady state, which was inferred from the stable oxygen and carbon dioxide concentrations in the fermentor off-gas and stable optical density in the effluent medium for at least twice as long as the residence ti ... me. The feed medium containing 2 g of unlabeled sodium acetate per liter was then replaced by an identical medium containing 1.8 g of sodium acetate labeled by natural abundance per liter and 0.2 g of [2-13C] sodium acetate (Wako Co., Osaka, Japan) per liter. In the case where glucose was used as a sole carbon source, the mixture of 0.3 g uniformly labeled glucose [U-13C], 0.3 g first carbon labeled glucose [1-13C] and 3.4 g of naturally labeled glucose per liter was used to replace the initial feed medium containing 4 g l−1 unlabeled glucose. Biomass samples for GC-MS analysis were taken after one residence time, and the labeling measurements were corrected for the remaining original (nonlabeled) biomass that was still present even at the end of the labeling experiment [1]. Sample preparation for analysis:
Samples were taken from the cultivation for the determination of absorbance at 600 nm using a spectrophotometer (V-530, JASCO Co., Japan). Thereafter, centrifugation of the remaining sample volume was carried out at 4 °C, 10 000 rpm for 10 min, and the filtrate was frozen at −30 °C for later use in extracellular metabolites analyses. After this, the cell pellet was resuspended in distilled water and centrifuged again. For the analysis of the fractional labeling of intracellular metabolites, about 20 mg of wet biomass was transferred to 1 ml of 6 M HCl. The closed tube was heated for 24 h at 110 °C for complete hydrolysis and, after cooling to room temperature, the solvent was evaporated under a stream of air. After this, about 1 ml of distilled water was added to the dried hydrolytes, which was then filtered through a 0.2 μm-pore-size filter for separation of the cell debris. The filtrate was dried again under a stream of air and redissolved in 0.5 ml of acetonitrile (chromatographic grade) for later GC-MS analysis. GC-MS analysis: Hundred microliter acetonitrile containing biomass hydrolysate was added to 100 μl of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBST-FA) (derivatization grade, Aldrich, USA). The mixture was incubated for 60 min at 110 °C for complete derivatization. After cooling to room temperature, aliquots of the solution containing the derivatives were used directly for GC. GC-MS analysis was carried out using AutoSystem XL GC (PerkinElmer Co., USA) equipped with a DB-5MS column (30 m×0.25 mm×0.25 μm, Agilent Co., USA) which was directly connected to a TurboMass Gold mass spectrometer (PerkinElmer Co.). The injection volume was 1 μl with flow mode in split control. The carrier gas flow was set at 1-ml min−1 helium. The oven temperature was initially held at 80 °C for 2 min. Hereafter, the temperature was raised with a gradient of 8 °C min−1 until the temperature reached to 290 °C. This temperature, 290 °C, was held for 3 min. Other settings were as follows: 250 °C interface temperature, 200 °C ion source temperature, and electron impact ionization (EI) at 70 eV. Mass spectra were analyzed by both full scan mode and selected ion monitoring (SIM) mode. Raw MS data were processed using the program TurboMass Gold V4.3 (PerkinElmer Co.) to obtain a purified spectrum by removing residual background contaminants, partially eluting peaks, and column bleed from the spectrum. Skewing effects of natural isotopes were corrected by developing a program using matlab language (Mathwork Co., USA).Please see more details in the original publication. -----------------------------------References---------------------------------
[1] M Dauner, J.E Bailey, U Sauer. Biotechnol. Bioeng., 76 (2001), pp. 144-156. http://doi.org/db9x4v