7.04 g/l Glucose monohydrate, 1.6 g/l Methanol, 0.915 g/l citric acid, 2 g/l (NH4)2 HPO4, 0.3 g/l MgSO4.7H2O, 1.4 g/l KH2PO4, 0.01 g/l CaCl2 · 2H2O, 0.5 ml/l trace salt solution, 0.3 ml/l biotin (0.2 g/l)

Sampling and measurement of metabolite concentrations - For quenching and extraction, the protocol recently described for P. pastoris growing on glucose [1] was used.
The metabolite extraction was performed with 75% (v/v) aqueous ethanol at 95 °C. Concentrations were m ... easured from two independent chemostat experiments in triplicate. The labelling enrichment was measured from one wash-in experiment by sampling at 20 time-points. The sampling times were selected based on the theoretical calculations by Nöh and co-workers [2], exponentially increasing intervals over a total of 6 hours to also achieve steady state in large intracellular pools like glutamate. The first sample is taken at 5 s after the switch to labelled material, which is the maximum velocity for manual sampling. All further sample processing steps were carried out as described previously [3].
Quantification of extracellular metabolites - Triplicate samples (5 ml) for extracellular metabolite analyses were centrifuged at 6,000 rpm for 3 min in a micro centrifuge (Minispin, Eppendorf) to remove the cells, and subsequently filtered through 0.45 mm-filters (Millipore type HAWP). Glucose, methanol, and other extracellular compounds were analyzed by HPLC analysis using an ionic exchange column, (ICSep ICE-COREGEL 87H3, Transgenomic). The mobile phase was 6 mM sulphuric acid.

------------References--------------
[1] Carnicer M, Canelas AB, Pierick A, Zeng Z, Dam J, Albiol J, Ferrer P, Heijnen JJ, Gulik W: Development of quantitative metabolomics for Pichia pastoris. Metabolomics 2011, 8:284–298. http://doi.org/dsg2kz
[2] Nöh K, Wahl A, Wiechert W: Computational tools for isotopically instationary 13C labeling experiments under metabolic steady state conditions. Metab Eng 2006, 8:554–577. http://doi.org/bxd49p
[3] Douma RD, de Jonge LP, Jonker CTH, Seifar RM, Heijnen JJ, van Gulik WM: Intracellular metabolite determination in the presence of extracellular abundance, Application to the penicillin biosynthesis pathway in Penicillium chrysogenum. Biotechnol Bioeng 2010, 107:105–115. http://doi.org/cdphfq