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)

Labelling experiment - After a minimum of five residence times of continuous cultivation, the feed was switched to the labelled medium. The composition of the labelling feed was based on the approach proposed by Nöh and Wiechert [1]. The feed contained for glucose, 80% [1-13 ... C1] and 20% [U-13C6] and for methanol, 100% [U-13C1]. Immediately after switching to labelled medium feed, more than 10 samples were taken during the first five minutes; thereafter, 10 more samples were taken evenly distributed until 6 hours of labelling feed. The labelling experiment was performed in two independent chemostat cultivation replicates.
13C-based metabolic flux analysis (13C-MFA) - The metabolic network used for 13C-based metabolic flux analyses was derived from the metabolic model described in [2] for P. pastoris. This initial model was further extended to include trehalose metabolism and the pyruvate dehydrogenase bypass reactions, as well as reversible reactions for Ala, Asp and Glu synthesis.

---------References----------
[1] Nöh K, Wiechert W: Experimental design principles for isotopically instationary 13C labeling experiments. Biotechnol Bioeng 2006, 94:234–251. http://doi.org/bxmkn8
[2] Jordà J, Jouhten P, Cámara E, Maaheimo H, Albiol J, Ferrer P: Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures. Microb Cell Fact 2012, 11:57. http://doi.org/s6q