Optimisation of substrate feeding in Pichia pastoris cultures. Pichia uses methanol as a carbon/energy source. Methanol, however, is toxic at high concentrations. A recombinant strain of Pichia pastoris GS115 was cultivated in 1 l baffled shake flasks with three side necks containing 200 ml of BMM medium at 30°C with 200 rpm. To maintain the expression of the product, 100 % of methanol was added to a final concentration of 0.5 % (v/v) either twice a day (a) or alternatively, at the time when the DOT level increased (b). Times for substrate feeding are indicated with vertical lines.