Two-phase fed-batch cultivation of P. pastoris GS115 carrying 8-copies of the HBsAg gene under the control of the AOX1 promoter. Cells were first grown in a batch phase with glycerol as carbon source followed by a methanol feeding phase to induce the production of HBsAg. (A) Concentrations of glycerol (filled squares) and biomass (optical density: filled circles; cell dry mass: filled triangles). (B) Concentration of methanol (solid line) and amount of methanol added to the bioreactor (dashed line). (C) Medium pH (solid line) and amount of ammonium hydroxide added to the bioreactor (dashed line). (D) Dissolved oxygen concentration (solid line), aeration rate (dotted line), and stirrer speed (dashed line). (E) Oxygen transfer (dashed line) and carbon dioxide evolution (solid line) rates and respiratory quotient (dotted line). Small arrows indicate removal of culture broth. (F) Cell growth (filled circles) and accumulation of HBsAg (total HBsAg: filled squares and soluble HBsAg: open squares). The dashed vertical line indicates the end of the glycerol batch and the start of the methanol feeding phase.