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P. pastoris Fermentation using a BioFlo 110 Benchtop Fermentor

DO probe was calibrated at 0%, (obtained by briefly disconnecting the cable), and at 100% (obtained using 1,000 rpm agitation and 5 L/m (1 vvm) airflow. After calibration, DO remained at approximately 100% until inoculation.

An agitation cascade was selected in the controller to maintain DO at setpoint through automatic adjustment of agitation speed. The agitation cascade increases agitation speed with increasing oxygen demand. To set up the cascade, we used the DO control display and keypad on the PCU to select:
Cascade : . . . . . . . . . . . . . . . . . . . .Agit
Minimum RPM : . . . . . . . . . . . . . . .300
Maximum RPM: . . . . . . . . . . . . .1,200


Nutrient Feed
Initial feed was 360 mL of 50% glycerol solution with 12 mL/L of PTM1 (trace metals). BioCommand began this feed automatically when the dissolved oxygen showed a sudden rise above the setpoint, a well-known carbonexhaustion indicator. After all the glycerol was consumed, we allowed a brief starvation phase, then changed the feed to 100% methanol solution with 12 mL/L of PTM1 (trace metals) solution.

Glycerol . . . . . . . . . .Pump 2 of the 4-Pump Module
Methanol . . . . . . . . .Pump 3 of the 4-Pump Module
Transfer tubing . . . .Silicone tubing as supplied (1.4mm inner diameter and 4.8mm outer diameter), P0740-2396
Vessel inlet . . . . . . . Triport adapter in the vessel headplate

Control Setup:
1) Pump 2 plugged into "Pump A" power-outlet of the Power Controller.
2) Pump 3 plugged into "Pump B" power-outlet of the Power Controller.
3) Pumps A & B: Manual mode, controlled by BioCommand


PH Control
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