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P. pastoris Fermentation using a BioFlo 110 Benchtop Fermentor


The inoculum was prepared using Pichia shake-flask growth medium:
Potassium phosphate monobasic (anhydrous) . . .11.5 g/L
Potassium phosphate dibasic (anhydrous) . . . . . . .2.7 g/L
Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 g/L
10X YNB solution. . . . . . . . .. . . . . . . . .. . . . . . . 10% by volume
10x YNB solution consists of 67 g/L YNB without amino acids. The solution is filter-sterilized and added to other media components after they are heat-sterilized and cooled.

The inoculum was cultivated for 40 hours at 28C in a rotary shaker (NBS model G25) running at 240 rpm. Optical Density at 600 nm (OD600) was 10.94 at inoculation.


Medium
The initial fermentor medium composition included:
Calcium sulfate dihydrate . . . . . . . . . . . . . . . . .. . . .0.93 g/L
Potassium sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . .18.2 g/L
Magnesium sulfate heptahydrate . . . . . . . . . .. . . . . .14.9 g/L
Potassium hydroxide . . . . . . . . . . . . . . . . . . . . . .. . .4.13 g/L
Phosphoric acid . . . . . . . . . . . . . . . . . . . . . . . . . . . .26.7 ml/L
Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .40.0 g/L
Antifoam (Breox Foam Control Agent FMT 30) . . .1.0 ml/L

To allow space in the 5.7 L (working volume) vessel for components added after sterilization, the initial medium volume was only 3.5 L. Post-sterilization medium components included:
Trace Metals solution, PTM1 . . . . . . . . . . .4.6 mL/L
Base, to adjust the initial pH . . . . . . . . . . . .25 mL/L
Inoculum . . . . . . . . . . . . . . . . . . .
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