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P. pastoris Fermentation using a BioFlo 110 Benchtop Fermentor

inal 5.7 L working volume. All BioFlo 110 fermentation vessels are configured with a 4-baffle stainless-steel insert, dual Rushton agitation impellers, and a high-speed, direct-drive agitation system with mechanical face-seal. Dissolved oxygen and pH probes (Mettler Toledo) are also included, as are a variety of items such as liquid addition bottle kits (3), cables, tubing and clamps.


Control System
The four control modules included with the Advanced Fermentation Kit were used for the first run.



Materials and Methods
Overview

This Pichia fermentation follows a well-established protocol in which glycerol is the initial carbon source, and after a brief carbon starvation, we switch to a methanol feed. The switch to methanol produces a metabolite of interest by triggering the AOX1 promoter in genetically engineered Pichia . These are fed-batch fermentations, since first glycerol and later methanol is added while the culture is growing.


Control Program
We created a feed control program using BioCommand Plus software, NBS Catalog Number M1291-0000. It turned on the glycerol feed-pump when the dissolved oxygen (DO) level rose above 40%. Each time DO exceeded 40%, the glycerol pump turned on; each time it fell below 40%, the pump turned off. 40% is a high DO level, indicative of reduced metabolism due to carbon exhaustion. The rationale for this strategy is that the DO increases due to reduced growth of the cells, which is a result of nutrient depletion. Approximately one hour after a rise in DO which indicated depletion of the supplementary glycerol, the program automatically turned on the methanol feed-pump.


Inoculum
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