The four control modules included with the Advanced Fermentation Kit were used for the first run.
Materials and Methods
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.
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.
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