The methylotrophic yeast Pichia pastoris is one of the most widely used recombinant protein expression systems, with applications from food to the pharmaceutical industry. It is capable of growing using methanol as the only carbon source, for which it uses alcohol oxidase (AOX) promoter, a strong inducer for recombinant protein expression. One of the critical aspects of methanol addition strategies in these cultures is the methanol induction moment and avoiding its excessive addition that leads to its accumulation, making it toxic for the yeast when above certain concentration values in the medium. There are limited on-line monitoring sensors for methanol levels at the process level in cultures with Pichia, which can also be scaled and integrated with the process control software and the substrate addition pumps. In this work, the evolution of exhaust gas composition in O2 and CO2 has been used as an alternative to on-line methanol sensors, mainly according to the evolution of exhaust CO2, to establish optimal strategies for the methanol feeding and induction moment. For this purpose, benchtop and pilot Bionet fermenters have been used with external modules for continuous analysis of exhausted O2 and CO2 integrated into the equipment software. This preliminary work will make it possible to implement future controls that optimize the substrate addition strategies in fermentation processes according to a direct measurement of cellular metabolism.
