Evaluation of three methanol feed strategies for recombinant Pichia pastoris Muts fermentation

Methylotrophic yeast Pichia pastoris is an ideal host organism for recombinant protein production. However, adequate methanol feed is still a critical point of successful product formation in P. pastoris Mut(S) fermentations. Three methanol feed strategies were tested: an organic vapor sensor, a dissolved oxygen controlled methanol addition and a pre-determined model, as well. The organic vapor sensor proved to be unsuitable for methanol concentration measurements when samples were taken from the head space of the bioreactor, but may have the potential to substitute expensive gas analyzers in methanol fed-batch with a suitable selector submerged into the fermentation media. Dissolved oxygen and substrate consumption did not show strict mathematical relation. However, drop of dissolved oxygen tension for periodic methanol addition may be applied for the determination of the substrate concentration in the media. The rate of methanol consumption shows peaks at 0.45 and 0.95% (v/v) substrate concentrations. The rate of the specific methanol consumption of our model organism was determined. Based on the value of 0.023 h(-1), which is significantly less than suggested by earlier experiments, a successful pre-determined methanol feed strategy was set up.