Mixed feeds of glycerol and methanol can improve the performance of Pichia pastoris cultures: A quantitative study based on concentration gradients in transient continuous cultures

Transient continuous cultures constitute a means to speed up strain characterization, by avoiding the need for many time-consuming steady-state experiments. In this study, mixed substrate growth on glycerol and methanol of a Pichia pastoris strain expressing and secreting recombinant avidin was characterized quantitatively by performing a nutrient gradient with linear increase of the methanol fraction in the feed medium from 0.5 to 0.93 C-mol C-mol−1 at a dilution rate of 0.06 h−1. The influence of the methanol fraction in the feed medium on recombinant avidin productivity and on specific alcohol oxidase activity were also examined.Results showed that, compared with cultures on methanol as sole carbon source, the specific recombinant avidin production rate was the same provided the methanol fraction in the feed medium was higher than 0.6 C-mol C-mol−1. The volumetric avidin production rate was even 1.1-fold higher with a methanol fraction in the feed medium of 0.62 C-mol C-mol−1 as a result of the higher biomass yield on mixed substrate growth compared with methanol alone. Moreover, since heat production and oxygen uptake rates are lower during mixed substrate growth on glycerol and methanol, mixed substrate cultures present technical advantages for the performance of high cell density P. pastoris cultures.Results obtained in a high cell density fed-batch culture with a mixed feed of 0.65 C-mol C-mol−1 methanol and 0.35 C-mol C-mol−1 glycerol were in agreement with results obtained during the transient nutrient gradient.