Pichia pastoris Continuous Cultivations for Strain Characterization and Continuous Biomanufacturing.

Continuous cultivation provides physiological and product-related data of the recombinant-producing strain, which is crucial for bioprocess design and optimization. In particular, continuous cultivation enables the study of one condition at a time, being also a suitable mode for systems biology. Besides, regulatory and technological bases are being set at the industrial scale to achieve continuous biomanufacturing in the near future. Therefore, this chapter will describe the process of conducting carbon-limiting continuous cultivations, also known as chemostat cultivations, the most used continuous strategy for Pichia pastoris (Komagataella phaffii). In this protocol, design equations and setup descriptions can be found in order to carry out chemostat cultivations in a benchtop bioreactor. Although the process is described for K. phaffii using glucose, glycerol, or methanol as the sole carbon source, the protocol can be adapted to other substrates and microorganisms.