The purpose of this chapter is to educate the reader about the basic equipment and strategies used in fermentations of P. pastoris in both bench-top and pilot-scale operations. A key element in expression of foreign proteins in this yeast is the need for sufficient aeration, which is achieved by proper mixing of the media and by blending gases to control dissolved oxygen content. Automatic pH control is essential for growth and expression in P. pastoris. Finally, fed-batch fermentations require the use of peristaltic pumps and tubing capable of low rates of delivery for the feeding of nutrients and base. Teflon tubing and peristaltic pump adapters are recommended for fed-batch operations. The information in this chapter should enable a reader with little or no experience to perform a high-cell density fermentation of a P. pastoris expression strain. Although most procedures described here are specifically for the BioFlo III (NBS), it should be possible to achieve high expression levels with almost any good-quality fermentor, modified to accommodate this organism.

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