Production of a Recombinant Protein Using Pichia Pastoris Grown in Kraft Evaporator Condensate, 2003 Environmental Conference Proceedings

The feasibility of cultivating a recombinant strain of the methylotrophic yeast, Pichia pastoris, on combined evaporator condensate from a Kraft mill was investigated. The use of a recombinant yeast in this application is novel and the reasoning behind this research is two-fold. Firstly, to facilitate complete methanol removal from a waste stream, rendering it suitable for re-use within the mill or discharge. Secondly, to generate a product of potential economic value in the pulp and paper industry. A literature review was conducted to generate a list of Pichia transformants expressing potentially useful products. Through a selection process that rated each candidate based on a criteria set, a recombinant Pichia strain expressing lipase from Geotrichum candidum was selected.

A series of shake flask experiments were performed to gauge the effect of various media compositions on yeast growth. This was followed by fed-batch cultivations in a 1.8 L reactor which was automated via a control program written in LabVIEW [National Instruments, Austin, Texas]. Cell densities of 8-12 g/L (dry weight) were reached within the reactor grown on a condensate/methanol feed. Lipase activity was determined titrimetrically and was produced in condensate-based media, however only in the presence of yeast-peptone.