Production of a recombinant protein using Pichia pastoris grown in evaporator condensate from a Kraft pulp mill

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 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 for 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 found to occur only in the presence of yeast peptone.