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.