| Summary: | Increasingly, environmental regulations are guiding industries to adopt practices
which minimize water use and associated effluent discharge. In the pulp and paper
industry, this has resulted in a trend to recycle waste streams as process water within
various mill operations. Since instituting environmentally sound practices is often a
balance between satisfying regulatory requirements and plant economics, any process
which may satisfy both goals should be investigated further.
In this study, 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 monitored and controlled via a program written in Lab VIEW
[National Instruments, Austin, Texas]. Feeding was automated using a feedback
algorithm loosely based on the SCF technique which used DO patterns to initiate cycling.
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. Maximum enzyme activities for the reactor trials were in the
range of 10.8 - 13.9 μmol/min/mL. Protein concentrations for the two final runs were
measured at 57 and 48 mg/L, yielding respective specific activity values of 220 and 140
jjmol/min/mg. These values are roughly 6-7 times lower than cited in literature and is
thought to have been resultant from various factors including proteolytic deactivation.
This study has demonstrated the possibility of growing a recombinant yeast in a
kraft waste effluent to produce a useful product.
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