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Growth of a genetically-modified Pichia pastoris and protein production in an industrial waste stream Yuen, Vivian Hoi Nga


This study demonstrated the possibility of growing a genetically-modified Pichia pastoris (Geotrichum candidum recombinant Pichia strain, GS115 (his4), harboring plasmid YpDC420) and producing a recombinant protein in the distillation column bottom stream of Methanex Corporation, Kitimat, B.C. The Pichia pastoris strain was grown in an automatically controlled reactor, which originally contained 1.6 L of 10 g/L yeast extract and 20 g/L peptone, running in continuous mode at a temperature of 30°C, pH of 5.6, and dissolved oxygen concentration of 3.0 g/L. The Methanex wastewater, which had a methanol concentration of 2.5 g/L, was continuously fed into the reactor at dilution rates (D) of 0.011, 0.017, 0.026, 0.034 and 0.042 1/h. The reactor reached steady state after about 120 hours for D = 0.011, 0.017 and 0.026 1/h. However, the reactor did not reach steady state even at extended running time (>150 hours) for D = 0.034 and 0.042 1/h, which were at or greater than the critical dilution rate, i.e., 0.034 1/h. The cell yield from methanol, product yield from cell, maximum specific growth rate and saturation constant for methanol were 3.3 ± 0.067 g/g, 0.015 + 0.0023 g/g, 0.034 1/h and 4.5 mg/L, respectively. At the optimum dilution rate of 0.033 1/h, Q[sub X] and Q[sub P] were maximized with values of 0.26 g/L/h and 4.0 mg/L/h. Steady state methanol concentrations in the reactor were almost zero when dilution rates were well below the critical dilution rate. The corresponding cell and protein concentrations were about 8 g/L (dry cell weight) and 125 mg/L. As dilution rates approached the critical value, steady state methanol concentration increased while cell and protein concentrations decreased. At the critical dilution rate, cell washout occurred and both steady state cell and protein concentrations reduced to zero. The corresponding methanol concentration in the reactor became the same as the methanol concentration in the feed. The reactor system model under study could be represented by 4 ordinary differential equations (ODEs) and the following parameters: maximum specific growth rate for methanol = 0.034 1/h, saturation constant for methanol = 4.5 mg/L, cell yield from methanol = 3.3 g/g, product yield from cell = 0.015 g/g, maximum specific growth rate for yeast extract and peptone = 0.20 1/h, saturation constant for yeast extract and peptone = 200 mg/L, and cell yield from yeast extract and peptone = 0.20 g/g. The ODEs were solved numerically and the resultant model predictions compared favourably to the reactor behaviour.

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