This paper provides an approach for optimizing the cell density (Xc) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon tau (INF-tau). The objective was to maximize the volumetric productivity (Q, mg INF-tau l(-1) h(-1)), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of Xc and D within the ranges 150< or = Xc < or =450 g cells (wet weight) l(-1) and 0.1 microm< or = D< or =0.9 microm (microm=0.0678 h(-1), the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor). The methanol and medium feed rates that resulted in the desired Xc and D were determined based on the mass balance. From the RSM model, the optimal Xc and D were 328.9 g l(-1) and 0.0333 h(-1) for a maximum Q of 2.73 mg l(-1) h(-1). The model of specific production rate (rho, mg INF-tau g(-1) cells h(-1)) was also established and showed the optimal Xc 287.7 g l(-1) and D=0.0361 h(-1) for the maximum rho (predicted to be 8.92 x 10(-3) mg(-1) g(-1) h(-1)). The methanol specific consumption rate (nu, g methanol g(-1) cells h(-1)) was calculated and shown to be independent of the cell density. The relationship between nu and mu (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.
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