Chemical and Biomolecular Engineering, Department of


Date of this Version



A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Chemical Engineering, Under the Supervision of Professor William H. Velander. Lincoln, Nebraska: December, 2012

Copyright (c) 2012 Frank M. Fabian


Pichia pastoris has demonstrated the ability to express high levels of recombinant heterologous proteins. Protein expression is enhanced during fermentation at high cell density. However, the level of expression is mainly regulated by fermentation operation factors. This research is directed to investigate the effect of methanol growth rate, temperature and pH in the expression of the total 5P12-Rantes concentration, expression of active 5P12-Rantes and Specific yield using the response surface methodology and central composite design.

The response surface methodology, RSM, has been used successfully used by Zhang, W. and Ian, M. to optimize the cell density and fermentation process. The RSM is equipped with statistical tools to determine the significance of a factor over a response. The evaluation of factors using the RSM uses experimental design in order to distribute the selected variables within the boundaries of the design.

The central composite design, CCD, allows allocation of Methanol growth rate, temperature, and pH to evaluate their effect in the expression of 5P12-Rantes. The effect of methanol growth rate is evaluated from a minimum value of 0.01 h-1 to a maximum of 0.4 h-1, 24.06 °C to 29.94 °C for temperature and 1.99 to 4.51 for pH.

The methanol growth rate has demonstrated to have no effect in any of the responses. In contrast, Temperature and pH has a significant effect in all the responses. However, the lack of fit of the proposed model doesn’t allow good estimations of predicted responses. In order to minimize the lack of fit of the propose models, the methanol growth rate has been excluded from all the three models. A new 2 factor second order model is proposed to analyze the significance of temperature and pH. The lack of fit decreased and the optimal operating conditions for temperature and pH was determined at 27.14 °C and 3.16 which results in a maximum of 26.52 g/L of active 5P12-Rantes and 0.16 mg Rantes/g dry cell.

Adviser: William H. Velander