Statistics-based experimental design was used to investigate the effect of medium components (starch, peptone, ammonium sulfate, yeast extract, and CaCl(2)center dot H2O) on hen's egg white lysozyme production by Aspergillus niger HEWL WT-13-16. A 2(5-1) fractional factorial design augmented with center points revealed that peptone, starch, and ammonium sulfate were the most significant factors, whereas the other factors were not important within the levels tested. The method of steepest ascent was used to approach the proximity of optimum. This task was followed by a central composite design to develop a response surface for medium optimization. The optimum medium composition for lysozyme production was found to be: starch 34 g L-1, peptone 34 g L-1, ammonium sulfate 11.9 g L-1, yeast extract 0.5 g L-1, and CaCl(2)center dot 2H(2)O 0.5 g L-1. This medium was projected to produce, theoretically, 212 mg L-1 lysozyme. Using this medium, an experimental maximum lysozyme concentration of 209 18 mg L-1 verified the applied methodology. (c) 2005 Wiley Periodicals, Inc.