Production of β-Glucosidase by Aspergillus niger on Wheat Bran and Glycerol in Submerged Culture: Factorial Experimental Design and Process Optimization

Abstract

The goal of this study was to develop a fermentation process for the production of β-glucosidase, an important enzyme in the hydrolysis of lignocellulose and has many applications in food and flavor industries, using low-cost agricultural residues as substrates. Based on statistical experimental design, high-titer production of β-glucosidase on wheat bran and glycerol by Aspergillus niger in a submerged culture was achieved. A 2-level Plackett-Burman design was first used to screen the bioprocess parameters affecting β-glucosidase production. Among the tested parameters, the concentrations of wheat bran, glycerol, corn steep liquor and KCl showed significant effects on β-glucosidase production. These four medium components were further optimized using a 3-level Box-Behnken design, and their optimal levels were found to be: wheat bran, 3.5 g/L; glycerol, 5 g/L; KCl, 0.1 g/L and corn steep liquor, 7.5 g/L, giving a high β- glucosidase titer of 9.37 IU/ml, 1.6-fold of the maximum level obtained in the screening experiment. With the optimized medium, 5.41 IU/ml of β-glucosidase was produced in a stirred-tank bioreactor inoculated with a spore suspension. When the reactor was inoculated with precultured cell pellets and operated at optimized agitation (450 rpm) and aeration rates (2 vvm), β-glucosidase production increased to 9.33 IU/ml, which was comparable to the level obtained in shake-flasks. This study demonstrated a scalable fermentation process for the production of β-glucosidase on low-cost wheat bran and glycerol, which should have important industrial applications. Keywords: Aspergillus niger, β-glucosidase, glycerol, plackett-burman design, stirred tank bioreactor, wheat bran.