Hybrid On-Line Optimal Control Strategy for Producing α-Amylase by Bacillus subtilis

Abstract

The combined effect of macronutrients in the extraction medium on α-amylase produced by Bacillus subtilis were studied by using response surface methodology in shaken flask cultures. The production of amylase was significantly affected by the interaction between wheat bran and the cotton seed extract in the extraction medium and by the interaction between the cotton seed extract and starch. The optimal combination in the extraction medium for maximum α-amylase production was determined as 10.80 g·L⁻¹ of wheat bran, 9.90 g·L⁻¹ of cotton seed extract, 0.5 g·L⁻¹ of starch, 2.0 g·L⁻¹ of yeast extract, 5.00 g·L⁻¹ of NaCl and 2.00 g·L⁻¹ of CaCl₂. A 12.55-fold increase of enzyme activity was recorded in the optimized medium compared to the result acquired in a minimum essential medium. The optimized medium was used to compare different cultivation strategies in fermenters. The pH-stat strategy for reducing cellular stress response and the substrate concentration-stat strategy for reducing substrate inhibition were independently investigated. The temperature-limited strategy has been proposed to solve the proteolytic digestion problem, although the high-pressure strategy resulted in high productivity. A hybrid strategy simultaneously controlling pH, temperature, substrate concentration and pO₂ was finally investigated to enhance the efficiency of the process. This hybrid strategy resulted in high activity of α-amylase, increasing the productivity almost three-fold as compared to an ordinary fed-batch culture.