Optimization and validation of keratinase production by Bacillus aerius NSMk2 in a stirred tank reactor using response surface methodology

Keratinase is a robust enzyme that is produced in the presence of keratin substrates. This enzyme has been recognized for its applications in waste management, leather and detergent industries. Our group has isolated a potential keratinase producing strain of Bacillus aerius NSMk2 from poultry dump soil, and its hide dehairing and stain removal applications have been studied. Considering commercial applicability of keratinase, the present study reports the keratinase production in a stirred tank reactor (5 l). Central composite rotatable design of response surface methodology (RSM) was employed to study the effect of most influencing process variables, i.e., aeration (0.5-1.5 vvm), agitation (150-350 rpm) and incubation period (24-48 h) on keratinase production. The quadratic model predicted 15 experimental runs, and the influence of independent variables and their interaction on keratinase production were interpreted using analysis of variance (ANOVA) and t-test statistics. Coefficient of determination (R-2) value close to 1 and Fisher F-value of 3743.77 showed good fit of experimental data to second- order polynomial equation. A reasonable agreement between experimental and predicted values showed the accuracy of deduced model. Applying the desirability function, aeration rate of 1.0 vvm, agitation rate of 276.88 rpm and incubation period of 33.68 h supported maximum keratinase production (318.38 U/ml). Confirmatory experiments were performed to evaluate the accuracy of desirability function. Maximum keratinase activity of 318.11 U/ml close to predicted value (318.38 U/ml) validates the model. The present study provides useful guidelines for large-scale production of keratinase that can be used for various commercial applications.