Alkaline Protease Production by an Isolated Bacillus circulans under Solid-State Fermentation Using Agroindustrial Waste: Process Parameters Optimization † R. S. Prakasham,* Ch. Subba Rao, R. Sreenivas Rao, and P. N. Sarma Bioengineering and Environmental Center, Indian Institute of Chemical Technology, Hyderbad 500 007, India Alkaline protease production using isolated Bacillus circulans under solid-state fermentation environment was optimized by using Taguchi orthogonal array (OA) experimental design (DOE) methodology to understand the interaction of a large number of variables spanned by factors and their settings with a small number of experiments in order to economize the process optimization. The software-designed experiments with an OA worksheet of L-27 was selected to optimize fermentation (temperature, particle size, moisture content and pH), nutrition (yeast extract and maltose), and biomaterial-related (inoculum size and incubation time) factors for the best production yields. Analysis of experimental data using Qualitek-4 methodology showed significant variation in enzyme production levels (32000-73000 units per gram material) and dependence on the selected factors and their assigned levels. Validation of experimental results on alkaline protease production by this bacterial strain based on DOE methodology revealed 51% enhanced protease production compared to average performance of the fermentation, indicating the importance of this methodology in the evaluation of main and interaction effects of the selected factors individually and in combination for bioprocess optimization. Introduction Proteases are one of the most important classes of biocatalysts from an industrial point of view, occupying a major share of 60% of the total enzyme market (1-3). These biocatalysts hydrolyze peptide bonds in proteins and hence are classified as hydrolases and categorized in the subclass peptide hydrolases or peptidases (4). Because of this functional property, they are widely used in laundry detergents, leather processing, protein recov- ery or solubilization, meat tenderization, and the biscuit and cracker industries (5). However, other application potentials of these enzymes depend on the nature of catalytic activity with respect to reactant medium, which led to the classification of proteases as acidic, neutral, and alkaline. Among these different biocatalysts, alkaline proteases have wide application spectra and novel prop- erties due to their exotic catalytic nature. Hence, these proteases and their producing organisms attracted at- tention of scientific community to understand the protein chemistry and protein engineering to enhance their utilization niche (6). Though several microorganisms such as bacteria, fungi, yeast, plant, and mammalian tissues are known to produce alkaline proteases (4, 7), with increasing industrial demand for proteases it is expected that hyperactive strains will emerge and that the enzymes produced by new exotic microbial strains could be used as biocatalysts in the presently growing biotechnological era. Available literature information indicates that, among all protease-producing microbial organisms, the Bacillus genus assumes importance because of its poten- tial for production in large amounts (2, 8, 9). Moreover, several medium components such as nitrogen and car- bon sources, physiological factors such as pH, incuba- tion temperature and incubation time, and biological factors such as the genetic nature of the organism influences the metabolic/biochemical behavior of the microbial strain and subsequent metabolite production pattern (2, 4, 7). Hence, in commercial practice, the optimization of medium composition is one of the es- sential steps to maintain a balance between the various medium components to minimize the amount of unuti- lized components at the end of fermentation and have cost-effective metabolite yield (2, 7). In general, no defined medium has been established for the best pro- duction of any metabolite because the genetic diversity present in different microbial sources causes each organ- ism or strain to have its own special conditions for maximum product production (4). Therefore, it is es- sential to have a detailed investigation on newly isolated microbial strain for production pattern under different environmental conditions and in an optimized pattern to achieve maximum production benefit. For effective triggering of alkaline protease production, it is highly imperative to optimize all fermentation conditions in- cluding medium composition, which further facilitates economic design of the full-scale operation system. How- ever, it is impractical to optimize all parameters and to establish the best possible conditions by interrelating all parameters, as this involves numerous experiments to be carried out with all possible combinations (7, 10). Experimental design based on statistical tools are known to provide economic and practical solutions in such cases. Optimization procedures developed to optimize the bio- technological processes consist of an empirical modeling system developed on a full factorial central composite * To whom correspondence should be addressed. Email: prakasam@iict.res.in. † IICT Communication no. 050318. 1380 Biotechnol. Prog. 2005, 21, 1380-1388 10.1021/bp050095e CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 08/26/2005