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To optimize the fermentation medium for the production of alkaline protease by Bacillus licheniformis MZK05M9 (BlM9) molasses as a carbon source, soybean meal as a nitrogen source, and the salts NaCl, MgSO4.7H2O, and K2HPO4 were selected by Plackett–Burman approach. The response surface methodology based on central composite design revealed that the optimum values for the tested variables were found as (% w/v) molasses (0.92%), soybean meal (0.79%), NaCl (0.125%), MgSO4 (0.125%), and K2HPO4 (0.59%) with the protease activity 761 U/ml predicted by statistical software Minitab Version 17. The experimental value was found as 765 U/ml. The granular size of soybean meal 4.7 mm supported the enzyme production 5% higher than that of the mixed sizes between 6 and 4 mm. Fermentation in 7 l bioreactor exhibited the enzyme activity 1020 U/ml after 28 h. The statistically optimized medium based on cost-effective agro-industrial C and N sources yielded a high productivity 36,428 U/l h of protease by the mutant strain of B. licheniformis.

Optimization of fermenting medium by statistical method for production of alkaline protease by Bacillus licheniformis MZK05M9