METHODS: Simultaneous optimization of biomass and protease biosynthesis by a local isolated Pseudomonas sp. — Response surface optimization using Box-Behnken design

Response surface methodology (RSM) was used to optimize the fermentation medium for enhancing protease production by a Pseudomonas sp. In other work (in press), temperature, agitation speed, and salinity were found to be factors that significantly affected protease production, while temperature, glucose content, and pH were observed to have a significant influence on microorganism growth. In the current study, a Box-Behnken design was applied to determine the optimal conditions to maximize both protease and biomass production. Empirical second-order models were determined by multiple regression analysis of the experimental data to describe the relationship between tested variables and the responses. The determination coefficients (R2) were above 95%. For each of the optimized medium conditions, both protease production and microbial growth kinetics were evaluated. Under the best conditions (20.07°C, 47.61 rpm, and 2.07 g NaCl/L), practical protease production increased fivefold after 10 h of incubation. This compares with the kinetics obtained using a biphasic system and two-stage fermentation medium. Initially, Pseudomonas sp. was inoculated under the optimal biomass conditions (25.29°C, 3.35 g glucose/L, and pH 7.46) and then transferred to optimal media for protease production (20.07°C, 47.61 rpm, and 2.07 g NaCl/L). The use of a two-stage culture medium did not improve protease production over that observed in best-established conditions.