Bioprocess Optimization of Alkaline Protease Production by Bacillus tropicus: Effects of Culture Conditions and Nutritional Factors

Abstract

This study investigated the isolation, characterization, and optimization of alkaline protease production by a bacterial isolate obtained from fish pond (FW) and poultry farm (PW) water samples. Serial dilutions 10⁻² and 10⁻³ were cultured on skim milk agar at 37 °C and pH 9 for 72 h. Among the isolates, PW10⁻²A2 exhibited the highest proteolytic activity with a zone of hydrolysis (ZOH) of 13 mm, compared to PW10⁻²A1 (4 mm) and PW10⁻²B2 (2 mm). Morphological and biochemical characterization identified the isolate as a Gram-positive, motile, endospore-forming rod with broad carbohydrate utilization and enzymatic capabilities, consistent with Bacillus tropicus. The Optimization studies revealed that maximum protease production occurred at 96 h (0.1420 Umol/mL; 5.6807 µmol tyrosine released; biomass 1.912 OD₆₀₀), while minimum production was observed at 48 h (0.1052 Umol/mL; 4.2075 µmol tyrosine; biomass 1.286 OD₆₀₀). Among carbon sources, rice bran yielded the highest activity (0.1567 Umol/mL), whereas maltose showed the lowest (0.1085 Umol/mL). Beef extract was the best organic nitrogen source (0.1671 Umol/mL), while ammonium nitrate (NH₄NO₃) was the most effective overall nitrogen source (0.17435 Umol/mL). Optimal enzyme production occurred at pH 9 (0.1425 Umol/mL) and 35 °C (0.0703 Umol/mL). Maximum yield was achieved with 2.5 mL inoculum size (0.1314 Umol/mL), 96 h inoculum age (0.1305 Umol/mL), and agitation speed of 220 rpm (0.0605 Umol/mL). One-way ANOVA demonstrated that incubation time, carbon and nitrogen sources, pH, temperature, inoculum size, inoculum age, and agitation significantly influenced protease production (p < 0.05). Maximum enzyme yield was obtained under optimized conditions (96 h, pH 9, 35 °C, rice bran, NH₄NO₃, 2.5 mL inoculum, 220 rpm). Correlation analysis indicated that enzyme production was not strictly growth-associated (r = 0.42). The study demonstrates that Bacillus tropicus PW10⁻²A2 is a potent alkaline protease producer with significant industrial potential, particularly for applications in detergent and leather processing industries.