Optimization of thermostable amylolytic enzyme production from Bacillus cereus isolated from a recreational warm spring via Box Behnken design and response surface methodology

This study aimed to find a source for local amylase-producing microbes. Sixteen isolates were obtained from the water samples from the warm spring and characterized based on morphological and biochemical tests. The 16S rRNA molecular identification technique confirmed the most potent isolate as Bacillus cereus. The thermophilic property of the bacterium demonstrated that it could withstand temperatures of up to 80 °C. One-factor-at-a-time (OFAT) and Box Behnken Design (BBD) coupled with response surface methodology (RSM) optimization techniques were used to improve amylase production. OFAT established optimal physical parameter conditions as the starch concentration of 5% w/v, inoculum volume of 2% v/v, pH of 8, incubation temperature of 45 °C, and 48 h of incubation, leading to amylase activity of 172.6 U/mL by the isolated B. cereus. A quadratic mathematical model with a coefficient of determination (R²) of 0.9957 was established for the amylase production process. Enhanced amylase activity of 196.02 U/mL was achieved with BBD-RSM under optimal growth conditions of pH of 7, incubation time of 48 h, substrate concentration of 5% w/v of starch, and at 45 °C, a 1.2-fold increase compared to the OFAT method. The B. cereus strain isolated from the warm spring was a mildly thermophilic bacterium with the potential for synthesizing amylolytic enzymes with characteristics beneficial for commercial utilization.