Optimization of newly isolated Bacillus cereus α-amylase production using orange peels and crab shells and application in wastewater treatment.

A newly isolated amylolytic strain was identified as Bacillus cereus spH1 based on 16S and 16-23S gene sequencing (Accession numbers OP811441.1 and OP819558, respectively), optimization strategies, using one variable at time (OVAT) and Plackett-Burman design, were employed to improve the alpha-amylase (α-amylase) production. Condition inferred revealed that the optimal physical parameters for maximum enzyme production were 30 °C, pH 7.5, and 12 h of incubation, using tryptone, malt extract, orange ( Citrus sinensis) peels, crab ( Portunus segnis) shells, calcium, and sodium chloride (NaCl) as culture medium. The full factorial design (FFD) model was observed to possess a predicted R2 and adjusted R 2 values of 0.9788 and 0.9862, respectively, and it can effectively predict the response variables ( p  = 0). Following such efforts, α-amylase activity was increased 141.6-folds, ranging from 0.06 to 8.5 U/mL. The ideal temperature and pH for the crude enzyme activity were 65 °C and 7.5, respectively. The enzyme exhibited significant stability, with residual activity over 90% at 55 °C. The maltose was the only product generated during the starch hydrolysis. Moreover, the Bacillus cereus spH1 strain and its α-amylase were used in the treatment of effluents from the pasta industry. Germination index percentages of 143% and 139% were achieved when using the treated effluent with α-amylase and the strain, respectively. This work proposes the valorization of agro-industrial residues to improve enzyme production and to develop a green and sustainable approach that holds great promise for environmental and economic challenges.