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

Abstract

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 12h 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 R2 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.