A marked enhancement in production of amylase by Bacillus amyloliquefaciens in flask fermentation using statistical methods

A total of 126 bacterial strains were isolated from soil samples. Among them, 11 isolates were found positive for amylase production. Strain YL produced the largest zone of clearance on plate assay. The isolate YL was identified as Bacillus sp. based on morphological and physiochemical characterization. According to 16S rRNA gene sequencing data, the closest phylogenetic neighbor of strain YL was Bacillus amyloliquefaciens (99.54%). After that, an optimization of culture conditions was carried out for the improvement of alpha-amylase production. Response surface methodology (RSM) was applied to evaluate the effect of medium components including wheat bran, cottonseed extract, yeast extract, starch, NaCl and CaCl2. Three variables (wheat bran, cottonseed extract, and starch), which were identified to significantly affect amylase production by Plackett-Burman design were further optimized using response surface methodology of Box-Behnken design (BBD). The optimal concentrations estimated for each variable related to the maximum of amylase activity (86 kU/mL) were 10.80 g/L wheat bran, 9.90 g/L cottonseed extract, 0.5 g/L starch, 2.0 g/L yeast extract, 5.00 g/L NaCl and 2.00 g/L CaCl2. The fermentation using optimized culture medium allowed a significant increase in amylase production (by 3-fold). The improvement in the alpha-amylase production after optimization process can be considered adequate for large-scale applications.