Process optimization for amylase production by Bacillus subtilis R4 under agro-waste solid-state fermentation using statistical tools

Abstract

Solid-state fermentation (SSF) is recognized as an efficient method for microbial enzyme production using low-cost agro-waste. In this study, 32 microbial isolates obtained from soil samples collected from ecological fields (rice, sweet potato, and elephant foot yam) were screened on starch agar plates for extracellular amylase production. Four promising isolates (R3, R4, SP8, and EFY11) were characterized based on morphological, biochemical, and amylolytic activity in liquid fermentation. Among them, isolate R4 exhibited the highest enzymatic activity and was selected for further investigation. Molecular identification using 16S rRNA sequencing confirmed R4 as Bacillus subtilis, and the sequence was deposited in GenBank (Accession No. PV934264). Amylase production by B. subtilis R4 was enhanced through SSF using wheat bran as the substrate. Initially, the one-factor-at-a-time (OFAT) approach was applied to evaluate the influence of various physicochemical parameters and media components on enzyme yield. Significant factors were subsequently identified using the Plackett–Burman (PB) design, which revealed fructose, pH, temperature, and moisture level as key variables. Further optimization using Central Composite Design under Response Surface Methodology (CCD-RSM) resulted in the development of a predictive model with an R² value of 0.8403. Validation under optimized conditions (fructose 0.075 g, pH 7.5, temperature 52 °C, and moisture level 10.5 mL) predicted an amylase activity of 36.98 U/gds, while experimental trials yielded 37.26 U/gds. The close agreement between predicted and observed values confirms the reliability of the model. These findings demonstrate the potential of B. subtilis R4 as a promising candidate for cost-effective amylase production from agricultural waste.