Integration of enzyme immobilization and multi-enzyme hydrolysis to enhance saccharification efficiency and ethanol yield in sorghum fermentation systems

Abstract

The suboptimal ethanol yield in “Burukutu,” a traditional sorghum-based alcoholic beverage, stems from the recalcitrance of the grain’s starch–protein matrix to enzymatic hydrolysis. This study compared the performance of free and alginate-entrapped (2% w/v) α-amylase, glucoamylase, and protease, applied in various combinations, to optimize saccharification kinetics and bioethanol productivity in red and white sorghum cultivars. Results indicated that the triple-enzyme cocktail (α-amylase + glucoamylase + protease) was significantly superior to individual or dual-enzyme systems (F = 3740.50, p < 0.001), emphasizing the critical role of protease in deconstructing the protein barrier to enhance starch accessibility. Red sorghum exhibited significantly higher saccharification depth and ethanol titers than the white cultivar (p < 0.001), likely due to cultivar-specific differences in grain architecture. Peak ethanol concentrations reached 35.3 ± 0.2% v/v in red sorghum using the free triple-enzyme system, while the immobilized system achieved 29.70 ± 0.36% v/v at 72 h. Notably, the immobilized biocatalysts demonstrated robust operational stability, maintaining high sugar utilization efficiencies (> 85%) through six consecutive fermentation cycles. While a gradual decline in efficiency was observed (F = 151.95, p < 0.001), the immobilized multi-enzyme framework retained substantial activity, particularly in the red sorghum matrix. These findings demonstrate that synergistic multi-enzyme immobilization substantially improves starch-to-ethanol conversion and process sustainability, offering a scalable model for intensifying sorghum-based bioethanol production.