Enzymatic hydrolysis of Chlorella sorokiniana biomass using fungal enzyme mixtures: screening, optimization, and proteomic analysis for enhanced microalgal biomass valorization

The sustainable production of microalgal biomass for food and feed applications requires efficient downstream processing methods, particularly for the disruption of recalcitrant microalgal cell walls. This study aimed to develop an enzymatic hydrolysis method using fungal enzyme mixtures for effective degradation of the cell wall of the green microalga Chlorella sorokiniana. Eight fungal strains were screened for their enzyme production and hydrolysis efficiency, with Aspergillus awamori, Aspergillus niger, and Ceratocystis paradoxa showing the highest performance and selected for detailed analysis. A Design of Experiment approach optimized hydrolysis parameters, revealing that all enzyme mixtures showed maximal sugar release at 60 °C and an enzyme-to-biomass ratio of 5% (v/w). Among these, A. awamori enzyme mixtures exhibited the highest sugar-to-protein ratio, indicating superior catalytic efficiency. Proteomic analysis highlighted distinct enzymatic profiles between A. awamori and a commercial reference enzyme preparation (Cellic CTec3), with A. awamori showing higher glucosidase, mannosidase, and chitinase activities, which correlated with enhanced hydrolysis of chitin-like polysaccharides in the microalgal cell wall. These findings suggest that enzyme mixtures tailored to the specific carbohydrate composition of microalgae, which combine cellulolytic, mannosidic, and chitinolytic activities, can enhance microalgal biomass hydrolysis efficiency. Such enzyme mixtures represent promising candidates for sustainable bioconversion and valorization of C. sorokiniana biomass in industrial bioprocesses.