Synergistic cellulase production from <i>Trichoderma reesei</i> and <i>Aspergillus niger</i> in a 3:1 mixed culture: Immobilization and reusability for enhanced cellulose hydrolysis

Cellulase enzymes are widely used for converting lignocellulosic biomass into fermentable sugars, but their industrial application is limited by high production costs and low reusability. In this study, cellulase was produced using a 3:1 mixed culture of Trichoderma reesei and Aspergillus niger, followed by immobilization on chitosan–glutaraldehyde beads to obtain a reusable crude enzyme preparation. The mixed culture produced an enzyme activity of 0.375 U/mL at 32 °C after 10 days of fermentation. The immobilized crude enzyme was applied in repeated hydrolysis of microfibrillated cellulose. Reducing sugar concentration increased with hydrolysis time in each cycle, with a maximum value of 1.046 g/L obtained after 60 min in the fourth reuse cycle. However, hydrolysis performance did not follow a consistent trend across reuse cycles, and negative apparent net sugar production was observed in intermediate cycles. These variations are attributed to residual sugar carryover, internal diffusion limitations, and incomplete removal of soluble products during washing, rather than actual sugar consumption. The relatively low conversion efficiency compared to optimized systems is attributed to the use of crude enzyme preparations, mass transfer limitations within the immobilized matrix, and the short hydrolysis duration. Despite these limitations, the results demonstrate that crude mixed-culture cellulase can be immobilized and reused across multiple cycles. Overall, this study highlights the importance of considering operational effects, including residual products and mass transfer constraints, when evaluating immobilized enzyme systems. The proposed workflow provides a simple and practical approach for developing reusable cellulase systems for biomass hydrolysis applications.