Intensified Production of Enzymes for Biofuels by Solid-state Fermentation: From the Bioreactor to the Formulation

This work investigated the intensified production of lignocellulolytic enzymes to enable the viability of second-generation bioethanol (BE2G) in biorefineries, aligned with principles of circular bioeconomy. Bioprocess is based on solid-state fermentation (SSF), using the by-products sugarcane bagasse and wheat bran as substrates for cellulase production by Myceliophthora thermophila I-1D3b in multilayer packed-bed bioreactor (MLPBB), operated in batch and continuously, covering stages from cultivation to enzyme extraction, concentration and characterization. The results demonstrated greater efficiency and feasibility of continuous operation, with superior cumulative CO2 production and strong potential for industrial scale-up and integration with extraction by water percolation, whose flow rate significantly influenced enzyme recovery. Ammonium sulfate precipitation achieved yields of around 80% and the enzymes showed optimal activity at 60 °C and pH 4.0, with high stability in acetate buffer, stored for up to 6 months under refrigeration. Encapsulation in alginate beads proved to be a way to handle this enzymatic formulation commercially. The integration of bioreactor engineering with downstream processing is essential to reduce costs and enhance BE2G sustainability.