Integrated cellulase continuous production and downstream processing using a packed-bed bioreactor for solid-state fermentation by thermophilic fungus

In situ cellulase production is a key strategy to enhance efficiency, sustainability, and robustness of second-generation bioethanol processes. However, limitations related to heat transfer, bioreactor operation, and downstream processing still hinder large-scale implementation of solid-state fermentation (SSF) for enzymes production. This study systematically evaluated the integrated production, extraction, recovery and stabilization of endoglucanase from the cultivation of Myceliophthora thermophila I-1D3b in a multilayer packed-bed bioreactor operated under batch and continuous modes, using sugarcane bagasse and wheat bran (7:3 w/w) as substrate. Fermentations were carried at 45 °C for 96 h, and the enzymatic extracts were characterized as a function of pH and temperature, as well as subjected to concentration by ammonium sulfate and ethanol precipitation. Maximum enzymatic activity was achieved at 60 °C and pH 4.0, confirming a thermoacidophilic profile suitable for industrial applications. Ammonium sulfate precipitation outperformed ethanol, achieving 78.7% of recovering of total units of enzymatic activity from the raw extract and an activity of 156 U·g.d.s⁻¹ (enzyme units per gram of dry substrate), whereas ethanol resulted in significant activity losses under the evaluated conditions. Within the bioreactor, maximum temperatures reached 48.2 °C (batch) and 48.9 °C (continuous), indicating effective mitigation of overheating. Increasing percolation water flow rate significantly enhanced enzyme recovery, highlighting mass transfer as a key limiting factor in downstream performance. Similar average activities were obtained for batch (95.63 ± 28.37 U·g.d.s⁻¹) and continuous operation (96.36 ± 4.63 U·g.d.s⁻¹), while continuous mode reduced variability among modules, indicating improved spatial homogeneity. Overall, process integration combining thermal control, percolation-based extraction, and downstream processing enhances robustness and industrial applicability of SSF-based cellulase production.