Optimizing acid pretreatment and enzymatic hydrolysis for enhanced valorization of Theobroma cacao pod husk: A study on cameroonian lignocellulosic biomass

In this study, the valorization of cocoa pod husks was investigated through dilute acid pretreatment followed by enzymatic hydrolysis. The pretreatment process was optimized using a Central Composite Design considering acid concentration, temperature and reaction time as independent variables. The optimum pretreatment conditions were obtained at 1.5% HCl, 150 °C and 29.6 min, leading to a maximum reducing-sugar concentration of 39.15 g/L. Structural modifications of the biomass were analyzed using elemental analysis, Fourier Transform Infrared Spectroscopy, X-ray diffraction and scanning electron microscopy, revealing significant disruption of the lignocellulosic matrix, partial removal of hemicellulose, partial disruption of crystalline cellulose ordering (apparent Segal CrI decreasing from ≈ 22% to ≈ 17%) and improved accessibility of the polysaccharide fraction. Enzymatic hydrolysis was subsequently optimized using CCD by varying enzyme loading, temperature, reaction time and pH. The highest glucose-equivalent concentration of 21.38 g/L was achieved under optimal conditions of 30 FPU g⁻¹ enzyme loading, 50 °C, 60 min and pH 5.0. The developed regression model showed high statistical significance, indicating good agreement between experimental and predicted values. Kinetic parameters were estimated by non-linear regression of the initial hydrolysis rate on substrate concentration, with consistency verification by Lineweaver–Burk and Hanes–Woolf linearization. The system exhibited a typical saturation profile, with apparent Vmax = 14.92 ± 0.57 mM/min and apparent Km = 5.53 ± 0.58 mM. Because cellulose is an insoluble substrate and a multi-enzyme cocktail was used, these parameters are reported as apparent operational descriptors rather than thermodynamic dissociation constants. The results demonstrate that acid pretreatment combined with enzymatic hydrolysis significantly enhances the conversion of cocoa pod husks into glucose-equivalent reducing sugars, highlighting the potential of this abundant agricultural residue for sustainable biomass valorization within emerging biorefinery systems.