The development of second-generation (2G) bioethanol from lignocellulosic sources, such as sugarcane bagasse, is
very important as a viable alternative to conventional fossil fuels. However, the high cost associated with enzymatic
hydrolysis, which breaks down cellulose into fermentable sugars, poses a key challenge. This study focused on
enhancing cellulase enzyme production by a novel, locally isolated strain, Trichoderma harzianum PP400831,
using statistical optimization BBD-RSM to improve enzyme activity. Optimization efforts resulted in maximal
endoglucanase and exoglucanase activities of 4.01 IU/mL and 2.64 IU/mL, respectively after 9 days at 2% cellulose
mixture concentration and 0.15% tween 80. After saccharifcation of pretreated (SCB) by the crude enzymes and
fermentation of produced reduced sugar by S. cerevisiae MN901244 yielded an ethanol concentration of 25.63
g/L. This work represents a signifcant step toward developing a cost-effective, sustainable, and high-performing
cellulase production process for second-generation bioethanol.