Optimization of Cellulase Production by Trichoderma viride Using Response Surface Methodology

Background: Trichoderma viride is a cellulose-biodegrading fungus which produces high amount of cellulases with a higher ratio of β-glucosidase/total cellulase in its enzyme mixture compared to that in the enzyme complex of T. reesei strains. The culture optimization for cellulase production by T. viride has not been previously studied. Consequently, investigation on the enhancement of its enzyme activity and productivity is a significant aim for research. Methods: A consecutive optimization approach based on statistical experimental designs was applied to improve the production of cellulase enzyme by T. viride through a submerged fermentation. Two-levels Plackett-Burman design (PBD) was employed to screen the significance effects of the different parameters on cellulase production. This was followed by carrying out three levels Box-Behnken design (BBD) to create a polynomial model in order to correlate the relationship between the three tested variables and the cellulase activity. Results: The highest cellulase activity (1.066 IU/mL) was achieved under pH (4), Tween® 80 concentration (0.3 g/L), growth medium volume (30%), CMC concentration (10 g/L), peptone concentration (0.5 g/L), urea concentration (0.2 g/L), harvesting time (4 days), lactose concentration (0.5 g/L) and KH2PO4 concentration (1 g/L). Among the nine variables tested, growth medium volume, peptone concentration and Tween® 80 concentration were selected to be further optimized and modelled via response surface methodology (RSM), due to their significant positive effects on cellulase production. The Maximum predicted and actual cellulase activity (2.8 and 2.9 IU/mL, respectively) was obtained in a 30% growth medium volume containing 1.5 g/L peptone and 0.4 g/L surfactant, with a relatively high correlation between the experimentally observed and predicted values (89%). Conclusion: The most significant experimental factors were observed to be surfactant concentration, growth medium volume and peptone concentration after running the PBD matrix, demonstrating the enhanced level of cellulase activity when any of these factors was increased from its lower to higher level. The revealing of significant variables by PBD and the consequent optimization of their expanded levels by the BBD allowed the enhancement of cellulase activity by 64% (from 1.066 to 2.99 IU/mL).