Trichoderma atroviride mutants with enhanced production of cellulase and β-glucosidase on pretreated willow

Abstract

Trichoderma atroviride mutants were developed by using N-methyl- N′-nitro- N-nitrosoguanidine (NTG) treatment and UV-light followed by a semiquantitative plate clearing assay on Walseth-cellulose/agar plates. The parent strain of the mutants was an isolate from the Amazonas basin (TUB F-1505), whose identity was established by ITS 1 and 2 and tef1 gene sequence analysis. Strain F-1505 proved to be the most promising extracellular cellulase producer among 150 wild-type Trichoderma in a screening program performed in shake flask fermentation on pretreated willow. Reducing sugar content, soluble protein, filter paper cellulase activity (FPA), β-glucosidase activity and endoglucanase activity of the fermentation broths of the mutant strains were measured in both shake flask and lab-scale fermenters and compared with Trichoderma reesei Rut C30. Also, hydrolytic capacities of fermentation supernatants of T. reesei Rut C30, the parent strain (F-1505) and the best mutants were compared on pretreated willow as carbon source and hydrolysis substrate. The T. atroviride mutants produced high levels of extracellular cellulases as well as β-glucosidase, rendering the need for β-glucosidase supplementation in hydrolysis of cellulose or pretreated willow unnecessary. On the contrary, β-glucosidase supplementations were essential in order to obtain good glucose yields for all other cellulase preparations tested.