Enzymes for improved hydrolysis of lignocellulosics Doctoral dissertation

The considerably low β-glucosidase activity : FPA ratio of 0.5-0.6 represented by the enzyme complex of Trichoderma reesei RUT C30 results in poor hydrolysis potential. To overcome this problem culture conditions were developed to enhance β-glucosidase production. Mandels’ medium supplemented with tris-maleate buffer proved to be efficient for cellulase production with elevated β-glucosidase activity. β-Glucosidase activity : FPA ratio of the obtained enzymes were around the optimal on Solka Floc, lactose and glucose carbon sources. Furthermore, there were no considerable changes in maleic acid concentration of cultivations throughout the fermentations, therefore it is tempting to speculate that maleic acid is a kind of β-glucosidase inductor. In addition, in cultures with tris-maleate buffer pH values varied in a narrow range, thus tris-maleate buffered cultures can be used in shake flask experiments for modeling fermentations running at constant pH. Tris-maleate buffer system was applied in shake flask experiments to test steam pretreated corn stover, spruce and willow for cellulase production. On steam pretreated corn stover higher cellulase activities could be obtained than on Solka Floc that was used as reference. However, the achieved β-glucosidase activity was higher on Solka Floc compared to steam pretreated corn stover. On the other two steam pretreated materials lower enzyme activities were reached than on Solka Floc. The produced enzymes and two commercial cellulases (Celluclast 1.5L and Econase CE) were tested in hydrolysis experiments using the three pretreated materials and Solka Floc as substrates. Generally, the highest yields were obtained using the enzyme produced on steam pretreated corn stover. Moreover, the highest sugar yield was reached on steam pretreated corn stover using the enzyme produced on steam pretreated corn stover. Similar results were obtained in the case of chemically pretreated corn fiber. Significantly higher sugar yields were obtained in the hydrolysis of corn fiber using the enzyme produced on the same substrate i.e. on corn fiber than applying commercial cellulases. It was shown that mannanase and endoglucanase expressed coordinatedly on the examined carbon sources, since a good correlation was observed between the two enzyme activities. This observation might be valuable information to understand the regulation mechanism of hemicellulase genes in Trichoderma reesei. Results of mixed cultivation of Aspergillus niger and Trichoderma reesei indicate that Trichoderma can be used to produce glucose for Aspergillus from lignocellulosic wastes, which might be advantageous if cheap raw material is needed for large-scale production of βglucosidase by Aspergillus species.