3.2.3.1 Development of a process for high-titer cellulase production

Representative mutants derived from KY-746 were batch cultured in a 5-L fermentor. Considerable enhancement of titer was observed in four strains: KDG-3, PC-3-7, PCD-10, and CDU-11 (Table 3-2). An evaluation of carbon source utilization by these microorganisms, suggested that the production of high-titer cellulases necessitated an increase in the concentration of Avicel, the carbon source. A somewhat lower than expected maximum cellulase titer was however obtained at Avicel concentrations greater than 6% in batch culture (Fig. 3-5). This relatively lower titer, may be attributed either to adsorption of the cellulase produced on to the Avicel, or rate limitation of aeration due to the high viscosity of the culture medium. In order to overcome this, semi-batch culturing was investigated, where it was found that an initial Avicel concentration of 6%, followed by subsequent addition of 4% Avicel together with other components after a few days, resulted in relatively higher titer cellulase than that obtained in the batch process (Table 3-3). Titer improvement by the semi-batch process was however dependent on the types of microorganisms used. While the titer of KY-746 was relatively unaffected by the semi-batch process, various degrees of improvement in titer were observed for KDG-3, PC-3-7, PCD-10, and CDU-11. This semi-batch process was further utilized in order to optimize the stirring rate, aeration volume, feed change, seed volume, and the seed culture period. T. reesei generally exhibits poor (3-glucosidase activity. Strain CDU-11 was therefore created in order to enhance p-glucosidase activity. P-glucosidase activity can also be induced by elaborately controlling culture conditions.

CMCase, Avicelase, and FPU activities exhibited a pH optimum of approximately 4, while the pH optimum of P-glucosidase was between pH 5 and 6. pH adjustment was therefore investigated with the objective of uniformly inducing all three activities. Culturing at pH 4 for 2 days, followed by a shift to either pH 5 or 6 on the third day, resulted in a marked increase in the p-glucosidase activity of PC-3-7, but no beneficial effect was observed in the case of CDU-11 and of KDG-3, which was already of high [3-glucosidase activity (Table 3-4). On the basis of these findings, a scale-up experiment was conducted in a 1-kL tank using the semi-batch culturing method with 10% Avicel as a carbon source and soybean curd as a nitrogen source. The pH was controlled and shifted as described above. Results are shown in Fig. 3-6. With a shortened culture period of 12 days, elevated activities of 530 U/ml and 8 U/ml were obtained for CMCase and p-glucosidase respectively, with an elevated protein content of 40 mg/ml in the culture broth. The soluble protein content of the culture broth was 53 mg/ml, which was considered favorable.

Table 3-2 Cellulase Productivity of T. reesei Mutants in Batch Culture

Table 3-3 Cellulase Productivity of T. reesei Mutants in Semi-Batch Culture

Table 3-4 Cellulase Productivity of T. reesei Mutants Using the pH Shifting System