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3.2.1 Cellulase
Successful utilization of cellulosic materials as renewable carbon sources is dependent on the development of economically feasible process technologies for cellulase production, and for the enzymatic hydrolysis of cellulosic materials to low molecular weight products such as hexoses and pentoses. Spano et al. (4) showed that cellulase production, was the most expensive step during ethanol production from cellulosic biomass, in that it accounted for approximately 40% of the total cost. Significant cost reduction is required in order to enhance the commercial viability of cellulase production technology. A cellulosic enzyme system consists of three major components: endo--glucanase (EC 3.2.1.4), exo--glucanase (EC 3.2.1.91) and -glucosidase (EC 3.2.1.21). The mode of action of each of these being: (1) Endo-p-glucanase, 1,4--D-glucan glucanohydrolase, CMCase, Cx: "random" scission of cellulose chains yielding glucose and cello-oligo saccharides. (2) Exo-P-glucanase, 1,4- - D-glucan cellobiohydrolase, Avicelase, C1: exo-attack on the non-reducing end of cellulase with cellobiose as the primary structure. (3) -glucosidase, cellobiase: hydrolysis of cellobiose to glucose. Reese et al. 1950 (5) proposed that exo--glucanase causes a disruption in cellulose hydrogen bonding, followed by hydrolysis of the accessible cellulose with endo-gucanase. Although cellulase isolation techniques have not been fully developed, the hypothesis depicted in Fig. 3-2 is now accepted. According to this hypothesis, in a synergistic sequence of events, endo--glucanase acts randomly on the cellulose chain, while exo--glucanase acts on exposed chain ends by splitting off cellobiose or glucose. Cellobiose is subsequently hydrolysed by p-glucosidase to glucose. This hypothesis is however the opposite of that proposed by Reese et al. (5), and indicates that three, rather that two enzymes are essential for the decomposition of cellulosic biomass.
Aspergillus niger Fusarium solani Irpex lacteus Penicillium funmiculosum Phanerochaete chrysosporium Fungi Schizophyllum commune Sclerotium rolfsii Sporotrichum cellulophilum Talaromyces emersonii Thielavia terrestris Trichoderma k oningii Trichoderma reesei Trichoderma viride
Bacteria
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. Figure 3.3 - Clearing zones formed by T. reesei mutants on Walseth's Cellulose Agar Plates
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. Figure 3.4 - Genealogy of artificial mutants of Trichoderma reesei
21 22.4 19
24 28 23
pH: 4.0 (adjusted with ammonia) Culture conditions: temperature: 28/C, agitation: 550 r.p.m.; aeration: 0.8 v.v.m.
Table 3-4 Cellulase Productivity of T. reesei Mutants Using the pH Shifting System
Strain KDG-3 PC-3-7 CDU11 Protein (mg/ml) 22.8 24.0 23.6 CMCase (U/ml) 332 345 328 -Glucosidase (U/ml) 8.7 12.3 17.6 Avicelase (U/ml) 20.1 23.6 22.0 FPU (U/ml) 19.5 22.5 19.1
pH: Initial: 4.0; 5.0 after 48 hours Culture period: 7 days Culture conditions: temperature: 28/C2 agitation: 550 r.p.m.; aeration: 0.8 v.v.m.
An evaluation of the use of cheese whey, bagasse, and rice straw, as potential substrates for cellulase production, confirmed that PC-3-7 was capable of fermenting these substrates and efficiently producing cellulase enzymes. PC-3-7 was created as a strain, capable of partially producing cellulase enzymes. Table 3-5 shows an example of cellulase production using cheese whey as the carbon source. Flaskscale production of cellulase using bagasse and rice straw, the main raw materials used for fuel alcohol production, is shown in Fig. 3-7. Alkali pre-treatment of biomass was observed to enhance cellulase production. This seems to suggest that cellulase production is directly proportional to the crystallinity of the biomass from which it is produced, i.e. the higher the crystallinity, the better the yield of cellulase. The use of soybean curd, a low-cost nitrogen source, which is applicable as a substitute for expensive yeast extract and polypeptone, was investigated with cellulase fermentation residues (mainly waste cells). Results obtained, revealed that addition of 5% of the fermentation residue induced cellulase activity similar to that induced by the use of either yeast extract or polypeptone. Figure 3.6 - Time course of cellulase production by T. reesei KDG-3 in a 1kL tank
Carbon source: 10% Cheese whey and 1% Avicel Nitrogen source: Soybean meal
Figure 3.7 -Effect of alkali concentration of the production of cellulase from biomass