Selection And Formulation Of The Cellulase Mixture Hydrolyzing Efficiently Glycerol Organosolv Pretreated Wheat Straw

Bioprocessing of lignocelluloses to bioenergy and biobased products has addressed widespread attention for decades. However, the utilization of lignocelluloses is still encountered with many bottlenecks, especially in its efficient hydrolysis into fermentable sugars. Given the complex composition and recalcitrant structure of natural lignocellulosic substrate, it is supposed to a different ration of hydrolytic enzymes for a diversity of substrate to realize an efficient hydrolysis. Based on these, in the thesis a cellulase secreting strain, Trichoderma reesei Rut-C30, was utilized to produce cellulase to customize the cellulase preparation. Besides, a cellulase mixture which can efficiently hydrolyze was made by supplementing with some accessary enzymes and additives.Firstly, the enzymatic capability of common commercial cellulases was assessed to provide a aim for formulated cellulase mixture. the results indicated that the new cellulase prepration, Cellic CTec2 was applicable, but not desirable, for hydrolyzing the glycerol organosolv pretreated wheat straw. In addtion, some additives such as Tween, PEG and BSA, presented an active influence on the substrate hydrolysis.Secondly, a cellulase producing strain, Trichoderma reesei Rut-C30, was used in the thesis to produce cellulase. Under optimized fermentation condition(microcrystalline cellulose 15 g·L-1, peanut flour 1 g·L-1, inoculum ratio 10%(v/v), initial p H4.5, broth loading 100 / 250 m L), the enzyme activities reached 0.356 FPU·m L-1(FPA), 0.454 U·m L-1(BG) and 15.133 U·m L-1(CMCase). In addition, with an atmospheric and room temperature plasma jet and ultraviolet mutagenesis, a genetically stable mutant(ZW15) with high cellulase activity(FPA and BG) was obtained. By using some additives(Cu2+, PEG10000, L-ascorbic acid and trehalose), ZW15 reached enzyme activities at 0.612 FPU·m L-1(FPA), 0.639 U·m L-1(BG) and 18.716 U·m L-1(CMCase), which was 70%, 40%, 20% higher than original strain, respectively.Finally, a cellulase mixture which efficiently hydrolyzes glycerol organosolv pretreated wheat straw was formulated by adding some accessory enzymes and additives. At a cellulase loading of 5 FPU·g-1 polysaccharide, the formula of cocktail was made as below: β-glucosidase 11.5 U·g-1 polysaccharide, xylanase 180 U·g-1 polysaccharide, lysosome 75 U·g-1 polysaccharide, tea saponin 8 mg·g-1 substrate, C-PAM 2 mg·g-1 substrate. Under the above mentioned condition, the enzymatic hydrolysis yield of formulated cellulase mixture reached 90%(24 h) on the 5% substrate concentration, which was 80% higher than that before the formulation. Furthermore, the formulated cellulase mixture showed a higher enzymatic hydrolysis than Cellic CTec2 on the glycerol organosolv pretreated wheat straw, reached 90%(24 h) at 5 FPU·g-1 polysaccharide, which is 50% higher than Cellic CTec2. However, its protein loading was unexpectedly higher(170 mg g-1 polysaccharide) than the latter, which was almost 20 times that of Cellic CTec2. The useless protein existed in fermentation cellulase adversed to the decrease of cellulase cost, so removing those unnecessary protein maybe the key to realize economic hydrolysis.