Screening Of Cellulose Degradation Fungi And The Study Of Its Degradation Characteristics

Microbe degradation is a most potential and ideal way to the application of cellulose. Because of low degradation activity of microbe and ambiguous inducement and biodegradation mechanism, farther application of cellulose is relatively deferrable. In order to utilize cellulose efficiently as a tremendous green resource, to screen microbes with high degradation activity and research inducement and biodegradation mechanism would appear important theory and practice value.Ninety-seven strains were achieved by enrichment and isolation from 57 soil samples. Two methods were used in the process. The primary is congo red culture and filter liquid culture, the second is measurement of cellulose activity. Two strains with high cellulase activity were obtained using the methods, identified to be Penicillium simplicissimum H-11 and Trichoderma viride C-08.H-11 and C-08 were fermented in liquid media and fermentation conditions were optimized. The highest level of cellulase activity was induced in the medium containing wheat bran and straw with the ration of 4:3. Soybean powder is the best nitrogen source. The ratio of C to N is 7:1 and the mass concentration for P. simplicissimum H-11 is 3.6%. The culture temperature, pH and time of cellulase production of P. simplicissimum H-11 are 30℃, 3.6 and 114h respectively. The highest level of cellulase activity was induced by the compound containing wheat bran and straw, and their ration is 5:2. Ammonium sulfate and soybean powder are the best nitrogen source, and their ration is 1:5. The ratio of carbon source to nitrogen source is 5:1 and the mass concentration for T. viride C-08 is 3.6%. The culture temperature, pH and time of cellulose production of T. viride C-08 are 30℃, 3.2 and 96h respectively.The ratio of stuff to water is 1:1 for H-11 CMCase production, 1:1.5 for Xylanase production, and the ratio of stuff and water is 1:2 for C-08 CMCase production, 1:2.5 for Xylanase production. The optimal rice straw granularity is 150⁴25μm for C-08 CMCase production. The activity of CMCase of H-11 increase with rice straw granularity,but the activity of Xylanase appear lowest when granularity is 150⁴25μm. Granularity between 150⁴25μm is optimal for C-08 CMCase production, while granularity below 150μm and above 425μm is best for Xylanase production. The optimal pH for C-08 fermentation is 4, and the optimal pH for H-11 CMCase and xylanase production are 3 and 4. The optimal conditions for cellulase and xylanase production as follows: inoculum size is 7⁸×107, incubation temperature is 28℃for H-11, lower 28℃for C-08, fermentation period is 60h for H-11, 72h for C-08.The immobilized cells were prepared through embedment of C-08 and H-11 with sodium alginate. The suitable culture concentration of sodium alginate is 5%. Initial pH value of the substrate is 3-4 for H-11 to product CMCase and Xylanase, while initial pH value is 7 for C-08. Comparing of the immobilized cells and the normal cells, the productivity of immobilized cell is the better. The addition of the surfactant tween-80 is helpful to enzyme production.Inducement characteristics of carbon sources to enzyme production were studied using liquid culture and washed mycelium inducement methods. The results indicated that D-mannose is beneficial in H-11 cellulase production, and glucose has better effect on C-08 cellulase production. Glucose can induce C-08 and H-11 to produce FPase, CMCase, C1 andβ-Gase, but enzyme quantities are different between two strains and within each strain.In order to study the mechanism of cellualase inducement by different carbon sources, the extracellular, plasm-membrane-bound and intracellular cellulases were made to transform cellobiose, and metabolites were analyzed by HPLC. The results showed that, in addition to glucose, the extracellular cellulases of H-11 and the intracellular cellulases of C-08 can also produce an unknown matter A. The plasm-membrane-bound cellulases have no effect on cellobiose. The intracellular cellulases of H-11 and the extracellular cellulases of C-08 can hydrolyze cellobiose, demonstrating that cellobiose is not the inducer of C-08 and H-11.The crude cellulases of H-11 were purified from liquid fermentation media by (NH4)2SO4 precipitation and SephadexG-100. Theβ-Gase and CMCase were purified and physical and chemical properties were studied. The molecular mass ofβ-Gaseâ… ,β-Gaseâ…¡and CMCase are 126.0kD, 77.8kD and 33.2kD, respectively. The optimum temperature are all 60℃, the three enzymes are stable when the tempture is below 40℃, 40℃and 50℃. The optimum pH value are 4.4⁵.2, 3.6⁴.0 and 2.8, respectively, and the three enzymes are stable when pH value is 4.4⁶.8, 2.8⁶.8 and 2.8⁶.8. Mn²⁺, Ca²⁺, Sn²⁺ and Li+ are able to enhance the activity ofβ-Gaseâ… , however, Mg²⁺, Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺ and Fe3+ can inhibit its activity. Mn²⁺, Sn²⁺ and Fe²⁺ are able to enhance the activity ofβ-Gaseâ…¡, whereas, Zn²⁺, Cu ²⁺, Co²⁺ and Fe3+ can inhibit the activity. Sn²⁺ are able to enhance activity of CMCase, however, Mn²⁺ and Cu²⁺ can inhibit the activity. Km and Vmax ofβ-Gaseâ… are 622.306 mg·mL^-1 and 15.480 mg·mL^-1·min^-1, respectively. Km and Vmax ofβ-Gaseâ…¡are 26.689 mg·mL^-1 and 0.508 mg·mL^-1·min^-1, respectively. Km and Vmax of CMCase are 1.744mg·mL^-1 and 14.124mg·mL^-1·min^-1, respectively.β-Gaseâ… andβ-Gaseâ…¡can hydrolysis Salicin obviously, and CMCase can hydrolysis CMC-Na obviously.The cellulase peak was showed in the FT-IR spectra ofβ-Gaseâ… ,β-Gaseâ…¡and CMCase. The infrared spectrum of the amide I and II bands of them showed that the secondary structure is mainly consist ofα-helix structures in solid phase.C1, CMCase andβ-Gase from the crude enzyme of H-11 can all adsorb and desorb MCC, whereasβ-Gase can't.β-Gaseâ… ,β-Gaseâ…¡, CMCase from H-11 and C1, CMCase,β-Gase from the crude enzyme of C-08 can also adsorb and desorb MCC.β-Gaseâ… andβ-Gaseâ…¡have strong promoting function for the hydrolyzing of cellulose, whereasβ-Gase,β-Gaseâ…¡and CMCase of H-11 don't have the function to crude enzyme of C-08.The solid fermentation, liquid fermentation of H-11 and the solid fermentation of C-08 all exist the mechanism of oxidizing hydrolysis.