| This study chosen Trichoderma viride L4 C as the material to shed light on the best condition to product cellulose in liquid fermentation by single factor designs and orthogonal designs. Ultraviolet radaiation, NTG mutation and complex mutation were used to filter the bacterial strain with high productivity and stable inheritance by cellulose degradation and DNS methods. The results are as follows:1. The Trichoderma viride strain L4 C was fermented in liquid media and fermentation conditions were optimized by single factor designs. The optimum conditions obtained were that the complexity of straw powder and cellulose powder could be used as the best carbon source and ammonium sulfate was the most appropriate nitrogen source. The inoculation amount of strain and incubation time were 10% and 4 days, respectively. And the best cultivation temperature was 28℃and the optimum initial p H was 4.5. Based on the result of single factor experiment, the optimum conditions were got by more through orthogonal test. The results showed that the highest productivity could be acquired when the inoculation amount of strain, incubation time, temperature and the p H were 15%, 5 days, 28℃, and 5.0, respectively. After that the highest activities of CMCase, FPA and hemicellulase were 921.56 U/m L, 745.02 U/m L, and 556.71 U/m L, respectively, which increased by 56.7%, 65.0%, and 66.8% contrasted with basal medium before optimum.2. The experiment of ultraviolet radiation showed the optimized conditions were the energy, radiant distance, radiation, and the dilution of spores suspension were 15 w, 30 cm 300s, and 10-6. The lethality rate reached 85.0%. A mutant strain named Z15 was gained after UV mutation treatment. The CMCase and FPA reached 1196.21 U/m L and 863.56 U/m L after fermenting in shaking flash. The enzymatic productivity was 1.30 and 1.16 times than the original strain. Further study indicated that the strain Z15 had a good genetic stability.3. The experiment of NTG demonstated that the optimized condition on NTG mutagenesis was that NTG concentration was 0.6mg/m L with 50 minutes. The result indicated mortality of conidia was 80.4% and the positive-mutation rate was 8.01%. After NTG treatment, a mutant strain named Trichoderma viride N12 was obtained. And the CMCase and FPA reached 1410.02 U/m L and 992.67U/m L. Compared with the original strain, the CMCase activity of N12 was improved by 18.5%,and FPA of strain N12 was improved by 9.7%. It suggested the strain was stable after several generations.4. After uninterrupted complex mutation experiment, an optimized condition was obtained, and the optimum UV+NTG treating conditions were as follows: the concentration of NTG was 0.6mg/m L and the 15 w UV lamp was used as UV light with 30 cm radiation distance and the treatment time was 120 s. Thus mortality of conidia was 80.8% and the positive-mutation rate was 8.76%. After UV+NTG treatment, a mutant strain named Trichoderma viride ZN4 was got. The CMCase and FPA reached 1678.78 U/m L and 1031.56 U/m L. Compared to the original strain, the CMCase activity of UN4 was improved by 19.1%. However, FPA of strain UN4 improved by 12.3%, suggesting that the strain has better stability and enzymatic productivity.5. After treating the scrop straw in fermention for 3 days, 4 days, and 5 days with L4 C and ZN4 strain, and the content of crude protein got to the top 81.69% by 5th day while the degradation rate of crude fiber came up to 39.98% by 5th day. |
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