Isolation And Identification Of Cellulase-producing Strains And Investigation Of Their Cellulase Productivity

Screening high yield cellulase producing strain is very important to the development of the biomass into fuel ethanol and other light-based product. Eight different species cellulase-producing fungi with wild-type characteristics were selected from the natural environment in this article.Then the Penicillium glaucum NS3 selected was as original strain, response surface design used to optimise its cellulase-producing condition. Afterwards it was mutated by synergistic mutation to raising its cellulase-producing capacity. Sum up above, all these work establish a basis for further development of whole-cell catalytic decomposition of cellulose and utilization in the industrialization production of fuel ethanol. This thesis's main work contents and research results are as follows:Screened by agar plate contained CMCNa and Congo red indicator, thirty five cellulase-producing strains were isolated from rotten wood, straw piles and soil sampled in NanChang area. After second screening based on their cellulase-producing activities, eight cellulase-producing strains with higher CMCase and FPA activities were obtained. Through the analysis and investigation about their colony, mycelium, spore morphology and physiological characteristics, the eight strains were classified as Ramularia NS1, Trichoderma koningii NS2, Penicillium glaucum NS3, white-rot fungi NS4, T. viride Persex Fx NS5, Aspergillus glaucus NS6, Aspergillus niger NS7, pythium graminicola NS8. Under shaken flasks culture conditions, their CMCase activities reached 297.97 IU/mL,300.11 IU/mL,154.83 IU/mL,146.68 IU/mL, 308.14 IU/mL,94.51 IU/mL,218.02 IU/mL,203.02 IU/mL and FPA activities were 6.56 IU/mL,5.63 IU/mL,4.29 IU/mL,9.63 IU/mL,8.02 IU/mL,1.12 IU/mL, 4.58 IU/mL,11.36 IU/mL, respectively. With the appropriate fermentation conditions: CMC 6g/L, wheat bran 6g/L, (NH4)SO4 4g/L, cultured 3ds at the temperature 30℃, the CMCase and FPAase produced by stain T. viride Persex Fx NS5 reached 352.11 IU/mL and 13.96 IU/mL, which increased 14.26% and 74.06% than that of the original activities 308.14 IU/mL and 8.02 IU/mL Response surface design was used to optimise the cellulase-producing condition of Penicillium glaucum NS3. There are 18 factors effect on the cellulase-producing fermentation, such as the fermentation time, shaking speed, concentration of carbon sources, nitrogen sources and inorganic salts. The influence of the 18 factors was evaluated by Plackett-Burman design. The result showed that wheat bran, CMC-Na and the fermentation time played important roles in the cellulase production. According to the result of Plackett-Burman design experiment, the range of the influential factors could be estimated, a Box-Behnken design was employed to optimize the significant factors and the results were shown in response surface graph. The optimum fermentation conditions were as follows:6.0g/L wheat bran,7.0g/L CMC-Na, and 96 hours feimentation time. Under the above optimum fermentation conditions, the activity of cellulase produced by Penicillium glaucum NS3 reached 309IU/ml, which had increased 227% than that of the original fermentation conditions produced.After fermetation conditions optimising of the Penicillium glaucum NS3, a several of synergistic mutation have been carry out for increasing its cellulase-producing capacity. A higher cellulase-producjng strain named NS3c1 had been gotten, which was breeded through inducing mutation treatmentt of ultraviolet light and sodium nitrite. The optimal dosage of mutant by UV and sodium nitrite was 9 minutes with 15W UV light in 30cm distance and 0.3% concentration. Under the repeated synergistic mutations, the CMCase and FPAase produced by mutant Penicillium glaucum NS3c1 reached 400.07 IU/mL and 9.47 IU/mL, which increased 29.47% and 28.35% than that of the original activities 309 IU/mL and 7.37 IU/mL...