Research On Screening And Mutation Of Cellulose Degrading Strains

Cellulose is the largest nature reserve and the most widespread natural organic matter. They exist in the form of wood, straw and so on, and produced several million tons annually by photosynthesis, and they are the most affordable and abundant renewable energy resources. But most were burned or discarded, resulting in a waste of resources and the environment pollution. With the increasingly serious energy crisis, its degradation and utilization has become a focus of attention. The fungal cellulase system produced a more full, and can be secreted from cells.This study was to screen the ability of a cellulose degrading fungus strains, and through optimization of fermentation conditions and mutagenesis to enhance the enzymatic activity. So the experiment was divided into three parts:First, the selection of fungi with cellulase strain. In this study, we collected soil samples from Jilin, Shandong and Guizhou, which is rich in cellulose. And than culture them with filter paper only as carbon source, and we isolated 53 fungal strains. Among them, Aspergillus and Penicillium are the largest share, respectively 33.96% and 20.75%. After cellulose-Congo red medium for screening these 53 strains,we obtained 10 strains that can produce transparent circle ,including Penicillium, Aspergillus, Trichoderma, and other species. Then we rescreen by the DNS method, and obtain two strains of high FPA and CMC activity. Combination of morphology and molecular identification of rDNA ITS sequences,we come to a conclusion, one is Aspergillus. fumigatus, numbered S69, the FPA activity reached 306.76 U/mL, and CMC activity to 548.17 U/mL.The other is Fusarium oxysporum, numbered S62, FPA activity to 281.55 U/mL, and CMC activity to 540.7 U/mL.Secondly, S69 Aspergillus fermentation conditions were optimized. We culture the strain S69 in different nutritional environments and culture conditions ,and by measuring the enzyme activity to make sure optimal conditions for enzyme production. This study discusses the influence of the basis for liquid fermentation medium, glucose, nitrogen source, surfactant, PH value, fermentation temperature, aeration rate and fermentation time. In the end,we determine the best enzyme production of strain S69: mixed with microcrystalline cellulose and wheat bran as the basic medium, add 0.1% Tween 80,0.2% (NH4) 2SO4, 0.2% urea, PH value of the initial set of 8, and 30℃. 6d in the highest activity when the FPA, as 484.13 U / mL, compared with 57.82% before optimization; in 5d, the highest CMC activity for 880.51 U / mL, compared with 62.85% before optimization.Finally, to further improve the enzyme production strains, application of ultraviolet radiation, EMS (ethyl methane sulphonate) composite processing and the role of both methods were mutated Aspergillus S69. In terms of other factors constant, by changing the role of time to observe the mutation effect. In terms of other factors constant, by changing the role of time to observe the mutation effect. UV-induced mutation in a single study, in accordance with the mortality of 90% and 99.9% of the principle of mutation is better to determine the best UV mutagenesis 10min time. And single EMS mutagenesis in the study, because the mortality 70% of mutation is better , so we determine the best mutation time of 180min. Combination of single factors in mutagenesis and pre-experimental results, we choose ultraviolet radiation of 8min and EMS of 150min. we get 24strains after cellulose-Congo red medium for screening. And then through DNS method, ultimately selected six enzyme activity increased in the mutant strain. strain S69-E7 of the FPA activity increased more than before the increase compared with mutation 16.5%, S69-U3 of the CMC enzyme activity increased up to more than 6.25% before mutation increases.This study was supported by the higher capacity of fungal degradation of cellulose strains Aspergillus fumigatus S69, and its optimal fermentation conditions were optimized, at the same time, we mutate the strain S69, and obtain higher enzyme activity strain,and provide production resources and experimental basis for future basic research.