| Cellulose is a major component of biomass, which is a source of renewable organic matter in large quantities. In recent years, there is a growing trend in using various renewable resources to develop novel eco-friendly green materials. Degradation by microorganisms is a most potential and ideal method to utilize cellulose, with a great diversity of cellulolytic microbes in nature and their various strategies to digest cellulose.The cellulolytic microorganisms are ubiquitous in nature,mainly including protozoa,fungi and bacteria. The cellulose-decomposing bacteria contains aerobic, anaerobic,mesophilic and thermophilic strains, inhabiting a great variety of environments,including those with the most extreme temperature, pressure and p H.However, due to low degradation activity of cellulolytic microbes and the limited research on their degradation mechanism, further application of cellulose is still restricted.As stated above,it is of great significance to isolate strains with high cellulose-degradation ability.The results of this research were as follows:1、Sixteen strains were isolated from soil samples.Primary screen were carried out using Congo red coloration and filter liquid culture. Three fungus were identified with high cellulose-degradtion activity, which can culture with each other.Then the three fungus were cultured by mixed fermentation and as complex microbial system F-7.The results showed that the complex microbial system F-7 has high degradation activity.The enzyme activity of three fungus mixed culture was revealed to be 9.52 IU/m L higher than F5 single fungus.2、Combined with scanning electron microscopy(SEM)analysis,it was shown that the decomposition of straw and corncob started from the damage of wax coat in the surface layer and the dense structure,inducing the extensive collape of cell wall.After that,the internal architecture inside straw and corncob began to degrade, and eventually the whole straw and corncob organization structure presented a loose state.Through morphological microscopic characteristics and sequence homology analysis, strain F1,F2,F5 were identified as Botryosphaeria,Rhizopus oryzae and Fusariumoxysporum,respectively.3、 In the fermentation of the complex microbial system F-7,the cellulose degradation rates were set as response values to optimized the fermentation process. The most excellent combinations of F1,F2,F5 were found to be 3 m L%,3.5 m L%and4.2m L%,respectively.The optimal fermentation medium wasconfirmed as follows: corncob4 g,straw 7.6g,(NH4)2SO4 4.1g,K2HPO4 2 g,Mg SO4·7H2O 1.2 g, KH2PO4 2 g, Na Cl 1.5g,peptone 1.5 g,yeast extract 2 g and H2 O 1000 m L.Under this fermentation condition,the cellulose enzyme activity of complex microbial system F-7 was revealed to have an enhancement of 9% compared with unoptimized cultures.4、The F-7 fermentation optimization process was designed on the basis of single factor expertment and Box-Behnken principle.The optimum cultivation conditions were found to be:p H 6.5,fermentation 6d,fermentation temperature of 28 ℃,and rotation speed180 r/min. We also explored the suitable environmental conditions for cellulose hydrolysis by cellolase produced by F-7. The result showed that within the range of p H3.0~7.0,the optimal reaction p H was 5.0.The enzyme was recovered after centrifugation at 4000 r/min for 10 min.Higher enzyme activity appeared at 40~60 ℃.The optimal reaction temperature was at 55 ℃,and enzyme activity rapidly decreased when temperature was higher than 60 ℃.The hydrolysis reaction time was standardized at 30 minutes.5、The CMC-case were separated and purified from cellulase solutions produced by F-7 cellulose-degrading microflora through(NH4)2SO4 fractional precipitation, dialysis and column chromatography equipped with Sephadex G-75. The protein was purified in first peak of Sephadex G-75 after SDS-PAGE,and its molecular weight was estimated to be 66.2 k D. The recovery ratio of cellulase reached 19% and its specific activity was increased by1.6 times after purification. |
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