Research On Screening And Characteristics Of Three Frigostabile Cellulose Degrading Strains

At present,cellulose resources could not be fully utilized in China,resulting in a great quantity after harvesting residues is discarded or open burned,discharging SO₂,CO₂,CO to the atmosphere.The long terms low temperature climate in some areas of China caused the difficulty in the application of thermostabel cellulose degrading strains.Therefore,screening frigostabile cellulose degrading strians could help in the utilization of straw and the application of frigostabile cellulose degrading strians.In this study,identification,enzymology characteristics study,and enzyme production optimization were carried out on the screened out fungus.The results are as follows:?1?Screening and identificationThe soil samples were collected from Daqing Zhalong Wetland and rich humus forests.3frigostable cellulase producing fungi were screened out.The strains were capable in degrading cellulose and filter paper.Mycelium and spore morphology were observed with electron microscope and scanning electron microscope.And the molecular biology identification were analysed with ITS mothed.3 fungus were identified most similar to Trichoderma harzianum,Penicillium olsonii and Neurospora,named as Trichoderma harzianum L8,Penicillium olsonii L11and Neurospora tetrasperma L14.?2?Preliminary investigation on enzymology propertiesThe effect of temperature,pH and metal ions on cellulase activity?CMCase?and filter paper enzyme activity?FPA?were investigated with single factor experiment.The optimum reaction temperature for the cellulase and filter paper enzyme produced by Trichoderma harzianum L8 were 30?.The optimum reaction pH for cellulase was 6.0,and that for filter paper enzyme was 5.0.The cellulase maintained best stability at 5?,where the relative activity was 95%after 2 h'preservation.Of the 6 metal ions,Mg²⁺,Fe²⁺and Co²⁺could promote the CMCase.The optimum content of Fe²⁺is 0.9 mg/L,the relative activity was 128%.The optimum content of Mg²⁺was 0.6 mg/L,the relative activity was 143%.The optimum content of Co²⁺was 0.6 mg/L,the relative activity was 125%.Optimum reaction temperature for the cellulase and filter paper enzyme of Penicillium olsonii L11 were 35?.The optimal reaction pH for cellulase and filter paper enzyme was 5.0 and 7.0respectively.The cellulase maintained best stability at 15?,where the relative activity was 94%after 2 h preservation.Fe²⁺,and Ca²⁺could promote the CMCase of Penicillium olsonii L11.The optimum content of Fe²⁺was 0.6 mg/L,where the relative enzyme activity was 135%.The optimum content of Ca²⁺was 0.6 mg/L,where the relative enzyme activity was 118%.Optimum reaction temperature for the cellulase and filter paper enzyme of Neurospora tetrasperma L14 was 40?.The optimal pH of cellulase was 5.0 and that for filter paper enzyme was 6.0.The cellulase maintained best stability at 5?,where the relative activity was 90%.Ca²⁺,Na⁺,Co²⁺and a small amount of Fe²⁺could promote the CMCase,the optimum content of Co²⁺was 0.6 mg/L,the relative activity was 135%.The optimum content of Na⁺was 0.3 mg/L,and the relative activity was 127%.The optimum content of Ca²⁺was 0.6 mg/L,the relative activity was121%.The optimum content of Fe²⁺was 0.6mg/L,the relative activity was 117%.?3?Enzyme production optimizationSingle factor experiments were conducted to investigate the optimal incubation time,culture temperature,initial pH,types and dosage of carbon and nitrogen sources.Plackett-Burman?PB?experimental design was used to seek 4 most significant factors based on the single factor experiment results.CMCase were took as the response index.Central Composite Design?CCD?experimental design and Response Surface Mothology?RSM?were used in the enzyme production optimization.The 4 main factors affecting the enzyme production of Trichoderma harzianum L8 were initial pH and the concentration of CMC-Na,ammonium sulfate,and lecithin.The optimal cellulase prodcing conditions of Trichoderma harzianum L8 were CMC-Na 10.57 g/L,ammonium sulphate 2.21 g/L,initial pH 5.20,and lecithin 5.20 g/L,and the CMCase reached 53.401 IU/mL under this condition.The 4 main factors affecting Penicillium olsonii L11 were were initial pH and the concentration of bran,soybean powder and lecithin.The optimum conditions for the cellulase production of Penicillium olsonii L11 were:bran 11.01 g/L,soybean powder 2.31 g/L,initial pH5.35,and lecithin 2.25 g/L,the CMCase reached 48.809 IU/mL under this condition.The 4 main factors affecting Neurospora tetrasperma L14 were initial pH,and the concentration of bran,yeast powder and lecithin.The optimum conditions for the cellulase production of Neurospora tetrasperma L14 were bran 10.16 g/L,soybean powder 4.06 g/L,initial pH 7.96 and lecithin 3.80 g/L,the CMCase reached 63.598 IU/mL under this condition.In order to verify the reliability of the model,three groups of experiments were carried out based on the optimated culture conditions.The verifiction results were close to the results of regression fittiny.The reliability of the model was verified.