Research On Screening And Applications Of High-efficient Cellulose Degrading Strains

Cellulosic biomass is the most affordable and abundant renewable energy resources, its degradation and utilization has become a hot research at home and abroad. This study was to search and exploitation of efficient cellulolytic strains and through optimization of fermentation conditions and mutagenesis to enhance the enzymatic activity.The result of this study was reported below:1. Screening of strains for cellulase productionThrough the experiments of identifying of cellulose-Congo red medium, decomposition of filter paper, and liquid fermentation. We selected and isolated 4 effective cellulase-producing strains from putrid citrus, straw, wood, and soil etc. They were judged as Penicillium oxalicum D1, Eupenicillium javanicumE1, Rhizomucor Variabilis H-1, Aspergillus aculeatusA1, respectively by its biological property and analysis of 18S rDNA sequences preliminarily.2.The optimization of enzyme production of Penicillium oxalicum D1The optimal culture conditions for the production of cellulases using D1 were investigated. Results indicated that the highest level of cellulose activity was induced in the compound of rice bran and wheat straw, and their optimal ration was 3∶7, and the optimal mass concentration was 2.5%. (NH₄)₂SO₄ was the best nitrogen source; its optimal mass concentration was 0.15%.The optimal culture temperature of cellulose production for D1 was 35℃. The optimal pH of cellulase production for D1 was natural pH and the optimal time of cellulose production for D1 was 4d. Under the optimal reaction conditions, its maximum activities of CMCase and FPA were obtained as 31.12IU/mL and 45.28 IU/mL.3. Mutation of D1 and Optimization of enzyme hydrolysis conditions of strawUsing Penicillium oxalicum D1 as the starting strain, a high cellulase yield strain named NU-H was obtained after treated with ultraviolet, nitrous acid and the compound mutation. In comparison with the original strain, the cellulose activity of NU–H was increased by 69.08% and the activity of filter paper enzyme was increased by 75.28%, in addition the activity of xylanase and the yield of reducing sugar were increased as well. The optimal enzyme hydrolysis conditions of straw using cellulases were investigated by the single factor experiments and orthogonal experiment. Results showed that the optimum enzyme hydrolysis conditions were as follows: temperature50℃, time 28h, enzyme concentration 60%, substrate concentration 3%, pH 4.8. Under these conditions, the yield of reducing sugar was up to 23.25%.4. Production of feed enzymes from citrus processing waste using Eupenicillium javanicum E1In order to increase the production of feed multienzyme, the effects of culture medium composition, initial pH value, culture temperature and culture time on the production were investigated in solid-state fermentation of citrus processing waste by E1 using the activity of cellulose, xylanase and pectinase as the determination indications.Results showed that the optimal condition for producing of above multiple enzymes was:moisture content (80%), temperature (30°C), wheat bran (1g), (NH₄)₂SO₄ (0.05 g), NaNO₃ (0.05 g), CaCl₂ (0.1 g), time (96 h) and natural pH. Under these conditions, when medium inoculated with 1ml of spore suspension (10⁶-10⁷ conidia/ml), the average activity of CMCase,β-glucosidase, pectinase and xylanase by Eupenicillium javanicum E1 could reach 46.80 U/g, 49.64 U/g, 51.87 U/g and 106.42 U/g, respectively5. Production of feed enzymes from Lotus seed husk using Rhizomucor Variabilis H-1In order to increase the production of feed multienzyme, the effects of culture medium composition, initial pH value, culture temperature and culture time on the production were investigated in solid-state fermentation of Lotus seed husk by H-1. The results showed the optimization condition was that Lotus seed husk 4g, defatted soybean 1g, straw1g, the water 5ml/5g dry basis, (NH₄)₂SO₄ 0.1g, NaH₂PO₄ 0.1g, the initial pH 4.0,temperature 28℃, time 84 h. Under this condition, inoculated with 1 ml of spore suspension (10⁶-10⁷ conidia/ml),the enzyme activities of CMC,β-glycosidase, xylanase and pectinase could achieve 1033.8U/g, 1131.5U/g, 1386.6U/g, 585.1U/g, respectively.The research results suggested that the cellulose-decomposing fungi we isolated could be potential candidater for enzyme production in the future industrial utilization,at the same time, we provide useful theoretical clues to the exploitation and utilization of agricultural waste.