Sceening Of Facultative Anaerobic Cellulase-Producing Strain And Appliction In Biogas Fermentation

With the development of society and growing of population, mankind is facing more and more serious environmental crisis and energy crisis. Cellulose is the most abundant renewable resources on the earth. In our country, most of crop culm was burned and abandoned except few reused to farmland so that it caused serious environmental pollution. Anaerobic fermentation is convenient way to use bioenergy which is stored in crop culm.Inoculate anaerobic cellulase-producing bacteria could enhance the utilization rate of crops culm, production and quality of biogas.In this thesis, the congo red double layer flat-plate method was used to screen cellulase-producing bacteria preliminary, and secondary screening by determination enzyme activity of anaerobic fermentation broth. Selected high-yielding cellulase mutant by separate or compound mutation with UV and nitrous acid (NA), determined the optimal fermentation medium and fermentation conditions of producing cellulase by single factor and orthogonal experiment, studied the composition and enzyme nature of cellulase produced by stain. Optimized the anaerobic fermentation conditions of producing biogas with crops culm. Applicated separately Cellulomonas sp. CR-14, acidogenic bacteria B2 and ammonification bacteria or mixture of them in producing biogas with crops culm fermentation.1. Respectively isolated 4, 34 and 22 strains which could produce transparent circle from cattle rumen contents, sludge of sewage treatment plant and lotus pool. Secondary screened a higher cellulose-producing facultative anaerobic strain by determining cellulase activity of fermentation broth.According to morphological characteristics, physiological and biochemical characteristics and 16SrDNA homology and phylogenetic tree analysis , identified it belonged to Celloulonmonas sp.,named it CR-14.2.Selected 87 mutant strains which ratio of transparent circle and colony diamate larger than original strains with UV and NA mutagenesis including 27 NA mutant, 42 UV mutant, 18 compound mutant. Secondary srceened 6 mutants which cellulase-producing are significantly different from wild strain by determining cellulase activity of anaerobic fermentation broth. They separately are Y-11, UV-17, UV-28, UV-35, UV-Y-9, UV-Y-16, the cellulase-producing activity of compound mutant UV-Y-9 is the highest, Cx activity was 11.3 U, serial passage 8 times, the genetic stability.3.The optimum fermentation medium of strain CR-14 were composed of 1.0% straw powder, 0.1% MgSO4, 0.3% nitrogen,0.3% soybean powder, 0.1% KH2PO4, 0.08% NaCl.The optimal fermentation conditions for strain CR-14 were that the initial pH value of fermentation medium 8.0,ferment 3 days under the conditions of 30℃. In facultative anaerobic conditions, the ventilation did not significantly affect the cellulase production of the CR-14 strains. However, in anaerobic conditions the cellulase activity was significantly lower than at the same fermentation time. The cellulase activity of anaerobic broth was 62.9% of aerobic fermentation. Surfactant Tween 80 did not affect enzyme production of strains CR-14.4.The main components of cellulase of strain CR-14 was CX, C1 andβ-glucosidase. The activity of Cx was higher, while activity of C1 andβ-glucosidase was lower. The optimal enzyme reaction conditions was 50℃, pH 6.0. Under the conditions of pH6.0-7.5 cellulase was stability, preserved the vitality of the original 80%. Thermal stability was poor at the condition of more than 60℃,heated 1h at 60℃, the activity was only 13% of activity which heated 1h at 40℃. Mn2+, Fe2+ could significantly promote the vitality of the enzyme, however ,Zn2+, Cu2+, Ca2+, K+ would inhibit the vitality of the enzyme.5.Considered that biogas production, methane content of biogas, biogasgas production rate during biogas fermentation.Organic loading rate 45g/L was better at the initial of fermentation.Fed-batch should be carried out in the late in order to ensure the quality and production of biogas,organic loading rate should be no more than 60g/L after feding;Regulate carbon-nitrogen ratio to 25/1 was better, that was, 2% of the total solid content.Added CR-14 strain can significantly enhance the production of methane fermentation;However, add acid bacteria B5 inhibite fermentation and reduced biogas production; Add J5 strain did not significantly enhance biogas production but can buffer the pH value of fermentation broth.