| Cellulose is the main component of plant raw materials, which can be converted to glucose through the saccharification of cellulase, and then it can be used for the production of bio-fuel and other biochemicals for resource recycling. But the low activity and the high production cost of cellulase are still the major limiting factors in its industrial applications. Therefore, it is imperative to breed the strains with enhanced cellulase production and optimize the fermentation process. This study was to screen out the high cellulase activity mutants after the heavy ion irradiation, and establish a high-throughput screening method which was quick, easy and reliable, and furtherly improve the cellulase activity by the optimization of fermentation conditions and mixed fermentation. It would provide a new method for the breeding of high cellulase-yielding strains and found the base for industrial production of cellulase. 1. The influence and mutant breeding of Aspergillus niger by heavy ion beamsirradiationThe curve of survival rate showed that the dose of 50% lethal was 160 Gy. It was good for screening the positive mutation mutants at the irradiation doses of 80 Gy and 250 Gy for the positive mutation rates were 16.28% and 19.64%, respectively, which were relatively higher compared with those at other doses. A good correlation was observed between the FPA after 24-square deep-well microliter plate(MTP) fermentation and shake flask fermentation. The optimum volume was 1.5 m L and the fermentation time was 60 h in MTP fermentation. The microplate reader could replace the ultraviolet spectrophoto- meter for detecting the cellulase activity with the detection wavelength of 520 nm and the DNS additive amount of 140 u L. The filter paper activity(FPA) and β-glucosidase(BGL) activity of A.niger H11201 had increased 38.74% and 63.23% separately compared with A.niger H11 with significantly difference(P< 0.01) after the shake flask fermentation. The mutant A.niger H11201 was stable in genetics and could produce cellulase with high activity. 2. Optimization of the cellulase fermentation conditons by Trichoderma virideThe fermentation process was optimized through orthogonal experiment, and the optimum fermentation conditions were as follows: the fermentation time was 96 h, the growth time of the slant was 6 d, the inoculum dose was 5%, the inoculum age was 16 h, the initial p H value was 6, the fermentation temperature was 31 ℃ and the shaking speed was 240 r/min. The FPA, EG activity and BGL activity had increased 46.64%, 49.27% and 66.79%, reapectively, with significant difference(P< 0.01) under optimum fermentation conditions compared with those under the original conditions. 3. Mixed fermentation for cellulase productionThe optimal inoculum ratio of T.viride to A.niger was 5:1 and the fermentation time was 96 h. The FPA and EG activity of the 5:1 inoculum ratio at 96 h were increased by 6.49% and 6.02%, respectively, compared with those of the 5:3 system and 11.13% and 6.60%, respectively, compared with those of 5:5 system. The BGL activity of the 5:1 system at 96 h was increased by 26.51% and 11.94% compared with that of the 5:3 and 5:5 systems, respectively, with significant difference(P< 0.01). The FPA, EG activity and BGL activity of mixed fermentation was improved by 68.87%, 6.17%, 80.73% separately compared with the mono-culture of T.viride, and it was improved by 54.14%, 35.59%, 20.94% compared with the mono-culture of A.niger.The high-throughput screening method was established for quick screening after heavy ion beams irradiation, and a mutant of A.niger with high cellulase activity was screened out. Cellulase activity can be improved through the optimization of fermentation process and mixed fermentation, and FPA was improved by 68.87%. |
PDF Full Text Request