Studies On Solid Fermentation Of Cellulase By Trichoderma Viride

Cellulose, the main composition of the vegetal cell wall, exists widely in the nature and is the most abundant reproducible bio-polymer in the earth. In view of the high efficiency and the environment protection, the degradation catalysed with cellulase is the most effective way to hydrolyze cellulose thoroughly and causes no pollution. Cellulase can be extensively obtained, but except for some particular research, its production with other life-form but microorganism is unpractical, and the microbial fermentation is considered as the most convenient way.This research was mainly aimed at the improvement of the cellulase production with solid fermentation by Trichoderma viride. Firstly, in order to detect the biomass of the filamentous fungi in the solid culture medium, a preferable process for extracting ergosterol from the fungi cell wall was determined. Based on this, the linear relation between ergosterol and biomass was established.The studies about cellulase are widely performed, and the measurement and the definition of the cellulase activity unit in one laboratory differ with the others, which brings some difficulty to the comparison of the cellulase activity. Therefor, in the beginning of this research the activity unit was redefined and the method for measuring the activity of cellulase produced by this strain was optimized.In order to extract the cellulase from the solid culture medium completely, the extraction process was studied, and finally determined as following: adding 15 mL distilled water to every one gram dried koji, then extracting cellulase at 40℃ with a rotate speed of 80 r/min for about an hour. The liquid obtained in this way was salt-outed with 35% saturated (NR₄)₂SO₄ solution, thus, about 90% of the cellulase activity was conserved and some of the other proteins were removed. Then the cellulase stabilities in this crude enzymatic liquid were studied, including thermal stability and acid stability. The optimum temperature and pH for the hydrolysis were also determined as 60℃ and 5.0 respectively. In addition, the effect of eight general inorganic positive ions on the cellulase activity was also observed.Finally, on the basis of the optimization of the nutrition and the circumstance condition for the fermentation, a set of preferable solid fermentation process was established as following: 5.3 g solid culture medium, including 5.0 g straw powder which had already been used twice and 0.3 g bean cake powder, was put into a 250 mL flask, then add in some nutritious solution, containing 2% sucrose, 3% (NH₄)₂SO₄, 0.6% lactose, 0.25% MgSO₄ and 2% Tween-80, with a qualitative ratio which is 2.2 to the solid material, then inoculating with 1 mL spore suspension whose concentration is 3×10⁵/mL and cultivated at 32 ℃ in the prophase and then adjusted to30*C for 6 days totally. Compared to the former process, after the process optimization the activities of CMCase and FPAse were increased by 80.8% and 88.9% respectively.