Experimental Study On The Production Of Cellulase By Mixed Culture

With the development of the world economy, oil, coal and other fossil fuels are in short supply, so energy and environmental issues are world problems, and the finding for new alternative energy has gain much attention around the world. Cellulosic materials is the most abundant and cheapest renewable resources in nature. While at present, the cellulose are not be fully utilized, and it caused a lot of wastage of resources and brought environmental pollution. Cellulase can decompose cellulosic materials into sugar, which makes it easy to use cellulose. Thus, cellulase activity is a key factor affecting the utilization of cellulose. Therefore, the study of producing highly active cellulase has become the important research subject in biomass energy fields.In this paper, we firstly screened two kinds of fungis (Trichoderma viride D12 and Aspergillus niger F-3), which can produce cellulase. Then, the optimum conditions for enzyme production were investigated by single-factor experiment and response surface methodology (RSM). Furthermore, the mixed culture experiments were performed, and the optimum medium and culture conditions were investigated by using Placket-Burman experiment design and the response surface methodology experiment design. Finally, the saccharification effects of fermentation liquid were verified. Conclusions are as follows:(1) Studies mainly focused on introducing the knowledge of cellulase and the screen method of highly active cellulase producing microbes and fermentation conditions.(2) Screening Trichoderma viride D12 by Congo red flat and liquid fermentation, the enzyme activity is 0.62 IU/mL. By using the single-factor experiment and response surface methodology, the optimum conditions of Trichoderma viride D12 are culture temperature 30.4℃, fermentation time 121.2 h, initial pH value 4.85, and inoculation amount 5%(volume fraction). Under the conditions, cellulase activity can reach to 1.20 IU/mL, increased by 93.55%.(3) Screening Aspergillus niger F-3 from the ten strains of Aspergillus niger, the P-glucosidase activity is 3.26 IU/mL. By using the response surface methodology, the optimum conditions of Aspergillus niger F-3 are culture temperature 29.1℃, fermentation time 96.2 h, initial pH value 4.91. Under the conditions, (3-glucosidase activity can reach to 4.97 IU/mL, increased by 52.45%.(4) By using the single-factor experiment, the optimum conditions of Trichoderma viride D12 and Aspergillus niger F-3 for liquid mixed fermentation are as follows: inoculated Trichoderma viride D12 for 48 h at 30℃, then inoculated Aspergillus niger F-3 72 h at 29℃; the ratio of inoculation quantity of Trichoderma viride D12 to Aspergillus niger F-3 is 3.0 mL:2.0 mL.(5) Considering experimental economics, the corncob, peptone, (NH4)2SO4 and yeast extract were selected as the most important influence factors by using the PB experiment design. Then, the optimum medium compositions for enzyme production by liquid mixed fermentation experiment with Trichoderma viride D12 and Aspergillus niger F-3 were investigated by using the response surface methodology. The optimum medium is corncob 20 g/L, peptone 1 g/L, (NH4)2SO4 4 g/L, yeast extract 0 g/L, bran 10 g/L,avicel 20 g/L, K2HPO4 4 g/L, CaCl2 0.5 g/L, Tween -80 5 g/L.(6) Under the optimum conditions, the enzyme production can be greatly improved. The FPA activity is 2.14 IU/mL and the (3-glucosidase activity is 6.52 IU/mL. Compared with the enzyme activities obtained from Trichoderma viride D12 alone, the FPA activity increased by 81.36%, and the (3-glucosidase activity increased by 359.15%, respectively. Compared with the enzyme activities obtained from Aspergillus niger F-3 alone, the FPA activity increased by 345.83 %, andβ-glucosidase activity increased by 32.06%, respectively. Under the optimum conditions, the saccharification effect of the liquid mixed fermentation experiment with Trichoderma viride D12 and Aspergillus niger F-3 were verified, and the hydrolysis rate can reach to the best results after 48 h.