| China produces more than 70 million tons of sugarcane every year, which results in nearly 7 million tons of sugarcane bagasse. Converting the sugarcane bagasse into green energy resource as a substitute for chemical petroleum could be a possible way to alliviate the energy shortage and reduce environmental pollution. But so far there is no effective way to utilize it. In this research, a consolidated bioprocessing(CBP) based on three kinds of recombinant Pichia pastoris was constructed. The alkali pretreatment of sugarcane bagasse and the fermentation techniques of CBP were opitimized. The pretreatment of sugarcane bagasse and CBP were integrated by using acid to neutralize and regenerate the sugarcane bagasse.The eg3 of Trichoderma reesei, cbh2 and bgl1 of T. viride were cloned to expression vector pGAPZαA of P. pastoris X-33 to construct pGAPZαA-eg3, pGAPZαA-cbh2 and pGAPZαA-bgl1, respectively. The three cellulase genes were then integrated to genome of P. pastoris by electroporation and identifed by PCR. The results showed that three recombinant strains named P. pastoris-eg3, P. pastoris-cbh2 and P. pastoris-bgl1 could successfully expressed cellulases respectively.In shaking culture system, the recombinant EG3 reached the highest enzymatic activity of 0.082 U/mL at 72 h. The CBH2 reached the highest activity of 0.05 U/mL at 96 h, while that of BGL1 was 5.91 U/mL at 168 h. The optimal temperature for EG3, CBH2 and BGL1 was between 50 to 60 ℃ and the optimal pH was 5 to 6. Under the ion concentration of 1 mM and 10 mM, Ca2+ and Zn2+ represented obvious promotion for all three cellulases.In order to decrease the inhibitors produced in alkali pretreatment, the conditions of alkali pretreatment were optimized including alkali concentration, temperature, time, mesh of sugarcane bagasse and solid-liquid ratio. The effects were evaluated by testing the concentration of cellulose after pretreatment, the total phenol concentration in treating fluid, cellulase enzyme activity and yeast growth. The optimized alkali pretreatment conditions were finally determined as 4%(w/v)NaOH, 37 ℃, 48 h, 200-mesh sugarcane bagasse and solid-liquid ratio of 1:10. Under this condition, the total phenol concentration was only 0.47 mg/mL, while it was 0.84 mg/mL before optimization. Thus the inhibitors would have less inhibition effects on both cellulase activity and yeast growth after optimization. The sugarcane bagasse samples after alkali pretreatment were neutralized and regenerated by different acids, and then the regeneration effects were evaluated by X-ray diffraction(XRD) and scanning electron microscope(SEM). The crystallinity index(CrI) analized by XRD of raw material was 51.59%, and it decreased after generation by inorganic acids, among which, the lowest CrI was abserved by sulfuric acid at 39.12 %. However, the CrI increased using organic acids. Under the SEM, the surface of the samples treated by organic acids was smooth, while it became loosed and disordered using inorganic acids, which was easier for the attachment and hydrolysis of cellulases.The filter paper activity reached the highest with the cellulases ratio of 1:3:3(EG:CBH:BGL). Thus, three recombinant strains were inoculated with the ratio of 1:3:3 or 1:1:1 both under the optimal temperature of 30 ℃. The ethanol production reached the highest concentration of 6.58 g/L(1:3:3) and 5.20 g/L(1:1:1) respectively, both at 108 h. So the optimal inoculating ratio increased the ethanol production for 26.53%. The effect of non-ionic surfactants on fermentation was also studied. The enzymatic hydrolysis efficiency was improved while adding PEG or Tween in the fermentation system. The PEG could increase 5% of ethanol production. The final ethanol production could reach 66.19% of the theoretical ethanol production under the optimal fermentation conditions.In conclusion, in this study, a CBP process was constructed and it was highly integrated and easy to handle. The pretreatment and fermatation system were also optimized. However, the ethanol production should be improved in the future studies. |
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