The Research On The Improvement Of Clostridium Beijerinckii And Butanol Fermentation Conditions

In recent years, with increasingly declining in fossil fuel reserves and more and more severe environmental pollution from excessive use of it, researchers from every country spurred to search for a renewable clean energy. Butanol have many advantages such as high fuel value, little corrosion and can blended with gasoline at any ratio, which is considered to be a potential new bioenergy and many researchers are interested in it.Butanol production from fermentation is more mild, better selectivity and catalyst free than the chemical method, which attracted people's considerable attention. However,butanol fermentation is also facing with some problems such as low product concentration and high costs of raw material and product purification. In this study, we transformed the gene Cbei₂073 knockout vector into Clostridium beijerinckii NCIMB 8052 according to the group II intron technology, and then screened high butanol producing strains. After that, the metabolism regulation of butanol fermentation was studied. At last, the hydrolyzates of Dioscorea zingiberensis C. H. Wright?DZW? were used as substrate for butanol fermentation in order to reduce the cost and optimize of media and culture conditions to improve butanol yield.The histidine kinases of Clostridium beijerinckii NCIMB 8052 was classified,screened and homologously compared with gene cac3319 which was identified from Clostridium acetobutylicum ATCC 824. The result showed that gene Cbei₂073 had the highest homologous rate of 48.54%, while other histidine kinases were less than 20%. The gene knockout vector of Cbei₂073 was successfully constructed by using the E. coli-Clostridium beijerinckii shuttle vector pMTL007C-E2 according to the group II intron technology. Then the vector was transformed into Clostridium beijerinckii using electroporation and screened a erythromycin resistant strain called as cb2073. After butanol fermentation by the P2 medium, the butanol and total solvent?means butanol ethanol and acetone? of strain cb2073 reached 12.47±0.54 g/L and 18.46±0.71 g/L,increased by 17.3% and 19.5% respectively compared to the wild-type strain.To further improve the butanol yield of strain cb2073, five regulators inclouding calcium carbonate?pH buffer?, L-cysteine?reducing agent?, sodium butyrate?organic acidsalt?, neutral red?electron carrier? and changeable temperature fermentation were studied to explore the effect of butanol yield. Calcium carbonate increased organic acids and strongly inhibited butanol and total solvent. Low concentration of L-cysteine had no significant in promoting the yield of butanol and total solvent, but with the increasing concentrations of L-Cysteine, the yield of butanol and total solvent were enhancedly inhibited. Sodium butyrate increased butanol and total solvent when it was added in the period of late logarithmic phase?16 h?. When added 0.3% sodium butyrate, the butanol and total solvent were the highest, increased by 12.9% and 8.9% respectively compared with the contral group. Neutral red inhibited strain growth and butanol yield. In changeable temperature fermentation, butanol and total solvent were increased when the temperature decreased in the period of late logarithmic phase?18 h?. When the temperature decreased into 28?, the butanol and total solvent were the highest, increased by 15.5% and 11.6%respectively compared with the contral group.The hydrolysate of DZW was replaced the glucose as carbon source to be used for fermentation of butanol by strain cb2073, and it was found that the hydrolysate of DZW could promote the butanol yield and total solvent which increased by 8.8% and 15.4%compared with the contral group. The optimum fermentation condition and medium attained from single-factor experiment and response surface methodology by the strain cb2073 were: the II?buffer?, III?vitamin? and IV?mineral? component of P2 medium were unchanged; initial glucose was 70.00 g/L, yeast powder was 0.55g/L; initial pH was 6.0;shaking speed was 150 rpm; fermentation to 18 h at 34?; then added 4.92 g/L sodium butyrate and changed the temperature into 28? to fermentation to 84 h. Under these conditions, the butanol and total solvent reached 14.94±0.13 g/L and 23.53±0.62 g/L,increased by 17.08% and 10.68% respectively compared to the the previous conditions.In conclusion, a high butanol producing strains was screened after strain improvement of Clostridium beijerinckii; the cost of fermentation raw materials was reduced and the yield of butanol was improvemed after optimizing fermentation conditions. The results of this thesis provided a theoretical basis for industrial production of butanol fermentation.