The Construction And Fermentation Of C. Acetobutylicum And Product Separation

Biobutanol as a new generation of biofuels,has excellent properties,such as high calorific value,miscibility with petrol which was more and more attentioned.It is a currently trend using renewable material to produce biobutanol.Jerusalem artichoke(J.A)has advantages such as drought and salt resistant,becoming a kind of very potential materials.J.A tubers hydrolysate is mainly made up of fructose and glucose(~ 4:1).Clostridium acetobutylicum L7 about can not effectively utilize fructose in ABE fermentation process,while Carbon Catabolite Repression inhibite fermentation running smoothly in the mixed sugar sugar system,so it caused low yield of butanol.Therefore,this study focuses on fructose phosphoenolpyruvate-dependent phosphotransferase systems(PTSFru),improving the using of fructose,mix sugar and actual materials by bacteria via gene engineering strategy.In the fermentation of butanol,the volume of butanol is no more than 2% of fermentation liquor volume,so effective means of separation is needed to separate butanol.And we select to separate butanol in the fermention liquid through the pervaporation.In nature,genes cac0231(fruR),cac0232(fruB)belong to PTSFru encoding genes,play an important role in fructose transport system,and cac0231 is a transcriptional regulator of sugar metabolism,cac0232 coding 1-phosphofructokinase.These two genes were knocked out using TargeTron gene knockout technology respectively,developing two engineered strains C.acetobutylicum L7-?0231~?0232.Fructose-based and glucose-based batch(acetone-butanol-ethanol)ABE fermentation was performed by these two strains respectively.Result show the fructose utilization ratio of two strains had decline,of which 0.56 g/L butanol produced from 16.18 g/L fructose by C.acetobutylicum L7-?0231 and no butanol was produced by C.acetobutylicum L7-?0232 with 10.63 g/L fructose consumed.It should be noted that no significantly difference was observed during glucose-based batch ABE fermentation by engineered strains C.acetobutylicum L7-?0231~?0232 compared to the strain L7.L7-?0231 utilized 50.11 g/L glucose and produce 10.56 g/L butanol,and L7-?0232 consumed 52.23 g/L glucose with 10.19 g/L butanol producing.Genes cac0231 and cac0232 were overexpressed respectively,developing engineered strains C.acetobutylicum L7-0231~0232.The batch ABE fermentation of fructose or glucose as sole carbon source and fructose: glucose(4:1)as mixed carbon source was performed by these two strains,then J.A hydrolysate was used for fermentation by engineered strains.Glucose-based batch fermentation indicated that the two strains have no significantly difference contrasted to control strain.L7-0231 consumed 59.87 g/L glucose to produce 11.77 g/L butanol,and L7-0232 utilized 59.86 g/L glucose to produce 11.63 g/L butanol.The fermentation performance of the two strains all enhanced when they utilize fructose.And L7-0231 used 51.04 g/L fructose to produce 7.46 g/L butanol,and the utilization ratio of fructose increased 119.85% and yield of butanol enhanced 121.36% compared the control stain;L7-0232 utilized 48.58 g/L fructose and produced 7.64 g/L butanol,and the utilization ratio of fructose increased 109.13% and yield of butanol enhanced 126.73% compared to the control strain.When mixed sugar as substrate,only 5.69g/L butanol was produced by L7-0231 and the fermentation performance is close to control strain.While L7-0232 can produce 10.34 g/L butanol.Compared to the control strain,the production increased 83.83%.In J.A.hydrolysate system,fermentation prefermance of L7-0231 does not enhance.And that there was 8.32 g/L butanol generation by L7-0232.It increased 55.51% compared to the control.Because of low butanol concentration in the fermention liquor,means of separation is needed to separate butanol.Pervaporation,which has high effective and environmental protection,is a good way of separation,and it is widely used in the separation of chemical products.The fermentation liquor which L7-0232 used mixed sugar and L7-0231~0232 utilized J.A.hydrolysate to separate butanol via the pervaporation.The average butanol concentration of pervaporation productions is 241.91 g/L,111.58 g/L,191.72 g/L respectively.The ratio of butanol in ABE also increased to 63.30%,68.96%,63.82% respectively,and the separation effect is excellent.