| Butanol has become one of the most promising biofuels because of its superior physical and chemical properties.Considering the shortage of fossil fuels and abundant agricultural waste resources,biobutanol production via microbial ABE(Acetone-Butanol-Ethanol)fermentation using these cheap lignocellulosic feedstocks can not only contribute to resource utilization of agricultural waste,but also alleviate current situation of energy shortage,which is of great significance for sustainable social and economic development.Clostridium acetobutylicum as a hyper butanol producer could not simultaneously utilize glucose and xylose due to glucose-mediated carbon catabolite repression(CCR)and complex xylose metabolism when using lignocellulosic hydrolysate.Besides,complex byproducts such as acids,furans and phenolics are also generated during acid pretreatment of lignocellulosic feedstocks,which exhibit severe toxicities on sugar utilization,exponential cell growth and solventogenic metabolism of Clostridium acetobutylicum.Following physical,chemical or biological pretreatment process,separate hydrolysis and fermentation(SHF)and simultaneous saccharification and fermentation(SSF)are the most widely used strategies for lignocellulosic butanol production.Compared with SHF,SSF contributes to reduce process steps,operating costs,contamination risk and cellulase inhibition by cellobiose and glucose as end-products of enzymatic hydrolysis.Considering the optimal temperature for enzymatic hydrolysis(45-50℃)and ABE fermentation(35-37℃),the major drawbacks for most SSFs lie in poor cellulase activity and butanol productivity at low operating temperature.Considering the poor tolerance of pretreatment-derived inhibitors and poor utilization of fermentable sugars when using non-detoxified lignocellulosic hydrolysate,engineered strains were used for developing high-efficient butanol production via genetic regulation of glucose transport,xylose transport and metabolism in C.acetobutylicum.When using the engineered strain overexpressing gene glc G,significant improvements on butanol production(18.3 g/L)and productivity(0.76 g/L/h)were achieved within 24 h during batch glucose culture with p H control and artificial electronic carrier addition,which contributed to boosted supplies of intracellular ATP and NADH associated with energy and redox metabolism.Compared to the control strain,butanol production and productivity were increased by 55%and 280%,respectively.Similar results were also observed within 36 h during non-detoxified corn stover hydrolysate culture,of which butanol production and productivity were increased by 55%and280%,respectively,compared to those of the control strain.On the other hand,When using the engineered strain overexpressing XylT,XylB and XylC,which were responsible for xylose transport,xylose conversion into xylonate,significant improvements on butanol production(12.0 g/L)and productivity(0.50 g/L/h)were also achieved within 24 h during batch glucose/xylose culture with p H control.Butanol production and productivity were increased by 300%and 610%during non-detoxified CS hydrolysate culture.These work significantly contributed to lignocellulosic butanol production.Furthermore,in order to solve the actual problem of the mismatch between the cellulase enzymatic hydrolysis in SSF and the ABE fermentation temperature,this thesis starts with the heat resistance of the strain,using glucose synthesis medium and non-detoxified acid hydrolysate,respectively.The emphasis is on the high temperature stress tolerance of the above-mentioned engineered strains.The experimental results show that the Glc G-overexpression strain has better high temperature tolerance and butanol fermentation performance at 39-45℃,and its butanol and ABE yield at 42℃ fermentation conditions is still maintained at 10.4 g/L and 18.2 g/L.It provides necessary conditions for the establishment of high-temperature SSF process.Under the conditions of 42℃ and 12-hour pre-hydrolysis at detoxified pre-treated straw,the butanol yield and average productivity are10.5 g/L and 0.18 g/L/h,respectively.Compared with the traditional SSF process,the production efficiency is greatly improved,which is of great significance for improving the production economy and competitiveness of cellulose butanol!... |
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