| Lignocellulosic biomass is abundant and renewable resource,such as agricultural and forestry waste,which can be used in the production of different bioprocessed products?ethanol,butanol,amino acids,etc.?.As a traditional agricultural country,with the harvest of three major food crops?rice,wheat,and corn?every year,a large amount of agricultural waste will be generated,such as rice straw,wheat straw,corn straw and corncob.The comprehensive utilization of agricultural waste in China is relatively under-developed,and most of the agricultural waste was incinerated,which causes a great waste of resources.In this study,a deep eutectic solvent?DES?was used to pretreat lignocellulosic biomass such as rice straw,wheat straw,and corn cob in this study.Corncob exhibiting the best pretreatment effect was selected as the raw material for subsequent high-value utilization.The pretreated corncob was hydrolyzed by cellulase to obtain hydrolysate with a high sugar concentration.Finally,the sugars in the hydrolysate were converted into butanol and trans-4-hydroxy-L-proline by Clostridium saccharobutylicum DSM 13864 and the metabolically modified Corynebacterium glutamicum.In this way,corncob was successfully transformed into a high value-added products.The main research contents and results are as follows:?1?DES?EaCl:Lac?synthesized from ethylamine hydrochloride and lactic acid was used to pretreat agricultural wastes such as wheat straw,rice straw,and corn cob.Among them,EaCl:Lac had the best pretreatment effect on corncob.Composition analysis experiments were performed and the removal rates of hemicellulose and lignin in corncob after EaCl:Lac pretreatment were 87.9%and 71.5%,respectively,which greatly improved the accessibility of cellulose in corncob.The pretreated corncob was then hydrolyzed by 50 FPU·g-1tot al-solid cellulase for 24 h,and the sugar concentration in the hydrolysate was 50 g·L-1.In order to further improve the efficiency of hydrolysis,0.5%Tween80 was added to the hydrolysis system,and the sugar concentration was increased to 53.8 g·L-1,representing 7.6%increase compared with that without Tween80.?2?Corncob hydrolysate was utilized for butanol fermentation by C.saccharobutylicum DSM 13864.To reduce the cost,the cellulase attached to corncob was reused for five times.For the control?without Tween80?,the sugar concentration in Cycle I was 49.5 g·L-1.For the experimental group?with 0.5%Tween80?,the sugar concentration in Cycle I was 52.9 g·L-1.The sugar concentration in Cycle VI of the control and the experimental groups were 54.9 g·L-1 and 58.8 g·L-1,respectively.The amount of cellulase was reduced from 50 FPU·g-1tot al-solid to 25FPU·g-1tot al-solid,which saved a total of 75 FPU·g-1tot al-solid.Cycle VI hydrolysate of the control and experimental groups were utilized in butanol fermentation by C.saccharobutylicum DSM13864,and butanol titers of 10.2 g·L-1 and 10.4 g·L-1 were achieved,slightly lower than the glucose control group(11.2 g·L-11 and 11.4 g·L-1).?3?Trans-4-hydroxy-L-proline producing Corynebacterium glutamicum strains were constructed.Taking ornithine-producing strain KBJ07?SNK118?arg R?argF?ncgl2228?as the starting strain.In order to construct a complete trans-4-hydroxy-L-proline synthesis pathway,ornithine cyclodeaminase?ocd?and proline-4-hydroxylase?p4h?was expressed in strain KBJ07.To reduce the metabolic consumption of proline,CRISPR-Cpf1 technology was used to knock out proline dehydrogenase?putA?gene.Following strains were constructed:LH02?LH01-pDXW-8?,LH03?LH01-pDXW-ocd/Dap4h?,LH04?LH01-pDXW-ocd/Ubp4h?,LH05?LH01-pDXW-Dap4h/ocd?,and LH06?LH01-pDXW-Ubp4h/ocd?,strain LH01 is KBJ07?putA.After92 h of fermentation using glucose as carbon source,LH06 produced the highest Hyp level of310 mg·L-1.When using hydrolysate as carbon source,Hyp of 327 mg·L-1 was achieved,suggesting strain LH06 could produce amino acids using corncob hydrolysate.This research provides a new idea for converting lignocellulose into other high-value products such as amino acids. |
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