The Study On Butyric Acid And Butanol Production By Clostridium Tyrobutyricum From Saccharina Japonica Hydrolysate

Butyric acid and butanol are important bulk chemicals with broad applications in the chemical,food,and pharmaceutical industries.Currently,they are mainly produced by chemosynthesis from petroleum-based feedstocks.Due to the gradually depletion of fossil resources and the rapidly growing issue of environmental pollution,more and more attention has been paid to microbial production of bio-based chemicals and biofuels from renewable biomass.However,butyric acid and butanol via fermentation cannot compete with chemical synthesis on the economy due to high cost of substrates,low titer and yield because of the existence of byproducts.Nicotinamide adenine dinucleotide(NADH)is an essential cofactor during butyric acid and butanol formation,many researchers demonstrated that the production of NADH-dependent products can be enhanced by increasing the availability of NADH with the supplement of electron carriers or more reduced substrates such as glycerol and mannitol.In this study,Saccharina japonica was selected as a cheap,abundant and renewable source of mannitol.In order to achieve efficient utilization of mannitol and Saccharina japonica hydrolysate for butyric acid and butanol production by Clostridium tyrobutyricum ATCC 25755,metabolic engineering and process engineering strategies were employed,which provides a promising approach for cost-effective production of bio-based butyric acid and butanol.As a result,mannitol/glucose co-fermentation with a ratio of 1:2 or 2:3 can effectively enhance the yield(0.44-0.46 g/g)and selectivity(up to 100%)of butyric acid by Clostridium tyrobutyricum ATCC 25755.To further improve the economic viability of butyric acid fermentation,S.japonica hydrolysate(supplemented with glucose to achieve a mannitol/glucose ratio of 1:2)was obtained after acid hydrolysis and enzyme saccharification,and then used as substrate for butyric acid production.As a result,the butyric acid selectivity and yield achieved 100% and 0.42 g/g,respectively.This study provids an economically competitive approach for bio-based butyric acid.Overexpressing the biofunctional aldehyde/alcoholdehy drogenasegenes adh E2 from C.acetobutylicum ATCC 824 in C.tyrobutyricum ATCC 25755 could significantly enhance its ability to use mannitol and make it became a hyper-butanol producer.The engineered strain was named Ct-p MA and achieved 12.44 g/L butanol with a yield of 0.26 g/g when mannitol was used as the carbon source in serum bottles,increased by 117% and 117%,respectively,when compared with glucose fermentation.Based on this result,to improve the butanol tolerance of this strain,the effect of the heat shock protein overexpression(Gro ESL from C.tyrobutyricum ATCC 25755,Clostridium acetobutylicum ATCC 824 and Deinococcus wulumuqiensis R12,or Dna K from D.wulumuqiensis R12)on the fermentation performance of Ct-p MA was studied.The results indicated that butanol tolerance of the engineered strain Ctp MA12 G and Ct-p MA12 D were significantly enhanced.The results of butanol fermentation using S.japonica hydrolysate by different pretreatment methods indicated that the effect of ultrasonic pretreatment was better than that of acid pretreatment and enzyme saccharification.When ～1.3-fold ultrasonic pretreated S.japonica hydrolysate was used as the substrate,butanol titer and yield of Ct-p MA12 G achieved 11.13 g/L and 0.31 g/g,respectively,increased by 23.53% and 19.23% when compared with Ct-p MA.The ability of mannitol utilization,butanol titer and ratio of solvents to acids were significantly improved by optimizing the p H in bioreactor and the p H of 6.5 was finally selected for butanol production by Ct-p MA.To further improve the butanol yield,butyric acid formation pathway of C.tyrobutyricum was blocked by replacing cat1 gene with adh E2 using CRISPRCas genome engineering system.In addition,repeated-batch fermentation with engineered C.tyrobutyricum Ct-Δcat1::adh E2 in a fibrous-bed bioreactor was performed at the optimum p H 6.5 to improve the butanol titer and productivity.Consequently,stable butanol production with high butanol titer(>18 g/L),yield(0.30 g/g)and productivity(～ 0.80g/L·h)was obtained.Finally,butanol production from 1.5-fold ultrasonic pretreated S.japonica extracts in serum bottles achieved a butanol titer of 14.99 g/L with a yield of 0.39 g/g and productivity of 0.25g/L·h,demonstrating the feasibility of butanol production from S.japonica.