Construction And Characterization Of Clostridium Tyrobutyrium/TAL~+ For Enhanced Butyric Acid Fermentation In Wheat Straw Hydrolyzate

Butyric acid and its derivatives are very important chemical raw materials and fine chemicals. They are widely used in food, chemical engineering, energy, medicine and agriculture. Currently, butyric acid is synthesized commercially via chemical systhesis.However, the demand for butyric acid from microbial fermentation increased due to a strong preference by consumers for bio-based natural ingredients, as well as concerns about the future scarcity, cost and environmental impact of fossil fuel. Thus it is of great interest to produce butyric acid by microbial fermentation from cheap biomass. In this work,C.tyrobutyricum ATCC25755 strain was metabolic engineered to improve butyric acid production from wheat straw hydrolysate, especially xylose.Clostridium.tyrobutyricum ATCC25755(Wide Type, WT) could not efficiently utilize xylose and it was expected that xylose utilization should be improved by overexpresing transaldolase which is a rate-limiting enzyme in the pentose phosphate pathway(PPP).C.tyrobutyricum ATCC25755 was metabolic engineered by transfering tal gene from three different strains, C.tyrobutyricum ATCC25755, Escherichia coli BL21( DE3),Thermoanaerobacterium aotearoense SCUT27 respectively. Transaldolase expression was verified by SDS-PAGE and the results showed that transformant with tal gene from T.aotearoense SCUT27 has higher transaldolase expression level. The xylose consumption,concentration and productivity of butyric acid of C. tyrobutyricum / TAL transformant were12.11 g/L, 4.82 g/L and 0.09 g/(L.h), respectively, increased by 6.79%,8.07%,28.57% with29.95 g/L of xylose as substrate compared to wild type strain. Simultaneously the xylose consumption, butyric acid concentration and productivity of the transformant increased by18.34%, 8.13%, 13.33% in mixture medium containing19.06 g/L xylose and 9.5 g/L glucose.The acid hydrolysis of wheat straw biomass was optimized through single factor and response surface methodology. The main chemical composition of wheat straw was measured as follows(dried basis): 38.21% cellulose, 28.27% hemicellulose, 20.26% lignin, 1.67% ash and 11.59% others. The optimum hydrolysis conditions were as follows: solid-to-liquid ratio1:12 and 2.01% of sulfuric acid for 67 min at 120 ℃.The final concentration and yield of reducing sugar were 27.75 g/L and 0.33 g/g.Performance of transformant was superior to wild strain in serum bottle and 5 L fermentor with free and immobilized cell fermentation. The concentration, and productivity of butyric acid of C.tyrobutyricum / TAL transformant was 5.25 g/L and 0.09 g/(L.h), which increased by 6.92% and 28.57% compared with wild type with original hydrolysate of xylose19.55g/L and glucose 3.61 g/L. In free cell batch fermentation, the butyric acid concentration and productivity increased by 14.67%, 34.61% respectively. And those also increased by4.07% and 9.75% in immobilized fermentation mode.