Fermentation Processes And Genetic Engineering Of The Microbe Converting Cellulosic Substrate Into Butanol

Bioenergy, as a renewable energy carrier, can effectively solve the problems in the fields of energy crisis, environmental pollution, sustainable economic development, and many other issues. Biobutanol, including n-butanol and isobutanol, has aroused more and more attention in recent decades. Compared with traditional biofuel ethanol, butanol has many superior characteristics such as higher energy destiny, lower vapor pressure, nonhygroscopic, broader prospects for application as raw material, and can be completely miscible with gasoline or diesel in any ratios. However, the key factor affecting the fermentative butanol production is the high cost. Thus, the current studies focus on the combination of fermentative biobutanol production and the usage of abundant, cheap and renewable cellulosic biomass as an alternative substrate. This paper aims at the main technical bottlenecks which restrict the process of convertion of cellulosic substrate into butanol, such as the high cost of cellulase and the low conversion efficiency. The performance of crude cellulase in the hydrolysis and n-butanol fermentation was investigated. Then co-culture systems with microbe synergy were established for cellulose direct microbial convertion to n-butanol. Besides, combining the principles of synthetic biology, a genetic engineered strain was constructed for cellulose direct microbial convertion of isobutanol, whose performance was analized in the following fermentations. The major achievements of this paper were as follows:Focusing on the problems of high cost and low efficiency of cellulase in the cellulosic hydrolysis process, crude enzyme from Trichoderma viride was introduced in to cellulosic enzymatic saccharification and the hydrolysis conditions were optimized to be p H 5.1, temperature of 51°C, cellulase dosage of 41.1 g/L, and substrate concentration of 38.1 U/g using response surface method. The saccharification efficiency of crude cellulase was 62.42%, with reducing sugar of 17.32 g/L, which was 82.3% of that using commercial cellulase R10. Seperated hydrolysis and fermentation experiments were then investigated, and the concentration of butanol production was 7.05 g/L, while the yield of 0.155 g/g substrate, and the productivity of 0.141 g/L·h. Simultaneous saccharification and fermentation using crude cellulase showed a n-butanol procudtion of of 5.05 g/L, while the n-butanol yield and productivity was 0.127 g/g substrate, and 0.080 g/L·h. These results confirmed the feasibility of using crude cellulase instead of commercial cellulase in the cellulosic saccharification and fermentative n-butanol production. They also provided a new insight in the cost reducing of biobutanol production using cellulosic substrate.According to the principles of microbe synergy and the problems of mismatch between the conditions for the growth and function within the co-culture system, a mesophilic anaerobic cellulolytic consortium N3 and cellulolytic strain Clostridium celevecrescens N3-2 were introdued in respectively, with n-butanol producing strain C. acetobutylicum ATCC824 for cellulose conversion into butanol. Substantially, a n-butanol production of 3.73 g /L were obtained in the co-culture system N3 + ATCC824, and butanol yield of 0.145 g/g substrate, while n-butanol production of 2.69 g/L and yield of 0.134 g/g substrate in the co-culture N3-2 + ATCC824. This study provides important theoretical basis and reference value on the bioconvertion from cellulose to n-butanol using consolidated bioprocess under mesophilic condition.As no original strain or engineered strain can directly convert cellulosic substrate into isobutanol, an engineered strain capable of producing isobutanol from cellulosic substrate was constructed. Firstly, a cellulolytic strain of bacillus W12 was isolated, and identified as Bacillus cereus W12. In addition, the isobutanol tolerance of W12 and other common host strains in genetic engineering were tested, and W12 has the high isobutanol tolerance among the host strains. Using E. coli-bacillus shuttle plasmid p MA5, genes aro10 and adh2 were transfered into W12 for stable expression resulting in isobutanol synthesis 0.126 g/L in W12-RD, which indicated the achievement of Ehrlich pathway for isobutanol synthesis. Studies also found that the different link order of the genes affects their expression levels, thereby affecting the isobutanol production. Further more, by adding different Ehrlich pathway precursors, W12-RD strain was able to synthesize the corresponding alcohols, which means the character of universal and competitive of the method.In order to further increase the production of isobutanol, the gene als S, which was important to the synthesis of 2-ketoisovalerate, was overexpressed. And the genes pfl B and pta were knocked out to block the comsuption of pyruvate. Engineered strain W12-C2 B was obtained and one-step fermentation of cellulose to isobutanol in consolidated bioprocessingwas carried out with final isobutanol production of 1.310 g/L, maximum productivity and yields of 0.074 g/g substrate and 0.007 g/L·h, respectively.