Screening Of Clostridium Strains Through Ribosome Engineering For Butanol Production Efficiency

As a second generation biofuel, butanol has its outstanding advantages meanwhile demonstrates great development potential, which attracts worldwide attention. Due to crises including continuing crude oil price increasing, energy shortage, environmental concern, global warming and climatic change, there is an increasing interest in butanol by the Acetone-Butanol-Ethanol (ABE) fermentation. At present, the main obstacles lay in butanol fermentation are low-production capability and toxicity, therefore constrain fast growing in biobutanol economy. Selecting a high butanol-production strain will contribute a lot in the microbial butanol fermentation industry, which makes it on a good wicket in fuel market competition. In this study, Clostridium acetobutylicum L7was manipulated by ribosome engineering, finally a novel mutant strain S3with high butanol production ability and tolerance was obtained.Within microbial cells, ribosomes functioning as places of protein synthesis are important organelles, which affect the intracellular metabolism. Ribosome engineering technology, in which antibiotic-resistant strains are resulted from mutations on microbial ribosome, was used to stimulate the expression of metabolites, thereby enhancing metabolite production. So four antibiotics-streptomycin, kanamycin, chloramphenicol, gentamicin-were used to treat on C. acetobutylicum L7and the MIC (Minimal Inhibitory Concentration) were ascertained. Combining with the screening strategy in which antibiotic concentration were improved successively after condition adjustment,12high butanol-production strains including six streptomycin mutants were obtained, whose production rate ranged from10.14%to12.68%. These results show that ribosome engineering technology for screening butanol-producing strain is a promising way, and streptomycin as a selection will be optimal.The genetic stability of streptomycin-resistance strain S3was favorable, whose butanol tolerance was increased from12g/L to14g/L. Studying the batch fermentation of glucose, it is found that final yield of butanol is12.48g/L and ethanol is1.70g/L,11.2%and50%, respectively higher than the parent strain, were achieved in S3. The conversion rate of glucose to butanol was increased from0.19to0.22whereas fermentation time was shortened by9hours. A increased butanol productivity by30.5%, reaching0.24g/(L-h) was observed. While the viscosity of fermentation broth was dramatically decreased to4mPa-s,60%lower than the parent strain, which became more convenient for the subsequent separation and reduced the whole fermentation cost. The data indicated that the metabolic flux of butanol and ethanol were changed. Comparing with the process of parent strain fermentation, it is assumed that the structures or activities of butanol dehydrogenase may change, leading to the increase of metabolite production. The key enzyme is correlated and responsible for butanol.The fermentation of fructose as raw material were also investigated, and the butanol of S3is12.42g/L, increased by10.6%; fermentation time was118h and butanol productivity reached0.11g/(L-h),17.83%higher than the parent strain; but in Jerusalem artichoke fermentation, S3produced8.05g/L butanol, close to parent strain, remaining many sugars.The high concentration of undissociated butyric acid leaded to the toxicity of organic acids in the fermentation broth, impaired cell growth and inhibited the glucose metabolism. In this study, it indicates in Clostridium acetobutylicum fermentation, the substrates had significantly effect on the conversion rate of glucose to butanol, butanol proportion in total solvent and also productivity.