| Along with the increase of both chemical and fuel markets needs, global energy is becoming less amd less, bio-butanol fermentation production was gradually attended. The interest in producing n-butanol using biological route has been rejuvenated to engineer an economical fermentation process, competing with the currently-dominant chemical synthesis. However, butanol yield of conventional fermentation method is poor because of the toxic effects of butanol on the cell. It has been an effective way to solve the problem, which is Screening high butanol tolerance strain to construct the new butanol product pathway or obtain improved strains by genetic engineering methods for producing bio-butanol. According to many reports, lactic acid bacteria possesses inherent butanol tolerance. In this paper, we obtained the relative high tolerant butanol lactic acid bacteria strains through natural selection, artificial domestication and identification, and then studied a series of biological Characteristics about the strain with the highest butanol tolerance. Main research content and conclusion as follows:(1) Screening the high butanol tolerant lactic acid bacteria strains: 10 samples were riched in lactic acid bacteria for separating source, with the butanol stress conditions, 159 strains, which can tolerate 1.5%(v/v) butanol, were screened. After that, we screened 14 strains from 159 strains in different butanol concentrations, that could grow in the concentration of 2%(v/v) butanol, and relative growth rate is more than 20%. Calculating each strain's relative growth rate at different concentrations, And then, in order to improve the butanol screence of strains, strains were domesticated by adding different concentrations of Sodium chloride and butanol. As a result, butanol tolerance of strains had a different degree of increase, 14 strains could grow substantially in 2.5%(v / v) butanol concentration, at 3%(v / v) butanol concentration, four lactobacillus relative growth rate of more than 10%,which is named A29, B11, C25, E4.(2) Identifying lactic acid bacteria:14 strains were inoculated MRS agar plate, then we observed colony morphology, microscopic shape using microscope, and tested physiological and biochemical characterization. According to the different results of strains, we could make preliminary speculation of them. Strains A16,A29,B27,C25,D10,E4 are in conformity with the physiological and biochemical characteristics of Lactobacillus brevis,A2,B2,B11,D19 are in conformity with the physiological and biochemical characteristics of Lactobacillus plantarum,B5,D3 is one of the few bacteria which can metabolise rhamnosus, maybe they are Lactobacillus rhamnosus. According to the test characteristics of strains D4,H7, thay can't be temporarily determined. By molecular identification, using Polymerase Chain Reaction propagated their 16 S rDNA sequences, using results established phylogenetic tree. Finally strains A16,A29,B27,C25,D10,E4 were determined as Lactobacillus brevis; A2,B2,B11,D19 were determined as Lactobacillus plantarum; B5,D3 were determined as Lactobacillus rhamnosus, D4 were determined as Lactobacillus casei;H7 were determined as Lactobacillus paracasei. Except for D4 and H7, the results are consistent.(3) The characteristic study of Lactobacillus plantarum(B11): the butanol tolerance of B11 is relatively high, so we choose B11 to study the biological characteristics, As the research object to lactic acid content to study the influence of the temperature and initial pH. According to the results, in the conditions of 35? and initial pH6~6.5, strains can grow fast. while in the conditions of the temperature and pH, fermentation result is relatively high. We measured the yield of lactic acid, growth and pH changes After enlargement culturing. Then optimizing the MRS culture medium, as a result, glucose, beef extract, yeast extract are the most important factors for fermentation. Ultimately, the optimal conditions of beef extract, yeast extract and glucose are about 30g/L,20g/L and 60g/L. |
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