The Research Of Butanol Tolerance Strain By Compound Mutation

One of the important factors to constraint traditional production of butanol fermentation was the lowability of strains to tolerate butanol.It could improve the butanol capacity effectively that selecting andconstructing new heterologous recombinant strains of butanol production and improving its ability to tolerancehigh concentrations of butanol. The screening strains often have not high butanol tolerance, so it is necessaryto screen in the secondary before have practical value. In this experiment, Lactobacillus fermentum by thelaboratory screening and preservation as the starting strain, conducted through microwave,LiCl,microwave-LiCl complex mutation, the purpose is to get a strain with high butanol tolerance.1. Mutagenizing Lactobacillus fermentum by microwave radiation method, research the microwavemutagenesis effect under the condition of the different microwave power and microwave processing time andthe different bacteria suspension concentration. Through single factor experiments to determine the optimumconditions of mutagenesis was that bacterial suspension concentration was107cfu/mL, microwave radiationintensity was80%, mutagenesis time was45s, obtained two mutant strains W-45and W-27after screening, therelative growth rate In3%(v/v) concentration of butanol set out by the strain of11.82%to15.42%and15.42%, and their heredity were stability and proved by genetic stability experiments.Proved this test usedmicrowave radiation mutagenes was effective.2. Mutagenizing Lactobacillus fermentum by LiCl, research the mutagenesis effect under the condition ofthe different concentration of LiCl, through the analysis of the fatality rate, strain mutagenesis of the optimumconditions were concentration of LiCl was1.2%, the mortality rate was83.20%, and obtained two mutantstrains L-87and L-19, with the relative growth rate increased to14.90%and15.35%, and through the geneticstability of L-19test results showed that the genetic stable performan and L-87was not stable. Proved this testused LiCl mutagenes was effective.3. In order to further screening strains with higher butanol tolerance, this experiment used the microwave-LiCl complex mutation to mutagenize Lactobacillus fermentum. Selected from the four factors of the bacterialsuspension concentration, microwave radiation intensity, microwave radiation time, concentration of LiCloptimum level to make orthogonal test. The results showed that the suspension concentration was107cfu/mL,microwave irradiation intensity was80%, the mutagenesis time was55s, concentration of LiCl was0.8%, thecomposite was the optimum conditions of compound mutagenesis, and the fatality rate was88.67%under thisconditions. Obtained two mutant strains F-84and F-25, the relative growth rate increased to16.08%and19.23%, the relative growth rate was slightly higher than single mutagenesis of microwave and LiCl, andfurther explained complex mutagenesis was better than a single mutation. And through the genetic stability testproved its genetic stability.4. The butanol tolerance of screened traits excellent strain compared with Lactobacillus brevis. Theresults showed that when the butanol concentration was3.0%（v/v）, the relative growth rate of mutant strainF-25was increased to2.29%. The results showed that the butanol tolerance of mutant strains F-25has reachedto3.0%.5. Finally to screen out the mutant strains F-25with good bacteria on culture conditions were optimized, studied the effect on different temperature, the initial pH. Determined by measuring the OD600value of thebroth, the strain of optimum growth temperature was34℃, the optimal initial pH value was5.