Screening And Optimization Of Fermentation Conditions For Saccharomyces Cerevisiae Which Can Produce Inulinase

The key composition of Jerusalem artichoke is inulin. Inulin is built as a straight linear chain of fructose units linked by β(2â†'1)glucoside bond with a glucose residue at its end. And branched chains are consisted of some microbial inulin. Compositae plants, such as Dahlia pinnata and Jerusalem artichoke, always contain higher synanthrin content. It exists in tubes of Jerusalem artichoke as the form of carbohydrate and accounts for about 75 percentage of the tube dry weight.Due to excellent bio-ecological specialties such as the salinity tolerance, leanness resistance, growing simple and well adaptation, Jerusalem artichoke has been widely studied since 1950s. Moreover, as a non-food source, it never competes with land and water for food production which is very important for relieving the stress of inadequate land resources. The Jerusalem artichoke can be used for preparation inulin and high fructose corn syrup. The glucose and fructose produced in the process of synanthrin hydrolysis can be used to make alcohol by fermentation technology. Therefore, Jerusalem artichoke has emerged as one of the most advantageous and promising energy plants in the 21st century. Inulinase, as the highly efficient and specific catalyst, can hydrolyzes inulin in to fructose. It has also widely been applied in industrial production areas.Traditionally, the Jerusalem artichoke tubers had to fermented by the two-step fermentation, which is time-consuming and expensive in hydrolysis of inulin to fructose and glucose with enzyme and acid. In order to reduce the cost of fermentation in inulin ethanol fermentation. In our previous study, we successfully achieved the goal, the enzyme which can produced from Saccharomyces cerevisiae strain and then hydrolysis the inulin to fructose and glucose for fermentation.Methods for optimizing multivariable systems called traditional ’single-factor experiment" is time-consuming and often ignore the effect between components. Recently, many new experimental design method has been employed in the biological process optimization. As a model, response surface method (RSM), including mathematical methods, statistical techniques are widely used to consider the impact of some variables, and find the best conditions for more than one system. This method has been successfully applied to optimize the alcoholic fermentation. detailed information of this method has been lists. Overall, this optimization process consists of three main steps:making statistics, experimental design, mathematical model to evaluate the factors and predict the response and checking the adequacy of the model.The object of this study was to optimize the fermentation medium for the Jerusalem artichoke raw extract. Hence, Plackett-Burman design and Box-Behnken design were used to identify the effects of important factors influencing ethanol production.(1) Using the prebuilt INU-PUG-6 vector to transforming yeast Saccharomyces cerevisiae 6525, after the transformation, we got a strain can produce a high dynamic Inulinase named Saccharomyces cerevisiae 6525-C3, through further investigation that this strain can really induce inulinase and fermentation, with inulin as substrate, pH 4.6, the optimum temperature is 55℃, the enzyme measured in this transformant is 1.8U and more stable, we intend to optimize this child to better fermentation next step.(2) According response surface experimental optimization results, the best fermentation conditions was established (g·L-1):pH 5-6.5, peptone 2-4(gL·-1), yeast 1-3 (g·L-1), temperature 28-32℃, inoculum yield 5-15(%), NaCl concentration 0.1-0.2g·L-1), MgSO4 · 7H2O concentration of 0.07-0.1(g·L-1), and fermentation time 72-108h. CCD experimental results revealed that yeast extract, inoculum, MgSO4 · 7H2O are the three significant factor. Using the steepest ascent method we got these three values of the significant factors range should be yeast 8.6-9.4(g-L’’), inoculum 3.75-4.25(%), MgSO4 · 7H2O0.4-0.5(g·L-1), the final step in determining the optimal value of the final result obtained, yeast extract 9.24 g·L-1, inoculum 39.8mL/L, MgSO4 · 7H2O 0.45 g·L-1.(3) Use of optimization medium to fermentation infermentor to investigate yeast Saccharomyces cerevisiae 6525-C3 alcohol fermentation capacity, and to get the best fermentation conditions in the next step, hoping to improve the physical foundation of crude sugar daisy artichoke. Fermenter experiment shows the main problem influence of alcohol was the a lower rate of crude inulin sugar content, so the best method is to increase the sugar content, in 5L fermentor experiment when sugar content is 12% ethanol yield is 5%,50L fermenter ethanol yield has not improved significantly. Experimental methods using solid state fermentation alcohol measured yield of 4.6%. Still has not improved apparently. Ultrasonic extraction and hot water bath also failed to achieve significant results, and then using the first enzymatic fermentation can increase the sugar content, but it will make the cost of the industry higher.According to the above experiment, theoretical guidance provided in the recombinant Saccharomyces cerevisiae fermenter in ethanol yield can be improved with other conditions fixed.