Genetic Improvement And Ethanol Tolerance Mechanism Study On Thermophilic Anaerobic Strains

With the purpose of producing relatively cheap bio-ethanol to reduce the consumption of the fossil energy and protect the environment, strains, used for bio-ethanol production, must be able to tolerate a higher ethanol concentration. One of the main direction of bio-ethanol researches is to using the thermophilic anaerobic strains, fermenting the lignocellulosic raw materials to produce ethanol. The thermophilic anaerobic strains have many benefits such as fermentation at a high temperature to reduce the possibility of other bacteria pollution and saving the energy consumption used to cool the fermentation tanks. And the lignocellulose has many advantages, not competing with food supplication and not competing with the land utilization, to attract researchers'attention.In this study, Thermoanearobacterium sp. Rxl which was screened from the environment was studied. Through traditional genetic improvement techniques, UV mutagenesis and chemical mutagenesis(DES) and protoplast fusion mutagenesis, Rxl's ethanol tolerance is finally improved. The results show that the best UV mutagenesis conditions are as follows:UV lamp15W, radiation distance45cm and exposure time60s in the anaerobic incubator; Through UV mutagenesis,16strains which can tolerate6%(V/V) ethanol concentration are selected. The best chemical mutagenesis conditions are as follows:DES concentration0.5%, treatment time5min, Na2S2O3concentration25%and treatment5min to terminate the reaction; Through DES mutagenesis,8strains which can tolerate6％(V/V) ethanol concentration are selected. Through the characters of fermenting glucose, U-16and E-6are chosen from the previous mutagenesis for the protoplast fusion mutagenesis. Ultimately, one strain which can tolerate8%ethanol concentration is selected. And the best protoplast fusion mutagenesis conditions are as follows:penicillin enzyme concentration0.8U/ml, the lysozyme concentration0.1mg/ml; UV lamp15W, radiation distance45cm and exposure time600s in the anaerobic incubator; water bath processing8min at92℃. Secondly, the characteristics of the stains, Rxl and U-16and E-6and P-2, were researched, in fermentation experiments by compare the facts using glucose and other materials such as corn cob and reed. The results show that:Rxl can use many kinds of glucose to produce ethanol and acetic acid and lactic acid. In2% xylan fermentation, Rxl can produce the highest concentrations of ethanol and acetic acid, respectively52.7mM and19.9mM; in2% galactose fermentation, the lactic acid production can be up to23mM. P-2have many distinguish traits such as:the ethanol tolerance is up to8%, growing fast and so on.Finally, RAPD methods was used to study the ethanol tolerance difference between the Rxl and mutant strains in gene level. The best reaction conditions are as follows: in20μl system, DNA template800ng, dNTP concentration0.4mM, primers concentration0.4μM, Taq enzyme1.5U, Mg2+concentration1.5mM. Subsequently, the primers of s18, s69, s93, s113and s141are selected from the80random primers, owing to they can create a better different brands.Then, the RAPD products of the different bands are purified and sequenced. And then, those sequences are submitted to the NCB1website and find that one of them is almost same with the sequences region encoding the sodium/calcium exchanger protein of Thermoanaerobacterium thermosaccharolyticum DSM571. Since the calcium is one of the most important second messengers in cell and regulates a great deal of important physiological functions, the different ethanol tolerance between the mutant and the original bacteria may be associated with the imbalance of the calcium ion concentration.A major factor limiting thermophilic anaerobic bacteria application in reality is its low ethanol tolerance. In this study, the best genetic improvement conditions for thermophilic anaerobic bacteria Rxl are determined, providing breeding methods and data support for other anaerobic bacteria's genetic improvement. Then with the method of RAPD, the ethanol tolerance in mutations is preliminary researched, showing the way for improving the ethanol production strains'performance.