| Confronted with the crises of environmental and energy crisis,developing of biofuel has become an important strategy.Butanol,as an alternative biofuel with many good virtues than ethanol,has attracted the view of many countries.Firstly,a stable genetic Bacillus strain with high butanol productivity was selected from the corn, which could grow under facultative anaerobic environment and was named as CM6.Then the morphological,physiological and biochemical characteristics of CM6 was studied.Sequencing results of 16S rRNA showed that the strain had 99%homology similarity with Bacillus licheniformis. Using glucose and starch as carbon source,the strain could produce butanol by fermentation.Static culture with corn medium,the strain could produce total solvent of 17.3 g·L-1 and the butanol concentration reached 10.36 g·L-1.In the culture of semisynthetic medium,the total solvent and butanol concentration reached 9.23 g·L-1 and 6.02 g·L-1 respectively.Analyzed by GC-MS,the main fermentation products were identified as butanol,ethanol,acetone.There also some other products such as acetic acid,butyric acid and 2-butanone with little amount were detected.By protoplasts mutation with 60Co radition,a mutant strain with higher butanol production ability was obtained.The optimal condition for protoplasts preparation was that the cell cultivating 15h was delt with 1 g·L-1 glycin and 0.3 U·L-1 penicillin for 2h and then digested with 1×104U·L-1 lysozyme at 37℃for 8h.By plate count method,the formation and the regeneration rate of protoplasts reached 99.82%and 10.12%respectively.The protoplasts were radiated by 60Co with the dose rate of 5Gy·min-1.The lethality of the protoplasts attained 99.3%when the radition dose was 500 Gy. Under such condition,a mutant strain R3 with higher butanol production ability and higher inherit stability was selected.In the culture of semisynthetic medium,the total solvent and butanol concentration reached 16.75 g·L-1 and 11.17g·L-1 respectively.Compared with the original strain,the total solvent concentration and the butanol concentration of the mutant strain R3 was increased 81.46%and 85.41%respectively. Cultivation condition and medium were optimized by single-factor experiment,fractional factorial experiment and response surface experiment.The optimal cultivation condition was that glucose concentration 53.47g·L-1,yeast extract 1 g·L-1,buffer solution 8.9ml·L-1,vitamin solution 10 ml·L -1,mineral solution 10 ml·L-1,natural pH,medium volume 55%of shake flask,seed culture time 24h,inoculation size 4%,culture temperature 39℃,static cultivation with a pin holed rubber plug. Under such condition,the butanol production reached 13.65 g·L-1.Based on the batch fermentation results,the dynamics model of the cell growth,glucose consumption and butanol production of the strain R3 were constructed.The fed-batch fermentation strategy of butanol production of the strain R3 was designed.The optimal strategy was that the glucose solution of 40 g·L-1 was fed to the exponential phase cell at the feeding rate of 0.708g·（L·h）-1.The butanol concentration reached 23.32 g·L-1,the butanol yield and productivity was 0.36g·g-1,and 0.2·（L·h）-1 respectively,which was increased 85.37%and 33.33%than that of batch fermentation.The key enzymes relative to solvent metabolism were analyzed,the results proved the existence of ethanol dehydrogenase,butyraldehyde/butanol dehydrogenase and acetoacetate decarboxylase.So, the major metabolic pathway of the strain R3 was similar with Clostridium acetobutylicum.Finally,the butanol production by the strain R3 with xylose and fiber hydrolyzeas subtrate were elementary explored.The results indicated that the strain R3 could utilize xylose as carbon source to produce butanol.The optimal culture medium was that xylose 26.6g·L-1,yeast extract 1.38g·L-1, vitamin solution 5.55ml·L-1,mineral solution 9.67ml·L-1.Under such condition,the butanol concentration was 6.69g·L-1.Innovations of this paper were as follows:（1） A facultative anaerobic Bacillus strain with high butanol productivity was isolated. Clostridium acetobutylicum or Clostridium beijerinckii were the traditional microorganism used for butanol fermentation,which could only survive and produce butanol under strictly anaerobic environment.However,the strain of Bacillus isolated in this study could produce butanol under facultative anaerobic condition and there was no report about that.（2） The butanol production was obviously enhanced by the 60Co irradiation on the protoplasts of Bacillus licheniformis,butanol production reached 11.17 g·L-1,which was 85.41%higher than the original strain.（3） The butanol production was significantly increased with the optimization of response surface methodology and the fed-batch strategy.In the batch fermentation,butanol production reached 13.65g·L-1,while to the fed batch,it was 23.32g/L higher. |
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