Metabolic Regulation Of (3R)-acetoin Fermentation By Bacillus Subtilis

Acetoin is one of the bio-based platform chemicals and its optically pure isomers are important potential intermediates and precursors in the synthesis of novel optically active materials.?3R?-acetoin could be synthesized via enzymatic catalysis,whole-cell catalysis and fermentation.Among them,the cost of fermentation is the lowest for industrial production.However,a native microorganism which could produce high concentration of?3R?or?3S?-acetoin with high optical purity has not been reported.In this thesis,a marine strain of Bacillus subtilis was isolated to produce?3R?-acetoin with glucose as carbon source.The effects of the factors on microbial metabolism,such as nutrients,dissolved oxygen,osmotic pressure and ionic liquid,were studied to increase the production of?3R?-acetoin.The results obtained in this work would provide experimental basis for industrial production of?3R?-acetoin.First,the strain was screened.The samples of sea mud were selected from Dalian sea area.The sample was enriched and cultured in the fermentation medium of acetoin.A strain with high yield of?3R?-acetoin was obtained by screening the plate and shaking flask.The strain was acclimated to enhance the acetoin tolerance of the strain,and finally the stable strain was obtained.The 16S rDNA sequence was sequenced and compared,the phylogenetic tree was established,and some physiological and biochemical experiments were carried out.The results showed this strain belongs to Bacillus subtilis and named as Bacillus subtilis DL11-11.At present,the strain was preserved in the China General Microbiological Culture Collection Center?CGMCC?,and the number was CGMCC No.13141.The fermentation performance of the bacteria was investigated in the next step.The initial glucose concentration was determined to be 100 g/L.The comparison experiment of two kinds of organic nitrogen sources?corn steep liquor powder and yeast extract?was carried out,and the optimum concentration of organic nitrogen source was investigated,as a result,The corn steep liquor powder was determined as organic nitrogen source and the concentration was 1.5 g/L.The fermentation performance of the Bacillus subtilis DL11-11 in the fermentor was investigated.Secondly,the effects of medium compositions on the fermentation of?3R?-acetoin were investigated.The first step is to optimize medium components.Plackett-Burman test showed that the factors of Mn²⁺,Co²⁺,HPO₄^2-,glucose,and corn steep liquor powder have significant effects on the formation of?3R?-acetoin,in which the effects of Mn²⁺,Co²⁺,HPO₄^2-and corn steep liquor powder on the formation of?3R?-acetoin were positive,and the effect of glucose on the formation of?3R?-acetoin was negative.PB experiments also showed that the factors(H₃BO₃,Mn²⁺,corn steep liquor powder,Co²⁺,NH₄⁺,Mo⁶⁺,and glucose)have significant influences on the conversion rate of glucose to?3R?-acetoin,in which Mn²⁺,corn steep liquor powder,Co²⁺,NH₄⁺,and glucose have positive correlation to the conversion rate of glucose to?3R?-acetoin,and the effect of H₃BO₃ and Mo⁶⁺on the conversion rate is negative.The most important factor of the above two response values is taken as the main optimization object.Mn²⁺and Co²⁺are selected as the optimization object,and the steepest climbing experiment and central combination experiment are used to determine the concentration of two metal ions,and the Design Expert software is used to analyze the model.The optimal results were Mn²⁺?0.053mmol/L?and Co²⁺?0.004 mmol/L?,and the conversion rate of glucose to?3R?-acetoin was0.4681.The conversion rate was 0.4752 g/g by the shaking flask experiment,which was close to the predicted value.Finally,the effect of fermentation conditions on the production of?3R?-acetoin was investigated.The effects of aeration rate,agitation speed and pH were investigated.The optimal condition was aeration rate of 0.2vvm,agitation speed of 170rpm without pH control.Under these conditions,?3R?-acetoin concentration was 72.53 g/L after 72-h fermentation,the conversion rate was 0.4773 g/g,the productivity was 1.012 g/L/h,and the optical purity of?3R?-acetoin surpassed 99%.The effects of 1-ethyl-3-methylimidazolium trifluoromethanesulfonate?[Emim]OTF?on the fermentation of?3R?-acetoin was studied.At present,the[Emim]OTF concentration of 5g/L and 10 g/L were investigated,and the effect of 5 g/L on the growth of the bacteria was less than that of 10 g/L.The addition of[Emim]OTF can obviously regulate the formation of organic acids,to a certain extent,it can improve the conversion rate of the acetoin and the optical purity of the acetoin in the fermentation process.