Optimization Of Fermentation Conditions For The Production Of Chiral 2,3-butanediol By Recombinant Bacillus Subtilis

The research of this paper is to construct a recombinant Bacillus subtilis,which can use hemicellulose to produce chiral 2,3-butanediol,and the medium was optimized by a statistical method in order to obtain higher yield of production.2,3-butanediol is a kind of bioenergy,an important food additive and the precursor of chiral drug molecules.Especially,it is the synthesis precursors of ink,spices,rubber and other chemicals.This paper overexpressed xylanase gene,inserted key genes of meso-2,3-butanediol in the genome and transferred plasmid pHP12-EBA in the meso-2,3-butanediol production strain.Finally,the engineering strain BSZ12 can use xylan as the sole carbon source to produce meso-2,3-butanediol.The M9 basic salt medium was used as the starting medium,and statistical methods was used to optimize the culture medium which include the analysis of the influencing factors by single factor experiment,the Plackett-Burman design,path of steepest ascent design,central combination design and response surface methodology.The single factor experiment indicated that seven factors influencing the meso-2,3-butanediol production were carbon source?xylan:glucose=9:1?,nitrogen source?yeast extract:tryptone=1:1?,pH=7,ammonium acetate,Mg²⁺,Fe²⁺,Cu²⁺,Mn²⁺.Those components were chosen for further optimization.The four main components of the initial medium and seven factors from single factor experiment were used to design the Plackett-Burman experiment.The positive effects of X1?carbon source?,X2?nitrogen source?,X4?CuCl₂?,X9?K₂HPO₄?were insignificant.Based on the steepest ascent experiment,the central point of central composite design was as follows:carbon source?xylan:glucose=9:1?-?15g/L?,nitrogen source?yeast extract:tryptone=1:1?-?10g/L?,CuCl₂-?3.75mg/L?,K₂H4PO₄-?3.9g/L?.The software of Design Expert 8.0 was used for central composite design and the value of BD/All was used as response value.Response surface methodology used in the last steps were useful to determine the optimum levels of the factors.The optimized concentrations of medium components are as follows:13g/L carbon source?xylan:glucose=9:1?,10g/L nitrogen source?yeast extract:tryptone=1:1?,4.0mg/L CuCl₂,3.9g/L K₂H4PO₄.The production of meso-2,3-butanediol by BSZ12 was carried out under the optimum conditions.The result of validation experiments is that production of meso-2,3-butanediol increased from 1.96g/L to 4.62g/L,which was 2.36-fold higher than the M9 basic salt medium,and the value of BD/All increased from 52%to 92%.The value of BD/All was very close to the predicted values 91.7%,and the model was successfully validated.The good agreement between the predicted and the experimental results verified the validity of the model,and the improvement of meso-2,3-butanediol production also indicated that response surface methodology was a powerful tool for determining the exact optimal values of the individual factors and the maximum respone value.