The Fermentation Of 3-hydroxy Butanone By Bacillus Strains

3-Hydroxy butanone is an important primary metabolite of microbial metabolism.It has been widely used as an important platform compounds in food,chemical,bio-fuel and other industries.There are many researches on the synthesis of 3-hydroxy butanone with biological method in recent years,but research on the genome sequence affecting the production of 3-hydroxy butanone in Bacillus is relatively fewer,and research on the production by thermophilic Bacilus is even more rarely.Based on two early screened new strains of Bacilus sp.H13-3 and H15-1 that have been used to produce 3-hydroxy butanone with fermentative method,the processes will be investigated and analyzed according to the following three aspects.First of all,before this research,there is no relevant report in regard of an online method for detecting acetoin,not only in the basic study but also in the permentation production.To resovle this problem,a rapid quantitative online detection system for the real-time monitoring content of 3-hydroxy butanone was established.The performance of the system has indicated the result of the system is highly accurate（RSD=0.02%）,this online system has a high average rate of recovery（99.42%）,the fitting degree of its standard curve is perfect and the range of the detection limits is wide（0.05¹10.00 g/L）.At the same time,the system have many other advantages,such as simple operation,lower price of monitor instrument,etc.This system has verified the feasibility of determination of 3-hydroxy butanone in fermentation solution online with the method of creatine colorimetry.The establishment of the system has a great value in application.Secondly,the fermentation process of Bacillus sp.H13-3 in the solution of cassava powder has been optimized.The optimized formula of fermentation medium achieved according to the method of Plackett-Burman:adding KH₂PO₄,30 g/L;CH₃COONa·3H₂O,0.3 g/L;CaCl₂,0.2 g/L;MgSO₄,0.1 g/L into the filtrated saccharification liquid of cassava powder.The purpose of high efficacy of production of 3-hydroxy butanone has been archived with the method of two stage pH controlling fermentation,which makes the highest yield of 3-hydroxy butanone to reach 67.03 g/L.To our knowledge,it is a highest yield of themophilic Bacilus.Finally,two novel byproducts,2-hydroxy pentanone and 3-hydroxy pentanone have been found and authenticated in the early permentation experiment,in order to explain the metabolic pathway of them from the enzymatic perspective,the whole genome of the Bacilus H15-1 has been measured,in order to explain the metabolic pathway of 2-hydroxy pentanone and 3-hydroxy pentanone from enzymatic perspective,the gene（alsS,alsD）of the key enzymesα-acetolactate syntheses andα-acetolactate that determine the synthesis of3-hydroxy pentanone have been cloned and expressed in Escherichia coli.It has been proved that the key enzymes of metabolic process of 3-hydroxy butanone also determine the synthesis of its homolog 2-hydroxy pentanone and 3-hydroxy pentanone by the interaction of purified enzyme with pyroracemic and 2-ketoacetic acid.Therefore,the function of the key enzymes in the synthesis of the 3-hydroxy butanone and its homolog has been analyzed from the enzymatic perspective.