Constructure And Fermentation Of Gernetically Engineered Escherichia Coli WS102 And Research Of Succinic Acid Separation

As a important C4 platform compound,succinic acid is the intermediate compound that used to make the products which is wildly applied in field such as industry,agriculture,food and medical.There is a great advantage compared with the method of traditional way with fossil energy that the production of succinic acid with the renewable resource using microorganism to ferment.This article is based on the genetically engineered Escherichia coli WS100 which is constructed by our laboratory.We use the method of ?Red homologous recombination to delete methylglyoxal synthase gene?mgsA?,and then culture the cells in anaerobic cube,we harvest the E.coli WS102 to decrease the acetic acid.Then we have the experiment on this strain WS102 in NBS broth media with 7L bioreactor.This strain WS102 produced 64.9g/L succinate with a yield of 0.731 g succinate per gram of metabolized glucose,no lactate can be detected by HPLC.The experiment of using CO₂ to ferment succinic acid based on the WS102.We found that the fermentation with CO₂ gas in whole process don't have a great performance,because in early stage of the anaerobic fermentation used to cause a unbalance status of metabolize with a lot of acetic acid.The result under this condition showed a production of 52.65g/L succinate and a yield of 0.689g/g.For this reason we designed a microaerobic-anaerobic process of fermentation by compared some single factor.In early time before stationary phase the growth of cell was maintained by adding KHCO₃,K₂CO₃ and KOH was acted as neutralizer and provide C that needed to be immobilization.When the cell was in stationary phase,the CO₂ gas was added into the NBS broth.With this process we got 58.3g succinate with a yield of 0.731g/g glucose.In this condition we carry on a fed-batch fermentation,at last 76.5g/L succinate was havested with a yield of 0.765g/g glucose and productivity of1.063g/L·h.At last fraction of this paper,we try to extraction succinic acid from broth.We chose to add 30% calcium chloride solution with equal volume ratio in 80? water bath for 30 minutes.The process line mainly include heat,filter by ceramic membrane to eliminate cells and partial protein,then the succinic acid was deposited by adding calcium chloride,at lase the succinic acid was harvested by acidolysis,decolor,ionexchange and concentration crystallization.The crystal succinic acid is about 99.6g and the total yield of succinic acid in whole process was up to 56.8%?...