Selection And Fermentation Of Enterbacter Aerogenes Mutant For Diacetyl Production

According to the theory of metabolic control fermentation,the dissertation mainly focuses on the breeding of high diacetyl-producing mutant,fenmentation optimization, enzymatic activities and mechanism of diacetyl accumulation.A mutant,designated as UV-3,was selected from wild-type Enterobacter aerogenes 10293 by application of UV irradiation in combination with cultivation in high glucose medium,leucine medium and diacetyl medium.Gas chromatography was employed to quantify the output of metabolization,and it was found that the yield of diacetyl for the mutant UV-3 was 18.7 fold to the wild-type 10293,up to 1.045g·L^-1.Acetoin and ethanol yields were decreased by 48.4%and 71.4%,respectively.The yield of acetate was increased by 34.6%.Based on the Logistic equation and Luedeking-Piret equation,three kinetic models were proposed for cell growth,product formation and substrate consumption.With the data from experiment to evaluate model parameters,the three kinetic models appeared to provide a reasonable description for the fermentation process.Optimum medium contained glucose10%,peptone 0.5%,yeast extract powder 0.5%, (NH₄)₂SO₄ 0.01%,citric 0.1%,MnSO₄ 0.2%,MgSO₄ 0.2%;Optimum fermentation conditions contained seed volume 10%,medium volume 150mL(500mL shake flask),temperature 37℃, pH 7.0,rotational speed 180r/min.After adopts this process,the production yield of diacetyl for UV-3 was 1.15±0.03 g·L^-1at the end of 60 h.Based on the results in shake flasks,diacetyl fermentation was scaled up in a 5L fermentor,the production yield of diacetyl reached 1.35 g·L^-1at the end of 60 h,which was increased by 17.4%compared to that of the shake flask fermentation.According to studies on these enzymes,lactate dehydrogenase(Ldh)activity,diacetyl reductase(Dr)activity andα-acetolactate decarboxylase(Ald)activity in mutant strain UV-3 was 0.024±0.002U/mg,0.036±0.002U/mg and 0.087±0.007U/mg,respectively,lower than that of the parental strain 10293.Acetoin dehydrogenase(Ad)activity both in mutant strain UV-3 and the parental strain 10293 were lower than 0.001U/mg.The activities of Ald,Ldh and Dr in the mutant UV-3 show the lowest at pH from 7.0 to 7.5,between 36℃and 39℃. Only Mn²⁺was inhibitor of the three enzyme activities at the same time.Fe²⁺,Zn²⁺and Mg²⁺ were stimulators for the three enzyme activities.Co²⁺inhibited both Ldh and Ald activities but stimulated Dr activity.Ba²⁺,Ca²⁺and Cu²⁺severely stimulated Dr and Ldh activities while Ba(2+)and Ca²⁺had almost no effect on Aid activity.Cu²⁺showed a significant inhibitor effect on Aid activity.These results show the level of diacetyl produced in Enterobacter aerogenes UV-3 would be dependent on the nonenzymatic oxidative decarboxylation of ALA.