| Bacillus coagulans has unique physiological characteristics,which can grow at high temperature to reduce the risk of microbial contamination,and meanwhile produce high optical purity L-lactic acid.Studies have found that by changing the fermentation conditions,B.coagulans exhibits obvious differences in phenotypes,which triggers growing interest of analysis of metabolic characteristics.In industrial lactic acid fermentation corn starch is used as the raw substrate,which results in a mixed carbon source after hydrolysis.At the end of fermentation,there will be a considerable amount of residual sugar that cannot be metabolized,which would reduce lactic acid production and increase the cost of subsequent purification.In this thesis,we analyzed metabolic characteristics of lactic acid fermentation process by B.coagulans 36D1 at different dilution rates and pH conditions in chemostat culture.Then we compared sugar utilization of B.coagulans 36D1,HL5 and BH1 using mixed carbon sources for lactic acid fermentation,especially glucose plus isomaltose.In the first study,glucose-limited chemostats were performed on chemically defined media MCDM3++ at different dilution rates of 0.05,0.10,0.15,0.20 and 0.25 h-1.It was found that ?=0.15 h-1 was the most suitable for lactic acid production according to the titer and yield of lactic acid.Using the chemostat data,the kinetic model of B.coagulans 36D1 growth,lactic acid production and glucose consumption was established by Monod,Luedeking-Piret and Luedking-Piret-Like equations,respectively.As a result,the maximum specific growth rate of 36D1 ?m=0.294 h-1 and Half saturation constant Km=0.4942 g/L were obtained,and the production of lactic acid was largely related to the growth rate.Based on the constructed 36D1 genome-scale metabolic network model iBag597,the fluxes of the central carbon metabolic pathway under different conditions were simulated.Two flux analysis methods,i.e.,pFBA and FVA,were used to simulate the fluxes at different specific growth rates,while sampling method was used to compare fluxes at different pH.It was found that the increase in specific growth rate and decrease in extracellular pH re-directed fluxes from glycolysis(EMP)pathway with low ATP yield toward phosphoketolase(XPK)pathway with high ATP yield.At the lowest specific growth rate,i.e.,0.05 h-1,the ATP production in B.coagulans 36D1 was enhanced by increasing the electron transport chain flux to maintain growth.So as shown above,ATP production pathway changed with various specific growth rates and pH conditions,which therefore affected lactic acid production.In the second study,the utilization of six sole and mixed carbon sources by B.coagulans BH1,HL5,and 36D1 was investigated.The results of the cultures with some sole carbon source showed that 36D1 efficiently used glucose,xylose and trehalose,but poorly maltose.BH1 and HL5 could efficiently use glucose and maltose,but poorly xylose and trehalose.All three strains only metabolized a small amount of isomaltose and had little consumption of sucrose.When cultured with glucose plus one of the other five carbon sources as a mixed carbon source,36D1 showed an obvious glucose catabolite repressive effect(CCR),while HL5 and BH1 did not show the CCR effect with maltose;By comparing the consumption of isomaltose before and after the time point glucose nearly exhaustion,the use of isomaltose by HL5 outperformed the other two strains.Using 45 g/L glucose plus 45 g/L isomaltose as a mixed carbon source in 5 L tank microaerobic fermentation,it was found that during the presence of glucose,36D1,HL5 and BHl metabolized isomaltose at a rate of 3.73 g/L/h,2.04 g/L/h and 1.02 g/L/h,respectively.From the glucose exhaustion to the end of the fermentation(72 h),the three strains metabolized isomaltose at a rate of 0.066 g/L/h,0.125 g/L/h and 0.042 g/L/h,respectively.During the entire fermentation process,compared to 36D1 and BHl,HL5 performed the best in using isomaltose. |
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