| Recently,amount of glycerol was produced as the by-product in the manufacture of biodiesel.Therefore,the effective use of glycerol has become a major factor to affect the development of biodiesel enterprises and how to make reasonable use of glycerol in the future has attracted widespread attention.3-hydroxypropionic acid(3-HP)can synthesize many important chemical intermediate because of its excellent properties,hence,3-HP fermentation from glycerol has broad application prospects.This study investigated a novel recombinant E.coli contained glycerol dehydratase and aldehyde dehydrogenase to improve the 3-HP production from glycerol.The basic culture conditions,the control strategy of the main fermentation parameters and the feeding process were studied.These results offered the fundamental data for the 3-hydroxypropionic acid production from glycerolFirstly,the fermentative culture condition of E.coli bio synthesizing 3-hydroxypropionic acid was optimized by single factor experiments.The results showed that the optimized culture media were composed of(g/L):glycerol 30,glucose 20,NaCl 1,MgSO4·7H2O 0.25,Na2HPO4·12H2022.7,KH2PO4 3,yeast extraction 4.8,(NH4)2S04 3.2,VB12 0.02;the optimal fermentation conditions were as follows:initial medium pH7.0,loading volume at 80mL per 500mL,inoculums size of 6%,seed culture time at 10 hour;selected IPTG as inducer,and its concentration was 1mM,the inducer temperature was 30? Under these optimized conditions,the maximal 3-hydroxypropionic acid production was 8.29 g/L,the biomass was 4.75 g/L,and they were improved 6-fold and 2.64-fold after the optimization experiments,respectively.Then the control strategy of the main factors during the fed-batch fermentation,such as pH,temperature and dissolved oxygen,was investigated on the basic of the above data.Conclusions are drawn as follows:The pH was controlled at 6.5 at the first 5 hour and 7.0 at the 5th-15th hour.Then pH was changed to 7.5 at the 15th-25th hour and 7.0 after the 25th hour.Under these conditions,biomass and 3-hydroxypropionic acid yield improved 21%and 18.54%,respectively,than that of pH7.0.The temperature was controlled at 37? at the first 5 hours,then changed to 30 ? after the 5th hour.Under these conditions,the biomass and 3-hydroxypropionic acid yield improved 18.8%and 6.8%than that of 30 ?,respectively.The research on dissolved oxygen showed that controlled the dissolved oxygen on 60%could make the biomass yield improve 4%and 18.47%than that of 20%and 40%,respectively;and 3-hydroxypropionic acid yield improved 5.7%and 7.2%than that of 20%and 40%,respectively.Finally,the feed-batch fermentation of 3-hydroxypropionic acid production was studied,as well as the effect of glycerol and glucose addition on the fermentation.This results showed the best time to feed glycerol and the glucose was at the concentration of glycerol decrease to 15 g/L and the concentration of glucose decrease to 3 g/L,respectively.When glycerol was fed at constant speed,the maximal 3-hydroxypropionic acid and biomass production were 16.45 g/L,and 11.84 g/L,respectively;When glucose was feed at constant speed,the maximal 3-hydroxypropionic acid and the biomass production were 15.02 g/L and 13.27 g/L,respectively.And when the glycerol and glucose were mixed fed to the fermentation.The optimal technology was follow:glucose was fed at constant speed after 8th hour;and glycerol was fed at constant after 14th hour,and the maximal 3-hydroxypropionic acid and the biomass production of 17.20 g/L and 13.80 g/L could obtained at this condition.This study founded theoretical bases for the characteristics of the production of 3-hydroxypropionic acid from glycerol by the E.coli,which is favorable to study the condition of production of 3-hydroxypropionic acid. |
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