Separation Of 3-Hydroxypropionic Acid From Fermentation Broth Of Recombinant K.Pneumoniae Strain

3-Hydroxypropionic acid(3-HP)is a commercially valuable bulk chemical manifesting broad applications.Currently,the main synthetic methods of 3-HP include chemical synthesis and microbial fermentation.Chemical synthesis of 3-HP suffers limitations in commercialization due to low yield,high cost and the complexity of separation and purification process.Impressive advances in microbial biosynthesis can mitigate these drawbacks.In this study,previously engineered Klebsiella pneumoniae strain was cultivated in bioreactor for high production of 3-HP,and the broth was subject to metabolic analysis.More importantly,separation route of 3-HP was preliminarily developed.Below are main experimental results:(1)Based on titer,glycerol conversion rate and productivity,the recombinant strain K.pneumoniae(3tac)was further shown to be high-yield strain for production of 3-HP.Results showed that the fermentation broth contained the following components(per liter):3-HP,90.01 g/L;lactic acid,7.36 g/L;acetic acid,13.31 g/L;1,3-propanediol,2.82 g/L and 2,3-butanediol,2.47 g/L.(2)Calcium chloride was used as flocculant to remove impurities such as bacterial debris and soluble proteins in the fermentation broth.Under optimal flocculation conditions,the flocculation rate of bacteria reached 99.99%.The removal rate of soluble proteins was 90.98%,and the loss rate of 3-HP was 1.6%.(3)Powdered activated carbon was recruited for decolorization and adsorption of 3-HP in the fermentation broth.At pH 7 and activated carbon dosage of 28 g/L,the decolorization rate of fermentation broth reached 90%and the loss rate of 3-HP was 5-8%at room temperature.Dynamic decolorization experiments were carried out using granular activated carbon as a decolorant.It was found that 1.34 mL/min was the ideal flow rate for decolorization,and the 732#cationic resin was not appropriate for decolorization of 3-HP fermentation broth.(4)In addition,we screened the resin used for desalination of fermentation.Results showed that 732#cation resin and D301R#anion resin could be appropriate desalting resin.The ideal volume flow rate of 732#cationic resin is 2 BV/h,and the ideal volume flow rate of D301R#anion resin is 1-2 BV/h.Therefore,711#anion resin and D290#anion resin were selected to adsorb 3-HP.(5)Finally,thin layer chromatography was harnessed to separate 3-HP from lactic acid.A small amount of pure 3-HP was isolated using methanol and dichloromethane(1:10)as developing agent.