| Poly malic acid(Poly malate,PMLA)is a high-molecular homopolymer which is assembled by malate.PMLA and its derivatives are a new macromolecule biomaterial,which can be used as biodegradable material,surgical suture,tissue engineering scaffolds and drug carrier.The chemical synthesis method for PMLA is complicated and has a lower productive rate.Therefore,the method of microbial fermentation for PMLA production is more and more interesting.However,the extraction of PMLA from fermentation liquor is very difficult because of the compositions of fermentation broth are complex.In this study,the PMLA from fermented liquid was extracted and purified by the combination of membrane technology and bioseparation technology.The results could provide technical support and theoretical guidance for coupling of fermentation and separation in the future.1)The fermentation process and the selection of membraneAfter fermentation,the parameters of fermented liquid were as followed: 80 g/L biomass,48.21 g/L PMLA with 9760 average molecular weight,and 1.16 dispersion coefficient.Meanwhile,the production of polysaccharide is 17 g/L and glucose consumption rate is 86.875%.These results are consistent with the previously reported.Therefore,the fermentation liquor can be used as samples for membrane separation process.Selection of film with appropriate pore sizes is a critical step in this study,and three microfiltration membranes with pore sizes of 1.4,0.2 and 0.14 ?m,and three ultrafiltration membranes with MWCO of 300,150 and 50 k Da were used for the separation of A.pullulans from PLMA broth,respectively.The results showed that the cell removal rate and PMLA recovery were high by using ultrafiltration membranes with 300 KDa and 150 KDa,and the membrane fouling was similar.However,the average flux of the ultrafiltration membranes with 300 KDa(91.91 Lm-2h-1)is higher than that of ultrafiltration membranes with 150 KDa(82.64 Lm-2h-1).As a result,the ultrafiltration membrane with 300 k Da was used as the most suitable membrane for removing the bacteria from fermentation broth.2)The optimization of membrane filtration conditions and the analysis of membrane foulingThe operating conditions were as followed: initial feed volume of 1300 m L,VRR of 7.2,cross-flow velocity of 6 m/s,TMP of 0.2 MPa,and a temperature of 40 ?.The results showed that fouling was mainly caused due to formation of the cake layer by macromolecular substances,which causing the pore blocking.3)Classification of PMLA and production analysisTo obtain the pure PMLA,the secondary separation and purification for PMLA was performed by a double-membrane-sandwich method the results showed that the double-membrane-sandwich method has better effect of separation and purification,which can purify PMLA step by step according to the different molecular weight,and the PMLA dispersion coefficient is close to 1.In order to further remove the small molecule sugar,macro-porous anion exchange resin D296 was used by anion exchange resin adsorption method.The results showed that the anion exchange resin could adsorb 158.40 mg PMLA per 1g resin,and removed 70.86% of sugar.It was indicated tha the anion exchange resin was better than other experimental resins.Therefore,the macroporous resin D296 was used to remove free sugars.The nuclear magnetic H spectrum analysis showed that the PMLA contains side chains of sugar.Enzymolysis showed that most of these sugars were connected by a-1,6 glucosidic bond.Furthermore,molecular weight of carbohydrate side chain is about 3534.5 Da.The infrared spectrum analysis showed that the PMLA is assembled by a beta polymerization.The results were consistent with previously reports. |
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