| Phenyllactic acid is a kind of phenolic acids with high value and widely found in natural honey and Chinese herbal medicine.This interesting compound has antimicrobial activities against a wide species of gram-positive and gram-negative bacteria by changing microorganism growth situation and quorum sensing systems.PLA can also be used as a pharmaceutical agent to treat coronary diseases or platelet inhibition as an analogue of "Danshensu".Moreover,it also could be used as the key monomer and building block chemical for the production of new bio-based plastics of poly(phenyllactic acid)s,which have high mechanical strength and high stability.Therefore,PLA has potential applications in chemical,pharmaceutical,food,and new material industries.There are several definitive chemical synthesis routes for preparing PLA.However,these synthesis methods have some disadvantages such as the complex procedures,the strict reaction conditions and multiple by-products.The biosynthesis method is a more sustainable method due to its advantages like the lower costs and more moderate reaction conditions.In this thesis,the preparation of PLA by microbial synthesis was achieved via the highly efficient strain screening,the catalytic process of bacterial strains and the immobilized cells.The biotransformation by using natural strains was suggested as the feasible direction of industrialization.The one-step method was developed and used for separating the PLA from crude feedstock by the chromatography using cryogels with double functional groups.The main results are summarized as follows:(1)The strain with high catalytic activity was screened.There were 60 strains screened from Chinese traditional pickle with the capacity of transforming phenylpyruvic acid(PPA)to PLA,and the strain GBS-3 was identified as Lactobacillus bucheri,which has the highest catalytic activity.The catalysis capability of L.buchneri GBS3 with different substrates was investigated,and the results showed that phenylalanine(Phe)and PPA could be the suitable substrates for PLA both in fermentation and biotransformation and PPA was better than Phe.Thus PPA as the substrate in PLA production using resting cells of L.buchneri GBS3 was confirmed for the subsequent experiments.(2)A new optimization method for the biotransformation process of PLA was developed.From the growth characteristic of L.buchneri GBS3 under different fermentation conditions,it was found that the dissolved oxygen content was positively correlated with the catalytic ability of the bacteria,but the relationship was not significant.Thus the static culture was selected to be the cultural method.Meanwhile,we proposed a new method,i.e.the uniform design with overlay sampling methodology,and the optimum condition of biotransformation was confirmed,i.e.,20 mg/mL glucose,270 mg/mL permeabilized cells,13 mg/mL PPA,pH 8.0 and the reaction time of 15 h.Finally,10.93 mg/mL PLA with the mole conversion ratio of 83.07%?from PPA to PLA was achieved in the biotransformtion system.(3)The immobilization of L.buchneri GBS3 was conducted and employed in the bioconversion.The poly hydroxyethyl methylacrylate(pHEMA)cryogel beads were fabricated to immobilize cells by the liquid droplet method,and the batch cycles of biotransformation and high density culture utilizing the cryogel beads with entrapped cells were investigated The results showed that the beads size was positivelycorrelated with the inner diameter of the microtube within a certain range,and the pHEMA cryogel beads had a supermacroporous structure.The batch cycle of biotransformation by using cryogel beads immobilized with L.buchneri GBS3 showed that the cells within beads still had the catalytic capacity after 10 times conversion,and the capacity was much better than the free cells.Meanwhile the molar conversion ratio increased with the beads size.As for the high density culture,the biomass increased with the beads size,and the highest dry cell weight reached 15.6 mg/mL which was six-fold compared with that under the suspended culture.Moreover,the sodium alginate was also used to immobilize L.buchneri GBS3 cells as the contrast,and the results showed that the stability and biotranformation capacity were much lower than that of the pHEMA cryogel beads.The L.buchneri GBS3 cells bound strongly to the matrix and had a higher acid resistance.(4)The separation of PLA by using cryogels with dual functional groups was investigated.The separation of PLA from the crude conversion broth was achieved by the chromatography using the pHEMA-based cryogel with a combination of anion-exchange and hydrophobic benzyl groups.The basic properties of column were desirable,and the pHEMA-based cryogel had a high capacity of PLA,i.e.,14.64 mg/mL cryogel and the adsorption of PLA was influenced by the salt concentration.By using deionized water as the running buffer,PLA with a high purity of 97.6%was obtained with one step elution using 0.3 M NaCl as the elution solution and the recovery was at the range of 80.2?90.8%from crude feedstock without any pretreatment at various flow velocities.These values were close to those obtained for the clarified broth,i.e.,the purity of 98.4%and the recovery of 92.3%under the same chromatography conditions at 1 cm/min.The cryogel was then applied to separate PLA from clarified feedstock,high purity(>96.7%)and recovery(>91.4%)of PLA were found with 20 cycles,which indicated the selectivity and robustness of the cryogels.Therefore,the chromatography using pHEMA-based cryogels with the.dual functional groups could be an effective approach for the isolation of PLA directly from the crude bioconversion broth and thus could be interesting in the separation anc production of high-purity PLA with one-step.(5)The non-adsorption breakthrough performance of phenyllactic acid in the cryogel was modeled.The capillary-based model was adopted to characterize the structure of cryogel and the non-adsorption breakthrough performance of small molecular organic acid.In this model,the cryogel is assumed to be made up of several groups of capillaries which have different diameters,lengths,tortuousness and the wall thickness.The results showed that for a given cryogel,by using the model based on the physical properties of the cyogel together with the PLA breakthrough curves,we can obtain a reasonable prediction and detailed characterization of the porous structure properties of cryogel matrix,particularly regarding the number of capillaries,the capillary tortuousness,the pore size distribution and the skeleton thickness.The model is also effective with regards to predicting the flow performance and the non-adsorption breakthrough profiles of PLA at different flow velocities.It is thus expected to be applicable for characterizing the properties of cryogels and predicting the chromatographic performance of small molecular organic acid with good accuracy.This thesis aimed to the synthesis of PLA.Based on strain screen,the optimization of biotransformation process and the immobilization of cells were achieved successfully.By the whole-cell biocatalyst of bacteria and the separation of PLA using the cryogel with dual function groups,PLA with high purity and recovery was obtained.Meanwhile,modeling the non-adsorption breakthrough performance of small molecular organic acid in the cryogel The results demonstrated the brilliant prospect of PLA production via biotransformation in future practice. |
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