| Biodiesel is a clean and sustainable fuel with the concept of recycling economy.Enzymatic synthesis of biodiesel has become a research hotspot due to its advantages of environmental friendliness,mild reaction conditions and easy extraction of product.However,the lack of long-term operational stability and effective recovery increases the reaction cost,which hinders the large-scale industrial application of enzymatic synthesis.In this thesis,intelligent hyperbranched polyglycerol hydrogels are prepared as the carrier to improve the stability and reusability of lipase in the process of biodiesel synthesis.The main researches are listed as follows:(1)Hyperbranched polyglycerol/polyacrylic acid hydrogel(HPG/PAA hydrogel)were prepared with methacrylated hyperbranched polyglycerol(HPG-MA)and acrylic acid(AA).The swelling properties and adsorption properties of the HPG/PAA hydrogels were studied.The hydrogels show a maximum swelling ratio of 580 g/g in deionized water.In five common used biological fluids(Hanks solution,Physiological saline,PBS,RPMI-1640 and MES),the swelling ratios of hydrogels were greater than 60 g/g.The hydrogels were responsive to the changes of pH and ionic strength of the solution.In strongly acidic solutions and high ionic strength solutions,the hydrogels present evident deswelling phenomenon.The methyl violet was chosen as a model compound,and HPG/PAA hydrogels reach a maximum adsoption of 405.7 mg/g at a feed concentration of 1000 mg/L and pH of 7.The adsorption processes fit to the pseudo-second-order model and Langmuir model.After five consecutive adsorption-desorption experiments,the hydrogels still remain about 90 percent of the initial dye adsorption capacity.(2)The immobilization of lipase in HPG/PAA hydrogels were performed with entrapment method.The maximum enzyme loading amount and relative enzyme activity of the hydrogels were 78.9 mg/g and 67.0%,respectively.The storage stability and thermal stability of free lipase and immobilized lipase were studied.At 4 ?,the half-life of free lipase was 5 d,and that of immobilized lipase were from 15 d to 20 d.At 60 ?,the half-life of free lipase was 7 h,and that of immobilized lipase were from 14 h to 20 h.The immobilized liapse were used to catalyze the transesterification of soybean oil and methanol to prepare biodiesel.The reaction conditions of biodiesel synthesis were optimized by response surface methodology.The biodiesel yield of 96%was obtained at a temperature of 35 ?,a water to oil mass ratio of 1:1,a methanol to oil molar ratio of 10.8:1,an enzyme to oil mass ratio of 0.15:1 and a solvent to oil mass ratio of 1.5:1.The immobilized lipase show good stability during the operation.After 5 repeat uses,the biodiesel yield still remain over 90%.By means of regulating the ionic strength of aqueous solution,the reversible binding of lipase to the hydrogel was achieved.The immobilization of lipase in HPG/PAA hydrogels was carried out by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride(EDC)and N-hydroxysuccinimide(NHS).The maximum enzyme loading amount and relative enzyme activity of 40.7 mg/g and 47.2%were obtained at an EDC to carboxyl group molar ratio of 3:1,a NHS to EDC molar ratio of 1:1,an enzyme concentration of 1 mg/mL.At 4 ? and 60 ?,the half-lives of the immobilized lipase were from 23 d to 29 d and from 12 h to 19 h,respectively.The immobilized liapse were used to catalyze the synthesis of biodiesel.The maximum biodiesel yield of 93%was obtained at a temperature of 37.5?,a water to oil mass ratio of 1.04:1,a methanol to oil molar ratio of 12.3:1,an enzyme to oil mass ratio of 0.15:1 and a solvent to oil mass ratio of 0.52:1.After 21 consecutive reactions,the biodiesel yield remains above 50%.According to the results,the entrapment method was easy operation,and the immobilized lipse have the advantages of high enzyme loading amount,high relative enzyme activity,excellent storage stability and desirable thermal stability.In the synthesis of biodiesel,a desirable biodiesel yield was achived and the immobilized lipase show good reusability.(3)The cationic hyperbranched polyglycerol microspheres were prepared by inverse suspension polymerization with HPG-MA,acrylamide(AM)and dimethyl diallyl ammonium chloride(DMDAAC)as monomers,ammonium persulphate(APS)as initiator,Span-80 as emulsifier and dodecane as dispersant.The structure and particle size of the microspheres are characterized by SEM and laser particle size analyzer.The stirring speed and HPG-MA content have a decisive influence on the average particle size and particle size distribution.The highest uniformity ofparicle size was obtained at the stirring speed of 800 RPM.The maximum swelling ratios of the microspheres in deionized water and PBS(0.01 M,pH 7.0)were 147.9 g/g and 120.3 g/g.The acid fuchsin dye was selected as a model compound to investigate the adsorption capacity of microsphere.A maximum dye adsorption of 882.2 mg/g was obtained at a dye concentration of 1500 mg/L and a pH of 3.The adsorption processes fit to the pseudo-second-order model and Langmuir model.(4)The immobilization of lipase in HPG/P(AM-DMDAAC)microspheres was performed with adsorption mehtod.The maximum enzyme loading amount and relative enzyme activity of 55.7 mg/g and 69.9%were observed at an enzyme concentration of 2 mg/mL and a loading time of 8 h.At4 ? and 60 ?,the half-lives of the immobilized enzyme were 18-25 d and 15-20 h.In aqueous solution with different ionic strength,the retention of immobilized lipase were all over 70%.M3 microsphere was selected as the carrier of lipase to catalyze the synthesis of biodeisel.The highest biodiesel yield reaches 95%at a temperature of 38 ?,a methanol to oil molar ratio of 7:1,a water to oil mass ratio of 0.38:1,an enzyme to oil mass ratio of 0.06:1 and a solvent to oil mass ratio of 0.36:1.After 10 consecutive reactions,the biodiesel yield still remain about 70%.Glutaric dialdehyde(GA)was used to immobilize lipase in HPG/P(AM-DMDAAC)microspheres.The maximum enzyme loading amount and relative enzyme activity of 47.3 mg/L and 63.6%were obtained at an enzyme concentration of 3 mg/mL,a cross-linking time of 6 h,and a GA concentration of 3%.The half-lives of the immobilized lipase were from 22 d to 27 d at 4 ? and from 17 to 22 h at 60 ?.The retention of immobilized lipase in aqueous solution with different ionic strength were greater than 97%.M6 microsphere was chosen to load lipase for the catalysis of biodiesel synthesis.The highest biodiesel yield reaches 96%at a temperature of 35.2 ?,a methanol to oil molar ratio of 6:1,a water to oil mass ratio of 0.36:1,an enzyme to oil mass ratio of 0.05:1 and a solvent to oil mass ratio of 0.25:1.After 7 consecutive reactions,the biodiesel yield still remain 90%approximately.The activity of the immobilized lipase was partially recovered by the remove of by-product glycerol.The results show that HPG/P(AM-DMDAAC)microsphere is a suitable carrier for lipase.High enzyme loading amount and relative enzyme activity can be obtained with both adsorption method and GA method.In the catalysis of biodiesel synthesis,two immobilized lipase can both achive desirable biodiesel yield above 95%,and good reusiblity were observed in continuous reaction process.Compared with lipase immobilized HPG/PAA hydrogel,lipase immobilized microspheres present higher catalytic activity and efficiency.The comsumption of raw materials in biodiesel synthesis decrease evidently,which is benefit to the reduction of reaction cost. |
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