| Laccase can catalyze and degrade chlorophenol organic pollutants,which has the advantages of high efficiency and no secondary pollution.The free laccase has poor stability and is difficult to recover.Through immobilization,its stability can be significantly improved and it can be reused.Chitosan/Layered double hydrotalcites,as an organic/inorganic composite material,can overcome the disadvantages of single organic and inorganic carriers,fuses their respective advantages,and has excellent application prospects in the fields of enzyme immobilization and environmental protection..In this study,Co-Fe-Layered double hydrotalcites(CFLDHs)were synthesized by low-saturation co-precipitation method,and chitosan molecules were introduced into the special layered structure of LDHs to synthesize Chitosan/Layered double hydrotalcites(CS/CFLDHs).The above two materials were characterized by SEM,FTIR and XRD.Adsorption properties of 2,4-dichlorophenol on the two kinds of materials were investigated,and the adsorption conditions were optimized.The Langmuir isotherm model and Freundlich isotherm model were used to fit the adsorption isotherms of the two materials.The adsorption kinetics of the two materials were also studied.In addition,glutaraldehyde was used as a cross-linking agent,and two kinds of materials were used to immobilize laccase through a cross-linking method.The influencing factors of the removal of 2,4-dichlorophenol by two kinds of immobilized laccases were studied;the reaction kinetics of the removal of 2,4-dichlorophenol by above immobilized laccases was further studied;and its stability and reusability were studied.Results as following:(1)Synthesis of CS/CFLDHs and characterization of its properties.The addition of macromolecule chitosan does not change the crystalline phase of the LDHs,but it slightly affects the crystallinity of the LDHs.LDHs and chitosan were intertwined in CS/CFLDHs,and chitosan molecules were dispersed between LDHs and LDHs.The dispersion of CS/CFLDHs was inferior to that of CFLDHs.The infrared spectra of CS/CFLDHs clearly show the characteristic infrared peaks of LDHs and chitosan,indicating that the composite of chitosan and LDHs is successful.Characterization by XRD can further prove the successful synthesis of CS/CFLDHs materials.(2)Adsorption properties of 2,4-dichlorophenol(20mg/L)on CFLDHs and CS/CFLDHs were studied.The optimal conditions for adsorption were obtained by the control variable method.The optimal conditions for the adsorption of 2,4-dichlorophenol by CS/CFLDHs: adsorption time t=90 min,adsorbent dosage 60 mg,p H=7;the optimum conditions of 4-dichlorophenol by CFLDHs: adsorption time t=60 min,adsorbent dosage 60 mg,p H=7.Under optimal conditions,the removal of 2,4-dichlorophenol by the two materials was 54%(CS/CFLDHs)and 43%(CFLDHs),respectively.The fitting of adsorption isotherms for above materials show that the adsorption isotherms of the two materials are highly compatible with the Langmuir model and the Freundlich isotherm model(R2>0.99).It can be judged from the value of n that the adsorption of 2,4-dichlorophenol on both materials is difficult.The adsorption capacities of the two materials were 1.9456 mg/g(CS/CFLDHs)and 1.3135 mg/g(CFLDHs),respectively.The adsorption capacities of the two materials were 1.9456 mg/g(CS/CFLDHs)and 1.3135 mg/g(CFLDHs),respectively.It can be judged from the value of n that the adsorption of 2,4-dichlorophenol on both materials is difficult.The adsorption capacities of the two materials were 1.9456 mg/g(CS/CFLDHs)and 1.3135 mg/g(CFLDHs),respectively.The adsorption capacity of CS/CFLDHs was better than that of CFLDHs.The second-order kinetic equations were used to study the adsorption process of 2,4-dichlorophenol adsorbed on the two materials.The experimental results showed that the adsorption rate of CS/CFLDHs was faster than that of CFLDHs,and the adsorption curves of the two materials were well fitted.For the pseudo-second-order kinetics model,the correlation coefficient between them is R2>0.99.(3)The immobilized laccases(L-CS/CFLDHs and L-CFLDHs)were prepared by cross-linking method and the immobilization conditions were optimized,using CFLDHs and CS/CFLDHs as carriers and glutaraldehyde as cross-linker.The optimal immobilization conditions for L-CS/CFLDHs: cross-linking time is 2h,immobilization time is 8h,glutaraldehyde concentration is 5%(v/v),p H=6,laccase concentration is 1.0 g/L.The optimal immobilization conditions for L-CFLDHs: cross-linking time is 3h,immobilizing time is 8h,glutaraldehyde concentration is 5%(v/v),p H=6,laccase concentration is 1.2 g/L.Due to the introduction of floccular chitosan,the contact area between glutaraldehyde and the material increases,and the contact sites increase,making the crosslinking time shorter than that of CFLDH.The immobilization efficiency of immobilized enzyme on inorganic LDHs is low,and the introduction of chitosan improves the disadvantage to some extent.The maximum enzyme activity of L-CS/CFLDHs was 1271.41 U/g,the maximum enzyme activity of L-CFLDHs was 1745.61 U/g,and the free enzyme activity was 9069.40 U/g.Fe3+ and Zn2+ have a slight promotion effect on the enzyme activity of laccase.Mg2+,Co2+,Ni2+ and Al3+ all have a certain inhibitory effect on the enzyme activity of laccase.The mixed salt solution of Co2+ and Fe3+ also has a certain inhibition on laccase.(4)The removal performance of 2,4-dichlorophenol(100 mg/L)by two immobilized laccases(L-CS/CFLDHs and L-CFLDHs)and the stability of above immobilized laccases were studied.The optimal conditions for removal of 2,4-dichlorophenol by two immobilized laccases: t=48h,T=55°C,p H=6,C2,4-dichlorophenol =100mg/L Under optimal conditions,the removal of 2,4-dichlorophenol by L-CS/CFLDHs was 81.55%,and the removal of 2,4-dichlorophenol by L-CFLDHs was 61.78%.The main structure of the two materials is LDHs,and its lamellar structure is composed of Co2+ and Fe3+,so the two materials themselves have a good catalytic oxidation for 2,4-dichlorophenol,and the removal of 2,4-dichlorophenol could reach 63.28%(CS/CFLDHs)and 50.03%(CFLDHs),respectively.The removal of 2,4-dichlorophenol by laccase from L-CS/CFLDHs immobilized laccase was 26.04%,and the removal of 2,4-dichlorophenol by laccase from L-CFLDHs immobilized laccase was 12.86%,which is much lower than free enzyme(39.48%).The first-order reaction kinetics of L-CS/CFLDHs are better than L-CFLDHs,and the reaction rate is faster.The two immobilized laccases have good stability between p H=6-9 and good stability below 55°C.After 5 cycles of repeated use,the removal of 2,4-dichlorophenol by the two immobilized laccases remained at 55.34%(L-CS/CFLDHs)and 32.73%(L-CFLDHs),respectively. |
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