Immobilization Of Enzyme/Cell By Light-Induced Surface Controlled/Living Graft Polymerization For Bioethanol Production

Bioethanol,as a renewable,clean,sustainable and environmental protective bioenergy,has attracted increasing research interests in rencent years.The common route to produce bioethanol is based on the conversion of cellulose,which is subject to the high cost in practical production.Immobilization of yeast cells and enzymes is an effective way to reduce the costs of bioethanol production and also can improve their environmental tolerance.Here we proposed several new strategies based on light-induced controlled/living radical polymerization(CLRP)to graft polymer brush or polyethylene glycol(PEG)3D net-cloth onto flexible polymer materials for the immobilization of cellulase and yeast cells for bioethanol production.The main results are listed as below:1.A new approach was developed to graft polymer brush onto polymeric material for immobilization of cellulase.Firstly,isopropyl thioxanthone semipinacol(ITXSP)dormant groups were seeded on the low density polyethylene(LDPE)surface.Then a poly(ethylene glycol)(PEG)polymer brush polymerized from poly(ethylene glycol)dimethacrylate(PEGMA)monomer was grafted onto LDPE surface under UV irradiation to act as a spacer.After graft polymerization,the dormant groups(ITXSP)were still retained on the end of PEG brush and can be reutilized for another surface initiated polymerization.Based on the living nature of this method,poly(glycidyl methacrylate)(PGMA)was grafted onto PEG polymer brushes to form block polymer.Then the epoxy groups of PGMA were converted into amino groups by the reaction with ?-polylysine(or ethylenediamine).Finally,glutaraldehyde(GA)was added and then polymer brushes were obtained with active end aldehyde groups.Cellulase was covalently immobilized on polymer brush by the coupling reaction of glutaraldehyde(GA)and amine group of enzymes.A variety of characterizations were used to verify that the block polymer brushes were successfully grafted on LDPE surface.The height of PEG spacer was 385 nm,which can increase the mobility of PGMA brush as well as the immobilized cellulase.The loading efficiencies of enzyme immobilized on s-polylysine or ethylenediamine treated surface were 2.58%and 1.98%,respectively.The immobilized cellulase was used in 9 consecutive batches for hydrolysis of filter paper,the maximum concentration of glucose was from 0.06 g·L-1 to 0.57 g·L-1.2.An extremely mild and effective method to use poly(ethylene glycol)(PEG)net-cloth grafted on polypropylene non-woven fabrics(PPF)as carriers to entrap living yeast cells via VSLGCP was developed.The method included two steps:seeding ITX semipinacol groups onto fabric surface under UV irradiation,and surface initiation of "living" crosslinking polymerization to graft PEG molecular net-cloth on PPF and simultaneously in situ immobilize yeast cells under visible light.Characterizations were used to confirm that majority of yeast cells were entrapped at the interface of net-cloth and could proliferate well after a month of fermentation with high viabily.The effect of the concentration of monomer(poly(ethylene glycol)diacrylate,PEGDA),temperature,rotation rate,the concentration of glucose on the immobilization and fermentation had been investigated and 50%(v/v)PEGDA,20 g·L-1 glucose,30 °C and 200 rpm were the optimum conditions with the maximum bioethanol yield of 88.2%.The visible light used in the experiment showed more obvious advantage than UV-light.The repeated 24 h batch fermentation results showed that immobilized yeast cells could retain 80.7±0.4%to 95.5±6.3%of ethanol yield after 25 cycles,displaying an excellent operation stability.With the advance of immobilized films,the system could be used for fermenting higher concentration of glucose and lay the basis for industrial application.3.An efficient approach was used to co-immobilize ?-glucosidase and yeast cells layer-by-layer on the surface of PPF for bioethanol simultaneous saccharification and fermentation(SSF).First,ITX semipinacol groups were grafted on the PPF under UV light.Second,(3-glucosidase and PEGDA mixture can be initiated under visible light to immobilize P-glucosidase in PEG net-cloth on PPF via VSLGCP.Based on the living nature of VSLGCP,yeast cells were entrapped on upper layer by the same method to realize layered co-immobilization.Characterizations were used to vertify the excellent viability and the distribution of yeast cells and ?-glucosidase.The thickness of whole system was 100 ?m with 40?m thickness of ?-glucosidase.The immobilization rate of ?-glucosidase was 98.09%and the activity of immobilized enzymes was 25%of free enzymes.The dense PEG net-cloth acted as a barrier,which efficiently separated ?-glucosidase from yeast cells to avoid enzymes leakage.The yeast cells preserved the activity of metabolism and fermentation.The bioethanol yield was more than 60%after 7 repeated batches,which exhibited better operational stability.