, Volume 24, Issue 12, pp 5541–5550 | Cite as

Preparation and characterization of magnetic Fe3O4–chitosan nanoparticles for cellulase immobilization

  • Yan Lin
  • Xi Liu
  • Zhaohui Xing
  • Yong Geng
  • Jeffrey Wilson
  • Deyi Wu
  • Hainan KongEmail author
Original Paper


Cellulase immobilization is an important issue for cellulose hydrolysis in bioethanol production. In this study, Fe3O4 nanoparticles were synthesized using a coprecipitation method and were coated with chitosan. Glutaraldehyde was used as cross-linking reagent for cellulase immobilization. The morphology, structure, and related physical and chemical properties of the supports were studied by X-ray diffraction, a physical property measurement system, Fourier transform infrared spectroscopy, and Transmission electron microscopy. Response surface methodology was used to determine the optimal cellulase loading efficiency (LE) and standard recovery ratio (RR). The verification tests show the immobilized particles had an LE of 99.6% and an RR value of 68.5%. It was found that immobilized cellulase had a wider range of adaptability to hydrolysis pH and temperature when compared with free cellulase. Five hydrolysis experiments demonstrated effective hydrolysis using immobilized cellulase while maintaining on average 80% of the free cellulase’s activity.


Magnetic nanoparticles Immobilization Cellulase Performance optimization 



The research project was sponsored by the Major Science and Technology Program for Water Pollution Control and Treatment (2009ZX07101-015-003). The authors would also like to thank Tyler Barzee at University of California, Davis for his invaluable support.


  1. Alftren J, Hobley TJ (2014) Immobilization of cellulase mixtures on magnetic particles for hydrolysis of lignocellulose and ease of recycling. Biomass Bioenergy 65:72–78. doi: 10.1016/j.biombioe.2014.03.009 CrossRefGoogle Scholar
  2. Ansari SA, Husain Q (2012) Potential applications of enzymes immobilized on/in nano materials: a review. Biotechnol Adv 30:512–523. doi: 10.1016/j.biotechadv.2011.09.005 CrossRefPubMedGoogle Scholar
  3. Chen G, Song W, Qi B, Lu J, Wan Y (2013) Recycling cellulase from enzymatic hydrolyzate of acid treated wheat straw by electroultrafiltration. Bioresour Technol 144(5):186–193. doi: 10.1016/j.biortech.2013.06.089 CrossRefPubMedGoogle Scholar
  4. Cipolatti EP, Silva MJA, Klein M, Feddern V, Feltes MMC, Oliveira JV, Ninowa JL, de Oliveira D (2014) Current status and trends in enzymatic nanoimmobilization. J Mol Catal B Enzym 99:56–67. doi: 10.1016/j.molcatb.2013.10.019 CrossRefGoogle Scholar
  5. Cuevas M, Sánchez S, García JF, Baeza J, Parra C, Freer J (2015) Enhanced ethanol production by simultaneous saccharification and fermentation of pretreated olive stones. Renew Energy 74:839–847. doi: 10.1016/j.renene.2014.09.004 CrossRefGoogle Scholar
  6. Datta S, Christena LR, Rajaram YRS (2013) Enzyme immobilization: an overview on techniques and support materials. 3. Biotech 3:1–9. doi: 10.1007/s13205-012-0071-7 CrossRefGoogle Scholar
  7. Dinçer A, Telefoncu A (2007) Improving the stability of cellulase by immobilization on modified polyvinyl alcohol coated chitosan beads. J Mol Catal B Enzym 45:10–14. doi: 10.1016/j.molcatb.2006.10.005 CrossRefGoogle Scholar
  8. Egüés I, Sanchez C, Mondragon I, Labidi J (2012) Effect of alkaline and autohydrolysis processes on the purity of obtained hemicelluloses from corn stalks. Bioresour Technol 103(1):239–248. doi: 10.1016/j.biortech.2011.09.139 CrossRefPubMedGoogle Scholar
  9. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268. doi: 10.1351/pac198759020257 CrossRefGoogle Scholar
  10. Godjevargova T, Gabrovska K (2006) Influence of matrix on external mass transfer resistance in immobilized urease membranes. Enzyme Microb Technol 38:338–342. doi: 10.1016/j.enzmictec.2004.10.011 CrossRefGoogle Scholar
  11. Guo M, Song W, Buhain J (2015) Bioenergy and biofuels: history, status, and perspective. Renew Sustain Energy Rev 42:712–725. doi: 10.1016/j.rser.2014.10.013 CrossRefGoogle Scholar
  12. Haven MØ, Lindedam J, Jeppesen MD, Elleskov M, Rodrigues AC, Gama M, Jørgensenb H, Felbyb C (2015) Continuous recycling of enzymes during production of lignocellulosic bioethanol in demonstration scale. Appl Energy 159:188–195. doi: 10.1016/j.apenergy.2015.08.062 CrossRefGoogle Scholar
  13. Hong G, Zou S, Liu B, Su R, Huang R, Wei Q, Zhang MH, He ZM (2015a) Reducing β-glucosidase supplementation during cellulase recovery using engineered strain for successive lignocellulose bioconversion. Bioresour Technol 187:362–368. doi: 10.1016/j.biortech.2015.03.105 CrossRefGoogle Scholar
  14. Hong J, Zhou J, Hong J (2015b) Comparative study of life cycle environmental and economic impact of corn- and corn stalk-based-ethanol production. J Renew Sustain Energy 7(023106):1–16. doi: 10.1063/1.4914008 CrossRefGoogle Scholar
  15. Igarashi K, Uchihashi T, Koivula A, Wada M, Kimura S, Okamoto T, Penttilä M, Ando T, Samejima M (2011) Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface. Science 333:1279–1282. doi: 10.1126/science.1208386 CrossRefPubMedGoogle Scholar
  16. Jiang J, Zhao J, He C, Cui B, Xiong J, Jiang H, Ao J, Xiang G (2017) Recyclable magnetic carboxymethyl chitosan/calcium alginate–cellulase bioconjugates for corn stalk hydrolysis. Carbohyd Polym 166:358–364. doi: 10.1016/j.carbpol.2017.03.003 CrossRefGoogle Scholar
  17. Kazi FK, Fortman JA, Anex RP, Hsu DD, Aden A, Dutta A, Kothandaramanc G (2010) Techno-economic comparison of process technologies for biochemical ethanol production from corn stover. Fuel 89:20–28. doi: 10.1016/j.fuel.2010.01.001 CrossRefGoogle Scholar
  18. Khan MJ, Husain Q, Azam A (2012) Immobilization of porcine pancreatic α-amylase on magnetic Fe2O3 nanoparticles: applications to the hydrolysis of starch. Biotechnol Bioprocess 17:377–384. doi: 10.1007/s12257-011-0105-8 CrossRefGoogle Scholar
  19. Kim DK, Zhang Y, Voit W, Rao KV, Muhammed M (2001) Synthesis and characterization of surfactant-coated superparamagnetic monodispersed iron oxide nanoparticles. J Magn Magn Mater 225:30–36. doi: 10.1016/s0304-8853(00)01224-5 CrossRefGoogle Scholar
  20. Kim IJ, Lee HJ, Choi IG, Kim KH (2014) Synergistic proteins for the enhanced enzymatic hydrolysis of cellulose by cellulase. Appl Microbiol Biotechnol 98(20):8469–8480. doi: 10.1007/s00253-014-6001-3 CrossRefPubMedGoogle Scholar
  21. Kim Y, Kreke T, Ko JK, Ladisch MR (2015) Hydrolysis-determining substrate characteristics in liquid hot water pretreated hardwood. Biotechnol Bioeng 112(4):677. doi: 10.1002/bit.25465 CrossRefPubMedGoogle Scholar
  22. Kneller EF, Luborsky FE (1963) Particle size dependence of coercivity and remanence of single-domain particles. J Appl Phys 34:656–658. doi: 10.1063/1.1729324 CrossRefGoogle Scholar
  23. Li B, Jia D, Zhou Y, Hu QL, Cai W (2006) In situ hybridization to chitosan/magnetite nanocomposite induced by the magnetic field. J Magn Magn Mater 306:223–227. doi: 10.1016/jjmmm.2006.01.250 CrossRefGoogle Scholar
  24. Liu J, Cao X (2014) Biodegradation of cellulose by β-glucosidase and cellulase immobilized on a pH-responsive copolymer. Biotechnol Bioprocess 19(5):829–837. doi: 10.1007/s12257-013-0716-3 CrossRefGoogle Scholar
  25. Lou HM, Li XL, Wang MX, Qiu XQ, Yang DJ, Deng YH (2013) Effect of pH value on cellulase adsorption on wheat straw alkaline lignin. J Central South Univ Technol 41(12):1–5. doi: 10.3969/j.issn.1000-565X.2013.12.001 CrossRefGoogle Scholar
  26. Mao X, Guo G, Huang J, Du Z, Huang Z, Ma L, Li P, Gu L (2006) A novel method to prepare chitosan powder and its application in cellulase immobilization. J Chem Technol Biotechnol 81:189–195. doi: 10.1002/jctb.1378 CrossRefGoogle Scholar
  27. Rahikainen JL, Martinsampedro R, Heikkinen H, Rovio S, Marjamaa K, Tamminen T, Rojas OJ, Kruus K (2013) Inhibitory effect of lignin during cellulose bioconversion: the effect of lignin chemistry on non-productive enzyme adsorption. Bioresour Technol 133(2):270–278. doi: 10.1016/j.biortech.2013.01.075 CrossRefPubMedGoogle Scholar
  28. Sánchez-Ramírez J, Martínez-Hernández JL, Segura- Ceniceros P, López G, Saade H, Medina-Morales MA, Ramos-González R, Aguilar CN, Ilyina A (2016) Cellulases immobilization on chitosan-coated magnetic nanoparticles: application for Agave atrovirens lignocellulosic biomass hydrolysis. Bioprocess Biosyst Eng 40:9–22. doi: 10.1007/s00449-016-1670-1 CrossRefPubMedGoogle Scholar
  29. Si S, Li C, Wang X, Yu D, Peng Q, Li Y (2005) Magnetic monodisperse Fe3O4 nanoparticles. Cryst Growth Des 5:391–393. doi: 10.1021/cg0497905 CrossRefGoogle Scholar
  30. Talbert JN, Goddard JM (2012) Enzymes on material surfaces. Colloid Surf B 93:8–19. doi: 10.1016/j.colsurfb.2012.01.003 CrossRefGoogle Scholar
  31. Taqieddin E, Amiji M (2004) Enzyme immobilization in novel alginate–chitosan core–shell microcapsules. Biomaterials 25:1937–1945. doi: 10.1016/j.biombioe.2014.03.009 CrossRefPubMedGoogle Scholar
  32. Trevorah RM, Othman MZ (2015) Alkali pretreatment and enzymatic hydrolysis of Australian timber mill sawdust for biofuel production. J Renew Energy 4:126–130. doi: 10.1155/2015/284250 CrossRefGoogle Scholar
  33. Ungurean M, Paul C, Peter F (2013) Cellulase immobilized by sol-gel entrapment for efficient hydrolysis of cellulose. Bioprocess Biosyst Eng 36:1327–1338. doi: 10.1007/s00449-012-0835-9 CrossRefPubMedGoogle Scholar
  34. Zang L, Qiu JH, Wu XL, Zhang WJ, Sakai E, Wei Y (2014) Preparation of magnetic chitosan nanoparticles as support for cellulase immobilization. Ind Eng Chem Res 53(9):3448–3454. doi: 10.1021/ie404072s CrossRefGoogle Scholar
  35. Zhang W, Lin Y, Zhang Q, Wang X, Wu D, Kong H (2013) Optimisation of simultaneous saccharification and fermentation of NaOH-pretreated wheat straw for ethanol production. Fuel 112:331–337. doi: 10.1016/j.fuel.2013.05.064 CrossRefGoogle Scholar
  36. Zhang Q, Lin Y, Shen S, Xing Z, Ruan X (2015a) Simulation and optimization on cellulase immobilization using response surface methodology. Int J Environ Sci Dev 6:664–667. doi: 10.7763/ijesd.2015.v6.677 CrossRefGoogle Scholar
  37. Zhang W, Qiu J, Zang L, Sakai E, Feng H (2015b) Preparation of functionalized magnetic silica nanospheres for the cellulase immobilization. Nano 10:3–9. doi: 10.1142/s1793292015500137 CrossRefGoogle Scholar
  38. Zhang J, Song Y, Wang B, Zhang X, Tan T (2016) Biomass to bio-ethanol: the evaluation of hybrid Pennisetum used as raw material for bio-ethanol production compared with corn stalk by steam explosion joint use of mild chemicals. Renew Energy 88:164–170. doi: 10.1016/j.renene.2015.11.034 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Yan Lin
    • 1
  • Xi Liu
    • 1
  • Zhaohui Xing
    • 1
  • Yong Geng
    • 1
  • Jeffrey Wilson
    • 1
  • Deyi Wu
    • 1
  • Hainan Kong
    • 1
    Email author
  1. 1.School of Environmental Science and EngineeringShanghai Jiao Tong UniversityShanghaiChina

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