, Volume 21, Issue 1, pp 407–416 | Cite as

Utilization of a biphasic oil/aqueous cellulose nanofiber membrane bioreactor with immobilized lipase for continuous hydrolysis of olive oil

  • Peng-Cheng Chen
  • Xiao-Jun Huang
  • Zhi-Kang Xu
Original Paper


A simple method is proposed to fabricate a biphasic lipase-immobilized cellulose membrane bioreactor with high enzyme loading and activity retention. This bioreactor was assembled with electrospun cellulose nanofiber membranes that were fixed in a spiral form and wound to increase their specific surface area. To improve the catalytic efficiency of the immobilized enzymes, the supports went through alkaline hydrolysis, NaIO4 oxidation and pentaethylenehexamine modification before covalently binding the lipase. Enzyme loading could reach 28.9 mg/g with the highest activity retention of 44.3 % for the immobilized lipases. The effects of the operational variables, namely the organic phase flow rate, aqueous phase flow rate and substrate concentration, on the performance of this bioreactor were investigated with continuous hydrolysis of olive oil. It was found that under optimum operational conditions, 100 % hydrolysis conversion of olive oil was achieved after 9 organic phase circulations at 10.5 mL/min organic phase flow rate, 600 mL/min aqueous phase flow rate and using a substrate of pure olive oil. Nanofiber membrane bioreactors offer potential as applications for various lipase-catalyzing reactions in industrial productions.


Cellulose Nanofiber membrane Enzyme immobilization Lipase Biphasic enzyme-immobilized membrane bioreactor 



The authors are grateful to the financial support from the Fundamental Research Funds for the Central Universities (Grant no. 2013QNA4090), the National Natural Science Foundation of China (Grant no. 21274126), and the National “Twelfth Five-Year” Plan for Science & Technology Support of China (Grant no. 2012BAI08B01).

Supplementary material

10570_2013_148_MOESM1_ESM.doc (109 kb)
Supplementary material 1 (DOC 109 kb)


  1. Agarwal S, Greiner A, Wendroff JH (2009) Electrospinning of manmade and biopolymer nanofibers-progress in techniques, materials, and applications. Adv Funct Mater 19:7198–7201CrossRefGoogle Scholar
  2. Bhardwaj N, Kundu SC (2010) Electrospinning: a fascinating fiber fabrication technique. Biotechnol Adv 28:1315–1338CrossRefGoogle Scholar
  3. Bradford M (1976) A rapid and sensitive method for the quantition of microgram quantities of protein utilizing the principle of dyebinding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  4. Brzozowski AM, Derewenda U, Derewenda ZS, Dodson GG, Lawson DM, Turkenburg JP, Bjorkling F, Huge-Jensen B, Patkar SA, Thim L (1991) A model for interfacial activation in lipases from the structure of a fungal lipase inhibitor complex. Nature 351:491–494CrossRefGoogle Scholar
  5. Chakraborty S, Drioli E, Giorno L (2012) Development of a two separate phase submerged biocatalytic membrane reactor for the production of fatty acids and glycerol from residual vegetable oil streams. Biomass Bioenergy 46:574–583CrossRefGoogle Scholar
  6. Chen PC, Huang XJ, Huang F, Ou Y, Chen MR, Xu ZK (2011) Immobilization of lipase onto cellulose nanofiber membrane for oil hydrolysis in high performance bioreactor. Cellulose 18:1563–1571CrossRefGoogle Scholar
  7. Crespy D, Friedemann K, Popa AM (2012) Colloid-electrospinning: fabrication of multicompartment nanofibers by the electrospinning of organic or/and inorganic dispersions and emulsions. Macromol Rapid Commun 33:1978–1995CrossRefGoogle Scholar
  8. Cunha AG, Gandini A (2010) Turning polysaccharides into hydrophobic materials: a critical review. Part 1. Cellulose. Cellulose 17:875–889CrossRefGoogle Scholar
  9. Deng HT, Xu ZK, Dai ZW, Wu J, Seta P (2005) Immobilization of Candida rugosa lipase on polypropylene microfiltration membrane modified by glycopolymer: hydrolysis of olive oil in biphasic bioreactor. Enzyme Microb Technol 36:996–1002CrossRefGoogle Scholar
  10. Deng M, Kumbar SG, Nair LS, Weikel AL, Allcock HR, Laurencin CT (2011) Biomimetic structures: biological implications of dipeptide-substituted polyphosphazene: polyester blend nanofiber matrices for load-bearing bone regeneration. Adv Funct Mater 21:2641–2651CrossRefGoogle Scholar
  11. Ferrario V, Ebert C, Knapic L, Fattor D, Basso A, Spizzo P, Gardossi L (2011) Conformational changes of lipases in aqueous media: a comparative computational study and experimental implication. Adv Synth Catal 353:2466–2480CrossRefGoogle Scholar
  12. Huang XJ, Yu AG, Ge D, Xu ZK (2008a) Immobilization and properties of lipase from Candida rugosa on electrospun nanofibrous membranes with biomimetic phospholipid moities. Chem Res Chinese U 24:231–237CrossRefGoogle Scholar
  13. Huang XJ, Yu AG, Xu ZK (2008b) Covalent immobilization of lipase from Candida rugosa onto ploy(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun fibrous membranes for potential bioreactor application. Bioresour Technol 99:5459–5465CrossRefGoogle Scholar
  14. Huang XJ, Chen PC, Huang Fu, Ou Y, Chen MR, Xu ZK (2011) Immobilization of Candida rugosa lipase on electrospun cellulose nanofiber membrane. J Mol Catal B 70:95–100CrossRefGoogle Scholar
  15. Jiao TF, Leca-Bouvier BD, Boullanger P, Blum LJ, Girard-Egrot AP (2010) A chemiluminescent langmuir-blodgett membrane as the sensing layer for the reagentless monitoring of an immobilized enzyme activity. Colloids Surf A 254:284–290CrossRefGoogle Scholar
  16. Keng PS, Basri M, Ariff AB, Rahman MBA, Rahman RNZA, Salleh AB (2008) Scale-up synthesis of lipase-catalyzed palm esters in stirred-tank reactor. Bioresour Technol 99:6097–6104CrossRefGoogle Scholar
  17. Kosaka PM, Kawano Y, El Seoud OA, Petri DFS (2007) Catalytic activity of lipase immobilized onto ultrathin films of cellulose esters. Langmuir 23:12167–12173CrossRefGoogle Scholar
  18. Lee SY, Lee J, Chang JH, Lee JH (2011) Inorganic nanomaterial-based biocatalysts. BMB Rep 44:77–86CrossRefGoogle Scholar
  19. Liu HQ, Hsieh YL (2002) Ultrafine fibrous cellulose membranes from electrospinning of cellulose acetate. J Polym Sci, Part B: Polym Phys 40:2119–2129CrossRefGoogle Scholar
  20. Liu HQ, Hsieh YL (2003) Surface methacrylation and graft copolymerization of ultrafine cellulose fibers. J Polym Sci, Part B: Polym Phys 41:953–964CrossRefGoogle Scholar
  21. Lu P, Hsieh YL (2010) Layer-by-layer self-assembly of Cibacron Blue F3GA and lipase on ultra-fine cellulose fibrous membrane. J Membr Sci 348:21–27CrossRefGoogle Scholar
  22. Monclus H, Zacharias S, Santos A, Pidou M, Judd S (2010) Criticality of flux and aeration for a hollow fiber membrane bioreactor. Sep Sci Technol 45:956–961CrossRefGoogle Scholar
  23. Pugazhemthi G, Kumar A (2004) Enzyme membrane reactor for hydrolysis of olive oil using lipase immobilized on modified PMMA composite membrane. J Membr Sci 228:187–197CrossRefGoogle Scholar
  24. Tang C, Ozcam AE, Stout B, Khan SA (2012) Effect of pH on protein distribution in electrospun PVA/BSA composite nanofibers. Biomacromolecules 13:1269–1278CrossRefGoogle Scholar
  25. Verger R (1997) Interfacial activation of lipase: facts and artifacts. Trends Biotechnol 15:32–38CrossRefGoogle Scholar
  26. Wait AF, Parkin A, Morley GM, dos Santos L, Armstrong FA (2010) Characteristics of enzyme-based hydrogen fuel cells using an oxygen-tolerant hydrogenase as the anodic catalyst. J Phys Chem C 114:12003–12009CrossRefGoogle Scholar
  27. Wang CS, Hartsuck JA (1993) Bile salt-activated lipase: a multiple function lipolytic enzyme. Biochim Biophys Acta 1166:1–19CrossRefGoogle Scholar
  28. Wang CS, Lee M (1985) Kinetic properties of human milk bile salt-activated lipases: studies using long chain triacylglycerol as substrate. J Lipid Res 26:824–830Google Scholar
  29. Wedberg R, Abildskov J, Peters GH (2012) Protein dynamics in organic media at varying water activity studied by molecular dynamics simulation. J Phys Chem B 116:2575–2585CrossRefGoogle Scholar
  30. Xie H, Wang ZR, Kong WJ, Wang L, Fu ZF (2013) A novel enzyme-immobilized flow cell used as end-column chemiluminescent detection interface in open-tubular capillary electrochromatography. Analyst 138:1107–1113CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and EngineeringZhejiang UniversityHangzhouChina

Personalised recommendations