, Volume 25, Issue 4, pp 2473–2485 | Cite as

Cellulase immobilized on mesoporous biochar synthesized by ionothermal carbonization of cellulose

  • Chang-hui Zhu
  • Zhen Fang
  • Tong-chao Su
  • Xing-kang Li
  • Qi-ying Liu
Original Paper


Cellulose-based biochar was prepared via ionothermal carbonization of cellulose in [Bmim]Cl with H2SO4and subsequent pyrolysis. The biochar was analyzed by a series of characterization methods, indicating that it was a kind of mesoporous carbon suitable for the adsorption of cellulase. Kinetic analysis showed that the immobilized cellulase exhibited higher affinity to carboxymethyl cellulose than free cellulase. The immobilized cellulase, at different pH and temperatures, was more stable than free cellulase. It was used to hydrolyze pretreated cellulose in [Bmim]Cl with a total reducing sugar (TRS) yield of 99.9%. The immobilized cellulase maintained activity of 74.8% after five cycles at an immobilized cellulase/cellulose weight ratio of 30:1. When the cellulose loading was increased by a factor of 5, the TRS yield decreased by only 27.5%.


Mesoporous biochar Ionothermal carbonization Immobilized cellulase Kinetics analysis Stability 



The authors wish to acknowledge the financial support from Nanjing Agricultural University (68Q-0603) and the National Science Foundation of China (51536009 and 51576199).


  1. Barrer R (1989) Clay minerals as selective and shape-selective sorbents. Pure Appl Chem 61:1903–1912CrossRefGoogle Scholar
  2. Chandra S, Chowdhury J, Ghosh M, Talapatra G (2011) Adsorption of 3-thiophene carboxylic acid on silver nanocolloids: FTIR, Raman, and SERS study aided by density functional theory. J Phys Chem C 115:14309–14324CrossRefGoogle Scholar
  3. Chen X, Dong S (2003) Sol-gel-derived titanium oxide/copolymer composite based glucose biosensor. Biosens Bioelectron 18:999–1004CrossRefGoogle Scholar
  4. Daoud FBO, Kaddour S, Sadoun T (2010) Adsorption of cellulase Aspergillus niger on a commercial activated carbon: kinetics and equilibrium studies. Colloids Surf. B 75:93–99CrossRefGoogle Scholar
  5. Dutta S, Wu KCW (2014) Enzymatic breakdown of biomass: enzyme active sites, immobilization, and biofuel production. Green Chem 16:4615–4626CrossRefGoogle Scholar
  6. Elzobair KA, Stromberger ME, Ippolito JA, Lentz RD (2016) Contrasting effects of biochar versus manure on soil microbial communities and enzyme activities in an Aridisol. Chemosphere 142:145–152CrossRefGoogle Scholar
  7. English BP, Wei M, Van Oijen AM, Lee KT, Luo G, Sun H, Cherayil BJ, Kou S, Xie XS (2006) Ever-fluctuating single enzyme molecules: Michaelis-Menten equation revisited. Nat Chem Biol 2:87CrossRefGoogle Scholar
  8. Fang Z (2015) How can we best solubilize lignocellulosic biomass for hydrolysis? Biofuel Bioprod Biorefin 9:621–622CrossRefGoogle Scholar
  9. Ghose T (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268CrossRefGoogle Scholar
  10. Hallett JP, Welton T (2011) Room-temperature ionic liquids: solvents for synthesis and catalysis. 2. Chem Rev 111:3508–3576CrossRefGoogle Scholar
  11. Heredia A, Fernández-Bolaños J, Guillén R (1990) Cellulase inhibition by polyphenols in olive fruits. Food Chem 38:69–73CrossRefGoogle Scholar
  12. Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315:804–807CrossRefGoogle Scholar
  13. Jabasingh SA, Nachiyar CV (2012) Immobilization of Aspergillus nidulans SU04 cellulase on modified activated carbon. J Therm Anal Calorim 109:193–202CrossRefGoogle Scholar
  14. Jia H, Zhu G, Wang P (2003) Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobility. Biotechnol Bioeng 84:406–414CrossRefGoogle Scholar
  15. Kumakura M (1997) Preparation of immobilized cellulase beads and their application to hydrolysis of cellulosic materials. Process Biochem 32:555–559CrossRefGoogle Scholar
  16. Lee SH, Doherty TV, Linhardt RJ, Dordick JS (2009) Ionic liquid-mediated selective extraction of lignin from wood leading to enhanced enzymatic cellulose hydrolysis. Biotechnol Bioeng 102:1368–1376CrossRefGoogle Scholar
  17. Lee JS, Mayes RT, Luo H, Dai S (2010) Ionothermal carbonization of sugars in a protic ionic liquid under ambient conditions. Carbon 48:3364–3368CrossRefGoogle Scholar
  18. Liu L, Chen H (2006) Enzymatic hydrolysis of cellulose materials treated with ionic liquid [Bmim] Cl. Chin Sci Bull 51:2432–2436CrossRefGoogle Scholar
  19. Liu WJ, Jiang H, Yu HQ (2015) Development of biochar-based functional materials: toward a sustainable platform carbon material. Chem Rev 115:12251–12285CrossRefGoogle Scholar
  20. Liu G, Xu Q, Dong X, Yang J, Pile LS, Geoff Wang G, Wang F (2016) Effect of protective gas and pyrolysis temperature on the biochar produced from three plants of Gramineae: physical and chemical characterization. Waste Biomass Valoriz. 7:1469–1480CrossRefGoogle Scholar
  21. Long J, Jiao A, Wei B, Wu Z, Zhang Y, Xu X, Jin Z (2014) A novel method for pullulanase immobilized onto magnetic chitosan/Fe3O4 composite nanoparticles by in situ preparation and evaluation of the enzyme stability. J Mol Catal B Enzym 109:53–61CrossRefGoogle Scholar
  22. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  23. Morris RE (2009) Ionothermal synthesis-ionic liquids as functional solvents in the preparation of crystalline materials. Chem Commun 21:2990–2998CrossRefGoogle Scholar
  24. Mubarak NM, Kundu A, Sahu JN, Abdullah EC, Jayakumar NS (2014a) Synthesis of palm oil empty fruit bunch magnetic pyrolytic char impregnating with FeCl3 by microwave heating technique. Biomass Bioenergy 61:265–275CrossRefGoogle Scholar
  25. Mubarak NM, Wong JR, Tan KW, Sahu JN, Abdullah E, Jayakumar N, Ganesan P (2014b) Immobilization of cellulase enzyme on functionalized multiwall carbon nanotubes. J Mol Catal B Enzym 107:124–131CrossRefGoogle Scholar
  26. Murmanis L, Highley TL, Palmer J (1987) Cytochemical localization of cellulases in decayed and nondecayed wood. Wood Sci Technol 21:101–109CrossRefGoogle Scholar
  27. Rui MFB, Dias AA (2004) Discrimination among eight modified Michaelis-Menten kinetics models of cellulose hydrolysis with a large range of substrate/enzyme ratios. Appl Biochem Biotech 112:173–184CrossRefGoogle Scholar
  28. Sankarraj N, Nallathambi G (2015) Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads. Biocatal Biotransform 33:81–88CrossRefGoogle Scholar
  29. Singh RK, Tiwari MK, Singh R, Lee JK (2013) From protein engineering to immobilization: promising strategies for the upgrade of industrial enzymes. Int J Mol Sci 14:1232CrossRefGoogle Scholar
  30. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2008) Determination of structural carbohydrates and lignin in biomass. Laboratory analytical procedure, Technical Report, NREL/TP-510-42618, 1617, 1-16, National Renewable Energy Laboratory (NREL), ColoradoGoogle Scholar
  31. Smith PK, Krohn RI, Hermanson G, Mallia A, Gartner F, Provenzano M, Fujimoto E, Goeke N, Olson B, Klenk D (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85CrossRefGoogle Scholar
  32. Sun X, Li Y (2004) Colloidal carbon spheres and their core/shell structures with noble-metal nanoparticles. Angew Chem Int Edit 43:597–601CrossRefGoogle Scholar
  33. Sun N, Rodríguez H, Rahman M, Rogers RD (2011) Where are ionic liquid strategies most suited in the pursuit of chemicals and energy from lignocellulosic biomass? Chem Commun 47:1405–1421CrossRefGoogle Scholar
  34. Sun Z, Tao M, Zhao Q, Guang H, Shi T, Wang X (2015) A highly active willow-derived sulfonated carbon material with macroporous structure for production of glucose. Cellulose 22:675–682CrossRefGoogle Scholar
  35. Thangalazhy-Gopakumar S, Al-Nadheri WMA, Jegarajan D, Sahu J, Mubarak N, Nizamuddin S (2015) Utilization of palm oil sludge through pyrolysis for bio-oil and bio-char production. Bioresource Technol 178:65–69CrossRefGoogle Scholar
  36. Thines KR, Abdullah EC, Mubarak NM (2017) Effect of process parameters for production of microporous magnetic biochar derived from agriculture waste biomass. Micropor Mesopor Mater 253:29–39CrossRefGoogle Scholar
  37. Tian XF, Fang Z, Jiang D, Sun XY (2011) Pretreatment of microcrystalline cellulose in organic electrolyte solutions for enzymatic hydrolysis. Biotechnol Biofuels 4:53. CrossRefGoogle Scholar
  38. Tian XF, Fang Z, Guo F (2012) Impact and prospective of fungal pre-treatment of lignocellulosic biomass for enzymatic hydrolysis. Biofuel Bioprod Biorefin 6:335–350CrossRefGoogle Scholar
  39. Tu M, Zhang X, Kurabi A, Gilkes N, Mabee W, Saddler J (2006) Immobilization of β-glucosidase on Eupergit C for lignocellulose hydrolysis. Biotechnol Lett 28:151–156CrossRefGoogle Scholar
  40. Van de Velden M, Baeyens J, Boukis I (2008) Modeling CFB biomass pyrolysis reactors. Biomass Bioenergy 32:128–139CrossRefGoogle Scholar
  41. Webb PA, Orr C (1997) Analytical methods in fine particle technology. Micromeritics Instrument Corp, Norcross, pp 30093–32901Google Scholar
  42. Weimer PJ, Weston WM (1985) Relationship between the fine structure of native cellulose and cellulose degradability by the cellulase complexes of Trichoderma reesei and Clostridium thermocellum. Biotechnol Bioeng 27:1540–1547CrossRefGoogle Scholar
  43. Xie ZL, White RJ, Weber J, Taubert A, Titirici MM (2011) Hierarchical porous carbonaceous materials via ionothermal carbonization of carbohydrates. J Mater Chem 21:7434–7442CrossRefGoogle Scholar
  44. Xu H, Pan W, Wang R, Zhang D, Liu C (2012) Understanding the mechanism of cellulose dissolution in 1-butyl-3-methylimidazolium chloride ionic liquid via quantum chemistry calculations and molecular dynamics simulations. J Comput Aided Mol Des 26:329–337CrossRefGoogle Scholar
  45. Zhang D, Zhang K, Yao YL, Xia XH, Chen HY (2004) Multilayer assembly of Prussian blue nanoclusters and enzyme-immobilized poly (toluidine blue) films and its application in glucose biosensor construction. Langmuir 20:7303–7307CrossRefGoogle Scholar
  46. Zhang P, Gong Y, Wei Z, Wang J, Zhang Z, Li H, Dai S, Wang Y (2014) Updating biomass into functional carbon material in ionothermal manner. ACS Appl Mater Interface 6:12515–12522CrossRefGoogle Scholar
  47. Zhao XS, Bao XY, Guo W, Fang YL (2006) Immobilizing catalysts on porous materials. Mater Today 9:32–39CrossRefGoogle Scholar
  48. Zhao H, Jones CL, Baker GA, Xia SQ, Olubajo O, Person VN (2009) Regenerating cellulose from ionic liquids for an accelerated enzymatic hydrolysis. J Biotechnol 139:47–54CrossRefGoogle Scholar
  49. Zhu CH, Guo F, Guo XQ, Li XK (2016) In situ saccharification of cellulose using a cellulase mixture and supplemental β-glucosidase in aqueous-ionic liquid media. BioResources 11:9068–9078Google Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Chinese Academy of Sciences, Guangzhou Institute of Energy ConversionGuangzhouChina
  2. 2.Biomass Group, College of EngineeringNanjing Agricultural UniversityNanjingChina
  3. 3.CAS Key Laboratory of Renewable EnergyGuangzhouChina
  4. 4.Chinese Academy of Sciences, Xishuangbanna Tropical Botanical GardenKunmingChina
  5. 5.University of Chinese Academy of SciencesBeijingChina

Personalised recommendations