Advertisement

Recovery of cellulase activity after ethanol stripping in a novel pilot-scale unit

  • Pernille Anastasia Skovgaard
  • Børge Holm Christensen
  • Claus Felby
  • Henning Jørgensen
Bioenergy/Biofuels/Biochemicals

Abstract

Recycling of enzymes has a potential interest during cellulosic bioethanol production as purchasing enzymes is one of the largest expenses in the process. By recycling enzymes after distillation, loss of sugars and ethanol are avoided, but depending on the distillation temperature, there is a potential risk of enzyme degradation. Studies of the rate of enzyme denaturation based on estimation of the denaturation constant K D was performed using a novel distillation setup allowing stripping of ethanol at 50–65 °C. Experiments were performed in a pilot-scale stripper, where the effect of temperature (55–65 °C) and exposure to gas–liquid and liquid–heat transmission interfaces were tested on a mesophilic and thermostable enzyme mixture in fiber beer and buffer. Lab-scale tests were included in addition to the pilot-scale experiments to study the effect of shear, ethanol concentration, and PEG on enzyme stability. When increasing the temperature (up to 65 °C) or ethanol content (up to 7.5 % w/v), the denaturation rate of the enzymes increased. Enzyme denaturation occurred slower when the experiments were performed in fiber beer compared to buffer only, which could be due to PEG or other stabilizing substances in fiber beer. However, at extreme conditions with high temperature (65 °C) and ethanol content (7.5 % w/v), PEG had no enzyme stabilizing effect. The novel distillation setup proved to be useful for maintaining enzyme activity during ethanol extraction.

Keywords

Thermostable cellulases Distillation Gas–liquid interfaces Enzyme recycling Ethanol Denaturation constant (KD

Notes

Acknowledgments

The fiber beer was a gift from Inbicon. The work was funded by the EU project HYPE, grant Agreement No. 213139, and the project Bio4Bio, which was financially supported by The Danish Council for Strategic Research.

References

  1. 1.
    Abdul-Fattah AM, Kalcinia DS, Pikal MI (2007) The challenge of drying method selection for protein pharmaceuticals: product quality implications. J Pharm Sci Us 96(8):1886–1916. doi: 10.1002/Jps.20842 CrossRefGoogle Scholar
  2. 2.
    Bailey MJ, Tahtiharju J (2003) Efficient cellulase production by Trichoderma reesei in continuous cultivation on lactose medium with a computer-controlled feeding strategy. Appl Microbiol Biotechnol 62(2–3):156–162. doi: 10.1007/s00253-003-1276-9 PubMedCrossRefGoogle Scholar
  3. 3.
    Börjesson J, Engqvist M, Sipos B, Tjerneld F (2007) Effect of poly(ethylene glycol) on enzymatic hydrolysis and adsorption of cellulase enzymes to pretreated lignocellulose. Enzym Microb Technol 41(1–2):186–195. doi: 10.1016/j.enzmictec.2007.01.003 CrossRefGoogle Scholar
  4. 4.
    Chylenski P, Felby C, Haven MØ, Gama M, Selig MJ (2012) Precipitation of T. reesei commercial cellulase preparations under standard enzymatic hydrolysis conditions for lignocelluloses. Biotechnol Lett 34(8):1475–1482. doi: 10.1007/s10529-012-0916-5 PubMedCrossRefGoogle Scholar
  5. 5.
    Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59(2):257–268Google Scholar
  6. 6.
    Gunjikar TP, Sawant SB, Joshi JB (2001) Shear deactivation of cellulase, exoglucanase, endoglucanase, and β-glucosidase in a mechanically agitated reactor. Biotechnol Prog 17(6):1166–1168. doi: 10.1021/Bp010114u PubMedCrossRefGoogle Scholar
  7. 7.
    Haki GD, Rakshit SK (2003) Developments in industrially important thermostable enzymes: a review. Biores Technol 89(1):17–34. doi: 10.1016/S0960-8524(03)00033-6 CrossRefGoogle Scholar
  8. 8.
    Hansen MAT, Jørgensen H, Laursen KH, Schjoerring JK, Felby C (2013) Structural and chemical analysis of process residue from biochemical conversion of wheat straw (Triticum aestivum L.) to ethanol. Biomass Bioenergy 56:572–581CrossRefGoogle Scholar
  9. 9.
    Haven MØ, Jørgensen H (2013) Adsorption of β-glucosidases in two commercial preparations onto pretreated biomass and lignin. Biotechnol Biofuels 6(1):165. doi: 10.1186/1754-6834-6-165 PubMedCrossRefGoogle Scholar
  10. 10.
    Holtzapple M, Cognata M, Shu Y, Hendrickson C (1990) Inhibition of T. reesei cellulase by sugars and solvents. Biotechnol Bioeng 36(3):275–287PubMedCrossRefGoogle Scholar
  11. 11.
    Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VG (2012) Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 5(1):45. doi: 10.1186/1754-6834-5-45 PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Jensen E (2011) Rectification apparatus using a heat pump. US Patent No. US7972423 B2Google Scholar
  13. 13.
    Jørgensen H, Vibe-Pedersen J, Larsen J, Felby C (2007) Liquefaction of lignocellulose at high-solids concentrations. Biotechnol Bioeng 96(5):862–870. doi: 10.1002/Bit.21115 PubMedCrossRefGoogle Scholar
  14. 14.
    Kim MH, Lee SB, Ryu DDY, Reese ET (1982) Surface deactivation of cellulase and its prevention. Enzym Microb Technol 4(2):99–103. doi: 10.1016/0141-0229(82)90090-4 CrossRefGoogle Scholar
  15. 15.
    Kristensen JB, Börjesson J, Bruun MH, Tjerneld F, Jørgensen H (2007) Use of surface active additives in enzymatic hydrolysis of wheat straw lignocellulose. Enzym Microb Technol 40(4):888–895. doi: 10.1016/j.enzmictec.2006.07.014 CrossRefGoogle Scholar
  16. 16.
    Kristensen JB, Felby C, Jørgensen H (2009) Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol Biofuels. doi: 10.1186/1754-6834-2-11 PubMedCentralPubMedGoogle Scholar
  17. 17.
    Larsen J, Petersen MØ, Thirup L, Li HW, Iversen FK (2008) The IBUS process: lignocellulosic bioethanol close to a commercial reality. Chem Eng Technol 31(5):765–772. doi: 10.1002/ceat.200800048 CrossRefGoogle Scholar
  18. 18.
    Li J, Li S, Fan C, Yan Z (2012) The mechanism of poly(ethylene glycol) 4,000 effect on enzymatic hydrolysis of lignocellulose. Coll Surf B Biointerf 89:203–210. doi: 10.1016/j.colsurfb.2011.09.019 CrossRefGoogle Scholar
  19. 19.
    Madsen P (2003) Ethanol distillation: the fundamentals. In: Jacques KA (ed) The alcohol textbook, 3rd edn. Notthingham University Press, UK, pp 319–336Google Scholar
  20. 20.
    Merino ST, Cherry J (2007) Progress and challenges in enzyme development for biomass utilization. Biofuels 108:95–120. doi: 10.1007/10_2007_066 CrossRefGoogle Scholar
  21. 21.
    Millqvist-Fureby A, Malmsten M, Bergenstahl B (1999) Spray-drying of trypsin: surface characterisation and activity preservation. Int J Pharm 188(2):243–253. doi: 10.1016/S0378-5173(99)00226-4 PubMedCrossRefGoogle Scholar
  22. 22.
    Rahikainen J, Mikander S, Marjamaa K, Tamminen T, Lappas A, Viikari L, Kruus K (2011) Inhibition of enzymatic hydrolysis by residual lignins from softwood—study of enzyme binding and inactivation on lignin-rich surface. Biotechnol Bioeng 108(12):2823–2834. doi: 10.1002/bit.23242 PubMedCrossRefGoogle Scholar
  23. 23.
    Sellami-Kamoun A, Haddar A, Ali NEH, Ghorbel-Frikha B, Kanoun S, Nasri M (2008) Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations. Microbiol Res 163(3):299–306. doi: 10.1016/j.micres.2006.06.001 PubMedCrossRefGoogle Scholar
  24. 24.
    Sipos B, Szilagyi M, Sebestyen Z, Perazzini R, Dienes D, Jakab E, Crestini C, Reczey K (2011) Mechanism of the positive effect of poly(ethylene glycol) addition in enzymatic hydrolysis of steam pretreated lignocelluloses. C R Biol 334(11):812–823. doi: 10.1016/j.crvi.2011.06.005 PubMedCrossRefGoogle Scholar
  25. 25.
    Skovgaard PA, Jørgensen H (2013) Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes. J Ind Microbiol Biotechnol 40(5):447–456. doi: 10.1007/s10295-013-1248-8 PubMedCrossRefGoogle Scholar
  26. 26.
    Szczodrak J, Targonski Z (1989) Simultaneous saccharification and fermentation of cellulose: effect of ethanol and cellulases on particular stages. Acta Biotechnol 9(6):555–564CrossRefGoogle Scholar
  27. 27.
    Thomas CR, Geer D (2011) Effects of shear on proteins in solution. Biotechnol Lett 33(3):443–456. doi: 10.1007/s10529-010-0469-4 PubMedCrossRefGoogle Scholar
  28. 28.
    Tu MB, Chandra RP, Saddler JN (2007) Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates. Biotechnol Progr 23(2):398–406. doi: 10.1021/Bp060354f CrossRefGoogle Scholar
  29. 29.
    Vane LM (2008) Separation technologies for the recovery and dehydration of alcohols from fermentation broths. Biofuel Bioprod Bior 2(6):553–588. doi: 10.1002/Bbb.108 CrossRefGoogle Scholar
  30. 30.
    Varnai A, Siika-Aho M, Viikari L (2010) Restriction of the enzymatic hydrolysis of steam-pretreated spruce by lignin and hemicellulose. Enzym Microb Technol 46(3–4):185–193. doi: 10.1016/j.enzmictec.2009.12.013 CrossRefGoogle Scholar
  31. 31.
    Viikari L, Alapuranen M, Puranen T, Vehmaanpera J, Siika-Aho M (2007) Thermostable enzymes in lignocellulose hydrolysis. Biofuels 108:121–145. doi: 10.1007/10_2007_065 CrossRefGoogle Scholar
  32. 32.
    Weiss N, Börjesson J, Pedersen LS, Meyer AS (2013) Enzymatic lignocellulose hydrolysis: improved cellulase productivity by insoluble solids recycling. Biotechnol Biofuels. doi: 10.1186/1754-6834-6-5 PubMedCentralPubMedGoogle Scholar
  33. 33.
    Xiao ZZ, Storms R, Tsang A (2004) Microplate-based filter paper assay to measure total cellulase activity. Biotechnol Bioeng 88(7):832–837. doi: 10.1002/Bit.20286 PubMedCrossRefGoogle Scholar
  34. 34.
    Zhang J, Viikari L (2012) Xylo-oligosaccharides are competitive inhibitors of cellobiohydrolase I from Thermoascus aurantiacus. Biores Technol 117:286–291. doi: 10.1016/j.biortech.2012.04.072 CrossRefGoogle Scholar
  35. 35.
    Zhang JH, Tuomainen P, Siika-aho M, Viikari L (2011) Comparison of the synergistic action of two thermostable xylanases from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis. Biores Technol 102(19):9090–9095. doi: 10.1016/j.biortech.2011.06.085 CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2014

Authors and Affiliations

  • Pernille Anastasia Skovgaard
    • 1
  • Børge Holm Christensen
    • 2
  • Claus Felby
    • 1
  • Henning Jørgensen
    • 1
    • 3
  1. 1.Department of Geosciences and Nature Resource Management, Faculty of ScienceUniversity of CopenhagenFrederiksberg CDenmark
  2. 2.Holm Christensen Biosystemer ApsÅlsgårdeDenmark
  3. 3.Department of Chemical and Biochemical Engineering, Center for Bioprocess EngineeringTechnical University of DenmarkLyngbyDenmark

Personalised recommendations