Applied Biochemistry and Biotechnology

, Volume 164, Issue 7, pp 1058–1070

Onsite Enzyme Production During Bioethanol Production from Biomass: Screening for Suitable Fungal Strains

  • Annette Sørensen
  • Philip J. Teller
  • Peter S. Lübeck
  • Birgitte K. Ahring


Cellulosic ethanol production from biomass raw materials involves process steps such as pre-treatment, enzymatic hydrolysis, fermentation, and distillation. Use of streams within cellulosic ethanol production was explored for onsite enzyme production as part of a biorefinery concept. Sixty-four fungal isolates were in plate assays screened for lignocellulolytic activities to discover the most suitable fungal strain with efficient hydrolytic enzymes for lignocellulose conversion. Twenty-five were selected for further enzyme activity studies using a stream derived from the bioethanol process. The filter cake left after hydrolysis and fermentation was chosen as substrate for enzyme production. Five of the 25 isolates were further selected for synergy studies with commercial enzymes, Celluclast 1.5L and Novozym 188. Finally, IBT25747 (Aspergillus niger) and strain AP (CBS 127449, Aspergillus saccharolyticus) were found as promising candidates for onsite enzyme production where the filter cake was inoculated with the respective fungus and in combination with Celluclast 1.5L used for hydrolysis of pre-treated biomass.


Onsite enzyme production Fungal screening Beta-glucosidase Cellulase Bioethanol 


  1. 1.
    NREL National Renewable Energy Laboratory. Biomass Research - Biochemical Conversion Projects 2009. Available at: Updated October 2009.
  2. 2.
    Knauf, M., & Moniruzzaman, M. (2004). Lignocellulosic biomass processing: a perspective. International Sugar Journal, 106, 147–150.Google Scholar
  3. 3.
    Ahring, B. K., & Westermann, P. (2007). Coproduction of bioethanol with other biofuels. Biofuels, 108, 289–302.CrossRefGoogle Scholar
  4. 4.
    Pandey, A., Selvakumar, P., Soccol, C. R., & Nigam, P. (1999). Solid state fermentation for the production of industrial enzymes. Current Science, 77, 149–162.Google Scholar
  5. 5.
    Pandey, A. (2003). Solid-state fermentation. Biochemical Engineering Journal, 13, 81–84.CrossRefGoogle Scholar
  6. 6.
    Mansfield, S. D., Mooney, C., & Saddler, J. N. (1999). Substrate and enzyme characteristics that limit cellulose hydrolysis. Biotechnology Progress, 15, 804–816.CrossRefGoogle Scholar
  7. 7.
    Zhang, Y.-P., Himmel, M. E., & Mielenz, J. R. (2006). Outlook for cellulase improvement: screening and selection strategies. Biotechnology Advances, 24, 452–481.CrossRefGoogle Scholar
  8. 8.
    Mathew, G. M., Sukumaran, R. K., Singhania, R. R., & Pandey, A. (2008). Progress in research on fungal cellulases for lignocellulose degradation. Journal of Scientific and Industrial Research, 67, 898–907.Google Scholar
  9. 9.
    NREL National Renewable Energy Laboratory. Biomass research - Standard Biomass Analytical procedures 2010. Available at: Updated September 2010.
  10. 10.
    Samson, R. A., Hoekstra, E. S., & Frisvad, J. C. (2004). Introduction to Food- and Airborne Fungi. Centralbureau voor Schimmelcultures (7th ed.). Utrecht, Netherlands: American Society Microbiology.Google Scholar
  11. 11.
    Wood, T. M., & Bhat, K. M. (1988). Methods for Measuring Cellulase Activities. Methods in Enzymology, 160, 87–112.CrossRefGoogle Scholar
  12. 12.
    Flachner, B., Brumbauer, A., & Reczey, K. (1999). Stabilization of beta-glucosidase in Aspergillus phoenicis QM 329 pellets. Enzyme and Microbial Technology, 24, 362–367.CrossRefGoogle Scholar
  13. 13.
    Ghose, T. K. (1987). Measurement of Cellulase Activities. Pure and Applied Chemistry, 59, 257–268.CrossRefGoogle Scholar
  14. 14.
    Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428.CrossRefGoogle Scholar
  15. 15.
    Berlin, A., Gilkes, N., Kurabi, A., Bura, R., Tu, M. B., Kilburn, D., et al. (2005). Weak lignin-binding enzymes—a novel approach to improve activity of cellulases for hydrolysis of lignocellulosics. Applied Biochemistry and Biotechnology, 121, 163–170.CrossRefGoogle Scholar
  16. 16.
    Jorgensen, H., Vibe-Pedersen, J., Larsen, J., & Felby, C. (2007). Liquefaction of lignocellulose at high-solids concentrations. Biotechnology and Bioengineering, 96, 862–870.CrossRefGoogle Scholar
  17. 17.
    Berlin, A., Balakshin, M., Gilkes, N., Kadla, J., Maximenko, V., Kubo, S., et al. (2006). Inhibition of cellulase, xylanase and beta-glucosidase activities by softwood lignin preparations. Journal of Biotechnology, 125, 198–209.CrossRefGoogle Scholar
  18. 18.
    Klinke, H. B., Thomsen, A. B., & Ahring, B. K. (2004). Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Applied Microbiology and Biotechnology, 66, 10–26.CrossRefGoogle Scholar
  19. 19.
    Pedersen, M., Hollensted, M., Lange, L., & Andersen, B. (2009). Screening for cellulose and hemicellulose degrading enzymes from the fungal genus Ulocladium. International Biodeterioration & Biodegradation, 63, 484–489.CrossRefGoogle Scholar
  20. 20.
    Alriksson, B., Rose, S. H., van Zyl, W. H., Sjode, A., Nilvebrant, N., & Jonsson, L. J. (2009). Cellulase production from spent lignocellulose hydrolysates by recombinant Aspergillus niger. Applied and Environmental Microbiology, 75, 2366–2374.CrossRefGoogle Scholar
  21. 21.
    Dien, B. S., Li, X., Iten, L. B., Jordan, D. B., O'Bryan, P. J., & Cotta, M. A. (2006). Enzymatic saccharification of hot-water pretreated corn fiber for production of monosaccharides. Enzyme and Microbial Technology, 39, 1137–1144.CrossRefGoogle Scholar
  22. 22.
    Doppelbauer, R., Esterbauer, H., Steiner, W., Lafferty, R. M., & Steinmuller, H. (1987). The use of lignocellulosic wastes for production of cellulase by Trichoderma reesei. Applied Microbiology and Biotechnology, 26, 485–494.CrossRefGoogle Scholar
  23. 23.
    Gupte, A., & Madamwar, D. (1997). Solid state fermentation of lignocellulosic waste for cellulase and beta-glucosidase production by cocultivation of Aspergillus ellipticus and Aspergillus fumigatus. Biotechnology Progress, 13, 166–169.CrossRefGoogle Scholar
  24. 24.
    Thygesen, A., Thomsen, A. B., Schmidt, A. S., Jorgensen, H., Ahring, B. K., & Olsson, L. (2003). Production of cellulose and hemicellulose-degrading enzymes by filamentous fungi cultivated on wet-oxidised wheat straw. Enzyme and Microbial Technology, 32, 606–615.CrossRefGoogle Scholar
  25. 25.
    Bhat, M. K., & Bhat, S. (1997). Cellulose degrading enzymes and their potential industrial applications. Biotechnology Advances, 15, 583–620.CrossRefGoogle Scholar
  26. 26.
    Beguin, P., & Aubert, J. P. (1994). The biological degradation of cellulose. FEMS Microbiology Reviews, 13, 25–58.CrossRefGoogle Scholar
  27. 27.
    Saha, B. C. (2003). Hemicellulose bioconversion. Journal of Industrial Microbiology & Biotechnology, 30, 279–291.CrossRefGoogle Scholar
  28. 28.
    Sørensen, A., Lübeck, P.S., Lübeck, M., Nielsen, K.F., Ahring, B.K., Teller, P.J., Frisvad, J.C. (2011). Aspergillus saccharolyticus sp. nov., a new black Aspergillus species isolated in Denmark. International Journal of Systematic and Evolutionary Microbiology (in press)Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Annette Sørensen
    • 1
  • Philip J. Teller
    • 2
  • Peter S. Lübeck
    • 1
  • Birgitte K. Ahring
    • 1
    • 2
  1. 1.Section for Sustainable BiotechnologyAalborg University CopenhagenBallerupDenmark
  2. 2.Center for Bioenergy and BioproductsWashington State UniversityRichlandUSA

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