Inhibitors Compounds on Sugarcane Bagasse Saccharification: Effects of Pretreatment Methods and Alternatives to Decrease Inhibition

  • Rafaela I. S. Ladeira-Ázar
  • Túlio Morgan
  • Gabriela Piccolo Maitan-AlfenasEmail author
  • Valéria M. Guimarães


Considering bioethanol production, extensive research has been performed to decrease inhibitors produced during pretreatments, to diminish energy input, and to decrease costs. In this study, sugarcane bagasse was pretreated with NaOH, H2SO4, and water. The higher concentration of phenols, 3.3 g/L, was observed in biomass liquid fraction after alkaline pretreatment. Acid pretreatment was responsible to release considerable acetic acid concentration, 2.3 g/L, while water-based pretreatment was the only to release formic acid, 0.02 g/L. Furans derivatives were not detected in liquid fractions regardless of pretreatment. Furthermore, washing step removed most of the phenols from pretreated sugarcane bagasse. Saccharification of alkali-pretreated biomass plus polyethylene glycol (PEG) at 0.4% (w/v) enhanced 8 and 26% the glucose and the xylose release, respectively, while polyvinylpyrrolidone (PVP) also at 0.4% (w/v) increased the release by 10 and 31% of these sugars, respectively, even without washing and filtration steps. Moreover, these polymers cause above 50% activation of endoglucanase and xylanase activities which are crucial for biomass hydrolysis.


Sugarcane bagasse pretreatment Inhibitors Enzymes Polyethylene glycol Polyvinylpyrrolidone 



The authors would like to thank the CNPq for the scholarship granted to the first author. This research was also supported by the Brazilian institutions FAPEMIG and CAPES.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Sun, S., Sun, S., Cao, X., & Sun, R. (2016). The role of pretreatment in improving the enzymatic hydrolysis of lignocellulosic materials. Bioresource Technology, 199, 49–58.CrossRefGoogle Scholar
  2. 2.
    McKendry, P. (2002). Energy production from biomass (part 1): overview of biomass. Bioresource Technology, 83(1), 37–46.CrossRefGoogle Scholar
  3. 3.
    Haghighi, S., Hossein, A., & Tabatabaei, M. (2013). Lignocellulosic biomass to bioethanol, a comprehensive review with a focus on pretreatment. Renewable & Sustainable Energy Reviews, 27, 77–93.CrossRefGoogle Scholar
  4. 4.
    Jönsson, L. J., & Martín, C. (2016). Pretreatment of lignocellulose: formation of inhibitory by-products and strategies for minimizing their effects. Bioresource Technology, 199, 103–112.CrossRefGoogle Scholar
  5. 5.
    Mood, S. H., Golfeshan, A. H., Tabatabaei, M., Jouzani, G. S., Najafi, G. H., Gholami, M., & Ardjmand, M. (2013). Lignocellulosic biomass to bioethanol, a comprehensive review with a focus on pretreatment. Renewable & Sustainable Energy Reviews, 27, 77–93.CrossRefGoogle Scholar
  6. 6.
    Florencio, C., Badino, A. C., & Farinas, C. S. (2016). Soybean protein as a cost-effective lignin- blocking additive for the saccharification of sugarcane bagasse. Bioresource Technology, 221, 172–180.CrossRefGoogle Scholar
  7. 7.
    Ximenes, E., Kim, Y., Mosier, N., Dien, B., & Ladisch, M. (2010). Inhibition of cellulases by phenols. Enzyme Microbial Technology, 46(3-4), 170–176.CrossRefGoogle Scholar
  8. 8.
    Kim, Y., Ximenes, E., Mosier, N. S., & Ladisch, M. R. (2011). Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass. Enzyme Microbial Technology, 48(4-5), 408–415.CrossRefGoogle Scholar
  9. 9.
    Ximenes, E., Kim, Y., Mosier, N., Dien, B., & Ladisch, M. (2011). Deactivation of cellulases by phenols. Enzyme Microbial Technology, 48(1), 54–60.CrossRefGoogle Scholar
  10. 10.
    Ladeira-Ázar, R. I. S., Morgan, T., dos Santos, A. C. F., Ximenes, E. A., Ladisch, M., & Guimarães, V. M. (2018). Deactivation and activation of lignocellulose degrading enzymes in the presence of laccase. Enzyme Microbial Technology, 109, 25–30.CrossRefGoogle Scholar
  11. 11.
    Borjesson, J., Peterson, R., & Tjernel, F. (2007). Enhanced enzymatic conversion of soft-wood lignocellulose by poly (ethylene glycol) addition. Enzyme Microbial Technology, 40(4), 754–762.CrossRefGoogle Scholar
  12. 12.
    Kristensen, J. B., Borjesson, J., Bruun, M., Tjerneld, F., & Jorgensen, H. (2007). Use of surface active additives in enzymatic hydrolysis of wheat straw lignocellulose. Enzyme Microbial Technology, 40(4), 888–895.CrossRefGoogle Scholar
  13. 13.
    Sipos, B., Szilágyi, M., Sebestyén, Z., Perazzini, R., Dienes, D., Jakab, E., Crestini, C., & Réczey, K. (2011). Mechanism of the positive effect of poly(ethylene glycol) addition in enzymatic hydrolysis of steam pretreated lignocelluloses. Comptes Rendus Biologies, 334(11), 812–823.CrossRefGoogle Scholar
  14. 14.
    Tejirian, A., & Xu, F. (2011). Inhibition of enzymatic cellulolysis by phenolic compounds. Enzyme Microbial Technology, 48(3), 239–247.CrossRefGoogle Scholar
  15. 15.
    Ghose, T. K. (1987). Measurement of cellulase activities. Pure and Applied Chemistry, 59(2), 257–268.CrossRefGoogle Scholar
  16. 16.
    Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31(3), 426–428.CrossRefGoogle Scholar
  17. 17.
    Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., & Crocker, D. (2008). Laboratory analytical procedure (LAP): determination of structural carbohydrates and lignin in biomass. Technical report: NREL/TP-510-42618. Golden: National Renewable Energy Laboratory.Google Scholar
  18. 18.
    Budini, R., Tonelli, D., & Girotti, S. (1980). Analysis of total phenols using the Prussian blue method. Journal of Agricultural and Food Chemistry, 28(6), 1236–1238.CrossRefGoogle Scholar
  19. 19.
    Nakagame, S., Chandra, R. P., Kadla, J. F., & Saddler, J. N. (2011). The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose. Bioresource Technology, 102(6), 4507–4517.CrossRefGoogle Scholar
  20. 20.
    Carvalheiro, F., Duarte, L. C., & Girio, F. M. (2008). Hemicellulose biorefineries: a review on biomass pretreatments. Journal of Scientific and Industrial Research, 67, 849–864.Google Scholar
  21. 21.
    Maitan-Alfenas, G. P., Visser, E. M., Alfenas, R. F., Nogueira, B. R. G., de Campos, G. G., Milagres, A. F., de Vries, R. P., & Guimaraes, V. M. (2015). The influence of pretreatment methods on saccharification of sugarcane bagasse by an enzyme extract from Chrysoporthe cubensis and commercial cocktails: a comparative study. Bioresource Technology, 192, 670–676.CrossRefGoogle Scholar
  22. 22.
    Lee, J. M., Shi, J., Venditti, R. A., & Jameel, H. (2009). Autohydrolysis pretreatment of coastal Bermuda grass for increased enzyme hydrolysis. Bioresource Technology, 100(24), 6434–6441.CrossRefGoogle Scholar
  23. 23.
    Pu, Y., Treasure, T., Gonzalez, R., Venditti, R., & Jameel, H. (2011). Autohydrolysis pretreatment of mixed hardwoods to extract value prior to combustion. Bioresources, 6, 4856–4870.Google Scholar
  24. 24.
    Falkoski, D. L., Guimaraes, V. M., de Almeida, M. N., Alfenas, A. C., Colodette, J. L., & de Rezende, S. T. (2013). Chrysoporthe cubensis: a new source of cellulases and hemicellulases to application in biomass saccharification processes. Bioresource Technology, 130, 296–305.CrossRefGoogle Scholar
  25. 25.
    Hendriks, A. T. W. M., & Zeeman, G. (2009). Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresource Technology, 100(1), 10–18.CrossRefGoogle Scholar
  26. 26.
    Harrison, M. D., Zhang, Z., Shand, K., Hara, I. M. O., Doherty, W. O. S., & Dale, J. L. (2013). Effect of pretreatment on saccharification of sugar cane bagasse by complex and simple enzyme mixtures. Bioresource Technology, 148, 105–113.CrossRefGoogle Scholar
  27. 27.
    Hu, F., & Ragauskas, A. (2012). Pretreatment and lignocellulosic chemistry. Bioenergy Research, 5(4), 1043–1066.CrossRefGoogle Scholar
  28. 28.
    Kim, Y., Kreke, T., Ko, J. K., & Ladisch, M. R. (2015). Hydrolysis-determining substrate characteristics in liquid hot water pretreated hardwood. Biotechnology and Bioengineering, 112(4), 677–687.CrossRefGoogle Scholar
  29. 29.
    Kim, Y., Kreke, T., Mosier, N. S., & Ladisch, M. R. (2014). Severity factor coefficients for subcritical liquid hot water pretreatment of hardwood chips. Biotechnology and Bioengineering, 111(2), 254–263.CrossRefGoogle Scholar
  30. 30.
    Jönsson, L. J., Alriksson, B., & Nilvebrant, N. (2013). Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnology for Biofuels, 6(1), 16–26.CrossRefGoogle Scholar
  31. 31.
    Mhlongo, S. I., Riaan, H., Viljoen-Blooma, M., & van Zyl, W. H. (2015). Lignocellulosic hydrolysate inhibitors selectively inhibit/deactivate cellulase performance. Enzyme Microbial Technology, 81, 16–22.CrossRefGoogle Scholar
  32. 32.
    Larsson, S., Palmqvist, E., Hahn-Hägerdal, B., Tengborg, C., Stenberg, K., & Zacchi, G. (1999). The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme Microbial Technology, 24(3-4), 151–159.CrossRefGoogle Scholar
  33. 33.
    Du, B., Sharma, L. N., Becker, C., Chen, S.-F., Mowery, R. A., van Walsum, G. P., & Chambliss, C. K. (2010). Effect of varying feedstock-pretreatment chemistry combinations on the formation and accumulation of potentially inhibitory degradation products in biomass hydrolysates. Biotechnology and Bioengineering, 107(3), 430–440.CrossRefGoogle Scholar
  34. 34.
    Kim, Y., Kreke, T., Hendrickson, R., Parenti, J., & Ladisch, M. R. (2013). Fractionation of cellulase and fermentation inhibitors from steam pretreated mixed hardwood. Bioresource Technology, 135, 30–38.CrossRefGoogle Scholar
  35. 35.
    Ko, J. K., Ximenes, E., Kim, Y., & Ladisch, M. R. (2015a). Adsorption of enzyme onto lignins of liquid hot water pretreated hardwoods. Biotechnology and Bioengineering, 112(3), 447–456.CrossRefGoogle Scholar
  36. 36.
    Guo, F., Shi, W., Sun, W., Li, X., Wang, F., Zhao, J., & Qu, Y. (2014). Differences in the adsorption of enzymes onto lignins from diverse types of lignocellulosic biomass and the underlying mechanism. Biotechnology for Biofuels, 7, 1–10.CrossRefGoogle Scholar
  37. 37.
    Ko, J. K., Kim, Y., Ximenes, E., & Ladisch, M. R. (2015b). Effect of liquid hot water pretreatment severity on properties of hardwood lignin and enzymatic hydrolysis of cellulose. Biotechnology and Bioengineering, 112(2), 252–262.CrossRefGoogle Scholar
  38. 38.
    Hodge, D. B., Karim, M. N., Schell, D. J., & McMillan, J. D. (2008). Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose. Bioresource Technology, 99(18), 8940–8948.CrossRefGoogle Scholar
  39. 39.
    Zhao, J., & Chen, H. (2014). Stimulation of cellulases by small phenolic compounds in pretreated stover. Journal of Agricultural and Food Chemistry, 62(14), 3223–3229.CrossRefGoogle Scholar
  40. 40.
    Moreno, A., Ibarra, D., Fernández, J. L., & Ballesteros, M. (2012). Different laccase detoxification strategies for ethanol production from lignocellulosic biomass by the thermotolerant yeast Kluyveromyces marxianus CECT 10875. Bioresource Technology, 106, 101–109.CrossRefGoogle Scholar
  41. 41.
    Galbe, M., & Zacchi, G. (2007). Pretreatment of lignocellulosic materials for efficient bioethanol production. Advances in Biochemical Engineering Biotechnology., 108, 41–65.Google Scholar
  42. 42.
    Bensah, E., & Mensah, M. (2013). Chemical pretreatment methods for the production of cellulosic ethanol: technologies and innovations. International Journal of Chemical Engineering, 1–21.CrossRefGoogle Scholar
  43. 43.
    González-Bautista, E., Santana-Morales, J. C., Ríos-Fránquez, F. X., Poggi-Varaldo, H. M., Ramos-Valdivia, A. C., Cristiani-Urbina, E., & Ponce-Noyola, T. (2017). Phenolic compounds inhibit cellulase and xylanase activities of Cellulomonas flavigena PR-22 during saccharification of sugarcane bagasse. Fuel, 196, 32–35.CrossRefGoogle Scholar
  44. 44.
    Toquero, C., & Bolado, S. (2014). Effect of four pretreatments on enzymatic hydrolysis and ethanol fermentation of wheat straw. Influence of inhibitors and washing. Bioresource Technology, 157, 68–76.CrossRefGoogle Scholar
  45. 45.
    Min, D., Xu, R., Hou, Z., Lv, J., Huang, C., Jin, Y., & Yong, Q. (2015). Minimizing inhibitors during pretreatment while maximizing sugar production in enzymatic hydrolysis through a two-stage hydrothermal pretreatment. Cellulose, 22(2), 1253–1261.CrossRefGoogle Scholar
  46. 46.
    García-Aparicio, M. P., Ballesteros, I., González, A., Oliva, J. M., Ballesteros, M., & Negro, M. J. (2006). Effect of inhibitors released during steam-explosion pretreatment of barley straw on enzymatic hydrolysis. Applied Biochemistry and Biotechnology, 129(1-3), 278–288.CrossRefGoogle Scholar
  47. 47.
    Cai, C., Qiu, X., Zheng, M., Lin, M., Lin, X., Lou, H., Zhan, X., Pang, Y., Huang, J., & Xie, L. (2017). Using polyvinylpyrrolidone to enhance the enzymatic hydrolysis of lignocelluloses by reducing the cellulase non-productive adsorption on lignin. Bioresource Technology, 227, 74–81.CrossRefGoogle Scholar
  48. 48.
    Quay, D. H. X., Bakar, F. D. A., Rabu, A., Said, M., Illias, R. M., Mahadi, N. M., Hassan, O., & Murad, A. M. A. (2011). Overexpression, purification and characterization of the Aspergillus niger endoglucanase, EglA, in Pichia pastoris. African Journal of Biotechnology, 10, 2101–2111.Google Scholar
  49. 49.
    Eriksson, T., Borjesson, J., & Tjerneld, F. (2002). Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme Microbial Technology, 31(3), 353–364.CrossRefGoogle Scholar
  50. 50.
    Lin, H., Hu, B., & Zhu, M. (2016). Enhanced hydrogen production and sugar accumulation from spent mushroom compost by Clostridium thermocellum supplemented with PEG8000 and JFC-E. International Journal of Hydrogen Energy, 41(4), 2383–2390.CrossRefGoogle Scholar
  51. 51.
    Cheng, J., Yu, Y., & Zhu, M. (2014). Enhanced biodegradation of sugarcane bagasse by Clostridium thermocellum with surfactant addition. Green Chemistry, 16(5), 2689–2695.CrossRefGoogle Scholar
  52. 52.
    Ouyang, J., Dong, Z., Song, X., Lee, X., Chen, M., & Yong, Q. (2010). Improved enzymatic hydrolysis of microcrystalline cellulose (Avicel PH101) by polyethylene glycol addition. Bioresource Technology, 101(17), 6685–6691.CrossRefGoogle Scholar
  53. 53.
    Kaar, W. E., & Holtzapple, M. T. (1998). Benefits from Tween during enzymatic hydrolysis of corn stover. Biotechnology and Bioengineering, 59(4), 419–427.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Rafaela I. S. Ladeira-Ázar
    • 1
  • Túlio Morgan
    • 1
  • Gabriela Piccolo Maitan-Alfenas
    • 2
    Email author
  • Valéria M. Guimarães
    • 1
  1. 1.Department of Biochemistry and Molecular Biology, BIOAGROFederal University of ViçosaViçosaBrazil
  2. 2.Department of Food and NutritionFederal University of Mato GrossoCuiabáBrazil

Personalised recommendations