Applied Biochemistry and Biotechnology

, Volume 187, Issue 2, pp 461–473 | Cite as

Alternative Low-Cost Additives to Improve the Saccharification of Lignocellulosic Biomass

  • Mariana G. Brondi
  • Vanessa M. Vasconcellos
  • Roberto C. Giordano
  • Cristiane S. FarinasEmail author


A potential strategy to mitigate problems related to unproductive adsorption of enzymes onto lignin during the saccharification of lignocellulosic biomass is the addition of lignin-blocking agents to the hydrolysis reaction medium. However, there is a clear need to find more cost-effective additives for use in large-scale processes. Here, selected alternative low-cost additives were evaluated in the saccharification of steam-exploded sugarcane bagasse using a commercial enzymatic cocktail. The addition of soybean protein, tryptone, peptone, and maize zein had positive effects on glucose release during the hydrolysis, with gains of up to 36% when 8% (w/w) soybean protein was used. These improvements were superior to those obtained using bovine serum albumin (BSA), a much more expensive protein that has been widely reported for such an application. Moreover, addition of soybean protein led to a saving of 48 h in the hydrolysis, corresponding to a 66% decrease in the reactor operation time required. In order to achieve the same hydrolysis yield without the soybean additive, the enzyme loading would need to be increased by 50%. FTIR spectroscopy and nitrogen elemental analysis revealed that the additives probably acted to reduce unproductive binding of cellulolytic enzymes onto the lignin portion of the sugarcane bagasse.


Additives Sugarcane bagasse Adsorption Lignin Lignocellulosic biomass Enzymatic hydrolysis 


Funding Information

The authors would like to thank Embrapa, CNPq (Process 401182/2014-2), CAPES, and FAPESP (Processes 2014/19000-3, 2016/10636-8, and 2017/13931-3) (all from Brazil) for financial support.

Compliance with Ethical Standards

Competing interests

The authors declare they have no competing interests.


  1. 1.
    Klein-Marcuschamer, D., Oleskowicz-Popiel, P., Simmons, B. A., & Blanch, H. W. (2012). The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnology and Bioengineering, 109(4), 1083–1087. Scholar
  2. 2.
    Valdivia, M., Galan, J. L., Laffarga, J., & Ramos, J. L. (2016). Biofuels 2020: Biorefineries based on lignocellulosic materials. Microbial Biotechnology, 9(5), 585–594. Scholar
  3. 3.
    Johnson, E. (2016). Integrated enzyme production lowers the cost of cellulosic ethanol. Biofuels, Bioproducts and Biorefining, 10(2), 164–174. Scholar
  4. 4.
    Liu, G., Zhang, J., & Bao, J. (2016). Cost evaluation of cellulase enzyme for industrial-scale cellulosic ethanol production based on rigorous aspen plus modeling. Bioprocess and Biosystems Engineering, 39(1), 133–140. Scholar
  5. 5.
    Hong, Y., Nizami, A. S., Pour Bafrani, M., Saville, B. A., & Maclean, H. L. (2013). Impact of cellulase production on environmental and financial metrics for lignocellulosic ethanol. Biofuels, Bioproducts and Biorefining, 7(3), 303–313. Scholar
  6. 6.
    Ko, J. K., Ximenes, E., Kim, Y., & Ladisch, M. R. (2015). Adsorption of enzyme onto lignins of liquid hot water pretreated hardwoods. Biotechnology and Bioengineering, 112(3), 447–456. Scholar
  7. 7.
    Rahikainen, J. L., Martin-Sampedro, R., Heikkinen, H., Rovio, S., Marjamaa, K., Tamminen, T., Rojas, O. J., & Kruus, K. (2013). Inhibitory effect of lignin during cellulose bioconversion: The effect of lignin chemistry on non-productive enzyme adsorption. Bioresource Technology, 133, 270–278. Scholar
  8. 8.
    Li, X., & Zheng, Y. (2017). Lignin-enzyme interaction: Mechanism, mitigation approach, modeling, and research prospects. Biotechnology Advances, 35(4), 466–489. Scholar
  9. 9.
    Lou, H., Wang, M., Lai, H., Lin, X., Zhou, M., Yang, D., & Qiu, X. (2013). Reducing non-productive adsorption of cellulase and enhancing enzymatic hydrolysis of lignocelluloses by noncovalent modification of lignin with lignosulfonate. Bioresource Technology, 146, 478–484. Scholar
  10. 10.
    Eriksson, T., Börjesson, J., & Tjerneld, F. (2002). Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme and Microbial Technology, 31(3), 353–364. Scholar
  11. 11.
    Zheng, Y., Pan, Z., Zhang, R., Wang, D., & Jenkins, B. (2008). Non-ionic surfactants and non-catalytic protein treatment on enzymatic hydrolysis of pretreated creeping wild ryegrass. Applied Biochemistry and Biotechnology, 146(1–3), 231–248. Scholar
  12. 12.
    Yang, B., & Wyman, C. E. (2006). BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechnology and Bioengineering, 94(4), 611–617. Scholar
  13. 13.
    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. Enzyme and Microbial Technology, 41(1–2), 186–195. Scholar
  14. 14.
    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. Scholar
  15. 15.
    Kaar, W. E., & Holtzapple, M. T. (1998). Benefits from tween during enzymic hydrolysis of corn Stover. Biotechnology and Bioengineering, 59(4), 419–427.Google Scholar
  16. 16.
    Cannella, D., & Jørgensen, H. (2014). Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol production? Biotechnology and Bioengineering, 111(1), 59–68. Scholar
  17. 17.
    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. Scholar
  18. 18.
    Gouveia, E. R., do Nascimento, R. T., Souto-Maior, A. M., & Rocha, G. J. d. M. (2009). Validation of methodology for the chemical characterization of sugar cane bagasse. Quimica Nova, 32(6), 1500–1503. Scholar
  19. 19.
    Wang, Z., Li, Y., Jiang, L., Qi, B., & Zhou, L. (2014). Relationship between secondary structure and surface hydrophobicity of soybean protein isolate subjected to heat treatment. Journal of Chemistry, 2014, 1–10. Scholar
  20. 20.
    Pereira, S. C., Maehara, L., Machado, C. M. M., & Farinas, C. S. (2016). Physical-chemical-morphological characterization of the whole sugarcane lignocellulosic biomass used for 2G ethanol production by spectroscopy and microscopy techniques. Renewable Energy, 87, 607–617. Scholar
  21. 21.
    Harrison, M. D., Zhang, Z., Shand, K., O’Hara, I. M., Doherty, W. O. S., & Dale, J. L. (2013). Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures. Bioresource Technology, 148, 105–113. Scholar
  22. 22.
    Militello, V., Casarino, C., Emanuele, A., Giostra, A., Pullara, F., & Leone, M. (2004). Aggregation kinetics of bovine serum albumin studied by FTIR spectroscopy and light scattering. Biophysical Chemistry, 107(2), 175–187. Scholar
  23. 23.
    Corrales, R. C. N. R., Mendes, F. M., Perrone, C., Sant’Anna, C., de Souza, W., Abud, Y., Bon, E. P. S., & Ferreira-Leitão, V. (2012). Structural evaluation of sugar cane bagasse steam pretreated in the presence of CO2 and SO2. Biotechnology for Biofuels, 5(1), 1–10. Scholar
  24. 24.
    Bhagia, S., Kumar, R., & Wyman, C. E. (2017). Effects of dilute acid and flowthrough pretreatments and BSA supplementation on enzymatic deconstruction of poplar by cellulase and xylanase. Carbohydrate Polymers, 157, 1940–1948. Scholar
  25. 25.
    Méndez Arias, J., de Oliveira Moraes, A., Modesto, L. F. A., de Castro, A. M., & Pereira, N. (2017). Addition of surfactants and non-hydrolytic proteins and their influence on enzymatic hydrolysis of pretreated sugarcane bagasse. Applied Biochemistry and Biotechnology, 181(2), 593–603. Scholar
  26. 26.
    Pauling, L., Corey, R. B., & Branson, H. R. (1951). The structure of proteins: Two hydrogen-bonded helical configurations of the polypeptide chain. Proceedings of the National Academy of Sciences, 37(4), 205–211. Scholar
  27. 27.
    Richardson, J. S. (1981). The anatomy and taxonomy of protein structure. Advances in Protein Chemistry, 34(C), 167–339. Scholar
  28. 28.
    Shahid, M., & Chawla, H. M. (2017). Dansylated adenine as a molecular probe for exploring hydrophobic pocket of bovine serum albumin (BSA) and its utility for mercury ion recognition. Journal of Luminescence, 188, 460–464. Scholar
  29. 29.
    Carter, D. C., & Ho, J. X. (1994). Structure of serum albumin. Advances in Protein Chemistry, 45(C), 153–203. Scholar
  30. 30.
    Badley, R. A., Atkinson, D., Hauser, H., Oldani, D., Green, J. P., & Stubbs, J. M. (1975). The structure, physical and chemical properties of the soy bean protein glycinin. Biochimica et Biophysica Acta (BBA) - Protein Structure, 412(2), 214–228. Scholar
  31. 31.
    Argos, P., Pedersen, K., Marks, M. D., & Larkins, B. A. (1982). A structural model for maize zein proteins. Journal of Biological Chemistry, 257(17), 9984–9990.Google Scholar
  32. 32.
    Guan, H., Diao, X., Jiang, F., Han, J., & Kong, B. (2018). The enzymatic hydrolysis of soy protein isolate by Corolase PP under high hydrostatic pressure and its effect on bioactivity and characteristics of hydrolysates. Food Chemistry, 245, 89–96. Scholar
  33. 33.
    Mahmoud, M. I., Malone, W. T., & Cordle, C. T. (1992). Enzymatic hydrolysis of casein: Effect of degree of hydrolysis on antigenicity and physical properties. Journal of Food Science, 57(5), 1223–1229. Scholar
  34. 34.
    Wu, W. U., Hettiarachchy, N. S., & Qi, M. (1998). Hydrophobicity, solubility, and emulsifying properties of soy protein peptides prepared by papain modification and ultrafiltration. Journal of the American Oil Chemists’ Society, 75(7), 845–850.Google Scholar
  35. 35.
    Kumar, R., & Wyman, C. E. (2009). Effect of additives on the digestibility of corn Stover solids following pretreatment by leading technologies. Biotechnology and Bioengineering, 102(6), 1544–1557. Scholar
  36. 36.
    Rocha-Martín, J., Martinez-Bernal, C., Pérez-Cobas, Y., Reyes-Sosa, F. M., & García, B. D. (2017). Additives enhancing enzymatic hydrolysis of lignocellulosic biomass. Bioresource Technology, 244(Pt 1), 48–56. Scholar
  37. 37.
    Wang, H., Kobayashi, S., & Mochidzuki, K. (2015). Effect of non-enzymatic proteins on enzymatic hydrolysis and simultaneous saccharification and fermentation of different lignocellulosic materials. Bioresource Technology, 190, 373–380. Scholar
  38. 38.
    Saini, J. K., Patel, A. K., Adsul, M., & Singhania, R. R. (2016). Cellulase adsorption on lignin: A roadblock for economic hydrolysis of biomass. Renewable Energy, 98, 29–42. Scholar
  39. 39.
    Ko, J. K., Kim, Y., Ximenes, E., & Ladisch, M. R. (2015). Effect of liquid hot water pretreatment severity on properties of hardwood lignin and enzymatic hydrolysis of cellulose. Biotechnology and Bioengineering, 112(2), 252–262. Scholar
  40. 40.
    Nakagame, S., Chandra, R. P., & Saddler, J. N. (2010). The effect of isolated lignins, obtained from a range of pretreated lignocellulosic substrates, on enzymatic hydrolysis. Biotechnology and Bioengineering, 105(5), 871–879. Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Embrapa InstrumentaçãoSão CarlosBrazil
  2. 2.Graduate Program of Chemical EngineeringFederal University of São CarlosSao CarlosBrazil

Personalised recommendations