Skip to main content
SpringerLink
Log in

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

  • Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H2SO4 with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, β-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 23 central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19–4.81%, w/w) and loadings of enzymes (1.9–38.1 FPU/g bagasse) and Tween 20 (0.0–0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Ahammed S, Prema P (2002) Influence of media nutrients on synthesis of lignin peroxidase from Aspergillus sp. Appl Biochem Biotechnol 102–103:327–336. doi:10.1385/ABAB:102-103:1-6:327

    Article  PubMed  Google Scholar 

  2. Alkasrawi M, Eriksson T, Borjesson J, Wingren A, Galbe M, Tjerneld F, Zacchi G (2003) The effect of Tween-20 on simultaneous saccharification and fermentation of softwood to ethanol. Enzyme Microb Technol 33:71–78. doi:10.1016/S0141-0229(03)00087-5

    Article  CAS  Google Scholar 

  3. Assavanig A, Amornkitticharoen B, Ekpaisal N, Meevootisom V, Flegel TW (1992) Isolation, characterization and function of laccase from Trichoderma. Appl Microbiol Biotechnol 38:198–202. doi:10.1007/BF00174468

    Article  CAS  Google Scholar 

  4. Bailey MJ, Biely P, Poutanen K (1992) International testing of methods for assay of xylanaseactivity. J Biotechnol 23:257–270. doi:10.1016/0168-1656(92)90074-J

    Article  CAS  Google Scholar 

  5. Berlin A, Gilkes N, Kurabi A, Bura R, Tu MB, Kilburn D, Saddler J (2005) Weak lignin-binding enzymes—a novel approach to improve activity of cellulases for hydrolysis of lignocellulosics. Appl Biochem Biotechnol 121:163–170. doi:10.1385/ABAB:121:1-3:0163

    Article  PubMed  Google Scholar 

  6. Bourbonnais R, Leech D, Paice MG (1998) Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochim Biophys Acta 1379:381–390. doi:10.1016/S0304-4165(97)00117-7

    PubMed  CAS  Google Scholar 

  7. Box GEP, Hunter WG, Hunter JS (1978) Statistics for experimenters: an introduction to design, data analysis and model building. Wiley, New York, p 653

    Google Scholar 

  8. Cara C, Moya M, Ballesteros I, Negro MJ, González A, Ruiz E (2007) Influence of solid loading on enzymatic hydrolysis of steam exploded or liquid hot water pretreated olive tree biomass. Process Biochem 42:1003–1009. doi:10.1016/j.procbio.2007.03.012

    Article  CAS  Google Scholar 

  9. Carvalho W, Canilha L, Silva SS (2007) Semi-continuous xylitol bioproduction in sugarcane bagasse hydrolysate: effect of nutritional supplementation. Braz J Pharm Sci 43:47–53. doi:10.1590/S1516-93322007000100006

    CAS  Google Scholar 

  10. Conesa A, van den Hondel CAMJJ, Punt PJ (2000) Studies on the production of fungal peroxidases in Aspergillus niger. Appl Environ Microbiol 66:3016–3023

    Article  PubMed  CAS  Google Scholar 

  11. Eriksson T, Börjesson J, Tjerneld F (2002) Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose. Enzyme Microb Technol 31:353–364. doi:10.1016/S0141-0229(02)00134-5

    Article  CAS  Google Scholar 

  12. Eriksson T, Karlsson J, Tjerneld F (2002) A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) of Trichoderma reesei. Appl Biochem Biotechn 101:41–60. doi:10.1385/ABAB:101:1:41

    Article  CAS  Google Scholar 

  13. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268

    Article  CAS  Google Scholar 

  14. Gochev VK, Krastanov AI (2007) Isolation of laccase producing Trichoderma spp. Bulg J Agric Sci 13:171–176

    Google Scholar 

  15. Gouveia ES, Nascimento RT, Souto-Maior AM, Rocha GJM (2009) Validação de metodologia para a caracterização química de bagaço de cana-de-açúcar. Quim Nova 32:1500–1503. doi:10.1590/S0100-40422009000600026

    Article  CAS  Google Scholar 

  16. Grohmman K, Himmel M, Rivard C, Tucker M, Torget R, Graboski M (1984) Chemical-mechanical methods for the enhanced utilization straw. Biotechnol Bioeng Symp 14:137–157

    Google Scholar 

  17. Hendriks ATWM, Zeeman G (2009) Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour Technol 100:10–18. doi:10.1016/j.biortech.2008.05.027

    Article  PubMed  CAS  Google Scholar 

  18. Holker U, Dohse J, Höfer M (2002) Extracellular laccases in ascomycetes Trichoderma atroviride and Trichoderma harzianum. Folia Microbiol 47:423–427. doi:10.1007/BF02818702

    Article  CAS  Google Scholar 

  19. Jorgensen H, Kristensen JB, Felby C (2007) Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities. Biofuels Bioprod Bioref 1:119–134. doi:10.1002/bbb.4

    Article  Google Scholar 

  20. Kaar WE, Holtzapple MT (1998) Benefits from Tween during enzymic hydrolysis of corn stover. Biotechnol Bioeng 59:419–427. doi:10.1002/(SICI)1097-0290(19980820)59:4<419::AID-BIT4>3.0.CO;2-J

    Article  PubMed  CAS  Google Scholar 

  21. Kanayama N, Suzuki T, Kawai K (2002) Purification and characterization of an alkaline manganese peroxidase from Aspergillus terreus LD-1. J Biosci Bioeng 93:405–410. doi:10.1016/S1389-1723(02)80075-5

    PubMed  CAS  Google Scholar 

  22. Khindaria A, Grover TA, Aust SD (1994) Oxalate-dependent reductive activity of manganese peroxidase from Phanerochaete chrysosporium. Arch Biochem Biophys 314:301–306. doi:10.1006/abbi.1994.1446

    Article  PubMed  CAS  Google Scholar 

  23. Kovacs K, Macrelli S, Szakacs G, Zacchi G (2009) Enzymatic hydrolysis of steam-pretreated lignocellulosic materials with Trichoderma atroviride enzymes produced in-house. Biotechnol Biofuels 2:14. doi:10.1186/1754-6834-2-14

    Article  PubMed  Google Scholar 

  24. Kumar S, Singh SP, Mishra IM, Adhikari DK (2009) Ethanol and xylitol production from glucose and xylose at high temperature by Kluyveromyces sp. IIPE453. J Ind Microbiol Biotechnol 36:1483–1489. doi:10.1007/s10295-009-0636-6

    Article  PubMed  CAS  Google Scholar 

  25. Law DJ, Timberlake WE (1980) Developmental regulation of laccase levels in Aspergillus nidulans. J Bacterol 144:509–517

    CAS  Google Scholar 

  26. Lu Y, Yang B, Gregg D, Saddler JN, Mansfield SD (2002) Cellulase adsorption and an evaluation of enzyme recycle during hydrolysis of steam-exploded softwood residues. Appl Biochem Biotechnol 98–100:641–654. doi:10.1385/ABAB:98-100:1-9:641

    Article  PubMed  Google Scholar 

  27. Mäkinen KK (2000) The rocky road of xylitol to its clinical application. J Dent Res 79:1352–1355. doi:10.1177/00220345000790060101

    Article  PubMed  Google Scholar 

  28. Martins LF, Kolling D, Camassola M, Dillon AJ, Ramos LP (2008) Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in relation to their activity against various cellulosic substrates. Bioresour Technol 99:1417–1424. doi:10.1016/j.biortech.2007.01.060

    Article  PubMed  CAS  Google Scholar 

  29. Matsushika A, Inoue H, Kodaki T, Sawayama S (2009) Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives. Appl Microbiol Biotechnol 84:37–53. doi:10.1007/s00253-009-2101-x

    Article  PubMed  CAS  Google Scholar 

  30. Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 96:673–686. doi:10.1016/j.biortech.2004.06.025

    Article  PubMed  CAS  Google Scholar 

  31. Mussato SI, Fernandes M, Milagres AMF, Roberto IC (2008) Effect of hemicellulose and lignin on enzymatic hydrolysis of cellulose from brewer’s spent grain. Enzyme Microb Technol 43:124–129. doi:10.1016/j.enzmictec.2007.11.006

    Google Scholar 

  32. Öhgren K, Bura R, Saddler J, Zacchi G (2007) Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover. Bioresour Technol 28:2503–2510. doi:10.1016/j.biortech.2006.09.003

    Article  Google Scholar 

  33. Panagiotou G, Olsson L (2007) Effect of compounds released during pretreatment of wheat straw on microbial growth and enzymatic hydrolysis rates. Biotechnol Bioeng 96:250–258. doi:10.1002/bit.21100

    Article  PubMed  CAS  Google Scholar 

  34. Saparrat MCN, Martínez MJ, Cabello MN, Arambarri M (2002) Screening for ligninolytic enzymes in autochthonous fungal strains from Argentina isolated from different substrata. Rev Iberoam Micol 19:181–185

    PubMed  Google Scholar 

  35. Sharrock K (1988) Cellulase assay methods: a review. J Biochem Biophys Methods 17:81–106

    Article  PubMed  CAS  Google Scholar 

  36. Silverstein RA, Chen Y, Sharma-Shirvappa RR, Boyette MD, Osborne J (2007) A comparison of chemical pretreatment methods for improving saccharification of cotton stalks. Bioresour Technol 98:3000–3011

    Article  PubMed  CAS  Google Scholar 

  37. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2008) Determination of structural carbohydrates and lignin in biomass. National Renewable Energy Laboratory, Golden, CO. http://www.nrel.gov/biomass/pdfs/42618.pdf. Accessed August 2010

  38. Tanaka H, Itakura S, Enoki A (2000) Phenol oxidase activity and one-electron oxidation activity in wood degradation by soft-rot deuteromycetes. Holzforschung 54:463–468. doi:10.1515/HF.2000.078

    Article  CAS  Google Scholar 

  39. Téllez-Jurado A, Arana-Cuenca A, González-Becerra AE, Viniegra-González G, Loera O (2006) Expression of a heterologous laccase by Aspergillus niger cultured by solid-state and submerged fermentations. Enzyme Microb Technol 38:665–669. doi:10.1016/j.enzmictec.2005.07.021

    Article  Google Scholar 

  40. Tien M, Kirk TK (1984) Lignin-degrading enzyme from Phanerochaete chrysosporium: purification, characterization, and catalytic properties of a unique H2O2-requiring oxygenase. Proc Natl Acad Sci USA 81:2280–2284

    Article  PubMed  CAS  Google Scholar 

  41. Tu M, Saddler JN (2010) Potencial enzyme cost reduction with the addition of surfactant during the hydrolysis of pretreated softwood. Appl Biochem Biotechnol 161:274–287. doi:10.1007/s12010-009-8869-4

    Article  PubMed  CAS  Google Scholar 

  42. Uhari M, Tapiainen T, Kontiokari T (2001) Xylitol in preventing acute otitis media. Vaccine 19:144–147. doi:10.1016/S0264-410X(00)00294-2

    Article  Google Scholar 

  43. Väljamäe P, Kipper K, Pettersson G, Johansson G (2003) Synergistic cellulose hydrolysis can be described in terms of fractal-like kinetics. Biotechnol Bioeng 84:254–257. doi:10.1002/bit.10775

    Article  PubMed  Google Scholar 

  44. Wood TM, Bath MK (1988) Methods for measuring cellulose activities. In: Wood WD, Kellogg ST (eds) Methods in enzymology. Academic, San Diego, pp 87–112

    Google Scholar 

  45. Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY (2005) Coordinated development of leading biomass pretreatment technologies. Bioresour Technol 96:1959–1966. doi:10.1016/j.biortech.2005.01.010

    Article  PubMed  CAS  Google Scholar 

  46. Yang B, Wyman CE (2006) BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechol Bioeng 94:611–617. doi:10.1002/bit.20750

    Article  CAS  Google Scholar 

  47. Zhao Y, Wu B, Yan B, Gao P (2004) Mechanism of cellobiose inhibition in cellulose hydrolysis by cellobiohydrolase. Sci China Ser C Life Sci 47:18–24. doi:10.1360/02yc0163

    Article  CAS  Google Scholar 

  48. Zheng Y, Pan Z, Zhang R, Wang D (2009) Enzymatic saccharification of dilute acid pretreated saline crops for fermentable sugar production. Appl Energy 86:2459–2467. doi:10.1016/j.apenergy.2009.03.012

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are grateful to FAPESP, CNPq, and CAPES for financial support.

Author information

Authors and Affiliations

  1. Department of Biotechnology, School of Engineering of Lorena, University of São Paulo (USP), Estrada Municipal do Campinho, s/n°, P.O. Box 116, 12602-810, Lorena, SP, Brazil

    Victor T. O. Santos, Paula J. Esteves, Adriane M. F. Milagres & Walter Carvalho

Authors
  1. Victor T. O. Santos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paula J. Esteves
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adriane M. F. Milagres
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Walter Carvalho
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Walter Carvalho.

Additional information

This article is based on a presentation at the 32nd Symposium on Biotechnology for Fuels and Chemicals.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Santos, V.T.O., Esteves, P.J., Milagres, A.M.F. et al. Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid. J Ind Microbiol Biotechnol 38, 1089–1098 (2011). https://doi.org/10.1007/s10295-010-0888-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-010-0888-1

Keywords

Search

Navigation