BioEnergy Research

, Volume 9, Issue 2, pp 559–565 | Cite as

Use of Ascomycete Extracts in Enzymatic Cocktail Formulations Increases Sugar Cane Bagasse Hydrolysis

  • Diogo RoblEmail author
  • Patrícia dos Santos Costa
  • Sarita Candida Rabelo
  • Priscila da Silva Delabona
  • Deise Juliana da Silva Lima
  • Gabriel Padilla
  • José Geraldo da Cruz Pradella


Hemicellulases and accessory enzymes are essential for supplementation of cellulolytic enzyme extracts, and combinations of these enzymes can lead to high performance in plant biomass hydrolysis. In this work, enzyme extracts rich in hemicellulases and β-glucosidase, produced by the unique ascomycete strains Annulohypoxylon stygium DR47 and Aspergillus niger DR02, were tested for use in formulations with Celluclast 1.5 L. Statistical analysis showed that a mixture based on these enzymes was able to increase the hydrolysis of hydrothermally pretreated sugar cane bagasse. The two A. stygium extracts only effectively increased glucose release when they were combined. These extracts had no positive effect when used together with the A. niger extract, and the findings suggested that a blend based on the commercial cellulose preparation and the xylanase-rich extract from A. niger provided the best carbohydrate solubilization. Supplementation at low cellulolytic loading resulted in 120 and 238 % increases in cellulose and hemicellulose hydrolysis yields.


Aspergillus niger Annulohypoxylon stygium Xylanase Accessory enzymes Hydrothermal pretreatment Sugar cane bagasse 



The authors thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support and the National Laboratory of Science and Technology of Bioethanol (CTBE) for the technical assistance.

Supplementary material

12155_2016_9721_MOESM1_ESM.docx (79 kb)
ESM 1 (DOCX 79 kb)


  1. 1.
    Macrelli S, Mogensen J, Zacchi G (2012) Techno-economic evaluation of 2nd generation bioethanol production from sugar cane bagasse and leaves integrated with the sugar-based ethanol process. Biotechnol Biofuels 5(1):1–18CrossRefGoogle Scholar
  2. 2.
    Goldbeck R, Damásio AR, Gonçalves TA, Machado CB, Paixão DA, Wolf LD, Mandelli F, Rocha GJ, Ruller R, Squina FM (2014) Development of hemicellulolytic enzyme mixtures for plant biomass deconstruction on target biotechnological applications. App Microbiol Biotechnol 1-13Google Scholar
  3. 3.
    Berlin A, Maximenko V, Gilkes N, Saddler J (2007) Optimization of enzyme complexes for lignocellulose hydrolysis. Biotechnol Bioeng 97(2):287–296. doi: 10.1002/bit.21238 CrossRefGoogle Scholar
  4. 4.
    Fortes Gottschalk LM, Oliveira RA, da Silva Bon EP (2010) Cellulases, xylanases, beta-glucosidase and ferulic acid esterase produced by Trichoderma and Aspergillus act synergistically in the hydrolysis of sugarcane bagasse. Biochem Eng J 51(1-2):72–78. doi: 10.1016/j.bej.2010.05.003 CrossRefGoogle Scholar
  5. 5.
    Gao D, Chundawat SP, Krishnan C, Balan V, Dale BE (2010) Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover. Bioresour Technol 101(8):2770–2781. doi: 10.1016/j.biortech.2009.10.056 CrossRefGoogle Scholar
  6. 6.
    Rocha GJM, Goncalves AR, Oliveira BR, Olivares EG, Rossell CEV (2012) Steam explosion pretreatment reproduction and alkaline delignification reactions performed on a pilot scale with sugarcane bagasse for bioethanol production. Ind Crop Prod 35(1):274–279. doi: 10.1016/j.indcrop.2011.07.010 CrossRefGoogle Scholar
  7. 7.
    de Souza AP, Leite DC, Pattathil S, Hahn MG, Buckeridge MS (2013) Composition and structure of sugarcane cell wall polysaccharides: implications for second-generation bioethanol production. Bioenergy Res 6(2):564–579CrossRefGoogle Scholar
  8. 8.
    Pel HJ, de Winde JH, Archer DB, Dyer PS, Hofmann G, Schaap PJ, Turner G, de Vries RP, Albang R, Albermann K (2007) Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat Biotechnol 25(2):221–231CrossRefGoogle Scholar
  9. 9.
    de Vries RP, Visser J (2001) Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiol Mol Biol Rev 65(4):497. doi: 10.1128/mmbr.65.4.497-522.2001 CrossRefGoogle Scholar
  10. 10.
    Robl D, da Silva Delabona P, dos Santos Costa P, da Silva Lima DJ, Rabelo SC, Pimentel IC, Büchli F, Squina FM, Padilla G, da Cruz Pradella JG (2015) Xylanase production by endophytic Aspergillus niger using pentose-rich hydrothermal liquor from sugarcane bagasse. Biocatal Biotransform 1-13Google Scholar
  11. 11.
    Robl D, Delabona Pda S, Mergel CM, Rojas JD, Costa Pdos S, Pimentel IC, Vicente VA, da Cruz Pradella JG, Padilla G (2013) The capability of endophytic fungi for production of hemicellulases and related enzymes. BMC Biotechnol 13:94. doi: 10.1186/1472-6750-13-94 CrossRefGoogle Scholar
  12. 12.
    Berlin A, Gilkes N, Kilburn D, Bura R, Markov A, Skomarovsky A, Okunev O, Gusakov A, Maximenko V, Gregg D (2005) Evaluation of novel fungal cellulase preparations for ability to hydrolyze softwood substrates—evidence for the role of accessory enzymes. Enzym Microb Technol 37(2):175–184CrossRefGoogle Scholar
  13. 13.
    Matias de Oliveira D, Finger-Teixeira A, Rodrigues Mota T, Salvador VH, Moreira‐Vilar FC, Correa Molinari HB, Mitchell C, Andrew R, Marchiosi R, Ferrarese‐Filho O (2014) Ferulic acid: a key component in grass lignocellulose recalcitrance to hydrolysis. Plant Biotechnol JGoogle Scholar
  14. 14.
    Delabona Pda S, Cota J, Hoffmam ZB, Paixao DA, Farinas CS, Cairo JP, Lima DJ, Squina FM, Ruller R, Pradella JG (2013) Understanding the cellulolytic system of Trichoderma harzianum P49P11 and enhancing saccharification of pretreated sugarcane bagasse by supplementation with pectinase and alpha-L-arabinofuranosidase. Bioresour Technol 131:500–507. doi: 10.1016/j.biortech.2012.12.105 CrossRefGoogle Scholar
  15. 15.
    Robl D, dos Santos Costa P, Büchli F, da Silva Lima DJ, da Silva Delabona P, Squina FM, Pimentel IC, Padilla G, da Cruz Pradella JG (2015) Enhancing of sugar cane bagasse hydrolysis by Annulohypoxylon stygium glycohydrolases. Bioresour Technol 177:247–254CrossRefGoogle Scholar
  16. 16.
    Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59(2):257–268. doi: 10.1351/pac198759020257 CrossRefGoogle Scholar
  17. 17.
    Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428. doi: 10.1021/ac60147a030 CrossRefGoogle Scholar
  18. 18.
    Mamma D, Kourtoglou E, Christakopoulos P (2008) Fungal multienzyme production on industrial by-products of the citrus-processing industry. Bioresour Technol 99(7):2373–2383. doi: 10.1016/j.biortech.2007.05.018 CrossRefGoogle Scholar
  19. 19.
    Tabka M, Herpoël-Gimbert I, Monod F, Asther M, Sigoillot J (2006) Enzymatic saccharification of wheat straw for bioethanol production by a combined cellulase xylanase and feruloyl esterase treatment. Enzym Microb Technol 39(4):897–902CrossRefGoogle Scholar
  20. 20.
    Bussamra BC, Freitas S, da Costa AC (2015) Improvement on sugar cane bagasse hydrolysis using enzymatic mixture designed cocktail. Bioresour Technol 187:173–181CrossRefGoogle Scholar
  21. 21.
    Karnchanatat A, Petsom A, Sangvanich P, Piaphukiew J, Whalley AJS, Reynolds CD, Sihanonth P (2007) Purification and biochemical characterization of an extracellular beta-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.: Fr.) Rehm. FEMS Microbiol Lett 270(1):162–170. doi: 10.1111/j.1574-6968.2007.00662.x CrossRefGoogle Scholar
  22. 22.
    Chauve M, Mathis H, Huc D, Casanave D, Monot F, Ferreira NL (2010) Comparative kinetic analysis of two fungal β-glucosidases. Biotechnol Biofuels 3(1):3CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Diogo Robl
    • 1
    • 2
    Email author
  • Patrícia dos Santos Costa
    • 2
  • Sarita Candida Rabelo
    • 2
  • Priscila da Silva Delabona
    • 2
  • Deise Juliana da Silva Lima
    • 2
  • Gabriel Padilla
    • 1
  • José Geraldo da Cruz Pradella
    • 2
  1. 1.Institute of Biomedical SciencesUniversity of São Paulo (USP)São PauloBrazil
  2. 2.Brazilian Bioethanol Science and Technology Laboratory (CTBE)Brazilian Centre of Research in Energy and Materials (CNPEM)CampinasBrazil

Personalised recommendations