Wood Science and Technology

, Volume 53, Issue 1, pp 49–69 | Cite as

Improving enzymatic saccharification of Eucalyptus grandis branches by ozone pretreatment

  • Silvia Layara Floriani Andersen
  • Rafael Castoldi
  • Jessica A. A. Garcia
  • Adelar Bracht
  • Rosely A. Peralta
  • Edson Alves de Lima
  • Cristiane Vieira Helm
  • Regina de Fátima Peralta Muniz MoreiraEmail author
  • Rosane Marina PeraltaEmail author


Ozonolysis is potentially an effective method for pretreating lignocellulosic biomass to improve the production of fermentable sugars via enzymatic hydrolysis. The eliminated branches from eucalyptus trees can represent a production of around 30 million m3 of lignocellulosic material annually only in Brazil. Attempts of developing strategies for a rational use of this biomass are, thus, welcome. In this study, Eucalyptus grandis branches were pretreated with ozone in an attempt to increase enzymatic saccharification. Ozonolysis resulted in the degradation of lignin with negligible losses of cellulose and small losses of hemicellulose. Reduction in the lignin content from 26.63 to 9.53% already resulted in the maximal improvement of the saccharification yield (from 20 to 68%). The results indicate that ozone pretreatment can be a promising way of increasing the enzymatic digestibility of eucalyptus sawdust from eliminated branches of trees for its conversion into fermentable sugars.

List of symbols


Crystallinity index (%)


Diffusion coefficient (m2 s−1)


Particle diameter (mm)


Reduction potential (V)


Intensity of the amorphous peak at 2θ = 18° (counts)


Intensity of the crystalline peak at 2θ = 22° (counts)


Bed length (m)


Peclet number (dimensionless)


Reynolds number (dimensionless)


Reducing sugars (mg g−1)


Saccharification yield (%)


Total content of holocellulose (mg g −1)


O3 velocity (m s−1)


Dynamic viscosity of O3 (kg m−1 s−1)


Density of O3 (kg m3)



The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (Proc. 477825/2012-5 and Proc. 3079/2015-8) for funding this study. A. Bracht, R .F. Peralta-Muniz-Moreira, R. A. Peralta and R. M. Peralta are research grant recipients of CNPq.


  1. Agnihotri S, Johnsen IA, Bøe MS, Øyaas K, Moe S (2015) Ethanol organosolv pretreatment of softwood (Picea abies) and sugarcane bagasse for biofuel and biorefinery applications. Wood Sci Technol 49:881–896CrossRefGoogle Scholar
  2. Barrera-Martínez A, Guzmán N, Peña E, Vázquez T, Cerón-Camacho R, Folch J, Salazar JAH, Aburto J (2016) Ozonolysis of alkaline lignin and sugarcane bagasse: structural changes and their effect on saccharification. Biomass Bioenergy 94:167–172CrossRefGoogle Scholar
  3. Ben’ko EM, Manisova OR, Murav’eva GP, Lunin VV (2013) Structural changes in wood during ozonation. Russ J Phys Chem A 87:097–1101Google Scholar
  4. Buzala KP, Kalinowska H, Przybysz P, Małachowska E (2017) Conversion of various types of lignocellulosic biomass to fermentable sugars using kraft pulping and enzymatic hydrolysis. Wood Sci Technol 51:873–885CrossRefGoogle Scholar
  5. Carrier M, Loppinet-Serani A, Denux D, Lasnier J-M, Ham-Pichavant F, Cansell F, Aymonier C (2011) Thermogravimetric analysis as a new method to determine the lignocellulosic composition of biomass. Biomass Bioenergy 35:298–307CrossRefGoogle Scholar
  6. Carvalho DM, Sevastyanova O, Penna LS, Silva BP, Lindström ME, Colodette JL (2015) Assessment of chemical transformations in eucalyptus, sugarcane bagasse and straw during hydrothermal, dilute acid, and alkaline pretreatments. Ind Crop Prod 73:118–126CrossRefGoogle Scholar
  7. Carvalho DM, Queiroz JH, Colodette JL (2016) Assessment of alkaline pretreatment for the production of bioethanol from eucalyptus, sugarcane bagasse and sugarcane straw. Ind Crop Prod 94:932–941CrossRefGoogle Scholar
  8. Casas MV, Alonso M, Oliet E, Rojo F, Rodríguez F (2012) FTIR analysis of lignin regenerated from Pinus radiata and Eucalyptus globulus woods dissolved in imidazolium-based ionic liquids. J Chem Technol Biotechnol 87:472–480CrossRefGoogle Scholar
  9. Castoldi R, Bracht A, Morais GR, Baesso ML, Correa RCG, Peralta RA, Moreira RFPM, Polizeli MLTM, Souza CGM, Peralta RM (2014) Biological pretreatment of Eucalyptus grandis sawdust with white-rot fungi: study of degradation patterns and saccharification kinetics. Chem Eng J 258:240–246CrossRefGoogle Scholar
  10. Castoldi R, Correa VG, Morais CR, Souza CGM, Bracht A, Peralta RA, Peralta-Muniz-Moreira RF, Peralta RM (2017) Liquid nitrogen pretreatment of eucalyptus sawdust and rice hull for enhanced enzymatic saccharification. Bioresour Technol 224:648–655CrossRefGoogle Scholar
  11. Da Silva JCG, Alves JLF, Galdino WVA, Andersen SLF, de Sena RF (2018) Pyrolysis kinetic evaluation by single-step for waste wood from reforestation. Waste Manag 72:265–273CrossRefGoogle Scholar
  12. Dong BY, Chen YF, Zhao CC, Zhang SJ, Guo XW, Xiao DG (2013) Simultaneous determination of furfural, acetic acid and 5-hydroxymethylfurfural in corncob hydrolysates using liquid chromatography with ultraviolet detection. J AOAC Int 96:1239–1244CrossRefGoogle Scholar
  13. Garcia-Cubero MT, Palacín LG, González-Benito G, Bolado S, Lucas S, Coca M (2012) An analysis of lignin removal in a fixed bed reactor by reaction of cereal straws with ozone. Bioresour Technol 107:229–234CrossRefGoogle Scholar
  14. Hemraj-Benny T, Bandosz TJ, Wong SS (2008) Effect of ozonolysis on the pore structure, surface chemistry, and bundling of single-walled carbon nanotubes. J Colloid Interface Sci 317:375–382CrossRefGoogle Scholar
  15. Jang J-H, Lee S-H, Endo T, Kim N-H (2013) Characteristics of microfibrillated cellulosic fibers and paper sheets from Korean white pine. Wood Sci Technol 47:925–937CrossRefGoogle Scholar
  16. Jönsson LJ, Martin C (2016) Pretreatment of lignocellulose: formation of inhibitory by-products and strategies for minimizing their effects. Bioresour Technol 199:103–112CrossRefGoogle Scholar
  17. Jönsson LJ, Abiksson B, Nilvebrant N-O (2013) Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnol Biofuels 6:16–25CrossRefGoogle Scholar
  18. Kojima Y, Yoon S-L (2008) Improved enzymatic hydrolysis of waste paper by ozone pretreatment. J Mater Cycles Waste Manag 10:134–139CrossRefGoogle Scholar
  19. Lee JM, Jameel H, Venditti RA (2010) Effect of ozone and autohydrolysis pretreatments on enzymatic digestibility of coastal Bermuda grass. BioResources 5:1084–1101Google Scholar
  20. Lienqueo ME, Ravanal MC, Pezoa-Conte R, Cortínez V, Martínez L, Niklitschek T, Salazar O, Carmona R, García A, Hyvärinen S, Mäki-Arvela P, Mikkola J-P (2016) Second generation bioethanol from Eucalyptus globulus Labill and Nothofagus pumilio: ionic liquid pretreatment boosts the yields. Ind Crop Prod 80:148–155CrossRefGoogle Scholar
  21. Mamleeva NA, Autlov SA, Bazarnova NG, Lunin VV (2009) Delignification of softwood by ozonation. Pure Appl Chem 81:2081–2091CrossRefGoogle Scholar
  22. Mamleeva NA, Autlov SA, Barzanova NG, Lunin VV (2016) Degradation of polysaccharides and lignin in wood ozonation. Russ J Bioorg Chem 42:694–699CrossRefGoogle Scholar
  23. Martino DC, Colodette JL, Chandra R, Saddler J (2017) Steam explosion pretreatment used to remove hemicellulose to enhance the production of a eucalyptus organosolv dissolving pulp. Wood Sci Technol 51:557–569CrossRefGoogle Scholar
  24. Martins A, Cardoso AL, Stahl JA, Diniz J (2007) Low temperature conversion of rice husks, eucalyptus sawdust and peach stones for the production of carbon-like adsorbent. Bioresour Technol 98:1095–1100CrossRefGoogle Scholar
  25. Martin-Sampedro R, Revilla E, Villar JC, Eugenio ME (2014) Enhancement of enzymatic saccharification of Eucalyptus globulus steam explosion versus steam treatment. Bioresour Technol 167:186–191CrossRefGoogle Scholar
  26. Matsushita Y, Yamauchi K, Takabe K, Awano T, Yoshinaga MA, Kato M, Kobayashi T, Asada T, Furujyo A, Fukushim K (2010) Enzymatic saccharification of Eucalyptus bark using hydrothermal pretreatment with carbon dioxide. Bioresour Technol 101:4936–4939CrossRefGoogle Scholar
  27. Miller GL (1959) Dinitrosalicylic acid reagent for determination of reducing sugars. Anal Chem 31:426–428CrossRefGoogle Scholar
  28. Miura T, Lee S-H, Inoue S, Endo T (2012) Combined pretreatment using ozonolysis and wet-disk milling to improve enzymatic saccharification of Japanese cedar. Bioresour Technol 126:182–186CrossRefGoogle Scholar
  29. Pandey KK, Pitman AJ (2003) FT-IR studies of the changes in wood chemistry following decay by brown-rot and white-rot fungi. Int Biodeterior Biodegrad 52:151–160CrossRefGoogle Scholar
  30. Peciulyte A, Karlstrom K, Larsson PT, Olsson L (2015) Impact of the supramolecular structure of cellulose on the efficiency of enzymatic hydrolysis. Biotechnol Biofuels 8:56–69CrossRefGoogle Scholar
  31. Peretz R, Gerchman Y, Mamame H (2017) Ozonation of tannic acid to model biomass pretreatment for bioethanol production. Bioresour Technol 241:1060–1066CrossRefGoogle Scholar
  32. Rastegara SO, Gu T (2017) Empirical correlations for axial dispersion coefficient and Peclet number in fixed-bed columns. J Chromatogr A 1490:33–137Google Scholar
  33. Rico A, Rencoret J, del Rio JC, Martinez AT, Gutiérrez A (2014) Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock. Biotechnol Biofuels 7:6–20CrossRefGoogle Scholar
  34. Rollin JA, Zhu Z, Sathitsuksanoh N, Zhang Y-HP (2011) Increasing cellulose accessibility is more important than removing lignin: a comparison of cellulose solvent-Based lignocellulose fractionation and soaking in aqueous ammonia. Biotechnol Bioeng 108:22–30CrossRefGoogle Scholar
  35. Romani A, Garrote G, Alonso JL, Parajó JC (2010) Bioethanol production from hydrothermally pretreated Eucalyptus globulus wood. Bioresour Technol 101:8706–8712CrossRefGoogle Scholar
  36. Segal L, Creely JJ, Martin AE Jr, Conrad CM (1962) An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Text Res J 29:786–794CrossRefGoogle Scholar
  37. Shi F, Xiang H, Li Y (2015) Combined pretreatment using ozonolysis and ball milling to improve enzymatic saccharification of corn straw. Bioresour Technol 179:414–451CrossRefGoogle Scholar
  38. Silva MR, Machado GO, Deiner J, Calil C Jr (2010) Permeability measurements of Brazilian Eucalyptus. Mater Res 13:281–286CrossRefGoogle Scholar
  39. Singh S, Varanasi P, Singh P, Adams PD, Auer M, Simmons BA (2013) Understanding the impact of ionic liquid pretreatment on cellulose and lignin via thermochemical analysis. Biomass Bioenergy 54:276–283CrossRefGoogle Scholar
  40. Singh S, Cheng G, Sathitsuksanoh N, Wu D, Varansi P, George A, Balan V, Gao X, Kumar R, Dale BE, Wyman CE, Simmons BA (2015) Comparison of different biomass pretreatment techniques and their impact on chemistry and structure. Front Energy Res 2:1–12CrossRefGoogle Scholar
  41. Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 16:144–158Google Scholar
  42. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2008) Determination of structural carbohydrates and lignin in biomass (LAP). NREL, Golden, CoGoogle Scholar
  43. Sudiyani Y, Imamura Y, Doi S, Yamauchi S (2003) Infrared spectroscopic investigations of weathering effects on the surface of tropical wood. J Wood Sci 49:86–92CrossRefGoogle Scholar
  44. Teramoto Y, Tanaka N, Lee S-H, Endo T (2008) Pretreatment of eucalyptus wood chips for enzymatic saccharification using combined sulfuric acid-free ethanol cooking and ball milling. Biotechnol Bioeng 99:75–85CrossRefGoogle Scholar
  45. Travaini R, Otero MDM, Coca M, Da-Silva R, Bolado S (2013) Sugarcane bagasse ozonolysis pretreatment: effect on enzymatic digestibility and inhibitory compound formation. Bioresour Technol 133:332–339CrossRefGoogle Scholar
  46. Travaini R, Martín-Juárez J, Lorenzo-Hernando A, Bolado-Rodríguez S (2016) Ozonolysis: an advantageous pretreatment for lignocellulosic biomass revisited. Bioresour Technol 199:2–12CrossRefGoogle Scholar
  47. Vidal PF, Molinier J (1988) Ozonolysis of lignin—improvement of in vitro digestibility of poplar sawdust. Biomass 16:1–17CrossRefGoogle Scholar
  48. Wei W, Wu S, Liu L (2012) Enzymatic saccharification of dilute acid pretreated eucalyptus chips for fermentable sugar production. Bioresour Technol 110:302–307CrossRefGoogle Scholar
  49. Ximenes E, Kim Y, Mosier NB, Dien B, Ladisch M (2011) Deactivation of cellulases by phenols. Enzyme Microb Technol 48:54–60CrossRefGoogle Scholar
  50. Xu F, Yu J, Tesso T, Dowell F, Wang D (2013) Qualitative and quantitative analysis of lignocellulosic biomass using infrared techniques: a mini-review. Appl Energy 104:801–809CrossRefGoogle Scholar
  51. Yáñez JSM, Rojas J, Castro J, Ragauskas A, Baeza J, Freer J (2013) Fuel ethanol production from Eucalyptus globulus wood by autocatalyzed organosolv pretreatment ethanol–water and SSF. J Chem Technol Biotechnol 88:39–48CrossRefGoogle Scholar
  52. Yu H, Zhang X, Song L, Ke J, Xu C, Du W, Zhang J (2010) Evaluation of white-rot fungi assisted alkaline/oxidative pretreatment of corn straw undergoing enzymatic hydrolysis by cellulase. J Biosci Bioeng 110:660–664CrossRefGoogle Scholar
  53. Yunos NSHM, Baharuddin AS, Yunos KFM, Naim MN, Nishida H (2012) Physicochemical property changes of oil palm mesocarp fibers treated with high-pressure steam. BioResources 7:5983–5994CrossRefGoogle Scholar
  54. Zhang X, Yu H, Huang H, Liu Y (2007) Evaluation of biological pretreatment with white rot fungi for the enzymatic hydrolysis of bamboo culms. Int Biodeterior Biodegrad 60:159–164CrossRefGoogle Scholar
  55. Zhang J, Feng L, Wang D, Zhang R, Liu G, Cheng G (2014) Thermogravimetric analysis of lignocellulosic biomass with ionic liquid pretreatment. Bioresour Technol 153:379–382CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Silvia Layara Floriani Andersen
    • 1
    • 2
  • Rafael Castoldi
    • 3
  • Jessica A. A. Garcia
    • 3
  • Adelar Bracht
    • 3
  • Rosely A. Peralta
    • 4
  • Edson Alves de Lima
    • 5
  • Cristiane Vieira Helm
    • 5
  • Regina de Fátima Peralta Muniz Moreira
    • 1
    Email author
  • Rosane Marina Peralta
    • 3
    Email author
  1. 1.Laboratory of Energy and the Environment, Chemical and Food Engineering DepartmentFederal University of Santa CatarinaFlorianópolisBrazil
  2. 2.Department of Renewable Energy Engineering, Center of Alternative and Renewable EnergyFederal University of ParaíbaJoão PessoaBrazil
  3. 3.Department of BiochemistryState University of MaringáMaringáBrazil
  4. 4.Chemical DepartmentFederal University of Santa CatarinaFlorianópolisBrazil
  5. 5.Embrapa-FlorestasColomboBrazil

Personalised recommendations