, Volume 25, Issue 6, pp 3423–3434 | Cite as

Water retention value predicts biomass recalcitrance for pretreated lignocellulosic materials across feedstocks and pretreatment methods

  • Noah D. WeissEmail author
  • Claus Felby
  • Lisbeth G. Thygesen
Original Paper


Understanding the causes of lignocellulosic biomass recalcitrance is necessary for developing robust biomass conversion processes for fuels and chemicals. A key factor in biomass recalcitrance is the physical and chemical relationship between biomass and water. Water is known to be important for enzymatic hydrolysis both because it is a co-substrate for cellulose hydrolysis, but also because it acts as a swelling agent that allows enzymes access to the substrate. It has been shown that the water retention value, and water constraint as measured by spin–spin low field NMR (T2 LFNMR) techniques, correlated to biomass recalcitrance for similar lignocellulosic materials pretreated at different severities. In this work, water retention and water constraint was measured across species and pretreatment methods and compared to the enzymatic digestibility of the cellulose fraction. There is an overall positive correlation between the water retention value and glucose hydrolysis yields. Average water constraint in the samples (as represented by monocomponent T2 decay times) could not be correlated to biomass recalcitrance; however a relationship was found between the relative amount of more highly constrained water measured and hydrolysis performance. Feedstock heterogeneity and differences in sample morphology may account for the variation in the sample set. Further research is needed to develop these predictive methods, but can be applied with good accuracy on specific feedstock types or for specific pretreatment methods.


Water retention value Biomass recalcitrance Pretreatment Water constraint Biomass-water interactions Pretreatment LFNMR 



We would like to thank the following people and institutions for kindly providing materials for this study. Erik Kuhn and Dan Schell at the National Renewable Energy Laboratory, Mads Pedersen from BioGasol A/S, Henning Jørgensen from University of Copenhagen, Jack Saddler and Richard Chandra from the University of British Columbia, Demi Tristan Djajadi at the Technical University of Denmark, and Mats Galbe and Christian Roslander from Lund University. We would also like to thank Novozymes A/S for providing the enzymes for this study, and the BioValue project for funding this research.


This research was funded by the Strategic Platforms for Innovation and Research fund, which is managed by the Danish Innovation Fund, as part of the project entitled BioValue.

Compliance with ethical standards

Conflict of interest

The Authors declare that they have no conflicts of interest. All authors are wholly employed by the University of Copenhagen.


  1. Agbor VB, Cicek N, Sparling R et al (2011) Biomass pretreatment: fundamentals toward application. Biotechnol Adv 29:675–685. CrossRefPubMedGoogle Scholar
  2. Alvira P, Tomás-Pejó E, Ballesteros M, Negro MJ (2010) Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour Technol 101:4851–4861. CrossRefPubMedGoogle Scholar
  3. Araujo CD, MacKay AL, Hailey JRT et al (1992) Proton magnetic resonance techniques for characterization of water in wood: application to white spruce. Wood Sci Technol 26:101–113. CrossRefGoogle Scholar
  4. Araujo CD, Mackay AL, Whittall KP, Hailey JRT (1993) A diffusion model for spin–spin relaxation of compartmentalized water in wood. J Magn Reson B 101:248–261. CrossRefGoogle Scholar
  5. Baroi GN, Skiadas IV, Westermann P, Gavala HN (2015) Continuous fermentation of wheat straw hydrolysate by clostridium tyrobutyricum with in-situ acids removal. Waste Biomass Valorization 6:317–326. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Beckham GT, Matthews JF, Peters B et al (2011) Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs. J Phys Chem B 115:4118–4127. CrossRefPubMedGoogle Scholar
  7. Carles JE, Scallan AM (1973) The determination of the amount of bound water within cellulosic gels by NMR spectroscopy. J Appl Polym Sci 17:1855–1865. CrossRefGoogle Scholar
  8. Cox J, McDonald PJ, Gardiner BA (2010) A study of water exchange in wood by means of 2D NMR relaxation correlation and exchange. Holzforschung 64:259–266. CrossRefGoogle Scholar
  9. Dasari RK, Eric Berson R (2007) The effect of particle size on hydrolysis reaction rates and rheological properties in cellulosic slurries. Appl Biochem Biotechnol 137–140:289–299. CrossRefPubMedGoogle Scholar
  10. Del Rio LF, Chandra RP, Saddler JN (2011) The effects of increasing swelling and anionic charges on the enzymatic hydrolysis of organosolv-pretreated softwoods at low enzyme loadings. Biotechnol Bioeng 108:1549–1558. CrossRefPubMedGoogle Scholar
  11. DeMartini JD, Pattathil S, Miller JS et al (2013) Investigating plant cell wall components that affect biomass recalcitrance in poplar and switchgrass. Energy Environ Sci 6:898–909. CrossRefGoogle Scholar
  12. Djajadi DT, Hansen AR, Jensen A et al (2017) Surface properties correlate to the digestibility of hydrothermally pretreated lignocellulosic Poaceae biomass feedstocks. Biotechnol Biofuels 10:49. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Felby C, Thygesen LG, Kristensen JB et al (2008) Cellulose–water interactions during enzymatic hydrolysis as studied by time domain NMR. Cellulose 15:703–710. CrossRefGoogle Scholar
  14. Flibotte S, Menon RS, MacKay AL, Hailey JRT (2007) Proton magnetic resonance of western red cedar. Wood Fiber Sci 22:362–376Google Scholar
  15. Foston M, Ragauskas AJ (2010) Changes in the structure of the cellulose fiber wall during dilute acid pretreatment in populus studied by 1H and 2H NMR. Energy Fuels 24:5677–5685. CrossRefGoogle Scholar
  16. Fredriksson M, Thygesen LG (2016) The states of water in Norway spruce (Picea abies (L.) Karst.) studied by low-field nuclear magnetic resonance (LFNMR) relaxometry: assignment of free-water populations based on quantitative wood anatomy. Holzforschung 71:77–90. CrossRefGoogle Scholar
  17. Froix MF, Nelson R (1975) The interaction of water with cellulose from nuclear magnetic resonance relaxation times. Macromolecules 8:726–730. CrossRefGoogle Scholar
  18. Grethlein HE (1985) The effect of pore size distribution on the rate of enzymatic hydrolysis of cellulosic substrates. Nat Biotechnol 3:155–160. CrossRefGoogle Scholar
  19. Haghighi Mood S, Hossein Golfeshan A, Tabatabaei M et al (2013) Lignocellulosic biomass to bioethanol, a comprehensive review with a focus on pretreatment. Renew Sustain Energy Rev 27:77–93. CrossRefGoogle Scholar
  20. Himmel ME, Ding S-Y, Johnson DK et al (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315:804–807. CrossRefPubMedGoogle Scholar
  21. Hsieh CC, Cannella D, Jørgensen H et al (2014) Cellulase inhibition by high concentrations of monosaccharides. J Agric Food Chem 62:3800–3805. CrossRefPubMedGoogle Scholar
  22. Iii JS, Kuhn EM, Nagle NJ et al (2014) Characterization of pilot-scale dilute acid pretreatment performance using deacetylated corn stover. Biotechnol Biofuels 7:23. CrossRefGoogle Scholar
  23. Kabel MA, Bos G, Zeevalking J et al (2007) Effect of pretreatment severity on xylan solubility and enzymatic breakdown of the remaining cellulose from wheat straw. Bioresour Technol 98:2034–2042. CrossRefPubMedGoogle Scholar
  24. Karimi K, Taherzadeh MJ (2016) A critical review on analysis in pretreatment of lignocelluloses: degree of polymerization, adsorption/desorption, and accessibility. Bioresour Technol 203:348–356. CrossRefPubMedGoogle Scholar
  25. Kristensen JB, Felby C, Jørgensen H (2009a) Determining yields in high solids enzymatic hydrolysis of biomass. Appl Biochem Biotechnol 156:127–132. CrossRefPubMedGoogle Scholar
  26. Kristensen JB, Felby C, Jørgensen H (2009b) Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnol Biofuels 2:11. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Luo XL, Zhu JY, Gleisner R, Zhan HY (2011) Effects of wet-pressing-induced fiber hornification on enzymatic saccharification of lignocelluloses. Cellulose 18:1055–1062. CrossRefGoogle Scholar
  28. Lv S, Yu Q, Zhuang X et al (2013) The influence of hemicellulose and lignin removal on the enzymatic digestibility from sugarcane bagasse. BioEnergy Res 6:1128–1134. CrossRefGoogle Scholar
  29. Meiboom S, Gill D (1958) Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 29:688–691. CrossRefGoogle Scholar
  30. Meng X, Ragauskas AJ (2014) Recent advances in understanding the role of cellulose accessibility in enzymatic hydrolysis of lignocellulosic substrates. Curr Opin Biotechnol 27:150–158. CrossRefPubMedGoogle Scholar
  31. Menon RS, MaCkay AL, Hailey JRT et al (1987) An NMR determination of the physiological water distribution in wood during drying. J Appl Polym Sci 33:1141–1155. CrossRefGoogle Scholar
  32. Mosier N (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 96:673–686. CrossRefPubMedGoogle Scholar
  33. Ohgren K, Bura R, Saddler J, Zacchi G (2007) Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover. Bioresour Technol 98:2503–2510. CrossRefPubMedGoogle Scholar
  34. Pan X, Xie D, Yu RW et al (2007) Pretreatment of lodgepole pine killed by mountain pine beetle using the ethanol organosolv process: fractionation and process optimization. Ind Eng Chem Res 46:2609–2617. CrossRefGoogle Scholar
  35. Pönni R, Vuorinen T, Kontturi E (2012) Proposed nano-scale coalescence of cellulose in chemical pulp fibers during technical treatments. BioResources 7:6077–6108. CrossRefGoogle Scholar
  36. Pu Y, Hu F, Huang F et al (2013) Assessing the molecular structure basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments. Biotechnol Biofuels 6:15. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Roberts KM, Lavenson DM, Tozzi EJ et al (2011) The effects of water interactions in cellulose suspensions on mass transfer and saccharification efficiency at high solids loadings. Cellulose 18:759–773. CrossRefGoogle Scholar
  38. Scallan AM, Carles JE (1972) The correlation of the water retention value with the fibre saturation point. Sven Papperstidning 75:699–703Google Scholar
  39. Selig MJ, Thygesen LG, Johnson DK et al (2013) Hydration and saccharification of cellulose Iβ, II and IIII at increasing dry solids loadings. Biotechnol Lett 35:1599–1607. CrossRefPubMedGoogle Scholar
  40. Selig MJ, Thygesen LG, Felby C (2014) Correlating the ability of lignocellulosic polymers to constrain water with the potential to inhibit cellulose saccharification. Biotechnol Biofuels 7:159. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Sluiter A, Hames B, Ruiz R et al (2008) Determination of structural carbohydrates and lignin in biomass. Lab Anal Proced 1617:1–16Google Scholar
  42. Tian D, Chandra RP, Lee J-S et al (2017) A comparison of various lignin-extraction methods to enhance the accessibility and ease of enzymatic hydrolysis of the cellulosic component of steam-pretreated poplar. Biotechnol Biofuels 10:157. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Tsuchida JE, Rezende CA, de Oliveira-Silva R et al (2014) Nuclear magnetic resonance investigation of water accessibility in cellulose of pretreated sugarcane bagasse. Biotechnol Biofuels 7:127. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Weiss ND, Thygesen LG, Felby C et al (2016) Biomass-water interactions correlate to recalcitrance and are intensified by pretreatment: an investigation of water constraint and retention in pretreated spruce using low field NMR and water retention value techniques. Biotechnol Prog. CrossRefPubMedGoogle Scholar
  45. Whittall KP, Bronskill MJ, Henkelman RM (1991) Investigation of analysis techniques for complicated NMR relaxation data. J Magn Reson (1969) 95:221–234. CrossRefGoogle Scholar
  46. Williams DL, Hodge DB (2014) Impacts of delignification and hot water pretreatment on the water induced cell wall swelling behavior of grasses and its relation to cellulolytic enzyme hydrolysis and binding. Cellulose 21:221–235. CrossRefGoogle Scholar
  47. Williams DL, Crowe JD, Ong RG, Hodge DB (2017) Water sorption in pretreated grasses as a predictor of enzymatic hydrolysis yields. Bioresour Technol. CrossRefPubMedGoogle Scholar
  48. Zhao X, Zhang L, Liu D (2012a) Biomass recalcitrance. Part I: the chemical compositions and physical structures affecting the enzymatic hydrolysis of lignocellulose. Biofuels Bioprod Biorefin 6:465–482. CrossRefGoogle Scholar
  49. Zhao X, Zhang L, Liu D (2012b) Biomass recalcitrance. Part II: fundamentals of different pre-treatments to increase the enzymatic digestibility of lignocellulose. Biofuels Bioprod Biorefin 6:561–579. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Geosciences and Natural Resource ManagementUniversity of CopenhagenFrederiksbergDenmark

Personalised recommendations