BioEnergy Research

, Volume 6, Issue 1, pp 211–221 | Cite as

Two-Dimensional NMR Evidence for Cleavage of Lignin and Xylan Substituents in Wheat Straw Through Hydrothermal Pretreatment and Enzymatic Hydrolysis

  • Daniel J. Yelle
  • Prasad Kaparaju
  • Christopher G. Hunt
  • Kolby Hirth
  • Hoon Kim
  • John Ralph
  • Claus Felby


Solution-state two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy of plant cell walls is a powerful tool for characterizing changes in cell wall chemistry during the hydrothermal pretreatment process of wheat straw for second-generation bioethanol production. One-bond 13C–1H NMR correlation spectroscopy, via an heteronuclear single quantum coherence experiment, revealed substantial lignin β-aryl ether cleavage, deacetylation via cleavage of the natural acetates at the 2-O- and 3-O-positions of xylan, and uronic acid depletion via cleavage of the (1 → 2)-linked 4-O-methyl-α-d-glucuronic acid of xylan. In the polysaccharide anomeric region, decreases in the minor β-d-mannopyranosyl, and α-l-arabinofuranosyl units were observed in the NMR spectra from hydrothermally pretreated wheat straw. The aromatic region indicated only minor changes to the aromatic structures during the process (e.g., further deacylation revealed by the depletion in ferulate and p-coumarate structures). Supplementary chemical analyses showed that the hydrothermal pretreatment increased the cellulose and lignin concentration with partial removal of extractives and hemicelluloses. The subsequent enzymatic hydrolysis incurred further deacetylation of the xylan, leaving approximately 10 % of acetate intact based on the weight of original wheat straw.


Wheat straw Hydrothermal Lignin Polysaccharides O-acetyls β-aryl ethers Uronic acids Cinnamates 



two-dimensional (solution state) nuclear magnetic resonance spectroscopy


heteronuclear single quantum coherence


β-d-xylopyranosyl units

2-O-Ac-β-d-Xylp, 3-O-Ac-β-d-Xylp

O-acetylated β-d-xylopyranosyl units


4-O-methyl-α-d-glucuronic acid units


β-d-mannopyranosyl units


α-l-arabinofuranosyl units


scanning electron microscope


  1. 1.
    Larsen J, Petersen MØ, Thirup L, Li HW, Iversen FK (2008) The IBUS process – lignocellulosic bioethanol close to a commercial reality. Chem Eng Technol 31(5):765–772CrossRefGoogle Scholar
  2. 2.
    Aspinall GO (1982) Isolation and fractionation of polysaccharides. In: Aspinall GO (ed) The Polysaccharides, vol 1. Academic, New York, pp 19–35Google Scholar
  3. 3.
    Lai YZ, Sarkanen KV (1971) Isolation and structural studies. In: Sarkanen KV, Ludwig CH (eds) Lignins. Occurrence, formation, structure and reactions. Wiley Interscience, New York, pp 165–240Google Scholar
  4. 4.
    Lu F, Ralph J (2003) Non-degradative dissolution and acetylation of ball-milled plant cell walls: high-resolution solution-state NMR. Plant J 35(4):535–544PubMedCrossRefGoogle Scholar
  5. 5.
    Forziati FH, Stone WK, Rowen JW, Appel WD (1950) Cotton powder for infrared transmission measurements. J Res Nat Bur Stand 45(2):109–113CrossRefGoogle Scholar
  6. 6.
    Schwanninger M, Rodrigues JC, Periera H, Hinterstoisser B (2004) Effects of short-time vibratory ball milling on the shape of FT-IR spectra of wood and cellulose. Vib Spectrosc 36:23–40CrossRefGoogle Scholar
  7. 7.
    Fujimoto A, Matsumoto Y, Chang HM, Meshitsuka G (2005) Quantitative evaluation of milling effects on lignin structure during the isolation process of milled wood lignin. J Wood Sci 51:89–91CrossRefGoogle Scholar
  8. 8.
    Ikeda T, Holtman K, Kadla JF, Chang HM, Jameel H (2002) Studies on the effect of ball milling on lignin structure using a modified DFRC method. J Agric Food Chem 50(1):129–135PubMedCrossRefGoogle Scholar
  9. 9.
    Yelle DJ, Ralph J, Frihart CR (2008) Characterization of nonderivatized plant cell walls using high-resolution solution-state NMR spectroscopy. Magn Reson Chem 46:508–517PubMedCrossRefGoogle Scholar
  10. 10.
    Kim H, Ralph J, Akiyama T (2008) Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6. Bioenerg Res 1:56–66CrossRefGoogle Scholar
  11. 11.
    Lapierre C (2011) Personal CommunicationGoogle Scholar
  12. 12.
    Lapierre C, Pollet B, Rolando C (1995) New insights into the molecular architecture of hardwood lignins by chemical degradative methods. Res Chem Intermed 21(3–5):397–412CrossRefGoogle Scholar
  13. 13.
    Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Ann Rev Plant Biol 54:519–546CrossRefGoogle Scholar
  14. 14.
    Crestini C, Argyropoulos DS (1997) Structural analysis of wheat straw lignin by quantitative 31P and 2D NMR spectroscopy. The occurrence of ester bonds and α-O-4 substructures. J Agr Food Chem 45:1212–1219CrossRefGoogle Scholar
  15. 15.
    Ralph J, Hatfield RD, Quideau S, Helm RF, Grabber JH, Jung HJG (1994) Pathway of p-coumaric acid incorporation into maize lignin as revealed by NMR. J Am Chem Soc 116(21):9448–9456CrossRefGoogle Scholar
  16. 16.
    Li J, Henriksson G, Gellerstedt G (2007) Lignin depolymerization/repolymerization and its critical role for delignification of aspen wood by steam explosion. Bioresour Technol 98:3061–3068PubMedCrossRefGoogle Scholar
  17. 17.
    Kaparaju P, Felby C (2010) Characterization of lignin during oxidative and hydrothermal pretreatment processes of wheat straw and corn stover. Bioresour Technol 101:3175–3181PubMedCrossRefGoogle Scholar
  18. 18.
    Buchala AJ, Wilkie KCB (1973) Total hemicelluloses from wheat at different stages of growth. Phytochemistry 12:499–505CrossRefGoogle Scholar
  19. 19.
    Garrote G, Domínguez H, Parajó JC (1999) Hydrothermal processing of lignocellulosic materials. Holz als Roh- und Werkst 57:191–202CrossRefGoogle Scholar
  20. 20.
    Scalbert A, Monties B, Lallemand J-Y, Guittet E, Rolando C (1985) Ether linkage between phenolic acids and lignin fractions from wheat straw. Phytochemistry 24(6):1359–1362CrossRefGoogle Scholar
  21. 21.
    Sjöström E (1993) Wood Chemistry: fundamentals and applications, 2nd edn. Academic Press, Inc., San DiegoGoogle Scholar
  22. 22.
    Fry SC, Miller JG (1989) Toward a working model of the growing plant cell wall: phenolic cross-linking reaction in the primary cell walls of dicotyledons. In: Lewis NG, Paice MG (eds) Plant cell wall polymers: biogenesis and biodegradation, vol ACS, Symposium Series 399. American Chemical Society, Washington, D.C., pp 33–46CrossRefGoogle Scholar
  23. 23.
    Ralph J (2010) Hydroxycinnamates in lignification. Phytochem Rev 9:65–83CrossRefGoogle Scholar
  24. 24.
    Ralph J, Grabber JH, Hatfield RD (1995) Lignin-ferulate cross-links in grasses – active incorporation of ferulate polysaccharide esters into ryegrass lignins. Carbohydr Res 275(1):167–178CrossRefGoogle Scholar
  25. 25.
    Ralph J, Quideau S, Grabber JH, Hatfield RD (1994) Identification and synthesis of new ferulic acid dehydrodimers present in grass cell-walls. J Chem Soc Perk T 1(23):3485–3498Google Scholar
  26. 26.
    Bunzel M, Heuermann B, Kim H, Ralph J (2008) Peroxidase-catalyzed oligomerization of ferulic acid esters. J Agr Food Chem 56:10368–10375CrossRefGoogle Scholar
  27. 27.
    Bunzel M, Ralph J, Funk C, Steinhart H (2003) Isolation and identification of a ferulic acid dehydrotrimer from saponified maize bran insoluble fiber. Eur Food Res Technol 217(2):128–133CrossRefGoogle Scholar
  28. 28.
    Quideau S, Ralph J (1997) Lignin-ferulate cross-links in grasses 4. Incorporation of 5-5-coupled dehydrodiferulate into synthetic lignin. J Chem Soc Perk T 1 1(16):2351–2358CrossRefGoogle Scholar
  29. 29.
    Hatfield RD, Ralph J, Grabber JH (1999) Cell wall cross-linking by ferulates and diferulates in grasses. J Sci Food Agr 79(3):403–407CrossRefGoogle Scholar
  30. 30.
    Mueller-Harvey I, Hartley RD, Harris PJ, Curzon EH (1986) Linkage of p-coumaryl and feruloyl groups to cell wall polysaccharides of barley straw. Carbohydr Res 148:71–85CrossRefGoogle Scholar
  31. 31.
    Bunzel M, Ralph J, Lu F, Hatfield RD, Steinhart H (2004) Lignins and ferulate-coniferyl alcohol cross-coupling products in cereal grains. J Agr Food Chem 52(21):6496–6502CrossRefGoogle Scholar
  32. 32.
    Grabber JH, Ralph J, Hatfield RD (2002) Model studies of ferulate-coniferyl alcohol cross-product formation in primary maize walls: implications for lignification in grasses. J Agr Food Chem 50(21):6008–6016CrossRefGoogle Scholar
  33. 33.
    Iiyama K, Lam TBT, Stone BA (1990) Phenolic acid bridges between polysaccharides and lignin in wheat internodes. Phytochemistry 29:733–737CrossRefGoogle Scholar
  34. 34.
    Jacquet B, Pollet B, Lapierre C, Mhamdi F, Rolando C (1995) New ether-linked ferulic acid-coniferyl alcohol dimers identified in grass straws. J Agr Food Chem 43:2746–2751CrossRefGoogle Scholar
  35. 35.
    Ralph J, Bunzel M, Marita JM, Hatfield R, Lu F, Kim H et al (2004) Peroxidase-dependent cross-linking reactions of p-hydroxycinnamates in plant cell walls. Phytochem Rev 3:79–96CrossRefGoogle Scholar
  36. 36.
    Bunzel M, Ralph J, Kim H, Lu FC, Ralph SA, Marita JM et al (2003) Sinapate dehydrodimers and sinapate-ferulate heterodimers in cereal dietary fiber. J Agr Food Chem 51(5):1427–1434CrossRefGoogle Scholar
  37. 37.
    Lu F, Ralph J (1999) Detection and determination of p-coumaroylated units in lignins. J Agr Food Chem 47(5):1988–1992CrossRefGoogle Scholar
  38. 38.
    Ralph J, Landucci L (2010) NMR of lignins. In: Heitner C, Dimmel DR, Schmidt JA (eds) Lignin and Lignans: advances in chemistry. CRC Press (Taylor & Francis Group), Boca Raton, pp 137–234CrossRefGoogle Scholar
  39. 39.
    Sluiter A (2004) Determination of structural carbohydrates and lignin in biomass. National Renewable Energy Laboratory (NREL) Analytical Procedures
  40. 40.
    Kristensen JB, Thygesen LG, Felby C, Jørgensen H, Elder T (2008) Cell-wall structural changes in wheat straw pretreated for bioethanol production. Biotechnol Biofuels 1(5):1–9Google Scholar
  41. 41.
    Ralph SA, Ralph J, Landucci LL (2004) NMR database of lignin and cell wall model compounds,
  42. 42.
    Kim H, Ralph J (2010) Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d6/pyridine-d5. Org Biomol Chem 8(3):576–591PubMedCrossRefGoogle Scholar
  43. 43.
    Sun XF, Sun R, Fowler P, Baird MS (2005) Extraction and characterization of original lignin and hemicelluloses from wheat straw. J Agr Food Chem 53:860–870CrossRefGoogle Scholar
  44. 44.
    Teleman A, Lundqvist J, Tjerneld F, Stalbrand H, Dahlman O (2000) Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy. Carbohydr Res 329(4):807–815PubMedCrossRefGoogle Scholar
  45. 45.
    Selig MJ, Viamajala S, Decker SR, Tucker MP, Himmel ME, Vinzant TB (2007) Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose. Biotechnol Prog 23:1333–1339PubMedCrossRefGoogle Scholar
  46. 46.
    Mosier NS, Hendrickson R, Brewer M, Ho N, Sedlak M, Dreshel R et al (2005) Industrial scale-up of pH-controlled liquid hot water pretreatment of corn fiber for fuel ethanol production. Appl Biochem Biotechnol 125:77–97PubMedCrossRefGoogle Scholar
  47. 47.
    Donohoe BS, Tucker MP, Davis M, Decker SR, Himmel ME, Vinzant TB (2007) Tracking lignin coalescence and migration through plant cell walls during pretreatment, vol 5B-01. 29th Symposium on Biotechnology for Fuels and Chemicals. Denver, COGoogle Scholar
  48. 48.
    Hansen MA, Kristensen JB, Felby C, Jørgensen H (2011) Pretreatment and enzymatic hydrolysis of wheat straw (Triticum aestivum L.) – the impact of lignin relocation and plant tissues on enzymatic accessibility. Bioresour Technol 102:2804–2811PubMedCrossRefGoogle Scholar
  49. 49.
    Kabel MA, Bos G, Zeevalking J, Voragen AGJ, Schols HA (2007) Effect of pretreatment severity on xylan solubility and enzymatic breakdown of the remaining cellulose from wheat straw. Bioresour Technol 98:2034–2042PubMedCrossRefGoogle Scholar
  50. 50.
    Grethlein HE (1985) The effect of pore size distribution on the rate of enzymatic hydrolysis of cellulosic substrates. Nat Biotechnol 3(2):155–160CrossRefGoogle Scholar
  51. 51.
    Chundawat SPS, Donohoe BS, da Costa SL, Elder T, Agarwal UP, Lu F et al (2011) Multi-scale visualization and characterization of lignocellulosic plant cell wall deconstruction during thermochemical pretreatment. Energy Environ Sci. doi:10.1039/c1030ee00574f
  52. 52.
    Han G, Deng J, Zhang S, Bicho P, Wu Q (2010) Effect of steam explosion treatment on characteristics of wheat straw. Ind Crop Prod 31(1):28–33CrossRefGoogle Scholar
  53. 53.
    Ralph J, Marita JM, Ralph SA, Hatfield R, Lu F, Ede RM et al (1999) Solution-state NMR of lignins. In: Argyropoulos DS, Rials T (eds) Advances in lignocellulosics characterization. TAPPI Press, Atlanta, pp 55–108Google Scholar
  54. 54.
    Akiyama T, Kim H, Dixon RA, Ralph J (2007) Dibenzodioxocin structures involving p-hydroxyphenyl units in C3H down-regulated lignin. In: 10th International Congress on Biotechnology in the Pulp and Paper Industry. Madison, WI, p 71Google Scholar
  55. 55.
    Ämmälahti E, Brunow G, Bardet M, Robert D, Kilpeläinen I (1998) Identification of side-chain structures in a poplar lignin using three-dimensional HMQC-HOHAHA NMR spectroscopy. J Agr Food Chem 46(12):5113–5117CrossRefGoogle Scholar
  56. 56.
    Karhunen P, Rummakko P, Sipilä J, Brunow G, Kilpeläinen I (1995) Dibenzodioxocins; a novel type of linkage in softwood lignins. Tetrahedron Lett 36:169–170CrossRefGoogle Scholar
  57. 57.
    Karhunen P, Rummakko P, Pujunen A, Brunow G (1996) Synthesis and crystal structure determination of model compounds for the dibenzodioxocine structure occurring in wood lignins. J Chem Soc Perk T 1(18):2303–2308Google Scholar
  58. 58.
    Stewart JJ, Akiyama T, Chapple C, Ralph J, Mansfield SD (2009) The effects on lignin structure of overexpression of ferulate 5-hydroxylase in hybrid poplar. Plant Physiol 150:621–635PubMedCrossRefGoogle Scholar
  59. 59.
    Wagner A, Donaldson L, Kim H, Phillips L, Flint H, Steward D et al (2009) Suppression of 4-coumarate-CoA ligase in the coniferous gymnosperm Pinus radiata. Plant Physiol 149(1):370–383PubMedCrossRefGoogle Scholar
  60. 60.
    Yelle DJ, Ralph J, Frihart CR (2011) Delineating pMDI model reactions with loblolly pine via solution-state NMR spectroscopy. Part 2. Non-catalyzed reactions with the wood cell wall. Holzforschung 65:145–154Google Scholar
  61. 61.
    Zhang LM, Gellerstedt G (2007) Quantitative 2D HSQC NMR determination of polymer structures by selecting suitable internal standard references. Magn Reson Chem 45:37–45PubMedCrossRefGoogle Scholar
  62. 62.
    Koskela H, Heikkilä O, Kilpeläinen I, Heikkinen S (2010) Quantitative two-dimensional HSQC experiment for high magnetic field NMR spectrometers. J Magn Reson 202:24–33PubMedCrossRefGoogle Scholar
  63. 63.
    Kupče E, Freeman R (2007) Compensated adiabatic inversion pulses: broadband INEPT and HSQC. J Magn Reson 187:258–265PubMedCrossRefGoogle Scholar
  64. 64.
    Lundquist K, Lundgren R (1972) Acid degradation of lignin. Part VII. The cleavage of ether bonds. Acta Chem Scand 26(5):2005–2023CrossRefGoogle Scholar
  65. 65.
    Reicher F, Corrêa JBC, Gorin PAJ (1984) Location of O-acetyl groups in the acidic d-xylan of Mimosa scabrella (bracatinga). A study of O-acetyl group migration. Carbohydr Res 135:129–140CrossRefGoogle Scholar
  66. 66.
    Çetinkol ÖP, Dibble DC, Cheng G, Kent MS, Knierim B, Auer M et al (2010) Understanding the impact of ionic liquid pretreatment on eucalyptus. Biofuels 1:33–46CrossRefGoogle Scholar
  67. 67.
    Ede RM, Brunow G, Poppius K, Sundquist J, Hortling B (1988) Formic acid/peroxyformic acid pulping. 1. Reactions of β-aryl ether model compounds with formic acid. Nord Pulp Pap Res J 3(3):119–123CrossRefGoogle Scholar
  68. 68.
    Nimz HH, Robert D (1985) 13C NMR spectra of acetic acid lignins. In: International Symposium on Wood and Pulping Chemistry. Vancouver, B.C., p 267Google Scholar
  69. 69.
    Sarkanen KV (1980) Acid catalyzed delignification of lignocellulosics in organic solvents. In: Sarkanen KV, Tillman DA (eds) Progress in biomass conversion, vol 2. Academic, New York, pp 127–144Google Scholar
  70. 70.
    Shimada K, Hosoya S, Tomimura Y (1991) Delignification with organic acids. In: International Symposium on Wood and Pulping Chemistry. TAPPI Press, Melbourne, pp 183–188Google Scholar

Copyright information

© Springer Science+Business Media, LLC (outside the USA) 2012

Authors and Affiliations

  • Daniel J. Yelle
    • 1
  • Prasad Kaparaju
    • 2
    • 4
  • Christopher G. Hunt
    • 1
  • Kolby Hirth
    • 1
  • Hoon Kim
    • 3
  • John Ralph
    • 3
  • Claus Felby
    • 2
  1. 1.U.S. Forest ServiceForest Products LaboratoryMadisonUSA
  2. 2.Forest & Landscape, Faculty of Life SciencesUniversity of CopenhagenFrederiksbergDenmark
  3. 3.Department of Biochemistry, DOE Great Lakes Bioenergy Research Center, and Wisconsin Bioenergy InitiativeUniversity of WisconsinMadisonUSA
  4. 4.Department of Biological and Environmental ScienceUniversity of JyväskyläJyväskyläFinland

Personalised recommendations