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Analysis of lignin–carbohydrate and lignin–lignin linkages after hydrolase treatment of xylan–lignin, glucomannan–lignin and glucan–lignin complexes from spruce wood

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Abstract

Xylan–lignin (XL), glucomannan–lignin (GML) and glucan–lignin (GL) complexes were isolated from spruce wood, hydrolyzed with xylanase or endoglucanase/β-glucosidase, and analyzed by analytical pyrolysis and 2D-NMR. The enzymatic hydrolysis removed most of the polysaccharide moieties in the complexes, and the lignin content and relative abundance of lignin–carbohydrate linkages increased. Analytical pyrolysis confirmed the action of the enzymatic hydrolysis, with strong decreases of levoglucosane and other carbohydrate-derived products. Unexpectedly it also revealed that the hydrolase treatment alters the pattern of lignin breakdown products, resulting in higher amounts of coniferyl alcohol. From the anomeric carbohydrate signals in the 2D-NMR spectra, phenyl glycoside linkages (undetectable in the original complexes) could be identified in the hydrolyzed GML complex. Lower amounts of glucuronosyl and benzyl ether linkages were also observed after the hydrolysis. From the 2D-NMR spectra of the hydrolyzed complexes, it was concluded that the lignin in GML is less condensed than in XL due to its higher content in β-O-4′ ether substructures (62 % of side chains in GML vs 53 % in XL) accompanied by more coniferyl alcohol end units (16 vs 13 %). In contrast, the XL lignin has more pinoresinols (11 vs 6 %) and dibenzodioxocins (9 vs 2 %) than the GML (and both have ~13 % phenylcoumarans and 1 % spirodienones). Direct 2D-NMR analysis of the hydrolyzed GL complex was not possible due to its low solubility. However, after sample acetylation, an even less condensed lignin than in the GML complex was found (with up to 72 % β-O-4′ substructures and only 1 % pinoresinols). The study provides evidence for the existence of structurally different lignins associated to hemicelluloses (xylan and glucomannan) and cellulose in spruce wood and, at the same time, offers information on some of the chemical linkages between the above polymers.

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Abbreviations

GL:

Glucan–lignin

GML:

Glucomannan–lignin

XL:

Xylan–lignin

LCC:

Lignin–carbohydrate complex

Py-GC/MS:

Pyrolysis–gas chromatography/mass spectrometry

2D-NMR:

Two-dimensional nuclear magnetic resonance spectroscopy

HSQC:

Heteronuclear single quantum coherence

MWL:

Milled wood lignin

Ara:

Arabinose

Xyl:

Xylose

Man:

Mannose

Gal:

Galactose

Glc:

Glucose

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Acknowledgments

This study was supported by the EU projects LIGNODECO (KBBE-2009-3-244362), the ProLignin project of the WoodWisdom-Bioenergy ERA-Net, and the Spanish project AGL2011-25379. J.R. thanks the CSIC for a JAE-DOC contract, cofinanced by Fondo Social Europeo. Alicia Prieto (CIB, Madrid) is acknowledged for the monosaccharide analysis of the enzymatically treated LCCs.

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Authors and Affiliations

  1. Department of Fibre and Polymer Technology, Royal Institute of Technology, KTH, Teknikringen 56-58, 10044, Stockholm, Sweden

    Xueyu Du & Jiebing Li

  2. Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain

    Marta Pérez-Boada, Carmen Fernández & Angel T. Martínez

  3. Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, PO Box 1052, 41080, Seville, Spain

    Jorge Rencoret, José C. del Río, Jesús Jiménez-Barbero & Ana Gutiérrez

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  1. Xueyu Du
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  2. Marta Pérez-Boada
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  3. Carmen Fernández
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  4. Jorge Rencoret
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  5. José C. del Río
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  7. Jiebing Li
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  9. Angel T. Martínez
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Correspondence to Jiebing Li.

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Du, X., Pérez-Boada, M., Fernández, C. et al. Analysis of lignin–carbohydrate and lignin–lignin linkages after hydrolase treatment of xylan–lignin, glucomannan–lignin and glucan–lignin complexes from spruce wood. Planta 239, 1079–1090 (2014). https://doi.org/10.1007/s00425-014-2037-y

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