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Laccase-Mediator Pretreatment of Wheat Straw Degrades Lignin and Improves Saccharification

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Abstract

Agricultural by-products such as wheat straw are attractive feedstocks for the production of second-generation bioethanol due to their high abundance. However, the presence of lignin in these lignocellulosic materials hinders the enzymatic hydrolysis of cellulose. The purposes of this work are to study the ability of a laccase-mediator system to remove lignin improving saccharification, as a pretreatment of wheat straw, and to analyze the chemical modifications produced in the remaining lignin moiety. Up to 48 % lignin removal from ground wheat straw was attained by pretreatment with Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) as mediator, followed by alkaline peroxide extraction. The lignin removal directly correlated with increases (∼60 %) in glucose yields after enzymatic saccharification. The pretreatment using laccase alone (without mediator) removed up to 18 % of lignin from wheat straw. Substantial lignin removal (37 %) was also produced when the enzyme-mediator pretreatment was not combined with the alkaline peroxide extraction. Two-dimensional nuclear magnetic resonance (2D NMR) analysis of the whole pretreated wheat straw material swollen in dimethylsulfoxide-d 6 revealed modifications of the lignin polymer, including the lower number of aliphatic side chains involved in main β-O-4′ and β-5′ inter-unit linkages per aromatic lignin unit. Simultaneously, the removal of p-hydroxyphenyl, guaiacyl, and syringyl lignin units and of p-coumaric and ferulic acids, as well as a moderate decrease of tricin units, was observed without a substantial change in the wood polysaccharide signals. Especially noteworthy was the formation of Cα-oxidized lignin units during the enzymatic treatment.

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Acknowledgments

This study was funded by the INDOX EU-project (KBBE-2013-7-613549); the LIGNOCELL, LIGNIN, NOESIS, and BIORENZYMERY Spanish MICINN (co-financed by FEDER funds) projects (AGL2011-25379, CTQ2014-60764-JIN, BIO2014-56388 R and AGL2014-53730-R); and the CSIC (201440E097) Project. A.P. thanks the Spanish MINECO for a FPI fellowship. A. Lomascolo and E. Record from INRA (Marseille, France) are acknowledged for the P. cinnabarinus laccase, and H. Lund and M. Tovborg from Novozymes (Bagsvaerd, Denmark) for Celluclast 1.5L and Novozyme 188.. The authors thank Dr. Angulo for performing the NMR analyses that were acquired on a Bruker AVANCE III 500-MHz instrument from the NMR facilities of the General Research Services of the University of Seville (SGI CITIUS).

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    1. Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Reina Mercedes 10, 41012, Seville, Spain

      Jorge Rencoret, Antonio Pereira, José C. del Río & Ana Gutiérrez

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

      Angel T. Martínez

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    1. Jorge Rencoret
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    Correspondence to Ana Gutiérrez.

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    Jorge Rencoret and Antonio Pereira contributed equally to this work.

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    Rencoret, J., Pereira, A., del Río, J.C. et al. Laccase-Mediator Pretreatment of Wheat Straw Degrades Lignin and Improves Saccharification. Bioenerg. Res. 9, 917–930 (2016). https://doi.org/10.1007/s12155-016-9745-z

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